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Sample records for h4iie transfectant insensitive

  1. Evaluation of antioxidant defense systems in H4IIE cells infected with a retroviral vector.

    PubMed

    Oh, Soo Jin; Chae, Jooyoung; Zhu, Hongmei; Hien, Tran Thi; Lee, Kiho; Kim, Hwan Mook; Kang, Keon Wook; Song, Gyu Yong; Kang, Jong Seong; Kim, Bong-Hee; Kwon, Kwang-il; Kim, Sang Kyum

    2010-06-01

    Retroviral gene transfer technology is frequently used to establish stable transgenic cell lines. However, no studies to date have evaluated antioxidant defense systems in cells infected with retroviral particles. In the present study, we examined the effects of retroviral infection on antioxidant defense systems using H4IIE cells infected with a retrovirus that overexpresses green fluorescent protein (retro-H4IIE cells). Total oxyradical scavenging capacity and glutathione (GSH), malondialdehyde, and peroxide levels were not significantly altered in retro-H4IIE cells; however, retro-H4IIE cells showed a higher resistance against cytotoxicity, GSH depletion, and malondialdehyde elevation under H(2)O(2)-induced oxidative stress conditions. Immunoblot analysis showed that alpha-class GSH S-transferase (GST) was increased 2.5-fold in retro-H4IIE cells as compared with H4IIE cells; however, catalase, GSH peroxidase-1, peroxiredoxin-1, and thioredoxin-1 remained unaltered or slightly decreased. l-Buthionine-(S,R)-sulfoximine, a GSH synthesis inhibitor, and 1-chloro-2,4-dinitrobenzene, a GST substrate and competitive inhibitor, decreased the difference in H(2)O(2) responses between the two cell types. These results support the hypothesis that the resistance of retro-H4IIE cells to H(2)O(2) can be attributed to an increase in alpha-class GST expression, as levels of GSH and GSH peroxidase-1 were not altered. The present study suggests that antioxidant enzyme expression may change during the establishment of stable transformed cell lines using retroviral techniques.

  2. The phytoestrogen daidzein affects the antioxidant enzyme system of rat hepatoma H4IIE cells.

    PubMed

    Röhrdanz, Elke; Ohler, Sandra; Tran-Thi, Quynh-Hoa; Kahl, Regine

    2002-03-01

    Phytoestrogens such as the soy isoflavonoid daidzein have potential health benefits. The antioxidant properties of phytoestrogens are considered to be responsible in part for their protective effects. The antioxidant enzyme (AOE) system plays an important role in the defense of cells against oxidative insults. To determine whether flavonoids can exert antioxidative effects not only directly but also indirectly by modulating the AOE system, we investigated the influence of the flavonoid daidzein on the expression of different AOE. Daidzein treatment of hepatoma H4IIE cells increased catalase mRNA expression two- to threefold. Expression levels of copper zinc superoxide dismutase (CuZnSOD) were not affected by exposure to daidzein. Manganese superoxide dismutase (MnSOD) mRNA expression levels decreased slightly and glutathione peroxidase (GPx) levels increased slightly after daidzein exposure. Changes in AOE mRNA expression levels were significant at 300 micromol/L daidzein. To elucidate the mechanisms underlying the strong increase in catalase mRNA, transfection experiments were performed. Transient transfection of hepatoma cells with reporter plasmids containing different parts of the upstream region of the catalase gene showed a significant one- to threefold increase in reporter gene activity after daidzein exposure. This indicates that daidzein can directly activate the rat catalase promoter region. Despite the increase in catalase mRNA, daidzein pretreatment of cells did not protect against oxidative stress resulting from H(2)O(2) exposure. On the contrary, daidzein itself exerted a mild oxidative stress. In conclusion, the changes in the AOE system provoked by daidzein affected the oxidant rather than the antioxidant properties of daidzein.

  3. Osmoregulated taurine transport in H4IIE hepatoma cells and perfused rat liver.

    PubMed Central

    Warskulat, U; Wettstein, M; Häussinger, D

    1997-01-01

    The effects of aniso-osmotic exposure on taurine transport were studied in H4IIE rat hepatoma cells. Hyperosmotic (405 mosmol/l) exposure of H4IIE cells stimulated Na+-dependent taurine uptake and led to an increase in taurine transporter (TAUT) mRNA levels, whereas hypo-osmotic (205 mosmol/l) exposure diminished both taurine uptake and TAUT mRNA levels when compared with normo-osmotic (305 mosmol/l) control incubations. Taurine uptake increased 30-40-fold upon raising the ambient osmolarity from 205 to 405 mosmol/l. When H4IIE cells and perfused livers were preloaded with taurine, hypo-osmotic cell swelling led to a rapid release of taurine from the cells. The taurine efflux, but not taurine uptake, was sensitive to 4,4'-di-isothiocyanatostilbene-2,2'-disulphonic acid (DIDS), suggestive of an involvement of DIDS-sensitive channels in mediating volume-regulatory taurine efflux. Whereas in both H4IIE rat hepatoma cells and primary hepatocytes TAUT mRNA levels were strongly dependent upon ambient osmolarity, mRNAs for other osmolyte transporters, i.e. the betaine transporter BGT-1 and the Na+/myo-inositol transporter SMIT, were not detectable. In line with this, myo-inositol uptake by H4IIE hepatoma cells was low and was not stimulated by hyperosmolarity. However, despite the absence of BGT-1 mRNA, a slight osmosensitive uptake of betaine was observed, but the rate was less than 10% of that of taurine transport. This study identifies a constitutively expressed and osmosensitive TAUT in H4IIE cells and the use of taurine as a main osmolyte, whereas betaine and myo-inositol play little or no role in the osmolyte strategy in these cells. This is in contrast with rat liver macrophages, in which betaine has been shown to be a major osmolyte. PMID:9032454

  4. The H4IIE cell bioassay as an indicator of dioxin-like chemicals in wildlife and the environment.

    PubMed

    Whyte, J J; Schmitt, C J; Tillitt, D E

    2004-01-01

    The H4IIE cell bioassay has proven utility as a screening tool for planar halogenated hydrocarbons (PHHs) and structurally similar chemicals accumulated in organisms from the wild. This bioassay has additional applications in hazard assessment of PHH exposed populations. In this review, the toxicological principles, current protocols, performance criteria, and field applications for the assay are described. The H4IIE cell bioassay has several advantages over the analytical measurement of PHHs in environmental samples, but conclusions from studies can be strengthened when both bioassay and analytical chemistry data are presented together. Often, the bioassay results concur with biological effects in organisms and support direct measures of PHHs. For biomonitoring purposes and prioritization of PHH-contaminated environments, the H4IIE bioassay may be faster and less expensive than analytical measurements. The H4IIE cell bioassay can be used in combination with other biomarkers such as in vivo measurements of CYP1A1 induction to help pinpoint the sources and identities of dioxin-like chemicals. The number of studies that measure H4IIE-derived TCDD-EQs continues to increase, resulting in subtle improvements over time. Further experiments are required to determine if TCDD-EQs derived from mammalian cells are adequate predictors of toxicity to non-mammalian species. The H4IIE cell bioassay has been used in over 300 published studies, and its combination of speed, simplicity, and ability to integrate the effects of complex contaminant mixtures makes it a valuable addition to hazard assessment and biomonitoring studies.

  5. Pancreatic Transdifferentiation and Glucose-Regulated Production of Human Insulin in the H4IIE Rat Liver Cell Line

    PubMed Central

    Ren, Binhai; Tao, Chang; Swan, Margaret Anne; Joachim, Nichole; Martiniello-Wilks, Rosetta; Nassif, Najah T.; O’Brien, Bronwyn A.; Simpson, Ann M.

    2016-01-01

    Due to the limitations of current treatment regimes, gene therapy is a promising strategy being explored to correct blood glucose concentrations in diabetic patients. In the current study, we used a retroviral vector to deliver either the human insulin gene alone, the rat NeuroD1 gene alone, or the human insulin gene and rat NeuroD1 genes together, to the rat liver cell line, H4IIE, to determine if storage of insulin and pancreatic transdifferentiation occurred. Stable clones were selected and expanded into cell lines: H4IIEins (insulin gene alone), H4IIE/ND (NeuroD1 gene alone), and H4IIEins/ND (insulin and NeuroD1 genes). The H4IIEins cells did not store insulin; however, H4IIE/ND and H4IIEins/ND cells stored 65.5 ± 5.6 and 1475.4 ± 171.8 pmol/insulin/5 × 106 cells, respectively. Additionally, several β cell transcription factors and pancreatic hormones were expressed in both H4IIE/ND and H4IIEins/ND cells. Electron microscopy revealed insulin storage vesicles in the H4IIE/ND and H4IIEins/ND cell lines. Regulated secretion of insulin to glucose (0–20 mmol/L) was seen in the H4IIEins/ND cell line. The H4IIEins/ND cells were transplanted into diabetic immunoincompetent mice, resulting in normalization of blood glucose. This data shows that the expression of NeuroD1 and insulin in liver cells may be a useful strategy for inducing islet neogenesis and reversing diabetes. PMID:27070593

  6. Pancreatic Transdifferentiation and Glucose-Regulated Production of Human Insulin in the H4IIE Rat Liver Cell Line.

    PubMed

    Ren, Binhai; Tao, Chang; Swan, Margaret Anne; Joachim, Nichole; Martiniello-Wilks, Rosetta; Nassif, Najah T; O'Brien, Bronwyn A; Simpson, Ann M

    2016-04-08

    Due to the limitations of current treatment regimes, gene therapy is a promising strategy being explored to correct blood glucose concentrations in diabetic patients. In the current study, we used a retroviral vector to deliver either the human insulin gene alone, the rat NeuroD1 gene alone, or the human insulin gene and rat NeuroD1 genes together, to the rat liver cell line, H4IIE, to determine if storage of insulin and pancreatic transdifferentiation occurred. Stable clones were selected and expanded into cell lines: H4IIEins (insulin gene alone), H4IIE/ND (NeuroD1 gene alone), and H4IIEins/ND (insulin and NeuroD1 genes). The H4IIEins cells did not store insulin; however, H4IIE/ND and H4IIEins/ND cells stored 65.5 ± 5.6 and 1475.4 ± 171.8 pmol/insulin/5 × 10⁶ cells, respectively. Additionally, several β cell transcription factors and pancreatic hormones were expressed in both H4IIE/ND and H4IIEins/ND cells. Electron microscopy revealed insulin storage vesicles in the H4IIE/ND and H4IIEins/ND cell lines. Regulated secretion of insulin to glucose (0-20 mmol/L) was seen in the H4IIEins/ND cell line. The H4IIEins/ND cells were transplanted into diabetic immunoincompetent mice, resulting in normalization of blood glucose. This data shows that the expression of NeuroD1 and insulin in liver cells may be a useful strategy for inducing islet neogenesis and reversing diabetes.

  7. Nitroreduction of 2,4-dinitrotoluene in vitro by cytochrome P-450 induced H4IIE cells.

    PubMed

    Mitchell, W R; Burrows, E P

    1995-07-01

    Conditions have been established for H4IIE rat hepatoma cell cultures in which effects of cytochrome P-450 induction on the metabolism of a munitions wastestream pollutant can be studied. Under these conditions, the polychlorinated hydrocarbon 2,3,4,7,8-pentachlorodibenzfuran (PCDBF) induced cytochrome P-450 (1A1) aryl hydrocarbon hydroxylase (AHH) activity over a wide range of concentrations without significant cytotoxic effects. The munition pollutant 2,4-dinitrotoluene (2,4-DNT) did not induce AHH activity itself, but its metabolism was considerably altered when applied to PCDBF induced cultures. Production of amino nitrotoluene isomers was greatly enhanced in induced cultures as compared to uninduced controls, as was the conversion of radiolabeled 2,4-DNT to relatively more polar metabolites. To some extent, the results with H4IIE cells parallel those reported for animals exposed to 2,4-DNT after induction of cytochrome P-450 AHH activity. The preliminary findings suggest that with further development and validation, H4IIE cultures could be of use in characterizing metabolites that result from exposure to chemical mixtures involving a P-450 (1A1) inducer.

  8. Resveratrol induces apoptotic cell death in rat H4IIE hepatoma cells but necrosis in C6 glioma cells.

    PubMed

    Michels, G; Wätjen, W; Weber, N; Niering, P; Chovolou, Y; Kampkötter, A; Proksch, P; Kahl, R

    2006-08-15

    Resveratrol (trans-3,5,4',-trihydroxystilbene) is assumed to possess cancer-preventive and cancer-therapeutic properties. The aim of this project was to analyze cellular effects of resveratrol in metabolically active H4IIE rat hepatoma cells in comparison to metabolically poorly active C6 rat glioma cells. Resveratrol is rapidly taken up by both cell types and acts as a potent intracellular antioxidant. On the other hand, resveratrol in higher concentrations is relatively toxic to both cell lines as measured by the neutral red accumulation assay. In H4IIE cells, resveratrol concentrations rapidly decline to very low levels during the first hours of incubation due to formation of resveratrol glucuronides. The first resveratrol effect found at 3h after the start of resveratrol treatment was the induction of mild DNA damage as detected by the comet assay. Cell death was caused via induction of apoptosis as detected by caspase activation, oligonucleosomal DNA fragmentation and formation of apoptotic nuclei. Following DNA damage, resveratrol led to an activation of caspases 2 and 8/10 at 6h and consequently of caspase 3 at 12h, but failed to activate caspase 9. In contrast to H4IIE cells, resveratrol is not metabolised in C6 glioma cells and accumulates to concentrations which are assumed to drive the cell into necrosis. This suggests that the mode of cell death caused by resveratrol and the usefulness of resveratrol for cancer prevention and treatment critically depends on the metabolic capacity of the tumor cell to be eradicated.

  9. Chenodeoxycholic acid increases the induction of CYP1A1 in HepG2 and H4IIE cells

    PubMed Central

    IBRAHIM, ZEIN SHABAN

    2015-01-01

    Bile acids are considered to promote carcinogenesis. Cytochrome P450 1A1 (CYP1A1) plays a critical role in the biotransformation of drugs and procarcinogens. This study aimed to investigate the ability of bile acids to modulate CYP1A1 expression. Treatment of HepG2 cells with chenodeoxycholic acid (CDCA) and Sudan III (S.III) upregulated CYP1A1 transcriptional activity in HepG2 cells and CYP1A1 mRNA expression in H4IIE cells. Pretreatment of the HepG2 and H4IIE cells with CDCA upregulated the S.III-induced CYP1A transcriptional activity and mRNA expression. The CDCA-induced enhancement of CYP1A1 was not abolished by the p38 inhibitor SB203580. However, exposure of the cells to the mitogen-activated protein kinase kinase (MEK)1/2 inhibitor PD98059 suppressed the CDCA-induced enhancement of CYP1A1. These results show the ability of CDCA to upregulate CYP1A1 transcription and expression, which may explain the hepatocarcinogenesis-inducing effect of cholestasis. The CDCA-induced upregulation of CYP1A1 most probably proceeded through MEK1/2 activation, indicating that this may be a therapeutic target to prevent the cancer-promoting effects of excessive amounts of bile acids. PMID:26640583

  10. Protective and detrimental effects of kaempferol in rat H4IIE cells: Implication of oxidative stress and apoptosis

    SciTech Connect

    Niering, Petra; Michels, Gudrun; Waetjen, Wim . E-mail: wim.waetjen@uni-duesseldorf.de; Ohler, Sandra; Steffan, Baerbel; Chovolou, Yvonni; Kampkoetter, Andreas; Proksch, Peter; Kahl, Regine

    2005-12-01

    Flavonoids are ubiquitous substances in fruits and vegetables. Among them, the flavonol kaempferol contributes up to 30% of total dietary flavonoid intake. Flavonoids are assumed to exert beneficial effects on human health, e.g., anticancer properties. For this reason, they are used in food supplements at high doses. The aim of this project was to determine the effects of kaempferol on oxidative stress and apoptosis in H4IIE rat hepatoma cells over a broad concentration range. Kaempferol is rapidly taken up and glucuronidated by H4IIE cells. The results demonstrate that kaempferol protects against H{sub 2}O{sub 2}-induced cellular damage at concentrations which lead to cell death and DNA strand breaks in the absence of H{sub 2}O{sub 2}-mediated oxidative stress. Preincubation with 50 {mu}M kaempferol exerts protection against the loss of cell viability induced by 500 {mu}M H{sub 2}O{sub 2} (2 h) while the same concentration of kaempferol reduces cell viability by 50% in the absence of H{sub 2}O{sub 2} (24 h). Preincubation with 50 {mu}M kaempferol ameliorates the strong DNA damage induced by 500 {mu}M H{sub 2}O{sub 2} while 50 {mu}M kaempferol leads to a significant increase of DNA breakage in the absence of H{sub 2}O{sub 2}. Preincubation with 50 {mu}M kaempferol reduces H{sub 2}O{sub 2}-mediated caspase-3 activity by 40% (4 h) while the same concentration of kaempferol leads to the formation of a DNA ladder in the absence of H{sub 2}O{sub 2} (24 h). It is concluded that the intake of high dose kaempferol in food supplements may not be advisable because in our cellular model protective kaempferol concentrations can also induce DNA damage and apoptosis by themselves.

  11. Carrier effects of dosing the H4IIE cells with 3,3',4,4'-tetrachlorobiphenyl (PCB77) in dimethyl sulfoxide or isooctane.

    PubMed

    Yu, K O; Fisher, J W; Burton, G A; Tillitt, D E

    1997-08-01

    A rat hepatoma cell line, H4IIE, serves as a bioassay tool to assess the potential toxicity of dioxin-like chemicals, including polychlorinated biphenyls (PCB) in environmental samples. PCB exposure to these cells induces cytochrome (CYP) P4501A1 activity in a dose-dependent fashion, thus allowing assessment of mixtures. The objective of this study was to determine the effect of different carriers, dimethyl sulfoxide (DMSO) and isooctane on the concentrations of PCBs in the H4IIE cells and induction of CYP1A1 activity as measured by ethoxyresorufin O-deethylase (EROD) activity. H4IIE cells were dosed with three micrograms of UL-14C-PCB77/plate dissolved in DMSO or isooctane, and were harvested at sequential time periods for 4 days. PCB77 concentration and EROD activity were measured in the cells. EROD activity was greater when using DMSO as compared to isooctane, while there was no difference in the distribution of PCB77-derived radioactivities within the cell culture system based upon the carrier solvent used to deliver PCB77.

  12. Relative induction potency of benzo(a)pyrene and dibenzo(a,h) anthracene in environmental samples using the H4IIE cell bioassay

    SciTech Connect

    Willett, K.; Gardinali, P.; Sericano, J.; Wade, T.; Safe, S.

    1995-12-31

    The induction of ethoxyresorufin O-deethylase (EROD) activity in H4IIE cells has proven to be a useful assay for assessing the toxic equivalents of complex mixtures of halogenated aromatic hydrocarbons and related compounds in environmental samples. However, there is often a discrepancy between bioassay induction results and toxic equivalents calculated from chemical analysis of samples; the former yielding higher I-TEQs (induction/toxic equivalents). Polynuclear aromatic hydrocarbons (PAHs) are a class of chemicals which can significantly contribute to I-TEQ estimates. Benzo(a)pyrene (BAP) and dibenzo(a,h)anthracene (DBA) are both carcinogenic PAHs found in environmental samples, specifically oysters (Crassostrea virginica) collected from Galveston Bay. Dose-response studies were conducted with H4IIE cells to determine the relative induction potency of these two PAHs relative to 2,3,7,8-TCDD. Using probit analysis, the I-TEFs were 0.00144 and 0.0095 for BAP and DBA, respectively. Reconstituted PAH mixtures (744 to 4,466 ng/g total PAHs) with constant percentages of BAP (4.5%) and DEA (3.5%) yielded bioassay derived I-TEQs which ranged from 0.52 to 1.44 ng/g. Oysters treated with the same PAH mix for 30 days differentially accumulated the DBA and BAP over the time course. The bioassay derived I-TEQs of extracts from these oysters ranged from 0.99 to 6.14 ng/g whereas the calculated values using only the I-TEQs of BAP and BDA ranged from 0.04 to 0.57 accounting for only 5--10% of the observed induction response. These results indicate that presence of other PAHs in the treated oyster extracts are highly active as inducers of EROD activity in the H4IIE bioassay.

  13. Carrier effects of dosing the h4iie cells with 3,3′,4,4tt´etrachlorobiphenyl (PCB77) in dimethyl sulfoxide or isooctane

    USGS Publications Warehouse

    Yu, Kyung O.; Fisher, Jeff W.; Burton, G. Allen; Tillitt, Donald E.

    1997-01-01

    A rat hepatoma cell line, H4IIE serves as a bioassay tool to assess the potential toxicity of dioxin-like chemicals, including polychlorinated biphenyls (PCB) in environmental samples. PCB exposure to these cells induces cytochrome (CYP) P4501A1 activity in a dose-dependent fashion, thus allowing assessment of mixtures. The objective of this study was to determine the effect of different carriers, dimethyl sulfoxide (DMSO) and isooctane on the concentrations of PCBs in the H411E cells and induction of CYPIA1 activity as measured by ethoxyresorufm O-deethylase (EROD) activity. H4IIE cells were dosed with three micrograms of UL-14C-PCB77/ plate dissolved in DMSO or isooctane, and were harvested at sequential time periods for 4 days. PCB77 concentration and EROD activity were measured in the cells. EROD activity was greater when using DMSO as compared to isooctane, while there was no difference in the distribution of PCB77-derived radioactivities within the cell culture system based upon the carrier solvent used to deliver PCB77.

  14. Evaluation of planar halogenated and polycyclic aromatic hydrocarbons in estuarine sediments using ethoxyresorufin-O-deethylase induction of H4IIE cells

    USGS Publications Warehouse

    Gale, R.W.; Long, E.R.; Schwartz, T.R.; Tillitt, D.E.

    2000-01-01

    Polycyclic aromatic hydrocarbons (PAHs) and planar halogenated hydrocarbons (PHHs), including polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and biphenyls (PCBs) were determined in fractionated sediment extracts from the Hudson-Raritan estuary and Newark Bay, New Jersey, USA, as part of a comprehensive risk assessment. Contributions of PCDDs/PCDFs, PCBs, and PAHs to the total toxic equivalents (TEQs) were measured using an H4IIE bioassay and calculated from instrumentally determined concentrations using international toxic equivalency factors. The H4IIE TEQs of whole and fractionated extracts were compared to calculated TEQs to investigate the applicability of the bioassay approach for evaluating 7-ethoxyresorufin-O-deethylease induction by PHHs and PAHs present together in complex mixtures. Although 2,3,7,8-tetrachlorodibenzo-p-dioxin contributed from 41 to 79% of the calculated TEQs from PCDDs/PCDFs and planar PCBs in all sediments sampled, the PAH-containing fractions accounted for >80% of the total TEQs determined either instrumentally or by bioassay. Calculated TEQs from PAHs, based on reported toxic equivalency factors for only seven PAHs, were severalfold greater than the bioassay-derived TEQs of PAH-only fractions of the sediment extracts. Significant correlations were observed between bioassay and instrumentally determined toxic equivalents in the more purified fractions but not in fractions only purified by size-exclusion or argentate chromatographies alone.

  15. Effects of oolong tea on gene expression of gluconeogenic enzymes in the mouse liver and in rat hepatoma H4IIE cells.

    PubMed

    Yasui, Kensuke; Miyoshi, Noriyuki; Tababe, Hiroki; Ishigami, Yoko; Fukutomi, Ryuuta; Imai, Shinjiro; Isemura, Mamoru

    2011-09-01

    Tea has many beneficial effects. We have previously reported that green tea and a catechin-rich green tea beverage modulated the gene expression of the gluconeogenic enzymes glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) in the normal murine liver. In the present study, we examined the effects of oral administration of oolong tea on the hepatic expression of gluconeogenesis-related genes in the mouse. The intake of oolong tea for 4 weeks reduced the hepatic expression of G6Pase and PEPCK together with that of the transcription factor hepatocyte nuclear factor (HNF) 4α. When rat hepatoma H4IIE cells were incubated in the presence of oolong tea, the expression of these genes was repressed in accordance with the findings in vivo. The reduced protein expression of PEPCK and HNF4α was also demonstrated. We then fractionated oolong tea by sequential extraction with three organic solvents to give three fractions and the residual fraction (Fraction IV). In addition to organic fractions, Fraction IV, which was devoid of low-molecular-weight catechins such as (-)-epigallocatechin gallate (EGCG), had effects similar to those of oolong tea on H4IIE cells. Fraction IV repressed the gene expression of insulin-like growth factor binding protein 1, as insulin did. This activity was different from that of EGCG. The present findings suggest that drinking oolong tea may help to prevent diabetes and that oolong tea contains a component or components with insulin-like activity distinguishable from EGCG. Identification of such component(s) may open the way to developing a new drug for diabetes.

  16. Development of stably transfected human and rat hepatoma cell lines for the species-specific assessment of xenobiotic response enhancer module (XREM)-dependent induction of drug metabolism.

    PubMed

    Fery, Yvonne; Mueller, Stefan O; Schrenk, Dieter

    2010-11-09

    Based on our current knowledge, PXR holds a key position in the induction of a selective battery of enzymes and transporters of drug metabolism. In order to prevent serious adverse drug effects or unpredicted drug-drug interactions (DDI), it is compulsory to investigate the possible inducing potency of drugs under development. Furthermore, analysis of the inducing potency of environmental pollutants and new or manufactured chemicals is part of toxicological risk assessment. In non-transfected human HepG2 and rat H4IIE hepatoma cells, we examined the characteristics of expression of 45 genes involved in drug metabolism. A few gene products such as CYP2B6 or CYP3A4 mRNA were prominent in HepG2 cells while their major rat counterparts were, e.g., CYP2B3 or CYP3A1/3A3. Furthermore, a number of xenobiotic receptors including PXR were expressed in both cell lines. A number of genes were regulated in a cell type and species-specific manner after incubation with the prototypical PXR agonists rifampicin or dexamethasone, respectively. Then, we established cell-based reporter gene assays for screening for PXR-dependent induction of drug metabolism. HepG2 and H4IIE cells were stably transfected with a reporter gene containing PXR responsive elements (XREMs) which mediate the induction of PXR target genes such as CYP3A enzymes. With both stable cell lines the CYP inducers clotrimazole, dexamethasone, omeprazole, phenobarbital, rifampicin, as well as the drug candidate EMD 392949 and the brominated flame retardants hexabromocylododecane (HBCD) and a pentabromodiphenyl ether (pentaBDE) mixture were screened. In the human HepG2-XREM3 and rat H4IIE-XREM3 cells, clotrimazole and HBCD were found as common activators of the human and rat PXR whereas pentaBDE was more effective with the human cell system. Omeprazole and phenobarbital did not induce the rat PXR-dependent reporter gene expression in H4IIE-XREM3 cells, while a moderate increase was found in HepG2-XREM3 cells. EMD 392949

  17. Insensitive HE EOS

    SciTech Connect

    Ree, F.H.; Viecelli, J.; van Thiel, M.

    1997-12-01

    A typical insensitive high explosive such as LX-17 has a large carbon-content and produces hydrogen fluoride (HF) as a detonation product. It is also characterized by slow energy release as indicated by a large curvature of the detonation front. We analyze these new physics issues which are needed to predict the performance of a insensitive high explosive. (U)

  18. Fibre based cellular transfection.

    PubMed

    Tsampoula, X; Taguchi, K; Cizmár, T; Garces-Chavez, V; Ma, N; Mohanty, S; Mohanty, K; Gunn-Moore, F; Dholakia, K

    2008-10-13

    Optically assisted transfection is emerging as a powerful and versatile method for the delivery of foreign therapeutic agents to cells at will. In particular the use of ultrashort pulse lasers has proved an important route to transiently permeating the cell membrane through a multiphoton process. Though optical transfection has been gaining wider usage to date, all incarnations of this technique have employed free space light beams. In this paper we demonstrate the first system to use fibre delivery for the optical transfection of cells. We engineer a standard optical fibre to generate an axicon tip with an enhanced intensity of the remote output field that delivers ultrashort (~ 800 fs) pulses without requiring the fibre to be placed in very close proximity to the cell sample. A theoretical model is also developed in order to predict the light propagation from axicon tipped and bare fibres, in both air and water environments. The model proves to be in good agreement with the experimental findings and can be used to establish the optimum fibre parameters for successful cellular transfection. We readily obtain efficiencies of up to 57 % which are comparable with free space transfection. This advance paves the way for optical transfection of tissue samples and endoscopic embodiments of this technique.

  19. Androgen insensitivity syndrome.

    PubMed

    Hughes, Ieuan A; Davies, John D; Bunch, Trevor I; Pasterski, Vickie; Mastroyannopoulou, Kiki; MacDougall, Jane

    2012-10-20

    Androgen insensitivity syndrome in its complete form is a disorder of hormone resistance characterised by a female phenotype in an individual with an XY karyotype and testes producing age-appropriate normal concentrations of androgens. Pathogenesis is the result of mutations in the X-linked androgen receptor gene, which encodes for the ligand-activated androgen receptor--a transcription factor and member of the nuclear receptor superfamily. This Seminar describes the clinical manifestations of androgen insensitivity syndrome from infancy to adulthood, reviews the mechanism of androgen action, and shows examples of how mutations of the androgen receptor gene cause the syndrome. Management of androgen insensitivity syndrome should be undertaken by a multidisciplinary team and include gonadectomy to avoid gonad tumours in later life, appropriate sex-hormone replacement at puberty and beyond, and an emphasis on openness in disclosure.

  20. Complete Androgen Insensitivity Syndrome.

    PubMed

    Hashmi, Asra; Hanif, Farha; Hanif, Shumaila Muhammad; Abdullah, Farhan Essa; Shamim, Muhammad Shahid

    2008-07-01

    The incidence of Complete Androgen Insensitivity Syndrome (CAIS) is about 1 in 20,000. People with CAIS are normal appearing females, despite the presence of testes and a 46, XY chromosome constitution. We came across a case in which a 17 years old girl presented with the complaint of inguinal hernia and amenorrhea. Subsequent investigations were done revealing absence of female internal genitalia and the presence of abdominal mass, possibly testes. Syndrome has been linked to mutations in AR, the gene for the human Androgen Receptor, located at Xq11-12 leading to the insensitivity of the receptor to testosterone. Gonadectomy was performed and life long Hormone replacement therapy was advised.

  1. Graphene based gene transfection

    NASA Astrophysics Data System (ADS)

    Feng, Liangzhu; Zhang, Shuai; Liu, Zhuang

    2011-03-01

    Graphene as a star in materials research has been attracting tremendous attentions in the past few years in various fields including biomedicine. In this work, for the first time we successfully use graphene as a non-toxic nano-vehicle for efficient gene transfection. Graphene oxide (GO) is bound with cationic polymers, polyethyleneimine (PEI) with two different molecular weights at 1.2 kDa and 10 kDa, forming GO-PEI-1.2k and GO-PEG-10k complexes, respectively, both of which are stable in physiological solutions. Cellular toxicity tests reveal that our GO-PEI-10k complex exhibits significantly reduced toxicity to the treated cells compared to the bare PEI-10k polymer. The positively charged GO-PEI complexes are able to further bind with plasmid DNA (pDNA) for intracellular transfection of the enhanced green fluorescence protein (EGFP) gene in HeLa cells. While EGFP transfection with PEI-1.2k appears to be ineffective, high EGFP expression is observed using the corresponding GO-PEI-1.2k as the transfection agent. On the other hand, GO-PEI-10k shows similar EGFP transfection efficiency but lower toxicity compared with PEI-10k. Our results suggest graphene to be a novel gene delivery nano-vector with low cytotoxicity and high transfection efficiency, promising for future applications in non-viral based gene therapy.Graphene as a star in materials research has been attracting tremendous attentions in the past few years in various fields including biomedicine. In this work, for the first time we successfully use graphene as a non-toxic nano-vehicle for efficient gene transfection. Graphene oxide (GO) is bound with cationic polymers, polyethyleneimine (PEI) with two different molecular weights at 1.2 kDa and 10 kDa, forming GO-PEI-1.2k and GO-PEG-10k complexes, respectively, both of which are stable in physiological solutions. Cellular toxicity tests reveal that our GO-PEI-10k complex exhibits significantly reduced toxicity to the treated cells compared to the bare PEI

  2. Transfection using DEAE-dextran.

    PubMed

    Selden, R F

    2001-05-01

    Two protocols for DEAE-dextran transfection of cells are provided in this unit. The Basic Protocol describes a procedure used to transfect adherent cells and the first Alternate Protocol presents a method used to transfect suspension cells. If an increase in transfection efficiency is needed, cells can be treated with chloroquine as described in the second Alternate Protocol.

  3. Insensitive control technology development

    NASA Technical Reports Server (NTRS)

    Harvey, C. A.; Pope, R. E.

    1978-01-01

    THe investigation of two insensitive controller synthesis techniques was reported. The finite dimensional inverse approach produces a time varying insensitive controller and/or parameter identifier by constructing inverse functions derived from a finite number of input output pair relationships. The MD/IM concept relies on the information matrix theory that was developed in the estimation and identification field. The MD/IM synthesis technique is based on the hypothesis that minimizing the information matrix will reduce system identifiability and consequently system sensitivity to uncertain parameters. The controllers designed with both techniques were evaluated on a realistic C-5A aircraft flight control problem. Results indicate that the FDI controller is more suited to trajectory type problems because of its time varying nature. The MD/IM controller performed as well as the top-rated controllers of the initial effort and has direct application to aircraft flight control problems.

  4. Androgen insensitivity syndrome.

    PubMed

    Hughes, Ieuan Arwel; Werner, Ralf; Bunch, Trevor; Hiort, Olaf

    2012-10-01

    The androgen insensitivity syndromes (AIS) fall within the generic category of 46,XY DSD (disorder of sex development) and present as phenotypes associated with complete or partial resistance to the action of androgens. Three categories are recognized: complete androgen insensitivity syndrome (CAIS), partial androgen insensitivity syndrome (PAIS), mild androgen insensitivity syndrome (MAIS). The androgen receptor (AR) is encoded by an 8 exon gene on the X chromosome long arm. More than 800 mutations in the AR gene have been reported in AIS patients (www.androgendb.mcgill.ca/). They are distributed throughout the gene with a preponderance located in the ligand binding domain. The most severe mutations are generally associated with a CAIS phenotype, but the correlation is less defined in PAIS. CAIS presents typically as primary amenorrhoea in an adolescent female and less commonly in infancy with bilateral inguinal/labial swellings due to testes. The differential diagnosis in CAIS is limited, whereas in PAIS, numerous other causes of DSD can also produce the typical phenotype of micropenis, severe hypospadias and bifid scrotum. Management issues in CAIS involve timing of gonadectomy, appropriate hormone replacement therapy and assessment of the need for vaginal dilation or rarely, vaginal surgery. The risk of gonadal germ cell tumor is low during childhood and adolescence but increases in later adulthood. Expert psychological counseling is mandatory to manage the disconnect between chromosomal, gonadal and phenotypic sex and to choreograph the evolving process of disclosure from late childhood through to maturity. It is implicit that management in AIS requires a multidisciplinary team and engagement with patient advocacy groups.

  5. [Congenital insensitivity to pain].

    PubMed

    Popko, Janusz; Karpiński, Michał; Guszczyn, Tomasz

    2014-02-01

    Congenital insensitivity to pain belongs to rare diseases called hereditary sensory neuropathy (HSN). The disturbance of sense and secondary harms are creating clinical picture. The aim of this report was to describe therapeutic problems with which we met with a three siblings with congenital insensitivity to pain. The authors have described three children with congenital insensitivity to pain. The disease was diagnosed at the age of 3-5. These children painlessly have broken their lower limbs. These fractures were late diagnosed what resulted in a badly healed deformation of legs. For this reason, the right knee of the oldest boy had to be stiffened. This boy had also late diagnosed the left hip luxation, and hematomas had arisen, which become filled with pus. The boy was in sepsis and a dramatic life-and-death struggle was performed. A purulent focuses were removed from abdomen and femoral head was also resected. The other two siblings had fractures and infections, but not such severe as the oldest boy. It is well known that a causal treatment of this disease in unknown. Patients must learn to avoid mechanical and thermal trauma. It is the only way to prevent complications of this disease.

  6. Transfection using DEAE-dextran.

    PubMed

    Gulick, T

    2001-05-01

    Transfection of cultured mammalian cells using diethylaminoethyl (DEAE)-dextran/DNA can be an attractive alternative to other transfection methods in many circumstances. The major advantages of the technique are its relative simplicity and speed, limited expense, and remarkably reproducible interexperimental and intraexperimental transfection efficiency. Disadvantages include inhibition of cell growth and induction of heterogeneous morphological changes in cells. Furthermore, the concentration of serum in the culture medium must be transiently reduced during the transfection. In general, DEAE-dextran DNA transfection is ideal for transient transfections with promoter/reporter plasmids in analyses of promoter and enhancer functions, and is suitable for overexpression of recombinant protein in transient transfections or for generation of stable cell lines using vectors designed to exist in the cell as episomes. This unit presents a general description of DEAE-dextran transfection, as well as two more specific protocols for typical experimental applications. The basic protocol is suitable for transfection of anchorage-dependent (attached) cells. For cells that grow in suspension, electroporation or lipofection is usually preferred, although DEAE-dextran-mediated transfection can be used.

  7. Transfection using DEAE-dextran.

    PubMed

    Gulick, Tod

    2003-08-01

    Transfection of cultured mammalian cells using diethylaminoethyl (DEAE)-dextran/DNA can be an attractive alternative to other transfection methods in many circumstances. The major advantages of the technique are its relative simplicity and speed, limited expense, and remarkably reproducible interexperimental and intraexperimental transfection efficiency. Disadvantages include inhibition of cell growth and induction of heterogeneous morphological changes in cells. Furthermore, the concentration of serum in the culture medium must be transiently reduced during the transfection. In general, DEAE-dextran DNA transfection is ideal for transient transfections with promoter/reporter plasmids in analyses of promoter and enhancer functions, and is suitable for overexpression of recombinant protein in transient transfections or for generation of stable cell lines using vectors designed to exist in the cell as episomes. This unit presents a general description of DEAE-dextran transfection, as well as two more specific protocols for typical experimental applications. The basic protocol is suitable for transfection of anchorage-dependent (attached) cells. For cells that grow in suspension, electroporation or lipofection is usually preferred, although DEAE-dextran-mediated transfection can be used.

  8. Transfection using DEAE-dextran.

    PubMed

    Gulick, T

    2001-05-01

    Transfection of cultured mammalian cells using diethylaminoethyl (DEAE)-dextran/DNA can be an attractive alternative to other transfection methods in many circumstances. The major advantages of the technique are its relative simplicity and speed, limited expense, and remarkably reproducible interexperimental and intraexperimental transfection efficiency. Disadvantages include inhibition of cell growth and induction of heterogeneous morphological changes in cells. Furthermore, the concentration of serum in the culture medium must be transiently reduced during the transfection. In general, DEAE-dextran DNA transfection is ideal for transient transfections with promoter/reporter plasmids in analyses of promoter and enhancer functions, and is suitable for overexpression of recombinant protein in transient transfections or for generation of stable cell lines using vectors designed to exist in the cell as episomes. This unit presents a general description of DEAE-dextran transfection, as well as two more specific protocols for typical experimental applications. The Basic Protocol is suitable for transfection of anchorage-dependent (attached) cells. For cells that grow in suspension, electroporation or lipofection is usually preferred, although DEAE-dextran-mediated transfection can be used.

  9. Androgen insensitivity syndrome.

    PubMed

    Mongan, Nigel P; Tadokoro-Cuccaro, Rieko; Bunch, Trevor; Hughes, Ieuan A

    2015-08-01

    Androgen insensitivity syndrome (AIS) results from androgen receptor dysfunction and is a common cause of disorder of sex development. The AIS phenotype largely depends on the degree of residual androgen receptor (AR) activity. This review describes the molecular action of androgens and the range of androgen receptor gene mutations, essential knowledge to understand the pathogenesis of the complete and partial forms of this syndrome. A multidisciplinary approach is recommended for clinical management from infancy through to adulthood. Hormone replacement therapy is needed following gonadectomy. Patients who choose to retain the gonads are at risk of developing germ cell tumors for which sensitive circulating tumor markers may soon become available. Whilst the contribution of AR dysfunction to complete AIS is well understood, the involvement of the AR and associated proteins as contributors to partial AIS is an area of active research. Disorders of sex development such as AIS which are related to AR dysfunction offer a breadth of manifestations for the clinician to manage and opportunities for further research on the mechanism of androgen action.

  10. Androgen insensitivity syndrome.

    PubMed

    Mendoza, Nicolás; Motos, Miguel Angel

    2013-01-01

    Androgen insensitivity syndrome (AIS) is a disorder caused by a mutation of the gene encoding the androgen receptor (AR; Xq11-q12). The prevalence of AIS has been estimated to be one case in every 20,000 to 64,000 newborn males for the complete syndrome (CAIS), and the prevalence is unknown for the partial syndrome (PAIS). The symptoms range from phenotypically normal males with impaired spermatogenesis to phenotypically normal women with primary amenorrhea. Various forms of ambiguous genitalia have been observed at birth. The diagnosis is confirmed by determining the exact mutation in the AR gene. PAIS individuals require precise diagnosis as early as possible so that the sex can be assigned, treatment can be recommended, and they can receive proper genetic counseling. After birth, differential diagnosis should be performed using other forms of abnormal sexual differentiation of primary amenorrhea. The treatment of AIS is based on reinforcement sexual identity, gonadectomy planning, and hormone replacement therapy. The prognosis for CAIS is good if the testicular tissue is removed at the appropriate time. For PAIS, the prognosis depends on the ambiguity of the genitalia and physical and psychosocial adjustment to the assigned sex.

  11. [Congenital insensitivity to pain].

    PubMed

    Danziger, N; Willer, J-C

    2009-02-01

    Congenital insensitivity to pain (CIP) is a rare syndrome with various clinical expressions, characterized by a dramatic impairment of pain perception since birth. In the 1980s, progress in nerve histopathology allowed to demonstrate that CIP was almost always a manifestation of hereditary sensory and autonomic neuropathies (HSAN) involving the small-calibre (A-delta and C) nerve fibres which normally transmit nociceptive inputs along sensory nerves. Identification of the genetic basis of several clinical subtypes has led to a better understanding of the mechanisms involved, emphasizing in particular the crucial role of nerve growth factor (NGF) in the development and survival of nociceptors. Recently, mutations of the gene coding for the sodium channel Nav1.7--a voltage-dependent sodium channel expressed preferentially on peripheral nociceptors and sympathetic ganglia--have been found to be the cause of CIP in patients showing a normal nerve biopsy. This radical impairment of nociception mirrors the hereditary pain syndromes associated with "gain of function" mutations of the same ion channel, such as familial erythromelalgia and paroxysmal extreme pain disorder. Future research with CIP patients may identify other proteins specifically involved in nociception, which might represent potential targets for chronic pain treatment. Moreover, this rare clinical syndrome offers the opportunity to address interesting neuropsychological issues, such as the role of pain experience in the construction of body image and in the empathic representation of others' pain.

  12. Transfection of Platyhelminthes

    PubMed Central

    Moguel, Bárbara; Bobes, Raúl J.; Carrero, Julio C.; Laclette, Juan P.

    2015-01-01

    Flatworms are one of the most diverse groups within Lophotrochozoa with more than 20,000 known species, distributed worldwide in different ecosystems, from the free-living organisms in the seas and lakes to highly specialized parasites living in a variety of hosts, including humans. Several infections caused by flatworms are considered major neglected diseases affecting countries in the Americas, Asia, and Africa. For several decades, a particular interest on free-living flatworms was due to their ability to regenerate considerable portions of the body, implying the presence of germ cells that could be important for medicine. The relevance of reverse genetics for this group is clear; understanding the phenotypic characteristics of specific genes will shed light on developmental traits of free-living and parasite worms. The genetic manipulation of flatworms will allow learning more about the mechanisms for tissue regeneration, designing new and more effective anthelmintic drugs, and explaining the host-parasite molecular crosstalk so far partially inaccessible for experimentation. In this review, availability of transfection techniques is analyzed across flatworms, from the initial transient achievements to the stable manipulations now developed for free-living and parasite species. PMID:26090388

  13. Transfection of Platyhelminthes.

    PubMed

    Moguel, Bárbara; Bobes, Raúl J; Carrero, Julio C; Laclette, Juan P

    2015-01-01

    Flatworms are one of the most diverse groups within Lophotrochozoa with more than 20,000 known species, distributed worldwide in different ecosystems, from the free-living organisms in the seas and lakes to highly specialized parasites living in a variety of hosts, including humans. Several infections caused by flatworms are considered major neglected diseases affecting countries in the Americas, Asia, and Africa. For several decades, a particular interest on free-living flatworms was due to their ability to regenerate considerable portions of the body, implying the presence of germ cells that could be important for medicine. The relevance of reverse genetics for this group is clear; understanding the phenotypic characteristics of specific genes will shed light on developmental traits of free-living and parasite worms. The genetic manipulation of flatworms will allow learning more about the mechanisms for tissue regeneration, designing new and more effective anthelmintic drugs, and explaining the host-parasite molecular crosstalk so far partially inaccessible for experimentation. In this review, availability of transfection techniques is analyzed across flatworms, from the initial transient achievements to the stable manipulations now developed for free-living and parasite species.

  14. Imperfection Insensitive Thin Shells

    NASA Astrophysics Data System (ADS)

    Ning, Xin

    The buckling of axially compressed cylindrical shells and externally pressurized spherical shells is extremely sensitive to even very small geometric imperfections. In practice this issue is addressed by either using overly conservative knockdown factors, while keeping perfect axial or spherical symmetry, or adding closely and equally spaced stiffeners on shell surface. The influence of imperfection-sensitivity is mitigated, but the shells designed from these approaches are either too heavy or very expensive and are still sensitive to imperfections. Despite their drawbacks, these approaches have been used for more than half a century. This thesis proposes a novel method to design imperfection-insensitive cylindrical shells subject to axial compression. Instead of following the classical paths, focused on axially symmetric or high-order rotationally symmetric cross-sections, the method in this thesis adopts optimal symmetry-breaking wavy cross-sections (wavy shells). The avoidance of imperfection sensitivity is achieved by searching with an evolutionary algorithm for smooth cross-sectional shapes that maximize the minimum among the buckling loads of geometrically perfect and imperfect wavy shells. It is found that the shells designed through this approach can achieve higher critical stresses and knockdown factors than any previously known monocoque cylindrical shells. It is also found that these shells have superior mass efficiency to almost all previously reported stiffened shells. Experimental studies on a design of composite wavy shell obtained through the proposed method are presented in this thesis. A method of making composite wavy shells and a photogrametry technique of measuring full-field geometric imperfections have been developed. Numerical predictions based on the measured geometric imperfections match remarkably well with the experiments. Experimental results confirm that the wavy shells are not sensitive to imperfections and can carry axial compression

  15. The dipeptide H-Trp-Arg-OH (WR) Is a PPARα agonist and reduces hepatic lipid accumulation in lipid-loaded H4IIE cells.

    PubMed

    Jia, Yaoyao; Kim, Jong-Ho; Nam, Bora; Kim, Jiyoung; Lee, Ji Hae; Kim, Kyung Ok; Hwang, Kwang Yeon; Lee, Sung-Joon

    2015-01-01

    Dipeptides absorbed by the intestinal epithelium are delivered to circulation, but their metabolic roles are not yet clearly understood. We investigated the biological activities of a dietary dipeptide, H-Trp-Arg-OH (WR), on the regulation of peroxisome proliferator-activated receptor (PPAR) α activity. Reporter gene assays revealed that WR dose-dependently induced PPARα transactivation. Surface plasmon resonance experiments demonstrated that WR interacts directly with the PPARα ligand binding domain, and time-resolved fluorescence energy transfer analyses revealed recruitment of a co-activator peptide, fluorescein-PGC1α, to PPARα, confirming the direct binding of WR to PPARα and occurrence of conformational changes. WR induced cellular fatty acid uptake and the expression of PPARα response genes in fatty acid oxidation, thus reducing intracellular triglyceride accumulation in lipid-loaded hepatocytes. In conclusion, the dietary dipeptide WR activates PPARα and reduces hepatic lipid accumulation in lipid-loaded hepatocytes.

  16. CHARACTERIZATION OF THE H4IIE RAT HEPATOMA CELL BIOASSAY AS A TOOL FOR ASSESSING TOXIC POTENCY OF PLANAR HALOGENATED HYDROCARBONS IN ENVIRONMENTAL SAMPLES. (R823881)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  17. Molecular biology of androgen insensitivity.

    PubMed

    Jääskeläinen, Jarmo

    2012-04-16

    Androgen insensitivity syndrome (AIS) is the most common specific cause of 46,XY disorder in sex development. The androgen signaling pathway is complex but so far, the only gene linked with AIS is the androgen receptor (AR). Mutations in the AR are found in most subjects with complete AIS but in partial AIS, the rate has varied 28-73%, depending on the case selection. More than 400 different mutations in AR leading to AIS have been reported. Most mutations are missense substitutions located in the ligand binding domain of the receptor. However, when systematically screened, a substantial amount of mutations can be detected also in the N-terminal domain encoded by exon 1. Within this exon lie two trinucleotide, CAG and GGN repeat regions which are polymorphic in length. Their role in androgen insensitivity is somewhat unclear. Recent advances in protein modeling have resulted in better understanding of the mechanism of known AR mutations.

  18. Neutron Detector Gamma Insensitivity Criteria

    SciTech Connect

    Kouzes, Richard T.; Ely, James H.; Lintereur, Azaree T.; Stephens, Daniel L.

    2009-10-21

    The shortage of 3He has triggered the search for an effective alternative neutron detection technology for radiation portal monitor applications. Any new detection technology must satisfy two basic criteria: 1) it must meet the neutron detection efficiency requirement, and 2) it must be insensitive to gamma ray interference at a prescribed level, while still meeting the neutron detection requirement. It is the purpose of this document to define this latter criterion.

  19. Insensitive fuze train for high explosives

    SciTech Connect

    Cutting, Jack L.; Lee, Ronald S.; Von Holle, William G.

    1994-01-01

    A generic insensitive fuze train to initiate insensitive high explosives, such as PBXW-124. The insensitive fuze train uses a slapper foil to initiate sub-gram quantities of an explosive, such as HNS-IV or PETN. This small amount of explosive drives a larger metal slapper onto a booster charge of an insensitive explosive, such as UF-TATB. The booster charge initiates a larger charge of an explosive, such as LX-17, which in turn, initiates the insensitive high explosive, such as PBXW-124.

  20. Insensitive fuze train for high explosives

    SciTech Connect

    Cutting, J.L.; Lee, R.S.; Von Holle, W.G.

    1994-01-04

    A generic insensitive fuze train to initiate insensitive high explosives, such as PBXW-124 is described. The insensitive fuze train uses a slapper foil to initiate sub-gram quantities of an explosive, such as HNS-IV or PETN. This small amount of explosive drives a larger metal slapper onto a booster charge of an insensitive explosive, such as UF-TATB. The booster charge initiates a larger charge of an explosive, such as LX-17, which in turn, initiates the insensitive high explosive, such as PBXW-124. 3 figures.

  1. SIP: Systematics-Insensitive Periodograms

    NASA Astrophysics Data System (ADS)

    Angus, Ruth

    2016-09-01

    SIP (Systematics-Insensitive Periodograms) extends the generative model used to create traditional sine-fitting periodograms for finding the frequency of a sinusoid by including systematic trends based on a set of eigen light curves in the generative model in addition to using a sum of sine and cosine functions over a grid of frequencies, producing periodograms with vastly reduced systematic features. Acoustic oscillations in giant stars and measurement of stellar rotation periods can be recovered from the SIP periodograms without detrending. The code can also be applied to detection other periodic phenomena, including eclipsing binaries and short-period exoplanet candidates.

  2. Calcium Phosphate Transfection of Primary Hippocampal Neurons

    PubMed Central

    DiBona, Victoria L.; Wu, Qian; Zhang, Huaye

    2013-01-01

    Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells. PMID:24300106

  3. Molecular basis of androgen insensitivity.

    PubMed

    Brinkmann, A O

    2001-06-20

    Androgens are important steroid hormones for expression of the male phenotype. They have characteristic roles during male sexual differentiation, during development and maintenance of secondary male characteristics, and during the initiation and maintenance of spermatogenesis. The two most important androgens in this respect are testosterone and 5 alpha-dihydrotestosterone. Each androgen has its own specific role during male sexual differentiation, testosterone is involved in the development and differentiation of Wolffian duct derived structures, whereas 5 alpha-dihydrotestosterone, a metabolite of testosterone, is the active ligand in the urogenital sinus and tubercle and their derived structures. The actions of androgens are mediated by the androgen receptor. This ligand dependent transcription factor belongs to the superfamily of nuclear receptors, including those for the other steroid hormones. The androgen receptor gene is located on the X-chromosome at Xq11--12 and codes for a protein with a molecular mass of approximately 110 kDa. Only one androgen receptor cDNA has been identified sofar, despite two different ligands. It is generally accepted that defects in the androgen receptor gene prevent the normal development of both internal and external male structures in 46, XY individuals. The end-organ resistance to androgens has been designated as androgen insensitivity syndrome (AIS) and is distinct from other forms of male pseudohermaphroditism like 17 beta-hydroxy-steroid dehydrogenase type 3 deficiency, leydig cell hypoplasia due to inactivating LH receptor mutations or 5 alpha-reductase type 2 deficiency. Furthermore, two additional pathological situations are associated with abnormal androgen receptor structure and function -- spinal and bulbar muscular atrophy (SBMA, or Kennedy's disease) and prostate cancer. In the AR gene, four different types of mutations have been detected in DNA from individuals with AIS -- (i) single point mutations resulting in

  4. Gamma-insensitive optical sensor

    DOEpatents

    Kruger, H.W.

    1994-03-15

    An ultraviolet/visible/infrared gamma-insensitive gas avalanche focal plane array is described comprising a planar photocathode and a planar anode pad array separated by a gas-filled gap and across which is applied an electric potential. Electrons ejected from the photocathode are accelerated sufficiently between collisions with the gas molecules to ionize them, forming an electron avalanche. The gap acts like a proportional counter. The array of anode pad are mounted on the front of an anode plate and are connected to matching contact pads on the back of the anode via feed through wires. Connection of the anode to signal processing electronics is made from the contact pads using standard indium bump techniques, for example. 6 figures.

  5. Gamma-insensitive optical sensor

    DOEpatents

    Kruger, Hans W.

    1994-01-01

    An ultra-violet/visible/infra-red gamma-insensitive gas avalanche focal plane array comprising a planar photocathode and a planar anode pad array separated by a gas-filled gap and across which is applied an electric potential. Electrons ejected from the photocathode are accelerated sufficiently between collisions with the gas molecules to ionize them, forming an electron avalanche. The gap acts like a proportional counter. The array of anode pad are mounted on the front of an anode plate and are connected to matching contact pads on the back of the anode via feed through wires. Connection of the anode to signal processing electronics is made from the contact pads using standard indium bump techniques, for example.

  6. Norrbottnian congenital insensitivity to pain.

    PubMed

    Minde, Jan K

    2006-04-01

    Congenital insensitivity to pain is a rare hereditary neuropathy. We present patients from a large family in Norrbotten, Sweden with a mutation in the nerve growth factor beta gene (NGFbeta). Using a model of recessive inheritance, we identified an 8.3-Mb region on chromosome 1p11.2-p13.2 shared by the affected individuals in the family. Analysis of candidate genes in the disease-critical region revealed a mutation in the coding region of the NGFbeta gene specific for the disease haplotype. All three severely affected individuals were homozygous for the mutation. The disease haplotype was also observed in both unaffected and mildly affected family members, but in heterozygote form. We have identified 43 patients, 3 homozygous and 40 heterozygous. The homozygous patients have a severe congenital form with onset of symptoms at an early age, most often affecting the lower extremities with insidious progressive joint swellings or painless fractures. Fracture healing was normal, but the arthropathy was progressive, resulting in disabling Charcot joints with gross deformity and instability. These patients lacked deep pain perception in bones and joints and had no protective reflexes, leading to gross bone and joint complications. They also had abnormal temperature perception but normal ability to sweat. There was no mental retardation. Clinically, they fit best into the group HSAN type V. Sural nerve biopsies showed a moderate loss of thin myelinated fibers (Adelta-fibers) and a severe reduction of unmyelinated fibers (C-fibers). 14 of the 40 heterozygous adult patients had mild or moderate problems with joint deformities, usually with only slight discomfort. Treatment was conservative with (if needed) different kinds of orthosis and in three cases joint replacement. Nine patients had neuropathy, and nine patients had no symptoms. In congenital disorders like these, it is important to evaluate the age and also the slowly progressive nature, when considering treatment

  7. Genetics Home Reference: congenital insensitivity to pain

    MedlinePlus

    ... have a complete loss of the sense of smell (anosmia). Congenital insensitivity to pain is considered a ... to cells that detect sensations such as touch, smell, and pain. Related Information What does it mean ...

  8. Congenital Insensitivity to Pain (HSNA type IV).

    PubMed

    Millichap, J Gordon

    2015-04-01

    Investigators from New York University, NY, studied 14 patients with congenital insensitivity to pain with anhidrosis (CIPA), compared to 10 patients with chronically deficient sympathetic activity (pure autonomic failure), and 15 normal age-matched controls.

  9. Genetics Home Reference: androgen insensitivity syndrome

    MedlinePlus

    ... typically raised as females and have a female gender identity. Affected individuals have male internal sex organs ( ... and may have a male or a female gender identity. People with mild androgen insensitivity are born ...

  10. Strain Insensitive Optical Phase Locked Loop

    NASA Technical Reports Server (NTRS)

    Egalon, Claudio O. (Inventor); Rogowski, Robert S. (Inventor)

    1998-01-01

    A strain sensor uses optical fibers including strain insensitive portions and a strain sensitive portion. The optical fibers form a sensitive arm of an optical phase locked loop (OPLL). The use of the OPLL allows for multimode optical fiber to be used in a strain insensitive configuration. Only strain information for the strain sensitive portion is monitored rather than the integrated strain measurements commonly made with optical fiber sensors.

  11. Optimization of Gene Transfection in Murine Myeloma Cell Lines using Different Transfection Reagents

    PubMed Central

    Shabani, Mahdi; Hemmati, Sheyda; Hadavi, Reza; Amirghofran, Zahra; Jeddi-Tehrani, Mahmood; Rabbani, Hodjatallah; Shokri, Fazel

    2010-01-01

    Purification and isolation of cellular target proteins for monoclonal antibody (MAb) production is a difficult and time-consuming process. Immunization of mice with murine cell lines stably transfected with genes coding for xenogenic target molecules is an alternative method for mouse immunization and MAb production. Here we present data on transfection efficiency of some commercial reagents used for transfection of murine myeloma cell lines. Little is known about transfectability of murine myeloma cell lines by different transfection reagents. Mouse myeloma cell lines (SP2/0, NS0, NS1, Ag8, and P3U1) were transfected with pEGFP-N1 vector using Lipofectamine 2000, jetPEI and LyoVec commercial transfection reagents in different combinations. The transfection permissible HEK293-FT cell line was used as a control in transfection procedure. Transfected cells, expressing the Enhanced Green Fluorescent Protein (EGFP), were analyzed by flow cytometry 48 hrs post transfection. Our results showed transfection efficiency of 71%, 57% and 22% for HEK293-FT, 5.5%, 3.4% and 1% for SP2/0, 55.7%, 21.1% and 9.3% for NS0, 8.2%, 6% and 5.5% for NS1, 22%, 49.2% and 5.5% for Ag8 and 6.3%, 21.5% and 4.6% for P3U1 cell lines after transfection with Lipofectamine 2000, jetPEI and LyoVec reagents, respectively. Our data indicate that NS0 and Ag8 are efficiently transfected by Lipofectamine 2000 and jetPEI reagents. Finally, we propose Ag8 and NS0 cell lines as suitable host cells for efficient expression of target genes which can be used for mouse immunization and MAb production. PMID:23408356

  12. Ballistic transfection of mammalian cells in vivo

    SciTech Connect

    Kolesnikov, V.A.; Zelenin, A.V.; Zelenina, I.A.

    1995-11-01

    The method of ballistic transfection initially proposed for genetic transformation of plants was used for animal cells in vitro and in situ. The method consists in bombarding the transfected cells with microparticles of heavy metals carrying foreign DNA. Penetrating the cell nucleus, the microparticles transport the introduced gene. Successful genetic transformation of the cultured mouse cells and fish embryos was realized, and this allowed the study of mammalian cells in situ. The performed studies allowed us to demonstrate expression of the reporter genes of chloramphenicol acetyltransferase, galactosidase, and neomycin phosphotransferase in the mouse liver, mammary gland and kidney explants, in the liver and cross-striated muscle of mouse and rat in situ, and in developing mouse embryos at the stages of two-cell embryo, morula, and blastocyst. All these genes were introduced by ballistic transfection. In the liver and cross-striated muscle the transgene activity was detected within two to three months after transfection. Thus, the ballistic introduction of the foreign genes in the cells in situ was demonstrated, and this opens possibilities for the use of this method in gene therapy. Methodical aspects of the bombarding and transfection are considered in detail, and the published data on transfection and genetic transformation of mammalian cells are discussed. 41 refs., 13 figs., 1 tab.

  13. Laparoscopic gonedectomy in a case of complete androgen insensitivity syndrome.

    PubMed

    Bhaskararao, G; Himabindu, Y; Nayak, Samir Rajan; Sriharibabu, M

    2014-07-01

    Complete Androgen insensitivity syndrome is a disorder of hormone resistance characterized by a female phenotype in an individual with an XY karyotype. The pathogenesis of CAIS involves a defective androgen receptor gene located on X-chromosome at Xq11-12and end organ insensitivity to androgens, although androgen concentrations are appropriate for the age of the patient. There are three major types of androgen insensitivity syndrome: Complete androgen insensitivity syndrome, minimal androgen insensitivity syndrome, and partial androgen insensitivity syndrome. Management of androgen insensitivity syndrome includes multidisciplinary approach and involves gonedectomy to avoid gonadal tumors in later life. Hormone replacement therapy (HRT) and psychological support are required in long-term basis.

  14. Equation of state of insensitive high explosives

    SciTech Connect

    Ree, F H; Van Thiel, M; Viecelli, J A

    1998-08-12

    Detonation of an insensitive high explosive formulated with a fluorine containing binder produces a large amount of condensed carbon and gaseous HF product, which transforms into CF{sub 4} as the pressure is increased. The former (carbon condensation) is characterized by slow energy release, while the latter (HF) has no shockwave data. We have identified that these two items are the key factors, which make reliable prediction of the performance of an insensitive high explosive very difficult. This paper describes physical models to address these issues and apply the models to analyze experimental data of LX-17.

  15. Phase-Insensitive Ultrasonic Testing System

    NASA Technical Reports Server (NTRS)

    Madaras, Eric I.

    1995-01-01

    Ultrasonic testing system developed for use in revealing hidden disbonds at rough, inaccessible interfaces between layers of material. Includes array of small piezoelectric transducers, receiving outputs electronically processed individually and combined in such way as to make system phase-insensitive, overcoming limitations imposed by phase-sensitivity. Development of present ultrasonic system and phase-insensitive-array technique which based motivated by need to detect disbonds under conditions of bondline inhibitor, liner, and fuel at ends of segments of solid rocket motor of space shuttle. Here, liner-to-fuel bondline very rough with respect to ultrasonic wavelength.

  16. Complete androgen insensitivity syndrome--a review.

    PubMed

    Oakes, Meghan B; Eyvazzadeh, Aimee D; Quint, Elisabeth; Smith, Yolanda R

    2008-12-01

    This review paper highlights important diagnostic and therapeutic concerns for girls with Complete Androgen Insensitivity Syndrome (CAIS). CAIS is an androgen receptor defect disorder associated with vaginal and uterine agenesis in women with a 46,XY karyotype. The major clinical issues surrounding this syndrome include timing of gonadectomy, hormone replacement, vaginal dilation, and attention to psychological issues.

  17. Optimizacion of Babesia bovis transfection methods

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The tick borne Babesia parasites remain an important limitation for development of cattle industries worldwide. A stable transfection of Babesia bovis will be useful for functional analysis of the recently sequenced B. bovis genome and to design improved methods to control Babesia infections. In thi...

  18. [Case of complete androgen insensitivity syndrome].

    PubMed

    Ohba, Kojiro; Hayashida, Yasushi; Hakariya, Hironobu; Ichinose, Syunsuke; Naitou, Shinji

    2009-05-01

    Androgen insensitivity syndrome, gonadectomy, estrogen supplementation a 23-year-old single female visited our gynecological clinic because of primary amenorrhea. The patient's breast development was good. However the patient had thin pubic hair and blind-ending vagina. Serum levels of estrogen E2, testosterone, luteinizing hormone (LH) and follide stimulating hormone (FSH) were 37.0, 497 pg/ml, 22.0 and 8.7 mIU/ml, respectively. Chromosomal analysis was a karyotype of 46, XY. There was no uterus and no ovaries. However, there were bilateral inguinal elastic masses which were gonads. The patient was diagnosed with complete androgen insensitivity syndrome and bilateral gonadectomy was performed. The postoperative course was good and the patient is receiving estrogen replacement therapy.

  19. Congenital insensitivity to pain in one family.

    PubMed

    Svec, Andrey; Feldinszka, Jana; Kokavec, Milan

    2016-12-09

    Congenital insensitivity to pain (CIP) is a rare autosomal recessive genetic disease caused by mutation in several different genes. The diagnosis requires the combined skills and cooperation of pediatricians, neurologists, radiologists, pathologists, and orthopedic surgeons. Orthopedic manifestations of CIP include delayed diagnosis of fractures, nonunions, Charcot arthropathy, avascular necrosis, osteomyelitis, joint dislocations, and heterotopic ossifications. We present case reports of two brothers with CIP with various orthopedic manifestations and methods of surgical treatment with 10 years of follow-up.

  20. Insensitive Munitions Development for General Purpose Bombs

    DTIC Science & Technology

    1990-08-01

    combat readiness. There has been a concerted effort by industry and A i r Force explosive development teams t o provide an energetic material w h...Insensitive Munitions Development for General Purpose Bombs 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d...house efforts, the Air Force has stimulated commercial industry involvement i n explosives research and development . output from this ef for t has

  1. Imaging characteristics of androgen insensitivity syndrome.

    PubMed

    Tank, Jay; Knoll, Abraham; Gilet, Anthony; Kim, Susanne

    2015-01-01

    Androgen insensitivity syndrome (AIS), also known as testicular feminization, is a genetic disorder which leads to lack of response to androgens caused by a defect in the androgen receptor. It is relatively uncommon and is usually diagnosed through clinical symptoms, laboratory findings, physical exam, radiological imaging, and genetic analysis. Our case is a middle-aged woman with complete AIS and demonstrates the importance of the various imaging modalities that are implemented in initially diagnosing and assisting in surgical management.

  2. The dipeptide H-Trp-Glu-OH (WE) shows agonistic activity to peroxisome proliferator-activated protein-α and reduces hepatic lipid accumulation in lipid-loaded H4IIE cells.

    PubMed

    Jia, Yaoyao; Kim, Jong-Ho; Nam, Bora; Kim, Jiyoung; Lee, Ji Hae; Hwang, Kwang-Yeon; Lee, Sung-Joon

    2014-07-01

    Dipeptides digested from dietary proteins can be directly absorbed by the intestine and delivered to the circulatory system. However, the dipeptides' metabolic roles and biological activities are largely unknown. Lipid-loaded HII4E cells stimulated with H-Trp-Glu-OH (WE) exhibited reduced lipid accumulation, of which the effect was abolished by peroxisome proliferator-activated receptor (PPAR) α gene knock down. A luciferase assay showed that the WE dipeptide induced PPARα transactivation in a dose-dependent manner. Surface plasmon resonance and time-resolved fluorescence resonance energy transfer analyses demonstrated that WE interacts directly with the PPARα ligand binding domain (KD, 120 μM; EC50, 83 μM). Cells stimulated with WE induced PPARα and its responsive genes and increased cellular fatty acid uptake. In conclusion, WE reduces hepatic lipid accumulation in lipid-loaded hepatocytes via the activation of PPARα by a direct interaction.

  3. Suppressive effects of caraway (Carum carvi) extracts on 2, 3, 7, 8-tetrachloro-dibenzo-p-dioxin-dependent gene expression of cytochrome P450 1A1 in the rat H4IIE cells.

    PubMed

    Naderi-Kalali, B; Allameh, A; Rasaee, M J; Bach, H-J; Behechti, A; Doods, K; Kettrup, A; Schramm, K-W

    2005-04-01

    Cytochrome P450 1A1 (CYP1A1) is among the cytochrome P450 classes known to convert xenobiotics and endogenous compounds to toxic and/or carcinogenic metabolites. Suppression of CYP1A1 over expression by certain compounds is implicated in prevention of cancer caused by chemical carcinogens. Chemopreventive agents containing high levels of flavonoids and steroids-like compounds are known to suppress CYP1A1. This study was carried out for assessment of the genomic and proteomic effects of caraway (Carum carvi) extracts containing high levels of both flavonoids and steroid-like substances on ethoxy resorufin dealkylation (EROD) activity and CYP1A1 at mRNA levels. Rat hepatoma cells co-treated with a CYP1A1 inducer i.e. TCDD (2, 3, 7, 8-tetrachlorodibenzo-p-dioxin) and different preparations of caraway extracts at concentrations of 0, 0.13, 1.3, and 13 microM in culture medium. After incubation (37 degrees C and 7% CO2 for 20 h), changes in EROD specific activity recorded and compared in cells under different treatments. The results show that caraway seed extract prepared in three different organic solvents suppressed the enzyme activity in hepatoma cells in a dose-dependent manner. The extracts added above 0.13 microM could significantly inhibit EROD activity and higher levels of each extract (1.3 and 13 microM) caused approximately 10-fold suppression in the enzyme activity. Accordingly, data obtained from the RT-PCR (TaqMan) clearly showed the suppressive effects of plant extract on CYP1A1-related mRNA expression. These data clearly show that substances in caraway seeds extractable in organic solvents can potentially reverse the TCDD-dependent induction in cytochrome P450 1A1.

  4. Bend-insensitive optical fibers for FTTH applications

    NASA Astrophysics Data System (ADS)

    Li, Ming-Jun

    2009-01-01

    This paper reviews recent development in bend-insensitive fibers for fiber-to-the-home (FTTH) applications. First, requirements for bend-insensitive fibers are discussed. Then different design approaches for reducing fiber bending loss are described and compared. A new bend-insensitive fiber using the nano-engineered ring design is presented in detail.

  5. Lipid-based Transfection Reagents Exhibit Cryo-induced Increase in Transfection Efficiency

    PubMed Central

    Sork, Helena; Nordin, Joel Z; Turunen, Janne J; Wiklander, Oscar PB; Bestas, Burcu; Zaghloul, Eman M; Margus, Helerin; Padari, Kärt; Duru, Adil D; Corso, Giulia; Bost, Jeremy; Vader, Pieter; Pooga, Margus; Smith, CI Edvard; Wood, Matthew JA; Schiffelers, Raymond M; Hällbrink, Mattias; Andaloussi, Samir EL

    2016-01-01

    The advantages of lipid-based transfection reagents have permitted their widespread use in molecular biology and gene therapy. This study outlines the effect of cryo-manipulation of a cationic lipid-based formulation, Lipofectamine 2000, which, after being frozen and thawed, showed orders of magnitude higher plasmid delivery efficiency throughout eight different cell lines, without compromising cell viability. Increased transfection efficiency with the freeze-thawed reagent was also seen with 2'-O-methyl phosphorothioate oligonucleotide delivery and in a splice-correction assay. Most importantly, a log-scale improvement in gene delivery using the freeze-thawed reagent was seen in vivo. Using three different methods, we detected considerable differences in the polydispersity of the different nucleic acid complexes as well as observed a clear difference in their surface spreading and sedimentation, with the freeze-thawed ones displaying substantially higher rate of dispersion and deposition on the glass surface. This hitherto overlooked elevated potency of the freeze-thawed reagent facilitates the targeting of hard-to-transfect cells, accomplishes higher transfection rates, and decreases the overall amount of reagent needed for delivery. Additionally, as we also saw a slight increase in plasmid delivery using other freeze-thawed transfection reagents, we postulate that freeze-thawing might prove to be useful for an even wider variety of transfection reagents. PMID:27111416

  6. Toward Contactless Biology: Acoustophoretic DNA Transfection

    PubMed Central

    Vasileiou, Thomas; Foresti, Daniele; Bayram, Adem; Poulikakos, Dimos; Ferrari, Aldo

    2016-01-01

    Acoustophoresis revolutionized the field of container-less manipulation of liquids and solids by enabling mixing procedures which avoid contamination and loss of reagents due to the contact with the support. While its applications to chemistry and engineering are straightforward, additional developments are needed to obtain reliable biological protocols in a contactless environment. Here, we provide a first, fundamental step towards biological reactions in air by demonstrating the acoustophoretic DNA transfection of mammalian cells. We developed an original acoustophoretic design capable of levitating, moving and mixing biological suspensions of living mammalians cells and of DNA plasmids. The precise and sequential delivery of the mixed solutions into tissue culture plates is actuated by a novel mechanism based on the controlled actuation of the acoustophoretic force. The viability of the contactless procedure is tested using a cellular model sensitive to small perturbation of neuronal differentiation pathways. Additionally, the efficiency of the transfection procedure is compared to standard, container-based methods for both single and double DNA transfection and for different cell types including adherent growing HeLa cancer cells, and low adhesion neuron-like PC12 cells. In all, this work provides a proof of principle which paves the way to the development of high-throughput acoustophoretic biological reactors. PMID:26828312

  7. Toward Contactless Biology: Acoustophoretic DNA Transfection

    NASA Astrophysics Data System (ADS)

    Vasileiou, Thomas; Foresti, Daniele; Bayram, Adem; Poulikakos, Dimos; Ferrari, Aldo

    2016-02-01

    Acoustophoresis revolutionized the field of container-less manipulation of liquids and solids by enabling mixing procedures which avoid contamination and loss of reagents due to the contact with the support. While its applications to chemistry and engineering are straightforward, additional developments are needed to obtain reliable biological protocols in a contactless environment. Here, we provide a first, fundamental step towards biological reactions in air by demonstrating the acoustophoretic DNA transfection of mammalian cells. We developed an original acoustophoretic design capable of levitating, moving and mixing biological suspensions of living mammalians cells and of DNA plasmids. The precise and sequential delivery of the mixed solutions into tissue culture plates is actuated by a novel mechanism based on the controlled actuation of the acoustophoretic force. The viability of the contactless procedure is tested using a cellular model sensitive to small perturbation of neuronal differentiation pathways. Additionally, the efficiency of the transfection procedure is compared to standard, container-based methods for both single and double DNA transfection and for different cell types including adherent growing HeLa cancer cells, and low adhesion neuron-like PC12 cells. In all, this work provides a proof of principle which paves the way to the development of high-throughput acoustophoretic biological reactors.

  8. Complete Androgen Insensitivity Syndrome in Three Sisters

    PubMed Central

    Verim, Levent

    2014-01-01

    Disorders of sexual development (DSD) are congenital anomalies due to atypical development of chromosomes, gonads and anatomy. Complete androgen insensitivity syndrome (CAIS), also known as testicular feminization (TF) is a rare DSD disease. The majority of CAIS patients apply to hospital with the complaint of primary amenorrhea or infertility. Given that CAIS patients are all phenotypically female while having 46, XY karyotypes, CAIS diagnosis should be disclosed in an age-appropriate manner preferably by a mental health professional. Cases are reported here for three 46XY siblings consistent with CAIS. PMID:24520507

  9. A General RNA Motif for Cellular Transfection

    PubMed Central

    Magalhães, Maria LB; Byrom, Michelle; Yan, Amy; Kelly, Linsley; Li, Na; Furtado, Raquel; Palliser, Deborah; Ellington, Andrew D; Levy, Matthew

    2012-01-01

    We have developed a selection scheme to generate nucleic acid sequences that recognize and directly internalize into mammalian cells without the aid of conventional delivery methods. To demonstrate the generality of the technology, two independent selections with different starting pools were performed against distinct target cells. Each selection yielded a single highly functional sequence, both of which folded into a common core structure. This internalization signal can be adapted for use as a general purpose reagent for transfection into a wide variety of cell types including primary cells. PMID:22233578

  10. Isolation, culture, and transfection of melanocytes.

    PubMed

    Godwin, Lauren S; Castle, Joanna T; Kohli, Jaskaren S; Goff, Philip S; Cairney, Claire J; Keith, W Nicol; Sviderskaya, Elena V; Bennett, Dorothy C

    2014-06-03

    Located in the basal epidermis and hair follicles, melanocytes of the integument are responsible for its coloration through production of melanin pigments. Melanin is produced in lysosomal-like organelles called melanosomes. In humans, this skin pigmentation acts as an ultraviolet radiation filter. Abnormalities in the division of melanocytes are quite common, with potentially oncogenic growth usually followed by cell senescence producing benign naevi (moles), or occasionally melanoma. Therefore, melanocytes are a useful model for studying melanoma, as well as pigmentation and organelle transport and the diseases affecting these mechanisms. This chapter focuses on the isolation, culture, and transfection of human and murine melanocytes. The first basic protocol describes the primary culture of melanocytes from human skin and the maintenance of growing cultures. The second basic protocol details the subculture and preparation of mouse keratinocyte feeder cells. The primary culture of melanocytes from mouse skin is described in the third basic protocol, and, lastly, the fourth basic protocol outlines a technique for transfecting melanocytes and melanoma cells.

  11. Optimization of conditions for transfection with the Sofast gene vector.

    PubMed

    Zhou, Lei; Liu, Fan; Qiao, Fang-Fang; Tong, Man-Li; Fu, Zuo-Gen; Dan, Bing; Yang, Tian-Ci; Zhang, Zhong-Ying

    2011-01-01

    We previously reported the synthesis and characterization of a novel cationic polymer gene vector. The present article further explored and optimized the working conditions of the Sofast gene vector both in vitro and in vivo, and improved its performance. The transfection conditions of Sofast, such as cell type, cell density, transfection time, N/P values and analysis time after transfection, were further explored. Moreover, the effects of the fusion peptide diINF-7 on transfection efficiency were examined. Sofast was successfully applied for the transfection of exogenous genes into more than 40 types of cell lines derived from humans, mice, monkeys and other species. When the cells were 50-80% confluent, Sofast possessed a better transfection efficiency. In most cases, Sofast also had a higher transfection efficiency when it was used to transfect cells that were seeded for several hours and had adhered to the substrate. The results from in vitro experiments indicate that the recommended Sofast to DNA mass ratio is 16:1, and the optimum analysis time after transfection is 48 h. The salt concentration in the Sofast working solution markedly affected the transfection efficiency. When conducting in vivo transfection, the working solution should be salt-free, whereas for in vitro transfection, it is more appropriate for the working solution to include certain salt concentrations. Finally, the results confirm that diINF-7 significantly promotes the transfection efficiency of Sofast. In conclusion, the present research not only established the optimal conditions for Sofast in the transfection of commonly used cells, but also built the foundations for in vivo and in vitro applications of Sofast, as well as its use in clinical practice.

  12. Birefringence insensitive optical coherence domain reflectometry system

    DOEpatents

    Everett, Matthew J.; Davis, Joseph G.

    2002-01-01

    A birefringence insensitive fiber optic optical coherence domain reflectometry (OCDR) system is provided containing non-polarization maintaining (non-PM) fiber in the sample arm and the reference arm without suffering from signal degradation caused by birefringence. The use of non-PM fiber significantly reduces the cost of the OCDR system and provides a disposable or multiplexed section of the sample arm. The dispersion in the reference arm and sample arm of the OCDR system are matched to achieve high resolution imaging. This system is useful in medical applications or for non-medical in situ probes. The disposable section of non-PM fiber in the sample arm can be conveniently replaced when contaminated by a sample or a patient.

  13. Partial Androgen Insensitivity Syndrome Presenting with Gynecomastia.

    PubMed

    Lee, Sung Won; Kwak, Dong Shin; Jung, In Sub; Kwak, Joo Hee; Park, Jung Hwan; Hong, Sang Mo; Lee, Chang Bum; Park, Yong Soo; Kim, Dong Sun; Choi, Woong Hwan; Ahn, You Hern

    2015-06-01

    Gynecomastia is a benign enlargement of the male breast caused by the proliferation of glandular breast tissue. Determining the various causes of gynecomastia such as physiological causes, drugs, systemic diseases, and endocrine disorders is important. Androgen insensitivity syndrome (AIS) is a rare endocrine disorder presenting with gynecomastia and is a disorder of male sexual differentiation caused by mutations within the androgen receptor gene. All individuals with AIS have the 46 XY karyotype, although AIS phenotypes can be classified as mild, partial or complete and can differ among both males and females including ambiguous genitalia or infertility in males. We experienced a case of partial AIS presenting with gynecomastia and identified the androgen receptor gene mutation.

  14. Congenital insensitivity to pain and anhidrosis.

    PubMed

    Sasnur, Ashok H; Sasnur, Prakash A; Ghaus-Ul, Raza Shamikh Muneer

    2011-05-01

    Congenital insensitivity to pain and anhidrosis (CIPA) is a rare reported entity characterised by disturbance in the pain and temperature perception due to involvement of the autonomic and sensory nervous system. It is an autosomal recessive trait with several defects of the gene NTRK1 coding for the neurotrophic tyrosine kinase - a nerve growth factor receptor on chromosome 1q21-q22. Traumatic fractures are common and, because of lack of pain, may go unrecognised for prolonged periods, resulting in nonunion or pseudoarthrosis. A Charcot joint may be the end result. Treatment complications are very common in these patients and range from infection to wound breakdown to failure of fixation. We report here a rare case of CIPA in a 9-year-old girl and her younger male sibling with generalised absence of pain, anhidrosis and its orthopaedic implications.

  15. Polarization-Insensitive Metalenses at Visible Wavelengths.

    PubMed

    Khorasaninejad, M; Zhu, A Y; Roques-Carmes, C; Chen, W T; Oh, J; Mishra, I; Devlin, R C; Capasso, F

    2016-11-09

    In this Letter, we demonstrate highly efficient, polarization-insensitive planar lenses (metalenses) at red, green, and blue wavelengths (λ = 660, 532, and 405 nm). Metalenses with numerical apertures (NA) of 0.85 and 0.6 and corresponding efficiencies as high as 60% and 90% are achieved. These metalenses are less than 600 nm-thick and can focus incident light down to diffraction-limited spots as small as ∼0.64λ and provide high-resolution imaging. In addition, the focal spots are very symmetric with high Strehl ratios. The single step lithography and compatibility with large-scale fabrication processes make metalenses highly promising for widespread applications in imaging and spectroscopy.

  16. Bend-insensitive fiber based vibration sensor

    NASA Astrophysics Data System (ADS)

    Xu, Yanping; Lu, Ping; Baset, Farhana; Bhardwaj, Vedula Ravi; Bao, Xiaoyi

    2014-05-01

    We report two novel fiber-optic vibration sensors based on standard telecom bend-insensitive fiber (BIF). A tapered BIF forming a fiber Mach-Zehnder interferometer could measure continuous and damped vibration from 1 Hz up to 500 kHz. An enclosed microcantilever is fabricated inside the BIF by chemical etching and fusion spliced with a readout singlemode fiber that exhibits a frequency range from 5 Hz to 10 kHz with high signal-to-noise ratio (SNR) up to 68 dB. The unique double cladding structure of the BIF ensures both sensors with advantages of compactness, high resistance to the external disturbance and stronger mechanical strength.

  17. Prenatal diagnosis of androgen insensitivity syndrome.

    PubMed

    Bianca, S; Cataliotti, A; Bartoloni, G; Torrente, I; Barrano, B; Boemi, G; Lo Presti, M; Indaco, L; Barone, C; Ettore, G

    2009-01-01

    Androgen insensitivity syndrome (AIS) (OMIM 300068) is an X-linked recessive genetic disorder with an XY karyotype that is caused by androgen receptor (AR) defects. We report a prenatal diagnosis case with clinical and molecular findings. The fetal phenotype was female, moreover the autopsy revealed the presence of abdominal testes confirmed by histopathological examination. The AR gene molecular analysis performed on the fetal DNA showed the presence of a c.2493C>T change in exon 4. The single nucleotide change resulted in a Q711X amino acid substitution within the AR ligand-binding domain of the protein that has never been described before in the literature. AIS is an important consideration in pregnancies that show sex discordance in ultrasonography and karyotype results with the opportunity to perform molecular analysis of the AR gene in order to confirm the diagnosis.

  18. Enhancement of the efficiency of femtosecond optical transfection

    NASA Astrophysics Data System (ADS)

    Praveen, Bavishna B.; Stevenson, David; Antkowiak, Maciej; Gunn-Moore, Frank J.; Dholakia, Kishan

    2010-12-01

    Cell transfection is the process in which extra cellular nucleic acids such as DNA, RNA, Si-RNA can be deliberately injected into the cytoplasm of the cell. This technique of cell transfection forms a central tool in the hands of a cell biologist to explore the mechanism within the cell. In optical transfection a well focused laser spot alters the permeability of the cell membrane so as to allow the entry of extra-nuclear materials into the cell. Femto-second optical transfection have proved to be better than other laser based cell transfection, owing to the three dimensionally confined multi-photon effects on the cell membrane thereby leaving the rest of the cell unaffected. Even though the femto-second optical transfection has proved to be sterile, non-invasive and highly selective, it has to improve in terms of efficiency, and throughput to address real life problems. We report here a method to achieve significant enhancement in the efficiency of femto-second optical transfection. The protocol of the transfection procedure is modified by adding a suitable biochemical reagent - Nupherin-neuron - into the cell medium during the transfection, which can assist the delivery of DNA into the nucleus once the DNA gets injected into the cytoplasm of the cell. We achieved a 3 fold enhancement in the transfection efficiency with this modified protocol. Also we report for the first time the transfection of recently trypsinised cells with a very high transfection efficiency, which would pave way to the development of high throughput microfluidic optical transfection devices.

  19. Enhancement of the efficiency of femtosecond optical transfection

    NASA Astrophysics Data System (ADS)

    Praveen, Bavishna B.; Stevenson, David; Antkowiak, Maciej; Gunn-Moore, Frank J.; Dholakia, Kishan

    2011-08-01

    Cell transfection is the process in which extra cellular nucleic acids such as DNA, RNA, Si-RNA can be deliberately injected into the cytoplasm of the cell. This technique of cell transfection forms a central tool in the hands of a cell biologist to explore the mechanism within the cell. In optical transfection a well focused laser spot alters the permeability of the cell membrane so as to allow the entry of extra-nuclear materials into the cell. Femto-second optical transfection have proved to be better than other laser based cell transfection, owing to the three dimensionally confined multi-photon effects on the cell membrane thereby leaving the rest of the cell unaffected. Even though the femto-second optical transfection has proved to be sterile, non-invasive and highly selective, it has to improve in terms of efficiency, and throughput to address real life problems. We report here a method to achieve significant enhancement in the efficiency of femto-second optical transfection. The protocol of the transfection procedure is modified by adding a suitable biochemical reagent - Nupherin-neuron - into the cell medium during the transfection, which can assist the delivery of DNA into the nucleus once the DNA gets injected into the cytoplasm of the cell. We achieved a 3 fold enhancement in the transfection efficiency with this modified protocol. Also we report for the first time the transfection of recently trypsinised cells with a very high transfection efficiency, which would pave way to the development of high throughput microfluidic optical transfection devices.

  20. Plasma-mediated transfection of RPE

    NASA Astrophysics Data System (ADS)

    Palanker, D.; Chalberg, T.; Vankov, A.; Huie, P.; Molnar, F. E.; Butterwick, A.; Calos, M.; Marmor, M.; Blumenkranz, M. S.

    2006-02-01

    A major obstacle in applying gene therapy to clinical practice is the lack of efficient and safe gene delivery techniques. Viral delivery has encountered a number of serious problems including immunological reactions and malignancy. Non-viral delivery methods (liposomes, sonoporation and electroporation) have either low efficiency in-vivo or produce severe collateral damage to ocular tissues. We discovered that tensile stress greatly increases the susceptibility of cellular membranes to electroporation. For synchronous application of electric field and mechanical stress, both are generated by the electric discharge itself. A pressure wave is produced by rapid vaporization of the medium. To prevent termination of electric current by the vapor cavity it is ionized thus restoring its electric conductivity. For in-vivo experiments with rabbits a plasmid DNA was injected into the subretinal space, and RPE was treated trans-sclerally with an array of microelectodes placed outside the eye. Application of 250-300V and 100-200 μs biphasic pulses via a microelectrode array resulted in efficient transfection of RPE without visible damage to the retina. Gene expression was quantified and monitored using bioluminescence (luciferase) and fluorescence (GFP) imaging. Transfection efficiency of RPE with this new technique exceeded that of standard electroporation by a factor 10,000. Safe and effective non-viral DNA delivery to the mammalian retina may help to materialize the enormous potential of the ocular gene therapy. Future experiments will focus on continued characterization of the safety and efficacy of this method and evaluation of long-term transgene expression in the presence of phiC31 integrase.

  1. Congenital Insensitivity to Pain: Novel SCN9A Missense and In-Frame Deletion Mutations

    PubMed Central

    Cox, James J; Sheynin, Jony; Shorer, Zamir; Reimann, Frank; Nicholas, Adeline K; Zubovic, Lorena; Baralle, Marco; Wraige, Elizabeth; Manor, Esther; Levy, Jacov; Woods, C Geoffery; Parvari, Ruti

    2010-01-01

    SCN9A encodes the voltage-gated sodium channel Nav1.7, a protein highly expressed in pain-sensing neurons. Mutations in SCN9A cause three human pain disorders: bi-allelic loss of function mutations result in Channelopathy-associated Insensitivity to Pain (CIP), whereas activating mutations cause severe episodic pain in Paroxysmal Extreme Pain Disorder (PEPD) and Primary Erythermalgia (PE). To date, all mutations in SCN9A that cause a complete inability to experience pain are protein truncating and presumably lead to no protein being produced. Here, we describe the identification and functional characterization of two novel non-truncating mutations in families with CIP: a homozygously-inherited missense mutation found in a consanguineous Israeli Bedouin family (Nav1.7-R896Q) and a five amino acid in-frame deletion found in a sporadic compound heterozygote (Nav1.7-ΔR1370-L1374). Both of these mutations map to the pore region of the Nav1.7 sodium channel. Using transient transfection of PC12 cells we found a significant reduction in membrane localization of the mutant protein compared to the wild type. Furthermore, voltage clamp experiments of mutant-transfected HEK293 cells show a complete loss of function of the sodium channel, consistent with the absence of pain phenotype. In summary, this study has identified critical amino acids needed for the normal subcellular localization and function of Nav1.7. © 2010 Wiley-Liss, Inc. PMID:20635406

  2. Moisture insensitive primer: A myth or truth

    PubMed Central

    Shukla, Chandresh; Maurya, Rajkumar; Jain, Upendra; Gupta, Ankur; Garg, Jayshree

    2014-01-01

    Objectives: To compare the mean shear bond strength (SBS) of moisture insensitive primer (MIP) used for orthodontic bonding in the presence and absence of saliva. Materials and Methods: A total of 60 human noncarious maxillary premolars with sound buccal surfaces, recently extracted were collected in two groups of each 30. Maxillary premolar brackets were bonded to the teeth using light cure (Transbond XT, 3M Unitek, Monrovia, CA, USA) and MIP (Transbond MIP 3M Unitek, Monrovia, CA, USA,) in the presence and absence of saliva. Operators’ saliva was used during the bonding under moist condition. After debonding, all the specimens were examined under a stereomicroscope (×40 magnification) for adhesive remnant using adhesive remnant index (ARI). The SBS tests were done using Instron universal testing machine at cross-head speed of 1 mm/min, force passing parallel to the buccal surface using custom rod and registered in Newtons later converted into Megapascals. Results: Light cure and MIP (Transbond MIP and Transbond XT, 3M Unitek, Monrovia, CA, USA) in the absence of saliva showed higher mean SBS than the presence of saliva. Group I (light cure and MIP) in the absence of saliva showed mean SBS of 9.65 ± 0.90 Mpa. Group II (light cure and MIP) with the presence of saliva showed mean SBS of 9.03 ± 1.14 Mpa. The difference between both the groups was statistically significant, as confirmed by paired t-test (P < 0.05). In-Group I, ARI scores showed that more than half of the adhesive was left over the tooth surface, and Group II showed that there was no or insignificant amount of adhesive left over the tooth surface. Chi-square test revealed significant difference in debonding characteristics among the test groups of ARI (P < 0.05). Failure occurred mainly in resin– bracket base and resin – adhesive interfaces (χ² = 10.04, df = 3, P = 0.031). Conclusion: Moisture insensitive primer is effective in the presence/absence of moisture and has shown SBS value of more

  3. Testicular cancer in androgen insensitivity syndrome in a Mexican population.

    PubMed

    Aguilar-Ponce, José; Chilaca Rosas, Fátima; Molina Calzada, Carlos; Granados García, Martín; Jiménez Ríos, Miguel Angel; De la Garza Salazar, Jaime

    2008-12-01

    Male pseudohermaphroditism and androgen insensitivity syndrome cases have an increased risk of developing testicular cancer due to many factors such as mutations, hormonal disturbances involving gonadotropins and cryptorchidism. We describe the clinical features, diagnosis and treatment of two cases with partial androgen insensitivity syndrome and testicular cancer development, which were handled at the National Cancer Institute of Mexico.

  4. Transfection of mammalian cells using block copolypeptide vesicles.

    PubMed

    Sun, Victor Z; Choe, Uh-Joo; Rodriguez, April R; Dai, Howard; Deming, Timothy J; Kamei, Daniel T

    2013-05-01

    An arginine-leucine block copolypeptide (R60 L20 ) is synthesized, which is capable of forming vesicles with controllable sizes, able to transport hydrophilic cargo across the cell membrane, and exhibit relatively low cytotoxicity. The R60 L20 vesicles also possess the ability to deliver DNA into mammalian cells for transfection. Although the transfection efficiency is lower than that of the commercially available transfection agent Lipofectamine 2000, the R60 L20 vesicles are able to achieve transfection with significantly lower cytotoxicity and immunogenicity. This behavior is potentially due to its stronger interaction with DNA which subsequently provides better protection against anionic heparin.

  5. Polarization insensitive imaging through polarization gratings.

    PubMed

    Nersisyan, Sarik R; Tabiryan, Nelson V; Hoke, Landa; Steeves, Diane M; Kimball, Brian R

    2009-02-02

    Liquid crystal polarization gratings exhibit high diffraction efficiency (approximately 100%) in thin material layers comparable to the radiation wavelength. We demonstrate that they can be combined for polarization-insensitive imaging and optical switching applications. A pair of closely spaced, parallel oriented, cycloidal polarization gratings is capable of canceling the diffractive property of an individual grating. As a result, the phase of the beam is not distorted, and holographic images can be formed through them. An anti-parallel arrangement results in a broader effective diffraction band and doubles the diffraction angle. Broadband diffraction spanning from 480 nm to beyond 900 nm wavelengths has been obtained for a pair of gratings with 500 nm and 633 nm peak diffraction wavelengths. Liquid crystal polymer cycloidal gratings were used in the study showing 98% diffraction efficiency over a large area, and allowed for the use of laser beams expanded to 25 mm. The characteristics of combined cycloidal gratings were tested with laser beams at both UV and red wavelengths.

  6. Pain insensitivity syndrome misinterpreted as inflicted burns.

    PubMed

    van den Bosch, Gerbrich E; Baartmans, Martin G A; Vos, Paul; Dokter, Jan; White, Tonya; Tibboel, Dick

    2014-05-01

    We present a case study of a 10-year-old child with severe burns that were misinterpreted as inflicted burns. Because of multiple injuries since early life, the family was under suspicion of child abuse and therefore under supervision of the Child Care Board for 2 years before the boy was burned. Because the boy incurred the burns without feeling pain, we conducted a thorough medical examination and laboratory testing, evaluated detection and pain thresholds, and used MRI to study brain morphology and brain activation patterns during pain between this patient and 3 healthy age- and gender-matched controls. We found elevated detection and pain thresholds and lower brain activation during pain in the patient compared with the healthy controls and reference values. The patient received the diagnosis of hereditary sensory and autonomic neuropathy type IV on the basis of clinical findings and the laboratory testing, complemented with the altered pain and detection thresholds and MRI findings. Hereditary sensory and autonomic neuropathy IV is a very rare congenital pain insensitivity syndrome characterized by the absence of pain and temperature sensation combined with oral mutilation due to unawareness, fractures, and anhidrosis caused by abnormalities in the peripheral nerves. Health care workers should be aware of the potential presence of this disease to prevent false accusations of child abuse.

  7. pH-insensitive glucose indicators.

    PubMed

    Garrett, Jared R; Wu, Xinxin; Jin, Sha; Ye, Kaiming

    2008-01-01

    There is an urgent need for developing a biosensor that can real-time and noninvasively determine glucose concentration within living cells. In our previous study, we have engineered a glucose indicator protein (GIP) that can provide continuous glucose monitoring through a conformation change-induced Förster resonance-energy transfer measurement. Because of the pH-sensitivity of the fluorescent proteins used in the GIP construction, the GIP made from these fluorescent proteins is less tolerant to a pH change, especially to the acidic environment. It has been well documented that intracellular pH does not always remain the same, and it fluctuates in metabolism and other cellular activities and also differs between cellular compartments. To address these issues, we developed a GIP that can tolerate to pH change. This GIP was constructed by flanking a glucose binding protein with a cyan fluorescent protein and a pH-insensitive yellow fluorescent protein. Our experimental results indicated that the new GIP is more tolerant to pH change. The glucose response of this new GIP kept almost unchanged from pH 7.3 to 5.3, suggesting its capability of tolerating to acidic environment. This capability is desirable for intracellular glucose measurement.

  8. Radiation-Insensitive Inverse Majority Gates

    NASA Technical Reports Server (NTRS)

    Manohara, Harish; Mojarradi, Mohammad

    2008-01-01

    To help satisfy a need for high-density logic circuits insensitive to radiation, it has been proposed to realize inverse majority gates as microscopic vacuum electronic devices. In comparison with solid-state electronic devices ordinarily used in logic circuits, vacuum electronic devices are inherently much less adversely affected by radiation and extreme temperatures. The proposed development would involve state-of-the-art micromachining and recent advances in the fabrication of carbon-nanotube-based field emitters. A representative three-input inverse majority gate would be a monolithic, integrated structure that would include three gate electrodes, six bundles of carbon nanotubes (serving as electron emitters) at suitable positions between the gate electrodes, and an overhanging anode. The bundles of carbon nanotubes would be grown on degenerately doped silicon substrates that would be parts of the monolithic structure. The gate electrodes would be fabricated as parts of the monolithic structure by means of a double-silicon-on-insulator process developed at NASA's Jet Propulsion Laboratory. The tops of the bundles of carbon nanotubes would lie below the plane of the tops of the gate electrodes. The particular choice of shapes, dimensions, and relative positions of the electrodes and bundles of carbon nanotubes would provide for both field emission of electrons from the bundles of carbon nanotubes and control of the electron current to obtain the inverse majority function, which is described in the paper.

  9. Punishment Insensitivity in Early Childhood: A Developmental, Dimensional Approach.

    PubMed

    Nichols, Sara R; Briggs-Gowan, Margaret J; Estabrook, Ryne; Burns, James L; Kestler, Jacqueline; Berman, Grace; Henry, David B; Wakschlag, Lauren S

    2015-08-01

    Impairment in learning from punishment ("punishment insensitivity") is an established feature of severe antisocial behavior in adults and youth but it has not been well studied as a developmental phenomenon. In early childhood, differentiating a normal: abnormal spectrum of punishment insensitivity is key for distinguishing normative misbehavior from atypical manifestations. This study employed a novel measure, the Multidimensional Assessment Profile of Disruptive Behavior (MAP-DB), to examine the distribution, dimensionality, and external validity of punishment insensitivity in a large, demographically diverse community sample of preschoolers (3-5 years) recruited from pediatric clinics (N = 1,855). Caregivers completed surveys from which a seven-item Punishment Insensitivity scale was derived. Findings indicated that Punishment Insensitivity behaviors are relatively common in young children, with at least 50 % of preschoolers exhibiting them sometimes. Item response theory analyses revealed a Punishment Insensitivity spectrum. Items varied along a severity continuum: most items needed to occur "Often" in order to be severe and behaviors that were qualitatively atypical or intense were more severe. Although there were item-level differences across sociodemographic groups, these were small. Construct, convergent, and divergent validity were demonstrated via association to low concern for others and noncompliance, motivational regulation, and a disruptive family context. Incremental clinical utility was demonstrated in relation to impairment. Early childhood punishment insensitivity varies along a severity continuum and is atypical when it predominates. Implications for understanding the phenomenology of emergent disruptive behavior are discussed.

  10. Punishment insensitivity in early childhood: A developmental, dimensional approach

    PubMed Central

    Nichols, Sara R.; Briggs-Gowan, Margaret; Estabrook, Ryne; Burns, James; Kestler, Jacqueline; Berman, Grace; Henry, David; Wakschlag, Lauren

    2014-01-01

    Impairment in learning from punishment ("punishment insensitivity") is an established feature of severe antisocial behavior in adults and youth but it has not been well studied as a developmental phenomenon. In early childhood, differentiating a normal:abnormal spectrum of punishment insensitivity is key for distinguishing normative misbehavior from atypical manifestations. This study employed a novel measure, the Multidimensional Assessment Profile of Disruptive Behavior (MAPDB), to examine the distribution, dimensionality, and external validity of punishment insensitivity in a large, demographically diverse community sample of preschoolers (three-five years) recruited from pediatric clinics (N=1,855). Caregivers completed surveys from which a seven-item Punishment Insensitivity scale was derived. Findings indicated that Punishment Insensitivity behaviors are relatively common in young children, with at least 50% of preschoolers exhibiting them sometimes. Item response theory analyses revealed a Punishment Insensitivity spectrum. Items varied along a severity continuum: most items needed to occur "Often" in order to be severe and behaviors that were qualitatively atypical or intense were more severe. Although there were item-level differences across sociodemographic groups, these were small. Construct, convergent, and divergent validity were demonstrated via association to low concern for others and noncompliance, motivational regulation, and a disruptive family context. Incremental clinical utility was demonstrated in relation to impairment. Early childhood punishment insensitivity varies along a severity continuum and is atypical when it predominates. Implications for understanding the phenomenology of emergent disruptive behavior are discussed. PMID:25425187

  11. Enhanced operation of femtosecond lasers and applications in cell transfection.

    PubMed

    Brown, Christian T A; Stevenson, David J; Tsampoula, Xanthi; McDougall, Craig; Lagatsky, Alexander A; Sibbett, Wilson; Gunn-Moore, Frank J; Dholakia, Kishan

    2008-08-01

    In this work we present a review and discussion on the enhancement of femtosecond (fs) lasers for use within biophotonics with a particular focus on their use in optical transfection techniques. We describe the broad range of source options now available for the generation of femtosecond pulses before briefly reviewing the application of fs laser in optical transfection studies. We show that major performance enhancements may be obtained by optimising the spatial and temporal performance of the laser source before considering possible future directions in this field. In relation to optical transfection we describe how such laser sources initiate a multiphoton process to permeate the cell membrane in a transient fashion. We look at aspects of this technique including the ability to combine transfection with optical trapping. For future implementation of such transfection we explore the role of new sources and "nondiffracting" light fields.

  12. Temperature-insensitive phase-matched optical harmonic conversion crystal

    DOEpatents

    Barker, C.E.; Eimerl, D.; Velsko, S.P.; Roberts, D.

    1993-11-23

    Temperature-insensitive, phase-matched harmonic frequency conversion of laser light at a preferred wavelength of 1.064 microns can be achieved by use of a crystal of deuterated l-arginine phosphate. The crystal is cut and oriented so that the laser light propagates inside the crystal along one of several required directions, which correspond to a temperature-insensitive, phase-matching locus. The method of measuring and calculating the temperature-insensitive, phase-matching angles can be extended to other fundamental wavelengths and other crystal compositions. 12 figures.

  13. Temperature-insensitive phase-matched optical harmonic conversion crystal

    DOEpatents

    Barker, Charles E.; Eimerl, David; Velsko, Stephan P.; Roberts, David

    1993-01-01

    Temperature-insensitive, phase-matched harmomic frequency conversion of laser light at a preferred wavelength of 1.064 microns can be achieved by use of a crystal of deuterated l-arginine phosphate. The crystal is cut and oriented so that the laser light propagates inside the crystal along one of several required directions, which correspond to a temperature-insensitive, phase-matching locus. The method of measuring and calculating the temperature-insensitive, phase-matching angles can be extended to other fundamental wavelengths and other crystal compositions.

  14. Thermal Decomposition of IMX-104: Ingredient Interactions Govern Thermal Insensitivity

    SciTech Connect

    Maharrey, Sean; Wiese-Smith, Deneille; Highley, Aaron M.; Steill, Jeffrey D.; Behrens, Richard; Kay, Jeffrey J.

    2015-04-01

    This report summarizes initial studies into the chemical basis of the thermal insensitivity of INMX-104. The work follows upon similar efforts investigating this behavior for another DNAN-based insensitive explosive, IMX-101. The experiments described demonstrate a clear similarity between the ingredient interactions that were shown to lead to the thermal insensitivity observed in IMX-101 and those that are active in IMX-104 at elevated temperatures. Specifically, the onset of decomposition of RDX is shifted to a lower temperature based on the interaction of the RDX with liquid DNAN. This early onset of decomposition dissipates some stored energy that is then unavailable for a delayed, more violent release.

  15. Insensitive detonator apparatus for initiating large failure diameter explosives

    SciTech Connect

    Perry, III, William Leroy

    2015-07-28

    A munition according to a preferred embodiment can include a detonator system having a detonator that is selectively coupled to a microwave source that functions to selectively prime, activate, initiate, and/or sensitize an insensitive explosive material for detonation. The preferred detonator can include an explosive cavity having a barrier within which an insensitive explosive material is disposed and a waveguide coupled to the explosive cavity. The preferred system can further include a microwave source coupled to the waveguide such that microwaves enter the explosive cavity and impinge on the insensitive explosive material to sensitize the explosive material for detonation. In use the preferred embodiments permit the deployment and use of munitions that are maintained in an insensitive state until the actual time of use, thereby substantially preventing unauthorized or unintended detonation thereof.

  16. Insensitive explosive composition of halogenated copolymer and triaminotrinitrobenzene

    DOEpatents

    Benziger, Theodore M.

    1976-01-01

    A highly insensitive and heat resistant plastic-bonded explosive containing 90 wt % triaminotrinitrobenzene and 10 wt % of a fully saturated copolymer of chlorotrifluoroethylene and vinylidene fluoride is readily manufactured by the slurry process.

  17. Hydrophobic Moiety of Cationic Lipids Strongly Modulates Their Transfection Activity

    SciTech Connect

    Koynova, Rumiana; Tenchov, Boris; Wang, Li; MacDonald, Robert C.

    2010-01-18

    Synthetic cationic lipids are widely used components of nonviral gene carriers, and the factors regulating their transfection efficiency are the subject of considerable interest. In view of the important role that electrostatic interactions with the polyanionic nucleic acids play in formation of lipoplexes, a common empirical approach to improving transfection has been the synthesis and testing of amphiphiles with new versions of positively charged polar groups, while much less attention has been given to the role of the hydrophobic lipid moieties. On the basis of data for {approx}20 cationic phosphatidylcholine (PC) derivatives, here we demonstrate that hydrocarbon chain variations of these lipids modulate by over 2 orders of magnitude their transfection efficiency. The observed molecular structure-activity relationship manifests in well-expressed dependences of activity on two important molecular characteristics, chain unsaturation and total number of carbon atoms in the lipid chains, which is representative of the lipid hydrophobic volume and hydrophilic-lipophilic ratio. Transfection increases with decrease of chain length and increase of chain unsaturation. Maximum transfection was found for cationic PCs with monounsaturated 14:1 chains. It is of particular importance that the high-transfection lipids strongly promote cubic phase formation in zwitterionic membrane phosphatidylethanolamine (PE). These remarkable correlations point to an alternative, chain-dependent process in transfection, not related to the electrostatic cationic-anionic lipid interactions.

  18. [Transfection of HL-60 cells by Venus lentiviral vector].

    PubMed

    Li, Zheng; Hu, Shao-Yan; Cen, Jian-Nong; Chen, Zi-Xing

    2013-06-01

    In order to study the potential of Venus, lentiviral vector, applied to acute myeloid leukemia, the recombinant vector Venus-C3aR was transfected into 293T packing cells by DNA-calcium phosphate coprecipitation. All virus stocks were collected and transfected into HL-60, the GFP expression in HL-60 cells was measured by flow cytometry. The expression level of C3aR1 in transfected HL-60 cells was identified by RT-PCR and flow cytometry. The lentiviral toxicity on HL-60 was measured by using CCK-8 method and the ability of cell differentiation was observed. The results indicated that the transfection efficacy of lentiviral vector on HL-60 cells was more than 95%, which meets the needs for further study. C3aR1 expression on HL-60 cells increased after being transfected with recombinant lentiviral vector. Before and after transfection, the proliferation and differentiation of cells were not changed much. It is concluded that the lentiviral vector showed a high efficacy to transfect AML cells and can be integrated in genome of HL-60 cells to realize the stable expression of interest gene. Meanwhile, lentiviral vector can not affect HL-60 cell ability to proliferate and differentiate.

  19. Transfection of Lactobacillus bulgaricus protoplasts by bacteriophage DNA.

    PubMed

    Boizet, B; Flickinger, J L; Chassy, B M

    1988-12-01

    A protoplast transfection system has been developed for Lactobacillus bulgaricus. The procedure involves a polyethylene glycol-mediated fusion of bacteriophage DNA encapsulated in liposomes into mutanolysin-treated cells. With L. bulgaricus B004 and DNA isolated from the phage phi c5004, transfection reached a maximum when at least 95% of the cells were osmotically fragile. The incorporation of phage DNA into liposomes was essential; no transfectants were detected in the absence of liposomes. The largest number of transfectants was observed after longer periods (20 min) of fusion of mutanolysin-treated cells and liposomes with polyethylene glycol. The maximum efficiency of 5 x 10(7) PFU/microgram of DNA was reached after a 24-h incubation in growth media prior to plating transfected cells in an agar overlay to detect the appearance of plaques. A minimum of 4 h of incubation in growth medium after fusion was required to detect the production and release of virions. The possibility that the high frequencies observed were due to bursting of transfected cells and subsequent infection of additional cells was found not to be a factor. The number of transfectants observed was directly proportional to the quantity of DNA added. These results define conditions appropriate for the introduction of DNA into L. bulgaricus.

  20. A reverse transfection technology to genetically engineer adult stem cells.

    PubMed

    Okazaki, Arimichi; Jo, Jun-Ichiro; Tabata, Yasuhiko

    2007-02-01

    A new non-viral method of gene transfection was designed to enhance the level of gene expression for rat mesenchymal stem cells (MSCs). Pullulan was cationized using chemical introduction of spermine to prepare cationized pullulan of non-viral carrier (spermine-pullulan). The spermine-pullulan was complexed with a plasmid deoxyribonucleic acid (DNA) of luciferase and coated on the surface of culture substrate together with Pronectin of artificial cell adhesion protein. MSCs were cultured and transfected on the complex-coated substrate (reverse transfection), and the level and duration of gene expression were compared with those of MSCs transfected by culturing in the medium containing the plasmid DNA-spermine-pullulan complex (conventional method). The reverse transfection method enhanced and prolonged gene expression significantly more than did the conventional method. The reverse method permitted the transfection culture of MSCs in the presence of serum, in contrast to the conventional method, which gave cells a good culture condition to lower cytotoxicity. The reverse transfection was carried out for a non-woven fabric of polyethylene terephthalate (PET) coated with the complex and Pronectin using agitation and stirring culture methods. The two methods enhanced the level and duration of gene expression for MSCs significantly more than did the static method. It is possible that medium circulation improves the culture conditions of cells in terms of oxygen and nutrition supply and waste excretion, resulting in enhanced gene expression.

  1. Neurophysiologic studies in congenital insensitivity to pain with anhidrosis.

    PubMed

    Shorer, Z; Moses, S W; Hershkovitz, E; Pinsk, V; Levy, J

    2001-11-01

    Thirteen patients with congenital insensitivity to pain and anhidrosis, carrying a mutation at the TRK-A gene, were studied. Neurologic examination revealed vestigial pain sensitivity, suggesting an incomplete involvement of the affected nerves. All 13 patients manifested normal electrophysiologic studies but striking absence of sympathetic skin responses. We suggest the use of the sympathetic skin response test in the clinical evaluation of patients suspected of having congenital insensitivity to pain and anhidrosis.

  2. [VEGF gene expression in transfected human multipotent stromal cells].

    PubMed

    Smirnikhina, S A; Lavrov, A V; Bochkov, N P

    2011-01-01

    Dynamics of VEGF gene expression in transfected multipotent stromal cells from adipose tissue was examined using electroporation and lipofection. Differences in the potency and dynamics of plasmid elimination (up to day 9) between cell cultures were observed. All cultures were divided into fast and slow plasmid-eliminating ones. Interculture differences in VEGF expression were detected. The possibility of a 5-6-fold increase of VEGF expression was shown. There were no differences in transfection potency, plasmid elimination dynamics, and VEGF expression after transfection by both nonviral methods.

  3. Lipid-based transfection reagents can interfere with cholesterol biosynthesis.

    PubMed

    Danielli, Mauro; Marinelli, Raúl A

    2016-02-15

    Lipid-based transfection reagents are widely used for delivery of small interfering RNA into cells. We examined whether the commonly used commercial transfection reagents DharmaFECT-4 and Lipofectamine 2000 can interfere with lipid metabolism by studying cholesterogenesis. Cholesterol de novo synthesis from [(14)C]acetate was assessed in human hepatocyte-derived Huh-7 cells. The results revealed that DharmaFECT, but not Lipofectamine, markedly inhibited cholesterol biosynthesis by approximately 70%. Cell viability was not significantly altered. These findings suggest that caution is required in the choice of certain lipid-based transfection reagents for gene silencing experiments, particularly when assessing cholesterol metabolism.

  4. Reverse transfected cell microarrays in infectious disease research.

    PubMed

    Konrad, Andreas; Jochmann, Ramona; Kuhn, Elisabeth; Naschberger, Elisabeth; Chudasama, Priya; Stürzl, Michael

    2011-01-01

    Several human pathogenic viruses encode large genomes with often more than 100 genes. Viral pathogenicity is determined by carefully orchestrated co-operative activities of several different viral genes which trigger the phenotypic functions of the infected cells. Systematic analyses of these complex interactions require high-throughput transfection technology. Here we have provided a laboratory manual for the reverse transfected cell microarray (RTCM; alternative name: cell chip) as a high-throughput transfection procedure, which has been successfully applied for the systematic analyses of single and combination effects of genes encoded by the human herpesvirus-8 on the NF-kappaB signal transduction pathway. In order to quantitatively determine the effects of viral genes in transfected cells, protocols for the use of GFP as an indicator gene and for indirect immunofluorescence staining of cellular target proteins have been included. RTCM provides a useful methodological approach to investigate systematically combination effects of viral genes on cellular functions.

  5. Congenital insensitivity to pain: novel SCN9A missense and in-frame deletion mutations.

    PubMed

    Cox, James J; Sheynin, Jony; Shorer, Zamir; Reimann, Frank; Nicholas, Adeline K; Zubovic, Lorena; Baralle, Marco; Wraige, Elizabeth; Manor, Esther; Levy, Jacov; Woods, C Geoffery; Parvari, Ruti

    2010-09-01

    SCN9Aencodes the voltage-gated sodium channel Na(v)1.7, a protein highly expressed in pain-sensing neurons. Mutations in SCN9A cause three human pain disorders: bi-allelic loss of function mutations result in Channelopathy-associated Insensitivity to Pain (CIP), whereas activating mutations cause severe episodic pain in Paroxysmal Extreme Pain Disorder (PEPD) and Primary Erythermalgia (PE). To date, all mutations in SCN9A that cause a complete inability to experience pain are protein truncating and presumably lead to no protein being produced. Here, we describe the identification and functional characterization of two novel non-truncating mutations in families with CIP: a homozygously-inherited missense mutation found in a consanguineous Israeli Bedouin family (Na(v)1.7-R896Q) and a five amino acid in-frame deletion found in a sporadic compound heterozygote (Na(v)1.7-DeltaR1370-L1374). Both of these mutations map to the pore region of the Na(v)1.7 sodium channel. Using transient transfection of PC12 cells we found a significant reduction in membrane localization of the mutant protein compared to the wild type. Furthermore, voltage clamp experiments of mutant-transfected HEK293 cells show a complete loss of function of the sodium channel, consistent with the absence of pain phenotype. In summary, this study has identified critical amino acids needed for the normal subcellular localization and function of Na(v)1.7.

  6. Histone H2A significantly enhances in vitro DNA transfection.

    PubMed Central

    Balicki, D.; Beutler, E.

    1997-01-01

    BACKGROUND: Gene transfer is a potential treatment modality of genetic disease. Efficient, practical methods of DNA transfection are currently under investigation. MATERIALS AND METHODS: A beta-galactosidase reporter plasmid interacted electrostatically with histones, poly-L-Lys, poly-L-Arg, and a combination of poly-L-Lys and poly-L-Arg. This complex was then used to transfect COS-7 cells. beta-galactosidase activity was quantified and used to compare the efficiency of gene transfection in vitro. A comparison was also made of DNA transfection with the most active histone subclass, i.e., histone H2A, in the absence and presence of an anionic liposome. RESULTS: There was a marked increase in DNA transfection in the presence of histone H2A when compared with the control, whereas each of the other histones and polycations showed little, if any, effect. The extent of activation depends strongly on the DNA/histone ratio and is also a function of the molarity of the final Tris-acetate, pH 8, solution. The anionic liposomes used demonstrated an inhibitory effect. CONCLUSIONS: Histone H2A significantly enhances in vitro DNA transfection whereas other histones and anionic liposomes do not. A study of the difference between histone H2A and other histone subclasses may serve to clarify some of the mechanisms and the essential components of efficient gene delivery. PMID:9407553

  7. Genotype versus phenotype in families with androgen insensitivity syndrome.

    PubMed

    Boehmer, A L; Brinkmann, O; Brüggenwirth, H; van Assendelft, C; Otten, B J; Verleun-Mooijman, M C; Niermeijer, M F; Brunner, H G; Rouwé, C W; Waelkens, J J; Oostdijk, W; Kleijer, W J; van der Kwast, T H; de Vroede, M A; Drop, S L

    2001-09-01

    Androgen insensitivity syndrome encompasses a wide range of phenotypes, which are caused by numerous different mutations in the AR gene. Detailed information on the genotype/phenotype relationship in androgen insensitivity syndrome is important for sex assignment, treatment of androgen insensitivity syndrome patients, genetic counseling of their families, and insight into the functional domains of the AR. The commonly accepted concept of dependence on fetal androgens of the development of Wolffian ducts was studied in complete androgen insensitivity syndrome (CAIS) patients. In a nationwide survey in The Netherlands, all cases (n = 49) with the presumptive diagnosis androgen insensitivity syndrome known to pediatric endocrinologists and clinical geneticists were studied. After studying the clinical phenotype, mutation analysis and functional analysis of mutant receptors were performed using genital skin fibroblasts and in vitro expression studies. Here we report the findings in families with multiple affected cases. Fifty-nine percent of androgen insensitivity syndrome patients had other affected relatives. A total of 17 families were studied, seven families with CAIS (18 patients), nine families with partial androgen insensitivity (24 patients), and one family with female prepubertal phenotypes (two patients). No phenotypic variation was observed in families with CAIS. However, phenotypic variation was observed in one-third of families with partial androgen insensitivity resulting in different sex of rearing and differences in requirement of reconstructive surgery. Intrafamilial phenotypic variation was observed for mutations R846H, M771I, and deletion of amino acid N682. Four newly identified mutations were found. Follow-up in families with different AR gene mutations provided information on residual androgen action in vivo and the development of the prepubertal and adult phenotype. Patients with a functional complete defective AR had some pubic hair, Tanner

  8. Slipped Distal Femoral Epiphysis in Congenital Insensitivity to Pain

    PubMed Central

    Shaikh, M Javed; Rex, C; Vignesh, R; Chavan, Madhav

    2016-01-01

    Introduction: Congenital insensitivity to pain (CIP) is a rare disorder, and often presents to an orthopaedic surgeon as recurrent fractures, dislocations, pseudoarthrosis, osteomyelitis etc. Here, we report a case of congenital insensitivity to pain presenting with distal femoral physeal separation in a child. Case Report: A 12-year-old girl child came with complaints of limp while walking and swelling in the left knee for past 5 weeks. Mother gave a history that the girl is a known case of congenital insensitivity to pain with clear history of no pain on intramuscular injection since birth. She was born of consanguineous marriage and had no significant trauma, fever, other joint involvement or any features of rheumatism. On local examination, she had no bony tenderness, mild warmth, and moderate knee effusion with restricted range of movement. Plain radiograph showed epiphysiolysis of distal femur with widening of physis. Examination under anaesthesia demonstrated gross movement indicating lower femoral physeal separation. This unstable distal femoral epiphysis was treated under general anaesthesia with closed reduction, percutaneous cross pinning and above knee plaster cast. Conclusion: Congenital insensitivity to pain (CIP) is a rare disorder to diagnose. Emphasis is given on early diagnosis of orthopaedic problems and prompt treatment, educating parents and prevention of accidents. Physeal separation without significant trauma must prompt an orthopaedic surgeon to think about congenital insensitivity to pain as a differential diagnosis. PMID:27703943

  9. Electroweak Symmetry Breaking via UV Insensitive Anomaly Mediation

    SciTech Connect

    Kitano, Ryuichiro; Kribs, Graham D.; Murayama, Hitoshi

    2004-02-19

    Anomaly mediation solves the supersymmetric flavor and CP problems. This is because the superconformal anomaly dictates that supersymmetry breaking is transmitted through nearly flavor-blind infrared physics that is highly predictive and UV insensitive. Slepton mass squareds, however, are predicted to be negative. This can be solved by adding D-terms for U(1)_Y and U(1)_{B-L} while retaining the UV insensitivity. In this paper we consider electroweak symmetry breaking via UV insensitive anomaly mediation in several models. For the MSSM we find a stable vacuum when tanbeta< 1, but in this region the top Yukawa coupling blows up only slightly above the supersymmetry breaking scale. For the NMSSM, we find a stable electroweak breaking vacuum but with a chargino that is too light. Replacing the cubic singlet term in the NMSSM superpotential with a term linear in the singlet wefind a stable vacuum and viable spectrum. Most of the parameter region with correct vacua requires a large superpotential coupling, precisely what is expected in the"Fat Higgs'" model in which the superpotential is generated dynamically. We have therefore found the first viable UV complete, UV insensitive supersymmetry breaking model that solves the flavor and CP problems automatically: the Fat Higgs model with UV insensitive anomaly mediation. Moreover, the cosmological gravitino problem is naturally solved, opening up the possibility of realistic thermal leptogenesis.

  10. Bichromatic state-insensitive trapping of caesium atoms

    NASA Astrophysics Data System (ADS)

    Metbulut, M. M.; Renzoni, F.

    2015-12-01

    State-insensitive dipole trapping of multilevel atoms can be achieved by an appropriate choice of the wavelength of the trapping laser, so that the interaction with the different transitions results in equal AC Stark shifts for the ground and excited states of interest. However, this approach is severely limited by the availability of coherent sources at the required wavelength and of appropriate power. This work investigates state-insensitive trapping of caesium atoms for which the required wavelength of 935.6 nm is inconvenient in terms of experimental realization. Bichromatic state-insensitive trapping is proposed to overcome the lack of suitable laser sources. We first consider pairs of laser wavelengths in the ratio 1:2 and 1:3, as obtained via second- and third-harmonic generation. We found that the wavelength combinations 931.8-1863.6 nm and 927.5-2782.5 nm are suitable for state-insensitive trapping of caesium atoms. In addition, we examine bichromatic state-insensitive trapping produced by pairs of laser wavelengths corresponding to currently available high-power lasers. These wavelength pairs were found to be in the range of 585-588 nm and 623-629 for one laser and 1064-1080 nm for the other.

  11. Recent developments in bend-insensitive and ultra-bend-insensitive fibers

    NASA Astrophysics Data System (ADS)

    Boivin, David; de Montmorillon, Louis-Anne; Provost, Lionel; Montaigne, Nelly; Gooijer, Frans; Aldea, Eugen; Jensma, Jaap; Sillard, Pierre

    2010-02-01

    Designed to overcome the limitations in case of extreme bending conditions, Bend- and Ultra-Bend-Insensitive Fibers (BIFs and UBIFs) appear as ideal solutions for use in FTTH networks and in components, pigtails or patch-cords for ever demanding applications such as military or sensing. Recently, however, questions have been raised concerning the Multi-Path-Interference (MPI) levels in these fibers. Indeed, they are potentially subject to interferences between the fundamental mode and the higher-order mode that is also bend resistant. This MPI is generated because of discrete discontinuities such as staples, bends and splices/connections that occur on distance scales that become comparable to the laser coherent length. In this paper, we will demonstrate the high MPI tolerance of all-solid single-trench-assisted BIFs and UBIFs. We will present the first comprehensive study combining theoretical and experimental points of view to quantify the impact of fusion splices on coherent MPI. To be complete, results for mechanical splices will also be reported. Finally, we will show how the single-trench- assisted concept combined with the versatile PCVD process allows to tightly control the distributions of fibers characteristics. Such controls are needed to massively produce BIFs and to meet the more stringent specifications of the UBIFs.

  12. Congenital insensitivity to pain with anhidrosis presenting with palmoplantar keratoderma.

    PubMed

    Sayyahfar, Shirin; Chavoshzadeh, Zahra; Khaledi, Mojdeh; Madadi, Firooz; Yeganeh, Mehrnoosh Hassas; Sawamura, Daisuke; Nakano, Hajime; Rezaei, Nima

    2013-01-01

    Congenital insensitivity to pain with anhidrosis (CIPA) is a rare autosomal-recessive disease caused by mutations in the NTRK1 gene. The disease is characterized by insensitivity to pain and absence of thermal perception. Herein a 6-year-old boy is presented with a large ulcer on the sole of his right foot and a thick, hyperkeratotic appearance of his palms and soles; there was also a medical history of hyperthermia, anhidrosis, recurrent bone fractures, osteomyelitis, injuries, mental retardation, dry and exfoliative skin, insensitivity to pain, and lack of thermal sensation. Genetic studies revealed a homozygote mutation in the NTRK1 gene. Although the patient initially presented with palmoplantar keratoderma, genetic studies confirmed the diagnosis of CIPA.

  13. Family of detuning-insensitive phase-shifting algorithms.

    PubMed

    Malacara-Doblado, D; Dorrío, B V

    2000-10-01

    The design of phase-shifting algorithms (PSA's) has been carried out with diverse strategies by different authors. A generalized algebraic approach is employed to obtain a family of detuning-insensitive PSA's; their behavior against a linear phase error is analyzed from a geometric point of view. The obtained results are compared with the conditions provided by the Fourier representation of the corresponding sampling reference functions. In our case, new equations as criteria for determining whether a PSA is detuning insensitive, new analytic expressions for the phase error, and new algorithms with interesting properties are achieved.

  14. Femtosecond cellular transfection using a non-diffracting beam

    NASA Astrophysics Data System (ADS)

    Tsampoula, X.; Garcés-Chávez, V.; Comrie, M.; Stevenson, D. J.; Agate, B.; Brown, C. T. A.; Gunn-Moore, F.; Dholakia, K.

    2008-02-01

    Efficient DNA delivery into single living cells would be a very powerful capability for cell biologists for elucidating basic cellular functions but also in other fields such as applied drug discovery and gene therapy. The ability to gently permeate the cell membrane and introduce foreign DNA with the assistance of lasers is a powerful methodology but requires exact focusing due to the required two-photon power density. Here, we demonstrate a laser-mediated delivery method of the red fluorescent protein DS-RED into Chinese hamster Ovary (CHO) cells. We used an elongated beam of light created by a Bessel beam (BB) which obviates the need to locate precisely the cell membrane, permitting two-photon excitation along a line leading to cell transfection. Assuming a threshold for transfection of 20%, the BB gives us transfection over twenty times the axial distance compared to the Gaussian beam of equivalent core diameter. In addition, by exploiting the BB property of reconstruction, we demonstrate successful transfection of CHO cells which involves the BB passing through an obstructive layer and re forming itself prior to reaching the cell membrane. In the light of this exciting result, one can envisage the possibility of achieving transfection through multiple cell monolayer planes and tissues using this novel light field, eliminating this way the stringent requirements for tight focusing.

  15. Synthesis of linear polyethylenimine derivatives for DNA transfection.

    PubMed

    Brissault, Blandine; Kichler, Antoine; Guis, Christine; Leborgne, Christian; Danos, Olivier; Cheradame, Hervé

    2003-01-01

    A series of linear polymers containing varying amounts of ethylenimine or N-propylethylenimine units were synthesized by hydrolysis and/or reduction of polyethyloxazolines. The pK(a)s of the polyamines were determined potentiometrically. Gel mobility shift assay showed that the efficiency of DNA complexation was related to the fraction of amino groups that are protonated at neutral pH. The effects of cationic charge density and molar weight of the polymers on the transfection efficiency were evaluated on HepG2 cells. The results obtained with different copolymers show that the transfection efficiency primarily depends on the fraction of ethylenimine units included in the polymer albeit the molar weight is also of importance. On the basis of the results obtained with poly(N-propylethylenimines), we also demonstrate that the high transfection efficiency of polyethylenimines does not solely rely on their capacity to capture protons which are transferred into the endo-lysosomes during acidification.

  16. In trans promoter activation by enhancers in transient transfection.

    PubMed

    Smirnov, N A; Akopov, S B; Didych, D A; Nikolaev, L G

    2017-03-01

    Earlier, it was reported that the strong cytomegalovirus enhancer can activate the cytomegalovirus promoter in trans, i.e. as a separate plasmid co-transfected with a promoter-reporter gene construct. Here we demonstrate that the ability of enhancers to activate promoters in trans in transient transfection experiments is a property of not only viral regulatory elements but also of various genomic enhancers and promoters. Enhancer-promoter activation in trans is promoter- and cell type-specific, and accompanied by physical interaction between promoter and enhancer as revealed by chromosome conformation capture assays. Thus, promoter activation in transient co-transfection of promoters and enhancers shares a number of important traits with long-distance promoter activation by enhancers in living cells and may therefore serve as a model of this fundamental cellular process.

  17. 96-well electroporation method for transfection of mammalian central neurons.

    PubMed

    Buchser, William J; Pardinas, Jose R; Shi, Yan; Bixby, John L; Lemmon, Vance P

    2006-11-01

    Manipulating gene expression in primary neurons has been a goal for many scientists for over 20 years. Vertebrate central nervous system neurons are classically difficult to transfect. Most lipid reagents are inefficient and toxic to the cells, and time-consuming methods such as viral infections are often required to obtain better efficiencies. We have developed an efficient method for the transfection of cerebellar granule neurons and hippocampal neurons with standard plasmid vectors. Using 96-well electroporation plates, square-wave pulses can introduce 96 different plasmids into neurons in a single step. The procedure results in greater than 20% transfection efficiencies and requires only simple solutions of nominal cost. In addition to enabling the rapid optimization of experimental protocols with multiple parameters, this procedure enables the use of high content screening methods to characterize neuronal phenotypes.

  18. 96-Well electroporation method for transfection of mammalian central neurons

    PubMed Central

    Buchser, William J.; Pardinas, Jose R.; Shi, Yan; Bixby, John L.; Lemmon, Vance P.

    2008-01-01

    Manipulating gene expression in primary neurons has been a goal for many scientists for over 20 years. Vertebrate central nervous system neurons are classically difficult to transfect. Most lipid reagents are inefficient and toxic to the cells, and time-consuming methods such as viral infections are often required to obtain better efficiencies. We have developed an efficient method for the transfection of cerebellar granule neurons and hippocampal neurons with standard plasmid vectors. Using 96-well electroporation plates, square-wave pulses can introduce 96 different plasmids into neurons in a single step. The procedure results in greater than 20% transfection efficiencies and requires only simple solutions of nominal cost. In addition to enabling the rapid optimization of experimental protocols with multiple parameters, this procedure enables the use of high content screening methods to characterize neuronal phenotypes. PMID:17140120

  19. Androgen insensitivity syndrome and Klinefelter's syndrome: sex and gender considerations.

    PubMed

    Diamond, Milton; Watson, Linda Ann

    2004-07-01

    Two of the most common intersex conditions, androgen insensitivity and Klinefelter's syndrome, are described with an emphasis on aspects that are of relevance to psychiatrists. Attention is focused on commonalities and differences between these syndromes and particular attention is given to how persons who have these conditions manifest sexual and gender adjustments to their situations. Tips on counseling and medical management are offered.

  20. Extrinsic Michelson interferometric fibre optic sensor with bend insensitive downlead

    NASA Astrophysics Data System (ADS)

    Hand, D. P.; Carolan, T. A.; Barton, J. S.; Jones, J. D. C.

    1993-04-01

    A novel optical arrangement is described for an interferometric optical fibre sensor of the extrinsic type. Based on a Michelson interferometer, it combines a bend insensitive downlead with the availability of antiphase outputs without insertion loss, and provides isolation of the source.

  1. Congenital insensitivity to pain: report of two cases.

    PubMed

    Lawoyin, J O; Lawoyin, D O

    2001-01-01

    Congenital indifference or insensitivity to pain (CIP) is a rare syndrome. It mimics a number of other syndromes categorized under peripheral sensory neuropathies, often making early diagnosis difficult. Two cases from the middle east are presented, highlighting possible diagnostic, and management difficulties.

  2. Scope insensitivity in contingent valuation of complex environmental amenities.

    PubMed

    Veisten, Knut; Fredrik Hoen, Hans; Navrud, Ståle; Strand, Jon

    2004-12-01

    It has been argued that respondents in contingent valuation (CV) surveys, asked to value complex environmental amenities, will state willingness to pay (WTP) independently of the scope of the project. Such insensitivity to scope would be at odds with rational choice, and could therefore imply that CV is not a theoretically valid method for biodiversity valuation. The scope test in the present CV study was applied to endangered species preservation. Respondents were split in four sub-samples facing different scopes of endangered species preservation. The design allowed for both external and internal scope tests. Furthermore, the tests were split according to elicitation format. Of four external tests of insensitivity to scope, one was rejected, two gave mixed results, depending on either the type of test or elicitation format, and for the last one the null hypothesis could not be rejected. Of five internal tests, insensitivity to scope was rejected in three cases, one test gave mixed results, and one could not be rejected. Survey design features of the CV study, especially an unfamiliar sub-group of endangered species, could explain the apparent insensitivity to scope observed.

  3. Punishment insensitivity and impaired reinforcement learning in preschoolers

    PubMed Central

    Briggs-Gowan, Margaret J.; Nichols, Sara R.; Voss, Joel; Zobel, Elvira; Carter, Alice S.; McCarthy, Kimberly J.; Pine, Daniel S.; Blair, James; Wakschlag, Lauren S.

    2013-01-01

    Background Youth and adults with psychopathic traits display disrupted reinforcement learning. Advances in measurement now enable examination of this association in preschoolers. The current study examines relations between reinforcement learning in preschoolers and parent ratings of reduced responsiveness to socialization, conceptualized as a developmental vulnerability to psychopathic traits. Methods 157 preschoolers (mean age 4.7 ±0.8 years) participated in a substudy that was embedded within a larger project. Children completed the “Stars-in-Jars” task, which involved learning to select rewarded jars and avoid punished jars. Maternal report of responsiveness to socialization was assessed with the Punishment Insensitivity and Low Concern for Others scales of the Multidimensional Assessment of Preschool Disruptive Behavior (MAP-DB). Results Punishment Insensitivity, but not Low Concern for Others, was significantly associated with reinforcement learning in multivariate models that accounted for age and sex. Specifically, higher Punishment Insensitivity was associated with significantly lower overall performance and more errors on punished trials (“passive avoidance”). Conclusions Impairments in reinforcement learning manifest in preschoolers who are high in maternal ratings of Punishment Insensitivity. If replicated, these findings may help to pinpoint the neurodevelopmental antecedents of psychopathic tendencies and suggest novel intervention targets beginning in early childhood. PMID:24033313

  4. Structure-activity relationship in cationic lipid mediated gene transfection.

    PubMed

    Niculescu-Duvaz, Dan; Heyes, James; Springer, Caroline J

    2003-07-01

    Non-viral synthetic vectors for gene delivery represent a safer alternative to viral vectors. Their main drawback is the low transfection efficiency, especially in vivo. Among the non-viral vectors currently in use, the cationic liposomes composed of cationic lipids are the most common. This review discusses the physicochemical properties of cationic lipids, the formation, macrostructure and specific parameters of the corresponding formulated liposomes, and the effect of all these parameters on transfection efficiency. The optimisation of liposomal vectors requires both the understanding of the biological variables involved in the transfection process, and the effect of the structural elements of the cationic lipids on these biological variables. The biological barriers relevant for in vitro and in vivo transfection are identified, and solutions to overcome them based on rational design of the cationic lipids are discussed. The review focuses on the relationship between the structure of the cationic lipid and the transfection activity. The structure is analysed in a modular manner. The hydrophobic domain, the cationic head group, the backbone that acts as a scaffold for the other domains, the linkers between backbone, hydrophobic domain and cationic head group, the polyethyleneglycol chains and the targeting moiety are identified as distinct elements of the cationic lipids used in gene therapy. The main chemical functionalities used to built these domains, as well as overall molecular features such as architecture and geometry, are presented. Studies of structure-activity relationships of each cationic lipid domain, including the authors', and the trends identified by these studies, help furthering the understanding of the mechanism governing the formation and behaviour of cationic liposomes in gene delivery, and therefore the rational design of new improved cationic lipids vectors capable of achieving clinical significance.

  5. Two novel SCN9A mutations causing insensitivity to pain.

    PubMed

    Nilsen, K B; Nicholas, A K; Woods, C G; Mellgren, S I; Nebuchennykh, M; Aasly, J

    2009-05-01

    The sensation of pain is important and there may be serious consequences if it is missing. Recently, the genetic basis for a channelopathy characterised by a congenital inability to experience pain has been described and channelopathy-associated insensitivity to pain has been proposed as a suitable name for this condition. Different mutations in the SCN9A gene causing loss of function of the voltage-gated sodium channel Nav1.7 have been reported in patients with this rare disease. Here we describe a woman with insensitivity to pain with two novel mutations in the SCN9A gene, coding for the Nav1.7 channel. We also discuss the finding of anosmia which apparently is a common feature in these patients.

  6. Gender dysphoria and gender change in androgen insensitivity or micropenis.

    PubMed

    Mazur, Tom

    2005-08-01

    This review article answers three questions relevant to the medical management and care of individuals born with complete androgen insensitivity syndrome (CAIS), partial androgen insensitivity syndrome (PAIS), or a micropenis: (1) Do any of these individuals reassign themselves from their initial gender assignment? (2) Do more reassign than the ones who do not? (3) Is there evidence of gender dysphoria in those who do not self-initiate reassignment? Reviewed were all articles on CAIS, PAIS, and micropenis cited in K. J. Zucker (1999) plus articles published through 2004. There were no documented cases of gender change in individuals with CAIS (N= 156 females) or micropenis (N= 89: 79 males, 10 females). Nine (9.1%) out of 99 individuals with PAIS changed gender. Thus, self-initiated gender reassignment was rare. Gender dysphoria also appears to be a rare occurrence. The best predictor of adult gender identity in CAIS, PAIS, and micropenis is initial gender assignment.

  7. Angle Insensitive Color Filters in Transmission Covering the Visible Region

    PubMed Central

    Mao, Kening; Shen, Weidong; Yang, Chenying; Fang, Xu; Yuan, Wenjia; Zhang, Yueguang; Liu, Xu

    2016-01-01

    Angle insensitive color filter based on Metal-SiOx-Metal structure is proposed in this paper, which can keep the same perceived transmitted color when the incidence angle changes from 0° to 60°, especially for p-polarization light. Various silicon oxide films deposited by reaction magnetron sputtering with a tunable refractive index from 1.97 to 3.84 is introduced to meet the strict angle insensitive resonance conditions. The angle resolved spectral filtering for both p-polarization light and s-polarization light are quite well, which can be attributed to the different physical origins for the high angular tolerance for two polarizations. Finally, the effect of SiOx absorption and Ag thickness on the peak transmittance are analyzed. PMID:26765544

  8. Tradeoff studies in multiobjective insensitive design of airplane control systems

    NASA Technical Reports Server (NTRS)

    Schy, A. A.; Giesy, D. P.

    1983-01-01

    A computer aided design method for multiobjective parameter-insensitive design of airplane control systems is described. Methods are presented for trading off nominal values of design objectives against sensitivities of the design objectives to parameter uncertainties, together with guidelines for designer utilization of the methods. The methods are illustrated by application to the design of a lateral stability augmentation system for two supersonic flight conditions of the Shuttle Orbiter. Objective functions are conventional handling quality measures and peak magnitudes of control deflections and rates. The uncertain parameters are assumed Gaussian, and numerical approximations of the stochastic behavior of the objectives are described. Results of applying the tradeoff methods to this example show that stochastic-insensitive designs are distinctly different from deterministic multiobjective designs. The main penalty for achieving significant decrease in sensitivity is decreased speed of response for the nominal system.

  9. Ultra-flexible polarization-insensitive multiband terahertz metamaterial absorber.

    PubMed

    Chen, Xu; Fan, Wenhui

    2015-03-20

    A thin-flexible and polarization-insensitive multiband terahertz metamaterial absorber (MMA) has been investigated. Each unit cell of the MMA consists of two metallic structures, which include the top metal resonator ring and the bottom metal ground plane, separated by a thin-flexible dielectric spacer. Finite element simulation indicates that this MMA has three high absorption peaks in the terahertz region, with absorptivities of 89% at 0.72 THz, 98% at 1.4 THz, and 85% at 2.3 THz. However, because of its rotationally symmetric structure, this MMA is polarization-insensitive and can perform very well at a wide range of incident angles, namely, 30° for transverse electric waves and 40° for transverse magnetic waves. The thin-flexible device structure and good performance shows that this MMA is very promising to disguise objects and make them less detectable to radar in the terahertz region.

  10. Congenital insensitivity to pain with anhidrosis in Sudanese children.

    PubMed

    Othman, Suhair A; Malik, Amel A

    2016-01-01

    Congenital insensitivity to pain with anhidrosis (CIPA), also called hereditary sensory and autonomic neuropathy type IV (HSAN-IV), is an extremely rare autosomal recessive disease. It is characterized by insensitivity to pain, inability to sweat, episodes of hyperpyrexia, and intellectual disability. These factors render the affected persons to repeatedly injure and traumatise themselves to the degree that they become disabled. No specific treatment to the moment, but it needs multidisciplinary approach, and certain life adaptations and education. Here we report 4 unrelated Sudanese children affected with this rare neurological disorder, to raise the awareness on this rare disease, reflecting its spectrum, and the challenges which patients and their families face, especially when living in a hot country.

  11. Progressive central puberty in a toddler with partial androgen insensitivity.

    PubMed

    Dougan, Grace C; Uli, Naveen; Shulman, Dorothy I

    2014-03-01

    A male infant was diagnosed with partial androgen insensitivity caused by a novel mutation in the androgen receptor. At 3.5 months of age, he received 100 mg of testosterone intramuscularly over the course of 3 months to increase phallic size. He developed pubic hair after 5 months and signs of progressive central precocious puberty when re-examined at 17.5 months, which subsequently was suppressed with depot leuprolide.

  12. Partial androgen insensitivity syndrome with thermolability in the androgen receptor.

    PubMed

    Hiraoka, Kenji; Kawauchi, Akihiro; Soh, Jintetsu; Ohe, Hiroshi; Shima, Hiroki; Miki, Tsuneharu

    2006-01-01

    We report case of partial androgen insensitivity syndrome in a 12-year-old boy referred to our clinic complaining of bilateral gynecomastia and left undescended testicle. Laparoscopy for undescended testicle and bilateral mastectomy were performed, and the left testicle was absent. When skin fibroblasts of the scrotum obtained during surgery were cultured to analyse the androgen receptors, a slight thermolability was observed. Genomic examination of the androgen receptor gene could not detect any mutations.

  13. VLSI Implementation of Stray Insensitive Switched Capacitor Composite Operational Amplifiers

    DTIC Science & Technology

    1993-12-01

    INSENSITIVE SWITCHED CAPACITOR COMPOSITE OPERATIONAL AMPLIFIERS 12. PERSONAL AUTHOR(S) Gregory A. Silvemagel 13a. TYPE OF REPORT 13b. TIME COVERED 14. DATE OF...impedance. These limitations must be considered in any applications that use the operational amplifier. In many cases these characteristics are the...track one another. In addition, the temperature and voltage coefficients of resistors and capacitors are not correlated. Therefore, the time

  14. A Model-Following Technique for Insensitive Aircraft Control Systems.

    DTIC Science & Technology

    1981-01-01

    Harvey and Pope(131 and Vinkler[301 compared several different methods in their works, while Shenkar [261 and Ashkenazi[2i extended the most promising...Following for In- sensitive Control works, let us consider the simple, first-order system used by Shenkar [261. The plant is described by x -(1 + Ar)x + u...representative of the methods of Vinkler, Asikenazi, and Shenkar ), and Model Following for Insensitive Control (MrIC). For the LQR design, we assume that our

  15. Molecular studies of a patient with complete androgen insensitivity and a 47,XXY karyotype.

    PubMed

    Girardin, C M; Deal, C; Lemyre, E; Paquette, J; Lumbroso, R; Beitel, L K; Trifiro, M A; Van Vliet, G

    2009-09-01

    A phenotypic female with complete androgen insensitivity from a maternally inherited mutation in the androgen receptor had a 47,XXY karyotype. Partial maternal X isodisomy explained the expression of androgen insensitivity despite the presence of 2 X chromosomes.

  16. Angle- and Polarization-Insensitive Metamaterial Absorber using Via Array

    PubMed Central

    Lim, Daecheon; Lee, Dongju; Lim, Sungjoon

    2016-01-01

    In this paper, we propose an angle- and polarization-insensitive metamaterial absorber. We design a metamaterial unit cell that is based on a split ring cross resonator (SRCR). We observe that the absorption frequency and absorption ratio are insensitive to incident angles when a via array surrounds the SRR. We demonstrate the effect of the via array using full-wave simulations by comparing the absorptivity of the SRCR with and without the via array. Because of the symmetric geometry, we also realize polarization insensitivity. We build the proposed absorber on a printed-circuit-board with 30 × 30 unit cells, and we demonstrate its performance experimentally in free space. Under normal incidence, the fabricated absorber shows 99.6% absorptivity at 11.3 GHz for all polarization angles, while for oblique incidence, the fabricated absorber maintains an absorptivity higher than 90% for incident angles up to 70° and 60° for transverse magnetic (TM) and transverse electric (TE) modes, respectively. PMID:28000770

  17. Tradeoff methods in multiobjective insensitive design of airplane control systems

    NASA Technical Reports Server (NTRS)

    Schy, A. A.; Giesy, D. P.

    1984-01-01

    The latest results of an ongoing study of computer-aided design of airplane control systems are given. Constrained minimization algorithms are used, with the design objectives in the constraint vector. The concept of Pareto optimiality is briefly reviewed. It is shown how an experienced designer can use it to find designs which are well-balanced in all objectives. Then the problem of finding designs which are insensitive to uncertainty in system parameters are discussed, introducing a probabilistic vector definition of sensitivity which is consistent with the deterministic Pareto optimal problem. Insensitivity is important in any practical design, but it is particularly important in the design of feedback control systems, since it is considered to be the most important distinctive property of feedback control. Methods of tradeoff between deterministic and stochastic-insensitive (SI) design are described, and tradeoff design results are presented for the example of the a Shuttle lateral stability augmentation system. This example is used because careful studies have been made of the uncertainty in Shuttle aerodynamics. Finally, since accurate statistics of uncertain parameters are usually not available, the effects of crude statistical models on SI designs are examined.

  18. Angle- and Polarization-Insensitive Metamaterial Absorber using Via Array.

    PubMed

    Lim, Daecheon; Lee, Dongju; Lim, Sungjoon

    2016-12-21

    In this paper, we propose an angle- and polarization-insensitive metamaterial absorber. We design a metamaterial unit cell that is based on a split ring cross resonator (SRCR). We observe that the absorption frequency and absorption ratio are insensitive to incident angles when a via array surrounds the SRR. We demonstrate the effect of the via array using full-wave simulations by comparing the absorptivity of the SRCR with and without the via array. Because of the symmetric geometry, we also realize polarization insensitivity. We build the proposed absorber on a printed-circuit-board with 30 × 30 unit cells, and we demonstrate its performance experimentally in free space. Under normal incidence, the fabricated absorber shows 99.6% absorptivity at 11.3 GHz for all polarization angles, while for oblique incidence, the fabricated absorber maintains an absorptivity higher than 90% for incident angles up to 70° and 60° for transverse magnetic (TM) and transverse electric (TE) modes, respectively.

  19. Angle- and Polarization-Insensitive Metamaterial Absorber using Via Array

    NASA Astrophysics Data System (ADS)

    Lim, Daecheon; Lee, Dongju; Lim, Sungjoon

    2016-12-01

    In this paper, we propose an angle- and polarization-insensitive metamaterial absorber. We design a metamaterial unit cell that is based on a split ring cross resonator (SRCR). We observe that the absorption frequency and absorption ratio are insensitive to incident angles when a via array surrounds the SRR. We demonstrate the effect of the via array using full-wave simulations by comparing the absorptivity of the SRCR with and without the via array. Because of the symmetric geometry, we also realize polarization insensitivity. We build the proposed absorber on a printed-circuit-board with 30 × 30 unit cells, and we demonstrate its performance experimentally in free space. Under normal incidence, the fabricated absorber shows 99.6% absorptivity at 11.3 GHz for all polarization angles, while for oblique incidence, the fabricated absorber maintains an absorptivity higher than 90% for incident angles up to 70° and 60° for transverse magnetic (TM) and transverse electric (TE) modes, respectively.

  20. Photoporation and cell transfection using a violet diode laser

    NASA Astrophysics Data System (ADS)

    Paterson, L.; Agate, B.; Comrie, M.; Ferguson, R.; Lake, T. K.; Morris, J. E.; Carruthers, A. E.; Brown, C. T. A.; Sibbett, W.; Bryant, P. E.; Gunn-Moore, F.; Riches, A. C.; Dholakia, Kishan

    2005-01-01

    The introduction and subsequent expression of foreign DNA inside living mammalian cells (transfection) is achieved by photoporation with a violet diode laser. We direct a compact 405 nm laser diode source into an inverted optical microscope configuration and expose cells to 0.3 mW for 40 ms. The localized optical power density of ~1200 MW/m2 is six orders of magnitude lower than that used in femtosecond photoporation (~104 TW/m2). The beam perforates the cell plasma membrane to allow uptake of plasmid DNA containing an antibiotic resistant gene as well as the green fluorescent protein (GFP) gene. Successfully transfected cells then expand into clonal groups which are used to create stable cell lines. The use of the violet diode laser offers a new and simple poration technique compatible with standard microscopes and is the simplest method of laser-assisted cell poration reported to date.

  1. Enhanced transfection of brain tumor suppressor genes by photochemical internalization

    NASA Astrophysics Data System (ADS)

    Chou, Chih H.; Sun, Chung-Ho; Zhou, Yi-Hong; Madsen, Steen J.; Hirschberg, Henry

    2011-03-01

    One of many limitations for cancer gene therapy is the inability of the therapeutic gene to transfect a sufficient number of tumor cells. Photochemical internalization (PCI) is a photodynamic therapy-based approach for improving the delivery of macromolecules and genes into the cell cytosol. The utility of PCI for the delivery of a tumor suppressor gene (PAX-6) was investigated in monolayers and spheroids consisting of F98 rat glioma cells.

  2. Nanothermite-Based Microsystem for Drug Delivery and Cell Transfection

    DTIC Science & Technology

    2008-12-01

    presented. 2. EXPERIMENTAL 2.1 Nanothermite Preparation Nanothermite mixtures consisting of Bi2O3 nanoparticles and Al nanoparticles were used...for the transfection experiments reported herein. The Bi2O3 Report Documentation Page Form ApprovedOMB No. 0704-0188 Public reporting burden for the...particles were physically mixed in isopropanol (IPA) using ultrasonic agitation. Batches were mixed by dispersing 200mg of Bi2O3 in 1.5 mL of IPA by

  3. Application of nanostructured biochips for efficient cell transfection microarrays

    NASA Astrophysics Data System (ADS)

    Akkamsetty, Yamini; Hook, Andrew L.; Thissen, Helmut; Hayes, Jason P.; Voelcker, Nicolas H.

    2007-01-01

    Microarrays, high-throughput devices for genomic analysis, can be further improved by developing materials that are able to manipulate the interfacial behaviour of biomolecules. This is achieved both spatially and temporally by smart materials possessing both switchable and patterned surface properties. A system had been developed to spatially manipulate both DNA and cell growth based upon the surface modification of highly doped silicon by plasma polymerisation and polyethylene grafting followed by masked laser ablation for formation of a pattered surface with both bioactive and non-fouling regions. This platform has been successfully applied to transfected cell microarray applications with the parallel expression of genes by utilising its ability to direct and limit both DNA and cell attachment to specific sites. One of the greatest advantages of this system is its application to reverse transfection, whereupon by utilising the switchable adsorption and desorption of DNA using a voltage bias, the efficiency of cell transfection can be enhanced. However, it was shown that application of a voltage also reduces the viability of neuroblastoma cells grown on a plasma polymer surface, but not human embryonic kidney cells. This suggests that the application of a voltage may not only result in the desorption of bound DNA but may also affect attached cells. The characterisation of a DNA microarray by contact printing has also been investigated.

  4. Graphene and carbon nanotube nanocomposite for gene transfection.

    PubMed

    Hollanda, L M; Lobo, A O; Lancellotti, M; Berni, E; Corat, E J; Zanin, H

    2014-06-01

    Graphene and carbon nanotube nanocomposite (GCN) was synthesised and applied in gene transfection of pIRES plasmid conjugated with green fluorescent protein (GFP) in NIH-3T3 and NG97 cell lines. The tips of the multi-walled carbon nanotubes (MWCNTs) were exfoliated by oxygen plasma etching, which is also known to attach oxygen content groups on the MWCNT surfaces, changing their hydrophobicity. The nanocomposite was characterised by high resolution scanning electron microscopy; energy-dispersive X-ray, Fourier transform infrared and Raman spectroscopies, as well as zeta potential and particle size analyses using dynamic light scattering. BET adsorption isotherms showed the GCN to have an effective surface area of 38.5m(2)/g. The GCN and pIRES plasmid conjugated with the GFP gene, forming π-stacking when dispersed in water by magnetic stirring, resulting in a helical wrap. The measured zeta potential confirmed that the plasmid was connected to the nanocomposite. The NIH-3T3 and NG97 cell lines could phagocytize this wrap. The gene transfection was characterised by fluorescent protein produced in the cells and pictured by fluorescent microscopy. Before application, we studied GCN cell viability in NIH-3T3 and NG97 line cells using both MTT and Neutral Red uptake assays. Our results suggest that GCN has moderate stability behaviour as colloid solution and has great potential as a gene carrier agent in non-viral based therapy, with low cytotoxicity and good transfection efficiency.

  5. Towards gene therapy based on femtosecond optical transfection

    NASA Astrophysics Data System (ADS)

    Antkowiak, M.; Torres-Mapa, M. L.; McGinty, J.; Chahine, M.; Bugeon, L.; Rose, A.; Finn, A.; Moleirinho, S.; Okuse, K.; Dallman, M.; French, P.; Harding, S. E.; Reynolds, P.; Gunn-Moore, F.; Dholakia, K.

    2012-06-01

    Gene therapy poses a great promise in treatment and prevention of a variety of diseases. However, crucial to studying and the development of this therapeutic approach is a reliable and efficient technique of gene and drug delivery into primary cell types. These cells, freshly derived from an organ or tissue, mimic more closely the in vivo state and present more physiologically relevant information compared to cultured cell lines. However, primary cells are known to be difficult to transfect and are typically transfected using viral methods, which are not only questionable in the context of an in vivo application but rely on time consuming vector construction and may also result in cell de-differentiation and loss of functionality. At the same time, well established non-viral methods do not guarantee satisfactory efficiency and viability. Recently, optical laser mediated poration of cell membrane has received interest as a viable gene and drug delivery technique. It has been shown to deliver a variety of biomolecules and genes into cultured mammalian cells; however, its applicability to primary cells remains to be proven. We demonstrate how optical transfection can be an enabling technique in research areas, such as neuropathic pain, neurodegenerative diseases, heart failure and immune or inflammatory-related diseases. Several primary cell types are used in this study, namely cardiomyocytes, dendritic cells, and neurons. We present our recent progress in optimizing this technique's efficiency and post-treatment cell viability for these types of cells and discuss future directions towards in vivo applications.

  6. Characterization of cell lines stably transfected with rubella virus replicons

    SciTech Connect

    Tzeng, Wen-Pin; Xu, Jie; Frey, Teryl K.

    2012-07-20

    Rubella virus (RUBV) replicons expressing a drug resistance gene and a gene of interest were used to select cell lines uniformly harboring the replicon. Replicons expressing GFP and a virus capsid protein GFP fusion (C-GFP) were compared. Vero or BHK cells transfected with either replicon survived drug selection and grew into a monolayer. However, survival was {approx}9-fold greater following transfection with the C-GFP-replicon than with the GFP-expressing replicon and while the C-GFP-replicon cells grew similarly to non-transfected cells, the GFP-replicon cells grew slower. Neither was due to the ability of the CP to enhance RNA synthesis but survival during drug selection was correlated with the ability of CP to inhibit apoptosis. Additionally, C-GFP-replicon cells were not cured of the replicon in the absence of drug selection. Interferon-alpha suppressed replicon RNA and protein synthesis, but did not cure the cells, explaining in part the ability of RUBV to establish persistent infections.

  7. DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

    PubMed Central

    DiFranco, Marino; Quinonez, Marbella; Capote, Joana; Vergara, Julio

    2009-01-01

    A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver1, skeletal muscle2,3, and even the brain4. We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP3, calpain, FKBP12, β2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and α-actinin; and membrane proteins, i.e. α1s-DHPR, RyR1, cardiac Na/Ca2+ exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical3 and functional evidence5. However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the

  8. DNA transfection of mammalian skeletal muscles using in vivo electroporation.

    PubMed

    DiFranco, Marino; Quinonez, Marbella; Capote, Joana; Vergara, Julio

    2009-10-19

    A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver, skeletal muscle, and even the brain. We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP, calpain, FKBP12, beta2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and alpha-actinin; and membrane proteins, i.e. alpha1s-DHPR, RyR1, cardiac Na/Ca(2+) exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical and functional evidence. However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the

  9. Collision of millimetre droplets induces DNA and protein transfection into cells

    PubMed Central

    Ikemoto, Kazuto; Sakata, Ichiro; Sakai, Takafumi

    2012-01-01

    Nonperturbing and simple transfection methods are important for modern techniques used in biotechnology. Recently, we reported that electrospraying can be applied to DNA transfection in cell lines, bacteria, and chicken embryos. However, the transfection efficiency was only about 2%. To improve the transfection rate, physical properties of the sprayed droplets were studied in different variations of the method. We describe a highly efficient technique (30–93%) for introduction of materials such as DNA and protein into living cells by electrospraying droplets of a high conductivity liquid onto cells incubated with the material for transfection. Electric conductivity has a sizable influence on the success of transfection. In contrast, molecular weight of the transfected material, types of ions in the electrospray solution, and the osmotic pressure do not influence transfection efficiency. The physical analysis revealed that collision of cells with millimetre-sized droplets activates intracellular uptake. PMID:22375250

  10. Memory Insensitive Simplification for View-Dependent Refinement

    SciTech Connect

    Lindstrom, P

    2002-04-03

    We present an algorithm for end-to-end out-of-core simplification and view-dependent visualization of large surfaces. The method consists of three phases: (1) memory insensitive simplification; (2) memory insensitive construction of a level-of-detail hierarchy; and (3) run-time, output sensitive, view-dependent rendering and navigation of the mesh. The first two off-line phases are performed entirely on disk, and use only a small, constant amount of memory, whereas the run-time component relies on memory mapping to page in only the rendered parts of the mesh in a cache coherent manner. As a result, we are able to process and visualize arbitrarily large meshes given a sufficient amount of disk space--a constant multiple of the size of the input mesh. Similar to recent work on out-of-core simplification, our memory insensitive method uses vertex clustering on a uniform octree grid to coarsen a mesh and create a hierarchy, and a quadric error mettic to choose vertex positions at all levels of resolution. We show how the quadric information can be used to concisely represent vertex position, surface normal, error, and curvature information for anisotropic view-dependent coarsening and silhouette preservation. The focus of this paper is on the out-of-core construction of a level-of-detail hierarchy---our framework is general enough to incorporate many different aspects of view-dependent rendering. We therefore emphasize the off-line phases of our method, and report on their theoretical and experimental memory and disk usage and execution time. Our results indicate on average one to two orders of magnitude improvement in processing speed over previous out-of-core methods. Meanwhile, all phases of the method are both disk and memory efficient, and are fairly straightforward to implement.

  11. Insensitivity of Control System Performance to Controller and System Parameters

    NASA Technical Reports Server (NTRS)

    Levine, William S.

    2001-01-01

    An experimental and theoretical study of the CONDUIT sensitivity tools was conducted. The literature on sensitivity in nonlinear programming was reviewed to see in what ways it could be applied to CONDUIT. One result of this was the conclusion that the current insensitivity measure in CONDUIT is the right one. The question of scaling of the specifications in CONDUIT was also studied. Many simple examples were created, analyzed and, in most cases, solved. However, no general solution to the scaling problem was found. Instead, the appropriate scaling needs to be determined by review of a reasonable number of real aircraft design problems.

  12. Suppression of Clock Shifts at Magnetic-Field-Insensitive Transitions

    NASA Astrophysics Data System (ADS)

    Arnold, K. J.; Barrett, M. D.

    2016-10-01

    We show that it is possible to significantly reduce rank 2 tensor shifts of a clock transition by operating at a judiciously chosen magnetic-field-insensitive point. In some cases shifts are almost completely eliminated making the transition an effective J =0 to J =0 candidate. This significantly improves the feasibility of a recent proposal for clock operation with large ion crystals. For such multi-ion clocks, geometric constraints and selection rules naturally divide clock operation into two categories based on the orientation of the magnetic field. We discuss the limitations imposed on each type and how calibrations might be carried out for clock operation.

  13. Hormonal management of complete androgen insensitivity syndrome from adolescence onward.

    PubMed

    Bertelloni, Silvano; Dati, Eleonora; Baroncelli, Giampiero I; Hiort, Olaf

    2011-01-01

    Complete androgen insensitivity syndrome (CAIS) represents a main disorder of sex development. Women with CAIS may have their gonads removed before, during or after adolescence, thus requiring hormonal replacement therapy to induce puberty and/or maintain secondary sexual characteristics, to optimize bone mass accrual, and to promote physical and social well-being. Usually estrogens are used for this purpose, but formulations and doses should be better defined in multicentric prospective studies. Some women started testostosterone as hormonal replacement therapy, but this practice remains anecdotal. Bone health remains a crucial aspect in the management of persons with CAIS, but few sound data are available to guide clinical practice.

  14. Delivery of episomal vectors into primary cells by means of commercial transfection reagents.

    PubMed

    Han, Na Rae; Lee, Hyun; Baek, Song; Yun, Jung Im; Park, Kyu Hyun; Lee, Seung Tae

    2015-05-29

    Although episomal vectors are commonly transported into cells by electroporation, a number of electroporation-derived problems have led to the search for alternative transfection protocols, such as the use of transfection reagents, which are inexpensive and easy to handle. Polyplex-mediated transport of episomal vectors into the cytoplasm has been conducted successfully in immortalized cell lines, but no report exists of successful transfection of primary cells using this method. Accordingly, we sought to optimize the conditions for polyplex-mediated transfection for effective delivery of episomal vectors into the cytoplasm of primary mouse embryonic fibroblasts. Episomal vectors were complexed with the commercially available transfection reagents Lipofectamine 2000, FuGEND HD and jetPEI. The ratio of transfection reagent to episomal vectors was varied, and the subsequent transfection efficiency and cytotoxicity of the complexes were analyzed using flow cytometry and trypan blue exclusion assay, respectively. No cytotoxicity and the highest transfection yield were observed when the ratio of transfection reagent to episomal vector was 4 (v/wt) in the cases of Lipofectamine 2000 and FuGENE HD, and 2 in the case of jetPEI. Of the three transfection reagents tested, jetPEI showed the highest transfection efficiency without any cytotoxicity. Thus, we confirmed that the transfection reagent jetPEI could be used to effectively deliver episomal vectors into primary cells without electroporation.

  15. Systematics-insensitive Periodic Signal Search with K2

    NASA Astrophysics Data System (ADS)

    Angus, Ruth; Foreman-Mackey, Daniel; Johnson, John A.

    2016-02-01

    From pulsating stars to transiting exoplanets, the search for periodic signals in K2 data, Kepler’s two-wheeled extension, is relevant to a long list of scientific goals. Systematics affecting K2 light curves due to the decreased spacecraft pointing precision inhibit the easy extraction of periodic signals from the data. We here develop a method for producing periodograms of K2 light curves that are insensitive to pointing-induced systematics; the Systematics-insensitive Periodogram (SIP). Traditional sine-fitting periodograms use a generative model to find the frequency of a sinusoid that best describes the data. We extend this principle by including systematic trends, based on a set of “eigen light curves,” following Foreman-Mackey et al., in our generative model as well as a sum of sine and cosine functions over a grid of frequencies. Using this method we are able to produce periodograms with vastly reduced systematic features. The quality of the resulting periodograms are such that we can recover acoustic oscillations in giant stars and measure stellar rotation periods without the need for any detrending. The algorithm is also applicable to the detection of other periodic phenomena such as variable stars, eclipsing binaries and short-period exoplanet candidates. The SIP code is available at https://github.com/RuthAngus/SIPK2.

  16. Androgen insensitivity syndrome: ten years of our experience.

    PubMed

    Subramaniam, Amudha; Singh, Rajender; Tilak, Preetha; Devi, Rema; Kulandaivelu, Mahalingam; Kumarasamy, Thangaraj

    2013-01-01

    Abnormalities of secondary sexual differentiation manifest in varying degrees depending upon the severity of the underlying cause. Primary amenorrhea in phenotypic females is caused by several different factors, including hormonal imbalance, nutritional deficiency and sex differentiation abnormalities. Androgen insensitivity syndrome (AIS) accounts for a large proportion of such cases in phenotypic females but genetically male individuals. Over the past 10 years, we have collected data related to androgen insensitivity from more than 150 cases. The research identified several important but neglected facts about this syndrome; including the identification of mutations in 39% of the cases and the establishment of the cause of pathogenesis in 60% of them. The most intriguing facts were uncovered in relation to late presentation of the AIS cases, little awareness among patients and family members, no consensus on the age of performing gonadectomy, and reluctance of the patients to undergo recommended surgery. These issues need immediate attention to improve healthcare and management of AIS cases. This article summarizes our observations about AIS with an aim to spread awareness among patients and clinicians.

  17. Tissue Engineering Using Transfected Growth-Factor Genes

    NASA Technical Reports Server (NTRS)

    Madry, Henning; Langer, Robert S.; Freed, Lisa E.; Trippel, Stephen; Vunjak-Novakovic, Gordana

    2005-01-01

    A method of growing bioengineered tissues includes, as a major component, the use of mammalian cells that have been transfected with genes for secretion of regulator and growth-factor substances. In a typical application, one either seeds the cells onto an artificial matrix made of a synthetic or natural biocompatible material, or else one cultures the cells until they secrete a desired amount of an extracellular matrix. If such a bioengineered tissue construct is to be used for surgical replacement of injured tissue, then the cells should preferably be the patient s own cells or, if not, at least cells matched to the patient s cells according to a human-leucocyteantigen (HLA) test. The bioengineered tissue construct is typically implanted in the patient's injured natural tissue, wherein the growth-factor genes enhance metabolic functions that promote the in vitro development of functional tissue constructs and their integration with native tissues. If the matrix is biodegradable, then one of the results of metabolism could be absorption of the matrix and replacement of the matrix with tissue formed at least partly by the transfected cells. The method was developed for articular chondrocytes but can (at least in principle) be extended to a variety of cell types and biocompatible matrix materials, including ones that have been exploited in prior tissue-engineering methods. Examples of cell types include chondrocytes, hepatocytes, islet cells, nerve cells, muscle cells, other organ cells, bone- and cartilage-forming cells, epithelial and endothelial cells, connective- tissue stem cells, mesodermal stem cells, and cells of the liver and the pancreas. Cells can be obtained from cell-line cultures, biopsies, and tissue banks. Genes, molecules, or nucleic acids that secrete factors that influence the growth of cells, the production of extracellular matrix material, and other cell functions can be inserted in cells by any of a variety of standard transfection techniques.

  18. Graphene for improved femtosecond laser based pluripotent stem cell transfection.

    PubMed

    Mthunzi, Patience; He, Kuang; Ngcobo, Sandile; Khanyile, Thulile; Warner, Jamie H

    2014-05-01

    Pluripotent stem cells are hugely attractive in the tissue engineering research field as they can self-renew and be selectively differentiated into various cell types. For stem cell and tissue engineering research it is important to develop new, biocompatible scaffold materials and graphene has emerged as a promising material in this area as it does not compromise cell proliferation and accelerates specific cell differentiation. Previous studies have shown a non-invasive optical technique for mouse embryonic stem (mES) cell differentiation and transfection using femtosecond (fs) laser pulses. To investigate cellular responses to the influence of graphene and laser irradiation, here we present for the first time a study of mES cell fs laser transfection on graphene coated substrates. First we studied the impact of graphene on Chinese Hamster Ovary (CHO-K1) cell viability and cell cytotoxicity in the absence of laser exposure. These were tested via evaluating the mitochondrial activity through adenosine triphosphates (ATP) luminescence and breakages on the cell plasma membrane assessed using cytosolic lactate dehydrogenase (LDH) screening. Secondly, the effects of fs laser irradiation on cell viability and cytotoxicity at 1064 and 532 nm for cells plated and grown on graphene and pure glass were assessed. Finally, optical transfection of CHO-K1 and mES cells was performed on graphene coated versus plain glass substrates. Our results show graphene stimulated cell viability whilst triggering a mild release of intracellular LDH. We also observed that compared to pure glass substrates; laser irradiation at 1064 nm on graphene plates was less cytotoxic. Finally, in mES cells efficient optical transfection at 1064 (82%) and 532 (25%) nm was obtained due to the presence of a graphene support as compared to pristine glass. Here we hypothesize an up-regulation of cell adhesion promoting peptides or laminin-related receptors of the extracellular matrix (ECM) in cell samples

  19. Enhanced gene transfection performance and biocompatibility of polyethylenimine through pseudopolyrotaxane formation with α-cyclodextrin.

    PubMed

    Hu, Li-Zhong; Wan, Ning; Ma, Xi-Xi; Jing, Zi-Wei; Zhang, Ya-Xuan; Li, Chen; Zhou, Si-Yuan; Zhang, Bang-Le

    2017-03-24

    Polyethylenimine (PEI), a commercially available gene transfection reagent, is a promising nonviral vector due to its inherent ability to efficiently condense genetic materials and its successful transfection performance in vitro. However, its low transfection efficiency in vivo, along with its high cytotoxicity, limit any further applications in gene therapy. To enhance the gene transfection performance and reduce the cytotoxicity of linear polyethylenimine, pseudopolyrotaxane PEI25k/CD and the polyrotaxanes PEI25k/CD-PA and PEI25k/CD-PB were prepared and their transfection efficiencies were then evaluated. The pseudopolyrotaxane PEI25k/CD exhibited better transfection efficiency and lower cytotoxicity than the transfection reagent linear PEI25k, even in the presence of serum. It also showed a remarkably higher cell viability, similar DNA protecting capability, and better DNA decondensation and release ability, and could be useful for the development of novel and safe nonviral gene delivery vectors for gene therapy.

  20. Enhanced gene transfection performance and biocompatibility of polyethylenimine through pseudopolyrotaxane formation with α-cyclodextrin

    NASA Astrophysics Data System (ADS)

    Hu, Li-Zhong; Wan, Ning; Ma, Xi-Xi; Jing, Zi-Wei; Zhang, Ya-Xuan; Li, Chen; Zhou, Si-Yuan; Zhang, Bang-Le

    2017-03-01

    Polyethylenimine (PEI), a commercially available gene transfection reagent, is a promising nonviral vector due to its inherent ability to efficiently condense genetic materials and its successful transfection performance in vitro. However, its low transfection efficiency in vivo, along with its high cytotoxicity, limit any further applications in gene therapy. To enhance the gene transfection performance and reduce the cytotoxicity of linear polyethylenimine, pseudopolyrotaxane PEI25k/CD and the polyrotaxanes PEI25k/CD-PA and PEI25k/CD-PB were prepared and their transfection efficiencies were then evaluated. The pseudopolyrotaxane PEI25k/CD exhibited better transfection efficiency and lower cytotoxicity than the transfection reagent linear PEI25k, even in the presence of serum. It also showed a remarkably higher cell viability, similar DNA protecting capability, and better DNA decondensation and release ability, and could be useful for the development of novel and safe nonviral gene delivery vectors for gene therapy.

  1. Towards optical cell transfection inside a micro flow cell

    NASA Astrophysics Data System (ADS)

    Breunig, H. G.; Uchugonova, A.; König, K.

    2014-03-01

    For optical transfection, cells are shortly subjected to intense, focused laser radiation which leads to a temporary opening in the cell membrane. Although the method is very efficient and ensures high cell viability, the targeting of single cells with laser pulses is a tedious and slow approach. We present first measurements aiming at an experimental setup which is suitable for high throughput and automated optical cell transfection. In our setup, cells flow through a micro flow cell where they are spatially confined. The laser radiation is focused into the cell in a way that an elongated focal region is realized. This makes the time consuming aiming of the laser beam at individual cells unnecessary and opens the possibility to develop a completely automated system. The elongated laser focal region is realized by a quasi-Bessel beam which is generated by an axicon lens setup and continuously scanned from side to side of the cell. We present test measurements of the newly employed setup and discuss its suitability to be fully integrated into a flow cell sequencing system.

  2. Multiple C4/Slp genes distinguished by expression after transfection.

    PubMed Central

    Robins, D M; Malissen, M; Hood, L; Ferreira, A; Walthall, D; Mitchell, M

    1986-01-01

    The S region of the murine major histocompatibility complex contains two closely related genes: C4, encoding the fourth component of complement, and Slp, encoding sex-limited protein. We cloned these genes from a cosmid library of the B10.W7R strain that does not show androgen regulation of the Slp protein. Restriction site polymorphisms revealed at least four C4-like genes within the Sw7 locus, indicating evolutionary amplification of this region. Transfection of these genes into L cells resulted in expression, processing, and secretion of immunologically correct C4 and Slp proteins. At least two different Slp genes and one C4 gene were capable, after transfection, of expressing C4 and Slp indistinguishable from macrophage-derived protein. A third Slp gene exists within this locus whose recombinant cognate did not express in L cells. Thus, the B10.W7R S region includes one C4 gene and at least three Slp-like genes. Images PMID:3023818

  3. Molecular genetic transfection of the coccidian parasite Sarcocystis neurona.

    PubMed

    Gaji, Rajshekhar Y; Zhang, Deqing; Breathnach, Cormac C; Vaishnava, Shipra; Striepen, Boris; Howe, Daniel K

    2006-11-01

    Sarcocystis neurona is an apicomplexan parasite that is the major cause of equine protozoal myeloencephalitis (EPM). The biology of this pathogen remains poorly understood in part due to unavailability of molecular genetic tools. Hence, with an objective to develop DNA transfection capabilities for S. neurona, the 5' flanking region of the SnSAG1 gene was isolated from a genomic library and used to construct expression plasmids. In transient assays, the reporter molecules beta-galactosidase (beta-gal) and yellow fluorescent protein (YFP) could be detected in electroporated S. neurona, thereby confirming the feasibility of transgene expression in this organism. Stable transformation of S. neurona was achieved using a mutant dihydrofolate reductase thymidylate synthase (DHFR-TS) gene of Toxoplasma gondii that confers resistance to pyrimethamine. This selection system was used to create transgenic S. neurona that stably express beta-gal and YFP. As shown in this study, these transgenic clones can be useful for analyzing growth rate of parasites in vitro and for assessing drug sensitivities. More importantly, the DNA transfection methods described herein should greatly facilitate studies examining intracellular parasitism by this important coccidian pathogen.

  4. Transfection-mediated recombination of influenza A virus.

    PubMed Central

    Bergmann, M; García-Sastre, A; Palese, P

    1992-01-01

    Several mechanisms, including a high mutation rate and reassortment of genes, have been found to be responsible for the variability of influenza A viruses. RNA recombination would be another mechanism leading to genetic variation; however, recombination has only rarely been reported to occur in influenza viruses. During ribonucleoprotein transfection experiments designed to generate viable influenza viruses from in vitro-synthesized RNA, we discovered several viruses which must have originated from recombination events. The ribonucleoprotein transfection system may enhance the formation of viruses which result from jumping of the viral polymerase between RNAs or from ligation of different viral RNAs. Five different recombinant viruses are described. Two of these, REC1 and REC2, contain a neuraminidase (NA) gene whose defective polyadenylation signal has been repaired via intergenic recombination; 124 and 95 nucleotides have been added, respectively. Another virus, REC5, must have originated by multiple recombination events since it contains a mosaic gene with sequences derived from the NA gene of influenza A/WSN/33 virus and the matrix, polymerase protein PB1, and NA genes of influenza A/PR/8/34 virus. Images PMID:1279208

  5. Comparison of nanoparticle-mediated transfection methods for DNA expression plasmids: efficiency and cytotoxicity

    PubMed Central

    2011-01-01

    Background Reproducibly high transfection rates with low methodology-induced cytotoxic side effects are essential to attain the required effect on targeted cells when exogenous DNA is transfected. Different approaches and modifications such as the use of nanoparticles (NPs) are being evaluated to increase transfection efficiencies. Several studies have focused on the attained transfection efficiency after NP-mediated approaches. However, data comparing toxicity of these novel approaches with conventional methods is still rare. Transfection efficiency and methodology-induced cytotoxicity were analysed after transfection with different NP-mediated and conventional approaches. Two eukaryotic DNA-expression-plasmids were used to transfect the mammalian cell line MTH53A applying six different transfection protocols: conventional transfection reagent (FuGENE HD, FHD), FHD in combination with two different sizes of stabilizer-free laser-generated AuNPs (PLAL-AuNPs_S1,_S2), FHD and commercially available AuNPs (Plano-AuNP), and two magnetic transfection protocols. 24 h post transfection efficiency of each protocol was analysed using fluorescence microscopy and GFP-based flow cytometry. Toxicity was assessed measuring cell proliferation and percentage of propidium iodide (PI%) positive cells. Expression of the respective recombinant proteins was evaluated by immunofluorescence. Results The addition of AuNPs to the transfection protocols significantly increased transfection efficiency in the pIRES-hrGFPII-eIL-12 transfections (FHD: 16%; AuNPs mean: 28%), whereas the magnet-assisted protocols did not increase efficiency. Ligand-free PLAL-AuNPs had no significant cytotoxic effect, while the ligand-stabilized Plano-AuNPs induced a significant increase in the PI% and lower cell proliferation. For pIRES-hrGFPII-rHMGB1 transfections significantly higher transfection efficiency was observed with PLAL-AuNPs (FHD: 31%; PLAL-AuNPs_S1: 46%; PLAL-AuNPs_S2: 50%), while the magnet

  6. Discordant measures of androgen-binding kinetics in two mutant androgen receptors causing mild or partial androgen insensitivity, respectively.

    PubMed

    Shkolny, D L; Beitel, L K; Ginsberg, J; Pekeles, G; Arbour, L; Pinsky, L; Trifiro, M A

    1999-02-01

    We have characterized two different mutations of the human androgen receptor (hAR) found in two unrelated subjects with androgen insensitivity syndrome (AIS): in one, the external genitalia were ambiguous (partial, PAIS); in the other, they were male, but small (mild, MAIS). Single base substitutions have been found in both individuals: E772A in the PAIS subject, and R871G in the MAIS patient. In COS-1 cells transfected with the E772A and R871G hARs, the apparent equilibrium dissociation constants (Kd) for mibolerone (MB) and methyltrienolone are normal. Nonetheless, the mutant hAR from the PAIS subject (E772A) has elevated nonequilibrium dissociation rate constants (k(diss)) for both androgens. In contrast, the MAIS subject's hAR (R871G) has k(diss) values that are apparently normal for MB and methyltrienolone; in addition, the R871G hAR's ability to bind MB resists thermal stress better than the hAR from the PAIS subject. The E772A and R871G hARs, therefore, confer the same pattern of discordant androgen-binding parameters in transfected COS-1 cells as observed previously in the subjects' genital skin fibroblasts. This proves their pathogenicity and correlates with the relative severity of the clinical phenotype. In COS-1 cells transfected with an androgen-responsive reporter gene, trans-activation was 50% of normal in cells containing either mutant hAR. However, mutant hAR-MB binding is unstable during prolonged incubation with MB, whereas normal hAR-MB binding increases. Thus, normal equilibrium dissociation constants alone, as determined by Scatchard analysis, may not be indicative of normal hAR function. An increased k(diss) despite a normal Kd for a given androgen suggests that it not only has increased egress from a mutant ligand-binding pocket, but also increased access to it. This hypothesis has certain implications in terms of the three-dimensional model of the ligand-binding domain of the nuclear receptor superfamily.

  7. Compact Multilayer Film Structure for Angle Insensitive Color Filtering

    PubMed Central

    Yang, Chenying; Shen, Weidong; Zhang, Yueguang; Li, Kan; Fang, Xu; Zhang, Xing; Liu, Xu

    2015-01-01

    Here we report a compact multilayer film structure for angle robust color filtering, which is verified by theoretical calculations and experiment results. The introduction of the amorphous silicon in the proposed unsymmetrical resonant cavity greatly reduces the angular sensitivity of the filters, which is confirmed by the analysis of the phase shift within the structure. The temperature of the substrate during the deposition is expressly investigated to obtain the best optical performance with high peak reflectance and good angle insensitive color filtering by compromising the refractive index of dielectric layer and the surface roughness of the multilayer film. And the outlayer of the structure, worked as the anti-reflection layer, have an enormous impact on the filtering performance. This method, described in this paper, can have enormous potential for diverse applications in display, colorful decoration, anti-counterfeiting and so forth. PMID:25788330

  8. Development and Testing of the Contaminant Insensitive Sublimator

    NASA Technical Reports Server (NTRS)

    Leimkuehler, Thomas O.; Stephan, Ryan A.; Westheimer, David T.

    2006-01-01

    Sublimators have been used for heat rejection for a variety of space applications including the Apollo Lunar Module and the Extravehicular Mobility Unit (EMU). Some of the attractive features of sublimators are that they are compact, lightweight, and self-regulating. One of the drawbacks of previous designs has been sensitivity to non-volatile contamination in the feedwater, which can clog relatively small pores (approx.3-6 microns) in the porous plates where ice forms and sublimates. A new design that is less sensitive to contaminants is being developed at the Johnson Space Center. This paper describes the design, fabrication, and testing of the Contaminant Insensitive Sublimator (CIS) Engineering Development Unit (EDU).

  9. Development and Testing of the Contaminant Insensitive Sublimator

    NASA Technical Reports Server (NTRS)

    Leimkuehler, Thomas O.; Stephan, Ryan A.

    2007-01-01

    Sublimators have been used for heat rejection for a variety of space applications including the Apollo Lunar Module and the Extravehicular Mobility Unit (EMU). Some of the attractive features of sublimators are that they are compact, lightweight, and self-regulating. One of the drawbacks of previous designs has been sensitivity to non-volatile contamination in the feedwater, which can clog relatively small pores (approx. 3-6 micrometers) in the porous plates where ice forms and sublimates. A new design that is less sensitive to contaminants is being developed at the Johnson Space Center (JSC). This paper describes the design, fabrication, and testing of the Contaminant Insensitive Sublimator (CIS) Engineering Development Unit (EDU).

  10. Evaluation of ingredients for the development of new insensitive munitions.

    SciTech Connect

    Maharrey, Sean P.; Johnston, Lois A.; Behrens, Richard, Jr.; Wiese-Smith, Deneille

    2004-12-01

    Several ingredients being considered by the U.S. Army for the development of new insensitive munitions have been examined. One set of ingredients consists of 2,4-dinitrophenylhydrazine (DNPH) and hexahydro-1,3,5-trinitro-s-triazine (RDX). In this set, the decomposition of the mixture was examined to determine whether adding DNPH to RDX would generate a sufficient quantity of gas to rupture the case of a munition prior to the onset of the rapid reaction of RDX, thus mitigating the violence of reaction. The second set of ingredients consists of three different reduced sensitivity RDX (RS-RDX) powders manufactured by SNPE and Dyno-Nobel. In this set, the objective was to determine properties of RS-RDX powders that may distinguish them from normal RDX powder and may account for their reduced shock sensitivity. The decomposition reactions and sublimation properties of these materials were examined using two unique instruments: the simultaneous thermogravimetric modulated beam mass spectrometry (STMBMS) instrument and the Fourier Transform ion cyclotron resonance (FTICR) mass spectrometry instrument. These instruments provide the capability to examine the details of decomposition reactions in energetic materials. DNPH does not appear to be a good candidate to mitigate the violence of the RDX reaction in a munition. DNPH decomposes between 170 C and 180 C. When mixed with RDX it decomposes between 155 C and 170 C. It decomposes to form 1,3-dintrobenzene (DNB), ammonia, water and nitrogen. Of these compounds only nitrogen and ammonia are capable of generating high pressures within a munition. When DNPH is mixed with RDX, the DNB formed in the decomposition of DNPH interacts with RDX on the surface of the RDX powder leading to a higher rate of formation of CH2O and N2O. The CH2O is consumed by reaction with DNPH to form 2-methylene-1-(2,4-dintrophenyl)hydrazine. As a result, DNPH does not generate a large quantity of gas that will lead to rupture of a munition case. Another

  11. Incomplete androgen insensitivity (Reifenstein syndrome) - a case report.

    PubMed

    Turan, Volkan; Yeniel, Ozgür; Ergenoğlu, Mete; Terek, Coşan; Ulukuş, Murat

    2010-01-01

    We report a 20 year old case of partial androgen insensitivity syndrome, referred to our clinic with complaints concerning external genital organs and left undescended testicle. The phenotypically male case was first evaluated for secondary sex development. Axillary hair was scanty and no pubic hair was found. There was no breast development. In the gynecological examination, the clitoris was hypertrophic (4.6 cm) and a blind vagina with intact hymen was seen. Abdominopelvic ultrasonography revealed the absence of an uterus and adnexes which was supported by magnetic resonance imaging (MRI). There was a palpable mass in the left inguinal canal (cryptorchism), seen as atrophic tissue under the skin in MRI. Although the other testis was in the labioscrotal fold, it was atrophic. The Karyotype was 46 XY after genetic investigation.

  12. [Androgen insensitivity syndrome. Clinical features and molecular genetics].

    PubMed

    Sólyom, J; Scheiber, D; Fekete, G

    2001-08-05

    Androgen insensitivity syndrome (AIS) is an X-linked hereditary disorder caused by the mutation of the androgen receptor gene leading to variable phenotypes according to the depth of the hormonal resistance. There is a lack of knowledge regarding the criteria used to decide the management of infants with partial AIS, particularly with respect to sex of rearing. Therefore a national survey of patients with AIS in Hungary has been decided to compose a database for analyzing current practice. Preliminary results of the analysis for the mutations in the androgen receptor gene of Hungarian patients with AIS has been presented. The authors suggest that guidelines for clinicians on appropriate diagnostic and management strategies for AIS patients, particularly in the case of suspected partial AIS, would be helpful.

  13. Insensitivity of tunneling anisotropic magnetoresistance to non-magnetic electrodes

    SciTech Connect

    Wang, Y. Y.; Song, C. Wang, G. Y.; Zeng, F.; Pan, F.

    2013-11-11

    Ferromagnetic electrodes play a crucial role in magnetoresistance effect and spin injection, whereas the essential features of non-magnetic metal electrodes in spintronics are commonly ignored except for their electrical conductivity. Here, we verify that the room-temperature tunneling anisotropic magnetoresistance (TAMR) behavior in antiferromagnet-based [Pt/Co]/IrMn/AlO{sub x}/metal (metal = Pt, Au, Cu, Al) junctions is insensitive to the top metal electrodes. Similar out-of-plane signals are detected for different electrodes, in contrast to the varied shapes of in-plane TAMR curves which are most likely attributed to the differences in the multidomain structure of the magnetic electrode. This would add a different dimension to spintronics.

  14. Dual-band polarization-/angle-insensitive metamaterial absorber

    SciTech Connect

    Xiong, Han; Zhong, Lin-Lin; Luo, Chao-Ming; Hong, Jing-Song

    2015-06-15

    A dual-band metamaterial absorber (MA) based on triangular resonators is designed and investigated in this paper. It is composed of a two-dimensional periodic metal-dielectric-metal sandwiches array on a dielectric substrate. The simulation results clearly show that this absorber has two absorption peaks at 14.9 and 18.9 GHz, respectively, and experiments are conducted to verify the proposed designs effectively. For each polarization, the dual-band absorber is insensitive to the incident angle (up to 60°) and the absorption peaks remain high for both transverse electric (TE) and transverse magnetic (TM) radiation. To study the physical mechanism of power loss, the current distribution at the dual absorption peaks is given. The MA proposed in this paper has potential applications in many scientific and martial fields.

  15. Incomplete androgen insensitivity (Reifenstein syndrome) - a case report

    PubMed Central

    Turan, Volkan; Yeniel, Özgür; Ergenoğlu, Mete; Terek, Coşan; Ulukuş, Murat

    2010-01-01

    We report a 20 year old case of partial androgen insensitivity syndrome, referred to our clinic with complaints concerning external genital organs and left undescended testicle. The phenotypically male case was first evaluated for secondary sex development. Axillary hair was scanty and no pubic hair was found. There was no breast development. In the gynecological examination, the clitoris was hypertrophic (4.6 cm) and a blind vagina with intact hymen was seen. Abdominopelvic ultrasonography revealed the absence of an uterus and adnexes which was supported by magnetic resonance imaging (MRI). There was a palpable mass in the left inguinal canal (cryptorchism), seen as atrophic tissue under the skin in MRI. Although the other testis was in the labioscrotal fold, it was atrophic. The Karyotype was 46 XY after genetic investigation. PMID:24591911

  16. Humoral immunodeficiency in congenital insensitivity to pain with anhidrosis.

    PubMed

    Kilic, Sara Sebnem; Ozturk, Rifatcan; Sarisozen, Bartu; Rotthier, Annelies; Baets, Jonathan; Timmerman, Vincent

    2009-04-01

    Congenital insensitivity to pain with anhidrosis (CIPA) is an autosomal recessive disorder caused by mutations in the neurotrophic tyrosine receptor kinase 1 (NTRK1) gene, which encodes the receptor for nerve growth factor. We report the clinical course of a 7-year-old girl with CIPA and proven NTRK1 mutation. In addition to recurrent dislocation of the left hip joint and avascular necrosis of the left talus, the patient also presented with recurrent infections secondary to hypogammaglobulinemia, a feature not previously known to be associated with CIPA. The patient was treated with regular administration of intravenous immunoglobulins. Conservative treatment of the recurrent left hip dislocation by cast immobilization and bracing was implemented to stabilize the joint. The implication of the immune system of the reported patient broadens the clinical phenotype associated with NTRK1 mutations.

  17. Congenital Insensitivity to Pain without Anhidrosis: Orodental Problems and Management.

    PubMed

    Abdullah, N; Fakhruddin, Kausar Sadia; Samsudin, A R

    2015-01-01

    This paper reports the case of a 4-year-old male patient who was brought by parents requesting for replacement of multiple missing anterior teeth. The patient suffered from congenital insensitivity to pain without anhidrosis and presented with full blown sequelae of the condition in the form of oral self-mutilation leading to loss of teeth, tongue tip amputation, finger tips destruction, and lower limb wound infections. Dental and orthopaedic treatment consists of local management of oral wound and prevention from further oral and finger injuries that takes the form of dental splints and finger sleeve splints, constant feet coverage with shoes, and behavioural medical therapy. The age of the patient and parents' education present challenges in managing this condition to avoid morbidity and premature mortality.

  18. Congenital Insensitivity to Pain with Anhidrosis (CIPA): A Case Report.

    PubMed

    Safari, A; Khaledi, A A; Vojdani, M

    2011-02-01

    Congenital insensitivity to pain with anhidrosis (CIPA) is a rare disorder characterized by episodes of fever and the inability to sense of pain despite the fact that all other sensory modalities remain intact or minimally impaired. The patient also may exhibit the signs of self-mutilation, mental retardation and little or no perspiration. We present a 10 years old Iranian patient diagnosed with CIPA with the above-mentioned clinical characteristics. The prosthetic treatment and the subsequent six month follow-up are discussed. Follow-up of the patient revealed that, with the use of this prosthesis, the patient's oral function and esthetics were established and the mouth lesions improved. Therefore especial dental management of CIPA patients according to their mental status, age, oral and dental condition is essential for solving the specific problems each case may present and the full mouth teeth extraction should be considered as the last treatment.

  19. Molecular design for moisture insensitivity of compositionally graded hybrid films.

    PubMed

    Giachino, Marta; Dubois, Geraud; Dauskardt, Reinhold H

    2015-04-01

    Effective bonding of organic/inorganic interfaces especially in high humidity environments is paramount to the structural reliability of modern multilayer device technologies, such as flexible electronics, photovoltaics, microelectronic devices, and fiber-metal laminates used in aerospace applications. We demonstrate the ability to design compositionally graded hybrid organic/inorganic films with an inorganic zirconium network capable of forming a moisture-insensitive bond at the interface between an oxide and organic material. By controlling the chemistry of the deposited films and utilizing time-dependent debonding studies, we were able to correlate the behavior of the hybrid films at high humidity to their underlying molecular structure. As a result, an outstanding threefold improvement in adhesion of silicon/epoxy interfaces can be obtained with the introduction of these films even in high humidity environments.

  20. Dual-band polarization-/angle-insensitive metamaterial absorber

    NASA Astrophysics Data System (ADS)

    Xiong, Han; Zhong, Lin-Lin; Luo, Chao-Ming; Hong, Jing-Song

    2015-06-01

    A dual-band metamaterial absorber (MA) based on triangular resonators is designed and investigated in this paper. It is composed of a two-dimensional periodic metal-dielectric-metal sandwiches array on a dielectric substrate. The simulation results clearly show that this absorber has two absorption peaks at 14.9 and 18.9 GHz, respectively, and experiments are conducted to verify the proposed designs effectively. For each polarization, the dual-band absorber is insensitive to the incident angle (up to 60°) and the absorption peaks remain high for both transverse electric (TE) and transverse magnetic (TM) radiation. To study the physical mechanism of power loss, the current distribution at the dual absorption peaks is given. The MA proposed in this paper has potential applications in many scientific and martial fields.

  1. Exceptionally tough and notch-insensitive magnetic hydrogels.

    PubMed

    Haider, Hussain; Yang, Can Hui; Zheng, Wen Jiang; Yang, Jian Hai; Wang, Mei Xiang; Yang, Sen; Zrínyi, Miklós; Osada, Yoshihito; Suo, Zhigang; Zhang, Qiqing; Zhou, Jinxiong; Chen, Yong Mei

    2015-11-14

    Most existing magnetic hydrogels are weak and brittle. The development of strong and tough magnetic hydrogels would extend their applications into uncultivated areas, such as in actuators for soft machines and guided catheters for magnetic navigation systems, which is still a big challenge. Here a facile and versatile approach to fabricating highly stretchable, exceptionally tough and notch-insensitive magnetic hydrogels, Fe(3)O(4)@Fe-alginate/polyacrylamide (PAAm), is developed, by dispersing alginate-coated Fe(3)O(4) nanoparticles into the interpenetrating polymer networks of alginate and PAAm, with hybrid physical and chemical crosslinks. A cantilever bending beam actuator as well as a proof-of-concept magnetically guided hydrogel catheter is demonstrated. The method proposed in this work can be integrated into other strong and tough magnetic hydrogels for the development of novel hydrogel nanocomposites with both desirable functionality and superior mechanical properties.

  2. Congenital Insensitivity to Pain without Anhidrosis: Orodental Problems and Management

    PubMed Central

    Abdullah, N.; Fakhruddin, Kausar Sadia; Samsudin, A. R.

    2015-01-01

    This paper reports the case of a 4-year-old male patient who was brought by parents requesting for replacement of multiple missing anterior teeth. The patient suffered from congenital insensitivity to pain without anhidrosis and presented with full blown sequelae of the condition in the form of oral self-mutilation leading to loss of teeth, tongue tip amputation, finger tips destruction, and lower limb wound infections. Dental and orthopaedic treatment consists of local management of oral wound and prevention from further oral and finger injuries that takes the form of dental splints and finger sleeve splints, constant feet coverage with shoes, and behavioural medical therapy. The age of the patient and parents' education present challenges in managing this condition to avoid morbidity and premature mortality. PMID:26457210

  3. Mutations in the amino-terminal domain of the human androgen receptor may be associated with partial androgen insensitivity and impaired transactivation in vitro.

    PubMed

    Holterhus, P-M; Werner, R; Struve, D; Hauffa, B P; Schroeder, C; Hiort, O

    2005-09-01

    The majority of genetic variations in the androgen receptor (AR) gene are point mutations leading to impairment of the DNA- or hormone-binding domains. The N-terminus encoded by the first exon of the AR-gene usually harbors disruptive mutations associated with complete androgen insensitivity syndrome (CAIS) while missense mutations related with partial androgen insensitivity syndrome (PAIS) are seemingly rare. We present a 46,XY male with scrotal hypospadias in whom we detected a S432 F point mutation within the N-terminus. Transient transfections of an AR expression plasmid carrying the S432 F mutation using Chinese Hamster Ovary (CHO) cells revealed a significant partial reduction in transactivation of the co-transfected androgen responsive (ARE) (2)TATA luciferase reporter gene thus confirming PAIS. In two further 46, XY patients with slight to moderate virilization defects, we detected an S411 N mutation, and a 9 base pair deletion leading to the loss of amino acids 409 to 411 (L-A-S), respectively. These mutations did not compromise AR-function under the chosen experimental settings. The S432 F-patient supports particular significance of the AR-N-terminus for mild forms of AIS while the functional role of the two further mutations remains unclear. The N-terminus is a species-specific AR-domain possibly also involved in contributing to target tissue selectivity of AR-actions via mediating co-regulator interactions. Therefore, mild molecular defects of the AR-N-terminus may not necessarily inhibit general transactivation properties using currently established reporter gene models.

  4. Protease-sensitive transfection of Bacillus subtilis with bacteriophage GA-1 DNA: a probable case of heterologous transfection.

    PubMed

    Arwert, F; Venema, G

    1974-03-01

    The host bacterium of bacteriophage GA-1, Bacillus sp. G1R, was compared with respect to its taxonomic relationship to Bacillus subtilis, B. licheniformis, and B. pumilis. The physiological-biochemical properties of Bacillus sp. G1R are equal to those of B. licheniformis, but the thermal denaturation midpoint of G1R DNA differs by 3 C and the buoyant density by 0.005 g/cm(3) from that of B. licheniformis. Transformation with G1R donor DNA was neither observed in B. licheniformis nor in B. subtilis-competent recipients. Bacteriophage GA-1 shows neither infectivity on B. licheniformis nor on B. subtilis. However, infection of competent B. subtilis cultures with phenol-extracted GA-1 DNA results in the production of infective GA-1 particles. The transfecting activity of GA-1 DNA is destroyed by treatment with proteolytic enzymes. Resistance of transfecting DNA to inactivation by trypsin develops earlier than that to inactivation by DNase. Protease-treated GA-1 DNA competes with transforming DNA to approximately the same extent as does untreated GA-1 DNA, suggesting that uptake of GA-1 DNA is not affected by protease treatment. CsCl density gradient centrifugation reveals that the density of trypsinized GA-1 DNA is 0.004 g/cm(3) greater than that of untreated DNA.

  5. Congenital insensitivity to pain with anhydrosis: report of a family case

    PubMed Central

    Labib, Smael; Adnane Berdai, Mohamed; Abourazzak, Sanae; Hida, Mustapha; Harandou, Mustapha

    2011-01-01

    Congenital Insensitivity to pain with anhydrosis (CIPA) is a rare inherited disease. It is classified as hereditary sensory and autonomic neuropathy type IV. Pain insensitivity and autonomic deficits are present, but touch and pressure sensitivity are unimpaired. Mental retardation is usually present. We report a family case of a 5 years old girl and 2 years old boy with congenital insensitivity to pain, while discussing the clinical features and the anesthetic strategy of such patients. Patients with Congenital Insensitivity to Pain with anhydrosis may undergo surgery because of susceptibility to trauma due to absence of pain. The clinical features may intrinsically possess anesthetic challenges. PMID:22355435

  6. Congenital insensitivity to pain with anhydrosis: report of a family case.

    PubMed

    Labib, Smael; Adnane Berdai, Mohamed; Abourazzak, Sanae; Hida, Mustapha; Harandou, Mustapha

    2011-01-01

    Congenital Insensitivity to pain with anhydrosis (CIPA) is a rare inherited disease. It is classified as hereditary sensory and autonomic neuropathy type IV. Pain insensitivity and autonomic deficits are present, but touch and pressure sensitivity are unimpaired. Mental retardation is usually present. We report a family case of a 5 years old girl and 2 years old boy with congenital insensitivity to pain, while discussing the clinical features and the anesthetic strategy of such patients. Patients with Congenital Insensitivity to Pain with anhydrosis may undergo surgery because of susceptibility to trauma due to absence of pain. The clinical features may intrinsically possess anesthetic challenges.

  7. Mouse in utero electroporation: controlled spatiotemporal gene transfection.

    PubMed

    Matsui, Asuka; Yoshida, Aya C; Kubota, Mayumi; Ogawa, Masaharu; Shimogori, Tomomi

    2011-08-15

    In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early

  8. Enhancing magnetic nanoparticle-based DNA transfection: Intracellular-active cassette features

    NASA Astrophysics Data System (ADS)

    Vernon, Matthew Martin

    Efficient plasmid DNA transfection of embryonic stem cells, mesenchymal stem cells, neural cell lines and the majority of primary cell lines is a current challenge in gene therapy research. Magnetic nanoparticle-based DNA transfection is a gene vectoring technique that is promising because it is capable of outperforming most other non-viral transfection methods in terms of both transfection efficiency and cell viability. The nature of the DNA vector implemented depends on the target cell phenotype, where the particle surface chemistry and DNA binding/unbinding kinetics of the DNA carrier molecule play a critical role in the many steps required for successful gene transfection. Accordingly, Neuromag, an iron oxide/polymer nanoparticle optimized for transfection of neural phenotypes, outperforms many other nanoparticles and lipidbased DNA carriers. Up to now, improvements to nanomagnetic transfection techniques have focused mostly on particle functionalization and transfection parameter optimization (cell confluence, growth media, serum starvation, magnet oscillation parameters, etc.). None of these parameters are capable of assisting the nuclear translocation of delivered plasmid DNA once the particle-DNA complex is released from the endosome and dissociates in the cell's cytoplasm. In this study, incorporation of a DNA targeting sequence (DTS) feature in the transfecting plasmid DNA confers improved nuclear translocation, demonstrating significant improvement in nanomagnetic transfection efficiency in differentiated SH-SY5Y neuroblastoma cells. Other parameters, such as days in vitro, are also found to play a role and represent potential targets for further optimization.

  9. Magnetic nanoparticles as gene delivery agents: enhanced transfection in the presence of oscillating magnet arrays

    NASA Astrophysics Data System (ADS)

    McBain, S. C.; Griesenbach, U.; Xenariou, S.; Keramane, A.; Batich, C. D.; Alton, E. W. F. W.; Dobson, J.

    2008-10-01

    Magnetic nanoparticle-based gene transfection has been shown to be effective in combination with both viral vectors and with non-viral agents. In these systems, therapeutic or reporter genes are attached to magnetic nanoparticles which are then focused to the target site/cells via high-field/high-gradient magnets. The technique has been shown to be efficient and rapid for in vitro transfection and compares well with cationic lipid-based reagents, producing good overall transfection levels with lower doses and shorter transfection times. In spite of its potential advantages (particularly for in vivo targeting), the overall transfection levels do not generally exceed those of other non-viral agents. In order to improve the overall transfection levels while maintaining the advantages inherent in this technique, we have developed a novel, oscillating magnet array system which adds lateral motion to the particle/gene complex in order to promote transfection. Experimental results indicate that the system significantly enhances overall in vitro transfection levels in human airway epithelial cells compared to both static field techniques (p<0.005) and the cationic lipids (p<0.001) tested. In addition, it has the previously demonstrated advantages of magnetofection—rapid transfection times and requiring lower levels of DNA than cationic lipid-based transfection agents. This method shows potential for non-viral gene delivery both in vitro and in vivo.

  10. Clickable Poly(ionic liquids): A Materials Platform for Transfection.

    PubMed

    Freyer, Jessica L; Brucks, Spencer D; Gobieski, Graham S; Russell, Sebastian T; Yozwiak, Carrie E; Sun, Mengzhen; Chen, Zhixing; Jiang, Yivan; Bandar, Jeffrey S; Stockwell, Brent R; Lambert, Tristan H; Campos, Luis M

    2016-09-26

    The potential applications of cationic poly(ionic liquids) range from medicine to energy storage, and the development of efficient synthetic strategies to target innovative cationic building blocks is an important goal. A post-polymerization click reaction is reported that provides facile access to trisaminocyclopropenium (TAC) ion-functionalized macromolecules of various architectures, which are the first class of polyelectrolytes that bear a formal charge on carbon. Quantitative conversions of polymers comprising pendant or main-chain secondary amines were observed for an array of TAC derivatives in three hours using near equimolar quantities of cyclopropenium chlorides. The resulting TAC polymers are biocompatible and efficient transfection agents. This robust, efficient, and orthogonal click reaction of an ionic liquid, which we term ClickabIL, allows straightforward screening of polymeric TAC derivatives. This platform provides a modular route to synthesize and study various properties of novel TAC-based polymers.

  11. DNA-poly(diallyldimethylammonium chloride) complexation and transfection efficiency.

    PubMed

    Alatorre-Meda, Manuel; Taboada, Pablo; Krajewska, Barbara; Willemeit, Markus; Deml, Alexander; Klösel, Roland; Rodríguez, Julio R

    2010-07-29

    The present work assesses the influence of the cationic charge density (CD) and the cationic valence of poly(diallyldimethylammonium chloride) (pDADMAC) on the DNA compaction and subsequent transfection. Four homopolymers (CD = 1, with different valences) and one copolymer, poly(acrylamide-co-diallyldimethylammonium chloride) (coDADMAC) (CD < 1, equivalent in valence to one of the homopolymers), were studied. The characterization of the DNA-pDADMAC complexes (polyplexes) as a function of the polycation nitrogen to DNA phosphate molar ratios, N/P, was done by means of conductometry, electrophoretic mobility (zeta-potential), dynamic light scattering (DLS), isothermal titration calorimetry (ITC), atomic force microscopy (AFM), and beta-galactosidase (ONPG) and luciferase expression assays at 25 degrees C and physiological pH. In general, all polyplexes rendered compact and stable structures (R(H) approximately 100 nm) with positive surface charges ( approximately 11 mV) but low transfection efficiencies. As revealed by ITC, the DNA-pDADMAC complexation was characterized by a high binding affinity, the process being entropically driven. In particular, two characteristic ratios ((N/P)c and (N/P)*) were detected. Conductometry and ITC data demonstrated that the DNA compaction ratio, (N/P)c, was mainly governed by CD. Meanwhile the ratio from which the polyplex size remained constant, (N/P)*, was found to be valence-dependent as revealed by DLS. On the other hand, the low transfer rate of the polyplexes appeared to be correlated with the high binding affinity observed throughout the complexation process and with a core-shell structure the complexes presumably adopt.

  12. Spatial and Temporal Control of Cavitation Allows High In Vitro Transfection Efficiency in the Absence of Transfection Reagents or Contrast Agents.

    PubMed

    Chettab, Kamel; Roux, Stéphanie; Mathé, Doriane; Cros-Perrial, Emeline; Lafond, Maxime; Lafon, Cyril; Dumontet, Charles; Mestas, Jean-Louis

    2015-01-01

    Sonoporation using low-frequency high-pressure ultrasound (US) is a non-viral approach for in vitro and in vivo gene delivery. In this study, we developed a new sonoporation device designed for spatial and temporal control of ultrasound cavitation. The regulation system incorporated in the device allowed a real-time control of the cavitation level during sonoporation. This device was evaluated for the in vitro transfection efficiency of a plasmid coding for Green Fluorescent Protein (pEGFP-C1) in adherent and non-adherent cell lines. The transfection efficiency of the device was compared to those observed with lipofection and nucleofection methods. In both adherent and non-adherent cell lines, the sonoporation device allowed high rate of transfection of pEGFP-C1 (40-80%), as determined by flow cytometry analysis of GFP expression, along with a low rate of mortality assessed by propidium iodide staining. The transfection efficiency and toxicity of sonoporation on the non-adherent cell lines Jurkat and K562 were similar to those of nucleofection, while these two cell lines were resistant to transfection by lipofection. Moreover, sonoporation was used to produce three stably transfected human lymphoma and leukemia lines. Significant transfection efficiency was also observed in two fresh samples of human acute myeloid leukemia cells. In conclusion, we developed a user-friendly and cost-effective ultrasound device, well adapted for routine in vitro high-yield transfection experiments and which does not require the use of any transfection reagent or gas micro-bubbles.

  13. Application of fluorescence spectroscopy and multispectral imaging for non-invasive estimation of GFP transfection efficiency

    NASA Astrophysics Data System (ADS)

    Tamošiūnas, M.; Jakovels, D.; Lihačovs, A.; Kilikevičius, A.; Baltušnikas, J.; Kadikis, R.; Šatkauskas, S.

    2014-10-01

    Electroporation and ultrasound induced sonoporation has been showed to induce plasmid DNA transfection to the mice tibialis cranialis muscle. It offers new prospects for gene therapy and cancer treatment. However, numerous experimental data are still needed to deliver the plausible explanation of the mechanisms governing DNA electro- or sono-transfection, as well as to provide the updates on transfection protocols for transfection efficiency increase. In this study we aimed to apply non-invasive optical diagnostic methods for the real time evaluation of GFP transfection levels at the reduced costs for experimental apparatus and animal consumption. Our experimental set-up allowed monitoring of GFP levels in live mice tibialis cranialis muscle and provided the parameters for DNA transfection efficiency determination.

  14. Oscillating Magnet Array−Based Nanomagnetic Gene Transfection: A Valuable Tool for Molecular Neurobiology Studies

    PubMed Central

    Subramanian, Mahendran; Tyler, Aimee-Jayne; Luther, Eva Maria; Daniel, Elena Di; Lim, Jenson; Dobson, Jon

    2017-01-01

    To develop treatments for neurodegenerative disorders, it is critical to understand the biology and function of neurons in both normal and diseased states. Molecular studies of neurons involve the delivery of small biomolecules into cultured neurons via transfection to study genetic variants. However, as cultured primary neurons are sensitive to temperature change, stress, and shifts in pH, these factors make biomolecule delivery difficult, particularly non-viral delivery. Herein we used oscillating nanomagnetic gene transfection to successfully transfect SH-SY5Y cells as well as primary hippocampal and cortical neurons on different days in vitro. This novel technique has been used to effectively deliver genetic material into various cell types, resulting in high transfection efficiency and viability. From these observations and other related studies, we suggest that oscillating nanomagnetic gene transfection is an effective method for gene delivery into hard-to-transfect neuronal cell types. PMID:28336862

  15. The development of an autonomous gust insensitive unmanned aerial vehicle

    NASA Astrophysics Data System (ADS)

    Pisano, William James

    The study of a small Unmanned Aerial Vehicle (UAV) that is designed towards eventual operation in harsh storm-like conditions is presented. Investigation of the aircraft equations of motion shows that the selection of certain aerodynamic derivatives has a significant effect on the gust response of a small unmanned aircraft. Analytical comparison of this newly formulated Autonomous Gust Insensitive Aircraft (AGIA) to a conventionally designed aircraft shows a significant reduction in undesirable roll motion caused by gusts. A simulation is presented showing that the AGIA is capable of operating in more extreme environments than a conventional aircraft, and puts less strain on the control system components in both extreme and calm environments. The role that aircraft size plays in gust response is also studied. Pilot instinct dictates that smaller aircraft are more difficult to fly in windy environments than larger ones. This phenomenon is investigated using an analytic approach, providing insight into why smaller aircraft are indeed more difficult to fly in more challenging environments. As an aircraft gets smaller, its natural aerodynamic modes and response get faster. In an ideal system, this does not limit small aircraft to poor performance (in fact it will be shown that idealized small aircraft theoretically perform better than their larger counterparts). A more realistic system is presented that includes not only aerodynamics, but also realistic sensor and actuator dynamics. It is shown that these additional dynamics become a limiting factor in control system performance, and thus limit the closed-loop flight performance of small aircraft in turbulent environments. It is shown that the AGIA design approach plays a more significant role the as an aircraft gets smaller. To provide experimental validation of the gust insensitive theory presented herein, a representative small conventional aircraft was built alongside a similar aircraft that incorporated the AGIA

  16. Immune Cells, if Rendered Insensitive to Transforming Growth Factorbeta, Can Cure Prostate Cancer

    DTIC Science & Technology

    2007-02-01

    insensitive bone marrow transplants have met with the same fate by developing autoimmune syndrome , although these animals were able to eliminate challenged......Rendered Insensitive to Transforming Growth Factor-beta, Can 5a. CONTRACT NUMBER Cure Prostate Cancer 5b. GRANT NUMBER W81XWH-04-1-0166 5c. PROGRAM

  17. Study of synthesis techniques for insensitive aircraft control systems

    NASA Technical Reports Server (NTRS)

    Harvey, C. A.; Pope, R. E.

    1977-01-01

    Insensitive flight control system design criteria was defined in terms of maximizing performance (handling qualities, RMS gust response, transient response, stability margins) over a defined parameter range. Wing load alleviation for the C-5A was chosen as a design problem. The C-5A model was a 79-state, two-control structure with uncertainties assumed to exist in dynamic pressure, structural damping and frequency, and the stability derivative, M sub w. Five new techniques (mismatch estimation, uncertainty weighting, finite dimensional inverse, maximum difficulty, dual Lyapunov) were developed. Six existing techniques (additive noise, minimax, multiplant, sensitivity vector augmentation, state dependent noise, residualization) and the mismatch estimation and uncertainty weighting techniques were synthesized and evaluated on the design example. Evaluation and comparison of these six techniques indicated that the minimax and the uncertainty weighting techniques were superior to the other six, and of these two, uncertainty weighting has lower computational requirements. Techniques based on the three remaining new concepts appear promising and are recommended for further research.

  18. Androgen Insensitivity Syndrome: Management Considerations from Infancy to Adulthood.

    PubMed

    Chen, Min-Jye; Vu, Bach-Mai K; Axelrad, Marni; Dietrich, Jennifer E; Gargollo, Patricio; Gunn, Sheila; Macias, Charles G; McCullough, Laurence B; Roth, David R; Sutton, V Reid; Karaviti, Lefkothea P

    2015-06-01

    Androgen insensitivity syndrome (AIS) is an undervirilization syndrome in individuals with 46, XY karyotype. The undervirilization can be complete feminization or incomplete virilization with grades of ambiguity. AIS is caused by mutations in the androgen receptor, resulting in resistance to the physiologic activities of androgens. Differing degrees of resistance lead to three phenotypes: a complete form with female-appearing external genitalia, a partial form with a wide range of virilization, and a mild form with only minor undervirilization. AIS presents different challenges depending on whether resistance is complete or partial. Challenges include sex assignment, which impacts other medical decisions such as gonadectomy, hormonal replacement, and other surgical interventions. This review describes medical, psychosocial, and ethical concerns for each stage of development in complete and partial AIS, from the neonatal period to adulthood. These aspects of care should be addressed within an ethical framework by a multidisciplinary team, with the patients and families being the stakeholders in the decision-making process. We use the GRADE system when appropriate to appraise the existing evidence and provide recommendations and guidelines for management of AIS and appropriate transition of patients from pediatric to adult care.

  19. Male gender identity in complete androgen insensitivity syndrome.

    PubMed

    T'Sjoen, Guy; De Cuypere, Griet; Monstrey, Stan; Hoebeke, Piet; Freedman, F Kenneth; Appari, Mahesh; Holterhus, Paul-Martin; Van Borsel, John; Cools, Martine

    2011-06-01

    Women and girls with complete androgen insensitivity syndrome (CAIS) invariably have a female typical core gender identity. In this case report, we describe the first case of male gender identity in a CAIS individual raised female leading to complete sex reassignment involving both androgen treatment and phalloplasty. CAIS was diagnosed at age 17, based on an unambiguously female phenotype, a 46,XY karyotype, and a 2660delT androgen receptor (AR) gene mutation, leading to a premature stop in codon 807. Bilateral gonadectomy was performed but a short period of estrogen treatment induced a negative emotional reaction and treatment was stopped. Since the age of 3, childhood-onset cross gender behavior had been noticed. After a period of psychotherapy, persisting male gender identity was confirmed. There was no psychiatric co-morbidity and there was an excellent real life experience. Testosterone substitution was started, however without inducing any of the desired secondary male characteristics. A subcutaneous mastectomy was performed and the patient received phalloplasty by left forearm free flap and scrotoplasty. Testosterone treatment was continued, without inducing virilization, and bone density remained normal. The patient qualifies as female-to-male transsexual and was treated according to the Standards of Care by the World Professional Association for Transgender Health with good outcome. However, we do not believe that female sex of rearing as a standard procedure should be questioned in CAIS. Our case challenges the role of a functional AR pathway in the development of male gender identity.

  20. The clinical and molecular spectrum of androgen insensitivity syndromes

    SciTech Connect

    Hiort, O.; Sinnecker, G.H.G.; Holterhus, P.M.; Nitsche, E.M.; Kruse, K.

    1996-05-03

    Androgen insensitivity syndromes (AIS) are due to end-organ resistance to androgenic steroids in males leading to defective virilization of the external genitalia. The phenotype encompasses a wide array of genital ambiguity and may range from completely female to undervirilized but unequivocally male with infertility. This disorder is caused by mutations of the androgen receptor and is an X-linked recessive trait. We have studied 47 patients with AIS and have characterized the underlying molecular abnormality in the androgen receptor gene. Twenty patients had complete AIS and twenty-seven had partial AIS. Of the latter, 11 were of predominantly female phenotypic appearance and gender was assigned accordingly, while 16 were raised as males. Within the group of complete AIS, two patients had gross deletions within the gene, one had a small deletion, and one had an insertion. In the other patients with complete AIS, as well as all individuals with partial AIS, single nucleotide substitutions within the coding region were detected, each leading to an amino acid alteration. Seven codons were involved in more than one mutation in different cases. In addition, in one patient with spinal and bulbar muscular atrophy, an elongation of a glutamine-repeat was characterized. We conclude that mutations in the androgen receptor gene may be present throughout the whole coding region. However, our study provides evidence that several mutational hot spots exist. 18 refs., 2 figs.

  1. Endonucleases induced TRAIL-insensitive apoptosis in ovarian carcinoma cells

    SciTech Connect

    Geel, Tessa M.; Meiss, Gregor; Gun, Bernardina T. van der; Kroesen, Bart Jan; Leij, Lou F. de; Zaremba, Mindaugas; Silanskas, Arunas; Kokkinidis, Michael; Ruiters, Marcel H.; McLaughlin, Pamela M.; Rots, Marianne G.

    2009-09-10

    TRAIL induced apoptosis of tumor cells is currently entering phase II clinical settings, despite the fact that not all tumor types are sensitive to TRAIL. TRAIL resistance in ovarian carcinomas can be caused by a blockade upstream of the caspase 3 signaling cascade. We explored the ability of restriction endonucleases to directly digest DNA in vivo, thereby circumventing the caspase cascade. For this purpose, we delivered enzymatically active endonucleases via the cationic amphiphilic lipid SAINT-18{sup Registered-Sign }:DOPE to both TRAIL-sensitive and insensitive ovarian carcinoma cells (OVCAR and SKOV-3, respectively). Functional nuclear localization after delivery of various endonucleases (BfiI, PvuII and NucA) was indicated by confocal microscopy and genomic cleavage analysis. For PvuII, analysis of mitochondrial damage demonstrated extensive apoptosis both in SKOV-3 and OVCAR. This study clearly demonstrates that cellular delivery of restriction endonucleases holds promise to serve as a novel therapeutic tool for the treatment of resistant ovarian carcinomas.

  2. A viable supersymmetric model with UV insensitive anomaly mediation

    SciTech Connect

    Ibe, Masahiro; Kitano, Ryuichiro; Murayama, Hitoshi

    2004-12-14

    We propose an electroweak model which is compatible with the UV insensitive anomaly mediated supersymmetry breaking. The model is an extension of the NMSSM by adding vector-like matter fields which can drive the soft scalar masses of the singlet Higgs field negative and the successful electroweak symmetry breaking is achieved. Viable parameter regions are found to preserve perturbativity of all the coupling constants up to the Planck scale. With this success, the model becomes a perfect candidate of physics beyond the standard model without the FCNC and CP problem. The cosmology is also quite interesting. The lightest neutralino is the wino which is a perfect cold dark matter candidate assuming the non-thermal production from the gravitino decay. There is no gravitino problem because it decays before the BBN era, and thus the thermal leptogenesis works. The cosmological domain wall problem inherent in the NMSSM is absent since the Z_3 symmetry is broken by the QCD instanton effect in the presence of the vector-like quarks. We also briefly comment on a possible solution to the strong CP problem a la the Nelson-Barr mechanism.

  3. Temperature insensitive measurements of displacement using fiber Bragg grating sensors

    NASA Astrophysics Data System (ADS)

    Yang, Shuang; Li, Jun; Xu, Shengming; Sun, Miao; Tang, Yuquan; Gao, Gang; Dong, Fengzhong

    2016-11-01

    Optical fiber Bragg grating (FBG) displacement sensors play an important role in various areas due to the high sensitivity to displacement. However, it becomes a serious problem of FBG cross-sensitivity of temperature and displacement in applications with FBG displacement sensing. This paper presents a method of temperature insensitive measurement of displacement via using an appropriate layout of the sensor. A displacement sensor is constructed with two FBGs mounted on the opposite surface of a cantilever beam. The wavelengths of the FBGs shift with a horizontal direction displacement acting on the cantilever beam. Displacement measurement can be achieved by demodulating the wavelengths difference of the two FBGs. In this case, the difference of the two FBGs' wavelengths can be taken in order to compensate for the temperature effects. Four cantilever beams with different shapes are designed and the FBG strain distribution is quite different from each other. The deformation and strain distribution of cantilever beams are simulated by using finite element analysis, which is used to optimize the layout of the FBG displacement sensor. Experimental results show that an obvious increase in the sensitivity of this change on the displacement is obtained while temperature dependence greatly reduced. A change in the wavelength can be found with the increase of displacement from 0 to 10mm for a cantilever beam. The physical size of the FBG displacement sensor head can be adjusted to meet the need of different applications, such as structure health monitoring, smart material sensing, aerospace, etc.

  4. Conjunctival mucin deficiency in complete androgen insensitivity syndrome (CAIS).

    PubMed

    Mantelli, Flavio; Moretti, Costanzo; Micera, Alessandra; Bonini, Stefano

    2007-06-01

    Sex steroid hormones are essential for a healthy ocular surface and the androgen receptor impairment found in patients with complete androgen insensitivity syndrome (CAIS) has been described to cause meibomian gland dysfunction and functional dry eye for lipid tear film layer instability. However, it has not been reported if the mucous layer is also affected. A 37-year-old CAIS patient with persistent symptoms of dry eye underwent ophthalmological examination and was evaluated for qualitative and quantitative tear function tests and conjunctival cytology. Samples obtained from the conjunctival epithelium were stained for histology and immunohistochemistry and compared with three age-matched female controls. Western blot and relative real-time RT-PCR for MUC1 and MUC5AC were also performed on these samples. Immunohistochemistry, Western blot and relative real-time RT-PCR showed a decrease in the expression of MUC1 and MUC5AC in CAIS. Changes in the tear film mucous layer were accompanied by a reduction in the tear film break up time test. This is the first report describing mucous layer alteration associated with androgen receptor impairment. Decreased mucin levels contribute in explaining the tear film instability in CAIS and should be considered an additional cause of dry eye in sex steroid hormone pathology.

  5. Demonstration of a reversible phase-insensitive optical amplifier

    SciTech Connect

    Yoshikawa, Jun-ichi; Miwa, Yoshichika; Furusawa, Akira; Filip, Radim

    2011-05-15

    We experimentally demonstrate phase-insensitive linear amplification of a continuous variable system in the optical regime, preserving the ancilla system at the output. Since our amplification operation is unitary up to small excess noise, it is reversible beyond the classical limit. Here, entanglement between the amplified output system and the ancilla system is the resource for the reversibility, and the amplification gain is G=2.0. In addition, combining this amplifier with a beamsplitter, we also demonstrate approximate cloning of coherent states where an anticlone is present. We investigate the reversibility by reconstructing the initial state from the output correlations, and the results are slightly beyond the cloning limit. Furthermore, full characterization of the amplifier and cloner is given by using coherent states with several different mean values as inputs. Our amplifier is based on linear optics, offline-prepared additional ancillas in nonclassical states, and homodyne measurements followed by feedforward. Squeezed states are used as the additional ancillas, and nonlinear optical effects are exploited only for their generation. They introduce nonclassicality into the amplifying operation, making entanglement at the output.

  6. Design, synthesis, and transfection biology of novel cationic glycolipids for use in liposomal gene delivery.

    PubMed

    Banerjee, R; Mahidhar, Y V; Chaudhuri, A; Gopal, V; Rao, N M

    2001-11-22

    The molecular structure of the cationic lipids used in gene transfection strongly influences their transfection efficiency. High transfection efficiencies of non-glycerol-based simple monocationic transfection lipids with hydroxyethyl headgroups recently reported by us (Banerjee et al. J. Med. Chem. 1999, 42, 4292-4299) are consistent with the earlier observations that the presence of hydroxyl functionalities in the headgroup region of a cationic lipid contributes favorably in liposomal gene delivery. Using simple sugar molecules as the source of multiple hydroxyl functionalities in the headgroup region of the transfection lipids, we have synthesized four novel simple monocationic transfection lipids, namely, 1-deoxy-1-[dihexadecyl(methyl)ammonio]-D-xylitol (1), 1-deoxy-1-[methyl(ditetradecyl)ammonio]-D-arabinitol (2), 1-deoxy-1-[dihexadecyl(methyl)ammonio]-D-arabinitol (3) and 1-deoxy-1-[methyl(dioctadecyl)ammonio]-D-arabinitol (4), containing hydrophobic aliphatic tails and the hydrophilic arabinosyl or xylose sugar groups linked directly to the positively charged nitrogen atom. Syntheses, chemical characterizations, and the transfection biology of these novel transfection lipids 1-4 are described in this paper. Lipid 1, the xylosyl derivative, showed maximum transfection on COS-1 cells. All the lipids showed transfection with cholesterol as colipid and not with dioleoylphosphatidylethanolamine (DOPE). Radioactive quantitation of free and complexed DNA combined with ethidium bromide exclusion measurements suggest that though nearly 70% of the DNA exists as complexed DNA, the DNA may not have condensed as was observed with other cationic lipids. Presence of additional (more than two) hydroxyl functionalities in the headgroup of the cationic lipids appears to have improved the transfection efficiency and made these lipids less cytotoxic compared to two-hydroxyl derivatives.

  7. Size Specific Transfection to Mammalian Cells by Micropillar Array Electroporation

    PubMed Central

    Zu, Yingbo; Huang, Shuyan; Lu, Yang; Liu, Xuan; Wang, Shengnian

    2016-01-01

    Electroporation serves as a promising non-viral gene delivery approach, while its current configuration carries several drawbacks associated with high-voltage electrical pulses and heterogeneous treatment on individual cells. Here we developed a new micropillar array electroporation (MAE) platform to advance the electroporation-based delivery of DNA and RNA probes into mammalian cells. By introducing well-patterned micropillar array texture on the electrode surface, the number of pillars each cell faces varies with its plasma membrane surface area, despite their large population and random locations. In this way, cell size specific electroporation is conveniently carried out, contributing to a 2.5~3 fold increase on plasmid DNA transfection and an additional 10–55% transgene knockdown with siRNA probes, respectively. The delivery efficiency varies with the number and size of micropillars as well as their pattern density. As MAE works like many single cell electroporation are carried out in parallel, the electrophysiology response of individual cells is representative, which has potentials to facilitate the tedious, cell-specific protocol screening process in current bulk electroporation (i.e., electroporation to a large population of cells). Its success might promote the wide adoption of electroporation as a safe and effective non-viral gene delivery approach needed in many biological research and clinical treatments. PMID:27924861

  8. Software-aided automatic laser optoporation and transfection of cells

    NASA Astrophysics Data System (ADS)

    Georg Breunig, Hans; Uchugonova, Aisada; Batista, Ana; König, Karsten

    2015-06-01

    Optoporation, the permeabilization of a cell membrane by laser pulses, has emerged as a powerful non-invasive and highly efficient technique to induce transfection of cells. However, the usual tedious manual targeting of individual cells significantly limits the addressable cell number. To overcome this limitation, we present an experimental setup with custom-made software control, for computer-automated cell optoporation. The software evaluates the image contrast of cell contours, automatically designates cell locations for laser illumination, centres those locations in the laser focus, and executes the illumination. By software-controlled meandering of the sample stage, in principle all cells in a typical cell culture dish can be targeted without further user interaction. The automation allows for a significant increase in the number of treatable cells compared to a manual approach. For a laser illumination duration of 100 ms, 7-8 positions on different cells can be targeted every second inside the area of the microscope field of view. The experimental capabilities of the setup are illustrated in experiments with Chinese hamster ovary cells. Furthermore, the influence of laser power is discussed, with mention on post-treatment cell survival and optoporation-efficiency rates.

  9. Software-aided automatic laser optoporation and transfection of cells

    PubMed Central

    Georg Breunig, Hans; Uchugonova, Aisada; Batista, Ana; König, Karsten

    2015-01-01

    Optoporation, the permeabilization of a cell membrane by laser pulses, has emerged as a powerful non-invasive and highly efficient technique to induce transfection of cells. However, the usual tedious manual targeting of individual cells significantly limits the addressable cell number. To overcome this limitation, we present an experimental setup with custom-made software control, for computer-automated cell optoporation. The software evaluates the image contrast of cell contours, automatically designates cell locations for laser illumination, centres those locations in the laser focus, and executes the illumination. By software-controlled meandering of the sample stage, in principle all cells in a typical cell culture dish can be targeted without further user interaction. The automation allows for a significant increase in the number of treatable cells compared to a manual approach. For a laser illumination duration of 100 ms, 7-8 positions on different cells can be targeted every second inside the area of the microscope field of view. The experimental capabilities of the setup are illustrated in experiments with Chinese hamster ovary cells. Furthermore, the influence of laser power is discussed, with mention on post-treatment cell survival and optoporation-efficiency rates. PMID:26053047

  10. Biolistic techniques for transfection of mosquito embryos (Anopheles gambiae).

    PubMed

    Mialhe, E; Miller, L H

    1994-05-01

    To compensate for the extremely low rates of transformation by DNA microinjection into mosquito embryos of Anopheles gambiae, biolistic techniques were evaluated for introduction of DNA into large numbers of mosquito embryos. Biolistic experiments were first performed with a commercially available instrument intended for this purpose, according to the recommended procedure. The amount of DNA delivered was measured by the expression of luciferase under the control of the Drosophila heat shock protein (hsp) 70 promoter. Despite attempts to optimize biolistic parameters, the level of luciferase activity was low and highly variable. Two other methods of biolistic delivery of DNA-coated particles in aqueous suspension were then evaluated. One method used the gas explosion of the commercially available instrument (mentioned above) to drive an aqueous suspension of DNA-coated particles at high pressure. This method reproducibly increased the level of expression about 100-fold without greatly reducing embryo viability. Another method, which was recently described for plant transfection, uses lower pressure to deliver the aqueous suspension of DNA-coated particles. The level of expression of luciferase and the survival of embryos were equivalent to that obtained with the instrument modified for aqueous delivery of particles. Thus, both aqueous methods offer the advantages of reproducibly delivering more DNA to the embryos. Moreover, these methods could be suitable for delivering DNA mixed with proteins, such as restriction endonucleases and integrases, that may be destroyed by ethanol precipitation used in the standard PDS-1000/He method.

  11. Coding potential of transfected human placental lactogen genes.

    PubMed Central

    Reséndez-Pérez, D; Ramírez-Solís, R; Varela-Echavarría, A; Martínez-Rodríguez, H G; Barrera-Saldaña, H A

    1990-01-01

    We have joined the promoter-less sequences of the three hPL genes (hPL-1, hPL-3 and hPL-4) to strong transcriptional control elements. in vivo 35S-labeled proteins from the culture medium of cells transfected with the genes were resolved on SDS-polyacrylamide gels. The presence of characteristic labeled bands, visualized by autoradiography, determined that hPL-4 and hPL-3, but not hPL-1, contribute to the production of mature hPL. In these experiments hPL-3 expressed more RNA and protein than hPL-4. By exchanging the first two exons among hPL and hGH genes, we determined that the abundance of chimeric proteins depended on the genetic origin of the first two exons. Finally, we found evidence indicating that the splice mutation (G----A) at the beginning of the second intron of hPL-1, is not the only cause of the apparent lack of inactivity of this gene, since its reversion does not restore expression. Images PMID:2395633

  12. Size Specific Transfection to Mammalian Cells by Micropillar Array Electroporation

    NASA Astrophysics Data System (ADS)

    Zu, Yingbo; Huang, Shuyan; Lu, Yang; Liu, Xuan; Wang, Shengnian

    2016-12-01

    Electroporation serves as a promising non-viral gene delivery approach, while its current configuration carries several drawbacks associated with high-voltage electrical pulses and heterogeneous treatment on individual cells. Here we developed a new micropillar array electroporation (MAE) platform to advance the electroporation-based delivery of DNA and RNA probes into mammalian cells. By introducing well-patterned micropillar array texture on the electrode surface, the number of pillars each cell faces varies with its plasma membrane surface area, despite their large population and random locations. In this way, cell size specific electroporation is conveniently carried out, contributing to a 2.5~3 fold increase on plasmid DNA transfection and an additional 10–55% transgene knockdown with siRNA probes, respectively. The delivery efficiency varies with the number and size of micropillars as well as their pattern density. As MAE works like many single cell electroporation are carried out in parallel, the electrophysiology response of individual cells is representative, which has potentials to facilitate the tedious, cell-specific protocol screening process in current bulk electroporation (i.e., electroporation to a large population of cells). Its success might promote the wide adoption of electroporation as a safe and effective non-viral gene delivery approach needed in many biological research and clinical treatments.

  13. Structure of the trypanosome cyanide-insensitive alternative oxidase

    PubMed Central

    Shiba, Tomoo; Kido, Yasutoshi; Sakamoto, Kimitoshi; Inaoka, Daniel Ken; Tsuge, Chiaki; Tatsumi, Ryoko; Takahashi, Gen; Balogun, Emmanuel Oluwadare; Nara, Takeshi; Aoki, Takashi; Honma, Teruki; Tanaka, Akiko; Inoue, Masayuki; Matsuoka, Shigeru; Saimoto, Hiroyuki; Moore, Anthony L.; Harada, Shigeharu; Kita, Kiyoshi

    2013-01-01

    In addition to haem copper oxidases, all higher plants, some algae, yeasts, molds, metazoans, and pathogenic microorganisms such as Trypanosoma brucei contain an additional terminal oxidase, the cyanide-insensitive alternative oxidase (AOX). AOX is a diiron carboxylate protein that catalyzes the four-electron reduction of dioxygen to water by ubiquinol. In T. brucei, a parasite that causes human African sleeping sickness, AOX plays a critical role in the survival of the parasite in its bloodstream form. Because AOX is absent from mammals, this protein represents a unique and promising therapeutic target. Despite its bioenergetic and medical importance, however, structural features of any AOX are yet to be elucidated. Here we report crystal structures of the trypanosomal alternative oxidase in the absence and presence of ascofuranone derivatives. All structures reveal that the oxidase is a homodimer with the nonhaem diiron carboxylate active site buried within a four-helix bundle. Unusually, the active site is ligated solely by four glutamate residues in its oxidized inhibitor-free state; however, inhibitor binding induces the ligation of a histidine residue. A highly conserved Tyr220 is within 4 Å of the active site and is critical for catalytic activity. All structures also reveal that there are two hydrophobic cavities per monomer. Both inhibitors bind to one cavity within 4 Å and 5 Å of the active site and Tyr220, respectively. A second cavity interacts with the inhibitor-binding cavity at the diiron center. We suggest that both cavities bind ubiquinol and along with Tyr220 are required for the catalytic cycle for O2 reduction. PMID:23487766

  14. Quantitative ultrasonic computed tomography using phase-insensitive pyroelectric detectors

    NASA Astrophysics Data System (ADS)

    Zeqiri, Bajram; Baker, Christian; Alosa, Giuseppe; Wells, Peter N. T.; Liang, Hai-Dong

    2013-08-01

    The principle of using ultrasonic computed tomography (UCT) clinically for mapping tissue acoustic properties was suggested almost 40 years ago. Despite strong research activity, UCT been unable to rival its x-ray counterpart in terms of the ability to distinguish tissue pathologies. Conventional piezoelectric detectors deployed in UCT are termed phase-sensitive (PS) and it is well established that this property can lead to artefacts related to refraction and phase-cancellation that mask true tissue structure, particularly for reconstructions involving attenuation. Equally, it has long been known that phase-insensitive (PI) detectors are more immune to this effect, although sufficiently sensitive devices for clinical use have not been available. This paper explores the application of novel PI detectors to UCT. Their operating principle is based on exploiting the pyroelectric properties of the piezoelectric polymer polyvinylidene difluoride. An important detector performance characteristic which makes it particularly suited to UCT, is the lack of directionality of the PI response, relative to the PS detector mode of operation. The performance of the detectors is compared to conventional PS detection methods, for quantitatively assessing the attenuation distribution within various test objects, including a two-phase polyurethane phantom. UCT images are presented for a range of single detector apertures; tomographic reconstruction images being compared with the known structure of phantoms containing inserts as small as 3 mm, which were readily imaged. For larger diameter inserts (>10 mm), the transmitter-detector combination was able to establish the attenuation coefficient of the insert to within ±10% of values determined separately from plane-wave measurements on representative material plaques. The research has demonstrated that the new PI detectors are significantly less susceptible to refraction and phase-cancellation artefacts, generating realistic images in

  15. Transfection with replicating DNA from the temperate Bacillus bacteriophage phi 105 and with T4-ligase treated phi105 DNA: the importance in transfection of being longer than genome-length.

    PubMed

    Flock, J I

    1978-07-06

    Replicating phage DNA extracted from Bacillus subtilis infected with phage phi 105 has a higher activity in transfection than mature DNA. By heteroduplex analysis it was shown that this DNA contains concatemeric molecules. Concatemers, constructed in vitro by treatment of mature DNA with T4-ligase also have an increased activity in transfection. DNA showing an increased activity in transfection does not have a requirement for more than one molecule per transfection event as is typically found for transfection with mature phi 105 DNA. An explanation is given for this difference suggesting that the structure of the ends of the transfecting molecules play an important role intransfection.

  16. Membrane fusion inducers, chloroquine and spermidine increase lipoplex-mediated gene transfection

    SciTech Connect

    Wong-Baeza, Carlos; Bustos, Israel; Serna, Manuel; Tescucano, Alonso; Alcantara-Farfan, Veronica; Ibanez, Miguel; Montanez, Cecilia; Wong, Carlos; Baeza, Isabel

    2010-05-28

    Gene transfection into mammalian cells can be achieved with viral and non-viral vectors. Non-viral vectors, such as cationic lipids that form lipoplexes with DNA, are safer and more stable than viral vectors, but their transfection efficiencies are lower. Here we describe that the simultaneous treatment with a membrane fusion inducer (chlorpromazine or procainamide) plus the lysosomotropic agent chloroquine increases lipoplex-mediated gene transfection in human (HEK293 and C-33 A) and rat (PC12) cell lines (up to 9.2-fold), as well as in situ in BALB/c mice spleens and livers (up to 6-fold); and that the polyamine spermidine increases lipoplex-mediated gene transfection and expression in cell cultures. The use of these four drugs provides a novel, safe and relatively inexpensive way to considerably increase lipoplex-mediated gene transfection efficiency.

  17. Role of cholesterol on the transfection barriers of cationic lipid/DNA complexes

    NASA Astrophysics Data System (ADS)

    Pozzi, Daniela; Cardarelli, Francesco; Salomone, Fabrizio; Marchini, Cristina; Amenitsch, Heinz; Barbera, Giorgia La; Caracciolo, Giulio

    2014-08-01

    Most lipid formulations need cholesterol for efficient transfection, but the precise motivation remains unclear. Here, we have investigated the effect of cholesterol on the transfection efficiency (TE) of cationic liposomes made of 1,2-dioleoyl-3-trimethylammonium-propane and dioleoylphosphocholine in Chinese hamster ovary cells. The transfection mechanisms of cholesterol-containing lipoplexes have been investigated by TE, synchrotron small angle X-ray scattering, and laser scanning confocal microscopy experiments. We prove that cholesterol-containing lipoplexes enter the cells using different endocytosis pathways. Formulations with high cholesterol content efficiently escape from endosomes and exhibit a lamellar-nonlamellar phase transition in mixture with biomembrane mimicking lipid formulations. This might explain both the DNA release ability and the high transfection efficiency. These studies highlight the enrichment in cholesterol as a decisive factor for transfection and will contribute to the rational design of lipid nanocarriers with superior TE.

  18. CL22 - a novel cationic peptide for efficient transfection of mammalian cells.

    PubMed

    Haines, A M; Irvine, A S; Mountain, A; Charlesworth, J; Farrow, N A; Husain, R D; Hyde, H; Ketteringham, H; McDermott, R H; Mulcahy, A F; Mustoe, T L; Reid, S C; Rouquette, M; Shaw, J C; Thatcher, D R; Welsh, J H; Williams, D E; Zauner, W; Phillips, R O

    2001-01-01

    Condensing peptide-DNA complexes have great potential as nonviral agents for gene delivery. To date, however, such complexes have given transfection activities greatly inferior to adenovirus and somewhat inferior to cationic lipid-DNA complexes, even for cell lines and primary cells in vitro. We report here the identification of a novel condensing peptide, CL22, which forms DNA complexes that efficiently transfect many cell lines, as well as primary dendritic and endothelial cells. We report studies with sequence and structure variants that define some properties of the peptide that contribute to efficient transfection. We demonstrate that the superior transfection activity of CL22 compared with other DNA condensing peptides is conferred at a step after uptake of the complexes into cells. We show that CL22-DNA complexes have transfection activity that is at least equivalent to the best available nonviral agents.

  19. Efficient non-viral gene delivery mediated by nanostructured calcium carbonate in solution-based transfection and solid-phase transfection.

    PubMed

    Chen, Si; Li, Feng; Zhuo, Ren-Xi; Cheng, Si-Xue

    2011-10-01

    Among different non-viral gene delivery methods, the technique of co-precipitation of Ca(2+) with DNA in the presence of inorganic anions is an attractive option because of the biocompatibility and biodegradability. In this study, nano-sized CaCO(3)/DNA co-precipitates for gene delivery were prepared. The effect of Ca(2+)/CO(3)(2-) molar ratio on the gene delivery was investigated. The mechanism of the transfection mediated by CaCO(3)/DNA co-precipitates was studied by treatment of the cells with chloroquine, wortmannin and cytochalasin D, respectively. The in vitro gene transfections in different cells were carried out for both solution-based transfection and solid-phase transfection. The gene expression of the calcium carbonate based approach is strongly affected by the Ca(2+)/CO(3)(2-) ratio because the size of CaCO(3)/DNA co-precipitates is mainly determined by the Ca(2+)/CO(3)(2-) ratio. In addition, the encapsulation efficiency of DNA increases with decreasing Ca(2+)/CO(3)(2-) ratio. With a suitable Ca(2+)/CO(3)(2-) ratio, CaCO(3)/DNA co-precipitates could effectively mediate gene transfection with the expression levels higher than that of Lipofectamine 2000 in the presence of serum. The mechanism study shows that CaCO(3)/DNA co-precipitates are internalized via endocytosis of the cells and macropinocytosis is the main route of internalization. Compared with the solution-based transfection, CaCO(3)/DNA co-precipitates in the solid-phase transfection exhibit a lower gene expression level. The calcium carbonate based approach has great potential in gene delivery.

  20. Lipid-mediated transfection of normal adult human hepatocytes in primary culture.

    PubMed

    Ourlin, J C; Vilarem, M J; Daujat, M; Harricane, M C; Domergue, J; Joyeux, H; Baulieux, J; Maurel, P

    1997-04-05

    The aim of this work was to develop a procedure for the lipid-mediated transfection of DNA into normal adult human hepatocytes in culture. Cells were plated in a serum-free culture medium at various cell densities, on plastic or collagen-coated dishes, both in the absence and in the presence of epidermal growth factor (EGF). The cells were incubated for various periods of time with mixtures of DNA-lipofectin or DNA-3 beta[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-chol) liposomes, and the efficiency of transfection was assessed by measuring the activity of reporter genes, beta-galactosidase or chloramphenicol acetyl-transferase (CAT). For comparison, similar experiments were carried out with human cell lines including HepG2, Caco-2, and WRL68. The efficiency of transfection (in percentage of cells) was not significantly different after transfection with lipofectin or DC-chol and comprised between 0.04 and 1.7% (extreme values) for different cultures. The efficiency of transfection decreased as the age or density of the culture increased and increased in cultures treated with EGF. Direct measurement of the rate of DNA synthesis suggested that the efficiency of transfection was related to the number of cells entering the S phase. Under the same conditions, the efficiency of transfection was one to two orders of magnitude greater in the three cell lines. A plasmid harboring 660 bp of the 5'-flanking region of CYP1A1 (containing two xenobiotic enhancer elements) fused upstream of the promoter of thymidine kinase and the CAT reporter gene was constructed. When this plasmid was transfected in human hepatocytes, CAT activity was induced as expected. We conclude that normal adult human hepatocytes can be transfected with exogenous DNA and that the transfected construct is regulated in the manner expected from in vivo studies.

  1. Complete androgen insensitivity syndrome or testicular feminization: review of literature based on a case report

    PubMed Central

    Souhail, Regragui; Amine, Slaoui; Nadia, Abounouh; Tarik, Karmouni; Khalid, El Khader; Abdellatif, Koutani; Ahmed, Ibn Attya

    2016-01-01

    Testicular feminization, or the androgen insensitivity syndrome, is a rare disease. Because of various abnormalities of the X chromosome, a male, genetically XY, has some physical characteristics of a woman or a full female phenotype. Indeed the androgen insensitivity syndrome occurs because of a resistance to the actions of the androgen hormones, which in turn switches the development towards the aspect of a woman. We report a case of complete androgen insensitivity syndrome in a 30 years old woman who presented primary amenorrhea. We aim to improve our knowledge of this illness from the data that provides us this study, and a review of the literature. PMID:28270903

  2. Old fractures in two patients with congenital insensitivity to pain with anhidrosis: radiological findings.

    PubMed

    Yang, Li; Ji, Shao F; Yue, Rui J; Cheng, Jing L; Niu, Jun J

    2013-01-01

    Congenital insensitivity to pain with anhidrosis (CIPA) is a rare autosomal recessive disease that is characterized by anhidrosis, insensitivity to noxious stimuli, and mental retardation. Patients who suffer from CIPA easily sustain injuries due to pain insensitivity. Radiological findings in two CIPA patients revealed several old fractures of which the patients were unaware of previous injury. An early diagnosis of CIPA is important for the prevention and treatment of various complications. Our data indicate that radiological findings may provide important information for the diagnosis of CIPA.

  3. Bichromatic state-insensitive trapping of cold 133Cs-87Rb atomic mixtures

    NASA Astrophysics Data System (ADS)

    Metbulut, M. M.; Renzoni, F.

    2015-12-01

    We investigate simultaneous state-insensitive trapping of a mixture of two different atomic species, Caesium and Rubidium. The magic wavelengths of the Caesium and Rubidium atoms are different, 935.6 and 789.9 nm respectively, thus single-frequency simultaneous state-insensitive trapping is not possible. We thus identify bichromatic trapping as a viable approach to tune the two magic wavelengths to a common value. Correspondingly, we present several common magic wavelength combinations appropriate for simultaneous state-insensitive trapping of the two atomic species.

  4. Characterization of Sugar Insensitive (sis) Mutants of Arabidopsis

    SciTech Connect

    Gibson, Susan I.

    2009-06-08

    Despite the fact that soluble sugar levels have been postulated to play an important role in the control of a wide variety of plant metabolic and developmental pathways, the mechanisms by which plants respond to soluble sugar levels remain poorly understood. Plant responses to soluble sugar levels are also important in bioenergy production, as plant sugar responses are believed to help regulate both carbon fixation and carbon partitioning. For example, accumulation of soluble sugars, such as sucrose and glucose, in source tissues leads to feedback inhibition of photosynthesis, thereby decreasing rates of carbon fixation. Soluble sugar levels can also affect sink strengths, affecting the rates of accumulation of carbon-based compounds into both particular molecular forms (e.g. carbohydrates versus lipids versus proteins) and particular plant organs and tissues. Mutants of Arabidopsis that are defective in the ability to respond to soluble sugar levels were isolated and used as tools to identify some of the factors involved in plant sugar response. These sugar insensitive (sis) mutants were isolated by screening mutagenized seeds for those that were able to germinate and develop relatively normal shoot systems on media containing 0.3 M glucose or 0.3 M sucrose. At these sugar concentrations, wild-type Arabidopsis germinate and produce substantial root systems, but show little to no shoot development. Twenty-eight sis mutants were isolated during the course of four independent mutant screens. Based on a preliminary characterization of all of these mutants, sis3 and sis6 were chosen for further study. Both of these mutations appear to lie in previously uncharacterized loci. Unlike many other sugar-response mutants, sis3 mutants exhibit a wild-type or near wild-type response in all phytohormone-response assays conducted to date. The sis6-1 mutation is unusual in that it appears to be due to overexpression of a gene, rather than representing a loss of function mutation

  5. Study of mechanisms of electric field-induced DNA transfection. II. Transfection by low-amplitude, low-frequency alternating electric fields.

    PubMed

    Xie, T D; Tsong, T Y

    1990-10-01

    Electroporation for DNA transfection generally uses short intense electric pulses (direct current of kilovolts per centimeter, microseconds to milliseconds), or intense dc shifted radio-frequency oscillating fields. These methods, while remarkably effective, often cause death of certain cell populations. Previously it was shown that a completely reversible, high ionic permeation state of membranes could be induced by a low-frequency alternating electric field (ac) with a strength one-tenth, or less, of the critical breakdown voltage of the cell membrane (Teissie, J., and T. Y. Tsong. 1981. J. Physiol. (Paris). 77:1043-1053). We report the transfection of E. coli (JM105) by plasmid PUC18 DNA, which carries an ampicillin-resistance gene, using low-amplitude, low-frequency ac fields. E. coli transformants confer the ampicillin resistance and the efficiency of the transfection can be conveniently assayed by counting colonies in a selection medium containing ampicillin. For the range of ac fields employed (peak-to-peak amplitude 50-200 V/cm, frequency 0.1 Hz-1 MHz, duration 1-100 s), 100% of the E. coli survived the electric field treatment. Transfection efficiencies varied with field strength and frequency, and as high as 1 x 10(5)/micrograms DNA was obtained with a 200 V/cm square wave, 1 Hz ac field, 30 s exposure time, when the DNA/cell ratio was 50-75. Control samples gave a background transfection of much less than 10/micrograms DNA. With a square wave ac field, the transfection efficiency showed a frequency window: the optimal frequency was 1 Hz with a 200 V/cm field, and was approximately 0.1 Hz with a 50 V/cm field. Transfection efficiency varied with the waveform: square wave > sine wave > triangle wave. If the DNA was added after the ac field was turned off, transfection efficiency was reduced to the background level within 1 min. The field intensity used in this study was low and insufficient to cause electric breakdown of cell membranes. Thus, DNA

  6. Two de novo mutations in the AR gene cause the complete androgen insensitivity syndrome in a pair of monozygotic twins.

    PubMed

    Mongan, Nigel P; Jääskeläinen, Jarmo; Green, Katherine; Schwabe, John W; Shimura, Naoto; Dattani, Mehul; Hughes, Ieuan A

    2002-03-01

    The androgen insensitivity syndrome (AIS) is the most common cause of male undermasculinization and is typically caused by mutations in the AR gene. Affected individuals may exhibit either complete external feminization (complete AIS) or a partial phenotype (partial AIS). Here we describe monozygotic twins diagnosed with complete AIS who each possess two substitutions (C-->G at position 2930 and T-->C at position 2955, both in exon 7), leading to Phe(856)Leu and Ser(865)Pro mutations, respectively. Neither parent was found to be a carrier for these mutations, indicating that the double mutation arose de novo. Both mutations were recreated by site-directed mutagenesis and compared functionally with the wild-type receptor. The Phe(856)Leu mutation did not affect androgen binding when expressed in COS-1 cells, nor did this mutation decrease androgen-dependent trans-activation in transfected HeLa cells. However, the Ser(865)Pro mutation completely ablated androgen binding and trans-activation. In this study we demonstrate that the replacement of serine by proline at position 865 is sufficient in itself to cause complete AIS in these twins. Analyses of nuclear receptor structures suggest that this mutation is likely to perturb the conformation of helix 10/11, which plays a role in ligand binding, dimerization, and receptor activation. To our knowledge this is the first confirmed instance of AIS (complete or partial) due to an AR mutation occurring in twins. Furthermore, the phenotype was associated with two mutations that were both novel in nature.

  7. Partial androgen insensitivity and correlations with the predicted three dimensional structure of the androgen receptor ligand-binding domain.

    PubMed

    Yong, E L; Tut, T G; Ghadessy, F J; Prins, G; Ratnam, S S

    1998-02-01

    Genetic defects of the human androgen receptor (AR) can cause a wide spectrum of androgen insensitivity syndromes (AIS) ranging from phenotypic females in those with complete AIS; ambiguous genitalia in partial AIS; to male infertility in minimal AIS. The majority of these defects are due to point mutations resulting in amino acid substitutions. It is however unclear why certain mutations result in partial AIS, whereas others in the same exon cause the complete syndrome. We present a case of partial AIS due to a point mutation affecting codon 758 of the AR ligand-binding domain (LBD) that changed the sense of the codon from asparagine to threonine (N758T). The mutant receptor displayed normal binding affinity to DHT but abnormal dissociation kinetics in both patient's fibroblasts and transfected COS-7 cells. The mutant AR was thermolabile, and resulted in approximately 50% reduction in receptor transactivation capacity when examined with a reporter gene incorporating an androgen-response-element. Although the 3-D structure of AR LBD is not known, the homologous region in a member of the steroid receptor superfamily, retinoid-X receptor (RXR-alpha), has been crystallized, allowing comparison of aligned amino-acid sequences of RXR-alpha and AR. The mutation, N758T, lies in a predicted linker region between the fifth alpha-helix (H5) and the first beta-strand (S1). Generally, mutations leading to partial AIS tend to cluster in the predicted linker regions located between the structural helices of the AR LBD. Most strikingly, the predicted linker regions contain over 70% of the mutant ARs associated with prostate cancer in the LBD. The occurrence of mutations associated with both partial AIS and prostate cancer in the same predicted linker regions, suggest that this clustering is not coincidental and that the predicted linker regions are likely to have important, but subtle, roles in defining androgen binding and ligand specificity.

  8. An examination of how different mutations at arginine 855 of the androgen receptor result in different androgen insensitivity phenotypes.

    PubMed

    Elhaji, Youssef A; Wu, Jian Hui; Gottlieb, Bruce; Beitel, Lenore K; Alvarado, Carlos; Batist, Gerald; Trifiro, Mark A

    2004-08-01

    Two substitutions at an identical location in the ligand-binding domain (LBD) of the human androgen receptor (AR), R855C and R855H, are associated with complete androgen insensitivity syndrome (AIS) and partial AIS, respectively. Kinetic analysis of the mutant receptors in genital skin fibroblasts and in transfected cells revealed very low total binding (Bmax) and increased rate constants of dissociation (k) for the R855C mutant; and normal Bmax and k, with slightly elevated equilibrium affinity constants (Kd), but decreased transactivational capacity for the R855H mutant. Further analysis of the R855H mutant revealed both thermolability and decreased N/C-terminal inter-actions in the presence and absence of the co-activator transcriptional intermediary factor 2. To establish the nature of these functional differences we have used molecular dynamic modeling to create four-dimensional models of each of the mutant receptors. Molecular dynamic modeling produced profoundly different models for each of the mutants: in modeling of R855C a surprisingly significant distant alteration in the position of helix 12 of the helix 12 positioning of the AR ligand binding domain (AR-LBD) occurs, which would predict severe ligand binding abnormalities and complete AIS; in modeling of R855H, no dramatic effect on the position of helix 12 was seen; thus, binding properties of the receptor are not compromised. Molecular dynamics four-dimensional modeling clearly supports the biochemical and kinetic studies of both mutants. Such novel computational modeling may lead to a better understanding of the structure-function relationships and the molecular mechanics of ligand binding not only of the AR-LBD but also of other nuclear receptors.

  9. Functional analysis of a novel androgen receptor mutation, Q902K, in an individual with partial androgen insensitivity.

    PubMed

    Umar, Arzu; Berrevoets, Cor A; Van, N Mai; van Leeuwen, Marije; Verbiest, Michael; Kleijer, Wim J; Dooijes, Dennis; Grootegoed, J Anton; Drop, Stenvert L S; Brinkmann, Albert O

    2005-01-01

    Androgen insensitivity syndrome (AIS) is caused by defects in the androgen receptor (AR) that render the AR partially or completely inactive. As a result, embryonic sex differentiation is impaired. Here, we describe a novel mutation in the AR found in a patient with partial AIS. The mutation results in a substitution of a glutamine (Q) by a lysine (K) residue at position 902, Q902K. The AR Q902K mutation was investigated in vitro with respect to its functional properties. The equilibrium dissociation constants (K(d)s) of AR Q902K in the presence of either the synthetic androgen R1881 or the natural ligand DHT were slightly elevated. The R1881 dissociation rate (t(1/2)) was increased 3-fold for AR Q902K compared with wild type. Transcriptional activity was decreased to 85% of wild type, and the dose-response curve revealed that the sensitivity to hormone was decreased due to the mutation. Furthermore, the 114-kDa androgen-induced phosphorylated AR protein band was not detectable in genital skin fibroblasts. However, it could be detected in transfected CHO cells expressing the mutant receptor in the presence of 10 and 100 nm R1881. Functional interaction assays and a GST pull-down assay showed that the interaction between the NH2 and COOH terminus of AR Q902K was reduced to 50% of wild type. Furthermore, the transactivation by the coactivator TIF2 (transcriptional intermediary factor 2) was decreased 2- to 3-fold. The half-maximal response in both assays was shifted to a higher hormone concentration compared with wild type. These results indicate that residue Q902 is involved in TIF2 and NH2/COOH interaction and that the Q to K mutation results in a mild impairment of AR function, which can explain the partial AIS phenotype of the patient.

  10. Munitions having an insensitive detonator system for initiating large failure diameter explosives

    SciTech Connect

    Perry, III, William Leroy

    2015-08-04

    A munition according to a preferred embodiment can include a detonator system having a detonator that is selectively coupled to a microwave source that functions to selectively prime, activate, initiate, and/or sensitize an insensitive explosive material for detonation. The preferred detonator can include an explosive cavity having a barrier within which an insensitive explosive material is disposed and a waveguide coupled to the explosive cavity. The preferred system can further include a microwave source coupled to the waveguide such that microwaves enter the explosive cavity and impinge on the insensitive explosive material to sensitize the explosive material for detonation. In use the preferred embodiments permit the deployment and use of munitions that are maintained in an insensitive state until the actual time of use, thereby substantially preventing unauthorized or unintended detonation thereof.

  11. Surfactant for dye-penetrant inspection is insensitive to liquid oxygen

    NASA Technical Reports Server (NTRS)

    1966-01-01

    LOX insensitive solvent is blended into a mixture of commercially available surfactants to clean metal surfaces which are to be investigated by the dye-penetrant method. The surfactant mixture is applied before and after application of the dye.

  12. ANALYSIS OF IN VITRO TRANSFECTION BY SONOPORATION USING CATIONIC AND NEUTRAL MICROBUBBLES

    PubMed Central

    Tlaxca, Jose L.; Anderson, Christopher R.; Klibanov, Alexander L.; Lowrey, Bryce; Hossack, John A.; Alexander, J. Steven; Lawrence, Michael B.; Rychak, Joshua J.

    2010-01-01

    The objective of the study was to examine the role of acoustic power intensity and microbubble and plasmid concentrations on transfection efficiency in HEK-293 cells using a sonoporator with a 1-MHz transducer. A green fluorescent protein (GFP) reporter plasmid was delivered in as much as 80% of treated cells, and expression of the GFP protein was observed in as much as 75% of cells, using a power intensity of 2 W/cm2 with a 25% duty cycle. In addition, the relative transfection abilities of a lipid noncationic and cationic microbubble platform were investigated. As a positive control, cells were transfected using Lipofectamine reagent. Cell survival and transfection efficiency were inversely proportional to acoustic power and microbubble concentration. Our results further demonstrated that high-efficiency transfection could be achieved, but at the expense of cell loss. Moreover, direct conjugation of plasmid to the microbubble did not appear to significantly enhance transfection efficiency under the examined conditions, although this strategy may be important for targeted transfection in vivo. PMID:20800945

  13. Effects of molecular size and chemical factor on plasma gene transfection

    NASA Astrophysics Data System (ADS)

    Ikeda, Yoshihisa; Motomura, Hideki; Kido, Yugo; Satoh, Susumu; Jinno, Masafumi

    2016-07-01

    In order to clarify the mechanism of plasma gene transfection, the relationship between transfection efficiency and transferred molecular size was investigated. Molecules with low molecular mass (less than 50 kDa; dye or dye-labeled oligonucleotide) and high molecular mass (more than 1 MDa; plasmid DNA or fragment of plasmid DNA) were transferred to L-929 cells. It was found that the transfection efficiency decreases with increasing in transferred molecular size and also depends on the tertiary structure of transferred molecules. Moreover, it was suggested the transfection mechanism is different between the molecules with low (less than 50 kDa) and high molecular mass (higher than 1 MDa). For the amount of gene transfection after plasma irradiation, which is comparable to that during plasma irradiation, it is shown that H2O2 molecules are the main contributor. The transfection efficiency decreased to 0.40 ± 0.22 upon scavenging the H2O2 generated by plasma irradiation using the catalase. On the other hand, when the H2O2 solution is dropped into the cell suspension without plasma irradiation, the transfection efficiency is almost 0%. In these results, it is also suggested that there is a synergetic effect of H2O2 with electrical factors or other reactive species generated by plasma irradiation.

  14. Stable transfection into rat bone marrow mesenchymal stem cells by lentivirus-mediated NT-3.

    PubMed

    Gong, Yu; Wang, Hongfei; Xia, Haijun

    2015-01-01

    Transplantation of bone marrow mesenchymal stem cells (BMSCs) is the most promising therapeutic strategy in the treatment of spinal cord injuries. BMSCs have a wide variety of sources and are characterized by being exempt from immune rejection, marked secretory functions and neuronal plasticity during differentiation. The lentiviral vector, namely PLV.Ex3d.P/neo-EF1A-NT3-internal ribosome entry site-enhanced green fluorescent protein, was constructed and subsequently transfected into Sprague Dawley (SD) rat BMSCs. The gene and protein expression levels of the nucleic acid neurotrophin-3 (NT-3) were then detected. The results demonstrated that the constructed NT-3 gene lentiviral expression vector matched the expected design and that the NT-3 gene was transfected into the BMSCs via the lentivirus‑mediated method at a transfection efficiency of 60‑70%. NT-3 gene expression was detected within the stably transfected positive cells at the nucleic acid and protein levels. The cell morphology and biological activity of BMSCs did not alter significantly following transfection with NT-3. NT-3-transfected SD BMSCs were successfully constructed and served as effective vector seed cells with stable expression. These results can be used as a reference for subsequent studies on the transplantation therapy of rat spinal cord injuries using lentivirus-mediated NT-3-transfected SD BMSCs.

  15. Mechanistic investigations and molecular medicine applications of gold nanoparticle mediated (GNOME) laser transfection

    NASA Astrophysics Data System (ADS)

    Schomaker, M.; Heinemann, D.; Kalies, S.; Willenbrock, S.; Murua Escobar, H.; Buch, A.; Sodeik, B.; Ripken, T.; Meyer, H.

    2014-03-01

    Alternative high throughput transfection methods are required to understand the molecular network of the cell, which is linked to the evaluation of target genes as therapeutic agents. Besides diagnostic purposes, the transfection of primary- and stem cells is of high interest for therapeutic use. Here, the cell release of trans- or exogene proteins is used to develop immune cancer therapies. The basic requirement to accomplish manipulation of cells is an efficient and gentle transfection method. Therefore, we developed an automatized cell manipulation platform providing high throughput by using GNOME laser transfection. Herein, the interaction of moderately focused laser pulses with gold nanoparticles in close vicinity to the cell membrane mediate transient membrane permeabilization. The exact nature of the involved permeabilization effects depends on the applied particles and laser parameters. Hereinafter, we describe investigations considering the parameter regime, the permeabilization mechanism and the safety profile of GNOME laser transfection. The experimental and calculated results imply a combined permeabilization mechanism consisting of both photochemical and photothermal effects. Furthermore, paramount spatial control achieved either by laser illumination with micrometer precision or targeted gold nanoparticle binding to the cells was demonstrated, allowing selective cell manipulation and destruction. Additionally, the possibility to manipulate difficult to transfect primary cells (neurons) is shown. These results give insights in the basic mechanisms involved in GNOME laser transfection and serve as a strong basis to deliver different molecules for therapeutic (e.g. proteins) and diagnostic (siRNA) use.

  16. Factors that affect the efficiency of antisense oligodeoxyribonucleotide transfection by insonated gas-filled lipid microbubbles

    NASA Astrophysics Data System (ADS)

    Zhao, Ying-Zheng; Lu, Cui-Tao

    2008-03-01

    Objective: To investigate the factors that affect the efficiency of antisense oligodeoxyribonucleotide(AS-ODNs) transfection by insonated gas-filled lipid microbubbles. Methods: Lipid microbubbles filled with two types of gases-air and C3F8, were prepared respectively. An AS-ODNs sequence HA824 and a breast cancer cell line SK-BR-3 were used to define the various operating variables determining the transfection efficiency of insonated microbubbles. Two mixing methods, three levels of mixing speed, different mixing durations and various ultrasound initiation time after mixing were examined respectively. Transfection efficiency was detected by fluorescence microscopy. Results: C3F8 microbubbles gave higher levels of AS-ODNs transfection efficiency than air microbubbles in all test conditions. Transfection efficiency resulted from mixing method A (incubation of HA824 and microbubbles before mixing cells) did not show significant difference with that of mixing method B (without incubation of HA824 and microbubbles before mixing cells). Mixing speed, duration of mixing and ultrasound initiation time after mixing were central to determining HA824 transfection efficiency in vitro. The optimum parameters for SK-BR-3 cells were found at a mixing speed of 40-50 rpm for 30-60 s with less than 60 s delay before ultrasound. Conclusion: Ultrasound-mediated AS-ODNs transfection enhanced by C3F8-filled lipid microbubbles represents an effective avenue for AS-ODNs transfer.

  17. Anesthetic considerations in a parturient with congenital insensitivity to pain with anhidrosis.

    PubMed

    Pirani, Z; Qasem, F; Katsiris, S

    2017-02-01

    Congenital insensitivity to pain is a rare autosomal recessive disease characterized by varying degrees of autonomic dysfunction and sensory loss, including nociceptive hyposensitivity. It is this autonomic dysfunction that makes both general and neuraxial anesthesia challenging. Testing the block during neuraxial anesthesia may be difficult and the patient may be prone to hypotension and bradycardia. This case report describes the anesthetic management of a parturient with congenital insensitivity to pain presenting for cesarean delivery.

  18. Time-division multiplexing of polarization-insensitive fiber-optic Michelson interferometric sensors

    NASA Astrophysics Data System (ADS)

    Huang, S. C.; Lin, W. W.; Chen, M. H.

    1995-06-01

    A system of time-division multiplexing of polarization-insensitive fiber-optic Michelson interferometric sensors that uses Faraday rotator mirror elements is demonstrated. This system is constructed with conventional low-birefringence single-mode fiber and is able to solve the polarization-fading problem by a combination of Faraday rotator mirrors with unbalanced Michelson interferometers. The system is lead-fiber insensitive and has potentials for practical field applications.

  19. Timing of Gonadectomy in Patients with Complete Androgen Insensitivity Syndrome-Current Recommendations and Future Directions.

    PubMed

    Patel, Vrunda; Casey, Rachel Kastl; Gomez-Lobo, Veronica

    2016-08-01

    This review highlights the controversy regarding timing of gonadectomy in patients with complete androgen insensitivity syndrome (CAIS). We will review the published literature regarding frequency of gonadal malignancy and summarize historical findings. Recent research suggests that gonadectomy may be deferred until adulthood due to the low risk of malignancy. An algorithm is also provided to help guide clinicians in management of patients with complete androgen insensitivity syndrome who have deferred gonadectomy.

  20. Migraine: possible role of platelet insensitivity to prostaglandin E1 (PGE1).

    PubMed

    Cerneca, F; de Luyk, S; Radillo, O; Simeone, R; Mangiarotti, M

    1993-01-01

    Platelet aggregation inhibition, induced by prostaglandin E1 (PGE1), was evaluated in 38 patients affected by migraine. Our data indicate a complete insensitivity to PGE1 in these subjects. The insensitivity to PGE1 leads to decreased cyclic-AMP (cAMP) levels, determining an imbalance in the inhibitory mechanism. From this observation we can suppose that the decreased affinity of PGE1-receptors, causing decreased cAMP levels, may be involved in pathogenesis of migraine.

  1. Metastatic potential of cloned murine melanoma cells transfected with activated c-Ha-ras.

    PubMed

    Price, J E; Aukerman, S L; Ananthaswamy, H N; McIntyre, B W; Schackert, G; Schackert, H K; Fidler, I J

    1989-08-01

    We sought to determine whether the transfection of tumorigenic but not metastatic cells with the activated c-Ha-ras oncogene was invariably associated with acquisition of the metastatic phenotype. Three clonally derived lines of the K-1735 murine melanoma, characterized as nonmetastatic or poorly metastatic, were transfected with plasmids containing the 6.6-kilobase BamHI fragment of the mutant human c-Ha-ras gene and the neo gene, that confers resistance to neomycin (pSV2neoEJ). Cells transfected with pSV2neo, a plasmid containing the neo gene, served as controls for the procedure of Polybrene-mediated transfection. All cell lines were injected into syngeneic C3H/HeN and into athymic mice, and the results were compared with those produced by highly metastatic K-1735 M-2 cells. Although the pSV2neoEJ-transfected cells produced more rapidly growing s.c. tumors than the control cell lines did, the incidence of spontaneous metastasis was not increased. Following i.v. inoculation, the c-Ha-ras transfectants were retained in lung vasculature in greater proportions than pSV2neo counterpart transfectants were. The c-Ha-ras transfectants also produced significantly more lung tumor colonies, which grew faster than the few lung tumor colonies in mice given injections of control melanoma cells. We concluded that transfection of the activated c-Ha-ras oncogene into nonmetastatic K-1735 melanoma cells leads to accelerated tumor growth in vivo and can confer the ability to form lung colonies after i.v. injection but not the ability to metastasize from a primary s.c. tumor.

  2. Combinatorial synthesis of new cationic lipids and high-throughput screening of their transfection properties.

    PubMed

    Lenssen, Karl; Jantscheff, Peter; von Kiedrowski, Günter; Massing, Ulrich

    2002-09-02

    Here we describe the first synthesis-screening approach for the identification and optimization of new cationic lipids for gene transfer in various cell lines. Combinatorial solid-phase chemistry was used to synthesize a library of new cationic lipids based on 3-methylamino-1,2-dihydroxypropane as the polar, cationic lipid part. As the nonpolar lipid part, different hydrocarbon chains were bound to the amino group of the scaffold and the amino group was further methylated to afford constantly cationic lipids. Lipids were synthesized in both configurations and as racemates, and the counter ions were also varied. By using a fully automated transfection screening method and COS-7 cells, the cationic lipid N,N-ditetradecyl-N-methyl-amino-2,3-propanediol (KL-1-14) was identified as a candidate lipid for the development of an improved transfection reagent. Screening the transfection properties of KL-1-14 in numerous combinations with the helper lipids dioleoylphosphatidylethanolamine (DOPE) and cholesterol (Chol) revealed that Chol is the most suitable helper lipid and the best KL-1-14/Chol ratio is 0.5-0.7. Compared to the standard transfection lipid N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate (DOTAP), transfection efficiency was improved by a factor of about 40. Furthermore, by using R- and S-configured KL-1-14, it could be shown that the configuration of the lipids had no significant influence on its transfection efficiency. The highest transfection efficiencies were achieved with chloride as the counter ion. The new lipofection reagent was further tested to transfect the cell lines MDA-MB-468, MCF-7, MDCK-C7, and primary dentritic cells (DC), which are important for the development of new anticancer gene therapy strategies. Even in these cells, KL-1-14/Chol (1:0.6) had improved transfection efficiencies, which were about two to four times higher than for DOTAP.

  3. Photoperiod insensitive Ppd-A1a mutations in tetraploid wheat (Triticum durum Desf.).

    PubMed

    Wilhelm, Edward P; Turner, Adrian S; Laurie, David A

    2009-01-01

    Variation in photoperiod response plays an important role in adapting crops to agricultural environments. In hexaploid wheat, mutations conferring photoperiod insensitivity (flowering after a similar time in short or long days) have been mapped on the 2B (Ppd-B1) and 2D (Ppd-D1) chromosomes in colinear positions to the 2H Ppd-H1 gene of barley. No A genome mutation is known. On the D genome, photoperiod insensitivity is likely to be caused by deletion of a regulatory region that causes misexpression of a member of the pseudo-response regulator (PRR) gene family and activation of the photoperiod pathway irrespective of day length. Photoperiod insensitivity in tetraploid (durum) wheat is less characterized. We compared pairs of near-isogenic lines that differ in photoperiod response and showed that photoperiod insensitivity is associated with two independent deletions of the A genome PRR gene that cause altered expression. This is associated with induction of the floral regulator FT. The A genome deletions and the previously described D genome deletion of hexaploid wheat remove a common region, suggesting a shared mechanism for photoperiod insensitivity. The identification of the A genome mutations will allow characterization of durum wheat germplasm and the construction of genotypes with novel combinations of photoperiod insensitive alleles.

  4. Structure-activity correlation in transfection promoted by pyridinium cationic lipids.

    PubMed

    Parvizi-Bahktar, P; Mendez-Campos, J; Raju, L; Khalique, N A; Jubeli, E; Larsen, H; Nicholson, D; Pungente, M D; Fyles, T M

    2016-03-21

    The efficiency of the transfection of a plasmid DNA encoding a galactosidase promoted by a series of pyridinium lipids in mixtures with other cationic lipids and neutral lipids was assessed in CHO-K1 cells. We identify key molecular parameters of the lipids in the mixture - clog P, lipid length, partial molar volume - to predict the morphology of the lipid-DNA lipoplex and then correlate these same parameters with transfection efficiency in an in vitro assay. We define a Transfection Index that provides a linear correlation with normalized transfection efficiency over a series of 90 different lipoplex compositions. We also explore the influence of the same set of molecular parameters on the cytotoxicity of the formulations.

  5. Oncogene transfection of mink lung cells: effect on growth characteristics in vitro and in vivo.

    PubMed

    Khan, M Z; Spandidos, D A; Kerr, D J; McNicol, A M; Lang, J C; De Ridder, L; Freshney, R I

    1991-01-01

    Three sublines have been derived from the parental line Mv1Lu by transfection with normal and mutated Ha-ras, and myc oncogenes, and subsequent cloning. All the oncogenes have increased the growth rate of the cell in vitro, increased their plating efficiency in monolayer and suspension, and reduced their serum dependence. Growth in vivo as xenografts in nude mice has also been increased. Very few tumours were generated from the parental line and those that did form did so after a prolonged lag period, while the transfected lines produced tumours with 100% efficiency, and a short lag period. In general the effects of ras transfection were more extreme, with the highest growth rates and plating efficiencies in vitro and the shortest lag period and doubling times in vivo. There was no increase in plasminogen activator activity as a result of transfection, and the invasive behaviour of the lines in organotypic culture was broadly similar.

  6. Femtosecond optical transfection as a tool for genetic manipulation of human embryonic stem cells

    NASA Astrophysics Data System (ADS)

    Torres-Mapa, M. L.; Gardner, J.; Bradburn, H.; King, J.; Dholakia, K.; Gunn-Moore, F.

    2013-03-01

    We demonstrate the use of femtosecond optical transfection for the genetic manipulation of human embryonic stem cells. Using a system with an SLM combined with a scanning mirror allows poration of both single-cell and colony-formed human embryonic stem cells in a rapid and targeted manner. In this work, we show successful transfection of plasmid DNA tagged with fluorescent reporters into human embryonic stem cells using three doses of focused femtosecond laser. A significant number of transfected cells retained their undifferentiated morphological feature of large nucleus with high nucleus to cytoplasmic ratio, 48h after photoporation. Furthermore, DNA constructs driven by different types of promoters were also successfully transfected into human embryonic stem cells using this technique.

  7. Minimally-Invasive Gene Transfection by Chemical and Physical Interaction of Atmospheric Pressure Plasma Flow

    NASA Astrophysics Data System (ADS)

    Kaneko, Toshiro

    2014-10-01

    Non-equilibrium atmospheric pressure plasma irradiated to the living-cell is investigated for medical applications such as gene transfection, which is expected to play an important role in molecular biology, gene therapy, and creation of induced pluripotent stem (iPS) cells. However, the conventional gene transfection using the plasma has some problems that the cell viability is low and the genes cannot be transferred into some specific lipid cells, which is attributed to the unknown mechanism of the gene transfection using the plasma. Therefore, the time-controlled atmospheric pressure plasma flow is generated and irradiated to the living-cell suspended solution for clarifying the transfection mechanism toward developing highly-efficient and minimally- invasive gene transfection system. In this experiment, fluorescent dye YOYO-1 is used as the simulated gene and LIVE/DEAD Stain is simultaneously used for cell viability assay. By the fluorescence image, the transfection efficiency is calculated as the ratio of the number of transferred and surviving cells to total cell count. It is clarified that the transfection efficiency is significantly increased by the short-time (<4 sec) and short-distance (<40 mm) plasma irradiation, and the high transfection efficiency of 53% is realized together with the high cell viability (>90%). This result indicates that the physical effects such as the electric field caused by the charged particles arriving at the surface of the cell membrane, and chemical effects associated with plasma-activated products in solution act synergistically to enhance the cell-membrane transport with low-damage. This work was supported by JSPS KAKENHI Grant Number 24108004.

  8. Activation of kinin B1 receptor increases the release of metalloproteases-2 and -9 from both estrogen-sensitive and -insensitive breast cancer cells.

    PubMed

    Ehrenfeld, Pamela; Conejeros, Ivan; Pavicic, Maria F; Matus, Carola E; Gonzalez, Carlos B; Quest, Andrew F G; Bhoola, Kanti D; Poblete, Maria T; Burgos, Rafael A; Figueroa, Carlos D

    2011-02-01

    The kinin B(1) receptor (B(1)R) agonist Lys-des[Arg(9)]-bradykinin (LDBK) increases proliferation of estrogen-sensitive breast cancer cells by a process involving activation of the epidermal growth factor receptor (EGFR) and downstream signaling via the ERK1/2 mitogen-activated protein kinase pathway. Here, we investigated whether B(1)R stimulation induced release of the extracellular matrix metalloproteases MMP-2 and MMP-9 via ERK-dependent pathway in both estrogen-sensitive MCF-7 and -insensitive MDA-MB-231 breast cancer cells. Cells were stimulated with 1-100nM of the B(1)R agonist for variable time-points. Western blotting and gelatin zymography were used to evaluate the presence of MMP-2 and MMP-9 in the extracellular medium. Stimulation of B(1)R with as little as 1 nM LDBK induced the accumulation of these metalloproteases in the medium within 5-30min of stimulation. In parallel, immunocytochemistry revealed that metalloprotease levels in the breast cancer cells declined after stimulation. This effect was blocked either by pre-treating the cells with a B(1)R antagonist or by transfecting with B(1)R-specific siRNA. Activation of the ERK1/2 pathway and EGFR transactivation was required for release of metalloproteases because both the MEK1 inhibitor, PD98059, and AG1478, an inhibitor of the EGFR-tyrosine kinase activity, blocked this event. The importance of EGFR-dependent signaling was additionally confirmed since transfection of cells with the dominant negative EGFR mutant HERCD533 blocked the release of metalloproteases. Thus, activation of B(1)R is likely to enhance breast cancer cells invasiveness by releasing enzymes that degrade the extracellular matrix and thereby favor metastasis.

  9. Ultrasound-mediated gene transfection: A comparison between cells irradiated in suspension and attachment status

    NASA Astrophysics Data System (ADS)

    Zhang, Yiwei; Azuma, Takashi; Sasaki, Akira; Yoshinaka, Kiyoshi; Takagi, Shu; Matsumoto, Yoichiro

    2012-10-01

    Sonoporation, in the presence of microbubbles, is a promising nonviral gene transfection method. Although the mechanism is not yet fully understood, shock waves emitted by cavitation bubbles have been known to play an important role in creating pores on cell membranes. This work investigates the gene transfection efficiency and influencing parameters of cells in two different statuses: attachment and suspension based on the fact that cells in suspension have more bubbles surrounding them and that shock wave has distinct effects on hit objects whether the object is attached to a rigid wall or not. Fibroblast cells (NIH3T3), both in attachment and suspension, and green fluorescent protein (GFP) plasmid were exposed to variations in acoustic pressure (0.6-1.2 MPa) and 10% duty cycle at fixed settings of 2 MHz central frequency, 5 kHz pulse repetition frequency and 1 minute insonation time, in the presence of 10% v/v microbubbles (Sonazoid, a commercialized product of ultrasound contrast agent). The transfection efficiency and cell viability are compared for two statuses and a distribution map of GFP transfected cells as well as viable cells over the well bottom is given for attachment status. The results show that cells irradiated in suspension status has higher transfection ratio as well as viability than those irradiated in attachment status with the same intensity and that the transfected cells of attachment status experiment are highly concentrated near the center of the well.

  10. Gene Transfection Method Using Atmospheric Pressure Dielectric-Barrier Discharge Plasmas

    NASA Astrophysics Data System (ADS)

    Sasaki, Shota; Kanzaki, Makoto; Kaneko, Toshiro

    2013-09-01

    Gene transfection which is the process of deliberately introducing nucleic acids into cells is expected to play an important role in medical treatment because the process is necessary for gene therapy and creation of induced pluripotent stem (iPS) cells. However, the conventional transfection methods have some problems, so we focus attention on promising transfection methods by atmospheric pressure dielectric-barrier discharge (AP-DBD) plasmas. AP-DBD He plasmas are irradiated to the living cell covered with genes. Preliminarily, we use fluorescent dye YOYO-1 instead of the genes and use LIVE/DEAD Stain for cell viability test, and we analyze the transfection efficiency and cell viability under the various conditions. It is clarified that the transfection efficiency is strongly dependence on the plasma irradiation time and cell viability rates is high rates (>90%) regardless of long plasma irradiation time. These results suggest that ROS (Reactive Oxygen Species) and electric field generated by the plasma affect the gene transfection. In addition to this (the plasma irradiation time) dependency, we now investigate the effect of the plasma irradiation under the various conditions.

  11. Cell Transfection with a β-Cyclodextrin-PEI-Propane-1,2,3-Triol Nanopolymer

    PubMed Central

    Lai, Wing-Fu; Jung, Han-Sung

    2014-01-01

    Successful gene therapy necessitates safe and efficient gene transfer. This article describes the use of a cationic polymer, which was synthesized by cross-linking low molecular weight branched poly(ethylenimine) (PEI) with both β-cyclodextrin and propane-1,2,3-triol, for efficient and safe non-viral gene delivery. Experimentation demonstrated that the polymer had a pH buffering capacity and DNA condensing ability comparable to those of PEI 25 kDa. In B16-F0 cells, the polymer increased the transfection efficiency of naked DNA by 700-fold and yielded better transfection efficiencies than Fugene HD (threefold higher) and PEI 25 kDa (fivefold higher). The high transfection efficiency of the polymer was not affected by the presence of serum during transfection. In addition to B16-F0 cells, the polymer enabled efficient transfection of HepG2 and U87 cells with low cytotoxicity. Our results indicated that our polymer is a safe and efficient transfection reagent that warrants further development for in vitro, in vivo and clinical applications. PMID:24956480

  12. Bacterial IMPDH gene used for the selection of mammalian cell transfectants.

    SciTech Connect

    Baccam, M.; Huberman, E.; Energy Systems

    2003-06-01

    Stable cell transfection is used for the expression of exogenous genes or cDNAs in eukaryotic cells. Selection of these transfectants requires a dominant selectable marker. A variety of such markers has been identified and is currently in use. However, many of these are not suitable for all cell types or require unique conditions. Here we describe a simple and versatile dominant selectable marker that involves bacterial IMP dehydrogenase (IMPDH), an enzyme essential for the replication of mammalian and bacterial cells. Although IMPDH is evolutionarily conserved, the bacterial enzyme is orders of magnitude more resistant to the toxic effect of the drug mycophenolic acid, which is an IMPDH inhibitor. We have demonstrated that transfection of human, monkey or Chinese hamster cell lines with an expression vector containing bacterial IMPDH and mycophenolic acid treatment resulted in the selection of colonies with a strikingly increased resistance to mycophenolic acid toxicity. Analysis of cells derived from these colonies indicated that the acquisition of this resistance was associated with bacterial IMPDH protein expression. As a proof of principle, we showed that mammalian cell transfection with a hicistronic IMPDH/GFP expression vector and mycophenolic acid treatment can he used to successfully select transfectants that express the fluorescent protein. These results indicate that bacterial IMPDH is a practical dominant selectable marker that can be used for the selection of transfectants that express exogenous genes or cDNAs in mammalian cells.

  13. Combined Pulse Electroporation – A Novel Strategy for Highly Efficient Transfection of Human and Mouse Cells

    PubMed Central

    Stroh, Thorsten; Erben, Ulrike; Kühl, Anja A.; Zeitz, Martin; Siegmund, Britta

    2010-01-01

    The type of a nucleic acid and the type of the cell to be transfected generally affect the efficiency of electroporation, the versatile method of choice for gene regulation studies or for recombinant protein expression. We here present a combined square pulse electroporation strategy to reproducibly and efficiently transfect eukaryotic cells. Cells suspended in a universal buffer system received an initial high voltage pulse that was continuously combined with a subsequent low voltage pulse with independently defined electric parameters of the effective field and the duration of each pulse. At comparable viable cell recoveries and transfection efficiencies of up to 95% of all cells, a wide variety of cells especially profited from this combined pulse strategy by high protein expression levels of individual cells after transfection. Long-term silencing of gene expression by transfected small interfering RNA was most likely due to the uptake of large nucleic acid amounts as shown by direct detection of fluorochromated small interfering RNA. The highly efficient combined pulse electroporation strategy enables for external regulation of the number of naked nucleic acid molecules taken up and can be easily adapted for cells considered difficult to transfect. PMID:20209146

  14. Fast targeted gene transfection and optogenetic modification of single neurons using femtosecond laser irradiation

    PubMed Central

    Antkowiak, Maciej; Torres-Mapa, Maria Leilani; Witts, Emily C.; Miles, Gareth B.; Dholakia, Kishan; Gunn-Moore, Frank J.

    2013-01-01

    A prevailing problem in neuroscience is the fast and targeted delivery of DNA into selected neurons. The development of an appropriate methodology would enable the transfection of multiple genes into the same cell or different genes into different neighboring cells as well as rapid cell selective functionalization of neurons. Here, we show that optimized femtosecond optical transfection fulfills these requirements. We also demonstrate successful optical transfection of channelrhodopsin-2 in single selected neurons. We extend the functionality of this technique for wider uptake by neuroscientists by using fast three-dimensional laser beam steering enabling an image-guided “point-and-transfect” user-friendly transfection of selected cells. A sub-second transfection timescale per cell makes this method more rapid by at least two orders of magnitude when compared to alternative single-cell transfection techniques. This novel technology provides the ability to carry out large-scale cell selective genetic studies on neuronal ensembles and perform rapid genetic programming of neural circuits. PMID:24257461

  15. Ultrasound-Mediated Mesenchymal Stem Cells Transfection as a Targeted Cancer Therapy Platform

    PubMed Central

    Haber, Tom; Baruch, Limor; Machluf, Marcelle

    2017-01-01

    Mesenchymal stem cells (MSCs) hold tremendous potential as a targeted cell-based delivery platform for inflammatory and cancer therapy. Genetic manipulation of MSCs, however, is challenging, and therefore, most studies using MSCs as therapeutic cell carriers have utilized viral vectors to transduce the cells. Here, we demonstrate, for the first time, an alternative approach for the efficient transfection of MSCs; therapeutic ultrasound (TUS). Using TUS with low intensities and moderate frequencies, MSCs were transfected with a pDNA encoding for PEX, a protein that inhibits tumor angiogenesis, and studied as a cell vehicle for in vivo tumor therapy. TUS application did not alter the MSCs’ stemness or their homing capabilities, and the transfected MSCs transcribed biologically active PEX. Additionally, in a mouse model, 70% inhibition of prostate tumor growth was achieved following a single I.V. administration of MSCs that were TUS-transfected with pPEX. Further, the repeated I.V. administration of TUS-pPEX transfected-MSCs enhanced tumor inhibition up to 84%. Altogether, these results provide a proof of concept that TUS-transfected MSCs can be effectively used as a cell-based delivery approach for the prospective treatment of cancer. PMID:28169315

  16. Transplantation of syngeneic transfected cells to probe the in vivo immune response to viral proteins

    SciTech Connect

    Nakayama, Hiroyuki; Shibata, Motohiro; Wohlenberg, C.; Rooney, J.F.; Notkins, A.L. )

    1991-01-01

    BALB/3T3 cells were transfected with the glycoprotein D (gD) gene of herpes simplex virus (HSV) and a cell line expressing gD on the cell surface was isolated. In vitro, {sup 51}Cr release tests showed that the transfected cells were destroyed by anti-HSV antibody in the presence of complement. To investigate in vivo immune response, the gD-transfected cells were transplanted into the footpads of syngeneic HSV-immunized or unimmunized BALB/c mice. In unimmunized mice, transfected cells remained intact for 7 days or longer, and the site of injection showed only slight lymphocyte infiltration. In contrast, in immunized mice, transfected cells elicited massive lymphocyte infiltration and were mostly destroyed by day 4. Analysis of infiltrating cells revealed that they were mainly Thy1{sup +} and CD8{sup +} lymphocytes along with small numbers of CD5{sup +}, CD4{sup +}, and B lymphocytes. These studies show that transfected murine cells expressing gD can be used to study the in vivo immune response to single viral proteins and they argue that the immune response contributes to the pathogenesis of HSV infection.

  17. Temperature- and wavelength-insensitive parametric amplification enabled by noncollinear achromatic phase-matching

    PubMed Central

    Tang, Daolong; Ma, Jingui; Wang, Jing; Zhou, Bingjie; Xie, Guoqiang; Yuan, Peng; Zhu, Heyuan; Qian, Liejia

    2016-01-01

    Optical parametric chirped-pulse amplification (OPCPA) has been demonstrated to be a promising approach for pushing femtosecond pulses towards ultra-high peak powers. However, the future success of OPCPA strongly relies on the ability to manipulate its phase-matching (PM) configuration. When a high average power pump laser is involved, the thermal effects in nonlinear crystals induce phase-mismatch distortions that pose an inherent limitation on the conversion efficiency. Here, we demonstrate that the noncollinear configuration previously adopted for wavelength-insensitive PM can be employed for temperature-insensitive PM when the noncollinear angle is properly reset. Simultaneous temperature- and wavelength-insensitive PM is realized for the first time by imposing such a temperature-insensitive noncollinear configuration with an angularly dispersed seed signal. Based on the lithium triborate crystal, the proposed noncollinear achromatic PM has a thermal acceptance 6 times larger than that of the conventional wavelength-insensitive noncollinear PM and has a sufficient spectral acceptance to support pulse durations of ~20 fs at 800 nm. These achievements open new possibilities for generating ultra-high peak power lasers with high average power. PMID:27786299

  18. Angle- and polarization-insensitive metamaterial absorber based on vertical and horizontal symmetric slotted sectors.

    PubMed

    Trung, Nguyen Toan; Lee, Dongju; Sung, Hyuk-Kee; Lim, Sungjoon

    2016-10-10

    This novel vertically and horizontally symmetric slotted-sector design aims to realize an angle- and polarization-insensitive metamaterial absorber. The unit-cell symmetries achieve polarization insensitivity, while an optimized slotted-sector inner angle enables angle insensitivity. Because the absorptivity of a metamaterial absorber depends on the incident angle and polarization, many researchers have studied angle- and polarization-insensitive unit cells. In this work, a novel vertically and horizontally symmetric slotted sector is proposed in order to realize an angle- and polarization-insensitive metamaterial absorber. The absorber performance is demonstrated with full-wave simulation and measurements. Angular sensitivity is studied for different slotted-sector inner angles. For an 85° inner angle, the simulated absorptivity exceeds 90% and the frequency variation is less than 1.2% up to 70° incidence. The measured absorptivity at 10.34 GHz is close to 98.5% for all polarization angles at normal incidence. As the incidence angle varies from 0° to 70°, the measured absorptivity at 10.34 GHz remains above 90% in the transverse electric mode.

  19. Temperature- and wavelength-insensitive parametric amplification enabled by noncollinear achromatic phase-matching

    NASA Astrophysics Data System (ADS)

    Tang, Daolong; Ma, Jingui; Wang, Jing; Zhou, Bingjie; Xie, Guoqiang; Yuan, Peng; Zhu, Heyuan; Qian, Liejia

    2016-10-01

    Optical parametric chirped-pulse amplification (OPCPA) has been demonstrated to be a promising approach for pushing femtosecond pulses towards ultra-high peak powers. However, the future success of OPCPA strongly relies on the ability to manipulate its phase-matching (PM) configuration. When a high average power pump laser is involved, the thermal effects in nonlinear crystals induce phase-mismatch distortions that pose an inherent limitation on the conversion efficiency. Here, we demonstrate that the noncollinear configuration previously adopted for wavelength-insensitive PM can be employed for temperature-insensitive PM when the noncollinear angle is properly reset. Simultaneous temperature- and wavelength-insensitive PM is realized for the first time by imposing such a temperature-insensitive noncollinear configuration with an angularly dispersed seed signal. Based on the lithium triborate crystal, the proposed noncollinear achromatic PM has a thermal acceptance 6 times larger than that of the conventional wavelength-insensitive noncollinear PM and has a sufficient spectral acceptance to support pulse durations of ~20 fs at 800 nm. These achievements open new possibilities for generating ultra-high peak power lasers with high average power.

  20. The Synergistic Effect between Electrical and Chemical Factors in Plasma Gene/Molecule-Transfection

    NASA Astrophysics Data System (ADS)

    Jinno, Masafumi

    2016-09-01

    This study has been done to know what kind of factors in plasma and processes on cells promote plasma gene/molecule transfection. We have discovered a new plasma source using a microcapillary electrode which enables high transfection efficiency and high cell survivability simultaneously. However, the mechanism of the transfection by plasma was not clear. To clarify the transfection mechanisms by micro plasma, we focused on the effects of electrical (current, charge, field, etc.) and chemical (radicals, RONS, etc.) factors generated by the micro plasma and evaluated the contribution weight of three groups of the effects and processes, i.e. electrical, chemical and biochemical ones. At first, the necessity of the electrical factors was estimated by the laser produced plasma (LPP). Mouse L-929 fibroblast cell was cultured on a 96-well plate or 12-well micro slide chamber. Plasmids pCX-EGFP in Tris-EDTA buffer was dropped on the cells and they were exposed to the capillary discharge plasma (CDP) or the LPP. In the case of the CDP, the plasma was generated between the tip of the capillary electrode and the cells so that both electrical and chemical factors were supplied to the cells. In this setup, about 20% of average transfection efficiency was obtained. In the case of the LPP, the plasma was generated apart from the cells so that electrical factors were not supplied to the cells. In this setup, no transfection was observed. These results show that the electrical factors are necessary for the plasma gene transfection. Next, the necessity of the chemical factors was estimated the effect of catalase to remove H2O2 in CDP. The transfection efficiency decreased to 0.4 by scavenging H2O2 with catalase. However, only the solution of H2O2 caused no gene transfection in cells. These results shows that H2O2 is important species to cause gene/molecule transfection but still needs a synergistic effect with electrical or other chemical factors. This work was partly supported by

  1. Rapid, in vivo, evaluation of antiangiogenic and antineoplastic gene products by nonviral transfection of tumor cells.

    PubMed

    Weiss, Jonathan M; Shivakumar, Rama; Feller, Stephanie; Li, Lin-Hong; Hanson, Art; Fogler, William E; Fratantoni, Joseph C; Liu, Linda N

    2004-05-01

    Using a nonviral, electroporation-based gene transfection approach, we demonstrate the efficient and consistent transfection of two poorly immunogenic tumor cell lines: B16F10 melanoma and renal carcinoma (RENCA). Three genes, IL-12, angiostatin (AS), and an endostatin:angiostatin fusion protein (ES:AS) were subcloned into a DNA plasmid containing EBNA1-OriP, which was then transfected into B16F10 and RENCA cells. Significant levels of protein were secreted into the culture supernatants of transfected cells in vitro. Transfected tumor cells were injected subcutaneously into mice. All the three transgenes were capable of significantly delaying and reducing the formation of primary B16F10 and RENCA tumors, as well as B16F10 lung metastases. By day 11 post-injection, all control mice that received either mock-transfected or empty vector DNA-transfected B16F10 tumor cells had developed large primary tumors. In contrast, mice that received IL-12-transfected B16F10 cells did not develop appreciable tumors until day 17, and these were significantly smaller than controls. Similar results were observed for the RENCA model, in which only one of the IL-12 mice had developed tumors out to day 31. Expression of AS or ES:AS also significantly delayed and reduced primary tumors. Overall, ES:AS was more effective than AS alone. Furthermore, 25% of the AS mice and 33% of the ES:AS mice remained tumor-free at day 17, by which point all control mice had significant tumors. Mouse survival rates also correlated with the extent of tumor burden. Importantly, no lung metastases were detected in the lungs of mice that had received either AS or ES:AS-transfected B16F10 tumor cells and significantly fewer metastases were found in the IL-12 group. The consistency of our transfection results highlight the feasibility of directly electroporating tumor cells as a means to screen, identify, and validate in vivo potentially novel antiangiogenic and/or antineoplastic genes.

  2. MAOHUZI6/ETHYLENE INSENSITIVE3-LIKE1 and ETHYLENE INSENSITIVE3-LIKE2 Regulate Ethylene Response of Roots and Coleoptiles and Negatively Affect Salt Tolerance in Rice.

    PubMed

    Yang, Chao; Ma, Biao; He, Si-Jie; Xiong, Qing; Duan, Kai-Xuan; Yin, Cui-Cui; Chen, Hui; Lu, Xiang; Chen, Shou-Yi; Zhang, Jin-Song

    2015-09-01

    Ethylene plays important roles in plant growth, development, and stress responses. The ethylene signaling pathway has been studied extensively, mainly in Arabidopsis (Arabidopsis thaliana). However, the molecular mechanism of ethylene signaling is largely unknown in rice (Oryza sativa). Previously, we have isolated a set of rice ethylene-response mutants. Here, we characterized the mutant maohuzi6 (mhz6). Through map-based cloning, we found that MHZ6 encodes ETHYLENE INSENSITIVE3-LIKE1 (OsEIL1), a rice homolog of ETHYLENE INSENSITIVE3 (EIN3), which is the master transcriptional regulator of ethylene signaling in Arabidopsis. Disruption of MHZ6/OsEIL1 caused ethylene insensitivity mainly in roots, whereas silencing of the closely related OsEIL2 led to ethylene insensitivity mainly in coleoptiles of etiolated seedlings. This organ-specific functional divergence is different from the functional features of EIN3 and EIL1, both of which mediate the incomplete ethylene responses of Arabidopsis etiolated seedlings. In Arabidopsis, EIN3 and EIL1 play positive roles in plant salt tolerance. In rice, however, lack of MHZ6/OsEIL1 or OsEIL2 functions improves salt tolerance, whereas the overexpressing lines exhibit salt hypersensitivity at the seedling stage, indicating that MHZ6/OsEIL1 and OsEIL2 negatively regulate salt tolerance in rice. Furthermore, this negative regulation by MHZ6/OsEIL1 and OsEIL2 in salt tolerance is likely attributable in part to the direct regulation of HIGH-AFFINITY K(+) TRANSPORTER2;1 expression and Na(+) uptake in roots. Additionally, MHZ6/OsEIL1 overexpression promotes grain size and thousand-grain weight. Together, our study provides insights for the functional diversification of MHZ6/OsEIL1 and OsEIL2 in ethylene response and finds a novel mode of ethylene-regulated salt stress response that could be helpful for engineering salt-tolerant crops.

  3. Congenital Insensitivity to Pain and Anhydrosis: Diagnostic and Therapeutic Dilemmas revisited

    PubMed Central

    Kandregula, Chaitanya Ram; Koya, Srikanth; Lakhotia, Disha

    2015-01-01

    ABSTRACT First described in 1932 by Dearborn as ‘congenital pure analgesia’, congenital insensitivity to pain and anhydrosis (CIPA) or hereditary sensory and autonomic neuropathy (HSAN) type IV is an extremely rare autosomal recessive disorder. A 7-year-old female child who is an established case of congenital insensitivity to pain and anhydrosis visited the department of pediatric medicine with osteoarthritic neuropathy. A multidisciplinary team approach was utilized to treat the child under general anesthesia. This article also discusses the diagnostic and therapeutic dilemmas involved in treating this type of children. How to cite this article: Ravichandra KS, Kandregula CR, Koya S, Lakhotia D. Congenital Insensitivity to Pain and Anhydrosis: Diagnostic and Therapeutic Dilemmas revisited. Int J Clin Pediatr Dent 2015;8(1):75-81. PMID:26124587

  4. [Morris syndrome: description of a case characterized by partial androgen insensitivity].

    PubMed

    Creta, Massimiliano; Smelzo, Salvatore; Di Vito, Concetta; De Stefano, Giacomo; Forchia, Francesco; Chiancone, Francesco; Imbimbo, Ciro

    2010-01-01

    The Morris syndrome is a X-linked recessive condition due to a complete or partial insensitivity to androgens, resulting in a failure of normal masculinization of the external genitalia in chromosomally male individuals. This failure of virilization can be either complete or partial depending on the amount of residual androgen receptor function. The phenotype of individuals with partial androgen insensitivity syndrome may range from mildly virilized female external genitalia to mildly undervirilized male external genitalia. We describe a case of Partial Androgen Insensitivity Syndrome in a 21-year-old patient with a 46, XY karyotype, bilateral inguinal masses, clitoral enlargement and partial posterior labial fusion. Surgical care consisted of bilateral orchiectomy and plastic surgery of external genitalia. The patient underwent estrogen replacement therapy.

  5. [Congenital insensitivity to pain: clinical and neurophysiological study in three sisters of a Moroccan family].

    PubMed

    Kissani, N; Krrati, H; Alarcon, G; Belaaidi, H; Ouazzani, R

    2013-11-01

    Congenital insensitivity to pain is a rare hereditary sensory and autonomic neuropathy (HSAN). This disorder is an autosomal recessive condition: since 1996, mutations attributed to this entity have been found in the neurotrophin tyrosine-kinase gene receptor on chromosome 1. The authors report 3 cases of congenital insensitivity to pain. In these 3 sisters of consanguineous parents, the clinical investigation showed total absence of pain and temperature sensations with preservation of all other sensory modalities, mental retardation, but in contrast to HSAN type IV, there was no anhidrosis. The neurophysiological investigation revealed an isolated axonal sensory polyneuropathy in the 3 patients. The clinical and neurophysiological investigations were normal in both parents and the brother. The physiopathology of this entity is discussed. We suggest a particular form of HSAN type IV with preservation of transpiration or a new entity of congenital insensitivity to pain, which should be analyzed genetically.

  6. Congenital Insensitivity to Pain and Anhydrosis: Diagnostic and Therapeutic Dilemmas revisited.

    PubMed

    Ravichandra, K S; Kandregula, Chaitanya Ram; Koya, Srikanth; Lakhotia, Disha

    2015-01-01

    First described in 1932 by Dearborn as 'congenital pure analgesia', congenital insensitivity to pain and anhydrosis (CIPA) or hereditary sensory and autonomic neuropathy (HSAN) type IV is an extremely rare autosomal recessive disorder. A 7-year-old female child who is an established case of congenital insensitivity to pain and anhydrosis visited the department of pediatric medicine with osteoarthritic neuropathy. A multidisciplinary team approach was utilized to treat the child under general anesthesia. This article also discusses the diagnostic and therapeutic dilemmas involved in treating this type of children. How to cite this article: Ravichandra KS, Kandregula CR, Koya S, Lakhotia D. Congenital Insensitivity to Pain and Anhydrosis: Diagnostic and Therapeutic Dilemmas revisited. Int J Clin Pediatr Dent 2015;8(1):75-81.

  7. Sequence diversity in the large subunit of RNA polymerase I contributes to Mefenoxam insensitivity in Phytophthora infestans.

    PubMed

    Randall, Eva; Young, Vanessa; Sierotzki, Helge; Scalliet, Gabriel; Birch, Paul R J; Cooke, David E L; Csukai, Michael; Whisson, Stephen C

    2014-09-01

    Phenylamide fungicides have been widely used for the control of oomycete-incited plant diseases for over 30 years. Insensitivity to this chemical class of fungicide was recorded early in its usage history, but the precise protein(s) conditioning insensitivity has proven difficult to determine. To determine the genetic basis of insensitivity and to inform strategies for the cloning of the gene(s) responsible, genetic crosses were established between Mefenoxam sensitive and intermediate insensitive isolates of Phytophthora infestans, the potato late blight pathogen. F1 progeny showed the expected semi-dominant phenotypes for Mefenoxam insensitivity and suggested the involvement of multiple loci, complicating the positional cloning of the gene(s) conditioning insensitivity to Mefenoxam. Instead, a candidate gene strategy was used, based on previous observations that the primary effect of phenylamide compounds is to inhibit ribosomal RNA synthesis. The subunits of RNA polymerase I (RNApolI) were sequenced from sensitive and insensitive isolates and F1 progeny. Single nucleotide polymorphisms (SNPs) specific to insensitive field isolates were identified in the gene encoding the large subunit of RNApolI. In a survey of field isolates, SNP T1145A (Y382F) showed an 86% association with Mefenoxam insensitivity. Isolates not showing this association belonged predominantly to one P. infestans genotype. The transfer of the 'insensitive' allele of RPA190 to a sensitive isolate yielded transgenic lines that were insensitive to Mefenoxam. These results demonstrate that sequence variation in RPA190 contributes to insensitivity to Mefenoxam in P. infestans.

  8. Basic Fourier properties for generalized phase shifting and some interesting detuning insensitive algorithms

    NASA Astrophysics Data System (ADS)

    Téllez-Quiñones, Alejandro; Malacara-Doblado, Daniel; García-Márquez, Jorge

    2011-07-01

    In this manuscript, some interesting properties for generalized or nonuniform phase-shifting algorithms are shown in the Fourier frequency space. A procedure to find algorithms with equal amplitudes for their sampling function transforms is described. We also consider in this procedure the finding of algorithms that are orthogonal for all possible values in the frequency space. This last kind of algorithms should closely satisfy the first order detuning insensitive condition. The procedure consists of the minimization of functionals associated with the desired insensitivity conditions.

  9. Use of fiber helical coils to obtain polarization insensitive fiber devices

    NASA Astrophysics Data System (ADS)

    Tentori, Diana; Garcia-Weidner, A.; Rodriguez-Garcia, J. A.

    2016-09-01

    Using a new model for the description of the birefringence of a helical coil, it is shown that the birefringence effect on the signal polarization introduced by a fiber device can be canceled out by introducing two helical coils at the required orientation. Experimental results obtained using this modification in a polarization insensitive device (optical isolator) and in a non-polarization insensitive device working at two different wavelengths (wavelength division multiplexer) are presented and discussed. Such modified devices were used in the construction of an erbium-doped fiber amplifier (EDFA) with a full control of the input signal and pump states of polarization.

  10. [Congenital insensitivity to pain with anhidrosis. Clinical diagnosis, evolution and complications: case report].

    PubMed

    Albuja Echeverría, Byron Orlando; Alvear Lozano, Mayra Bersabeth; Ordóñez Paredes, Carla Patricia

    2014-10-01

    The congenital insensitivity to pain with anhidrosis is a rare autosomal recessive disease caused by mutations in NTRK1 gene (neurotrophic tyrosine kinase receptor 1) located in chromosome 1q21-22, encoding the tyrosinase domain receptor high affinity nerve growth factor. It is characterized by anhidrosis, insensitivity to painful stimuli and mental retardation. Given their low prevalence and the few reported cases, it is important to know its main features to be considered in the differential diagnosis in pediatric practice. We describe the clinical diagnosis, complications, sequelae and symptomatic treatment administered to a 3 years and 6 months old girl in the Hospital Asdrubal de la Torre, Cotacachi, Ecuador.

  11. Evidence of Nicotine-Induced, Curare-Insensitive, Behavior in Planarians.

    PubMed

    Pagán, Oné R; Montgomery, Erica; Deats, Sean; Bach, Daniel; Baker, Debra

    2015-10-01

    Planarians are rapidly developing into very useful research subjects in pharmacology and neuroscience research. Here we report that curare, a cholinergic nicotinic receptor antagonist, alleviates the nicotine-induced planarian seizure-like movements (pSLM) by up to 50 % at equimolar concentrations of nicotine and curare (1 mM), while curare alone does not induce significant pSLMs. The simplest interpretation of our data is that there are nicotine induced behaviors insensitive to curare in our experimental organism. To the best of our knowledge, this is the first report on curare-insensitive, nicotine-induced effects in any organism.

  12. Basic Fourier properties for generalized phase shifting and some interesting detuning insensitive algorithms.

    PubMed

    Téllez-Quiñones, Alejandro; Malacara-Doblado, Daniel; García-Márquez, Jorge

    2011-07-20

    In this manuscript, some interesting properties for generalized or nonuniform phase-shifting algorithms are shown in the Fourier frequency space. A procedure to find algorithms with equal amplitudes for their sampling function transforms is described. We also consider in this procedure the finding of algorithms that are orthogonal for all possible values in the frequency space. This last kind of algorithms should closely satisfy the first order detuning insensitive condition. The procedure consists of the minimization of functionals associated with the desired insensitivity conditions.

  13. Specificity of gap junction communication among human mammary cells and connexin transfectants in culture

    PubMed Central

    1993-01-01

    In a previous paper (Lee et al., 1992), it was shown that normal human mammary epithelial cells (NMEC) express two connexin genes, Cx26 and Cx43, whereas neither gene is transcribed in a series of mammary tumor cell lines (TMEC). In this paper it is shown that normal human mammary fibroblasts (NMF) communicate and express Cx43 mRNA and protein. Transfection of either Cx26 or Cx43 genes into a tumor line, 21MT-2, induced the expression of the corresponding mRNAs and proteins as well as communication via gap junctions (GJs), although immunofluorescence demonstrated that the majority of Cx26 and Cx43 proteins present in transfected TMEC was largely cytoplasmic. Immunoblotting demonstrated that NMEC, NMF, and transfected TMEC each displayed a unique pattern of posttranslationally modified forms of Cx43 protein. The role of different connexins in regulating gap junction intercellular communication (GJIC) was examined using a novel two-dye method to assess homologous and heterologous communication quantitatively. The recipient cell population was prestained with a permanent non-toxic lipophilic dye that binds to membranes irreversibly (PKH26, Zynaxis); and the donor population is treated with a GJ-permeable dye Calcein, a derivative of fluorescein diacetate (Molecular Probes). After mixing the two cell populations under conditions promoting GJ formation, cells were analyzed by flow cytometry to determine the percentage of cells containing both dyes. It is shown here that Cx26 and Cx43 transfectants display strong homologous communication, as do NMEC and NMF. Furthermore, NMEC mixed with NMF communicate efficiently, Cx26 transfectants communicate with NMEC but not with NMF, and Cx43 transfectants communicate with NMF. Communication between Cx26 TMEC transfectants and NMEC was asymetrical with preferential movement of calcein from TMEC to NMEC. Despite the presence of Cx43 as well as Cx26 encoded proteins in the GJs of NMEC, few Cx43 transfectants communicated with NMEC

  14. The development of mechanically formed stable nanobubbles intended for sonoporation-mediated gene transfection.

    PubMed

    Abdalkader, Rodi; Kawakami, Shigeru; Unga, Johan; Higuchi, Yuriko; Suzuki, Ryo; Maruyama, Kazuo; Yamashita, Fumiyoshi; Hashida, Mitsuru

    2017-11-01

    In this study, stable nano-sized bubbles (nanobubbles [NBs]) were produced using the mechanical agitation method in the presence of perfluorocarbon gases. NBs made with perfluoropropane had a smaller size (around 400 nm) compared to that of those made with perfluorobutane or nitrogen gas. The lipid concentration in NBs affected both their initial size and post-formulation stability. NBs formed with a final lipid concentration of 0.5 mg/ml tended to be more stable, having a uniform size distribution for 24 h at room temperature and 50 h at 4 °C. In vitro gene expression revealed that NBs/pDNA in combination with ultrasound (US) irradiation had significantly higher transfection efficacy in colon C26 cells. Moreover, for in vivo gene transfection in mice left limb muscles, there was notable local transfection activity by NBs/pDNA when combined with US irradiation. In addition, the aged NBs kept at room temperature or 4 °C were still functional at enhancing gene transfection in mice. We succeeded in preparing stable NBs for efficient in vivo gene transfection, using the mechanical agitation method.

  15. Ultrasound-mediated interferon {beta} gene transfection inhibits growth of malignant melanoma

    SciTech Connect

    Yamaguchi, Kazuki; Feril, Loreto B.; Tachibana, Katsuro; Takahashi, Akira; Matsuo, Miki; Endo, Hitomi; Harada, Yoshimi; Nakayama, Juichiro

    2011-07-22

    Highlights: {yields} Successful ultrasound-mediated transfection of melanoma (C32) cells with IFN-{beta} genes both in vitro and in vivo. {yields} Ultrasound-mediated IFN-{beta} transfection inhibited proliferation of melanoma cells in vitro. {yields} Ultrasound-mediated IFN-{beta} transfection inhibited melanoma tumor growth in vivo. -- Abstract: We investigated the effects of ultrasound-mediated transfection (sonotransfection) of interferon {beta} (IFN-{beta}) gene on melanoma (C32) both in vitro and in vivo. C32 cells were sonotransfected with IFN-{beta} in vitro. Subcutaneous C32 tumors in mice were sonicated weekly immediately after intra-tumor injection with IFN-{beta} genes mixed with microbubbles. Successful sonotransfection with IFN-{beta} gene in vitro was confirmed by ELISA, which resulted in C32 growth inhibition. In vivo, the growth ratio of tumors transfected with IFN-{beta} gene was significantly lower than the other experimental groups. These results may lead to a new method of treatment against melanoma and other hard-to-treat cancers.

  16. Imidazole and dimethyl aminopropyl-functionalized hyperbranched polymers for nucleic acid transfection.

    PubMed

    Germershaus, Oliver; Pickaert, Guillaume; Konrad, Juliane; Krüger, Ute; Kissel, Thomas; Haag, Rainer

    2010-09-09

    In order to mimic the histidine binding motives of naturally occurring histones as DNA complexing proteins, hyperbranched poly(ethylene imine) and polyglycerol were functionalized with imidazole or 3-dimethylamino propyl groups. These new polycationic polymers were tested for interaction with dye-labelled oligonucleotide and DNA using UV and fluorescence spectroscopy and gel electrophoresis. Formation of stable complexes was observed above N/P ratios of 4 for unfunctionalized and 8 for functionalized PEIs. No stable complexes were formed with polyglycerol-based polyamines up to N/P 16. Cytotoxicity determined by MTT assay of all functionalized PEI polymers was found to be significantly lower than for unfunctionalized PEI. PG-based polymers showed no toxicity in the tested concentration range. Dynamic light scattering showed that only for PEI(21)-Imidaz polyplexes hydrodynamic diameters below 250 nm could be reached.The influence of functionalization and polymer type on transfection efficiency was evaluated in L929, NIH/3T3 and HeLa cells. Only imidazole-functionalized PEIs reached similar transfection efficiencies as unfunctionalized PEIs, while 3-dimethylamino propyl modification resulted in lower transfection efficiencies. We also demonstrated that the polymer plays an important role for transfection properties since, regardless of the modifications of polyglycerol, only low transfection efficiencies were observed at functionalization levels below 50%.

  17. Multifunctional oligomer incorporation: a potent strategy to enhance the transfection activity of poly(l-lysine).

    PubMed

    Liu, Shuai; Yang, Jixiang; Ren, Hongqi; O'Keeffe-Ahern, Jonathan; Zhou, Dezhong; Zhou, Hao; Chen, Jiatong; Guo, Tianying

    2016-03-01

    Natural polycations, such as poly(l-lysine) (PLL) and chitosan (CS), have inherent superiority as non-viral vectors due to their unparalleled biocompatibility and biodegradability. However, the application was constrained by poor transfection efficiency and safety concerns. Since previous modification strategies greatly weakened the inherent advantages of natural polycations, developing a strategy for functional group introduction with broad applicability to enhance the transfection efficiency of natural polycations without compromising their cationic properties is imperative. Herein, two uncharged functional diblock oligomers P(DMAEL-b-NIPAM) and P(DMAEL-b-Vlm) were prepared from a lactose derivative, N-iso-propyl acrylamide (NIPAM) as well as 1-vinylimidazole (Vlm) and further functionalized with four small ligands folate, glutathione, cysteine and arginine, respectively, aiming to enhance the interactions of complexes with cells, which were quantified utilizing a quartz crystal microbalance (QCM) biosensor, circumventing the tedious material screening process of cell transfection. Upon incorporation with PLL and DNA, the multifunctional oligomers endow the formulated ternary complexes with great properties suitable for transfection, such as anti-aggregation in serum, destabilized endosome membrane, numerous functional sites for promoted endocytosis and therefore robust transfection activity. Furthermore, different from the conventional strategy of decreasing cytotoxicity by reducing the charge density, the multifunctional oligomer incorporation strategy maintains the highly positive charge density, which is essential for efficient cellular uptake. This system develops a new platform to modify natural polycations towards clinical gene therapy.

  18. Synthesis and DNA transfection properties of new head group modified malonic acid diamides.

    PubMed

    Wölk, Christian; Heinze, Martin; Kreideweiss, Patrick; Dittrich, Matthias; Brezesinski, Gerald; Langner, Andreas; Dobner, Bodo

    2011-05-16

    Malonic acid diamides with two long hydrophobic alkyl chains and a basic polar head group as a new class of non-viral gene transferring compounds have shown high transfection efficiency and moderate toxicity. Based on the results obtained with saturated and unsaturated alkyl residues new derivatives with a more complex head group structure have been synthesized. For this purpose, cationic respectively basic groups were introduced by one or two lysine residues bound via tris(aminoethyl)amine spacer to the malonic acid diamide backbone. By studying in vitro gene delivery an increase of transfection efficacy was observed when using lipids with at least one unsaturated alkyl chain. This leads to cationic lipids exhibiting comparable or even higher transfection efficacies compared to the commercially available transfection agents LipofectAmine™ and SuperFect™. Phase transitions and phase structures of selected compounds have been analyzed and discussed in terms of transfection abilities. Particle size and zeta potential of liposomes and lipoplexes were also determined.

  19. Transfection efficiency and transgene expression kinetics of mRNA delivered in naked and nanoparticle format.

    PubMed

    Phua, Kyle K L; Leong, Kam W; Nair, Smita K

    2013-03-28

    Transfection efficiencies and transgene expression kinetics of messenger RNA (mRNA), an emerging class of nucleic acid-based therapeutics, have been poorly characterized. In this study, we evaluated transfection efficiencies of mRNA delivered in naked and nanoparticle format in vitro and in vivo using GFP and luciferase as reporters. While mRNA nanoparticles transfect primary human and mouse dendritic cells (DCs) efficiently in vitro, naked mRNA could not produce any detectable gene product. The protein expression of nanoparticle-mediated transfection in vitro peaks rapidly within 5-7h and decays in a biphasic manner. In vivo, naked mRNA is more efficient than mRNA nanoparticles when administered subcutaneously. In contrast, mRNA nanoparticle performs better when administered intranasally and intravenously. Gene expression is most transient when delivered intravenously in nanoparticle format with an apparent half-life of 1.4h and lasts less than 24h, and most sustained when delivered in the naked format subcutaneously at the base of tail with an apparent half-life of 18h and persists for at least 6days. Notably, exponential decreases in protein expression are consistently observed post-delivery of mRNA in vivo regardless of the mode of delivery (naked or nanoparticle) or the site of administration. This study elucidates the performance of mRNA transfection and suggests a niche for mRNA therapeutics when predictable in vivo transgene expression kinetics is imperative.

  20. Analysis of CCR7 mediated T cell transfectant migration using a microfluidic gradient generator.

    PubMed

    Wu, Xun; Wu, Jiandong; Li, Hongzhao; Legler, Daniel F; Marshall, Aaron J; Lin, Francis

    2015-04-01

    T lymphocyte migration is crucial for adaptive immunity. Manipulation of signaling molecules controlling cell migration combined with in-vitro cell migration analysis provides a powerful research approach. Microfluidic devices, which can precisely configure chemoattractant gradients and allow quantitative single cell analysis, have been increasingly applied to cell migration and chemotaxis studies. However, there are a very limited number of published studies involving microfluidic migration analysis of genetically manipulated immune cells. In this study, we describe a simple microfluidic method for quantitative analysis of T cells expressing transfected chemokine receptors and other cell migration signaling probes. Using this method, we demonstrated chemotaxis of Jurkat transfectants expressing wild-type or C-terminus mutated CCR7 within a gradient of chemokine CCL19, and characterized the difference in transfectant migration mediated by wild-type and mutant CCR7. The EGFP-tagged CCR7 allows identification of CCR7-expressing transfectants in cell migration analysis and microscopy assessment of CCR7 dynamics. Collectively, our study demonstrated the effective use of the microfluidic method for studying CCR7 mediated T cell transfectant migration. We envision this developed method will provide a useful platform to functionally test various signaling mechanisms at the cell migration level.

  1. Strategy for increased efficiency of transfection in human cell lines using radio frequency electroporation.

    PubMed

    Zald, P B; Cotter, M A; Robertso, E S

    2001-02-01

    Traditional electroporation devices use direct current electric fields to stimulate the uptake of oligonucleotides, plasmids, short peptides, and proteins into a variety of cell types. A variation of this widely used technique is now available which relies on radio frequency (RF) electrical pulses. This oscillating type of electrical field reportedly elicits greater uptake of plasmid DNA across the plasma membrane. We evaluated a protocol for RF electroporation of the a human embryonic kidney cell line and a Burkitt's lymphoma (BL) cell line for effeciency of transfection by RF electroporation. The plasmid EGFP, which codes for the widely used fusion protein, enhanced green fluorescent protein (EGFP), was used as a reporter of plasmid uptake after transfections. Transfection efficiency consistently increased approximately 30% from that typically obtained with conventional DC type electroporation and was accompanied by greater survivability of cells. Additionally, in some instances, percent transfection efficiency increased to over 70%. Thus, RF electroporation represents an improved methodology for transfection of human cell lines. Moreover, the RF protocol is simple to incorporate in laboratories already utilizing conventional electroporation devices and techniques.

  2. Alpha 2-adrenergic receptor stimulation of phospholipase A2 and of adenylate cyclase in transfected Chinese hamster ovary cells is mediated by different mechanisms

    SciTech Connect

    Jones, S.B.; Halenda, S.P.; Bylund, D.B. )

    1991-02-01

    The effect of alpha 2-adrenergic receptor activation on adenylate cyclase activity in Chinese hamster ovary cells stably transfected with the alpha 2A-adrenergic receptor gene is biphasic. At lower concentrations of epinephrine forskolin-stimulated cyclic AMP production is inhibited, but at higher concentrations the inhibition is reversed. Both of these effects are blocked by the alpha 2 antagonist yohimbine but not by the alpha 1 antagonist prazosin. Pretreatment with pertussis toxin attenuates inhibition at lower concentrations of epinephrine and greatly potentiates forskolin-stimulated cyclic AMP production at higher concentrations of epinephrine. alpha 2-Adrenergic receptor stimulation also causes arachidonic acid mobilization, presumably via phospholipase A2. This effect is blocked by yohimbine, quinacrine, removal of extracellular Ca2+, and pretreatment with pertussis toxin. Quinacrine and removal of extracellular Ca2+, in contrast, have no effect on the enhanced forskolin-stimulated cyclic AMP production. Thus, it appears that the alpha 2-adrenergic receptor in these cells can simultaneously activate distinct signal transduction systems; inhibition of adenylate cyclase and stimulation of phospholipase A2, both via G1, and potentiation of cyclic AMP production by a different (pertussis toxin-insensitive) mechanism.

  3. Use of cryopreserved transiently transfected cells in high-throughput pregnane X receptor transactivation assay.

    PubMed

    Zhu, Zhengrong; Puglisi, Jaime; Connors, David; Stewart, Jeremy; Herbst, John; Marino, Anthony; Sinz, Michael; O'Connell, Jonathan; Banks, Martyn; Dickinson, Kenneth; Cacace, Angela

    2007-03-01

    Cryopreserved, transiently transfected HepG2 cells were compared to freshly transfected HepG2 cells for use in a pregnane X receptor (PXR) transactivation assay. Assay performance was similar for both cell preparations; however, cryopreserved cells demonstrated less interassay variation. Validation with drugs of different PXR activation potencies and efficacies demonstrated an excellent correlation (r(2) > 0.95) between cryopreserved and fresh cells. Cryopreservation did not change the effect of known CYP3A4 inducers that have poor cell permeability, indicating that cryopreservation had little effect on membrane permeability. In addition, cryopreserved HepG2 cells did not exhibit enhanced susceptibility to cytotoxic compounds compared to transiently transfected control cells. The use of cryopreserved cells enables this assay to run with enhanced efficiency.

  4. Transfected cell lines as tools for high throughput screening: a call for standards.

    PubMed

    Pagliaro, L; Praestegaard, M

    2001-06-01

    During 1999, Journal of Biomolecular Screening presented a series of Point-Counterpoint articles that addressed a question posed by editor Bill Janzen: "What is the future of HTS?" These articles discussed many of the global issues involved in HTS, such as target identification and library size, as well as the scientific and technical challenges facing the field. In this perspective we address a related, but very focused, issue that is increasingly important for many of us in the HTS community: the use of stably transfected cell lines as an integral part of screening strategies. Transfected cell lines provide powerful tools for assay design, but at the same time they introduce complex variables into the screening system. Although it is difficult to develop precise definitions and standards for biologicals such as cell lines, we propose that the development of guidelines for the nomenclature and use of transfected cell lines is essential for their use in HTS.

  5. Comparation of enhanced green fluorescent protein gene transfected and wild-type porcine neural stem cells.

    PubMed

    Zheng, Yue-Mao; An, Zhi-Xing; Zhao, Xiao-E; Quan, Fu-Sheng; Zhao, Hui-Ying; Zhang, Ya-Rong; Liu, Jun; He, Xiao-Ying; He, Xiao-Ning

    2010-02-01

    The aim of this study was to transfect and express the enhanced green fluorescence protein (EGFP) gene into porcine neural stem cells (NSCs) to determine whether EGFP can be used as a marker to monitor NSCs. NSCs were isolated from embryonic day 30 fetal pig brain and transfected with EGFP gene using lipofection. Transfected and wild-type NSCs were induced to differentiate into cells of neuronal and myogenic lineages. Markers of passage three NSCs and their differentiated cells were tested by reverse transcription polymerase chain reaction. The results showed that EGFP could be expressed in NSCs and the differentiated cells. NSCs expressed Nestin, NogoA, DCX, Hes1, Oct4, CD-90 and Sox2. NSCs could differentiated into astrocyte (GFAP(+)), oligodendrocyte (GalC(+)), neuron (NF(+), NSE(+) and MAP2(+)) and myocyte (myf-6(+) and myoD(+)). We concluded that EGFP can be used as a marker in monitoring NSCs.

  6. Structural and transfection properties of amine-substituted gemini surfactant-based nanoparticles

    SciTech Connect

    Wettig,S.; Badea, I.; Donkuru, M.; Verrall, R.; Foldvari, M.

    2007-01-01

    Increases in DNA transfection efficiencies for non-viral vectors can be achieved through rational design of novel cationic building blocks. Based on previous results examining DNA condensation by polyamines, novel gemini surfactants have been designed that incorporate aza or imino substituents within the spacer group in order to increase interactions with DNA and potentially improve their DNA transfection ability. Transfection efficiencies and cell toxicity of gemini nanoparticles constructed from plasmid DNA, gemini surfactant, and a neutral lipid were measured in COS7 cells using a luciferase assay. Structural properties of nanoparticles were examined by using circular dichroism, particle size, zeta potential, and small-angle X-ray scattering (SAXS) measurements. The incorporation of aza and imino substituents within the spacer group was observed to enhance the transfection ability of gemini surfactants. Incorporation of an imino group in the structure of the 1,9-bis(dodecyl)-1,1,9,9-tetramethyl-5-imino-1,9-nonanediammonium dibromide surfactant (12-7NH-12) resulted in a statistically significant (p < 0.01) 9-fold increase in transfection compared to an unsubstituted gemini surfactant and a 3-fold increase compared to the corresponding aza-substituted compound. A pH-dependent transition in size and zeta potential was observed to occur at pH 5.5 for complexes formed from the 12-7NH-12 compound. SAXS results show weakly ordered structures and the presence of multiple phases. The incorporation of a pH-active imino group within the spacer of the gemini surfactant results in a significant increase in transfection efficiency that can be related to both pH-induced changes in nanoparticle structure and the formation of multiple phases that more readily allow for membrane fusion that may facilitate DNA release.

  7. Transfection of microRNA Mimics Should Be Used with Caution

    PubMed Central

    Jin, Hyun Yong; Gonzalez-Martin, Alicia; Miletic, Ana V.; Lai, Maoyi; Knight, Sarah; Sabouri-Ghomi, Mohsen; Head, Steven R.; Macauley, Matthew S.; Rickert, Robert C.; Xiao, Changchun

    2015-01-01

    Transient transfection of chemically synthesized microRNA (miRNA) mimics is being used extensively to study the functions and mechanisms of endogenous miRNAs. However, it remains unclear whether transfected miRNAs behave similarly to endogenous miRNAs. Here we show that transient transfection of miRNA mimics into HeLa cells by a commonly used method led to the accumulation of high molecular weight RNA species and a few hundred fold increase in mature miRNA levels. In contrast, expression of the same miRNAs through lentiviral infection or plasmid transfection of HeLa cells, transgenic expression in primary lymphocytes, and endogenous overexpression in lymphoma and leukemia cell lines did not lead to the appearance of high molecular weight RNA species. The increase of mature miRNA levels in these cells was below 10-fold, which was sufficient to suppress target gene expression and to drive lymphoma development in mice. Moreover, transient transfection of miRNA mimics at high concentrations caused non-specific alterations in gene expression, while at low concentrations achieved expression levels comparable to other methods but failed to efficiently suppress target gene expression. Small RNA deep sequencing analysis revealed that the guide strands of miRNA mimics were frequently mutated, while unnatural passenger strands of some miRNA mimics accumulated to high levels. The high molecular weight RNA species were a heterogeneous mixture of several classes of RNA species generated by concatemerization, 5′- and 3′-end tailing of miRNA mimics. We speculate that the supraphysiological levels of mature miRNAs and these artifactual RNA species led to non-specific changes in gene expression. Our results have important implications for the design and interpretation of experiments primarily employing transient transfection of miRNA mimics. PMID:26697058

  8. Gene Transfection in High Serum Levels: Case Studies with New Cholesterol Based Cationic Gemini Lipids

    PubMed Central

    Misra, Santosh K.; Biswas, Joydeep; Kondaiah, Paturu; Bhattacharya, Santanu

    2013-01-01

    Background Six new cationic gemini lipids based on cholesterol possessing different positional combinations of hydroxyethyl (-CH2CH2OH) and oligo-oxyethylene -(CH2CH2O)n- moieties were synthesized. For comparison the corresponding monomeric lipid was also prepared. Each new cationic lipid was found to form stable, clear suspensions in aqueous media. Methodology/Principal Findings To understand the nature of the individual lipid aggregates, we have studied the aggregation properties using transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential measurements and X-ray diffraction (XRD). We studied the lipid/DNA complex (lipoplex) formation and the release of the DNA from such lipoplexes using ethidium bromide. These gemini lipids in presence of a helper lipid, 1, 2-dioleoyl phophatidyl ethanol amine (DOPE) showed significant enhancements in the gene transfection compared to several commercially available transfection agents. Cholesterol based gemini having -CH2-CH2-OH groups at the head and one oxyethylene spacer was found to be the most effective lipid, which showed transfection activity even in presence of high serum levels (50%) greater than Effectene, one of the potent commercially available transfecting agents. Most of these geminis protected plasmid DNA remarkably against DNase I in serum, although the degree of stability was found to vary with their structural features. Conclusions/Significance -OH groups present on the cationic headgroups in combination with oxyethylene linkers on cholesterol based geminis, gave an optimized combination of new genera of gemini lipids possessing high transfection efficiency even in presence of very high percentage of serum. This property makes them preferential transfection reagents for possible in vivo studies. PMID:23861884

  9. New Insight into the Angle Insensitivity of Ultrathin Planar Optical Absorbers for Broadband Solar Energy Harvesting.

    PubMed

    Liu, Dong; Yu, Haitong; Duan, Yuanyuan; Li, Qiang; Xuan, Yimin

    2016-09-01

    Two challenging problems still remain for optical absorbers consisting of an ultrathin planar semiconductor film on top of an opaque metallic substrate. One is the angle-insensitive mechanism and the other is the system design needed for broadband solar energy harvesting. Here, first we theoretically demonstrates that the high refractive index, instead of the ultrathin feature as reported in previous studies, is the physical origin of the angle insensitivity for ultrathin planar optical absorbers. They exhibit omnidirectional resonance for TE polarization due to the high complex refractive index difference between the semiconductor and the air, while for TM polarization the angle insensitivity persists up to an incident angle related to the semiconductor refractive index. These findings were validated by fabricating and characterizing an 18 nm Ge/Ag absorber sample (representative of small band gap semiconductors for photovoltaic applications) and a 22 nm hematite/Ag sample (representative of large band gap semiconductors for photoelectrochemical applications). Then, we took advantage of angle insensitivity and designed a spectrum splitting configuration for broadband solar energy harvesting. The cascaded solar cell and unassisted solar water splitting systems have photovoltaic and photoelectrochemical cells that are also spectrum splitters, so an external spectrum splitting element is not needed.

  10. New Insight into the Angle Insensitivity of Ultrathin Planar Optical Absorbers for Broadband Solar Energy Harvesting

    PubMed Central

    Liu, Dong; Yu, Haitong; Duan, Yuanyuan; Li, Qiang; Xuan, Yimin

    2016-01-01

    Two challenging problems still remain for optical absorbers consisting of an ultrathin planar semiconductor film on top of an opaque metallic substrate. One is the angle-insensitive mechanism and the other is the system design needed for broadband solar energy harvesting. Here, first we theoretically demonstrates that the high refractive index, instead of the ultrathin feature as reported in previous studies, is the physical origin of the angle insensitivity for ultrathin planar optical absorbers. They exhibit omnidirectional resonance for TE polarization due to the high complex refractive index difference between the semiconductor and the air, while for TM polarization the angle insensitivity persists up to an incident angle related to the semiconductor refractive index. These findings were validated by fabricating and characterizing an 18 nm Ge/Ag absorber sample (representative of small band gap semiconductors for photovoltaic applications) and a 22 nm hematite/Ag sample (representative of large band gap semiconductors for photoelectrochemical applications). Then, we took advantage of angle insensitivity and designed a spectrum splitting configuration for broadband solar energy harvesting. The cascaded solar cell and unassisted solar water splitting systems have photovoltaic and photoelectrochemical cells that are also spectrum splitters, so an external spectrum splitting element is not needed. PMID:27582317

  11. Can Parents' Involvement in Children's Education Offset the Effects of Early Insensitivity on Academic Functioning?

    ERIC Educational Resources Information Center

    Monti, Jennifer D.; Pomerantz, Eva M.; Roisman, Glenn I.

    2014-01-01

    Data from the National Institute of Child Health and Human Development Study of Early Child Care and Youth Development (N = 1,312) were analyzed to examine whether the adverse effects of early insensitive parenting on children's academic functioning can be offset by parents' later involvement in children's education. Observations of mothers' early…

  12. Novel NTRK1 Frameshift Mutation in Congenital Insensitivity to Pain With Anhidrosis.

    PubMed

    Liu, Sen; Wu, Nan; Liu, Jiaqi; Ming, Xuan; Chen, Jun; Pavelec, Derek; Su, Xinlin; Qiu, Guixing; Tian, Ye; Giampietro, Philip; Wu, Zhihong

    2015-09-01

    Congenital insensitivity to pain with anhidrosis is a rare autosomal recessive disorder. It has been reported that the defect in the NTRK1 gene encoding tropomyosin-related kinase A (TrkA) can cause congenital insensitivity to pain with anhidrosis. Nerve growth factor (NGF), the product of NGFB, mediates biological effects by binding to and activating tropomyosin-related kinase A. In addition, necdin (encoded by NDN) is also essential in nerve growth factor-tropomyosin-related kinase A pathway. We performed mutation analysis in NTRK1, NGFB, and NDN genes in a Chinese Han 17-year-old female patient with congenital insensitivity to pain with anhidrosis and her healthy family members. As a result, the patient was found to have a novel insertion in exon 7 (c.727insT) of NTRK1, which causes premature termination, and a single nucleotide polymorphism (rs2192206 G>A) in NDN. Our findings imply that the genetic variations of the nerve growth factor-tropomyosin-related kinase A pathway play an important role in congenital insensitivity to pain with anhidrosis.

  13. Assessment of Cognitive and Adaptive Behaviour among Individuals with Congenital Insensitivity to Pain and Anhidrosis

    ERIC Educational Resources Information Center

    Erez, Daniella Levy; Levy, Jacov; Friger, Michael; Aharoni-Mayer, Yael; Cohen-Iluz, Moran; Goldstein, Esther

    2010-01-01

    Aim: Individuals with congenital insensitivity to pain with anhidrosis (CIPA) are reported to have mental retardation but to our knowledge no detailed study on the subject has ever been published. The present study assessed and documented cognitive and adaptive behaviour among Arab Bedouin children with CIPA. Methods: Twenty-three Arab Bedouin…

  14. Congenital insensitivity to pain and anhydrosis: a report of two cases.

    PubMed

    Karthikeyan, Maharajan; Sreenivas, Thimmaiah; Menon, Jagdish; Patro, Dilip Kumar

    2013-04-01

    We report on 2 patients with congenital insensitivity to pain and anhydrosis. The first one was a 7-month-old boy who presented with non-traumatic, haematogenous septic dislocation of the right elbow with physeal separation of the distal humerus. The second one was a 3-year-old girl suspected to have Job syndrome with an altered immunological profile.

  15. New Insight into the Angle Insensitivity of Ultrathin Planar Optical Absorbers for Broadband Solar Energy Harvesting

    NASA Astrophysics Data System (ADS)

    Liu, Dong; Yu, Haitong; Duan, Yuanyuan; Li, Qiang; Xuan, Yimin

    2016-09-01

    Two challenging problems still remain for optical absorbers consisting of an ultrathin planar semiconductor film on top of an opaque metallic substrate. One is the angle-insensitive mechanism and the other is the system design needed for broadband solar energy harvesting. Here, first we theoretically demonstrates that the high refractive index, instead of the ultrathin feature as reported in previous studies, is the physical origin of the angle insensitivity for ultrathin planar optical absorbers. They exhibit omnidirectional resonance for TE polarization due to the high complex refractive index difference between the semiconductor and the air, while for TM polarization the angle insensitivity persists up to an incident angle related to the semiconductor refractive index. These findings were validated by fabricating and characterizing an 18 nm Ge/Ag absorber sample (representative of small band gap semiconductors for photovoltaic applications) and a 22 nm hematite/Ag sample (representative of large band gap semiconductors for photoelectrochemical applications). Then, we took advantage of angle insensitivity and designed a spectrum splitting configuration for broadband solar energy harvesting. The cascaded solar cell and unassisted solar water splitting systems have photovoltaic and photoelectrochemical cells that are also spectrum splitters, so an external spectrum splitting element is not needed.

  16. Wavelength-insensitive radiation coupling for multi-quantum well sensor based on intersubband absorption

    NASA Technical Reports Server (NTRS)

    Gunapala, Sarath D. (Inventor); Bandara, Sumith V. (Inventor); Liu, John K. (Inventor)

    2006-01-01

    Devices and techniques for coupling radiation to intraband quantum-well semiconductor sensors that are insensitive to the wavelength of the coupled radiation. At least one reflective surface is implemented in the quantum-well region to direct incident radiation towards the quantum-well layers.

  17. A Constitutive Model for Long Time Duration Mechanical Behavior in Insensitive High Explosives

    SciTech Connect

    Darnell, I M; Oh, S; Hrousis, C A; Cunningham, B J; Gagliardi, F J

    2010-03-09

    An anisotropic constitutive model for the long term dimensional stability of insensitive high explosives is proposed. Elastic, creep, thermal, and ratchet growth strains are developed. Pressure and temperature effects are considered. The constitutive model is implemented in an implicit finite element code and compared to a variety of experimental data.

  18. Androgen insensitivity syndrome in a Thoroughbred mare (64, XY — testicular feminization)

    PubMed Central

    2004-01-01

    Abstract A Thoroughbred mare was presented for stallion-like behavior. Reproductive and ultrasonographic evaluation, testosterone assays, and karyotyping confirmed a diagnosis of androgen insensitivity syndrome (64, XY — testicular feminization). Surgery to remove abdominal testicles was successful in alleviating the behavioral abnormality. This condition is discussed with reference to the current literature. PMID:15283519

  19. A transfection reporter for the prevention of false-negative results in molecular beacon experiments.

    PubMed

    Toga, Tatsuya; Kuraoka, Isao; Yasui, Akira; Iwai, Shigenori

    2013-09-01

    We previously developed a molecular beacon-type probe to detect the strand scission in cellular base excision repair and found that the phosphodiester linkages in the fluorophore/quencher linkers were cleaved. This reaction was applied to a transfection reporter, which contained the unmodified phosphodiester in the linker to another type of fluorophore. After cotransfection of cells with the probe and the reporter, the signals were used to detect the incision and to confirm the proper transfection, respectively. This method will contribute to the prevention of false-negative results in experiments using molecular beacon-type probes.

  20. Highly efficient and minimally invasive transfection using time-controlled irradiation of atmospheric-pressure plasma

    NASA Astrophysics Data System (ADS)

    Sasaki, Shota; Kanzaki, Makoto; Kaneko, Toshiro

    2014-02-01

    To develop a highly efficient and minimally invasive gene transfection method, the cells after direct plasma irradiation under various conditions are evaluated by simultaneous analysis of transfection efficiency and cell viability. As a result, the efficiency has a maximal value at a short plasma irradiation time (3-5 s) while maintaining a very high cell viability, and the volume of irradiated cell suspension changes the time dependence of the efficiency, which could be caused by the competition between the synergetic effects of reactive oxygen species and electric field stimulation, and membrane transport such as exocytosis which is the process of excretion.

  1. Quantification of plasmid DNA copies in the nucleus after lipoplex and polyplex transfection.

    PubMed

    Cohen, Richard N; van der Aa, Marieke A E M; Macaraeg, Nichole; Lee, Ai Ping; Szoka, Francis C

    2009-04-17

    Nuclear uptake of plasmid DNA is one of the many cellular barriers that limit the efficiency of non-viral gene delivery systems. We have determined the number of plasmids that reach the nucleus of a transfected cell using an internally standardized quantitative PCR (qPCR) assay. We isolated nuclei using two different protocols: a density gradient technique and a detergent-based method. The density gradient procedure yielded nuclei with substantially less adhering plasmids on the outside of the nuclei. Using the density gradient protocol we determined that cells transfected with Lipofectamine lipoplexes or polyethylenimine polyplexes contained between 75 and 50,000 plasmids/nucleus, depending on the applied plasmid dose. Any increase above 3000 plasmids/nucleus resulted in only marginal increases in transgene expression. Furthermore, lipoplex-delivered plasmids were more efficiently expressed, on the basis of protein expression per plasmid number in the nucleus, than polyplex-delivered plasmids. This indicates that polymer may remain bound to some plasmids in the nucleus. Lastly, by sorting transfected cells into high- and low-expressing sub-populations, we observe that a sub-population of cells contain 3x greater plasmids/nucleus but express nearly 100x more transgene than other cells within a single transfection reaction. Taken together these results suggest the importance of considering the processes downstream from nuclear entry for strategies to improve the efficiency of gene transfer reagents.

  2. Single Cell Transfection through Precise Microinjection with Quantitatively Controlled Injection Volumes

    PubMed Central

    Chow, Yu Ting; Chen, Shuxun; Wang, Ran; Liu, Chichi; Kong, Chi-wing; Li, Ronald A.; Cheng, Shuk Han; Sun, Dong

    2016-01-01

    Cell transfection is a technique wherein foreign genetic molecules are delivered into cells. To elucidate distinct responses during cell genetic modification, methods to achieve transfection at the single-cell level are of great value. Herein, we developed an automated micropipette-based quantitative microinjection technology that can deliver precise amounts of materials into cells. The developed microinjection system achieved precise single-cell microinjection by pre-patterning cells in an array and controlling the amount of substance delivered based on injection pressure and time. The precision of the proposed injection technique was examined by comparing the fluorescence intensities of fluorescent dye droplets with a standard concentration and water droplets with a known injection amount of the dye in oil. Injection of synthetic modified mRNA (modRNA) encoding green fluorescence proteins or a cocktail of plasmids encoding green and red fluorescence proteins into human foreskin fibroblast cells demonstrated that the resulting green fluorescence intensity or green/red fluorescence intensity ratio were well correlated with the amount of genetic material injected into the cells. Single-cell transfection via the developed microinjection technique will be of particular use in cases where cell transfection is challenging and genetically modified of selected cells are desired. PMID:27067121

  3. Calcium-activated gene transfection from DNA/poly(amic acid-co-imide) complexes.

    PubMed

    Wu, Szu-Yuan; Chang, Li-Ting; Peng, Sydeny; Tsai, Hsieh-Chih

    2015-01-01

    In this study, we synthesized a water-soluble poly(amic acid-co-imide) (PA-I) from ethylenediaminetetraacetic dianhydride (EDTA) and 2,2'-(ethylenedioxy)bis(ethylamine) that possesses comparable transfection efficiency to that of polyethylenimine (PEI), when prepared in combination with divalent calcium cations. The polycondensation of monomers afforded poly(amic acid) (PA) precursors, and subsequent thermal imidization resulted in the formation of PA-I. At a polymer/DNA ratio (indicated by the molar ratio of nitrogen in the polymer to phosphate in DNA) of 40, complete retardation of the DNA band was observed by gel electrophoresis, indicating the strong association of DNA with PA-I. A zeta potential of -22 mV was recorded for the PA-I polymer solution, and no apparent cytotoxicity was observed at concentrations up to 500 μg·mL(-1). In the presence of divalent Ca(2+), the transfection efficiency of PA-I was higher than that of PA, due to the formation of a copolymer/Ca(2+)/DNA polyplex and the reduction in negative charge due to thermal cyclization. Interestingly, a synergistic effect of Ca(2+) and the synthesized copolymer on DNA transfection was observed. The use of Ca(2+) or copolymer alone resulted in unsatisfactory delivery, whereas the formation of three-component polyplexes synergistically increased DNA transfection. Our findings demonstrated that a PA-I/Ca(2+)/DNA polyplex could serve as a promising candidate for gene delivery.

  4. Effects of menstrual cycle on gene transfection through mouse vagina for DNA vaccine.

    PubMed

    Kanazawa, T; Takashima, Y; Hirayama, S; Okada, H

    2008-08-06

    Human immunodeficiency virus (HIV) infections mainly occur through the vaginal and rectal mucosal membranes. In the present study, to develop a DNA vaginal vaccine against viral and bacterial infections, the effects of the menstrual cycle on DNA transfection through the vaginal mucosa in female mice and transfection enhancement by electroporation, a chelating agent, cell-penetrating peptides (CPP) and nuclear localizing signals (NLS) were investigated. The transfection efficiencies of a marker plasmid DNA (pDNA), pCMV-Luc, on the vaginal mucosal membrane in mice at the stages of metestrus and diestrus were significantly higher than those at the stages of proestrus and estrus. The gene expression was markedly enhanced by electroporation and by pretreatment with the chelating agent. The highest level of expression was obtained by 2h pretreatment with 5% citric acid solution combined with electroporation with 15 pulses at 250 V/cm for 5 milliseconds (ms). Furthermore, a synergistic promoting effect on pDNA transfection was obtained by co-administration of CPP, the Tat peptide analog, and NLS, the NF-kappaB analog. These results indicate that effective DNA vaccination administered through the vaginal tract is possible by selecting the menstrual stage and overcoming the mucosal barrier using a combination of methods that promotes uptake.

  5. Direct and sustained intracellular delivery of exogenous molecules using acoustic-transfection with high frequency ultrasound

    PubMed Central

    Yoon, Sangpil; Kim, Min Gon; Chiu, Chi Tat; Hwang, Jae Youn; Kim, Hyung Ham; Wang, Yingxiao; Shung, K. Kirk

    2016-01-01

    Controlling cell functions for research and therapeutic purposes may open new strategies for the treatment of many diseases. An efficient and safe introduction of membrane impermeable molecules into target cells will provide versatile means to modulate cell fate. We introduce a new transfection technique that utilizes high frequency ultrasound without any contrast agents such as microbubbles, bringing a single-cell level targeting and size-dependent intracellular delivery of macromolecules. The transfection apparatus consists of an ultrasonic transducer with the center frequency of over 150 MHz and an epi-fluorescence microscope, entitled acoustic-transfection system. Acoustic pulses, emitted from an ultrasonic transducer, perturb the lipid bilayer of the cell membrane of a targeted single-cell to induce intracellular delivery of exogenous molecules. Simultaneous live cell imaging using HeLa cells to investigate the intracellular concentration of Ca2+ and propidium iodide (PI) and the delivery of 3 kDa dextran labeled with Alexa 488 were demonstrated. Cytosolic delivery of 3 kDa dextran induced via acoustic-transfection was manifested by diffused fluorescence throughout whole cells. Short-term (6 hr) cell viability test and long-term (40 hr) cell tracking confirmed that the proposed approach has low cell cytotoxicity. PMID:26843283

  6. Development of a confocal ultrasound device using an inertial cavitation control for transfection in-vitro

    NASA Astrophysics Data System (ADS)

    Mestas, J. L.; Chettab, K.; Roux, S.; Prieur, F.; Lafond, M.; Dumontet, C.; Lafon, C.

    2015-12-01

    Sonoporation using low-frequency high-pressure ultrasound (US) is a non-viral approach for in vitro and in vivo gene delivery. We developed a new sonoporation device designed for spatial and temporal control of ultrasound cavitation. This device was evaluated for the in vitro transfection efficiency of a plasmid coding for Green Fluorescent Protein (peGFP- C1) in adherent and non-adherent cell lines. The frequency spectrum of the signal receive by a hydrophone is used to compute a cavitation index (CI) representative of the inertial cavitation activity. The influence of the CI on transfection efficiency, as well as reproducibility were determined. A real-time feedback loop control on CI was integrated in the process to regulate the cavitation level during sonoporation. In both adherent and non-adherent cell lines, the sonoporation device produced a highly efficient transfection of peGFP-C1 (40-80%), as determined by flow cytometry analysis of GFP expression, along with a low rate of mortality assessed by propidium iodide staining. Moreover, the sonoporation of non-adherent cell lines Jurkat and K562 was found to be equivalent to nucleofection in terms of efficiency and toxicity while these two cell lines were resistant to transfection with lipofection.

  7. Effects of Circular DNA Length on Transfection Efficiency by Electroporation into HeLa Cells

    PubMed Central

    Hornstein, Benjamin D.; Roman, Dany; Arévalo-Soliz, Lirio M.; Engevik, Melinda A.

    2016-01-01

    The ability to produce extremely small and circular supercoiled vectors has opened new territory for improving non-viral gene therapy vectors. In this work, we compared transfection of supercoiled DNA vectors ranging from 383 to 4,548 bp, each encoding shRNA against GFP under control of the H1 promoter. We assessed knockdown of GFP by electroporation into HeLa cells. All of our vectors entered cells in comparable numbers when electroporated with equal moles of DNA. Despite similar cell entry, we found length-dependent differences in how efficiently the vectors knocked down GFP. As vector length increased up to 1,869 bp, GFP knockdown efficiency per mole of transfected DNA increased. From 1,869 to 4,257 bp, GFP knockdown efficiency per mole was steady, then decreased with increasing vector length. In comparing GFP knockdown with equal masses of vectors, we found that the shorter vectors transfect more efficiently per nanogram of DNA transfected. Our results rule out cell entry and DNA mass as determining factors for gene knockdown efficiency via electroporation. The length-dependent effects we have uncovered are likely explained by differences in nuclear translocation or transcription. These data add an important step towards clinical applications of non-viral vector delivery. PMID:27918590

  8. Direct and sustained intracellular delivery of exogenous molecules using acoustic-transfection with high frequency ultrasound

    NASA Astrophysics Data System (ADS)

    Yoon, Sangpil; Kim, Min Gon; Chiu, Chi Tat; Hwang, Jae Youn; Kim, Hyung Ham; Wang, Yingxiao; Shung, K. Kirk

    2016-02-01

    Controlling cell functions for research and therapeutic purposes may open new strategies for the treatment of many diseases. An efficient and safe introduction of membrane impermeable molecules into target cells will provide versatile means to modulate cell fate. We introduce a new transfection technique that utilizes high frequency ultrasound without any contrast agents such as microbubbles, bringing a single-cell level targeting and size-dependent intracellular delivery of macromolecules. The transfection apparatus consists of an ultrasonic transducer with the center frequency of over 150 MHz and an epi-fluorescence microscope, entitled acoustic-transfection system. Acoustic pulses, emitted from an ultrasonic transducer, perturb the lipid bilayer of the cell membrane of a targeted single-cell to induce intracellular delivery of exogenous molecules. Simultaneous live cell imaging using HeLa cells to investigate the intracellular concentration of Ca2+ and propidium iodide (PI) and the delivery of 3 kDa dextran labeled with Alexa 488 were demonstrated. Cytosolic delivery of 3 kDa dextran induced via acoustic-transfection was manifested by diffused fluorescence throughout whole cells. Short-term (6 hr) cell viability test and long-term (40 hr) cell tracking confirmed that the proposed approach has low cell cytotoxicity.

  9. The intracellular trafficking mechanism of Lipofectamine-based transfection reagents and its implication for gene delivery.

    PubMed

    Cardarelli, Francesco; Digiacomo, Luca; Marchini, Cristina; Amici, Augusto; Salomone, Fabrizio; Fiume, Giuseppe; Rossetta, Alessandro; Gratton, Enrico; Pozzi, Daniela; Caracciolo, Giulio

    2016-05-11

    Lipofectamine reagents are widely accepted as "gold-standard" for the safe delivery of exogenous DNA or RNA into cells. Despite this, a satisfactory mechanism-based explanation of their superior efficacy has remained mostly elusive thus far. Here we apply a straightforward combination of live cell imaging, single-particle tracking microscopy, and quantitative transfection-efficiency assays on live cells to unveil the intracellular trafficking mechanism of Lipofectamine/DNA complexes. We find that Lipofectamine, contrary to alternative formulations, is able to efficiently avoid active intracellular transport along microtubules, and the subsequent entrapment and degradation of the payload within acidic/digestive lysosomal compartments. This result is achieved by random Brownian motion of Lipofectamine-containing vesicles within the cytoplasm. We demonstrate here that Brownian diffusion is an efficient route for Lipofectamine/DNA complexes to avoid metabolic degradation, thus leading to optimal transfection. By contrast, active transport along microtubules results in DNA degradation and subsequent poor transfection. Intracellular trafficking, endosomal escape and lysosomal degradation appear therefore as highly interdependent phenomena, in such a way that they should be viewed as a single barrier on the route for efficient transfection. As a matter of fact, they should be evaluated in their entirety for the development of optimized non-viral gene delivery vectors.

  10. The intracellular trafficking mechanism of Lipofectamine-based transfection reagents and its implication for gene delivery

    PubMed Central

    Cardarelli, Francesco; Digiacomo, Luca; Marchini, Cristina; Amici, Augusto; Salomone, Fabrizio; Fiume, Giuseppe; Rossetta, Alessandro; Gratton, Enrico; Pozzi, Daniela; Caracciolo, Giulio

    2016-01-01

    Lipofectamine reagents are widely accepted as “gold-standard” for the safe delivery of exogenous DNA or RNA into cells. Despite this, a satisfactory mechanism-based explanation of their superior efficacy has remained mostly elusive thus far. Here we apply a straightforward combination of live cell imaging, single-particle tracking microscopy, and quantitative transfection-efficiency assays on live cells to unveil the intracellular trafficking mechanism of Lipofectamine/DNA complexes. We find that Lipofectamine, contrary to alternative formulations, is able to efficiently avoid active intracellular transport along microtubules, and the subsequent entrapment and degradation of the payload within acidic/digestive lysosomal compartments. This result is achieved by random Brownian motion of Lipofectamine-containing vesicles within the cytoplasm. We demonstrate here that Brownian diffusion is an efficient route for Lipofectamine/DNA complexes to avoid metabolic degradation, thus leading to optimal transfection. By contrast, active transport along microtubules results in DNA degradation and subsequent poor transfection. Intracellular trafficking, endosomal escape and lysosomal degradation appear therefore as highly interdependent phenomena, in such a way that they should be viewed as a single barrier on the route for efficient transfection. As a matter of fact, they should be evaluated in their entirety for the development of optimized non-viral gene delivery vectors. PMID:27165510

  11. Optical reprogramming of human cells in an ultrashort femtosecond laser microfluidic transfection platform.

    PubMed

    Uchugonova, Aisada; Breunig, Hans Georg; Batista, Ana; König, Karsten

    2016-09-01

    Induced pluripotent stem cell (iPS cell) technology can be used to produce unlimited numbers of functional cells for both research and therapeutic purposes without ethical controversy. Typically, viruses are applied for efficient intracellular delivery of genes/transcription factors to generate iPS cells. However, the viral genomic integration may cause a risk of mutation as well as tumor formation therefore limits its clinical application. Here we demonstrate that spatially shaped extreme ultrashort laser pulses of sub-20 femtoseconds induce transient membrane permeabilisation which enables contamination-free transfection of cells in a microfluidic tube with multiple genes at the individual cell level in order to achieve optical reprogramming of large cell populations. We found that the ultrashort femtosecond laser-microfluidic cell transfection platform enhanced the efficacy of iPS-like colony-forming following merely a single transfection. Illustration of the spatially shaped femtosecond laser-assisted microfluidic cell transfection platform for production of iPS cell colonies.

  12. Single Cell Transfection through Precise Microinjection with Quantitatively Controlled Injection Volumes

    NASA Astrophysics Data System (ADS)

    Chow, Yu Ting; Chen, Shuxun; Wang, Ran; Liu, Chichi; Kong, Chi-Wing; Li, Ronald A.; Cheng, Shuk Han; Sun, Dong

    2016-04-01

    Cell transfection is a technique wherein foreign genetic molecules are delivered into cells. To elucidate distinct responses during cell genetic modification, methods to achieve transfection at the single-cell level are of great value. Herein, we developed an automated micropipette-based quantitative microinjection technology that can deliver precise amounts of materials into cells. The developed microinjection system achieved precise single-cell microinjection by pre-patterning cells in an array and controlling the amount of substance delivered based on injection pressure and time. The precision of the proposed injection technique was examined by comparing the fluorescence intensities of fluorescent dye droplets with a standard concentration and water droplets with a known injection amount of the dye in oil. Injection of synthetic modified mRNA (modRNA) encoding green fluorescence proteins or a cocktail of plasmids encoding green and red fluorescence proteins into human foreskin fibroblast cells demonstrated that the resulting green fluorescence intensity or green/red fluorescence intensity ratio were well correlated with the amount of genetic material injected into the cells. Single-cell transfection via the developed microinjection technique will be of particular use in cases where cell transfection is challenging and genetically modified of selected cells are desired.

  13. In vitro transfection of plasmid DNA by cationized gelatin prepared from different amine compounds.

    PubMed

    Kushibiki, Toshihiro; Tomoshige, Ryuji; Iwanaga, Kazunori; Kakemi, Masawo; Tabata, Yasuhiko

    2006-01-01

    The objective of this paper is to compare the in vitro transfection efficiency of a luciferase plasmid DNA using cationized gelatin prepared from different amine compounds. The compounds used here were ethylenediamine, putrescine, spermidine and spermine, chemically introduced to the carboxyl group of gelatin for the cationization. Complexation of the cationized gelatin with the plasmid DNA was performed by simply mixing the two materials at various N+/P- mixing ratios (the molar number ratio of amino groups of gelatin to the phosphate groups of DNA) in aqueous solution. Gel retardation studies revealed that the formation of cationized-gelatin-plasmid DNA complexes depended on the N+/P- mixing ratio. The stronger interaction of plasmid DNA with the cationized gelatin of spermine compared to the other cationized gelatins was observed by an ethidium bromide intercalation assay and Scatchard binding analysis. When the transfection efficiency of plasmid DNA complexed with the various cationized gelatins at different N+/P- mixing ratios was evaluated for mouse L929 fibroblasts, the highest transfection efficiency was observed for the complex prepared from the cationized gelatin of spermine at a N+/P- mixing ratio of 2. The present study indicates that there is an optimal N+/P- mixing ratio and a type of amine compound or cationization extent of cationized gelatin to enhance the transfection efficiency of plasmid DNA.

  14. Cationic Liposomes Modified with Polyallylamine as a Gene Carrier: Preparation, Characterization and Transfection Efficiency Evaluation

    PubMed Central

    Kazemi Oskuee, Reza; Mahmoudi, Asma; Gholami, Leila; Rahmatkhah, Alireza; Malaekeh-Nikouei, Bizhan

    2016-01-01

    Purpose: Cationic polymers and cationic liposomes have shown to be effective non-viral gene delivery vectors. In this study, we tried to improve the transfection efficiency by employing the advantages of both. Methods: For this purpose, modified polyallylamines (PAAs) were synthesized. These modifications were done through the reaction of PAA (15 KDa) with acrylate and 6-bromoalkanoic acid derivatives. Liposomes comprising of these cationic polymers and cationic lipid were prepared and extruded through polycarbonate filters to obtain desired size. Liposome-DNA nanocomplexes were prepared in three carrier to plasmid (C/P) ratios. Size, zeta potential and DNA condensation ability of each complex were characterized separately and finally transfection efficiency and cytotoxicity of prepared vectors were evaluated in Neuro2A cell line. Results: The results showed that mean particle size of all these nanocomplexes was lower than 266 nm with surface charge of 22.0 to 33.9 mV. Almost the same condensation pattern was observed in all vectors and complete condensation was occurred at C/P ratio of 1.5. The lipoplexes containing modified PAA 15 kDa with 10% hexyl acrylate showed the highest transfection efficacy and lowest cytotoxicity in C/P ratio of 0.5. Conclusion: In some cases nanocomplexes consisting of cationic liposome and modified PAA showed better transfection activity and lower cytotoxicity compared to PAA. PMID:28101458

  15. Fluid Phase Endocytosis Contributes to Transfection of DNA by PEI-25

    PubMed Central

    Hufnagel, Hansjörg; Hakim, Parvez; Lima, Aline; Hollfelder, Florian

    2009-01-01

    The understanding of internalization pathways of lipo- or polyplexes is crucial for engineering successful reagents for nonviral gene transfection. A known inhibitor of fluid phase endocytosis (FPE), rottlerin, was used to quantify the contribution of this pathway by flow cytometric and fluorescence assays. Rottlerin was shown to be a specific inhibitor of transfection by polyethylene imine (PEI-25)/DNA complexes, leading to a decrease in the amount of transfected HeLa and CHO-K1 cells and a decrease in the expression of enhanced green fluorescent protein (EGFP) reporter gene by up to 50%. Experiments using fluorescently labeled polyplexes result in a decrease of uptake by up to 40%. Additionally, rottlerin does not cross-inhibit clathrin- and caveolin-mediated endocytotic pathways of internalization, consistent with direct uptake inhibition by rottlerin. Nonspecific effects as a result of toxicity were ruled out by control experiments at concentrations where rottlerin inhibition was specific. These findings suggest that for CHO-K1 and HeLa cells, internalization of PEI-25/DNA complexes by FPE plays a decisive role in gene transfection. The establishment of an additional pathway that is independent of clathrin- and caveolin-mediated endocytotic uptake may have an impact on the design of future reagents of nonviral gene therapy and investigations of the uptake pathways and intracellular trafficking involved. PMID:19532143

  16. Transfection efficiency of chitosan and thiolated chitosan in retinal pigment epithelium cells: A comparative study

    PubMed Central

    Oliveira, Ana V.; Silva, Andreia P.; Bitoque, Diogo B.; Silva, Gabriela A.; Rosa da Costa, Ana M.

    2013-01-01

    OBJECTIVE: Gene therapy relies on efficient vector for a therapeutic effect. Efficient non-viral vectors are sought as an alternative to viral vectors. Chitosan, a cationic polymer, has been studied for its gene delivery potential. In this work, disulfide bond containing groups were covalently added to chitosan to improve the transfection efficiency. These bonds can be cleaved by cytoplasmic glutathione, thus, releasing the DNA load more efficiently. MATERIALS AND METHODS: Chitosan and thiolated chitosan nanoparticles (NPs) were prepared in order to obtain a NH3+:PO4− ratio of 5:1 and characterized for plasmid DNA complexation and release efficiency. Cytotoxicity and gene delivery studies were carried out on retinal pigment epithelial cells. RESULTS: In this work, we show that chitosan was effectively modified to incorporate a disulfide bond. The transfection efficiency of chitosan and thiolated chitosan varied according to the cell line used, however, thiolation did not seem to significantly improve transfection efficiency. CONCLUSION: The apparent lack of improvement in transfection efficiency of the thiolated chitosan NPs is most likely due to its size increase and charge inversion relatively to chitosan. Therefore, for retinal cells, thiolated chitosan does not seem to constitute an efficient strategy for gene delivery. PMID:23833516

  17. GAP JUNCTION COMMUNICATON IN A TRANSFECTED HUMAN CELL LINE: ACTION OF MELATONIN AND MAGNETIC FIELDS

    EPA Science Inventory

    GAP JUNCTION COMMUNICTION IN TRANSFECTED HUMAN CELL LINE: ACTION OF MELATONIN AND MAGNETIC FIELDS.

    OBJECTIVE: We previously showed that functional gap junction communication (GJC), as monitored by dye transfer (DT), could be enhanced in mouse C3H 10T112 cells and in mouse...

  18. Transfected Cell Microarrays for the Expression of Membrane-Displayed Single-Chain Antibodies

    DTIC Science & Technology

    2011-01-01

    Appli- cations of single-chain variable fragment antibodies in therapeutics and diagnostics. Biotechnology Adv 27, 502–520. 6. Denzin , L. K...4-20. J Biol Chem 266, 14095–14103. Transfected Cell Microarrays 137 7. Denzin , L. K., Gulliver, G. A., Voss, E. W., Jr. (1993) Mutational analysis of

  19. Parameters affecting organization and transfection efficiency of amphiphilic copolymers/DNA carriers.

    PubMed

    Roques, Caroline; Bouchemal, Kawthar; Ponchel, Gilles; Fromes, Yves; Fattal, Elias

    2009-08-19

    Amphiphilic block copolymers are attracting increasing interest in the field of gene therapy, especially for transfection of striated muscles. However, little is known about the parameters affecting their transfection efficiency in vivo. These copolymers can self-assemble as micelles in certain conditions. Since micellization strongly depends on the temperature and ionic content of the preparation medium, the present paper aimed at investigating the influence of these parameters in the context of gene delivery. We first assessed the micellization of pluronic L64 and tetronic 304 at various temperatures in water, saline or Tyrode's salts solution. Pluronic L64 can form micelles at temperatures above 37 degrees C in water or at 37 degrees C in the Tyrode's salts solution, in the range of concentration investigated. For tetronic 304, CMC was found to be far below the concentrations used to transfer DNA. Pluronic L64 interacted with DNA only in the presence of micelles. Moreover, in vivo evaluation demonstrated that significantly improved transfection efficiency was obtained at 37 degrees C in Tyrode's salts solution for pluronic L64 based formulations, compared to 4 degrees C and 20 degrees C. Such differences were not recorded with tetronic 304. Finally, optimized formulations of both tetronic 304 and pluronic L64 were able to mediate efficient transfection in dystrophic muscles.

  20. Transfecting deoxyribonucleic acid of Bacillus bacteriophage phi 29 that is protease sensitive.

    PubMed

    Hirokawa, H

    1972-06-01

    The transfecting activity of Bacillus phage varphi29 DNA, extracted either by sodium lauroyl sarcosine-phenol or by 2 M perchlorate, was destroyed by treatment with proteolytic enzymes, although these enzymes did not effect transfecting DNAs of SPP1, SPO1, and SP50. These facts suggest that a protein is associated with transfective varphi29 DNA. Stabilization of protease-resistance during transfection appeared earlier than that of DNaseresistance, indicating that the protein associated with varphi29 DNA is necessary for initiation of the incorporation of DNA molecules into competent cells. The physical nature of varphi29 DNA before and after the trypsin treatment was investigated by sucrose and CsCl density gradient centrifugations. The trypsin treatment did not alter the sedimentation rate of the unit varphi29 DNA; however, it did convert the sedimentation rate of the aggregated material in the untreated DNA to that of the unit varphi29 DNA. The density of the trypsinized DNA was 0.009 g/cm(3) greater than that of the untreated DNA. The possible location of the protein on the DNA is discussed.

  1. Purification of transfection-grade plasmid DNA from bacterial cells with superparamagnetic nanoparticles

    NASA Astrophysics Data System (ADS)

    Chiang, Chen-Li; Sung, Ching-Shan

    2006-07-01

    The functionalized magnetic nanobeads were used to develop a rapid protocol for extracting and purifying transfection-grade plasmid DNA from bacterial culture. Nanosized superparamagnetic nanoparticles (Fe 3O 4) were prepared by chemical coprecipitation method using Fe 2+, Fe 3+ salt, and ammonium hydroxide under a nitrogen atmosphere. The surface of Fe 3O 4 nanoparticles was modified by coating with the multivalent cationic agent, polyethylenimine (PEI). The PEI-modified magnetic nanobeads were employed to simplify the purification of plasmid DNA from bacterial cells. We demonstrated a useful plasmid, pRSETB-EGFP, encoding the green fluorescent protein with T7 promoter, was amplified in DE3 strain of Escherichia coli. The loaded nanobeads are recovered by magnetically driven separation and regenerated by exposure to the elution buffer with optimal ionic strength (1.25 M) and pH (9.0). Up to approximately 819 μg of high-purity (A 260/A 280 ratio=1.86) plasmid DNA was isolated from 100 ml of overnight bacterial culture. The eluted plasmid DNA was used directly for restriction enzyme digestion, bacterial cell transformation and animal cell transfection applications with success. The PEI-modified magnetic nanobead delivers significant time-savings, overall higher yields and better transfection efficiencies compared to anion-exchange and other methods. The results presented in this report show that PEI-modified magnetic nanobeads are suitable for isolation and purification of transfection-grade plasmid DNA.

  2. Poly(ethyleneimine) functionalized carbon nanotubes as efficient nano-vector for transfecting mesenchymal stem cells.

    PubMed

    Moradian, Hanieh; Fasehee, Hamidreza; Keshvari, Hamid; Faghihi, Shahab

    2014-10-01

    For gene and drug delivery applications, carbon nanotubes (CNTs) have to be functionalized in order to become compatible with aqueous media and bind with genetic materials. In this study, combination of polyethyleneimine (PEI) grafted multi-walled carbon nanotubes (PEI-g-MWCNTs) and chitosan substrate is used as an efficient gene delivery system for transfection of hard-to-transfect bone marrow mesenchymal stem cells (BMSCs) with enhanced green fluorescent protein (EGFP) gene. Fourier transform infrared (FT-IR) spectra, dynamic light scattering (DLS) analysis and zeta potential measurements are used to characterize binding of PEI, particle size distribution and colloidal stability of the functionalized CNTs, respectively. DNA binding affinity, cellular uptake, transfection efficiency and possible cytotoxicity are also tested by agarose gel electrophoresis, flow cytometry, cytochemisty and MTT assay. The results demonstrate that cytotoxic effect of PEI-g-MWCNTs is negligible under optimal transfection condition. In consistency with high cellular uptake (>82%), PEI-g-MWCNTs give higher delivery of EGFP into the BMSCs which results in a more sustained expression of the model gene (EGFP) in short-term culture. These results suggest that PEI-g-MWCNTs in corporation with chitosan substrates would be a promising delivery system for BMSCs, a cell type with relevancy in the regenerative medicine and clinical applications.

  3. Electroporation, an alternative to biolistics for transfection of Bombyx mori embryos and larval tissues

    PubMed Central

    Thomas, Jean-luc

    2003-01-01

    There are few powerful techniques available to transfect insect tissues. We previously used biolistics to transfect Bombyx mori embryos, and larval and pupal tissues (Thomas J-L et al. 2001. Journal of Insect Science 1/9, Kravariti L et al. 2001. Insect Biochemistry and Molecular Biology 31: 473–479). As the main limitation was the irregularity in results we explored electroporation as an alternative technique by adapting techniques used for chicken embryos to B. mori embryos. By injecting the DNA solution into the hemocoel of late embryos that were finishing organogenesis, we expressed marker genes in numerous tissues following electroporation. With some adaptation of the method this was also achieved for early embryos lacking a hemocoel. Some larval tissues were also transfected. During these technical studies we found that optimizing parameters such as electrical voltage, number of pulses and their frequency, and conductivity of the buffer was important. These results confirmed that electroporation is a reliable technique for transfecting B. mori tissues. Abbreviation: GFP Green Fluorescent Protein CCD Charged Coupled Device PMID:15841233

  4. Single-Cell-State Culture of Human Pluripotent Stem Cells Increases Transfection Efficiency

    PubMed Central

    Nii, Takenobu; Kohara, Hiroshi; Marumoto, Tomotoshi; Sakuma, Tetsushi; Yamamoto, Takashi; Tani, Kenzaburo

    2016-01-01

    Abstract Efficient gene transfer into human pluripotent stem cells (hPSCs) holds great promise for regenerative medicine and pharmaceutical development. In the past decade, various methods were developed for gene transfer into hPSCs; however, hPSCs form tightly packed colonies, making gene transfer difficult. In this study, we established a stable culture method of hPSCs at a single-cell state to reduce cell density and investigated gene transfection efficiency followed by gene editing efficiency. hPSCs cultured in a single-cell state were transfected using nonliposomal transfection reagents with plasmid DNA or mRNA encoding enhanced green fluorescent protein. We found that most cells (DNA > 90%; mRNA > 99%) were transfected without the loss of undifferentiated PSC marker expression or pluripotency. Moreover, we demonstrated an efficient gene editing method using transcription activator-like effector nucleases (TALENs) targeting the adenomatous polyposis coli (APC) gene. Our new method may improve hPSC gene transfer techniques, thus facilitating their use for human regenerative medicine. PMID:27257519

  5. [Establishment of murine cell line transfected with human CD14 gene].

    PubMed

    Ning, Bo-Tao; Tang, Yong-Min; Xu, Yan; Chen, Yan-Fei; Cao, Jiang

    2006-04-01

    This study was aimed to construct the CD14 eukaryotic expression vector, establish the transgeneic CD14 positive cell line in order to facilitate the establishment of a mouse model of antibody targeting therapy for human acute monocytic leukemia (AML-M(5)). Total RNA extracted from peripheral blood mononuclear cells was treated with RNAase-free DNAase, the human CD14 gene was cloned and sequenced through the RT-PCR and T-A clone techniques. Eukaryotic expressional vector pcDNA3.1(+)/CD14 was constructed by cleaving with double restriction endonuleases and ligating with T4 ligase. A murine melanoma cell line B16 was transfected with the pcDNA3.1(+)/CD14 recombinant with Superfect transfection reagent. Positive clones were selected by G418 and the expression of human CD14 on the transfectant was confirmed by flow cytometry (FCM). The results indicated that the sequence of the human CD14 cDNA cloned was exact to be same as the one from GenBank database. The recombinant pcDNA3.1(+)/CD14 was identified with double-enzyme cleaving. The expression of the human CD14 on the transfectant (B16/CD14) was confirmed by FCM. In conclusion, the murine cell line B16/CD14 fransfected with human CD14 gene has been established which can be used for the study of human AML-M(5) antibody targeting therapy with mouse model.

  6. Inclusion of the helper lipid dioleoyl-phosphatidylethanolamine in solid lipid nanoparticles inhibits their transfection efficiency.

    PubMed

    de Jesus, Marcelo B; Radaic, Allan; Hinrichs, Wouter L J; Ferreira, Carmen V; de Paula, Eneida; Hoekstra, Dick; Zuhorn, Inge S

    2014-02-01

    Solid lipid nanoparticles (SLNs) are a promising system for the delivery of lipophilic and hydrophilic drugs. They consist of a solid lipid core that is stabilized by a layer of surfactants. By the incorporation of cationic lipids in the formulation, positively charged SLNs can be generated, that are suitable carriers for nucleic acids (DNA, siRNA). Considering the beneficial effect of helper lipids on the transfection efficiency with cationic liposomes, the effect of the helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) on transfection with cationic lipid-containing solid lipid nanoparticles was investigated in PC3 prostate cancer cells. The inclusion of DOPE in SLN formulations, instead of promoted, strongly inhibited SLN transfection efficiency, by frustrating the accommodation of DNA by the particles, as was revealed by biochemical analysis. SLNs devoid of DOPE maintained a homogenous size distribution of approximately 150 nm following lipoplex assembly and cellular delivery, and showed transfection efficiency comparable to that of Lipofectamine 2000' (LF2k). Moreover, the SLNs maintain their high transfection efficiency after lyophilization and long-term storage (1-2 years), an important asset for biomedical applications. There is even the possibility to lyophilize the SLN carrier together with its DNA cargo, which represents an interesting pharmaceutical advantage of the SLN formulations over LF2k. These results reflect marked differences between the physicochemical properties of cationic liposomes and SLNs, the latter requiring more critical lipid-depending properties for effective 'packaging' of DNA but displaying a higher storage stability than cationic lipid based carriers like LF2k.

  7. Comparative nucleic acid transfection efficacy in primary hepatocytes for gene silencing and functional studies

    PubMed Central

    2011-01-01

    Background Primary hepatocytes are the best resource for in vitro studies directed at understanding hepatic processes at the cellular and molecular levels, necessary for novel drug development to treat highly prevalent diseases such as non-alcoholic steatohepatitis, cardiovascular disease and type 2 diabetes. There is a need to identify simple methods to genetically manipulate primary hepatocytes and conduct functional studies with plasmids, small interfering RNA (siRNA) or microRNA (miRNA). New lipofection reagents are available that have the potential to yield higher levels of transfection with reduced toxicity. Findings We have tested several liposome-based transfection reagents used in molecular biology research. We show that transfection efficiency with one of the most recently developed formulations, Metafectene Pro, is high with plasmid DNA (>45% cells) as well as double stranded RNA (>90% with siRNA or microRNA). In addition, negligible cytotoxicity was present with all of these nucleic acids, even if cells were incubated with the DNA:lipid complex for 16 hours. To provide the proof of concept that these conditions can be used not only for overexpression of a gene of interest, but also in RNA interference applications, we targeted two liver expressed genes, Sterol Regulatory Element-Binding Protein-1 and Fatty Acid Binding Protein 5 using plasmid-mediated short hairpin RNA expression. In addition, similar transfection conditions were used to optimally deliver siRNA and microRNA. Conclusions We have identified a lipid-based reagent for primary hepatocyte transfection of nucleic acids currently used in molecular biology laboratories. The conditions described here can be used to expedite a large variety of research applications, from gene function studies to microRNA target identification. PMID:21244687

  8. Single-cell Transfection by Electroporation Using an Electrolyte/Plasmid-Filled Capillary

    PubMed Central

    Wang, Manyan; Orwar, Owe; Weber, Stephen G.

    2009-01-01

    Single-cell transfection of adherent cells has been accomplished using single-cell electroporation (SCEP) with a pulled capillary. HEPES-buffered physiological saline solution containing pEGFP plasmid at a low concentration (0.16 ∼ 0.78 µg/µL) filled a 15 cm long capillary with a tip opening of 2 µm. The electric field is applied to individual cells by bringing the tip close to the cell and subsequently applying one or two brief electric pulses. Many individual cells can thus be transfected with a small volume of plasmid-containing solution (∼ 1 µL). The extent of electroporation is determined by measuring the percentage loss of freely diffusing thiols (chiefly reduced glutathione) that have been derivatized with the fluorogenic ThioGlo 1. A mass transport model is used to fit the time-dependent fluorescence intensity decay in the target cells. The fits, which are excellent, yield the electroporation-induced fluorescence loss at steady state and the mass transfer rate through the electroporated cell membrane. Steady-state fluorescence loss ranged approximately from 0 to about 80% (based on the fluorescence intensity before electroporation). For the cells having a loss of thiol-ThioGlo 1 fluorescence intensity greater than 10%, and mass transfer rate greater than 0.03 s−1, EGFP fluorescence is observed after 24 hours. The EGFP fluorescence is increased at 48 hours. With a loss smaller than 10% and a mass transfer rate smaller than 0.03 s−1, no EGFP fluorescence is detected. Thus, transfection success is closely related to the small molecule mass transport dynamics as indicated by the loss of fluorescence from thiol-ThioGlo 1 conjugates. The EGFP expression is weaker than bulk lipid-mediated transfection, as indicated by the EGFP fluorescence intensities. However, the success with the single-cell approach is considerably greater than lipid-mediated transfection. PMID:19351139

  9. Bioreducible polyether-based pDNA ternary polyplexes: balancing particle stability and transfection efficiency.

    PubMed

    Lai, Tsz Chung; Kataoka, Kazunori; Kwon, Glen S

    2012-11-01

    Polyplex particles formed with plasmid DNA (pDNA) and Pluronic P85-block-poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} (P85-b-P[Asp(DET)]) demonstrated highly effective transfection ability compared to PEG-based block cationomer, PEG-b-P[Asp(DET)]. Ternary polyplexes comprising PEG-b-P[Asp(DET)], poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide)-b-P[Asp(DET)] (P(EPE)-b-P[Asp(DET)]) used as an analog of P85-b-P[Asp(DET)], and pDNA were prepared in this work aiming at maintaining adequate transfection efficiency while solving the stability issues of the P85-b-P[Asp(DET)] polyplexes. Furthermore, a bioreducible P(EPE)-SS-P[Asp(DET)] possessing a redox potential-sensitive disulfide linkage between the P(EPE) polymer and the cationic block was used as a substitute for P(EPE)-b-P[Asp(DET)] during ternary complex formation to investigate whether the transfection ability of the ternary polyplex system could be enhanced by triggered release of P(EPE) polymers from the polyplexes. The ternary complexes showed significant improvement in terms of stability against salt-induced aggregation compared to binary complexes, although the gene delivery ability dropped with the amount of PEG-b-P[Asp(DET)] used for complexation. By manipulating the difference in redox potential between the extracellular and intracellular environments, the reducible ternary complexes achieved higher transfection compared to the non-reducible polyplexes; moreover, the reducible polyplexes exhibited comparable stability to the non-reducible ones. These results suggest that reducible ternary complexes could provide satisfactory transfection efficiency without comprising the colloidal stability of the particles.

  10. An alternative in vitro drug screening test using Leishmania amazonensis transfected with red fluorescent protein✩

    PubMed Central

    Rocha, Marcele N.; Corrêa, Célia M.; Melo, Maria N.; Beverley, Stephen M.; Martins-Filho, Olindo Assis; Madureira, Ana Paula; Soares, Rodrigo P.

    2013-01-01

    Fluorescent and colorimetric reporter genes are valuable tools for drug screening models, since microscopy is labor intensive and subject to observer variation. In this work, we propose a fluorimetric method for drug screening using red fluorescent parasites. Fluorescent Leishmania amazonensis were developed after transfection with integration plasmids containing either red (RFP) or green fluorescent protein (GFP) genes. After transfection, wild-type (LaWT) and transfected (LaGFP and LaRFP) parasites were subjected to flow cytometry, macrophage infection, and tests of susceptibility to current antileishmanial agents and propranolol derivatives previously shown to be active against Trypanosoma cruzi. Flow cytometry analysis discriminated LaWT from LaRFP and LaGFP parasites, without affecting cell size or granulosity. With microscopy, transfection with antibiotic resistant genes was not shown to affect macrophage infectivity and susceptibility to amphotericin B and propranolol derivatives. Retention of fluorescence remained in the intracellular amastigotes in both LaGFP and LaRFP transfectants. However, detection of intracellular RFP parasites was only achieved in the fluorimeter. Murine BALB/c macrophages were infected with LaRFP parasites, exposed to standard (meglumine antimoniate, amphotericin B, Miltefosine, and allopurinol) and tested molecules. Although it was possible to determine IC50 values for 4 propranolol derivatives (1, 2b, 3, and 4b), all compounds were considered inactive. This study is the first to develop a fluorimetric drug screening test for L. amazonensis RFP. The fluorimetric test was comparable to microscopy with the advantage of being faster and not requiring manual counting. PMID:23312610

  11. Biophysical and transfection studies of the diC(14)-amidine/DNA complex.

    PubMed Central

    Cherezov, Vadim; Qiu, Hong; Pector, Veronique; Vandenbranden, Michel; Ruysschaert, Jean-Marie; Caffrey, Martin

    2002-01-01

    Liposomes of the synthetic cationic lipid, N-t-butyl-N'-tetradecylamino-propionamidine (diC(14)-amidine), efficiently ports DNA into mammalian cells in the absence of other (neutral) lipids. The compositional simplicity of this transfection mix makes it attractive from a formulation perspective. We have used low- and wide-angle x-ray diffraction and polarized light microscopy to characterize the thermotropic phase behavior and microstructure of diC(14)-amidine and of the lipid/DNA (circular plasmid, 5.4 kb) complex with a view to understanding the structure of the complex and its role in transfection. Upon heating, the lipid in buffer undergoes a lamellar crystalline (L(c), d(001) = 41.7 A)-to-lamellar liquid crystal (L(c)(alpha), d(001) depends on hydration and T) transition at approximately 40 degrees C. Sonicated lipid vesicles with a reported transition temperature of approximately 23 degrees C complex with DNA. Complex formation is complete at a DNA/lipid mole ratio (rho) of 0.8. Adding DNA to the lipid causes d(001) of the multilayered complex to drop from 52 to 49 A as rho rises from 0.03 to 1.64. The minimal DNA-DNA duplex separation observed is 26 A, consistent with the close packing of B-DNA. Lipid bilayers in the complex undergo a lamellar gel (L(c)(beta))-to-L(c)(alpha) (superscript c refers to complex) transition at approximately 23 degrees C. Transfection efficiency was maximized at rho = 0.4. The structure and transfection data combined suggest that densely packaged DNA in a net positively charged complex is essential for transfection. PMID:12023234

  12. Dye and pigment-free structural colors and angle-insensitive spectrum filters

    DOEpatents

    Guo, Lingjie Jay; Hollowell, Andrew E.; Wu, Yi-Kuei

    2017-01-17

    Optical spectrum filtering devices displaying minimal angle dependence or angle insensitivity are provided. The filter comprises a localized plasmonic nanoresonator assembly having a metal material layer defining at least one nanogroove and a dielectric material disposed adjacent to the metal material layer. The dielectric material is disposed within the nanogroove(s). The localized plasmonic nanoresonator assembly is configured to funnel and absorb a portion of an electromagnetic spectrum in the at least one nanogroove via localized plasmonic resonance to generate a filtered output having a predetermined range of wavelengths that displays angle insensitivity. Thus, flexible, high efficiency angle independent color filters having very small diffraction limits are provided that are particularly suitable for use as pixels for various display devices or for use in anti-counterfeiting and cryptography applications. The structures can also be used for colored print applications and the elements can be rendered as pigment-like particles.

  13. Courant Number and Mach Number Insensitive CE/SE Euler Solvers

    NASA Technical Reports Server (NTRS)

    Chang, Sin-Chung

    2005-01-01

    It has been known that the space-time CE/SE method can be used to obtain ID, 2D, and 3D steady and unsteady flow solutions with Mach numbers ranging from 0.0028 to 10. However, it is also known that a CE/SE solution may become overly dissipative when the Mach number is very small. As an initial attempt to remedy this weakness, new 1D Courant number and Mach number insensitive CE/SE Euler solvers are developed using several key concepts underlying the recent successful development of Courant number insensitive CE/SE schemes. Numerical results indicate that the new solvers are capable of resolving crisply a contact discontinuity embedded in a flow with the maximum Mach number = 0.01.

  14. Partial androgen insensitivity syndrome with R840H mutation in androgen receptor: report of one case.

    PubMed

    Yen, Jui-Lung; Chang, Kuang-Huey; Sheu, Jin-Cherng; Lee, Yann-Jinn; Tsai, Li-Ping

    2005-01-01

    Androgen insensitivity syndrome (AIS) is the major cause of male pseudohermaphroditism. The severity of the disorders varies widely, ranging from the phenotypic women with female external genitalia in cases of complete AIS to the phenotype of ambiguous genitalia in partial androgen insensitivity syndrome (PAIS) and a rare group of phenotypic normal males with azoospermia. Here, we report an infant of PAIS with a missense mutation at position 2881 (G-->A) in exon 7, encoding substitution of histidine for arginine at codon 840 of the androgen receptor (AR) gene. Both the biochemical and molecular studies are presented. Establishing the diagnosis of PAIS is very important for gender assignment to an infant of ambiguous genitalia. The molecular analysis will facilitate genetic counselling to the maternal side relatives for carrier detection and prenatal diagnosis.

  15. A rare case of large epididymal cyst in androgen insensitivity syndrome removed laparoscopically.

    PubMed

    Kriplani, Alka; Savithrisowmya, S; Agarwal, Nutan; Garg, Pradeep; Maheswari, Deepa; Ammini, A C; Rajan, K D Anand

    2009-01-01

    Androgen insensitivity syndrome is a disorder of sexual differentiation with 46XY karyotype. The gonad is at risk (33% by 50 years of age) for development of malignant tumors. Hence, gonadectomy is warranted. We present a case of a 22-year-old woman diagnosed with androgen insensitivity syndrome during investigation of primary amenorrhea. Ultrasonography showed intraabdominally-located gonads, with a large, nontender cyst of 9.2 x 5.6 x 5.4 cm size, with limited mobility, to the right of the midline. There was also a partial septum, with a wall thickness of 1 to 2 mm and containing clear fluid. Because of suspicion of malignancy, complete surgery including laparoscopic peritoneal cytologic study, gonadectomy, lymphadenectomy, and omentectomy were performed. Histopathologic study showed testis with an epididymal cyst. Formation of epididymal cyst is rare in these cases. The patient did well in the postoperative period. She was put on hormone replacement therapy and is doing well.

  16. An effective method for designing insensitive resonator of continuous-wave passively mode-locked laser.

    PubMed

    Wen, Qiao; Sun, Liqun; Wang, Yonggang; Zhang, Enyao; Tian, Qian

    2009-05-25

    An effective method for designing the insensitive resonator of a continuous-wave passively mode-locked laser is firstly presented in this paper. This method, using resonator transform circle graphic theory, is both intuitive and reliable. Theoretical results show that the resonator is suitable to obtain highly stable mode locking operation when the following two conditions are fulfilled. First, the transform circle of the terminal mirror opposite the semiconductor saturable absorber mirror (SESAM) propagates through a series of lens (including the thermal lens of the gain medium) and a small transform circle in the image space is obtained, which ensures the small spot size at the SESAM. Second, the resonator transform circles orthogonally (or nearly orthogonally) intersect at the SESAM, which ensures the spot size at the SESAM is insensitive to the external perturbation. The experimental results of the mode locking lasers show good agreement with the theoretical studies very well.

  17. Multipath interference characterization of bend-insensitive optical fibers and short jumpers.

    PubMed

    Olivero, Massimo; Greborio, Laura; Orta, Renato; Pellegrino, Paolo; Perrone, Guido; Regio, Paola

    2016-04-10

    Multipath interference (MPI) in bend-insensitive optical fibers is investigated by analyzing different aspects, ranging from a review of the theoretical background, through the analysis of measurement issues, to the characterization of short patch cords. Two setups for the characterization of MPI are analyzed, highlighting their advantages and limitations. Then, a number of commercial bend-insensitive fibers are compared, showing that they generally exhibit a level below -30  dB in the range of 1250-1350 nm. The investigation also includes short offset-spliced fiber segments, with lengths to 1 cm, to simulate the behavior of field-installable connectors and isolate their contribution to the MPI. The results show a step-like increment of MPI when two or more cm-long fiber segments are cascaded.

  18. Refractive index insensitive temperature sensor based on waist-enlarged few mode fiber bitapers

    NASA Astrophysics Data System (ADS)

    Liu, Qiang; Wang, Si-wen; Fu, Xing-hu; Fu, Guang-wei; Jin, Wa; Bi, Wei-hong

    2017-01-01

    A refractive index insensitive temperature sensor based on waist-enlarged few mode fiber (FMF) bitapers is presented. The first section of FMF is spliced between two single-mode fibers. In fusion process, the waist-enlarged FMF bitapers can be obtained by large current discharging repeatedly. The refractive index and temperature sensing mechanisms are analyzed. For the sensors with different sizes, the refractive index and temperature experiments have been performed. The results show that in the refractive index ranges of 1.335 0—1.346 6 and 1.348 2—1.419 3, the refractive index insensitivity is verified. In a temperature range of 31.9—90 °C, the sensor sensitivity can be up to 85.57 pm/°C. In addition, it has a compact structure. Therefore, the sensor can avoid the cross sensitivity for measuring the refractive index and temperature simultaneously.

  19. Polarization insensitive wide-angle triple-band metamaterial bandpass filter

    NASA Astrophysics Data System (ADS)

    Fu, Wenyue; Han, Yuchen; Li, Jiandong; Wang, Haoshen; Li, Haipeng; Han, Kui; Shen, Xiaopeng; Cui, Tiejun

    2016-07-01

    In this letter, we report the design, fabrication and measurement of a polarization insensitive wide-angle triple-band metamaterial bandpass filter (BPF) in the microwave frequency. The proposed BPF consists of two identical metal resonant units which have three concentric square slots separated by a dielectric layer. Experimental results show that the BPF has three distinctive transmission bands centered at frequencies 6.22 GHz, 9.46 GHz and 12.14 GHz with transmission rates of  -0.40 dB, -0.71 dB and  -1.40 dB, respectively, agreeing well with simulation results. By introducing the substrate integrated waveguide, the filter is valid to a wide range of incident angles for both transverse electric and transverse magnetic polarizations. The triple-band metamaterial BPF can be used as multiband filters and radomes owing to its multiband transmissions, polarizations insensitive and wide-angle responses.

  20. Traumatic lesions from congenital insensitivity to pain with anhidrosis in a pediatric patient: dental management.

    PubMed

    Neves, Beatriz Gonçalves; Roza, Rosemere Teixeira; Castro, Gloria Fernanda

    2009-10-01

    Congenital insensitivity to pain with anhidrosis is a rare autosomal-recessive disorder characterized by unexplained fever episodes, anhidrosis, pain insensitivity, self-mutilating behavior, and mental retardation. The lack of sensitivity to pain results in traumatic lesions, such as ulcers, fractures, burns, bites, scars, and digital amputations. Several methods have been suggested to treat these patients; however, appropriate management is difficult, especially when the mutilation is particularly severe. This report describes the case of a 2-year-old female patient who had severe self-mutilating injuries to her tongue, hands, lips, and oral mucosa caused by biting. The patient presented digital amputation and also a premature loss of a permanent tooth germ during the treatment. The dental management is described and discussed. It is important to include the dentist on the multidisciplinary team to reduce the frequency and severity of the self-inflicted lesions in these patients, also to prevent complications.

  1. Congenital insensitivity to pain in a child attending a paediatric fracture clinic.

    PubMed

    Abdulla, Mohamed; Khaled, Sari S; Khaled, Yazan S; Kapoor, Harish

    2014-09-01

    Congenital insensitivity to pain is a rare condition that is often undiagnosed until patients present with a variety of musculoskeletal problems. A major sequel of these orthopaedic manifestations is the development of heterotopic ossification and callus formation following fractures, eventually leading to the development of a Charcot's joint. This case reports on a 7-year-old child who was diagnosed with congenital insensitivity to pain type V, after he presented in our clinic with fractures of the metatarsals in his left foot while continuing to weight bear, without any discomfort. The patient failed to attend the follow-up in paediatric neurology clinic despite multiple invitations. This case highlights the importance of establishing an early diagnosis and keeping a close eye on this rare entity, which can present for the first time in a paediatric fracture clinic.

  2. Observing Local Realism Violations with a Combination of Sensitive and Insensitive Detectors

    SciTech Connect

    Yurke, B.; Stoler, D.

    1997-12-01

    Several systems are exhibited in which the establishment of local realism violation is achieved even when some of the detectors employed are insensitive. The insensitive detectors are homodyne detectors whose task is to determine if the amplitude of a coherent state is {alpha} or {minus}{alpha} . This determination can be achieved with low quantum efficiency detectors when {vert_bar}{alpha}{vert_bar} is large. The other detectors employed determine whether there is an even number or an odd number of photons in a state. These detectors are sensitive since they are required to resolve the difference of one photon. For the examples given, the local realism violation is not diminished as {vert_bar}{alpha}{vert_bar} is increased. {copyright} {ital 1997} {ital The American Physical Society}

  3. Complete androgen insensitivity syndrome with concomitant seminoma and Sertoli cell adenoma: an unusual combination.

    PubMed

    Thirunavukkarasu, Balamurugan; Mridha, Asit Ranjan; Malhotra, Neena; Chandrashekhara, Sheragaru Hanumanthappa

    2016-12-30

    Androgen insensitivity syndrome is a rare disorder of sex development and its clinical manifestations vary from subtle male infertility to an overt complete androgen insensitivity syndrome (CAIS) with a female phenotype. CAIS is often diagnosed at puberty or in adolescence during investigation for primary amenorrhoea. Undiagnosed patients have an increased risk of development of malignancy in the harboured testes. Inguinal hernia is the commonest mode of presentation of CAIS in childhood and various screening methods are available during the initial herniorrhaphy procedure. Controversy exists in the need to screen and the methods of screening in all cases of premenstrual girls with inguinal hernia. Abnormal observation in a suspicious case requires karyotyping for confirmation. We describe a case of CAIS with simultaneous presence of seminoma and a Sertoli cell adenoma in a 17-year-old patient who had a history of surgery for inguinal hernia at age of 5 years.

  4. A case of complete androgen insensitivity syndrome with a novel androgen receptor mutation.

    PubMed

    Chin, Vivian L; Sheffer-Babila, Sharone; Lee, Ting A; Tanaka, Kathryn; Zhou, Ping

    2012-01-01

    We report a case of a 14-year-old girl with primary amenorrhea and phenotypic as well as hormonal features of complete androgen insensitivity syndrome (CAIS), who tested positive for a novel missense androgen receptor gene mutation resulting in serine-to-isoleucine change at position 703 in exon 4 in the ligand-binding domain. The interesting features of this case include a persistence of Müllerian derivatives, Sertoli cell adenoma, Tanner III pubic hair, and a normal bone mineral density. These features are not typically described in CAIS. This novel mutation associated with a unique clinical presentation serves to significantly enrich the literature on this rare and fascinating disorder of androgen insensitivity syndrome.

  5. Matrix-insensitive protein assays push the limits of biosensors in medicine.

    PubMed

    Gaster, Richard S; Hall, Drew A; Nielsen, Carsten H; Osterfeld, Sebastian J; Yu, Heng; Mach, Kathleen E; Wilson, Robert J; Murmann, Boris; Liao, Joseph C; Gambhir, Sanjiv S; Wang, Shan X

    2009-11-01

    Advances in biosensor technologies for in vitro diagnostics have the potential to transform the practice of medicine. Despite considerable work in the biosensor field, there is still no general sensing platform that can be ubiquitously applied to detect the constellation of biomolecules in diverse clinical samples (for example, serum, urine, cell lysates or saliva) with high sensitivity and large linear dynamic range. A major limitation confounding other technologies is signal distortion that occurs in various matrices due to heterogeneity in ionic strength, pH, temperature and autofluorescence. Here we present a magnetic nanosensor technology that is matrix insensitive yet still capable of rapid, multiplex protein detection with resolution down to attomolar concentrations and extensive linear dynamic range. The matrix insensitivity of our platform to various media demonstrates that our magnetic nanosensor technology can be directly applied to a variety of settings such as molecular biology, clinical diagnostics and biodefense.

  6. Design of polarization-insensitive superconducting single photon detectors with high-index dielectrics

    NASA Astrophysics Data System (ADS)

    Redaelli, L.; Zwiller, V.; Monroy, E.; Gérard, J. M.

    2017-03-01

    In this paper, the design of superconducting-nanowire single-photon detectors which are insensitive to the polarization of the incident light is investigated. By using high-refractive-index dielectrics, the index mismatch between the nanowire and the surrounding media is reduced. This enhances the absorption of light with electric field vector perpendicular to the nanowire segments, which is generally hindered in these kind of detectors. Building on this principle and focusing on NbTiN nanowire devices, we present several easy-to-realize cavity architectures which allow high absorption efficiency (in excess of 90%) and polarization insensitivity simultaneously. Designs based on ultranarrow nanowires, for which the polarization sensitivity is much more marked, are also presented. Finally, we briefly discuss the specific advantages of this approach in the case of WSi or MoSi nanowires.

  7. Polyethyleneimine-poly(ethylene glycol)-star-copolymers as efficient and biodegradable vectors for mammalian cell transfection.

    PubMed

    Ladewig, Katharina; Xu, Zhi Ping; Gray, Peter; Max Lu, G Q

    2014-07-01

    High molecular weight (MW) polyethyleneimine (PEI) has been successfully used for the transfection of a broad variety of cell lines. In contrast to low MW PEI, which exhibits low transfection efficiencies but also low cytotoxicity, high MW PEI-mediated transfection achieves much higher efficiencies but at the cost of cell viability; therefore its use in commercial scale transfection and clinical application is limited. In this work we address this problem by constructing biodegradable high MW PEI mimics built from low MW PEI building blocks. The end-groups of small 5-arm star polyethylene glycol (PEG) prepolymers were decorated with linear oligo-ethyleneimine (OEI)/PEI arms of various MW via azomethine linkages. The resultant PEI-PEG-star-copolymers were investigated for their ability to complex plasmid DNA. Polymer/DNA complexes were characterized using techniques such as dynamic light scattering and transmission electron microscopy. Having established their cytotoxicity limits, they were tested as gene delivery vehicles for the transfection of suspension adapted Chinese hamster ovary (CHO-S) cells under serum-free conditions and adherent human embryonic kidney cells (HEK293T) in serum containing medium. Our PEI-PEG-star-copolymers showed a reduced cytotoxicity compared to high MW PEI while maintaining the ability to complex plasmid DNA and transfect mammalian cells, with significant transfection efficiencies. The effects of the optimum parameters on the transfection of mammalian cells using such novel polymers are discussed.

  8. Bidirectional cytoplasmic incompatibility induced by cross-order transfection of Wolbachia: implications for control of the host population.

    PubMed

    Zhong, Yong; Li, Zheng-Xi

    2014-10-01

    Wolbachia are widespread endosymbionts in arthropods and some nematodes. This genus of bacteria is known to manipulate host reproduction by inducing cytoplasmic incompatibility (CI). This important phenotype is implicated in the control of host populations since Wolbachia can suppress host populations through the induction of CI in a way similar to the sterile insect technique. Here, we identified a candidate CI-inducing Wolbachia strain from the parasitic wasp Scleroderma guani (wSguBJ) by sequencing and phylogenetic analysis. This Wolbachia strain was then isolated, purified, and artificially transfected into the new whitefly host Bemisia tabaci through nymphal microinjection. Infection frequency monitoring by molecular detection showed that 60-80 % of the offspring from transfected whitefly populations was infected with wSguBJ six generations after the transfer. Laboratory rearing experiments indicated that the artificial transfection caused no significant difference in the numbers of offspring between the transfected and naturally infected populations and had no significant detrimental effects on the development of transfected males, although the development of transfected females was delayed. Reciprocal crossings revealed that bidirectional CI was induced between the transfected and naturally infected whiteflies. These data indicated that the cross-order transfer of the heterologous Wolbachia strain by nymphal microinjection was successful. Mass release of the transfected males that could stably carry the heterologous Wolbachia without significant compromise of fecundity/development may provide an alternative approach to control of host populations.

  9. The use of size-defined DNA-functionalized calcium phosphate nanoparticles to minimise intracellular calcium disturbance during transfection.

    PubMed

    Neumann, Sebastian; Kovtun, Anna; Dietzel, Irmgard D; Epple, Matthias; Heumann, Rolf

    2009-12-01

    Calcium phosphate-based transfection methods are frequently used to transfer DNA into living cells. However, it has so far not been studied in detail to what extend the different transfection methods lead to a net calcium uptake. Upon subsequent resolution of the calcium phosphate, intracellular free ionic calcium-surges could result, inducing as side effect various physiological responses that may finally result in cell death. Here we investigated the overall calcium uptake by the human bladder carcinoma cell line T24 during the standard calcium phosphate transfection method and also during transfection with custom-made calcium phosphate/DNA nanoparticles by isotope labelling with (45)calcium. (45)Calcium uptake was strongly increased after 7h of standard calcium phosphate transfection but not if the transfection was performed with calcium phosphate nanoparticles. Time lapse imaging microscopy using the calcium-sensitive dye Fura-2 revealed large transient increases of the intracellular free calcium level during the standard calcium phosphate transfection but not if calcium phosphate nanoparticles were used. Consistently, the viability of cells transfected by calcium phosphate/DNA nanoparticles was not changed, in remarkable contrast to the standard method where considerable cell death occurred.

  10. Inner Ear Gene Transfection in Neonatal Mice Using Adeno-Associated Viral Vector: A Comparison of Two Approaches

    PubMed Central

    Xia, Li; Yin, Shankai; Wang, Jian

    2012-01-01

    Local gene transfection is a promising technique for the prevention and/or correction of inner ear diseases, particularly those resulting from genetic defects. Adeno-associated virus (AAV) is an ideal viral vector for inner ear gene transfection because of its safety, stability, long-lasting expression, and its high tropism for many different cell types. Recently, a new generation of AAV vectors with a tyrosine mutation (mut-AAV) has demonstrated significant improvement in transfection efficiency. A method for inner ear gene transfection via the intact round window membrane (RWM) has been developed in our laboratory. This method has not been tested in neonatal mice, an important species for the study of inherited hearing loss. Following a preliminary study to optimize the experimental protocol in order to reduce mortality, the present study investigated inner ear gene transfection in mice at postnatal day 7. We compared transfection efficiency, the safety of the scala tympani injection via RWM puncture, and the trans-RWM diffusion following partial digestion with an enzyme technique. The results revealed that approximately 47% of inner hair cells (IHCs) and 17% of outer hair cells (OHCs) were transfected via the trans-RWM approach. Transfection efficiency via RWM puncture (58% and 19% for IHCs and OHCs, respectively) was slightly higher, but the difference was not significant. PMID:22912830

  11. Inner ear gene transfection in neonatal mice using adeno-associated viral vector: a comparison of two approaches.

    PubMed

    Xia, Li; Yin, Shankai; Wang, Jian

    2012-01-01

    Local gene transfection is a promising technique for the prevention and/or correction of inner ear diseases, particularly those resulting from genetic defects. Adeno-associated virus (AAV) is an ideal viral vector for inner ear gene transfection because of its safety, stability, long-lasting expression, and its high tropism for many different cell types. Recently, a new generation of AAV vectors with a tyrosine mutation (mut-AAV) has demonstrated significant improvement in transfection efficiency. A method for inner ear gene transfection via the intact round window membrane (RWM) has been developed in our laboratory. This method has not been tested in neonatal mice, an important species for the study of inherited hearing loss. Following a preliminary study to optimize the experimental protocol in order to reduce mortality, the present study investigated inner ear gene transfection in mice at postnatal day 7. We compared transfection efficiency, the safety of the scala tympani injection via RWM puncture, and the trans-RWM diffusion following partial digestion with an enzyme technique. The results revealed that approximately 47% of inner hair cells (IHCs) and 17% of outer hair cells (OHCs) were transfected via the trans-RWM approach. Transfection efficiency via RWM puncture (58% and 19% for IHCs and OHCs, respectively) was slightly higher, but the difference was not significant.

  12. Frequency-resolved noise figure measurements of phase (in)sensitive fiber optical parametric amplifiers.

    PubMed

    Malik, R; Kumpera, A; Lorences-Riesgo, A; Andrekson, P A; Karlsson, M

    2014-11-17

    We measure the frequency-resolved noise figure of fiber optical parametric amplifiers both in phase-insensitive and phase-sensitive modes in the frequency range from 0.03 to 3 GHz. We also measure the variation in noise figure due to the degradation in pump optical signal to noise ratio and also as a function of the input signal powers. Noise figure degradation due to stimulated Brillouin scattering is observed.

  13. Regulation of the voltage-insensitive step of HERG activation by extracellular pH.

    PubMed

    Zhou, Qinlian; Bett, Glenna C L

    2010-06-01

    Human ether-à-go-go-related gene (HERG, Kv11.1, KCNH2) voltage-gated K(+) channels dominate cardiac action potential repolarization. In addition, HERG channels play a role in neuronal and smooth cell excitability as well as cancer pathology. Extracellular pH (pH(o)) is modified during myocardial ischemia, inflammation, and respiratory alkalosis, so understanding the response of HERG channels to changes in pH is of clinical significance. The relationship between pH(o) and HERG channel gating appears complex. Acidification has previously been reported to speed, slow, or have no effect on activation. We therefore undertook comprehensive analysis of the effect of pH(o) on HERG channel activation. HERG channels have unique and complex activation gating characteristics with both voltage-sensitive and voltage-insensitive steps in the activation pathway. Acidosis decreased the activation rate, suppressed peak current, and altered the sigmoidicity of gating near threshold potentials. At positive voltages, where the voltage-insensitive transition is rate limiting, pH(o) modified the voltage-insensitive step with a pK(a) similar to that of histidine. Hill coefficient analysis was incompatible with a coefficient of 1 but was well described by a Hill coefficient of 4. We derived a pH(o)-sensitive term for a five-state Markov model of HERG channel gating. This model demonstrates the mechanism of pH(o) sensitivity in HERG channel activation. Our experimental data and mathematical model demonstrate that the pH(o) sensitivity of HERG channel activation is dominated by the pH(o) sensitivity of the voltage-insensitive step, in a fashion that is compatible with the presence of at least one proton-binding site on each subunit of the channel tetramer.

  14. Insensitive explosive composition and method of fracturing rock using an extrudable form of the composition

    SciTech Connect

    Davis, Lloyd L

    2013-11-05

    Insensitive explosive compositions were prepared by reacting di-isocyanate and/or poly-isocyanate monomers with an explosive diamine monomer. Prior to a final cure, the compositions are extrudable. The di-isocyanate monomers tend to produce tough, rubbery materials while polyfunctional monomers (i.e. having more than two isocyanate groups) tend to form rigid products. The extrudable form of the composition may be used in a variety of applications including rock fracturing.

  15. Insensitive explosive composition and method of fracturing rock using an extrudable form of the composition

    DOEpatents

    Davis, Lloyd L.

    2015-07-28

    Insensitive explosive compositions were prepared by reacting di-isocyanate and/or poly-isocyanate monomers with an explosive diamine monomer. Prior to a final cure, the compositions are extrudable. The di-isocyanate monomers tend to produce tough, rubbery materials while polyfunctional monomers (i.e. having more than two isocyanate groups) tend to form rigid products. The extrudable form of the composition may be used in a variety of applications including rock fracturing.

  16. Mineral nutrient remobilization during corolla senescence in ethylene-sensitive and -insensitive flowers

    PubMed Central

    Jones, Michelle L.

    2013-01-01

    The flower has a finite lifespan that is controlled largely by its role in sexual reproduction. Once the flower has been pollinated or is no longer receptive to pollination, the petals are programmed to senesce. A majority of the genes that are up-regulated during petal senescence, in both ethylene-sensitive and -insensitive flowers, encode proteins involved in the degradation of nucleic acids, proteins, lipids, fatty acids, and cell wall and membrane components. A smaller subset of these genes has a putative role in remobilizing nutrients, and only a few of these have been studied in detail. During senescence, carbohydrates (primarily sucrose) are transported from petals, and the degradation of macromolecules and organelles also allows the plant to salvage mineral nutrients from the petals before cell death. The remobilization of mineral nutrients from a few species has been investigated and will be reviewed in this article. Ethylene's role in nutrient remobilization is discussed by comparing nutrient changes during the senescence of ethylene-sensitive and -insensitive flowers, and by studies in transgenic petunias (Petunia × hybrida) that are insensitive to ethylene. Gene expression studies indicate that remobilization is a key feature of senescence, but some senescence-associated genes have different expression in leaves and petals. These gene expression patterns, along with differences in the nutrient content of leaves and petals, suggest that there are differences in the mechanisms of cellular degradation and nutrient transport in vegetative and floral organs. Autophagy may be the mechanism for large-scale degradation that allows for recycling during senescence, but it is unclear if this causes cell death. Future research should focus on autophagy and the regulation of ATG genes by ethylene during both leaf and petal senescence. We must identify the mechanisms by which individual mineral nutrients are transported out of senescing corollas in both ethylene

  17. Fundamental quantum limits on phase-insensitive linear amplification and phase conjugation in a practical framework

    SciTech Connect

    Namiki, Ryo

    2011-04-15

    We present experimentally testable quantum limitations on the phase-insensitive linear amplification and phase conjugation with respect to the transformation of a Gaussian-distributed set of coherent states following the footing to assess the success of continuous-variable quantum teleportation and quantum memory devices. The results enable us to compare the real device with the quantum-limited device via the feasible input of coherent states.

  18. Probing activation/deactivation of the BRASSINOSTEROID INSENSITIVE1 receptor kinase by immunoprecipitation

    PubMed Central

    Martins, Sara; Vert, Grégory; Jaillais, Yvon

    2017-01-01

    Summary Brassinosteroids are plant sterol-derived hormones that control plant growth and development. The BR receptor complex is encoded by the BRASSINOSTEROID INSENSITIVE1 (BRI1) and members of the SOMATIC EMBRYOGENESIS RECEPTOR KINASE family. BR receptor complex activation and deactivation uses different post-translational modifications and recruitment of partner proteins. In this chapter, we describe optimized immunoprecipitation protocols and variants for biochemical analyses of BRI1 post-translational modification and protein-protein interaction. PMID:28124254

  19. A phase insensitive all-optical router based on nonlinear lenslike planar waveguides.

    PubMed

    Mateo, Eduardo; Liñares, Jesús

    2005-05-02

    We present the design of an all-optical router based on the properties of both propagation and interaction of Gaussian beams in lenslike planar guides. Variational results of single co- and counterpropagation are derived and used to design three integrated optical devices, that is, a header extraction device, an optical bistable device and a data routing device, which perform an ultrafast, phase-insensitive and fiber compatible routing operation in the optical domain.

  20. Insensitive unification of gauge couplings with three vector-like families

    SciTech Connect

    Dermisek, Radovan

    2013-05-23

    The standard model extended by three vector-like families with masses of order 1 TeV - 100 TeV allows for unification of gauge couplings. The values of gauge couplings at the electroweak scale are highly insensitive to fundamental parameters. The grand unification scale is large enough to avoid the problem with fast proton decay. The electroweak minimum of the Higgs potential is stable.

  1. The influence of chitosan valence on the complexation and transfection of DNA: the weaker the DNA-chitosan binding the higher the transfection efficiency.

    PubMed

    Alatorre-Meda, Manuel; Taboada, Pablo; Hartl, Florian; Wagner, Tobias; Freis, Michael; Rodríguez, Julio R

    2011-01-01

    The DNA-chitosan polyplexes have attracted for some years now the attention of physical-chemists and biologists for their potential use in gene therapy, however, the correlation between the physicochemical properties of these polyplexes with their transfection efficiency remains still unclear. In a recent paper we demonstrated by means of DLS that the DNA-chitosan complexation is favored at acidic conditions considering that fewer amounts of chitosan were required to compact the DNA. As a second study, in the present work we analyze the influence of chitosan valence on the complexation and transfection of DNA. Three chitosans of different molecular weights (three different valences) are characterized as gene carriers at 25°C and pH 5 over a wide range of chitosan-Nitrogen to DNA-Phosphate molar ratios, N/P, by means of conductometry, electrophoretic mobility, isothermal titration calorimetry (ITC), transmission electron microscopy (TEM), atomic force microscopy (AFM), and β-galactosidase and luciferase expression assays.

  2. An insensitive acetylcholinesterase confers resistance to methomyl in the beet armyworm Spodoptera exigua (Lepidoptera: Noctuidae).

    PubMed

    Byrne, F J; Toscano, N C

    2001-04-01

    Two forms of acetylcholinesterase were identified in field populations of the beet armyworm, Spodoptera exigua (Hübner), collected from cotton in San Joaquin Valley, CA. Strains (BESS and BKRR) homogeneous for each variant were isolated and their relative susceptibilities to methomyl, chlorpyrifos, and chlorpyrifos-oxon assessed by topical application bioassay. In comparisons with a laboratory susceptible strain (DOW), BKRR and BESS expressed 68-fold and sevenfold resistance, respectively, to the carbamate methomyl. Neither strain was cross-resistant to chlorpyrifos or its oxygen analog (chlorpyrifos-oxon). In biochemical studies, the BKRR AChE enzyme was approximately 30-fold and sixfold more insensitive to methomyl and chlorpyrifos-oxon, respectively, compared with the DOW enzyme. The correlation between the toxicological and biochemical studies provides strong evidence that target-site insensitivity is an important mechanism of resistance to methomyl. The lack of significant cross-resistance to chlorpyrifos suggests that the insensitive AChE in these field populations was selected by methomyl alone and not by the organophosphate.

  3. Laser ignitibility of insensitive secondary explosive 1,1-diamino-2,2-dinitroethene (FOX-7).

    PubMed

    Fang, Xiao; McLuckie, Warren G

    2015-03-21

    An experimental investigation into laser ignitibility of insensitive secondary explosives, 1,1-diamino-2,2-dinitroethene (FOX-7) has been carried out, using a diode laser of continuous wave at the laser wavelength of 974 nm. The direct optical ignition of an insensitive explosive will add more safety features to insensitive munitions (IM) or explosive devices. In this study, effects of laser parameters on the ignitibility were analysed in terms of laser ignition threshold, the times to initiate the ignition and full combustion, and burning sustainability. The results have shown that carbon black (CB) as an optical sensitizer is compatible with FOX-7, and significantly enhances laser ignitibility of the explosive when a small amount of CB is uniformly doped in FOX-7. The delay times for ignition and subsequent development of sustainable burning of the material are mainly determined by ignition laser power, although the other laser parameters have effects. The minimum laser power required to ignite the optically sensitized FOX-7 was found below 10 W and a fast ignition was initiated in as short as 70 μs by a laser power of 40 W. Also the effect of the mixture uniformity of FOX-7/CB on laser ignition performance was evaluated in this study.

  4. Incident Angle- and Polarization-Insensitive Metamaterial Absorber using Circular Sectors

    PubMed Central

    Lee, Dongju; Hwang, Jung Gyu; Lim, Daecheon; Hara, Tadayoshi; Lim, Sungjoon

    2016-01-01

    In this paper, an incident angle- and polarization-insensitive metamaterial absorber is proposed for X-band applications. A unit cell of the proposed absorber has a square patch at the centre and four circular sectors are rotated around the square patch. The vertically and horizontally symmetric structure of the unit cell enables polarization-insensitivity. The circular sector of the unit cell enables an angle-insensitivity. The performances of the proposed absorber are demonstrated with a full-wave simulation and measurements. The angular sensitivity is studied at different inner angles of the circular sector. When the inner angle of the circular sector is 90°, the simulated absorptivity is higher than 90%, and the frequency variation is less than 0.96% for incident angles up to 70°. The measured absorptivity at 10.44 GHz is close to 100% for all the polarization angles under normal incidence. When the incident angles are varied from 0°– 60°, the measured absorptivity is maintained above 90% for both the transverse electric (TE) and the transverse magnetic (TM) modes. PMID:27257089

  5. Incident Angle- and Polarization-Insensitive Metamaterial Absorber using Circular Sectors

    NASA Astrophysics Data System (ADS)

    Lee, Dongju; Hwang, Jung Gyu; Lim, Daecheon; Hara, Tadayoshi; Lim, Sungjoon

    2016-06-01

    In this paper, an incident angle- and polarization-insensitive metamaterial absorber is proposed for X-band applications. A unit cell of the proposed absorber has a square patch at the centre and four circular sectors are rotated around the square patch. The vertically and horizontally symmetric structure of the unit cell enables polarization-insensitivity. The circular sector of the unit cell enables an angle-insensitivity. The performances of the proposed absorber are demonstrated with a full-wave simulation and measurements. The angular sensitivity is studied at different inner angles of the circular sector. When the inner angle of the circular sector is 90°, the simulated absorptivity is higher than 90%, and the frequency variation is less than 0.96% for incident angles up to 70°. The measured absorptivity at 10.44 GHz is close to 100% for all the polarization angles under normal incidence. When the incident angles are varied from 0°– 60°, the measured absorptivity is maintained above 90% for both the transverse electric (TE) and the transverse magnetic (TM) modes.

  6. Incident Angle- and Polarization-Insensitive Metamaterial Absorber using Circular Sectors.

    PubMed

    Lee, Dongju; Hwang, Jung Gyu; Lim, Daecheon; Hara, Tadayoshi; Lim, Sungjoon

    2016-06-03

    In this paper, an incident angle- and polarization-insensitive metamaterial absorber is proposed for X-band applications. A unit cell of the proposed absorber has a square patch at the centre and four circular sectors are rotated around the square patch. The vertically and horizontally symmetric structure of the unit cell enables polarization-insensitivity. The circular sector of the unit cell enables an angle-insensitivity. The performances of the proposed absorber are demonstrated with a full-wave simulation and measurements. The angular sensitivity is studied at different inner angles of the circular sector. When the inner angle of the circular sector is 90°, the simulated absorptivity is higher than 90%, and the frequency variation is less than 0.96% for incident angles up to 70°. The measured absorptivity at 10.44 GHz is close to 100% for all the polarization angles under normal incidence. When the incident angles are varied from 0°- 60°, the measured absorptivity is maintained above 90% for both the transverse electric (TE) and the transverse magnetic (TM) modes.

  7. Nerve growth factor, pain, itch and inflammation: lessons from congenital insensitivity to pain with anhidrosis.

    PubMed

    Indo, Yasuhiro

    2010-11-01

    NGF is a well-known neurotrophic factor essential for the survival and maintenance of primary afferent neurons and sympathetic neurons. NGF is also an inflammatory mediator associated with pain and itch. Congenital insensitivity to pain with anhidrosis is a genetic disorder due to loss-of-function mutations in the NTRK1 gene encoding TrkA, a receptor tyrosine kinase for NGF. Since patients with congenital insensitivity to pain with anhidrosis lack NGF-dependent unmyelinated (C-) and thinly myelinated (Aδ-) fibers, and their dermal sweat glands are without innervation, they exhibit no pain, itch, signs of neurogenic inflammation or sympathetic skin responses. Based on the pathophysiology of congenital insensitivity to pain with anhidrosis, this article indicates how NGF-dependent neurons are essential for the establishment of neural networks for interoception and homeostasis, and play crucial roles in brain-immune-endocrine interactions in pain, itch and inflammation. In addition, it refers to involvements of the NGF-TrkA system in various disease states, and potential pharmacological effects when this system is targeted.

  8. Variation of muscular structure in congenital insensitivity to pain and anhidrosis.

    PubMed

    Shorer, Zamir; Shaco-Levy, Ruth; Pinsk, Vered; Kachko, Leonid; Levy, Jacov

    2013-04-01

    Congenital insensitivity to pain with anhidrosis is a rare disease affecting the nervous system. The patients present with unexplained fever from poor thermoregulation and inability to sweat. Because of the indifference to pain, they manifest frequent traumatic and infectious injuries. Evaluations of these patients include investigation of the hypotonia and weakness evident in this group of patients. We report four patients presenting characteristic features of congenital insensitivity to pain with anhidrosis who carry an identical mutation in the TRK-A gene and who underwent nerve and skeletal muscle biopsies. All four patients had normal sensory and motor conduction studies but lacked sympathetic skin responses. Examination of the skeletal muscles biopsies obtained from two of the patients disclosed marked myopathic changes. The muscle biopsy of a third patient showed mild variation in muscle fibers and the fourth patient's muscle biopsy showed type 1 fiber predominance. Electron microscopy studies revealed remarkable decrease in the number of small caliber-myelinated and unmyelinated nerve fibers. We assume that the variable histological findings in the muscle biopsies of these patients reflect a variation in congenital insensitivity to pain with anhidrosis patients that is not related to their genetic mutation.

  9. Congenital insensitivity to pain and anhidrosis: a case report from South India.

    PubMed

    Udayashankar, Carounanidy; Oudeacoumar, P; Nath, Amiya Kumar

    2012-11-01

    Congenital insensitivity to pain with anhidrosis, also known as hereditary sensory and autonomic neuropathy type IV, is an autosomal recessive disorder characterized by the congenital lack of pain sensation, inability to sweat, episodes of recurrent hyperpyrexia, mental retardation, and self-mutilating behavior. It is an extremely rare disorder with only a handful of reports from India. A five- year- old boy, born to second-degree consanguineous parents after uneventful antenatal period, presented to us with history of recurrent unexplained fever, recurrent ulcers in the lower limbs, insensitivity to painful stimuli (like injections, vaccination) and self-mutilating behavior from early childhood. Cutaneous examination showed multiple ulcers, loss of teeth, loss of tip of the tongue (due to biting), scarring of finger tips, xerosis and lichenification. Sensory examination showed complete loss of pain and temperature sensations, but fine touch and vibration were preserved. Deep tendon reflexes were normal. Evaluation for Hansen's disease was non-contributory. An intradermal injection of histamine did not show any flare response. Based on clinical as well as compatible histological features a diagnosis of congenital insensitivity to pain with anhidrosis was made. The ulcers were treated with appropriate antibiotics and daily dressings. The parents were counseled about appropriate care of the child.

  10. Temperature-insensitive vertical-cavity surface-emitting lasers and method for fabrication thereof

    DOEpatents

    Chow, Weng W.; Choquette, Kent D.; Gourley, Paul L.

    1998-01-01

    A temperature-insensitive vertical-cavity surface-emitting laser (VCSEL) and method for fabrication thereof. The temperature-insensitive VCSEL comprises a quantum-well active region within a resonant cavity, the active region having a gain spectrum with a high-order subband (n.gtoreq.2) contribution thereto for broadening and flattening the gain spectrum, thereby substantially reducing any variation in operating characteristics of the VCSEL over a temperature range of interest. The method for forming the temperature-insensitive VCSEL comprises the steps of providing a substrate and forming a plurality of layers thereon for providing first and second distributed Bragg reflector (DBR) mirror stacks with an active region sandwiched therebetween, the active region including at least one quantum-well layer providing a gain spectrum having a high-order subband (n.gtoreq.2) gain contribution, and the DBR mirror stacks having predetermined layer compositions and thicknesses for providing a cavity resonance within a predetermined wavelength range substantially overlapping the gain spectrum.

  11. Temperature-insensitive vertical-cavity surface-emitting lasers and method for fabrication thereof

    DOEpatents

    Chow, W.W.; Choquette, K.D.; Gourley, P.L.

    1998-01-27

    A temperature-insensitive vertical-cavity surface-emitting laser (VCSEL) and method for fabrication thereof are disclosed. The temperature-insensitive VCSEL comprises a quantum-well active region within a resonant cavity, the active region having a gain spectrum with a high-order subband (n {>=} 2) contribution thereto for broadening and flattening the gain spectrum, thereby substantially reducing any variation in operating characteristics of the VCSEL over a temperature range of interest. The method for forming the temperature-insensitive VCSEL comprises the steps of providing a substrate and forming a plurality of layers thereon for providing first and second distributed Bragg reflector (DBR) mirror stacks with an active region sandwiched therebetween, the active region including at least one quantum-well layer providing a gain spectrum having a high-order subband (n {>=} 2) gain contribution, and the DBR mirror stacks having predetermined layer compositions and thicknesses for providing a cavity resonance within a predetermined wavelength range substantially overlapping the gain spectrum. 12 figs.

  12. A novel ABA insensitive mutant of Lotus japonicus with a wilty phenotype displays unaltered nodulation regulation.

    PubMed

    Biswas, Bandana; Chan, Pick Kuen; Gresshoff, Peter M

    2009-05-01

    An ABA insensitive mutant, Beyma, was isolated in Lotus japonicus MG-20 from an EMS mutagenesis population using root growth inhibition to applied ABA as the screening criterion. (The name 'Beyma' was taken from the Australian Aboriginal language, Wagiman, beyma, meaning 'drying up'.) The stable mutant that segregates as a dominant Mendelian mutation is insensitive to ABA induced inhibition of germination, vegetative growth, stomatal opening, as well as nodulation. Tissue ABA levels were normal, suggesting a sensitivity rather than biosynthesis mutation. It is slow-growing (50-70% of wild-type MG-20) and has a near-constitutive wilty phenotype associated with its inability to regulate stomatal opening. Whilst showing a wide range of ABA insensitive phenotypes, Beyma did not show alteration of nodule number control, as, in the absence of added ABA, the number and patterning (but not size) of nodules formed in the mutant were similar to that of MG-20. Split root experiments on MG-20 showed that application of ABA on one side of the root inhibited nodulation locally but not systemically. We propose that ABA is not involved directly in systemic autoregulation of nodulation (AON).

  13. Miniature temperature insensitive fiber optic sensors for minimally invasive surgical devices

    NASA Astrophysics Data System (ADS)

    Rajan, Ginu; Callaghan, Dean; Semenova, Yuliya; Farrell, Gerald

    2011-05-01

    This paper presents the concept of implementing miniature temperature insensitive optical fiber sensors into minimally invasive surgical devices such as graspers, staplers and scissors. The lack of temperature insensitive and accurate force feedback end effectors make the current minimally invasive surgeries (MIS) less effective especially in the area of electrosurgery. The failure to provide accurate force feedback information reduces the user's sense of immersion in the operating procedure. In this paper we present fiber sensors based on photonic crystal fibers (PCF) for force feedback from the end effectors. Two types of miniature temperature insensitive PCF sensors can be implemented for MIS applications; a Fabry-Perot interferometric sensor based on hollow core PCF and a tapered modal interferometric sensor based on a solid core PCF. A concept for interrogating these sensors effectively at minimal cost is also presented. The integration of sensors onto the end effectors is also important as one has to find an optimum position for maximum strain/force transfer to the fiber sensor without interfering with the operation of the surgical tool. We have also presented the methodology for incorporating the sensors into surgical end-effectors in this paper.

  14. Comparison of Different Electroporation Parameters on Transfection Efficiency of Sheep Testicular Cells

    PubMed Central

    Niakan, Sarah; Heidari, Banafsheh; Akbari, Ghasem; Nikousefat, Zahra

    2016-01-01

    Objective Electroporation can be a highly efficient method for introducing the foreign genetic materials into the targeted cells for transient and/or permanent genetic modification. Considering the application of this technique as a very efficient method for drug, oligonucleotide, antibody and plasmid delivery for clinical applications and production of transgenic animals, the present study aimed to optimize the transfection efficiency of sheep testicular cells including spermatogonial stem cells (SSCs) via electroporation. Materials and Methods This study is an experimental research conducted in Biotechnology Research Center (Avicenna Research Institute, Tehran, Iran) from September 2013 to March 2014. Following isolation and propagation of one-month lamb testicular cells (SSCs and somatic testicular cells including; Sertoli, Leydig, and myoid cells), the effect of different electroporation parameters including total voltages (280, 320, and 350 V), burst durations (10, 8, and 5 milliseconds), burst modes (single or double) and addition of dimethyl sulfoxide (DMSO) were evaluated on transfection efficiency, viability rate and mean fluorescent intensity (MFI) of sheep testicular cells. Results The most transfection efficiency was obtained in 320 V/8 milliseconds/single burst group in transduction medium with and without DMSO. There was a significantly inverse correlation between transfection efficiency with application of both following parameters: addition of DMSO and double burst. After transfection, the highest and lowest viability rates of testicular cells were demonstrated in 320 V/8 milliseconds with transduction medium without DMSO and 350 V/5 milliseconds in medium containing DMSO. Ad- dition of DMSO to transduction medium in all groups significantly decreased the viability rate. The comparison of gene expression indicated that Sertoli and SSCs had the most fluorescence intensity in 320 V/double burst/DMSO positive. However, myoid and Leydig cells showed the

  15. Efficient rescue of foot-and-mouth disease virus in cultured cells transfected with RNA extracted from clinical samples.

    PubMed

    Bisht, Punam; Mohapatra, Jajati K; Subramaniam, Saravanan; Das, Biswajit; Pande, Veena; Biswal, Jitendra K; Sharma, Gaurav K; Rout, Manoranjan; Ranjan, Rajeev; Dash, Bana B; Sanyal, Aniket; Pattnaik, Bramhadev

    2014-02-01

    In this study, an RNA transfection was used to rescue infectious foot-and-mouth disease (FMD) virus from clinical samples in BHK-21 cell line for diagnosis of FMD. Tissue samples (n=190) were subjected to FMD virus isolation by conventional cell culture and also by RNA transfection. FMD virus was isolated from 62% of the clinical samples by RNA transfection, whereas virus was isolated only from 16% of the clinical samples in conventional cell culture method, suggesting better performance of the RNA transfection. Virus was rescued from 67% and 10% of ELISA negative but multiplex PCR positive samples by RNA transfection and conventional cell culture, respectively. The efficiency of transfection was studied on clinical samples subjected to temperature as high as 37°C and varying pH (pH 4-9). Except up to 1 week of storage at 4°C at pH 7.5, virus isolation was not possible by cell culture. Virus was rescued by transfection from samples stored at 4°C for any of the applied pH up to 4 weeks, and when stored at 37°C virus could be rescued up to 4 weeks at pH 7.5 suggesting the fitness of transfection to isolate virus from clinical samples stored under inappropriate conditions. The sequence data and antigenic relationships with the vaccine strains, between virus rescued by transfection and conventional cell culture, were comparable. The RNA transfection will help to increase the efficiency of virus isolation, diagnosis and molecular epidemiological studies.

  16. Evaluation of pulsed laser ablation in liquids generated gold nanoparticles as novel transfection tools: efficiency and cytotoxicity

    NASA Astrophysics Data System (ADS)

    Willenbrock, Saskia; Durán, María. Carolina; Barchanski, Annette; Barcikowski, Stephan; Feige, Karsten; Nolte, Ingo; Murua Escobar, Hugo

    2014-03-01

    Varying transfection efficiencies and cytotoxicity are crucial aspects in cell manipulation. The utilization of gold nanoparticles (AuNP) has lately attracted special interest to enhance transfection efficiency. Conventional AuNP are usually generated by chemical reactions or gas pyrolysis requiring often cell-toxic stabilizers or coatings to conserve their characteristics. Alternatively, stabilizer- and coating-free, highly pure, colloidal AuNP can be generated by pulsed laser ablation in liquids (PLAL). Mammalian cells were transfected efficiently by addition of PLAL-AuNP, but data systematically evaluating the cell-toxic potential are lacking. Herein, the transfection efficiency and cytotoxicity of PLAL AuNP was evaluated by transfection of a mammalian cell line with a recombinant HMGB1/GFP DNA expression vector. Different methods were compared using two sizes of PLAL-AuNP, commercialized AuNP, two magnetic NP-based protocols and a conventional transfection reagent (FuGENE HD; FHD). PLAL-AuNP were generated using a Spitfire Pro femtosecond laser system delivering 120 fs laser pulses at a wavelength of 800 nm focusing the fs-laser beam on a 99.99% pure gold target placed in ddH2O. Transfection efficiencies were analyzed after 24h using fluorescence microscopy and flow cytometry. Toxicity was assessed measuring cell proliferation and percentage of necrotic, propidium iodide positive cells (PI %). The addition of PLAL-AuNP significantly enhanced transfection efficiencies (FHD: 31 %; PLAL-AuNP size-1: 46 %; size-2: 50 %) with increased PI% but no reduced cell proliferation. Commercial AuNP-transfection showed significantly lower efficiency (23 %), slightly increased PI % and reduced cell proliferation. Magnetic NP based methods were less effective but showing also lowest cytotoxicity. In conclusion, addition of PLAL-AuNP provides a novel tool for transfection efficiency enhancement with acceptable cytotoxic side-effects.

  17. Establishment of the cell line, HeLa-CD14, transfected with the human CD14 gene.

    PubMed

    Ning, Bo-Tao; Tang, Yong-Min

    2012-04-01

    CD14 is the pivotal molecule in the diagnosis and therapy of CD14-associated diseases, and is important in bacteremia. The HeLa cell line is regarded as immortal due to its prolific character. The HeLa cell line is derived from human cervical cancer cells and has been widely used in cancer research and gene transfection. In the present study, we established the expression plasmid pcDNA3.1(+)-CD14, and transfected it into the human cervical cancer cell line HeLa to establish a stable cell line (HeLa-CD14) expressing human CD14 antigen on the membrane. After the human CD14 gene was cloned and sequenced through RT-PCR and T-A cloning techniques, the eukaryotic expression vector pcDNA3.1(+)-CD14 was constructed by cleaving with double restriction endonucleases and ligating with T4 ligase. HeLa cells were transfected with the pcDNA3.1(+)-CD14 recombinant plasmid using Superfect transfection reagent. The cells were selected using G418 and the expression of human CD14 on the transfectant was confirmed by RT-PCR and immunohistochemistry. The expression of CD14 mRNA was significantly different between the blank pcDNA3.1(+)-transfected cell group and the pcDNA3.1(+)-CD14-transfected cell group (p<0.01). The fluorescence was significantly stronger on the established stable cell line than on the transiently transfected HeLa cells, and no visible fluorescence was observed in blank pcDNA3.1(+)-transfected cells. In this study, the human CD14 transfectant, stable cell line HeLa-CD14, was successfully established, which may be used to study CD14 and cervical cancer in vitro and in vivo.

  18. Incorporating mesh-insensitive structural stress into the fatigue assessment procedure of common structural rules for bulk carriers

    NASA Astrophysics Data System (ADS)

    Kim, Seong-Min; Kim, Myung-Hyun

    2015-01-01

    This study introduces a fatigue assessment procedure using mesh-insensitive structural stress method based on the Common Structural Rules for Bulk Carriers by considering important factors, such as mean stress and thickness effects. The fatigue assessment result of mesh-insensitive structural stress method have been compared with CSR procedure based on equivalent notch stress at major hot spot points in the area near the ballast hold for a 180 K bulk carrier. The possibility of implementing mesh-insensitive structural stress method in the fatigue assessment procedure for ship structures is discussed.

  19. Targeted gene transfection from microbubbles into vascular smooth muscle cells using focused, ultrasound-mediated delivery

    PubMed Central

    Phillips, Linsey C.; Klibanov, Alexander L.; Wamhoff, Brian R.; Hossack, John A.

    2010-01-01

    We investigate a method for gene delivery to vascular smooth muscle cells using ultrasound triggered delivery of plasmid DNA from electrostatically coupled cationic microbubbles. Microbubbles carrying reporter plasmid DNA were acoustically ruptured in the vicinity of smooth muscle cells in vitro under a range of acoustic pressures (0–950 kPa) and pulse durations (0–100 cycles). No effect on gene transfection or viability was observed from application of microbubbles, DNA, or ultrasound alone. Microbubbles in combination with ultrasound (500 kPa, 1MHz, 50 cycle bursts at a Pulse Repetition Frequency [PRF] of 100 Hz) significantly reduced viability both with DNA (53 +/− 27%) and without (19 +/− 8%). Maximal gene transfection (~1% of cells) occurred using 50 cycle, 1 MHz pulses at 300 kPa which resulted in 40% viability of cells. We demonstrated that we can locally deliver DNA to vascular smooth muscle cells in vitro using microbubble carriers and focused ultrasound. PMID:20800174

  20. Evaluation of transfection efficiency in skeletal muscle using nano/microbubbles and ultrasound.

    PubMed

    Kodama, Tetsuya; Aoi, Atsuko; Watanabe, Yukiko; Horie, Sachiko; Kodama, Mizuho; Li, Li; Chen, Rui; Teramoto, Noriyoshi; Morikawa, Hidehiro; Mori, Shiro; Fukumoto, Manabu

    2010-07-01

    Recent studies have revealed that ultrasound contrast agents with low-intensity ultrasound, namely, sonoporation, can noninvasively deliver therapeutic molecules into target sites. However, the efficiency of molecular delivery is relatively low and the methodology requires optimization. Here, we investigated three types of nano/microbubbles (NMBs)-human albumin shell bubbles, lipid bubbles and acoustic liposomes-to evaluate the efficiency of gene expression in skeletal muscle as a function of their physicochemical properties and the number of bubbles in solution. We found that acoustic liposomes showed the highest transfection and gene expression efficiency among the three types of NMBs under ultrasound-optimized conditions. Liposome transfection efficiency increased with bubble volume concentration; however, neither bubble volume concentration nor their physicochemical properties were related to the tissue damage detected in the skeletal muscle, which was primarily caused by needle injection.

  1. Structure-activity investigation on the gene transfection properties of cardiolipin mimicking gemini lipid analogues.

    PubMed

    Bajaj, Avinash; Paul, Bishwajit; Kondaiah, Paturu; Bhattacharya, Santanu

    2008-06-01

    A structure-activity relationship has been explored on the gene transfection efficiencies of cardiolipin mimicking gemini lipid analogues upon variation of length and hydrophilicity of the spacer between the cationic ammonium headgroups and lipid hydrocarbon chain lengths. All the gemini lipids were found to be highly superior in gene transfer abilities as compared to their monomeric lipid and a related commercially available formulation. Pseudoglyceryl gemini lipids bearing an oxyethylene (-CH2-(CH2-O-CH2)m-CH2-) spacer were found to be superior gene transfecting agents as compared to those bearing polymethylene (-CH2)m-) spacers. The major characteristic feature of the present set of gemini lipids is their serum compatibility, which is most often the major hurdle in liposome-mediated gene delivery.

  2. An improved transfection assay for evaluating the effects of heavy metals.

    PubMed

    Suzuki, Kaoru; Koizumi, Shinji

    2009-08-01

    The transfection assay is an important tool for evaluating the health effects of industrial chemicals, with the reporter gene expression as an indicator. However, heavy metals often influence the expression of the reference plasmids used to correct variations in transfection efficiency between assay plates, reducing the reliability of this assay. We found that the target of heavy metals is the reporter, rather than the promoter used in the reference plasmid. Of the reporters we tested, luciferase (Luc) enzyme activity was affected by heavy metals, whereas gene product levels of the chloramphenicol acetyltransferase (CAT) or beta-galactosidase (betaGal) gene were not. Neither heavy metals nor extracts from cells exposed to heavy metals showed an effect when directly added to the Luc enzyme, suggesting that heavy metals act through an indirect mechanism. These data indicate that the use of CAT or betaGal as a reporter is appropriate for a reliable assay of heavy metal effects.

  3. Octaarginine-modified chitosan as a nonviral gene delivery vector: properties and in vitro transfection efficiency

    NASA Astrophysics Data System (ADS)

    Zhao, Xiaoli; Li, Zhaoyang; Liu, Wenguang; Lam, Wingmoon; Sun, Peng; Kao, Richard Y. T.; Luk, Keith D. K.; Lu, William W.

    2011-02-01

    Protein transduction domains (PTD) have been identified to have the capacity to facilitate molecular cargo to translocate through cell membrane. This study aims to utilize the cell membrane penetrating ability of octaarginine oligopeptide, a simplified prototype of the PTD, to enhance the transfection efficiency of chitosan. Octaarginine-modified chitosan (R8-CS) was synthesized as a gene transfer carrier by carbodiimide chemistry. The structure and composition of R8-CSs were characterized using FTIR and 1H NMR. Agarose gel electrophoresis assay showed that R8-CS could efficiently condense the DNA. The particle size of R8-CS/DNA complexes were determined to be around 100-200 nm. The nanoparticle complexes exhibited a spherical and compact morphology. R8-CS demonstrated higher transfection activity and lower cytotoxicity as compared to the unmodified chitosan and also showed good serum resistance.

  4. KLF7-transfected Schwann cell graft transplantation promotes sciatic nerve regeneration.

    PubMed

    Wang, Ying; Li, Wen-Yuan; Jia, Hua; Zhai, Feng-Guo; Qu, Wen-Rui; Cheng, Yong-Xia; Liu, Yan-Cui; Deng, Ling-Xiao; Guo, Su-Fen; Jin, Zai-Shun

    2017-01-06

    Our former study demonstrated that Krüppel-like Factor 7 (KLF7) is a transcription factor that stimulates axonal regeneration after peripheral nerve injury. Currently, we used a gene therapy approach to overexpress KLF7 in Schwann cells (SCs) and assessed whether KLF7-transfected SCs graft could promote sciatic nerve regeneration. SCs were transfected by adeno-associated virus 2 (AAV2)-KLF7 in vitro. Mice were allografted by an acellular nerve (ANA) with either an injection of DMEM (ANA group), SCs (ANA+SCs group) or AAV2-KLF7-transfected SCs (ANA+KLF7-SCs group) to assess repair of a sciatic nerve gap. The results indicate that KLF7 overexpression promoted the proliferation of both transfected SCs and native SCs. The neurite length of the dorsal root ganglia (DRG) explants was enhanced. Several beneficial effects were detected in the ANA+KLF7-SCs group including an increase in the compound action potential amplitude, sciatic function index score, enhanced expression of PKH26-labeling transplant SCs, peripheral myelin protein 0, neurofilaments, S-100, and myelinated regeneration nerve. Additionally, HRP-labeled motoneurons in the spinal cord, CTB-labeled sensory neurons in the DRG, motor endplate density and the weight ratios of target muscles were increased by the treatment while thermal hyperalgesia was diminished. Finally, expression of KLF7, NGF, GAP43, TrkA and TrkB were enhanced in the grafted SCs, which may indicate that several signal pathways may be involved in conferring the beneficial effects from KLF7 overexpression. We concluded that KLF7-overexpressing SCs promoted axonal regeneration of the peripheral nerve and enhanced myelination, which collectively proved KLF-SCs as a novel therapeutic strategy for injured nerves.

  5. Enhanced photo-transfection efficiency of mammalian cells on graphene coated substrates

    NASA Astrophysics Data System (ADS)

    Mthunzi, Patience; He, Kuang; Ngcobo, Sandile; Warner, Jamie W.

    2014-03-01

    Literature reports graphene, an atomic-thick sheet of carbon atoms as one of the promising biocompatible scaffolds that promotes cellular proliferation in human mesenchymal stem cells. On the other hand, different mammalian cell lines including the induced pluripotent stem cells exhibited an accelerated proliferation rate when cultured on graphene or graphene oxide coated substrates. These findings provide strong motivation to explore the full capability of graphene in further pluripotent stem cell research activities as there exists an urgent requirement to preserve their therapeutic potential. This therefore calls for non-invasive procedures for handling stem cells in-vitro. For example, resent literature has shown successful laser light driven transfection in both multipotent and pluripotent stem cells. In order to explore the non-invasive nature of optical transfection alongside biocompatible qualities of graphene, in this work we investigated the impact of optically transfecting mouse embryonic stem (mES) cells plated on graphene coated sample chambers. Using Chinese Hamster Ovary cells (CHO-K1), we further studied the influence of graphene on cell viability as well as cell cytotoxicity through assessing changes in levels of mitochondrial adenosine triphosphate (ATP) activity and the release of cytosolic lactate dehydrogenase (LHD) respectively. Our results showed that compared to those treated on plain glass, CHO-K1 cells optically treated while plated on graphene coated substrates exhibited a higher production of ATP and a milder release of LDH. In addition there was enhanced photo-transfection efficiency in both CHO-K1 and mES cells irradiated on graphene sample chambers.

  6. Calcium-activated gene transfection from DNA/poly(amic acid-co-imide) complexes

    PubMed Central

    Wu, Szu-Yuan; Chang, Li-Ting; Peng, Sydeny; Tsai, Hsieh-Chih

    2015-01-01

    In this study, we synthesized a water-soluble poly(amic acid-co-imide) (PA-I) from ethylenediaminetetraacetic dianhydride (EDTA) and 2,2′-(ethylenedioxy)bis(ethylamine) that possesses comparable transfection efficiency to that of polyethylenimine (PEI), when prepared in combination with divalent calcium cations. The polycondensation of monomers afforded poly(amic acid) (PA) precursors, and subsequent thermal imidization resulted in the formation of PA-I. At a polymer/DNA ratio (indicated by the molar ratio of nitrogen in the polymer to phosphate in DNA) of 40, complete retardation of the DNA band was observed by gel electrophoresis, indicating the strong association of DNA with PA-I. A zeta potential of −22 mV was recorded for the PA-I polymer solution, and no apparent cytotoxicity was observed at concentrations up to 500 μg·mL−1. In the presence of divalent Ca2+, the transfection efficiency of PA-I was higher than that of PA, due to the formation of a copolymer/Ca2+/DNA polyplex and the reduction in negative charge due to thermal cyclization. Interestingly, a synergistic effect of Ca2+ and the synthesized copolymer on DNA transfection was observed. The use of Ca2+ or copolymer alone resulted in unsatisfactory delivery, whereas the formation of three-component polyplexes synergistically increased DNA transfection. Our findings demonstrated that a PA-I/Ca2+/DNA polyplex could serve as a promising candidate for gene delivery. PMID:25767385

  7. Transfection Studies with Colloidal Systems Containing Highly Purified Bipolar Tetraether Lipids from Sulfolobus acidocaldarius

    PubMed Central

    Pinnapireddy, Shashank Reddy; Baghdan, Elias; Jedelská, Jarmila

    2017-01-01

    Lipid vectors are commonly used to facilitate the transfer of nucleic acids into mammalian cells. In this study, two fractions of tetraether lipids from the archaea Sulfolobus acidocaldarius were extracted and purified using different methods. The purified lipid fractions polar lipid fraction E (PLFE) and hydrolysed glycerol-dialkyl-nonitol tetraether (hGDNT) differ in their structures, charge, size, and miscibility from conventional lipids. Liposomes were prepared by mixing tetraether lipids with cholesterol (CH) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) resulting in stable vectors for gene delivery. Lipoplexes were prepared by complexation of liposomes with a luciferase expressing plasmid (pCMV-luc) at certain nitrogen-to-phosphorus (N/P) ratios and optimised for the transient transfection of ovarian adenocarcinoma cells (SK-OV-3). Complexation efficacy was investigated by gel-red fluorescence assay. Biophysical properties, like size, surface charge, and morphology, were investigated by differential light scattering (DLS), atomic force microscopy (AFM), and scanning electron microscopy (Cryo-SEM), respectively, revealing structural differences between liposomes and lipoplexes. A range of stable transfecting agents containing tetraether lipids were obtained by incorporating 5 mol% of tetraether lipids. Lipoplexes showed a decrease in free gel-red with increasing N/P ratios indicating efficient incorporation of plasmid DNA (pDNA) and remarkable stability. Transfection experiments of the lipoplexes revealed successful and superior transfection of SK-OV-3 cell line compared to the commercially available DOTAP and branched polyethyleneimine (25 kDa bPEI). PMID:28239294

  8. Transfection Studies with Colloidal Systems Containing Highly Purified Bipolar Tetraether Lipids from Sulfolobus acidocaldarius.

    PubMed

    Engelhardt, Konrad H; Pinnapireddy, Shashank Reddy; Baghdan, Elias; Jedelská, Jarmila; Bakowsky, Udo

    2017-01-01

    Lipid vectors are commonly used to facilitate the transfer of nucleic acids into mammalian cells. In this study, two fractions of tetraether lipids from the archaea Sulfolobus acidocaldarius were extracted and purified using different methods. The purified lipid fractions polar lipid fraction E (PLFE) and hydrolysed glycerol-dialkyl-nonitol tetraether (hGDNT) differ in their structures, charge, size, and miscibility from conventional lipids. Liposomes were prepared by mixing tetraether lipids with cholesterol (CH) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) resulting in stable vectors for gene delivery. Lipoplexes were prepared by complexation of liposomes with a luciferase expressing plasmid (pCMV-luc) at certain nitrogen-to-phosphorus (N/P) ratios and optimised for the transient transfection of ovarian adenocarcinoma cells (SK-OV-3). Complexation efficacy was investigated by gel-red fluorescence assay. Biophysical properties, like size, surface charge, and morphology, were investigated by differential light scattering (DLS), atomic force microscopy (AFM), and scanning electron microscopy (Cryo-SEM), respectively, revealing structural differences between liposomes and lipoplexes. A range of stable transfecting agents containing tetraether lipids were obtained by incorporating 5 mol% of tetraether lipids. Lipoplexes showed a decrease in free gel-red with increasing N/P ratios indicating efficient incorporation of plasmid DNA (pDNA) and remarkable stability. Transfection experiments of the lipoplexes revealed successful and superior transfection of SK-OV-3 cell line compared to the commercially available DOTAP and branched polyethyleneimine (25 kDa bPEI).

  9. Partial androgen insensitivity with phenotypic variation caused by androgen receptor mutations that disrupt activation function 2 and the NH(2)- and carboxyl-terminal interaction.

    PubMed

    Quigley, Charmian A; Tan, Jiann-an; He, Bin; Zhou, Zhong-xun; Mebarki, Farida; Morel, Yves; Forest, Maguelone G; Chatelain, Pierre; Ritzén, E Martin; French, Frank S; Wilson, Elizabeth M

    2004-01-01

    Partial androgen insensitivity with sex phenotype variation in two unrelated families was associated with missense mutations in the androgen receptor (AR) gene that disrupted the AR NH(2)-terminal/carboxy terminal interaction. Each mutation caused a single amino acid change within the region of the ligand-binding domain that forms activation function 2 (AF2). In one family, the mutation I737T was in alpha helix 4 and in the other F725L was between helices 3 and 4. Neither mutation altered androgen binding as determined by assays of mutant AR in the patient's cultured genital skin fibroblasts or of recombinant mutant receptors transfected into COS cells. In transient cotransfection assays in CV1 cells, transactivation with the AR mutants at low concentrations of DHT was reduced several fold compared with wild-type AR but increased at higher concentrations. Defects in NH(2)-terminal/carboxy terminal interactions were identified in mammalian two hybrid assays. In similar assays, there was reduced binding of the p160 coactivators TIF2/SRC2 and SRC1 to the mutant AR ligand binding domains (LBD). In the family with AR I737T, sex phenotype varied from severely defective masculinization in the proband to a maternal great uncle whose only manifestation of AIS was severe gynecomastia. He was fertile and passed the mutation to two daughters. The proband of the F725L family was also incompletely masculinized but was raised as a male while his half-sibling by a different father was affected more severely and reared as a female. These studies indicate that the function of an AR AF2 mutant in male development can vary greatly depending on the genetic background.

  10. Construction and application of a microprojectile system for the transfection of organotypic brain slices.

    PubMed

    Ibrahim, M; Si-Ammour, A; Celio, M R; Mauch, F; Menoud, P

    2000-09-15

    In this study we outline a method for constructing an inexpensive chamber used in the transfection of organotypic brain slices. This chamber differs from most commercially available chambers in that DNA-coated gold microcarriers are directly carried by a flow of helium at low pressure (26 psi). Most other chambers employ macrocarriers onto which DNA-coated gold is first loaded, and then released by a shock of helium onto the reverse side of the macrocarriers. This home constructed device has been successfully employed in the transfection of organotypic brain slices cultured using the air-medium interface method. Mammalian expression vectors containing cytomegalovirus (CMV) and simian virus (SV40) enhancers/promoters were used to express enhanced green fluorescence protein (EGFP). DNA was coated onto 0.6-microm gold microcarriers. Transfected cells were visualised under a fluorescence microscope and included identifiable neurones and oligodendrocytes. Also included in this study are step-by-step methods for the preparation of gold microcarriers and organotypic brain slices.

  11. Photochemical internalization-mediated nonviral gene transfection: polyamine core-shell nanoparticles as gene carrier

    NASA Astrophysics Data System (ADS)

    Zamora, Genesis; Wang, Frederick; Sun, Chung-Ho; Trinidad, Anthony; Kwon, Young Jik; Cho, Soo Kyung; Berg, Kristian; Madsen, Steen J.; Hirschberg, Henry

    2014-10-01

    The overall objective of the research was to investigate the utility of photochemical internalization (PCI) for the enhanced nonviral transfection of genes into glioma cells. The PCI-mediated introduction of the tumor suppressor gene phosphatase and tensin homolog (PTEN) or the cytosine deaminase (CD) pro-drug activating gene into U87 or U251 glioma cell monolayers and multicell tumor spheroids were evaluated. In the study reported here, polyamine-DNA gene polyplexes were encapsulated in a nanoparticle (NP) with an acid degradable polyketal outer shell. These NP synthetically mimic the roles of viral capsid and envelope, which transport and release the gene, respectively. The effects of PCI-mediated suppressor and suicide genes transfection efficiency employing either "naked" polyplex cores alone or as NP-shelled cores were compared. PCI was performed with the photosensitizer AlPcS2a and λ=670-nm laser irradiance. The results clearly demonstrated that the PCI can enhance the delivery of both the PTEN or CD genes in human glioma cell monolayers and multicell tumor spheroids. The transfection efficiency, as measured by cell survival and inhibition of spheroid growth, was found to be significantly greater at suboptimal light and DNA levels for shelled NPs compared with polyamine-DNA polyplexes alone.

  12. Glyoxalase I in detoxification: studies using a glyoxalase I transfectant cell line.

    PubMed Central

    Ranganathan, S; Walsh, E S; Tew, K D

    1995-01-01

    The glyoxalase system (glyoxalase I, glyoxalase II and GSH as cofactor) is involved in the detoxification of methylglyoxal (a byproduct of the glycolytic pathway) and other alpha-oxoaldehydes. We have transfected a 622 bp cDNA encoding human glyoxalase I into murine NIH3T3 cells. The recipient cells were shown to express elevated transcript and protein levels and a 10-fold increase in glyoxalase I enzyme activity. This was accompanied by an increased tolerance for exogenous methylglyoxal and enhanced resistance to the cytotoxic effects of two glyoxalase I inhibitors (s-p-bromobenzylglutathione diethyl ester and s-p-bromobenzylglutathione dicyclopentyl ester), a glutathione analogue [gamma-glutamyl-(S)-(benzyl)cysteinyl-(R)-(-)-phenylglycine diethyl ester] and the anti-cancer drugs mitomycin C and adriamycin. Steady-state levels of GSH were significantly lower in the transfected cells, perhaps reflecting increased flux as a consequence of elevated glyoxalase activity. This decrease did not alter the sensitivity to the alkylating agent chlorambucil. Although transfection did not affect the growth or doubling time of the NIH3T3 cells, analysis of glyoxalase I activity showed a consistent increase in tumour tissue when compared with pair-matched controls. Thus increased glyoxalase I is associated with the malignant phenotype and may also contribute to protection against the cytotoxicity of certain anti-cancer drugs. Images Figure 1 PMID:7619046

  13. Matrix attachment region combinations increase transgene expression in transfected Chinese hamster ovary cells.

    PubMed

    Zhao, Chun-Peng; Guo, Xiao; Chen, Si-Jia; Li, Chang-Zheng; Yang, Yun; Zhang, Jun-He; Chen, Shao-Nan; Jia, Yan-Long; Wang, Tian-Yun

    2017-02-20

    Matrix attachment regions (MARs) are cis-acting DNA elements that can increase transgene expression levels in a CHO cell expression system. To investigate the effects of MAR combinations on transgene expression and the underlying regulatory mechanisms, we generated constructs in which the enhanced green fluorescent protein (eGFP) gene flanked by different combinations of human β-interferon and β-globin MAR (iMAR and gMAR, respectively), which was driven by the cytomegalovirus (CMV) or simian virus (SV) 40 promoter. These were transfected into CHO-K1 cells, which were screened with geneticin; eGFP expression was detected by flow cytometry. The presence of MAR elements increased transfection efficiency and transient and stably expression of eGFP expression under both promoters; the level was higher when the two MARs differed (i.e., iMAR and gMAR) under the CMV but not the SV40 promoter. For the latter, two gMARs showed the highest activity. We also found that MARs increased the ratio of stably transfected positive colonies. These results indicate that combining the CMV promoter with two different MAR elements or the SV40 promoter with two gMARs is effective for inducing high expression level and stability of transgenes.

  14. Gene Therapy Vectors with Enhanced Transfection Based on Hydrogels Modified with Affinity Peptides

    PubMed Central

    Shepard, Jaclyn A.; Wesson, Paul J.; Wang, Christine E.; Stevans, Alyson C.; Holland, Samantha J.; Shikanov, Ariella; Grzybowski, Bartosz A.; Shea, Lonnie D.

    2011-01-01

    Regenerative strategies for damaged tissue aim to present biochemical cues that recruit and direct progenitor cell migration and differentiation. Hydrogels capable of localized gene delivery are being developed to provide a support for tissue growth, and as a versatile method to induce the expression of inductive proteins; however, the duration, level, and localization of expression isoften insufficient for regeneration. We thus investigated the modification of hydrogels with affinity peptides to enhance vector retention and increase transfection within the matrix. PEG hydrogels were modified with lysine-based repeats (K4, K8), which retained approximately 25% more vector than control peptides. Transfection increased 5- to 15-fold with K8 and K4 respectively, over the RDG control peptide. K8- and K4-modified hydrogels bound similar quantities of vector, yet the vector dissociation rate was reduced for K8, suggesting excessive binding that limited transfection. These hydrogels were subsequently applied to an in vitro co-culture model to induce NGF expression and promote neurite outgrowth. K4-modified hydrogels promoted maximal neurite outgrowth, likely due to retention of both the vector and the NGF. Thus, hydrogels modified with affinity peptides enhanced vector retention and increased gene delivery, and these hydrogels may provide a versatile scaffold for numerous regenerative medicine applications. PMID:21514659

  15. Role of liposome and peptide in the synergistic enhancement of transfection with a lipopolyplex vector

    NASA Astrophysics Data System (ADS)

    Munye, Mustafa M.; Ravi, Jascindra; Tagalakis, Aristides D.; McCarthy, David; Ryadnov, Maxim G.; Hart, Stephen L.

    2015-03-01

    Lipopolyplexes are of widespread interest for gene therapy due to their multifunctionality and high transfection efficiencies. Here we compared the biological and biophysical properties of a lipopolyplex formulation with its lipoplex and polyplex equivalents to assess the role of the lipid and peptide components in the formation and function of the lipopolyplex formulation. We show that peptide efficiently packaged plasmid DNA forming spherical, highly cationic nanocomplexes that are taken up efficiently by cells. However, transgene expression was poor, most likely due to endosomal degradation since the polyplex lacks membrane trafficking properties. In addition the strong peptide-DNA interaction may prevent plasmid release from the complex and so limit plasmid DNA availability. Lipid/DNA lipoplexes, on the other hand, produced aggregated masses that showed poorer cellular uptake than the polyplex but contrastingly greater levels of transgene expression. This may be due to the greater ability of lipoplexes relative to polyplexes to promote endosomal escape. Lipopolyplex formulations formed spherical, cationic nanocomplexes with efficient cellular uptake and significantly enhanced transfection efficiency. The lipopolyplexes combined the optimal features of lipoplexes and polyplexes showing optimal cell uptake, endosomal escape and availability of plasmid for transcription, thus explaining the synergistic increase in transfection efficiency.

  16. Efficient gene transfection using novel cationic polymers poly(hydroxyalkylene imines).

    PubMed

    Zaliauskiene, Lolita; Bernadisiute, Ula; Vareikis, Ausvydas; Makuska, Ricardas; Volungeviciene, Ieva; Petuskaite, Agne; Riauba, Laurynas; Lagunavicius, Arunas; Zigmantas, Sarunas

    2010-09-15

    A series of novel cationic polymers poly(hydroxyalkylene imines) were synthesized and tested for their ability to transfect cells in vitro and in vivo. Poly(hydroxyalkylene imines), in particular, poly(2-hydroxypropylene imine) (pHP), poly(2-hydroxypropylene imine ethylene imine) (pHPE), and poly(hydroxypropylene imine propylene imine) (pHPP) were synthesized by polycondensation reaction from 1,3-diamino-2-propanol and the appropriate dibromide. Electron microscopic examination demonstrated that the resulting polymers condensed DNA into toroid shape complexes of 100-150 nm in size. Transfection studies showed that all three polymers were able to deliver genetic material into the cell, with pHP being superior to pHPP and pHPE. pHP acted as an efficient gene delivery agent in a variety of different cell lines and outcompeted most of the widely used polymer or lipid based transfection reagents. Intravenous administration of pHP-DNA polyplexes in mice followed by the reporter gene analysis showed that the reagent was suitable for in vivo applications. In summary, the results indicate that pHP is a new efficient reagent for gene delivery in vitro and in vivo.

  17. Alteration of some cellular function in amikacin resistant Pseudomonas aeruginosa transfected macrophages: a time dependent approach

    PubMed Central

    Chakraborty, Subhankari Prasad; KarMahapatra, Santanu; Das, Sabyasachi; Roy, Somenath

    2011-01-01

    Objective To evaluate the free radical generation and antioxidant enzymes status in murine peritoneal macrophage during in vitro amikacin resistant Pseudomonas aeruginosa (ARPA) treatment with different time interval. Methods Peritoneal macrophages were treated with 1×108 CFU/mL ARPA cell suspension in vitro for different time interval (1, 2, 3, 6, 12, and 24 h) and super oxide anion generation, NO generation, reduced glutathione level and antioxidant enzymes status were analyzed. Results Super oxide anion generation and NO generation got peak at 12 h, indicating maximal free radical generation through activation of NADPH oxidase in murine peritoneal macrophages during ARPA transfection. Reduced glutathione level and antioxidant enzymes status were decreased significantly (P<0.05) with increasing time of ARPA transfection. All the changes in peritoneal macrophages after 12 h in vitro ARPA transfection had significant difference (P<0.05). Conclusions From this study, it may be summarized that in vitro ARPA infection not only generates excess free radical but also affects the antioxidant system and glutathione cycle in murine peritoneal macrophage. PMID:23569818

  18. Efficient Gene Knockdown in Mouse Oocytes through Peptide Nanoparticle-Mediated SiRNA Transfection

    PubMed Central

    Jin, Zhen; Li, Ruichao; Zhou, Chunxiang; Shi, Liya; Zhang, Xiaolan; Yang, Zhixia; Zhang, Dong

    2016-01-01

    The use of mouse oocytes as a model for studying female meiosis is very important in reproductive medicine. Gene knockdown by specific small interfering RNA (siRNA) is usually the first step in the study of the function of a target gene in mouse oocytes during in vitro maturation. Traditionally, the only way to introduce siRNA into mouse oocytes is through microinjection, which is certainly less efficient and strenuous than siRNA transfection in somatic cells. Recently, in research using somatic cells, peptide nanoparticle-mediated siRNA transfection has been gaining popularity over liposome nanoparticle-mediated methods because of its high efficiency, low toxicity, good stability, and strong serum compatibility. However, no researchers have yet tried transfecting siRNA into mouse oocytes because of the existence of the protective zona pellucida surrounding the oocyte membrane (vitelline membrane). We therefore tested whether peptide nanoparticles can introduce siRNA into mouse oocytes. In the present study, we showed for the first time that our optimized program can efficiently knock down a target gene with high specificity. Furthermore, we achieved the expected meiotic phenotypes after we knocked down a test unknown target gene TRIM75. We propose that peptide nanoparticles may be superior for preliminary functional studies of unknown genes in mouse oocytes. PMID:26974323

  19. Liver suction-mediated transfection in mice using a pressure-controlled computer system.

    PubMed

    Shimizu, Kazunori; Zhang, Guangyuan; Kawakami, Shigeru; Taniguchi, Yota; Hayashi, Kouji; Hashida, Mitsuru; Konishi, Satoshi

    2014-01-01

    We previously developed an in vivo tissue suction-mediated transfection method (denoted as the tissue suction method) for naked nucleic acids, such as plasmid DNA (pDNA) and small interfering RNA (siRNA), in mice. However, it remains unclear whether the suction pressure conditions affect the results of this method. Therefore, in the present study, we assembled a computer system to control the suction pressure and investigate the effects of the suction pressure conditions on the efficiency of the liver suction transfection of naked pDNA that encodes luciferase in mice. Using the developed system, we examined the effects of the minimum magnitude of the suction pressure, suction pressure waveform, and suction times of the luciferase expression level in mice livers. We determined that the liver suction method at 5 kPa was not only effective but also caused the lowest hepatic toxicity in mice. Additionally, the results indicated that the suction pressure waveform affects the luciferase expression levels, and a single period of suction on the targeted portion of the liver is sufficient for transfection. Thus, the developed system is useful for performing the tissue suction method with high accuracy and safety.

  20. Matrix attachment region combinations increase transgene expression in transfected Chinese hamster ovary cells

    PubMed Central

    Zhao, Chun-Peng; Guo, Xiao; Chen, Si-Jia; Li, Chang-Zheng; Yang, Yun; Zhang, Jun-He; Chen, Shao-Nan; Jia, Yan-Long; Wang, Tian-Yun

    2017-01-01

    Matrix attachment regions (MARs) are cis-acting DNA elements that can increase transgene expression levels in a CHO cell expression system. To investigate the effects of MAR combinations on transgene expression and the underlying regulatory mechanisms, we generated constructs in which the enhanced green fluorescent protein (eGFP) gene flanked by different combinations of human β-interferon and β-globin MAR (iMAR and gMAR, respectively), which was driven by the cytomegalovirus (CMV) or simian virus (SV) 40 promoter. These were transfected into CHO-K1 cells, which were screened with geneticin; eGFP expression was detected by flow cytometry. The presence of MAR elements increased transfection efficiency and transient and stably expression of eGFP expression under both promoters; the level was higher when the two MARs differed (i.e., iMAR and gMAR) under the CMV but not the SV40 promoter. For the latter, two gMARs showed the highest activity. We also found that MARs increased the ratio of stably transfected positive colonies. These results indicate that combining the CMV promoter with two different MAR elements or the SV40 promoter with two gMARs is effective for inducing high expression level and stability of transgenes. PMID:28216629

  1. Efficient Gene Knockdown in Mouse Oocytes through Peptide Nanoparticle-Mediated SiRNA Transfection.

    PubMed

    Jin, Zhen; Li, Ruichao; Zhou, Chunxiang; Shi, Liya; Zhang, Xiaolan; Yang, Zhixia; Zhang, Dong

    2016-01-01

    The use of mouse oocytes as a model for studying female meiosis is very important in reproductive medicine. Gene knockdown by specific small interfering RNA (siRNA) is usually the first step in the study of the function of a target gene in mouse oocytes during in vitro maturation. Traditionally, the only way to introduce siRNA into mouse oocytes is through microinjection, which is certainly less efficient and strenuous than siRNA transfection in somatic cells. Recently, in research using somatic cells, peptide nanoparticle-mediated siRNA transfection has been gaining popularity over liposome nanoparticle-mediated methods because of its high efficiency, low toxicity, good stability, and strong serum compatibility. However, no researchers have yet tried transfecting siRNA into mouse oocytes because of the existence of the protective zona pellucida surrounding the oocyte membrane (vitelline membrane). We therefore tested whether peptide nanoparticles can introduce siRNA into mouse oocytes. In the present study, we showed for the first time that our optimized program can efficiently knock down a target gene with high specificity. Furthermore, we achieved the expected meiotic phenotypes after we knocked down a test unknown target gene TRIM75. We propose that peptide nanoparticles may be superior for preliminary functional studies of unknown genes in mouse oocytes.

  2. Proliferation and Differentiation of Rat Osteoporosis Mesenchymal Stem Cells (MSCs) after Telomerase Reverse Transcriptase (TERT) Transfection

    PubMed Central

    Li, Chao; Wei, Guojun; Gu, Qun; Wang, Qiang; Tao, Shuqin; Xu, Liang

    2015-01-01

    Background The aim of this study was to determine whether MSC are excellent materials for MSCs transplantation in the treatment of osteoporosis. Material/Methods We studied normal, osteoporosis, and TERT-transfected MSC from normal and osteoporosis rats to compare the proliferation and osteogenic differentiation using RT-PCR and Western blot by constructing an ovariectomized rat model of osteoporosis (OVX). The primary MSC from model rats were extracted and cultured to evaluate the proliferation and differentiation characteristics. Results MSCs of osteoporosis rats obviously decreased in proliferation ability and osteogenic differentiation compared to that of normal rats. In contrast, in TERT-transfected MSC, the proliferation and differentiation ability, and especially the ability of osteogenic differentiation, were significantly higher than in osteoporosis MSC. Conclusions TERT-transfected MSCs can help osteoporosis patients in whom MSC proliferation and osteogenic differentiation ability are weak, with an increase in both bone mass and bone density, becoming an effective material for autologous transplantation of MSCs in further treatment of osteoporosis. However, studies are still needed to prove the in vivo effect, biological safety, and molecular mechanism of TERT-osteoporosis treatment. Additionally, because the results are from an animal model, more research is needed in generalizing rat model findings to human osteoporosis patients. PMID:25796354

  3. Intracellular ROS mediates gas plasma-facilitated cellular transfection in 2D and 3D cultures

    PubMed Central

    Xu, Dehui; Wang, Biqing; Xu, Yujing; Chen, Zeyu; Cui, Qinjie; Yang, Yanjie; Chen, Hailan; Kong, Michael G.

    2016-01-01

    This study reports the potential of cold atmospheric plasma (CAP) as a versatile tool for delivering oligonucleotides into mammalian cells. Compared to lipofection and electroporation methods, plasma transfection showed a better uptake efficiency and less cell death in the transfection of oligonucleotides. We demonstrated that the level of extracellular aqueous reactive oxygen species (ROS) produced by gas plasma is correlated with the uptake efficiency and that this is achieved through an increase of intracellular ROS levels and the resulting increase in cell membrane permeability. This finding was supported by the use of ROS scavengers, which reduced CAP-based uptake efficiency. In addition, we found that cold atmospheric plasma could transfer oligonucleotides such as siRNA and miRNA into cells even in 3D cultures, thus suggesting the potential for unique applications of CAP beyond those provided by standard transfection techniques. Together, our results suggest that cold plasma might provide an efficient technique for the delivery of siRNA and miRNA in 2D and 3D culture models. PMID:27296089

  4. Structural mediation on polycation nanoparticles by sulfadiazine to enhance DNA transfection efficiency and reduce toxicity.

    PubMed

    Long, Xingwen; Zhang, Zhihui; Han, Shangcong; Tang, Minjie; Zhou, Junhui; Zhang, Jianhua; Xue, Zhenyi; Li, Yan; Zhang, Rongxin; Deng, Liandong; Dong, Anjie

    2015-04-15

    Reducing the toxicity while maintaining high transfection efficiency is an important issue for cationic polymers as gene carriers in clinical application. In this paper, a new zwitterionic copolymer, polycaprolactone-g-poly(dimethylaminoethyl methyacrylate-co-sulfadiazine methacrylate) (PC-SDZ) with unique pH-sensitivity, was designed and prepared. The incorporation of sulfadiazine into poly(dimethylaminoethyl methacrylate) (PDMAEMA) chains successfully mediates the surface properties including compacter shell structure, lower density of positive charges, stronger proton buffer capability, and enhanced hydrophobicity, which lead to reduction in toxicity and enhancements in stability, cellular uptake, endosome escape, and transfection efficiency for the PC-SDZ2 nanoparticles (NPs)/DNA complexes. Excellent transfection efficiency at the optimal N/P ratio of 10 was observed for PC-SDZ2 NPs/DNA complexes, which was higher than that of the commercial reagent-branched polyethylenimine (PEI). The cytotoxicity was evaluated by CCK8 measurement, and the results showed significant reduction in cytotoxicity even at high concentration of complexes after sulfadiazine modification. Therefore, this work may demonstrate a new way of structural mediation of cationic polymer carriers for gene delivery with high efficiency and low toxicity.

  5. Stabilizing in vitro ultrasound-mediated gene transfection by regulating cavitation.

    PubMed

    Lo, Chia-Wen; Desjouy, Cyril; Chen, Shing-Ru; Lee, Jyun-Lin; Inserra, Claude; Béra, Jean-Christophe; Chen, Wen-Shiang

    2014-03-01

    It is well known that acoustic cavitation can facilitate the inward transport of genetic materials across cell membranes (sonoporation). However, partially due to the unstationary behavior of the initiation and leveling of cavitation, the sonoporation effect is usually unstable, especially in low intensity conditions. A system which is able to regulate the cavitation level during sonication by modulating the applied acoustic intensity with a feedback loop is implemented and its effect on in vitro gene transfection is tested. The regulated system provided better time stability and reproducibility of the cavitation levels than the unregulated conditions. Cultured hepatoma cells (BNL) mixed with 10 μg luciferase plasmids are exposed to 1-MHz pulsed ultrasound with or without cavitation regulation, and the gene transfection efficiency and cell viability are subsequently assessed. Experimental results show that for all exposure intensities (low, medium, and high), stable and intensity dependent, although not higher, gene expression could be achieved in the regulated cavitation system than the unregulated conditions. The cavitation regulation system provides a better control of cavitation and its bioeffect which are crucial important for clinical applications of ultrasound-mediated gene transfection.

  6. Transfection of Actinomyces spp. by genomic DNA of bacteriophages from human dental plaque.

    PubMed

    Yeung, M K; Kozelsky, C S

    1997-01-01

    Bacteriophages that produced turbid or clear zones of lysis in strains of Actinomyces were isolated from 22 of 124 samples of fresh human dental plaque. All human and nonhuman strains of Actinomyces viscosus or Actinomyces naeslundii tested in this study were sensitive to infection by one or more of these phages. In contrast, none of the Actinomyces odontolyticus, Actinomyces israelii, or Actinomyces bovis strains tested were susceptible. Results of restriction endonuclease analyses indicated that the genomes of these phages consisted of double-stranded DNA molecules ranging in size between 16 and 60 kbp. Sequence homology under hybridization conditions of high stringency was observed among a few of the isolated phages. A lysogenized isolate of A. viscosus MG-1 was obtained following infection with a temperate phage, designated phi 225. Results of Southern blot analyses indicated that phi 225 replicated as a plasmid in the lysogenized strain. Genomic DNA from several lytic phages was used to establish conditions for transfection by electroporation of strains of Actinomyces spp. Efficiencies of DNA transfer ranged from 10(2) to 10(5) plaque-forming units per microgram of DNA were obtained under optimal transfection conditions. The results of these studies demonstrate that transfer of genetic information in Actinomyces spp. can be achieved by transfection.

  7. Alteration of glycolipids in ras-transfected NIH 3T3 cells

    SciTech Connect

    Matyas, G.R.; Aaronson, S.A.; Brady, R.O.; Fishman, P.H.

    1987-09-01

    Glycosphingolipid alterations upon viral transformation are well documented. Transformation of mouse 3T3 cells with murine sarcoma viruses results in marked decreases in the levels of gangliosides GM1 and GD1a and an increase in gangliotriaosylceramide. The transforming oncogenes of these viruses have been identified as members of the ras gene family. The authors analyzed NIH 3T3 cells transfected with human H-, K- and N-ras oncogenes for their glycolipid composition and expression of cell surface gangliosides. Using conventional thin-layer chromatographic analysis, they found that the level of GM3 was increased and that of GD1a was slightly decreased or unchanged, and GM1 was present but not in quantifiable levels. Cell surface levels of GM1 were determined by /sup 125/I-labeled cholera toxin binding to intact cells. GD1a was determined by cholera toxin binding to cells treated with sialidase prior to toxin binding. All ras-transfected cells had decreased levels of surface GM1 and GD1 as compared to logarithmically growing normal NIH 3T3 cells. Levels of GM1 and, to a lesser extent, GD1a increased as the latter cells became confluent. Using a monoclonal antibody assay, they found that gangliotriaosylceramide was present in all ras-transfected cells studied but not in logarithmically growing untransfected cells. These results indicated that ras oncogenes derived form human tumors are capable of inducing alterations in glycolipid composition.

  8. Mechanisms of Nanoparticle Mediated siRNA Transfection by Melittin-Derived Peptides

    PubMed Central

    Hou, Kirk K.; Pan, Hua; Ratner, Lee; Schlesinger, Paul H.; Wickline, Samuel A.

    2014-01-01

    Traditional peptide-mediated siRNA transfection via peptide transduction domains exhibits limited cytoplasmic delivery of siRNA due to endosomal entrapment. This work overcomes these limitations with the use of membrane-destabilizing peptides derived from melittin for the knockdown of NFkB signaling in a model of adult T-Cell leukemia/lymphoma. While the mechanism of siRNA delivery into the cytoplasmic compartment by peptide transduction domains has not been well studied, our analysis of melittin derivatives indicates that concurrent nanocomplex disassembly and peptide-mediated endosomolysis are crucial to siRNA transfection. Importantly, in the case of the most active derivative, p5RHH, this process is initiated by acidic pH, indicating that endosomal acidification after macropinocytosis can trigger siRNA release into the cytoplasm. These data provide general principles regarding nanocomplex response to endocytosis which may guide the development of peptide/siRNA nanocomplex-based transfection. PMID:24053333

  9. In vitro transfection of bone marrow-derived dendritic cells with TATp-liposomes.

    PubMed

    Pappalardo, Juan Sebastián; Langellotti, Cecilia A; Di Giacomo, Sebastián; Olivera, Valeria; Quattrocchi, Valeria; Zamorano, Patricia I; Hartner, William C; Levchenko, Tatyana S; Torchilin, Vladimir P

    2014-01-01

    Dendritic cells (DC) are antigen-presenting cells uniquely capable of priming naïve T cells and cross-presenting antigens, and they determine the type of immune response elicited against an antigen. TAT peptide (TATp), is an amphipathic, arginine-rich, cationic peptide that promotes penetration and translocation of various molecules and nanoparticles into cells. TATp-liposomes (TATp-L) used for DC transfection were prepared using TATp derivatized with a lipid-terminated polymer capable of anchoring in the liposomal membrane. Here, we show that the addition of TATp to DNA-loaded liposomes increased the uptake of DNA in DC. DNA-loaded TATp-L increased the in vitro transfection efficiency in DC cultures as evidenced by a higher expression of the enhanced green fluorescent protein and bovine herpes virus type 1 glycoprotein D (gD). The de novo synthesized gD protein was immunologically stimulating when transfections were performed with TATp-L, as indicated by the secretion of interleukin 6.

  10. Extended gene expression by medium exchange and repeated transient transfection for recombinant protein production enhancement.

    PubMed

    Cervera, Laura; Gutiérrez-Granados, Sonia; Berrow, Nicholas Simon; Segura, Maria Mercedes; Gòdia, Francesc

    2015-05-01

    Production of recombinant products in mammalian cell cultures can be achieved by stable gene expression (SGE) or transient gene expression (TGE). The former is based on the integration of a plasmid DNA into the host cell genome allowing continuous gene expression. The latter is based on episomal plasmid DNA expression. Conventional TGE is limited to a short production period of usually about 96 h, therefore limiting productivity. A novel gene expression approach termed extended gene expression (EGE) is explored in this study. The aim of EGE is to prolong the production period by the combination of medium exchange and repeated transfection of cell cultures with plasmid DNA to improve overall protein production. The benefit of this methodology was evaluated for the production of three model recombinant products: intracellular GFP, secreted GFP, and a Gag-GFP virus-like particles (VLPs). Productions were carried out in HEK 293 cell suspension cultures grown in animal-derived component free media using polyethylenimine (PEI) as transfection reagent. Transfections were repeated throughout the production process using different plasmid DNA concentrations, intervals of time, and culture feeding conditions in order to identify the best approach to achieve sustained high-level gene expression. Using this novel EGE strategy, the production period was prolonged between 192 and 240 h with a 4-12-fold increase in production levels, depending on the product type considered.

  11. Functional identification of the stable transfection C5aR cell line Molt-4.

    PubMed

    Zhang, Chunmei; Xu, Ruonan; Wang, Jianan; Han, Gencheng; Chen, Guojiang; Wang, Renxi; Wei, Huawei; Shen, Beifen; Ma, Yuanfang; Li, Yan

    2007-12-01

    The complement C5 anaphylatoxin receptor is a member of the seven transmembrane-spanning G protein-coupled receptor superfamily that signals through Galphai and Galpha16. C5aR is mostly expressed on neutrophils, macrophages and endothelial cells. C5a and C5aR interaction plays an important role in numerous biological effects such as in vivo cytokine storm which results in inflammatory damage. Considering the limitation of collection of human peripheral blood neutrophils and their short half life, the stably transfected cell line for studying the biological effects of C5aR is needed. In this study, we transfected C5aR gene into Molt-4 cell line and examined the function of ectopic C5aR. Our results showed stable expression of the C5aR in Molt-4 cell line and their interaction with human C5a induced ERK1/2 phosphorylation, Ca++ influx. This stable transfected cell line may provide a useful tool for studying signal pathways related to C5a and C5aR interplay and antibody development specific for C5aR.

  12. Inhibitors of the mitochondrial cytochrome b-c1 complex inhibit the cyanide-insensitive respiration of Trypanosoma brucei.

    PubMed

    Turrens, J F; Bickar, D; Lehninger, A L

    1986-06-01

    The cyanide-insensitive respiration of bloodstream trypomastigote forms of Trypanosoma brucei (75 +/- 8 nmol O2 min-1(mg protein)-1) is completely inhibited by the mitochondrial ubiquinone-like inhibitors 2-hydroxy-3-undecyl-1,4-naphthoquinone (UHNQ) and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT). The Ki values for UHDBT (30 nM) and UHNQ (2 microM) are much lower than the reported Ki for salicylhydroxamic acid (SHAM) (5 microM), a widely used inhibitor of the cyanide-insensitive oxidase. UHNQ also stimulated the glycerol-3-phosphate-dependent reduction of phenazine methosulfate, demonstrating that the site of UHNQ inhibition is on the terminal oxidase of the cyanide-insensitive respiration of T. brucei. These results suggest that a ubiquinone-like compound may act as an electron carrier between the two enzymatic components of the cyanide-insensitive glycerol-3-phosphate oxidase.

  13. Cell fusion-mediated improvement in transfection competence for repair-deficient mutant of mouse T cell line

    SciTech Connect

    Shiomi, T.; Hieda-Shiomi, N.; Sato, K.; Yoshizumi, T.; Nakazawa, T.

    1988-03-01

    A multiple mutagen-sensitive mutant (XUM1) of mouse T-cell lymphoma line, L5178Y, is hypersensitive to ionizing radiation, ultraviolet (UV) light, and cross-linking agents (such as mitomycin C). The frequency of transfection for XUM1 cells after exposure to calcium phosphate-coprecipitated pSV2neo DNA was more than 10(4)-fold less effective than that for Ltk-aprt- (LTA) cells. Other transfection methods (DEAE-dextran and polybrene-DMSO) were not effective for L5178Y and XUM1 cells. The transfection-proficient trait of LTA cells was demonstrated to be genetically dominant by examining the the transfection frequency in hybrid clones constructed between XUM1 and LTA cells. To circumvent the problem with XUM1, the LTA genes necessary for transformation processes were introduced into XUM1 cells by constructing hybrids between XUM1 and LTA cells irradiated with X-rays which causes directional chromosome elimination for hybrid cells. Four of 194 hybrid clones tested were transfection-proficient and hypersensitive to UV (XL102, XL107, XL215, and XL216). All four clones were not hypersensitive to X-rays or mitomycin C. The frequencies of transfection for XL102 and XL216 were nearly the same level as that for LTA cells. The efficiency of transfection for XL107 and XL215 was 10 to 100-fold lower than that for LTA cells.

  14. Charge Density and Molecular Weight of Polyphosphoramidate Gene Carrier Are Key Parameters Influencing Its DNA Compaction Ability and Transfection Efficiency

    PubMed Central

    Ren, Yong; Jiang, Xuan; Pan, Deng; Mao, Hai-Quan

    2011-01-01

    A series of polyphosphoramidates (PPA) with different molecular weights (MWs) and charge densities were synthesized and examined for their DNA compaction ability and transfection efficiency. A strong correlation was observed between the transfection efficiency of PPA/DNA nanoparticles and the MW and net positive charge density of the PPA gene carriers in three different cell lines (HeLa, HEK293 and HepG2 cells). An increase in MW and/or net positive charge density of PPA carrier yielded higher DNA compaction capacity, smaller nanoparticles with higher surface charges and higher complex stability against challenges by salt and polyanions. These favorable physicochemical properties of nanoparticles led to enhanced transfection efficiency. PPA/DNA nanoparticles with the highest complex stability showed comparable transfection efficiency as PEI/DNA nanoparticles likely by compensating the low buffering capacity with higher cellular uptake and affording higher level of protection to DNA in endolysosomal compartment. The differences in transfection efficiency were not attributed by any difference in cytotoxicity among the carriers, as all nanoparticles showed minimal level of cytotoxicity under the transfection conditions. Using PPA as a model system, we demonstrated the structural dependence of transfection efficiency of polymer gene carrier. These results offer more insights into nanoparticle engineering for non-viral gene delivery. PMID:21067136

  15. Effects of AC/DC magnetic fields, frequency, and nanoparticle aspect ratio on cellular transfection of gene vectors

    NASA Astrophysics Data System (ADS)

    Ford, Kris; Mair, Lamar; Fisher, Mike; Rowshon Alam, Md.; Juliano, Rudolph; Superfine, Richard

    2008-10-01

    In order to make non-viral gene delivery a useful tool in the study and treatment of genetic disorders, it is imperative that these methodologies be further refined to yield optimal results. Transfection of magnetic nanoparticles and nanorods are used as non-viral gene vectors to transfect HeLa EGFP-654 cells that stably express a mutated enhanced green fluorescent protein (EGFP) gene. We deliver antisense oligonucleotides to these cells designed to correct the aberrant splicing caused by the mutation in the EGFP gene. We also transfect human bronchial endothelial cells and immortalized WI-38 lung cells with pEGFP-N1 vectors. To achieve this we bind the genes to magnetic nanoparticles and nanorods and introduce magnetic fields to effect transfection. We wish to examine the effects of magnetic fields on the transfection of these particles and the benefits of using alternating (AC) magnetic fields in improving transfection rates over direct (DC) magnetic fields. We specifically look at the frequency dependence of the AC field and particle aspect ratio as it pertains to influencing transfection rate. We posit that the increase in angular momentum brought about by the AC field and the high aspect ratio of the nanorod particles, is vital to generating the force needed to move the particle through the cell membrane.

  16. In vivo osteogenic activity of bone marrow stromal stem cells transfected with Ad-GFP-hBMP-2.

    PubMed

    Wang, G X; Hu, L; Hu, H X; Zhang, Z; Liu, D P

    2014-06-16

    The aim of this study was to investigate the expression of bone morphogenetic protein-2 (BMP-2) in bone marrow stromal stem cells (BMSCs) and the in vivo and in vitro osteogenic activity of BMSCs transfected with the adenovirus plasmid, Ad-GFP-hBMP-2. The Ad-GFP-hBMP-2 plasmid was packaged and transfected into rabbit BMSCs to determine the transfection rate. The alkaline phosphatase (ALP) activities of Ad-GFP-hBMP-2-transfected BMSCs (experimental group) and untransfected BMSCs (control group) were detected. In situ hybridization of type I collagen and Western blot were used to determine the BMP-2 gene and protein expressions. The transfected and untransfected BMSCs were respectively inoculated into nude mice to observe in vivo osteogenesis. The decalcified bovine cancellous bone scaffold was respectively combined with transfected and untransfected BMSCs and implanted into ulnar defects in rabbits to repair the bone. The adenovirus titer was 1.2x10(10) pfu/mL. Green fluorescent protein expression appeared 48 h after transfection with the adenovirus plasmid, and the transfection rate was 71.1%. The ALP activity was higher in the experimental group than the control group at each time point after transfection. The gene and protein expressions of BMP-2 were higher in the experimental group than the control group. The positive rates of in vivo osteogenesis in the experimental and control groups were 90% and 40%, respectively. The bone defect repair effects differed markedly between the two groups. The BMP-2 gene can be highly expressed in BMSCs to successfully induce osteogenic differentiation. BMSCs can be used as seed cells for bone tissue engineering.

  17. Transfection effect of microbubbles on cells in superposed ultrasound waves and behavior of cavitation bubble.

    PubMed

    Kodama, Tetsuya; Tomita, Yukio; Koshiyama, Ken-Ichiro; Blomley, Martin J K

    2006-06-01

    The combination of ultrasound and ultrasound contrast agents (UCAs) is able to induce transient membrane permeability leading to direct delivery of exogenous molecules into cells. Cavitation bubbles are believed to be involved in the membrane permeability; however, the detailed mechanism is still unknown. In the present study, the effects of ultrasound and the UCAs, Optison on transfection in vitro for different medium heights and the related dynamic behaviors of cavitation bubbles were investigated. Cultured CHO-E cells mixed with reporter genes (luciferase or beta-gal plasmid DNA) and UCAs were exposed to 1 MHz ultrasound in 24-well plates. Ultrasound was applied from the bottom of the well and reflected at the free surface of the medium, resulting in the superposition of ultrasound waves within the well. Cells cultured on the bottom of 24-well plates were located near the first node (displacement node) of the incident ultrasound downstream. Transfection activity was a function determined with the height of the medium (wave traveling distance), as well as the concentration of UCAs and the exposure time was also determined with the concentration of UCAs and the exposure duration. Survival fraction was determined by MTT assay, also changes with these values in the reverse pattern compared with luciferase activity. With shallow medium height, high transfection efficacy and high survival fraction were obtained at a low concentration of UCAs. In addition, capillary waves and subsequent atomized particles became significant as the medium height decreased. These phenomena suggested cavitation bubbles were being generated in the medium. To determine the effect of UCAs on bubble generation, we repeated the experiments using crushed heat-treated Optison solution instead of the standard microbubble preparation. The transfection ratio and survival fraction showed no additional benefit when ultrasound was used. These results suggested that cavitation bubbles created by the

  18. Ultrasound-Mediated Vascular Gene Transfection by Cavitation of Endothelial-Targeted Cationic Microbubbles

    PubMed Central

    Xie, Aris; Belcik, Todd; Qi, Yue; Morgan, Terry K.; Champaneri, Shivam A.; Taylor, Sarah; Davidson, Brian P.; Zhao, Yan; Klibanov, Alexander L.; Kuliszewski, Michael A.; Leong-Poi, Howard; Ammi, Azzdine; Lindner, Jonathan R.

    2013-01-01

    OBJECTIVES Ultrasound-mediated gene delivery can be amplified by acoustic disruption of microbubble carriers that undergo cavitation. We hypothesized that endothelial targeting of microbubbles bearing cDNA is feasible and, through optimizing proximity to the vessel wall, increases the efficacy of gene transfection. BACKGROUND Contrast ultrasound-mediated gene delivery is a promising approach for site-specific gene therapy, although there are concerns with the reproducibility of this technique and the safety when using high-power ultrasound. METHODS Cationic lipid-shelled decafluorobutane microbubbles bearing a targeting moiety were prepared and compared with nontargeted microbubbles. Microbubble targeting efficiency to endothelial adhesion molecules (P-selectin or intercellular adhesion molecule [ICAM]-1) was tested using in vitro flow chamber studies, intravital microscopy of tumor necrosis factor-alpha (TNF-α)–stimulated murine cremaster muscle, and targeted contrast ultrasound imaging of P-selectin in a model of murine limb ischemia. Ultrasound-mediated transfection of luciferase reporter plasmid charge coupled to microbubbles in the post-ischemic hindlimb muscle was assessed by in vivo optical imaging. RESULTS Charge coupling of cDNA to the microbubble surface was not influenced by the presence of targeting ligand, and did not alter the cavitation properties of cationic microbubbles. In flow chamber studies, surface conjugation of cDNA did not affect attachment of targeted microbubbles at microvascular shear stresses (0.6 and 1.5 dyne/cm2). Attachment in vivo was also not affected by cDNA according to intravital microscopy observations of venular adhesion of ICAM-1–targeted microbubbles and by ultrasound molecular imaging of P-selectin–targeted microbubbles in the post-ischemic hindlimb in mice. Transfection at the site of high acoustic pressures (1.0 and 1.8 MPa) was similar for control and P-selectin–targeted microbubbles but was associated with

  19. High efficiency, site-specific transfection of adherent cells with siRNA using microelectrode arrays (MEA).

    PubMed

    Patel, Chetan; Muthuswamy, Jit

    2012-09-13

    The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for silencing specific genes for functional genomics and therapeutics. A major challenge involved in RNAi based studies is the delivery of RNAi agents to targeted cells. Traditional non-viral delivery techniques, such as bulk electroporation and chemical transfection methods often lack the necessary spatial control over delivery and afford poor transfection efficiencies. Recent advances in chemical transfection methods such as cationic lipids, cationic polymers and nanoparticles have resulted in highly enhanced transfection efficiencies. However, these techniques still fail to offer precise spatial control over delivery that can immensely benefit miniaturized high-throughput technologies, single cell studies and investigation of cell-cell interactions. Recent technological advances in gene delivery have enabled high-throughput transfection of adherent cells, a majority of which use microscale electroporation. Microscale electroporation offers precise spatio-temporal control over delivery (up to single cells) and has been shown to achieve high efficiencies. Additionally, electroporation based approaches do not require a prolonged period of incubation (typically 4 hours) with siRNA and DNA complexes as necessary in chemical based transfection methods and lead to direct entry of naked siRNA and DNA molecules into the cell cytoplasm. As a consequence gene expression can be achieved as early as six hours after transfection. Our lab has previously demonstrated the use of microelectrode arrays (MEA) for site-specific transfection in adherent mammalian cell cultures. In the MEA based approach, delivery of genetic payload is achieved via localized micro-scale electroporation of cells. An application of electric pulse to selected electrodes generates local electric field that leads to electroporation of cells present in the region

  20. Regulation of GLUT transporters by flavonoids in androgen-sensitive and -insensitive prostate cancer cells.

    PubMed

    Gonzalez-Menendez, Pedro; Hevia, David; Rodriguez-Garcia, Aida; Mayo, Juan C; Sainz, Rosa M

    2014-09-01

    Cancer cells show different metabolic requirements from normal cells. In prostate cancer, particularly, glycolytic metabolism differs in androgen-responsive and nonresponsive cells. In addition, some natural compounds with antiproliferative activities are able to modify glucose entry into cells by either modulating glucose transporter (GLUT) expression or by altering glucose binding. The aim of this work was to study the regulation of some GLUTs (GLUT1 and GLUT4) in both androgen-sensitive (LNCaP) and -insensitive (PC-3) prostate cancer cells by 4 structurally different flavonoids (ie, genistein, phloretin, apigenin, and daidzein). Glucose uptake was measured using nonradiolabeled 2-deoxyglucose. The evaluation of protein levels as well as subcellular distribution of GLUT1/4 were analyzed by Western blot and immunocytochemistry, respectively. Androgen-insensitive LNCaP-R and androgen-sensitive PC-3-AR cells were used to study the effect of androgen signaling. Additionally, a docking simulation was employed to compare interactions between flavonoids and XylE, a bacterial homolog of GLUT1 to -4. Results show for the first time the presence of functionally relevant GLUT4 in prostate cancer cells. Furthermore, differences in GLUT1 and GLUT4 levels and glucose uptake were found, without differences on subcellular distribution, after incubation with flavonoids. Docking simulation showed that all compounds interact with the same location of transporters. More importantly, differences between androgen-sensitive and -insensitive prostate cancer cells were found in both GLUT protein levels and glucose uptake. Thus, phenotypic characteristics of prostate cancer cells are responsible for the different effects of these flavonoids in glucose uptake and in GLUT expression rather than their structural differences, with the most effective in reducing cell growth being the highest in modifying glucose uptake and GLUT levels.

  1. Identification of an AR Mutation-Negative Class of Androgen Insensitivity by Determining Endogenous AR Activity

    PubMed Central

    Ukat, M.; Schweikert, H. U.; Hiort, O.; Werner, R.; Drop, S. L. S.; Cools, M.; Hughes, I. A.; Audi, L.; Ahmed, S. F.; Demiri, J.; Rodens, P.; Worch, L.; Wehner, G.; Kulle, A. E.; Dunstheimer, D.; Müller-Roßberg, E.; Reinehr, T.; Hadidi, A. T.; Eckstein, A. K.; van der Horst, C.; Seif, C.; Siebert, R.; Ammerpohl, O.; Holterhus, P.-M.

    2016-01-01

    Context: Only approximately 85% of patients with a clinical diagnosis complete androgen insensitivity syndrome and less than 30% with partial androgen insensitivity syndrome can be explained by inactivating mutations in the androgen receptor (AR) gene. Objective: The objective of the study was to clarify this discrepancy by in vitro determination of AR transcriptional activity in individuals with disorders of sex development (DSD) and male controls. Design: Quantification of DHT-dependent transcriptional induction of the AR target gene apolipoprotein D (APOD) in cultured genital fibroblasts (GFs) (APOD assay) and next-generation sequencing of the complete coding and noncoding AR locus. Setting: The study was conducted at a university hospital endocrine research laboratory. Patients: GFs from 169 individuals were studied encompassing control males (n = 68), molecular defined DSD other than androgen insensitivity syndrome (AIS; n = 18), AR mutation-positive AIS (n = 37), and previously undiagnosed DSD including patients with a clinical suspicion of AIS (n = 46). Intervention(s): There were no interventions. Main Outcome Measure(s): DHT-dependent APOD expression in cultured GF and AR mutation status in 169 individuals was measured. Results: The APOD assay clearly separated control individuals (healthy males and molecular defined DSD patients other than AIS) from genetically proven AIS (cutoff < 2.3-fold APOD-induction; 100% sensitivity, 93.3% specificity, P < .0001). Of 46 DSD individuals with no AR mutation, 17 (37%) fell below the cutoff, indicating disrupted androgen signaling. Conclusions: AR mutation-positive AIS can be reliably identified by the APOD assay. Its combination with next-generation sequencing of the AR locus uncovered an AR mutation-negative, new class of androgen resistance, which we propose to name AIS type II. Our data support the existence of cellular components outside the AR affecting androgen signaling during sexual differentiation with high

  2. Insensitivity of leptogenesis with flavor effects to low energy leptonic CP violation.

    PubMed

    Davidson, Sacha; Garayoa, Julia; Palorini, Federica; Rius, Nuria

    2007-10-19

    If the baryon asymmetry of the Universe is produced by leptogenesis, CP violation is required in the lepton sector. In the seesaw extension of the standard model with three hierarchical right-handed neutrinos, we show that the baryon asymmetry is insensitive to the Pontecorvo-Maki-Nagakawa-Sakata phases: thermal leptogenesis can work for any value of the observable phases. This result was well known when there were no flavor effects in leptogenesis; we show that it remains true when flavor effects are included.

  3. Complete androgen insensitivity syndrome: a rare case of disorder of sex development.

    PubMed

    Pizzo, Alfonsa; Laganà, Antonio Simone; Borrielli, Irene; Dugo, Nella

    2013-01-01

    Androgen Insensitivity Syndrome (AIS) could be considered as a disease that causes resistance to androgens actions, influencing both the morphogenesis and differentiation of the body structures, and systems in which this hormone exerts its effects. It depends on an X-linked mutations in the Androgen Receptor (AR) gene that express a variety of phenotypes ranging from male infertility to completely normal female external genitalia. The clinical phenotypes of AIS could vary and be classified into three categories, as complete (CAIS), partial (PAIS), and mild (MAIS) forms, according to the severity of androgen resistance. We will describe a case of CAIS in a 16-year-old patient.

  4. Polarization insensitive all-fiber mode-lockers functioned by carbon nanotubes deposited onto tapered fibers

    NASA Astrophysics Data System (ADS)

    Song, Yong-Won; Morimune, Keiyo; Set, Sze Y.; Yamashita, Shinji

    2007-01-01

    The authors demonstrate a nonblocked all-fiber mode locker operated by the interaction of carbon nanotubes with the evanescent field of propagating light in a tapered fiber. Symmetric cross section of the device with the randomly oriented nanotubes guarantees the polarization insensitive operation of the pulse formation. In order to minimize the scattering, the carbon nanotubes are deposited within a designed area around the tapered waist. The demonstrated passively pulsed laser has the repetition rate of 7.3MHz and the pulse width of 829fs.

  5. Injection-insensitive lateral divergence in broad-area diode lasers achieved by spatial current modulation

    NASA Astrophysics Data System (ADS)

    Wang, Tao; Tong, Cunzhu; Wang, Lijie; Zeng, Yugang; Tian, Sicong; Shu, Shili; Zhang, Jian; Wang, Lijun

    2016-11-01

    High-power broad-area (BA) diode lasers often suffer from low beam quality, broad linewidth, and a widened slow-axis far field with increasing current. In this paper, a two-dimensional current-modulated structure is proposed and it is demonstrated that it can reduce not only the far-field sensitivity to the injection current but also the linewidth of the lasing spectra. Injection-insensitive lateral divergence was realized, and the beam parameter product (BPP) was improved by 36.5%. At the same time, the linewidth was decreased by about 45% without significant degradations of emission power and conversion efficiency.

  6. Isolation and characterization of mutants defective in the cyanide-insensitive respiratory pathway of Pseudomonas aeruginosa.

    PubMed

    Cunningham, L; Williams, H D

    1995-01-01

    The branched respiratory chain of Pseudomonas aeruginosa contains at least two terminal oxidases which are active under normal physiological conditions. One of these, cytochrome co, is a cytochrome c oxidase which is completely inhibited by concentrations of the respiratory inhibitor potassium cyanide as low as 100 microM. The second oxidase, the cyanide-insensitive oxidase, is resistant to cyanide concentrations in excess of 1 mM as well as to sodium azide. In this work, we describe the isolation and characterization of a mutant of P. aeruginosa defective in cyanide-insensitive respiration. This insertion mutant was isolated with mini-D171 (a replication-defective derivative of the P. aeruginosa phage D3112) as a mutagen and by screening the resulting tetracycline-resistant transductants for the loss of ability to grow in the presence of 1 mM sodium azide. Polarographic studies on the NADH-mediated respiration rate of the mutant indicated an approximate 50% loss of activity, and titration of this activity against increasing cyanide concentrations gave a monophasic curve clearly showing the complete loss of cyanide-insensitive respiration. The mutated gene for a mutant affected in the cyanide-insensitive, oxidase-terminated respiratory pathway has been designated cio. We have complemented the azide-sensitive phenotype of this mutant with a wild-type copy of the gene by in vivo cloning with another mini-D element, mini-D386, carried on plasmid pADD386. The complemented cio mutant regained the ability to grow on medium containing 1 mM azide, titration of its NADH oxidase activity with cyanide gave a biphasic curve similar to that of the wild-type organism, and the respiration rate returned to normal levels. Spectral analysis of the cytochrome contents of the membranes of the wild type, the cio mutant, and the complemented mutant suggests that the cio mutant is not defective in any membrane-bound cytochromes and that the complementing gene does not encode a heme

  7. Congenital insensitivity to pain: a case report and review of the literature.

    PubMed

    Peddareddygari, Leema Reddy; Oberoi, Kinsi; Grewal, Raji P

    2014-01-01

    Congenital insensitivity to pain (CIP) is a rare autosomal recessive genetic disease caused by mutations in the SCN9A gene. We report a patient with the clinical features consistent with CIP in whom we detected a novel homozygous G2755T mutation in exon 15 of this gene. Routine electrophysiological studies are typically normal in patients with CIP. In our patient, these studies were abnormal and could represent the consequences of secondary complications of cervical and lumbosacral spine disease and associated severe Charcot's joints.

  8. Design and simulation of temperature-insensitive arrayed waveguide gratings based on silicon nanowires

    NASA Astrophysics Data System (ADS)

    Chen, Yan; Lang, Tingting; Zou, Jun; He, J.-J.

    2011-12-01

    The design and simulation results of temperature-insensitive arrayed waveguide gratings based on silicon nanowires are presented. The temperature dependent wavelength shift is minimized by using negative thermo-optic coefficient material SU-8 as the upper-cladding. Simulation results show that by using an appropriate thickness and width of the waveguide, quasi-athermal operation can be achieved. For temperature varying from 0°C to 80°C, the TD-CWS can be controlled down to 0.036nm with little polarization dependence for 272nm×253nm waveguide.

  9. Insensitivity of Leptogenesis with Flavor Effects to Low Energy Leptonic CP Violation

    SciTech Connect

    Davidson, Sacha; Palorini, Federica; Garayoa, Julia; Rius, Nuria

    2007-10-19

    If the baryon asymmetry of the Universe is produced by leptogenesis, CP violation is required in the lepton sector. In the seesaw extension of the standard model with three hierarchical right-handed neutrinos, we show that the baryon asymmetry is insensitive to the Pontecorvo-Maki-Nagakawa-Sakata phases: thermal leptogenesis can work for any value of the observable phases. This result was well known when there were no flavor effects in leptogenesis; we show that it remains true when flavor effects are included.

  10. Polarization insensitive silicon photonic ROADM with selectable communication direction for radio access networks.

    PubMed

    Sorianello, Vito; Angelis, Gabriele De; Cassese, Tommaso; Preite, Massimo Valerio; Velha, Philippe; Bianchi, Alberto; Romagnoli, Marco; Testa, Francesco

    2016-12-15

    We report on an experimental prototype of a low-cost silicon photonic reconfigurable optical add/drop multiplexer (ROADM). The device is able to operate with up to 12 wavelength division multiplexing channels. In order to control the polarization of the multi-wavelength signal at the input of the device, an integrated polarization controller is investigated as an alternative to the polarization diversity device architecture. The integrated ROADM is equipped with optical switches for the selection of the path direction and variable optical attenuators for optical power control. We demonstrate the polarization insensitive routing of 10 Gb/s channels between two ROADM nodes with error-free transmission.

  11. Novel androgen receptor gene mutation in patient with complete androgen insensitivity syndrome.

    PubMed

    Ning, Ye; Zhang, Feng; Zhu, Yong; Chen, Huixing; Lu, Jianqi; Li, Zheng

    2012-07-01

    To present a rare case of a patient probably with complete androgen insensitivity syndrome (CAIS) and studied its potential genetic cause. A 24-year-old woman with a normal-appearing vulva and vagina presented to us because of primary amenorrhea. Imaging studies showed no uterus or ovary development but inguinal cryptorchism. Histopathologic examination revealed normal testicular structures. Sequencing the CAIS-associated androgen receptor gene revealed a novel missense mutation of T to G (F698L). A novel androgen receptor gene mutation in the ligand binding domain was detected in the present patient with CAIS, supporting the important role of an androgen receptor defect in the etiology of CAIS.

  12. Complete androgen insensitivity syndrome: factors influencing gonadal histology including germ cell pathology.

    PubMed

    Kaprova-Pleskacova, Jana; Stoop, Hans; Brüggenwirth, Hennie; Cools, Martine; Wolffenbuttel, Katja P; Drop, Stenvert L S; Snajderova, Marta; Lebl, Jan; Oosterhuis, J Wolter; Looijenga, Leendert H J

    2014-05-01

    Patients with complete androgen insensitivity syndrome are at an increased risk for the development of gonadal germ cell cancer. Residual androgen receptor (AR) activity and abnormal gonadal location may influence the survival of atypical germ cells and the development of other histopathological features. To assess this, we evaluated 37 gonads from 19 patients with complete androgen insensitivity (ranging in age from 3 months to 18 years). Histological abnormalities were examined using hematoxylin and eosin-stained sections and sections stained for POU5F1 and KITLG, markers of early changes in germ cells at risk for malignant transformation. Hamartomatous nodules (HNs), Leydig cell hyperplasia (LCH), decreased germ cells, tubular atrophy and stromal fibrosis were more pronounced as age increased (P<0.001). Expected residual AR activity acted as a positive predictor only for non-malignant germ cell survival in (post)pubertal patients (P<0.05). Immunohistochemical studies indicated that delayed maturation of germ cells was present in three patients, whereas intermediate changes that occurred between delayed maturation and intratubular germ cell neoplasia, designated pre-intratubular germ cell neoplasia, were identified in four cases. Intratubular germ cell neoplasia was observed in one patient. Neither POU5F1 nor KITLG expression was dependent on expected residual AR activity. An independent effect of inguinal versus abdominal position of the gonads was difficult to assess because inguinal gonads were present primarily in the youngest individuals. In conclusion, many histological changes occur increasingly with age. Expected residual AR activity contributes to better survival of the general germ cell population in (post)pubertal age; however, it did not seem to have an important role in the survival of the germ cells at risk for malignant transformation (defined by POU5F1 positivity and KITLG overexpression) in complete androgen insensitivity. Comparison of the high

  13. Enhancing NMR of insensitive nuclei by transfer of SABRE spin hyperpolarization

    NASA Astrophysics Data System (ADS)

    Pravdivtsev, Andrey N.; Yurkovskaya, Alexandra V.; Zimmermann, Herbert; Vieth, Hans-Martin; Ivanov, Konstantin L.

    2016-09-01

    We describe the performance of methods for enhancing NMR (Nuclear Magnetic Resonance) signals of "insensitive", but important NMR nuclei, which are based on the SABRE (Signal Amplification By Reversible Exchange) technique, i.e., on spin order transfer from parahydrogen (H2 molecule in its nuclear singlet spin state) to a substrate in a transient organometallic complex. Here such transfer is performed at high magnetic fields by INEPT-type NMR pulse sequences, modified for SABRE. Signal enhancements up to three orders of magnitude are obtained for 15N nuclei; the possibility of sensitive detection of 2D-NMR 1H-15N spectra of SABRE complexes and substrates is demonstrated.

  14. Common Low-cost IM Explosive Program. Development of Next Generation Insensitive Munitions: A Success Story

    DTIC Science & Technology

    2011-11-30

    to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data...Insensitive Munitions Business Case Analysis (BCA) Filter 1 Criteria Cheetah Calculations Standard Safety Tests Electrostatic Friction Impact...Cook‐off V Heating rate is 3.3°C/hr Bullet Impact into HE IV Type V if scored to criteria that  existed  at program start Fragment Impact into HE V  2,532

  15. Temperature-insensitive pressure or strain sensing technology with fiber optic hybrid Sagnac interferometer

    NASA Astrophysics Data System (ADS)

    Yang, Yuanhong; Lu, Lin; Liu, Shuo; Jin, Wei; Han, Zonghu; Cao, Yaohui

    2016-05-01

    The transmission spectrum characteristic of two-segment polarization maintaining fibers Sagnac interferometer was investigated and simulated in detail and a temperature-insensitive pressure or strain sensing technology was proposed. An experimental hybrid Sagnac interferometer was built and the solid core polarization maintaining photonic crystal fiber was taken as the sensing probe. The side pressure sensitive coefficients and the temperature crosstalk drift were measured and compared. The experimental results show that the side pressure sensitive coefficient was ~0.2877 nm/N and the temperature drift was less than 0.1 pm/°C.

  16. Photonic crystals with broadband, wide-angle, and polarization-insensitive transparency.

    PubMed

    Yao, Zhongqi; Luo, Jie; Lai, Yun

    2016-11-01

    Photonic crystals (PhCs) are well-known band gap materials that can block the propagation of electromagnetic waves within certain frequency regimes. Here, we demonstrate that PhCs can also exhibit the contrary property: broadband, wide-angle, and polarization-insensitive transparency beyond normal dielectric solids. Such high transparency attributes to robust impedance matching between a large group of eigen-states in PhCs and propagating waves in free space. As a demonstration, a transparent wall for broadband microwaves is designed for enhancing the transmittance of WiFi and 4G signals.

  17. Genetics of flowering time in bread wheat Triticum aestivum: complementary interaction between vernalization-insensitive and photoperiod-insensitive mutations imparts very early flowering habit to spring wheat.

    PubMed

    Kumar, Sushil; Sharma, Vishakha; Chaudhary, Swati; Tyagi, Anshika; Mishra, Poonam; Priyadarshini, Anupama; Singh, Anupam

    2012-01-01

    Time to flowering in the winter growth habit bread wheat is dependent on vernalization (exposure to cold conditions) and exposure to long days (photoperiod). Dominant Vrn-1 (Vrn-A1, Vrn-B1 and Vrn-D1) alleles are associated with vernalization independent spring growth habit. The semidominant Ppd-D1a mutation confers photoperiod-insensitivity or rapid flowering in wheat under short day and long day conditions. The objective of this study was to reveal the nature of interaction between Vrn-1 and Ppd-D1a mutations (active alleles of the respective genes vrn-1 and Ppd-D1b). Twelve Indian spring wheat cultivars and the spring wheat landrace Chinese Spring were characterized for their flowering times by seeding them every month for five years under natural field conditions in New Delhi. Near isogenic Vrn-1 Ppd-D1 and Vrn-1 Ppd-D1a lines constructed in two genetic backgrounds were also phenotyped for flowering time by seeding in two different seasons. The wheat lines of Vrn-A1a Vrn-B1 Vrn-D1 Ppd-D1a, Vrn-A1a Vrn-B1 Ppd-D1a and Vrn-A1a Vrn-D1 Ppd-D1a (or Vrn-1 Ppd-D1a) genotypes flowered several weeks earlier than that of Vrn-A1a Vrn-B1 Vrn-D1 Ppd-D1b, Vrn-A1b Ppd-D1b and Vrn-D1 Ppd-D1b (or Vrn-1 Ppd-D1b) genotypes. The flowering time phenotypes of the isogenic vernalization-insensitive lines confirmed that Ppd-D1a hastened flowering by several weeks. It was concluded that complementary interaction between Vrn-1 and Ppd-D1a active alleles imparted super/very-early flowering habit to spring wheats. The early and late flowering wheat varieties showed differences in flowering time between short day and long day conditions. The flowering time in Vrn-1 Ppd-D1a genotypes was hastened by higher temperatures under long day conditions. The ambient air temperature and photoperiod parameters for flowering in spring wheat were estimated at 25°C and 12 h, respectively.

  18. Immobilization of gold nanoparticles on cell culture surfaces for safe and enhanced gold nanoparticle-mediated laser transfection

    NASA Astrophysics Data System (ADS)

    Kalies, Stefan; Heinemann, Dag; Schomaker, Markus; Gentemann, Lara; Meyer, Heiko; Ripken, Tammo

    2014-07-01

    In comparison to standard transfection methods, gold nanoparticle-mediated laser transfection has proven to be a versatile alternative. This is based on its minor influence on cell viability and its high efficiency, especially for the delivery of small molecules like small interfering RNA. However, in order to transfer it to routine usage, a safety aspect is of major concern: The avoidance of nanoparticle uptake by the cells is desired. The immobilization of the gold nanoparticles on cell culture surfaces can address this issue. In this study, we achieved this by silanization of the appropriate surfaces and the binding of gold nanoparticles to them. Comparable perforation efficiencies to the previous approaches of gold nanoparticle-mediated laser transfection with free gold nanoparticles are demonstrated. The uptake of the immobilized particles by the cells is unlikely. Consequently, these investigations offer the possibility of bringing gold nanoparticle-mediated laser transfection closer to routine usage.

  19. Synthesis and aggregation properties of dissymmetric phytanyl-gemini surfactants for use as improved DNA transfection vectors.

    PubMed

    Wang, Haitang; Wettig, Shawn D

    2011-01-14

    Improvements in transfection efficiency are required in order to make the goal of cellular gene delivery by non-viral vectors realizable. Novel derivatives of gemini surfactants having dissymmetric tail groups have been designed specifically as a means to improve DNA transfection; the micelle and interfacial properties are reported herein. The effect of these substitutions on the aggregation properties of the gemini surfactants is discussed in the context of results for the m-3-m gemini series, previously reported in the literature. Phytanyl substitution results in lower cmc and higher micelle ionization. In addition, the phytanyl substituted gemini surfactants form vesicles at room temperature. Preliminary in vitro transfection assays showed the phytanyl substituted gemini surfactants to be more efficient transfection vectors as compared to symmetric gemini surfactants.

  20. Dynamics of elimination of plasmids and expression of VEGF121 gene transfected into human mesenchymal stem cells by different methods.

    PubMed

    Smirnikhina, S A; Lavrov, A V; Bochkov, N P

    2011-05-01

    We compared two methods of transfection (lipofection and electroporation) with plasmid containing VEGF121 gene in four cultures of mesenchymal stem cells from the human adipose tissue. The efficacy of transfection after 1 day, the dynamics of plasmid elimination after 3, 6, 9 days, and expression of the target gene were evaluated. Transfection by both methods failed in one of 4 cultures. Analysis of the plasmid elimination dynamics showed that the content of plasmids introduced by both methods decreased by 30-69% in all cultures by day 3 and then remained unchanged from day 3 to day 9. The expression of the target gene did not correlate with the content of plasmids in cells and varied by 2-10 times in control cells and cells transfected by both methods. Fluctuation of VEGF121 expression was not related to methylation.

  1. Immobilization of gold nanoparticles on cell culture surfaces for safe and enhanced gold nanoparticle-mediated laser transfection.

    PubMed

    Kalies, Stefan; Heinemann, Dag; Schomaker, Markus; Gentemann, Lara; Meyer, Heiko; Ripken, Tammo

    2014-01-01

    In comparison to standard transfection methods, gold nanoparticle-mediated laser transfection has proven to be a versatile alternative. This is based on its minor influence on cell viability and its high efficiency, especially for the delivery of small molecules like small interfering RNA. However, in order to transfer it to routine usage, a safety aspect is of major concern: The avoidance of nanoparticle uptake by the cells is desired. The immobilization of the gold nanoparticles on cell culture surfaces can address this issue. In this study, we achieved this by silanization of the appropriate surfaces and the binding of gold nanoparticles to them. Comparable perforation efficiencies to the previous approaches of gold nanoparticle-mediated laser transfection with free gold nanoparticles are demonstrated. The uptake of the immobilized particles by the cells is unlikely. Consequently, these investigations offer the possibility of bringing gold nanoparticle-mediated laser transfection closer to routine usage.

  2. Non-viral bone morphogenetic protein 2 transfection of rat dental pulp stem cells using calcium phosphate nanoparticles as carriers.

    PubMed

    Yang, Xuechao; Walboomers, X Frank; van den Dolder, Juliette; Yang, Fang; Bian, Zhuan; Fan, Mingwen; Jansen, John A

    2008-01-01

    Calcium phosphate nanoparticles have shown potential as non-viral vectors for gene delivery. The aim of this study was to induce bone morphogenetic protein (Bmp)2 transfection in rat dental pulp stem cells using calcium phosphate nanoparticles as a gene vector and then to evaluate the efficiency and bioactivity of the transfection. We also intended to investigate the behavior of transfected cells when seeded on 3-dimensional titanium fiber mesh scaffolds. Nanoparticles of calcium phosphate encapsulating plasmid deoxyribonucleic acid (DNA) (plasmid enhanced green fluorescent protein-BMP2) were prepared. Then, STRO-1-selected rat dental pulp stem cells were transfected using these nanoparticles. Transfection and bioactivity of the secreted BMP2 were examined. Thereafter, the transfected cells were cultured on a fibrous titanium mesh. The cultures were investigated using scanning electron microscipy and evaluated for cell proliferation, alkaline phosphatase activity and calcium content. Finally, real-time polymerase chain reaction was performed for odontogenesis-related gene expression. The results showed that the size of the DNA-loaded particles was approximately 100 nm in diameter. Nanoparticles could protect the DNA encapsulated inside from external DNase and release the loaded DNA in a low-acid environment (pH 3.0). In vitro, nanoparticle transfection was shown to be effective and to accelerate or promote the odontogenic differentiation of rat dental pulp stem cells when cultured in the 3-dimensional scaffolds. Based on our results, plasmid DNA-loaded calcium phosphate nanoparticles appear to be an effective non-viral vector for gene delivery and functioned well for odontogenic differentiation through Bmp2 transfection.

  3. Cell penetrating peptide-based polyplexes shelled with polysaccharide to improve stability and gene transfection

    NASA Astrophysics Data System (ADS)

    Li, Wenyu; Liu, Yajie; Du, Jianwei; Ren, Kefeng; Wang, Youxiang

    2015-04-01

    Cell-penetrating peptides (CPP) have been widely developed as a strategy to enhance cell penetrating ability and transfection. In this work, octa-arginine modified dextran gene vector with pH-sensitivity was developed via host-guest interactions. α-Cyclodextrin was modified with octa-arginine (CDR), which had excellent cell penetrating ability. Dextran was selected as a backbone and modified with azobenzene as guest units by acid-labile imine bonds (Az-I-Dex). The supramolecular polymer CDR/Az-I-Dex with high a C/A molar ratio (molar ratio of CD on CDR to Az on Az-I-Dex) was unfavorable for DNA condensation. The dextran shell of CDR/Az-I-Dex/DNA polyplexes improved the stability under physiological conditions. However, once treated with acetate buffer (pH 5.4) for 3 h, large aggregates formed rapidly due to the cleavage of the dextran shell. As expected, the vector had cell viability of 80% even when the CDR concentration increased to 100 μg mL-1. Moreover, due to the effective cellular uptake efficiency, CDR/Az-I-Dex/DNA polyplexes had 6-300 times higher transfection efficiency than CDR/DNA polyplexes. It was even higher than high molecular weight PLL-based polyplexes of HEK293 T cells. Importantly, chloroquine as an endosomal escape agent could not improve the transfection of CDR/Az-I-Dex/DNA polyplexes, which indicated that the CDR/Az-I-Dex supramolecular polymer had its own ability for endosomal escape. These results suggested that the CPP-based polyplexes shelled with polysaccharide can be promising non-viral gene delivery carriers.Cell-penetrating peptides (CPP) have been widely developed as a strategy to enhance cell penetrating ability and transfection. In this work, octa-arginine modified dextran gene vector with pH-sensitivity was developed via host-guest interactions. α-Cyclodextrin was modified with octa-arginine (CDR), which had excellent cell penetrating ability. Dextran was selected as a backbone and modified with azobenzene as guest units by acid

  4. Mutational analysis of the androgen receptor gene in two Indian families with partial androgen insensitivity syndrome.

    PubMed

    Nagaraja, M R; Rastogi, Amit; Raman, Rajiva; Gupta, Dinesh K; Singh, S K

    2009-12-01

    Mutation in the androgen receptor gene (AR) is known to cause androgen insensitivity syndrome (AIS). In an X-linked recessive manner, an AR mutation gets transmitted to the offspring through carrier mothers in 70% of cases, the other 30% arising de novo. However, reports on AR mutations amongst Indian patients with AIS are scarce in the literature. This study reports mutations in AR from two Indian families, each having a proband with partial androgen insensitivity syndrome (PAIS) phenotype. Clinical, endocrine and cytogenetic evaluation of these affected children was performed. Mutational analysis was carried out by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis followed by sequencing. The two point mutations were in exon 5: p.M742I, familial in patient 1 and p.V746M de novo in patient 2. These are hitherto unrecognized mutations in our population. Similar mutational studies are suggested in patients with AIS, in order to identify their frequency and clinical severity in our population.

  5. Immobilized Biocatalyst for Detection and Destruction of the Insensitive Explosive, 2,4-Dinitroanisole (DNAN).

    PubMed

    Karthikeyan, Smruthi; Kurt, Zohre; Pandey, Gunjan; Spain, Jim C

    2016-10-18

    Accurate and convenient detection of explosive components is vital for a wide spectrum of applications ranging from national security and demilitarization to environmental monitoring and restoration. With the increasing use of DNAN as a replacement for 2,4,6-trinitrotoluene (TNT) in insensitive explosive formulations, there has been a growing interest in strategies to minimize its release and to understand and predict its behavior in the environment. Consequently, a convenient tool for its detection and destruction could enable development of more effective decontamination and demilitarization strategies. Biosensors and biocatalysts have limited applicability to the more traditional explosives because of the inherent limitations of the relevant enzymes. Here, we report a highly specific, convenient and robust biocatalyst based on a novel ether hydrolase enzyme, DNAN demethylase (that requires no cofactors), from a Nocardioides strain that can mineralize DNAN. Biogenic silica encapsulation was used to stabilize the enzyme and enable it to be packed into a model microcolumn for application as a biosensor or as a bioreactor for continuous destruction of DNAN. The immobilized enzyme was stable and not inhibited by other insensitive munitions constituents. An alternative method for DNAN detection involved coating the encapsulated enzyme on cellulose filter paper. The hydrolase based biocatalyst could provide the basis for a wide spectrum of applications including detection, identification, destruction or inertion of explosives containing DNAN (demilitarization operations), and for environmental restorations.

  6. Update Review and Clinical Presentation in Congenital Insensitivity to Pain and Anhidrosis

    PubMed Central

    Pérez-López, L. M.; Cabrera-González, M.; Gutiérrez-de la Iglesia, D.; Ricart, S.; Knörr-Giménez, G.

    2015-01-01

    Introduction. Congenital insensitivity to pain and anhidrosis (CIPA) or hereditary sensory and autonomic neuropathy type IV is an extremely rare syndrome. Three clinical findings define the syndrome: insensitivity to pain, impossibility to sweat, and mental retardation. This pathology is caused by a genetic mutation in the NTRK1 gene, which encodes a tyrosine receptor (TrkA) for nerve growth factor (NGF). Methods. The consultation of a child female in our center with CIPA and a tibia fracture in pseudoarthrosis encouraged us to carefully review literature and examine the therapeutic possibilities. A thorough review of literature published in Pubmed was done about CIPA and other connected medical issues mentioned in the paper. Conclusions. The therapeutic approach of CIPA remains unclear. The preventive approach remains the only possible treatment of CIPA. We propose two new important concepts in the therapeutic approach for these patients: (1) early surgical treatment for long bone fractures to prevent pseudoarthrosis and to allow early weight bearing, decreasing the risk of further osteopenia, and (2) bisphosphonates to avoid the progression of osteopenia and to reduce the number of consecutive fractures. PMID:26579324

  7. Transient receptor potential melastatin 4 channel contributes to migration of androgen-insensitive prostate cancer cells

    PubMed Central

    Kilch, Tatiana; Jochum, Marcus Martin; Urban, Sabine Katharina; Jung, Volker; Stöckle, Michael; Rother, Karen; Greiner, Markus; Peinelt, Christine

    2015-01-01

    Impaired Ca2+ signaling in prostate cancer contributes to several cancer hallmarks, such as enhanced proliferation and migration and a decreased ability to induce apoptosis. Na+ influx via transient receptor potential melastatin 4 channel (TRPM4) can reduce store-operated Ca2+ entry (SOCE) by decreasing the driving force for Ca2+. In patients with prostate cancer, gene expression of TRPM4 is elevated. Recently, TRPM4 was identified as a cancer driver gene in androgen-insensitive prostate cancer. We investigated TRPM4 protein expression in cancer tissue samples from 20 patients with prostate cancer. We found elevated TRPM4 protein levels in prostatic intraepithelial neoplasia (PIN) and prostate cancer tissue compared to healthy tissue. In primary human prostate epithelial cells (hPEC) from healthy tissue and in the androgen-insensitive prostate cancer cell lines DU145 and PC3, TRPM4 mediated large Na+ currents. We demonstrated significantly increased SOCE after siRNA targeting of TRPM4 in hPEC and DU145 cells. In addition, knockdown of TRPM4 reduced migration but not proliferation of DU145 and PC3 cells. Taken together, our data identify TRPM4 as a regulator of SOCE in hPEC and DU145 cells, demonstrate a role for TRPM4 in cancer cell migration and suggest that TRPM4 is a promising potential therapeutic target. PMID:26496025

  8. Ferrite phase shifters using stress insensitive materials. Final report, July 1991-July 1993

    SciTech Connect

    Vaughn, T.; Cox, P.; Harrison, G.; Rodrigue, P.

    1993-10-01

    This RD program sponsored by the Naval Research Laboratory and being conducted by EMS Technologies, Inc., Norcross, Georgia, is focused toward achieving improved performance in microwave switching components via use of stress insensitive' microwave ferrite materials for applications where stable hysteresis characteristics of the materials are critical to the RF performance. The program, therefore, primarily addresses how to relieve or improve the magnetostrictive characteristics of the materials with emphasis on the specific application and demonstration of these materials in microwave switching components, particularly ferrite toroidal phase shifters. Material investigations were focused on Mn+3 substitutions in Yttrium-gadolinium iron garnet. These compounds were evaluated in dual toroid waveguide phase shifter structures with temperature, pressure Rf power as variables Manganese substitution per formula unit for Fe+3 of 0.11 to 0.13 (2.2 to 2.6%) produced compounds which exhibited stable performance from magnetostrictive stresses in phaser structures. Ferrite phase shifters, Stress insensitive materials Manganese substitution in garnets, Magnetostrictive, Stresses in ferrite phasers.

  9. Ultrathin triple-band polarization-insensitive wide-angle compact metamaterial absorber

    NASA Astrophysics Data System (ADS)

    Shang, Shuai; Yang, Shizhong; Tao, Lu; Yang, Lisheng; Cao, Hailin

    2016-07-01

    In this study, the design, realization, and characterization of an ultrathin triple-band polarization-insensitive wide-angle metamaterial absorber are reported. The metamaterial absorber comprises a periodic array of modified six-fold symmetric snowflake-shaped resonators with strip spiral line load, which is printed on a dielectric substrate backed by a metal ground plane. It is shown that the absorber exhibits three distinct near-unity absorption peaks, which are distributed across C, X, Ku bands, respectively. Owing to the six-fold symmetry, the absorber is insensitive to the polarization of the incident radiation. In addition, the absorber shows excellent absorption performance over wide oblique incident angles for both transverse electric and transverse magnetic polarizations. Simulated surface current and field distributions at the three absorption peaks are demonstrated to understand the absorption mechanism. Particularly, the absorption modes come from the fundamental and high-order dipole resonances. Furthermore, the experimental verification of the designed absorber is conducted, and the measured results are in reasonable agreement with the simulated ones. The proposed ultrathin (˜0.018λ0, λ0 corresponding to the lowest peak absorption frequency) compact (0.168λ0×0.168λ0 corresponding to the area of a unit cell) absorber enables potential applications such as stealth technology, electromagnetic interference and spectrum identification.

  10. Compatibility Study of DNTF with Some Insensitive Energetic Materials and Inert Materials

    NASA Astrophysics Data System (ADS)

    Li, Xi; Wang, Bo-Liang; Lin, Qiu-Han; Chen, Li-Ping

    2016-10-01

    The compatibility of 3,4-dinitrofurazanfuroxan (DNTF) with insensitive energetic materials and inert materials was studied in detail using differential scanning calorimetry (DSC). 2,4,6-Trinitrotoluene (TNT), 2,4,6-triamino-1,3,5-trinitrobenzene (TATB), 3-nitro-1,2,4-triazol-5-one (NTO), 2,6-diamino-3,5-dinitropyrazine-1-oxide (LLM-105), 2,6-diamino-3,5-dinitropyridine-1-oxide (ANPyO), and 5-amino-1H-tetrazole nitrate (5-ATEZN) are used as insensitive energetic materials, and polymer(vinyl acetate) (PVAC), hydroxyl-terminated polybutadiene (HTPB), dinoctylsebacate (DOS), 2,4-dinitrotoluene (DNT), and wax are used as inert materials. The results show that DNTF/TNT and DNTF/5-ATEZN possess good compatibility, DNTF/NTO and DNTF/TATB have moderate compatibility, and the compatibility of DNTF/LLM-105 and DNTF/PVAC is poor; in addition, DNTF/ANPyO, DNTF/HTPB, DNTF/DNT, DNTF/DOS, and DNTF/wax have bad compatibility.

  11. Clinical and genetic analysis of Korean patients with congenital insensitivity to pain with anhidrosis.

    PubMed

    Lee, Seung-Tae; Lee, Jeehun; Lee, Munhyang; Kim, Jong-Won; Ki, Chang-Seok

    2009-11-01

    Congenital insensitivity to pain with anhidrosis (CIPA) is a rare autosomal recessive disease characterized by anhidrosis, insensitivity to noxious stimuli, and mental retardation. Mutations in the NTRK1 gene are associated with the pathogenesis of CIPA. In this study, we performed a clinical and genetic analysis on the NTRK1 gene in four Korean patients with CIPA. All patients had typical clinical manifestations of CIPA, including anhidrosis, recurrent fever, absent pain perception, and developmental delay. Sequencing analysis revealed one predominant mutation, c.851-33T>A, in four affected alleles and three novel mutations, including c.287+2dupT, c.2155G>A (p.Glu719Lys), and c.1218delC (p.Pro407ArgfsX), in each affected allele. For one patient, who was heterozygous for c.851-33T>A, another mutation could not be identified, suggesting that a possible hidden intronic or large genomic mutation may have been present. This study extends the spectrum of mutations in the NTRK1 gene and confirms that Korean patients with CIPA have the same genetic background as other ethnicities.

  12. Update Review and Clinical Presentation in Congenital Insensitivity to Pain and Anhidrosis.

    PubMed

    Pérez-López, L M; Cabrera-González, M; Gutiérrez-de la Iglesia, D; Ricart, S; Knörr-Giménez, G

    2015-01-01

    Introduction. Congenital insensitivity to pain and anhidrosis (CIPA) or hereditary sensory and autonomic neuropathy type IV is an extremely rare syndrome. Three clinical findings define the syndrome: insensitivity to pain, impossibility to sweat, and mental retardation. This pathology is caused by a genetic mutation in the NTRK1 gene, which encodes a tyrosine receptor (TrkA) for nerve growth factor (NGF). Methods. The consultation of a child female in our center with CIPA and a tibia fracture in pseudoarthrosis encouraged us to carefully review literature and examine the therapeutic possibilities. A thorough review of literature published in Pubmed was done about CIPA and other connected medical issues mentioned in the paper. Conclusions. The therapeutic approach of CIPA remains unclear. The preventive approach remains the only possible treatment of CIPA. We propose two new important concepts in the therapeutic approach for these patients: (1) early surgical treatment for long bone fractures to prevent pseudoarthrosis and to allow early weight bearing, decreasing the risk of further osteopenia, and (2) bisphosphonates to avoid the progression of osteopenia and to reduce the number of consecutive fractures.

  13. Congenital insensitivity to pain with anhidrosis: a case report of a 33-year-old patient.

    PubMed

    Kosmidis, Ilias; Krallis, Panagiotis; Tsiamasfirou, Damiani; Filiopoulos, Konstantinos

    2013-01-01

    Congenital insensitivity to pain with anhidrosis is a type IV hereditary sensory and autonomic neuropathy, presenting early in life. This disorder results from defective neural crest differentiation with loss of the first-order afferent system, which is responsible for sensations of pain and temperature; a neuronal loss in the sympathetic ganglia is also present. A case of a 33-year-old patient with congenital insensitivity to pain with anhidrosis is presented. From the time of birth, he did not sweat and did not respond to painful stimuli, although unexplained bouts of fever were often observed in infancy; an extensive workup during childhood helped establish the diagnosis. Throughout childhood and adulthood, the patient presented multiple infections and fractures in various sites of his body, growth disturbances, and avascular necrosis, and Charcot arthropathies and joint dislocations mainly affected his elbow and hip joint. At the final follow-up, at the age of 33 years, he was found to be obese, with a limited social life. A Charcot elbow restricted the activity of his left upper limb, and the dislocated hips combined with the instability of the ankle joints limited the ambulation distance. A specific treatment protocol has not been established in the literature; the main principles that can be applied in patients with normal intelligence include training programs to prevent self-mutilation and accidental injuries and an early diagnosis and treatment of the infections.

  14. Non-truncating LIFR mutation: causal for prominent congenital pain insensitivity phenotype with progressive vertebral destruction?

    PubMed

    Elsaid, M F; Chalhoub, N; Kamel, H; Ehlayel, M; Ibrahim, N; Elsaid, A; Kumar, P; Khalak, H; Ilyin, V A; Suhre, K; Abdel Aleem, A

    2016-02-01

    We present a Qatari family with two children who displayed a characteristic phenotype of congenital marked pain insensitivity with hypohidrosis and progressive aseptic destruction of joints and vertebrae resembling that of hereditary sensory and autonomic neuropathies (HSANs). The patients, aged 10 and 14, remained of uncertain genetic diagnosis until whole genome sequencing was pursued. Genome sequencing identified a novel homozygous C65S mutation in the LIFR gene that is predicted to markedly destabilize and alter the structure of a particular domain and consequently to affect the functionality of the whole multi-domain LIFR protein. The C65S mutant LIFR showed altered glycosylation and an elevated expression level that might be attributed to a slow turnover of the mutant form. LIFR mutations have been reported in Stüve-Wiedemann syndrome (SWS), a severe autosomal recessive skeletal dysplasia often resulting in early death. Our patients share some clinical features of rare cases of SWS long-term survivors; however, they also phenocopy HSAN due to the marked pain insensitivity phenotype and progressive bone destruction. Screening for LIFR mutations might be warranted in genetically unresolved HSAN phenotypes.

  15. Broadband Tunability of Polarization-Insensitive Absorber Based on Frequency Selective Surface

    PubMed Central

    Wang, Han; Kong, Peng; Cheng, Wentao; Bao, Wenzong; Yu, Xiaowei; Miao, Ling; Jiang, Jianjun

    2016-01-01

    An innovative tunable and polarization-insensitive 1.6–8 GHz frequency selective surface (FSS) absorber was investigated in this study. The proposed FSS, which is in 4-axial symmetrical form, includes a novel array of PIN diodes with biasing lines including inductors. A gradually reduced equivalent resistor of PIN diodes can be achieved with increasing DC voltage, which characterizes tunable, multi-resonance absorption peaks. Via this simplified design, small value resistor and equivalent capacitance of the gap between patterns can improve the absorber’s performance in low frequencies; an active tunable absorber can be realized in a broad frequency range by employing adjustable devices. Changing the working state of the PIN diode allows the user to obtain strong absorption within the desired frequency. We analyzed the performance of the proposed absorber and found that it indeed shows very favorable absorption performance in low frequency (−10 dB in 1.6−4.3 GHz) and wideband (−8 dB in 4.3−5.4 GHz and −10 dB in 5.4−8.0 GHz) conditions. Calculation and simulation results also illustrated that the absorber is entirely polarization-insensitive. PMID:26983804

  16. Sorghum DW1 positively regulates brassinosteroid signaling by inhibiting the nuclear localization of BRASSINOSTEROID INSENSITIVE 2.

    PubMed

    Hirano, Ko; Kawamura, Mayuko; Araki-Nakamura, Satoko; Fujimoto, Haruka; Ohmae-Shinohara, Kozue; Yamaguchi, Miki; Fujii, Akihiro; Sasaki, Hiroaki; Kasuga, Shigemitsu; Sazuka, Takashi

    2017-12-01

    Semi-dwarf traits have been widely introgressed into cereal crops to improve lodging resistance. In sorghum (Sorghum bicolor L. Moench), four major unlinked dwarfing genes, Dw1-Dw4, have been introduced to reduce plant height, and among them, Dw3 and Dw1 have been cloned. Dw3 encodes a gene involved in auxin transport, whereas, Dw1 was recently isolated and identified as a gene encoding a protein of unknown function. In this study, we show that DW1 is a novel component of brassinosteroid (BR) signaling. Sorghum possessing the mutated allele of Dw1 (dw1), showed similar phenotypes to rice BR-deficient mutants, such as reduced lamina joint bending, attenuated skotomorphogenesis, and insensitivity against feedback regulation of BR-related genes. Furthermore, DW1 interacted with a negative regulator of BR signaling, BRASSINOSTEROID INSENSITIVE 2 (BIN2), and inhibited its nuclear localization, indicating that DW1 positively regulates BR signaling by inhibiting the function of BIN2. In contrast to rice and wheat breeding which used gibberellin (GA) deficiency to reduce plant height, sorghum breeding modified auxin and BR signaling. This difference may result from GA deficiency in rice and wheat does not cause deleterious side effects on plant morphology, whereas in sorghum it leads to abnormal culm bending.

  17. RNASeq-derived transcriptome comparisons reveal neuromodulatory deficiency in the CO2 insensitive brown Norway rat.

    PubMed

    Puissant, Madeleine M; Echert, Ashley E; Yang, Chun; Mouradian, Gary C; Novotny, Tyler; Liu, Pengyuan; Liang, Mingyu; Hodges, Matthew R

    2014-11-10

    Raphé-derived serotonin (5-HT) and thyrotropin-releasing hormone (TRH) play important roles in fundamental, homeostatic control systems such as breathing and specifically the ventilatory CO2 chemoreflex. Brown Norway (BN) rats exhibit an inherent and severe ventilatory insensitivity to hypercapnia but also exhibit relatively normal ventilation at rest and during other conditions, similar to multiple genetic models of 5-HT system dysfunction in mice. Herein, we tested the hypothesis that the ventilatory insensitivity to hypercapnia in BN rats is due to altered raphé gene expression and the consequent deficiencies in raphé-derived neuromodulators such as TRH. Medullary raphé transcriptome comparisons revealed lower expression of multiple 5-HT neuron-specific genes in BN compared to control Dahl salt-sensitive rats, predictive of reduced central nervous system monoamines by bioinformatics analyses and confirmed by high-performance liquid chromatography measurements. In particular, raphé Trh mRNA and peptide levels were significantly reduced in BN rats, and injections of the stable TRH analogue Taltirelin (TAL) stimulated breathing dose-dependently, with greater effects in BN versus control Sprague-Dawley rats. Importantly, TAL also effectively normalized the ventilatory CO2 chemoreflex in BN rats, but TAL did not affect CO2 sensitivity in control Sprague-Dawley rats. These data establish a molecular basis of the neuromodulatory deficiency in BN rats, and further suggest an important functional role for TRH signalling in the mammalian CO2 chemoreflex.

  18. RNASeq-derived transcriptome comparisons reveal neuromodulatory deficiency in the CO2 insensitive brown Norway rat.

    PubMed

    Puissant, Madeleine M; Echert, Ashley E; Yang, Chun; Mouradian, Gary C; Novotny, Tyler; Liu, Pengyuan; Liang, Mingyu; Hodges, Matthew R

    2015-01-15

    Raphé-derived serotonin (5-HT) and thyrotropin-releasing hormone (TRH) play important roles in fundamental, homeostatic control systems such as breathing and specifically the ventilatory CO2 chemoreflex. Brown Norway (BN) rats exhibit an inherent and severe ventilatory insensitivity to hypercapnia but also exhibit relatively normal ventilation at rest and during other conditions, similar to multiple genetic models of 5-HT system dysfunction in mice. Herein, we tested the hypothesis that the ventilatory insensitivity to hypercapnia in BN rats is due to altered raphé gene expression and the consequent deficiencies in raphé-derived neuromodulators such as TRH. Medullary raphé transcriptome comparisons revealed lower expression of multiple 5-HT neuron-specific genes in BN compared to control Dahl salt-sensitive rats, predictive of reduced central nervous system monoamines by bioinformatics analyses and confirmed by high-performance liquid chromatography measurements. In particular, raphé Trh mRNA and peptide levels were significantly reduced in BN rats, and injections of the stable TRH analogue Taltirelin (TAL) stimulated breathing dose-dependently, with greater effects in BN versus control Sprague-Dawley rats. Importantly, TAL also effectively normalized the ventilatory CO2 chemoreflex in BN rats, but TAL did not affect CO2 sensitivity in control Sprague-Dawley rats. These data establish a molecular basis of the neuromodulatory deficiency in BN rats, and further suggest an important functional role for TRH signalling in the mammalian CO2 chemoreflex.

  19. Computational Study of 3-D Hot-Spot Initiation in Shocked Insensitive High-Explosive

    NASA Astrophysics Data System (ADS)

    Najjar, F. M.; Howard, W. M.; Fried, L. E.

    2011-06-01

    High explosive shock sensitivity is controlled by a combination of mechanical response, thermal properties, and chemical properties. The interplay of these physical phenomena in realistic condensed energetic materials is currently lacking. A multiscale computational framework is developed investigating hot spot (void) ignition in a single crystal of an insensitive HE, TATB. Atomistic MD simulations are performed to provide the key chemical reactions and these reaction rates are used in 3-D multiphysics simulations. The multiphysics code, ALE3D, is linked to the chemistry software, Cheetah, and a three-way coupled approach is pursued including hydrodynamics, thermal and chemical analyses. A single spherical air bubble is embedded in the insensitive HE and its collapse due to shock initiation is evolved numerically in time; while the ignition processes due chemical reactions are studied. Our current predictions showcase several interesting features regarding hot spot dynamics including the formation of a ``secondary'' jet. Results obtained with hydro-thermo-chemical processes leading to ignition growth will be discussed for various pore sizes and different shock pressures. LLNL-ABS-471438. This work performed under the auspices of the U.S. Department of Energy by LLNL under Contract DE-AC52-07NA27344.

  20. Mutational analysis of the androgen receptor gene in two Chinese families with complete androgen insensitivity syndrome.

    PubMed

    Wang, Song; Xu, Haikun; An, Wei; Zhu, Dechun; Li, Dejun

    2016-06-01

    Androgens are essential for normal male sex differentiation and are responsible for the normal development of male secondary sexual characteristics at puberty. The physiological effects of androgens are mediated by the androgen receptor (AR). Mutations in the AR gene are the most common cause of androgen insensitivity syndrome. The present study undertook a genetic analysis of the AR gene in two unrelated families affected by complete androgen insensitivity syndrome (CAIS) in China. In family 1, a previously reported nonsense mutation (G-to-A; p.W751X) was identified in exon 5 of the AR gene. In addition, a novel missense mutation was detected in exon 6 of the AR gene from family 2; this mutation resulted in a predicted amino acid change from phenylalanine to serine at codon 804 (T-to-C; p.F804S) in the ligand-binding domain (LBD) of AR. Computer simulation of the structural changes generated by the p.F804S substitution revealed marked conformational alterations in the hydrophobic core responsible for the stability and function of the AR-LBD. In conclusion, the present study identified two mutations from two unrelated Chinese families affected by CAIS. The novel mutation (p.F804S) may provide insights into the molecular mechanism underlying CAIS. Furthermore, it expands on the number of mutational hot spots in the international AR mutation database, which may be useful in the future for prenatal diagnosis and genetic counseling.

  1. Insensitive high-energy energetic structural material of tungsten-polytetrafluoroethylene-aluminum composites

    SciTech Connect

    Wang, Liu; Liu, Jinxu Zhang, Xinbo; Li, Shukui

    2015-11-15

    Energetic structural material is a kind of materials that are inert under normal conditions but could produce exothermic chemical reaction when subjected to impact. This report shows a kind of energetic structural material of tungsten (W)-polytetrafluoroethylene (PTFE)-aluminum (Al) with density of 4.12 g/cm{sup 3}, excellent ductility and dynamic compressive strength of 96 MPa. Moreover, 50W-35PTFE-15Al (wt%) can exhibit a high reaction energy value of more than 2 times of TNT per unit mass and 5 times of TNT per unit volume, respectively, but with excellent insensitivity compared with traditional explosives. Under thermal conditions, the W-PTFE-Al composite can keep stable at 773 K. Under impact loading, when the strain rate up to ∼4820 s{sup −1} coupled with the absorbed energy per unit volume of 120 J/cm{sup 3}, deflagration occurs and combustion lasts for 500 μs. During impact compressive deformation, the PTFE matrix is elongated into nano-fibers, thus significantly increases the reaction activity of W-PTFE-Al composites. The nano-fiber structure is necessary for the reaction of W-PTFE-Al composites. The formation of PTFE nano-fibers must undergo severe plastic deformation, and therefore the W-PTFE-Al composites exhibit excellent insensitivity and safety. Furthermore, the reaction mechanisms of W-PTFE-Al composites in argon and in air are revealed.

  2. Growth hormone insensitivity syndrome (Laron syndrome): main characteristics and effects of IGF1 treatment.

    PubMed

    Carel, J C; Chaussain, J L; Chatelain, P; Savage, M O

    1996-07-01

    Growth hormone (GH) insensitivity is a pathological state characterized by a disturbance of the physiological relationship between GH secretion, synthesis of insulin-like growth-factor I (IGF-1) and the biological actions of GH. Laron syndrome, the prototype for GH insensitivity, is most often due to GH receptor deficiency. However, this syndrome is heterogeneous in terms of growth characteristics, bio-chemical features and, most importantly, genetic defects. Recent data have indicated that partial GH receptor deficiency could be involved in children with apparently idiopathic short stature. Laron syndrome, because of extreme growth deficiency and a lack of alternative treatment, was the first clinical situation in which recombinant human IGF-1 was used. IGF-1 accelerates growth rate in most patients, induces subtle modifications of the craniofacies and decreases fat mass. However, it is still too early to evaluate the long-term effects of IGF-1 on final height. Tolerance to the drug has been excellent in all reported trials. The major (but rare) side effects are transient intracranial hypertension and hypokalemia. Generalization of data obtained in Laron syndrome to other clinical situations should take account of the profound alterations in IGF-1 pharmacokinetics resulting from a deficiency in IGF-binding proteins.

  3. Distinct mechanosensitive properties of capsaicin-sensitive and -insensitive sensory neurons.

    PubMed

    Drew, Liam J; Wood, John N; Cesare, Paolo

    2002-06-15

    Mechanical stimulation of the somata of cultured neonatal rat dorsal root ganglia (DRG) neurons evoked inward cationic currents that displayed distinct properties between different subsets of cells. The presumptive nociceptor population, defined by capsaicin sensitivity, showed higher thresholds for the induction of an inward current and lower peak currents than other mechanosensitive neurons. A subset of capsaicin-sensitive IB4-positive sensory neurons was refractory to mechanical stimulation. All mechanically activated currents were blocked by gadolinium (IC50 approximately 8 microm) and ruthenium red (IC50 approximately 3 microm). Disruption of the actin cytoskeleton by acute application of 10 microm cytochalasin B inhibited currents much more effectively in capsaicin-insensitive (61%) than capsaicin-sensitive neurons (20%). Extracellular calcium also attenuated mechanosensitive currents and to a greater degree in capsaicin-insensitive neurons than capsaicin-sensitive neurons. These data demonstrate that the somata of different types of cultured sensory neurons have distinct mechanosensitive phenotypes that retain properties associated with nerve terminal mechanosensors in vivo.

  4. A Robust, Gravity-Insensitive, High-Temperature Condenser for Water Recovery

    NASA Technical Reports Server (NTRS)

    Chen, Weibo; Conboy, Thomas; Ewert, Michael

    2016-01-01

    Regenerative life support systems are vital for NASA's future long-duration human space exploration missions. A Heat Melt Compactor (HMC) system is being developed by NASA to dry and compress trash generated during space missions. The resulting water vapor is recovered and separated from the process gas flow by a gravity-insensitive condenser. Creare is developing a high-temperature condenser for this application. The entire condenser is constructed from metals that have excellent resistance to chemical attack from contaminants and is suitable for high-temperature operation. The metal construction and design configuration also offer greatest flexibility for potential coating and regeneration processes to reduce biofilm growth and thus enhancing the reliability of the condenser. The proposed condenser builds on the gravity-insensitive phase separator technology Creare developed for aircraft and spacecraft applications. This paper will first discuss the design requirements for the condenser in an HMC system that will be demonstrated on the International Space Station (ISS). Then, it will present the overall design of the condenser and the preliminary thermal test results of a subscale condenser. Finally, this paper will discuss the predicted performance of the full-size condenser and the development plan to mature the technology and enhance its long-term reliability for a flight system.

  5. Cationic nanoparticles with quaternary ammonium-functionalized PLGA-PEG-based copolymers for potent gene transfection

    NASA Astrophysics Data System (ADS)

    Wang, Yan-Hsung; Fu, Yin-Chih; Chiu, Hui-Chi; Wang, Chau-Zen; Lo, Shao-Ping; Ho, Mei-Ling; Liu, Po-Len; Wang, Chih-Kuang

    2013-11-01

    The objective of the present work was to develop new cationic nanoparticles (cNPs) with amphiphilic cationic copolymers for the delivery of plasmid DNA (pDNA). Cationic copolymers were built on the synthesis of quaternary ammonium salt compounds from diethylenetriamine (DETA) to include the positively charged head group and amphiphilic multi-grafts. PLGA- phe-PEG- qDETA (PPD), phe-PEG- qDETA-PLGA (PDP), and PLGA- phe-PEG- qDETA-PLGA (PPDP) cationic copolymers were created by this moiety of DETA quaternary ammonium, heterobifunctional polyethylene glycol (COOH-PEG-NH2), phenylalanine ( phe), and poly(lactic- co-glycolic acid) (PLGA). These new cNPs were prepared by the water miscible solvent displacement method. They exhibit good pDNA binding ability, as shown in a retardation assay that occurred at a particle size of 217 nm. The zeta potential was approximately +21 mV when the cNP concentration was 25 mg/ml. The new cNPs also have a better buffering capacity than PLGA NPs. However, the pDNA binding ability was demonstrated starting at a weight ratio of approximately 6.25 cNPs/pDNA. Gene transfection results showed that these cNPs had transfection effects similar to those of Lipofectamine 2000 in 293T cells. Furthermore, cNPs can also transfect human adipose-derived stem cells. The results indicate that the newly developed cNP is a promising candidate for a novel gene delivery vehicle.

  6. One pyrimidine dimer inactivates expression of a transfected gene in xeroderma pigmentosum cells

    SciTech Connect

    Protic-Sabljic, M.; Kraemer, K.H.

    1985-10-01

    The authors have developed a host cell reactivation assay of DNA repair utilizing UV-treated plasmid vectors. The assay primarily reflects cellular repair of transcriptional activity of damaged DNA measured indirectly as enzyme activity of the transfected genes. They studied three plasmids (pSV2cat, 5020 base pairs; pSV2catSVgpt, 7268 base pairs; and pRSVcat, 5027 base pairs) with different sizes and promoters carrying the bacterial cat gene (CAT, chloramphenicol acetyltransferase) in a construction that permits cat expression in human cells. All human simian virus 40-transformed cells studied expressed high levels of the transfected cat gene. UV treatment of the plasmids prior to transfection resulted in differential decrease in CAT activity in different cell lines. With pSV2catSVgpt, UV inactivation of CAT expression was greater in the xeroderma pigmentosum group A and D lines than in the other human cell lines tested. The D0 of the CAT inactivation curve was 50 J X m-2 for pSV2cat and for pRSVcat in the xeroderma pigmentosum group A cells. The similarity of the D0 data in the xeroderma pigmentosum group A cells for three plasmids of different size and promoters implies they all have similar UV-inactivation target size. UV-induced pyrimidine dimer formation in the plasmids was quantified by assay of the number of UV-induced T4 endonuclease V-sensitive sites. In the most sensitive xeroderma pigmentosum cells, with all three plasmids, one UV-induced pyrimidine dimer inactivates a target of about 2 kilobases, close to the size of the putative CAT mRNA.

  7. Design of a microchannel-nanochannel-microchannel array based nanoelectroporation system for precise gene transfection.

    PubMed

    Gao, Keliang; Li, Lei; He, Lingna; Hinkle, Kevin; Wu, Yun; Ma, Junyu; Chang, Lingqian; Zhao, Xi; Perez, Daniel Gallego; Eckardt, Sigrid; McLaughlin, John; Liu, Boyu; Farson, Dave F; Lee, L James

    2014-03-12

    A micro/nano-fabrication process of a nanochannel electroporation (NEP) array and its application for precise delivery of plasmid for non-viral gene transfection is described. A dip-combing device is optimized to produce DNA nanowires across a microridge array patterned on the polydimethylsiloxane (PDMS) surface with a yield up to 95%. Molecular imprinting based on a low viscosity resin, 1,4-butanediol diacrylate (1,4-BDDA), adopted to convert the microridge-nanowire-microridge array into a microchannel-nanochannel-microchannel (MNM) array. Secondary machining by femtosecond laser ablation is applied to shorten one side of microchannels from 3000 to 50 μm to facilitate cell loading and unloading. The biochip is then sealed in a packaging case with reservoirs and microfluidic channels to enable cell and plasmid loading, and to protect the biochip from leakage and contamination. The package case can be opened for cell unloading after NEP to allow for the follow-up cell culture and analysis. These NEP cases can be placed in a spinning disc and up to ten discs can be piled together for spinning. The resulting centrifugal force can simultaneously manipulate hundreds or thousands of cells into microchannels of NEP arrays within 3 minutes. To demonstrate its application, a 13 kbp OSKM plasmid of induced pluripotent stem cell (iPSC) is injected into mouse embryonic fibroblasts cells (MEFCs). Fluorescence detection of transfected cells within the NEP biochips shows that the delivered dosage is high and much more uniform compared with similar gene transfection carried out by the conventional bulk electroporation (BEP) method.

  8. Boron nitride nanotubes chemically functionalized with glycol chitosan for gene transfection in eukaryotic cell lines.

    PubMed

    Ferreira, T H; Hollanda, L M; Lancellotti, M; de Sousa, E M B

    2015-06-01

    Nanostructured materials have been widely studied concerning their potential biomedical applications, primarily to selectively carry specific drugs or molecules within a tissue or organ. In this context, boron nitride nanotubes (BNNTs) have generated considerable interest in the scientific community because of their unique properties, presenting good chemical inertness and high thermal stability. Among the many applications proposed for BNNTs in the biomedical field in recent years, the most important include their use as biosensors, nanovectors for the delivery of proteins, drugs, and genes. In the present study, BNNTs were synthesized, purified, and functionalized with glycol chitosan through a chemical process, yielding the BNNT-GC. The size of BNNT-GC was reduced using an ultrasound probe. Two samples with different sizes were selected for in vitro assays. The nanostructures were characterized by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), thermal analysis (TGA), and dynamic light scattering (DLS). The in vitro assays MTT and neutral red (NR) were performed with NIH-3T3 and A549 cell lines and demonstrated that this material is not cytotoxic. Furthermore, the BNNT-GC was applied in gene transfection of plasmid pIRES containing a gene region that express a green fluorescent protein (GFP) in NIH-3T3 and A549 cell lines. The gene transfection was characterized by fluorescent protein produced in the cells and pictured by fluorescent microscopy. Our results suggest that BNNT-GC has moderate stability and presents great potential as a gene carrier agent in nonviral-based therapy, with low cytotoxicity and good transfection efficiency.

  9. Modulation of pyridinium cationic lipid-DNA complex properties by pyridinium gemini surfactants and its impact on lipoplex transfection properties.

    PubMed

    Sharma, Vishnu Dutt; Lees, Julia; Hoffman, Nicholas E; Brailoiu, Eugen; Madesh, Muniswamy; Wunder, Stephanie L; Ilies, Marc A

    2014-02-03

    The study presents the effects of blending a cationic gemini surfactant into cationic lipid bilayers and its impact on the plasmid DNA compaction and delivery process. Using nanoDSC, dynamic light scattering, zeta potential, and electrophoretic mobility measurements, together with transfection (2D- and 3D-) and viability assays, we identified the main physicochemical parameters of the lipid bilayers, liposomes, and lipoplexes that are affected by the gemini surfactant addition. We also correlated the cationic bilayer composition with the dynamics of the DNA compaction process and with transfection efficiency, cytotoxicity, and the internalization mechanism of the resultant nucleic acid complexes. We found that the blending of gemini surfactant into the cationic bilayers fluidized the supramolecular assemblies, reduced the amount of positive charge required to fully compact the plasmid DNA and, in certain cases, changed the internalization mechanism of the lipoplexes. The transfection efficiency of select ternary lipoplexes derived from cationic gemini surfactants and lipids was several times superior to the transfection efficiency of corresponding binary lipoplexes, also surpassing standard transfection systems. The overall impact of gemini surfactants into the formation and dynamic of cationic bilayers was found to depend heavily on the presence of colipids, their nature, and amount present in lipoplexes. The study confirmed the possibility of combining the specific properties of pyridinium gemini surfactants and cationic lipids synergistically to obtain efficient synthetic transfection systems with negligible cytotoxicity useful for therapeutic gene delivery.

  10. In vivo gene transfection with heat shock protein 70 enhances myocardial tolerance to ischemia-reperfusion injury in rat.

    PubMed Central

    Suzuki, K; Sawa, Y; Kaneda, Y; Ichikawa, H; Shirakura, R; Matsuda, H

    1997-01-01

    Heat shock protein 70 (HSP70) has been reported to be involved in the myocardial self-preservation system. To obtain the evidence that HSP70 plays a direct role in the protection from myocardial ischemia-reperfusion injury, rat hearts were transfected with human HSP70 gene by intracoronary infusion of hemagglutinating virus of Japan (HVJ)-liposome containing human HSP70 gene. The control hearts were infused with HVJ-liposome without the HSP70 gene. The hearts from whole-body heat-stressed or nontreated rats were also examined. Western blot and immunohistochemical analysis showed that apparent overexpression of HSP70 occurred in the gene transfected hearts and that gene transfection might be more effective for HSP70 induction than heat stress. In Langendorff perfusion, better functional recovery as well as less creatine phosphokinase leakage after ischemia were obtained in the gene transfected hearts with HSP70 than in the control or nontreated hearts. Furthermore, the gene transfected hearts showed better functional recovery than the heat-stressed hearts. These results indicated that overexpressed HSP70 plays a protective role in myocardial injury, suggesting the possibility that gene transfection with HSP70 may become a novel method for myocardial protection through enforcing the self-preservation systems. PMID:9120008

  11. Size matters for in vitro gene delivery: investigating the relationships among complexation protocol, transfection medium, size and sedimentation

    PubMed Central

    Pezzoli, Daniele; Giupponi, Elisa; Mantovani, Diego; Candiani, Gabriele

    2017-01-01

    Although branched and linear polyethylenimines (bPEIs and lPEIs) are gold standard transfectants, a systematic analysis of the effects of the preparation protocol of polyplexes and the composition of the transfection medium on their physicochemical behaviour and effectiveness in vitro have been much neglected, undermining in some way the identification of precise structure-function relationships. This work aimed to address these issues. bPEI/DNA and lPEI/DNA, prepared using two different modes of addition of reagents, gave rise to polyplexes with exactly the same chemical composition but differing in dimensions. Upon dilution in serum-free medium, the size of any kind of polyplex promptly rose over time while remained invariably stable in complete DMEM. Of note, the bigger the dimension of polyplexes (in the nano- to micrometer range), the greater their efficiency in vitro. Besides, centrifugal sedimentation of polyplexes displaying different dimensions to speed up and enhance their settling onto cells boosted transfection efficiencies. Conversely, transgene expression was significantly blunted in cells held upside-down and transfected, definitively pointing out the impact of gravitational sedimentation of polyplexes on their transfection efficiency. Overall, much more attention must be paid to the actual polyplex size that relies on the complexation conditions and the transfection medium. PMID:28272487

  12. A convenient method of preparing gene vector for real time monitoring transfection process based on the quantum dots

    SciTech Connect

    Zhang, Hai-Li; Zhang, Ming-Zhen; Li, Xiang-Yong; Wan, Min; Li, Yong-Qiang; Zhang, Rong-Ying; Zhao, Yuan-Di

    2012-11-15

    Highlights: ► An easy and direct way to prepare QDs–DNA complexes was developed. ► Surface charge of QDs was tuned with different ratio of amino and glycolate. ► Transfection efficiency was dependent on the surface zeta potentials of QDs. ► Cellular toxicity of this gene vectors is much lower than commercial liposome. ► Whole intracellular behavior of QDs–DNA complexes can be monitored in real time. -- Abstract: Nanoparticle carrier has been developed by combining water-soluble quantum dots and plasmid DNA expressed enhanced green fluorescent protein (EGFP) in a convenient and direct way. First the QDs with different surface charges were obtained by coating with amino and carboxyl terminals at different ratios. Then plasmid DNA was conjugated to QDs via electrostatic interaction. The resultant QDs–DNA complexes showed enhanced resistance to DNase I digestion. The following transfection experiments demonstrated that the transfection efficiency was dependent on the surface charges on QDs. The real time imaging of the transfection process showed that the nanoparticles experienced binding, penetrating the cell membrane and entering cytoplasm in the first 6 h of transfection. The green fluorescence of EGFP began to appear after 18 h transfection and plasmid DNA was fully expressed in the following 6 h. This new QDs–DNA platform showed great potential as new gene delivery carrier.

  13. Inclusion of high molecular weight dextran in calcium phosphate-mediated transfection significantly improves gene transfer efficiency.

    PubMed

    Wu, C; Lu, Y

    2007-05-15

    Calcium phosphate-based mammalian cell transfection is a widely used gene transfer technology. To facilitate the efficiency of this gene transfer method, several polysaccharide compounds were tested and evaluated for their effectiveness in enhancing DNA transfection. Using a HIV-1-derived lentivirus vector plasmid as a gene transfer indicator, we demonstrated that the addition of high molecular weight dextran-500 at 0.6-1.2% in the 2x Hepes buffered saline (HBS) increased transfection efficiency by over 50% (as reflected by the number of GFP-positive cells) and increased the titer of resulting lentivirus vector particles even more (up to 4-fold). This enhancement of transfection efficiency was further increased when higher molecular weight dextran formulations were used in place of dextran-500, and also when dextran was used in combination with polybrene, another polycationic chemical compound. Examination of transfected cells showed that dextran had no apparent adverse effect on cell viability and growth. Our data represent the first report showing that dextran can be used to enhance calcium phosphate-mediated gene transfer; this may be useful in applications for the generation of high-titer virus vector stocks using transient transfection technology.

  14. Transfection of the IHH gene into rabbit BMSCs in a simulated microgravity environment promotes chondrogenic differentiation and inhibits cartilage aging

    PubMed Central

    Liu, Peng-Cheng; Liu, Kuan; Liu, Jun-Feng; Xia, Kuo; Chen, Li-Yang; Wu, Xing

    2016-01-01

    The effect of overexpressing the Indian hedgehog (IHH) gene on the chondrogenic differentiation of rabbit bone marrow-derived mesenchymal stem cells (BMSCs) was investigated in a simulated microgravity environment. An adenovirus plasmid encoding the rabbit IHH gene was constructed in vitro and transfected into rabbit BMSCs. Two large groups were used: conventional cell culture and induction model group and simulated microgravity environment group. Each large group was further divided into blank control group, GFP transfection group, and IHH transfection group. During differentiation induction, the expression levels of cartilage-related and cartilage hypertrophy-related genes and proteins in each group were determined. In the conventional model, the IHH transfection group expressed high levels of cartilage-related factors (Coll2 and ANCN) at the early stage of differentiation induction and expressed high levels of cartilage hypertrophy-related factors (Coll10, annexin 5, and ALP) at the late stage. Under the simulated microgravity environment, the IHH transfection group expressed high levels of cartilage-related factors and low levels of cartilage hypertrophy-related factors at all stages of differentiation induction. Under the simulated microgravity environment, transfection of the IHH gene into BMSCs effectively promoted the generation of cartilage and inhibited cartilage aging and osteogenesis. Therefore, this technique is suitable for cartilage tissue engineering. PMID:27802423

  15. Size matters for in vitro gene delivery: investigating the relationships among complexation protocol, transfection medium, size and sedimentation.

    PubMed

    Pezzoli, Daniele; Giupponi, Elisa; Mantovani, Diego; Candiani, Gabriele

    2017-03-08

    Although branched and linear polyethylenimines (bPEIs and lPEIs) are gold standard transfectants, a systematic analysis of the effects of the preparation protocol of polyplexes and the composition of the transfection medium on their physicochemical behaviour and effectiveness in vitro have been much neglected, undermining in some way the identification of precise structure-function relationships. This work aimed to address these issues. bPEI/DNA and lPEI/DNA, prepared using two different modes of addition of reagents, gave rise to polyplexes with exactly the same chemical composition but differing in dimensions. Upon dilution in serum-free medium, the size of any kind of polyplex promptly rose over time while remained invariably stable in complete DMEM. Of note, the bigger the dimension of polyplexes (in the nano- to micrometer range), the greater their efficiency in vitro. Besides, centrifugal sedimentation of polyplexes displaying different dimensions to speed up and enhance their settling onto cells boosted transfection efficiencies. Conversely, transgene expression was significantly blunted in cells held upside-down and transfected, definitively pointing out the impact of gravitational sedimentation of polyplexes on their transfection efficiency. Overall, much more attention must be paid to the actual polyplex size that relies on the complexation conditions and the transfection medium.

  16. Optimization of input parameters of acoustic-transfection for the intracellular delivery of macromolecules using FRET-based biosensors

    NASA Astrophysics Data System (ADS)

    Yoon, Sangpil; Wang, Yingxiao; Shung, K. K.

    2016-03-01

    Acoustic-transfection technique has been developed for the first time. We have developed acoustic-transfection by integrating a high frequency ultrasonic transducer and a fluorescence microscope. High frequency ultrasound with the center frequency over 150 MHz can focus acoustic sound field into a confined area with the diameter of 10 μm or less. This focusing capability was used to perturb lipid bilayer of cell membrane to induce intracellular delivery of macromolecules. Single cell level imaging was performed to investigate the behavior of a targeted single-cell after acoustic-transfection. FRET-based Ca2+ biosensor was used to monitor intracellular concentration of Ca2+ after acoustic-transfection and the fluorescence intensity of propidium iodide (PI) was used to observe influx of PI molecules. We changed peak-to-peak voltages and pulse duration to optimize the input parameters of an acoustic pulse. Input parameters that can induce strong perturbations on cell membrane were found and size dependent intracellular delivery of macromolecules was explored. To increase the amount of delivered molecules by acoustic-transfection, we applied several acoustic pulses and the intensity of PI fluorescence increased step wise. Finally, optimized input parameters of acoustic-transfection system were used to deliver pMax-E2F1 plasmid and GFP expression 24 hours after the intracellular delivery was confirmed using HeLa cells.

  17. High‐throughput screening of clinically approved drugs that prime polyethylenimine transfection reveals modulation of mitochondria dysfunction response improves gene transfer efficiencies

    PubMed Central

    Nguyen, Albert; Beyersdorf, Jared; Riethoven, Jean‐Jack

    2016-01-01

    Abstract Nonviral gene delivery methods are advantageous over viral vectors in terms of safety, cost, and flexibility in design and application, but suffer from lower gene transfer efficiency. In addition to modifications to nucleic acid design and nonviral carriers, new tools are sought to enhance transfection. Priming is the pharmacological modulation of transfection efficiency and transgene expression, and has demonstrated transfection increase in several compounds, for example, chloroquine and glucocorticoids. To develop a library of transfection priming compounds, a high‐throughput screen was performed of the NIH Clinical Collection (NCC) to identify clinical compounds that prime polyethylenimine (PEI) transfection. HEK293T cells were treated with priming compounds, then transfected with enhanced green fluorescent protein (EGFP)‐encoding plasmid by PEI. After 48‐hr culture, primed and transfected cells were assayed for transfection, cell proliferation, and cell viability by fluorescence measurement of EGFP reporter, Hoechst 33342 nuclei stain, and resazurin metabolic assay. From the microscope image analysis and microplate measurements, transfection fold‐changes were determined, and compounds resulting in statistically significant transfection fold‐change were identified. NCC compounds were clustered using PubChem fingerprint similarity by Tanimoto coefficients in ChemmineTools. Fold‐changes for each compound were linked to drug clusters, from which drug classes that prime transfection were identified. Among the identified drugs classes that primed transfection increases were antioxidants, GABAA receptor modulators, and glucocorticoids. Resveratrol and piceid, stilbenoid antioxidants found in grapes, and zolpidem, a GABAA modulator, increased transfection nearly three‐fold. Literature indicate interaction of the identified transfection priming drug clusters with mitochondria, which may modulate mitochondrial dysfunction known to be associated

  18. Imaging of transfection and intracellular release of intact, non-labeled DNA using fluorescent nanodiamonds

    NASA Astrophysics Data System (ADS)

    Petrakova, V.; Benson, V.; Buncek, M.; Fiserova, A.; Ledvina, M.; Stursa, J.; Cigler, P.; Nesladek, M.

    2016-06-01

    Efficient delivery of stabilized nucleic acids (NAs) into cells and release of the NA payload are crucial points in the transfection process. Here we report on the fabrication of a nanoscopic cellular delivery carrier that is additionally combined with a label-free intracellular sensor device, based on biocompatible fluorescent nanodiamond particles. The sensing function is engineered into nanodiamonds by using nitrogen-vacancy color centers, providing stable non-blinking luminescence. The device is used for monitoring NA transfection and the payload release in cells. The unpacking of NAs from a poly(ethyleneimine)-terminated nanodiamond surface is monitored using the color shift of nitrogen-vacancy centers in the diamond, which serve as a nanoscopic electric charge sensor. The proposed device innovates the strategies for NA imaging and delivery, by providing detection of the intracellular release of non-labeled NAs without affecting cellular processing of the NAs. Our system highlights the potential of nanodiamonds to act not merely as labels but also as non-toxic and non-photobleachable fluorescent biosensors reporting complex molecular events.Efficient delivery of stabilized nucleic acids (NAs) into cells and release of the NA payload are crucial points in the transfection process. Here we report on the fabrication of a nanoscopic cellular delivery carrier that is additionally combined with a label-free intracellular sensor device, based on biocompatible fluorescent nanodiamond particles. The sensing function is engineered into nanodiamonds by using nitrogen-vacancy color centers, providing stable non-blinking luminescence. The device is used for monitoring NA transfection and the payload release in cells. The unpacking of NAs from a poly(ethyleneimine)-terminated nanodiamond surface is monitored using the color shift of nitrogen-vacancy centers in the diamond, which serve as a nanoscopic electric charge sensor. The proposed device innovates the strategies for

  19. Ultraviolet light-resistant primary transfectants of xeroderma pigmentosum cells are also DNA repair-proficient

    SciTech Connect

    Stark, M.; Naiman, T.; Canaani, D. )

    1989-08-15

    In a previous work, an immortal xeroderma pigmentosum cell line belonging to complementation group C was complemented to a UV-resistant phenotype by transfection with a human cDNA clone library. We now report that the primary transformants selected for UV-resistance also acquired normal levels of DNA repair. This was assessed both by measurement of UV-induced ({sup 3}H)thymidine incorporation and by equilibrium sedimentation analysis of repair-DNA synthesis. Therefore, the transduced DNA element which confers normal UV-resistance also corrects the excision repair defect of the xeroderma pigmentosum group C cell line.

  20. Transfected Babesia bovis Expressing a Tick GST as a Live Vector Vaccine

    PubMed Central

    Oldiges, Daiane P.; Laughery, Jacob M.; Tagliari, Nelson Junior; Leite Filho, Ronaldo Viana; Davis, William C.; da Silva Vaz, Itabajara; Termignoni, Carlos; Knowles, Donald P.; Suarez, Carlos E.

    2016-01-01

    The Rhipicephalus microplus tick is a notorious blood-feeding ectoparasite of livestock, especially cattle, responsible for massive losses in animal production. It is the main vector for transmission of pathogenic bacteria and parasites, including Babesia bovis, an intraerythrocytic apicomplexan protozoan parasite responsible for bovine Babesiosis. This study describes the development and testing of a live B. bovis vaccine expressing the protective tick antigen glutathione-S-transferase from Haemaphysalis longicornis (HlGST). The B. bovis S74-T3B parasites were electroporated with a plasmid containing the bidirectional Ef-1α (elongation factor 1 alpha) promoter of B. bovis controlling expression of two independent genes, the selectable marker GFP-BSD (green fluorescent protein–blasticidin deaminase), and HlGST fused to the MSA-1 (merozoite surface antigen 1) signal peptide from B. bovis. Electroporation followed by blasticidin selection resulted in the emergence of a mixed B. bovis transfected line (termed HlGST) in in vitro cultures, containing parasites with distinct patterns of insertion of both exogenous genes, either in or outside the Ef-1α locus. A B. bovis clonal line termed HlGST-Cln expressing intracellular GFP and HlGST in the surface of merozoites was then derived from the mixed parasite line HlGST using a fluorescent activated cell sorter. Two independent calf immunization trials were performed via intravenous inoculation of the HlGST-Cln and a previously described control consisting of an irrelevant transfected clonal line of B. bovis designated GFP-Cln. The control GFP-Cln line contains a copy of the GFP-BSD gene inserted into the Ef-1α locus of B. bovis in an identical fashion as the HIGST-Cln parasites. All animals inoculated with the HlGST-Cln and GFP-Cln transfected parasites developed mild babesiosis. Tick egg fertility and fully engorged female tick weight was reduced significantly in R. microplus feeding on HlGST-Cln-immunized calves

  1. A cost-effective approach to microporate mammalian cells with the Neon Transfection System.

    PubMed

    Brees, Chantal; Fransen, Marc

    2014-12-01

    Electroporation is one of the most efficient nonviral methods for transferring exogenous DNA into mammalian cells. However, the relatively high costs of electroporation kits and reagents temper the routine use of this fast and easy to perform technique in many laboratories. Several years ago, a new flexible and easy to operate electroporation device was launched under the name Neon Transfection System. This device uses specialized pipette tips containing gold-plated electrodes as electroporation chamber. Here we report a protocol to regenerate these expensive tips as well as some other Neon kit accessories, thereby reducing the cost of electroporation at least 10-fold.

  2. Characterisation of human thyroid epithelial cells immortalised in vitro by simian virus 40 DNA transfection.

    PubMed Central

    Lemoine, N. R.; Mayall, E. S.; Jones, T.; Sheer, D.; McDermid, S.; Kendall-Taylor, P.; Wynford-Thomas, D.

    1989-01-01

    Human primary thyroid follicular epithelial cells were transfected with a plasmid containing an origin-defective SV40 genome (SVori-) to produce several immortal cell lines. Two of the 10 cell lines analysed expressed specific features of thyroid epithelial function (iodide-trapping and thyroglobulin production). These two lines were characterised in detail and found to be growth factor-independent, capable of anchorage-independent growth at low frequency but non-tumorigenic in nude mice. These differentiated, These differentiated, partially transformed cell lines were shown to be suitable for gene transfer at high frequency using simple coprecipitation techniques. Images Figure 2 Figure 3 Figure 4 PMID:2557880

  3. Spatially optimized gene transfection by laser-induced breakdown of optically trapped nanoparticles

    NASA Astrophysics Data System (ADS)

    Arita, Yoshihiko; Torres-Mapa, Maria Leilani; Lee, Woei Ming; Čižmár, Tomáš; Campbell, Paul; Gunn-Moore, Frank J.; Dholakia, Kishan

    2011-02-01

    We demonstrate laser-induced breakdown of an optically trapped nanoparticle with a nanosecond laser pulse. Controllable cavitation within a microscope sample was achieved, generating shear stress to monolayers of live cells. This efficiently permeabilize their plasma membranes. We show that this technique is an excellent tool for plasmid-DNA transfection of cells with both reduced energy requirements and reduced cell lysis compared to previously reported approaches. Simultaneous multisite targeted nanosurgery of cells is also demonstrated using a spatial light modulator for parallelizing the technique.

  4. 3D nanochannel electroporation for high-throughput cell transfection with high uniformity and dosage control

    NASA Astrophysics Data System (ADS)

    Chang, Lingqian; Bertani, Paul; Gallego-Perez, Daniel; Yang, Zhaogang; Chen, Feng; Chiang, Chiling; Malkoc, Veysi; Kuang, Tairong; Gao, Keliang; Lee, L. James; Lu, Wu

    2015-12-01

    Of great interest to modern medicine and biomedical research is the ability to inject individual target cells with the desired genes or drug molecules. Some advances in cell electroporation allow for high throughput, high cell viability, or excellent dosage control, yet no platform is available for the combination of all three. In an effort to solve this problem, here we show a ``3D nano-channel electroporation (NEP) chip'' on a silicon platform designed to meet these three criteria. This NEP chip can simultaneously deliver the desired molecules into 40 000 cells per cm2 on the top surface of the device. Each 650 nm pore aligns to a cell and can be used to deliver extremely small biological elements to very large plasmids (>10 kbp). When compared to conventional bulk electroporation (BEP), the NEP chip shows a 20 fold improvement in dosage control and uniformity, while still maintaining high cell viability (>90%) even in cells such as cardiac cells which are characteristically difficult to transfect. This high-throughput 3D NEP system provides an innovative and medically valuable platform with uniform and reliable cellular transfection, allowing for a steady supply of healthy, engineered cells.Of great interest to modern medicine and biomedical research is the ability to inject individual target cells with the desired genes or drug molecules. Some advances in cell electroporation allow for high throughput, high cell viability, or excellent dosage control, yet no platform is available for the combination of all three. In an effort to solve this problem, here we show a ``3D nano-channel electroporation (NEP) chip'' on a silicon platform designed to meet these three criteria. This NEP chip can simultaneously deliver the desired molecules into 40 000 cells per cm2 on the top surface of the device. Each 650 nm pore aligns to a cell and can be used to deliver extremely small biological elements to very large plasmids (>10 kbp). When compared to conventional bulk

  5. Transfection using hydroxyapatite nanoparticles in the inner ear via an intact round window membrane in chinchilla

    NASA Astrophysics Data System (ADS)

    Wu, Xuewen; Ding, Dalian; Jiang, Haiyan; Xing, Xiaowei; Huang, Suping; Liu, Hong; Chen, Zhedong; Sun, Hong

    2012-01-01

    Hydroxyapatite nanoparticles (nHAT) are known to have excellent biocompatibility, and have attracted increasing attention as new candidates of non-viral vectors for gene therapy. In our previous studies, nHAT carrying a therapeutic gene and a reporter gene were successfully transfected into the spiral ganglion neurons in the inner ear of guinea pigs in vivo as well as in the cultured cell lines, although the transfection efficiencies were never higher than 30%. In this study, the surface modification of nHAT with polyethylenimine (PEI) was made (PEI-nHAT, diameter = 73.09 ± 27.32 nm) and a recombinant plasmid carrying enhanced green fluorescent protein (EGFP) gene and neurotrophin-3 (NT-3) gene was constructed as pEGFPC2-NT3. The PEI modified nHAT and the recombinant plasmid was then connected to form the nHAT-based vector-gene complex (PEI-nHAT-pEGFPC2-NT3). This complex was then placed onto the intact round window membranes of the chinchillas for inner ear transfection. Auditory brainstem response (ABR) was tested to evaluate auditory function. Green fluorescence of EGFP was observed using confocal microscopy 48 h after administering vector-gene complexes. There was no significant threshold shift in tone burst-evoked ABR at any tested frequency. Abundant, condensed green fluorescence was found in dark cells on both sides of the crista and around the macula of the utricle. Scattered EGFP signals were also detected in vestibular hair cells, some Schwann cells in the cochlear spiral ganglion region, some outer pillar cells in the organ of Corti, and a few cells in the stria vascularis. The density of green fluorescence-marked cells was obviously higher in the vestibular dark cell area than in other areas of the inner ear, suggesting that vestibular dark cells may have the ability to actively engulf the nHAT-based vector-gene complexes. Considering the high transfection efficiency in the vestibular system, PEI-nHAT may be a potential vector for gene therapy of inner

  6. Bioreducible cross-linked nanoshell enhances gene transfection of polycation/DNA polyplex in vivo.

    PubMed

    Piao, Ji-Gang; Ding, Sheng-Gang; Yang, Lu; Hong, Chun-Yan; You, Ye-Zi

    2014-08-11

    In this study, we have prepared a self-cross-linking PEG-based branched polymer, which easily forms a bioreducible nanoshell around polyplexes of cationic polymer and DNA, simply via heating the polyplex dispersions in the presence of this self-cross-linking branched polymer. This nanoshell can prevent the polyplex from dissociation and aggregation in physiological fluids without inhibiting the electrostatic interactions between the polymer and DNA. Furthermore, glutathione (GSH) can act as a stimulus to open the nanoshell after it has entered the cell. The polyplexes coated with the bioreducible nanoshell show an obvious enhancement in gene transfection in vivo compared with bare polyplexes.

  7. High-pressure crystal structures of an insensitive energetic crystal: 1,1-diamino-2,2-dinitroethene

    DOE PAGES

    Dreger, Zbigniew A.; Stash, Adam I.; Yu, Zhi -Gang; ...

    2015-12-03

    Understanding the insensitivity/stability of insensitive high explosive crystals requires detailed structural information at high pressures and high temperatures of interest. Synchrotron single crystal x-ray diffraction experiments were used to determine the high-pressure structures of 1,1-diamino-2,2-dinitroethene (FOX-7), a prototypical insensitive high explosive. The phase transition around 4.5 GPa was investigated and the structures were determined at 4.27 GPa (α’-phase) and 5.9 GPa (ε-phase). The α’-phase (monoclinic, P21/n), structurally indistinguishable from the ambient α-phase, transforms to the new ε-phase (triclinic, P1). The most notable features of the ε-phase, compared to the α’-phase, are: formation of planar layers and flattening of molecules. Densitymore » functional theory (DFT-D2) calculations complemented the experimental results. Furthermore, the results presented here are important for understanding the molecular and crystalline attributes governing the high-pressure insensitivity/stability of insensitive high explosive crystals.« less

  8. DNA methylation pattern of Photoperiod-B1 is associated with photoperiod insensitivity in wheat (Triticum aestivum).

    PubMed

    Sun, Han; Guo, Zhiai; Gao, Lifeng; Zhao, Guangyao; Zhang, Wenping; Zhou, Ronghua; Wu, Yongzhen; Wang, Haiyang; An, Hailong; Jia, Jizeng

    2014-11-01

    As one of the three key components of the 'Green Revolution', photoperiod insensitivity is vital for improved adaptation of wheat (Triticum aestivum) cultivars to a wider geographical range. Photoperiod-B1a (Ppd-B1a) is one of the major genes that confers photoperiod insensitivity in 'Green Revolution' varieties, and has made a significant contribution to wheat yield improvement. In this study, we investigated the mechanisms underlying the photoperiod insensitivity of Ppd-B1a alleles from an epigenetic perspective using a combination of bisulfite genomic sequencing, orthologous comparative analysis, association analysis, linkage analysis and gene expression analysis. Based on the study of a large collection of wheat germplasm, we report two methylation haplotypes of Ppd-B1 and demonstrate that the higher methylation haplotype (haplotype a) was associated with increased copy numbers and higher expression levels of the Ppd-B1 gene, earlier heading and photoperiod insensitivity. Furthermore, assessment of the distribution frequency of the different methylation haplotypes suggested that the methylation patterns have undergone selection during the wheat breeding process. Our study suggests that DNA methylation in the regulatory region of the Ppd-B1 alleles, which is closely related to copy number variation, plays a significant role in wheat breeding, to confer photoperiod insensitivity and better adaptation to a wider geographical range.

  9. Repeated functional convergent effects of NaV1.7 on acid insensitivity in hibernating mammals.

    PubMed

    Liu, Zhen; Wang, Wei; Zhang, Tong-Zuo; Li, Gong-Hua; He, Kai; Huang, Jing-Fei; Jiang, Xue-Long; Murphy, Robert W; Shi, Peng

    2014-02-07

    Hibernating mammals need to be insensitive to acid in order to cope with conditions of high CO2; however, the molecular basis of acid tolerance remains largely unknown. The African naked mole-rat (Heterocephalus glaber) and hibernating mammals share similar environments and physiological features. In the naked mole-rat, acid insensitivity has been shown to be conferred by the functional motif of the sodium ion channel NaV1.7. There is now an opportunity to evaluate acid insensitivity in other taxa. In this study, we tested for functional convergence of NaV1.7 in 71 species of mammals, including 22 species that hibernate. Our analyses revealed a functional convergence of amino acid sequences, which occurred at least six times independently in mammals that hibernate. Evolutionary analyses determined that the convergence results from both parallel and divergent evolution of residues in the functional motif. Our findings not only identify the functional molecules responsible for acid insensitivity in hibernating mammals, but also open new avenues to elucidate the molecular underpinnings of acid insensitivity in mammals.

  10. Repeated functional convergent effects of NaV1.7 on acid insensitivity in hibernating mammals

    PubMed Central

    Liu, Zhen; Wang, Wei; Zhang, Tong-Zuo; Li, Gong-Hua; He, Kai; Huang, Jing-Fei; Jiang, Xue-Long; Murphy, Robert W.; Shi, Peng

    2014-01-01

    Hibernating mammals need to be insensitive to acid in order to cope with conditions of high CO2; however, the molecular basis of acid tolerance remains largely unknown. The African naked mole-rat (Heterocephalus glaber) and hibernating mammals share similar environments and physiological features. In the naked mole-rat, acid insensitivity has been shown to be conferred by the functional motif of the sodium ion channel NaV1.7. There is now an opportunity to evaluate acid insensitivity in other taxa. In this study, we tested for functional convergence of NaV1.7 in 71 species of mammals, including 22 species that hibernate. Our analyses revealed a functional convergence of amino acid sequences, which occurred at least six times independently in mammals that hibernate. Evolutionary analyses determined that the convergence results from both parallel and divergent evolution of residues in the functional motif. Our findings not only identify the functional molecules responsible for acid insensitivity in hibernating mammals, but also open new avenues to elucidate the molecular underpinnings of acid insensitivity in mammals. PMID:24352952

  11. High-pressure crystal structures of an insensitive energetic crystal: 1,1-diamino-2,2-dinitroethene

    SciTech Connect

    Dreger, Zbigniew A.; Stash, Adam I.; Yu, Zhi -Gang; Chen, Yu -Sheng; Tao, Yuchuan; Gupta, Yogendra M.

    2015-12-03

    Understanding the insensitivity/stability of insensitive high explosive crystals requires detailed structural information at high pressures and high temperatures of interest. Synchrotron single crystal x-ray diffraction experiments were used to determine the high-pressure structures of 1,1-diamino-2,2-dinitroethene (FOX-7), a prototypical insensitive high explosive. The phase transition around 4.5 GPa was investigated and the structures were determined at 4.27 GPa (α’-phase) and 5.9 GPa (ε-phase). The α’-phase (monoclinic, P21/n), structurally indistinguishable from the ambient α-phase, transforms to the new ε-phase (triclinic, P1). The most notable features of the ε-phase, compared to the α’-phase, are: formation of planar layers and flattening of molecules. Density functional theory (DFT-D2) calculations complemented the experimental results. Furthermore, the results presented here are important for understanding the molecular and crystalline attributes governing the high-pressure insensitivity/stability of insensitive high explosive crystals.

  12. Role of hypoxia-inducible factor-{alpha} in hepatitis-B-virus X protein-mediated MDR1 activation

    SciTech Connect

    Han, Hyo-Kyung; Han, Chang Yeob; Cheon, Eun-Pa; Lee, Jaewon; Kang, Keon Wook . E-mail: kwkang@chosun.ac.kr

    2007-06-01

    The transition from chemotherapy-responsive cancer cells to chemotherapy-resistant cancer cells is mainly accompanied by the increased expression of multi-drug resistance 1 (MDR1). We found that hepatitis-B-virus X protein (HBx) increases the transcriptional activity and protein level of MDR1 in a hepatoma cell line, H4IIE. In addition, HBx overexpression made H4IIE cells more resistant to verapamil-uptake. HBx stabilized hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) and induced the nuclear translocation of C/EBP{beta}. Reporter gene analyses showed that HBx increased the reporter activity in the cells transfected with the reporter containing MDR1 gene promoter. Moreover, the luciferase reporter gene activity was significantly inhibited by HIF-1{alpha} siRNA but not by overexpression of C/EBP dominant negative mutant. These results imply that HBx increases the MDR1 transporter activity through the transcriptional activation of the MDR1 gene with HIF-1{alpha} activation, and suggest HIF-1{alpha} for the therapeutic target of HBV-mediated chemoresistance.

  13. Subcellular localization of RNAs in transfected cells: role of sequences at the 5' terminus.

    PubMed Central

    Alfonso-Pizarro, A; Carlson, D P; Ross, J

    1984-01-01

    We have investigated the intracellular location of RNAs transcribed from transfected DNA. COS cells transfected with a clone containing the human adult beta globin gene contain three classes of globin RNAs. Their 3' termini and splice sites are indistinguishable from those of mature reticulocyte beta globin mRNA, and they are polyadenylated. However, as determined by S1 mapping, their 5' sequences are different. The 5' terminus of one is the same as that of mature beta globin mRNA (+1, cap site). The presumed 5' terminus of the second is located 30 nucleotides downstream from the cap site (+30). The third class contains additional nucleotides transcribed from sequences located 5' to the cap site (5' upstream RNA). The 5' upstream RNA molecules are restricted to the nucleus and are more stable than heterogeneous nuclear RNA. The +30 and +1 RNAs are located primarily in the cytoplasm. The data support the notion that nucleotide sequences and/or secondary modifications in the 5' region determine if an RNA is to be transported. Images PMID:6209612

  14. Cholesterol-conjugated supramolecular assemblies of low generations polyamidoamine dendrimers for enhanced EGFP plasmid DNA transfection

    NASA Astrophysics Data System (ADS)

    Golkar, Nasim; Samani, Soliman Mohammadi; Tamaddon, Ali Mohammad

    2016-05-01

    Aimed to prepare an enhanced gene delivery system with low cytotoxicity and high transfection efficiency, various cholesterol-conjugated derivates of low generation polyamidoamine (PAMAM) dendrimers were prepared. The conjugates were characterized by TNBS assay, FTIR, and 1H-NMR spectroscopy. Self-assembly of the dendrimer conjugates (G1-Chol, G2-Chol, and G3-Chol) was investigated by pyrene assay. Following formation of the complexes between enhanced green fluorescence protein plasmid and the dendrimer conjugates at various N (primary amine)/P (phosphate) mole ratios, plasmid condensation, biologic stability, cytotoxicity, and protein expression were investigated. The conjugates self-assembled into micellar dispersions with the critical micelle concentration values (<50 µg/ml) depending on the dendrimer generation and cholesterol/amine mole ratio. Cholesterol conjugation resulted in higher resistance of the condensed plasmid DNA in a competition assay with heparin sulfate. Also, the transfection efficiency was determined higher for the cholesterol conjugates than unmodified dendrimers in HepG2 cells, showing the highest for G2-Chol at 40 % degree of cholesterol modification (G2-Chol40 %) among various dendrimer generations. Interestingly, such conjugate showed a complete protection of plasmid against serum nucleases. Our results confirmed that the cholesterol conjugation to PAMAM dendrimers of low generations bearing little cytotoxicity improves their several physicochemical and biological characteristics required for an enhanced delivery of plasmid DNA into cells.

  15. High-throughput continuous flow femtosecond laser-assisted cell optoporation and transfection.

    PubMed

    Breunig, Hans Georg; Uchugonova, Aisada; Batista, Ana; König, Karsten

    2014-12-01

    We present a femtosecond-laser based nanoprocessing system for transient optical cell membrane poration to allow the introduction of foreign molecules into the interior of a cell with very high throughput. In the setup, cells flow through a micro-flow tube for spatial confinement and are simultaneously targeted by fs laser radiation. Beam-shaping generates a focal geometry along a line which is scanned across the micro-flow cell to increase the number of reachable cells. Successful cell membrane poration was observed indirectly by cell transfection even with cell-light interaction times in the millisecond range. The system was characterized by experiments with Chinese hamster ovary cells regarding cell viability, the uptake of extrinsic molecules and cell transfection efficiency. The continuous flow of cells enables a tremendous increase of cell throughput compared to previous nonflow approaches by treating millions of cells, although with only limited efficiency. The setup opens the possibility to realize a completely automated high-throughput laser-assisted cell-poration system which could be integrated in lab-on-a-chip devices.

  16. Preparation of cationized polysaccharides as gene transfection carrier for bone marrow-derived mesenchymal stem cells.

    PubMed

    Jo, Jun-ichiro; Okazaki, Arimichi; Nagane, Kentaro; Yamamoto, Masaya; Tabata, Yasuhiko

    2010-01-01

    The objective of this study is to prepare a non-viral carrier of gene transfection from various polysaccharides and evaluate the feasibility in gene expression for mesenchymal stem cells (MSCs). Various amounts of spermine were chemically introduced into pullulan, dextran and mannan with a molecular weight of around 40 000 or pullulan with different molecular weights to prepare cationized polysaccharides with different extents of spermine introduced (spermine-polysaccharide). Each cationized polysaccharide was complexed with a plasmid DNA at various ratios and in vitro gene transfection was investigated for rat bone marrow-derived MSCs. The level of gene expression depended on the type of cationized polysaccharide. The highest level was observed for the complex of spermine-pullulan and plasmid DNA. Additionally, the level also depended on the molecular weight of pullulan and the extent of spermine introduced to pullulan. Suppression of gene expression with chlorpromazine and methyl-beta-cyclodextrin of endocytosis inhibitors demonstrated that the cellular uptake of spermine-pullulan-plasmid DNA complexes was mediated by clathrin- and raft/caveolae-dependent endocytic pathways. The cationized pullulan is a promising non-viral carrier of plasmid DNA for MSCs.

  17. Luminescent-Activated Transfected Killer Cells to Monitor Leukocyte Trafficking During Systemic Bacterial and Fungal Infection

    PubMed Central

    Lin, Lin; Ibrahim, Ashraf S.; Baquir, Beverlie; Palosaari, Andrew

    2012-01-01

    Background. Activated transfected killer (ATAK) cells are immortal phagocytes transfected with a luminescence reporter that effectively treat lethal infections in neutropenic mice. Their in vivo trafficking, lifespan, and immunogenicity are unknown. Methods. Mice were made neutropenic; infected or not with Staphylococcus aureus, Acinetobacter baumannii, Candida albicans, or Aspergillus fumigatus; and treated intraperitoneally with ATAK cells. Cell trafficking and lifespan were assessed by in vivo imaging and reverse transcription–polymerase chain reaction. Results. In uninfected neutropenic mice, ATAK cells spread from the mesentery into visceral organs on days 1–3. Splenic accumulation of ATAK cells increased at day 1 after infection with S. aureus and A. baumannii, and kidney accumulation increased in mice infected with C. albicans. Lung accumulation was seen at day 3 in mice infected by inhalation with A. fumigatus. By day 8, coincident with increasing anti-ATAK antibodies, luminescence signal was lost and there was no detectable mRNA transcription from ATAK cells. Conclusions. ATAK cells accumulated in target organs with distinct profiles, depending on the microbial etiology of infection. Finally, generation of an anti-ATAK immune response may provide an important safety mechanism that helps clear the cells from the host as the marrow recovers. PMID:22124127

  18. Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation.

    PubMed

    Itaka, Keiji; Uchida, Satoshi; Matsui, Akitsugu; Yanagihara, Kayoko; Ikegami, Masaru; Endo, Taisuke; Ishii, Takehiko; Kataoka, Kazunori

    2015-07-31

    To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 µm diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

  19. Imaging of transfection and intracellular release of intact, non-labeled DNA using fluorescent nanodiamonds.

    PubMed

    Petrakova, V; Benson, V; Buncek, M; Fiserova, A; Ledvina, M; Stursa, J; Cigler, P; Nesladek, M

    2016-06-09

    Efficient delivery of stabilized nucleic acids (NAs) into cells and release of the NA payload are crucial points in the transfection process. Here we report on the fabrication of a nanoscopic cellular delivery carrier that is additionally combined with a label-free intracellular sensor device, based on biocompatible fluorescent nanodiamond particles. The sensing function is engineered into nanodiamonds by using nitrogen-vacancy color centers, providing stable non-blinking luminescence. The device is used for monitoring NA transfection and the payload release in cells. The unpacking of NAs from a poly(ethyleneimine)-terminated nanodiamond surface is monitored using the color shift of nitrogen-vacancy centers in the diamond, which serve as a nanoscopic electric charge sensor. The proposed device innovates the strategies for NA imaging and delivery, by providing detection of the intracellular release of non-labeled NAs without affecting cellular processing of the NAs. Our system highlights the potential of nanodiamonds to act not merely as labels but also as non-toxic and non-photobleachable fluorescent biosensors reporting complex molecular events.

  20. Transfection of normal human bronchial epithelial cells with the bcl-2 oncogene

    SciTech Connect

    Kennedy, C.H.; Kenyon, K.D.; Tesfaigzi, J.

    1995-12-01

    In vitro, studies examining the transformation of virus-immortalized human bronchial epithelial (HBE) cells after exposure to chemical and physical carcinogens have contributed to our understanding of the mechanisms that underlie the development of lung cancer. Virus-immortalized HBE cells have been used because of both the limited life span of normal human bronchial epithelial (NHBE) cells in culture (approximately 30-35 population doublins) and their resistance to in vitro malignant transformation. For example, human papillomavirus (HPV)-immortalized HBE cells have been used to study the genetic changes that occur after exposure to {alpha}-particles in vitro. Although this model may prove to be useful for studying the 18% or less of bronchogenic carcinomas found to contain HPV sequences, it is not an appropriate model for studying the majority of lung epithelial malignancies in which HPV DNA is not detected. This view is supported by the fact that HPV-immortalized cell lines commonly exhibit aneuploidy. This results of this study suggest that: (1) NHBE cells can be transiently transfected with the pCMV{Beta} vector; and (2) the antibiotic hygromycin-resistant transfected cells.