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Sample records for h4iie transfectant insensitive

  1. 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

  2. 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

    2016-01-01

    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

  3. 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

  4. Osmotic regulation of betaine homocysteine-S-methyltransferase expression in H4IIE rat hepatoma cells.

    PubMed

    Schäfer, Christine; Hoffmann, Lars; Heldt, Katrin; Lornejad-Schäfer, Mohammad Reza; Brauers, Gernot; Gehrmann, Thor; Garrow, Timothy A; Häussinger, Dieter; Mayatepek, Ertan; Schwahn, Bernd C; Schliess, Freimut

    2007-04-01

    Cell hydration changes critically affect liver metabolism and gene expression. In the course of gene expression studies using nylon cDNA-arrays we found that hyperosmolarity (405 mosmol/l) suppressed the betaine-homocysteine methyltransferase (Bhmt) mRNA expression in H4IIE rat hepatoma cells. This was confirmed by Northern blot and real-time quantitative RT-PCR analysis, which in addition unraveled a pronounced induction of Bhmt mRNA expression by hypoosmotic (205 mosmol/l) swelling. Osmotic regulation of Bhmt mRNA expression was largely paralleled at the levels of Bhmt protein and enzymatic activity. Like hyperosmotic NaCl, hyperosmotic raffinose but not hyperosmotic urea suppressed Bhmt mRNA expression, suggesting that cell shrinkage rather than increased ionic strength or hyperosmolarity per se is the trigger. Hypoosmolarity increased the expression of a reporter gene driven by the entire human BHMT promoter, whereas destabilization of BHMT mRNA was observed under hyperosmotic conditions. Osmosensitivity of Bhmt mRNA expression was impaired by inhibitors of tyrosine kinases and cyclic nucleotide-dependent kinases. The osmotic regulation of BHMT may be part of a cell volume-regulatory response and additionally lead to metabolic alterations that depend on the availability of betaine-derived methyl groups. PMID:17218476

  5. Elevation in Tanis expression alters glucose metabolism and insulin sensitivity in H4IIE cells.

    PubMed

    Gao, Yuan; Walder, Ken; Sunderland, Terry; Kantham, Lakshmi; Feng, Helen C; Quick, Melissa; Bishara, Natalie; de Silva, Andrea; Augert, Guy; Tenne-Brown, Janette; Collier, Gregory R

    2003-04-01

    Increased hepatic glucose output and decreased glucose utilization are implicated in the development of type 2 diabetes. We previously reported that the expression of a novel gene, Tanis, was upregulated in the liver during fasting in the obese/diabetic animal model Psammomys obesus. Here, we have further studied the protein and its function. Cell fractionation indicated that Tanis was localized in the plasma membrane and microsomes but not in the nucleus, mitochondria, or soluble protein fraction. Consistent with previous gene expression data, hepatic Tanis protein levels increased more significantly in diabetic P. obesus than in nondiabetic controls after fasting. We used a recombinant adenovirus to increase Tanis expression in hepatoma H4IIE cells and investigated its role in metabolism. Tanis overexpression reduced glucose uptake, basal and insulin-stimulated glycogen synthesis, and glycogen content and attenuated the suppression of PEPCK gene expression by insulin, but it did not affect insulin-stimulated insulin receptor phosphorylation or triglyceride synthesis. These results suggest that Tanis may be involved in the regulation of glucose metabolism, and increased expression of Tanis could contribute to insulin resistance in the liver.

  6. Metabolic regulation of Na(+)/P(i)-cotransporter-1 gene expression in H4IIE cells.

    PubMed

    Xie, Z; Li, H; Liu, L; Kahn, B B; Najjar, S M; Shah, W

    2000-04-01

    We showed that the rat Na(+)/P(i) cotransporter-1 (RNaPi-1) gene was regulated by insulin and glucose in rat hepatocytes. The aim of this work was to elucidate signaling pathways of insulin-mediated metabolic regulation of the RNaPi-1 gene in H4IIE cells. Insulin increased RNaPi-1 mRNA abundance in the presence of glucose and decreased RNaPi-1 mRNA in the absence of glucose, clearly establishing an involvement of metabolic signals for insulin-induced upregulation of the RNaPi-1 gene. Pyruvate and insulin increased RNaPi-1 expression but downregulated L-pyruvate kinase, indicating the existence of gene-specific metabolic signals. Although fructose, glycerol, and lactate could support insulin-induced upregulation of the RNaPi-1 gene, compounds entering metabolism beyond pyruvate oxidation, such as acetate and citrate, could not, suggesting that RNaPi-1-specific metabolic signals are generated at or above pyruvate oxidation. Wortmannin, LY-294002, and rapamycin abolished the insulin effect on the RNaPi-1 gene, whereas expression of dominant negative Asn(17) Ras and mitogen-activating protein kinase (MAPK) kinase (MEK) inhibitor PD-98059 exhibited no effect. Thus we herein propose that metabolic regulation of RNaPi-1 expression by insulin is mediated through the phosphatidylinositol 3-kinase/p70 ribosomal S6 kinase pathways, but not the Ras/MAPK pathway. PMID:10751198

  7. Isoquercitrin activates the AMP–activated protein kinase (AMPK) signal pathway in rat H4IIE cells

    PubMed Central

    2014-01-01

    Background Isoquercitrin, a flavonoid compound that is widely distributed in medicinal and dietary plants, possesses many biological activities, including inhibition of adipocyte differentiation. In this study, we investigated the effect of isoquercitrin on lipid accumulation and its molecular mechanisms in rat hepatoma H4IIE cells. Methods To investigate the effect of isoquercitrin on lipid accumulation, H4IIE cells were induced by FFA and the total lipid levels were detected by Oil Red O staining. Furthermore, The protein levels of AMPK and acetyl-CoA carboxylase (ACC), the gene expressions of transcriptional factor, lipogenic genes, and adiponectin receptor 1 (AdipoR1) were analyzed by Western blotting and quantitative real-time PCR. To further confirm the pathway of isoquercitrin-mediated hepatic lipid metabolism, H4IIE cells were treated with an AMPK inhibitor and AdipoR1 siRNA. Results Isoquercitrin significantly enhances AMPK phosphorylation, downregulates sterol regulatory element binding protein transcription factor 1 (SREBP-1) and fatty acid synthase (FAS) gene expressions. Pretreatment with AMPK inhibitor, significantly decreased the AMPK phosphorylation and increased FAS expression stimulated by isoquercitrin. Isoquercitrin might also upregulate the expression of AdipoR1 dose-dependently via AMPK in the presence of an AMPK inhibitor and AdipoR1 siRNA. Conclusions Isoquercitrin appears to regulate AMPK activation, thereby enhancing AdipoR1 expression, suppressing SREBP-1 and FAS expressions, and resulting in the regulation of lipid accumulation. These results suggest that isoquercitrin is a novel dietary compound that can be potentially be used to prevent lipid metabolic disorder and nonalcoholic fatty liver disease. PMID:24490657

  8. 3-Methylcholanthrene elicits DNA adduct formation in the CYP1A1 promoter region and attenuates reporter gene expression in rat H4IIE cells

    SciTech Connect

    Moorthy, Bhagavatula . E-mail: bmoorthy@bcm.tmc.edu; Muthiah, Kathirvel; Fazili, Inayat S.; Kondraganti, Sudha R.; Wang Lihua; Couroucli, Xanthi I.; Jiang Weiwu

    2007-03-23

    Cytochrome CYP1A (CYP1A) enzymes catalyze bioactivation of 3-methylcholanthrene (MC) to genotoxic metabolites. Here, we tested the hypothesis that CYP1A2 catalyzes formation of MC-DNA adducts that are preferentially formed in the promoter region of CYP1A1, resulting in modulation of CYP1A1 gene expression. MC bound covalently to plasmid DNA (50 {mu}g) containing human CYP1A1 promoter (pGL3-1A1), when incubated with wild-type (WT) liver microsomes (2 mg) and NAPPH 37 {sup o}C for 2 h, giving rise to 9 adducts, as determined by {sup 32}P-postlabeling. Eighty percent of adducts was located in the promoter region. Transient transfection of the adducted plasmids into rat hepatoma (H4IIE) cells for 16 h, followed by MC (1 {mu}M) treatment for 24 h inhibited reporter (luciferase) gene expression by 75%, compared to unadducted controls. Our results suggest that CYP1A2 plays a key role in sequence-specific MC-DNA adduct formation in the CYP1A1 promoter region, leading to attenuation of CYP1A1 gene expression.

  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. Sodium 2-propenyl thiosulfate derived from garlic induces phase II detoxification enzymes in rat hepatoma H4IIE cells.

    PubMed

    Chang, Hye-Sook; Ko, Miyan; Ishizuka, Mayumi; Fujita, Shoichi; Yabuki, Akira; Hossain, Mohammad Alamgir; Yamato, Osamu

    2010-06-01

    There is evidence that onions and garlic protect against cancer in humans. It has been suggested that this effect is partly due to the organosulfur compounds in Allium vegetables and that these substances act through induction of phase II detoxification enzymes. Here, we hypothesized that alk(en)yl thiosulfates, sodium n-propyl thiosulfate (NPTS), and sodium 2-propenyl thiosulfate (2PTS), which were identified in onions and garlic, respectively, may induce phase II enzymes. Therefore, rat hepatoma cells (H4IIE) were cultured with 1 to 100 micromol/L of NPTS or 2PTS for 48 hours at 37 degrees C; and the activities and messenger RNA (mRNA) expression levels of phase II enzymes in H4IIE cells were investigated. The effects of diallyl trisulfide and tert-butylhydroquinone, known as phase II inducers, were also examined as positive controls and compared with the responses of NPTS and 2PTS. Quinone reductase (QR) activity and mRNA expression levels of QR and epoxide hydrolase 1 were significantly increased by 2PTS (P < .05-.005). In particular, QR activity was increased at a relatively low concentration of 2PTS (10 micromol/L). However, glutathione S-transferase activity and mRNA expression levels of glutathione S-transferase A5 and uridine diphosphate glucuronosyl transferase 1A1 were not changed by 2PTS. In contrast, NPTS did not affect the activities and mRNA expression levels of these phase II enzymes. These results show that 2PTS can induce phase II enzymes, and its inductive effect is comparable or superior to that of diallyl trisulfide and tert-butylhydroquinone. PMID:20650352

  12. Effects of methylated derivatives of Luteolin isolated from Cyperus alopecuroides in rat H4IIE hepatoma cells*.

    PubMed

    Michels, Gudrun; Mohamed, Gamal A; Weber, Nadine; Chovolou, Yvonni; Kampkötter, Andreas; Wätjen, Wim; Proksch, Peter

    2006-02-01

    Polyphenols are ubiquitous substances in human diet. Their antioxidative, antiinflammatory and antiviral effects are of interest for human health, and polyphenols such as luteolin are used at high concentrations in food supplements. Luteolin is metabolized to glucuronides, but also to methylated derivatives. For example, O-methylation of the catechol group mediated by the catechol-O-methyl transferase, is an important step in flavonoid metabolism. The aim of this project was to determine the effect of O-methylation on antioxidative capacity and cytotoxicity of luteolin in H4IIE rat hepatoma cells. Therefore we analyzed the effects of luteolin 5,3'-dimethylether, isolated from the flowers of foxtail flatsedge (Cyperus alopecuroides) and luteolin 5,7,3',4'-tetramethylether compared to the non-methylated flavonoid luteolin. The antioxidative potential of luteolin was lowered by methylation, an effect that seems to be mediated by masking of the catechol moiety in the B ring. The cytotoxic potential of luteolin 5,3'-dimethylether is comparable to luteolin, but the tetramethylether showed no cytotoxic effect. The cytotoxic effect of luteolin but not luteolin 5,3'-dimethylether was mediated via apoptosis (caspase-3 activation). We conclude that the O-methylation of luteolin led to a decreased radical-scavenging activity and to a reduction in the apoptotic potential of the flavonoid.

  13. Ethoxyresorufin-O-deethylase induction potency of polychlorinated diphenyl ethers in H4IIE rat hepatoma cells

    SciTech Connect

    Koistinen, J.; Sanderson, J.T.; Giesy, J.P.; Nevalainen, T.; Paasivirta, J.

    1996-11-01

    Polychlorinated diphenyl ethers (PCDEs) are structurally similar to polychlorinated biphenyls (PCBs), and some PCDE congeners have been reported to cause toxic responses similar to those caused by some of the non-ortho-substituted PCBs, which are mediated by the aryl hydrocarbon receptor (AhR). Twenty-nine PCDEs were tested for their potency as AhR agonists relative to 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) by measuring their ability to induce the cytochrome P-450 1A1-associated enzyme activity, ethoxyresorufin-O-deethylase (EROD), in the H4IIE rat hepatoma cell bioassay. All PCDE congeners tested were found to be inactive as EROD inducers except for PCDE 156, which was a weak EROD inducer with a 2,3,7,8-TCDD equivalency factor of about 1.2 {times} 10{sup {minus}5}. During this study the authors determined that small amounts of polychlorinated dibenzofurans (PCDFs) that occurred as impurities in the PCDE preparations were the cause of the apparent EROD induction initially measured in their experiments. Once the PCDF impurities were removed by purification on florisil, little or no activity could be attributed to the PCDEs.

  14. Low concentrations of flavonoids are protective in rat H4IIE cells whereas high concentrations cause DNA damage and apoptosis.

    PubMed

    Wätjen, Wim; Michels, Gudrun; Steffan, Bärbel; Niering, Petra; Chovolou, Yvonni; Kampkötter, Andreas; Tran-Thi, Quynh-Hoa; Proksch, Peter; Kahl, Regine

    2005-03-01

    Dietary flavonoids possess a wide spectrum of biochemical and pharmacological actions and are assumed to protect human health. These actions, however, can be antagonistic, and some health claims are mutually exclusive. The antiapoptotic actions of flavonoids may protect against neurodegenerative diseases, whereas their proapoptotic actions could be used for cancer chemotherapy. This study was undertaken to determine whether a cytoprotective dose range of flavonoids could be differentiated from a cytotoxic dose range. Seven structurally related flavonoids were tested for their ability to protect H4IIE rat hepatoma cells against H(2)O(2)-induced damage on the one hand and to induce cellular damage on their own on the other hand. All flavonoids proved to be good antioxidants in a cell-free assay. However, their pharmacologic activity did not correlate with in vitro antioxidant potential but rather with cellular uptake. For quercetin and fisetin, which were readily taken up into the cells, protective effects against H(2)O(2)-induced cytotoxicity, DNA strand breaks, and apoptosis were detected at concentrations as low as 10-25 micromol/L. On the other hand, these flavonoids induced cytotoxicity, DNA strand breaks, oligonucleosomal DNA fragmentation, and caspase activation at concentrations between 50 and 250 micromol/L. Published data on quercetin pharmacokinetics in humans suggest that a dietary supplement of 1-2 g of quercetin may result in plasma concentrations between 10 and 50 micromol/L. Our data suggest that cytoprotective concentrations of some flavonoids are lower by a factor of 5-10 than their DNA-damaging and proapoptotic concentrations.

  15. Metabolic chiral inversion of 2-arylpropionates in rat H4IIE and human Hep G2 hepatoma cells. Relationship to in vivo metabolism.

    PubMed

    Menzel-Soglowek, S; Geisslinger, G; Mollenhauer, J; Brune, K

    1992-04-01

    The inversion of 2-arylpropionic acids (2-APAs) has been investigated in vitro using rat H4IIE and human Hep G2 hepatoma cells in continuous culture. The effect of substrate concentration (15-150 micrograms/mL), cell density (1.5-12 x 10(6) cells/dish) and serum content of the culture medium (0-20%) on inversion was examined in rat H4IIE hepatoma cells using R-ibuprofen as model compound. Increasing R-ibuprofen concentrations and decreasing serum content of the medium resulted in increased inversion whereas variation of cell density had no effect. Furthermore, rat H4IIE and human Hep G2 hepatoma cells were incubated with the individual enantiomers of ibuprofen, ketoprofen and flurbiprofen under optimized culture conditions (serum-free culture medium). The elimination rate constants (kel) and fractions inverted (Fi) were determined. Although inversion occurred slowly in the tumor cells and thus long incubation periods (120 hr) were required, the hepatoma cells were nevertheless able to mimic qualitatively the species and substance specificity of inversion of 2-APAs as observed in vivo.

  16. 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.

  17. Differential expression of CYP1A1 and CYP1A2 genes in H4IIE rat hepatoma cells exposed to TCDD and PAHs.

    PubMed

    Kaisarevic, Sonja; Dakic, Vanja; Hrubik, Jelena; Glisic, Branka; Lübcke-von Varel, Urte; Pogrmic-Majkic, Kristina; Fa, Svetlana; Teodorovic, Ivana; Brack, Werner; Kovacevic, Radmila

    2015-01-01

    Rat hepatoma cells H4IIE were treated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and polycyclic aromatic hydrocarbons (PAHs) (dibenz(a,h)anthracene, benzo(a)pyrene, benz(a)anthracene, chrysene), low-concentration mixtures of PAHs and TCDD, and environmental mixtures contaminated by PAHs and their derivatives. Expression of the gene battery comprising cytochrome P450 Cyp1a1, Cyp1a2, Cyp1b1, and glutathione-s-transferase Gsta2 and Gstp was investigated using quantitative real time polymerase chain reaction (qRT-PCR) analysis. The results revealed that TCDD induce Cyp1a1>Cyp1a2>Cyp1b1, while PAHs and PAH-containing environmental mixtures induce Cyp1a2>Cyp1a1>Cyp1b1 gene expression pattern. While low-concentration mixtures elicited a more pronounced response in comparison to single treatments, the typical gene expression patterns were not observed. In all samples, Gsta2 was predominantly expressed relative to Gstp. These findings indicate that differential Cyp1a1 and Cyp1a2 expression in the H4IIE cells might be used for detection of PAHs in highly contaminated environmental mixtures, but not in low-concentration mixtures of these compounds.

  18. 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.

  19. 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.

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

    SciTech Connect

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

    2000-05-01

    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 that 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.

  1. Dietary exposure to polychlorinated dibenzo-p-dioxins and dibenzofurans via fish consumption and dioxin-like activity in fish determined by H4IIE-luc bioassay.

    PubMed

    Chan, Janet Kit Yan; Man, Yu Bon; Xing, Guan Hua; Wu, Sheng Chun; Murphy, Margaret B; Xu, Ying; Wong, Ming H

    2013-10-01

    Dietary exposure to polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) via fish consumption in two major electronic (e) waste sites: Guiyu (GY), Guangdong Province and Taizhou (TZ), Zhejiang Province, and dioxin-like activity in fish determined by H4IIE-luc bioassay. In the present study, all fish were below EU's maximum allowable concentration in muscle of fish (4 pg WHO-TEQ/g wet wt), except crucian (4.28 pg WHO-TEQ/g wet wt) and silver carps (7.49 pg WHO-TEQ/g wet wt) collected from GY rivers. Moreover, the residual concentration in bighead carp collected from GY (2.15 pg WHO-TEQ/g wet wt) was close to the EU's action level (3 pg WHO-TEQ/g wet wt) which gives "early warning" to the competent authorities and operators to take measures to eliminate contamination. In addition, results indicated that the maximum human intake of PCDD/Fs via freshwater fish consumption in GY was 4.31 pg WHO-TEQ/kg bw/day, which exceeds the higher end of the tolerable daily intake recommended by the WHO, EC-SCF and JECFA (1-4, 2 and 2.3 pg WHO-TEQ/kg bw/day respectively). Furthermore, H4IIE-luc cell bioassay provides a very sensitive and cost-efficient screening tool for assessing the overall dioxin-like toxicity in the study, and is therefore valuable for high-throughput environmental monitoring studies.

  2. Dietary exposure to polychlorinated dibenzo-p-dioxins and dibenzofurans via fish consumption and dioxin-like activity in fish determined by H4IIE-luc bioassay.

    PubMed

    Chan, Janet Kit Yan; Man, Yu Bon; Xing, Guan Hua; Wu, Sheng Chun; Murphy, Margaret B; Xu, Ying; Wong, Ming H

    2013-10-01

    Dietary exposure to polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) via fish consumption in two major electronic (e) waste sites: Guiyu (GY), Guangdong Province and Taizhou (TZ), Zhejiang Province, and dioxin-like activity in fish determined by H4IIE-luc bioassay. In the present study, all fish were below EU's maximum allowable concentration in muscle of fish (4 pg WHO-TEQ/g wet wt), except crucian (4.28 pg WHO-TEQ/g wet wt) and silver carps (7.49 pg WHO-TEQ/g wet wt) collected from GY rivers. Moreover, the residual concentration in bighead carp collected from GY (2.15 pg WHO-TEQ/g wet wt) was close to the EU's action level (3 pg WHO-TEQ/g wet wt) which gives "early warning" to the competent authorities and operators to take measures to eliminate contamination. In addition, results indicated that the maximum human intake of PCDD/Fs via freshwater fish consumption in GY was 4.31 pg WHO-TEQ/kg bw/day, which exceeds the higher end of the tolerable daily intake recommended by the WHO, EC-SCF and JECFA (1-4, 2 and 2.3 pg WHO-TEQ/kg bw/day respectively). Furthermore, H4IIE-luc cell bioassay provides a very sensitive and cost-efficient screening tool for assessing the overall dioxin-like toxicity in the study, and is therefore valuable for high-throughput environmental monitoring studies. PMID:22959899

  3. Determination of the CYP1A-inducing potential of single substances, mixtures and extracts of samples in the micro-EROD assay with H4IIE cells.

    PubMed

    Schiwy, Andreas; Brinkmann, Markus; Thiem, Ines; Guder, Gabriele; Winkens, Kerstin; Eichbaum, Kathrin; Nüßer, Leonie; Thalmann, Beat; Buchinger, Sebastian; Reifferscheid, Georg; Seiler, Thomas-Benjamin; Thoms, Brigitte; Hollert, Henner

    2015-11-01

    This protocol describes a quantitative and robust 96-well-plate-reader-based assay for the measurement of ethoxyresorufin-O-deethylase (EROD) activity using the rat hepatoma cell line H4IIE. The assay can be used to determine the cytochrome P450 subfamily 1A (CYP1A)-inducing potential of single substances, as well as of mixtures and extracts of samples. It is based on the aryl hydrocarbon receptor (AhR)-mediated induction of cytochrome P450 enzymes (subfamily 1A) in cells after exposure to dioxins and dioxin-like compounds. One enzymatic reaction catalyzed by CYP1A is the deethylation of the exogenous substrate 7-ethoxyresorufin to the fluorescent product resorufin, which is measured as EROD activity in the assay. The CYP1A-inducing potential of a sample can be reliably quantified by comparing the EROD activity with the concentration-response curve of the standard substance 2,3,7,8-tetrachlorodibenzo-p-dioxin, which can be detected at concentrations down to the picogram per liter range. A researcher familiar with the procedure can process up to 160 samples with four wells each within 3 d. The series described uses four plates with three concentrations per sample, which can be easily scaled to accommodate different sample sizes. PMID:26448361

  4. Cytotoxic effects of 109 reference compounds on rat H4IIE and human HepG2 hepatocytes. III: Mechanistic assays on oxygen consumption with MitoXpress and NAD(P)H production with Alamar Blue™.

    PubMed

    Schoonen, Willem G E J; Stevenson, Joe C R; Westerink, Walter M A; Horbach, G Jean

    2012-04-01

    In vitro toxicity screening can reduce the attrition rate of drug candidates in the pharmaceutical industry in the early development process. The focus in this study is to compare the sensitivity for cytotoxicity of a time-resolved fluoro metric oxygen probe with that of a fluoro metric Alamar Blue™ (AB) assay. Both assays measure mitochondrial activity by either oxygen consumption (LUX-A65N-1 (MitoXpress, Luxcel) probe) or NADH/FADH conversion (AB). Both assays were carried out with increasing concentrations of 109 reference compounds using rat H4IIE and human HepG2 hepatocytes at incubation periods of 24, 48 and 72 h. Prior to this study, the influence on medium with either glucose or galactose was studied to analyze the rate of glycolysis and oxygen consumption, which latter process may be impaired in hepatoma cells. Inhibitors of oxygen consumption in combination with a glucose up-take inhibitor showed the largest consumption rate differences in the presence of 5mM of glucose. The choice for the 109 reference compounds was based on the so-called Multicentre Evaluation for In vitro Cytotoxicity (MEIC) and on diverse drug categories. For 59 toxic reference compounds, an evaluation for both assays was carried up to 10(-3)M. Toxicity was demonstrated with MitoXpress for 23 (39%) and 36 (61%) compounds in H4IIE and HepG2 cells, respectively, and with AB for 44 (75%) and 40 (68%) compounds. For 50 more pharmaceutical drugs more physiological concentrations were used up to 3.16×10(-5)M, and only 19 (38%) of these compounds appeared to be toxic in both assays. In conclusion, overall 63 (58%) and 60 (55%) compounds showed toxic effects with the MitoXpress and AB assays on rat H4IIE and human HepG2 hepatocytes, respectively. AB assays were more sensitive with respect to H4IIE cells and MitoXpress assays with respect to HepG2 cells. At all tested time intervals, MitoXpress showed its sensitivity, while AB is more sensitive at 48 and 72 h. With AB more toxic compounds

  5. 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

    2010-11-01

    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

  6. Insensitive explosive

    SciTech Connect

    Lee, Kien-yin; Storm, C.B.

    1991-12-31

    This invention relates to the field of chemistry and, more particularly, to explosives. This invention is the result of a contract with the Department of Energy (Contract No. W-7405-ENG-36). It is desirable to use explosives in weapons and other applications which are less sensitive than the common explosives RDX, TNT, and HMX, since there have been catastrophic explosions of munitions which use these compounds. In preliminary characterization and sensitivity testing, it has been found that 3-amino-5-nitro-1,2,4-triazole (ANTA) is a promising insensitive high explosive. This report details the safety, production, and physical properties of ANTA.

  7. Single cell optical transfection.

    PubMed

    Stevenson, David J; Gunn-Moore, Frank J; Campbell, Paul; Dholakia, Kishan

    2010-06-01

    The plasma membrane of a eukaryotic cell is impermeable to most hydrophilic substances, yet the insertion of these materials into cells is an extremely important and universal requirement for the cell biologist. To address this need, many transfection techniques have been developed including viral, lipoplex, polyplex, capillary microinjection, gene gun and electroporation. The current discussion explores a procedure called optical injection, where a laser field transiently increases the membrane permeability to allow species to be internalized. If the internalized substance is a nucleic acid, such as DNA, RNA or small interfering RNA (siRNA), then the process is called optical transfection. This contactless, aseptic, single cell transfection method provides a key nanosurgical tool to the microscopist-the intracellular delivery of reagents and single nanoscopic objects. The experimental possibilities enabled by this technology are only beginning to be realized. A review of optical transfection is presented, along with a forecast of future applications of this rapidly developing and exciting technology. PMID:20064901

  8. 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.

  9. 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

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. Insensitive fuze train for high explosives

    DOEpatents

    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.

  17. Insensitive fuze train for high explosives

    DOEpatents

    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.

  18. 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.

  19. Integrated Electrowetting Nanoinjector for Single Cell Transfection

    PubMed Central

    Shekaramiz, Elaheh; Varadarajalu, Ganeshkumar; Day, Philip J.; Wickramasinghe, H. Kumar

    2016-01-01

    Single cell transfection techniques are essential to understand the heterogeneity between cells. We have developed an integrated electrowetting nanoinjector (INENI) to transfect single cells. The high transfection efficiency, controlled dosage delivery and ease of INENI fabrication promote the widespread application of the INENI in cell transfection assays. PMID:27374766

  20. 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...

  1. INSENSITIVE HIGH-NITROGEN COMPOUNDS

    SciTech Connect

    D. CHAVEZ; ET AL

    2001-03-01

    The conventional approach to developing energetic molecules is to chemically place one or more nitro groups onto a carbon skeleton, which is why the term ''nitration'' is synonymous to explosives preparation. The nitro group carries the oxygen that reacts with the skeletal carbon and hydrogen fuels, which in turn produces the heat and gaseous reaction products necessary for driving an explosive shock. These nitro-containing energetic molecules typically have heats of formation near zero and therefore most of the released energy is derived from the combustion process. Our investigation of the tetrazine, furazan and tetrazole ring systems has offered a different approach to explosives development, where a significant amount of the chemical potential energy is derived from their large positive heats of formation. Because these compounds often contain a large percentage of nitrogen atoms, they are usually regarded as high-nitrogen fuels or explosives. A general artifact of these high-nitrogen compounds is that they are less sensitive to initiation (e.g. by impact) when compared to traditional nitro-containing explosives of similar performances. Using the precursor, 3,6-bis-(3,5-dimethylpyrazol-1-yl)-s-tetrazine, several useful energetic compounds based on the s-tetrazine system have been synthesized and studied. Some of the first compounds are 3,6-diamino-s-tetrazine-1,4-dioxide (LAX-112) and 3,6-dihydrazino-s-tetrazine (DHT). LAX-112 was once extensively studied as an insensitive explosive by Los Alamos; DHT is an example of a high-nitrogen explosive that relies entirely on its heat of formation for sustaining a detonation. Recent synthesis efforts have yielded an azo-s-tetrazine, 3,3'-azobis(6-amino-s-tetrazine) or DAAT, which has a very high positive heat of formation. The compounds, 4,4'-diamino-3,3'-azoxyfurazan (DAAF) and 4,4'-diamino-3,3'-azofurazan (DAAzF), may have important future roles in insensitive explosive applications. Neither DAAF nor DAAzF can be

  2. 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.

  3. 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.

  4. Transient transfection of purified Babesia bovis merozoites

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transient transfection of intraerythrocytic Babesia bovis parasites has been previously reported. In this study, we describe the development and optimization of methods for transfection of purified B. bovis merozoites using either nucleofection (Amaxa) or conventional electroporation (Gene Pulser II...

  5. Congenital insensitivity to pain with neuroparalytic keratitis.

    PubMed

    Biedner, B; Dagan, M; Gedalia, A; David, R

    1990-08-01

    Congenital insensitivity to pain is a well-defined entity in the group of sensory deficiency syndromes. To the best of our knowledge, unilateral neuroparalytic keratitis associated with congenital insensitivity to pain has not been reported. We report such a case to alert clinicians to this potentially blinding problem.

  6. 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 ...

  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. Transfection in the third dimension

    PubMed Central

    Dhaliwal, Anandika; Oshita, Victor; Segura, Tatiana

    2013-01-01

    An understanding of parameters that modulate gene transfer in 3-D will assist in the formation of gene delivery systems and scaffolds, which can mediate efficient non-viral delivery for guiding in-vivo tissue regeneration and therapy. We have previously demonstrated the cell area and length, integrin expression, and RhoGTPases mediated signalling to be pivotal parameters that guide gene transfer to mouse mesenchymal stem cells (mMSCs) cultured in 2-D and are modulated by ECM proteins. In this study, we were interested in determining if cationic polymer mediated gene transfer to cells seeded in 3-D would occur through different mechanisms as compared to 2-D. In particular, we examined the endocytosis pathways used to internalize polyplexes, and the role of cytoskeletal dynamics and RhoGTPases on non-viral gene transfer for cells seeded in 2-D and 3-D. Inhibition of clathrin- and caveolae- mediated endocytosis resulted in more drastic decrease in overall transgene expression for cells seeded in 3-D than those in 2-D. In addition, polyplex internalization was only significantly decreased in 3-D when clathrin-mediated endocytosis was inhibited, while caveolae-mediated endocytosis inhibition for cells seeded in 2-D resulted in the strongest polyplex internalization inhibition. Actin and microtubule polymerization affected 2-D and 3-D transfection differently. Microtubule depolymerization enhanced transgene expression in 2-D, but inhibited transgene expression in 3-D. Last, inhibition of RhoGTPases also affected 2-D and 3-D transfection differently. The inhibition of ROCK effector resulted in a decrease of transgene expression and internalization for cells seeded in 3-D, but not 2-D and the inhibition of effector PAK1 resulted in an increase of transgene expression for both 2-D and 3-D. Overall, our study suggests that the process of gene transfer occurs through different mechanisms for cells seeded in 2-D compared to those seeded in 3-D. PMID:23929354

  9. 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.

  10. 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.

  11. Modal insensitivity with optimality. [in feedback control

    NASA Technical Reports Server (NTRS)

    Calise, A. J.; Raman, K. V.

    1984-01-01

    This paper deals with the design of a constant gain, feedback controller which results in selected modal insensitivity, and at the same time optimizes a quadratic performance index representative of desired system performance for nominal plant parameter values. Both full state and output feedback control are considered. A constraint is established for the feedback gain matrix that results in modal insensitivity, and necessary conditions for optimality subject to this constraint are given. This forms the basis for a numerical algorithm to compute the optimal feedback gain. To illustrate the procedure, a design is carried out using the lateral dynamics of an L-1011 aircraft.

  12. Growth hormone insensitivity syndrome: unusual oral manifestations.

    PubMed

    Borges, Alvaro Henrique; Siqueira, Carlos Rodrigo Barros; Pedro, Fábio Luis Miranda; Palma, Vinícius Canavarros; Sakai, Vivien Thiemy; Volpato, Luiz Evaristo Ricci

    2013-01-01

    Children with significant growth retardation and normal levels of growth hormone are diagnosed with growth hormone insensitivity. The main oral findings observed in patients with growth hormone insensitivity syndrome (GHIS) are underdeveloped jaws, crowded teeth and delayed eruption of permanent teeth. This manuscript describes a 9-year-old child diagnosed with GHIS, who had delayed eruption of permanent teeth and 14 unerupted supernumerary teeth. All supernumerary teeth were extracted except for two maxillary and one mandibular teeth which were difficult to identify and access. Multiple supernumerary teeth have never been reported before in patients with GHIS.

  13. 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

  14. Root formation in ethylene-insensitive plants.

    PubMed

    Clark, D G; Gubrium, E K; Barrett, J E; Nell, T A; Klee, H J

    1999-09-01

    Experiments with ethylene-insensitive tomato (Lycopersicon esculentum) and petunia (Petunia x hybrida) plants were conducted to determine if normal or adventitious root formation is affected by ethylene insensitivity. Ethylene-insensitive Never ripe (NR) tomato plants produced more below-ground root mass but fewer above-ground adventitious roots than wild-type Pearson plants. Applied auxin (indole-3-butyric acid) increased adventitious root formation on vegetative stem cuttings of wild-type plants but had little or no effect on rooting of NR plants. Reduced adventitious root formation was also observed in ethylene-insensitive transgenic petunia plants. Applied 1-aminocyclopropane-1-carboxylic acid increased adventitious root formation on vegetative stem cuttings from NR and wild-type plants, but NR cuttings produced fewer adventitious roots than wild-type cuttings. These data suggest that the promotive effect of auxin on adventitious rooting is influenced by ethylene responsiveness. Seedling root growth of tomato in response to mechanical impedance was also influenced by ethylene sensitivity. Ninety-six percent of wild-type seedlings germinated and grown on sand for 7 d grew normal roots into the medium, whereas 47% of NR seedlings displayed elongated tap-roots, shortened hypocotyls, and did not penetrate the medium. These data indicate that ethylene has a critical role in various responses of roots to environmental stimuli.

  15. Cell biology: Targeted transfection by femtosecond laser

    NASA Astrophysics Data System (ADS)

    Tirlapur, Uday K.; König, Karsten

    2002-07-01

    The challenge for successful delivery of foreign DNA into cells in vitro, a key technique in cell and molecular biology with important biomedical implications, is to improve transfection efficiency while leaving the cell's architecture intact. Here we show that a variety of mammalian cells can be directly transfected with DNA without perturbing their structure by first creating a tiny, localized perforation in the membrane using ultrashort (femtosecond), high-intensity, near-infrared laser pulses. Not only does this superior optical technique give high transfection efficiency and cell survival, but it also allows simultaneous evaluation of the integration and expression of the introduced gene.

  16. Cationic phospholipids: structure transfection activity relationships

    SciTech Connect

    Koynova, Rumiana; Tenchov, Boris

    2010-01-18

    Synthetic cationic lipids are presently the most widely used non-viral gene carriers. Examined here is a particularly attractive cationic lipid class, triester phosphatidylcholines (PCs) exhibiting low toxicities and good transfection efficiency. Similarly to other cationic lipids, they form stable complexes (lipoplexes) with the polyanionic nucleic acids. A summary of studies on a set of {approx}30 cationic PCs reveals the existence of a strong, systematic dependence of their transfection efficiency on the lipid hydrocarbon chain structure: transfection activity increases with increase of chain unsaturation from 0 to 2 double bonds per lipid and decreases with increase of chain length in the range {approx}30-50 total number of chain carbon atoms. Maximum transfection was observed for ethyl phosphate PCs (EPCs) with monounsaturated 14:1 chains (total of 2 double bonds and 30 chain carbon atoms). Lipid phase behavior is known to depend strongly on the chain molecular structure and the above relationships thus substantiate a view that cationic PC phase propensities are an important determinant of their activity. Indeed, X-ray structural studies show that the rate of DNA release from lipoplexes as well as transfection activity well correlate with non-lamellar phase progressions observed in cationic PC mixtures with membrane lipids. These findings appear to be of considerable interest because, according to current views, key processes in lipid-mediated transfection such as lipoplex disassembly and DNA release within the cells are believed to take place upon cationic lipid mixing with cellular lipids.

  17. State-insensitive bichromatic optical trapping

    SciTech Connect

    Arora, Bindiya; Safronova, M. S.; Clark, Charles W.

    2010-08-15

    We study a scheme for state-insensitive trapping of neutral atoms by using light with two independent wavelengths. In particular, we describe the use of trapping and control lasers to minimize the variance of the potential experienced by a trapped Rb atom in ground and excited states. We present calculated values of wavelength pairs for which the 5s and 5p{sub 3/2} levels have the same ac Stark shifts in the presence of two laser fields.

  18. Insensitive TATP Training Aid by Microencapsulation

    NASA Astrophysics Data System (ADS)

    Oxley, J. C.; Smith, J. L.; Canino, J. N.

    2015-07-01

    There is a need in the explosives detection community for an insensitive, storage-stable source of triacetone triperoxide (TATP). To achieve this, the solvent evaporation microencapsulation technique was used to disperse TATP in a plastic matrix. This lowered the shock sensitivity greatly and prevented loss of TATP at room temperature, allowing for easy long-term storage. It was then demonstrated that pure TATP vapor was released on demand from the matrix by heating.

  19. Polarization insensitive phase modulator for quantum cryptosystems.

    PubMed

    Qi, Bing; Huang, Lei-Lei; Lo, Hoi-Kwong; Qian, Li

    2006-05-15

    In this paper, we propose a polarization-insensitive phase modulation scheme based on frequency modulation of light waves using either one or a pair of acousto-optic modulators. A stable Sagnac quantum key distribution (QKD) system employing this technique is also proposed. The interference visibility for a 40km and a 10km fiber loop is 96% and 99% respectively, at single-photon level. We ran standard BB84 QKD protocol in a simplified Sagnac setup (40km fiber loop) continuously for one hour and the measured quantum bit error rate stayed within 2%-5% range. PMID:19516579

  20. 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.

  1. Partial Androgen Insensitivity Syndrome Presenting with Gynecomastia

    PubMed Central

    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

    2015-01-01

    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. PMID:25433660

  2. 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

  3. 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

  4. 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.

  5. 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

  6. 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.

  7. Dissolution of three insensitive munitions formulations.

    PubMed

    Taylor, Susan; Park, Eileen; Bullion, Katherine; Dontsova, Katerina

    2015-01-01

    The US military fires live munitions during training. To save soldiers lives both during training and war, the military is developing insensitive munitions (IM) that minimize unintentional detonations. Some of the compounds in the IM formulation are, however, very soluble in water, raising environmental concerns about their fate and transport. We measured the dissolution of three of these IM formulations, IMX101, IMX104 and PAX21 using laboratory drip tests and studied the accompanying changes in particle structure using micro computed tomography. Our laboratory drip tests mimic conditions on training ranges, where spatially isolated particles of explosives scattered by partial detonations are dissolved by rainfall. We found that the constituents of these IM formulations dissolve sequentially and in the order predicted by their aqueous solubility. The order of magnitude differences in solubility among their constituents produce water solutions whose compositions and concentrations vary with time. For IMX101 and IMX104, that contain 3-nitro-1,2,4-triazol-5-one (NTO), the solutions also vary in pH. The good mass balances measured for the drip tests indicate that the formulations are not being photo-or bio-transformed under laboratory conditions. PMID:25043961

  8. Numerical Model of Detonation for Insensitive HE

    NASA Astrophysics Data System (ADS)

    Klimenko, Vladimir

    2011-06-01

    Most of modern munitions are filled by insensitive HE. However, mechanism of initiation of these HE is still unknown. IHE have not any pores and, therefore, hot spot mechanism does not work here. What is a mechanism working in this case? We have used 3D hydrocode to study process of shock wave loading of mixture of HMX grains with different binders (HMX/binder=88/12) and have determined formation of surface layers with increased plastic deformation. According to the dislocation mechanism of detonation (V. Klimenko, I. Kozyreva, J. Energetic Materials, 2010, v. 28, pp. 249-262) plastic deformation generates definite concentration of radicals. Surface layers have also increased temperature due to viscous work. So, these activated layers have increased temperature and number of radicals in comparison with values inside grains. Kinetic calculation has shown fast decomposition of these layers. As a result, the activated layer is ignited and this gives beginning of grain burning process. The developed two-stages mechanism has been incorporated into 2D hydrocode. The developed numerical model demonstrates high accuracy in simulation of detonation processes in IHE (in particular, PBXN-110 and B2241).

  9. Dissolution of three insensitive munitions formulations.

    PubMed

    Taylor, Susan; Park, Eileen; Bullion, Katherine; Dontsova, Katerina

    2015-01-01

    The US military fires live munitions during training. To save soldiers lives both during training and war, the military is developing insensitive munitions (IM) that minimize unintentional detonations. Some of the compounds in the IM formulation are, however, very soluble in water, raising environmental concerns about their fate and transport. We measured the dissolution of three of these IM formulations, IMX101, IMX104 and PAX21 using laboratory drip tests and studied the accompanying changes in particle structure using micro computed tomography. Our laboratory drip tests mimic conditions on training ranges, where spatially isolated particles of explosives scattered by partial detonations are dissolved by rainfall. We found that the constituents of these IM formulations dissolve sequentially and in the order predicted by their aqueous solubility. The order of magnitude differences in solubility among their constituents produce water solutions whose compositions and concentrations vary with time. For IMX101 and IMX104, that contain 3-nitro-1,2,4-triazol-5-one (NTO), the solutions also vary in pH. The good mass balances measured for the drip tests indicate that the formulations are not being photo-or bio-transformed under laboratory conditions.

  10. 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.

  11. H4IIE hepatoma cell bioassay-derived 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents in colonial fish-eating waterbird eggs from the Great Lakes

    SciTech Connect

    Tillitt, D.E.; Ankley, G.T.; Verbrugge, D.A.; Giesy, J.P.; Ludwig, J.P.

    1991-01-01

    Fish-eating waterbirds from the Great Lakes of North America have shown symptoms of poisoning similar to those observed in laboratory exposures of various avian species to planar halogenated hydrocarbons(PHHs). PHHs, include among others, polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs), and polychlorinated dibenzofurans (PCDFs) and have been implicated in some of the reproductive problems of Great Lakes waterbirds.

  12. 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. PMID:25425187

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. Insensitive detonator apparatus for initiating large failure diameter explosives

    DOEpatents

    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.

  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. Optimization of Transfection Conditions for siRNA Screening.

    PubMed

    Montoya, Justin J; Azorsa, David O

    2016-01-01

    RNAi screening of mammalian cells is often performed using siRNAs and cationic lipids as transfection reagents. Efficiency of transfection depends on growth characteristics of the cells and the cationic lipid used. With a large selection of cationic lipids available, it can often be difficult to select the optimal lipid and lipid:siRNA (vol:wt) ratio. Here, we describe the process of optimizing siRNA transfection conditions for efficient reverse transfection of mammalian cells using specific positive and negative siRNA controls. PMID:27581281

  2. Mammalian cell transfection: the present and the future

    PubMed Central

    Kim, Tae Kyung

    2010-01-01

    Transfection is a powerful analytical tool enabling study of the function of genes and gene products in cells. The transfection methods are broadly classified into three groups; biological, chemical, and physical. These methods have advanced to make it possible to deliver nucleic acids to specific subcellular regions of cells by use of a precisely controlled laser-microcope system. The combination of point-directed transfection and mRNA transfection is a new way of studying the function of genes and gene products. However, each method has its own advantages and disadvantages so the optimum method depends on experimental design and objective. PMID:20549496

  3. Defective membrane expression of human growth hormone (GH) receptor causes Laron-type GH insensitivity syndrome.

    PubMed Central

    Duquesnoy, P; Sobrier, M L; Amselem, S; Goossens, M

    1991-01-01

    Mutations in the growth hormone receptor (GHR) gene can cause growth hormone (GH) resistance. Given the sequence homology between the extracellular domain of the GHR and a soluble GH-binding protein (GH-BP), it is remarkable that GH-BP binding activity is absent from the serum of patients with Laron-type GH insensitivity, a hereditary form of severe dwarfism. We have previously identified a mutation within the extracellular domain of this receptor, replacing phenylalanine by serine at position 96 of the mature protein, in a patient with Laron syndrome. We have now investigated the effect of this Phe----Ser substitution on hormone binding activity by expressing the total human GHR cDNA and mutant form in eukaryotic cells. The wild-type protein expressed was able to bind GH but no plasma membrane binding was detectable on cells transfected with the mutant cDNA; this was also the case of cells transfected with a Phe96----Ala mutant cDNA, suggesting that the lack of binding activity is not due to a posttranslational modification of serine. Examination of the variant proteins in subcellular fractions revealed the presence of specific GH binding activity in the lysosomal fraction, whereas immunofluorescence studies located mutant proteins in the cytosol. Our findings suggest that these mutant GHRs fail to follow the correct intracellular transport pathway and underline the potential importance of this phenylalanine residue, which is conserved among the GH, prolactin, and erythropoietin receptors that belong to the same cytokine receptor superfamily. Images PMID:1719554

  4. 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.

  5. 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

  6. 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.

  7. Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

    PubMed Central

    Maeß, Marten B.; Wittig, Berith; Lorkowski, Stefan

    2014-01-01

    Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings. PMID:25226503

  8. 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.

  9. Transfection of isolated rainbow trout, Oncorhynchus mykiss, granulosa cells through chemical transfection and electroporation at 12°C.

    PubMed

    Marivin, E; Mourot, B; Loyer, P; Rime, H; Bobe, J; Fostier, A

    2015-09-15

    Over-expression or inhibition of gene expression can be efficiently used to analyse the functions and/or regulation of target genes. Modulation of gene expression can be achieved through transfection of exogenous nucleic acids into target cells. Such techniques require the development of specific protocols to transfect cell cultures with nucleic acids. The aim of this study was to develop a method of transfection suitable for rainbow trout granulosa cells in primary culture. After the isolation of rainbow trout granulosa cells, chemical transfection of cells with a fluorescent morpholino oligonucleotide (MO) was tested using FuGENE HD at 12 °C. Electroporation was also employed to transfect these cells with either a plasmid or MO. Transfection was more efficient using electroporation (with the following settings: 1200 V/40 ms/1p) than chemical transfection, but electroporation by itself was deleterious, resulting in a decrease of the steroidogenic capacity of the cells, measured via estradiol production from its androgenic substrate. The disturbance of cell biology induced by the transfection method per se should be taken into account in data interpretation when investigating the effects of under- or over-expression of candidate genes.

  10. 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.

  11. Role of transfection and clonal selection in mediating radioresistance

    SciTech Connect

    Pardo, F.S.; Taghia, A. ); Bristow, R.G. ); Ong, A.; Borek, C. New England Medical Center, Boston, MA )

    1991-12-01

    Transfected oncogenes have been reported to increase the radioresistance of rodent cells Whether transfected nononcogenic DNA sequences and subsequent clonal selection can result in radioresistant cell populations is unknown. The present set of experiments describe the in vitro radiosensitivity and tumorigenicity of selected clones of primary rat embryo cells and human glioblastoma cells, after transfection with a neomycin-resistance marker (pSV2neo or pCMVneo) and clonal selection. Radiobiological data comparing the surviving fraction at 2 Gy (SF{sub 2}) and the mean inactivation dose shown the induction of radioresistance in two rat embryo cell clones and one glioblastoma clone, as compared to untransfected cells. Wild-type and transfectant clones were injected into three strains of immune-deficient mice (scid, NIH, and nu/nu) to assay for tumorigenicity and metastatic potential. only the glioblastoma parent line and its transfectant clones were tumorigenic. The results show that transfection of a neomycin-resistance marker and clonal selection can impart radioresistance on both normal and tumor cells. The work also indicates that altered radiation sensitivity does not necessarily correlate with changes in cell-cycle kinetics at the time of irradiation, tumorigenicity, or altered metastatic potential. The findings have critical implications for transfection studies investigating determinants of cellular radiosensitivity.

  12. Role of transfection and clonal selection in mediating radioresistance.

    PubMed Central

    Pardo, F S; Bristow, R G; Taghian, A; Ong, A; Borek, C

    1991-01-01

    Transfected oncogenes have been reported to increase the radioresistance of rodent cells. Whether transfected nononcogenic DNA sequences and subsequent clonal selection can result in radioresistant cell populations is unknown. The present set of experiments describe the in vitro radiosensitivity and tumorigenicity of selected clones of primary rat embryo cells and human glioblastoma cells, after transfection with a neomycin-resistance marker (pSV2neo or pCMVneo) and clonal selection. Radiobiological data comparing the surviving fraction at 2 Gy (SF2) and the mean inactivation dose show the induction of radioresistance in two rat embryo cell clones and one glioblastoma clone, as compared to untransfected cells. Wild-type and transfectant clones were injected into three strains of immune-deficient mice (scid, NIH, and nu/nu) to assay for tumorigenicity and metastatic potential. Only the glioblastoma parent line and its transfectant clones were tumorigenic. None of the cells produced spontaneous or experimentally induced metastases. Flow cytometric analyses indicated that the induction of radioresistance could not be attributed to changes in cell kinetics at the time of irradiation. Our results show that transfection of a neomycin-resistance marker and clonal selection can impart radioresistance on both normal and tumor cells. The work also indicates that altered radiation sensitivity does not necessarily correlate with changes in cell-cycle kinetics at the time of irradiation, tumorigenicity, or altered metastatic potential. Our findings have critical implications for transfection studies investigating determinants of cellular radiosensitivity. PMID:1961732

  13. [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.

  14. 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.

  15. Functional characterisation of a natural androgen receptor missense mutation (N771H) causing human androgen insensitivity syndrome.

    PubMed

    Cai, J; Cai, L-Q; Hong, Y; Zhu, Y-S

    2012-05-01

    Androgen insensitivity syndrome (AIS) is an X-linked disorder due to mutations of androgen receptor (AR) gene. Various AR mutations have been identified, and the characterisation of these mutations greatly facilitates our understanding of AR structure-function. In this study, we have analysed an AR missense mutation (N771H) identified in patients with AIS. Functional analysis of the mutant AR was performed by in vitro mutagenesis-cotransfection assays. Compared to the wild-type AR, the dose-response curve of dihydrotestosterone-induced transactivation activity in the mutant AR was greatly shifted to the right and significantly decreased. However, the maximal efficacy of transactivation activity in the mutant AR was similar to that of the wild type. Receptor binding assay indicated that the mutant AR had an approximately 2.5-fold lower binding affinity to dihydrotestosterone compared to the wild type. Western blot analysis showed that the size and the expression level of mutant AR in transfected cells were comparable to the wild type. These data underscore the importance of asparagine at amino acid position 771 of human AR in normal ligand binding and normal receptor function, and a mutation at this position results in androgen insensitivity in affected subjects.

  16. Transfection of insect cell in suspension for efficient baculovirus generation.

    PubMed

    Roest, S; Kapps-Fouthier, S; Klopp, J; Rieffel, S; Gerhartz, B; Shrestha, B

    2016-01-01

    Baculovirus (BV) mediated insect cell expression system utilizes transfection as a first step to introduce recombinant baculovirus DNA into insect cells. Many labs are still relying on the conventional liposome based transfection method in adherent culture. Here we describe a more efficient method that can replace the existing method. This method is economical and does not require any special adjustment in existing labs. •An innovative method of transfecting insect cells in suspension using polyethyleneimine (PEI) is described here.•The beauty of this method is minimal intermediate manipulation of culture during transfection and virus generation.•The method significantly reduces the chances of cross contamination of viruses while handling multiple targets and constructs as well as the other microbial contamination. PMID:27222826

  17. Cationic Phospholipids Forming Cubic Phases: Lipoplex Structure and Transfection Efficiency

    SciTech Connect

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

    2008-10-29

    The transfection activity and the phase behavior of two novel cationic O-alkyl-phosphatidylcholines, 1,2-dioleoyl-sn-glycero-3-hexylphosphocholine (C6-DOPC) and 1,2-dierucoyl-sn-glycero-3-ethylphosphocholine (di22:1-EPC), have been examined with the aim of more completely understanding the mechanism of lipid-mediated DNA delivery. Both lipids form cubic phases: C6-DOPC in the entire temperature range from -10 to 90 C, while di22:1-EPC exhibits an irreversible lamellar-cubic transition between 50 and 70 C on heating. The lipoplexes formed by C6-DOPC arrange into hexagonal phase, while the lipoplexes of di22:1-EPC are lamellar. Both lipids exhibit lower transfection activity than the lamellar-forming 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EDOPC). Thus, for the studied cationic phospholipid-DNA systems, the lipoplex phase state is a factor that does not seem to correlate with transfection activity. The parameter that exhibits better correlation with the transfection activity within the present data set is the phase state of the lipid dispersion prior to the addition of DNA. Thus, the lamellar lipid dispersion (EDOPC) produces more efficient lipoplexes than the dispersion with coexisting lamellar and cubic aggregates (diC22:1-EPC), which is even more efficient than the purely cubic dispersions (C6-DOPC; diC22:1-EPC after heating). It could be inferred from these data and from previous research that cubic phase lipid aggregates are unlikely to be beneficial to transfection. The lack of correlation between the phase state of lipoplexes and their transfection activity observed within the present data set does not mean that lipid phase state is generally unimportant for lipofection: a viewpoint now emerging from our previous studies is that the critical factor in lipid-mediated transfection is the structural evolution of lipoplexes within the cell, upon interacting and mixing with cellular lipids.

  18. Optimization of lentiviral vector production using polyethylenimine-mediated transfection.

    PubMed

    Tang, Yong; Garson, Kenneth; Li, Li; Vanderhyden, Barbara C

    2015-01-01

    The aim of the present study was to optimize the polyethylenimine (PEI)-mediated transfection method in order to simplify the efficient production of lentiviral vectors (LvVs), and to compare the CaPO4- and PEI-mediated transfection methods for producing LvVs. Different titration methods of LvV stocks, as well as different culture media, culture durations, cell densities and DNA quantities were compared to obtain an optimized procedure for the production of LvVs. Optimization of the production method for LvVs was achieved using PEI-mediated transient transfections. Serum-free Opti-MEM(®) was used to directly produce LvVs that could be harvested 48 h after transfection. Furthermore, a cell density of 15×10(6) cells/10-cm plate and a DNA concentration of 1X were selected for the optimum production of LvVs. The optimized LvV titration method was simple and direct; it involved LvVs carrying fluorescent reporters, which proved to be faster than the standard methods but equally as sensitive. In conclusion, a scalable process for production of LvVs by PEI-mediated transfection was established and optimized. The optimized PEI-mediated transfection method was easy to use, as well as providing greater reliability with a higher degree of reproducibility and consistency. Despite using less DNA, the PEI-mediated transfection method resulted in viral titers that were the same as those achieved using the CaPO4-mediated method.

  19. 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. PMID:25968524

  20. Integrated Experiment and Modeling of Insensitive High Explosives

    NASA Astrophysics Data System (ADS)

    Stewart, D. Scott; Lambert, David E.; Yoo, Sunhee; Lieber, Mark; Holman, Steven

    2009-12-01

    New design paradigms for insensitive high explosives are being sought for use in munitions applications that require enhanced safety, reliability and performance. We describe recent work of our group that uses an integrated approach to develop predictive models, guided by experiments. Insensitive explosive can have relatively longer detonation reaction zones and slower reaction rates than their sensitive counterparts. We employ reactive flow models that are constrained by detonation shock dynamics (DSD) to pose candidate predictive models. We discuss the variation of the pressure dependent reaction rate exponent and reaction order on the length of the supporting reaction zone, the detonation velocity curvature relation, the computed critical energy required for initiation, the relation between the diameter effect curve and the corresponding normal detonation velocity curvature relation.

  1. Integrated Experiment and Modeling of Insensitive High Explosives

    NASA Astrophysics Data System (ADS)

    Stewart, D. Scott; Lambert, David E.; Yoo, Sunhee; Lieber, M.; Holman, Steven

    2009-06-01

    New design paradigms for insensitive high explosives are being sought for use in munitions applications that require enhanced, safety, reliability and performance. We describe recent work of our group that uses an integrated approach to develop predictive models, guided by experiments. Insensitive explosive can have relatively longer detonation reaction zones and slower reaction rates than their sensitive counterparts. We employ reactive flow models that are constrained by detonation shock dynamics to pose candidate predictive models. We discuss variation of the pressure dependent reaction rate exponent and reaction order, on the length of the supporting reaction zone, the detonation velocity curvature relation, computed critical energy required for initiation, the relation between the diameter effect curve and the corresponding normal detonation velocity curvature relation. We discuss representative characterization experiments carried out at Eglin, AFB and the constraints imposed on models by a standardized experimental characterization sequence.

  2. 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.

  3. Hig Resolution Seismometer Insensitive to Extremely Strong Magnetic Fields

    SciTech Connect

    Abramovich, Igor A

    2009-07-14

    A highly sensitive broadband seismic sensor has been developed successfully to be used in beam focusing systems of particale accelerators. The sensor is completely insensitive to extremely strong magnetic fields and to hard radiation conditions that exist at the place of their installation. A unique remote sensor calibration method has been invented and implemented. Several such sensors were sold to LAPP (LAPP-IN2P3/CNRS-Université de Savoie; Laboratoire d'Annecy-le-Vieux de Physique des Particules)

  4. Broadband polarization-insensitive absorber based on gradient structure metamaterial

    NASA Astrophysics Data System (ADS)

    Yang, Guo-Hui; Liu, Xiao-Xin; Lv, Yue-Long; Fu, Jia-Hui; Wu, Qun; Gu, Xuemai

    2014-05-01

    Metamaterial absorber (MA) is a hot spot in the research on electromagnetic absorbers. In this paper, a metamaterial based broadband polarization-insensitive absorber is proposed. The absorber is fabricated with FR-4 dielectric substrate foiled with copper. The top layer of the unit cell of the MA is composed of resistors mounted crosswire and gradient split ring resonator (SRR) with a square metal patch (SMP) in it. The overall structure is symmetrical, which makes the MA polarization-insensitive. The gradient SRRs and SMPs resonate at adjacent frequencies resulting in broadband property. The absorption rates of the MA for TE and TM wave are calculated through the simulated S-parameters. The bandwidth is 9.9 GHz, where the absorption rate maintains 60% up to 98.28% in both cases and the relative bandwidth is 57.13%. Both broadband and polarization-insensitivity properties are achieved, which demonstrate promising application prospect of the proposed MA in shielding and stealth technology.

  5. 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.

  6. Evaluation of the magnetic field requirements for nanomagnetic gene transfection

    PubMed Central

    Fouriki, A.; Farrow, N.; Clements, M.A.; Dobson, J.

    2010-01-01

    The objective of this work was to examine the effects of magnet distance (and by proxy, field strength) on nanomagnetic transfection efficiency. Methods non-viral magnetic nanoparticle-based transfection was evaluated using both static and oscillating magnet arrays. Results Fluorescence intensity (firefly luciferase) of transfected H292 cells showed no increase using a 96-well NdFeB magnet array when the magnets were 5 mm from the cell culture plate or nearer. At 6 mm and higher, fluorescence intensity decreased systematically. Conclusion In all cases, fluorescence intensity was higher when using an oscillating array compared to a static array. For distances closer than 5 mm, the oscillating system also outperformed Lipofectamine 2000™. PMID:22110859

  7. Transient transfection of mammalian cells using a violet diode laser

    NASA Astrophysics Data System (ADS)

    Torres-Mapa, Maria Leilani; Angus, Liselotte; Ploschner, Martin; Dholakia, Kishan; Gunn-Moore, Frank J.

    2010-07-01

    We demonstrate the first use of the violet diode laser for transient mammalian cell transfection. In contrast to previous studies, which showed the generation of stable cell lines over a few weeks, we develop a methodology to transiently transfect cells with an efficiency of up to ~40%. Chinese hamster ovary (CHO-K1) and human embryonic kidney (HEK293) cells are exposed to a tightly focused 405-nm laser in the presence of plasmid DNA encoding for a mitochondrial targeted red fluorescent protein. We report transfection efficiencies as a function of laser power and exposure time for our system. We also show, for the first time, that a continuous wave laser source can be successfully applied to selective gene silencing experiments using small interfering RNA. This work is a major step towards an inexpensive and portable phototransfection system.

  8. Physicochemical and transfection properties of cationic Hydroxyethylcellulose/DNA nanoparticles.

    PubMed

    Fayazpour, Farzaneh; Lucas, Bart; Alvarez-Lorenzo, Carmen; Sanders, Niek N; Demeester, Jo; De Smedt, Stefaan C

    2006-10-01

    In this study the physicochemical and transfection properties of cationic hydroxyethylcellulose/plasmid DNA (pDNA) nanoparticles were investigated and compared with the properties of DNA nanoparticles based on polyethylene imine (PEI), which is widely investigated as a gene carrier. The two types of cationic hydroxyethylcelluloses studied, polyquaternium-4 (PQ-4) and polyquaternium-10 (PQ-10), are already commonly used in cosmetic and topical drug delivery devices. Both PQ-4 and PQ-10 spontaneously interact with pDNA with the formation of nanoparticles approximately 200 nm in size. Gel electrophoresis and fluorescence dequenching experiments indicated that the interactions between pDNA and the cationic celluloses were stronger than those between pDNA and PEI. The cationic cellulose/pDNA nanoparticles transfected cells to a much lesser extent than the PEI-based pDNA nanoparticles. The low transfection property of the PQ-4/pDNA nanoparticles was attributed to their neutrally charged surface, which does not allow an optimal binding of PQ-4/pDNA nanoparticles to cellular membranes. Although the PQ-10/pDNA nanoparticles were positively charged and thus expected to be taken up by cells, they were also much less efficient in transfecting cells than were PEI/pDNA nanoparticles. Agents known to enhance the endosomal escape were not able to improve the transfection properties of PQ-10/pDNA nanoparticles, indicating that a poor endosomal escape is, most likely, not the major reason for the low transfection activity of PQ-10/pDNA nanoparticles. We hypothesized that the strong binding of pDNA to PQ-10 prohibits the release of pDNA from PQ-10 once the PQ-10/pDNA nanoparticles arrive in the cytosol of the cells. Tailoring the nature and extent of the cationic side chains on this type of cationic hydroxyethylcellulose may be promising to further enhance their DNA delivery properties.

  9. Physicochemical and transfection properties of cationic Hydroxyethylcellulose/DNA nanoparticles.

    PubMed

    Fayazpour, Farzaneh; Lucas, Bart; Alvarez-Lorenzo, Carmen; Sanders, Niek N; Demeester, Jo; De Smedt, Stefaan C

    2006-10-01

    In this study the physicochemical and transfection properties of cationic hydroxyethylcellulose/plasmid DNA (pDNA) nanoparticles were investigated and compared with the properties of DNA nanoparticles based on polyethylene imine (PEI), which is widely investigated as a gene carrier. The two types of cationic hydroxyethylcelluloses studied, polyquaternium-4 (PQ-4) and polyquaternium-10 (PQ-10), are already commonly used in cosmetic and topical drug delivery devices. Both PQ-4 and PQ-10 spontaneously interact with pDNA with the formation of nanoparticles approximately 200 nm in size. Gel electrophoresis and fluorescence dequenching experiments indicated that the interactions between pDNA and the cationic celluloses were stronger than those between pDNA and PEI. The cationic cellulose/pDNA nanoparticles transfected cells to a much lesser extent than the PEI-based pDNA nanoparticles. The low transfection property of the PQ-4/pDNA nanoparticles was attributed to their neutrally charged surface, which does not allow an optimal binding of PQ-4/pDNA nanoparticles to cellular membranes. Although the PQ-10/pDNA nanoparticles were positively charged and thus expected to be taken up by cells, they were also much less efficient in transfecting cells than were PEI/pDNA nanoparticles. Agents known to enhance the endosomal escape were not able to improve the transfection properties of PQ-10/pDNA nanoparticles, indicating that a poor endosomal escape is, most likely, not the major reason for the low transfection activity of PQ-10/pDNA nanoparticles. We hypothesized that the strong binding of pDNA to PQ-10 prohibits the release of pDNA from PQ-10 once the PQ-10/pDNA nanoparticles arrive in the cytosol of the cells. Tailoring the nature and extent of the cationic side chains on this type of cationic hydroxyethylcellulose may be promising to further enhance their DNA delivery properties. PMID:17025362

  10. ERBB2 increases metastatic potentials specifically in androgen-insensitive prostate cancer cells.

    PubMed

    Tome-Garcia, Jessica; Li, Dan; Ghazaryan, Seda; Shu, Limin; Wu, Lizhao

    2014-01-01

    Despite all the blood-based biomarkers used to monitor prostate cancer patients, prostate cancer remains as the second common cause of cancer mortality in men in the United States. This is largely due to a lack of understanding of the molecular pathways that are responsible for the aggressive forms of prostate cancers, the castrate-resistant prostate cancer and the metastatic prostate cancer. Cell signaling pathways activated by the ERBB2 oncogene or the RAS oncogene are frequently found to be altered in metastatic prostate cancers. To evaluate and define the role of the ERBB2/RAS pathway in prostate cancer metastasis, we have evaluated the impact of ERBB2- or RAS-overexpression on the metastatic potentials for four prostate cancer cell lines derived from tumors with different androgen sensitivities. To do so, we transfected the human DU145, LnCaP, and PC3 prostate cancer cells and the murine Myc-CaP prostate cancer cells with the activated form of ERBB2 or H-RAS and assessed their metastatic potentials by three complementary assays, a wound healing assay, a transwell motility assay, and a transwell invasion assay. We showed that while overexpression of ERBB2 increased the metastatic potential of the androgen-insensitive prostate cancer cells (i.e. PC3 and DU145), it did not affect metastatic potentials of the androgen-sensitive prostate cancer cells (i.e. LnCaP and Myc-CaP). In contrast, overexpression of H-RAS only increased the cell motility of Myc-CaP cells, which overexpress the human c-MYC oncogene. Our data suggest that ERBB2 collaborates with androgen signaling to promote prostate cancer metastasis, and that although RAS is one of the critical downstream effectors of ERBB2, it does not phenocopy ERBB2 for its impact on the metastatic potentials of prostate cancer cell lines. PMID:24937171

  11. 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.

  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. Resonant coupling in trenched bend-insensitive optical fiber.

    PubMed

    Ren, Guobin; Lin, Zhen; Zheng, Siwen; Jian, Shuisheng

    2013-03-01

    We report in this Letter the resonant coupling mechanism in bending trenched bend-insensitive fiber (BIF). It is found that among the trench parameters, the core-trench distance is predominant for optimized BIF design. We reveal that resonant coupling is an intrinsic characteristic of bending trenched BIF, and resonant coupling between the fiber core and the innermost cladding would limit the ultimate bending loss of BIF under tight bend. Resonant coupling is also present in double-trenched BIF, and would impair its bending performance.

  14. [Cashmere goat bacterial artificial chromosome recombination and cell transfection system].

    PubMed

    Huang, Tian; Cao, Zhongyang; Yang, Yaohui; Cao, Gengsheng

    2016-03-01

    The Cashmere goat is mainly used to produce cashmere, which is very popular for its delicate fiber, luscious softness and natural excellent warm property. Keratin associated protein (KAP) and bone morphogenetic protein (BMP) of the Cashmere goat play an important role in the proliferation and development of cashmere fiber follicle cells. Bacterial artificial chromosome containing kap6.3, kap8.1 and bmp4 genes were used to increase the production and quality of Cashmere. First, we constructed bacterial artificial chromosomes by homology recombination. Then Tol2 transposon was inserted into bacterial artificial chromosomes that were then transfected into Cashmere goat fibroblasts by Amaxa Nucleofector technology according to the manufacture's instructions. We successfully constructed the BAC-Tol2 vectors containing target genes. Each vector contained egfp report gene with UBC promoter, Neomycin resistant gene for cell screening and two loxp elements for resistance removing after transfected into cells. The bacterial artificial chromosome-Tol2 vectors showed a high efficiency of transfection that can reach 1% to 6% with a highest efficiency of 10%. We also obtained Cashmere goat fibroblasts integrated exogenous genes (kap6.3, kap8.1 and bmp4) preparing for the clone of Cashmere goat in the future. Our research demonstrates that the insertion of Tol2 transposons into bacterial artificial chromosomes improves the transfection efficiency and accuracy of bacterial artificial chromosome error-free recombination.

  15. Small Interfering RNA Transfection Across a Phospholipid Membrane

    NASA Astrophysics Data System (ADS)

    Ngo, Van; Choubey, Amit; Kalia, Rajiv; Nakano, Aiichiro; Vashishta, Priya

    2012-02-01

    Small interfering RNA (siRNA) molecules play a pivotal role in silencing gene expression via the RNA interference mechanism. We have performed steered MD simulations to study the transfection of a bare siRNA and siRNA/Oleic Acid (OA) complex across the dipalmitoylphosphatidycholine (DPPC) bilayer at T = 323 K. Bare siRNA induces the formation of frustrated lipid gel domains, whereas in the presence of siRNA/OA complex the membrane is found to be in the liquid-ordered phase. In both cases the stress profiles across the membrane indicate that the membrane is under tension near the head groups and highly compressed at the water-hydrophobic interface. During transfection, the membrane is deformed and the lateral stress is significantly lowered for the bare siRNA and siRNA/OA complex. The bare siRNA transfects through a lipid-nanopore of hydrophilic head-groups and hydrophobic carbon chains, whereas the siRNA/OA complex transfects through a lipid-nanopore of hydrophilic head groups.

  16. Optical transfection using an endoscope-like system

    NASA Astrophysics Data System (ADS)

    Ma, Nan; Gunn-Moore, Frank; Dholakia, Kishan

    2011-02-01

    Optical transfection is a powerful method for targeted delivery of therapeutic agents to biological cells. A tightly focused pulsed laser beam may transiently change the permeability of a cell membrane to facilitate the delivery of foreign genetic material into cells. We report the first realization of an endoscope-like integrated system for optical transfection. An imaging fiber (coherent optical fiber bundle) with ~6000 cores (pixels) embedded in a fiber cladding of ~300 μm in diameter, produces an image circle (area) of ~270 μm diam. This imaging fiber, with an ordered axicon lens array chemically etched at its exit face, is used for the delivery of a femtosecond laser to the cell membrane for optical transfection along with subcellular resolution imaging. A microcapillary-based microfluidic system for localized drug delivery was also combined in this miniature, flexible system. Using this novel system, a plasmid transfection efficiency up to ~72% was obtained for CHO-K1 cells. This endoscope-like system opens a range of exciting applications, in particular, in the targeted in vivo optical microsurgery area.

  17. Carbon nanoparticles for gene transfection in eukaryotic cell lines.

    PubMed

    Zanin, H; Hollanda, L M; Ceragioli, H J; Ferreira, M S; Machado, D; Lancellotti, M; Catharino, R R; Baranauskas, V; Lobo, A O

    2014-06-01

    For the first time, oxygen terminated cellulose carbon nanoparticles (CCN) was synthesised and applied in gene transfection of pIRES plasmid. The CCN was prepared from catalytic of polyaniline by chemical vapour deposition techniques. This plasmid contains one gene that encodes the green fluorescent protein (GFP) in eukaryotic cells, making them fluorescent. This new nanomaterial and pIRES plasmid formed π-stacking when dispersed in water by magnetic stirring. The frequencies shift in zeta potential confirmed the plasmid strongly connects to the nanomaterial. In vitro tests found that this conjugation was phagocytised by NG97, NIH-3T3 and A549 cell lines making them fluorescent, which was visualised by fluorescent microscopy. Before the transfection test, we studied CCN in cell viability. Both MTT and Neutral Red uptake tests were carried out using NG97, NIH-3T3 and A549 cell lines. Further, we use metabolomics to verify if small amounts of nanomaterial would be enough to cause some cellular damage in NG97 cells. We showed two mechanisms of action by CCN-DNA complex, producing an exogenous protein by the transfected cell and metabolomic changes that contributed by better understanding of glioblastoma, being the major finding of this work. Our results suggested that this nanomaterial has great potential as a gene carrier agent in non-viral based therapy, with low cytotoxicity, good transfection efficiency, and low cell damage in small amounts of nanomaterials in metabolomic tests.

  18. [Cashmere goat bacterial artificial chromosome recombination and cell transfection system].

    PubMed

    Huang, Tian; Cao, Zhongyang; Yang, Yaohui; Cao, Gengsheng

    2016-03-01

    The Cashmere goat is mainly used to produce cashmere, which is very popular for its delicate fiber, luscious softness and natural excellent warm property. Keratin associated protein (KAP) and bone morphogenetic protein (BMP) of the Cashmere goat play an important role in the proliferation and development of cashmere fiber follicle cells. Bacterial artificial chromosome containing kap6.3, kap8.1 and bmp4 genes were used to increase the production and quality of Cashmere. First, we constructed bacterial artificial chromosomes by homology recombination. Then Tol2 transposon was inserted into bacterial artificial chromosomes that were then transfected into Cashmere goat fibroblasts by Amaxa Nucleofector technology according to the manufacture's instructions. We successfully constructed the BAC-Tol2 vectors containing target genes. Each vector contained egfp report gene with UBC promoter, Neomycin resistant gene for cell screening and two loxp elements for resistance removing after transfected into cells. The bacterial artificial chromosome-Tol2 vectors showed a high efficiency of transfection that can reach 1% to 6% with a highest efficiency of 10%. We also obtained Cashmere goat fibroblasts integrated exogenous genes (kap6.3, kap8.1 and bmp4) preparing for the clone of Cashmere goat in the future. Our research demonstrates that the insertion of Tol2 transposons into bacterial artificial chromosomes improves the transfection efficiency and accuracy of bacterial artificial chromosome error-free recombination. PMID:27349114

  19. 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.

  20. Stimulation of human Jurkat cells by monoclonal antibody crosslinking of transfected-Ly-6A.2 (TAP) molecules.

    PubMed

    McGrew, J T; Rock, K L

    1991-10-01

    Monoclonal antibody crosslinking of phosphatidylinositol-anchored Ly-6A.2 molecules on the surface of murine T lymphocytes leads to cell activation and secretion of IL-2. To examine the potential activity of these molecules in human T cells we transfected the Ly-6A.2 gene into Jurkat cells. Transfection of Jurkat cells with genomic Ly-6A.2 sequences results in low levels of Ly-6A.2 on the cell surface. However, linking the Ly-6A.2 sequences to the enhancer from the human CD2 gene results in greatly increased expression of Ly-6A.2. These molecules are anchored to the membrane via a phosphatidylinositol linkage. Crosslinking of Ly-6A.2 molecules with soluble mAb stimulates the transfected Jurkat cells to produce IL-2. This stimulation is abrogated by treatment with phosphatidylinositol-specific phospholipase C. The transfected human T cells displayed the same unusual crosslinking requirements for stimulation with anti-Ly-6A.2 mAbs as previously observed for murine T cells. Crosslinking of Ly-6A.2 with soluble antibodies is stimulatory, whereas immobilized antibodies are inactive. The crosslinking requirements for antiCD3 mAb stimulation display a reciprocal pattern. These data demonstrate that the Ly-6A.2 pathway for T cell activation is conserved between human and murine T cells.

  1. Study of mechanisms of electric field-induced DNA transfection. III. Electric parameters and other conditions for effective transfection.

    PubMed

    Xie, T D; Tsong, T Y

    1992-07-01

    Electric parameters, osmolality, temperature, and pH of the suspending medium and the growth phase of cells, etc., are known to influence the efficiency of the pulsed electric field (PEF)-induced DNA transfection of cells. PEF-induced transfection of Escherichia coli JM105 by plasmid DNA PUC18, PUC19, PBR322, and PMSG has been used as a model system to establish quantitative relationships between these parameters and transfection efficiency. The main findings are summarized for experiments using unipolar square wave PEF. (a) For a given field strength (up to 6 kV/cm), the transfection efficiency (TE) was linearly dependent on the pulse width (up to 1 ms). (b) When field strength is fixed, Log [TE] correlated with the number of pulses applied. Similarly, when field duration was fixed, Log [TE] correlated with the number of pulses. (c) In the absence of MgCl2, TE showed a maximal value at 50 mM sucrose and was reduced by several fold at lower and higher sucrose concentrations. Cell survival was nearly constant in the range 1-300 mM sucrose. (d) E. coli in the early and mid-exponential growth phases was more susceptible to PEF for DNA transfection than it was in the stationary phase. (e) For a given set of electric parameters, TE was the highest at neutral pH and was greatly reduced at acidic and alkaline pH. (f) Increasing the temperature from 0 to 37 degrees C resulted in the reduction of TE by three orders of magnitude. This could reflect a rapid shrinking of pores at higher temperatures. (g) TE was inversely proportional to the square of the size of the plasmid DNA. By adjusting the above parameters to optimize transfection, a TE of 1010 1microg-1 DNA (PUC18) has been recorded. Further improvement in percent cell transfection may be expected by a more exhaustive search of conditions than the present study has done.

  2. 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.

  3. Surface modified gold nanowires for mammalian cell transfection

    NASA Astrophysics Data System (ADS)

    Kuo, Chiung-Wen; Lai, Jun-Jung; Wei, Kung Hwa; Chen, Peilin

    2008-01-01

    Aminothiol modified gold nanowires have been used as vectors for the delivery of plasmid DNA into two different types of mammalian cells: 3T3 and HeLa. It was measured that positively charged gold nanowires with a diameter of 200 nm and a length around 5 µm were capable of carrying 1 pg of plasmid DNA per nanowire into cells. Compared with other transfection reagents, the gold nanowires exhibited the highest transfection efficiency while almost no cytotoxicity was observed. In addition, it has been shown that individual nanowires can be visualized with sub-micrometer resolution, which may allow the use of functionalized multi-segment nanowires as local probes for the investigation of the microenvironment inside cells.

  4. 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

  5. Pleasure-related analgesia activates opioid-insensitive circuits.

    PubMed

    Kut, Elvan; Candia, Victor; von Overbeck, Jan; Pok, Judit; Fink, Daniel; Folkers, Gerd

    2011-03-16

    Recent findings suggest that pain and pleasure share common neurochemical circuits, and studies in animals and humans show that opioid-mediated descending pathways can inhibit or facilitate pain. We explored the role of endogenous opioid neurotransmission in pleasure-related analgesia. μ-Opioidergic activity was blocked with 0.2 mg/kg naloxone to assess its effects on hedonic responses to pleasant emotional pictures (International Affective Picture System) and its modulating effects on heat pain tolerance. Naloxone did not alter subjective and autonomous reactions to pleasure induction or overall mood of participants. In addition, pleasure-related increases in pain tolerance persisted after reversal of endogenous μ-opioidergic neurotransmission. Subjective pain intensity and unpleasantness ratings increased after naloxone administration. These findings suggest that, in addition to opioid-sensitive circuits, mainly opioid-insensitive pain-modulating circuits are activated during pleasure-related analgesia. PMID:21411655

  6. 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.

  7. Asynchronous and timing jitter insensitive data echo cancellation

    NASA Astrophysics Data System (ADS)

    Messerschmitt, David G.

    1986-12-01

    An approach to the implementation of asynchronous and timing jitter insensitive data echo cancellation is described. This approach introduces a small amount of jitter in the transmitted data signal, or alternatively in the received signal sampling, and uses a simple digital phase-locked loop together with the storage of two sets of echo canceller coefficients. The effect of derived timing jitter on the echo cancellation accuracy is completely eliminated for a loop timed transceiver (as in a digital subscriber loop network termination transceiver), and is easily reduced to negligible levels for a nonloop timed transceiver (as in a digital subscriber loop line card transceiver or a voiceband data modem). In the case of a voiceband data modem, this approach is one method to achieve asynchronous echo cancellation without the need to recover and resample a continuous-time far-end data signal.

  8. Analytical Methods for Detonation Residues of Insensitive Munitions

    NASA Astrophysics Data System (ADS)

    Walsh, Marianne E.

    2016-01-01

    Analytical methods are described for the analysis of post-detonation residues from insensitive munitions. Standard methods were verified or modified to obtain the mass of residues deposited per round. In addition, a rapid chromatographic separation was developed and used to measure the mass of NTO (3-nitro-1,2,4-triazol-5-one), NQ (nitroguanidine) and DNAN (2,4-dinitroanisole). The HILIC (hydrophilic-interaction chromatography) separation described here uses a trifunctionally-bonded amide phase to retain the polar analytes. The eluent is 75/25 v/v acetonitrile/water acidified with acetic acid, which is also suitable for LC/MS applications. Analytical runtime was three minutes. Solid phase extraction and LC/MS conditions are also described.

  9. 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.

  10. 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

  11. Application of composite materials to impact-insensitive munitions

    NASA Technical Reports Server (NTRS)

    Neradka, Vincent F.; Chang, Yale; Grady, Joseph E.; Trowbridge, Daniel A.

    1992-01-01

    An approach is outlined for developing bullet-impact-insensitive munitions based on composite materials that provide rapid venting of the rocket-motor case. Impact experiments are conducted with test specimens of hybrid laminates of graphite/epoxy and epoxy reinforcing with woven glass fibers. The dynamic strain response and initial impact force are measured with strain gauges, and perforation damage is examined in the plates. The results show that impact damage can be designed by means of parametric variations of the fiber, matrix, and ply orientations. It is suggested that rocket-motor cases can be designed with composite materials to provide rapid venting during the failure mode. The experimental ballistic testing performed provides data that can be used comparatively with analytical data on composite materials.

  12. 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).

  13. 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).

  14. High Throughput siRNA Screening Using Reverse Transfection.

    PubMed

    von Schantz, Carina; Saarela, Jani

    2016-01-01

    RNA interference (RNAi) is a commonly used technique to knockdown gene function. Here, we describe a high throughput screening method for siRNA mediated gene silencing of the breast cancer cell line MDA-MB-231 using reverse transfection. Furthermore, we describe the setup for two separate methods for detecting viable and dead cells using either homogenous assays or image-based analysis. PMID:27581282

  15. Ultrasonic enhancement of gene transfection in murine melanoma tumors.

    PubMed

    Miller, D L; Bao, S; Gies, R A; Thrall, B D

    1999-11-01

    The enhancement of gene transfection by ultrasound (US) was evaluated in vitro and in vivo using the B16 mouse melanoma model. Cultured cells were either exposed in suspensions in vitro or implanted subcutaneously in female C57BL/6 mice for 10-14 days and, subsequently exposed, in vivo. For comparison to results with a luciferase plasmid, a reporter plasmid for green fluorescent protein (GFP) was used to evaluate transfection efficiency. US was supplied by a system, similar to a Dornier HM-3 lithotripter, that produced shock waves (SW) of 24.4 MPa peak positive and 5.2 MPa peak negative pressure amplitudes at the focus. The plasmids were mixed with the suspensions to achieve 20 ,microL mL(-1), or were injected intratumorally to provide 0.2 mg DNA per mL of tumor. Acoustic cavitation was promoted by retaining 0.2 mL of air in the 1.2-mL exposure chambers in vitro and by injecting air at 10% of tumor volume in vivo. In vitro, cell counts declined to 5.3% of shams after 800 SW exposure, with 1.4% of the cells expressing GFP after 2 days of culture. In vivo, 2 days after 400 SW exposure, viable-cell recovery from excised tumors was reduced to 4.2% of shams and cell transfection was enhanced by a factor of about 8, reaching 2.5% of cell counts (p < 0.005 in t-test). These results show that strong tumor ablation induced by US shock wave treatment can be coupled with simultaneous enhancement of gene transfection. PMID:10626630

  16. 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.

  17. Isolation and Transfection of Primary Culture Bovine Retinal Pericytes.

    PubMed

    Primo, Vincent A; Arboleda-Velasquez, Joseph F

    2016-01-01

    This protocol describes an enzymatic approach for isolating homogeneous cultures of pericytes from retinas of bovine source. In summary, retinas are dissected, washed, digested, filtered, cultured in specific media to select for pericytes, and finally expanded for a low passage culture of about 14 million bovine retinal pericytes (BRP) within 4-6 weeks. This protocol also describes a liposomal-based technique for transfection of BRPs. PMID:27172949

  18. 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.

  19. DNA uptake, intracellular trafficking and gene transfection after ultrasound exposure.

    PubMed

    Liu, Ying; Yan, Jing; Santangelo, Philip J; Prausnitz, Mark R

    2016-07-28

    Ultrasound has been studied as a promising tool for intracellular gene delivery. In this work, we studied gene transfection of a human prostate cancer cell line exposed to megahertz pulsed ultrasound in the presence of contrast agent and assessed the efficiency of fluorescently labelled DNA delivery into cell nuclei, which is necessary for gene transfection. At the sonication conditions studied, ~30% of cells showed DNA uptake 30min after sonication, but that fraction decreased over time to ~10% of cells after 24h. Most cells containing DNA had DNA in their nuclei, but the amount varied significantly. Transfection efficiency peaked at ~10% at 8h post sonication. Among those cells containing DNA, ~30% of DNA was localized in the cell nuclei, ~30% was in autophagosomes/autophagolysosomes and the remainder was "free" in the cytoplasm 30min after sonication. At later times up to 24h, ~30% of DNA continued to be found in the nuclei and most or all of the rest of the DNA was in autophagosomes/autophagolysosomes. These results demonstrate that ultrasound can deliver DNA into cell nuclei shortly after sonication and that the rest of the DNA can be cleared by autophagosomes/autophagolysosomes. PMID:27165808

  20. Hormonal induction of transfected genes depends on DNA topology.

    PubMed

    Piña, B; Haché, R J; Arnemann, J; Chalepakis, G; Slater, E P; Beato, M

    1990-02-01

    Plasmids containing the hormone regulatory element of mouse mammary tumor virus linked to the thymidine kinase promoter of herpes simplex virus and the reporter gene chloramphenicol acetyltransferase of Escherichia coli respond to glucocorticoids and progestins when transfected into appropriate cells. In the human mammary tumor cell line T47D, the response to progestins, but not to glucocorticoids, is highly dependent on the topology of the transfected DNA. Although negatively supercoiled plasmids respond optimally to the synthetic progestin R5020, their linearized counterparts exhibit markedly reduced progestin inducibility. This is not due to changes in the efficiency of DNA transfection, since the amount of DNA incorporated into the cell nucleus is not significantly dependent on the initial topology of the plasmids. In contrast, cotransfection experiments with glucocorticoid receptor cDNA in the same cell line show no significant influence of DNA topology on induction by dexamethasone. A similar result was obtained with fibroblasts that contain endogenous glucocorticoid receptors. When the distance between receptor-binding sites or between the binding sites and the promoter was increased, the dependence of progestin induction on DNA topology was more pronounced. In contrast to the original plasmid, these constructs also revealed a similar topological dependence for induction by glucocorticoids. The differential influence of DNA topology is not due to differences in the affinity of the two hormone receptors for DNA of various topologies, but probably reflects an influence of DNA topology on the interaction between different DNA-bound receptor molecules and between receptors and other transcription factors.

  1. Cell transfection as a tool to study growth hormone action

    SciTech Connect

    Norstedt, G.; Enberg, B.; Francis, S.

    1994-12-31

    The isolation of growth hormone receptor (GHR) cDNA clones has made possible the transfection of GHRs into cultured cells. Our aim in this minireview is to show how the application of such approaches have benefited GHR research. GH stimulation of cells expressing GHR cDNAs can cause an alteration of cellular function that mimic those of the endogenous GHR. GHR cDNA transfected cells also offer a system where the mechanism of GH action can be studied. Such a system has been used to demonstrate that the GHR itself becomes tyrosine phosphorylated and that further phosphorylation of downstream proteins is important in GH action. The GH signals are transmitted to the nucleus and GH regulated genes have now begun to be characterized. The ability to use cell transfection for mechanistic studies of GH action will be instrumental to define domains within the receptor that are of functional importance and to determined pathways whereby GH signals are conveyed within the cell. 33 refs., 2 tabs.

  2. 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.

  3. Enhanced apoptotic response to photodynamic therapy after bcl-2 transfection.

    PubMed

    Kim, H R; Luo, Y; Li, G; Kessel, D

    1999-07-15

    Apoptosis is a cellular death process involving the sequential activation of a series of caspases, endonucleases, and other enzymes. The initiation of apoptosis can be inhibited by overexpression of bcl-2 and certain other members of a related family of proteins. We examined the effects of bcl-2 overexpression on the apoptotic response to photodynamic therapy (PDT), using aluminum phthalocyanine as the photosensitizing agent. In this study, we compared the immortalized human breast epithelial cell line MCF10A with a subline (MCF10A/bcl-2) transfected with the human bcl-2 gene. The latter was approximately 2-fold more sensitive to the phototoxic effects of PDT. At a 50 mJ/cm2 light dose, photodamage to MCF-10A/bcl-2 resulted in a greater loss of the mitochondrial membrane potential (delta(psi)m), enhanced release of mitochondrial cytochrome c, a more rapid and greater activation of caspase-3, and a greater apoptotic response. Western blot analysis revealed that the transfected cell line showed overexpression of both bcl-2 and bax, and that PDT caused selective destruction of bcl-2, leaving bax unaffected. The greater apoptotic response by the transfected line is, therefore, attributed to the higher bax:bcl-2 ratio after photodamage.

  4. 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

  5. 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. PMID:22355435

  6. 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.

  7. 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

  8. A nanoparticle formulation that selectively transfects metastatic tumors in mice

    PubMed Central

    Yang, Jian; Hendricks, William; Liu, Guosheng; McCaffery, J. Michael; Kinzler, Kenneth W.; Huso, David L.; Vogelstein, Bert; Zhou, Shibin

    2013-01-01

    Nanoparticle gene therapy holds great promise for the treatment of malignant disease in light of the large number of potent, tumor-specific therapeutic payloads potentially available for delivery. To be effective, gene therapy vehicles must be able to deliver their therapeutic payloads to metastatic lesions after systemic administration. Here we describe nanoparticles comprised of a core of high molecular weight linear polyethylenimine (LPEI) complexed with DNA and surrounded by a shell of polyethyleneglycol-modified (PEGylated) low molecular weight LPEI. Compared with a state-of-the-art commercially available in vivo gene delivery formulation, i.v. delivery of the core/PEGylated shell (CPS) nanoparticles provided more than a 16,000-fold increase in the ratio of tumor to nontumor transfection. The vast majority of examined liver and lung metastases derived from a colorectal cancer cell line showed transgene expression after i.v. CPS injection in an animal model of metastasis. Histological examination of tissues from transfected mice revealed that the CPS nanoparticles selectively transfected neoplastic cells rather than stromal cells within primary and metastatic tumors. However, only a small fraction of neoplastic cells (<1%) expressed the transgene, and the extent of delivery varied with the tumor cell line, tumor site, and host mouse strain used. Our results demonstrate that these CPS nanoparticles offer substantial advantages over previously described formulations for in vivo nanoparticle gene therapeutics. At the same time, they illustrate that major increases in the effectiveness of such approaches are needed for utility in patients with metastatic cancer. PMID:23959886

  9. 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.

  10. High precision fabrication of polarization insensitive resonant grating filters

    NASA Astrophysics Data System (ADS)

    Boye, R. R.; Peters, D. W.; Wendt, J. R.; Samora, S.; Stevens, J.; Shul, R. J.; Hunker, J.; Kellogg, R. A.; Kemme, S. A.

    2012-03-01

    Resonant subwavelength gratings have been designed and fabricated as wavelength-specific reflectors for application as a rotary position encoder utilizing ebeam based photolithography. The first grating design used a two-dimensional layout to provide polarization insensitivity with separate layers for the grating and waveguide. The resulting devices had excellent pattern fidelity and the resonance peaks and widths closely matched the expected results. Unfortunately, the gratings were particularly angle sensitive and etch depth errors led to shifts in the center wavelength of the resonances. A second design iteration resulted in a double grating period to reduce the angle sensitivity as well as different materials and geometry; the grating and waveguide being the same layer. The inclusion of etch stop layers provided more accurate etch depths; however, the tolerance to changes in the grating duty cycle was much tighter. Results from these devices show the effects of small errors in the pattern fidelity. The fabrication process flows for both iterations of devices will be reviewed as well as the performance of the fabricated devices. A discussion of the relative merits of the various design choices provides insight into the importance of fabrication considerations during the design stage.

  11. 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.

  12. 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.

  13. Bacteriophage-insensitive mutants for high quality Crescenza manufacture

    PubMed Central

    Chirico, Donatella; Gorla, Arianna; Verga, Viola; Pedersen, Per D.; Polgatti, Eliseo; Cava, Antonio; Dal Bello, Fabio

    2014-01-01

    Streptococcus thermophilus is a thermophilic lactic acid bacterium used as starter culture for the manufacture of fermented dairy products. For the production of Crescenza and other soft cheeses, Sacco has developed and provides dairies with three different defined blends of S. thermophilus strains. Each blend contains two different S. thermophilus strains. The strains were selected based on their unique technological properties as well as different phage profiles. Analysis of 133 whey samples collected in 2009–2010 from Italian dairies showed a high prevalence (about 50%) of bacteriophage attacks on the blend ST020. More specifically, the strain S. thermophilus ST1A was found to be the preferred target of the bacteriophages. A bacteriophage insensitive mutant (BIM5) of the phage-sensitive strain ST1A was successfully developed and used to substitute strain ST1A in the Crescenza starter culture ST020. The strain BIM5 showed identical technological and industrial traits as those of the phage-sensitive strain ST1A. The improved resistance of the modified Crescenza starter culture ST020R was confirmed at Italian dairies, and its effectiveness monitored on 122 whey samples collected in 2011–2012. Compared to the previous values (2009–2010), the use of the phage-hardened blend ST020R allowed reducing of frequency of phage attacks from about 50 to less than 5% of the whey samples investigated. PMID:24834065

  14. Single-shot, optical-phase-insensitive interferometry with BECs

    NASA Astrophysics Data System (ADS)

    Griffin, Paul; Robertson, Billy; MacKellar, Andrew; Halket, James; Arnold, Aidan; Riis, Erling

    2016-05-01

    Atom interferometers allow the measurement of forces through detection of the differential phase shifts induced in the atomic wavefunction by the interaction. The atomic phase can then be readout against a lab-frame reference, typically the spatial phase of an optical standing wave. This readout is a leading limitation to practical measurement, requiring long temporal stability of the optical phase, without which the resolution of the atomic signal can be lost. We have built an atom interferometer that is inherently insensitive to the phase noise of the readout system. Here, we will describe new features developed in our Bose-Einstein condensate system, including tuneable, high-fidelity, symmetric atomic-beamsplitters through a multi-pulse Kapitza-Dirac scheme. We use an atomic homodyne detection that transfers the atomic phase into a temporal atomic beat-note, and show how the entire interferometric signal can be readout in a single shot. Results from the system include measurement of small-angle projection of the gravitational force, as well as the sensitivity of the atomic phase to gradients of magnetic fields.

  15. Viable supersymmetric model with UV insensitive anomaly mediation

    SciTech Connect

    Ibe, Masahiro; Kitano, Ryuichiro; Murayama, Hitoshi

    2005-04-01

    We propose an electroweak model which is compatible with the UV insensitive anomaly-mediated supersymmetry breaking. The model is an extension of the next to minimal supersymmetric standard model (NMSSM) by adding vectorlike 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 flavor changing neutral current 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 nonthermal production from the gravitino decay. There is no gravitino problem because it decays before the big-bang nucleosynthesis era, and thus the thermal leptogenesis works. The cosmological domain wall problem inherent in the NMSSM is absent since the Z{sub 3} symmetry is broken by the QCD instanton effect in the presence of the vectorlike quarks. We also briefly comment on a possible solution to the strong CP problem a la the Nelson-Barr mechanism.

  16. 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.

  17. 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.

  18. High and low velocity detonation in a highly insensitive explosive

    NASA Astrophysics Data System (ADS)

    Sandusky, H. W.; Hayden, H. F.

    2014-05-01

    Low-velocity detonation (LVD) in a solid explosive from input shocks below the threshold for high-velocity detonation (HVD) had been previously reported for PBXN-109 in two gap tests with sample diameters of 36.5 and 73.0 mm. Similar phenomenon has now been observed for the highly insensitive PBXIH-140, whose critical diameter of ~100 mm required an even larger gap test with a sample diameter of 178 mm. When just exceeding the critical gap for HVD, LVD propagated at similar velocities as in PBXN-109 and would punch clean holes in a witness plate like HVD. For somewhat greater gaps, there was enough shock reaction to drive LVD at constant but reduced velocities as the input shock decreased to ~ ½ of critical. With a different formulation now exhibiting LVD, it may be more prevalent than previously realized. It is speculated to occur in various confinements when small percentages of easily detonable ingredients fail to initiate the remainder of less shock sensitive ingredients.

  19. 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.

  20. 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.

  1. Resolution enhanced T1-insensitive steady-state imaging.

    PubMed

    Derakhshan, Jamal J; Nour, Sherif G; Sunshine, Jeffrey L; Griswold, Mark A; Duerk, Jeffrey L

    2012-08-01

    Resolution enhanced T(1)-insensitive steady-state imaging (RE-TOSSI) is a new MRI pulse sequence for the generation of rapid T(2) contrast with high spatial resolution. TOSSI provides T(2) contrast by using nonequally spaced inversion pulses throughout a balanced steady-state free precession (SSFP) acquisition. In RE-TOSSI, these energy and time intensive adiabatic inversion pulses and associated magnetization preparation are removed from TOSSI after acquisition of the data around the center of k-space. Magnetization evolution simulations demonstrate T(2) contrast in TOSSI as well as reduction in the widening of the point spread function width (by up to a factor of 4) to a near ideal case for RE-TOSSI. Phantom experimentation is used to characterize and compare the contrast and spatial resolution properties of TOSSI, RE-TOSSI, balanced SSFP, Half-Fourier Acquisition Single-Shot Turbo Spin Echo (HASTE), and turbo spin echo and to optimize the fraction of k-space acquired using TOSSI. Comparison images in the abdomen and brain demonstrate similar contrast and improved spatial resolution in RE-TOSSI compared with TOSSI; comparison balanced SSFP, HASTE, and turbo spin echo images are provided. RE-TOSSI is capable of providing high spatial resolution T(2)-weighted images in 1 s or less per image.

  2. 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.

  3. Transient and stable transfection in the protozoan parasite Entamoeba invadens.

    PubMed

    Ehrenkaufer, Gretchen M; Singh, Upinder

    2012-07-01

    Entamoeba histolytica is an important human pathogen and a major health problem worldwide. Many aspects of parasite biology can be studied with the exception of stage conversion, which cannot be reproduced adequately in E. histolytica. The reptile parasite Entamoeba invadens is a vital model system for studying stage conversion since it can be induced to undergo both encystation and excystation with high efficiency in vitro. However, functional studies using E. invadens have been limited by the lack of genetic tools in this species. Here, we report a new method for both transient and stable transfection of E. invadens. These new tools will greatly enhance research into Entamoeba development.

  4. Preparation of Gene Gun Bullets and Biolistic Transfection of Neurons in Slice Culture

    PubMed Central

    Woods, Georgia; Zito, Karen

    2008-01-01

    Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed protocol for biolistic transfection of rat hippocampal slices, from the initial preparation of DNA coated bullets to the final shooting of the organotypic slice cultures using a gene gun. Gene gun transfection is an efficient and easy means of transfecting neurons and is especially useful for fluorescently labeling a small subset of cells in tissue slice. In this video, we first outline the steps required to coat gold particles with DNA. We next demonstrate how to line the inside of plastic tubing with the gold/DNA bullets, and how to cut this tubing to obtain the plastic cartridges for loading into the gene gun. Finally, we perform biolistic transfection of rat hippocampal slice cultures, demonstrating handling of the Bio-Rad Helios gene gun, and offering trouble shooting advice to obtain healthy and optimally transfected tissue slices. PMID:19066564

  5. Improvement of efficiency and viability in plasma gene transfection by plasma minimization and optimization electrode configuration

    NASA Astrophysics Data System (ADS)

    Jinno, Masafumi; Tachibana, Kunihide; Motomura, Hideki; Saeki, Noboru; Satoh, Susumu

    2016-07-01

    Plasma gene transfection is expected as a safe and useful method of gene transfection. However, in this method, there is difficulty in keeping both high transfection efficiency and less cell damage simultaneously. The authors have evaluated transfection efficiency and cell viability using four different plasma sources, such as arc discharge, plasma jet, dielectric barrier discharge (DBD), and microplasma. A high transfection efficiency was achieved by discharge forms in which the electric current flows via the cells. This suggested that an electric current plays an important role in plasma gene transfection. The total volume of gas flow must be small or zero and the area in which the cells are directly irradiated by plasma must be small in order to achieve a higher cell viability. The microplasma that satisfies these conditions achieved both the highest transfection efficiency and the highest cell viability simultaneously.

  6. Preparation of gene gun bullets and biolistic transfection of neurons in slice culture.

    PubMed

    Woods, Georgia; Zito, Karen

    2008-02-13

    Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed protocol for biolistic transfection of rat hippocampal slices, from the initial preparation of DNA coated bullets to the final shooting of the organotypic slice cultures using a gene gun. Gene gun transfection is an efficient and easy means of transfecting neurons and is especially useful for fluorescently labeling a small subset of cells in tissue slice. In this video, we first outline the steps required to coat gold particles with DNA. We next demonstrate how to line the inside of plastic tubing with the gold/DNA bullets, and how to cut this tubing to obtain the plastic cartridges for loading into the gene gun. Finally, we perform biolistic transfection of rat hippocampal slice cultures, demonstrating handling of the Bio-Rad Helios gene gun, and offering trouble shooting advice to obtain healthy and optimally transfected tissue slices.

  7. Down-regulation of rat mitochondrial branched-chain 2-oxoacid dehydrogenase kinase gene expression by glucocorticoids.

    PubMed Central

    Huang, Y S; Chuang, D T

    1999-01-01

    The mammalian mitochondrial branched-chain 2-oxoacid dehydrogenase (BCOD) complex is regulated by a reversible phosphorylation (inactivation)/dephosphorylation (activation) cycle. In the present study, the effects of glucocorticoids on the level of BCOD kinase mRNA were investigated in rat hepatoma cell lines (H4IIE and FTO-2B), as well as in the rat. In H4IIE cells, dexamethasone was found to significantly reduce steady-state concentrations of BCOD kinase mRNA after a 48 h culture, and this was correlated with a 2-fold increase in the dephosphorylated form of the BCOD complex. The half-life of the kinase mRNA in H4IIE cells was not affected by dexamethasone treatment. Therefore, the decrease in the steady-state kinase mRNA level resulting from dexamethasone treatment was not caused by changes in mRNA stability, which raised the possibility of regulation at the level of gene transcription. To identify the negative glucocorticoid-responsive element in the kinase promoter, nested deletion constucts in the 3.0 kb promoter region were examined in H4IIE cells cultured in the presence or absence of dexamethasone. No significant differences in promoter activity were observed on either transient or stable transfection. The data showed that the glucocorticoid-responsive element was located outside the 3. 0 kb promoter region. At the physiological level, hepatic BCOD kinase mRNA levels were reduced in rats injected intraperitoneally with dexamethasone. This effect was liver-specific, and was not detected in other tissues. These results suggest that the down-regulation of kinase gene expression by glucocorticoids is mediated through a liver-specific or -enriched transcription factor(s). PMID:10215586

  8. In vitro transformation of mouse testis cells by oncogene transfection.

    PubMed

    Morimoto, Hiroko; Lee, Jiyoung; Tanaka, Takashi; Ishii, Kei; Toyokuni, Shinya; Kanatsu-Shinohara, Mito; Shinohara, Takashi

    2012-05-01

    Germ cell tumors (GCTs) are unique in that they exhibit diverse biological characteristics and pathological features. Although several in vivo GCT models are available, studies on GCTs are hampered because in vivo development of GCTs is time consuming and prevents a detailed molecular analysis of the transformation process. Here we developed a novel strategy to transform mouse testis cells in vitro. Lentivirus-mediated transfection of dominant negative Trp53, Myc, and activated Hras1 into a CD9-expressing testis cells caused tumorigenic conversion in vitro. Although these cells resembled embryonic stem (ES) cells, they were aneuploid and lacked Nanog expression, which is involved in the maintenance of the undifferentiated state in ES cells. Euploid ES-like cells were produced by transfecting the Yamanaka factors (Pou5f1, Myc, Klf4, and Sox2) into the same cell population. Although these cells expressed Nanog, they were distinct from ES cells in that they expressed CD44, a cancer stem cell antigen. Both treatments induced similar changes in the DNA methylation patterns in differentially methylated regions of imprinted genes. Moreover, despite the differences in their phenotype and karyotype, both cell types similarly produced mixed GCTs on transplantation, which were composed of teratomas, seminomas, and embryonal carcinomas. Thus, in vitro testis cell transformation facilitates an analysis of the GCT formation process, and our results also suggest the close similarity between GCT formation and reprogramming. PMID:22357549

  9. Lipophosphoramidate-based bipolar amphiphiles: their syntheses and transfection properties.

    PubMed

    Berchel, Mathieu; Le Gall, Tony; Lozach, Olivier; Haelters, Jean-Pierre; Montier, Tristan; Jaffrès, Paul-Alain

    2016-03-14

    Six new cationic bolaamphiphiles (also called bipolar amphiphiles, bolaform amphiphiles, or bolalipids) were readily prepared by a thiol-ene click reaction that engaged a mercapto-alcohol (mercapto-ethanol or mercapto-hexanol) and a cationic based lipophosphoramidate. The cationic lipophosphoramidates contain two lipid chains that end in an alkene group and a selected cationic polar head group (trimethylammonium, dimethyl hydroxyethyl ammonium, or methylimidazolium). These compounds were formulated in water (with or without DOPE as a colipid) to produce supramolecular aggregates. These aggregates, before (i.e. bolasomes) and after (i.e. bolaplexes) mixing with plasmid DNA (pDNA) at various charge ratios, were characterized with regard to their sizes and zeta potentials. In the case of bolasomes, the suspensions were unstable since precipitation occurred after only a few hours at room temperature. On the other hand, bolaplex formulations exhibited clearly a better colloidal stability. Then, the gene delivery properties of the cationic bolasomes were investigated using two human-derived epithelial cell lines (A549 and 16HBE). Compared to the commercially available lipofection reagent (Lipofectamine), most of the cationic bolaamphiphiles were able to efficiently transfect these cells when they were formulated with DOPE in a 1 : 1 molar ratio. We report herein that bolaamphiphiles possessing a trimethylammonium or a dimethyl hydroxyethyl ammonium head group were the most efficient in terms of transfection efficiency while exhibiting no significant cytotoxicity.

  10. A robust transfection reagent for the transfection of CHO and HEK293 cells and production of recombinant proteins and lentiviral particles - PTG1.

    PubMed

    Gonçalves, Cristine; Gross, Fabian; Guégan, Philippe; Cheradame, Hervé; Midou, Patrick

    2014-11-01

    Bioproduction of recombinant proteins (r-proteins) and recombinant lentiviral particles (r-lentiviral particles) requires robust transfections consisting of efficient protocols that are easy to implement, with good reproducibility for a maximum production of proteins and lentiviral particles in a short time with low cytotoxicity. This study evaluates the capacity of histidinylated polyethyleneimine I (PTG1) to facilitate robust DNA transfection, with low cytotoxicity, of Chinese hamster ovary (CHO) and human embryonic kidney (HEK293T) cells for the production of r-proteins and r-lentiviral particles. We report that PTG1 transfection of cells in suspension with a plasmid DNA encoding enhanced green fluorescent protein leads to 72 and 97% of transfected CHO and HEK293T cells respectively, and does not significantly affect cell viability. PTG1 transfection of 100 mL of CHO-S cell culture in suspension at a cell density of 2 × 10(6) cells /mL resulted in a high level of transfected cells and protein expression after transfection with 0.75 μg/mL plasmid DNA. Transfection with PTG1 is more efficient than LipofectAmine2000™, and gene expression is higher than observed with FreeStyle™ and JetPEI®. Tri-transfection of HEK293T packaging cells leads to the production of a higher level of r-lentiviral particles compared to the calcium phosphate method, and permits two harvests of viral particles within three days. These results show that PTG1 is a powerful new transfection reagent for cell lines frequently used for recombinant protein and lentiviral particle production. PTG1 could be used in protocols for bioproduction of therapeutic proteins such as antibodies for cancer treatments and viral vectors for gene therapy applications. PMID:25215936

  11. Testosterone modulates mitochondrial aconitase in the full-length human androgen receptor-transfected PC-3 prostatic carcinoma cells.

    PubMed

    Juang, H-H; Hsieh, M-L; Tsui, K-H

    2004-08-01

    In vitro studies indicated that dihydrotestosterone (DHT) stimulates the enzymatic activity of the mitochondrial aconitase (mACON) in androgen-sensitive prostatic carcinoma cells, LNCaP. Cell proliferation assay determined that DHT doubles the optimal proliferation response of LNCaP cells. The androgen-insensitive human prostatic carcinoma cells, PC-3, were overexpressed in the human androgen receptor to assess the involvement of the native androgen receptor in the regulation by DHT of mACON gene expression. A stable-transfected clone that expresses the full-length androgen receptor was selected and termed PCAR9. The results revealed that DHT-treated PCAR9 cells paradoxically not only reduced the enzymatic activity of mACON but also blocked the biosynthesis of intracellular ATP attenuating cell proliferation. Transient gene expression assay indicated that DHT divergently regulates the promoter activity of the mACON gene in LNCaP and PCAR9 cells. This study suggested that DHT regulates mACON gene expression and the proliferation of cells in a receptor-dependent model through modulation by unidentified non-receptor factors. PMID:15291747

  12. 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

  13. 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.

  14. 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.

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

    PubMed

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

    2011-01-01

    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

  16. Immune Monitoring Using mRNA-Transfected Dendritic Cells.

    PubMed

    Borch, Troels Holz; Svane, Inge Marie; Met, Özcan

    2016-01-01

    Dendritic cells are known to be the most potent antigen presenting cell in the immune system and are used as cellular adjuvants in therapeutic anticancer vaccines using various tumor-associated antigens or their derivatives. One way of loading antigen into the dendritic cells is by mRNA electroporation, ensuring presentation of antigen through major histocompatibility complex I and potentially activating T cells, enabling them to kill the tumor cells. Despite extensive research in the field, only one dendritic cell-based vaccine has been approved. There is therefore a great need to elucidate and understand the immunological impact of dendritic cell vaccination in order to improve clinical benefit. In this chapter, we describe a method for performing immune monitoring using peripheral blood mononuclear cells and autologous dendritic cells transfected with tumor-associated antigen-encoding mRNA. PMID:27236804

  17. 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. PMID:27578602

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

    PubMed Central

    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. PMID:26274324

  19. 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.

  20. 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

  1. Gold Nanoparticles Enhanced Electroporation for Mammalian Cell Transfection

    PubMed Central

    Zu, Yingbo; Huang, Shuyan; Liao, Wei-Ching; Lu, Yang; Wang, Shengnian

    2015-01-01

    Electroporation figured prominently as an effective nonviral gene delivery approach for its balance on the transfection efficiency and cell viability, no restrictions of probe or cell type, and operation simplicity. The commercial electroporation systems have been widely adopted in the past two decades while still carry drawbacks associated with the high applied electric voltage, unsatisfied delivery efficiency, and/or low cell viability. By adding highly conductive gold nanoparticles (AuNPs) in electroporation solution, we demonstrated enhanced electroporation performance (i.e. better DNA delivery efficiency and higher cell viability) on mammalian cells from two different aspects: the free, naked AuNPs reduce the resistance of the electroporation solution so that the local pulse strength on cells was enhanced; targeting AuNPs (e.g., Tf-AuNPs) were brought to the cell membrane to work as virtual microelectrodes to porate cells with limited area from many different sites. The enhancement was confirmed with leukemia cells in both a commercial batch electroporation system and a home-made flow-through system using pWizGFP plasmid DNA probes. Such enhancement depends on the size, concentration, and the mixing ratio of free AuNPs/Tf-AuNPs. An equivalent mixture of free AuNPs and Tf-AuNPs exhibited the best enhancement with the transfection efficiency increased 2-3 folds at minimum sacrifice of cell viability. This new delivery concept, the combination of nanoparticles and electroporation technologies, may stimulate various in vitro and in vivo biomedical applications which rely on the efficient delivery of nucleic acids, anticancer drugs, or other therapeutic materials. PMID:24749393

  2. 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.

  3. Gold nanoparticles enhanced electroporation for mammalian cell transfection.

    PubMed

    Zu, Yingbo; Huang, Shuyan; Liao, Wei-Ching; Lu, Yang; Wang, Shengnian

    2014-06-01

    Electroporation figured prominently as an effective nonviral gene delivery approach for its balance on the transfection efficiency and cell viability, no restrictions of probe or cell type, and operation simplicity. The commercial electroporation systems have been widely adopted in the past two decades while still carry drawbacks associated with the high applied electric voltage, unsatisfied delivery efficiency, and/or low cell viability. By adding highly conductive gold nanoparticles (AuNPs) in electroporation solution, we demonstrated enhanced electroporation performance (i.e., better DNA delivery efficiency and higher cell viability) on mammalian cells from two different aspects: the free, naked AuNPs reduce the resistance of the electroporation solution so that the local pulse strength on cells was enhanced; targeting AuNPs (e.g., Tf-AuNPs) were brought to the cell membrane to work as virtual microelectrodes to porate cells with limited area from many different sites. The enhancement was confirmed with leukemia cells in both a commercial batch electroporation system and a home-made flow-through system using pWizGFP plasmid DNA probes. Such enhancement depends on the size, concentration, and the mixing ratio of free AuNPs/Tf-AuNPs. An equivalent mixture of free AuNPs and Tf-AuNPs exhibited the best enhancement with the transfection efficiency increased 2-3 folds at minimum sacrifice of cell viability. This new delivery concept, the combination of nanoparticles and electroporation technologies, may stimulate various in vitro and in vivo biomedical applications which rely on the efficient delivery of nucleic acids, anticancer drugs, or other therapeutic materials.

  4. Gold nanoparticle-enhanced electroporation for leukemia cell transfection.

    PubMed

    Huang, Shuyan; Zu, Yingbo; Wang, Shengnian

    2014-01-01

    Electroporation serves as an attractive nonviral gene delivery approach for its effectiveness, operational simplicity, and no restrictions of probe or cell type. The commercial electroporation systems have been widely adopted in research and clinics with protocols usually compromising appropriate transfection efficiency and cell viability. By introducing gold nanoparticles (AuNPs), we demonstrated greatly enhanced performance of electroporation from two aspects: the highly conductive, naked AuNPs help reduce the potential drop consumed by the electroporation solution so that the majority of the applied voltage of an electric pulse is truly imposed on cells with enhanced field strength; AuNPs with targeting ligands (e.g., transferrin-AuNPs or Tf-AuNPs) are bound to the cell membrane, working as virtual microelectrodes to create pores on cells with limited opening area while from many different sites. The addition of AuNPs during electroporation therefore benefits not only quicker recovery and better survival of cells but also more efficient uptake of the subjected probes. Such enhancement was successfully confirmed on a chronic myeloid leukemia cell line (i.e., K562 cells) in both a commercial batch electroporation system and a homemade flow system with pWizGFP plasmid DNA probes. The efficiency was found to be dependent on the size, concentration, and mixing ratio of free AuNPs/Tf-AuNPs. An equivalent mixture of free AuNPs and Tf-AuNPs exhibited the best enhancement with the transfection efficiency increase of two to threefold at a minimum sacrifice of the cell viability.

  5. Targeted Surface Expression of an Exogenous Antigen in Stably Transfected Babesia bovis

    PubMed Central

    Laughery, Jacob M.; Knowles, Donald P.; Schneider, David A.; Bastos, Reginaldo G.; McElwain, Terry F.; Suarez, Carlos E.

    2014-01-01

    Babesia bovis is a tick-borne intraerythocytic protozoan responsible for acute disease in cattle which can be controlled by vaccination with attenuated B. bovis strains. Emerging B. bovis transfection technologies may increase the usefulness of these live vaccines. One use of transfected B. bovis parasites may be as a vaccine delivery platform. Previous transfection methods for B. bovis were limited by single expression sites and intracellular expression of transfected antigens. This study describes a novel transfection system in which two exogenous genes are expressed: one for selection and the other for a selected antigen designed to be delivered to the surface of the parasites. The strategy for duplicating the number of transfected genes was based on the use of the putative bidirectional promoter of the B. bovis 1.4 Kb ef-1α intergenic region. The ability of this region to regulate two independent expression sites was demonstrated using a luciferase assay on transiently transfected B. bovis parasites and then incorporated into a stable transfection plasmid to control independent expression of the selectable marker GFP-BSD and another gene of interest. A chimeric gene was synthetized using sequences from the protective B-cell epitopes of Rhipicephalus microplus tick antigen Bm86 along with sequences from the surface exposed B. bovis major surface antigen-1. This chimeric gene was then cloned into the additional expression site of the transfection plasmid. Transfection of the B. bovis Mo7 strain with this plasmid resulted in stable insertion into the ef-1α locus and simultaneous expression of both exogenous genes. Expression of the Bm86 epitopes on the surface of transfected merozoites was demonstrated using immunofluorescence analyses. The ability to independently express multiple genes by the inclusion of a bidirectional promoter and the achievement of surface expression of foreign epitopes advances the potential of transfected B. bovis as a future vaccine

  6. Evaluating the role of low-speed centrifugation towards transfecting human peripheral blood mononuclear cell culture.

    PubMed

    Majumdar, M; Ratho, R; Chawla, Y; Singh, M P

    2014-01-01

    The conventional method of transfection of suspension cells by chemical has proven to be very difficult. We present a new transfection protocol, wherein, low-speed centrifugation of cell culture plates immediately after adding the lipid: DNA complex significantly enhances the transfection efficiency. Peripheral blood mononuclear cells (PBMCs) were transfected with BLOCK-iT™ Fluorescent Oligo (scrambled siRNA) and lipofectamine complex using conventional and low-speed centrifugation modified transfection protocols. The efficiency of transfection was determined using flowcytometer and cell viability was checked using MTT assay. Incorporation of low-speed centrifugation significantly enhances the transfection efficiency of BLOCK-iT™ in the suspension culture of PBMCs as compared to conventional transfection method (99.8% vs 28.3%; P < 0.0001), even at a low concentration of 40 picomoles without affecting the cell viability. Centrifugation enhanced transfection (CET) technique is simple, time-saving and novel application without compromising the cell viability in the context of recently popular RNA interference in suspension cultures of PBMCs. This undemanding modification might be applicable to a wide variety of cell lines and solve crucial problem of researchers working with RNA interference in suspension cultures. PMID:24713904

  7. 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.

  8. 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.

  9. Polarization-insensitive PAM-4-carrying free-space orbital angular momentum (OAM) communications.

    PubMed

    Liu, Jun; Wang, Jian

    2016-02-22

    We present a simple configuration incorporating single polarization-sensitive phase-only liquid crystal spatial light modulator (SLM) to facilitate polarization-insensitive free-space optical communications employing orbital angular momentum (OAM) modes. We experimentally demonstrate several polarization-insensitive optical communication subsystems by propagating a single OAM mode, multicasting 4 and 10 OAM modes, and multiplexing 8 OAM modes, respectively. Free-space polarization-insensitive optical communication links using OAM modes that carry four-level pulse-amplitude modulation (PAM-4) signal are demonstrated in the experiment. The observed optical signal-to-noise ratio (OSNR) penalties are less than 1 dB in both polarization-insensitive N-fold OAM modes multicasting and multiple OAM modes multiplexing at a bit-error rate (BER) of 2e-3 (enhanced forward-error correction (EFEC) threshold).

  10. Theoretical study of polarization insensitivity of carrier-induced refractive index change of multiple quantum well.

    PubMed

    Miao, Qingyuan; Zhou, Qunjie; Cui, Jun; He, Ping-An; Huang, Dexiu

    2014-12-29

    Characteristics of polarization insensitivity of carrier-induced refractive index change of 1.55 μm tensile-strained multiple quantum well (MQW) are theoretically investigated. A comprehensive MQW model is proposed to effectively extend the application range of previous models. The model considers the temperature variation as well as the nonuniform distribution of injected carrier in MQW. Tensile-strained MQW is expected to achieve polarization insensitivity of carrier-induced refractive index change over a wide wavelength range as temperature varies from 0°C to 40°C, while the magnitude of refractive index change keeps a large value (more than 3 × 10-3). And that the polarization insensitivity of refractive index change can maintain for a wide range of carrier concentration. Multiple quantum well with different material and structure parameters is anticipated to have the similar polarization insensitivity of refractive index change, which shows the design flexibility.

  11. Theoretical study of polarization insensitivity of carrier-induced refractive index change of multiple quantum well.

    PubMed

    Miao, Qingyuan; Zhou, Qunjie; Cui, Jun; He, Ping-An; Huang, Dexiu

    2014-12-29

    Characteristics of polarization insensitivity of carrier-induced refractive index change of 1.55 μm tensile-strained multiple quantum well (MQW) are theoretically investigated. A comprehensive MQW model is proposed to effectively extend the application range of previous models. The model considers the temperature variation as well as the nonuniform distribution of injected carrier in MQW. Tensile-strained MQW is expected to achieve polarization insensitivity of carrier-induced refractive index change over a wide wavelength range as temperature varies from 0°C to 40°C, while the magnitude of refractive index change keeps a large value (more than 3 × 10-3). And that the polarization insensitivity of refractive index change can maintain for a wide range of carrier concentration. Multiple quantum well with different material and structure parameters is anticipated to have the similar polarization insensitivity of refractive index change, which shows the design flexibility. PMID:25607157

  12. Polarization-insensitive PAM-4-carrying free-space orbital angular momentum (OAM) communications.

    PubMed

    Liu, Jun; Wang, Jian

    2016-02-22

    We present a simple configuration incorporating single polarization-sensitive phase-only liquid crystal spatial light modulator (SLM) to facilitate polarization-insensitive free-space optical communications employing orbital angular momentum (OAM) modes. We experimentally demonstrate several polarization-insensitive optical communication subsystems by propagating a single OAM mode, multicasting 4 and 10 OAM modes, and multiplexing 8 OAM modes, respectively. Free-space polarization-insensitive optical communication links using OAM modes that carry four-level pulse-amplitude modulation (PAM-4) signal are demonstrated in the experiment. The observed optical signal-to-noise ratio (OSNR) penalties are less than 1 dB in both polarization-insensitive N-fold OAM modes multicasting and multiple OAM modes multiplexing at a bit-error rate (BER) of 2e-3 (enhanced forward-error correction (EFEC) threshold). PMID:26907073

  13. 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.

  14. Transplantation of human telomerase reverse transcriptase gene-transfected Schwann cells for repairing spinal cord injury

    PubMed Central

    Zhang, Shu-quan; Wu, Min-fei; Liu, Jia-bei; Li, Ye; Zhu, Qing-san; Gu, Rui

    2015-01-01

    Transfection of the human telomerase reverse transcriptase (hTERT) gene has been shown to increase cell proliferation and enhance tissue repair. In the present study, hTERT was transfected into rat Schwann cells. A rat model of acute spinal cord injury was established by the modified free-falling method. Retrovirus PLXSN was injected at the site of spinal cord injury as a vector to mediate hTERT gene-transfected Schwann cells (1 × 1010/L; 10 μL) or Schwann cells (1 × 1010/L; 10 μL) without hTERT gene transfection. Between 1 and 4 weeks after model establishment, motor function of the lower limb improved in the hTERT-transfected group compared with the group with non-transfected Schwann cells. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and reverse transcription-polymerase chain reaction results revealed that the number of apoptotic cells, and gene expression of aquaporin 4/9 and matrix metalloproteinase 9/2 decreased at the site of injury in both groups; however, the effect improved in the hTERT-transfected group compared with the Schwann cells without hTERT transfection group. Hematoxylin and eosin staining, PKH26 fluorescent labeling, and electrophysiological testing demonstrated that compared with the non-transfected group, spinal cord cavity and motor and sensory evoked potential latencies were reduced, while the number of PKH26-positive cells and the motor and sensory evoked potential amplitude increased at the site of injury in the hTERT-transfected group. These findings suggest that transplantation of hTERT gene-transfected Schwann cells repairs the structure and function of the injured spinal cord. PMID:26889196

  15. Transfection in Pneumococcus: Single-Strand Intermediates in the Formation of Infective Centers

    PubMed Central

    Porter, Ronald D.; Guild, Walter R.

    1978-01-01

    Transfection has been found and characterized in pneumococcus. For replicating ω3 phage DNA extracted from infected cells, transfection was relatively efficient and rose linearly with DNA concentration and quadratically with time, according to T(T - 3.5) min2. For mature DNA extracted from phage particles, transfection was hardly detectable below 1 μg/ml but increased about as the cube of the DNA concentration up to 100 μg/ml, and was still rising at concentrations over 200 μg/ml. The kinetics suggest a dependence on a mixed cubic function of the time of exposure of cells to mature DNA. Cell and phage DNAs competed with each other for transformation and transfection. Transfection was reduced much more strongly than transformation in cells that were deficient in the membrane-bound endonuclease required for conversion of donor duplex DNA to intracellular single strands; these data agree with the kinetic data in implying that independent entry of segments of two strands is necessary for transfection by replicating ω3 phage DNA and entry of at least three strands is necessary for transfection by mature DNA. To reconcile differing DNA concentration dependences of transfection and transformation with a common entry path, it was necessary to reexamine data on transformation and to recognize that this process continued to rise slowly through the concentration region usually described as “plateau.” These results and the transfection data reflect multiple binding and nicking events that occurred on the cell surface before entry. Our conclusion is that transfection in pneumococcus occurs by association inside the cell of segments of single strands of phage DNA that have entered independently, creating gapped structures that need repair synthesis to create infective centers. Physical recombination is therefore automatically a prerequisite to transfection. PMID:23440

  16. Acute and persistent infection by a transfected Mo7 strain of Babesia bovis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Stable transfection of the Mo7 strain of Babesia bovis and expression of an exogenous gene has been demonstrated in long term culture. However, the use of transfected parasites as marker vaccines or vehicles for expressing exogenous antigens in vivo requires demonstration of acute and persistent inf...

  17. Synergistic effect of a biosurfactant and protamine on gene transfection efficiency.

    PubMed

    Inoh, Yoshikazu; Furuno, Tadahide; Hirashima, Naohide; Kitamoto, Dai; Nakanishi, Mamoru

    2013-04-11

    Several barriers need to be overcome to ensure successful gene transfection, including passing of the foreign gene through the plasma membrane, escape of this material from lysosomal degradation, and its translocation into the nucleus. We previously showed that the biosurfactant mannosylerythritol lipid-A (MEL-A) enhanced the efficiency of gene transfection mediated by cationic liposomes by facilitating rapid delivery of foreign genes into target cells through membrane fusion between liposomes and the plasma membrane. Moreover, using MEL-A-containing cationic liposomes, the foreign gene was efficiently delivered into the nucleus because it was released directly into the cytosol and thus escaped lysosomal degradation. Here we investigated the effect of pre-condensation of plasmid DNA by a cationic polymer, protamine, on gene transfection. We found that the efficiency of pre-condensed DNA transfection mediated by MEL-A-containing OH liposomes was >10 times higher than that of non-condensed DNA transfection. In contrast, the efficiency of pre-condensed DNA transfection mediated by OH liposomes was only 1.5 times higher than that of non-condensed DNA transfection. MEL-A did not influence plasmid DNA encapsulation by cationic liposomes, but it greatly accelerated the nuclear delivery of pre-condensed plasmid DNA. Our findings indicate that MEL-A and protamine synergistically accelerate the nuclear delivery of foreign gene and consequently promote gene transfection efficiency. PMID:23422688

  18. Non-Viral Transfection Methods Optimized for Gene Delivery to a Lung Cancer Cell Line

    PubMed Central

    Salimzadeh, Loghman; Jaberipour, Mansooreh; Hosseini, Ahmad; Ghaderi, Abbas

    2013-01-01

    Background Mehr-80 is a newly established adherent human large cell lung cancer cell line that has not been transfected until now. This study aims to define the optimal transfection conditions and effects of some critical elements for enhancing gene delivery to this cell line by utilizing different non-viral transfection Procedures. Methods In the current study, calcium phosphate (CaP), DEAE-dextran, superfect, electroporation and lipofection transfection methods were used to optimize delivery of a plasmid construct that expressed Green Fluorescent Protein (GFP). Transgene expression was detected by fluorescent microscopy and flowcytometry. Toxicities of the methods were estimated by trypan blue staining. In order to evaluate the density of the transfected gene, we used a plasmid construct that expressed the Stromal cell-Derived Factor-1 (SDF-1) gene and measured its expression by real-time PCR. Results Mean levels of GFP-expressing cells 48 hr after transfection were 8.4% (CaP), 8.2% (DEAE-dextran), 4.9% (superfect), 34.1% (electroporation), and 40.1% (lipofection). Lipofection had the highest intense SDF-1 expression of the analyzed methods. Conclusion This study has shown that the lipofection and electroporation methods were more efficient at gene delivery to Mehr-80 cells. The quantity of DNA per transfection, reagent concentration, and incubation time were identified as essential factors for successful transfection in all of the studied methods. PMID:23799175

  19. Nucleic acid transfection and transgenesis in parasitic nematodes.

    PubMed

    Lok, James B

    2012-04-01

    Transgenesis is an essential tool for assessing gene function in any organism, and it is especially crucial for parasitic nematodes given the dwindling armamentarium of effective anthelmintics and the consequent need to validate essential molecular targets for new drugs and vaccines. Two of the major routes of gene delivery evaluated to date in parasitic nematodes, bombardment with DNA-coated microparticles and intragonadal microinjection of DNA constructs, draw upon experience with the free-living nematode Caenorhabditis elegans. Bombardment has been used to transiently transfect Ascaris suum, Brugia malayi and Litomosoides sigmodontis with both RNA and DNA. Microinjection has been used to achieve heritable transgenesis in Strongyloides stercoralis, S. ratti and Parastrongyloides trichosuri and for additional transient expression studies in B. malayi. A third route of gene delivery revisits a classic method involving DNA transfer facilitated by calcium-mediated permeabilization of recipient cells in developing B. malayi larvae and results in transgene inheritance through host and vector passage. Assembly of microinjected transgenes into multi-copy episomal arrays likely results in their transcriptional silencing in some parasitic nematodes. Methods such as transposon-mediated transgenesis that favour low-copy number chromosomal integration may remedy this impediment to establishing stable transgenic lines. In the future, stable transgenesis in parasitic nematodes could enable loss-of-function approaches by insertional mutagenesis, in situ expression of inhibitory double-stranded RNA or boosting RNAi susceptibility through heterologous expression of dsRNA processing and transport proteins. PMID:21880161

  20. 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

  1. Asymmetric partitioning of transfected DNA during mammalian cell division

    PubMed Central

    Wang, Xuan; Le, Nhung; Denoth-Lippuner, Annina; Barral, Yves; Kroschewski, Ruth

    2016-01-01

    Foreign DNA molecules and chromosomal fragments are generally eliminated from proliferating cells, but we know little about how mammalian cells prevent their propagation. Here, we show that dividing human and canine cells partition transfected plasmid DNA asymmetrically, preferentially into the daughter cell harboring the young centrosome. Independently of how they entered the cell, most plasmids clustered in the cytoplasm. Unlike polystyrene beads of similar size, these clusters remained relatively immobile and physically associated to endoplasmic reticulum-derived membranes, as revealed by live cell and electron microscopy imaging. At entry of mitosis, most clusters localized near the centrosomes. As the two centrosomes split to assemble the bipolar spindle, predominantly the old centrosome migrated away, biasing the partition of the plasmid cluster toward the young centrosome. Down-regulation of the centrosomal proteins Ninein and adenomatous polyposis coli abolished this bias. Thus, we suggest that DNA clustering, cluster immobilization through association to the endoplasmic reticulum membrane, initial proximity between the cluster and centrosomes, and subsequent differential behavior of the two centrosomes together bias the partition of plasmid DNA during mitosis. This process leads to their progressive elimination from the proliferating population and might apply to any kind of foreign DNA molecule in mammalian cells. Furthermore, the functional difference of the centrosomes might also promote the asymmetric partitioning of other cellular components in other mammalian and possibly stem cells. PMID:27298340

  2. Transfection of eggs in the bivalve mollusc Chamelea gallina (Bivalvia, Veneridae).

    PubMed

    Guerra, R; Esponda, P

    2006-04-01

    Eggs from the bivalve mollusc Chamelea gallina were transfected in vitro. The p-GeneGrip gene construction that expresses the green fluorescent protein (GFP) was employed. It was necessary to remove the jelly coat which covers the egg surface for a successful transfection, and then 44.2% of gametes appeared transfected after using naked DNA. On the other hand, cationic liposomes (Lipofectamine) and neutral lipids (GenePORTER) were employed as gene vectors. After the employ of Lipofectamine 35.6% of eggs were transfected and 41.4% after using GenePORTER. Fluorescence analysis showed that the foreign gene appeared principally located in the egg cytoplasm, but laser confocal microscopy showed that it was also present in the nucleus. Furthermore, PCR analysis demonstrated that the foreign DNA appeared in the DNA extracted from the treated eggs. This simple method for the transfection of mollusc eggs would be interesting for future biotechnological applications in species of commercial interest. PMID:17283962

  3. 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.

  4. NBD-conjugated biosurfactant (MEL-A) shows a new pathway for transfection.

    PubMed

    Ueno, Yoshinobu; Inoh, Yoshikazu; Furuno, Tadahide; Hirashima, Naohide; Kitamoto, Dai; Nakanishi, Mamoru

    2007-11-20

    Gene transfection is a fundamental technology for molecular and cell biology, and also clinical gene therapy. A variety of non-viral vectors have been investigated for gene transfection, but their gene delivery had remained an inefficient process. Recently, we found that a biosurfactant, mannosylerythritol lipid (MEL)-A, dramatically increased the efficiency in transfection of plasmid DNA mediated by cationic liposomes. However, its mechanism has not been understood yet. Here we examined the mechanism of the transfection mediated by cationic liposomes with NBD-conjugated MEL-A. We found that MEL-A first gradually distributed on the intracellular membranes through the plasma membranes of target cells, while the cationic liposomes with MEL-A fused to the plasma membranes in 20-35 min. Thereafter, the oligonucleotide released from the vesicles was immediately transferred to the nucleus. The present results showed a new role of non-viral vectors in transfection. PMID:17884224

  5. A High-Throughput Microfluidic Platform for Mammalian Cell Transfection and Culturing

    PubMed Central

    Woodruff, Kristina; Maerkl, Sebastian J.

    2016-01-01

    Mammalian synthetic biology could be augmented through the development of high-throughput microfluidic systems that integrate cellular transfection, culturing, and imaging. We created a microfluidic chip that cultures cells and implements 280 independent transfections at up to 99% efficiency. The chip can perform co-transfections, in which the number of cells expressing each protein and the average protein expression level can be precisely tuned as a function of input DNA concentration and synthetic gene circuits can be optimized on chip. We co-transfected four plasmids to test a histidine kinase signaling pathway and mapped the dose dependence of this network on the level of one of its constituents. The chip is readily integrated with high-content imaging, enabling the evaluation of cellular behavior and protein expression dynamics over time. These features make the transfection chip applicable to high-throughput mammalian protein and synthetic biology studies. PMID:27030663

  6. Fetal feminization and female gender identity in the testicular feminizing syndrome of androgen insensitivity.

    PubMed

    Masica, D N; Money, J; Ehrhardt, A A

    1971-06-01

    Interview data on aspects of sex and eroticism from a sample of 10 chromosomally male (XY) patients with the complete testicular feminization (androgen insensitivity) syndrome and from a sample of 23 patients with the late-treated adrenogenital syndrome showed marked differences. Homosexual experiences and/or dreams were lacking in the androgen-insensitive group as compared with the adrenogenital group (p[Symbol: see text]0.01).The androgen-insensitive group reported lower frequency of sexual arousal from visual stimuli than did the adrenogenital group (p[Symbol: see text]0.05).Findings on different aspects of sexual behavior suggest a tendency for the androgen-insensitive patients as a group to have a lower sex drive, to be less keenly aware of their sex drive, to be less assertive in heterosexual relations, and to be less versatile in coitus than the adrenogenital patients. No case of exclusive lesbianism, transsexualism, or transvestism was reported from either patient group. Although the two groups differed in sexual and erotic behavior, both were within the range of what in our culture is accepted as feminine. Nonetheless, the androgen-insensitive patients conformed more closely to the conventional feminine stereotype. Regarding explicit satisfaction with female sex role and with cosmetic and clothing interests, the androgen-insensitive group was characteristically feminine. Findings on the Draw-a-Person Test and the Guilford-Zimmerman Temperament Survey are compatible with the results of normal females, but are in sharp contrast with those of normal males. Interview and psychometric data thus concur in showing the androgen-insensitive patients to be unmistakably feminine in behavior and outlook. Their femininity is best conceived of as a product of hormonal nonandrogenization, prenatally and later, in combination with the social experiences of rearing and development, after initial assignment as a girl. PMID:24179056

  7. Differentiation of human induced pluripotent stem cells into insulin-like cell clusters with miR-186 and miR-375 by using chemical transfection.

    PubMed

    Shaer, Anahita; Azarpira, Negar; Karimi, Mohammad Hosein

    2014-09-01

    Diabetes mellitus is characterized by either the inability to produce insulin or insensitivity to insulin secreted by the body. Islet cell replacement is an effective approach for diabetes treatment; however, it is not sufficient for all the diabetic patients. MicroRNAs (miRNAs) are a class of small noncoding RNAs that play an important role in mediating a broad and expanding range of biological activities, such as pancreas development. The present study aimed to develop a protocol to efficiently differentiate human induced pluripotent stem (iPS) cells into islet-like cell clusters (ILCs) in vitro by using miR-186 and miR-375. The human iPS colonies were transfected with hsa-miR-186 and hsa-miR-375 by using siPORT™ NeoFX™ Transfection Agent, and the differentiation was compared to controls. Total RNA was extracted 24 and 48 h after transfection. The gene expressions of insulin, NGN3, GLUT2, PAX4, PAX6, KIR6.2, NKX6.1, PDX1, Glucagon, and OCT4 were then evaluated through real-time qPCR. On the third day, the potency of the clusters was assessed in response to high glucose levels. Dithizone (DTZ) was used to identify the existence of the β-cells. Besides, the presence of insulin and NGN3 proteins was investigated by immunocytochemistry. Morphological changes were observed on the first day after the chemical transfection, and cell clusters were formed on the third day. The expression of pancreatic specific transcription factors was increased on the first day and significantly increased on the second day. The ILCs were positive for insulin and NGN3 proteins in the immunocytochemistry. Besides, the clusters were stained with DTZ and secreted insulin in glucose challenge test. Overexpression of miR-186 and miR-375 can be an alternative strategy for producing ILCs from the iPS cells in a short time. This work provides a new approach by using patient-specific iPSCs for β-cell replacement therapy in diabetic patients. PMID:25059983

  8. Differentiation of human induced pluripotent stem cells into insulin-like cell clusters with miR-186 and miR-375 by using chemical transfection.

    PubMed

    Shaer, Anahita; Azarpira, Negar; Karimi, Mohammad Hosein

    2014-09-01

    Diabetes mellitus is characterized by either the inability to produce insulin or insensitivity to insulin secreted by the body. Islet cell replacement is an effective approach for diabetes treatment; however, it is not sufficient for all the diabetic patients. MicroRNAs (miRNAs) are a class of small noncoding RNAs that play an important role in mediating a broad and expanding range of biological activities, such as pancreas development. The present study aimed to develop a protocol to efficiently differentiate human induced pluripotent stem (iPS) cells into islet-like cell clusters (ILCs) in vitro by using miR-186 and miR-375. The human iPS colonies were transfected with hsa-miR-186 and hsa-miR-375 by using siPORT™ NeoFX™ Transfection Agent, and the differentiation was compared to controls. Total RNA was extracted 24 and 48 h after transfection. The gene expressions of insulin, NGN3, GLUT2, PAX4, PAX6, KIR6.2, NKX6.1, PDX1, Glucagon, and OCT4 were then evaluated through real-time qPCR. On the third day, the potency of the clusters was assessed in response to high glucose levels. Dithizone (DTZ) was used to identify the existence of the β-cells. Besides, the presence of insulin and NGN3 proteins was investigated by immunocytochemistry. Morphological changes were observed on the first day after the chemical transfection, and cell clusters were formed on the third day. The expression of pancreatic specific transcription factors was increased on the first day and significantly increased on the second day. The ILCs were positive for insulin and NGN3 proteins in the immunocytochemistry. Besides, the clusters were stained with DTZ and secreted insulin in glucose challenge test. Overexpression of miR-186 and miR-375 can be an alternative strategy for producing ILCs from the iPS cells in a short time. This work provides a new approach by using patient-specific iPSCs for β-cell replacement therapy in diabetic patients.

  9. Robust polarization-insensitive strip-slot waveguide mode converter based on symmetric multimode interference.

    PubMed

    Deng, Qingzhong; Yan, Qiaojing; Liu, Lu; Li, Xinbai; Michel, Jurgen; Zhou, Zhiping

    2016-04-01

    Strip-slot waveguide mode converters for TE0 have been widely investigated. Here we demonstrate a polarization-insensitive converter numerically and experimentally. The polarization-insensitive performance is achieved by matching the optical field distribution of the 2-fold image of the Multimode Interference (MMI) and the TE0 (TM0) mode of a slot waveguide. The working principle for this MMI-based mode converter is thoroughly analyzed with the quantitatively evaluated optical field overlap ratio that is theoretically derived from the orthonormal relation of eigenmodes. Based on the analysis, the MMI-based polarization-insensitive converters are then simulated and fabricated. The simulation and measurement results indicate that the proposed scheme is a robust design since it is not only polarization-insensitive but also wavelength-insensitive and fabrication-tolerant. Moreover, the mode converter is as small as 1.22 μm × 4 μm while the measured conversion efficiencies are 95.9% for TE0 and 96.6% for TM0. All these excellent properties make the proposed mode converter an ideal solution for coupling light between strip and slot waveguides when both TE and TM polarizations are considered.

  10. 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

  11. In vivo transfection of melanoma cells by lithotripter shock waves.

    PubMed

    Bao, S; Thrall, B D; Gies, R A; Miller, D L

    1998-01-15

    The potential for gene transfection during shock wave tumor therapy was evaluated by searching for shock wave-induced DNA transfer in mouse tumor cells. B16 mouse melanoma cells were cultured by standard methods and implanted s.c. in female C57BL/6 mice 10-14 days before treatment. A luciferase reporter vector was used as the DNA plasmid for intratumoral injection at 0.2 mg/ml tumor. Air at 10% of tumor volume was injected after the DNA in some tumors to enhance acoustic cavitation activity. The shock wave generation system was similar to a Dornier HM-3 lithotripter with pressure amplitudes of 24.4 MPa peak positive and 5.2 MPa peak negative. Luciferase production in isolated tumor cells was measured with a luminometer 1 day after treatment to assess gene transfer and expression. Exposure to 800 shock waves, followed by immediate isolation and culture of tumor cells for 1 day, yielded 1.1 (0.43 SE) pg/10(6) cells for plasmid injection only and 7.5 (2.5 SE) pg/10(6) cells for plasmid plus air injection. Significantly increased luciferase production, relative to shams, occurred for 200-, 400-, 800-, and 1200-shock wave treatments with plasmid and air injection. Exposure with the isolation of tumor cells delayed for a day to allow gene expression within the growing tumors gave increased luciferase production for 100- and 400-shock wave exposures without and with air injection. Gene transfer therefore can be induced during lithotripter shock wave treatment in vivo, particularly with enhanced acoustic cavitation, which supports the concept that gene and shock wave therapy might be advantageously merged. PMID:9443395

  12. 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.

  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. DNA Targeting Sequence Improves Magnetic Nanoparticle-Based Plasmid DNA Transfection Efficiency in Model Neurons

    PubMed Central

    Vernon, Matthew M.; Dean, David A.; Dobson, Jon

    2015-01-01

    Efficient non-viral plasmid DNA transfection of most stem cells, progenitor cells and primary cell lines currently presents an obstacle for many applications within gene therapy research. From a standpoint of efficiency and cell viability, magnetic nanoparticle-based DNA transfection is a promising gene vectoring technique because it has demonstrated rapid and improved transfection outcomes when compared to alternative non-viral methods. Recently, our research group introduced oscillating magnet arrays that resulted in further improvements to this novel plasmid DNA (pDNA) vectoring technology. Continued improvements to nanomagnetic transfection techniques have focused primarily on magnetic nanoparticle (MNP) functionalization and transfection parameter optimization: cell confluence, growth media, serum starvation, magnet oscillation parameters, etc. Noting that none of these parameters can assist in the nuclear translocation of delivered pDNA following MNP-pDNA complex dissociation in the cell’s cytoplasm, inclusion of a cassette feature for pDNA nuclear translocation is theoretically justified. In this study incorporation of a DNA targeting sequence (DTS) feature in the transfecting plasmid improved transfection efficiency in model neurons, presumably from increased nuclear translocation. This observation became most apparent when comparing the response of the dividing SH-SY5Y precursor cell to the non-dividing and differentiated SH-SY5Y neuroblastoma cells. PMID:26287182

  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. siRNA transfection in larvae of the barnacle Amphibalanus amphitrite.

    PubMed

    Zhang, Gen; He, Li-Sheng; Wong, Yue Him; Yu, Li; Qian, Pei-Yuan

    2015-08-01

    RNA interference (RNAi) provides an efficient and specific technique for functional genomic studies. Yet, no successful application of RNAi has been reported in barnacles. In this study, siRNA against p38 MAPK was synthesized and then transfected into A. amphitrite larvae at either the nauplius or cyprid stage, or at both stages. Effects of siRNA transfection on the p38 MAPK level were hardly detectable in the cyprids when they were transfected at the nauplius stage. In contrast, larvae that were transfected at the cyprid stage showed lower levels of p38 MAPK than the blank and reagent controls. However, significantly decreased levels of phosphorylated p38 MAPK (pp38 MAPK) and reduced settlement rates were observed only in 'double transfections', in which larvae were exposed to siRNA solution at both the nauplius and cyprid stages. A relatively longer transfection time and more larval cells directly exposed to siRNA might explain the higher efficiency of double transfection experiments.

  17. DNA Targeting Sequence Improves Magnetic Nanoparticle-Based Plasmid DNA Transfection Efficiency in Model Neurons.

    PubMed

    Vernon, Matthew M; Dean, David A; Dobson, Jon

    2015-01-01

    Efficient non-viral plasmid DNA transfection of most stem cells, progenitor cells and primary cell lines currently presents an obstacle for many applications within gene therapy research. From a standpoint of efficiency and cell viability, magnetic nanoparticle-based DNA transfection is a promising gene vectoring technique because it has demonstrated rapid and improved transfection outcomes when compared to alternative non-viral methods. Recently, our research group introduced oscillating magnet arrays that resulted in further improvements to this novel plasmid DNA (pDNA) vectoring technology. Continued improvements to nanomagnetic transfection techniques have focused primarily on magnetic nanoparticle (MNP) functionalization and transfection parameter optimization: cell confluence, growth media, serum starvation, magnet oscillation parameters, etc. Noting that none of these parameters can assist in the nuclear translocation of delivered pDNA following MNP-pDNA complex dissociation in the cell's cytoplasm, inclusion of a cassette feature for pDNA nuclear translocation is theoretically justified. In this study incorporation of a DNA targeting sequence (DTS) feature in the transfecting plasmid improved transfection efficiency in model neurons, presumably from increased nuclear translocation. This observation became most apparent when comparing the response of the dividing SH-SY5Y precursor cell to the non-dividing and differentiated SH-SY5Y neuroblastoma cells. PMID:26287182

  18. siRNA transfection in larvae of the barnacle Amphibalanus amphitrite.

    PubMed

    Zhang, Gen; He, Li-Sheng; Wong, Yue Him; Yu, Li; Qian, Pei-Yuan

    2015-08-01

    RNA interference (RNAi) provides an efficient and specific technique for functional genomic studies. Yet, no successful application of RNAi has been reported in barnacles. In this study, siRNA against p38 MAPK was synthesized and then transfected into A. amphitrite larvae at either the nauplius or cyprid stage, or at both stages. Effects of siRNA transfection on the p38 MAPK level were hardly detectable in the cyprids when they were transfected at the nauplius stage. In contrast, larvae that were transfected at the cyprid stage showed lower levels of p38 MAPK than the blank and reagent controls. However, significantly decreased levels of phosphorylated p38 MAPK (pp38 MAPK) and reduced settlement rates were observed only in 'double transfections', in which larvae were exposed to siRNA solution at both the nauplius and cyprid stages. A relatively longer transfection time and more larval cells directly exposed to siRNA might explain the higher efficiency of double transfection experiments. PMID:26113139

  19. Dielectrophoresis-assisted 3D nanoelectroporation for non-viral cell transfection in adoptive immunotherapy.

    PubMed

    Chang, Lingqian; Gallego-Perez, Daniel; Zhao, Xi; Bertani, Paul; Yang, Zhaogang; Chiang, Chi-Ling; Malkoc, Veysi; Shi, Junfeng; Sen, Chandan K; Odonnell, Lynn; Yu, Jianhua; Lu, Wu; Lee, L James

    2015-08-01

    Current transfection technologies lead to significant inter-clonal variations. Previously we introduced a unique electrotransfection technology, Nanochannel-Electroporation (NEP), which can precisely and benignly transfect small cell populations (~100-200 cells) with single-cell resolution. Here we report on the development of a novel 3D NEP system for large scale transfection. A properly-engineered array of nanochannels, capable of handling/transfecting ~60 000 cells cm(-2), was fabricated using cleanroom technologies. Positive dielectrophoresis was used to selectively position cells on the nanochannels, thus allowing highly efficient transfection. Single-cell dosage control was demonstrated using both small and large molecules, and different cell types. The potential clinical relevance of this system was tested with difficult-to-transfect natural killer cell suspensions, and plasmids encoding for the chimeric antigen receptor (CAR), a model of high relevance for adoptive immunotherapy. Our results show significantly higher CAR transfection efficiencies for the DEP-NEP system (>70% vs. <30%), as well as enhanced cell viabilities.

  20. Recording, labeling, and transfection of single neurons in deep brain structures

    PubMed Central

    Dempsey, Bowen; Turner, Anita J.; Le, Sheng; Sun, Qi‐Jian; Bou Farah, Lama; Allen, Andrew M.; Goodchild, Ann K.; McMullan, Simon

    2015-01-01

    Abstract Genetic tools that permit functional or connectomic analysis of neuronal circuits are rapidly transforming neuroscience. The key to deployment of such tools is selective transfection of target neurons, but to date this has largely been achieved using transgenic animals or viral vectors that transduce subpopulations of cells chosen according to anatomical rather than functional criteria. Here, we combine single‐cell transfection with conventional electrophysiological recording techniques, resulting in three novel protocols that can be used for reliable delivery of conventional dyes or genetic material in vitro and in vivo. We report that techniques based on single cell electroporation yield reproducible transfection in vitro, and offer a simple, rapid and reliable alternative to established dye‐labeling techniques in vivo, but are incompatible with targeted transfection in deep brain structures. In contrast, we show that intracellular electrophoresis of plasmid DNA transfects brainstem neurons recorded up to 9 mm deep in the anesthetized rat. The protocols presented here require minimal, if any, modification to recording hardware, take seconds to deploy, and yield high recovery rates in vitro (dye labeling: 89%, plasmid transfection: 49%) and in vivo (dye labeling: 66%, plasmid transfection: 27%). They offer improved simplicity compared to the juxtacellular labeling technique and for the first time offer genetic manipulation of functionally characterized neurons in previously inaccessible brain regions. PMID:25602013

  1. Investigation of plasma induced electrical and chemical factors and their contribution processes to plasma gene transfection.

    PubMed

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

    2016-09-01

    This study has been done to know what kind of factors in plasmas and processes on cells induce plasma gene transfection. We evaluated the contribution weight of three groups of the effects and processes, i.e. electrical, chemical and biochemical ones, inducing gene transfection. First, the laser produced plasma (LPP) was employed to estimate the contribution of the chemical factors. Second, liposomes were fabricated and employed to evaluate the effects of plasma irradiation on membrane under the condition without biochemical reaction. Third, the clathrin-dependent endocytosis, one of the biochemical processes was suppressed. It becomes clear that chemical factors (radicals and reactive oxygen/nitrogen species) do not work by itself alone and electrical factors (electrical current, charge and field) are essential to plasma gene transfection. It turned out the clathrin-dependent endocytosis is the process of the transfection against the 60% in all the transfected cells. The endocytosis and electrical poration are dominant in plasma gene transfection, and neither permeation through ion channels nor chemical poration is dominant processes. The simultaneous achievement of high transfection efficiency and high cell survivability is attributed to the optimization of the contribution weight among three groups of processes by controlling the weight of electrical and chemical factors. PMID:27136710

  2. 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

  3. Temperature insensitive refractive index sensor based on concatenated long period fiber gratings

    NASA Astrophysics Data System (ADS)

    Tripathi, Saurabh M.; Bock, Wojtek J.; Mikulic, Predrag

    2013-10-01

    We propose and demonstrate a temperature immune biosensor based on two concatenated LPGs incorporating a suitable inter-grating-space (IGS). Compensating the thermal induced phase changes in the grating region by use of an appropriate length of the IGS the temperature insensitivity has been achieved. Using standard telecommunication grade single-mode fibers we show that a length ratio of ~8.2 is sufficient to realize the proposed temperature insensitivity. The resulting sensor shows a refractive index sensitivity of 423.28 nm/RIU displaying the capability of detecting an index variation of 2.36 × 10-6 RIU in the bio-samples. The sensor can also be applied as a temperature insensitive WMD channel isolation filter in the optical communication systems, removing the necessity of any external thermal insulation packaging.

  4. Transfection-mediated cell synchronization: acceleration of G1-S phase transition by gamma irradiation.

    PubMed

    Jung, E J; Flemington, E K

    2001-11-01

    We have previously provided evidence that the uptake of DNA into cells is cell cycle specific following transfection. We show here that, immediately after transfection, successfully transfected cells are greatly enriched for cells in early G1 or G0 phase and that, upon removal of the DNA precipitates, cells progress through G1 and enter S phase in a synchronous fashion. We also demonstrate that this approach can be utilized in meaningful cell-cycle experiments, and we show that gamma irradiation accelerates the G1-S phase transition in a cell line with a functionally inactive p53 protein. PMID:11730009

  5. Assay of insulator enhancer-blocking activity with the use of transient transfection.

    PubMed

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

    2013-08-01

    We used a transient transfection of cultured cells with linearized plasmids to analyze the enhancer-blocking activity of potential insulators including the standard cHS4 chicken beta-globin insulator and several DNA fragments selected from the human genome sequence. About 60-80% of the potential insulators do reveal the enhancer-blocking activity when probed by the transient transfection assay. The activity of different sequences is characterized by certain tissue specificity and by dependence on the orientation of the fragments relative to the promoter. Thus, the transfection model may be used for quantitative analysis of the enhancer-blocking activity of the potential insulators. PMID:24228877

  6. 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.

  7. 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.

  8. Increased shock sensitivity of the insensitive explosive LX-17 at high temperatures

    SciTech Connect

    Lee, R.S.; Chau, H.H.

    1994-05-01

    Explosive formulations based on TATB (1.3.5-trichloro-2,4,6-trinitrobenzene) have proven to be remarkably insensitive to shock and thermal stimuli. However, hazards to an insensitive high explosive (IHE) charge do not always confine themselves to a single stimulus. In the study reported here, we have investigated the response of the LLNL explosive LX-17 (92.5%/7.5% TATB/Kel-F 800) to shock when the explosive is at an elevated temperature. The motivation for the work was to learn the extent to which the shock initiation threshold and critical initiation area of LX-17 are lowered by exposure to elevated temperature.

  9. Transfected adenosine A1 receptor-mediated modulation of thrombin-stimulated phospholipase C and phospholipase A2 activity in CHO cells.

    PubMed

    Dickenson, J M; Hill, S J

    1997-02-19

    Thrombin receptor activation in Chinese hamster ovary (CHO) cells stimulates the hydrolysis of inositol phospholipids and the release of arachidonic acid. Our previous studies have shown that activation of the human transfected adenosine A1 receptor in CHO cells (CHO-A1) potentiates the accumulation of inositol phosphates elicited by endogenous P2U purinoceptors and CCKA receptors. In this study we have investigated whether adenosine A1 receptor activation can modulate thrombin-stimulated arachidonic acid release and/or inositol phospholipid hydrolysis in CHO-A1 cells. Thrombin stimulated [3H]arachidonic acid release and total [3H]inositol phosphate accumulation in CHO-A1 cells. Both these responses to thrombin were were insensitive to pertussis toxin. The protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), potentiated thrombin-stimulated [3H]arachidonic acid. In marked contrast, PMA inhibited thrombin-stimulated [3H]inositol phosphate accumulation. The selective protein kinase C inhibitor Ro 31-8220 (3-¿1-[3-(2-isothioureido)propyl] indol-3-yl¿-4-(1-methylindol-3-yl)-3-pyrrolin-2,5-dione) had no effect on thrombin-stimulated [3H]arachidonic acid release but reversed the potentiation of thrombin-stimulated [3H]arachidonic acid release elicited by PMA. The selective adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) augmented the release of [3H]arachidonic acid produced by thrombin. Co-activation of the adenosine A1 receptor also potentiated thrombin-stimulated [3H]inositol phosphate accumulation. The synergistic interactions between the adenosine A1 receptor and thrombin were abolished in pertussis-toxin-treated cells. The potentiation of [3H]arachidonic acid release by CPA was blocked by the protein kinase C inhibitors Ro 31-8220 and GF 109203X (3-[1-[3-(dimethylamino)propyl]-1 H-indol-3-yl]-4-(1 H-indol-3-yl)- 1H-pyrrole-2,5-dione). In conclusion, thrombin receptor activation in CHO-A1 cells stimulates the accumulation of [3H

  10. 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.

  11. Anti-epileptic effects of neuropeptide Y gene transfection into the rat brain☆

    PubMed Central

    Dong, Changzheng; Zhao, Wenqing; Li, Wenling; Lv, Peiyuan; Dong, Xiufang

    2013-01-01

    Neuropeptide Y gene transfection into normal rat brain tissue can provide gene overexpression, which can attenuate the severity of kainic acid-induced seizures. In this study, a recombinant adeno-associated virus carrying the neuropeptide Y gene was transfected into brain tissue of rats with kainic acid-induced epilepsy through stereotactic methods. Following these transfections, we verified overexpression of the neuropeptide Y gene in the epileptic brain. Electroencephalograms showed that seizure severity was significantly inhibited and seizure latency was significantly prolonged up to 4 weeks after gene transfection. Moreover, quantitative fluorescent PCR and western blot assays revealed that the mRNA and protein expression of the N-methyl-D-aspartate receptor subunits NR1, NR2A, and NR2B was inhibited in the hippocampus of epileptic rats. These findings indicate that neuropeptide Y may inhibit seizures via down-regulation of the functional expression of N-methyl-D-aspartate receptors. PMID:25206425

  12. Characterization of biosurfactant-containing liposomes and their efficiency for gene transfection.

    PubMed

    Ueno, Yoshinobu; Hirashima, Naohide; Inoh, Yoshikazu; Furuno, Tadahide; Nakanishi, Mamoru

    2007-01-01

    Recently we showed significance of biosurfactants in the field of non-viral vectors for gene transfection. There, a biosurfactant, mannosylerythritol lipid A (MEL-A), especially increased the efficiency of gene transfection mediated with cationic liposomes. However, the molecular mechanism has not been well-understood yet. Here, through the examination of the ability of cationic liposomes containing an MEL (MEL-A, MEL-B or MEL-C) for important transfectional processes of the DNA capsulation and the membrane fusion with anionic liposomes, we found that MEL-A-containing liposomes increased both processes, but that MEL-B and MEL-C-containing liposomes just increased either of them. The results indicated that these kinds of the physicochemical properties in MEL-A-containing liposomes are able to increase the efficiency of liposome-mediated gene transfection. PMID:17202680

  13. DEVELOPMENT OF AN ENVIRONMENTAL ESTROGEN SCREEN USING TRANSIENTLY TRANSFECTED RAINBOW TROUT CELL LINES

    EPA Science Inventory

    Rainbow troutp hepatoma (RTH-149) and gonad cells (RTG-2) were used to develop a screening protocol for estrogen disrupting chemicals. Transfection of an estrogen-responsive luciferase reporter plasmid into...

  14. Lipoplexes of dicationic gemini surfactants with DNA: Structural features of DNA compaction and transfection efficiency.

    PubMed

    Faizullin, D A; Zuev, Yu F; Zakharova, L Ya; Pokrovsky, A G; Korobeinikov, V A; Mukhametzyanov, T A; Konovalov, A I

    2015-01-01

    The internal structure of DNA lipoplexes with hydroxyethylated alkylammonium gemini surfactants (GS) with high transfection activity was studied by circular dichroism. It was shown that the efficiency of transfection of HEK293T cells with the pEGFP-N1 circular plasmid was different from zero only in the region of existence of chiral supramolecular DNA-GS complexes and reaches a maximum at concentrations at which the spontaneous aggregation of components is observed.

  15. 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.

  16. Construction of Hsp90β gene specific silencing plasmid and its transfection efficiency.

    PubMed

    Ji, Yewei; Nie, Bin; Li, Ping; Xu, Xiaoyu; Zhou, Yuanguo

    2007-07-01

    The purpose of this work was to construct the plasmid that could direct the synthesis of siRNA-like transcripts and thus mediate strong and specific repression of human heat shock protein 90β (Hsp90β) gene expression and to compare the transfection efficiency of the plasmids in varying conditions of transfection. Three 64 nt oligos corresponding to different regions of the target gene were chemically synthesized and annealed and were then ligated with pSUPER EGFP1 plasmid and double-digested with HindIII and BglII. Recombinant plasmids were transformed into Escherichia coli, DH5a, and the colonies were picked and grown in the Amp-agarose. The presence of positive clones was checked by the means of endodigestion and sequencing. Three cell strains, HepG2, Human umbilicus vein endothelium cells (HUVEC) and HeK293, were cultured. Then the plasmids were transfected into the cells at different ratios of plasmid to Lipofectamine. The transfection efficiency was measured by detection of enhanced green fluorescence protein (EGFP). The presence of positive recombinant clones were verified by double-digestion and sequencing. The bases inserted into the plasmids were correct and the positive colonies were named pSuper-Hsp90β1, pSuper-Hsp90β2 and pSuper-Hsp90β3. After optimizing the ratio of plasmid to Lipofectamine, we achieved high transfection efficiency in HeK293 cells. Transfection efficiency was still low in the HepG2 cells. In conclusion, the si-RNA-synthesizing plasmids targeting Hsp90β were constructed and transfected into cells with different transfection efficiency.

  17. Rice tungro spherical virus resistance into photoperiod-insensitive japonica rice by marker-assisted selection.

    PubMed

    Shim, Junghyun; Torollo, Gideon; Angeles-Shim, Rosalyn B; Cabunagan, Rogelio C; Choi, Il-Ryong; Yeo, Un-Sang; Ha, Woon-Goo

    2015-09-01

    Rice tungro disease (RTD) is one of the destructive and prevalent diseases in the tropical region. RTD is caused by Rice tungro spherical virus (RTSV) and Rice tungro bacilliform virus. Cultivation of japonica rice (Oryza sativa L. ssp japonica) in tropical Asia has often been restricted because most japonica cultivars are sensitive to short photoperiod, which is characteristic of tropical conditions. Japonica1, a rice variety bred for tropical conditions, is photoperiod-insensitive, has a high yield potential, but is susceptible to RTD and has poor grain quality. To transfer RTD resistance into Japonica1, we made two backcrosses (BC) and 8 three-way crosses (3-WC) among Japonica1 and RTSV-resistant cultivars. Among 8,876 BC1F2 and 3-WCF2 plants, 342 were selected for photoperiod-insensitivity and good grain quality. Photoperiod-insensitive progenies were evaluated for RTSV resistance by a bioassay and marker-assisted selection (MAS), and 22 BC1F7 and 3-WCF7 lines were selected based on the results of an observational yield trial. The results demonstrated that conventional selection for photoperiod-insensitivity and MAS for RTSV resistance can greatly facilitate the development of japonica rice that is suitable for cultivation in tropical Asia. PMID:26366118

  18. 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…

  19. 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.

  20. 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

    2016-01-01

    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

  1. 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)

    2003-01-01

    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.

  2. 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.

  3. 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.

  4. 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

  5. The Relationship between Multicultural Counseling Training and the Evaluation of Culturally Sensitive and Culturally Insensitive Counselors.

    ERIC Educational Resources Information Center

    Steward, Robbie J.; Wright, Doris J.; Jackson, James D.; Jo, Han Ik

    1998-01-01

    In this study multicultural counseling training contributed significantly to the variance in ratings of a culturally sensitive counselor. No significant contribution was made to the variance in ratings of the culturally insensitive counselor. Possible explanations and implications of these results in multicultural counseling supervision are…

  6. 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.

  7. A new insensitive explosive that has moderate performance and is low cost: 2,4-Dinitroimidazole

    SciTech Connect

    Simpson, R.; Coon, C.; Foltz, M.

    1995-01-01

    Shock loading experiments were performed on 2,4-Dinitroimidazole. Results indicate that it is a very shock insensitive material. The performance of the material is expected to be 60% greater than TNT. Costs appear to be low but are unresolved at this time. 2,4-DNI may be a realistic alternative to TNT for mass-use bombs.

  8. Synthesis of Amino- and Nitro-Substituted Heterocycles as Insensitive Energetic Materials

    SciTech Connect

    Pagoria, P F; Lee, G S; Mitchell, A R; Schmidt, R D

    2001-08-23

    In this paper we will describe the synthesis of several amino- and nitro-substituted heterocycles, examples from a continuing research project targeted at the synthesis of new, insensitive energetic materials that possess at least 80% the power of HMX (28% more power than TATB). Recently we reported the synthesis and scale-up of the insensitive energetic material, 2,6-diamino-3,5-dinitropyrazine-1-oxide (LLM-105). The energy content (81% the power of HMX) and thermal stability of LLM-105 make it a viable candidate material for insensitive boosters and deep oil perforation. We will report on recent synthetic improvements and several performance and safety tests performed on LLM-105, including a 1 in. cylinder shot and plate dent. We will also report on the synthesis and characterization of 4-amino-3,5-dinitropyrazole (LLM-116), an interesting new insensitive energetic material with a measured crystal density of 1.90 g/cc, to our knowledge the highest density yet measured for a five-membered heterocycle containing amino- and nitro-substituents. LLM-116 was synthesized by reacting 3,5-dinitropyrazole with 1,1,1-trimethylhydrazinium iodide (TMHI) in DMSO in the presence of base. The synthesis and characterization of 4-amino-5-nitro-1,2,3-triazole (ANTZ) and 43-dinitro-1,2,3-triazole (DNTZ), first described by Baryshnikov and coworkers, will also be presented along with the synthesis of several new energetic materials derived from ANTZ and DNTZ.

  9. 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…

  10. 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.

  11. Malate Oxidation and Cyanide-Insensitive Respiration in Avocado Mitochondria during the Climacteric Cycle.

    PubMed

    Moreau, F; Romani, R

    1982-11-01

    After preparation on self-generated Percoll gradients, avocado (Persea americana Mill, var. Fuerte and Hass) mitochondria retain a high proportion of cyanide-insensitive respiration, especially with alpha-ketoglutarate and malate as substrates. Whereas alpha-ketoglutarate oxidation remains unchanged, the rate of malate oxidation increases as ripening advances through the climacteric. An enhancement of mitochondrial malic enzyme activity, measured by the accumulation of pyruvate, closely parallels the increase of malate oxidation. The capacity for cyanide-insensitive respiration is also considerably enhanced while respiratory control decreases (from 3.3 to 1.7), leading to high state 4 rates.Both malate dehydrogenase and malic enzyme are functional in state 3, but malic enzyme appears to predominate before the addition of ADP and after its depletion. In the presence of cyanide, a membrane potential is generated when the alterntive pathway is operating. Cyanide-insensitive malate oxidation can be either coupled to the first phosphorylation site, sensitive to rotenone, or by-pass this site. In the absence of phosphate acceptor, malate oxidation is mainly carried out via malic enzyme and the alternative pathway. Experimental modification of the external mitochondrial environment in vitro (pH, NAD(+), glutamade) results in changes in malate dehydrogenase and malic enzyme activities, which also modify cyanide resistance. It appears that a functional connection exists between malic enzyme and the alternative pathway via a rotenone-insensitive NADH dehydrogenase and that this pathway is responsible, in part, for nonphosphorylating respiratory activity during the climacteric.

  12. Out-of-band insensitive polymer-bound PAG for EUV resist

    NASA Astrophysics Data System (ADS)

    Iwashita, Jun; Hirayama, Taku; Matsuzawa, Kensuke; Utsumi, Yoshiyuki; Ohmori, Katsumi

    2012-03-01

    Out of band (OoB) radiation has been regarded as one of the key issues on Extreme Ultra Violet Lithography (EUVL). OoB light especially in the deep ultraviolet (DUV) region have a negative impact on image contrast and resist profile, since general photo acid generator (PAG) used in chemically amplified EUV resist are also sensitive for DUV. It is reported that a Spectral Purify Filter (SPF) would eliminate OoB radiation. However it expense a large reduction in EUV power and hence throughput, so it is reported that HVM EUV exposure tool would not employ SPF. Therefore, both EUV sensitive and DUV insensitive are required property to overcome OoB radiation issue by resist material itself. Consideration of PAG cation structure was proceeded to control absorption for DUV. Based on the concept, OoB insensitivity was investigated both on blend resist platform and Polymer Bound PAG (PBP) platform. OoB insensitive concept was confirmed with UV spectrum and sensitivity for KrF and ArF. The OoB insensitive PAG cation worked well on PBP, while dark loss are seen on blend resist platform due to lack of inhibition effect. Lithographic performance would be exhibited using Alpha Demo Tool (ADT) and NXE3100. Outgassing property on witness sample (WS) and Residual Gas Analysis (RGA ) will be also discussed.

  13. A general strategy to achieve ultra-high gene transfection efficiency using lipid-nanoparticle composites.

    PubMed

    Vankayala, Raviraj; Chiang, Chi-Shiun; Chao, Jui-I; Yuan, Chiun-Jye; Lin, Shyr-Yeu; Hwang, Kuo Chu

    2014-09-01

    Gene therapy provides a new hope for previously "incurable" diseases. Low gene transfection efficiency, however, is the bottle-neck to the success of gene therapy. It is very challenging to develop non-viral nanocarriers to achieve ultra-high gene transfection efficiencies. Herein, we report a novel design of "tight binding-but-detachable" lipid-nanoparticle composite to achieve ultrahigh gene transfection efficiencies of 60∼82%, approaching the best value (∼90%) obtained using viral vectors. We show that Fe@CNPs nanoparticles coated with LP-2000 lipid molecules can be used as gene carriers to achieve ultra-high (60-80%) gene transfection efficiencies in HeLa, U-87MG, and TRAMP-C1 cells. In contrast, Fe@CNPs having surface-covalently bound N,N,N-trimethyl-N-2-methacryloxyethyl ammonium chloride (TMAEA) oligomers can only achieve low (23-28%) gene transfection efficiencies. Similarly ultrahigh gene transfection/expression was also observed in zebrafish model using lipid-coated Fe@CNPs as gene carriers. Evidences for tight binding and detachability of DNA from lipid-nanoparticle nanocarriers will be presented. PMID:24973297

  14. Targeted microbubbles for ultrasound mediated gene transfection and apoptosis induction in ovarian cancer cells

    PubMed Central

    Zhu, Shenyin; Yan, Yu; Zhu, Yi; Li, Min; Wang, Zhigang; Xu, Ronald X.

    2015-01-01

    Ultrasound-targeted microbubble destruction (UTMD) technique can be potentially used for non-viral delivery of gene therapy. Targeting wild-type p53 (wtp53) tumor suppressor gene may provide a clinically promising treatment for patients with ovarian cancer. However, UTMD mediated gene therapy typically uses non-targeted microbubbles with suboptimal gene transfection efficiency. We synthesized a targeted microbubble agent for UTMD mediated wtp53 gene therapy in ovarian cancer cells. Lipid micro-bubbles were conjugated with a Luteinizing Hormone–Releasing Hormone analog (LHRHa) via an avidin– biotin linkage to target the ovarian cancer A2780/DDP cells that express LHRH receptors. The microbubbles were mixed with the pEGFP-N1-wtp53 plasmid. Upon exposure to 1 MHz pulsed ultrasound beam (0.5 W/cm2) for 30 s, the wtp53 gene was transfected to the ovarian cancer cells. The transfection efficiency was (43.90 ± 6.19)%. The expression of wtp53 mRNA after transfection was (97.08 ± 12.18)%. The cell apoptosis rate after gene therapy was (39.67 ± 5.95)%. In comparison with the other treatment groups, ultrasound mediation of targeted microbubbles yielded higher transfection efficiency and higher cell apoptosis rate (p < 0.05). Our experiment verifies the hypothesis that ultrasound mediation of targeted microbubbles will enhance the gene transfection efficiency in ovarian cancer cells. PMID:22841613

  15. 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.

  16. Development of a semi-automated high throughput transient transfection system.

    PubMed

    Bos, Aaron B; Duque, Joseph N; Bhakta, Sunil; Farahi, Farzam; Chirdon, Lindsay A; Junutula, Jagath R; Harms, Peter D; Wong, Athena W

    2014-06-20

    Transient transfection of mammalian cells provides a rapid method of producing protein for research purposes. Combining the transient transfection protein expression system with new automation technologies developed for the biotechnology industry would enable a high throughput protein production platform that could be utilized to generate a variety of different proteins in a short amount of time. These proteins could be used for an assortment of studies including proof of concept, antibody development, and biological structure and function. Here we describe such a platform: a semi-automated process for PEI-mediated transient protein production in tubespins at a throughput of 96 transfections at a time using a Biomek FX(P) liquid handling system. In one batch, 96 different proteins can be produced in milligram amounts by PEI transfection of HEK293 cells cultured in 50 mL tubespins. Methods were developed for the liquid handling system to automate the different processes associated with transient transfections such as initial cell seeding, DNA:PEI complex activation and DNA:PEI complex addition to the cells. Increasing DNA:PEI complex incubation time resulted in lower protein expression. To minimize protein production variability, the methods were further optimized to achieve consistent cell seeding, control the DNA:PEI incubation time and prevent cross-contamination among different tubespins. This semi-automated transfection process was applied to express 520 variants of a human IgG1 (hu IgG1) antibody. PMID:24704608

  17. Development of a semi-automated high throughput transient transfection system.

    PubMed

    Bos, Aaron B; Duque, Joseph N; Bhakta, Sunil; Farahi, Farzam; Chirdon, Lindsay A; Junutula, Jagath R; Harms, Peter D; Wong, Athena W

    2014-06-20

    Transient transfection of mammalian cells provides a rapid method of producing protein for research purposes. Combining the transient transfection protein expression system with new automation technologies developed for the biotechnology industry would enable a high throughput protein production platform that could be utilized to generate a variety of different proteins in a short amount of time. These proteins could be used for an assortment of studies including proof of concept, antibody development, and biological structure and function. Here we describe such a platform: a semi-automated process for PEI-mediated transient protein production in tubespins at a throughput of 96 transfections at a time using a Biomek FX(P) liquid handling system. In one batch, 96 different proteins can be produced in milligram amounts by PEI transfection of HEK293 cells cultured in 50 mL tubespins. Methods were developed for the liquid handling system to automate the different processes associated with transient transfections such as initial cell seeding, DNA:PEI complex activation and DNA:PEI complex addition to the cells. Increasing DNA:PEI complex incubation time resulted in lower protein expression. To minimize protein production variability, the methods were further optimized to achieve consistent cell seeding, control the DNA:PEI incubation time and prevent cross-contamination among different tubespins. This semi-automated transfection process was applied to express 520 variants of a human IgG1 (hu IgG1) antibody.

  18. Optimizing electroporation conditions in primary and other difficult-to-transfect cells.

    PubMed

    Jordan, Elizabeth T; Collins, Michelle; Terefe, Joseph; Ugozzoli, Luis; Rubio, Teresa

    2008-12-01

    Electroporation is a valuable tool for nucleic acid delivery because it can be used for a wide variety of cell types. Many scientists are shifting toward the use of cell types that are more relevant to in vivo applications, including primary cells, which are considered difficult to transfect. The ability to electroporate these cell types with nucleic acid molecules of interest at a relatively high efficiency while maintaining cell viability is essential for elucidating the pathway(s) in which a gene product is involved. We present data demonstrating that by optimizing electroporation parameters, nucleic acid molecules can be delivered in a highly efficient manner. We display transfection results for primary and difficult-to-transfect cell types including human primary fibroblasts, human umbilical vein endothelial cells, Jurkat cells, and two neuroblastoma cell lines [SK-N-SH (human) and Neuro-2A (mouse)] with plasmid DNAs and siRNAs. Our data demonstrate that by determining proper electroporation conditions, glyceraldehyde phosphate dehydrogenase mRNA was silenced in Jurkat cells when compared with negative control siRNA electroporations as early as 4 h post-transfection. Other experiments demonstrated that optimized electroporation conditions using a fluorescently labeled transfection control siRNA resulted in 75% transfection efficiency for Neuro-2A, 93% for human primary fibroblasts, and 94% for HUVEC cells, as analyzed by flow cytometry. PMID:19183796

  19. Novel mechanism of gene transfection by low-energy shock wave

    PubMed Central

    Hoon Ha, Chang; Cheol Lee, Seok; Kim, Sunghyen; Chung, Jihwa; Bae, Hasuk; Kwon, Kihwan

    2015-01-01

    Extracorporeal shock wave (SW) therapy has been studied in the transfection of naked nucleic acids into various cell lines through the process of sonoporation, a process that affects the permeation of cell membranes, which can be an effect of cavitation. In this study, siRNAs were efficiently transfected into primary cultured cells and mouse tumor tissue via SW treatment. Furthermore SW-induced siRNA transfection was not mediated by SW-induced sonoporation, but by microparticles (MPs) secreted from the cells. Interestingly, the transfection effect of the siRNAs was transferable through the secreted MPs from human umbilical vein endothelial cell (HUVEC) culture medium after treatment with SW, into HUVECs in another culture plate without SW treatment. In this study, we suggest for the first time a mechanism of gene transfection induced by low-energy SW through secreted MPs, and show that it is an efficient physical gene transfection method in vitro and represents a safe therapeutic strategy for site-specific gene delivery in vivo. PMID:26243452

  20. 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.

  1. Single-Tailed Lipidoids Enhance the Transfection Activity of Their Double-Tailed Counterparts.

    PubMed

    Wu, Yihang; Li, Linxian; Chen, Qing; Su, Yi; Levkin, Pavel A; Davidson, Gary

    2016-01-11

    Cationic lipid-like molecules (lipidoids) are widely used for in vitro and in vivo gene delivery. Nearly all lipidoids developed to date employ double-tail or multiple-tail structures for transfection. Single-tail lipidoids are seldom considered for transfection as they have low efficiency in gene delivery. So far, there is no detailed study on the contribution to transfection efficiency of single-tail lipidoids when combined with standard double-tail lipidoids. Here, we use combinatorial chemistry to synthesize 17 double-tail and 17 single-tail lipidoids using thiol-yne and thiol-ene click chemistry, respectively. HEK 293T cells were used to analyze transfection efficiency by fluorescence microscopy and calculated based on the percentage of cells transfected. The size and zeta potential of liposomes and lipoplexes were characterized by dynamic light scattering (DLS). Intracellular DNA delivery and trafficking was further examined using confocal microscopy. Our study shows that combining single with double-tail lipidoids increases uptake of lipoplexes, as well as cellular transfection efficiency.

  2. A general strategy to achieve ultra-high gene transfection efficiency using lipid-nanoparticle composites.

    PubMed

    Vankayala, Raviraj; Chiang, Chi-Shiun; Chao, Jui-I; Yuan, Chiun-Jye; Lin, Shyr-Yeu; Hwang, Kuo Chu

    2014-09-01

    Gene therapy provides a new hope for previously "incurable" diseases. Low gene transfection efficiency, however, is the bottle-neck to the success of gene therapy. It is very challenging to develop non-viral nanocarriers to achieve ultra-high gene transfection efficiencies. Herein, we report a novel design of "tight binding-but-detachable" lipid-nanoparticle composite to achieve ultrahigh gene transfection efficiencies of 60∼82%, approaching the best value (∼90%) obtained using viral vectors. We show that Fe@CNPs nanoparticles coated with LP-2000 lipid molecules can be used as gene carriers to achieve ultra-high (60-80%) gene transfection efficiencies in HeLa, U-87MG, and TRAMP-C1 cells. In contrast, Fe@CNPs having surface-covalently bound N,N,N-trimethyl-N-2-methacryloxyethyl ammonium chloride (TMAEA) oligomers can only achieve low (23-28%) gene transfection efficiencies. Similarly ultrahigh gene transfection/expression was also observed in zebrafish model using lipid-coated Fe@CNPs as gene carriers. Evidences for tight binding and detachability of DNA from lipid-nanoparticle nanocarriers will be presented.

  3. Preparation of plasmid DNA in transfection complexes for fluorescence and electron spectroscopic imaging.

    PubMed

    Malecki, M

    1996-01-01

    The aim of this project was to develop procedures necessary to study mechanisms of receptor mediated gene transfer by means of integrated microscopy. Plasmid DNA was incorporated into a transfection complex consisting of poly(L)lysine and transferrin to which the nuclear localization signal was conjugated. This complex was presented to cultured glioma cells. Preparation of the transfected DNA for imaging was pursued by two methods. In the first method tetramethylrhodamine, nanogold, and ferritin were linked through streptavidin to the biotinylated plasmid DNA. Trafficking of the fluorescent derivatives was studied in living cells with fluorescence microscopy. Then, selected cells were rapidly cryo-immobilized. Ultra-structural distribution of the transfected DNA was imaged with energy filtering transmission electron microscopy. In the second method, the unmodified transfected DNA was detected in cryo-immobilized cells by in situ polymerase chain reaction and in situ hybridization. For laser scanning fluorescence microscopy probes were labeled with tetramethylrhodamine. For ultrastructural analysis by electron spectroscopic imaging, probes containing incorporated digoxigenin were labeled with anti-digoxigenin boronated antibodies. Based upon the developed procedures, it has been demonstrated that the presence of the nuclear localization signal in the transfection complex resulted in rapid nuclear import of the transfected DNA. PMID:9601525

  4. Helios(®) Gene Gun-Mediated Transfection of the Inner Ear Sensory Epithelium: Recent Updates.

    PubMed

    Belyantseva, Inna A

    2016-01-01

    The transfection of vertebrate inner ear hair cells has proven to be challenging. Therefore, many laboratories attempt to use and improve different transfection methods. Each method has its own advantages and disadvantages. A particular researcher's skills in addition to available equipment and the type of experiment (in vivo or in vitro) likely determine the transfection method of choice. Biolistic delivery of exogenous DNA, mRNA, or siRNA, also known as Helios(®) Gene Gun-mediated transfection, uses the mechanical energy of compressed helium gas to bombard tissue with micron- or submicron-sized DNA or RNA-coated gold particles, which can penetrate and transfect cells in vitro or in vivo. Helios(®) Gene Gun-mediated transfection has several advantages: (1) it is simple enough to learn in a short time; (2) it is designed to overcome cell barriers even as tough as plant cell membrane or stratum corneum in the epidermis; (3) it can transfect cells deep inside a tissue such as specific neurons within a brain slice; (4) it can accommodate mRNA, siRNA, or DNA practically of any size to be delivered; and (5) it works well with various cell types including non-dividing, terminally differentiated cells that are difficult to transfect, such as neurons or mammalian inner ear sensory hair cells. The latter advantage is particularly important for inner ear research. The disadvantages of this method are: (1) low efficiency of transfection due to many variables that have to be adjusted and (2) potential mechanical damage of the tissue if the biolistic shot parameters are not optimal. This chapter provides a step-by-step protocol and critical evaluation of the Bio-Rad Helios(®) Gene Gun transfection method used to deliver green fluorescent protein (GFP)-tagged full-length cDNAs of myosin 15a, whirlin, β-actin, and Clic5 into rodent hair cells of the postnatal inner ear sensory epithelia in culture.

  5. Enhancement of reverse transfection efficiency by combining stimulated DNA surface desorption and electroporation

    NASA Astrophysics Data System (ADS)

    Creasey, Rhiannon; Hook, Andrew; Thissen, Helmut; Voelcker, Nicolas H.

    2007-12-01

    Transfection cell microarrays (TCMs) are a high-throughput, miniaturised cell-culture system utilising reverse transfection, in which cells are seeded onto a DNA array resulting in localised regions of transfected cells. TCMs are useful for the analysis of gene expression, and can be used to identify genes involved in many cellular processes. This is of significant interest in fields such as tissue engineering, diagnostic screening, and drug testing [1, 2]. Low transfection efficiency has so far limited the application and utility of this technique. Recently, the transfection efficiency of TCMs was improved by an application of a high voltage for a short period of time to the DNA array resulting in the electroporation of cells attached to the surface [3, 4]. Furthermore, application of a low voltage for a longer period of time to the DNA array was shown to improve the transfection efficiency by stimulating the desorption of attached DNA, increasing the concentration of DNA available for cellular uptake [5]. In the present study, the optimisation of the uptake of adsorbed DNA vectors by adherent cells, utilising a voltage bias without compromising cell viability was investigated. This was achieved by depositing negatively charged DNA plasmids onto a positively charged allylamine plasma polymer (ALAPP) layer deposited on highly doped p-type silicon wafers either using a pipettor or a microarray contact printer. Surface-dependant human embryonic kidney (HEK 293 line) cells were cultured onto the DNA vector loaded ALAPP spots and the plasmid transfection events were detected by fluorescence microscopy. Cell viability assays, including fluorescein diacetate (FDA) / Hoechst DNA labelling, were carried out to determine the number of live adherent cells before and after application of a voltage. A protocol was developed to screen for voltage biases and exposure times in order to optimise transfection efficiency and cell viability. Cross-contamination between the microarray

  6. Helios(®) Gene Gun-Mediated Transfection of the Inner Ear Sensory Epithelium: Recent Updates.

    PubMed

    Belyantseva, Inna A

    2016-01-01

    The transfection of vertebrate inner ear hair cells has proven to be challenging. Therefore, many laboratories attempt to use and improve different transfection methods. Each method has its own advantages and disadvantages. A particular researcher's skills in addition to available equipment and the type of experiment (in vivo or in vitro) likely determine the transfection method of choice. Biolistic delivery of exogenous DNA, mRNA, or siRNA, also known as Helios(®) Gene Gun-mediated transfection, uses the mechanical energy of compressed helium gas to bombard tissue with micron- or submicron-sized DNA or RNA-coated gold particles, which can penetrate and transfect cells in vitro or in vivo. Helios(®) Gene Gun-mediated transfection has several advantages: (1) it is simple enough to learn in a short time; (2) it is designed to overcome cell barriers even as tough as plant cell membrane or stratum corneum in the epidermis; (3) it can transfect cells deep inside a tissue such as specific neurons within a brain slice; (4) it can accommodate mRNA, siRNA, or DNA practically of any size to be delivered; and (5) it works well with various cell types including non-dividing, terminally differentiated cells that are difficult to transfect, such as neurons or mammalian inner ear sensory hair cells. The latter advantage is particularly important for inner ear research. The disadvantages of this method are: (1) low efficiency of transfection due to many variables that have to be adjusted and (2) potential mechanical damage of the tissue if the biolistic shot parameters are not optimal. This chapter provides a step-by-step protocol and critical evaluation of the Bio-Rad Helios(®) Gene Gun transfection method used to deliver green fluorescent protein (GFP)-tagged full-length cDNAs of myosin 15a, whirlin, β-actin, and Clic5 into rodent hair cells of the postnatal inner ear sensory epithelia in culture. PMID:27259918

  7. Demonstration of polarization-insensitive spatial light modulation using a single polarization-sensitive spatial light modulator

    PubMed Central

    Liu, Jun; Wang, Jian

    2015-01-01

    We present a simple configuration incorporating a single polarization-sensitive phase-only liquid crystal spatial light modulator (LC-SLM) to facilitate polarization-insensitive spatial light modulation. The polarization-insensitive configuration is formed by a polarization beam splitter (PBS), a polarization-sensitive phase-only LC-SLM, a half-wave plate (HWP), and a mirror in a loop structure. We experimentally demonstrate polarization-insensitive spatial light modulations for incident linearly polarized beams with different polarization states and polarization-multiplexed beams. Polarization-insensitive spatial light modulations generating orbital angular momentum (OAM) beams are demonstrated in the experiment. The designed polarization-insensitive configuration may find promising applications in spatial light modulations accommodating diverse incident polarizations. PMID:26146032

  8. BRASSINOSTEROID INSENSITIVE2 interacts with ABSCISIC ACID INSENSITIVE5 to mediate the antagonism of brassinosteroids to abscisic acid during seed germination in Arabidopsis.

    PubMed

    Hu, Yanru; Yu, Diqiu

    2014-11-01

    Seed germination and postgerminative growth are regulated by a delicate hormonal balance. Abscisic acid (ABA) represses Arabidopsis thaliana seed germination and postgerminative growth, while brassinosteroids (BRs) antagonize ABA-mediated inhibition and promote these processes. However, the molecular mechanism underlying BR-repressed ABA signaling remains largely unknown. Here, we show that the Glycogen Synthase Kinase 3-like kinase BRASSINOSTEROID INSENSITIVE2 (BIN2), a critical repressor of BR signaling, positively regulates ABA responses during seed germination and postgerminative growth. Mechanistic investigation revealed that BIN2 physically interacts with ABSCISIC ACID INSENSITIVE5 (ABI5), a bZIP transcription factor. Further genetic analysis demonstrated that the ABA-hypersensitive phenotype of BIN2-overexpressing plants requires ABI5. BIN2 was found to phosphorylate and stabilize ABI5 in the presence of ABA, while application of epibrassinolide (the active form of BRs) inhibited the regulation of ABI5 by BIN2. Consistently, the ABA-induced accumulation of ABI5 was affected in BIN2-related mutants. Moreover, mutations of the BIN2 phosphorylation sites on ABI5 made the mutant protein respond to ABA improperly. Additionally, the expression of several ABI5 regulons was positively modulated by BIN2. These results provide evidence that BIN2 phosphorylates and stabilizes ABI5 to mediate ABA response during seed germination, while BRs repress the BIN2-ABI5 cascade to antagonize ABA-mediated inhibition.

  9. BRASSINOSTEROID INSENSITIVE2 Interacts with ABSCISIC ACID INSENSITIVE5 to Mediate the Antagonism of Brassinosteroids to Abscisic Acid during Seed Germination in Arabidopsis[W

    PubMed Central

    Hu, Yanru; Yu, Diqiu

    2014-01-01

    Seed germination and postgerminative growth are regulated by a delicate hormonal balance. Abscisic acid (ABA) represses Arabidopsis thaliana seed germination and postgerminative growth, while brassinosteroids (BRs) antagonize ABA-mediated inhibition and promote these processes. However, the molecular mechanism underlying BR-repressed ABA signaling remains largely unknown. Here, we show that the Glycogen Synthase Kinase 3-like kinase BRASSINOSTEROID INSENSITIVE2 (BIN2), a critical repressor of BR signaling, positively regulates ABA responses during seed germination and postgerminative growth. Mechanistic investigation revealed that BIN2 physically interacts with ABSCISIC ACID INSENSITIVE5 (ABI5), a bZIP transcription factor. Further genetic analysis demonstrated that the ABA-hypersensitive phenotype of BIN2-overexpressing plants requires ABI5. BIN2 was found to phosphorylate and stabilize ABI5 in the presence of ABA, while application of epibrassinolide (the active form of BRs) inhibited the regulation of ABI5 by BIN2. Consistently, the ABA-induced accumulation of ABI5 was affected in BIN2-related mutants. Moreover, mutations of the BIN2 phosphorylation sites on ABI5 made the mutant protein respond to ABA improperly. Additionally, the expression of several ABI5 regulons was positively modulated by BIN2. These results provide evidence that BIN2 phosphorylates and stabilizes ABI5 to mediate ABA response during seed germination, while BRs repress the BIN2-ABI5 cascade to antagonize ABA-mediated inhibition. PMID:25415975

  10. Efficient transfection of DNA into primarily cultured rat sertoli cells by electroporation.

    PubMed

    Li, Fuping; Yamaguchi, Kohei; Okada, Keisuke; Matsushita, Kei; Enatsu, Noritoshi; Chiba, Koji; Yue, Huanxun; Fujisawa, Masato

    2013-03-01

    The expression of exogenous DNA in Sertoli cells is essential for studying its functional genomics, pathway analysis, and medical applications. Electroporation is a valuable tool for nucleic acid delivery, even in primarily cultured cells, which are considered difficult to transfect. In this study, we developed an optimized protocol for electroporation-based transfection of Sertoli cells and compared its efficiency with conventional lipofection. Sertoli cells were transfected with pCMV-GFP plasmid by square-wave electroporation under different conditions. After transfection of plasmid into Sertoli cells, enhanced green fluorescent protein (EGFP) expression could be easily detected by fluorescent microscopy, and cell survival was evaluated by dye exclusion assay using Trypan blue. In terms of both cell survival and the percentage expressing EGFP, 250 V was determined to produce the greatest number of transiently transfected cells. Keeping the voltage constant (250 V), relatively high cell survival (76.5% ± 3.4%) and transfection efficiency (30.6% ± 5.6%) were observed with a pulse length of 20 μm. The number of pulses significantly affected cell survival and EGFP expression (P < 0.001). Cell survival clearly decreased following one to three pulses, from 83.9% ± 6.1% to 3.2% ± 1.1%, with EGFP expression increasing from 41.8% ± 9.4% to 66.7% ± 5.2%. The yield of positive cells increased with increasing concentration of plasmid DNA (range, 10-50 μg/ml), from 14.0% ± 2.8% to 35.0% ± 6.3%, but cell viability steadily decreased following 20 μg/ml plasmid DNA, from 73.1% ± 4.9% to 57.0% ± 6.6%. Compared with two popular cationic lipid transfection methods, the transfection efficiency of electroporation (21.5% ± 5.7%) was significantly higher than those of Lipofectamine 2000 (2.9% ± 1.0%) and Effectene (1.9% ± 0.8%) in this experiment (P < 0.001). We describe the process of optimizing electroporation conditions, and the successful electroporation of plasmid

  11. Hydrophobic modification of low molecular weight polyethylenimine for improved gene transfection.

    PubMed

    Teo, Pei Yun; Yang, Chuan; Hedrick, James L; Engler, Amanda C; Coady, Daniel J; Ghaem-Maghami, Sadaf; George, Andrew J T; Yang, Yi Yan

    2013-10-01

    Hydrophobic modification of low molecular weight (LMW) polyethylenimine (PEI) is known to increase gene transfection efficiency of LMW PEI. However, few studies have explored how the conjugated hydrophobic groups influence the properties of the modified LMW PEI mainly due to difficulties in obtaining well defined final product compositions and limitations in current chemical synthesis routes. The aim of this study was to modify LMW PEI (Mn 1.8 kDa, PEI-1.8) judiciously with different hydrophobic functional groups and to investigate how hydrophobicity, molecular structure and inclusion of hydrogen bonding properties in the conjugated side groups as well as the conjugation degree (number of primary amine groups of PEI-1.8 modified with hydrophobic groups) influence PEI-1.8 gene transfection efficiency. The modified polymers were characterized for DNA binding ability, particle size, zeta potential, in vitro gene transfection efficiency and cytotoxicity in SKOV-3 human ovarian cancer and HepG2 human liver carcinoma cell lines. The study shows that modified PEI-1.8 polymers are able to condense plasmid DNA into cationic nanoparticles, of sizes ~100 nm, whereas unmodified polymer/DNA complexes display larger particle sizes of 2 μm. Hydrophobic modification also increases the zeta potential of polymer/DNA complexes. Importantly, modified PEI-1.8 shows enhanced transfection efficiency over the unmodified counterpart. Higher transfection efficiency is obtained when PEI-1.8 is modified with shorter hydrophobic groups (MTC-ethyl) as opposed to longer ones (MTC-octyl and MTC-deodecyl). An aromatic structured functional group (MTC-benzyl) also enhances transfection efficiency more than an alkyl functional group (MTC-octyl). An added hydrogen-bonding urea group in the conjugated functional group (MTC-urea) does not enhance transfection efficiency over one without urea (MTC-benzyl). The study also demonstrates that modification degree greatly influences gene transfection, and

  12. Hyaluronidase and collagenase increase the transfection efficiency of gene electrotransfer in various murine tumors.

    PubMed

    Cemazar, Maja; Golzio, Muriel; Sersa, Gregor; Escoffre, Jean-Michel; Coer, Andrej; Vidic, Suzana; Teissie, Justin

    2012-01-01

    One of the applications of electroporation/electropulsation in biomedicine is gene electrotransfer, the wider use of which is hindered by low transfection efficiency in vivo compared with viral vectors. The aim of our study was to determine whether modulation of the extracellular matrix in solid tumors, using collagenase and hyaluronidase, could increase the transfection efficiency of gene electrotransfer in histologically different solid subcutaneous tumors in mice. Tumors were treated with enzymes before electrotransfer of plasmid DNA encoding either green fluorescent protein or luciferase. Transfection efficiency was determined 3, 9, and 15 days posttransfection. We demonstrated that pretreatment of tumors with a combination of enzymes significantly increased the transfection efficiency of electrotransfer in tumors with a high extracellular matrix area (LPB fibrosarcoma). In tumors with a smaller extracellular matrix area and less organized collagen lattice, the increase was not so pronounced (SA-1 fibrosarcoma and EAT carcinoma), whereas in B16 melanoma, in which only traces of collagen are present, pretreatment of tumors with hyaluronidase alone was more efficient than pretreatment with both enzymes. In conclusion, our results suggest that modification of the extracellular matrix could improve distribution of plasmid DNA in solid subcutaneous tumors, demonstrated by an increase in transfection efficiency, and thus have important clinical implications for electrogene therapy. PMID:21797718

  13. Cadherin transfection of Xenopus XTC cells downregulates expression of substrate adhesion molecules.

    PubMed

    Finnemann, S; Kühl, M; Otto, G; Wedlich, D

    1995-09-01

    Cadherins are discussed not in terms of their adhesive function but rather as morphoregulatory proteins. Changes in gene expression following cadherin transfection of cells in culture or by overexpression in embryos have, until now, not been reported. We established a protocol for stable transfection of Xenopus XTC cells and generated cells bearing high levels of membrane-integrated mouse uvomorulin (E-cadherin) or Xenopus XB-cadherin. These cell lines showed drastically impaired substrate adhesion on fibronectin and laminin. In immunoblot and radioimmunoprecipitation experiments, we found that fibronectin and alpha 3/beta 1 integrin are downregulated. The reduced amounts of proteins result from a decrease of the respective mRNAs as proven by RNase protection assays. Coprecipitations revealed that transfected cadherin molecules are complexed with alpha-catenin and beta-catenin at plasma membranes. However, the alpha-catenin present in the XB-cadherin complex differs immunologically from that found in the uvomorulin complex. When a truncated form of XB-cadherin lacking 38 of the most C-terminal amino acids was expressed in XTC cells, complex formation with endogenous catenins was abolished. In these transfectants, substrate adhesion was not affected. These results prove that complex formation of transfected cadherins in XTC cells with endogenous beta-catenin correlates with altered synthesis of certain substrate adhesion molecules.

  14. Cyclic Amphipathic Peptide-DNA Complexes Mediate High-Efficiency Transfection of Adherent Mammalian Cells

    NASA Astrophysics Data System (ADS)

    Legendre, Jean-Yves; Szoka, Francis C., Jr.

    1993-02-01

    A DNA transfection protocol has been developed that makes use of the cyclic cationic amphipathic peptide gramicidin S and dioleoyl phosphatidylethanolamine. The DNA complex is formed by mixing gramicidin S with DNA at a 1:1 charge ratio and then adding phosphatidylethanolamine at a lipid/peptide molar ratio of 5:1. The complex mediates rapid association of DNA with cells and leads to transient expression levels of β-galactosidase ranging from 1 to 30% of the transfected cells, with long-term expression being about an order of magnitude lower. The respective roles of peptide and phospholipid are not yet resolved but optimal transfection requires both the cyclic peptide and the hexagonal phase-competent phospholipid PtdEtn. Transfection in CV-1 cells is not affected by lysomotrophic agents, which suggests that DNA entry into the cell is via the plasma membrane. This technique that is simple, economical, and reproducible mediates transfection levels up to 20-fold higher than cationic liposomes in adherent mammalian cells.

  15. Transfection of immortalized keratinocytes by low toxic poly(2-(dimethylamino)ethyl methacrylate)-based polymers.

    PubMed

    Van Overstraeten-Schlögel, Nancy; Ho-Shim, Yong; Tevel, Virginie; Bontems, Sébastien; Dubois, Philippe; Raes, Martine

    2012-01-01

    Skin carcinoma are among the most spread diagnosed tumours in the world. In this study, we investigated the transfection of immortalized keratinocytes, used as an in vitro model for skin carcinoma, using antisense technology and poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA)-based polymers, with original architecture and functionalities. We tested PDMAEMA polymers with different structures: linear, with two (DEA-PDMAEMA) or three (TEA-PDMAEMA) arms. The cytotoxicity of these polymers was assessed over a wide range of apparent M n (from 7600 to 64 600). At a N/P ratio of 7.38, cytotoxicity increases with the M n. Keratinocytes were transfected with a fluorescent oligonucleotide and then analyzed by flow cytometry. For the three architectures tested, the percentage of transfected cells and abundance of internalized oligonucleotide were closely related to the M n of the polymer. Confocal microscopy and FACS analyses showed a wide spread fine granular distribution of the oligonucleotide up to 3 days post-transfection. Then, we assessed the silencing efficiency of the polymers, targeting GFP in GFP expressing keratinocytes. The maximal silencing effect (±40%) was obtained using a DEA-PDMAEMA polymer (M n = 30 300). These results suggest that PDMAEMA-based polymers can be efficiently used to transfect immortalized keratinocytes and, thus, open new perspectives in the therapy of skin carcinoma.

  16. 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.

  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. PMID:26797493

  18. Characterization and crystal structure of lysine insensitive Corynebacterium glutamicum dihydrodipicolinate synthase (cDHDPS) protein

    SciTech Connect

    Rice, E.A.; Bannon, G.A.; Glenn, K.C.; Jeong, S.S.; Sturman, E.J.; Rydel, T.J.

    2008-11-21

    The lysine insensitive Corynebacterium glutamicum dihydrodipicolinate synthase enzyme (cDHDPS) was recently successfully introduced into maize plants to enhance the level of lysine in the grain. To better understand lysine insensitivity of the cDHDPS, we expressed, purified, kinetically characterized the protein, and solved its X-ray crystal structure. The cDHDPS enzyme has a fold and overall structure that is highly similar to other DHDPS proteins. A noteworthy feature of the active site is the evidence that the catalytic lysine residue forms a Schiff base adduct with pyruvate. Analyses of the cDHDPS structure in the vicinity of the putative binding site for S-lysine revealed that the allosteric binding site in the Escherichia coli DHDPS protein does not exist in cDHDPS due to three non-conservative amino acids substitutions, and this is likely why cDHDPS is not feedback inhibited by lysine.

  19. Efficient design of polarization insensitive polymer optical waveguide devices considering stress-induced effects.

    PubMed

    Hossain, Md Faruque; Chan, Hau Ping; Kouzani, Abbas Z

    2014-04-21

    We present an approach for the efficient design of polarization insensitive polymeric optical waveguide devices considering stress-induced effects. In this approach, the stresses induced in the waveguide during the fabrication process are estimated first using a more realistic model in the finite element analysis. Then we determine the perturbations in the material refractive indices caused by the stress-optic effect. It is observed that the stresses cause non-uniform optical anisotropy in the waveguide materials, which is then incorporated in the modal analysis considering a multilayer structure of waveguide. The approach is exploited in the design of a Bragg grating on strip waveguide. Excellent agreement between calculated and published experimental results confirms the feasibility of our approach in the accurate design of polarization insensitive polymer waveguide devices.

  20. 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.

  1. 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.

  2. A Stress-Activated Transposon in Arabidopsis Induces Transgenerational Abscisic Acid Insensitivity

    PubMed Central

    Ito, Hidetaka; Kim, Jong-Myong; Matsunaga, Wataru; Saze, Hidetoshi; Matsui, Akihiro; Endo, Takaho A.; Harukawa, Yoshiko; Takagi, Hiroki; Yaegashi, Hiroki; Masuta, Yukari; Masuda, Seiji; Ishida, Junko; Tanaka, Maho; Takahashi, Satoshi; Morosawa, Taeko; Toyoda, Tetsuro; Kakutani, Tetsuji; Kato, Atsushi; Seki, Motoaki

    2016-01-01

    Transposable elements (TEs), or transposons, play an important role in adaptation. TE insertion can affect host gene function and provides a mechanism for rapid increases in genetic diversity, particularly because many TEs respond to environmental stress. In the current study, we show that the transposition of a heat-activated retrotransposon, ONSEN, generated a mutation in an abscisic acid (ABA) responsive gene, resulting in an ABA-insensitive phenotype in Arabidopsis, suggesting stress tolerance. Our results provide direct evidence that a transposon activated by environmental stress could alter the genome in a potentially positive manner. Furthermore, the ABA-insensitive phenotype was inherited when the transcription was disrupted by an ONSEN insertion, whereas ABA sensitivity was recovered when the effects of ONSEN were masked by IBM2. These results suggest that epigenetic mechanisms in host plants typically buffered the effect of a new insertion, but could selectively “turn on” TEs when stressed. PMID:26976262

  3. Parsing the construct of maternal insensitivity: distinct longitudinal pathways associated with early maternal withdrawal.

    PubMed

    Lyons-Ruth, Karlen; Bureau, Jean-Francois; Easterbrooks, M Ann; Obsuth, Ingrid; Hennighausen, Kate; Vulliez-Coady, Lauriane

    2013-01-01

    The current paper expands on Ainsworth's seminal construct of maternal sensitivity by exploring the developmental pathways associated with one particular form of insensitivity: maternal withdrawal. Drawing on longitudinal data from infancy to age 20 in a high-risk cohort, we highlight how maternal withdrawal over the first eight years of life is associated with child caregiving behavior and with maternal role confusion, as well as with features of borderline and antisocial personality disorders. We also present evidence for the specificity of this pathway in relation to other aspects of maternal insensitivity and other aspects of child adaptation. To illuminate these pathways we both review recent published work and report new findings on the middle childhood and adolescent components of these trajectories. Finally, we consider the implications for assessment of maternal behavior in high-risk samples and indicate directions for productive future work. PMID:24299135

  4. Polarization insensitive metamaterial absorber based on E-shaped all-dielectric structure

    NASA Astrophysics Data System (ADS)

    Li, Liyang; Wang, Jun; Ma, Hua; Wang, Jiafu; Du, Hongliang; Qu, Shaobo

    2015-04-01

    In this paper, we designed a metamaterial absorber performed in microwave frequency band. This absorber is composed of E-shaped dielectrics which are arranged along different directions. The E-shaped all-dielectric structure is made of microwave ceramics with high permittivity and low loss. Within about 1 GHz frequency band, more than 86% absorption efficiency was observed for this metamaterial absorber. This absorber is polarization insensitive and is stable for incident angles. It is figured out that the polarization insensitive absorption is caused by the nearly located varied resonant modes which are excited by the E-shaped all-dielectric resonators with the same size but in the different direction. The E-shaped dielectric absorber contains intensive resonant points. Our research work paves a way for designing all-dielectric absorber.

  5. 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}

  6. Design and characteristics of polarization-insensitive resonant gratings for color filtering

    NASA Astrophysics Data System (ADS)

    Xu, Banglian; Zhang, Dawei; Wang, Yinping; Huang, Yuanshen; Wang, Qi

    2013-12-01

    A method of designing polarization-insensitive color filters with guided-mode resonance grating is presented. The influence of incident conditions on exciting waveguide mode is investigated, and we find that polarization insensitivity may occur at the full conical incidence. Using rigorous coupled-wave analysis, we mainly analyze the effects of waveguide thickness and fill factor on the diffracted efficiency of the grating filter. The final structural parameters of the devices for three primary colors are collected after optimization, and the calculated results show that the spectral reflectance of each color filter is basically identical for different polarization states of incident light. Moreover, field analysis by the finite-difference time-domain technique indicates that two symmetric modes are excited between the waveguide layer and substrate under full conical incidence, which is coincident with the previous theoretical study. The reported work will eliminate the restriction of polarization-dependence of color filters and greatly expand their application range.

  7. 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

  8. 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.

  9. 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.

  10. Insensitive explosive composition and method of fracturing rock using an extrudable form of the composition

    DOEpatents

    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.

  11. 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.

  12. 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.

  13. Development of porcine embryos and offspring after intracytoplasmic sperm injection with liposome transfected or non-transfected sperm into in vitro matured oocytes.

    PubMed

    Lai, L; Sun, Q; Wu, G; Murphy, C N; Kühholzer, B; Park, K W; Bonk, A J; Day, B N; Prather, R S

    2001-11-01

    The objective of this study was to evaluate in vitro and in vivo development of porcine in vitro matured (IVM) porcine oocytes fertilised by intracytoplasmic sperm injection (ICSI) and the possibility of producing transgenic embryos and offspring with this procedure. Activated ICSI oocytes had a higher pronuclear formation than non-activated ICSI oocytes (mean 64.8+/-17.3% vs 28.5+/-3.4%, p<0.05). When the zygotes with two pronuclei were cultured to day 2, there was no difference (p<0.05) in the cleavage rate (mean 60.0+/-7.0% vs 63.3+/-12.7%) between the two groups. The blastocyst rate in the activation group was significantly higher than that in the non-activation group (mean 30.0+/-11.6% vs 4.6+/-4.2%, p<0.05). After injection of the sperm transfected with DNA/liposome complex, destabilised enhanced green fluorescent protein (d2EGFP) expression was not observed on day 2 in either cleaved or uncleaved embryos. But from day 3, some of the embryos at the 2-cell to 4-cell stage started to express d2EGFP. On day 7, about 30% of cleaved embryos, which were in the range of 2-cell to blastocyst stage, expressed d2EGFP. However, for the IVF oocytes inseminated with sperm transfected with DNA/liposome complex, and for oocytes injected with sperm transfected with DNA/liposome complex, and for oocytes injected with DNA/liposome complex following insemination with sperm not treated with DNA/liposome complex, none of the embryos expressed d2EGFP. Sixteen day 4 ICSI embryos derived from sperm not treated with DNA/liposome complex were transferred into a day 3 recipient. One recipient delivered a female piglet with normal birthweight. After transfer of the ICSI embryos derived from sperm transfected with DNA/liposome complex, none of the four recipients maintained pregnancy. PMID:11771901

  14. 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.

  15. The N-acetylcysteine-insensitive acetic acid-induced yeast programmed cell death occurs without macroautophagy.

    PubMed

    Antonacci, Lucia; Guaragnella, Nicoletta; Ždralevic, Maša; Passarella, Salvatore; Marra, Ersilia; Giannattasio, Sergio

    2012-12-01

    Programmed cell death can occur through two separate pathways caused by treatment of Saccharomyces cerevisiae with acetic acid (AA-PCD), which differ from one another essentially with respect to their sensitivity to N-acetylcysteine (NAC) and to the role played by cytochrome c and metacaspase YCA1. Moreover, yeast can also undergo macroautophagy which occurs in NAC-insensitive manner. In order to gain some insight into the relationship between AA-PCD and macroautophagy use was made of WT and knock-out cells lacking YCA1 and/or cytochrome c. We show that i. macroautophagy is modulated by YCA1 and by cytochrome c in a negative and positive manner, respectively, ii. the NAC-insensitive AA-PCD and macroautophagy differ from one another and iii. NAC-insensitive AA-PCD pathway takes place essentially without macroautophagy, even if the shift of extracellular pH to acidic values required for AA-PCD to occur leads itself to increased or decreased macroautophagy in YCA1 or cytochrome c-lacking cells. PMID:23072389

  16. Major QTLs associated with green stem disorder insensitivity of soybean (Glycine max (L.) Merr.).

    PubMed

    Yamada, Tetsuya; Shimada, Shinji; Hajika, Makita; Hirata, Kaori; Takahashi, Koji; Nagaya, Taiko; Hamaguchi, Hideo; Maekawa, Tomiya; Sayama, Takashi; Hayashi, Takeshi; Ishimoto, Masao; Tanaka, Junichi

    2014-12-01

    Green stem disorder (GSD) is one of the most serious syndromes affecting soybean (Glycine max) cultivation in Japan. In GSD, stems remain green even when pods mature. When soybean plants develop GSD, seed surfaces are soiled by tissue fluid and seed quality is deteriorated during machine harvesting. We performed quantitative trait locus (QTL) analyses for GSD insensitivity using recombinant inbred lines (RILs; n = 154) derived from a cross between an insensitive line ('Touhoku 129') and a sensitive leading cultivar ('Tachinagaha') during a 6-year evaluation. Three effective QTLs were detected. The influences of these QTLs were in the following order: qGSD1 (LG_H) > qGSD2 (LG_F) > qGSD3 (LG_L). At these three QTLs, 'Touhoku 129' genotypes exhibited more GSD insensitivity than 'Tachinagaha' genotypes. The lower incidence of GSD for 'Touhoku129' was attributable primarily to these three QTLs because RILs harboring a 'Touhoku 129' genotype at the three QTLs exhibited a GSD incidence similar to that of 'Touhoku 129.' Although a limitation of this study is that only one mapping population was evaluated, this QTL information and the flanking markers of these QTLs would be effective tools for resolving GSD in soybean breeding programs.

  17. 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

  18. 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. PMID:25528237

  19. Characterization of a state-insensitive dipole trap for cesium atoms

    SciTech Connect

    Phoonthong, P.; Douglas, P.; Wickenbrock, A.; Renzoni, F.

    2010-07-15

    In this work we characterize a state-insensitive dipole trap for cold cesium atoms, as realized by tightly focusing a single running laser beam at the magic wavelength. The use of trapping light at the magic wavelength of 935.6 nm resulted in the same ac Stark shift for the {sup 6}S{sub 1/2} ground state and the {sup 6}P{sub 3/2} excited state. A complete characterization of the trap is given, which includes the dependence of the lifetime on the trap depth, an analysis of the important role played by a depumper beam, and a comparison with dipole trapping at different (nonmagic) wavelengths. In particular, we measured the differential light shift of the relevant optical transition as a function of the trapping light wavelength, and showed that it becomes zero at the magic wavelength. Our results are compared to previous realizations of state-insensitive dipole traps for cesium atoms. We also discuss the possible role of the state-insensitive trap, its limitations, and possible developments for the study of ground-state quantum coherence phenomena and related applications.

  20. Joint photomicrobial process for the degradation of the insensitive munition N-guanylurea-dinitramide (FOX-12).

    PubMed

    Perreault, Nancy N; Halasz, Annamaria; Thiboutot, Sonia; Ampleman, Guy; Hawari, Jalal

    2013-05-21

    N-Guanylurea-dinitramide (FOX-12) is a very insensitive energetic material intended to be used in the composition of next-generation insensitive munitions. To help predict the environmental behavior and fate of FOX-12, we conducted a study to determine its photodegradability and biodegradability. When dissolved in water, FOX-12, a guanylurea-dinitramide salt, also named GUDN, dissociated instantly to produce the dinitramide moiety and guanylurea, as demonstrated by high-performance liquid chromatography (HPLC) analysis. When an aqueous solution of FOX-12 was subjected to photolysis using a solar-simulated photoreactor, we found a rapid removal of the dinitramide with concurrent formation of N₂O, NO₂(-), and NO₃(-). The second component, guanylurea, was photostable. However, when FOX-12 was incubated aerobically with the soil isolate Variovorax strain VC1 and protected from light, the dinitramide component of FOX-12 was recalcitrant but guanylurea degraded effectively to ammonia, guanidine, and presumably CO₂. When FOX-12 was incubated with strain VC1 in the presence of light, both components of FOX-12 degraded, giving similar products to those described above. We concluded that the new insensitive explosive FOX-12 can be effectively degraded by a joint photomicrobial process and, therefore, should not cause persistent contamination of surface waters.

  1. Micropenis. III. Primary hypogonadism, partial androgen insensitivity syndrome, and idiopathic disorders.

    PubMed

    Lee, P A; Danish, R K; Mazur, T; Migeon, C J

    1980-11-01

    This paper, the third in a series, presents data from 28 patients with micropenis categorized as primary hypogonadism (11 subjects), partial androgen insensitivity (1 subject), idiopathic (6 subjects), and etiology undetermined (10 subjects). Among the 11 patients with primary hypogonadism, 8 presented with various degrees of gonadal dysgenesis, 1 was a true hermaphrodite and 2 had the Robinow syndrome. Nine of the 28 patients were raised as females: 5 with primary hypogonadism and 4 with undetermined etiology. Eleven of the 19 patients raised as males received androgen stimulation during prepubertal years and responded with penile growth. However, this growth response was temporary and did not appear to be predictive of eventual adult size of the penis. Generally, the prestimulation size of this group of patients is more predictive of adult penile size. Only 7 of the patients raised as males have attained adult somatic growth. Three out of the three with primary hypogonadism, the subject with partial androgen insensitivity, and one of three with idiopathic micropenis have below-normal adult penile length. These limited data suggest that growth potential of the micropenis may be greater among the patients with an idiopathic state than among those with primary hypogonadism and partial androgen insensitivity.

  2. Joint photomicrobial process for the degradation of the insensitive munition N-guanylurea-dinitramide (FOX-12).

    PubMed

    Perreault, Nancy N; Halasz, Annamaria; Thiboutot, Sonia; Ampleman, Guy; Hawari, Jalal

    2013-05-21

    N-Guanylurea-dinitramide (FOX-12) is a very insensitive energetic material intended to be used in the composition of next-generation insensitive munitions. To help predict the environmental behavior and fate of FOX-12, we conducted a study to determine its photodegradability and biodegradability. When dissolved in water, FOX-12, a guanylurea-dinitramide salt, also named GUDN, dissociated instantly to produce the dinitramide moiety and guanylurea, as demonstrated by high-performance liquid chromatography (HPLC) analysis. When an aqueous solution of FOX-12 was subjected to photolysis using a solar-simulated photoreactor, we found a rapid removal of the dinitramide with concurrent formation of N₂O, NO₂(-), and NO₃(-). The second component, guanylurea, was photostable. However, when FOX-12 was incubated aerobically with the soil isolate Variovorax strain VC1 and protected from light, the dinitramide component of FOX-12 was recalcitrant but guanylurea degraded effectively to ammonia, guanidine, and presumably CO₂. When FOX-12 was incubated with strain VC1 in the presence of light, both components of FOX-12 degraded, giving similar products to those described above. We concluded that the new insensitive explosive FOX-12 can be effectively degraded by a joint photomicrobial process and, therefore, should not cause persistent contamination of surface waters. PMID:23594309

  3. 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.

  4. 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.

  5. 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.

  6. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure.

    PubMed Central

    Felgner, P L; Gadek, T R; Holm, M; Roman, R; Chan, H W; Wenz, M; Northrop, J P; Ringold, G M; Danielsen, M

    1987-01-01

    A DNA-transfection protocol has been developed that makes use of a synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). Small unilamellar liposomes containing DOTMA interact spontaneously with DNA to form lipid-DNA complexes with 100% entrapment of the DNA, DOTMA facilitates fusion of the complex with the plasma membrane of tissue culture cells, resulting in both uptake and expression of the DNA. The technique is simple, highly reproducible, and effective for both transient and stable expression of transfected DNA. Depending upon the cell line, lipofection is from 5- to greater than 100-fold more effective than either the calcium phosphate or the DEAE-dextran transfection technique. Images PMID:2823261

  7. Lipofection: A Highly Efficient, Lipid-Mediated DNA-Transfection Procedure

    NASA Astrophysics Data System (ADS)

    Felgner, Philip L.; Gadek, Thomas R.; Holm, Marilyn; Roman, Richard; Chan, Hardy W.; Wenz, Michael; Northrop, Jeffrey P.; Ringold, Gordon M.; Danielsen, Mark

    1987-11-01

    A DNA-transfection protocol has been developed that makes use of a synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). Small unilamellar liposomes containing DOTMA interact spontaneously with DNA to form lipid-DNA complexes with 100% entrapment of the DNA. DOTMA facilitates fusion of the complex with the plasma membrane of tissue culture cells, resulting in both uptake and expression of the DNA. The technique is simple, highly reproducible, and effective for both transient and stable expression of transfected DNA. Depending upon the cell line, lipofection is from 5- to >100-fold more effective than either the calcium phosphate or the DEAE-dextran transfection technique.

  8. Transfection of cells mediated by biodegradable polymer materials with surface-bound polyethyleneimine.

    PubMed

    Zheng, J; Manuel, W S; Hornsby, P J

    2000-01-01

    Poly(epsilon-CBZ-L-lysine) can be mixed with biodegradable polymers such as poly(D,L-lactic-co-glycolic acid) or poly(L-lactic acid) and formed into films, foams, or microspheres. Surface amino groups may then be deprotected with acid or lithium/liquid ammonia. The amino groups serve as a method to modify the surface by attachment of other molecules. In the present experiments, we show that these polymer materials, as films or foams, may be surface modified by the attachment of polyethyleneimine (PEI). Plasmid DNA attached to the PEI can transfect cells plated on the surface over several days. Covalent atachment of PEI was required for transfection to be efficient. PEI was also attached to surface-bound collagen on cell culture plates and was shown to mediate transfection.

  9. 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. PMID:19580981

  10. 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.

  11. The effect of transfected MHC class I genes on sensitivity to natural killer cells.

    PubMed Central

    Holscher, M; Givan, A L; Brooks, C G

    1991-01-01

    To test the hypothesis that major histocompatibility complex (MHC) molecules protect target cells from lysis by natural killer cells (NKC), we transfected the MHC- B16 melanoma line F10 with the class I genes encoding Dd, Kb, and Kk. Only low levels of Dd expression could be obtained and there was no protection against NKC. By contrast, Kb and Kk transfectants were obtained which displayed significant resistance to NKC, and with the latter transfectants resistance was clearly related to the level of transgene expression. Various mutants of the F10 line with altered patterns of MHC expression were also obtained. These mutant lines provided evidence that (i) the Db molecule is also capable of inducing resistance to NKC and (ii) high MHC class I expression does not by itself guarantee lowered susceptibility to NKC. PMID:1904402

  12. A fluorinated dendrimer achieves excellent gene transfection efficacy at extremely low nitrogen to phosphorus ratios

    NASA Astrophysics Data System (ADS)

    Wang, Mingming; Liu, Hongmei; Li, Lei; Cheng, Yiyun

    2014-01-01

    Polymers have shown great promise in the design of high efficient and low cytotoxic gene vectors. Here we synthesize fluorinated dendrimers for use as gene vectors. Fluorinated dendrimers achieve excellent gene transfection efficacy in several cell lines (higher than 90% in HEK293 and HeLa cells) at extremely low N/P ratios. These polymers show superior efficacy and biocompatibility compared with several commercial transfection reagents such as Lipofectamine 2000 and SuperFect. Fluorination enhances the cellular uptake of the dendrimer/DNA polyplexes and facilitates their endosomal escape. In addition, the fluorinated dendrimer shows excellent serum resistance and exhibits high gene transfection efficacy even in medium containing 50% FBS. The results suggest that fluorinated dendrimers are a new class of highly efficient gene vectors and fluorination is a promising strategy to design gene vectors without involving sophisticated syntheses.

  13. B35 neuroblastoma cells: an easily transfected, cultured cell model of central nervous system neurons.

    PubMed

    Otey, Carol A; Boukhelifa, Malika; Maness, Patricia

    2003-01-01

    A panel of neuronal cell lines was derived from tumors of the neonatal rat central nervous system (CNS) in 1974, and two of these lines are in wide use today. Both the B35 and B50 lines offer a number of advantages to researchers who study CNS neurons in culture: they are simple to grow, to differentiate, and to transfect. B50 cells have been used extensively in the study of neuronal cell death, toxicology, and differentiation, whereas B35 cells have proven useful in the molecular analysis of endocytosis and of signaling pathways, in particular those that guide axonal outgrowth and cell motility. This chapter provides protocols for growing and transfecting B35 cells, selecting stable transfectants, exploring protein function using an antisense approach, and assaying cell motility in a Transwell chamber. All of these protocols have been written for researchers who have some skill in basic cell culture techniques, but previous experience with cultured neurons is not required.

  14. 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.

  15. 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

  16. Transfection of microRNA Mimics Should Be Used with Caution.

    PubMed

    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

  17. [Transfection efficiency of adenoviral vector AD5/F35 to malignant hematopoietic cells of different origins].

    PubMed

    Wabg, Kai; Peng, Jian-Qinag; Yuan, Zhen-Hua; Wu, Xiao-Bin

    2006-06-01

    This study was aimed to investigate the transfection efficiency of adenoviral vector AD5/F35 to hematopoietic malignant cells lines of various origins and AD5/F35 cytotoxicity. The hematologic malignant cell lines of various origins were transfected by AD5/F35-EGFP at different multiple of infection (MOI) and AD5-EGFP was used as control; the proportion of fluorescence positive cells was detected by flow cytometry; the killing effect of virus on infective target cells was assayed by MTT and observed by fluorescence microscopy. The results showed that the transfection efficiency of AD5/F35 vector to cell line of myeloid origin was > 99% at MOI = 30, the transfective efficiency of AD5 vector was 26.4% at MOI = 1,000; the transfection efficiency of AD5/F35 vector and AD5 vector to cell line of B cell origin were 11.7% and 5.7%, respectively, at MOI = 1,000. AD5/F35 and AD5 vectors could not effectively transfect cells of T cell origin, no fluorescence positive cells were detected at MOI = 1,000; no significant killing effect of AD5/F35 vector on infective target cells was observed at MOI = 1,000. It is concluded that AD5/F35 vector infection has definite selectivity to hematologic malignant cells of various origin, the infection ability of AD5/F35 vector to cells of myeloid origin is stronger than that to cells of B cell origin, the cytotoxicity of AD5/F35 vector to infective target cells is small. The AD5/F35 vector is preferable to AD5 vector in respect of infection ability and offers good prospects of application in gene therapy for myeloid leukemia cells as target cells.

  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. Application of Polyethylenimine-Grafted Silicon Nanowire Arrays for Gene Transfection.

    PubMed

    Wang, Hongwei; Pan, Jingjing; Chen, Hong; Yuan, Lin

    2016-01-01

    Polyplexes are one of the most important and promising approaches to deliver exogenous DNA into cells. However, it is severely restricted by the aggregation of polyplexes. Surface-tethered polyplexes can inhibit the aggregation effect and increase the local concentrations of DNA, exhibiting an excellent potential in gene transfection. Since silicon nanowires have the ability to penetrate the cell membrane, branched polyethylenimine (bPEI)-grafted silicon nanowire arrays (SiNWAs) can stimulate gene transfection to a great extent. Herein, the method for the preparation of bPEI-grafted SiNWAs, as an example of surface-tethered polyplexes, is introduced in detail. PMID:27436326

  20. 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.

  1. Fast, Efficient, and Gentle Transfection of Human Adherent Cells in Suspension.

    PubMed

    Agrawal, Pranav; Ingle, Nilesh P; Boyle, William S; Ward, Emily; Tolar, Jakub; Dorfman, Kevin D; Reineke, Theresa M

    2016-04-13

    We demonstrate a highly efficient method for gene delivery into clinically relevant human cell types, such as induced pluripotent stem cells (iPSCs) and fibroblasts, reducing the protocol time by one full day. To preserve cell physiology during gene transfer, we designed a microfluidic strategy, which facilitates significant gene delivery in a short transfection time (<1 min) for several human cell types. This fast, optimized and generally applicable cell transfection method can be used for rapid screening of different delivery systems and has significant potential for high-throughput cell therapy applications. PMID:27035392

  2. Ultrasound-Mediated Transfection Enhanced by Cavitation Facilitation and Membrane Modification

    NASA Astrophysics Data System (ADS)

    Ogawa, Ryohei; Nozaki, Tetsuo; Kagiya, Go; Feril, Loreto B.; Fuse, Hideki; Kondo, Takashi

    2005-03-01

    The mechanism of ultrasound-mediated transfection (USMT) was explored to get a better understanding and improvement of the efficiency. Efficiency of USMT was linked to cavitational effects and also to plasma membrane conditions. These results indicate that the mechanism underlying USMT is possibly explained by the interaction between the cavitational effect and a plasma membrane response. When both were combined, transfection efficiency was significantly enhanced in vitro and in vivo. This enhancement could be useful for ultrasound-mediated gene therapy in the future.

  3. Functionalized nanomaterials: are they effective to perform gene delivery to difficult-to-transfect cells with no cytotoxicity?

    NASA Astrophysics Data System (ADS)

    Tonelli, F. M. P.; Lacerda, S. M. S. N.; Paiva, N. C. O.; Pacheco, F. G.; Scalzo Junior, S. R. A.; de Macedo, F. H. P.; Cruz, J. S.; Pinto, M. C. X.; Junior, J. D. Corrêa; Ladeira, L. O.; França, L. R.; Guatimosim, S.; Resende, R. R.

    2015-10-01

    Nanodiamonds (NDs), multiwalled carbon nanotubes (MWCNTs) and gold nanorods (NRs) can be functionalized to promote gene delivery to hard-to-transfect cells with higher transfection efficiency than cationic lipids, and inducing less cell death.Nanodiamonds (NDs), multiwalled carbon nanotubes (MWCNTs) and gold nanorods (NRs) can be functionalized to promote gene delivery to hard-to-transfect cells with higher transfection efficiency than cationic lipids, and inducing less cell death. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr04173b

  4. 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.

  5. The shape and size effects of polycation functionalized silica nanoparticles on gene transfection.

    PubMed

    Lin, Xinyi; Zhao, Nana; Yan, Peng; Hu, Hao; Xu, Fu-Jian

    2015-01-01

    Silica nanoparticles are attractive candidates for the development of safe and efficient non-viral gene carriers, owing to their controlled morphologies, potential of facile surface modification and excellent biocompatibility as well as in vivo biodegradability. Conversely, the size and shape of nanoparticles are considered to have an intense influence on their interaction with cells and biological systems, but the effects of particle size and shape on gene transfection are poorly understood. In this work, a series of novel gene carriers were designed employing polycation modified silica nanoparticles with five different morphologies, while keeping uniform zeta potential and surface functionality. Then the effects of particle size and shape of these five different carriers on gene transfection were investigated. The morphology of silica nanoparticles is demonstrated to play an important role in gene transfection, especially when the amount of polycation is low. Chiral nanorods with larger aspect ratio were found to fabricate the most efficient gene carriers with compromised cytotoxicity. It was also noted that hollow nanosphere-based carriers exhibited better gene transfection performance than did solid counterparts. These results may provide new strategies to develop promising gene carriers and useful information for the application of nanoparticles in biomedical areas. PMID:25219349

  6. Safety and Efficacy of Activated Transfected Killer Cells for Neutropenic Fungal Infections

    PubMed Central

    Lin, Lin; Ibrahim, Ashraf S.; Baquir, Beverlie; Fu, Yue; Applebaum, David; Schwartz, Julie; Wang, Amy; Avanesian, Valentina; Spellberg, Brad

    2010-01-01

    Background Invasive fungal infections cause considerable morbidity and mortality in neutropenic patients. White blood cell transfusions are a promising treatment for such infections, but technical barriers have prevented their widespread use. Methods To recapitulate white blood cell transfusions, we are developing a cell-based immunotherapy using a phagocytic cell line, HL-60. We sought to stably transfect HL-60 cells with a suicide trap (herpes simplex virus thymidine kinase), to enable purging of the cells when desired, and a bioluminescence marker, to track the cells in vivo in mice. Results Transfection was stable despite 20 months of continuous culture or storage in liquid nitrogen. Activation of these transfected cells with retinoic acid and dimethyl sulfamethoxazole enhanced their microbicidal effects. Activated transfected killer (ATAK) cells were completely eliminated after exposure to ganciclovir, confirming function of the suicide trap. ATAK cells improved the survival of neutropenic mice with lethal disseminated candidiasis and inhalational aspergillosis. Bioluminescence and histopathologic analysis confirmed that the cells were purged from surviving mice after ganciclovir treatment. Comprehensive necropsy, histopathology, and metabolomic analysis revealed no toxicity of the cells. Conclusions These results lay the groundwork for continued translational development of this promising, novel technology for the treatment of refractory infections in neutropenic hosts. PMID:20397927

  7. Transfection of an immunoglobulin kappa gene into mature human B lymphocytes

    SciTech Connect

    Bich-Thuy, L.T.; Queen, C.

    1988-01-01

    The authors show in this report that the transcription induced by interleukin-2 or pokeweed mitogens of the kappa MOPC 41 immunoglobulin light-chain gene transfected into primary human or murine B lymphocytes initiates from a previously unobserved start site about 26 base pairs upstream of the start site used in myeloma cell lines.

  8. Immunotherapy for neuroblastoma using syngeneic fibroblasts transfected with IL-2 and IL-12.

    PubMed

    Barker, S E; Grosse, S M; Siapati, E K; Kritz, A; Kinnon, C; Thrasher, A J; Hart, S L

    2007-07-16

    Cytokine-modified tumour cells have been used in clinical trials for immunotherapy of neuroblastoma, but primary tumour cells from surgical biopsies are difficult to culture. Autologous fibroblasts, however, are straightforward to manipulate in culture and easy to transfect using nonviral or viral vectors. Here we have compared the antitumour effect of fibroblasts and tumour cells transfected ex vivo to coexpress interleukin-2 (IL-2) and IL-12 in a syngeneic mouse model of neuroblastoma. Coinjection of cytokine-modified fibroblasts with Neuro-2A tumour cells abolished their in vivo tumorigenicity. Treatment of established tumours with three intratumoral doses of transfected fibroblasts showed a significant therapeutic effect with reduced growth or complete eradication of tumours in 90% of mice, associated with extensive leukocyte infiltration. Splenocytes recovered from vaccinated mice showed enhanced IL-2 production following Neuro-2A coculture, and increased cytotoxicity against Neuro-2A targets compared with controls. Furthermore, 100% of the tumour-free mice exhibited immune memory against tumour cells when rechallenged three months later. The potency of transfected fibroblasts was equivalent to that of tumour cells in all experiments. We conclude that syngeneic fibroblasts cotransfected with IL-2 and IL-12 mediate therapeutic effects against established disease, and are capable of generating immunological memory. Furthermore, as they are easier to recover and manipulate than autologous tumour cells, fibroblasts provide an attractive alternative immunotherapeutic strategy for the treatment of neuroblastoma.

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

    PubMed

    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

  10. 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.

  11. Mesomorphic imidazolium salts: new vectors for efficient siRNA transfection.

    PubMed

    Dobbs, William; Heinrich, Benoît; Bourgogne, Cyril; Donnio, Bertrand; Terazzi, Emmanuel; Bonnet, Marie-Elise; Stock, Fabrice; Erbacher, Patrick; Bolcato-Bellemin, Anne-Laure; Douce, Laurent

    2009-09-23

    The preparation of chloride (1(n)) and bromide (2(n)) derivatives of 1-methyl-3-[3,4-bis(alkoxy)benzyl]-4H-imidazolium with n = 6, 12, 16, 18 is described. The two series of salts possess a rich thermotropic mesomorphism, chain-length dependent. Thus, a lamellar smectic A phase, a bicontinuous cubic Ia3d phase, and a columnar hexagonal liquid crystalline mesophase are induced as a function of increasing chain length. The mesomorphic properties were studied by polarizing optical microscopy, differential scanning calorimetry, and X-ray diffraction, and with the support of dilatometry and molecular dynamics, models for the various supramolecular arrangements of the salts are proposed. Such cationic amphiphiles were expected to be candidate molecules to design a new delivery reagent for nucleic acid transfection, particularly for short interfering RNA (siRNA). The use of an RNA interference mechanism, by introduction into cells by transfection of chemically synthesized siRNAs, is a powerful method for gene silencing studies. To exploit the potential of these amphilic imidazolium salts, these molecules were formulated with cohelper lipids and tested for their efficacy to deliver active siRNAs. Our results show high transfection efficacy of our formulated compounds and high silencing efficiency with more than 80% inhibition of the targeted gene at 10 nM siRNA concentration. Taken together our results show the potency of amphiphilic imidazolium salts as a new generation of transfection reagents for RNA interference. PMID:19715309

  12. 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.

  13. 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.

  14. 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.

  15. Delivery of proteins to mammalian cells via gold nanoparticle mediated laser transfection

    NASA Astrophysics Data System (ADS)

    Heinemann, D.; Kalies, S.; Schomaker, M.; Ertmer, W.; Murua Escobar, H.; Meyer, H.; Ripken, T.

    2014-06-01

    Nanoparticle laser interactions are in widespread use in cell manipulation. In particular, molecular medicine needs techniques for the directed delivery of molecules into mammalian cells. Proteins are the final mediator of most cellular cascades. However, despite several methodical approaches, the efficient delivery of proteins to cells remains challenging. This paper presents a new protein transfection technique via laser scanning of cells previously incubated with gold nanoparticles. The laser-induced plasmonic effects on the gold nanoparticles cause a transient permeabilization of the cellular membrane, allowing proteins to enter the cell. Applying this technique, it was possible to deliver green fluorescent protein into mammalian cells with an efficiency of 43%, maintaining a high level of cell viability. Furthermore, a functional delivery of Caspase 3, an apoptosis mediating protein, was demonstrated and evaluated in several cellular assays. Compared to conventional protein transfection techniques such as microinjection, the methodical approach presented here enables high-throughput transfection of about 10 000 cells per second. Moreover, a well-defined point in time of delivery is guaranteed by gold nanoparticle mediated laser transfection, allowing the detailed temporal analysis of cellular pathways and protein trafficking.

  16. 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.

  17. 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. PMID:26530487

  18. Enhanced gene transfection by photochemical internalization of protomine sulfate/DNA complexes

    NASA Astrophysics Data System (ADS)

    Hirschberg, Henry; Mathews, Marlon B.; Shih, En-Chung; Madsen, Steen J.; Kwon, Young Jik

    2012-02-01

    Introduction: 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 the GFP indicator gene on the same plasmid as a tumor suppressor gene (PTEN) was investigated in monolayers of U251 human glioma cells. Materials and Methods: U251 monolayers were incubated in AlPcS2a for 18 h. The monolayers were incubated with non-viral vectors for either 4 or 18 hrs. In all cases, light treatment was performed with a diode laser at a wavelength of 670 nm. The non-viral transfection agents, branched PEI or protomine sulfate (PS), were used with the plasmid construct (GFP-PTEN). Results: PS was much less toxic to the gliomas cells compared to BPEI but was highly inefficient at gene transfection. PCI resulted in a 5-10 fold increase in GFP protein expression compared to controls. Conclusions: Collectively, the results suggest that AlPcS2a-mediated PCI can be used to enhance transfection of tumor suppressor genes in glioma cells.

  19. 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

  20. 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...

  1. 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

  2. Cell transfection in vitro and in vivo with nontoxic TAT peptide-liposome-DNA complexes

    NASA Astrophysics Data System (ADS)

    Torchilin, Vladimir P.; Levchenko, Tatyana S.; Rammohan, Ram; Volodina, Natalia; Papahadjopoulos-Sternberg, Brigitte; D'Souza, Gerard G. M.

    2003-02-01

    Liposomes modified with TAT peptide (TATp-liposomes) showed fast and efficient translocation into the cell cytoplasm with subsequent migration into the perinuclear zone. TATp-liposomes containing a small quantity (10 mol %) of a cationic lipid formed firm noncovalent complexes with DNA. Here, we present results demonstrating both in vitro and in vivo transfection with TATp-liposome-DNA complexes. Mouse NIH/3T3 fibroblasts and rat H9C2 cardiomyocytes were transfected with such complexes in vitro. The transfection with the TATp-liposome-associated pEGFP-N1 plasmid encoding for the green fluorescent protein (GFP) was high, whereas the cytotoxicity was lower than that of commonly used cationic lipid-based gene-delivery systems. Intratumoral injection of TATp-liposome-DNA complexes into the Lewis lung carcinoma tumor of mice also resulted in an expression of GFP in tumor cells. This transfection system should be useful for various protocols of cell treatment in vitro or ex vivo as well as for localized in vivo gene therapy.

  3. Delivery of proteins to mammalian cells via gold nanoparticle mediated laser transfection.

    PubMed

    Heinemann, D; Kalies, S; Schomaker, M; Ertmer, W; Murua Escobar, H; Meyer, H; Ripken, T

    2014-06-20

    Nanoparticle laser interactions are in widespread use in cell manipulation. In particular, molecular medicine needs techniques for the directed delivery of molecules into mammalian cells. Proteins are the final mediator of most cellular cascades. However, despite several methodical approaches, the efficient delivery of proteins to cells remains challenging. This paper presents a new protein transfection technique via laser scanning of cells previously incubated with gold nanoparticles. The laser-induced plasmonic effects on the gold nanoparticles cause a transient permeabilization of the cellular membrane, allowing proteins to enter the cell. Applying this technique, it was possible to deliver green fluorescent protein into mammalian cells with an efficiency of 43%, maintaining a high level of cell viability. Furthermore, a functional delivery of Caspase 3, an apoptosis mediating protein, was demonstrated and evaluated in several cellular assays. Compared to conventional protein transfection techniques such as microinjection, the methodical approach presented here enables high-throughput transfection of about 10 000 cells per second. Moreover, a well-defined point in time of delivery is guaranteed by gold nanoparticle mediated laser transfection, allowing the detailed temporal analysis of cellular pathways and protein trafficking.

  4. 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

  5. Induction by glucose of an antimycin-insensitive, azide-sensitive respiration in the yeast Kluyveromyces lactis.

    PubMed

    Ferrero, I; Viola, A M; Goffeau, A

    1981-03-01

    Increasing the glucose concentration from 0.1 to 10% in exponentially growing cultures of Kluyveromyces lactis CBS 2359 does not repress the antimycin-sensitive respiration (QO2 of 80 microliter O2 . h-1 . mg-1 dry weight) but raises the antimycin-insensitive respiration from 3 to 12 microliter O2 . h-2 . mg-1 dry weight. Antimycin A inhibits the growth of K. lactis on a variety of substrates with the exception of glucose at concentrations equal to or higher than 1% where substantial antimycin-insensitive respiratory rates are induced. It can be concluded that a minimal antimycin-insensitive QO2 is necessary for cellular growth when the normal respiratory pathway is not functional. The antimycin-insensitive respiration elicited by growth in high glucose concentrations is poorly inhibited by hydroxamate and is inhibited by 50% by 90 microM azide or 1 mM cyanide. These concentrations are much higher than those necessary to inhibit cytochrome c oxidase which is not involved in the antimycin-insensitive respiration as was demonstrated by spectral measurements. A pigment absorbing at 555 nm is specifically reduced after addition of glucose to antimycin-inhibited cells. The same pigment is reoxidized by further addition of high concentrations of sodium azide indicating its participation in the antimycin-insensitive, azide-sensitive respiration.

  6. The gene transfection efficiency of a folate-PEI600-cyclodextrin nanopolymer.

    PubMed

    Yao, Hong; Ng, Samuel S; Tucker, Wesley O; Tsang, Yuk-Kai-Tiu; Man, Kwan; Wang, Xiao-Mei; Chow, Billy K C; Kung, Hsiang-Fu; Tang, Gu-Ping; Lin, Marie C

    2009-10-01

    The success of gene therapy relies on a safe and effective gene delivery system. In this communication, we describe the use of folate grafted PEI(600)-CyD (H(1)) as an effective polyplex-forming plasmid delivery agent with low toxicity. The structures of the polymer and polyplex were characterized, and the in vitro transfection efficiency, cytotoxicity, and in vivo transfection of H(1) were examined. We found that folate molecules were successfully grafted to PEI(600)-CyD. At N/P ratios between 5 and 30, the resulting H(1)/DNA polyplexes had diameters less than 120 nm and zeta potentials less than 10 mV. In various tumor cell lines examined (U138, U87, B16, and Lovo), the in vitro transfection efficiency of H(1) was more than 50%, which could be improved by the presence of fetal bovine serum or albumin. The cytotoxicity of H(1) was significantly less than high molecular weight PEI-25 kDa. Importantly, in vivo optical imaging showed that the efficiency of H(1)-mediated transfection (50 microg luciferase plasmid (pLuc), N/P ratio=20/1) was comparable to that of adenovirus-mediated luciferase transduction (1 x 10(9) pfu) in melanoma-bearing mice, and it did not induce any toxicity in the tumor tissue. These results clearly show that H(1) is a safe and effective polyplex-forming agent for both in vitro and in vivo transfection of plasmid DNA and its application warrants further investigation. PMID:19615741

  7. Effect of TIMP1 transfection on PTEN expression in human kidney proximal tubular cells.

    PubMed

    Chen, J X; Cai, G Y; Chen, X M; Liu, H; Chen, X; Peng, Y M; Liu, F Y; Li, Z; Shi, S Z

    2015-12-21

    To explore the role of metalloproteinase-1 (TIMP-1) tissue inhibitor in the mechanisms of kidney aging, we observed the effects of sense and antisense transfection of TIMP-1 and of metalloproteinase (MMP) inhibitors on phosphatase and tensin homolog (PTEN), vascular endothelial growth factor (VEGF), and Flk-1 expression in TIMP-1 transgenic human proximal tubular epithelial cells (HKCs). Transfected HKCs were co-incubated with 100 μM MMP-2 and MMP-9 inhibitor III for 24 h to affect enzyme inhibition. TIMP-1, MMP-2, MMP-9, PTEN, VEGF, and Flk-1 mRNA expression was detected by reverse transcription-polymerase chain reaction. PTEN, VEGF, and Flk-1 protein expression in cells of each experimental group was measured by indirect immunofluorescence. We found that PTEN expression was up-regulated (P < 0.05) in the sense TIMP-1-transfected group (P < 0.05) compared with the non-transfected and empty vector groups, and that expression of VEGF and Flk-1 was down-regulated (P < 0.05). In contrast, the antisense TIMP-1 transgenic group showed the opposite results (P < 0.05). No significant differences in expression of PTEN, VEGF, or Flk-1 were observed among the MMP- 2/MMP-9 inhibitor III, non-transfected, and empty vector groups (P > 0.05). These results suggest that in the progression of renal aging, high expression of TIMP-1 up-regulates PTEN expression through an MMP-independent pathway, and subsequently down-regulates the expression of VEGF and Flk-1, indicating that PTEN and TIMP-1 are involved in the aging-associated impairment of renal angiogenesis. Our study provides a theoretical basis for further exploration of the mechanism underlying TIMP- 1 participation in renal aging progression.

  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. Novel dextran-spermine conjugates as transfecting agents: comparing water-soluble and micellar polymers.

    PubMed

    Eliyahu, H; Makovitzki, A; Azzam, T; Zlotkin, A; Joseph, A; Gazit, D; Barenholz, Y; Domb, A J

    2005-03-01

    Recently, a novel cationic polymer, dextran-spermine (D-SPM) was developed for gene delivery. An efficient transfection was obtained using this polycation for a variety of genes and cell lines in serum-free or serum-poor medium. However, transfection using the water-soluble D-SPM-based polyplexes decreased with increasing serum concentration in cell culture in a concentration-dependent manner, reaching 95% inhibition at 50% serum in the cell growth medium. In order to overcome this obstacle, oleyl derivatives of D-SPM (which form micelles in aqueous phase) were synthesized at 1, 10, and 20 mol% of oleyl moiety to polymer epsilon-NH2 to form N-oleyl-D-SPM (ODS). Polyplexes based on ODS transfected well in medium containing 50% serum. Comparison with polyplexes based on well-established polymers (branched and linear polyethyleneimine) and with DOTAP/Cholesterol lipoplexes showed that regarding beta-galactosidase transgene expression level and cytotoxicity in tissue culture, the D-SPM and ODS compare well with the above polyplexes and lipoplexes. Intracellular trafficking using FITC-labeled ODS and Rhodamine-labeled pGeneGrip plasmid cloned with hBMP2 monitored by confocal microscopy revealed that during the transfection process the fluorescent-labeled polymer concentrates in the Golgi apparatus and around the nucleus, while the cell cytoplasm was free of fluorescent particles, suggesting that the polyplexes move in the cell toward the nucleus by vesicular transport through the cytoplasm and not by a random diffusion. We found that the plasmids penetrate the cell nucleus without the polymer. Preliminary results in zebra fish and mice demonstrate the potential of ODS to serve as an efficient nonviral vector for in vivo transfection.

  10. 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

  11. Plasmid transfection in mammalian cells spatiotemporally tracked by a gold nanoparticle.

    PubMed

    Muroski, Megan E; Carnevale, Kate J F; Riskowski, Ryan A; Strouse, Geoffrey F

    2015-01-27

    Recent advances in cell transfection have suggested that delivery of a gene on a gold nanoparticle (AuNP) can enhance transfection efficiency. The mechanism of transfection is poorly understood, particularly when the gene is appended to a AuNP, as expression of the desired exogenous protein is dependent not only on the efficiency of the gene being taken into the cell but also on efficient endosomal escape and cellular processing of the nucleic acid. Design of a multicolor surface energy transfer (McSET) molecular beacon by independently dye labeling a linearized plasmid and short duplex DNA (sdDNA) appended to a AuNP allows spatiotemporal profiling of the transfection events, providing insight into package uptake, disassembly, and final plasmid expression. Delivery of the AuNP construct encapsulated in Lipofectamine2000 is monitored in Chinese hamster ovary cells using live-cell confocal microscopy. The McSET beacon signals the location and timing of the AuNP release and endosomal escape events for the plasmid and the sdDNA discretely, which are correlated with plasmid transcription by fluorescent protein expression within the cell. It is observed that delivery of the construct leads to endosomal release of the plasmid and sdDNA from the AuNP surface at different rates, prior to endosomal escape. Slow cytosolic diffusion of the nucleic acids is believed to be the limiting step for transfection, impacting the time-dependent expression of protein. The overall protein expression yield is enhanced when delivered on a AuNP, possibly due to better endosomal escape or lower degradation prior to endosomal escape.

  12. Assessing the concept of structure sensitivity or insensitivity for sub-nanometer catalyst materials

    NASA Astrophysics Data System (ADS)

    Crampton, Andrew S.; Rötzer, Marian D.; Ridge, Claron J.; Yoon, Bokwon; Schweinberger, Florian F.; Landman, Uzi; Heiz, Ueli

    2016-10-01

    The nature of the nano-catalyzed hydrogenation of ethylene, yielding benchmark information pertaining to the concept of structure sensitivity/insensitivity and its applicability at the bottom of the catalyst particle size-range, is explored with experiments on size-selected Ptn (n = 7-40) clusters soft-landed on MgO, in conjunction with first-principles simulations. As in the case of larger particles both the direct ethylene hydrogenation channel and the parallel hydrogenation-dehydrogenation ethylidyne-producing route must be considered, with the fundamental uncovering that at the < 1 nm size-scale the reaction exhibits characteristics consistent with structure sensitivity, in contrast to the structure insensitivity found for larger particles. In this size-regime, the chemical properties can be modulated and tuned by a single atom, reflected by the onset of low temperature hydrogenation at T > 150 K catalyzed by Ptn (n ≥ 10) clusters, with maximum room temperature reactivity observed for Pt13 using a pulsed molecular beam technique. Structure insensitive behavior, inherent for specific cluster sizes at ambient temperatures, can be induced in the more active sizes, e.g. Pt13, by a temperature increase, up to 400 K, which opens dehydrogenation channels leading to ethylidyne formation. This reaction channel was, however found to be attenuated on Pt20, as catalyst activity remained elevated after the 400 K step. Pt30 displayed behavior which can be understood from extrapolating bulk properties to this size range; in particular the calculated d-band center. In the non-scalable sub-nanometer size regime, however, precise control of particle size may be used for atom-by-atom tuning and manipulation of catalyzed hydrogenation activity and selectivity.

  13. Specificity of a retinoic acid response element in the phosphoenolpyruvate carboxykinase gene promoter: consequences of both retinoic acid and thyroid hormone receptor binding.

    PubMed Central

    Lucas, P C; Forman, B M; Samuels, H H; Granner, D K

    1991-01-01

    The ability of a retinoic acid (RA) response element (RARE) in the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter to mediate effects of either RA or thyroid hormone (T3) on gene expression was studied. Fusion gene constructs consisting of PEPCK promoter sequences ligated to the chloramphenicol acetyltransferase (CAT) reporter gene were used for this analysis. While T3 induced CAT expression to a small degree (about twofold) when such constructs were transiently transfected into H4IIE rat hepatoma cells, along with an expression vector encoding the alpha subtype of the T3 receptor (TR), this effect was mediated by promoter sequences distinct from the PEPCK RARE. Although TRs were capable of binding the PEPCK RARE in the form of putative monomers, dimers, and heterodimers with RA receptors (RARs), this element failed to mediate any positive effect of T3 on gene expression. In contrast, the PEPCK RARE mediated six- to eightfold induction of CAT expression by RA. When TRs were coexpressed along with RARs in transfected H4IIE cells, this RA induction was substantially blunted in a T3-independent manner. This inhibitory effect may be due to the binding of nonfunctional TRs or TR-RAR heterodimers to the PEPCK RARE. A model is proposed to explain the previously observed in vivo effects of T3 on PEPCK gene expression. Images PMID:1656224

  14. Full-field spatially incoherent illumination interferometry: a spatial resolution almost insensitive to aberrations

    NASA Astrophysics Data System (ADS)

    Xiao, Peng; Fink, Mathias; Boccara, A. Claude

    2016-09-01

    We show that with spatially incoherent illumination, the point spread function width of an imaging interferometer like that used in full-field optical coherence tomography (FFOCT) is almost insensitive to aberrations that mostly induce a reduction of the signal level without broadening. This is demonstrated by comparison with traditional scanning OCT and wide-field OCT with spatially coherent illuminations. Theoretical analysis, numerical calculation as well as experimental results are provided to show this specific merit of incoherent illumination in full-field OCT. To the best of our knowledge, this is the first time that such result has been demonstrated.

  15. 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.

  16. Congenital Insensitivity to Pain: A Case Report and Review of the Literature

    PubMed Central

    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. PMID:25309764

  17. Theory of magic optical traps for Zeeman-insensitive clock transitions in alkali-metal atoms

    SciTech Connect

    Derevianko, Andrei

    2010-05-15

    Precision measurements and quantum-information processing with cold atoms may benefit from trapping atoms with specially engineered, 'magic' optical fields. At the magic trapping conditions, the relevant atomic properties remain immune to strong perturbations by the trapping fields. Here we develop a theoretical analysis of magic trapping for especially valuable Zeeman-insensitive clock transitions in alkali-metal atoms. The involved mechanism relies on applying a magic bias B field along a circularly polarized trapping laser field. We map out these B fields as a function of trapping laser wavelength for all commonly used alkalis. We also highlight a common error in evaluating Stark shifts of hyperfine manifolds.

  18. Polarization insensitive all-optical wavelength conversion of polarization multiplexed signals using co-polarized pumps.

    PubMed

    Anthur, Aravind P; Zhou, Rui; O'Duill, Sean; Walsh, Anthony J; Martin, Eamonn; Venkitesh, Deepa; Barry, Liam P

    2016-05-30

    We study and experimentally validate the vector theory of four-wave mixing (FWM) in semiconductor optical amplifiers (SOA). We use the vector theory of FWM to design a polarization insensitive all-optical wavelength converter, suitable for advanced modulation formats, using non-degenerate FWM in SOAs and parallelly polarized pumps. We demonstrate the wavelength conversion of polarization-multiplexed (PM)-QPSK, PM-16QAM and a Nyquist WDM super-channel modulated with PM-QPSK signals at a baud rate of 12.5 GBaud, with total data rates of 50 Gbps, 100 Gbps and 200 Gbps respectively. PMID:27410100

  19. Synthesis of Insensitive N-2,4-dinitrophenyl-N1-nitroguanidine

    NASA Astrophysics Data System (ADS)

    Warner, Kirstin F.; Bellitto, Victor J.; Sorensen, Daniel N.; Remmers, Daniel L.

    2016-10-01

    A new, insensitive nitroguanidyl functionalized nitrogen-rich material derived from 2,4-dinitrophenyl derivative was synthesized. N-2,4-dinitrophenyl-N1-nitroguanidine (DNP-NQ) has a crystal density of 1.74 g/cm3. The synthesis and properties of DNP-NQ are described. DNP-NQ was characterized by nuclear magnetic resonance (NMR), infrared spectroscopy, and gas pycnometry. Thermal properties were studied by differential scanning calorimetry (DSC). Results from small-scale sensitivity tests indicate that DNP-NQ is less sensitive than Cyclotrimethylenetrinitramine (RDX). DNP-NQ can be prepared in high yield.

  20. 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.

  1. Polarization-insensitive unidirectional spoof surface plasmon polaritons coupling by gradient metasurface

    NASA Astrophysics Data System (ADS)

    Hong-yu, Shi; An-xue, Zhang; Jian-zhong, Chen; Jia-fu, Wang; Song, Xia; Zhuo, Xu

    2016-07-01

    A polarization-insensitive unidirectional spoof surface plasmon polariton (SPP) coupler mediated by a gradient metasurface is proposed. The field distributions and average Poynting vector of the coupled spoof SPPs are analyzed. The simulated and experimental results support the theoretical analysis and indicate that the designed gradient metasurface can couple both the parallel-polarized and normally-polarized incident waves to the spoof SPPs propagating in the same direction at about 5 GHz. Project supported by the China Postdoctoral Science Foundation (Grant No. 2015M580849) and the National Natural Science Foundation of China (Grant Nos. 61471292, 61501365, 61471388, 61331005, 41404095, and 41390454).

  2. 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.

  3. Vibration sensing using a tapered bend-insensitive fiber based Mach-Zehnder interferometer.

    PubMed

    Xu, Yanping; Lu, Ping; Qin, Zengguang; Harris, Jeremie; Baset, Farhana; Lu, Ping; Bhardwaj, Vedula Ravi; Bao, Xiaoyi

    2013-02-11

    In this study, a novel fiber-optic sensor consisting of a tapered bend-insensitive fiber based Mach-Zehnder interferometer is presented to realize damped and continuous vibration measurement. The double cladding structure and the central coating region of the in-fiber interferometer ensure an enhanced mechanical strength, reduced external disturbance, and a more uniform spectrum. A damped vibration frequency range of 29-60 Hz as well as continuous vibration disturbances ranging from 1 Hz up to 500 kHz are successfully demonstrated.

  4. N-Acetyl glycals are tight-binding and environmentally insensitive inhibitors of hexosaminidases.

    PubMed

    Santana, A G; Vadlamani, G; Mark, B L; Withers, S G

    2016-06-28

    Mono-, di- and trisaccharide derivatives of 1,2-unsaturated N-acetyl-d-glucal have been synthesized and shown to function as tight-binding inhibitors/slow substrates of representative hexosaminidases. Turnover is slow and not observed in the thioamide analogue, allowing determination of the 3-dimensional structure of the complex. Inhibition is insensitive to pH and to mutation of key catalytic residues, consistent with the uncharged character of the inhibitor. These properties could render this inhibitor class less prone to development of resistance. PMID:27253678

  5. Embryonal carcinoma in two cases of androgen insensitivity syndrome: clinical, endocrinological and pathological features.

    PubMed

    Lecca, U; Parodo, G; Fiore, R; Martino, E

    1988-01-01

    Embryonal carcinoma in two cases of complete androgen insensitivity syndrome (CAIS) is reported. In both cases gonadectomy carried out for prophylactic purposes led to the discovery of a localized embryonal carcinoma with areas of anaplastic seminoma in one case. In non-neoplastic tissue, gonad morphology in both cases was typical of AIS. Prevalently hypotrophic aspects, especially in the interstitial gland, were found in case 2. This may explain the different endocrine profile in the two cases before gonadectomy. Our study, aside from series of psycho-sexual problems, shows, according to all Authors, that the most serious complication is the high risk of malignancy after puberty in patients with AIS. PMID:3148467

  6. 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

  7. 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.

  8. [Optimizing the operating variables that affect the transfection experiment of antisense oligodeoxyribonucleotide by gas-filled microbubbles].

    PubMed

    Zhao, Ying-zheng; Luo, Yu-kun; Lu, Cui-tao; Xu, Jing-feng; Mei, Xing-guo; Wang, Hu-jun; Zhang, Mei

    2007-12-01

    To optimize the operating variables that affect the transfection of antisense oligodeoxyribonucleotide (AS-ODNs) by insonated gas-filled lipid microbubbles, SF6-filled microbubbles were prepared by sonication-lyophilization method. An AS-ODNs sequence and a breast cancer cell line SK-BR-3 were used to define the various operating variables determining the transfection efficiency of SF6-filled microbubbles. Three levels of mixing speed, different durations of mixing and various delay time before ultrasound were examined, separately. Transfection efficiency was detected by fluorescence microscopy. Transfection results with and without incubation of AS-ODNs and microbubbles before mixing cells were compared. From the results, there is no significant difference between the transinfection efficiency with or without incubation of AS-ODNs and microbubbles before mixing cells. AS-ODNs transfection efficiency showed an increasing trend with mixing speed and mixing duration, but there is a negative relationship with delay time before ultrasound. The optimum parameters for AS-ODNs transfection by SF6-filled microbubbles were found at a mixing speed of 40-50 r x min(-1) for 30-60 s with less than 60 s delay before ultrasound. For a successful transfection, long time of incubation with gene is essential for normal nonviral vectors such as liposomes or cationic lipid-polymer hybrids, because these vectors depend on endocytosis and membrane fusion to realize transfection. Unlike liposomes and cationic lipid-polymer hybrids, gas-filled lipid microbubbles depend on sonorporation effect to realize transfection. Therefore, the incubation of gene and microbubbles before mixing cells may not be necessary. Ultrasound-mediated AS-ODNs transfection enhanced by gas-filled lipid microbubbles represents an effective avenue for gene transfer.

  9. 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.

  10. 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

  11. Omnidirectional polarization insensitive tunable absorption in graphene metamaterial of nanodisk structure

    NASA Astrophysics Data System (ADS)

    Ning, Renxia; Bao, Jie; Jiao, Zheng; Xu, Yuan

    2015-11-01

    Tunable absorption based on graphene metamaterial with nanodisk structure at near-infrared frequency was investigated using the finite difference time domain method. The absorption of the nanodisk structure which consisting of Au-MgF2-graphene-Au-polyimide (from bottom to top) can be tuned by the chemical potential of graphene at certain diameter of nanodisk. The permittivity of graphene is discussed with different chemical potential to obtain tunable absorption. It is shown that the increased value of the chemical potential of graphene can lead to blue-shifted of the absorption peaks and the values decreased. Moreover, dual-band and triple-band absorption can be achieved for resonance frequencies at normal incidence. Compared with diameter of nanodisks, the multilayer structure shows multi-band absorber, and an omnidirectional absorption at 195.25 THz is insensitive to TE/TM polarization. This omnidirectional polarization insensitive absorption may be applied by optical communications such as optical absorber, near infrared stealth, and filter.

  12. 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

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. 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.

  18. Leishmania amazonensis: characterization of an ouabain-insensitive Na+-ATPase activity.

    PubMed

    de Almeida-Amaral, Elmo Eduardo; Caruso-Neves, Celso; Pires, Vanessa Maria Pereira; Meyer-Fernandes, José Roberto

    2008-02-01

    We characterized ouabain-insensitive Na+-ATPase activity present in the plasma membrane of Leishmania amazonensis and investigated its possible role in the growth of the parasite. An increase in Na+ concentration in the presence of 1mM ouabain, increased the ATPase activity with a V(max) of 154.1+/-13.5nmol Pi x h(-1) x mg(-1) and a K0.5 of 28.9+/-7.7mM. Furosemide and sodium orthovanadate inhibited the Na+-stimulated ATPase activity with an IC(50) of 270microM and 0.10microM, respectively. Furosemide inhibited the growth of L. amazonensis after 48h incubation, with maximal effect after 96h. The IC50 for furosemide was 840. On the other hand, ouabain (1mM) did not change the growth of the parasite. Taken together, these results show that L. amazonensis expresses a P-type, ouabain-insensitive Na+-ATPase that could be involved with the growth of the parasite. PMID:17825292

  19. Temperature-insensitive frequency tripling for generating high-average power UV lasers.

    PubMed

    Zhong, Haizhe; Yuan, Peng; Wen, Shuangchun; Qian, Liejia

    2014-02-24

    Aimed for generating high-average power ultraviolet (UV) lasers via third-harmonic generation (THG) consisting of frequency doubling and tripling stages, we numerically and experimentally demonstrate a novel frequency tripling scheme capable of supporting temperature-insensitive phase-matching (PM). Two cascaded tripling crystals, with opposite signs of the temperature derivation of phase-mismatch, are proposed and theoretically studied for improving the temperature-acceptance of PM. The proof-of-principle tripling experiment using two crystals of LBO and BBO shows that the temperature acceptance can be ~1.5 times larger than that of using a single tripling crystal. In addition, the phase shift caused by air dispersion, along with its influence on the temperature-insensitive PM, are also discussed. To illustrate the potential applications of proposed two-crystal tripling design in the high-average-power regime, full numerical simulations for the tripling process, are implemented based on the realistic crystals. The demonstrated two-crystal tripling scheme may provide a promising route to high-average-power THG in the UV region.

  20. Insensitive high-energy energetic structural material of tungsten-polytetrafluoroethylene-aluminum composites

    NASA Astrophysics Data System (ADS)

    Wang, Liu; Liu, Jinxu; Li, Shukui; Zhang, Xinbo

    2015-11-01

    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/cm3, 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-1 coupled with the absorbed energy per unit volume of 120 J/cm3, 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.

  1. Mutational analysis of the androgen receptor gene in two Chinese families with complete androgen insensitivity syndrome

    PubMed Central

    WANG, SONG; XU, HAIKUN; AN, WEI; ZHU, DECHUN; LI, DEJUN

    2016-01-01

    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. PMID:27284311

  2. Broadband Tunability of Polarization-Insensitive Absorber Based on Frequency Selective Surface

    NASA Astrophysics Data System (ADS)

    Wang, Han; Kong, Peng; Cheng, Wentao; Bao, Wenzong; Yu, Xiaowei; Miao, Ling; Jiang, Jianjun

    2016-03-01

    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.

  3. Design and optimization of broadband and polarization-insensitive dual-core photonic crystal fiber coupler.

    PubMed

    Lou, Shuqin; Tang, Zunwei; Wang, Liwen

    2011-05-10

    In this paper, we propose a novel (to our knowledge) broadband and polarization-insensitive dual-core photonic crystal fiber (PCF) coupler through the introduction of an elliptical-shaped central air hole to offset the slight birefringence arising from the dual core. With a full vectorial finite element method and anisotropic perfectly matched layers as the external boundaries, the impact of several fiber parameters on the coupling characteristics of dual-core PCF is investigated in detail. Through optimizing the main fiber parameters, including core diameter, size and ellipticity of the central air hole, and refractive index difference, broadband and polarization-insensitive characteristics are achieved in the wavelength range from 0.8 to 1.7 μm. The variation of the coupling ratio is stabilized at 50±1%, and the coupling ratio difference between x polarization and y polarization is less than 2% over the wavelength range. This dual-core PCF makes it easier to develop a 3 dB coupler over a wide wavelength for passive optical networks and large optical systems.

  4. Broadband Tunability of Polarization-Insensitive Absorber Based on Frequency Selective Surface.

    PubMed

    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

  5. 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.

  6. A new method for pulse oximetry possessing inherent insensitivity to artifact.

    PubMed

    Hayes, M J; Smith, P R

    2001-04-01

    A new method for pulse oximetry is presented that possesses an inherent insensitivity to corruption by motion artifact, a primary limitation in the practical accuracy and clinical applicability of current technology. Artifact corruption of the underlying photoplethysmographic signals is reduced in real time, using an electronic processing methodology that is based upon inversion of a physical artifact model. This fundamental approach has the potential to provide uninterrupted output and superior accuracy under conditions of sustained subject motion, therefore, widening the clinical scope of this useful measurement. A new calibration technique for oxygen saturation is developed for use with these processed signals, which is shown to be a generalization of the classical interpretation. The detailed theoretical and practical issues of implementation are then explored, highlighting important engineering simplifications implicit in this new approach. A quantitative investigation of the degree of insensitivity to artifact is also undertaken, with the aid of a custom electronic system and commercial pulse oximeter probes, which is compared and contrasted with the performance of a conventional implementation. It is demonstrated that this new methodology results in a reduced sensitivity to common classes of motion artifact, while retaining the generality to be combined with conventional signal processing techniques.

  7. Molecular characterization of an ABA insensitive 5 orthologue in Brassica oleracea.

    PubMed

    Zhou, Xiaona; Yuan, Feifei; Wang, Mengyao; Guo, Aiguang; Zhang, Yanfeng; Xie, Chang Gen

    2013-01-18

    ABI5 (ABA insensitive 5), a bZIP (Basic leucine zipper) transcription factor, has been shown to be a major mediator of plant ABA responses during seed germination. Although the molecular basis of ABI5-modulated processes has been well demonstrated in Arabidopsis thaliana, its identity and function in cabbage (Brassica oleracea var. capitata L.) remain elusive. Here, we describe our identification of BolABI5 (an ABI5 orthologue in B.oleracea) as a functional bZIP transcription factor in the modulation of plant ABA responses. Expression of BolABI5 was dramatically induced by drought stress and exogenous ABA. Heterogeneous expression of BolABI5 rescued the insensitive phenotype of Arabidopsis abi5-1 to ABA during seed germination. Subcellular localization and trans-activation assays revealed that BolABI5 was localized in the nucleus and possessed DNA binding and trans-activation activities. Deletion of the bZIP domain generated BolABI5ΔbZIP, which no longer localized exclusively in the nucleus and had almost no detectable DNA-binding or trans-activation activities. Overall, these results suggest that BolABI5 may function as ABI5 in the positive regulation of plant ABA responses. PMID:23246838

  8. Polarization-insensitive ultra-thin quasi-metasurface based on the spoof surface plasmon polaritons

    NASA Astrophysics Data System (ADS)

    Li, Si-Jia; Cao, Xiang-Yu; Gao, Jun; Han, Jiang-Feng; Zhang, Zhao; Huang, Fa; Zhang, Chen; Liu, Xiao

    2016-09-01

    We theoretically and experimentally propose a novel polarization-insensitive ultra-thin quasi-metasurface composed of the twelve gradually increasing two-sided metallic grooves, an ultra-thin flexible dielectric film, and a circular copper surface to induce the spoof surface plasmon polaritons in the microwave frequency. The evolution of dispersion characteristic is systematically analyzed in simulation, and we interestingly reveal that the quasi-metasurface provides a way to convert spatial propagating waves to the spoof surface plasmon polariton waves not only for transverse magnetic incidence but also for transverse electric waves. Hence, the proposed quasi-metasurface converts the scattering direction from incident wave direction to the spoof surface plasmon polaritons wave direction and the far-field scattering patterns can be controlled due to the conversion and transmission. A quasi-metasurface device is easily implemented using the common printed circuit board method for demonstration. Both numerical simulations and experimental results verify the polarization-insensitive ultra-thin quasi-metasurface.

  9. 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.

  10. Classical ethylene insensitive mutants of the Arabidopsis EIN2 orthologue lack the expected 'hypernodulation' response in Lotus japonicus.

    PubMed

    Chan, Pick Kuen; Biswas, Bandana; Gresshoff, Peter M

    2013-04-01

    Three independent ethylene insensitive mutants were selected from an EMS- mutagenized population of Lotus japonicus MG-20 (Miyakojima). The mutants, called 'Enigma', were mutated in the LjEIN2a gene from Lotus chromosome 1, sharing significant homology with Arabidopsis EIN2 (ethylene-insensitive2). All three alleles showed classical ethylene insensitivity phenotypes (e.g., Triple Response), but lacked the increased nodulation phenotype commonly associated with ethylene insensitivity. Indeed, all showed a marginal reduction in nodule number per plant, a phenotype that is enigmatic to sickle, an ethylene-insensitive EIN2 mutant in Medicago truncatula. In contrast to wild type, but similar to an ETR1-1 ethylene ethylene-insensitive transgenic of L. japonicus, enigma mutants formed nodules in between the protoxylem poles, demonstrating the influence of ethylene on radial positioning. Suppression of nodule numbers by nitrate and colonisation by mycorrhizal fungi in the enigma-1 mutant were indistinguishable from the wild-type MG-20. However, reflecting endogenous ethylene feedback, the enigma-1 mutant released more than twice the wild-type amount of ethylene. enigma-1 had a moderate reduction in growth, greater root mass (and lateral root formation), delayed flowering and ripening, smaller pods and seeds. Expression analysis of ethylene-regulated genes, such as ETR1, NRL1 (neverripe-like 1), and EIL3 in shoots and roots of enigma-1 and MG-20 illustrated that the ethylene-insensitive mutation strongly affected transcriptional responses in the root. These mutants open the possibility that EIN2 in L. japonicus, a determinate nodulating legume, acts in a more complex fashion possibly through the presence of a duplicated copy of LjEIN2.

  11. 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

  12. 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

  13. HygR and PurR plasmid vectors for episomal transfection of Trypanosoma cruzi.

    PubMed

    Nóbrega, Otávio T; Santana, Jaime M; Sturm, Nancy R; Teixeira, Antônio R L; Campbell, David A

    2004-08-01

    This work describes the development and functional testing of two episomes for stable transfection of Trypanosoma cruzi. pHygD contained the 5'- and 3'- flanking regions of the gene encoding the cathepsin B-like protease of T. cruzi as functional trans-splicing and polyadenylation signals for the hygR ORF. Evidence is presented to support extrachromosomal maintenance and organization as tandem repeats in transfected parasites. pPac was derived from pHygD by replacement of the entire hygR ORF with a purR coding region. The ability to modify pHygD and the availability of the complete DNA sequence make these plasmids useful tools for the genetic manipulation of T. cruzi.

  14. Acquisition of telomere repeat sequences by transfected DNA integrated at the site of a chromosome break

    SciTech Connect

    Murnane, J.P.; Lohchung Yu )

    1993-02-01

    Rearrangement of the human genome is an important element in both cancer biology and genetic disease. Rearrangements that have been observed include deletions, translocations, chromosome breakage or loss, and gene amplification. Transfection of the DNA into mammalian cells can created instability in the genome. The characterization of DNA rearrangement associated with transfected DNA may provide information about the general mechanisms involved in genomic instability. This genomic instability is an important aspect of tumor cell progression. This research examines chromosome breakage and rearrangement that results in interstitial telomere repeat sequences within the human genome. These sequences could promote genomic instability because short repeat sequences can be recombination hotspots. Also, DNA rearrangements involving telomere repeat sequences can be associated with chromosome breaks. The introduction of telomere repeat sequences at spontaneous or ionizing radiation-induced DNA strand breaks may therefore also be a mechanism of chromosome fragmentation. 52 refs., 7 figs.

  15. 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.

  16. Impact of Polyethylenimine Conjugation Mode on the Cell Transfection Efficiency of Silica Nanovectors.

    PubMed

    Wang, Xiaolin; Masse, Sylvie; Laurent, Guillaume; Hélary, Christophe; Coradin, Thibaud

    2015-10-13

    The conjugation of polyethylenimine (PEI) to silica nanoparticles has emerged as a useful strategy in gene delivery. Here we investigate the influence of the PEI conjugation mode on the transfection ability of plain silica nanoparticles. Surface functionalization with sulfonate- and chloride-bearing silanes modulates the amount and conformation of PEI and therefore the particles' affinity for the plasmid, without impacting on cytotoxicity. However, transfection efficiency in both immortalized and primary cells is more directly correlated to the nature and strength of the particle-PEI interactions. It suggests that PEI detachment from the particle surface at the stage of endosomal escape is a key event in the plasmid delivery process. These data should provide fruitful guidelines for the fine tuning of colloidal surfaces intended for intracellular delivery of bioactive molecules.

  17. 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.

  18. 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

    2014-02-01

    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.

  19. Chitosan as a non-viral co-transfection system in a cystic fibrosis cell line.

    PubMed

    Fernández Fernández, Elena; Santos-Carballal, Beatriz; Weber, Wolf-Michael; Goycoolea, Francisco M

    2016-04-11

    Successful gene therapy requires the development of suitable vehicles for the selective and efficient delivery of genes to specific target cells at the expense of minimal toxicity. In this work, we investigated a non-viral gene delivery system based on chitosan (CS) to specifically address cystic fibrosis (CF). Thus, electrostatic self-assembled CS-pEGFP and CS-pEGFP-siRNA complexes were prepared from high-pure fully characterized CS (Mw ∼ 20 kDa and degree of acetylation ∼ 30%). The average diameter of positively-charged complexes (i.e. ζ ∼+25 mV) was ∼ 200 nm. The complexes were found relatively stable over 14h in Opti-MEM. Cell viability study did not show any significant cytotoxic effect of the CS-based complexes in a human bronchial cystic fibrosis cell line (CFBE41o-). We evaluated the transfection efficiency of this cell line with both CS-pEGFP and co-transfected with CS-pEGFP-siRNA complexes at (N/P) charge ratio of 12. We reported an increase in the fluorescence intensity of CS-pEGFP and a reduction in the cells co-transfected with CS-pEGFP-siRNA. This study shows proof-of-principle that co-transfection with chitosan might be an effective delivery system in a human CF cell line. It also offers a potential alternative to further develop therapeutic strategies for inherited disease treatments, such as CF. PMID:26875537

  20. 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.

  1. Connexin32 gap junction channels in stably transfected cells: unitary conductance.

    PubMed Central

    Moreno, A P; Eghbali, B; Spray, D C

    1991-01-01

    Pairs of SKHep1 cells, which are derived from a highly metastatic human hepatoma, were studied using the whole cell voltage clamp technique with patch-type electrodes containing CsCl as the major ionic species. In 12 of 81 cell pairs, current flow through junctional membranes was detectable; in the remaining 69 cell pairs, junctional conductance was less than the noise limit of our recording apparatus (worst case: 10 pS). Macroscopic junctional conductance (gj) in the small percentage of pairs where it was detectable ranged from 100 to 600 pS. Unitary junctional conductance (gamma j) determined in the lowest conductance pairs or after reducing conductance with a short exposure to the uncoupling agent halothane was 25-35 pS. To study properties of gap junction channels formed of connexin32, the parental SKHep1 cell line was stably transfected with a plasmid containing cDNA that encodes connexin32, the major gap junction protein of rat liver cells. In 85 of 98 pairs of voltage clamped connexin32-transfected SKHep1 cells, macroscopic gj was greater than 1 nS; gj increased with time after dissociation (from 1.8 +/- 0.6 [mean +/- SE; n = 7] nS at 2 h after plating to 9.3 +/- 2.2 [n = 9] nS, the maximal value, at 24 h). Unitary conductance of gap junction channels between pairs of transfected SKHep1 cells was measured in low conductance pairs and after reducing gj by exposure to halothane or heptanol. Histograms of gamma j values in transfected cells, in 10 experiments where greater than 100 transitions were measurable, displayed two peaks; 120-130 pS and 25-35 pS. The smaller size corresponded to channels that were occasionally detected in the parental cells. We therefore conclude that connexin32 forms gap junctions channels of the 120-130 pS size class. PMID:1722119

  2. 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

  3. Reduced PMA enhances the responsiveness of transfected THP-1 macrophages to polarizing stimuli.

    PubMed

    Maeß, Marten B; Wittig, Berith; Cignarella, Andrea; Lorkowski, Stefan

    2014-01-15

    Macrophages are versatile cells of the immune system which react to various external stimuli through different polarization patterns which adjust the cells to the required function whether it is removal of pathogens or necrotic cells, tissue repair or propagation of inflammation. As much of macrophage behavior is determined by their polarization, appropriate models to study macrophage polarization are required. Previously we have published a protocol for transfection of THP-1 macrophages, which in brief pre-differentiates THP-1 monocytes for 48h using 100ng/ml PMA, followed by detachment of the cells and eletroporation using Lonza nucleofector technology and finally includes further 48h of differentiation with 100ng/ml PMA. When we applied this protocol to study interleukin (IL) 10 dependent polarization, the cells were inert to the IL10 stimulus, as indicated by a failure to induce IL10 target genes such as SOCS3. Further investigation revealed that the cells were classically activated by the differentiation agent phorbol 12-myristate 13-acetate (PMA) as shown by induction of chemokine receptor CCR7. Subsequent reduction of PMA concentration during THP-1 macrophage differentiation significantly improved their response to IL10 as SOCS3 increased more than 40-fold. This increased responsiveness of the THP-1 macrophages was also confirmed for polarization with LPS and IFNγ. Up-regulation of classical activation markers CCL3, CCR7 and TNFα was enhanced from 18-, 21- and 70-fold, respectively, to 48-, 222- and 951-fold, respectively. Reduction of PMA concentration did not negatively affect macrophage differentiation or transfection efficiency. Expression of macrophage differentiation markers CD11b and CD68 as well as cell morphology remained unchanged. In addition transfection efficiency and rates of apoptosis and necrosis remained unaffected. Thus our revised protocol combines high transfection efficiency and cell vitality with a strong response to polarizing

  4. Lipid-mediated DNA and siRNA Transfection Efficiency Depends on Peptide Headgroup.

    PubMed

    Zhang, Xiao-Xiang; Lamanna, Caroline M; Kohman, Richie E; McIntosh, Thomas J; Han, Xue; Grinstaff, Mark W

    2013-05-01

    A series of amphiphiles with differing cationic tri- and di- peptide headgroups, designed and synthesized based on lysine (K), ornithine (O), arginine (R), and glycine (G), have been characterized and evaluated for DNA and siRNA delivery. DNA-lipoplexes formed from the tri- and di- lipopeptides possessed lipid:nucleic acid charge ratios of 7:1 to 10:1, diameters of ~200 nm to 375 nm, zeta potentials of 23 mV to 41 mV, melting temperatures of 12 °C to 46 °C, and lamellar repeat periods of 6 nm to 8 nm. These lipid-DNA complexes formed supramolecular structures in which DNA is entrapped at the surface between multilamellar liposomal vesicles. Compared to their DNA counterparts, siRNA-lipoplexes formed slightly larger complexes (348 nm to 424 nm) and required higher charge ratios to form stable structures. Additionally, it was observed that lipids with multivalent, tripeptide headgroups (i.e., KGG, OGG, and RGG) were successful at transfecting DNA in vitro, whereas DNA transfection with the dipeptide lipids proved ineffective. Cellular uptake of DNA was more effective with the KGG compared to the KG lipopeptide. In siRNA knockdown experiments, both tri- and di- peptide lipids (i.e., RGG, GGG, KG, OG, RG, GG) showed some efficacy, but total cellular uptake of siRNA complexes was not indicative of knockdown outcomes and suggested that the intracellular fate of lipoplexes may be a factor. Overall, this lipopeptide study expands the library of efficient DNA transfection vectors available for use, introduces new vectors for siRNA delivery, and begins to address the structure-activity relationships which influence delivery and transfection efficacy. PMID:24391676

  5. Lipid-mediated DNA and siRNA Transfection Efficiency Depends on Peptide Headgroup

    PubMed Central

    Zhang, Xiao-Xiang; LaManna, Caroline M.; Kohman, Richie E.; McIntosh, Thomas J.; Han, Xue; Grinstaff, Mark W.

    2013-01-01

    A series of amphiphiles with differing cationic tri- and di- peptide headgroups, designed and synthesized based on lysine (K), ornithine (O), arginine (R), and glycine (G), have been characterized and evaluated for DNA and siRNA delivery. DNA-lipoplexes formed from the tri- and di- lipopeptides possessed lipid:nucleic acid charge ratios of 7:1 to 10:1, diameters of ~200 nm to 375 nm, zeta potentials of 23 mV to 41 mV, melting temperatures of 12 °C to 46 °C, and lamellar repeat periods of 6 nm to 8 nm. These lipid-DNA complexes formed supramolecular structures in which DNA is entrapped at the surface between multilamellar liposomal vesicles. Compared to their DNA counterparts, siRNA-lipoplexes formed slightly larger complexes (348 nm to 424 nm) and required higher charge ratios to form stable structures. Additionally, it was observed that lipids with multivalent, tripeptide headgroups (i.e., KGG, OGG, and RGG) were successful at transfecting DNA in vitro, whereas DNA transfection with the dipeptide lipids proved ineffective. Cellular uptake of DNA was more effective with the KGG compared to the KG lipopeptide. In siRNA knockdown experiments, both tri- and di- peptide lipids (i.e., RGG, GGG, KG, OG, RG, GG) showed some efficacy, but total cellular uptake of siRNA complexes was not indicative of knockdown outcomes and suggested that the intracellular fate of lipoplexes may be a factor. Overall, this lipopeptide study expands the library of efficient DNA transfection vectors available for use, introduces new vectors for siRNA delivery, and begins to address the structure-activity relationships which influence delivery and transfection efficacy. PMID:24391676

  6. 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.

  7. 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

  8. Further evaluation of a novel nano-scale gene vector for in vivo transfection of siRNA.

    PubMed

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

    2011-05-01

    In this research, a lipid-cationic polymer (LCP) containing the side-chain branching of brassidic acid was synthesized using chemical methods. As a gene vector for small interfering ribonucleic acid (siRNA) transfection, the efficiency and biosafety of LCP were preliminarily evaluated to investigate its possible application on tumor gene therapy. The toxicity, side-effects, and biosafety of LCP were investigated in animals based on the results of in vitro experiments. The siRNA against cyclooxygenase-2 (COX-2) was transfected by LCP to interfere with the COX-2 expression in nude-transplanted tumors. Hematoxylin and eosin stains, immunohistochemistry, reverse transcription-polymerase chain reaction, and Western blot were performed to evaluate the efficiency of LCP for siRNA transfection. The animal toxicity experiment showed that a high concentration of LCP had a low toxic effect on animals and did not induce allergic or pyrogenic reactions. The results from the in vivo transfection indicated that LCP could efficiently transfect siRNA and silence the target gene expression. The LCP gene vector for siRNA transfection is highly efficient during in vivo transfection and had low toxicity. From all aspects of tumor gene therapy and basic research, LCP is valuable for scientific research and medical applications.

  9. Multivalent dendritic polyglycerolamine with arginine and histidine end groups for efficient siRNA transfection

    PubMed Central

    Sheikhi Mehrabadi, Fatemeh; Zeng, Hanxiang; Johnson, Mark; Schlesener, Cathleen

    2015-01-01

    Summary The success of siRNA-based therapeutics highly depends on a safe and efficient delivery of siRNA into the cytosol. In this study, we post-modified the primary amines on dendritic polyglycerolamine (dPG-NH2) with different ratios of two relevant amino acids, namely, arginine (Arg) and histidine (His). To investigate the effects from introducing Arg and His to dPG, the resulting polyplexes of amino acid functionalized dPG-NH2s (AAdPGs)/siRNA were evaluated regarding cytotoxicity, transfection efficiency, and cellular uptake. Among AAdPGs, an optimal vector with (1:3) Arg to His ratio, showed efficient siRNA transfection with minimal cytotoxicity (cell viability ≥ 90%) in NIH 3T3 cells line. We also demonstrated that the cytotoxicity of dPG-NH2 decreased as a result of amino acid functionalization. While the incorporation of both cationic (Arg) and pH-responsive residues (His) are important for safe and efficient siRNA transfection, this study indicates that AAdPGs containing higher degrees of His display lower cytotoxicity and more efficient endosomal escape. PMID:26124878

  10. 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.

  11. Intracellular ROS mediates gas plasma-facilitated cellular transfection in 2D and 3D cultures.

    PubMed

    Xu, Dehui; Wang, Biqing; Xu, Yujing; Chen, Zeyu; Cui, Qinjie; Yang, Yanjie; Chen, Hailan; Kong, Michael G

    2016-06-14

    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.

  12. 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.

  13. 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

  14. In vitro transfection of bone marrow-derived dendritic cells with TATp-liposomes

    PubMed Central

    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. PMID:24611012

  15. 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. PMID:26974323

  16. New Transfection Agents Based on Liposomes Containing Biosurfactant MEL-A

    PubMed Central

    Nakanishi, Mamoru; Inoh, Yoshikazu; Furuno, Tadahide

    2013-01-01

    Nano vectors are useful tools to deliver foreign DNAs, oligonucleotides, and small interfering double-stranded RNAs (siRNAs) into mammalian cells with gene transfection and gene regulation. In such experiments we have found the liposomes with a biosurfacant mannosylerythriol lipid (MEL-A) are useful because of their high transfer efficiency, and their unique mechanism to transfer genes to target cells with the lowest toxicity. In the present review we will describe our current work, which may contribute to the great advance of gene transfer to target cells and gene regulations. For more than two decades, the liposome technologies have changed dramatically and various methods have been proposed in the fields of biochemistry, cell biology, biotechnology, and so on. In addition, they were towards to pharmaceutics and clinical applications. The liposome technologies were expected to use gene therapy, however, they have not reached a requested goal as of yet. In the present paper we would like to present an approach using a biosurfactant, MEL-A, which is a surface-active compound produced by microorganisms growing on water-insoluble substrates and increases efficiency in gene transfection. The present work shows new transfection agents based on liposomes containing biosurfactant MEL-A. PMID:24300514

  17. Single-cell optoporation and transfection using femtosecond laser and optical tweezers

    PubMed Central

    Waleed, Muhammad; Hwang, Sun-Uk; Kim, Jung-Dae; Shabbir, Irfan; Shin, Sang-Mo; Lee, Yong-Gu

    2013-01-01

    In this paper, we demonstrate a new single-cell optoporation and transfection technique using a femtosecond Gaussian laser beam and optical tweezers. Tightly focused near-infrared (NIR) femtosecond laser pulse was employed to transiently perforate the cellular membrane at a single point in MCF-7 cancer cells. A distinct technique was developed by trapping the microparticle using optical tweezers to focus the femtosecond laser precisely on the cell membrane to puncture it. Subsequently, an external gene was introduced in the cell by trapping and inserting the same plasmid-coated microparticle into the optoporated cell using optical tweezers. Various experimental parameters such as femtosecond laser exposure power, exposure time, puncture hole size, exact focusing of the femtosecond laser on the cell membrane, and cell healing time were closely analyzed to create the optimal conditions for cell viability. Following the insertion of plasmid-coated microparticles in the cell, the targeted cells exhibited green fluorescent protein (GFP) under the fluorescent microscope, hence confirming successful transfection into the cell. This new optoporation and transfection technique maximizes the level of selectivity and control over the targeted cell, and this may be a breakthrough method through which to induce controllable genetic changes in the cell. PMID:24049675

  18. 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

  19. 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.

  20. Role of liposome and peptide in the synergistic enhancement of transfection with a lipopolyplex vector.

    PubMed

    Munye, Mustafa M; Ravi, Jascindra; Tagalakis, Aristides D; McCarthy, David; Ryadnov, Maxim G; Hart, Stephen L

    2015-03-19

    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.

  1. Characterization of insulin-producing cells derived from PDX-1-transfected neural stem cells.

    PubMed

    Wang, Hailan; Jiang, Zesheng; Li, Aihui; Gao, Yi

    2012-12-01

    Islet cell transplantation is a promising treatment strategy for type-1 diabetes. However, functional islet cells are hard to obtain for transplantation and are in short supply. Directing the differentiation of stem cells into insulin‑producing cells, which serve as islet cells, would overcome this shortage. Bone marrow contains hematopoietic stem cells and mesenchymal stem cells. The present study used bone marrow cells isolated from rats and neural stem cells (NSCs) that were derived from bone marrow cells in culture. Strong nestin staining was detected in NSCs, but not in bone marrow stromal cells (BMSCs). In vitro transfection of the pancreatic duodenal homeobox-1 (PDX-1) gene into NSCs generated insulin‑producing cells. Reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) analysis confirmed that PDX-1-transfected NSCs expressed insulin mRNA and released insulin protein. However, insulin release from PDX-1-transfected NSCs did not respond to the challenge of glucose and glucagon-like peptide-1. These results support the use of bone marrow-derived NSCs as a renewable source of insulin-producing cells for autologous transplantation to treat type-1 diabetes.

  2. 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

  3. Pressure-Mediated Oligonucleotide Transfection of Rat and Human Cardiovascular Tissues

    NASA Astrophysics Data System (ADS)

    Mann, Michael J.; Gibbons, Gary H.; Hutchinson, Howard; Poston, Robert S.; Hoyt, E. Grant; Robbins, Robert C.; Dzau, Victor J.

    1999-05-01

    The application of gene therapy to human disease is currently restricted by the relatively low efficiency and potential hazards of methods of oligonucleotide or gene delivery. Antisense or transcription factor decoy oligonucleotides have been shown to be effective at altering gene expression in cell culture expreriments, but their in vivo application is limited by the efficiency of cellular delivery, the intracellular stability of the compounds, and their duration of activity. We report herein the development of a highly efficient method for naked oligodeoxynucleotide (ODN) transfection into cardiovascular tissues by using controlled, nondistending pressure without the use of viral vectors, lipid formulations, or exposure to other adjunctive, potentially hazardous substances. In this study, we have documented the ability of ex vivo, pressure-mediated transfection to achieve nuclear localization of fluorescent (FITC)-labeled ODN in approximately 90% and 50% of cells in intact human saphenous vein and rat myocardium, respectively. We have further documented that pressure-mediated delivery of antisense ODN can functionally inhibited target gene expression in both of these tissues in a sequence-specific manner at the mRNA and protein levels. This oligonucleotide transfection system may represent a safe means of achieving the intraoperative genetic engineering of failure-resistant human bypass grafts and may provide an avenue for the genetic manipulation of cardiac allograft rejection, allograft vasculopathy, or other transplant diseases.

  4. 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. PMID:24611012

  5. Bacteriophage Mediates Efficient Gene Transfer in Combination with Conventional Transfection Reagents.

    PubMed

    Donnelly, Amanda; Yata, Teerapong; Bentayebi, Kaoutar; Suwan, Keittisak; Hajitou, Amin

    2015-12-01

    The development of commercially available transfection reagents for gene transfer applications has revolutionized the field of molecular biology and scientific research. However, the challenge remains in ensuring that they are efficient, safe, reproducible and cost effective. Bacteriophage (phage)-based viral vectors have the potential to be utilized for general gene transfer applications within research and industry. Yet, they require adaptations in order to enable them to efficiently enter cells and overcome mammalian cellular barriers, as they infect bacteria only; furthermore, limited progress has been made at increasing their efficiency. The production of a novel hybrid nanocomplex system consisting of two different nanomaterial systems, phage vectors and conventional transfection reagents, could overcome these limitations. Here we demonstrate that the combination of cationic lipids, cationic polymers or calcium phosphate with M13 bacteriophage-derived vectors, engineered to carry a mammalian transgene cassette, resulted in increased cellular attachment, entry and improved transgene expression in human cells. Moreover, addition of a targeting ligand into the nanocomplex system, through genetic engineering of the phage capsid further increased gene expression and was effective in a stable cell line generation application. Overall, this new hybrid nanocomplex system (i) provides enhanced phage-mediated gene transfer; (ii) is applicable for laboratory transfection processes and (iii) shows promise within industry for large-scale gene transfer applications. PMID:26670247

  6. Bacteriophage Mediates Efficient Gene Transfer in Combination with Conventional Transfection Reagents

    PubMed Central

    Donnelly, Amanda; Yata, Teerapong; Bentayebi, Kaoutar; Suwan, Keittisak; Hajitou, Amin

    2015-01-01

    The development of commercially available transfection reagents for gene transfer applications has revolutionized the field of molecular biology and scientific research. However, the challenge remains in ensuring that they are efficient, safe, reproducible and cost effective. Bacteriophage (phage)-based viral vectors have the potential to be utilized for general gene transfer applications within research and industry. Yet, they require adaptations in order to enable them to efficiently enter cells and overcome mammalian cellular barriers, as they infect bacteria only; furthermore, limited progress has been made at increasing their efficiency. The production of a novel hybrid nanocomplex system consisting of two different nanomaterial systems, phage vectors and conventional transfection reagents, could overcome these limitations. Here we demonstrate that the combination of cationic lipids, cationic polymers or calcium phosphate with M13 bacteriophage-derived vectors, engineered to carry a mammalian transgene cassette, resulted in increased cellular attachment, entry and improved transgene expression in human cells. Moreover, addition of a targeting ligand into the nanocomplex system, through genetic engineering of the phage capsid further increased gene expression and was effective in a stable cell line generation application. Overall, this new hybrid nanocomplex system (i) provides enhanced phage-mediated gene transfer; (ii) is applicable for laboratory transfection processes and (iii) shows promise within industry for large-scale gene transfer applications. PMID:26670247

  7. Biosurfactant MEL-A enhances cellular association and gene transfection by cationic liposome.

    PubMed

    Igarashi, Saki; Hattori, Yoshiyuki; Maitani, Yoshie

    2006-05-30

    Mannnosylerythritol lipid A (MEL-A), a biosurfactant produced by microorganisms, has many biological activities. To enhance the gene transfection efficiency of a cationic liposome, we prepared a MEL-liposome (MEL-L) composed of 3beta-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol), dioleoyl phosphatidylethanolamine (DOPE) and MEL-A, and investigated its transfection efficiency in human cervix carcinoma Hela cells. MEL-L was about 40 nm in size, and the MEL-L/plasmid DNA complex (MEL-lipoplex) remained an injectable size (169 nm). MEL-A induced a significantly higher level of gene expression, compared to commercially available Tfx20 and the liposome without MEL-A (Cont-L). Analysis of flow cytometric profiles clearly indicated that the amount of DNA associated with the cells was rapidly increased and sustained by addition of MEL-A to the liposome. Confocal microscopic observation indicated that the MEL-lipoplex distributed widely in the cytoplasm, and the DNA was detected strongly in the cytoplasm and around the nucleus, compared with Cont-L. These results suggested that MEL-A increased gene expression by enhancing the association of the lipoplexes with the cells in serum. MEL-L might prove a remarkable non-viral vector for gene transfection and gene therapy. PMID:16624437

  8. The ratio of unsaturated fatty acids in biosurfactants affects the efficiency of gene transfection.

    PubMed

    Inoh, Yoshikazu; Furuno, Tadahide; Hirashima, Naohide; Kitamoto, Dai; Nakanishi, Mamoru

    2010-10-15

    An unsaturated hydrocarbon chain in phospholipid was reported to affect a phase transition and a fusogenic activity after mixing membranes, and consequently to achieve a high DNA transfection efficiency. We previously showed that a biosurfactant mannosylerythritol lipid-A (MEL-A) enhances the gene transfection efficiency of cationic liposomes. Here, we have studied the effects of unsaturated fatty acid ratio of MEL-A on the physicochemical properties and gene delivery into cells of cationic liposomes using MEL-A with three different unsaturated fatty acid ratios (9.1%, 21.5%, and 46.3%). The gene transfer efficiency of cationic liposomes containing MEL-A (21.5%) was much higher than that of those containing MEL-A (9.1%) and MEL-A (46.3%). MEL-A (21.5%)-containing cationic liposomes induced highly efficient membrane fusion after addition of anionic liposomes and led to subsequent DNA release. Imaging analysis revealed that MEL-A (21.5%)-containing liposomes fused with the plasma membrane and delivered DNA into the nucleus of NIH-3T3 cells, MEL-A (46.3%)-containing liposomes fused with the plasma membrane did not deliver DNA into the nucleus, and MEL-A (9.1%)-containing liposomes neither fused with the plasma membrane nor delivered DNA into the nucleus. Thus, it is understandable that the unsaturated fatty acid ratio of MEL-A strongly influences the gene transfection efficiency of cationic liposomes. PMID:20674726

  9. Gene Transfection of Human Turbinate Mesenchymal Stromal Cells Derived from Human Inferior Turbinate Tissues

    PubMed Central

    Kwon, Jin Seon; Park, Seung Hun; Baek, Ji Hye; Dung, Truong Minh; Kim, Sung Won; Min, Byoung Hyun; Kim, Jae Ho; Kim, Moon Suk

    2016-01-01

    Human turbinate mesenchymal stromal cells (hTMSCs) are novel stem cells derived from nasal inferior turbinate tissues. They are easy to isolate from the donated tissue after turbinectomy or conchotomy. In this study, we applied hTMSCs to a nonviral gene delivery system using polyethyleneimine (PEI) as a gene carrier; furthermore, the cytotoxicity and transfection efficiency of hTMSCs were evaluated to confirm their potential as resources in gene therapy. DNA-PEI nanoparticles (NPs) were generated by adding the PEI solution to DNA and were characterized by a gel electrophoresis and by measuring particle size and surface charge of NPs. The hTMSCs were treated with DNA-PEI NPs for 4 h, and toxicity of NPs to hTMSCs and gene transfection efficiency were monitored using MTT assay, fluorescence images, and flow cytometry after 24 h and 48 h. At a high negative-to-positive charge ratio, DNA-PEI NPs treatment led to cytotoxicity of hTMSCs, but the transfection efficiency of DNA was increased due to the electrostatic effect between the NPs and the membranes of hTMSCs. Importantly, the results of this research verified that PEI could deliver DNA into hTMSCs with high efficiency, suggesting that hTMSCs could be considered as untapped resources for applications in gene therapy. PMID:26783402

  10. Polarized endocytosis by Madin-Darby canine kidney cells transfected with functional chicken liver glycoprotein receptor

    PubMed Central

    1989-01-01

    We have studied the expression of the chicken hepatic glycoprotein receptor (chicken hepatic lectin [CHL]) in Madin-Darby canine kidney (MDCK) cells, by transfection of its cDNA under the control of a retroviral promotor. Transfected cell lines stably express 87,000 surface receptors/cell with a kd = 13 nM. In confluent monolayers, approximately 40% of CHL is localized at the plasma membrane. 98% of the surface CHL is expressed at the basolateral surface where it performs polarized endocytosis and degradation of glycoproteins carrying terminal N-acetylglucosamine at a rate of 50,000 ligand molecules/h. Studies of the half-life of metabolically labeled receptor and of the stability of biotinylated cell surface receptor after internalization indicate that transfected CHL performs several rounds of uptake and recycling before it gets degraded. The successful expression of a functional basolateral receptor in MDCK cells opens the way for the characterization of the mechanisms that control targeting and recycling of proteins to the basolateral membrane of epithelial cells. PMID:2687287

  11. Transfection, expression, and DNA sequence of a gene encoding a BoLA-A11 antigen

    SciTech Connect

    Sawhney, S.M.S.; Hasima, N.N.; Glass, E.J.; Al-Murrani, S.W.K.; Nichani, A.K.; Spooner, R.L.; Williams, J.L.; Russell, G.C.

    1995-02-01

    Cattle major histocompatibility complex [(MHC) (BoLA)] class I molecules are heterodimeric glycoproteins which present endogenous antigenic peptides to CD8{sup +} T lymphocytes, initiating a cellular immune response. The MHC-encoded heavy chains are highly polymorphic and, in cattle, have been characterized mainly by using allo-antisera raised by reciprocal calf/dam immunizations. This has enabled the identification of about 50 serlogical specificities, most of which behave as alleles of a single highly polymorphic class I locus. However, evidence from biochemical and molecular biological studies suggest that more than one BoLA class I locus is expressed. These loci are apparently in linkage disequilibrium, making them difficult to distinguish by conventional methods. In order to investigate the expression and function of individual class I locus products, we are correlating BoLA class I gene sequences with the expressed products by the transfection and characterization of genomic class I clones. Shotgun transfection and expression of BoLA class I molecules has been described previously, but the genes involved were not isolated. In this paper we report the isolation, DNA sequencing, transfection, and expression of a genomic clone encoding a BoLA-A11 determinant from an animal expressing A10 and A11 serological specificities. 23 refs., 3 figs.

  12. siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts.

    PubMed

    Lokossou, Adjimon Gatien; Toufaily, Chirine; Vargas, Amandine; Barbeau, Benoit

    2016-01-01

    Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend diseases related to pregnancy. In this protocol, human primary villous cytotrophoblasts freshly isolated from placentas through a standard DNase/trypsin protocol are microporated with small interfering RNA (siRNA). This approach provided greater efficiency for siRNA transfection when compared to a lipofection-based method. Transfected cells can subsequently be analyzed by standard Western blot within a time frame of 3-4 days post-transfection. In addition, using cultured primary villous cytotrophoblasts, Electrophoretic Mobility Shift Assay (EMSA) analysis was optimized and performed on extracts from days 1 to 4. The use of these cultured primary cells and the protocol described allow for an evaluation of the implication of specific genes and transcription factors in the process of villous cytotrophoblast differentiation into a syncytiotrophoblast-like cell layer. However, the limited time span allowable in culture precludes the use of methods requiring more time, such as generation of a stable cell population. Therefore testing of this cell population requires highly optimized gene transfer protocols. PMID:27685614

  13. 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

  14. Enhanced Apoptotic Response to Photodynamic Therapy after bcl-2 Transfection1

    PubMed Central

    Kim, Hyeong-Reh Choi; Luo, Yu; Li, Gangyong; Kessel, David

    2015-01-01

    Apoptosis is a cellular death process involving the sequential activation of a series of caspases, endonucleases, and other enzymes. The initiation of apoptosis can be inhibited by overexpression of bcl-2 and certain other members of a related family of proteins. We examined the effects of bcl-2 overexpression on the apoptotic response to photodynamic therapy (PDT), using aluminum phthalocyanine as the photosensitizing agent. In this study, we compared the immortalized human breast epithelial cell line MCF10A with a subline (MCF10A/bcl-2) transfected with the human bcl-2 gene. The latter was ~2-fold more sensitive to the phototoxic effects of PDT. At a 50 mJ/cm2 light dose, photodamage to MCF-10A/bcl-2 resulted in a greater loss of the mitochondrial membrane potential (ΔΨm), enhanced release of mitochondrial cytochrome c, a more rapid and greater activation of caspase-3, and a greater apoptotic response. Western blot analysis revealed that the transfected cell line showed overexpression of both bcl-2 and bax, and that PDT caused selective destruction of bcl-2, leaving bax unaffected. The greater apoptotic response by the transfected line is, therefore, attributed to the higher bax:bcl-2 ratio after photodamage. PMID:10416606

  15. Experimental research of RB94 gene transfection into retinoblastoma cells using ultrasound-targeted microbubble destruction.

    PubMed

    Zheng, Min-Ming; Zhou, Xi-Yuan; Wang, Li-Ping; Wang, Zhi-Gang

    2012-06-01

    The purpose of this study was to explore the transfection of the recombinant expression plasmid pEGFP-C1/RB94 into human retinoblastoma cells (HXO-Rb44) using ultrasound-targeted microbubble destruction (UTMD). pEGFP-C1/RB94 was transfected into HXO-Rb44 in vitro by UTMD, with liposome as the positive control. After 24 to 72 h, the expression of the reporter gene enhanced green fluorescent protein (EGFP) was observed using fluorescent microscopy and flow cytometry. The cell viability of HXO-Rb44 was measured by a MTT assay. The mRNA and proteins of RB94, caspase-3 and Bax were analyzed by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Moreover, the apoptosis rate and cell cycle progression of the cells were detected by flow cytometry. This study demonstrated that UTMD can enhance the transfection efficiency of RB94, which has an obvious impact on the inhibition of the growth process of retinoblastoma cells, suggesting that the combination of UTMD and RB94 compounds might be a useful tool for use in the gene therapy of retinoblastoma.

  16. Transfection efficiency of TDL compound in HUVEC enhanced by ultrasound-targeted microbubble destruction.

    PubMed

    Ren, Jian-Li; Wang, Zhi-Gang; Zhang, Yong; Zheng, Yuan-Yi; Li, Xing-Sheng; Zhang, Qun-Xia; Wang, Zhao-Xia; Xu, Chuan-Shan

    2008-11-01

    The aim of the present study was to explore the gene transfection efficiency of Tat peptide/plasmid DNA/ liposome (TDL) compound combined with ultrasound-targeted microbubble destruction (UTMD) in human umbilical vein endothelial cell (HUVEC). Tat peptide, plasmid DNA (pIRES2-EGFP-HGF) and Lipofectamine 2000 were used to prepare the TDL compound. Microbubbles were prepared using mechanic vibration. The expression of the report gene enhanced green fluorescent protein (EGFP) was observed using fluorescent microscopy and flow cytometry. The viability of HUVEC was measured by MTT assay. mRNA and protein of HGF was analyzed by reverse transcription-polymerase chain reaction and Western Blot. The intensity of green fluorescence and the gene transfection efficiency of TDL compound + microbubbles + ultrasound group were higher than those of other groups, and no significantly different viability was found between TDL compound + microbubbles + ultrasound group and the other groups. The HGF mRNA and HGF protein of TDL compound + microbubbles + ultrasound group were higher than those of other groups. Our finding demonstrated that UTMD could enhance the transfection efficiency of TDL compound without obvious effects on the cell viability of HUVEC, suggesting that the combination of UTMD and TDL compound might be a useful tool for the gene therapy of ischemic heart disease.

  17. Effects of microbubble size on ultrasound-mediated gene transfection in auditory cells.

    PubMed

    Liao, Ai-Ho; Hsieh, Yi-Lei; Ho, Hsin-Chiao; Chen, Hang-Kang; Lin, Yi-Chun; Shih, Cheng-Ping; Chen, Hsin-Chien; Kuo, Chao-Yin; Lu, Ying-Jui; Wang, Chih-Hung

    2014-01-01

    Gene therapy for sensorineural hearing loss has recently been used to insert genes encoding functional proteins to preserve, protect, or even regenerate hair cells in the inner ear. Our previous study demonstrated a microbubble- (MB-)facilitated ultrasound (US) technique for delivering therapeutic medication to the inner ear. The present study investigated whether MB-US techniques help to enhance the efficiency of gene transfection by means of cationic liposomes on HEI-OC1 auditory cells and whether MBs of different sizes affect such efficiency. Our results demonstrated that the size of MBs was proportional to the concentration of albumin or dextrose. At a constant US power density, using 0.66, 1.32, and 2.83 μm albumin-shelled MBs increased the transfection rate as compared to the control by 30.6%, 54.1%, and 84.7%, respectively; likewise, using 1.39, 2.12, and 3.47 μm albumin-dextrose-shelled MBs increased the transfection rates by 15.9%, 34.3%, and 82.7%, respectively. The results indicate that MB-US is an effective technique to facilitate gene transfer on auditory cells in vitro. Such size-dependent MB oscillation behavior in the presence of US plays a role in enhancing gene transfer, and by manipulating the concentration of albumin or dextrose, MBs of different sizes can be produced.

  18. Effects of Microbubble Size on Ultrasound-Mediated Gene Transfection in Auditory Cells

    PubMed Central

    Liao, Ai-Ho; Hsieh, Yi-Lei; Ho, Hsin-Chiao; Chen, Hang-Kang; Lin, Yi-Chun; Kuo, Chao-Yin; Lu, Ying-Jui

    2014-01-01

    Gene therapy for sensorineural hearing loss has recently been used to insert genes encoding functional proteins to preserve, protect, or even regenerate hair cells in the inner ear. Our previous study demonstrated a microbubble- (MB-)facilitated ultrasound (US) technique for delivering therapeutic medication to the inner ear. The present study investigated whether MB-US techniques help to enhance the efficiency of gene transfection by means of cationic liposomes on HEI-OC1 auditory cells and whether MBs of different sizes affect such efficiency. Our results demonstrated that the size of MBs was proportional to the concentration of albumin or dextrose. At a constant US power density, using 0.66, 1.32, and 2.83 μm albumin-shelled MBs increased the transfection rate as compared to the control by 30.6%, 54.1%, and 84.7%, respectively; likewise, using 1.39, 2.12, and 3.47 μm albumin-dextrose-shelled MBs increased the transfection rates by 15.9%, 34.3%, and 82.7%, respectively. The results indicate that MB-US is an effective technique to facilitate gene transfer on auditory cells in vitro. Such size-dependent MB oscillation behavior in the presence of US plays a role in enhancing gene transfer, and by manipulating the concentration of albumin or dextrose, MBs of different sizes can be produced. PMID:25254216

  19. Intracellular ROS mediates gas plasma-facilitated cellular transfection in 2D and 3D cultures.

    PubMed

    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

  20. Glucocorticoid receptor isoforms alpha and beta in in vitro cytokine-induced glucocorticoid insensitivity.

    PubMed

    Torrego, Alfons; Pujols, Laura; Roca-Ferrer, Jordi; Mullol, Joaquim; Xaubet, Antoni; Picado, César

    2004-08-15

    We stimulated peripheral blood mononuclear cells from 14 healthy subjects, 14 patients with stable asthma, and 13 patients with unstable asthma with interleukin (IL)-2 and IL-4 to induce glucocorticoid insensitivity and we examined the relationship between insensitivity and the expression of glucocorticoid receptor (GR) isoforms. Results are expressed as IC(50) (nanomolar) values (means +/- SD) in proliferation assays and as 10(3) cDNA molecules per microgram of total RNA (means +/- SD) in real-time polymerase chain reaction analysis. Cells from patients with unstable asthma were less sensitive (316 +/- 7 nM) to dexamethasone antiproliferative effects than those from healthy control subjects (102 +/- 4 nM, p < 0.05) and patients with stable asthma (107 +/- 2 nM, p < 0.05). Coincubation with IL-2 and IL-4 repressed the inhibitory effect of dexamethasone on proliferation in all groups (unstable: 851 +/- 47 nM, p < 0.01; stable: 912 +/- 52 nM, p = 0.001; control subjects: 537 +/- 45 nM, p = 0.001). GR-alpha mRNA baseline expression was higher in patients with unstable asthma [(1.95 +/- 0.40) x 10(3) cDNA molecules/microg total RNA, p < 0.05] than in patients with stable asthma [(1.46 +/- 0.35) x 10(3) cDNA molecules/microg total RNA] and healthy subjects [(1.35 +/- 0.25) x 10(3) cDNA molecules/microg total RNA]. GR-beta mRNA was 600 times lower than GR-alpha in the three groups. Coincubation with IL-2 and IL-4 significantly increased GR-alpha mRNA expression in the three groups (p < 0.01), but caused no significant change in GR-beta mRNA. GR-alpha, but not GR-beta, protein was detected at baseline and after cytokine exposure. Our data do not support the hypothesis that increased GR-beta expression can contribute to cytokine-induced glucocorticoid insensitivity. PMID:15184204

  1. TRPM5-dependent amiloride- and benzamil-insensitive NaCl chorda tympani taste nerve response

    PubMed Central

    Ren, ZuoJun; Rhyu, Mee-Ra; Phan, Tam-Hao T.; Mummalaneni, Shobha; Murthy, Karnam S.; Grider, John R.; DeSimone, John A.

    2013-01-01

    Transient receptor potential (TRP) subfamily M member 5 (TRPM5) cation channel is involved in sensing sweet, bitter, umami, and fat taste stimuli, complex-tasting divalent salts, and temperature-induced changes in sweet taste. To investigate if the amiloride- and benzamil (Bz)-insensitive NaCl chorda tympani (CT) taste nerve response is also regulated in part by TRPM5, CT responses to 100 mM NaCl + 5 μM Bz (NaCl + Bz) were monitored in Sprague-Dawley rats, wild-type (WT) mice, and TRP vanilloid subfamily member 1 (TRPV1) and TRPM5 knockout (KO) mice in the presence of resiniferatoxin (RTX), a TRPV1 agonist. In rats, NaCl + Bz + RTX CT responses were also monitored in the presence of triphenylphosphine oxide, a specific TRPM5 blocker, and capsazepine and N-(3-methoxyphenyl)-4-chlorocinnamid (SB-366791), specific TRPV1 blockers. In rats and WT mice, RTX produced biphasic effects on the NaCl + Bz CT response, enhancing the response at 0.5–1 μM and inhibiting it at >1 μM. The NaCl + Bz + SB-366791 CT response in rats and WT mice and the NaCl + Bz CT response in TRPV1 KO mice were inhibited to baseline level and were RTX-insensitive. In rats, blocking TRPV1 by capsazepine or TRPM5 by triphenylphosphine oxide inhibited the tonic NaCl + Bz CT response and shifted the relationship between RTX concentration and the magnitude of the tonic CT response to higher RTX concentrations. TRPM5 KO mice elicited no constitutive NaCl + Bz tonic CT response. The relationship between RTX concentration and the magnitude of the tonic NaCl + Bz CT response was significantly attenuated and shifted to higher RTX concentrations. The results suggest that pharmacological or genetic alteration of TRPM5 activity modulates the Bz-insensitive NaCl CT response and its modulation by TRPV1 agonists. PMID:23639808

  2. Biological effects of eukaryotic recombinant plasmid pReceiver-M61-BAI-1 transfection on T24 cells and HUVECs

    PubMed Central

    Tian, Da-Wei; Hu, Hai-Long; Sun, Yan; Tang, Yang; Lei, Ming-De; Liu, Li-Wei; Han, Rui-Fa; Wu, Chang-Li

    2016-01-01

    The aim of the current study was to investigate the biological effect on T24 cells and human umbilical vein endothelial cells (HUVECs) of transfection with brain-specific angiogenesis inhibitor-1 (BAI-1). The recombinant plasmid pReceiver-M61-BAI-1 was transfected into human superficial bladder tumor cells (T24) and HUVECs, in parallel with the vector control. mRNA and protein expression levels of BAI-1 were then detected by quantitative polymerase chain reaction (qPCR) and western blotting, respectively. Cell apoptosis of T24 cells and HUVECs prior and subsequent to transfection with BAI-1 was analyzed by flow cytometric analysis. Proliferation of T24 cells and HUVECs prior and subsequent to transfection of BAI-1 was assessed by the MTT method. T24 cells and HUVECs transfected with pReceiver-M61-BA1-1 were classed as the experimental group; T24 cells and HUVECs transfected with p-Receiver-M61 were the control group. qPCR and western blotting methods confirmed that there was positive expression of BAI-1 in T24 cells and HUVECs transfected with pReceiver-M61-BAI-1, however BAI-1 was not expressed in T24 cells and HUVECs transfected with pReceiver-M61. The results of the MTT assay demonstrated that absorbance was markedly reduced in HUVECs at 12, 48 and 72 h subsequent to transfection with pReceiver-M61-BAI-1 when compared with that of the control group and in T24 cells transfected with p-Receiver-M61-BAI-1. Furthermore, flow cytometry results also indicated that the apoptotic rate of HUVECs transfected with p-Receiver-M61-BAI-1 was significantly increased compared with that of the control group and T24 cells transfected with p-Receiver-M61-BAI-1. BAI-1 was observed to markedly inhibit the proliferation of vascular endothelial cells in vitro, however, no direct inhibition by BAI-1 was observed in T24 cells. In conclusion, BAI-1 is suggested to be a potential novel therapautic target for the inhibition of tumor neovascularization. PMID:27356780

  3. All-optical, polarization-insensitive light tuning properties in silver nanorod arrays covered with photoresponsive liquid crystals.

    PubMed

    Si, Guangyuan; Leong, Eunice S P; Jiang, Xiaoxiao; Lv, Jiangtao; Lin, Jiao; Dai, Haitao; Liu, Yan Jun

    2015-05-28

    Active plasmonics has been an interesting and important topic recently. Here we demonstrate the all-optical, polarization-insensitive tunable manipulation of a hybrid system that integrates a silver nanorod array with photoresponsive liquid crystals. The large-area plasmonic nanorod arrays are fabricated by laser interference lithography and ion milling. By covering a layer of photoresponsive liquid crystals, tunable control of plasmon resonance is achieved under an external light pump. The silver nanorod array also enables the homeotropic alignment of the liquid crystals, which makes the all-optical tuning behavior polarization-insensitive. With its advantages of cost-effective fabrication, easy integration, all-optical control, and polarization-insensitivity, the hybrid system could be valuable in many nanophotonic applications. PMID:25758775

  4. Sodium Kinetics of Na,K-ATPase α Isoforms in Intact Transfected HeLa Cells

    PubMed Central

    Zahler, Raphael; Zhang, Zhong-Ting; Manor, Mira; Boron, Walter F.

    1997-01-01

    By participating in the regulation of ion and voltage gradients, the Na-K pump (i.e., Na,K-ATPase) influences many aspects of cellular physiology. Of the four α isoforms of the pump, α1 is ubiquitous, α2 is predominant in skeletal muscle, and α3 is found in neurons and the cardiac conduction system. To determine whether the isoforms have different intracellular Na+ affinities, we used the Na+-sensitive dye sodium-binding benzofuran isophthalate (SBFI) to measure pump-mediated Na+ efflux as a function of [Na+]i in human HeLa cells stably transfected with rat Na-K pump isoforms. We Na+-loaded the cells, and then monitored the time course of the decrease in [Na+]i after removing external Na+. All transfected rat α subunits were highly ouabain resistant: the α1 isoform is naturally resistant, whereas the α2 and α3 isoforms had been mutagenized to render them resistant. Thus, the Na+ efflux mediated by endogenous and transfected pumps could be separated by studying the cells at low (1 μM) and high (4 mM) ouabain concentrations. We found that the apparent Km for Na+ efflux attributable to the native human α1 isoform was 12 mM, which was similar to the Km of rat α1. The α2 and α3 isoforms had apparent Km's of 22 and 33 mM, respectively. The cells expressing α3 had a high resting [Na+]i. The maximal activity of native α1 in the α3-transfected cells was only ∼56% of native α1 activity in untransfected HeLa cells, suggesting that transfection with α3 led to a compensatory decrease in endogenous α1 pumps. We conclude that the apparent Km(Na+) for rat Na-K pump isoforms increases in the sequence α1 < α2 < α3. The α3 isoform may be suited for handling large Na+ loads in electrically active cells. PMID:9236212

  5. Full-field spatially incoherent illumination interferometry: a spatial resolution almost insensitive to aberrations.

    PubMed

    Xiao, Peng; Fink, Mathias; Boccara, A Claude

    2016-09-01

    We show that with spatially incoherent illumination, the point spread function (PSF) width/spatial resolution of an imaging interferometer like that used in full-field optical coherence tomography (OCT) is almost insensitive to aberrations. In these systems, aberrations mostly induce a reduction of the signal level that leads to a loss of the signal-to-noise ratio without broadening the system PSF. This is demonstrated by comparison with traditional scanning OCT and wide-field OCT with spatially coherent illuminations. Theoretical analysis and numerical calculation as well as experimental results are provided to show this specific merit of incoherent illumination in full-field OCT. To the best of our knowledge, this is the first time that such a result has been demonstrated. PMID:27607937

  6. Design of a polarization-insensitive superconducting nanowire single photon detector with high detection efficiency

    NASA Astrophysics Data System (ADS)

    Zheng, Fan; Xu, Ruiying; Zhu, Guanghao; Jin, Biaobing; Kang, Lin; Xu, Weiwei; Chen, Jian; Wu, Peiheng

    2016-03-01

    Superconducting nanowire single photon detectors (SNSPDs) deliver superior performance over their competitors in the near-infrared regime. However, these detectors have an intrinsic polarization dependence on the incident wave because of their one-dimensional meander structure. In this paper, we propose an approach to eliminate the polarization sensitivity of SNSPDs by using near-field optics to increase the absorption of SNSPDs under transverse magnetic (TM) illumination. In addition, an optical cavity is added to our SNSPD to obtain nearly perfect absorption of the incident wave. Numerical simulations show that the maximum absorption of a designed SNSPD can reach 96% at 1550 nm, and indicate that the absorption difference between transverse electric (TE) and TM polarization is less than 0.5% across a wavelength window of 300 nm. Our work provides the first demonstration of the possibility of designing a polarization-insensitive and highly efficient SNSPD without performing device symmetry improvements.

  7. Clinical features for diagnosis and management of patients with PRDM12 congenital insensitivity to pain

    PubMed Central

    Zhang, Stella; Malik Sharif, Saghira; Chen, Ya-Chun; Valente, Enza-Maria; Ahmed, Mushtaq; Sheridan, Eamonn; Bennett, Christopher; Woods, Geoffrey

    2016-01-01

    Background Congenital insensitivity to pain (CIP) is a rare extreme phenotype characterised by an inability to perceive pain present from birth due to lack of, or malfunction of, nociceptors. PRDM12 has recently been identified as a new gene that can cause CIP. The full phenotype and natural history have not yet been reported. Methods We have ascertained five adult patients and report their clinical features. Results Based on our findings, and those of previous patients, we describe the natural history of the PRDM12-CIP disorder, and derive diagnostic and management features to guide the clinical management of patients. Conclusions PRDM12-CIP is a distinct and diagnosable disorder, and requires specific clinical management to minimise predictable complications. PMID:26975306

  8. [Cyto B dependent and ouabain insensitive regulatory volume decrease in bicellular mouse embryo].

    PubMed

    Pogorelova, M A; Golichenkov, V A; Tarasov, A V; Pogorelova, V N; Panait, A I; Pogorelov, A G

    2012-01-01

    Mouse single-cell embryos exhibit robust Regulatory Volume Decrease (RVD). In what manner the very early mammalian embryo following zygote stage is appreciably altered by the anisotonic extracellular solution is, as yet, totally unclear. Little attention was paid to this direction since there was no way to determine the blastomere volume. This work has served to quantitatively investigate the osmotic response of bicellular mouse embryos employing Laser Scanning Microtomography (LSM) followed with three-dimensional reconstruction (3 DR). We have shown that bicellular mouse embryos in hypotonic Dulbecco's experience RVD. Embryonic cells subjected to hyposmolar exhibit rapid osmotic swelling followed by gradual shrinking back toward their original volume. The van't Hoff law defines swelling phase with the effective hydraulic conductivity of 0.3 micron x min(-1) x atm(-1). Water release during RVD in bicellular mouse embryos is abolished by Cytochalasin B (Cyto B) and the volume recovery is insensitive to ouabain treatment. PMID:22650075

  9. Congenital Insensitivity to Pain and Anhydrosis (CIPA) Syndrome; A Report of 4 Cases

    PubMed Central

    Daneshjou, Khadije; Jafarieh, Hanieh; Raaeskarami, Seyed-Reza

    2012-01-01

    Background Background: Congenital insensitivity to pain with anhidrosis (CIPA) is characterized by recurrent episodes of infections and unexplained fever, anhidrosis (inability to sweat), and absence of reaction to noxious stimuli, self-mutilating behavior, mental retardation and damages to oral structures. Case Presentation In this article, we have demonstrated the signs and symptoms of 4 children that refer to the pediatrics department of the Imam Khomeini hospital and assay about their complications with this disease. They mostly presented by recurrent osteomyelitis in their feet that severely controlled by antibiotic therapy and even surgery. They had no pain sensation in spite of deep sore and infection. Conclusion This syndrome can be diagnosed by clinical and paraclinical tests together but it would be better to confirm by genetic test. The diagnosis of this syndrome helps us to try for the better quality of life for the patients and avoid unnecessary amputations. PMID:23400697

  10. Effect of slow energy releasing on divergent detonation of Insensitive High Explosives

    NASA Astrophysics Data System (ADS)

    Hu, Xiaomian; Pan, Hao; Huang, Yong; Wu, Zihui

    2014-03-01

    There exists a slow energy releasing (SER) process in the slow reaction zone located behind the detonation wave due to the carbon cluster in the detonation products of Insensitive High Explosives (IHEs), and the process will affect the divergent detonation wave's propagation and the driving process of the explosives. To study the potential effect, a new artificial burn model including the SER process based on the programmed burn model is proposed in the paper. Quasi-steady analysis of the new model indicates that the nonlinearity of the detonation speed as a function of front curvature owes to the significant change of the reaction rate and the reaction zone length at the sonic state. What's more, in simulating the detonation of IHE JB-9014, the new model including the slow reaction can predict a slower jump-off velocity, in good agreement with the result of the test.

  11. Transmission and Reflection Terahertz Spectroscopy of Insensitive Melt-Cast High-Explosive Materials

    NASA Astrophysics Data System (ADS)

    Palka, Norbert; Szala, Mateusz

    2016-10-01

    Currently, artillery shells and grenades that are introduced into the market are based on melt-castable insensitive high explosives (IHEs), which do not explode while they run a risk of impact, heat or shrapnel. Particles of explosives (such as hexogen, nitroguanidine and nitrotriazolone) are suspended in different proportions in a matrix of 2.4-dinitroanisole. In this paper, we investigated samples of commonly used IHEs: PAX-41, IMX-104 and IMX-101, whose internal structures were determined by a scanning electron microscope. Terahertz time domain spectroscopy was applied in both transmission and reflection configurations. At first, the complex refraction indices of four pure constituents creating IHEs were determined and became the basis of further calculations. Next, the experimentally determined transmission and reflection spectra of IHEs and pure constituents were compared with theoretical considerations. The influence of the grain size of constituent material and scattering on the reflection spectra was analysed, and good agreement between the experimental and theoretical data was achieved.

  12. A no adhesive and temperature-insensitive package design of fiber Bragg grating pressure sensor

    NASA Astrophysics Data System (ADS)

    Wang, Hui; Zhu, Jun; Yin, Hao; Zhang, Zhao; Tang, Haiyu; Yu, Benli

    2012-11-01

    In this study, an innovative temperature-insensitive and no adhesive package for FBG pressure sensor was designed. We presents an package construction to compensate for temperature deviation of a FBG by mechanical properties of different metal materials; the package realize no adhesive utilizing combined technique of electroless plating NI-P and electroplating NI and laser spot welding technology. On the basis of the study on the Material structural properties and the compensation principles analysis of the FBG, the finite element method is used to analysis of the feasibility of the construction and optimize the construction parameters. The results show that this compensation construction can effectively reduce the temperature sensitivity of the FBG.

  13. Phase-insensitive storage of coherences by reversible mapping onto long-lived populations

    NASA Astrophysics Data System (ADS)

    Mieth, Simon; Genov, Genko T.; Yatsenko, Leonid P.; Vitanov, Nikolay V.; Halfmann, Thomas

    2016-01-01

    We theoretically develop and experimentally demonstrate a coherence population mapping (CPM) protocol to store atomic coherences in long-lived populations, enabling storage times far beyond the typically very short decoherence times of quantum systems. The amplitude and phase of an atomic coherence is written onto the populations of a three-state system by specifically designed sequences of radiation pulses from two coupling fields. As an important feature, the CPM sequences enable a retrieval efficiency, which is insensitive to the phase of the initial coherence. The information is preserved in every individual atom of the medium, enabling applications in purely homogeneously or inhomogeneously broadened ensembles even when stochastic phase jumps are the main source of decoherence. We experimentally confirm the theoretical predictions by applying CPM for storage of atomic coherences in a doped solid, reaching storage times in the regime of 1 min.

  14. Vibration-insensitive temperature sensing system based on fluorescence decay and using a digital processing approach

    NASA Astrophysics Data System (ADS)

    Dong, H.; Zhao, W.; Sun, T.; Grattan, K. T. V.; Al-Shamma'a, A. I.; Wei, C.; Mulrooney, J.; Clifford, J.; Fitzpatrick, C.; Lewis, E.; Degner, M.; Ewald, H.; Lochmann, S. I.; Bramann, G.; Merlone Borla, E.; Faraldi, P.; Pidria, M.

    2006-07-01

    A fluorescence-based temperature sensor system using a digital signal processing approach has been developed and evaluated in operation on a working automotive engine. The signal processing approach, using the least-squares method, makes the system relatively insensitive to intensity variations in the probe and thus provides more precise measurements when compared to a previous system designed using analogue phase-locked detection. Experiments carried out to determine the emission temperatures of a running car engine have demonstrated the effectiveness of the sensor system in monitoring exhaust temperatures up to 250 °C, and potentially higher. This paper was presented at the 13th International Conference on Sensors and Their Applications, held in Chatham, Kent, on 6-7 September 2005.

  15. Design of a polarization-insensitive superconducting nanowire single photon detector with high detection efficiency.

    PubMed

    Zheng, Fan; Xu, Ruiying; Zhu, Guanghao; Jin, Biaobing; Kang, Lin; Xu, Weiwei; Chen, Jian; Wu, Peiheng

    2016-01-01

    Superconducting nanowire single photon detectors (SNSPDs) deliver superior performance over their competitors in the near-infrared regime. However, these detectors have an intrinsic polarization dependence on the incident wave because of their one-dimensional meander structure. In this paper, we propose an approach to eliminate the polarization sensitivity of SNSPDs by using near-field optics to increase the absorption of SNSPDs under transverse magnetic (TM) illumination. In addition, an optical cavity is added to our SNSPD to obtain nearly perfect absorption of the incident wave. Numerical simulations show that the maximum absorption of a designed SNSPD can reach 96% at 1550 nm, and indicate that the absorption difference between transverse electric (TE) and TM polarization is less than 0.5% across a wavelength window of 300 nm. Our work provides the first demonstration of the possibility of designing a polarization-insensitive and highly efficient SNSPD without performing device symmetry improvements. PMID:26948672

  16. Broadband, polarization-insensitive and wide-angle terahertz metamaterial absorber

    NASA Astrophysics Data System (ADS)

    Wang, Ben-Xin; Wang, Ling-Ling; Wang, Gui-Zhen; Huang, Wei-Qing; Zhai, Xiang

    2014-11-01

    We present a broadband and polarization-insensitive terahertz metamaterial absorber formed from double-layered same-sized patterned square patches on top of a metallic ground plane. The combination of the two same-sized metallic patches gives rise to an absorption bandwidth of 380 GHz with absorption over 90%. The full width at half maximum of this device with respect to the central resonance frequency can be up to 33%, which is double that of a single-layered structure. The mechanism of the broadband absorber is attributed to longitudinal coupling between layers, and a hybridized charge distribution model is proposed for analyzing the origin of the resonance bandwidth. The proposed absorber has potential applications in detection, imaging and stealth technology.

  17. Ultra-broadband and polarization-insensitive wide-angle terahertz metamaterial absorber

    NASA Astrophysics Data System (ADS)

    Li, Xinwang; Liu, Hongjun; Sun, Qibing; Huang, Nan

    2015-06-01

    In this paper, an ultra-broadband and polarization insensitive terahertz (THz) metamaterial absorber is presented and investigated. With the optimization of the structural parameters, resonant peaks are merged into a broadband absorption spectrum. The simulation results demonstrate that a wide bandwidth of 3.1 THz is obtained in the range from 2.6 to 5.7 THz, where the absorption is higher than 90% for the normal incident THz waves. The full width at half maximum (FWHM) of absorption spectrum is 95% with respect to central frequency (∼4 THz), which is five times greater than the FWHM of a single layer structure. Furthermore, this structure can keep the absorption above 88% over a large frequency range (>2.5 THz) when the incident angle is smaller than 50°. This metamaterial absorber can find potential applications in terahertz imaging and stealth technology.

  18. Development of a Gravity-Insensitive Heat Pump for Lunar Applications

    NASA Technical Reports Server (NTRS)

    Cole, Gregory S.; Scaringe, Robert P.; Grzyll, Lawrence R.; Ewert, Michael K.

    2006-01-01

    Mainstream Engineering Corporation is developing a gravity-insensitive system that will allow a vapor-compression-cycle heat pump to be used in both microgravity (10(exp -6)g) and lunar (10(exp -6)g) environments. System capacity is 5 kW to 15 kW at design refrigerant operating conditions of 4.44 C and 60 C evaporating and condensing temperatures, respectively. The current program, performed for NASA Johnson Space Center (JSC) and presented in this paper, includes compressor performance analysis, detailed system design, and thermal analysis. Future efforts, including prototype fabrication, integration of a solar power source and controls, ground-testing, and flight-testing support, are also discussed.

  19. Enforced Layer-by-Layer Stacking of Energetic Salts towards High-Performance Insensitive Energetic Materials.

    PubMed

    Zhang, Jiaheng; Mitchell, Lauren A; Parrish, Damon A; Shreeve, Jean'ne M

    2015-08-26

    Development of modern high-performance insensitive energetic materials is significant because of the increasing demands for both military and civilian applications. Here we propose a rapid and facile strategy called the "layer hydrogen bonding pairing approach" to organize energetic molecules via layer-by-layer stacking, which grants access to tunable energetic materials with targeted properties. Using this strategy, an unusual energetic salt, hydroxylammonium 4-amino-furazan-3-yl-tetrazol-1-olate, with good detonation performances and excellent sensitivities, was designed, synthesized, and fully characterized. In addition, the expected unique layer-by-layer structure with a high crystal packing coefficient was confirmed by single-crystal X-ray crystallography. Calculations indicate that the layer-stacking structure of this material can absorb the mechanical stimuli-induced kinetic energy by converting it to layer sliding, which results in low sensitivity.

  20. Silicon-based broadband antenna for high responsivity and polarization-insensitive photodetection at telecommunication wavelengths.

    PubMed

    Lin, Keng-Te; Chen, Hsuen-Li; Lai, Yu-Sheng; Yu, Chen-Chieh

    2014-01-01

    Although the concept of using local surface plasmon resonance based nanoantenna for photodetection well below the semiconductor band edge has been demonstrated previously, the nature of local surface plasmon resonance based devices cannot meet many requirements of photodetection applications. Here we propose the concept of deep-trench/thin-metal (DTTM) active antenna that take advantage of surface plasmon resonance phenomena, three-dimensional cavity effects, and large-area metal/semiconductor junctions to effectively generate and collect hot electrons arising from plasmon decay and, thereby, increase photocurrent. The DTTM-based devices exhibited superior photoelectron conversion ability and high degrees of detection linearity under infrared light of both low and high intensity. Therefore, these DTTM-based devices have the attractive properties of high responsivity, extremely low power consumption, and polarization-insensitive detection over a broad bandwidth, suggesting great potential for use in photodetection and on-chip Si photonics in many applications of telecommunication fields.

  1. Psychosexual outcomes in three siblings with partial androgen insensitivity syndrome: impact of nature versus nurture.

    PubMed

    Joseph, Angela Ann; Shabir, Iram; Marumadi, Eunice; Dada, Reema; Ammini, Ariachery C; Mehta, Manju

    2013-01-01

    There are few reports of adults with disorders of sexual development (DSD). Here we describe the clinical profile and results of psychological assessment of three siblings with 46, XY DSD caused by partial androgen insensitivity syndrome (PAIS). The elder sibling (aged 22 years) was reared as female, while the middle and youngest siblings (17 and 18 years of age), were reared as males. The gender identity was concordant with the sex of rearing. There was no gender dysphoria. The psychological distress that our patients experienced was due to the limitations placed on them by their medical condition. It did not permit them to experience various facets of being either male or female completely. The younger siblings reared as males had additional problems of gynecomastia and lack of male secondary sexual development. PMID:23729553

  2. Buffer for a gamma-insensitive optical sensor with gas and a buffer assembly

    DOEpatents

    Kruger, Hans W.

    1994-01-01

    A buffer assembly for a gamma-insensitive gas avalanche focal plane array operating in the ultra-violet/visible/infrared energy wavelengths and using a photocathode and an avalanche gas located in a gap between an anode and the photocathode. The buffer assembly functions to eliminate chemical compatibility between the gas composition and the materials of the photocathode. The buffer assembly in the described embodiment is composed of two sections, a first section constructed of glass honeycomb under vacuum and a second section defining a thin barrier film or membrane constructed, for example, of Al and Be, which is attached to and supported by the honeycomb. The honeycomb section, in turn, is supported by and adjacent to the photocathode.

  3. Buffer for a gamma-insensitive optical sensor with gas and a buffer assembly

    DOEpatents

    Kruger, H.W.

    1994-05-10

    A buffer assembly is disclosed for a gamma-insensitive gas avalanche focal plane array operating in the ultra-violet/visible/infrared energy wavelengths and using a photocathode and an avalanche gas located in a gap between an anode and the photocathode. The buffer assembly functions to eliminate chemical compatibility between the gas composition and the materials of the photocathode. The buffer assembly in the described embodiment is composed of two sections, a first section constructed of glass honeycomb under vacuum and a second section defining a thin barrier film or membrane constructed, for example, of Al and Be, which is attached to and supported by the honeycomb. The honeycomb section, in turn, is supported by and adjacent to the photocathode. 7 figures.

  4. Coupled thermal/chemical/mechanical modeling of insensitive explosives in thermal environments

    SciTech Connect

    Nichols, A.L. III

    1996-05-01

    The ability to predict the response of a weapon system that contains insensitive explosives to elevated temperatures is important in understanding its safety characteristics. To model such a system at elevated temperatures in a finite element computer code requires a variety of capabilities. These modeling capabilities include thermal diffusion and convection to transport the heat to the explosives in the weapon system, temperature based chemical reaction modeling of the decomposition of the explosive materials, and mechanical modeling of both the metal casing and the unreacted and decomposed explosive. The Chemical TOPAZ code has been developed to model coupled thermal/chemical problems where we do not need to model the mass motion. We have also developed the LYNX2D code, based on PALM2D and Chemical TOPAZ, which is an implicit, two-dimensional coupled thermal/chemical/mechanical finite element model computer code. Some representative examples are shown. {copyright} {ital 1996 American Institute of Physics.}

  5. Degradation of high energetic and insensitive munitions compounds by Fe/Cu bimetal reduction.

    PubMed

    Koutsospyros, Agamemnon; Pavlov, Julius; Fawcett, Jacqueline; Strickland, David; Smolinski, Benjamin; Braida, Washington

    2012-06-15

    A reductive technology based on a completely mixed two-phase reactor (bimetallic particles and aqueous stream) was developed for the treatment of aqueous effluents contaminated with nitramines and nitro-substituted energetic materials. Experimental degradation studies were performed using solutions of three high energetics (RDX, HMX, TNT) and three insensitive-munitions components (NTO, NQ, DNAN). The study shows that, on laboratory scale, these energetic compounds are easily degraded in solution by suspensions of bimetallic particles (Fe/Ni and Fe/Cu) prepared by electro-less deposition. The type of bimetal pair (Fe/Cu or Fe/Ni) does not appear to affect the degradation kinetics of RDX, HMX, and TNT. The degradation of all components followed apparent first-order kinetics. The half-lives of all compounds except NTO were under 10 min. Additional parameters affecting the degradation processes were solids loading and initial pH.

  6. Outdoor dissolution of detonation residues of three insensitive munitions (IM) formulations.

    PubMed

    Taylor, Susan; Dontsova, Katerina; Walsh, Marianne E; Walsh, Michael R

    2015-09-01

    We seek to understand the environmental fate of three new insensitive munitions, explosive formulations developed to reduce the incidence of unintended detonations. To this end, we measured the size distribution of residues from low order detonations of IMX 101, IMX 104, and PAX 21-filled munitions and are studying how these three formulations weather and dissolve outdoors. The largest pieces collected from the detonations were centimeter-sized and we studied 12 of these in the outdoors test. We found that the particles break easily and that the dissolution of 2,4-dinitroanisole (DNAN) is quasi-linear as a function of water volume. DNAN is the matrix and the least soluble major constituent of the three formulations. We used DNAN's linear dissolution rate to estimate the life span of the pieces. Particles ranging in mass from 0.3 to 3.5 g will completely dissolve in 3-21 years given 100 cm y(-1) precipitation rates.

  7. Multiphysics Simulations of Hot-Spot Initiation in Shocked Insensitive High-Explosive

    NASA Astrophysics Data System (ADS)

    Najjar, Fady; Howard, W. M.; Fried, L. E.

    2010-11-01

    Solid plastic-bonded high-explosive materials consist of crystals with micron-sized pores embedded. Under mechanical or thermal insults, these voids increase the ease of shock initiation by generating high-temperature regions during their collapse that might lead to ignition. Understanding the mechanisms of hot-spot initiation has significant research interest due to safety, reliability and development of new insensitive munitions. Multi-dimensional high-resolution meso-scale simulations are performed using the multiphysics software, ALE3D, to understand the hot-spot initiation. The Cheetah code is coupled to ALE3D, creating multi-dimensional sparse tables for the HE properties. The reaction rates were obtained from MD Quantum computations. Our current predictions showcase several interesting features regarding hot spot dynamics including the formation of a "secondary" jet. We will discuss the results obtained with hydro-thermo-chemical processes leading to ignition growth for various pore sizes and different shock pressures.

  8. Physicochemical properties of an insensitive munitions compound, N-methyl-4-nitroaniline (MNA).

    PubMed

    Boddu, Veera M; Abburi, Krishnaiah; Maloney, Stephen W; Damavarapu, Reddy

    2008-06-30

    Accurate information on physicochemical properties of an organic contaminant is essential for predicting its environmental impact and fate. These properties also provide invaluable information for the overall understanding of environmental distribution, biotransformation, and potential treatment processes. In this study the aqueous solubility (Sw), octanol-water partition coefficient (Kow), and Henry's law constant (K(H)) were determined for an insensitive munitions (IM) compound, N-methyl-4-nitroaniline (MNA), at 298.15, 308.15, and 318.15 K. Effect of ionic strength on solubility, using electrolytes such as NaCl and CaCl2, was also studied. The data on the physicochemical parameters were correlated using the standard Van't Hoff equation. All three properties exhibited a linear relationship with reciprocal temperature. The enthalpy and entropy of phase transfer were derived from the experimental data.

  9. Humidity insensitive step-index polymer optical fibre Bragg grating sensors

    NASA Astrophysics Data System (ADS)

    Woyessa, G.; Fasano, A.; Stefani, A.; Markos, C.; Nielsen, K.; Rasmussen, H. K.; Bang, O.

    2015-09-01

    We have fabricated and characterised a humidity insensitive step index(SI) polymer optical fibre(POF) Bragg grating sensors. The fibre was made based on the injection molding technique, which is an efficient method for fast, flexible and cost effective preparation of the fibre preform. The fabricated SIPOF has a core made from TOPAS with a glass transition temperature of 134°C and a cladding from ZEONEX with a glass transition temperature of 138°C. The main advantages of the proposed SIPOF are the low water absorption and good chemical resistance compared to the conventional poly-methyl-methacrylate (PMMA) based SIPOFs. The fibre has a minimum loss of ~6dB/m at 770nm.

  10. Design of a polarization-insensitive superconducting nanowire single photon detector with high detection efficiency

    PubMed Central

    Zheng, Fan; Xu, Ruiying; Zhu, Guanghao; Jin, Biaobing; Kang, Lin; Xu, Weiwei; Chen, Jian; Wu, Peiheng

    2016-01-01

    Superconducting nanowire single photon detectors (SNSPDs) deliver superior performance over their competitors in the near-infrared regime. However, these detectors have an intrinsic polarization dependence on the incident wave because of their one-dimensional meander structure. In this paper, we propose an approach to eliminate the polarization sensitivity of SNSPDs by using near-field optics to increase the absorption of SNSPDs under transverse magnetic (TM) illumination. In addition, an optical cavity is added to our SNSPD to obtain nearly perfect absorption of the incident wave. Numerical simulations show that the maximum absorption of a designed SNSPD can reach 96% at 1550 nm, and indicate that the absorption difference between transverse electric (TE) and TM polarization is less than 0.5% across a wavelength window of 300 nm. Our work provides the first demonstration of the possibility of designing a polarization-insensitive and highly efficient SNSPD without performing device symmetry improvements. PMID:26948672

  11. Psychosexual outcomes in three siblings with partial androgen insensitivity syndrome: impact of nature versus nurture.

    PubMed

    Joseph, Angela Ann; Shabir, Iram; Marumadi, Eunice; Dada, Reema; Ammini, Ariachery C; Mehta, Manju

    2013-01-01

    There are few reports of adults with disorders of sexual development (DSD). Here we describe the clinical profile and results of psychological assessment of three siblings with 46, XY DSD caused by partial androgen insensitivity syndrome (PAIS). The elder sibling (aged 22 years) was reared as female, while the middle and youngest siblings (17 and 18 years of age), were reared as males. The gender identity was concordant with the sex of rearing. There was no gender dysphoria. The psychological distress that our patients experienced was due to the limitations placed on them by their medical condition. It did not permit them to experience various facets of being either male or female completely. The younger siblings reared as males had additional problems of gynecomastia and lack of male secondary sexual development.

  12. Design of polarization insensitive filters with micro- and nano-grating structures

    NASA Astrophysics Data System (ADS)

    Wang, Wen-liang; Rong, Xiao-hong

    2014-03-01

    For isotropic dielectric thin films, polarization effect is an inherent characteristic. As it will make the performance of optical-electric system go to bad, such polarization-dependent properties are often intolerable and should be eliminated in many applications. In this paper, based on a micro- and nano-optical structure whose period consists of four parts, a polarization insensitive filter is obtained by combining rigorous wave theory and multi-objective immune optimization algorithm. Its working wavelength is 1315 nm which is often used in laser systems. The results of our design show that TE and TM polarized waves have reflectivities of 0.482 and 0.485, respectively at designed wavelength of 1315 nm. And it denotes that two values are both close to the design values, their difference is only 0.003, and polarization deviation is also very little. Therefore, the designed filter can eliminate the effect of polarization deviation very well at 1315 nm wavelength.

  13. Complete androgen insensitivity syndrome due to X chromosome inversion: a clinical report.

    PubMed

    Xu, Weizhen; Robert, Claudia; Thornton, Paul S; Spinner, Nancy B

    2003-07-30

    We have studied a patient with complete androgen insensitivity syndrome (CAIS) and a 46, inv(X),Y karyotype. The patient's mother and maternal aunt also carry the inverted X, and the mother is phenotypically normal, with a 46, inv(X),X karyotype, while a maternal aunt has CAIS with a 46,inv(X),Y karyotype. Molecular cytogenetic studies demonstrate that one of the X inversion breakpoints occurs within Xq11.2-12, which is the locus for the androgen receptor. FISH analysis demonstrated that a BAC clone containing the androgen receptor gene was disrupted by the inversion. We therefore hypothesize that disruption of the androgen receptor gene causes CAIS in this patient. This is the first report of CAIS caused by a chromosome inversion.

  14. The predictability of bleeding by prothrombin times sensitive or insensitive to PIVKA during intensive oral anticoagulation.

    PubMed

    Arnesen, H; Smith, P

    1991-02-01

    To evaluate the effect of PIVKA (Proteins Induced by Vitamin K Absence or Antagonism) on the bleeding tendency during oral anticoagulation, we studied consecutive patients intensively treated with warfarin (INR greater than 4.8). The level of anticoagulation was measured with the PIVKA-insensitive Normotest (NT) as well as with the PIVKA-sensitive Thrombotest (TT), and the results are expressed as per cent coagulant activity. The NT/TT ratio was determined. Twenty patients with bleeding episodes had a mean NT/TT ratio of 2.06 as compared to 2.20 in 143 patients without bleeding episodes (p = 0.08). As the NT/TT ratio was not higher in patients with bleedings, we conclude that PIVKA are of no importance for bleeding during anticoagulation with vitamin K antagonists.

  15. TOR-inhibitor insensitive-1 (TRIN1) regulates cotyledons greening in Arabidopsis.

    PubMed

    Li, Linxuan; Song, Yun; Wang, Kai; Dong, Pan; Zhang, Xueyan; Li, Fuguang; Li, Zhengguo; Ren, Maozhi

    2015-01-01

    Target of Rapamycin (TOR) is an eukaryotic protein kinase and evolutionally conserved from the last eukaryotic common ancestor (LECA) to humans. The growing evidences have shown that TOR signaling acts as a central controller of cell growth and development. The downstream effectors of TOR have been well-identified in yeast and animals by using the immunosuppression agent rapamycin. However, less is known about TOR in plants. This is largely due to the fact that plants are insensitive to rapamycin. In this study, AZD8055 (AZD), the novel ATP-competitive inhibitor of TOR, was employed to decipher the downstream effectors of TOR in Arabidopsis. One AZD insensitive mutant, T O R - i nhibitor i n sensitive- 1 (trin1), was screened from 10,000 EMS-induced mutation seeds. The cotyledons of trin1 can turn green when its seeds were germinated on ½ MS medium supplemented with 2 μM AZD, whereas the cotyledons greening of wild-type (WT) can be completely blocked at this concentration. Through genetic mapping, TRIN1 was mapped onto the long arm of chromosome 2, between markers SGCSNP26 and MI277. Positional cloning revealed that TRIN1 was an allele of ABI4, which encoded an ABA-regulated AP2 domain transcription factor. Plants containing P35S::TRIN1 or P35S::TRIN1-GUS were hypersensitive to AZD treatment and displayed the opposite phenotype observed in trin1. Importantly, GUS signaling was significantly enhanced in P35S::TRIN1-GUS transgenic plants in response to AZD treatment, indicating that suppression of TOR resulted in the accumulation of TRIN1. These observations revealed that TOR controlled seed-to-seedling transition by negatively regulating the stability of TRIN1 in Arabidopsis. For the first time, TRIN1, the downstream effector of TOR signaling, was identified through a chemical genetics approach.

  16. Inositol metabolism in WRK-1 cells. Relationship of hormone-sensitive to -insensitive pools of phosphoinositides

    SciTech Connect

    Monaco, M.E.

    1987-09-25

    Previous studies have indicated the existence of two separate pools of phosphoinositides in WRK-1 cells; one is labile and hormone-sensitive with respect to turnover, while the other is stable. Hormonal stimulation results in a rapid increase in /sup 32/Pi incorporation into the sensitive pool, while in the absence of hormone, incorporation of /sup 32/Pi into this pool is slow. Results are quite different when (/sup 3/H)inositol is the precursor utilized. Incorporation of (/sup 3/H)inositol into hormone-sensitive phosphoinositides is not stimulated in the presence of hormone, suggesting entry of this exogenous precursor into the cycle by a route other than the resynthetic phase of the cycle. Furthermore, failure of hormone to induce loss of (/sup 3/H)phosphoinositide in pulse-chase experiments in the absence of lithium suggests reutilization of the (/sup 3/H)inositol moiety generated by phosphodiesteratic cleavage of hormone-sensitive phosphoinositide. Time course studies indicate that the relative rates of incorporation of (/sup 3/H)inositol into sensitive and insensitive phosphoinositide remain constant from 2 to 24 h. Several factors are capable of increasing (/sup 3/H)inositol incorporation into hormone-insensitive phosphoinositide including vasopressin, calcium ionophores, and manganese. On the other hand, vasopressin treatment appears to decrease incorporation of (/sup 3/H)inositol into the hormone-sensitive pool, probably by shifting the equilibrium between phosphoinositides and inositol phosphates, since the decrease in radioactivity observed in the phosphoinositides is equaled by the increase observed in that in the inositol phosphates.

  17. Mutation of Oryza sativa CORONATINE INSENSITIVE 1b (OsCOI1b) delays leaf senescence.

    PubMed

    Lee, Sang-Hwa; Sakuraba, Yasuhito; Lee, Taeyoung; Kim, Kyu-Won; An, Gynheung; Lee, Han Yong; Paek, Nam-Chon

    2015-06-01

    Jasmonic acid (JA) functions in plant development, including senescence and immunity. Arabidopsis thaliana CORONATINE INSENSITIVE 1 encodes a JA receptor and functions in the JA-responsive signaling pathway. The Arabidopsis genome harbors a single COI gene, but the rice (Oryza sativa) genome harbors three COI homologs, OsCOI1a, OsCOI1b, and OsCOI2. Thus, it remains unclear whether each OsCOI has distinct, additive, synergistic, or redundant functions in development. Here, we use the oscoi1b-1 knockout mutants to show that OsCOI1b mainly affects leaf senescence under senescence-promoting conditions. oscoi1b-1 mutants stayed green during dark-induced and natural senescence, with substantial retention of chlorophylls and photosynthetic capacity. Furthermore, several senescence-associated genes were downregulated in oscoi1b-1 mutants, including homologs of Arabidopsis thaliana ETHYLENE INSENSITIVE 3 and ORESARA 1, important regulators of leaf senescence. These results suggest that crosstalk between JA signaling and ethylene signaling affects leaf senescence. The Arabidopsis coi1-1 plants containing 35S:OsCOI1a or 35S:OsCOI1b rescued the delayed leaf senescence during dark incubation, suggesting that both OsCOI1a and OsCOI1b are required for promoting leaf senescence in rice. oscoi1b-1 mutants showed significant decreases in spikelet fertility and grain weight, leading to severe reduction of grain yield, indicating that OsCOI1-mediated JA signaling affects spikelet fertility and grain filling.

  18. Ethanol modulates the VR-1 variant amiloride-insensitive salt taste receptor. I. Effect on TRC volume and Na+ flux.

    PubMed

    Lyall, Vijay; Heck, Gerard L; Phan, Tam-Hao T; Mummalaneni, Shobha; Malik, Shahbaz A; Vinnikova, Anna K; DeSimone, John A

    2005-06-01

    The effect of ethanol on the amiloride- and benzamil (Bz)-insensitive salt taste receptor was investigated by the measurement of intracellular Na(+) activity ([Na(+)](i)) in polarized rat fungiform taste receptor cells (TRCs) using fluorescence imaging and by chorda tympani (CT) taste nerve recordings. CT responses were monitored during lingual stimulation with ethanol solutions containing NaCl or KCl. CT responses were recorded in the presence of Bz (a specific blocker of the epithelial Na(+) channel [ENaC]) or the vanilloid receptor-1 (VR-1) antagonists capsazepine or SB-366791, which also block the Bz-insensitive salt taste receptor, a VR-1 variant. CT responses were recorded at 23 degrees C or 42 degrees C (a temperature at which the VR-1 variant salt taste receptor activity is maximally enhanced). In the absence of permeable cations, ethanol induced a transient decrease in TRC volume, and stimulating the tongue with ethanol solutions without added salt elicited only transient phasic CT responses that were insensitive to elevated temperature or SB-366791. Preshrinking TRCs in vivo with hypertonic mannitol (0.5 M) attenuated the magnitude of the phasic CT response, indicating that in the absence of mineral salts, transient phasic CT responses are related to the ethanol-induced osmotic shrinkage of TRCs. In the presence of mineral salts, ethanol increased the Bz-insensitive apical cation flux in TRCs without a change in cell volume, increased transepithelial electrical resistance across the tongue, and elicited CT responses that were similar to salt responses, consisting of both a transient phasic component and a sustained tonic component. Ethanol increased the Bz-insensitive NaCl CT response. This effect was further enhanced by elevating the temperature from 23 degrees C to 42 degrees C, and was blocked by SB-366791. We conclude that in the presence of mineral salts, ethanol modulates the Bz-insensitive VR-1 variant salt taste receptor. PMID:15928403

  19. The viral RNA-based transfection of enhanced green fluorescent protein (EGFP) in the parasitic protozoan Trichomonas vaginalis.

    PubMed

    Li, Wei; Ding, He; Zhang, Xinxin; Cao, Lili; Li, Jianhua; Gong, Pengtao; Li, He; Zhang, Guocai; Li, Shuhong; Zhang, Xichen

    2012-03-01

    Here we have developed methods to transiently and stably transfect the human pathogenic protist Trichomonas vaginalis. The viral RNA-based transfection vector pTVV-EGFP/NEO was constructed by using enhanced green fluorescent protein gene (EGFP) and neomycin resistance gene (NEO) in tandem to replace the whole gene encoding region of T. vaginalis virus (TVV). The in vitro transcripts of linearized pTVV-EGFP/NEO were electroporated into trophozoites and the transfectants transiently expressed EGFP after 16 h postincubation. Stable expression of EGFP was persistently detected by fluorescence microscopy and by RT-PCR in transfected trophozoites under G418 selection. Our study provides a novel and valuable approach for genetic study of T. vaginalis.

  20. Class I (H-2Kb) gene transfection reduces susceptibility of YAC-1 lymphoma targets to natural killer cells.

    PubMed

    Carlow, D A; Payne, U; Hozumi, N; Roder, J C; Czitrom, A A

    1990-04-01

    A "hybrid gene" (MTKb) comprised of the human metallothionein IIA promoter ligated to the genomic sequence of the major histocompatibility complex class I (H-2Kb) gene was subcloned into the expression vector pSV2neo and transfected into the natural killer (NK) cell-sensitive YAC-1 lymphoma. The Kb gene product was readily detectable on the cell surface of G418-resistant transfectants using both Kb-specific monoclonal antibodies and H-2b-specific cytolytic T cells. Unlike control pSV2neo transfectants, MTKb-pSV2neo transfectants were relatively resistant to lysis by NK cells from H-2a, H-2b, H-2k or H-2 (a x b)F1 haplotype mice. These data strongly suggest that the effects of MHC expression on susceptibility to NK cells can be mediated by a single and well-defined class I molecule, Kb.

  1. The viral RNA-based transfection of enhanced green fluorescent protein (EGFP) in the parasitic protozoan Trichomonas vaginalis.

    PubMed

    Li, Wei; Ding, He; Zhang, Xinxin; Cao, Lili; Li, Jianhua; Gong, Pengtao; Li, He; Zhang, Guocai; Li, Shuhong; Zhang, Xichen

    2012-03-01

    Here we have developed methods to transiently and stably transfect the human pathogenic protist Trichomonas vaginalis. The viral RNA-based transfection vector pTVV-EGFP/NEO was constructed by using enhanced green fluorescent protein gene (EGFP) and neomycin resistance gene (NEO) in tandem to replace the whole gene encoding region of T. vaginalis virus (TVV). The in vitro transcripts of linearized pTVV-EGFP/NEO were electroporated into trophozoites and the transfectants transiently expressed EGFP after 16 h postincubation. Stable expression of EGFP was persistently detected by fluorescence microscopy and by RT-PCR in transfected trophozoites under G418 selection. Our study provides a novel and valuable approach for genetic study of T. vaginalis. PMID:21861063

  2. 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.

  3. Contrasting signaling pathways of alpha1A- and alpha1B-adrenergic receptor subtype activation of phosphatidylinositol 3-kinase and Ras in transfected NIH3T3 cells.

    PubMed

    Hu, Z W; Shi, X Y; Lin, R Z; Hoffman, B B

    1999-01-01

    Activation of protein kinases is an important intermediate step in signaling pathways of many G protein-coupled receptors including alpha1-adrenergic receptors. The present study was designed to investigate the capacity of the three cloned subtypes of human alpha1-receptors, namely, alpha1A, alpha1B and alpha1D to activate phosphatidylinositol 3-kinase (PI 3-kinase) and p21ras in transfected NIH3T3 cells. Norepinephrine activated PI 3-kinase in cells expressing human alpha1A and alpha1B via pertussis toxin-insensitive G proteins; alpha1D-receptors did not detectably activate this kinase. Transient transfection of NIH 3T3 cells with the alpha-subunit of the G protein transducin (alpha(t)) a scavenger of betagamma-subunits released from activated G proteins, inhibited alpha1B-receptor but not alpha1A-receptor-stimulated PI 3-kinase activity. Stimulation of both alpha1A- and alpha1B-receptors activated p21ras and stimulated guanine nucleotide exchange on Ras protein. Overexpression of a dominant negative mutant of p21ras attenuated alpha1B-receptor but not alpha1A-receptor activation of PI 3-kinase. Overexpression of a dominant negative mutant of PI 3-kinase attenuated alpha1A- but not alpha1B-receptor-stimulated mitogen-activated protein kinase activity. These results demonstrate the capacity for heterologous signaling of the alpha1-adrenergic receptor subtypes in promoting cellular responses in NIH3T3 cells.

  4. 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.

  5. 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.

  6. 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.

  7. Selection and optimization of transfection enhancer additives for increased virus-like particle production in HEK293 suspension cell cultures.

    PubMed

    Cervera, Laura; Fuenmayor, Javier; González-Domínguez, Irene; Gutiérrez-Granados, Sonia; Segura, Maria Mercedes; Gòdia, Francesc

    2015-12-01

    The manufacturing of biopharmaceuticals in mammalian cells typically relies on the use of stable producer cell lines. However, in recent years, transient gene expression has emerged as a suitable technology for rapid production of biopharmaceuticals. Transient gene expression is particularly well suited for early developmental phases, where several potential therapeutic targets need to be produced and tested in vivo. As a relatively new bioprocessing modality, a number of opportunities exist for improving cell culture productivity upon transient transfection. For instance, several compounds have shown positive effects on transient gene expression. These transfection enhancers either facilitate entry of PEI/DNA transfection complexes into the cell or nucleus or increase levels of gene expression. In this work, the potential of combining transfection enhancers to increase Gag-based virus-like particle production levels upon transfection of suspension-growing HEK 293 cells is evaluated. Using Plackett-Burman design of experiments, it is first tested the effect of eight transfection enhancers: trichostatin A, valproic acid, sodium butyrate, dimethyl sulfoxide (DMSO), lithium acetate, caffeine, hydroxyurea, and nocodazole. An optimal combination of compounds exhibiting the highest effect on gene expression levels was subsequently identified using a surface response experimental design. The optimal consisted on the addition of 20 mM lithium acetate, 3.36 mM valproic acid, and 5.04 mM caffeine which increased VLP production levels 3.8-fold, while maintaining cell culture viability at 94%. PMID:26278533

  8. 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

  9. 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.

  10. Quantitative study of effects of free cationic chains on gene transfection in different intracellular stages.

    PubMed

    Cai, Jinge; Yue, Yanan; Wang, Yanjing; Jin, Zhenyu; Jin, Fan; Wu, Chi

    2016-09-28

    Previously, we revealed that in the application of using cationic polymer chains, polyethylenimine (PEI), to condense anionic plasmid DNA chains (pDNA) to form the DNA/polymer polyplexes, after all the pDNAs are complexed with PEI, further added PEIs exist individual chains and free in the solution mixture. It is those uncomplexed polycation chains that dramatically promote the gene transfection. In the current study, we studied how those free cationic chains with different lengths and topologies affect the intracellular trafficking of the polyplexes, the translocation of pDNA through the nuclear membrane, the transcription of pDNA to mRNA and the translocation of mRNA from nucleus to cytosol in HepG2 cells by using a combination of the three-dimensional confocal microscope and TaqMan real-time PCR. We found that free branched PEI chains with a molar mass of 25,000g/mol and a total concentration of 1.8×10(-6)g/mL promote the overall gene transfection efficiency by a factor of ~500 times. Our results quantitatively reveal that free chains help little in the cellular uptake, but clearly reduce the lysosomal entrapment of those internalized polyplexes (2-3 folds); assist the translocation of pDNA through nuclear membrane after it is released from the polyplexes in the cytosol (~5 folds); enhance the pDNA-to-mRNA transcription efficiency (~4 folds); and facilitate the nucleus-to-cytosol translocation of mRNA (7-8 folds). The total enhancement of those steps agrees well with the overall efficiency, demonstrating, for the first time, how free cationic polymer chains quantitatively promote the gene transfection in each step in the intracellular space. PMID:27448443

  11. Tumor priming enhances siRNA delivery and transfection in intraperitoneal tumors.

    PubMed

    Wang, Jie; Lu, Ze; Yeung, Bertrand Z; Wientjes, M Guillaume; Cole, David J; Au, Jessie L-S

    2014-03-28

    Cancers originating from the digestive system account for 290,000 or ~20% of all new cancer cases annually in the US. We previously developed paclitaxel-loaded tumor-penetrating microparticles (TPM) for intraperitoneal (IP) treatment of peritoneal tumors (Lu et al., 2008; Tsai et al., 2007; Tsai et al., 2013). TPM is undergoing NIH-supported IND-enabling studies for clinical evaluation. The present study evaluated the hypothesis that TPM, via inducing apoptosis and expanding the interstitial space, promotes the delivery and transfection of lipid vectors containing siRNA. The in vivo model was the metastatic human Hs766T pancreatic tumor that, upon IP injection, produced widely distributed solid tumors and ascites in the peritoneal cavity in 100% of animals. The target gene was survivin, an anti-apoptotic protein induced by chemotherapy and associated with metastases and poor prognosis of patients with gastric and colorectal cancers. The siRNA carrier was pegylated liposomes comprising cationic and neutral lipids plus a fusogenic lipid (PCat). PCat-loaded with survivin siRNA (PCat-siSurvivin) was active in cultured cells (decreased survivin mRNA and protein levels, reduced cell clonogenicity, enhanced paclitaxel activity), but lost its activity in vivo; this difference is consistent with the well-known problem of inadequate delivery and transfection of siRNA in vivo. In comparison, single agent TPM prolonged animal survival and, as expected, induced survivin expression in tumors. Addition of PCat-siSurvivin reversed the TPM-induced survivin expression and enhanced the antitumor activity of TPM. The finding that in vivo survivin knockdown by PCat-siSurvivin was successful only when it was given in combination with TPM provides the proof-of-concept that tumor priming promotes the delivery and transfection of liposomal siRNA. The data further suggest the TPM/PCat-siSurvivin combination as a potentially useful chemo-gene therapy for peritoneal cancer.

  12. Cell penetrating peptide-based polyplexes shelled with polysaccharide to improve stability and gene transfection.

    PubMed

    Li, Wenyu; Liu, Yajie; Du, Jianwei; Ren, Kefeng; Wang, Youxiang

    2015-05-14

    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. PMID:25893559

  13. 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. PMID:24173879

  14. Optimized heterologous transfection of viable adult organotypic brain slices using an enhanced gene gun

    PubMed Central

    2013-01-01

    Background Organotypic brain slices (OTBS) are an excellent experimental compromise between the facility of working with cell cultures and the biological relevance of using animal models where anatomical, morphological, and cellular function of specific brain regions can be maintained. The biological characteristics of OTBS can subsequently be examined under well-defined conditions. They do, however, have a number of limitations; most brain slices are derived from neonatal animals, as it is difficult to properly prepare and maintain adult OTBS. There are ample problems with tissue integrity as OTBS are delicate and frequently become damaged during the preparative stages. Notwithstanding these obstacles, the introduced exogenous proteins into both neuronal cells, and cells imbedded within tissues, have been consistently difficult to achieve. Results Following the ex vivo extraction of adult mouse brains, mounted inside a medium-agarose matrix, we have exploited a precise slicing procedure using a custom built vibroslicer. To transfect these slices we used an improved biolistic transfection method using a custom made low-pressure barrel and novel DNA-coated nanoparticles (40 nm), which are drastically smaller than traditional microparticles. These nanoparticles also minimize tissue damage as seen by a significant reduction in lactate dehydrogenase activity as well as propidium iodide (PI) and dUTP labelling compared to larger traditional gold particles used on these OTBS. Furthermore, following EYFP exogene delivery by gene gun, the 40 nm treated OTBS displayed a significantly larger number of viable NeuN and EYFP positive cells. These OTBS expressed the exogenous proteins for many weeks. Conclusions Our described methodology of producing OTBS, which results in better reproducibility with less tissue damage, permits the exploitation of mature fully formed adult brains for advanced neurobiological studies. The novel 40 nm particles are ideal for the viable

  15. Single cell transfection by laser-induced breakdown of an optically trapped gold nanoparticle

    NASA Astrophysics Data System (ADS)

    Arita, Yoshihiko; Ploschner, Martin; Antkowiak, Maciej; Gunn-Moore, Frank; Dholakia, Kishan

    2014-03-01

    Cell selective introduction of therapeutic agents remains a challenging problem. Cavitation-based therapies including ultrasound-induced sonoporation and laser-induced optoporation have led the way for novel approaches to provide the potential of sterility and cell selectivity compared with viral or biochemical counterparts. Acoustic streaming, shockwaves and liquid microjets associated with the cavitation dynamics are implicated in gene and drug delivery. These approaches, however, often lead to non-uniform and sporadic molecular uptake that lacks refined spatial control and suffers from a significant loss of cell viability. Here we demonstrate spatially controlled cavitation instigated by laser-induced breakdown of an optically trapped single gold nanoparticle. Our unique approach employs optical tweezers to trap a single nanoparticle, which when irradiated by a nanosecond laser pulse is subject to laser-induced breakdown followed by cavitation. Using this method for laser-induced cavitation, we can gain additional degrees of freedom for the cavitation process - the particle material, its size, and its position relative to cells or tissues. We show the energy breakdown threshold of gold nanoparticles of l00nm with a single nanosecond laser pulse at 532 nm is three orders of magnitude lower than that for water, which leads to gentle nanocavitation enabling single cell transfection. We optimize the shear stress to the cells from the expanding bubble to be in the range of 1-10 kPa for transfection by precisely positioning a trapped gold nanoparticle, and thus nanobubble, relative to a cell of interest. The method shows transfection of plasmid-DNA into individual mammalian cells with an efficiency of 75%.

  16. Characterization of 2,6-Diamino-3,5-Dinitropyrazine-1-Oxide (LLM-105) as an Insensitive High Explosive Material

    SciTech Connect

    Tran, T D; Pagoria, P F; Hoffman, D M; Cutting, J L; Lee, R S; Simpson, R L

    2002-04-09

    LLM-105 (2,6-diamino-3,5-dinitropyrazine-1-oxide) is a new molecule which has performance and insensitivity between those of HMX and TATB. Its calculated energy content is about 85% that of HMX and 15% more than that of TATB. It is thermally stable, insensitive to shock, spark and friction and has impact insensitivity level approaching that of TATB. These combined properties make it a realistic high-performance IHE material, attractive for applications that require moderate performance and insensitivity. Several morphologies of LLM-105 and plastic-bonded formulations containing these materials and another binder were prepared and characterized. Their physical properties and detonation spreading characteristics are compared to those of ultrafine TATB. The impact sensitivity (drop hammer results) is sensitive to particle morphologies. Detonation-spreading, spot-size tests on LLM-105 compositions showed higher energy output and superior divergence behavior than is observed for ultrafine TATB. The small-scale safety data, pressing characteristics and results from divergence experiments will be summarized.

  17. Stomatal behavior of ozone-sensitive and -insensitive coneflowers (Rudbeckia laciniata var. digitata) in Great Smoky Mountains National Park.

    PubMed

    Grulke, N E; Neufeld, H S; Davison, A W; Roberts, M; Chappelka, A H

    2007-01-01

    * Morphological and physiological attributes were assessed to elucidate the underlying mechanisms of ozone (O(3)) sensitivity in a highly sensitive species, cutleaf coneflower (Rudbeckia laciniata var. digitata). * Foliage at the same height in the canopy on paired O(3)-sensitive and -insensitive cutleaf coneflowers was assessed for level of foliar symptoms, stomatal density, stomatal responsiveness to dynamic changes in light and leaf-to-air vapor pressure deficit (VPD), steady-state responses to light and CO(2), intrinsic transpirational efficiency, and plant water balance. * There were no morphological differences between the sensitivity types that might have contributed to greater O(3) uptake in sensitive individuals. Stomata of sensitive plants were less responsive than those of insensitive plants to experimentally increased and decreased light intensities, and to increased VPD. O(3)-insensitive plants had greater intrinsic transpirational efficiencies, greater maximum assimilation rates under saturating CO(2) and light, and greater carboxylation rates. * Different physiological attributes vary independently within an individual plant, which collectively confer sensitivity or insensitivity to O(3) injury. PMID:17176397

  18. Wide-angle, polarization-insensitive and broadband absorber based on eight-fold symmetric SRRs metamaterial

    NASA Astrophysics Data System (ADS)

    Wu, Dong; Liu, Yumin; Yu, Zhongyuan; Chen, Lei; Ma, Rui; Li, Yutong; Li, Ruifang; Ye, Han

    2016-12-01

    In this paper, we propose a novel three dimensional metamaterial design with eight-fold rotational symmetry that shows a polarization-insensitive, wide-angle and broadband perfect absorption in the microwave band. By simulation, the polarization-insensitive absorption is over 90% between 26.9 GHz to 32.9 GHz, and the broadband absorption remains a good absorption performance to a wide incident angles for both TE and TM polarizations. The magnetic field distribution are investigated to interpret the physical mechanism of broadband absorption. The broadband absorption is based on overlapping the multiple magnetic resonances at the neighboring frequencies by coupling effects of multiple metallic split-ring resonators (SRRs). Moreover, it is demonstrate that the designed structure can be extended to other frequencies by scale down the size of the unit cell, such as the visible frequencies. The simulated results show that the absorption of the smaller absorber is above 90% in the frequency range from 467 THz to 765 THz(392-642 nm), which include orange to purple light in visible region(400-760nm). The wide-angle and polarization-insensitive stabilities of the smaller absorber is also demonstrated at visible region. The proposed work provides a new design of realization of a polarization-insensitive, wide-angle and broadband absorber ranging different frequency bands, and such a structure has potential application in the fields of solar cell, imaging and detection.

  19. Stabilized plasmid-lipid particles for regional gene therapy: formulation and transfection properties.

    PubMed

    Zhang, Y P; Sekirov, L; Saravolac, E G; Wheeler, J J; Tardi, P; Clow, K; Leng, E; Sun, R; Cullis, P R; Scherrer, P

    1999-08-01

    Previous work (Wheeler et al, Gene Therapy 1999; 6: 271-281) has shown that plasmid DNA can be entrapped in 'stabilized plasmid-lipid particles' (SPLP) containing the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE), low levels (5-10 mol%) of cationic lipid, and stabilized by a polyethyleneglycol (PEG) coating. The PEG moieties are attached to a ceramide anchor containing an arachidoyl acyl group (PEG-CerC20). These SPLP exhibit low transfection potencies in vitro, due in part to the long residence time of the PEG-CerC20 on the SPLP surface. In this work we employed SPLP stabilized by PEG attached to ceramide containing an octanoyl acyl group (PEG-CerC8), which is able to quickly exchange out of the SPLP, to develop systems that give rise to optimized in vitro and in vivo (regional) transfection. A particular objective was to achieve cationic lipid contents that give rise to maximum transfection levels. It is shown that by performing the dialysis procedure in the presence of increasing concentrations of citrate, SPLP containing up to 30 mol% of the cationic lipid dioleoydimethylammonium chloride (DODAC) could be generated. The SPLP produced could be isolated from empty vesicles by sucrose density gradient centrifugation, and exhibited a narrow size distribution (62 +/- 8 nm, as determined by freeze-fracture electron microscopy) and a high plasmid-to-lipid ratio of 65 microg/micromol (corresponding to one plasmid per particle) regardless of the DODAC content. It was found that isolated SPLP containing 20-24 mol% DODAC resulted in optimum transfection of COS-7 and HepG2 cells in vitro, with luciferase expression levels comparable to those achieved for plasmid DNA-cationic lipid complexes. In vivo studies employing an intraperitoneal B16 tumor model and intraperitoneal administration of SPLP also demonstrated maximum luciferase expression for DODAC contents of 20-24 mol% and significantly improved gene expression in tumor tissue as compared with complexes. We

  20. 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