Phase II Study of Autologous Monocyte-Derived mRNA Electroporated Dendritic Cells (TriMixDC-MEL) Plus Ipilimumab in Patients With Pretreated Advanced Melanoma.

Science.gov (United States)

Wilgenhof, Sofie; Corthals, Jurgen; Heirman, Carlo; van Baren, Nicolas; Lucas, Sophie; Kvistborg, Pia; Thielemans, Kris; Neyns, Bart

2016-04-20

Autologous monocyte-derived dendritic cells (DCs) electroporated with synthetic mRNA (TriMixDC-MEL) are immunogenic and have antitumor activity as a monotherapy in patients with pretreated advanced melanoma. Ipilimumab, an immunoglobulin G1 monoclonal antibody directed against the cytotoxic T-lymphocyte-associated protein 4 receptor that counteracts physiologic suppression of T-cell function, improves the overall survival of patients with advanced melanoma. This phase II study investigated the combination of TriMixDC-MEL and ipilimumab in patients with pretreated advanced melanoma. Thirty-nine patients were treated with TriMixDC-MEL (4 × 10(6) cells administered intradermally and 20 × 10(6) cells administered intravenously) plus ipilimumab (10 mg/kg every 3 weeks for a total of four administrations, followed by maintenance therapy every 12 weeks in patients who remained progression free). Six-month disease control rate according to the immune-related response criteria served as the primary end point. The 6-month disease control rate was 51% (95% CI, 36% to 67%), and the overall tumor response rate was 38% (including eight complete and seven partial responses). Seven complete responses and one partial tumor response are ongoing after a median follow-up time of 36 months (range, 22 to 43 months). The most common treatment-related adverse events (all grades) consisted of local DC injection site skin reactions (100%), transient post-DC infusion chills (38%) and flu-like symptoms (84%), dermatitis (64%), hepatitis (13%), hypophysitis (15%), and diarrhea/colitis (15%). Grade 3 or 4 immune-related adverse events occurred in 36% of patients. There was no grade 5 adverse event. The combination of TriMixDC-MEL and ipilimumab is tolerable and results in an encouraging rate of highly durable tumor responses in patients with pretreated advanced melanoma. © 2016 by American Society of Clinical Oncology.

Messenger RNA surveillance: neutralizing natural nonsense

DEFF Research Database (Denmark)

Weischelfeldt, Joachim Lütken; Lykke-Andersen, Jens; Porse, Bo

2005-01-01

Messenger RNA transcripts that contain premature stop codons are degraded by a process termed nonsense-mediated mRNA decay (NMD). Although previously thought of as a pathway that rids the cell of non-functional mRNAs arising from mutations and processing errors, new research suggests a more general...

Radiation sensitivity of messenger RNA

International Nuclear Information System (INIS)

Ponta, H.; Pfennig-Yeh, M.L.; Herrlich, P.; Karlsruhe Univ.; Wagner, E.F.; Schweiger, M.

1979-01-01

Messenger RNA function is inactivated by irradiation with ultraviolet light. A unit length mRNA (in bases) is 2-3 times more sensitive than a unit length of DNA (in base pairs) with respect to the inactivation of template function. These data stem from four experimental systems all of which do not repair DNA: the translation of E. coli mRNA in rifampicin-treated cells, of T7 mRNA in infected E.coli, of f2 phage RNA in vivo, and of stable mRNA in chromosomeless minicells. The comparison of relative sensitivities to UV is relevant to the technique of UV mapping of transcription units which enjoys increasing popularity in pro- and eukaryotic genetic research. (orig.) [de

Radiation sensitivity of messenger RNA

Energy Technology Data Exchange (ETDEWEB)

Ponta, H; Pfennig-Yeh, M L; Herrlich, P [Kernforschungszentrum Karlsruhe G.m.b.H. (Germany, F.R.). Inst. fuer Genetik und Toxikologie von Spaltstoffen; Karlsruhe Univ. (TH) (Germany, F.R.). Inst. fuer Genetik); Wagner, E F; Schweiger, M [Innsbruck Univ. (Austria). Inst. fuer Biochemie

1979-08-01

Messenger RNA function is inactivated by irradiation with ultraviolet light. A unit length mRNA (in bases) is 2-3 times more sensitive than a unit length of DNA (in base pairs) with respect to the inactivation of template function. These data stem from four experimental systems all of which do not repair DNA: the translation of E. coli mRNA in rifampicin-treated cells, of T7 mRNA in infected E.coli, of f2 phage RNA in vivo, and of stable mRNA in chromosomeless minicells. The comparison of relative sensitivities to UV is relevant to the technique of UV mapping of transcription units which enjoys increasing popularity in pro- and eukaryotic genetic research.

Direct and efficient transfection of mouse neural stem cells and mature neurons by in vivo mRNA electroporation.

Science.gov (United States)

Bugeon, Stéphane; de Chevigny, Antoine; Boutin, Camille; Coré, Nathalie; Wild, Stefan; Bosio, Andreas; Cremer, Harold; Beclin, Christophe

2017-11-01

In vivo brain electroporation of DNA expression vectors is a widely used method for lineage and gene function studies in the developing and postnatal brain. However, transfection efficiency of DNA is limited and adult brain tissue is refractory to electroporation. Here, we present a systematic study of mRNA as a vector for acute genetic manipulation in the developing and adult brain. We demonstrate that mRNA electroporation is far more efficient than DNA electroporation, and leads to faster and more homogeneous protein expression in vivo Importantly, mRNA electroporation allows the manipulation of neural stem cells and postmitotic neurons in the adult brain using minimally invasive procedures. Finally, we show that this approach can be efficiently used for functional studies, as exemplified by transient overexpression of the neurogenic factor Myt1l and by stably inactivating Dicer nuclease in vivo in adult born olfactory bulb interneurons and in fully integrated cortical projection neurons. © 2017. Published by The Company of Biologists Ltd.

Transfecting Human Monocytes with RNA.

Science.gov (United States)

Dannull, Jens; Nair, Smita K

2016-01-01

Targeting monocytes as a delivery system for drugs or nucleic acids, and thereby harnessing their natural tissue-infiltrating capacity, has become an area of intense investigation in both basic and clinical research. Herein we describe an efficient method to deliver mRNA (messenger RNA) or siRNA (small interfering RNA) into human monocytes by electroporation. This method can be applied in the laboratory to monocytes isolated via magnetic bead-based techniques, or in a clinical setting using monocytes that were collected via counterflow centrifugation elutriation using the Elutra(®) Cell Separation System. We further demonstrate that electroporation of monocytes with RNA represents a robust and highly relevant approach to modify monocytes for cell-based therapies. Last, the procedure described can readily be adapted to monocytes from different species, hence facilitating research in animal models.

Novel T cells with improved in vivo anti-tumor activity generated by RNA electroporation

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Xiaojun Liu

2017-05-01

Full Text Available ABSTRACT The generation of T cells with maximal anti-tumor activities will significantly impact the field of T-cell-based adoptive immunotherapy. In this report, we found that OKT3/IL-2-stimulated T cells were phenotypically more heterogeneous, with enhanced anti-tumor activity in vitro and when locally administered in a solid tumor mouse model. To further improve the OKT3/IL-2-based T cell manufacturing procedure, we developed a novel T cell stimulation and expansion method in which peripheral blood mononuclear cells were electroporated with mRNA encoding a chimeric membrane protein consisting of a single-chain variable fragment against CD3 and the intracellular domains of CD28 and 4-1BB (OKT3-28BB. The expanded T cells were phenotypically and functionally similar to T cells expanded by OKT3/IL-2. Moreover, co-electroporation of CD86 and 4-1BBL could further change the phenotype and enhance the in vivo anti-tumor activity. Although T cells expanded by the co-electroporation of OKT3-28BB with CD86 and 4-1BBL showed an increased central memory phenotype, the T cells still maintained tumor lytic activities as potent as those of OKT3/IL-2 or OKT3-28BB-stimulated T cells. In different tumor mouse models, T cells expanded by OKT3-28BB RNA electroporation showed anti-tumor activities superior to those of OKT3/IL-2 T cells. Hence, T cells with both a less differentiated phenotype and potent tumor killing ability can be generated by RNA electroporation, and this T cell manufacturing procedure can be further optimized by simply co-delivering other splices of RNA, thus providing a simple and cost-effective method for generating high-quality T cells for adoptive immunotherapy.

Bifurcations in the interplay of messenger RNA, protein and nonprotein coding RNA

International Nuclear Information System (INIS)

Zhdanov, Vladimir P

2008-01-01

The interplay of messenger RNA (mRNA), protein, produced via translation of this RNA, and nonprotein coding RNA (ncRNA) may include regulation of the ncRNA production by protein and (i) ncRNA-protein association resulting in suppression of the protein regulatory activity or (ii) ncRNA-mRNA association resulting in degradation of the miRNA-mRNA complex. The kinetic models describing these two scenarios are found to predict bistability provided that protein suppresses the ncRNA formation

Immunogenicity of DNA vaccines encoding simian immunodeficiency virus antigen targeted to dendritic cells in rhesus macaques.

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Matthias Tenbusch

Full Text Available BACKGROUND: Targeting antigens encoded by DNA vaccines to dendritic cells (DCs in the presence of adjuvants enhances their immunogenicity and efficacy in mice. METHODOLOGY/PRINCIPAL FINDINGS: To explore the immunogenicity of this approach in non-human primates, we generated a single chain antibody to the antigen uptake receptor DEC-205 expressed on rhesus macaque DCs. DNA vaccines encoding this single chain antibody fused to the SIV capsid protein were delivered to six monkeys each by either intramuscular electroporation or conventional intramuscular injection co-injected or not with poly ICLC, a stabilized poly I: C analogue, as adjuvant. Antibodies to capsid were induced by the DC-targeting and non-targeting control DNA delivered by electroporation while conventional DNA immunization at a 10-fold higher dose of DNA failed to induce detectable humoral immune responses. Substantial cellular immune responses were also observed after DNA electroporation of both DNAs, but stronger responses were induced by the non-targeting vaccine. Conventional immunization with the DC-targeting DNA at a 10-fold higher dose did not give rise to substantial cellular immune responses, neither when co-injected with poly ICLC. CONCLUSIONS/SIGNIFICANCE: The study confirms the potent immunogenicity of DNA vaccines delivered by electroporation. Targeting the DNA via a single chain antibody to DEC-205 expressed by DCs, however, does not improve the immunogenicity of the antigens in non-human primates.

Localization of calcium-binding proteins and GABA transporter (GAT-1) messenger RNA in the human subthalamic nucleus

International Nuclear Information System (INIS)

Augood, S.J.; Waldvogel, H.J.; Muenkle, M.C.; Faull, R.L.M.; Emson, P.C.

1999-01-01

The distribution of messenger RNA encoding the human GAT-1 (a high-affinity GABA transporter) was investigated in the subthalamic nucleus of 10 neurologically normal human post mortem cases. Further, the distribution of messenger RNA and protein encoding the three neuronally expressed calcium-binding proteins (calbindin D28k, parvalbumin and calretinin) was similarly investigated using in situ hybridization and immunohistochemical techniques. Cellular sites of calbindin D28k, parvalbumin, calretinin and GAT-1 messenger RNA expression were localized using human-specific oligonucleotide probes radiolabelled with [ 35 S]dATP. Sites of protein localization were visualized using specific anti-calbindin D28k, anti-parvalbumin and anti-calretinin antisera. Examination of emulsion-coated tissue sections processed for in situ hybridization revealed an intense signal for GAT-1 messenger RNA within the human subthalamic nucleus, indeed the majority of Methylene Blue-counterstained cells were enriched in this transcript. Further, a marked heterogeneity was noted with regard to the expression of the messenger RNA's encoding the three calcium-binding proteins; this elliptical nucleus was highly enriched in parvalbumin messenger RNA-positive neurons and calretinin mRNA-positive cells but not calbindin messenger RNA-positive cells. Indeed, only an occasional calbindin messenger RNA-positive cell was detected within the mediolateral extent of the nucleus. In marked contrast, numerous parvalbumin messenger RNA-positive cells and calretinin messenger RNA-positive cells were detected and they were topographically distributed; parvalbumin messenger RNA-positive cells were highly enriched in the dorsal subthalamic nucleus extending mediolaterally; calretinin messenger RNA-positive cells were more enriched ventrally although some degree of overlap was apparent. Computer-assisted analysis of the average cross-sectional somatic area of parvalbumin, calretinin and GAT-1 messenger RNA

Phenotypic characterization of neurotensin messenger RNA-expressing cells in the neuroleptic-treated rat striatum: a detailed cellular co-expression study

Energy Technology Data Exchange (ETDEWEB)

Emson, P C; Westmore, K; Augood, S J [MRC Molecular Neuroscience Group, The Department of Neurobiology, The Babraham Institute, Babraham, Cambridge (United Kingdom)

1996-12-11

The chemical phenotype of proneurotensin messenger RNA-expressing cells was determined in the acute haloperidol-treated rat striatum using a combination of [{sup 35}S]-labelled and alkaline phosphatase-labelled oligonucleotides. Cellular sites of proneurotensin messenger RNA expression were visualized simultaneously on tissue sections processed to reveal cellular sites of preproenkephalin A messenger RNA or the dopamine and adenylate cyclase phosphoprotein-32, messenger RNA. The cellular co-expression of preproenkepahlin A and preprotachykinin messenger RNA was also examined within forebrain structures. Cellular sites of preproenkephalin A and dopamine and adenylate cyclase phosphoprotein-32 messenger RNAs were visualized using alkaline phosphatase-labelled oligonucleotides whilst sites of preprotachykinin and proneurotensin messenger RNA expression were detected using [{sup 35}S]-labelled oligos. Cellular sites of enkephalin and dopamine and adenylate cyclase phosphoprotein-32 gene expression were identified microscopically by the concentration of purple alkaline phosphatase reaction product within the cell cytoplasm, whereas sites of substance P and proneurotensin gene expression were identified by the dense clustering of silver grains overlying cells.An intense hybridization signal was detected for all three neuropeptide messenger RNAs in the striatum, the nucleus accumbens and septum. Dopamine and adenylate cyclase phosphoprotein-32 messenger RNA was detected within the neostriatum but not within the septum. In all forebrain regions examined, with the exception of the islands of Cajella, the cellular expression of enkephalin messenger RNA and substance P messenger RNA was discordant; the two neuropeptide messenger RNAs were detected essentially in different cells, although in the striatum and nucleus accumbens occasional isolated cells were detected which contained both hybridization signals; dense clusters of silver grains overlay alkaline phosphatase

Phenotypic characterization of neurotensin messenger RNA-expressing cells in the neuroleptic-treated rat striatum: a detailed cellular co-expression study

International Nuclear Information System (INIS)

Emson, P.C.; Westmore, K.; Augood, S.J.

1996-01-01

The chemical phenotype of proneurotensin messenger RNA-expressing cells was determined in the acute haloperidol-treated rat striatum using a combination of [ 35 S]-labelled and alkaline phosphatase-labelled oligonucleotides. Cellular sites of proneurotensin messenger RNA expression were visualized simultaneously on tissue sections processed to reveal cellular sites of preproenkephalin A messenger RNA or the dopamine and adenylate cyclase phosphoprotein-32, messenger RNA. The cellular co-expression of preproenkepahlin A and preprotachykinin messenger RNA was also examined within forebrain structures. Cellular sites of preproenkephalin A and dopamine and adenylate cyclase phosphoprotein-32 messenger RNAs were visualized using alkaline phosphatase-labelled oligonucleotides whilst sites of preprotachykinin and proneurotensin messenger RNA expression were detected using [ 35 S]-labelled oligos. Cellular sites of enkephalin and dopamine and adenylate cyclase phosphoprotein-32 gene expression were identified microscopically by the concentration of purple alkaline phosphatase reaction product within the cell cytoplasm, whereas sites of substance P and proneurotensin gene expression were identified by the dense clustering of silver grains overlying cells.An intense hybridization signal was detected for all three neuropeptide messenger RNAs in the striatum, the nucleus accumbens and septum. Dopamine and adenylate cyclase phosphoprotein-32 messenger RNA was detected within the neostriatum but not within the septum. In all forebrain regions examined, with the exception of the islands of Cajella, the cellular expression of enkephalin messenger RNA and substance P messenger RNA was discordant; the two neuropeptide messenger RNAs were detected essentially in different cells, although in the striatum and nucleus accumbens occasional isolated cells were detected which contained both hybridization signals; dense clusters of silver grains overlay alkaline phosphatase-positive cells

ESTRADIOL-INDUCED SYNTHESIS OF VITELLOGENIN .3. ISOLATION AND CHARACTERIZATION OF VITELLOGENIN MESSENGER-RNA FROM AVIAN LIVER

NARCIS (Netherlands)

AB, G.; Roskam, W. G.; Dijkstra, J.; Mulder, J.; Willems, M.; van der Ende, A.; Gruber, M.

1976-01-01

The messenger RNA of the hormone-induced protein vitellogenin was isolated from the liver of estrogen-treated roosters. Starting from total polysomal RNA, the vitellogenin messenger was purified 67-fold by oligo (dT)-cellulose chromatography and sizing on a sucrose gradient. The messenger was

Effects of ionizing radiation and partial hepatectomy on messenger RNA synthesis

International Nuclear Information System (INIS)

Abdel-Halim, M.N.

1979-01-01

Newly synthesized messenger RNA, as measured by a 40 min uptake of the radioactive precursor (6- 14 C) orotic acid, was studied in the regenerating livers of non-irradiated and gamma-irradiated (1800 rad) adrenal-intact and adrenalectomized rats 24 and 48 hours after partial hepatectomy. Two groups of rats, one with and one without adrenal glands were each divided into four subgroups: (1) control rats, (2) irradiated rats, (3) partially hepatectomized rats and (4) irradiated, partially hepatectomized rats. The radioactive profile of polyribosome formation and distribution was determined by sucrose density gradient centrifugation (10 to 40 per cent). The result of this study indicates that ionizing radiation decreases the synthesis of newly formed messenger RNA in regenerating livers of adrenal-intact rats. However, adrenalectomy largely abolished that inhibition. These data suggest that the decrease in messenger RNA synthesis may be explained by the disturbance of adrenal hormones induced by partial hepatectomy and ionizing radiation. (author)

Thomson scattering measurements on an atmospheric Ar dc discharge lamp

NARCIS (Netherlands)

Zhu, Xiao-Yan; Redwitz, M.; Kieft, E.R.; Sande, van de M.J.; Mullen, van der J.J.A.M.

2004-01-01

Thomson scattering (TS) experiments have been performed in the region near the electrodes of a dc powered model lamp filled with 1-2 bar argon gas. In order to suppress the false stray light and Rayleigh scattered photons, a triple grating spectrograph was used. In this way the electron density and

[Lentivirus-mediated shRNA silencing of LAMP2A inhibits the proliferation of multiple myeloma cells].

Science.gov (United States)

Li, Lixuan; Li, Jia

2015-05-01

To study the effects of lentivirus-mediated short hairpin RNA (shRNA) silencing of lysosome-associated membrane protein type 2A (LAMP2A) expression on the proliferation of multiple myeloma cells. The constructed shRNA lentiviral vector was applied to infect human multiple myeloma cell line MM.1S, and stable expression cell line was obtained by puromycin screening. Western blotting was used to verify the inhibitory effect on LAMP2A protein expression. MTT assay was conducted to detect the effect of knocked-down LAMP2A on MM.1S cell proliferation, and the anti-tumor potency of suberoylanilide hydroxamic acid (SAHA) against the obtained MM.1S LAMP2A(shRNA) stable cell line. Lactate assay was performed to observe the impact of low LAMP2A expression on cell glycolysis. The stable cell line with low LAMP2A expression were obtained with the constructed human LAMP2A-shRNA lentiviral vector. Down-regulation of LAMP2A expression significantly inhibited MM.1S cell proliferation and enhanced the anti-tumor activity of SAHA. Interestingly, decreased LAMP2A expression also inhibited MM.1S cell lactic acid secretion. Down-regulation of LAMP2A expression could inhibit cell proliferation in multiple myeloma cells.

Heterosubtypic protection against pathogenic human and avian influenza viruses via in vivo electroporation of synthetic consensus DNA antigens.

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Dominick J Laddy

Full Text Available BACKGROUND: The persistent evolution of highly pathogenic avian influenza (HPAI highlights the need for novel vaccination techniques that can quickly and effectively respond to emerging viral threats. We evaluated the use of optimized consensus influenza antigens to provide broad protection against divergent strains of H5N1 influenza in three animal models of mice, ferrets, and non-human primates. We also evaluated the use of in vivo electroporation to deliver these vaccines to overcome the immunogenicity barrier encountered in larger animal models of vaccination. METHODS AND FINDINGS: Mice, ferrets and non-human primates were immunized with consensus plasmids expressing H5 hemagglutinin (pH5HA, N1 neuraminidase (pN1NA, and nucleoprotein antigen (pNP. Dramatic IFN-gamma-based cellular immune responses to both H5 and NP, largely dependent upon CD8+ T cells were seen in mice. Hemaggutination inhibition titers classically associated with protection (>1:40 were seen in all species. Responses in both ferrets and macaques demonstrate the ability of synthetic consensus antigens to induce antibodies capable of inhibiting divergent strains of the H5N1 subtype, and studies in the mouse and ferret demonstrate the ability of synthetic consensus vaccines to induce protection even in the absence of such neutralizing antibodies. After challenge, protection from morbidity and mortality was seen in mice and ferrets, with significant reductions in viral shedding and disease progression seen in vaccinated animals. CONCLUSIONS: By combining several consensus influenza antigens with in vivo electroporation, we demonstrate that these antigens induce both protective cellular and humoral immune responses in mice, ferrets and non-human primates. We also demonstrate the ability of these antigens to protect from both morbidity and mortality in a ferret model of HPAI, in both the presence and absence of neutralizing antibody, which will be critical in responding to the

A Contemporary, Laboratory-Intensive Course on Messenger RNA Transcription and Processing

Science.gov (United States)

Carson, Sue; Miller, Heather

2012-01-01

Messenger ribonucleic acid (mRNA) plays a pivotal role in the central dogma of molecular biology. Importantly, molecular events occurring during and after mRNA synthesis have the potential to create multiple proteins from one gene, leading to some of the remarkable protein diversity that genomes hold. The North Carolina State University…

Efficiency of cellular delivery of antisense peptide nucleic acid by electroporation depends on charge and electroporation geometry

DEFF Research Database (Denmark)

Joergensen, Mette; Agerholm-Larsen, Birgit; Nielsen, Peter E

2011-01-01

Electroporation is potentially a very powerful technique for both in vitro cellular and in vivo drug delivery, particularly relating to oligonucleotides and their analogs for genetic therapy. Using a sensitive and quantitative HeLa cell luciferase RNA interference mRNA splice correction assay...... with a functional luciferase readout, we demonstrate that parameters such as peptide nucleic acid (PNA) charge and the method of electroporation have dramatic influence on the efficiency of productive delivery. In a suspended cell electroporation system (cuvettes), a positively charged PNA (+8) was most efficiently...... transferred, whereas charge neutral PNA was more effective in a microtiter plate electrotransfer system for monolayer cells. Surprisingly, a negatively charged (-23) PNA did not show appreciable activity in either system. Findings from the functional assay were corroborated by pulse parameter variations...

Tissue distribution of human acetylcholinesterase and butyrylcholinesterase messenger RNA

Energy Technology Data Exchange (ETDEWEB)

Jbilo, O.; Barteles, C.F.; Chatonnet, A.; Toutant, J.P.; Lockridge, O.

1994-12-31

Tissue distribution of human acetyicholinesterase and butyryicholinesterase messenger RNA. 1 Cholinesterase inhibitors occur naturally in the calabar bean (eserine), green potatoes (solanine), insect-resistant crab apples, the coca plant (cocaine) and snake venom (fasciculin). There are also synthetic cholinesterase inhibitors, for example man-made insecticides. These inhibitors inactivate acetyicholinesterase and butyrylcholinesterase as well as other targets. From a study of the tissue distribution of acetylcholinesterase and butyrylcholinesterase mRNA by Northern blot analysis, we have found the highest levels of butyrylcholinesterase mRNA in the liver and lungs, tissues known as the principal detoxication sites of the human body. These results indicate that butyrylcholinesterase may be a first line of defense against poisons that are eaten or inhaled.

RNA-Based Vaccines in Cancer Immunotherapy

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Megan A. McNamara

2015-01-01

Full Text Available RNA vaccines traditionally consist of messenger RNA synthesized by in vitro transcription using a bacteriophage RNA polymerase and template DNA that encodes the antigen(s of interest. Once administered and internalized by host cells, the mRNA transcripts are translated directly in the cytoplasm and then the resulting antigens are presented to antigen presenting cells to stimulate an immune response. Alternatively, dendritic cells can be loaded with either tumor associated antigen mRNA or total tumor RNA and delivered to the host to elicit a specific immune response. In this review, we will explain why RNA vaccines represent an attractive platform for cancer immunotherapy, discuss modifications to RNA structure that have been developed to optimize mRNA vaccine stability and translational efficiency, and describe strategies for nonviral delivery of mRNA vaccines, highlighting key preclinical and clinical data related to cancer immunotherapy.

Immune monitoring using mRNA-transfected dendritic cells

DEFF Research Database (Denmark)

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 m......RNA 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....

Alterations in messenger RNA and small nuclear RNA metabolism resulting from fluorouracil incorporation

International Nuclear Information System (INIS)

Armstrong, R.D.; Cadman, E.C.

1985-01-01

Studies were completed to examine the effect of 5-fluorouracil (FUra) incorporation on messenger RNA (mRNA) and small molecular weight nuclear RNA (SnRNA) metabolism. Studies of mRNA were completed using cDNA-mRNA hybridization methods to specifically examine dihydrofolate reductase (DHFR) mRNA. C 3 -L5178Y murine leukemia cells which are gene-amplified for DHFR, were exposed to FUra for 6, 12 or 24 hr, and the nuclear and cytoplasmic levels of DHFR-mRNA determined by hybridization with 32 P-DHFR-cDNA. FUra produced a dose-dependent increase in nuclear DHFR-mRNA levels, while total cytoplasmic DHFR-mRNA levels appeared to be unchanged. To examine only mRNA synthesized during FUra exposure, cells were also treated concurrently with [ 3 H] cytidine, and the [ 3 H]mRNA-cDNA hybrids measured following S 1 -nuclease treatment. FUra produced a concentration-dependent increase in nascent nuclear DHFR-mRNA levels, and a decrease in nascent cytoplasmic DHFR-mRNAs levels. These results suggest that FUra produces either an inhibition of mRNA processing, or an inhibition of nuclear-cytoplasmic transport. Preliminary experiments to examine ATP-dependent mRNA transport were completed with isolated nuclei from cells treated with FUra for 1 or 24 hr and then pulse-labeled for 1 hr with [ 3 H] cytidine. The results demonstrate a FUra-concentration and time-dependent inhibition of ATP-mediated mRNA efflux

Identification of messenger RNA of fetoplacental source in maternal plasma of women with normal pregnancies and pregnancies with intrauterine growth restriction.

Science.gov (United States)

Ayala Ramírez, Paola; García Robles, Reggie; Rojas, Juan Diego; Bermúdez, Martha; Bernal, Jaime

2012-07-01

to quantify placenta-specific RNA in plasma of women carrying foetuses with intrauterine growth restriction and pregnant women with normal pregnancies. 8 pregnant women with foetuses with intrauterine growth restriction were studied as well as 18 women with uncomplicated pregnancies in the third pregnancy trimester. Total free RNA was quantified in maternal plasma by spectrophotometry and the gene expression of hPL (Human Placental Lactogen) at the messenger RNA level through technical Real Time-Chain Reaction Polymerase. plasma RNA of fetoplacental origin was successfully detected in 100% of pregnant women. There were no statistically significant differences between the values of total RNA extracted from plasma (p= 0.5975) nor in the messenger RNA expression of hPL gene (p= 0.5785) between cases and controls. messenger RNA of fetoplacental origin can be detected in maternal plasma during pregnancy.

Electroporation ablation: A new energy modality for ablation of arrhythmogenic cardiac substrate

NARCIS (Netherlands)

van Driel, VJHM

2016-01-01

At the very end of the Direct Current (DC) era, low-energy DC ablation was demonstrated to cause myocardial lesions by non-thermal irreversible electroporation (IRE) (permanent formation of pores in the cell membrane, leading to cell death), without arcing and/or barotrauma. To eliminate rather

Postage for the messenger: Designating routes for Nuclear mRNA Export

Science.gov (United States)

Natalizio, Barbara J.; Wente, Susan R.

2013-01-01

Transcription of messenger(m) RNA occurs in the nucleus, making the translocation of mRNA across the nuclear envelope (NE) boundary a critical determinant of proper gene expression and cell survival. A major mRNA export route occurs via the NXF1-dependent pathway through the nuclear pore complexes (NPCs) embedded in the NE. However, recent findings have discovered new evidence supporting the existence of multiple mechanisms for crossing the NE, including both NPC-mediated and NE budding-mediated pathways. An analysis of the trans-acting factors and cis components that define these pathways reveals shared elements as well as mechanistic differences. We review here the current understanding of the mechanisms that characterize each pathway and highlight the determinants that influence mRNA transport fate. PMID:23583578

Dendritic cells take up and present antigens from viable and apoptotic polymorphonuclear leukocytes.

Directory of Open Access Journals (Sweden)

Carlos Alfaro

Full Text Available Dendritic cells (DC are endowed with the ability to cross-present antigens from other cell types to cognate T cells. DC are poised to meet polymorphonuclear leukocytes (PMNs as a result of being co-attracted by interleukin-8 (IL-8, for instance as produced by tumor cells or infected tissue. Human monocyte-derived and mouse bone marrow-derived DC can readily internalize viable or UV-irradiated PMNs. Such internalization was abrogated at 4°C and partly inhibited by anti-CD18 mAb. In mice, DC which had internalized PMNs containing electroporated ovalbumin (OVA protein, were able to cross-present the antigen to CD8 (OT-1 and CD4 (OT-2 TCR-transgenic T cells. Moreover, in humans, tumor cell debris is internalized by PMNs and the tumor-cell material can be subsequently taken up from the immunomagnetically re-isolated PMNs by DC. Importantly, if human neutrophils had endocytosed bacteria, they were able to trigger the maturation program of the DC. Moreover, when mouse PMNs with E. coli in their interior are co-injected in the foot pad with DC, many DC loaded with fluorescent material from the PMNs reach draining lymph nodes. Using CT26 (H-2(d mouse tumor cells, it was observed that if tumor cells are intracellularly loaded with OVA protein and UV-irradiated, they become phagocytic prey of H-2(d PMNs. If such PMNs, that cannot present antigens to OT-1 T cells, are immunomagnetically re-isolated and phagocytosed by H-2(b DC, such DC productively cross-present OVA antigen determinants to OT-1 T cells. Cross-presentation to adoptively transferred OT-1 lymphocytes at draining lymph nodes also take place when OVA-loaded PMNs (H-2(d are coinjected in the footpad of mice with autologous DC (H-2(b. In summary, our results indicate that antigens phagocytosed by short-lived PMNs can be in turn internalized and productively cross-presented by DC.

Simple Genome Editing of Rodent Intact Embryos by Electroporation.

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Takehito Kaneko

Full Text Available The clustered regularly interspaced short palindromic repeat (CRISPR/CRISPR-associated (Cas system is a powerful tool for genome editing in animals. Recently, new technology has been developed to genetically modify animals without using highly skilled techniques, such as pronuclear microinjection of endonucleases. Technique for animal knockout system by electroporation (TAKE method is a simple and effective technology that produces knockout rats by introducing endonuclease mRNAs into intact embryos using electroporation. Using TAKE method and CRISPR/Cas system, the present study successfully produced knockout and knock-in mice and rats. The mice and rats derived from embryos electroporated with Cas9 mRNA, gRNA and single-stranded oligodeoxynucleotide (ssODN comprised the edited targeted gene as a knockout (67% of mice and 88% of rats or knock-in (both 33%. The TAKE method could be widely used as a powerful tool to produce genetically modified animals by genome editing.

Enhanced Delivery and Potency of Self-Amplifying mRNA Vaccines by Electroporation in Situ

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Kaustuv Banerjee

2013-08-01

Full Text Available Nucleic acid-based vaccines such as viral vectors, plasmid DNA (pDNA, and mRNA are being developed as a means to address limitations of both live-attenuated and subunit vaccines. DNA vaccines have been shown to be potent in a wide variety of animal species and several products are now licensed for commercial veterinary but not human use. Electroporation delivery technologies have been shown to improve the generation of T and B cell responses from synthetic DNA vaccines in many animal species and now in humans. However, parallel RNA approaches have lagged due to potential issues of potency and production. Many of the obstacles to mRNA vaccine development have recently been addressed, resulting in a revival in the use of non-amplifying and self-amplifying mRNA for vaccine and gene therapy applications. In this paper, we explore the utility of EP for the in vivo delivery of large, self-amplifying mRNA, as measured by reporter gene expression and immunogenicity of genes encoding HIV envelope protein. These studies demonstrated that EP delivery of self-amplifying mRNA elicited strong and broad immune responses in mice, which were comparable to those induced by EP delivery of pDNA.

Processivity and coupling in messenger RNA transcription.

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Stuart Aitken

2010-01-01

Full Text Available The complexity of messenger RNA processing is now being uncovered by experimental techniques that are capable of detecting individual copies of mRNA in cells, and by quantitative real-time observations that reveal the kinetics. This processing is commonly modelled by permitting mRNA to be transcribed only when the promoter is in the on state. In this simple on/off model, the many processes involved in active transcription are represented by a single reaction. These processes include elongation, which has a minimum time for completion and processing that is not captured in the model.In this paper, we explore the impact on the mRNA distribution of representing the elongation process in more detail. Consideration of the mechanisms of elongation leads to two alternative models of the coupling between the elongating polymerase and the state of the promoter: Processivity allows polymerases to complete elongation irrespective of the promoter state, whereas coupling requires the promoter to be active to produce a full-length transcript. We demonstrate that these alternatives have a significant impact on the predicted distributions. Models are simulated by the Gillespie algorithm, and the third and fourth moments of the resulting distribution are computed in order to characterise the length of the tail, and sharpness of the peak. By this methodology, we show that the moments provide a concise summary of the distribution, showing statistically-significant differences across much of the feasible parameter range.We conclude that processivity is not fully consistent with the on/off model unless the probability of successfully completing elongation is low--as has been observed. The results also suggest that some form of coupling between the promoter and a rate-limiting step in transcription may explain the cell's inability to maintain high mRNA levels at low noise--a prediction of the on/off model that has no supporting evidence.

Common acute lymphoblastic leukemia antigen: partial characterization by in vivo labeling and isolation of its messenger RNA

International Nuclear Information System (INIS)

Heinsohn, S.; Kabisch, H.

1987-01-01

Common acute lymphoblastic leukemia (ALL) antigen (CALLA)-like proteins were detected by in vivo labeling experiments carried out with human lymphoblastoid cell line KM3 and also in cell-free translation, directed by CALLA-specific mRNA prepared from immunoadsorbed KM3 polysomes. The CALLA-like structure found in both systems shows an Mr of 95kDa. Additional CALLA-like proteins could be identified in the in vivo experiments with calculated Mrs of 40kDa in the cells and 85 and 38kDa in the culture medium. In the cell-free translation system, an additional product of Mr 80kDa could be detected

Avoiding neuromuscular stimulation in liver irreversible electroporation using radiofrequency electric fields

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Castellví, Quim; Mercadal, Borja; Moll, Xavier; Fondevila, Dolors; Andaluz, Anna; Ivorra, Antoni

2018-02-01

Electroporation-based treatments typically consist of the application of high-voltage dc pulses. As an undesired side effect, these dc pulses cause electrical stimulation of excitable tissues such as motor nerves. The present in vivo study explores the use of bursts of sinusoidal voltage in a frequency range from 50 kHz to 2 MHz, to induce irreversible electroporation (IRE) whilst avoiding neuromuscular stimulation. A series of 100 dc pulses or sinusoidal bursts, both with an individual duration of 100 µs, were delivered to rabbit liver through thin needles in a monopolar electrode configuration, and thoracic movements were recorded with an accelerometer. Tissue samples were harvested three hours after treatment and later post-processed to determine the dimensions of the IRE lesions. Thermal damage due to Joule heating was ruled out via computer simulations. Sinusoidal bursts with a frequency equal to or above 100 kHz did not cause thoracic movements and induced lesions equivalent to those obtained with conventional dc pulses when the applied voltage amplitude was sufficiently high. IRE efficacy dropped with increasing frequency. For 100 kHz bursts, it was estimated that the electric field threshold for IRE is about 1.4 kV cm-1 whereas that of dc pulses is about 0.5 kV cm-1.

DNA Vaccine Electroporation and Molecular Adjuvants

Science.gov (United States)

2016-03-16

Suschak and Schmaljohn DNA Vaccine Electroporation and Molecular Adjuvants 1 Abstract To date, there is no protective vaccine for Ebola virus...the formulation of DNA launched virus-like particles (VLP). In this case, the antigen is encoded in one DNA plasmid, while structural proteins are...Virol, 2010. 155(12): p. 2083-103. 2. Feldmann, H. and T.W. Geisbert, Ebola haemorrhagic fever. Lancet, 2011. 377(9768): p. 849-62. 3. Hart, M.K

Expression of μ, κ, and δ opioid receptor messenger RNA in the human CNS: a 33P in situ hybridization study

International Nuclear Information System (INIS)

Peckys, D.; Landwehrmeyer, G.B.

1999-01-01

The existence of at least three opioid receptor types, referred to as μ, κ, and δ, is well established. Complementary DNAs corresponding to the pharmacologically defined μ, κ, and δ opioid receptors have been isolated in various species including man. The expression patterns of opioid receptor transcripts in human brain has not been established with a cellular resolution, in part because of the low apparent abundance of opioid receptor messenger RNAs in human brain. To visualize opioid receptor messenger RNAs we developed a sensitive in situ hybridization histochemistry method using 33 P-labelled RNA probes. In the present study we report the regional and cellular expression of μ, κ, and δ opioid receptor messenger RNAs in selected areas of the human brain. Hybridization of the different opioid receptor probes resulted in distinct labelling patterns. For the μ and κ opioid receptor probes, the most intense regional signals were observed in striatum, thalamus, hypothalamus, cerebral cortex, cerebellum and certain brainstem areas as well as the spinal cord. The most intense signals for the δ opioid receptor probe were found in cerebral cortex. Expression of opioid receptor transcripts was restricted to subpopulations of neurons within most regions studied demonstrating differences in the cellular expression patterns of μ, κ, and δ opioid receptor messenger RNAs in numerous brain regions. The messenger RNA distribution patterns for each opioid receptor corresponded in general to the distribution of opioid receptor binding sites as visualized by receptor autoradiography. However, some mismatches, for instance between μ opioid receptor receptor binding and μ opioid receptor messenger RNA expression in the anterior striatum, were observed. A comparison of the distribution patterns of opioid receptor messenger RNAs in the human brain and that reported for the rat suggests a homologous expression pattern in many regions. However, in the human brain, κ

Use of electroporation and reverse iontophoresis for extraction of transdermal multibiomarkers

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Ching CTS

2012-02-01

Full Text Available Congo Tak-Shing Ching1,2, Lin-Shien Fu3-5, Tai-Ping Sun1, Tzu-Hsiang Hsu1, Kang-Ming Chang21Department of Electrical Engineering, National Chi Nan University, Puli, Nantou County, 2Department of Photonics and Communication Engineering, Asia University, Wufeng, Taichung, 3Department of Pediatrics, National Yang Ming University, Taipei, 4Institute of Technology, National Chi Nan University, Puli, 5Department of Pediatrics, Taichung Veterans General Hospital, Taichung City, TaiwanBackground: Monitoring of biomarkers, like urea, prostate-specific antigen (PSA, and osteopontin, is very important because they are related to kidney disease, prostate cancer, and ovarian cancer, respectively. It is well known that reverse iontophoresis can enhance transdermal extraction of small molecules, and even large molecules if reverse iontophoresis is used together with electroporation. Electroporation is the use of a high-voltage electrical pulse to create nanochannels within the stratum corneum, temporarily and reversibly. Reverse iontophoresis is the use of a small current to facilitate both charged and uncharged molecule transportation across the skin. The objectives of this in vitro study were to determine whether PSA and osteopontin are extractable transdermally and noninvasively and whether urea, PSA, and osteopontin can be extracted simultaneously by electroporation and reverse iontophoresis.Methods: All in vitro experiments were conducted using a diffusion cell assembled with the stratum corneum of porcine skin. Three different symmetrical biphasic direct currents (SBdc, five various electroporations, and a combination of the two techniques were applied to the diffusion cell via Ag/AgCl electrodes. The three different SBdc had the same current density of 0.3 mA/cm2, but different phase durations of 0 (ie, no current, control group, 30, and 180 seconds. The five different electroporations had the same pulse width of 1 msec and number of pulses per second

Phase-resolved response of a metal-halide lamp

International Nuclear Information System (INIS)

Flikweert, A J; Beks, M L; Nimalasuriya, T; Kroesen, G M W; Van der Mullen, J J A M; Stoffels, W W

2009-01-01

The metal-halide (MH) lamp sometimes shows unwanted colour segregation, caused by a combination of convection and diffusion. In the past we investigated the lamp, running on a switched dc ballast of 120 Hz, using a dc approximation for the distribution of the radiating species. Here we present phase-resolved intensity measurements to verify this approximation. The MH lamp contains Hg as buffer gas and DyI 3 as salt additive; we measure the light emitted by Dy and by Hg atoms. An intensity fluctuation of ∼25% close to the electrodes is found only. The observed fluctuations are explained by the cataphoresis effect and temperature fluctuations; the time scales are in the same order. Furthermore, measurements at higher gravity in a centrifuge (up to 10g) show that the effect becomes smaller at increasing gravity levels. From these results it is concluded that a dc approximation, which is generally assumed by lamp developers, is allowed for this MH lamp.

Comparison of direct and alternating current vacuum ultraviolet lamps in atmospheric pressure photoionization.

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Vaikkinen, Anu; Haapala, Markus; Kersten, Hendrik; Benter, Thorsten; Kostiainen, Risto; Kauppila, Tiina J

2012-02-07

A direct current induced vacuum ultraviolet (dc-VUV) krypton discharge lamp and an alternating current, radio frequency (rf) induced VUV lamp that are essentially similar to lamps in commercial atmospheric pressure photoionization (APPI) ion sources were compared. The emission distributions along the diameter of the lamp exit window were measured, and they showed that the beam of the rf lamp is much wider than that of the dc lamp. Thus, the rf lamp has larger efficient ionization area, and it also emits more photons than the dc lamp. The ionization efficiencies of the lamps were compared using identical spray geometries with both lamps in microchip APPI mass spectrometry (μAPPI-MS) and desorption atmospheric pressure photoionization-mass spectrometry (DAPPI-MS). A comprehensive view on the ionization was gained by studying six different μAPPI solvent compositions, five DAPPI spray solvents, and completely solvent-free DAPPI. The observed reactant ions for each solvent composition were very similar with both lamps except for toluene, which showed a higher amount of solvent originating oxidation products with the rf lamp than with the dc lamp in μAPPI. Moreover, the same analyte ions were detected with both lamps, and thus, the ionization mechanisms with both lamps are similar. The rf lamp showed a higher ionization efficiency than the dc lamp in all experiments. The difference between the lamp ionization efficiencies was greatest when high ionization energy (IE) solvent compositions (IEs above 10 eV), i.e., hexane, methanol, and methanol/water, (1:1 v:v) were used. The higher ionization efficiency of the rf lamp is likely due to the larger area of high intensity light emission, and the resulting larger efficient ionization area and higher amount of photons emitted. These result in higher solvent reactant ion production, which in turn enables more efficient analyte ion production. © 2012 American Chemical Society

Systemic RNA delivery to dendritic cells exploits antiviral defence for cancer immunotherapy

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Kranz, Lena M.; Diken, Mustafa; Haas, Heinrich; Kreiter, Sebastian; Loquai, Carmen; Reuter, Kerstin C.; Meng, Martin; Fritz, Daniel; Vascotto, Fulvia; Hefesha, Hossam; Grunwitz, Christian; Vormehr, Mathias; Hüsemann, Yves; Selmi, Abderraouf; Kuhn, Andreas N.; Buck, Janina; Derhovanessian, Evelyna; Rae, Richard; Attig, Sebastian; Diekmann, Jan; Jabulowsky, Robert A.; Heesch, Sandra; Hassel, Jessica; Langguth, Peter; Grabbe, Stephan; Huber, Christoph; Türeci, Özlem; Sahin, Ugur

2016-06-01

Lymphoid organs, in which antigen presenting cells (APCs) are in close proximity to T cells, are the ideal microenvironment for efficient priming and amplification of T-cell responses. However, the systemic delivery of vaccine antigens into dendritic cells (DCs) is hampered by various technical challenges. Here we show that DCs can be targeted precisely and effectively in vivo using intravenously administered RNA-lipoplexes (RNA-LPX) based on well-known lipid carriers by optimally adjusting net charge, without the need for functionalization of particles with molecular ligands. The LPX protects RNA from extracellular ribonucleases and mediates its efficient uptake and expression of the encoded antigen by DC populations and macrophages in various lymphoid compartments. RNA-LPX triggers interferon-α (IFNα) release by plasmacytoid DCs and macrophages. Consequently, DC maturation in situ and inflammatory immune mechanisms reminiscent of those in the early systemic phase of viral infection are activated. We show that RNA-LPX encoding viral or mutant neo-antigens or endogenous self-antigens induce strong effector and memory T-cell responses, and mediate potent IFNα-dependent rejection of progressive tumours. A phase I dose-escalation trial testing RNA-LPX that encode shared tumour antigens is ongoing. In the first three melanoma patients treated at a low-dose level, IFNα and strong antigen-specific T-cell responses were induced, supporting the identified mode of action and potency. As any polypeptide-based antigen can be encoded as RNA, RNA-LPX represent a universally applicable vaccine class for systemic DC targeting and synchronized induction of both highly potent adaptive as well as type-I-IFN-mediated innate immune mechanisms for cancer immunotherapy.

The synthesis of polyadenylated messenger RNA in herpes simplex type I virus infected BHK cells.

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Harris, T J; Wildy, P

1975-09-01

The pattern of polyadenylated messenger RNA (mRNA) synthesis in BHK cell monolayers, infected under defined conditions with herpes simplex type I virus has been investigated by polyacrylamide gel electrophoresis or pulse-labelled RNA isolated by oligo dT-cellulose chromatography. Two classes of mRNA molecules were synthesized in infected cells; these were not detected in uninfected cells. The rate of synthesis of the larger, 18 to 30S RNA class reached a maximum soon after injection and then declined, whereas the rate of synthesis of the 7 to 11 S RNA class did not reach a maximum until much later and did not decline. In the presence of cytosine arabinoside, the rate of mRNA synthesis in infected cells was reduced but the electrophoretic pattern remained the same.

Early changes of placenta-derived messenger RNA in maternal plasma – potential value for preeclampsia prediction?

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Surugiu Sebastian

2015-12-01

Full Text Available Objective: the pourpose of the study was to determine if there are any differences between placenta derived plasmatic levels of messenger RNA in normal and future preeclamptic pregnancies and if these placental transcripts can predict preeclampsia long before clinical onset

Global inhibition of DC priming capacity in the spleen of self-antigen vaccinated mice requires IL-10

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Douglas Matthew Marvel

2014-02-01

Full Text Available DC in the spleen are highly activated following intravenous vaccination with a foreign antigen, promoting expansion of effector T cells, but remain phenotypically and functionally immature after vaccination with a self-antigen. Up-regulation or suppression of expression of a cohort of pancreatic enzymes 24-72 hours post-vaccination can be used as a biomarker of stimulatory versus toleragenic DC, respectively. Here we show, using MUC1 transgenic mice (MUC1.Tg and a vaccine based on the MUC1 peptide which these mice perceive as a self-antigen, that the difference in enzyme expression that predicts whether DC will promote immune response or immune tolerance, is seen as early as 4-8 hours following vaccination. We also identify early production of IL-10 as a predominant factor that both correlates with this early time point and controls DC function. Pre-treating mice with an antibody against the IL-10 receptor (IL-10R prior to vaccination results in DC that up-regulate CD40, CD80, and CD86 and promote stronger IFNγ+ T cell responses. This study suggests that transient inhibition of IL-10 prior to vaccination could improve responses to cancer vaccines that utilize self-tumor antigens.

Characterization of long noncoding RNA and messenger RNA signatures in melanoma tumorigenesis and metastasis.

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Siqi Wang

Full Text Available The incidence of melanoma, the most aggressive and life-threatening form of skin cancer, has significantly risen over recent decades. Therefore, it is essential to identify the mechanisms that underlie melanoma tumorigenesis and metastasis and to explore novel and effective melanoma treatment strategies. Accumulating evidence s uggests that aberrantly expressed long noncoding RNAs (lncRNAs have vital functions in multiple cancers. However, lncRNA functions in melanoma tumorigenesis and metastasis remain unclear. In this study, we investigated lncRNA and messenger RNA (mRNA expression profiles in primary melanomas, metastatic melanomas and normal skin samples from the Gene Expression Omnibus database. We used GSE15605 as the training set (n = 74 and GSE7553 as the validation set (n = 58. In three comparisons (primary melanoma versus normal skin, metastatic melanoma versus normal skin, and metastatic melanoma versus primary melanoma, 178, 295 and 48 lncRNAs and 847, 1758, and 295 mRNAs were aberrantly expressed, respectively. We performed Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses to examine the differentially expressed mRNAs, and potential core lncRNAs were predicted by lncRNA-mRNA co-expression networks. Based on our results, 15 lncRNAs and 144 mRNAs were significantly associated with melanoma tumorigenesis and metastasis. A subsequent analysis suggested a critical role for a five-lncRNA signature during melanoma tumorigenesis and metastasis. Low expression of U47924.27 was significantly associated with decreased survival of patients with melanoma. To the best of our knowledge, this study is the first to explore the expression patterns of lncRNAs and mRNAs during melanoma tumorigenesis and metastasis by re-annotating microarray data from the Gene Expression Omnibus (GEO microarray dataset. These findings reveal potential roles for lncRNAs during melanoma tumorigenesis and metastasis and provide a rich candidate

Targeted siRNA Delivery and mRNA Knockdown Mediated by Bispecific Digoxigenin-binding Antibodies

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Britta Schneider

2012-01-01

Full Text Available Bispecific antibodies (bsAbs that bind to cell surface antigens and to digoxigenin (Dig were used for targeted small interfering RNA (siRNA delivery. They are derivatives of immunoglobulins G (IgGs that bind tumor antigens, such as Her2, IGF1-R, CD22, and LeY, with stabilized Dig-binding variable domains fused to the C-terminal ends of the heavy chains. siRNA that was digoxigeninylated at its 3′end was bound in a 2:1 ratio to the bsAbs. These bsAb–siRNA complexes delivered siRNAs specifically to cells that express the corresponding antigen as demonstrated by flow cytometry and confocal microscopy. The complexes internalized into endosomes and Dig-siRNAs separated from bsAbs, but Dig-siRNA was not released into the cytoplasm; bsAb-targeting alone was thus not sufficient for effective mRNA knockdown. This limitation was overcome by formulating the Dig-siRNA into nanoparticles consisting of dynamic polyconjugates (DPCs or into lipid-based nanoparticles (LNPs. The resulting complexes enabled bsAb-targeted siRNA-specific messenger RNA (mRNA knockdown with IC50 siRNA values in the low nanomolar range for a variety of bsAbs, siRNAs, and target cells. Furthermore, pilot studies in mice bearing tumor xenografts indicated mRNA knockdown in endothelial cells following systemic co-administration of bsAbs and siRNA formulated in LNPs that were targeted to the tumor vasculature.

Administration of HPV DNA vaccine via electroporation elicits the strongest CD8+ T cell immune responses compared to intramuscular injection and intradermal gene gun delivery

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Best, Simon R.; Peng, Shiwen; Juang, Chi-Mou; Hung, Chien-Fu; Hannaman, Drew; Saunders, John R.; Wu, T.-C.; Pai, Sara I.

2009-01-01

DNA vaccines are an attractive approach to eliciting antigen-specific immunity. Intracellular targeting of tumor antigens through its linkage to immunostimulatory molecules such as calreticulin (CRT) can improve antigen processing and presentation through the MHC Class I pathway and increase cytotoxic CD8+ T cell production. However, even with these enhancements, the efficacy of such immunotherapeutic strategies is dependent on the identification of an effective route and method of DNA administration. Electroporation and gene gun-mediated particle delivery are leading methods of DNA vaccine delivery that can generate protective and therapeutic levels of immune responses in experimental models. In this study, we perform a head-to-head comparison of three methods of vaccination – conventional intramuscular injection, electroporation mediated intramuscular delivery, and epidermal gene gun-mediated particle delivery - in the ability to generate antigen specific cytotoxic CD8+ T cell responses as well as anti-tumor immune responses against an HPV-16 E7 expressing tumor cell line using the pNGVL4a-CRT/E7(detox) DNA vaccine. Vaccination via electroporation generated the highest number of E7-specific cytotoxic CD8+ T cells, which correlated to improved outcomes in the treatment of growing tumors. In addition, we demonstrate that electroporation results in significantly higher levels of circulating protein compared to gene gun or intramuscular vaccination, which likely enhances calreticulin’s role as a local tumor anti-angiogenesis agent. We conclude that electroporation is a promising method for delivery of HPV DNA vaccines and should be considered for DNA vaccine delivery in human clinical trials. PMID:19622402

Regulated-current dc power supply for gaseous-discharge lamps

Science.gov (United States)

Freeman, W.; Huguenin, D.

1970-01-01

Controlled current source having a high output resistance feeds continuous-flow hydrogen lamps in vacuum-ultraviolet photometric equipment. The power supply, also used with low-pressure sealed lamps, has a short recovery time and smooth regulation without overshoot.

The microRNA and messengerRNA profile of the RNA-induced silencing complex in human primary astrocyte and astrocytoma cells.

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Moser, Joanna J; Fritzler, Marvin J

2010-10-18

GW/P bodies are cytoplasmic ribonucleoprotein-rich foci involved in microRNA (miRNA)-mediated messenger RNA (mRNA) silencing and degradation. The mRNA regulatory functions within GW/P bodies are mediated by GW182 and its binding partner hAgo2 that bind miRNA in the RNA-induced silencing complex (RISC). To date there are no published reports of the profile of miRNA and mRNA targeted to the RISC or a comparison of the RISC-specific miRNA/mRNA profile differences in malignant and non-malignant cells. RISC mRNA and miRNA components were profiled by microarray analysis of malignant human U-87 astrocytoma cells and its non-malignant counterpart, primary human astrocytes. Total cell RNA as well as RNA from immunoprecipitated RISC was analyzed. The novel findings were fourfold: (1) miRNAs were highly enriched in astrocyte RISC compared to U-87 astrocytoma RISC, (2) astrocytoma and primary astrocyte cells each contained unique RISC miRNA profiles as compared to their respective cellular miRNA profiles, (3) miR-195, 10b, 29b, 19b, 34a and 455-3p levels were increased and the miR-181b level was decreased in U-87 astrocytoma RISC as compared to astrocyte RISC, and (4) the RISC contained decreased levels of mRNAs in primary astrocyte and U-87 astrocytoma cells. The observation that miR-34a and miR-195 levels were increased in the RISC of U-87 astrocytoma cells suggests an oncogenic role for these miRNAs. Differential regulation of mRNAs by specific miRNAs is evidenced by the observation that three miR34a-targeted mRNAs and two miR-195-targeted mRNAs were downregulated while one miR-195-targeted mRNA was upregulated. Biological pathway analysis of RISC mRNA components suggests that the RISC plays a pivotal role in malignancy and other conditions. This study points to the importance of the RISC and ultimately GW/P body composition and function in miRNA and mRNA deregulation in astrocytoma cells and possibly in other malignancies.

The microRNA and messengerRNA profile of the RNA-induced silencing complex in human primary astrocyte and astrocytoma cells.

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Joanna J Moser

2010-10-01

Full Text Available GW/P bodies are cytoplasmic ribonucleoprotein-rich foci involved in microRNA (miRNA-mediated messenger RNA (mRNA silencing and degradation. The mRNA regulatory functions within GW/P bodies are mediated by GW182 and its binding partner hAgo2 that bind miRNA in the RNA-induced silencing complex (RISC. To date there are no published reports of the profile of miRNA and mRNA targeted to the RISC or a comparison of the RISC-specific miRNA/mRNA profile differences in malignant and non-malignant cells.RISC mRNA and miRNA components were profiled by microarray analysis of malignant human U-87 astrocytoma cells and its non-malignant counterpart, primary human astrocytes. Total cell RNA as well as RNA from immunoprecipitated RISC was analyzed. The novel findings were fourfold: (1 miRNAs were highly enriched in astrocyte RISC compared to U-87 astrocytoma RISC, (2 astrocytoma and primary astrocyte cells each contained unique RISC miRNA profiles as compared to their respective cellular miRNA profiles, (3 miR-195, 10b, 29b, 19b, 34a and 455-3p levels were increased and the miR-181b level was decreased in U-87 astrocytoma RISC as compared to astrocyte RISC, and (4 the RISC contained decreased levels of mRNAs in primary astrocyte and U-87 astrocytoma cells.The observation that miR-34a and miR-195 levels were increased in the RISC of U-87 astrocytoma cells suggests an oncogenic role for these miRNAs. Differential regulation of mRNAs by specific miRNAs is evidenced by the observation that three miR34a-targeted mRNAs and two miR-195-targeted mRNAs were downregulated while one miR-195-targeted mRNA was upregulated. Biological pathway analysis of RISC mRNA components suggests that the RISC plays a pivotal role in malignancy and other conditions. This study points to the importance of the RISC and ultimately GW/P body composition and function in miRNA and mRNA deregulation in astrocytoma cells and possibly in other malignancies.

Calcium Electroporation

DEFF Research Database (Denmark)

Frandsen, Stine Krog; Gibot, Laure; Madi, Moinecha

2015-01-01

BACKGROUND: Calcium electroporation describes the use of high voltage electric pulses to introduce supraphysiological calcium concentrations into cells. This promising method is currently in clinical trial as an anti-cancer treatment. One very important issue is the relation between tumor cell kill...... efficacy-and normal cell sensitivity. METHODS: Using a 3D spheroid cell culture model we have tested the effect of calcium electroporation and electrochemotherapy using bleomycin on three different human cancer cell lines: a colorectal adenocarcinoma (HT29), a bladder transitional cell carcinoma (SW780......), and a breast adenocarcinoma (MDA-MB231), as well as on primary normal human dermal fibroblasts (HDF-n). RESULTS: The results showed a clear reduction in spheroid size in all three cancer cell spheroids three days after treatment with respectively calcium electroporation (p

Human Langerhans cells use an IL-15R-α/IL-15/pSTAT5-dependent mechanism to break T-cell tolerance against the self-differentiation tumor antigen WT1.

Science.gov (United States)

Romano, Emanuela; Cotari, Jesse W; Barreira da Silva, Rosa; Betts, Brian C; Chung, David J; Avogadri, Francesca; Fink, Mitsu J; St Angelo, Erin T; Mehrara, Babak; Heller, Glenn; Münz, Christian; Altan-Bonnet, Gregoire; Young, James W

2012-05-31

Human CD34(+) progenitor-derived Langerhans-type dendritic cells (LCs) are more potent stimulators of T-cell immunity against tumor and viral antigens in vitro than are monocyte-derived DCs (moDCs). The exact mechanisms have remained elusive until now, however. LCs synthesize the highest amounts of IL-15R-α mRNA and protein, which binds IL-15 for presentation to responder lymphocytes, thereby signaling the phosphorylation of signal transducer and activator of transcription 5 (pSTAT5). LCs electroporated with Wilms tumor 1 (WT1) mRNA achieve sufficiently sustained presentation of antigenic peptides, which together with IL-15R-α/IL-15, break tolerance against WT1 by stimulating robust autologous, WT1-specific cytolytic T-lymphocytes (CTLs). These CTLs develop from healthy persons after only 7 days' stimulation without exogenous cytokines and lyse MHC-restricted tumor targets, which include primary WT1(+) leukemic blasts. In contrast, moDCs require exogenous rhuIL-15 to phosphorylate STAT5 and attain stimulatory capacity comparable to LCs. LCs therefore provide a more potent costimulatory cytokine milieu for T-cell activation than do moDCs, thus accounting for their superior stimulation of MHC-restricted Ag-specific CTLs without need for exogenous cytokines. These data support the use of mRNA-electroporated LCs, or moDCs supplemented with exogenous rhuIL-15, as vaccines for cancer immunotherapy to break tolerance against self-differentiation antigens shared by tumors.

Self-Amplifying Replicon RNA Vaccine Delivery to Dendritic Cells by Synthetic Nanoparticles

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Kenneth C. McCullough

2014-10-01

Full Text Available Dendritic cells (DC play essential roles determining efficacy of vaccine delivery with respect to immune defence development and regulation. This renders DCs important targets for vaccine delivery, particularly RNA vaccines. While delivery of interfering RNA oligonucleotides to the appropriate intracellular sites for RNA-interference has proven successful, the methodologies are identical for RNA vaccines, which require delivery to RNA translation sites. Delivery of mRNA has benefitted from application of cationic entities; these offer value following endocytosis of RNA, when cationic or amphipathic properties can promote endocytic vesicle membrane perturbation to facilitate cytosolic translocation. The present review presents how such advances are being applied to the delivery of a new form of RNA vaccine, replicons (RepRNA carrying inserted foreign genes of interest encoding vaccine antigens. Approaches have been developed for delivery to DCs, leading to the translation of the RepRNA and encoded vaccine antigens both in vitro and in vivo. Potential mechanisms favouring efficient delivery leading to translation are discussed with respect to the DC endocytic machinery, showing the importance of cytosolic translocation from acidifying endocytic structures. The review relates the DC endocytic pathways to immune response induction, and the potential advantages for these self-replicating RNA vaccines in the near future.

Treatment planning of electroporation-based medical interventions: electrochemotherapy, gene electrotransfer and irreversible electroporation

International Nuclear Information System (INIS)

Zupanic, Anze; Kos, Bor; Miklavcic, Damijan

2012-01-01

In recent years, cancer electrochemotherapy (ECT), gene electrotransfer for gene therapy and DNA vaccination (GET) and tissue ablation with irreversible electroporation (IRE) have all entered clinical practice. We present a method for a personalized treatment planning procedure for ECT, GET and IRE, based on medical image analysis, numerical modelling of electroporation and optimization with the genetic algorithm, and several visualization tools for treatment plan assessment. Each treatment plan provides the attending physician with optimal positions of electrodes in the body and electric pulse parameters for optimal electroporation of the target tissues. For the studied case of a deep-seated tumour, the optimal treatment plans for ECT and IRE require at least two electrodes to be inserted into the target tissue, thus lowering the necessary voltage for electroporation and limiting damage to the surrounding healthy tissue. In GET, it is necessary to place the electrodes outside the target tissue to prevent damage to target cells intended to express the transfected genes. The presented treatment planning procedure is a valuable tool for clinical and experimental use and evaluation of electroporation-based treatments. (paper)

Flow-through electroporation based on constant voltage for large-volume transfection of cells.

Science.gov (United States)

Geng, Tao; Zhan, Yihong; Wang, Hsiang-Yu; Witting, Scott R; Cornetta, Kenneth G; Lu, Chang

2010-05-21

Genetic modification of cells is a critical step involved in many cell therapy and gene therapy protocols. In these applications, cell samples of large volume (10(8)-10(9)cells) are often processed for transfection. This poses new challenges for current transfection methods and practices. Here we present a novel flow-through electroporation method for delivery of genes into cells at high flow rates (up to approximately 20 mL/min) based on disposable microfluidic chips, a syringe pump, and a low-cost direct current (DC) power supply that provides a constant voltage. By eliminating pulse generators used in conventional electroporation, we dramatically lowered the cost of the apparatus and improved the stability and consistency of the electroporation field for long-time operation. We tested the delivery of pEFGP-C1 plasmids encoding enhanced green fluorescent protein into Chinese hamster ovary (CHO-K1) cells in the devices of various dimensions and geometries. Cells were mixed with plasmids and then flowed through a fluidic channel continuously while a constant voltage was established across the device. Together with the applied voltage, the geometry and dimensions of the fluidic channel determined the electrical parameters of the electroporation. With the optimal design, approximately 75% of the viable CHO cells were transfected after the procedure. We also generalize the guidelines for scaling up these flow-through electroporation devices. We envision that this technique will serve as a generic and low-cost tool for a variety of clinical applications requiring large volume of transfected cells. Copyright 2010 Elsevier B.V. All rights reserved.

The ribosome uses two active mechanisms to unwind messenger RNA during translation.

Science.gov (United States)

Qu, Xiaohui; Wen, Jin-Der; Lancaster, Laura; Noller, Harry F; Bustamante, Carlos; Tinoco, Ignacio

2011-07-06

The ribosome translates the genetic information encoded in messenger RNA into protein. Folded structures in the coding region of an mRNA represent a kinetic barrier that lowers the peptide elongation rate, as the ribosome must disrupt structures it encounters in the mRNA at its entry site to allow translocation to the next codon. Such structures are exploited by the cell to create diverse strategies for translation regulation, such as programmed frameshifting, the modulation of protein expression levels, ribosome localization and co-translational protein folding. Although strand separation activity is inherent to the ribosome, requiring no exogenous helicases, its mechanism is still unknown. Here, using a single-molecule optical tweezers assay on mRNA hairpins, we find that the translation rate of identical codons at the decoding centre is greatly influenced by the GC content of folded structures at the mRNA entry site. Furthermore, force applied to the ends of the hairpin to favour its unfolding significantly speeds translation. Quantitative analysis of the force dependence of its helicase activity reveals that the ribosome, unlike previously studied helicases, uses two distinct active mechanisms to unwind mRNA structure: it destabilizes the helical junction at the mRNA entry site by biasing its thermal fluctuations towards the open state, increasing the probability of the ribosome translocating unhindered; and it mechanically pulls apart the mRNA single strands of the closed junction during the conformational changes that accompany ribosome translocation. The second of these mechanisms ensures a minimal basal rate of translation in the cell; specialized, mechanically stable structures are required to stall the ribosome temporarily. Our results establish a quantitative mechanical basis for understanding the mechanism of regulation of the elongation rate of translation by structured mRNAs. ©2011 Macmillan Publishers Limited. All rights reserved

Perspectives on Transdermal Electroporation

Science.gov (United States)

Ita, Kevin

2016-01-01

Transdermal drug delivery offers several advantages, including avoidance of erratic absorption, absence of gastric irritation, painlessness, noninvasiveness, as well as improvement in patient compliance. With this mode of drug administration, there is no pre-systemic metabolism and it is possible to increase drug bioavailability and half-life. However, only a few molecules can be delivered across the skin in therapeutic quantities. This is because of the hindrance provided by the stratum corneum. Several techniques have been developed and used over the last few decades for transdermal drug delivery enhancement. These include sonophoresis, iontophoresis, microneedles, and electroporation. Electroporation, which refers to the temporary perturbation of the skin following the application of high voltage electric pulses, has been used to increase transcutaneous flux values by several research groups. In this review, transdermal electroporation is discussed and the use of the technique for percutaneous transport of low and high molecular weight compounds described. This review also examines our current knowledge regarding the mechanisms of electroporation and safety concerns arising from the use of this transdermal drug delivery technique. Safety considerations are especially important because electroporation utilizes high voltage pulses which may have deleterious effects in some cases. PMID:26999191

Alterations in polyribosome and messenger ribonucleic acid metabolism and messenger ribonucleoprotein utilization in osmotically stressed plant seedlings

International Nuclear Information System (INIS)

Mason, H.S.

1986-01-01

Polyribosome aggregation state in growing tissues of barley and wheat leaf of stems of pea and squash was studied in relation to seedling growth and water status of the growing tissue in plants at various levels of osmotic stress. It was found to be highly correlated with water potential and osmotic potential of the growing tissue and with leaf of stem elongation rate. Stress rapidly reduced polyribosome content and water status in growing tissues of barley leaves; changes were slow and slight in the non-growing leaf blade. Membrane-bound and free polyribosomes were equally sensitive to stress-induced disaggregation. Incorporation of 32 PO 4 3- into ribosomal RNA was rapidly inhibited by stress, but stability of poly(A) + RNA relative to ribosomal RNA was similar in stressed and unstressed tissues, with a half-life of about 12 hours. Stress also caused progressive loss of poly(A) + RNA from these tissues. Quantitation of poly(A) and in vitro messenger template activity in polysome gradient fractions showed a shift of activity from the polysomal region to the region of 20-60 S in stressed plants. Messenger RNA in the 20-60 S region coded for the same peptides as mRNA found in the polysomal fraction. Nonpolysomal and polysome-derived messenger ribonucleoprotein complexes (mRNP) were isolated, and characteristic proteins were found associated with either fraction. Polysomal mRNP from stressed or unstressed plants were translated with similar efficiency in a wheat germ cell-free system. It was concluded that no translational inhibitory activity was associated with nonpolysomal mRNP from barley prepared as described

In vivo expression of ß-galactosidase by rat oviduct exposed to naked DNA or messenger RNA

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MARIANA RIOS

2002-01-01

Full Text Available Intra-oviductal administration of RNA obtained from oviducts of estradiol-treated rats resulted in accelerated egg transport (Ríos et al., 1997. It is probable that estradiol-induced messenger RNA (mRNA entered oviductal cells and was translated into the proteins involved in accelerated egg transport. In order to test this interpretation we deposited in vivo 50 µg of pure ß-galactosidase (ß-gal mRNA, 50 µg of pure DNA from the reporter gene ß-gal under SV40 promoter or the vehicle (control oviducts into the oviductal lumen of rats. Twenty four hours later the ß-gal activity was assayed in oviductal tissue homogenates using o-nitrophenyl-ß-D-galactopyranoside as a substrate. The administration of ß-gal mRNA and pSVBgal plasmid increased ß-gal activity by 71% and 142%, respectively, over the control oviducts. These results indicate that naked DNA and mRNA coding for ß-gal can enter oviductal cells and be translated into an active enzyme. They are consistent with the interpretation that embryo transport acceleration caused by the injection of estradiol-induced RNA in the oviduct involves translation of the injected mRNA

Primary structure of the α-subunit of Na+, K+-ATPase. II. Isolation, reverse transcription, and cloning of messenger RNA

International Nuclear Information System (INIS)

Petrukhin, K.E.; Broude, N.E.; Arsenyan, S.G.; Grishin, A.V.; Dzhandzhugazyan, K.N.; Modyanov, N.N.

1986-01-01

The messenger RNA coding the α-subunit of Na + ,K + -ATPase has been isolated from the outer medullary layer of porcine kidneys. The mRNA gives a specific hybridization band in the 25S-26S region with three oligonucleotide probes synthesized on the basis of information on the structure of three peptides isolated from a tryptic hydrolyzate of the α-subunit of Na + ,K + -ATPase. The translation of the mRNA in Xenopus laevis oocytes followed by immunochemical identification of the products of synthesis confirmed the presence of the mRNA of the α-subunit of Na + ,K + -ATPase in an enriched fraction of poly(A + )-RNA. This preparation has been used for the synthesis of cloning of double-stranded cDNA

Electroporation in food processing and biorefinery.

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Mahnič-Kalamiza, Samo; Vorobiev, Eugène; Miklavčič, Damijan

2014-12-01

Electroporation is a method of treatment of plant tissue that due to its nonthermal nature enables preservation of the natural quality, colour and vitamin composition of food products. The range of processes where electroporation was shown to preserve quality, increase extract yield or optimize energy input into the process is overwhelming, though not exhausted; e.g. extraction of valuable compounds and juices, dehydration, cryopreservation, etc. Electroporation is--due to its antimicrobial action--a subject of research as one stage of the pasteurization or sterilization process, as well as a method of plant metabolism stimulation. This paper provides an overview of electroporation as applied to plant materials and electroporation applications in food processing, a quick summary of the basic technical aspects on the topic, and a brief discussion on perspectives for future research and development in the field. The paper is a review in the very broadest sense of the word, written with the purpose of orienting the interested newcomer to the field of electroporation applications in food technology towards the pertinent, highly relevant and more in-depth literature from the respective subdomains of electroporation research.

Development of mRNA-based body fluid identification using reverse transcription loop-mediated isothermal amplification.

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Satoh, Tetsuya; Kouroki, Seiya; Ogawa, Keita; Tanaka, Yorika; Matsumura, Kazutoshi; Iwase, Susumu

2018-04-25

Identifying body fluids from forensic samples can provide valuable evidence for criminal investigations. Messenger RNA (mRNA)-based body fluid identification was recently developed, and highly sensitive parallel identification using reverse transcription polymerase chain reaction (RT-PCR) has been described. In this study, we developed reverse transcription loop-mediated isothermal amplification (RT-LAMP) as a simple, rapid assay for identifying three common forensic body fluids, namely blood, semen, and saliva, and evaluated its specificity and sensitivity. Hemoglobin beta (HBB), transglutaminase 4 (TGM4), and statherin (STATH) were selected as marker genes for blood, semen, and saliva, respectively. RT-LAMP could be performed in a single step including both reverse transcription and DNA amplification under an isothermal condition within 60 min, and detection could be conveniently performed via visual fluorescence. Marker-specific amplification was performed in each assay, and no cross-reaction was observed among five representative forensically relevant body fluids. The detection limits of the assays were 0.3 nL, 30 nL, and 0.3 μL for blood, semen, and saliva, respectively, and their sensitivities were comparable with those of RT-PCR. Furthermore, RT-LAMP assays were applicable to forensic casework samples. It is considered that RT-LAMP is useful for body fluid identification.

Assessment of flhDC mRNA levels in Serratia liquefaciens swarm cells

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Tolker-Nielsen, Tim; Christensen, Allan Beck; Holmstrøm, K.

2000-01-01

We reported previously that artificial overexpression of the flhDC operon in liquid-grown Serratia liquefaciens resulted in the formation of filamentous, multinucleated, and hyperflagellated cells that were indistinguishable from surface-induced swarm cells (L. Eberl, G. Christiansen, S. Molin, a......, vegetative cells. This suggests that surface-induced S. liquefaciens swarm cell differentiation, although dependent on flhDC gene expression, does not occur through elevated flhDC mRNA levels....

Altered expression of estrogen receptor-α variant messenger RNAs between adjacent normal breast and breast tumor tissues

International Nuclear Information System (INIS)

Leygue, Etienne; Dotzlaw, Helmut; Watson, Peter H; Murphy, Leigh C

2000-01-01

Using semiquantitative reverse transcription-polymerase chain reaction assays, we investigated the expression of variant messenger RNAs relative to wild-type estrogen receptor (ER)-α messenger RNA in normal breast tissues and their adjacent matched breast tumor tissues. Higher ER variant truncated after sequences encoding exon 2 of the wild-type ER-α (ERC4) messenger RNA and a lower exon 3 deleted ER-α variant (ERD3) messenger RNA relative expression in the tumor compartment were observed in the ER-positive/PR-positive and the ER-positive subsets, respectively. A significantly higher relative expression of exon 5 deleted ER-α varient (ERD5) messenger RNA was observed in tumor components overall. These data demonstrate that changes in the relative expression of ER-α variant messenger RNAs occur between adjacent normal and neoplastic breast tissues. We suggest that these changes might be involved in the mechanisms that underlie breast tumorigenesis. Estrogen receptor (ER)-α and ER-β are believed to mediate the action of estradiol in target tissues. Several ER-α and ER-β variant messenger RNAs have been identified in both normal and neoplastic human tissues. Most of these variants contain a deletion of one or more exons of the wild-type (WT) ER messenger RNAs. The putative proteins that are encoded by these variant messenger RNAs would therefore be missing some functional domains of the WT receptors, and might interfere with WT-ER signaling pathways. The detection of ER-α variants in both normal and neoplastic human breast tissues raised the question of their possible role in breast tumorigenesis. We have previously reported an increased relative expression of exon 5 deleted ER-α variant (ERD5) messenger RNA and of another ER-α variant truncated of all sequences following the exon 2 of the WT ER-α (ERC4) messenger RNA in breast tumor samples versus independent normal breast tissues. In contrast, a decreased relative expression of exon 3 deleted ER

Optimised electroporation mediated DNA vaccination for treatment of prostate cancer.

LENUS (Irish Health Repository)

Ahmad, Sarfraz

2010-01-01

ABSTRACT: BACKGROUND: Immunological therapies enhance the ability of the immune system to recognise and destroy cancer cells via selective killing mechanisms. DNA vaccines have potential to activate the immune system against specific antigens, with accompanying potent immunological adjuvant effects from unmethylated CpG motifs as on prokaryotic DNA. We investigated an electroporation driven plasmid DNA vaccination strategy in animal models for treatment of prostate cancer. METHODS: Plasmid expressing human PSA gene (phPSA) was delivered in vivo by intra-muscular electroporation, to induce effective anti-tumour immune responses against prostate antigen expressing tumours. Groups of male C57 BL\\/6 mice received intra-muscular injections of phPSA plasmid. For phPSA delivery, quadriceps muscle was injected with 50 mug plasmid. After 80 seconds, square-wave pulses were administered in sequence using a custom designed pulse generator and acustom-designed applicator with 2 needles placed through the skin central to the muscle. To determine an optimum treatment regimen, three different vaccination schedules were investigated. In a separate experiment, the immune potential of the phPSA vaccine was further enhanced with co- administration of synthetic CpG rich oligonucleotides. One week after last vaccination, the mice were challenged subcutaneously with TRAMPC1\\/hPSA (prostate cancer cell line stably expressing human PSA) and tumour growth was monitored. Serum from animals was examined by ELISA for anti-hPSA antibodies and for IFNgamma. Histological assessment of the tumours was also carried out. In vivo and in vitro cytotoxicity assays were performed with splenocytes from treated mice. RESULTS: The phPSA vaccine therapy significantly delayed the appearance of tumours and resulted in prolonged survival of the animals. Four-dose vaccination regimen provided optimal immunological effects. Co - administration of the synthetic CpG with phPSA increased anti-tumour responses

Covalent Strategies for Targeting Messenger and Non-Coding RNAs: An Updated Review on siRNA, miRNA and antimiR Conjugates

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Santiago Grijalvo

2018-02-01

Full Text Available Oligonucleotide-based therapy has become an alternative to classical approaches in the search of novel therapeutics involving gene-related diseases. Several mechanisms have been described in which demonstrate the pivotal role of oligonucleotide for modulating gene expression. Antisense oligonucleotides (ASOs and more recently siRNAs and miRNAs have made important contributions either in reducing aberrant protein levels by sequence-specific targeting messenger RNAs (mRNAs or restoring the anomalous levels of non-coding RNAs (ncRNAs that are involved in a good number of diseases including cancer. In addition to formulation approaches which have contributed to accelerate the presence of ASOs, siRNAs and miRNAs in clinical trials; the covalent linkage between non-viral vectors and nucleic acids has also added value and opened new perspectives to the development of promising nucleic acid-based therapeutics. This review article is mainly focused on the strategies carried out for covalently modifying siRNA and miRNA molecules. Examples involving cell-penetrating peptides (CPPs, carbohydrates, polymers, lipids and aptamers are discussed for the synthesis of siRNA conjugates whereas in the case of miRNA-based drugs, this review article makes special emphasis in using antagomiRs, locked nucleic acids (LNAs, peptide nucleic acids (PNAs as well as nanoparticles. The biomedical applications of siRNA and miRNA conjugates are also discussed.

A statistical model describing combined irreversible electroporation and electroporation-induced blood-brain barrier disruption.

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Sharabi, Shirley; Kos, Bor; Last, David; Guez, David; Daniels, Dianne; Harnof, Sagi; Mardor, Yael; Miklavcic, Damijan

2016-03-01

Electroporation-based therapies such as electrochemotherapy (ECT) and irreversible electroporation (IRE) are emerging as promising tools for treatment of tumors. When applied to the brain, electroporation can also induce transient blood-brain-barrier (BBB) disruption in volumes extending beyond IRE, thus enabling efficient drug penetration. The main objective of this study was to develop a statistical model predicting cell death and BBB disruption induced by electroporation. This model can be used for individual treatment planning. Cell death and BBB disruption models were developed based on the Peleg-Fermi model in combination with numerical models of the electric field. The model calculates the electric field thresholds for cell kill and BBB disruption and describes the dependence on the number of treatment pulses. The model was validated using in vivo experimental data consisting of rats brains MRIs post electroporation treatments. Linear regression analysis confirmed that the model described the IRE and BBB disruption volumes as a function of treatment pulses number (r(2) = 0.79; p disruption, the ratio increased with the number of pulses. BBB disruption radii were on average 67% ± 11% larger than IRE volumes. The statistical model can be used to describe the dependence of treatment-effects on the number of pulses independent of the experimental setup.

RNA decay by messenger RNA interferases

DEFF Research Database (Denmark)

Christensen-Dalsgaard, Mikkel; Overgaard, Martin; Winther, Kristoffer Skovbo

2008-01-01

Two abundant toxin-antitoxin (TA) gene families, relBE and mazEF, encode mRNA cleaving enzymes whose ectopic overexpression abruptly inhibits translation and thereby induces a bacteriostatic condition. Here we describe and discuss protocols for the overproduction, purification, and analysis of mR...... cleaving enzymes such as RelE of Escherichia coli and the corresponding antitoxin RelB. In particular, we describe a set of plasmid vectors useful for the detailed analysis of cleavage sites in model mRNAs.......Two abundant toxin-antitoxin (TA) gene families, relBE and mazEF, encode mRNA cleaving enzymes whose ectopic overexpression abruptly inhibits translation and thereby induces a bacteriostatic condition. Here we describe and discuss protocols for the overproduction, purification, and analysis of mRNA...

mRNA: From a chemical blueprint for protein production to an off-the-shelf therapeutic.

Science.gov (United States)

Van Lint, Sandra; Heirman, Carlo; Thielemans, Kris; Breckpot, Karine

2013-02-01

Two decades ago, mRNA became the focus of research in molecular medicine and was proposed as an active pharmaceutical ingredient for the therapy of cancer. In this regard, mRNA has been mainly used for ex vivo modification of antigen-presenting cells (APCs), such as dendritic cells (DCs). This vaccination strategy has proven to be safe, well tolerated and capable of inducing tumor antigen-specific immune responses. Recently, the direct application of mRNA for in situ modification of APCs, hence immunization was shown to be feasible and at least as effective as DC-based immunization in pre-clinical models. It is believed that application of mRNA as an off-the-shelf vaccine represents an important step in the development of future cancer immunotherapeutic strategies. Here, we will discuss the use of ex vivo mRNA-modified DCs and "naked mRNA" for cancer immunotherapy focusing on parameters such as the employed DC subtype, DC activation stimulus and route of immunization. In addition, we will provide an overview on the clinical trials published so far, trying to link their outcome to the aforementioned parameters.

DNA Methylation of MMP9 Is Associated with High Levels of MMP-9 Messenger RNA in Periapical Inflammatory Lesions.

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Campos, Kelma; Gomes, Carolina Cavalieri; Farias, Lucyana Conceição; Silva, Renato Menezes; Letra, Ariadne; Gomez, Ricardo Santiago

2016-01-01

Matrix metalloproteinases (MMPs) are the major class of enzymes responsible for degradation of extracellular matrix components and participate in the pathogenesis of periapical inflammatory lesions. MMP expression may be regulated by DNA methylation. The purpose of the present investigation was to analyze the expression of MMP2 and MMP9 in periapical granulomas and radicular cysts and to test the hypothesis that, in these lesions, their transcription may be modulated by DNA methylation. Methylation-specific polymerase chain reaction was used to evaluate the DNA methylation pattern of the MMP2 gene in 13 fresh periapical granuloma samples and 10 fresh radicular cyst samples. Restriction enzyme digestion was used to assess methylation of the MMP9 gene in 12 fresh periapical granuloma samples and 10 fresh radicular cyst samples. MMP2 and MMP9 messenger RNA transcript levels were measured by quantitative real-time polymerase chain reaction. All periapical lesions and healthy mucosa samples showed partial methylation of the MMP2 gene; however, periapical granulomas showed higher MMP2 mRNA expression levels than healthy mucosa (P = .014). A higher unmethylated profile of the MMP9 gene was found in periapical granulomas and radicular cysts compared with healthy mucosa. In addition, higher MMP9 mRNA expression was observed in the periapical lesions compared with healthy tissues. The present study suggests that the unmethylated status of the MMP9 gene in periapical lesions may explain the observed up-regulation of messenger RNA transcription in these lesions. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Skeletal muscle microRNA and messenger RNA profiling in cofilin-2 deficient mice reveals cell cycle dysregulation hindering muscle regeneration.

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Sarah U Morton

Full Text Available Congenital myopathies are rare skeletal muscle diseases presenting in early age with hypotonia and weakness often linked to a genetic defect. Mutations in the gene for cofilin-2 (CFL2 have been identified in several families as a cause of congenital myopathy with nemaline bodies and cores. Here we explore the global messenger and microRNA expression patterns in quadriceps muscle samples from cofillin-2-null mice and compare them with sibling-matched wild-type mice to determine the molecular pathways and mechanisms involved. Cell cycle processes are markedly dysregulated, with altered expression of genes involved in mitotic spindle formation, and evidence of loss of cell cycle checkpoint regulation. Importantly, alterations in cell cycle, apoptosis and proliferation pathways are present in both mRNA and miRNA expression patterns. Specifically, p21 transcript levels were increased, and the expression of p21 targets, such as cyclin D and cyclin E, was decreased. We therefore hypothesize that deficiency of cofilin-2 is associated with interruption of the cell cycle at several checkpoints, hindering muscle regeneration. Identification of these pathways is an important step towards developing appropriate therapies against various congenital myopathies.

Electroporation-induced electrosensitization.

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Olga N Pakhomova

Full Text Available BACKGROUND: Electroporation is a method of disrupting the integrity of cell membrane by electric pulses (EPs. Electrical modeling is widely employed to explain and study electroporation, but even most advanced models show limited predictive power. No studies have accounted for the biological consequences of electroporation as a factor that alters the cell's susceptibility to forthcoming EPs. METHODOLOGY/PRINCIPAL FINDINGS: We focused first on the role of EP rate for membrane permeabilization and lethal effects in mammalian cells. The rate was varied from 0.001 to 2,000 Hz while keeping other parameters constant (2 to 3,750 pulses of 60-ns to 9-µs duration, 1.8 to 13.3 kV/cm. The efficiency of all EP treatments was minimal at high rates and started to increase gradually when the rate decreased below a certain value. Although this value ranged widely (0.1-500 Hz, it always corresponded to the overall treatment duration near 10 s. We further found that longer exposures were more efficient irrespective of the EP rate, and that splitting a high-rate EP train in two fractions with 1-5 min delay enhanced the effects severalfold. CONCLUSIONS/SIGNIFICANCE: For varied experimental conditions, EPs triggered a delayed and gradual sensitization to EPs. When a portion of a multi-pulse exposure was delivered to already sensitized cells, the overall effect markedly increased. Because of the sensitization, the lethality in EP-treated cells could be increased from 0 to 90% simply by increasing the exposure duration, or the exposure dose could be reduced twofold without reducing the effect. Many applications of electroporation can benefit from accounting for sensitization, by organizing the exposure either to maximize sensitization (e.g., for sterilization or, for other applications, to completely or partially avoid it. In particular, harmful side effects of electroporation-based therapies (electrochemotherapy, gene therapies, tumor ablation include convulsions

Detailed mapping of serotonin 5-HT1B and 5-HT1D receptor messenger RNA and ligand binding sites in guinea-pig brain and trigeminal ganglion: clues for function

International Nuclear Information System (INIS)

Leysen, J.E.; Schotte, A.; Jurzak, M.; Luyten, W.H.M.L.; Voorn, P.; Bonaventure, P.

1997-01-01

The similar pharmacology of the 5-HT 1B and 5-HT 1D receptors, and the lack of selective compounds sufficiently distinguishing between the two receptor subtypes, have hampered functional studies on these receptors. In order to provide clues for differential functional roles of the two subtypes, we performed a parallel localization study throughout the guinea-pig brain and the trigeminal ganglia by means of quantitative in situ hybridization histochemistry (using [ 35 S]-labelled riboprobes probes for receptor messenger RNA) and receptor autoradiography (using a new radioligand [ 3 H]alniditan).The anatomical patterns of 5-HT 1B and 5-HT 1D receptor messenger RNA were quite different. While 5-HT 1B receptor messenger RNA was abundant throughout the brain (with highest levels in the striatum, nucleus accumbens, olfactory tubercle, cortex, hypothalamus, hippocampal formation, amygdala, thalamus, dorsal raphe and cerebellum), 5-HT 1D receptor messenger RNA exhibited a more restricted pattern; it was found mainly in the olfactory tubercle, entorhinal cortex, dorsal raphe, cerebellum, mesencephalic trigeminal nucleus and in the trigeminal ganglion. The density of 5-HT 1B/1D binding sites (combined) obtained with [ 3 H]alniditan autoradiography was high in the substantia nigra, superior colliculus and globus pallidus, whereas lower levels were detected in the caudate-putamen, hypothalamus, hippocampal formation, amygdala, thalamus and central gray. This distribution pattern was indistinguishable from specific 5-HT 1B receptor labelling in the presence of ketanserin under conditions to occlude 5-HT 1D receptor labelling; hence the latter were below detection level. Relationships between the regional distributions of the receptor messenger RNAs and binding sites and particular neuroanatomical pathways are discussed with respect to possible functional roles of the 5-HT 1B and 5-HT 1D receptors. (Copyright (c) 1997 Elsevier Science B.V., Amsterdam. All rights reserved.)

Reprogramming human umbilical cord mesenchymal stromal cells to islet-like cells with the use of in vitro-synthesized pancreatic-duodenal homebox 1 messenger RNA.

Science.gov (United States)

Wang, Xiao Li; Hu, Pei; Guo, Xing Rong; Yan, Ding; Yuan, Yahong; Yan, Shi Rong; Li, Dong Sheng

2014-11-01

Human umbilical cord mesenchymal stromal cells (hUC-MSCs) hold great potential as a therapeutic candidate to treat diabetes, owing to their unlimited source and ready availability. In this study, we differentiated hUC-MSCs with in vitro-synthesized pancreatic-duodenal homebox 1 (PDX1) messenger (m)RNA into islet-like cell clusters. hUC-MSCs were confirmed by both biomarker detection and functional differentiation. In vitro-synthesized PDX1 messenger RNA can be transfected into hUC-MSCs efficiently. The upregulated expression of PDX1 protein can be detected 4 h after transfection and remains detectable for 36 h. The induction of islet-like structures was confirmed by means of morphology and dithizone staining. Reverse transcriptase-polymerase chain reaction results revealed the expression of some key pancreatic transcription factors, such as PDX1, NeuroD, NKX6.1, Glut-2 and insulin in islet-like cell clusters. Immunofluorescence analysis showed that differentiated cells express both insulin and C-peptide. Enzyme-linked immunosorbent assay analysis validated the insulin secretion of islet-like cell clusters in response to the glucose stimulation. Our results demonstrate the use of in vitro-synthesized PDX1 messenger RNA to differentiate hUC-MSCs into islet-like cells and pave the way toward the development of reprogramming and directed-differentiation methods for the expression of encoded proteins. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

FcepsilonRI-alpha siRNA inhibits the antigen-induced activation of mast cells.

Science.gov (United States)

Safaralizadeh, Reza; Soheili, Zahra-Soheila; Deezagi, Abdolkhaleg; Pourpak, Zahra; Samiei, Shahram; Moin, Mostafa

2009-12-01

FcepsilonRI, The high affinity receptor for IgE plays a critical role in triggering the allergic reactions. It is responsible for inducing mast cell degranulation and deliberation of allergy mediators when it is aggregated by allergen and IgE complexes. FcepsilonRI on the mast cells consists of three subunits; alpha chain directly binds IgE, beta chain and dimmer of gamma chains together mediate intracellular signaling. Cross-linking of IgE-bound FcepsilonRI on the surface of mast cells and basophils by the multivalent antigen induces release of chemical mediators. The present in vitro study was designed to investigate the effect of synthetic FcepsilonRI-alpha siRNA on the antigen-induced activation of MC/9 cells. MC/9 cells which are murine mast cells were transfected by FcepsilonRI-alpha siRNA and negative control siRNA. After 6 h, anti-DNP (Dinitrophenyl) IgE was used for the cells sensitization. Then the cells were challenged with Dinitrophenyl-Human Serum Albumin (DNP-HSA) for mast cell degranulation induction before collection of supernatants. The amount of mRNA and protein expression was measured by Real Time PCR and western blot analysis, respectively. Determination of the expression rate of FcepsilonRI-alpha on cell surface was achieved by flow cytometry. ELISA and spectrophotometry methods were used subsequently for measuring the effects of FcepsilonRI-alpha siRNA on antigen-induced histamine and beta-hexosaminidase release. FcepsilonRI-alpha siRNA treated cells showed significant decrease in FcepsilonRI-alpha mRNA and protein expression in comparison to control cells. FcepsilonRI-mediated mast cell release of beta-hexosaminidase and histamine were also inhibited. In this study it was shown that FcepsilonRI-alpha siRNA could suppress FcepsilonRI-alpha expression and inhibited degranulation and histamine release in antigen-stimulated MC/9 cells. In conclusion, knock-down of FcepsilonRI-alpha by siRNA could be a promising method for inhibition of the mast

Electrochemical Branched-DNA Assay for Polymerase Chain Reaction-Free Detection and Quantification of Oncogenes in Messenger RNA

Energy Technology Data Exchange (ETDEWEB)

Lee, Ai Cheng; Dai, Ziyu; Chen, Baowei; Wu, Hong; Wang, Jun; Zhang, Aiguo; Zhang, Lurong; Lim, Tit-Meng; Lin, Yuehe

2008-12-01

We describe a novel electrochemical branched-DNA (bDNA) assay for polymerase chain reaction (PCR)-free detection and quantification of p185 BCR-ABL leukemia fusion transcript in the population of messenger RNA (mRNA) extracted from cell lines. The bDNA amplifier carrying high loading of alkaline phosphatase (ALP) tracers was used to amplify targets signal. The targets were captured on microplate well surfaces through cooperative sandwich hybridization prior to the labeling of bDNA. The activity of captured ALP was monitored by square-wave voltammetric (SWV) analysis of the electroactive enzymatic product in the presence of 1-napthyl-phosphate. The specificity and sensitivity of assay enabled direct detection of target transcript in as little as 4.6 ng mRNA without PCR amplification. In combination with the use of a well-quantified standard, the electrochemical bDNA assay was capable of direct use for a PCR-free quantitative analysis of target transcript in total mRNA population. The approach thus provides a simple, sensitive, accurate and quantitative tool alternate to the RQ-PCR for early disease diagnosis.

A full scale comparative study of methods for generation of functional Dendritic cells for use as cancer vaccines

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Kvalheim Gunnar

2007-07-01

Full Text Available Background Dendritic cells (DCs are professional antigen-presenting cells with the ability to induce primary T-cell responses and are commonly produced by culturing monocytes in the presence of IL-4 and GM-CSF for 5–7 days (Standard DC. Recently, Dauer and co-workers presented a modified protocol for differentiation of human monocytes into mature DCs within 48 hours (Fast DC. Here we report a functional comparison of the two strategies for generation of DCs from human monocytes with adaptions for large-scale clinical use. Methods The Elutra Cell Selection System was used to isolate monocytes after collection of leukapheresis product. The enriched monocytes were cultured in gas permeable Teflon bags with IL-4 and GM-CSF for 24 hours (Fast DC or 5 days (Standard DC to obtain immature DCs. The cells were then transfected with mRNA from the leukemia cell line Jurkat E6 by electroporation and incubated for additional 24 h or 2 days in the presence of pro-inflammatory cytokines (TNFα, IL-1β, IL-6 and PGE2 to obtain mature DCs. Results Mature Fast DC and Standard DC displayed comparable levels of many markers expressed on DC, including HLA-DR, CD83, CD86, CD208 and CCR7. However, compared to Standard DC, mature Fast DC was CD14high CD209low. Fast DC and Standard DC transfected with Jurkat E6-cell mRNA were equally able to elicit T cell specifically recognizing transfected DCs in vitro. IFNγ-secreting T cells were observed in both the CD4+ and CD8+ subsets. Conclusion Our results indicate that mature Fast DC are functional antigen presenting cells (APCs capable of inducing primary T-cell responses, and suggest that these cells may be valuable for generation of anti-tumor vaccines.

A full scale comparative study of methods for generation of functional Dendritic cells for use as cancer vaccines.

Science.gov (United States)

Jarnjak-Jankovic, Silvija; Hammerstad, Hege; Saebøe-Larssen, Stein; Kvalheim, Gunnar; Gaudernack, Gustav

2007-07-03

Dendritic cells (DCs) are professional antigen-presenting cells with the ability to induce primary T-cell responses and are commonly produced by culturing monocytes in the presence of IL-4 and GM-CSF for 5-7 days (Standard DC). Recently, Dauer and co-workers presented a modified protocol for differentiation of human monocytes into mature DCs within 48 hours (Fast DC). Here we report a functional comparison of the two strategies for generation of DCs from human monocytes with adaptions for large-scale clinical use. The Elutra Cell Selection System was used to isolate monocytes after collection of leukapheresis product. The enriched monocytes were cultured in gas permeable Teflon bags with IL-4 and GM-CSF for 24 hours (Fast DC) or 5 days (Standard DC) to obtain immature DCs. The cells were then transfected with mRNA from the leukemia cell line Jurkat E6 by electroporation and incubated for additional 24 h or 2 days in the presence of pro-inflammatory cytokines (TNFalpha, IL-1beta, IL-6 and PGE2) to obtain mature DCs. Mature Fast DC and Standard DC displayed comparable levels of many markers expressed on DC, including HLA-DR, CD83, CD86, CD208 and CCR7. However, compared to Standard DC, mature Fast DC was CD14high CD209low. Fast DC and Standard DC transfected with Jurkat E6-cell mRNA were equally able to elicit T cell specifically recognizing transfected DCs in vitro. IFNgamma-secreting T cells were observed in both the CD4+ and CD8+ subsets. Our results indicate that mature Fast DC are functional antigen presenting cells (APCs) capable of inducing primary T-cell responses, and suggest that these cells may be valuable for generation of anti-tumor vaccines.

RNA interference of two glutathione S-transferase genes, Diaphorina citri DcGSTe2 and DcGSTd1, increases the susceptibility of Asian citrus psyllid (Hemiptera: Liviidae) to the pesticides fenpropathrin and thiamethoxam.

Science.gov (United States)

Yu, Xiudao; Killiny, Nabil

2018-03-01

The Asian citrus psyllid, Diaphorina citri Kuwayama, is an important agricultural pest of citrus globally. Foliar application of chemical insecticides is the most widely used option for reducing D. citri populations. Knockdown of glutathione S-transferase (GST) in several insect species leads to increased susceptibility to insecticides; however, information about the detoxifying role of GST genes in D. citri is unavailable. Via a sequence homology search, we isolated and characterized three DcGST genes (DcGSTd1, DcGSTe1 and DcGSTe2) from D. citri. Phylogenetic analysis grouped DcGSTd1 into the delta class of GST genes, whereas DcGSTe1 and DcGSTe2 were clustered in the epsilon clade. Gene expression analysis revealed that chlorpyrifos treatment increased the mRNA levels of DcGSTe1 and fenpropathrin enhanced the expression level of DcGSTd1, while DcGSTe2 was significantly up-regulated after exposure to thiamethoxam at a dose of 30% lethal concentration (LC30). RNA interference (RNAi) of DcGSTe2 and DcGSTd1 followed by an insecticide bioassay increased the mortalities of thiamethoxam-treated psyllids by 23.0% and fenpropathrin-treated psyllids by 15.0%. In contrast, knockdown of DcGSTe1 did not significantly increase the susceptibility of D. citri to any of these three insecticides. Further, feeding with double-stranded RNA (dsDcGSTe2-d1) interfusion co-silenced DcGSTe2 and DcGSTd1 expression in D. citri, and led to an increase of susceptibility to both fenpropathrin and thiamethoxam. The findings suggest that DcGSTe2 and DcGSTd1 play unique roles in detoxification of the pesticides thiamethoxam and fenpropathrin. In addition, co-silencing by creating a well-designed dsRNA interfusion against multiple genes was a good RNAi strategy in D. citri. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Electroporation Enhanced Effect of Dystrophin Splice Switching PNA Oligomers in Normal and Dystrophic Muscle

Directory of Open Access Journals (Sweden)

Camilla Brolin

2015-01-01

Full Text Available Peptide nucleic acid (PNA is a synthetic DNA mimic that has shown potential for discovery of novel splice switching antisense drugs. However, in vivo cellular delivery has been a limiting factor for development, and only few successful studies have been reported. As a possible modality for improvement of in vivo cellular availability, we have investigated the effect of electrotransfer upon intramuscular (i.m. PNA administration in vivo. Antisense PNA targeting exon 23 of the murine dystrophin gene was administered by i.m. injection to the tibialis anterior (TA muscle of normal NMRI and dystrophic mdx mice with or without electroporation. At low, single PNA doses (1.5, 3, or 10 µg/TA, electroporation augmented the antisense exon skipping induced by an unmodified PNA by twofold to fourfold in healthy mouse muscle with optimized electric parameters, measured after 7 days. The PNA splice switching was detected at the RNA level up to 4 weeks after a single-dose treatment. In dystrophic muscles of the MDX mouse, electroporation increased the number of dystrophin-positive fibers about 2.5-fold at 2 weeks after a single PNA administration compared to injection only. In conclusion, we find that electroporation can enhance PNA antisense effects in muscle tissue.

CALiPER Report 22.1: Photoelectric Performance of LED MR16 Lamps

Energy Technology Data Exchange (ETDEWEB)

None, None

2015-08-31

This report looks at the photoelectric performance of the same set of lamps assessed in Report 22, using commercially available transformers and dimmers as well as laboratory power supplies providing either AC or DC. The investigation explores several issues related to the testing and use of MR16 lamps in lighting systems and examines the range of performance that is possible for a given lamp model, based on the system to which it is connected.

Primary structure of the. cap alpha. -subunit of Na/sup +/, K/sup +/-ATPase. II. Isolation, reverse transcription, and cloning of messenger RNA

Energy Technology Data Exchange (ETDEWEB)

Petrukhin, K.E.; Broude, N.E.; Arsenyan, S.G.; Grishin, A.V.; Dzhandzhugazyan, K.N.; Modyanov, N.N.

1986-10-01

The messenger RNA coding the ..cap alpha..-subunit of Na/sup +/,K/sup +/-ATPase has been isolated from the outer medullary layer of porcine kidneys. The mRNA gives a specific hybridization band in the 25S-26S region with three oligonucleotide probes synthesized on the basis of information on the structure of three peptides isolated from a tryptic hydrolyzate of the ..cap alpha..-subunit of Na/sup +/,K/sup +/-ATPase. The translation of the mRNA in Xenopus laevis oocytes followed by immunochemical identification of the products of synthesis confirmed the presence of the mRNA of the ..cap alpha..-subunit of Na/sup +/,K/sup +/-ATPase in an enriched fraction of poly(A/sup +/)-RNA. This preparation has been used for the synthesis of cloning of double-stranded cDNA.

Blockade of OX40/OX40 ligand to decrease cytokine messenger RNA expression in acute renal allograft rejection in vitro.

Science.gov (United States)

Wang, Y-L; Li, G; Fu, Y-X; Wang, H; Shen, Z-Y

2013-01-01

The aim of this study was to investigate cytokine messenger RNA (mRNA) expression by peripheral blood mononuclear cells (PBMCs) from renal recipients experiencing acute rejection by blocking OX40-OX40L interactions with recombinant human OX40-Fc fusion protein (rhOX40Fc) in vitro. PBMCs were isolated from 20 recipients experiencing acute rejection episodes (rejection group) and 20 recipients with stable graft function (stable group). Levels of Th1 (interferon [IFN]-γ) and Th2 (interleukin [IL]-4) mRNA expressions by PBMCs were measured using real-time reverse transcriptase-polymerase chain reactions. IFN-γ mRNA expression levels were significantly higher in the rejection than the stable group (P rejection group, rhOX40Fc reduced significantly the expression of IFN-γ and IL-4 mRNA by anti-CD3-monoclonal antibody stimulated PBMCs (P type cytokines. Copyright © 2013 Elsevier Inc. All rights reserved.

High hydrostatic pressure affects antigenic pool in tumor cells: Implication for dendritic cell-based cancer immunotherapy.

Science.gov (United States)

Urbanova, Linda; Hradilova, Nada; Moserova, Irena; Vosahlikova, Sarka; Sadilkova, Lenka; Hensler, Michal; Spisek, Radek; Adkins, Irena

2017-07-01

High hydrostatic pressure (HHP) can be used to generate dendritic cell (DC)-based active immunotherapy for prostate, lung and ovarian cancer. We showed here that HHP treatment of selected human cancer cell lines leads to a degradation of tumor antigens which depends on the magnitude of HHP applied and on the cancer cell line origin. Whereas prostate or ovarian cell lines displayed little protein antigen degradation with HHP treatment up to 300MPa after 2h, tumor antigens are hardly detected in lung cancer cell line after treatment with HHP 250MPa at the same time. On the other hand, quick reduction of tumor antigen-coding mRNA was observed at HHP 200MPa immediately after treatment in all cell lines tested. To optimize the DC-based active cellular therapy protocol for HHP-sensitive cell lines the immunogenicity of HHP-treated lung cancer cells at 150, 200 and 250MPa was compared. Lung cancer cells treated with HHP 150MPa display characteristics of immunogenic cell death, however cells are not efficiently phagocytosed by DC. Despite induction of the highest number of antigen-specific CD8 + T cells, 150 MPa-treated lung cancer cells survive in high numbers. This excludes their use in DC vaccine manufacturing. HHP of 200MPa treatment of lung cancer cells ensures the optimal ratio of efficient immunogenic killing and delivery of protein antigens in DC. These results represent an important pre-clinical data for generation of immunogenic killed lung cancer cells in ongoing NSCLC Phase I/II clinical trial using DC-based active cellular immunotherapy (DCVAC/LuCa). Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Analysis of antigen-specific B-cell memory directly ex vivo.

Science.gov (United States)

McHeyzer-Williams, Louise J; McHeyzer-Williams, Michael G

2004-01-01

Helper T-cell-regulated B-cell memory develops in response to initial antigen priming as a cellular product of the germinal center (GC) reaction. On antigen recall, memory response precursors expand rapidly with exaggerated differentiation into plasma cells to produce the high-titer, high-affinity antibody(Ab) that typifies the memory B-cell response in vivo. We have devised a high-resolution flow cytometric strategy to quantify the emergence and maintenance of antigen-specific memory B cells directly ex vivo. Extended cell surface phenotype establishes a level of cellular diversity not previously appreciated for the memory B-cell compartment. Using an "exclusion transfer" strategy, we ascertain the capacity of two distinct memory B-cell populations to transfer antigen-specific memory into naive adoptive hosts. Finally, we sequence expressed messenger ribonucleic acid (mRNA) from single cells within the population to estimate the level of somatic hypermutation as the best molecular indicator of B-cell memory. In this chapter, we describe the methods used in each of these four sections that serve to provide high-resolution quantification of antigen-specific B-cell memory responses directly ex vivo.

Tutorial: Electroporation of cells in complex materials and tissue

Science.gov (United States)

Rems, L.; Miklavčič, D.

2016-05-01

Electroporation is being successfully used in biology, medicine, food processing, and biotechnology, and in some environmental applications. Recent applications also include in addition to classical electroporation, where cells are exposed to micro- or milliseconds long pulses, exposures to extremely short nanosecond pulses, i.e., high-frequency electroporation. Electric pulses are applied to cells in different structural configurations ranging from suspended cells to cells in tissues. Understanding electroporation of cells in tissues and other complex environments is a key to its successful use and optimization in various applications. Thus, explanation will be provided theoretically/numerically with relation to experimental observations by scaling our understanding of electroporation from the molecular level of the cell membrane up to the tissue level.

Porphyromonas gingivalis evasion of autophagy and intracellular killing by human myeloid dendritic cells involves DC-SIGN-TLR2 crosstalk.

Science.gov (United States)

El-Awady, Ahmed R; Miles, Brodie; Scisci, Elizabeth; Kurago, Zoya B; Palani, Chithra D; Arce, Roger M; Waller, Jennifer L; Genco, Caroline A; Slocum, Connie; Manning, Matthew; Schoenlein, Patricia V; Cutler, Christopher W

2015-02-01

Signaling via pattern recognition receptors (PRRs) expressed on professional antigen presenting cells, such as dendritic cells (DCs), is crucial to the fate of engulfed microbes. Among the many PRRs expressed by DCs are Toll-like receptors (TLRs) and C-type lectins such as DC-SIGN. DC-SIGN is targeted by several major human pathogens for immune-evasion, although its role in intracellular routing of pathogens to autophagosomes is poorly understood. Here we examined the role of DC-SIGN and TLRs in evasion of autophagy and survival of Porphyromonas gingivalis in human monocyte-derived DCs (MoDCs). We employed a panel of P. gingivalis isogenic fimbriae deficient strains with defined defects in Mfa-1 fimbriae, a DC-SIGN ligand, and FimA fimbriae, a TLR2 agonist. Our results show that DC-SIGN dependent uptake of Mfa1+P. gingivalis strains by MoDCs resulted in lower intracellular killing and higher intracellular content of P. gingivalis. Moreover, Mfa1+P. gingivalis was mostly contained within single membrane vesicles, where it survived intracellularly. Survival was decreased by activation of TLR2 and/or autophagy. Mfa1+P. gingivalis strain did not induce significant levels of Rab5, LC3-II, and LAMP1. In contrast, P. gingivalis uptake through a DC-SIGN independent manner was associated with early endosomal routing through Rab5, increased LC3-II and LAMP-1, as well as the formation of double membrane intracellular phagophores, a characteristic feature of autophagy. These results suggest that selective engagement of DC-SIGN by Mfa-1+P. gingivalis promotes evasion of antibacterial autophagy and lysosome fusion, resulting in intracellular persistence in myeloid DCs; however TLR2 activation can overcome autophagy evasion and pathogen persistence in DCs.

Dendritic cell mediated delivery of plasmid DNA encoding LAMP/HIV-1 Gag fusion immunogen enhances T cell epitope responses in HLA DR4 transgenic mice.

Directory of Open Access Journals (Sweden)

Gregory G Simon

2010-01-01

Full Text Available This report describes the identification and bioinformatics analysis of HLA-DR4-restricted HIV-1 Gag epitope peptides, and the application of dendritic cell mediated immunization of DNA plasmid constructs. BALB/c (H-2d and HLA-DR4 (DRA1*0101, DRB1*0401 transgenic mice were immunized with immature dendritic cells transfected by a recombinant DNA plasmid encoding the lysosome-associated membrane protein-1/HIV-1 Gag (pLAMP/gag chimera antigen. Three immunization protocols were compared: 1 primary subcutaneous immunization with 1x10(5 immature dendritic cells transfected by electroporation with the pLAMP/gag DNA plasmid, and a second subcutaneous immunization with the naked pLAMP/gag DNA plasmid; 2 primary immunization as above, and a second subcutaneous immunization with a pool of overlapping peptides spanning the HIV-1 Gag sequence; and 3 immunization twice by subcutaneous injection of the pLAMP/gag DNA plasmid. Primary immunization with pLAMP/gag-transfected dendritic cells elicited the greatest number of peptide specific T-cell responses, as measured by ex vivo IFN-gamma ELISpot assay, both in BALB/c and HLA-DR4 transgenic mice. The pLAMP/gag-transfected dendritic cells prime and naked DNA boost immunization protocol also resulted in an increased apparent avidity of peptide in the ELISpot assay. Strikingly, 20 of 25 peptide-specific T-cell responses in the HLA-DR4 transgenic mice contained sequences that corresponded, entirely or partially to 18 of the 19 human HLA-DR4 epitopes listed in the HIV molecular immunology database. Selection of the most conserved epitope peptides as vaccine targets was facilitated by analysis of their representation and variability in all reported sequences. These data provide a model system that demonstrates a the superiority of immunization with dendritic cells transfected with LAMP/gag plasmid DNA, as compared to naked DNA, b the value of HLA transgenic mice as a model system for the identification and evaluation

CRY 1AB trangenic cowpea obtained by nodal electroporation ...

African Journals Online (AJOL)

Electroporation-mediated genetic transformation was used to introduce Cry 1 Ab insecticidal gene into cowpea. Nodal buds were electroporated in planta with a plasmid carrying the Cry 1Ab and antibiotic resistance npt II genes driven by a 35S CaMV promoter. T1 seeds derived from electroporated branches were selected ...

Maternal provision of non-sex-specific transformer messenger RNA in sex determination of the wasp Asobara tabida.

Science.gov (United States)

Geuverink, E; Verhulst, E C; van Leussen, M; van de Zande, L; Beukeboom, L W

2018-02-01

In many insect species maternal provision of sex-specifically spliced messenger RNA (mRNA) of sex determination genes is an essential component of the sex determination mechanism. In haplodiploid Hymenoptera, maternal provision in combination with genomic imprinting has been shown for the parasitoid Nasonia vitripennis, known as maternal effect genomic imprinting sex determination (MEGISD). Here, we characterize the sex determination cascade of Asobara tabida, another hymenopteran parasitoid. We show the presence of the conserved sex determination genes doublesex (dsx), transformer (tra) and transformer-2 (tra2) orthologues in As. tabida. Of these, At-dsx and At-tra are sex-specifically spliced, indicating a conserved function in sex determination. At-tra and At-tra2 mRNA is maternally provided to embryos but, in contrast to most studied insects, As. tabida females transmit a non-sex-specific splice form of At-tra mRNA to the eggs. In this respect, As. tabida sex determination differs from the MEGISD mechanism. How the paternal genome can induce female development in the absence of maternal provision of sex-specifically spliced mRNA remains an open question. Our study reports a hitherto unknown variant of maternal effect sex determination and accentuates the diversity of insect sex determination mechanisms. © 2017 The Authors. Insect Molecular Biology published by John Wiley & Sons Ltd on behalf of Royal Entomological Society.

Paralleled comparison of vectors for the generation of CAR-T cells.

Science.gov (United States)

Qin, Di-Yuan; Huang, Yong; Li, Dan; Wang, Yong-Sheng; Wang, Wei; Wei, Yu-Quan

2016-09-01

T-lymphocytes genetically engineered with the chimeric antigen receptor (CAR-T) have shown great therapeutic potential in cancer treatment. A variety of preclinical researches and clinical trials of CAR-T therapy have been carried out to lay the foundation for future clinical application. In these researches, several gene-transfer methods were used to deliver CARs or other genes into T-lymphocytes, equipping CAR-modified T cells with a property of recognizing and attacking antigen-expressing tumor cells in a major histocompatibility complex-independent manner. Here, we summarize the gene-transfer vectors commonly used in the generation of CAR-T cell, including retrovirus vectors, lentivirus vectors, the transposon/transposase system, the plasmid-based system, and the messenger RNA electroporation system. The following aspects were compared in parallel: efficiency of gene transfer, the integration methods in the modified T cells, foreground of scale-up production, and application and development in clinical trials. These aspects should be taken into account to generate the optimal CAR-gene vector that may be suitable for future clinical application.

Detailed mapping of serotonin 5-HT{sub 1B} and 5-HT{sub 1D} receptor messenger RNA and ligand binding sites in guinea-pig brain and trigeminal ganglion: clues for function

Energy Technology Data Exchange (ETDEWEB)

Leysen, J.E. [Graduate School Neurosciences, Amsterdam (Netherlands); Schotte, A.; Jurzak, M.; Luyten, W.H.M.L. [Department of Biochemical Pharmacology, Janssen Research Foundation, Beerse (Belgium); Voorn, P.; Bonaventure, P. [Graduate School Neurosciences, Amsterdam (Netherlands)

1997-10-17

The similar pharmacology of the 5-HT{sub 1B} and 5-HT{sub 1D} receptors, and the lack of selective compounds sufficiently distinguishing between the two receptor subtypes, have hampered functional studies on these receptors. In order to provide clues for differential functional roles of the two subtypes, we performed a parallel localization study throughout the guinea-pig brain and the trigeminal ganglia by means of quantitative in situ hybridization histochemistry (using [{sup 35}S]-labelled riboprobes probes for receptor messenger RNA) and receptor autoradiography (using a new radioligand [{sup 3}H]alniditan).The anatomical patterns of 5-HT{sub 1B} and 5-HT{sub 1D} receptor messenger RNA were quite different. While 5-HT{sub 1B} receptor messenger RNA was abundant throughout the brain (with highest levels in the striatum, nucleus accumbens, olfactory tubercle, cortex, hypothalamus, hippocampal formation, amygdala, thalamus, dorsal raphe and cerebellum), 5-HT{sub 1D} receptor messenger RNA exhibited a more restricted pattern; it was found mainly in the olfactory tubercle, entorhinal cortex, dorsal raphe, cerebellum, mesencephalic trigeminal nucleus and in the trigeminal ganglion. The density of 5-HT{sub 1B/1D} binding sites (combined) obtained with [{sup 3}H]alniditan autoradiography was high in the substantia nigra, superior colliculus and globus pallidus, whereas lower levels were detected in the caudate-putamen, hypothalamus, hippocampal formation, amygdala, thalamus and central gray. This distribution pattern was indistinguishable from specific 5-HT{sub 1B} receptor labelling in the presence of ketanserin under conditions to occlude 5-HT{sub 1D} receptor labelling; hence the latter were below detection level. Relationships between the regional distributions of the receptor messenger RNAs and binding sites and particular neuroanatomical pathways are discussed with respect to possible functional roles of the 5-HT{sub 1B} and 5-HT{sub 1D} receptors. (Copyright (c

Effect of Thymine Starvation on Messenger Ribonucleic Acid Synthesis in Escherichia coli

Science.gov (United States)

Luzzati, Denise

1966-01-01

Luzzati, Denise (Institut de Biologie Physico-Chimique, Paris, France). Effect of thymine starvation on messenger ribonucleic acid synthesis in Escherichia coli. J. Bacteriol. 92:1435–1446. 1966.—During the course of thymine starvation, the rate of synthesis of messenger ribonucleic acid (mRNA, the rapidly labeled fraction of the RNA which decays in the presence of dinitrophenol or which hybridizes with deoxyribonucleic acid) decreases exponentially, in parallel with the viability of the thymine-starved bacteria. The ability of cell-free extracts of starved bacteria to incorporate ribonucleoside triphosphates into RNA was determined; it was found to be inferior to that of extracts from control cells. The analysis of the properties of cell-free extracts of starved cells shows that their decreased RNA polymerase activity is the consequence of a modification of their deoxyribonucleic acid, the ability of which to serve as a template for RNA polymerase decreases during starvation. PMID:5332402

A nonlinear electromechanical coupling model for electropore expansion in cell electroporation

KAUST Repository

Deng, Peigang

2014-10-15

Under an electric field, the electric tractions acting on a cell membrane containing a pore-nucleus are investigated by using a nonlinear electromechanical coupling model, in which the cell membrane is treated as a hyperelastic material. Iterations between the electric field and the structure field are performed to reveal the electrical forces exerting on the pore region and the subsequent pore expansion process. An explicit exponential decay of the membrane\\'s edge energy as a function of pore radius is defined for a hydrophilic pore and the transition energy as a hydrophobic pore converts to a hydrophilic pore during the initial stage of pore formation is investigated. It is found that the edge energy for the creation of an electropore edge plays an important role at the atomistic scale and it determines the hydrophobic-hydrophilic transition energy barrier. Various free energy evolution paths are exhibited, depending on the applied electric field, which provides further insight towards the electroporation (EP) phenomenon. In comparison with previous EP models, the proposed model has the ability to predict the metastable point on the free energy curve that is relevant to the lipid ion channel. In addition, the proposed model can also predict the critical transmembrane potential for the activation of an effective electroporation that is in a good agreement with previously published experimental data.

Recruitment of PfSET2 by RNA polymerase II to variant antigen encoding loci contributes to antigenic variation in P. falciparum.

Directory of Open Access Journals (Sweden)

Uchechi E Ukaegbu

2014-01-01

Full Text Available Histone modifications are important regulators of gene expression in all eukaryotes. In Plasmodium falciparum, these epigenetic marks regulate expression of genes involved in several aspects of host-parasite interactions, including antigenic variation. While the identities and genomic positions of many histone modifications have now been cataloged, how they are targeted to defined genomic regions remains poorly understood. For example, how variant antigen encoding loci (var are targeted for deposition of unique histone marks is a mystery that continues to perplex the field. Here we describe the recruitment of an ortholog of the histone modifier SET2 to var genes through direct interactions with the C-terminal domain (CTD of RNA polymerase II. In higher eukaryotes, SET2 is a histone methyltransferase recruited by RNA pol II during mRNA transcription; however, the ortholog in P. falciparum (PfSET2 has an atypical architecture and its role in regulating transcription is unknown. Here we show that PfSET2 binds to the unphosphorylated form of the CTD, a property inconsistent with its recruitment during mRNA synthesis. Further, we show that H3K36me3, the epigenetic mark deposited by PfSET2, is enriched at both active and silent var gene loci, providing additional evidence that its recruitment is not associated with mRNA production. Over-expression of a dominant negative form of PfSET2 designed to disrupt binding to RNA pol II induced rapid var gene expression switching, confirming both the importance of PfSET2 in var gene regulation and a role for RNA pol II in its recruitment. RNA pol II is known to transcribe non-coding RNAs from both active and silent var genes, providing a possible mechanism by which it could recruit PfSET2 to var loci. This work unifies previous reports of histone modifications, the production of ncRNAs, and the promoter activity of var introns into a mechanism that contributes to antigenic variation by malaria parasites.

Analysis of a cDNA clone expressing a human autoimmune antigen: full-length sequence of the U2 small nuclear RNA-associated B antigen

International Nuclear Information System (INIS)

Habets, W.J.; Sillekens, P.T.G.; Hoet, M.H.; Schalken, J.A.; Roebroek, A.J.M.; Leunissen, J.A.M.; Van de Ven, W.J.M.; Van Venrooij, W.J.

1987-01-01

A U2 small nuclear RNA-associated protein, designated B'', was recently identified as the target antigen for autoimmune sera from certain patients with systemic lupus erythematosus and other rheumatic diseases. Such antibodies enabled them to isolate cDNA clone λHB''-1 from a phage λgt11 expression library. This clone appeared to code for the B'' protein as established by in vitro translation of hybrid-selected mRNA. The identity of clone λHB''-1 was further confirmed by partial peptide mapping and analysis of the reactivity of the recombinant antigen with monospecific and monoclonal antibodies. Analysis of the nucleotide sequence of the 1015-base-pair cDNA insert of clone λHB''-1 revealed a large open reading frame of 800 nucleotides containing the coding sequence for a polypeptide of 25,457 daltons. In vitro transcription of the λHB''-1 cDNA insert and subsequent translation resulted in a protein product with the molecular size of the B'' protein. These data demonstrate that clone λHB''-1 contains the complete coding sequence of this antigen. The deduced polypeptide sequence contains three very hydrophilic regions that might constitute RNA binding sites and/or antigenic determinants. These findings might have implications both for the understanding of the pathogenesis of rheumatic diseases as well as for the elucidation of the biological function of autoimmune antigens

A theoretical analysis of the feasibility of a singularity-induced micro-electroporation system.

Directory of Open Access Journals (Sweden)

Gregory D Troszak

Full Text Available Electroporation, the permeabilization of the cell membrane lipid bilayer due to a pulsed electric field, has important implications in the biotechnology, medicine, and food industries. Traditional macro and micro-electroporation devices have facing electrodes, and require significant potential differences to induce electroporation. The goal of this theoretical study is to investigate the feasibility of singularity-induced micro-electroporation; an electroporation configuration aimed at minimizing the potential differences required to induce electroporation by separating adjacent electrodes with a nanometer-scale insulator. In particular, this study aims to understand the effect of (1 insulator thickness and (2 electrode kinetics on electric field distributions in the singularity-induced micro-electroporation configuration. A non-dimensional primary current distribution model of the micro-electroporation channel shows that while increasing insulator thickness results in smaller electric field magnitudes, electroporation can still be performed with insulators thick enough to be made with microfabrication techniques. Furthermore, a secondary current distribution model of the singularity-induced micro-electroporation configuration with inert platinum electrodes and water electrolyte indicates that electrode kinetics do not inhibit charge transfer to the extent that prohibitively large potential differences are required to perform electroporation. These results indicate that singularity-induced micro-electroporation could be used to develop an electroporation system that consumes minimal power, making it suitable for remote applications such as the sterilization of water and other liquids.

Hantavirus Gc induces long-term immune protection via LAMP-targeting DNA vaccine strategy.

Science.gov (United States)

Jiang, Dong-Bo; Zhang, Jin-Peng; Cheng, Lin-Feng; Zhang, Guan-Wen; Li, Yun; Li, Zi-Chao; Lu, Zhen-Hua; Zhang, Zi-Xin; Lu, Yu-Chen; Zheng, Lian-He; Zhang, Fang-Lin; Yang, Kun

2018-02-01

Hemorrhagic fever with renal syndrome (HFRS) occurs widely throughout Eurasia. Unfortunately, there is no effective treatment, and prophylaxis remains the best option against the major pathogenic agent, hantaan virus (HTNV), which is an Old World hantavirus. However, the absence of cellular immune responses and immunological memory hampers acceptance of the current inactivated HFRS vaccine. Previous studies revealed that a lysosome-associated membrane protein 1 (LAMP1)-targeting strategy involving a DNA vaccine based on the HTNV glycoprotein Gn successfully conferred long-term immunity, and indicated that further research on Gc, another HTNV antigen, was warranted. Plasmids encoding Gc and lysosome-targeted Gc, designated pVAX-Gc and pVAX-LAMP/Gc, respectively, were constructed. Proteins of interest were identified by fluorescence microscopy following cell line transfection. Five groups of 20 female BALB/c mice were subjected to the following inoculations: inactivated HTNV vaccine, pVAX-LAMP/Gc, pVAX-Gc, and, as the negative controls, pVAX-LAMP or the blank vector pVAX1. Humoral and cellular immunity were assessed by enzyme-linked immunosorbent assays (ELISAs) and 15-mer peptide enzyme-linked immunospot (ELISpot) epitope mapping assays. Repeated immunization with pVAX-LAMP/Gc enhanced adaptive immune responses, as demonstrated by the specific and neutralizing antibody titers and increased IFN-γ production. The inactivated vaccine induced a comparable humoral reaction, but the negative controls only elicited insignificant responses. Using a mouse model of HTNV challenge, the in vivo protection conferred by the inactivated vaccine and Gc-based constructs (with/without LAMP recombination) was confirmed. Evidence of pan-epitope reactions highlighted the long-term cellular response to the LAMP-targeting strategy, and histological observations indicated the safety of the LAMP-targeting vaccines. The long-term protective immune responses induced by pVAX-LAMP/Gc may be

CALiPER Report 22.1: Photoelectric Performance of LED MR16 Lamps

Energy Technology Data Exchange (ETDEWEB)

Royer, Michael P. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Poplawski, Michael E. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Brown, Charles C. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Merzouk, Massine B. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

2015-09-01

This report is a follow-up to CALiPER Application Summary Report 22, which investigated the photometric performance of LED MR16 lamps. The initial report found that many of the LED MR16 lamps did not perform as required by ENERGY STAR based on their equivalency claims, although they generally did provide substantial efficacy advantages compared to halogen MR16 lamps. All testing was completed using laboratory power supplies, with all but one product tested at 12 V AC. In contrast, this report examined the photoelectric performance of the same set of lamps, using commercially available transformers and dimmers as well as laboratory power supplies providing both AC and DC power.

Guardian of Genetic Messenger-RNA-Binding Proteins

Directory of Open Access Journals (Sweden)

Antje Anji

2016-01-01

Full Text Available RNA in cells is always associated with RNA-binding proteins that regulate all aspects of RNA metabolism including RNA splicing, export from the nucleus, RNA localization, mRNA turn-over as well as translation. Given their diverse functions, cells express a variety of RNA-binding proteins, which play important roles in the pathologies of a number of diseases. In this review we focus on the effect of alcohol on different RNA-binding proteins and their possible contribution to alcohol-related disorders, and discuss the role of these proteins in the development of neurological diseases and cancer. We further discuss the conventional methods and newer techniques that are employed to identify RNA-binding proteins.

Direct-current converter for gas-discharge lamps

Science.gov (United States)

Lutus, P.

1980-01-01

Metal/halide and similar gas-discharge lamps are powered from low-voltage dc source using small efficient converter. Converter is useful whenever 60-cycle ac power is not available or where space and weight allocations are limited. Possible applications are offshore platforms, mobile homes, and emergency lighting. Design innovations give supply high reliability and efficiency up to 75 percent.

Membrane and envelope virus proteins co-expressed as lysosome associated membrane protein (LAMP fused antigens: a potential tool to develop DNA vaccines against flaviviruses

Directory of Open Access Journals (Sweden)

Rafael Dhalia

2009-12-01

Full Text Available Vaccination is the most practical and cost-effective strategy to prevent the majority of the flavivirus infection to which there is an available vaccine. However, vaccines based on attenuated virus can potentially promote collateral side effects and even rare fatal reactions. Given this scenario, the developent of alternative vaccination strategies such as DNA-based vaccines encoding specific flavivirus sequences are being considered. Endogenous cytoplasmic antigens, characteristically plasmid DNA-vaccine encoded, are mainly presented to the immune system through Major Histocompatibility Complex class I - MHC I molecules. The MHC I presentation via is mostly associated with a cellular cytotoxic response and often do not elicit a satisfactory humoral response. One of the main strategies to target DNA-encoded antigens to the MHC II compartment is expressing the antigen within the Lysosome-Associated Membrane Protein (LAMP. The flavivirus envelope protein is recognized as the major virus surface protein and the main target for neutralizing antibodies. Different groups have demonstrated that co-expression of flavivirus membrane and envelope proteins in mammalian cells, fused with the carboxyl-terminal of LAMP, is able to induce satisfactory levels of neutralizing antibodies. Here we reviewed the use of the envelope flavivirus protein co-expression strategy as LAMP chimeras with the aim of developing DNA vaccines for dengue, West Nile and yellow fever viruses.A vacinação é a estratégia mais prática e o melhor custo-benefício para prevenir a maioria das infecções dos flavivirus, para os quais existe vacina disponível. Entretanto, as vacinas baseadas em vírus atenuados podem potencialmente promover efeitos colaterais e, mais raramente, reações fatais. Diante deste cenário, o desenvolvimento de estratégias alternativas de vacinação, como vacinas baseadas em DNA codificando seqüências específicas dos flavivirus, está sendo considerado

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

Science.gov (United States)

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

2016-01-01

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

Self-amplifying mRNA vaccines.

Science.gov (United States)

Brito, Luis A; Kommareddy, Sushma; Maione, Domenico; Uematsu, Yasushi; Giovani, Cinzia; Berlanda Scorza, Francesco; Otten, Gillis R; Yu, Dong; Mandl, Christian W; Mason, Peter W; Dormitzer, Philip R; Ulmer, Jeffrey B; Geall, Andrew J

2015-01-01

This chapter provides a brief introduction to nucleic acid-based vaccines and recent research in developing self-amplifying mRNA vaccines. These vaccines promise the flexibility of plasmid DNA vaccines with enhanced immunogenicity and safety. The key to realizing the full potential of these vaccines is efficient delivery of nucleic acid to the cytoplasm of a cell, where it can amplify and express the encoded antigenic protein. The hydrophilicity and strong net negative charge of RNA impedes cellular uptake. To overcome this limitation, electrostatic complexation with cationic lipids or polymers and physical delivery using electroporation or ballistic particles to improve cellular uptake has been evaluated. This chapter highlights the rapid progress made in using nonviral delivery systems for RNA-based vaccines. Initial preclinical testing of self-amplifying mRNA vaccines has shown nonviral delivery to be capable of producing potent and robust innate and adaptive immune responses in small animals and nonhuman primates. Historically, the prospect of developing mRNA vaccines was uncertain due to concerns of mRNA instability and the feasibility of large-scale manufacturing. Today, these issues are no longer perceived as barriers in the widespread implementation of the technology. Currently, nonamplifying mRNA vaccines are under investigation in human clinical trials and can be produced at a sufficient quantity and quality to meet regulatory requirements. If the encouraging preclinical data with self-amplifying mRNA vaccines are matched by equivalently positive immunogenicity, potency, and tolerability in human trials, this platform could establish nucleic acid vaccines as a versatile new tool for human immunization. Copyright © 2015 Elsevier Inc. All rights reserved.

A nonlinear electromechanical coupling model for electropore expansion in cell electroporation

KAUST Repository

Deng, Peigang; Lee, Yi Kuen; Zhang, Tong Yi

2014-01-01

the electroporation (EP) phenomenon. In comparison with previous EP models, the proposed model has the ability to predict the metastable point on the free energy curve that is relevant to the lipid ion channel. In addition, the proposed model can also predict

Diagnostic accuracy of circulating thyrotropin receptor messenger RNA combined with neck ultrasonography in patients with Bethesda III-V thyroid cytology.

Science.gov (United States)

Aliyev, Altay; Patel, Jinesh; Brainard, Jennifer; Gupta, Manjula; Nasr, Christian; Hatipoglu, Betul; Siperstein, Allan; Berber, Eren

2016-01-01

The aim of this study was to analyze the usefulness of thyrotropin receptor messenger RNA (TSHR-mRNA) combined with neck ultrasonography (US) in the management of thyroid nodules with Bethesda III-V cytology. Cytology slides of patients with a preoperative fine needle aspiration (FNA) and TSHR-mRNA who underwent thyroidectomy between 2002 and 2011 were recategorized based on the Bethesda classification. Results of thyroid FNA, TSHR-mRNA, and US were compared with the final pathology. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated. There were 12 patients with Bethesda III, 112 with Bethesda IV, and 58 with Bethesda V cytology. The sensitivity of TSHR-mRNA in predicting cancer was 33%, 65%, and 79 %, and specificity was 67%, 66%, and 71%, for Bethesda III, IV, and V categories, respectively. For the same categories, the PPV of TSHR-mRNA was 25%, 33%, and 79%, respectively; whereas the NPV was 75%, 88%, and 71%, respectively. The addition of neck US to TSHR-mRNA increased the NPV to 100% for Bethesda III, and 86%, for Bethesda IV, and 82% for Bethesda V disease. This study documents the potential usefulness of TSHR-mRNA for thyroid nodules with Bethesda III-V FNA categories. TSHR-mRNA may be used to exclude Bethesda IV disease. A large sample analysis is needed to determine its accuracy for Bethesda category III nodules. Copyright © 2016 Elsevier Inc. All rights reserved.

Electroporation-Induced Cell Modifications Detected with THz Time-Domain Spectroscopy

Science.gov (United States)

Romeo, Stefania; Vernier, P. Thomas; Zeni, Olga

2018-04-01

Electroporation (electropermeabilization) increases the electrical conductivity of biological cell membranes and lowers transport barriers for normally impermeant materials. Molecular simulations suggest that electroporation begins with the reorganization of water and lipid head group dipoles in the phospholipid bilayer interface, driven by an externally applied electric field, and the evolution of the resulting defects into water-filled, lipid pores. The interior of the electroporated membrane thus contains water, which should provide a signature for detection of the electropermeabilized state. In this feasibility study, we use THz time-domain spectroscopy, a powerful tool for investigating biomolecular systems and their interactions with water, to detect electroporation in human cells subjected to permeabilizing pulsed electric fields (PEFs). The time-domain response of electroporated human monocytes was acquired with a commercial THz, time-domain spectrometer. For each sample, frequency spectra were calculated, and the absorption coefficient and refractive index were extracted in the frequency range between 0.2 and 1.5 THz. This analysis reveals a higher absorption of THz radiation by PEF-exposed cells, with respect to sham-exposed ones, consistent with the intrusion of water into the cell through the permeabilized membrane that is presumed to be associated with electroporation.

Antibody-antigen-adjuvant conjugates enable co-delivery of antigen and adjuvant to dendritic cells in cis but only have partial targeting specificity

NARCIS (Netherlands)

Kreutz, M.; Giquel, B.; Hu, Q.; Abuknesha, R.; Uematsu, S.; Akira, S.; Nestle, F.O.; Diebold, S.S.

2012-01-01

Antibody-antigen conjugates, which promote antigen-presentation by dendritic cells (DC) by means of targeted delivery of antigen to particular DC subsets, represent a powerful vaccination approach. To ensure immunity rather than tolerance induction the co-administration of a suitable adjuvant is

Equine infectious anemia virus-infected dendritic cells retain antigen presentation capability

International Nuclear Information System (INIS)

Rivera, Julie A.; McGuire, Travis C.

2005-01-01

To determine if equine monocyte-derived dendritic cells (DC) were susceptible to equine infectious anemia virus (EIAV) infection, ex vivo-generated DC were infected with virus in vitro. EIAV antigen was detected by immunofluorescence 3 days post-infection with maximum antigen being detected on day 4, whereas there was no antigen detected in DC incubated with the same amount of heat-inactivated EIAV. No cytolytic activity was observed after EIAV WSU5 infection of DC. These monocyte-derived DC were more effective than macrophages and B cells in stimulating allogenic T lymphocytes. Both infected macrophages and DC stimulated similar levels of memory CTL responses in mixtures of CD8+ and CD4+ cells as detected with 51 Cr-release assays indicating that EIAV infection of DC did not alter antigen presentation. However, EIAV-infected DC were more effective than infected macrophages when used to stimulate memory CTL in isolated CD8+ cells. The maintenance of antigen processing and presenting function by EIAV-infected DC in vitro suggests that this function is maintained during in vivo infection

Handbook of electroporation

CERN Document Server

2017-01-01

This major reference work is a one-shot knowledge base on electroporation and the use of pulsed electric fields of high intensity and their use in biology, medicine, biotechnology, and food and environmental technologies. The Handbook offers a widespread and well-structured compilation of 156 chapters ranging from the foundations to applications in industry and hospital. It is edited and written by most prominent researchers in the field. With regular updates and growing in its volume it is suitable for academic readers and researchers regardless of their disciplinary expertise, and will also be accessible to students and serious general readers. The Handbook's 276 authors have established scholarly credentials and come from a wide range of disciplines. This is crucially important in a highly interdisciplinary field of electroporation and the use of pulsed electric fields of high intensity and its applications in different fields from medicine, biology, food proce ssing, agriculture, process engineering, en...

Natural RNA circles function as efficient microRNA sponges

DEFF Research Database (Denmark)

Hansen, Thomas Birkballe; Jensen, Trine I; Clausen, Bettina Hjelm

2013-01-01

MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression that act by direct base pairing to target sites within untranslated regions of messenger RNAs. Recently, miRNA activity has been shown to be affected by the presence of miRNA sponge transcripts, the so-called comp......MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression that act by direct base pairing to target sites within untranslated regions of messenger RNAs. Recently, miRNA activity has been shown to be affected by the presence of miRNA sponge transcripts, the so......-called competing endogenous RNA in humans and target mimicry in plants. We previously identified a highly expressed circular RNA (circRNA) in human and mouse brain. Here we show that this circRNA acts as a miR-7 sponge; we term this circular transcript ciRS-7 (circular RNA sponge for miR-7). ciRS-7 contains more...... sponge, suggesting that miRNA sponge effects achieved by circRNA formation are a general phenomenon. This study serves as the first, to our knowledge, functional analysis of a naturally expressed circRNA....

Antigen processing and remodeling of the endosomal pathway: requirements for antigen cross-presentation.

Science.gov (United States)

Compeer, Ewoud Bernardus; Flinsenberg, Thijs Willem Hendrik; van der Grein, Susanna Geertje; Boes, Marianne

2012-01-01

Cross-presentation of endocytosed antigen as peptide/class I major histocompatibility complex complexes plays a central role in the elicitation of CD8(+) T cell clones that mediate anti-viral and anti-tumor immune responses. While it has been clear that there are specific subsets of professional antigen presenting cells capable of antigen cross-presentation, identification of mechanisms involved is still ongoing. Especially amongst dendritic cells (DC), there are specialized subsets that are highly proficient at antigen cross-presentation. We here present a focused survey on the cell biological processes in the endosomal pathway that support antigen cross-presentation. This review highlights DC-intrinsic mechanisms that facilitate the cross-presentation of endocytosed antigen, including receptor-mediated uptake, maturation-induced endosomal sorting of membrane proteins, dynamic remodeling of endosomal structures and cell surface-directed endosomal trafficking. We will conclude with the description of pathogen-induced deviation of endosomal processing, and discuss how immune evasion strategies pertaining endosomal trafficking may preclude antigen cross-presentation.

Antigen processing and remodeling of the endosomal pathway: requirements for antigen cross-presentation.

Directory of Open Access Journals (Sweden)

Ewoud Bernardus Compeer

2012-03-01

Full Text Available The cross-presentation of endocytosed antigen as peptide/class I MHC complexes plays a central role in the elicitation of CD8+ T cell clones that mediate anti-viral and anti-tumor immune responses. While it has been clear that there are specific subsets of professional antigen presenting cells (APC capable of antigen cross-presentation, description of mechanisms involved is still ongoing. Especially amongst dendritic cells (DC, there are specialized subsets that are highly proficient at antigen cross-presentation. We here present a focused survey on the cell biological processes in the endosomal pathway that support antigen cross-presentation. This review highlight DC-intrinsic mechanisms that facilitate the cross-presentation of endocytosed antigen, including receptor-mediated uptake, recycling and maturation including the sorting of membrane proteins, dynamic remodeling of endosomal structures and cell-surface directed endosomal trafficking. We will conclude with description of pathogen-induced deviation of endosomal processing, and discuss how immune evasion strategies pertaining endosomal trafficking may preclude antigen cross-presentation.

High Expression of LAMP3 Is a Novel Biomarker of Poor Prognosis in Patients with Esophageal Squamous Cell Carcinoma

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Xiaoyu Liao

2015-07-01

Full Text Available Lysosomal-associated membrane protein 3 (LAMP3, identified as a molecular marker of mature dendritic cells, is one of the LAMP family members. Its expression was induced by hypoxia, and was associated with hypoxia mediated metastasis in breast and cervical cancers. However, epithelial expression of LAMP3 and its prognostic value in esophageal squamous cell carcinoma (ESCC is still unknown. In the current study, mRNA expression of LAMP3 in 157 ESCC tissues and 50 adjacent normal tissues was detected by quantitative real-time PCR (qRT-PCR. LAMP3 protein expression in 46 paired cancerous and normal tissues was detected by immunohistochemistry (IHC. Then, DNA copy number was examined to observe its potential correlation with mRNA expression. The results showed that both mRNA and protein expression level of LAMP3 was significantly higher in cancerous tissues compared with normal controls (p < 0.001. LAMP3 DNA copy number was amplified in 70% of ESCC tissues and positive correlated with mRNA expression (p = 0.037. Furthermore, patients with higher LAMP3 expression had worse overall survival (HR = 1.90, 95% CI = 1.17–3.09, p = 0.010 and disease-free survival (HR = 1.80, 95% CI = 1.18–2.74, p = 0.006. In conclusion, our results suggest that epithelial LAMP3 expression is an independent prognostic biomarker for ESCC.

Network for development of electroporation-based technologies and treatments: COST TD1104.

Science.gov (United States)

Miklavčič, Damijan

2012-10-01

Exposure of biological cells to a sufficiently strong external electric field results in increased permeability of cell membranes, referred to as "electroporation." Since all types of cells (animal, plant and microorganism) can be effectively electroporated, electroporation is considered to be a universal method and a platform technology. Electroporation has become a widely used technology applicable to, e.g., cancer treatment, gene transfection, food and biomass processing and microbial inactivation. However, despite significant progress in electroporation-based applications, there is a lack of coordination and interdisciplinary exchange of knowledge between researchers from different scientific domains. Thus, critical mass for new major breakthroughs is missing. This is why we decided to establish cooperation between research groups working in different fields of electroporation. Cooperation in Science and Technology (COST), which funds networking and capacity-building activities, presents a perfect framework for such scientific cooperation. This COST action aims at (1) providing necessary steps toward EU cooperation of science and technology to foster basic understanding of electroporation; (2) improving communication between research groups, resulting in streamlining European research and development activities; and (3) enabling development of new and further development of existing electroporation-based applications by integrating multidisciplinary research teams, as well as providing comprehensive training for early-stage researchers. Results of this COST action will provide multiple societal, scientific and technological benefits from improving existing electroporation-based applications to adding new ones in the fields of medicine, biotechnology and environmental preservation.

Fox-2 protein regulates the alternative splicing of scleroderma-associated lysyl hydroxylase 2 messenger RNA.

Science.gov (United States)

Seth, Puneet; Yeowell, Heather N

2010-04-01

Scleroderma (systemic sclerosis [SSc]) is a complex connective tissue disorder characterized by hardening and thickening of the skin. One hallmark of scleroderma is excessive accumulation of collagen accompanied by increased levels of pyridinoline collagen crosslinks derived from hydroxylysine residues in the collagen telopeptide domains. Lysyl hydroxylase 2 (LH2), an important alternatively spliced enzyme in collagen biosynthesis, acts as a collagen telopeptide hydroxylase. Changes in the pattern of LH2 alternative splicing, favoring increased inclusion of the alternatively spliced LH2 exon 13A, thereby increasing the levels of the long transcript of LH2 (LH2[long]), are linked to scleroderma disease. This study was undertaken to examine the role played by RNA binding protein Fox-2 in regulating exon 13A inclusion, which leads to the generation of scleroderma-associated LH2(long) messenger RNA (mRNA). Phylogenetic sequence analysis of introns flanking exon 13A was performed. A tetracycline-inducible system in T-Rex 293 cells was used to induce Fox-2 protein, and endogenous LH2(long) mRNA was determined by reverse transcriptase-polymerase chain reaction. An LH2 minigene was designed, validated, and used in Fox-2 overexpression and mutagenesis experiments. Knockdown of Fox-2 was performed in mouse embryonic fibroblasts and in fibroblasts from SSc patients. Overexpression of Fox-2 enhanced the inclusion of exon 13A and increased the generation of LH2(long) mRNA, whereas knockdown of Fox-2 decreased LH2(long) transcripts. Mutational analysis of an LH2 minigene demonstrated that 2 of the 4 Fox binding motifs flanking LH2 exon 13A are required for inclusion of exon 13A. In early passage fibroblasts derived from patients with scleroderma, the knockdown of Fox-2 protein significantly decreased the endogenous levels of LH2(long) mRNA. Our findings indicate that Fox-2 plays an integral role in the regulation of LH2 splicing. Knockdown of Fox-2 and other methods to decrease

Shielding the messenger (RNA): microRNA-based anticancer therapies

Science.gov (United States)

Sotillo, Elena; Thomas-Tikhonenko, Andrei

2011-01-01

It has been a decade since scientists realized that microRNAs (miRNAs) are not an oddity invented by worms to regulate gene expression at post-transcriptional levels. Rather, many of these 21–22-nucleotide-short RNAs exist in invertebrates and vertebrates alike and some of them are in fact highly conserved. miRNAs are now recognized as an important class of non-coding small RNAs that inhibit gene expression by targeting mRNA stability and translation. In the last ten years, our knowledge of the miRNAs world was expanding at vertiginous speed, propelled by the development of computational engines for miRNA identification and target prediction, biochemical tools and techniques to modulate miRNA activity, and last but not least, the emergence of miRNA-centric animal models. One important conclusion that has emerged from this effort is that many microRNAs and their cognate targets are strongly implicated in cancer, either as oncogenes or tumor and metastasis suppressors. In this review we will discuss the diverse role that miRNAs play in cancer initiation and progression and also the tools with which miRNA expression could be corrected in vivo. While the idea of targeting microRNAs towards therapeutic ends is getting considerable traction, basic, translational, and clinical research done in the next few years will tell whether this promise is well-founded. PMID:21514318

Pulsed Electromagnetic Field Assisted in vitro Electroporation: A Pilot Study

Science.gov (United States)

Novickij, Vitalij; Grainys, Audrius; Lastauskienė, Eglė; Kananavičiūtė, Rūta; Pamedytytė, Dovilė; Kalėdienė, Lilija; Novickij, Jurij; Miklavčič, Damijan

2016-09-01

Electroporation is a phenomenon occurring due to exposure of cells to Pulsed Electric Fields (PEF) which leads to increase of membrane permeability. Electroporation is used in medicine, biotechnology, and food processing. Recently, as an alternative to electroporation by PEF, Pulsed ElectroMagnetic Fields (PEMF) application causing similar biological effects was suggested. Since induced electric field in PEMF however is 2-3 magnitudes lower than in PEF electroporation, the membrane permeabilization mechanism remains hypothetical. We have designed pilot experiments where Saccharomyces cerevisiae and Candida lusitaniae cells were subjected to single 100-250 μs electrical pulse of 800 V with and without concomitant delivery of magnetic pulse (3, 6 and 9 T). As expected, after the PEF pulses only the number of Propidium Iodide (PI) fluorescent cells has increased, indicative of membrane permeabilization. We further show that single sub-millisecond magnetic field pulse did not cause detectable poration of yeast. Concomitant exposure of cells to pulsed electric (PEF) and magnetic field (PMF) however resulted in the increased number PI fluorescent cells and reduced viability. Our results show increased membrane permeability by PEF when combined with magnetic field pulse, which can explain electroporation at considerably lower electric field strengths induced by PEMF compared to classical electroporation.

The cataphoretic emitter effect exhibited in high intensity discharge lamp electrodes

Science.gov (United States)

Mentel, Juergen

2018-01-01

A mono-layer of atoms, electropositive with respect to the substrate atoms, forms a dipole layer, reducing its work function. Such a layer is generated by diffusion of emitter material from the interior of the substrate, by vapour deposition or by deposition of emitter material onto arc electrodes by cataphoresis. This cataphoretic emitter effect is investigated within metal halide lamps with transparent YAG ceramic burners, and within model lamps. Within the YAG lamps, arcs are operated with switched-dc current between rod shaped tungsten electrodes in high pressure Hg vapour seeded with metal iodides. Within the model lamps, dc arcs are operated between rod-shaped tungsten electrodes—one doped—in atmospheric pressure Ar. Electrode temperatures are determined by 1λ -pyrometry, combined with simulation of the electrode heat balance. Plasma temperatures, atom and ion densities of emitter material are determined by emission and absorption spectroscopy. Phase resolved measurements in YAG lamps seeded with CeI3, CsI, DyI3, TmI3 and LaI3 show, within the cathodic half period, a reduction of the electrode temperature and an enhanced metal ion density in front of the electrode, and an opposite behavior after phase reversal. With increasing operating frequency, the state of the cathode overlaps onto the anodic phase—except for Cs, being low in adsorption energy. Generally, the phase averaged electrode tip temperature is reduced by seeding a lamp with emitter material; its height depends on admixtures. Measurements at tungsten electrodes doped with ThO2, La2O3 and Ce2O3 within the model lamp show that evaporated emitter material is redeposited by an emitter ion current onto the electrode surface. It reduces the work function of tungsten cathodes above the evaporation temperature of the emitter material, too; and also of cold anodes, indicating a field reversal in front of them. The formation of an emitter spot at low cathode temperature and high emitter material

Feasibility of Parylene Coating for Planar Electroporation Copper Electrodes

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Vitalij NOVICKIJ

2017-08-01

Full Text Available This paper is focused on the feasibility study of parylene as a biocompatible coating for planar electroporation microelectrodes. The planar parallel and the circular interdigitated electrodes are applied in the analysis. The electrodes feature 100 μm width with a 300 μm gap between anode and cathode. The parylene coating thickness was varied in the 250 nm – 2 μm range. The resultant electric field distribution evaluation has been performed using the finite element method. The electrodes have been applied in electroporation experiments with Saprolegnia parasitica. For reference the additional experiments using conventional electroporation cuvette (1 mm gap have been performed. It has been determined that the parylene coating with hydrophobic properties has limited applicability for the passivation of the planar electroporation electrodes.DOI: http://dx.doi.org/10.5755/j01.ms.23.2.14953

Recovery of viral RNA and infectious foot-and-mouth disease virus from positive lateral-flow devices.

Science.gov (United States)

Fowler, Veronica L; Bankowski, Bartlomiej M; Armson, Bryony; Di Nardo, Antonello; Valdazo-Gonzalez, Begoña; Reid, Scott M; Barnett, Paul V; Wadsworth, Jemma; Ferris, Nigel P; Mioulet, Valérie; King, Donald P

2014-01-01

Foot-and-mouth disease Virus (FMDV) is an economically important, highly contagious picornavirus that affects both wild and domesticated cloven hooved animals. In developing countries, the effective laboratory diagnosis of foot-and-mouth disease (FMD) is often hindered by inadequate sample preservation due to difficulties in the transportation and storage of clinical material. These factors can compromise the ability to detect and characterise FMD virus in countries where the disease is endemic. Furthermore, the high cost of sending infectious virus material and the biosecurity risk it presents emphasises the need for a thermo-stable, non-infectious mode of transporting diagnostic samples. This paper investigates the potential of using FMDV lateral-flow devices (LFDs) for dry transportation of clinical samples for subsequent nucleic acid amplification, sequencing and recovery of infectious virus by electroporation. FMDV positive samples (epithelial suspensions and cell culture isolates) representing four FMDV serotypes were applied to antigen LFDs: after which it was possible to recover viral RNA that could be detected using real-time RT-PCR. Using this nucleic acid, it was also possible to recover VP1 sequences and also successfully utilise protocols for amplification of complete FMD virus genomes. It was not possible to recover infectious FMDV directly from the LFDs, however following electroporation into BHK-21 cells and subsequent cell passage, infectious virus could be recovered. Therefore, these results support the use of the antigen LFD for the dry, non-hazardous transportation of samples from FMD endemic countries to international reference laboratories.

Antigen-specific IL-23/17 pathway activation by murine semi-mature DC-like cells

Energy Technology Data Exchange (ETDEWEB)

Nagasaka, Shinya; Iwasaki, Takumi; Okano, Tomoko [Department of Biological Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba 278-8510 (Japan); Chiba, Joe, E-mail: chibaj@rs.noda.tus.ac.jp [Department of Biological Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba 278-8510 (Japan)

2009-09-11

We analyzed the phenotype and function of bone marrow-derived dendritic cells (DCs) induced in vitro without using any serum during the late stage of cultivation. These 'serum-free' DCs (SF-DCs) possessed the ability to induce T cell proliferation as well as antibody responses, indicating that they were functional DCs. Surprisingly, the SF-DCs akin to semi-mature DCs in terms of both phenotypic and functional characteristics. The SF-DCs did not produce IL-12 but produced large amounts of IL-23 following lipopolysaccharide stimulation. The antigen-specific production of IL-17 by CD4{sup +} T cells co-cultured with OVA-loaded SF-DCs was significantly higher than that with OVA-loaded conventional DCs. These results suggest that SF-DCs tend to produce IL-23 and can consequently induce the IL-17 producing CD4{sup +} T cells. The semi-mature DC-like cells reported here will be useful vehicles for DC immunization and might contribute to studies on the possible involvement of semi-mature DCs in Th17 cell differentiation.

DC Grids for Smart LED-Based Lighting: The EDISON Solution

Directory of Open Access Journals (Sweden)

Steffen Thielemans

2017-09-01

Full Text Available This paper highlights the benefits and possible drawbacks of a DC-based lighting infrastructure for powering Light Emitting Diode (LED-lamps. It also evaluates the efforts needed for integrating the so called smart lighting and other sensor/actuator based control systems, and compares existing and emerging solutions. It reviews and discusses published work in this field with special focus on the intelligent DC-based infrastructure named EDISON that is primarily dedicated to lighting, but is applicable to building automation in general. The EDISON “PowerLAN” consists of a DC-based infrastructure that offers telecommunication abilities and can be applied to lighting retrofitting scenarios for buildings. Its infrastructure allows simple and efficient powering of DC-oriented devices like LED lamps, sensors and microcontrollers, while offering a wired communication channel. This paper motivates the design choices for organizing DC lighting grids and their associated communication possibilities. It also shows how the EDISON based smart lighting solution is evolving today to include new communication technologies and to further integrate other parts of building management solutions through the OneM2M (Machine to Machine service bus.

Simulation of micro/nano electroporation for cell transfection

Science.gov (United States)

Zhang, Guocheng; Fan, Na; Jiang, Hai; Guo, Jian; Peng, Bei

2018-03-01

The 3D micro/nano electroporation for transfection has become a powerful biological cell research technique with the development of micro-nano manufacturing technology. The micro channels connected the cells with transfection reagents on the chip were important to the transmemnbrane potentical, which directly influences the electroporation efficiency. In this study, a two-dimensional model for electroporation of cells was designed to address the effects of channels’ sizes and number on transmembrane potential. The simulation results indicated that the transmembrane potential increased with increasing size of channels’ entrances. Moreover, compared with single channel entrance, the transmembrane potential was higher when the cells located at multiple channels entrances. These results suggest that it IS required to develop higher micro manufacturing technology to create channels as we expected size.

RNA Binding of T-cell Intracellular Antigen-1 (TIA-1) C-terminal RNA Recognition Motif Is Modified by pH Conditions*

Science.gov (United States)

Cruz-Gallardo, Isabel; Aroca, Ángeles; Persson, Cecilia; Karlsson, B. Göran; Díaz-Moreno, Irene

2013-01-01

T-cell intracellular antigen-1 (TIA-1) is a DNA/RNA-binding protein that regulates critical events in cell physiology by the regulation of pre-mRNA splicing and mRNA translation. TIA-1 is composed of three RNA recognition motifs (RRMs) and a glutamine-rich domain and binds to uridine-rich RNA sequences through its C-terminal RRM2 and RRM3 domains. Here, we show that RNA binding mediated by either isolated RRM3 or the RRM23 construct is controlled by slight environmental pH changes due to the protonation/deprotonation of TIA-1 RRM3 histidine residues. The auxiliary role of the C-terminal RRM3 domain in TIA-1 RNA recognition is poorly understood, and this work provides insight into its binding mechanisms. PMID:23902765

75 FR 11939 - Arkansas Lamp Manufacturing, Including On-Site Leased Workers From TEC, Van Buren, AR; Notice of...

Science.gov (United States)

2010-03-12

... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-71,714] Arkansas Lamp... a petition filed on July 17, 2009, by a company official on behalf of workers of Arkansas Lamp.... Signed at Washington, DC, this 4th day of January 2010. Del Min Amy Chen, Certifying Officer, Division of...

mRNA transfection of mouse and human neural stem cell cultures.

Directory of Open Access Journals (Sweden)

Samuel McLenachan

Full Text Available The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages.

mRNA Transfection of Mouse and Human Neural Stem Cell Cultures

Science.gov (United States)

McLenachan, Samuel; Zhang, Dan; Palomo, Ana Belén Alvarez; Edel, Michael J.; Chen, Fred K.

2013-01-01

The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages. PMID:24386231

mRNA transfection of mouse and human neural stem cell cultures.

Science.gov (United States)

McLenachan, Samuel; Zhang, Dan; Palomo, Ana Belén Alvarez; Edel, Michael J; Chen, Fred K

2013-01-01

The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages.

DC-STAMP Is an Osteoclast Fusogen Engaged in Periodontal Bone Resorption.

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Wisitrasameewong, W; Kajiya, M; Movila, A; Rittling, S; Ishii, T; Suzuki, M; Matsuda, S; Mazda, Y; Torruella, M R; Azuma, M M; Egashira, K; Freire, M O; Sasaki, H; Wang, C Y; Han, X; Taubman, M A; Kawai, T

2017-06-01

Dendritic cell-specific transmembrane protein (DC-STAMP) plays a key role in the induction of osteoclast (OC) cell fusion, as well as DC-mediated immune regulation. While DC-STAMP gene expression is upregulated in the gingival tissue with periodontitis, its pathophysiological roles in periodontitis remain unclear. To evaluate the effects of DC-STAMP in periodontitis, anti-DC-STAMP-monoclonal antibody (mAb) was tested in a mouse model of ligature-induced periodontitis ( n = 6-7/group) where Pasteurella pneumotropica ( Pp)-reactive immune response activated T cells to produce receptor activator of nuclear factor kappa-B ligand (RANKL), which, in turn, promotes the periodontal bone loss via upregulation of osteoclastogenesis. DC-STAMP was expressed on the cell surface of mature multinuclear OCs, as well as immature mononuclear OCs, in primary cultures of RANKL-stimulated bone marrow cells. Anti-DC-STAMP-mAb suppressed the emergence of large, but not small, multinuclear OCs, suggesting that DC-STAMP is engaged in the late stage of cell fusion. Anti-DC-STAMP-mAb also inhibited pit formation caused by RANKL-stimulated bone marrow cells. Attachment of ligature to a second maxillary molar induced DC-STAMP messenger RNA and protein, along with elevated tartrate-resistant acid phosphatase-positive (TRAP+) OCs and alveolar bone loss. As we expected, systemic administration of anti-DC-STAMP-mAb downregulated the ligature-induced alveolar bone loss. Importantly, local injection of anti-DC-STAMP-mAb also suppressed alveolar bone loss and reduced the total number of multinucleated TRAP+ cells in mice that received ligature attachment. Attachment of ligature induced significantly elevated tumor necrosis factor-α, interleukin-1β, and RANKL in the gingival tissue compared with the control site without ligature ( P < 0.05), which was unaffected by local injection with either anti-DC-STAMP-mAb or control-mAb. Neither in vivo anti- Pp IgG antibody nor in vitro anti- Pp T

Immunological quality and performance of tumor vessel-targeting CAR-T cells prepared by mRNA-EP for clinical research.

Science.gov (United States)

Inoo, Kanako; Inagaki, Ryo; Fujiwara, Kento; Sasawatari, Shigemi; Kamigaki, Takashi; Nakagawa, Shinsaku; Okada, Naoki

2016-01-01

We previously reported that tumor vessel-redirected T cells, which were genetically engineered with chimeric antigen receptor (CAR) specific for vascular endothelial growth factor receptor 2 (VEGFR2), demonstrated significant antitumor effects in various murine solid tumor models. In the present study, we prepared anti-VEGFR2 CAR-T cells by CAR-coding mRNA electroporation (mRNA-EP) and analyzed their immunological characteristics and functions for use in clinical research. The expression of anti-VEGFR2 CAR on murine and human T cells was detected with approximately 100% efficiency for a few days, after peaking 6-12 hours after mRNA-EP. Triple transfer of murine anti-VEGFR2 CAR-T cells into B16BL6 tumor-bearing mice demonstrated an antitumor effect comparable to that for the single transfer of CAR-T cells engineered with retroviral vector. The mRNA-EP did not cause any damage or defects to human T-cell characteristics, as determined by viability, growth, and phenotypic parameters. Additionally, two kinds of human anti-VEGFR2 CAR-T cells, which expressed different CAR construction, differentiated to effector phase with cytokine secretion and cytotoxic activity in antigen-specific manner. These results indicate that our anti-VEGFR2 CAR-T cells prepared by mRNA-EP have the potential in terms of quality and performance to offer the prospect of safety and efficacy in clinical research as cellular medicine.

Immunological quality and performance of tumor vessel-targeting CAR-T cells prepared by mRNA-EP for clinical research

Directory of Open Access Journals (Sweden)

Kanako Inoo

2016-01-01

Full Text Available We previously reported that tumor vessel-redirected T cells, which were genetically engineered with chimeric antigen receptor (CAR specific for vascular endothelial growth factor receptor 2 (VEGFR2, demonstrated significant antitumor effects in various murine solid tumor models. In the present study, we prepared anti-VEGFR2 CAR-T cells by CAR-coding mRNA electroporation (mRNA-EP and analyzed their immunological characteristics and functions for use in clinical research. The expression of anti-VEGFR2 CAR on murine and human T cells was detected with approximately 100% efficiency for a few days, after peaking 6–12 hours after mRNA-EP. Triple transfer of murine anti-VEGFR2 CAR-T cells into B16BL6 tumor-bearing mice demonstrated an antitumor effect comparable to that for the single transfer of CAR-T cells engineered with retroviral vector. The mRNA-EP did not cause any damage or defects to human T-cell characteristics, as determined by viability, growth, and phenotypic parameters. Additionally, two kinds of human anti-VEGFR2 CAR-T cells, which expressed different CAR construction, differentiated to effector phase with cytokine secretion and cytotoxic activity in antigen-specific manner. These results indicate that our anti-VEGFR2 CAR-T cells prepared by mRNA-EP have the potential in terms of quality and performance to offer the prospect of safety and efficacy in clinical research as cellular medicine.

PLK-1 Silencing in Bladder Cancer by siRNA Delivered With Exosomes.

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Greco, Kristin A; Franzen, Carrie A; Foreman, Kimberly E; Flanigan, Robert C; Kuo, Paul C; Gupta, Gopal N

2016-05-01

To use exosomes as a vector to deliver small interfering ribonucleic acid (siRNA) to silence the polo-like kinase 1 (PLK-1) gene in bladder cancer cells. Exosomes were isolated from both human embryonic kidney 293 (HEK293) cell and mesenchymal stem cell (MSC) conditioned media. Fluorescently labeled exosomes were co-cultured with bladder cancer and normal epithelial cells and uptake was quantified by image cytometry. PLK-1 siRNA and negative control siRNA were loaded into HEK293 and MSC exosomes using electroporation. An invasive bladder cancer cell line (UMUC3) was co-cultured with the electroporated exosomes. Quantitative reverse transcriptase polymerase chain reaction was performed. Protein analysis was performed by Western blot. Annexin V staining and MTT assays were used to investigate effects on apoptosis and viability. Bladder cancer cell lines internalize an increased percentage of HEK293 exosomes when compared to normal bladder epithelial cells. Treatment of UMUC3 cells with exosomes electroporated with PLK-1 siRNA achieved successful knockdown of PLK-1 mRNA and protein when compared to cells treated with negative control exosomes. HEK293 and MSC exosomes were effectively used as a delivery vector to transport PLK-1 siRNA to bladder cancer cells in vitro, resulting in selective gene silencing of PLK-1. The use of exosomes as a delivery vector for potential intravesical therapy is attractive. Copyright © 2016 Elsevier Inc. All rights reserved.

Impact of tuberculosis treatment on CD4 cell count, HIV RNA, and p24 antigen in patients with HIV and tuberculosis

DEFF Research Database (Denmark)

Wejse, Christian; Furtado, A.; Camara, C.

2013-01-01

To describe HIV RNA levels during tuberculosis (TB) infection in patients co-infected with TB and HIV. Moreover, to examine the p24 antigen profile during TB treatment.......To describe HIV RNA levels during tuberculosis (TB) infection in patients co-infected with TB and HIV. Moreover, to examine the p24 antigen profile during TB treatment....

Ultra-sensitive DNA assay based on single-molecule detection coupled with fluorescent quantum dot-labeling and its application to determination of messenger RNA

International Nuclear Information System (INIS)

Li Li; Li Xincang; Li Lu; Wang Jinxing; Jin Wenrui

2011-01-01

An ultra-sensitive single-molecule detection (SMD) method for quantification of DNA using total internal reflection fluorescence microscopy (TIRFM) coupled with fluorescent quantum dot (QD)-labeling was developed. In this method, the target DNA (tDNA) was captured by the capture DNA immobilized on the silanized coverslip blocked with ethanolamine and bovine serum albumin. Then, the QD-labeled probe DNA was hybridized to the tDNA. Ten fluorescent images of the QD-labeled sandwich DNA hybrids on the coverslip were taken by a high-sensitive CCD. The tDNA was quantified by counting the bright spots on the images using a calibration curve. The LOD of the method was 1 x 10 -14 mol L -1 . Several key factors, including image acquirement, fluorescence probe, substrate preparation, noise elimination from solutions and glass coverslips, and nonspecific adsorption and binding of solution-phase detection probes were discussed in detail. The method could be applied to quantify messenger RNA (mRNA) in cells. In order to determine mRNA, double-stranded RNA-DNA hybrids consisting of mRNA and corresponding cDNA were synthesized from the cellular mRNA template using reverse transcription in the presence of reverse transcriptase. After removing the mRNA in the double-stranded hybrids using ribonuclease, cDNA was quantified using the SMD-based TIRFM. Osteopontin mRNA in decidual stromal cells was chosen as the model analyte.

BAD-LAMP controls TLR9 trafficking and signalling in human plasmacytoid dendritic cells.

Science.gov (United States)

Combes, Alexis; Camosseto, Voahirana; N'Guessan, Prudence; Argüello, Rafael J; Mussard, Julie; Caux, Christophe; Bendriss-Vermare, Nathalie; Pierre, Philippe; Gatti, Evelina

2017-10-13

Toll-like receptors (TLR) are essential components of the innate immune system. Several accessory proteins, such as UNC93B1, are required for transport and activation of nucleic acid sensing Toll-like receptors in endosomes. Here, we show that BAD-LAMP (LAMP5) controls TLR9 trafficking to LAMP1 + late endosomes in human plasmacytoid dendritic cells (pDC), leading to NF-κB activation and TNF production upon DNA detection. An inducible VAMP3 +/ LAMP2 +/ LAMP1 - endolysosome compartment exists in pDCs from which TLR9 activation triggers type I interferon expression. BAD-LAMP-silencing enhances TLR9 retention in this compartment and consequent downstream signalling events. Conversely, sustained BAD-LAMP expression in pDCs contributes to their lack of type I interferon production after exposure to a TGF-β-positive microenvironment or isolation from human breast tumours. Hence, BAD-LAMP limits interferon expression in pDCs indirectly, by promoting TLR9 sorting to late endosome compartments at steady state and in response to immunomodulatory cues.TLR9 is highly expressed by plasmacytoid dendritic cells and detects nucleic acids, but to discriminate between host and microbial nucleic acids TLR9 is sorted into different endosomal compartments. Here the authors show that BAD-LAMP limits type 1 interferon responses by sorting TLR9 to late endosomal compartments.

An "Off-the-Shelf" System for Intraprocedural Electrical Current Evaluation and Monitoring of Irreversible Electroporation Therapy.

Science.gov (United States)

Neal, Robert E; Kavnoudias, Helen; Thomson, Kenneth R

2015-06-01

Irreversible electroporation (IRE) ablation uses a series of brief electric pulses to create nanoscale defects in cell membranes, killing the cells. It has shown promise in numerous soft-tissue tumor applications. Larger voltages between electrodes will increase ablation volume, but exceeding electrical limits may risk damage to the patient, cause ineffective therapy delivery, or require generator restart. Monitoring electrical current for these conditions in real-time enables managing these risks. This capacity is not presently available in clinical IRE generators. We describe a system using a Tektronix TCP305 AC/DC Current Probe connected to a TCPA300 AC/DC Current Probe Amplifier, which is read on a computer using a Protek DSO-2090 USB computer-interfacing oscilloscope. Accuracy of the system was tested with a resistor circuit and by comparing measured currents with final outputs from the NanoKnife clinical electroporation pulse generator. Accuracy of measured currents was 1.64 ± 2.4 % relative to calculations for the resistor circuit and averaged 0.371 ± 0.977 % deviation from the NanoKnife. During clinical pulse delivery, the system offers real-time evaluation of IRE procedure progress and enables a number of methods for identifying approaching issues from electrical behavior of therapy delivery, facilitating protocol changes before encountering therapy delivery issues. This system can monitor electrical currents in real-time without altering the electric pulses or modifying the pulse generator. This facilitates delivering electric pulse protocols that remain within the optimal range of electrical currents-sufficient strength for clinically relevant ablation volumes, without the risk of exceeding safe electric currents or causing inadequate ablation.

An optimized electroporation approach for efficient CRISPR/Cas9 genome editing in murine zygotes.

Directory of Open Access Journals (Sweden)

Simon E Tröder

Full Text Available Electroporation of zygotes represents a rapid alternative to the elaborate pronuclear injection procedure for CRISPR/Cas9-mediated genome editing in mice. However, current protocols for electroporation either require the investment in specialized electroporators or corrosive pre-treatment of zygotes which compromises embryo viability. Here, we describe an easily adaptable approach for the introduction of specific mutations in C57BL/6 mice by electroporation of intact zygotes using a common electroporator with synthetic CRISPR/Cas9 components and minimal technical requirement. Direct comparison to conventional pronuclear injection demonstrates significantly reduced physical damage and thus improved embryo development with successful genome editing in up to 100% of living offspring. Hence, our novel approach for Easy Electroporation of Zygotes (EEZy allows highly efficient generation of CRISPR/Cas9 transgenic mice while reducing the numbers of animals required.

Rescue of foot-and-mouth disease viruses that are pathogenic for cattle from preserved viral RNA samples.

Directory of Open Access Journals (Sweden)

Graham J Belsham

Full Text Available BACKGROUND: Foot and mouth disease is an economically important disease of cloven-hoofed animals including cattle, sheep and pigs. It is caused by a picornavirus, foot-and-mouth disease virus (FMDV, which has a positive sense RNA genome which, when introduced into cells, can initiate virus replication. PRINCIPAL FINDINGS: A system has been developed to rescue infectious FMDV from RNA preparations generated from clinical samples obtained under experimental conditions and then applied to samples collected in the "field". Clinical samples from suspect cases of foot-and-mouth disease (FMD were obtained from within Pakistan and Afghanistan. The samples were treated to preserve the RNA and then transported to National Veterinary Institute, Lindholm, Denmark. Following RNA extraction, FMDV RNA was quantified by real-time RT-PCR and samples containing significant levels of FMDV RNA were introduced into susceptible cells using electroporation. Progeny viruses were amplified in primary bovine thyroid cells and characterized using antigen ELISA and also by RT-PCR plus sequencing. FMD viruses of three different serotypes and multiple lineages have been successfully rescued from the RNA samples. Two of the rescued viruses (of serotype O and Asia 1 were inoculated into bull calves under high containment conditions. Acute clinical disease was observed in each case which spread rapidly from the inoculated calves to in-contact animals. Thus the rescued viruses were highly pathogenic. The availability of the rescued viruses enabled serotyping by antigen ELISA and facilitated genome sequencing. CONCLUSIONS: The procedure described here should improve the characterization of FMDVs circulating in countries where the disease is endemic and thus enhance disease control globally.

Magnetic resonance electrical impedance tomography for determining electric field distribution during electroporation

International Nuclear Information System (INIS)

Kranjc, Matej; Miklavcic, Damijan; Bajd, Franci; Serša, Igor

2013-01-01

Electroporation is a phenomenon caused by externally applied electric field to cells that results in an increase of cell membrane permeability to various molecules. Accurate coverage of the tissue with a sufficiently large electric field presents one of the most important conditions for successful membrane permeabilization. Applications based on electroporation would greatly benefit with a method for monitoring the electric field, especially if it could be done in situ. As the membrane electroporation is a consequence of an induced transmembrane potential, which is directly proportional to the local electric field, we have been investigating current density imaging and magnetic resonance electrical impedance tomography techniques to determine the electric field distribution during electroporation. In this paper, we present comparison of current density and electric field distribution in an agar phantom and in a liver tissue exposed to electroporation pulses. As expected, a region of increased electrical conductivity was observed in the liver tissue exposed to sufficiently high electric field but not in agar phantom.

Egr2 induced during DC development acts as an intrinsic negative regulator of DC immunogenicity.

Science.gov (United States)

Miah, Mohammad Alam; Byeon, Se Eun; Ahmed, Md Selim; Yoon, Cheol-Hee; Ha, Sang-Jun; Bae, Yong-Soo

2013-09-01

Early growth response gene 2 (Egr2), which encodes a zinc finger transcription factor, is rapidly and transiently induced in various cell types independently of de novo protein synthesis. Although a role for Egr2 is well established in T-cell development, Egr2 expression and its biological function in dendritic cells (DCs) have not yet been described. Here, we demonstrate Egr2 expression during DC development, and its role in DC-mediated immune responses. Egr2 is expressed in the later stage of DC development from BM precursor cells. Even at steady state, Egr2 is highly expressed in mouse splenic DCs. Egr2-knockdown (Egr2-KD) DCs showed increased levels of major histocompatability complex (MHC) class I and II and co-stimulatory molecules, and enhanced antigen uptake and migratory capacities. Furthermore, Egr2-KD abolished SOCS1 expression and signal transducer and activator of transcription 5 (STAT5) activation during DC development, probably resulting in the enhancement of IL-12 expression and Th1 immunogenicity of a DC vaccine. DC-mediated cytotoxic T lymphocyte (CTL) activation and antitumor immunity were significantly enhanced by Egr2-KD, and impaired by Egr2 overexpression in antigen-pulsed DC vaccines. These data suggest that Egr2 acts as an intrinsic negative regulator of DC immunogenicity and can be an attractive molecular target for DC vaccine development. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Determination of HID electrode falls in a model lamp I: Pyrometric measurements

International Nuclear Information System (INIS)

Dabringhausen, L.; Nandelstaedt, D.; Luhmann, J.; Mentel, J.

2002-01-01

To verify models describing the near-electrode regions electrodes of pure and doped tungsten for high intensity discharge lamps are investigated in a special model lamp. It can be operated with arc currents of 1 A to 10 A, DC or AC with arbitrary waveforms up to a few kHz. Argon and xenon, at pressures from 0.1 MPa to 1 MPa, are used as fill gases. A large variety of electrodes can be inserted. To perform spatially resolved measurements they are displaced reproducibly within the discharge tube during lamp operation. Spatially resolved pyrometric measurements of the electrode surface temperature in the case of DC operation are presented. From the temperature distribution the power loss of the electrodes by thermal radiation and heat conduction is determined. It increases almost linearly with the arc current at the anode and less than linear at the cathode. A relation is deduced between the cathode fall and the power fed into the cathode setting up the power balance of the cathodic current transfer zone. The resulting cathode falls show a strong dependence on the electrode diameter. Electrical measurements of separate cathode and anode falls are given in a subsequent paper. The outcomes of both methods and of modelling are compared in a third paper. (author)

A novel method of modifying immune responses by vaccination with lipiodol-siRNA mixtures

Directory of Open Access Journals (Sweden)

Yijian Li

2006-01-01

Full Text Available Abstract The dendritic cell (DC possesses the ability to stimulate both T helper 1 (Th1 and Th2 responses depending on activation stimuli. Although it is known that chemically or genetically modified DC can be used therapeutically to steer immune responses towards either Th1 or Th2, cellular therapy with ex vivo manipulated DC is clinically difficult. Here we demonstrate a novel method of switching immune responses from Th1 to Th2 through in vivo immune modulation by administration of siRNA. We demonstrate that siRNA targeting of the IL-12p35 gene leads to a Th2 bias in vitro through an IL-10 dependent mechanism. In vivo administration of siRNA admixed with the oil-based contrast agent lipiodol in the presence of antigen and adjuvant induced a deviation in recall response to reduced production of IFN-γ and augmented IL-4 response using either KLH or ovalbumin. This simple method of in vivo modification of immune response possesses therapeutic potential in Th1-mediated diseases such as multiple sclerosis and autoimmune diabetes.

Direct therapeutic applications of calcium electroporation to effectively induce tumor necrosis

DEFF Research Database (Denmark)

Frandsen, Stine Krog; Gissel, Hanne; Hojman, Pernille

2012-01-01

in vivo. Calcium electroporation elicited dramatic antitumor responses in which 89% of treated tumors were eliminated. Histologic analyses indicated complete tumor necrosis. Mechanistically, calcium electroporation caused acute ATP depletion likely due to a combination of increased cellular use of ATP......, decreased production of ATP due to effects on the mitochondria, as well as loss of ATP through the permeabilized cell membrane. Taken together, our findings offer a preclinical proof of concept for the use of electroporation to load cancer cells with calcium as an efficient anticancer treatment...

Development Ground Fault Detecting System for D.C Voltage Line

Energy Technology Data Exchange (ETDEWEB)

Kim Taek Soo; Song Ung Il; Gwon, Young Dong; Lee Hyoung Kee [Korea Electric Power Research Institute, Taejon (Korea, Republic of)

1996-12-31

It is necessary to keep the security of reliability and to maximize the efficiency of maintenance by prompt detection of a D.C feeder ground fault point at the built ed or a building power plants. At present, the most of the power plants are set up the ground fault indicator lamp in the monitor room. If a ground fault occurs on DC voltage feeder, a current through the ground fault relay is adjusted and the lamps have brightened while the current flows the relay coil. In order to develop such a system, it is analyzed a D.C feeder ground circuit theoretically and studied a principles which can determine ground fault point or a polarity discrimination and a phase discrimination of the line. So, the developed system through this principles can compute a resistance ground fault current and a capacitive ground fault current. It shows that the system can defect a ground fault point or a bad insulated line by measuring a power plant D.C feeder insulation resistance at the un interruptible power status, and therefore the power plant could protect an unexpected service interruption . (author). 18 refs., figs.

Recovery of viral RNA and infectious foot-and-mouth disease virus from positive lateral-flow devices.

Directory of Open Access Journals (Sweden)

Veronica L Fowler

Full Text Available Foot-and-mouth disease Virus (FMDV is an economically important, highly contagious picornavirus that affects both wild and domesticated cloven hooved animals. In developing countries, the effective laboratory diagnosis of foot-and-mouth disease (FMD is often hindered by inadequate sample preservation due to difficulties in the transportation and storage of clinical material. These factors can compromise the ability to detect and characterise FMD virus in countries where the disease is endemic. Furthermore, the high cost of sending infectious virus material and the biosecurity risk it presents emphasises the need for a thermo-stable, non-infectious mode of transporting diagnostic samples. This paper investigates the potential of using FMDV lateral-flow devices (LFDs for dry transportation of clinical samples for subsequent nucleic acid amplification, sequencing and recovery of infectious virus by electroporation. FMDV positive samples (epithelial suspensions and cell culture isolates representing four FMDV serotypes were applied to antigen LFDs: after which it was possible to recover viral RNA that could be detected using real-time RT-PCR. Using this nucleic acid, it was also possible to recover VP1 sequences and also successfully utilise protocols for amplification of complete FMD virus genomes. It was not possible to recover infectious FMDV directly from the LFDs, however following electroporation into BHK-21 cells and subsequent cell passage, infectious virus could be recovered. Therefore, these results support the use of the antigen LFD for the dry, non-hazardous transportation of samples from FMD endemic countries to international reference laboratories.

Theory and in vivo application of electroporative gene delivery.

Science.gov (United States)

Somiari, S; Glasspool-Malone, J; Drabick, J J; Gilbert, R A; Heller, R; Jaroszeski, M J; Malone, R W

2000-09-01

Efficient and safe methods for delivering exogenous genetic material into tissues must be developed before the clinical potential of gene therapy will be realized. Recently, in vivo electroporation has emerged as a leading technology for developing nonviral gene therapies and nucleic acid vaccines (NAV). Electroporation (EP) involves the application of pulsed electric fields to cells to enhance cell permeability, resulting in exogenous polynucleotide transit across the cytoplasmic membrane. Similar pulsed electrical field treatments are employed in a wide range of biotechnological processes including in vitro EP, hybridoma production, development of transgenic animals, and clinical electrochemotherapy. Electroporative gene delivery studies benefit from well-developed literature that may be used to guide experimental design and interpretation. Both theory and experimental analysis predict that the critical parameters governing EP efficacy include cell size and field strength, duration, frequency, and total number of applied pulses. These parameters must be optimized for each tissue in order to maximize gene delivery while minimizing irreversible cell damage. By providing an overview of the theory and practice of electroporative gene transfer, this review intends to aid researchers that wish to employ the method for preclinical and translational gene therapy, NAV, and functional genomic research.

Irreversible electroporation: state of the art

Directory of Open Access Journals (Sweden)

Wagstaff PGK

2016-04-01

Full Text Available Peter GK Wagstaff,1 Mara Buijs,1 Willemien van den Bos,1 Daniel M de Bruin,2 Patricia J Zondervan,1 Jean JMCH de la Rosette,1 M Pilar Laguna Pes1 1Department of Urology, 2Department of Biomedical Engineering and Physics, Academic Medical Center, Amsterdam, the Netherlands Abstract: The field of focal ablative therapy for the treatment of cancer is characterized by abundance of thermal ablative techniques that provide a minimally invasive treatment option in selected tumors. However, the unselective destruction inflicted by thermal ablation modalities can result in damage to vital structures in the vicinity of the tumor. Furthermore, the efficacy of thermal ablation intensity can be impaired due to thermal sink caused by large blood vessels in the proximity of the tumor. Irreversible electroporation (IRE is a novel ablation modality based on the principle of electroporation or electropermeabilization, in which electric pulses are used to create nanoscale defects in the cell membrane. In theory, IRE has the potential of overcoming the aforementioned limitations of thermal ablation techniques. This review provides a description of the principle of IRE, combined with an overview of in vivo research performed to date in the liver, pancreas, kidney, and prostate. Keywords: irreversible electroporation, IRE, tumor, ablation, focal therapy, cancer

Model study of DC ignition of fluorescent tubes

International Nuclear Information System (INIS)

Brok, W J M; Gendre, M F; Mullen, J J A M van der

2007-01-01

Breakdown in a discharge tube is investigated by means of a fluid model. The discharge tube is similar to a compact fluorescent lamp tube, containing argon at 3 Torr and mercury at a few millitorr. It was found that the minimum breakdown voltage is decreased substantially compared with a tube containing pure argon. Penning ionization of mercury via an argon metastable state plays an important role in this effect. This is illustrated for a lamp operated on a DC voltage, where significant Penning ionization takes place in the wake of the ionization front. Furthermore, contrary to what is suggested in earlier literature, the development of the surface potential of the lamp is shown to be not only determined by surface charges, but also by volume charges

Electroporation-delivered transdermal neostigmine in rats: equivalent action to intravenous administration.

Science.gov (United States)

Berkó, Szilvia; Szűcs, Kálmán F; Balázs, Boglárka; Csányi, Erzsébet; Varju, Gábor; Sztojkov-Ivanov, Anita; Budai-Szűcs, Mária; Bóta, Judit; Gáspár, Róbert

2016-01-01

Transdermal electroporation has become one of the most promising noninvasive methods for drug administration, with greatly increased transport of macromolecules through the skin. The cecal-contracting effects of repeated transdermal electroporation delivery and intravenous administration of neostigmine were compared in anesthetized rats. The cecal contractions were detected with implantable strain gauge sensors, and the plasma levels of neostigmine were followed by high-performance liquid chromatography. Both intravenously and EP-administered neostigmine (0.2-66.7 μg/kg) increased the cecal contractions in a dose-dependent manner. For both the low doses and the highest dose, the neostigmine plasma concentrations were the same after the two modes of administration, while an insignificantly higher level was observed at a dose of 20 μg/kg after intravenous administration as compared with the electroporation route. The contractile responses did not differ significantly after the two administration routes. The results suggest that electroporation-delivered neostigmine elicits action equivalent to that observed after intravenous administration as concerning both time and intensity. Electroporation permits the delivery of even lower doses of water-soluble compounds through the skin, which is very promising for clinical practice.

Use of electroporation to study the cytotoxic effects of fluorodeoxyuridylate in intact cells.

Science.gov (United States)

Jastreboff, M M; Sokoloski, J A; Bertino, J R; Narayanan, R

1987-04-15

The introduction of 2'-deoxyuridine 5'-monophosphate and its analog, 5-fluoro-2'-deoxyuridine 5'-monophosphate, into intact CCRF-CEM and NIH3T3 cells was achieved by electroporation. Following electroporation, cells were shown to be fully functional as monitored by the incorporation of deoxyuridylate, after conversion to thymidylate, into DNA. Pretreatment of cells with fluorodeoxyuridine completely abolished this effect. In contrast, introduction of the fluoro analog into cells by electroporation markedly inhibited both DNA synthesis and cell growth in a time-dependent manner. Thus, electroporation offers a powerful tool to permeabilize cells to a variety of cellular metabolites and antimetabolites.

DC grid for home applications

Science.gov (United States)

Elangovan, D.; Archana, R.; Jayadeep, V. J.; Nithin, M.; Arunkumar, G.

2017-11-01

More than fifty percent Indian population do not have access to electricity in daily lives. The distance between the power generating stations and the distribution centers forms one of the main reasons for lack of electrification in rural and remote areas. Here lies the importance of decentralization of power generation through renewable energy resources. In the present world, electricity is predominantly powered by alternating current, but most day to day devices like LED lamps, computers and electrical vehicles, all run on DC power. By directly supplying DC to these loads, the number of power conversion stages was reduced, and overall system efficiency increases. Replacing existing AC network with DC is a humongous task, but with power electronic techniques, this project intends to implement DC grid at a household level in remote and rural areas. Proposed work was designed and simulated successfully for various loads amounting to 250 W through appropriate power electronic convertors. Maximum utilization of the renewable sources for domestic and commercial application was achieved with the proposed DC topology.

Regulation of HIV-Gag Expression and Targeting to the Endolysosomal/Secretory Pathway by the Luminal Domain of Lysosomal-Associated Membrane Protein (LAMP-1) Enhance Gag-Specific Immune Response

Science.gov (United States)

Lucas, Carolina Gonçalves de Oliveira; Rigato, Paula Ordonhez; Gonçalves, Jorge Luiz Santos; Sato, Maria Notomi; Maciel, Milton; Peçanha, Ligia Maria Torres; August, J. Thomas; de Azevedo Marques, Ernesto Torres; de Arruda, Luciana Barros

2014-01-01

We have previously demonstrated that a DNA vaccine encoding HIV-p55gag in association with the lysosomal associated membrane protein-1 (LAMP-1) elicited a greater Gag-specific immune response, in comparison to a DNA encoding the native gag. In vitro studies have also demonstrated that LAMP/Gag was highly expressed and was present in MHCII containing compartments in transfected cells. In this study, the mechanisms involved in these processes and the relative contributions of the increased expression and altered traffic for the enhanced immune response were addressed. Cells transfected with plasmid DNA constructs containing p55gag attached to truncated sequences of LAMP-1 showed that the increased expression of gag mRNA required p55gag in frame with at least 741 bp of the LAMP-1 luminal domain. LAMP luminal domain also showed to be essential for Gag traffic through lysosomes and, in this case, the whole sequence was required. Further analysis of the trafficking pathway of the intact LAMP/Gag chimera demonstrated that it was secreted, at least in part, associated with exosome-like vesicles. Immunization of mice with LAMP/gag chimeric plasmids demonstrated that high expression level alone can induce a substantial transient antibody response, but targeting of the antigen to the endolysosomal/secretory pathways was required for establishment of cellular and memory response. The intact LAMP/gag construct induced polyfunctional CD4+ T cell response, which presence at the time of immunization was required for CD8+ T cell priming. LAMP-mediated targeting to endolysosomal/secretory pathway is an important new mechanistic element in LAMP-mediated enhanced immunity with applications to the development of novel anti-HIV vaccines and to general vaccinology field. PMID:24932692

An “Off-the-Shelf” System for Intraprocedural Electrical Current Evaluation and Monitoring of Irreversible Electroporation Therapy

Energy Technology Data Exchange (ETDEWEB)

Neal, Robert E., E-mail: Robert.Neal@alfred.org.au; Kavnoudias, Helen; Thomson, Kenneth R. [The Alfred Hospital, Radiology Research Unit, Department of Radiology (Australia)

2015-06-15

IntroductionIrreversible electroporation (IRE) ablation uses a series of brief electric pulses to create nanoscale defects in cell membranes, killing the cells. It has shown promise in numerous soft-tissue tumor applications. Larger voltages between electrodes will increase ablation volume, but exceeding electrical limits may risk damage to the patient, cause ineffective therapy delivery, or require generator restart. Monitoring electrical current for these conditions in real-time enables managing these risks. This capacity is not presently available in clinical IRE generators.MethodsWe describe a system using a Tektronix TCP305 AC/DC Current Probe connected to a TCPA300 AC/DC Current Probe Amplifier, which is read on a computer using a Protek DSO-2090 USB computer-interfacing oscilloscope. Accuracy of the system was tested with a resistor circuit and by comparing measured currents with final outputs from the NanoKnife clinical electroporation pulse generator.ResultsAccuracy of measured currents was 1.64 ± 2.4 % relative to calculations for the resistor circuit and averaged 0.371 ± 0.977 % deviation from the NanoKnife. During clinical pulse delivery, the system offers real-time evaluation of IRE procedure progress and enables a number of methods for identifying approaching issues from electrical behavior of therapy delivery, facilitating protocol changes before encountering therapy delivery issues.ConclusionsThis system can monitor electrical currents in real-time without altering the electric pulses or modifying the pulse generator. This facilitates delivering electric pulse protocols that remain within the optimal range of electrical currents—sufficient strength for clinically relevant ablation volumes, without the risk of exceeding safe electric currents or causing inadequate ablation.

Ultra-sensitive DNA assay based on single-molecule detection coupled with fluorescent quantum dot-labeling and its application to determination of messenger RNA

Energy Technology Data Exchange (ETDEWEB)

Li Li [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Li Xincang [School of Life Sciences, Shandong University, Jinan 250100 (China); Li Lu [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Wang Jinxing [School of Life Sciences, Shandong University, Jinan 250100 (China); Jin Wenrui, E-mail: jwr@sdu.edu.cn [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China)

2011-01-24

An ultra-sensitive single-molecule detection (SMD) method for quantification of DNA using total internal reflection fluorescence microscopy (TIRFM) coupled with fluorescent quantum dot (QD)-labeling was developed. In this method, the target DNA (tDNA) was captured by the capture DNA immobilized on the silanized coverslip blocked with ethanolamine and bovine serum albumin. Then, the QD-labeled probe DNA was hybridized to the tDNA. Ten fluorescent images of the QD-labeled sandwich DNA hybrids on the coverslip were taken by a high-sensitive CCD. The tDNA was quantified by counting the bright spots on the images using a calibration curve. The LOD of the method was 1 x 10{sup -14} mol L{sup -1}. Several key factors, including image acquirement, fluorescence probe, substrate preparation, noise elimination from solutions and glass coverslips, and nonspecific adsorption and binding of solution-phase detection probes were discussed in detail. The method could be applied to quantify messenger RNA (mRNA) in cells. In order to determine mRNA, double-stranded RNA-DNA hybrids consisting of mRNA and corresponding cDNA were synthesized from the cellular mRNA template using reverse transcription in the presence of reverse transcriptase. After removing the mRNA in the double-stranded hybrids using ribonuclease, cDNA was quantified using the SMD-based TIRFM. Osteopontin mRNA in decidual stromal cells was chosen as the model analyte.

Electroporation of cells using EM induction of ac fields by a magnetic stimulator

International Nuclear Information System (INIS)

Chen, C; Robinson, M P; Evans, J A; Smye, S W; O'Toole, P

2010-01-01

This paper describes a method of effectively electroporating mammalian cell membranes with pulsed alternating-current (ac) electric fields at field strengths of 30-160 kV m -1 . Although many in vivo electroporation protocols entail applying square wave or monotonically decreasing pulses via needles or electrode plates, relatively few have explored the use of pulsed ac fields. Following our previous study, which established the effectiveness of ac fields for electroporating cell membranes, a primary/secondary coil system was constructed to produce sufficiently strong electric fields by electromagnetic induction. The primary coil was formed from the applicator of an established transcranial magnetic stimulation (TMS) system, while the secondary coil was a purpose-built device of a design which could eventually be implanted into tissue. The effects of field strength, pulse interval and cumulative exposure time were investigated using microscopy and flow cytometry. Results from experiments on concentrated cell suspensions showed an optimized electroporation efficiency of around 50%, demonstrating that electroporation can be practicably achieved by inducing such pulsed ac fields. This finding confirms the possibility of a wide range of in vivo applications based on magnetically coupled ac electroporation.

Electroporation of cells using EM induction of ac fields by a magnetic stimulator

Energy Technology Data Exchange (ETDEWEB)

Chen, C; Robinson, M P [Department of Electronics, University of York, Heslington, York YO10 5DD (United Kingdom); Evans, J A [Academic Unit of Medical Physics, University of Leeds, Leeds LS2 9JT (United Kingdom); Smye, S W [Department of Medical Physics and Engineering, Leeds Teaching Hospitals, St. James' s University Hospital, Leeds LS9 7TF (United Kingdom); O' Toole, P [Department of Biology, University of York, Heslington, York YO10 5DD (United Kingdom)

2010-02-21

This paper describes a method of effectively electroporating mammalian cell membranes with pulsed alternating-current (ac) electric fields at field strengths of 30-160 kV m{sup -1}. Although many in vivo electroporation protocols entail applying square wave or monotonically decreasing pulses via needles or electrode plates, relatively few have explored the use of pulsed ac fields. Following our previous study, which established the effectiveness of ac fields for electroporating cell membranes, a primary/secondary coil system was constructed to produce sufficiently strong electric fields by electromagnetic induction. The primary coil was formed from the applicator of an established transcranial magnetic stimulation (TMS) system, while the secondary coil was a purpose-built device of a design which could eventually be implanted into tissue. The effects of field strength, pulse interval and cumulative exposure time were investigated using microscopy and flow cytometry. Results from experiments on concentrated cell suspensions showed an optimized electroporation efficiency of around 50%, demonstrating that electroporation can be practicably achieved by inducing such pulsed ac fields. This finding confirms the possibility of a wide range of in vivo applications based on magnetically coupled ac electroporation.

Single exponential decay waveform; a synergistic combination of electroporation and electrolysis (E2 for tissue ablation

Directory of Open Access Journals (Sweden)

Nina Klein

2017-04-01

Full Text Available Background Electrolytic ablation and electroporation based ablation are minimally invasive, non-thermal surgical technologies that employ electrical currents and electric fields to ablate undesirable cells in a volume of tissue. In this study, we explore the attributes of a new tissue ablation technology that simultaneously delivers a synergistic combination of electroporation and electrolysis (E2. Method A new device that delivers a controlled dose of electroporation field and electrolysis currents in the form of a single exponential decay waveform (EDW was applied to the pig liver, and the effect of various parameters on the extent of tissue ablation was examined with histology. Results Histological analysis shows that E2 delivered as EDW can produce tissue ablation in volumes of clinical significance, using electrical and temporal parameters which, if used in electroporation or electrolysis separately, cannot ablate the tissue. Discussion The E2 combination has advantages over the three basic technologies of non-thermal ablation: electrolytic ablation, electrochemical ablation (reversible electroporation with injection of drugs and irreversible electroporation. E2 ablates clinically relevant volumes of tissue in a shorter period of time than electrolysis and electroporation, without the need to inject drugs as in reversible electroporation or use paralyzing anesthesia as in irreversible electroporation.

The Effects of Irreversible Electroporation on the Achilles Tendon: An Experimental Study in a Rabbit Model.

Directory of Open Access Journals (Sweden)

Yue Song

Full Text Available To evaluate the potential effects of irreversible electroporation ablation on the Achilles tendon in a rabbit model and to compare the histopathological and biomechanical changes between specimens following electroporation ablation and radiofrequency ablation.A total of 140 six-month-old male New Zealand rabbits were used. The animals were randomly divided into two groups, 70 in the radiofrequency ablation group and 70 in the electroporation group. In situ ablations were applied directly to the Achilles tendons of rabbits using typical electroporation (1800 V/cm, 90 pulses and radiofrequency ablation (power control mode protocols. Histopathological and biomechanical evaluations were performed to examine the effects of electroporation ablation and radiofrequency ablation over time.Both electroporation and radiofrequency ablation produced complete cell ablation in the target region. Thermal damage resulted in tendon rupture 3 days post radiofrequency ablation. In contrast, electroporation-ablated Achilles tendons preserved their biomechanical properties and showed no detectable rupture at this time point. The electroporation-ablated tendons exhibited signs of recovery, including tenoblast regeneration and angiogenesis within 2 weeks, and the restoration of their integral structure was evident within 12 weeks.When applying electroporation to ablate solid tumors, major advantage could be that collateral damage to adjacent tendons or ligaments is minimized due to the unique ability of electroporation ablation to target the cell membrane. This advantage could have a significant impact on the field of tumor ablation near vital tendons or ligaments.

The Effects of Irreversible Electroporation on the Achilles Tendon: An Experimental Study in a Rabbit Model.

Science.gov (United States)

Song, Yue; Zheng, Jingjing; Yan, Mingwei; Ding, Weidong; Xu, Kui; Fan, Qingyu; Li, Zhao

2015-01-01

To evaluate the potential effects of irreversible electroporation ablation on the Achilles tendon in a rabbit model and to compare the histopathological and biomechanical changes between specimens following electroporation ablation and radiofrequency ablation. A total of 140 six-month-old male New Zealand rabbits were used. The animals were randomly divided into two groups, 70 in the radiofrequency ablation group and 70 in the electroporation group. In situ ablations were applied directly to the Achilles tendons of rabbits using typical electroporation (1800 V/cm, 90 pulses) and radiofrequency ablation (power control mode) protocols. Histopathological and biomechanical evaluations were performed to examine the effects of electroporation ablation and radiofrequency ablation over time. Both electroporation and radiofrequency ablation produced complete cell ablation in the target region. Thermal damage resulted in tendon rupture 3 days post radiofrequency ablation. In contrast, electroporation-ablated Achilles tendons preserved their biomechanical properties and showed no detectable rupture at this time point. The electroporation-ablated tendons exhibited signs of recovery, including tenoblast regeneration and angiogenesis within 2 weeks, and the restoration of their integral structure was evident within 12 weeks. When applying electroporation to ablate solid tumors, major advantage could be that collateral damage to adjacent tendons or ligaments is minimized due to the unique ability of electroporation ablation to target the cell membrane. This advantage could have a significant impact on the field of tumor ablation near vital tendons or ligaments.

Effects of in ovo electroporation on endogenous gene expression: genome-wide analysis

Directory of Open Access Journals (Sweden)

Chambers David

2011-04-01

Full Text Available Abstract Background In ovo electroporation is a widely used technique to study gene function in developmental biology. Despite the widespread acceptance of this technique, no genome-wide analysis of the effects of in ovo electroporation, principally the current applied across the tissue and exogenous vector DNA introduced, on endogenous gene expression has been undertaken. Here, the effects of electric current and expression of a GFP-containing construct, via electroporation into the midbrain of Hamburger-Hamilton stage 10 chicken embryos, are analysed by microarray. Results Both current alone and in combination with exogenous DNA expression have a small but reproducible effect on endogenous gene expression, changing the expression of the genes represented on the array by less than 0.1% (current and less than 0.5% (current + DNA, respectively. The subset of genes regulated by electric current and exogenous DNA span a disparate set of cellular functions. However, no genes involved in the regional identity were affected. In sharp contrast to this, electroporation of a known transcription factor, Dmrt5, caused a much greater change in gene expression. Conclusions These findings represent the first systematic genome-wide analysis of the effects of in ovo electroporation on gene expression during embryonic development. The analysis reveals that this process has minimal impact on the genetic basis of cell fate specification. Thus, the study demonstrates the validity of the in ovo electroporation technique to study gene function and expression during development. Furthermore, the data presented here can be used as a resource to refine the set of transcriptional responders in future in ovo electroporation studies of specific gene function.

Electroporation-based DNA delivery technology

DEFF Research Database (Denmark)

Gothelf, A; Gehl, Julie

2014-01-01

DNA delivery to for example skin and muscle can easily be performed with electroporation. The method is efficient, feasible, and inexpensive and the future possibilities are numerous. Here we present our protocol for gene transfection to mouse skin using naked plasmid DNA and electric pulses....

Visual loop-mediated isothermal amplification (LAMP) for the rapid diagnosis of Enterocytozoon hepatopenaei (EHP) infection.

Science.gov (United States)

T, Sathish Kumar; A, Navaneeth Krishnan; J, Joseph Sahaya Rajan; M, Makesh; K P, Jithendran; S V, Alavandi; K K, Vijayan

2018-05-01

The emerging microsporidian parasite Enterocytozoon hepatopenaei (EHP), the causative agent of hepatopancreatic microsporidiosis, has been widely reported in shrimp-farming countries. EHP infection can be detected by light microscopy observation of spores (1.7 × 1 μm) in stained hepatopancreas (HP) tissue smears, HP tissue sections, and fecal samples. EHP can also be detected by polymerase chain reaction (PCR) targeting the small subunit (SSU) ribosomal RNA (rRNA) gene or the spore wall protein gene (SWP). In this study, a rapid, sensitive, specific, and closed tube visual loop-mediated isothermal amplification (LAMP) protocol combined with FTA cards was developed for the diagnosis of EHP. LAMP primers were designed based on the SSU rRNA gene of EHP. The target sequence of EHP was amplified at constant temperature of 65 °C for 45 min and amplified LAMP products were visually detected in a closed tube system by using SYBR™ green I dye. Detection limit of this LAMP protocol was ten copies. Field and clinical applicability of this assay was evaluated using 162 field samples including 106 HP tissue samples and 56 fecal samples collected from shrimp farms. Out of 162 samples, EHP could be detected in 62 samples (47 HP samples and 15 fecal samples). When compared with SWP-PCR as the gold standard, this EHP LAMP assay had 95.31% sensitivity, 98.98% specificity, and a kappa value of 0.948. This simple, closed tube, clinically evaluated visual LAMP assay has great potential for diagnosing EHP at the farm level, particularly under low-resource circumstances.

The effects of metallic implants on electroporation therapies: feasibility of irreversible electroporation for brachytherapy salvage.

Science.gov (United States)

Neal, Robert E; Smith, Ryan L; Kavnoudias, Helen; Rosenfeldt, Franklin; Ou, Ruchong; Mclean, Catriona A; Davalos, Rafael V; Thomson, Kenneth R

2013-12-01

Electroporation-based therapies deliver brief electric pulses into a targeted volume to destabilize cellular membranes. Nonthermal irreversible electroporation (IRE) provides focal ablation with effects dependent on the electric field distribution, which changes in heterogeneous environments. It should be determined if highly conductive metallic implants in targeted regions, such as radiotherapy brachytherapy seeds in prostate tissue, will alter treatment outcomes. Theoretical and experimental models determine the impact of prostate brachytherapy seeds on IRE treatments. This study delivered IRE pulses in nonanimal, as well as in ex vivo and in vivo tissue, with and in the absence of expired radiotherapy seeds. Electrical current was measured and lesion dimensions were examined macroscopically and with magnetic resonance imaging. Finite-element treatment simulations predicted the effects of brachytherapy seeds in the targeted region on electrical current, electric field, and temperature distributions. There was no significant difference in electrical behavior in tissue containing a grid of expired radiotherapy seeds relative to those without seeds for nonanimal, ex vivo, and in vivo experiments (all p > 0.1). Numerical simulations predict no significant alteration of electric field or thermal effects (all p > 0.1). Histology showed cellular necrosis in the region near the electrodes and seeds within the ablation region; however, there were no seeds beyond the ablation margins. This study suggests that electroporation therapies can be implemented in regions containing small metallic implants without significant changes to electrical and thermal effects relative to use in tissue without the implants. This supports the ability to use IRE as a salvage therapy option for brachytherapy.

From Cell to Tissue Properties-Modeling Skin Electroporation With Pore and Local Transport Region Formation.

Science.gov (United States)

Dermol-Cerne, Janja; Miklavcic, Damijan

2018-02-01

Current models of tissue electroporation either describe tissue with its bulk properties or include cell level properties, but model only a few cells of simple shapes in low-volume fractions or are in two dimensions. We constructed a three-dimensional model of realistically shaped cells in realistic volume fractions. By using a 'unit cell' model, the equivalent dielectric properties of whole tissue could be calculated. We calculated the dielectric properties of electroporated skin. We modeled electroporation of single cells by pore formation on keratinocytes and on the papillary dermis which gave dielectric properties of the electroporated epidermis and papillary dermis. During skin electroporation, local transport regions are formed in the stratum corneum. We modeled local transport regions and increase in their radii or density which affected the dielectric properties of the stratum corneum. The final model of skin electroporation accurately describes measured electric current and voltage drop on the skin during electroporation with long low-voltage pulses. The model also accurately describes voltage drop on the skin during electroporation with short high-voltage pulses. However, our results indicate that during application of short high-voltage pulses additional processes may occur which increase the electric current. Our model connects the processes occurring at the level of cell membranes (pore formation), at the level of a skin layer (formation of local transport region in the stratum corneum) with the tissue (skin layers) and even level of organs (skin). Using a similar approach, electroporation of any tissue can be modeled, if the morphology of the tissue is known.

DC Pollution of AC Mains due to modern compact fluorescent light lamps and LED lamps

NARCIS (Netherlands)

Keyer, Cornelis H.A.; Timens, R.B.; Buesink, Frederik Johannes Karel; Leferink, Frank Bernardus Johannes

2013-01-01

Modern so-called energy efficient equipment often draw current only during a very short period of the period of a power supply mains. This is causing unwanted non-sinusoidal and harmonic currents. In some cases even a single diode is used for rectification causing direct current (DC) in the mains

Immunization of neonatal mice with LAMP/p55 HIV gag DNA elicits robust immune responses that last to adulthood

International Nuclear Information System (INIS)

Ordonhez Rigato, Paula; Maciel, Milton; Goldoni, Adriana Leticia; Piubelli, Orlando; Alves de Brito, Cyro; Fusaro, Ana Elisa; Eurico de Alencar, Liciana Xavier; August, Thomas; Torres Azevedo Marques, Ernesto; Silva Duarte, Alberto Jose da; Sato, Maria Notomi

2010-01-01

Successful T cell priming in early postnatal life that can generate effective long-lasting responses until adulthood is critical in HIV vaccination strategies because it prevents early sexual initiation and breastfeeding transmission of HIV. A chimeric DNA vaccine encoding p55 HIV gag associated with lysosome-associated membrane protein 1 (LAMP-1; which drives the antigen to the MIIC compartment), has been used to enhance cellular and humoral antigen-specific responses in adult mice and macaques. Herein, we investigated LAMP-1/gag vaccine immunogenicity in the neonatal period in mice and its ability to generate long-lasting effects. Neonatal vaccination with chimeric LAMP/gag generated stronger Gag-specific immune responses, as measured by the breadth of the Gag peptide-specific IFN-γ, proliferative responsiveness, cytokine production and antibody production, all of which revealed activation of CD4+ T cells as well as the generation of a more robust CTL response compared to gag vaccine alone. To induce long-lived T and B cell memory responses, it was necessary to immunize neonates with the chimeric LAMP/gag DNA vaccine. The LAMP/gag DNA vaccine strategy could be particularly useful for generating an anti-HIV immune response in the early postnatal period capable of inducing long-term immunological memory.

On electrode erosion in fluorescent lamps during instant start

Energy Technology Data Exchange (ETDEWEB)

Hadrath, S.

2006-09-15

A fluorescent lamp driven with an 'instant start electronic control gear' starts in a glow mode. In the glow mode, which lasts typically for tens of milliseconds, the cathode fall exceeds hundreds of volts. This causes high energy ion bombardment of the electrode which heats the electrode, and induces a transition from glow to arc mode. In the arc mode the electrode emits thermionically and the cathode fall drops to the 12 - 15 V range. Unfortunately, the high energy ion bombardment during the glow mode leads also to intense sputtering of electrode material, including tungsten as well as emitter. Thus, instant started fluorescent lamps often suffer from early failures due to coil fracture. Therefore, the investigation of tungsten erosion during instant start is necessary and was the main goal of this work. The density of neutral atomic tungsten is determined by laser-induced fluorescence (LIF) and optical emission spectroscopy measurements (OES). Investigations are performed on a low-pressure argon dc discharge and on commercial fluorescent lamps. To include the entire temperature profile along the electrode the diffuse and spot operation modes of the dc lamp are studied experimentally and theoretically. The measured dependencies of the cathode temperature along the coil on the discharge and heating parameters are compared with the calculated results. For the first time the tungsten erosion during instant start of commercial fluorescent lamps was experimentally investigated in this work. The erosion process could be related to sputtering. A reconstruction of the temporal evolution of the absolute tungsten population density of the ground state during the glow mode was presented. The sputtered tungsten density increases immediately with the ignition, reaches a maximum where the discharge contracts at the end of the glow mode, and decreases some milliseconds before the glow-to-arc transition takes place. The maximum tungsten density was observed within a

TargetRNA: a tool for predicting targets of small RNA action in bacteria

OpenAIRE

Tjaden, Brian

2008-01-01

Many small RNA (sRNA) genes in bacteria act as posttranscriptional regulators of target messenger RNAs. Here, we present TargetRNA, a web tool for predicting mRNA targets of sRNA action in bacteria. TargetRNA takes as input a genomic sequence that may correspond to an sRNA gene. TargetRNA then uses a dynamic programming algorithm to search each annotated message in a specified genome for mRNAs that evince basepair-binding potential to the input sRNA sequence. Based on the calculated basepair-...

DC-STAMP, a novel multimembrane-spanning molecule preferentially expressed by dendritic cells.

NARCIS (Netherlands)

Hartgers, F.C.; Vissers, J.L.M.; Looman, M.W.G.; Zoelen, C. van; Huffine, C.; Figdor, C.G.; Adema, G.J.

2000-01-01

Dendritic cells (DC) are unique in their ability to present antigen to naive T cells, and therefore play a central role in the initiation of immune responses. Characterization of DC-specific genes may help to unravel the mechanism underlying their potent antigen presenting capacity. Here we describe

Comparison of a newly developed automated and quantitative hepatitis C virus (HCV) core antigen test with the HCV RNA assay for clinical usefulness in confirming anti-HCV results.

Science.gov (United States)

Kesli, Recep; Polat, Hakki; Terzi, Yuksel; Kurtoglu, Muhammet Guzel; Uyar, Yavuz

2011-12-01

Hepatitis C virus (HCV) is a global health care problem. Diagnosis of HCV infection is mainly based on the detection of anti-HCV antibodies as a screening test with serum samples. Recombinant immunoblot assays are used as supplemental tests and for the final detection and quantification of HCV RNA in confirmatory tests. In this study, we aimed to compare the HCV core antigen test with the HCV RNA assay for confirming anti-HCV results to determine whether the HCV core antigen test may be used as an alternative confirmatory test to the HCV RNA test and to assess the diagnostic values of the total HCV core antigen test by determining the diagnostic specificity and sensitivity rates compared with the HCV RNA test. Sera from a total of 212 treatment-naive patients were analyzed for anti-HCV and HCV core antigen both with the Abbott Architect test and with the molecular HCV RNA assay consisting of a reverse transcription-PCR method as a confirmatory test. The diagnostic sensitivity, specificity, and positive and negative predictive values of the HCV core antigen assay compared to the HCV RNA test were 96.3%, 100%, 100%, and 89.7%, respectively. The levels of HCV core antigen showed a good correlation with those from the HCV RNA quantification (r = 0.907). In conclusion, the Architect HCV antigen assay is highly specific, sensitive, reliable, easy to perform, reproducible, cost-effective, and applicable as a screening, supplemental, and preconfirmatory test for anti-HCV assays used in laboratory procedures for the diagnosis of hepatitis C virus infection.

Seeing the electroporative uptake of cell-membrane impermeable fluorescent molecules and nanoparticles

Science.gov (United States)

Kim, Kisoo; Kim, Jeong Ah; Lee, Soon-Geul; Lee, Won Gu

2012-07-01

This paper presents direct visualization of uptake directionality for cell-membrane impermeant fluorescent molecules and fluorescence-doped nanoparticles at a single-cell level during electroporation. To observe directly the uptake direction, we used microchannel-type electroporation that can generate a relatively symmetric and uniform electric field. For all the image frames during electroporation, fluorescence intensities that occurred at cell membranes in both uptake directions toward the electrodes have been sequentially recorded and quantitatively analyzed pixel by pixel. In our experiments, we found that fluorescent molecules, even not labeled to target biomolecules, had their own uptake direction with different intensities. It is also observed that the uptake intensity toward the cell membrane had a maximal value at a certain electric voltage, not at the highest value of voltages applied. The results also imply that the uptake direction of fluorescence-doped nanoparticles can be determined by a net surface charge of uptake materials and sizes in the electroporative environments. In summary, we performed a quantitative screening and direct visualization of uptake directionality for a set of fluorescent molecules and fluorescence-doped nanoparticles using electric-pulsation. Taking a closer look at the uptake direction of exogenous materials will help researchers to understand an unknown uptake phenomenon in which way foreign materials are inclined to move, and furthermore to design functional nanoparticles for electroporative gene delivery.This paper presents direct visualization of uptake directionality for cell-membrane impermeant fluorescent molecules and fluorescence-doped nanoparticles at a single-cell level during electroporation. To observe directly the uptake direction, we used microchannel-type electroporation that can generate a relatively symmetric and uniform electric field. For all the image frames during electroporation, fluorescence intensities

Electroporation and use of hepatitis B virus envelope L proteins as bionanocapsules.

Science.gov (United States)

Yamada, Tadanori; Jung, Joohee; Seno, Masaharu; Kondo, Akihiko; Ueda, Masakazu; Tanizawa, Katsuyuki; Kuroda, Shun'ichi

2012-06-01

Hepatitis B virus (HBV) envelope L proteins, when synthesized in yeast cells, form a hollow bionanocapsule (BNC) in which genes (including large plasmids up to 40 kbp), small interfering RNA (siRNA), drugs, and proteins can be enclosed by electroporation. BNCs made from L proteins have several advantages as a delivery system: Because they display a human liver-specific receptor (the pre-S region of the L protein) on their surface, BNCs can efficiently and specifically deliver their contents to human liver-derived cells and tissues ex vivo (in cell culture) and in vivo (in a mouse xenograft model). Retargeting can be achieved simply by substituting other biorecognition molecules such as antibodies, ligands, receptors, and homing peptides for the pre-S region. In addition, BNCs have already been proven to be safe for use in humans during their development as an immunogen of hepatitis B vaccine. This protocol describes the loading of BNCs and their use in cell culture and in vivo.

49 CFR 393.24 - Requirements for head lamps, auxiliary driving lamps and front fog lamps.

Science.gov (United States)

2010-10-01

... Devices, and Electrical Wiring § 393.24 Requirements for head lamps, auxiliary driving lamps and front fog lamps. (a) Headlamps. Every bus, truck and truck tractor shall be equipped with headlamps as required by...

Hypoxic activation of the unfolded protein response (UPR) induces expression of the metastasis-associated gene LAMP3

International Nuclear Information System (INIS)

Mujcic, Hilda; Rzymski, Tomasz; Rouschop, Kasper M.A.; Koritzinsky, Marianne; Milani, Manuela; Harris, Adrian L.; Wouters, Bradly G.

2009-01-01

Background and purpose: Tumour hypoxia contributes to failure of cancer treatment through its ability to protect against therapy and adversely influence tumour biology. In particular, several studies suggest that hypoxia promotes metastasis. Hypoxia-induced cellular changes are mediated by oxygen-sensitive signaling pathways that activate downstream transcription factors. We have investigated the induction and transcriptional regulation of a novel metastasis-associated gene, LAMP3 during hypoxia. Materials and methods: Microarray, quantitative PCR, Western blot analysis and immunohistochemistry were used to investigate hypoxic regulation of LAMP3. The mechanism for LAMP3 induction was investigated using transient RNAi and stable shRNA targeting components of the hypoxic response. Endoplasmic reticulum stress inducing agents, including proteasome inhibitors were assessed for their ability to regulate LAMP3. Results: LAMP3 is strongly induced by hypoxia at both the mRNA and protein levels in a large panel of human tumour cell lines. Induction of LAMP3 occurs as a consequence of the activation of the PERK/eIF2α/ATF4 arm of the unfolded protein response (UPR) and is independent of HIF-1α. LAMP3 is expressed heterogeneously within the microenvironment of tumours, overexpressed in breast cancer, and increases in tumours treated with avastin. Conclusions: These data identify LAMP3 as a novel hypoxia-inducible gene regulated by the UPR. LAMP3 is a new candidate biomarker of UPR activation by hypoxia in tumours and is a potential mediator of hypoxia-induced metastasis.

Translation of a nonpolyadenylated viral RNA is enhanced by binding of viral coat protein or polyadenylation of the RNA.

Science.gov (United States)

Neeleman, L; Olsthoorn, R C; Linthorst, H J; Bol, J F

2001-12-04

On entering a host cell, positive-strand RNA virus genomes have to serve as messenger for the translation of viral proteins. Efficient translation of cellular messengers requires interactions between initiation factors bound to the 5'-cap structure and the poly(A) binding protein bound to the 3'-poly(A) tail. Initiation of infection with the tripartite RNA genomes of alfalfa mosaic virus (AMV) and viruses from the genus Ilarvirus requires binding of a few molecules of coat protein (CP) to the 3' end of the nonpolyadenylated viral RNAs. Moreover, infection with the genomic RNAs can be initiated by addition of the subgenomic messenger for CP, RNA 4. We report here that extension of the AMV RNAs with a poly(A) tail of 40 to 80 A-residues permitted initiation of infection independently of CP or RNA 4 in the inoculum. Specifically, polyadenylation of RNA 1 relieved an apparent bottleneck in the translation of the viral RNAs. Translation of RNA 4 in plant protoplasts was autocatalytically stimulated by its encoded CP. Mutations that interfered with CP binding to the 3' end of viral RNAs reduced translation of RNA 4 to undetectable levels. Possibly, CP of AMV and ilarviruses stimulates translation of viral RNAs by acting as a functional analogue of poly(A) binding protein or other cellular proteins.

Electroporation increases antitumoral efficacy of the bcl-2 antisense G3139 and chemotherapy in a human melanoma xenograft

Directory of Open Access Journals (Sweden)

Baldi Alfonso

2011-07-01

Full Text Available Abstract Background Nucleic acids designed to modulate the expression of target proteins remain a promising therapeutic strategy in several diseases, including cancer. However, clinical success is limited by the lack of efficient intracellular delivery. In this study we evaluated whether electroporation could increase the delivery of antisense oligodeoxynucleotides against bcl-2 (G3139 as well as the efficacy of combination chemotherapy in human melanoma xenografts. Methods Melanoma-bearing nude mice were treated i.v. with G3139 and/or cisplatin (DDP followed by the application of trains of electric pulses to tumors. Western blot, immunohistochemistry and real-time PCR were performed to analyze protein and mRNA expression. The effect of electroporation on muscles was determined by histology, while tumor apoptosis and the proliferation index were analyzed by immunohistochemistry. Antisense oligodeoxynucleotides tumor accumulation was measured by FACS and confocal microscopy. Results The G3139/Electroporation combined therapy produced a significant inhibition of tumor growth (TWI, more than 50% accompanied by a marked tumor re-growth delay (TRD, about 20 days. The efficacy of this treatment was due to the higher G3139 uptake in tumor cells which led to a marked down-regulation of bcl-2 protein expression. Moreover, the G3139/EP combination treatment resulted in an enhanced apoptotic index and a decreased proliferation rate of tumors. Finally, an increased tumor response was observed after treatment with the triple combination G3139/DDP/EP, showing a TWI of about 75% and TRD of 30 days. Conclusions These results demonstrate that electroporation is an effective strategy to improve the delivery of antisense oligodeoxynucleotides within tumor cells in vivo and it may be instrumental in optimizing the response of melanoma to chemotherapy. The high response rate observed in this study suggest to apply this strategy for the treatment of melanoma patients.

Exosomes as divine messengers: are they the Hermes of modern molecular oncology?

Science.gov (United States)

Braicu, C; Tomuleasa, C; Monroig, P; Cucuianu, A; Berindan-Neagoe, I; Calin, G A

2015-01-01

Exosomes are cell-derived vesicles that convey key elements with the potential to modulate intercellular communication. They are known to be secreted from all types of cells, and are crucial messengers that can regulate cellular processes by ‘trafficking' molecules from cells of one tissue to another. The exosomal content has been shown to be broad, composed of different types of cytokines, growth factors, proteins, or nucleic acids. Besides messenger RNA (mRNA) they can also contain noncoding transcripts such as microRNAs (miRNAs), which are small endogenous cellular regulators of protein expression. In diseases such as cancer, exosomes can facilitate tumor progression by altering their vesicular content and supplying the tumor niche with molecules that favor the progression of oncogenic processes such as proliferation, invasion and metastasis, or even drug resistance. The packaging of their molecular content is known to be tissue specific, a fact that makes them interesting tools in clinical diagnostics and ideal candidates for biomarkers. In the current report, we describe the main properties of exosomes and explain their involvement in processes such as cell differentiation and cell death. Furthermore, we emphasize the need of developing patient-targeted treatments by applying the conceptualization of exosomal-derived miRNA-based therapeutics. PMID:25236394

Solar fed DC-DC single ended primary inductance converter for low power applications

Science.gov (United States)

Narendranath, K. V.; Viswanath, Y.; Babu, K. Suresh; Arunkumar, G.; Elangovan, D.

2017-11-01

This paper presents 34 to 36 volts. SEPIC converter for solar fed applications. Now days, there has been tremendous increase in the usage of solar energy and this solar energy is most valuable energy source available all around the world. The solar energy system require a Dc-Dc converter in order to modulate and govern the changing output of the panel. In this paper, a system comprising of Single Ended Primary Inductance Converter [SEPIC] integrated with solar panel is proposed. This paper proposes SEPIC power converter design that will secure high performance and cost efficiency while powering up a LAMP load. This power converter designed with low output ripple voltage, higher efficiency and less electrical pressure on the power switching elements. The simulation and prototype hardware results are presented in this paper.

microRNA-independent recruitment of Argonaute 1 to nanos mRNA through the Smaug RNA-binding protein.

Science.gov (United States)

Pinder, Benjamin D; Smibert, Craig A

2013-01-01

Argonaute (Ago) proteins are typically recruited to target messenger RNAs via an associated small RNA such as a microRNA (miRNA). Here, we describe a new mechanism of Ago recruitment through the Drosophila Smaug RNA-binding protein. We show that Smaug interacts with the Ago1 protein, and that Ago1 interacts with and is required for the translational repression of the Smaug target, nanos mRNA. The Ago1/nanos mRNA interaction does not require a miRNA, but it does require Smaug. Taken together, our data suggest a model whereby Smaug directly recruits Ago1 to nanos mRNA in a miRNA-independent manner, thereby repressing translation.

Effect of escitalopram versus placebo on GRα messenger RNA expression in peripheral blood cells of healthy individuals with a family history of depression - a secondary outcome analysis from the randomized AGENDA trial

DEFF Research Database (Denmark)

Knorr, Ulla; Koefoed, Pernille; Gluud, Christian

2016-01-01

Background Selective serotonin reuptake inhibitors (SSRIs) are widely prescribed as first-line drugs for the treatment of depression. However, the mechanisms of action for SSRIs are unclear and besides neurotransmitter modulation may depend on modulation of the hypothalamic-pituitary-adrenal (HPA......) system. The glucocorticoid receptor (GR) isoform α plays an important role in the negative feedback regulation of the HPA axis and reduced GRα messenger RNA (mRNA) expression has been shown in mood disorder patients and first-degree relatives compared to healthy individuals with no family history...

Effective plasmid DNA and small interfering RNA delivery to diseased human brain microvascular endothelial cells.

Science.gov (United States)

Slanina, H; Schmutzler, M; Christodoulides, M; Kim, K S; Schubert-Unkmeir, A

2012-01-01

Expression of exogenous DNA or small interfering RNA (siRNA) in vitro is significantly affected by the particular delivery system utilized. In this study, we evaluated the transfection efficiency of plasmid DNA and siRNA into human brain microvascular endothelial cells (HBMEC) and meningioma cells, which constitute the blood-cerebrospinal fluid barrier, a target of meningitis-causing pathogens. Chemical transfection methods and various lipofection reagents including Lipofectamin™, FuGene™, or jetPRIME®, as well as physical transfection methods and electroporation techniques were applied. To monitor the transfection efficiencies, HBMEC and meningioma cells were transfected with the reporter plasmid pTagGFP2-actin vector, and efficiency of transfection was estimated by fluorescence microscopy and flow cytometry. We established protocols based on electroporation using Cell Line Nucleofector® Kit V with the Amaxa® Nucleofector® II system from Lonza and the Neon® Transfection system from Invitrogen resulting in up to 41 and 82% green fluorescent protein-positive HBMEC, respectively. Optimal transfection solutions, pulse programs and length were evaluated. We furthermore demonstrated that lipofection is an efficient method to transfect meningioma cells with a transfection efficiency of about 81%. Finally, we applied the successful electroporation protocols to deliver synthetic siRNA to HBMEC and analyzed the role of the actin-binding protein cortactin in Neisseria meningitidis pathogenesis. Copyright © 2012 S. Karger AG, Basel.

A high efficiency gene disruption strategy using a positive-negative split selection marker and electroporation for Fusarium oxysporum.

Science.gov (United States)

Liang, Liqin; Li, Jianqiang; Cheng, Lin; Ling, Jian; Luo, Zhongqin; Bai, Miao; Xie, Bingyan

2014-11-01

The Fusarium oxysporum species complex consists of fungal pathogens that cause serial vascular wilt disease on more than 100 cultivated species throughout the world. Gene function analysis is rapidly becoming more and more important as the whole-genome sequences of various F. oxysporum strains are being completed. Gene-disruption techniques are a common molecular tool for studying gene function, yet are often a limiting step in gene function identification. In this study we have developed a F. oxysporum high-efficiency gene-disruption strategy based on split-marker homologous recombination cassettes with dual selection and electroporation transformation. The method was efficiently used to delete three RNA-dependent RNA polymerase (RdRP) genes. The gene-disruption cassettes of three genes can be constructed simultaneously within a short time using this technique. The optimal condition for electroporation is 10μF capacitance, 300Ω resistance, 4kV/cm field strength, with 1μg of DNA (gene-disruption cassettes). Under these optimal conditions, we were able to obtain 95 transformants per μg DNA. And after positive-negative selection, the transformants were efficiently screened by PCR, screening efficiency averaged 85%: 90% (RdRP1), 85% (RdRP2) and 77% (RdRP3). This gene-disruption strategy should pave the way for high throughout genetic analysis in F. oxysporum. Copyright © 2014 Elsevier GmbH. All rights reserved.

Intracellular Protein Delivery and Gene Transfection by Electroporation Using a Microneedle Electrode Array

Science.gov (United States)

Choi, Seong-O; Kim, Yeu-Chun; Lee, Jeong Woo; Park, Jung-Hwan

2012-01-01

The impact of many biopharmaceuticals, including protein- and gene-based therapies, has been limited by the need for better methods of delivery into cells within tissues. Here, we present intracellular delivery of molecules and transfection with plasmid DNA by electroporation using a novel microneedle electrode array designed for targeted treatment of skin and other tissue surfaces. The microneedle array is molded out of polylactic acid. Electrodes and circuitry required for electroporation are applied to the microneedle array surface by a new metal-transfer micromolding method. The microneedle array maintains mechanical integrity after insertion into pig cadaver skin and is able to electroporate human prostate cancer cells in vitro. Quantitative measurements show that increasing electroporation pulse voltage increases uptake efficiency of calcein and bovine serum albumin, whereas increasing pulse length has lesser effects over the range studied. Uptake of molecules by up to 50 % of cells and transfection of 12 % of cells with a gene for green fluorescent protein is demonstrated at high cell viability. We conclude that the microneedle electrode array is able to electroporate cells, resulting in intracellular uptake of molecules, and has potential applications to improve intracellular delivery of proteins, DNA and other biopharmaceuticals. PMID:22328093

Insulin-like growth factor II messenger RNA-binding protein-3 is an independent prognostic factor in uterine leiomyosarcoma.

Science.gov (United States)

Yasutake, Nobuko; Ohishi, Yoshihiro; Taguchi, Kenichi; Hiraki, Yuka; Oya, Masafumi; Oshiro, Yumi; Mine, Mari; Iwasaki, Takeshi; Yamamoto, Hidetaka; Kohashi, Kenichi; Sonoda, Kenzo; Kato, Kiyoko; Oda, Yoshinao

2018-04-01

The aim of this study was to identify the prognostic factors of uterine leiomyosarcoma (ULMS). We reviewed 60 cases of surgically resected ULMSs and investigated conventional clinicopathological factors, together with the expression of insulin-like growth factor II messenger RNA-binding protein-3 (IMP3), hormone receptors and cell cycle regulatory markers by immunohistochemistry. Mediator complex subunit 12 (MED12) mutation analysis was also performed. Univariate analyses revealed that advanced stage (P < 0.0001), older age (P = 0.0244) and IMP3 expression (P = 0.0011) were significant predictors of a poor outcome. Multivariate analysis revealed advanced stage (P < 0.0001) and IMP3 (P = 0.0373) as independent predictors of a poor prognosis. Expressions of cell cycle markers and hormone receptors, and MED12 mutations (12% in ULMSs) were not identified as prognostic markers in this study. IMP3 expression in ULMS could be a marker of a poor prognosis. © 2017 John Wiley & Sons Ltd.

Use of electroporation for high-molecular-weight DNA-mediated gene transfer.

Science.gov (United States)

Jastreboff, M M; Ito, E; Bertino, J R; Narayanan, R

1987-08-01

Electroporation was used to introduce high-molecular-weight DNA into murine hematopoietic cells and NIH3T3 cells. CCRF-CEM cells were stably transfected with SV2NEO plasmid and the genomic DNA from G-418-resistant clones (greater than 65 kb) was introduced into mouse bone marrow and NIH3T3 cells by electroporation. NEO sequences and expression were detected in the hematopoietic tissues of lethally irradiated mice, with 24% of individual spleen colonies expressing NEO. The frequency of genomic DNA transfer into NIH3T3 cells was 0.25 X 10(-3). Electroporation thus offers a powerful mode of gene transfer not only of cloned genes but also of high-molecular-weight DNA into cells.

Targeted knock down of CCL22 and CCL17 by siRNA during DC differentiation and maturation affects the recruitment of T subsets.

Science.gov (United States)

Kang, Shijun; Xie, Jianmin; Ma, Shudong; Liao, Wangjun; Zhang, Junyi; Luo, Rongcheng

2010-01-01

Chemokines secreted by DC are instrumental for DC to regulate their own migratory capacities and to recruit T lymphocytes during local tumour immune response. Using the recently developed chemokine protein arrays, we analyzed 38 chemokines associated with monocyte-derived DC (MoDC), including the CC family (CCL2, CCL3, CCL4, CCL17, CCL18, CCL22, CCL23, CCL24, CCL27) and the CXC family (CXCL3, CXCL5, CXCL7, CXCL8, CXCL16) chemokines. Our results indicate that MoDC largely inherit the chemokines constitutively expressed by monocytes, with a significant induction of CCL17, CCL22 and CCL23. Spent culture supernatant collected from MoDC exhibited chemotatic abilities to activate CD4(+), CD8(+), and CD25(+) Foxp3(+) regulatory T cells (Tregs). Selective knock down of CCL22 and CCL17 expression by siRNA decreased the ratios of CD4(+) to CD8(+), as well as the frequency of Tregs recruited by MoDC. Intratumoural injection of MoDC transfected with siCCL22 and siCCL17, significantly reduced the number of Tregs while increasing the number of infiltrating CD8(+) T cells in human tumour xenografts in athymic nude mice. This study demonstrates that chemokine expression of MoDC is complex and may change dynamically. Using siRNA to selectively knock down chemokines which are highly chemoattractive to Tregs may consequentially alter the lymphocyte populations recruited into the tumour microenvironment, therefore has the potency to provide insight into cellular interactions in cancer immunology. This may lead to a new strategy for DC vaccine development to improve cancer immunobiotherapy.

Lentivirus-Induced Dendritic Cells (iDC for Immune-Regenerative Therapies in Cancer and Stem Cell Transplantation

Directory of Open Access Journals (Sweden)

Renata Stripecke

2014-08-01

Full Text Available Conventional dendritic cells (cDC are ex vivo differentiated professional antigen presenting cells capable of potently stimulating naïve T cells and with vast potential for immunotherapeutic applications. The manufacture of clinical-grade cDC is relatively complex and requires several days for completion. Clinical trials showed poor trafficking of cDC from subcutaneous injection sites to lymph nodes (LN, where DC can optimally stimulate naïve lymphocytes for long-lasting memory responses. We demonstrated in mouse and human systems that a single overnight ex vivo lentiviral (LV gene transfer into DC precursors for production of combination of cytokines and antigens was capable to induce autonomous self-differentiation of antigen-loaded DC in vitro and in vivo. These highly viable induced DC (iDC effectively migrated from the injected skin to LN, where they effectively activated de novo antigen-specific effector memory T cells. Two iDC modalities were validated in relevant animal models and are now in clinical development: Self-differentiated Myeloid-derived Antigen-presenting-cells Reactive against Tumors co-expressing GM-CSF/IL-4/TRP2 for melanoma immunotherapy in the autologous setting (SmartDCtrp2, and Self-differentiated Myeloid-derived Lentivirus-induced against human cytomegalovirus as an allogeneic matched adoptive cell after stem cell transplantation (SmyleDCpp65. The lentiviral vector design and packaging methodology has “evolved” continuously in order to simplify and optimize function and biosafety of in vitro and in vivo genetic reprogramming of iDC. Here, we address the challenges seeking for new creations of genetically programmed iDC and integrase-defective LV vaccines for immune regeneration.

Analysis of RNA metabolism in fission yeast

DEFF Research Database (Denmark)

Wise, Jo Ann; Nielsen, Olaf

2017-01-01

Here we focus on the biogenesis and function of messenger RNA (mRNA) in fission yeast cells. Following a general introduction that also briefly touches on other classes of RNA, we provide an overview of methods used to analyze mRNAs throughout their life cycles....

Magnetic resonance electrical impedance tomography for measuring electrical conductivity during electroporation

International Nuclear Information System (INIS)

Kranjc, M; Miklavčič, D; Bajd, F; Serša, I

2014-01-01

The electroporation effect on tissue can be assessed by measurement of electrical properties of the tissue undergoing electroporation. The most prominent techniques for measuring electrical properties of electroporated tissues have been voltage–current measurement of applied pulses and electrical impedance tomography (EIT). However, the electrical conductivity of tissue assessed by means of voltage–current measurement was lacking in information on tissue heterogeneity, while EIT requires numerous additional electrodes and produces results with low spatial resolution and high noise. Magnetic resonance EIT (MREIT) is similar to EIT, as it is also used for reconstruction of conductivity images, though voltage and current measurements are not limited to the boundaries in MREIT, hence it yields conductivity images with better spatial resolution. The aim of this study was to investigate and demonstrate the feasibility of the MREIT technique for assessment of conductivity images of tissues undergoing electroporation. Two objects were investigated: agar phantoms and ex vivo liver tissue. As expected, no significant change of electrical conductivity was detected in agar phantoms exposed to pulses of all used amplitudes, while a considerable increase of conductivity was measured in liver tissue exposed to pulses of different amplitudes. (paper)

Establishment of Lipofection for Studying miRNA Function in Human Adipocytes

OpenAIRE

Enlund, Eveliina; Fischer, Simon; Handrick, René; Otte, Kerstin; Debatin, Klaus-Michael; Wabitsch, Martin; Fischer-Posovszky, Pamela

2014-01-01

miRNA dysregulation has recently been linked to human obesity and its related complications such as type 2 diabetes. In order to study miRNA function in human adipocytes, we aimed for the modulation of mature miRNA concentration in these cells. Adipocytes, however, tend to be resistant to transfection and there is often a need to resort to viral transduction or electroporation. Our objective therefore was to identify an efficient, non-viral transfection reagent capable of delivering small RNA...

Dynamical Messengers for Gauge Mediation

Energy Technology Data Exchange (ETDEWEB)

Hook, Anson; Torroba, Gonzalo; /SLAC /Stanford U., Phys. Dept.

2011-08-17

We construct models of indirect gauge mediation where the dynamics responsible for breaking supersymmetry simultaneously generates a weakly coupled subsector of messengers. This provides a microscopic realization of messenger gauge mediation where the messenger and hidden sector fields are unified into a single sector. The UV theory is SQCD with massless and massive quarks plus singlets, and at low energies it flows to a weakly coupled quiver gauge theory. One node provides the primary source of supersymmetry breaking, which is then transmitted to the node giving rise to the messenger fields. These models break R-symmetry spontaneously, produce realistic gaugino and sfermion masses, and give a heavy gravitino.

Application of a three-dimensional model for a study of the energy transfer of a high-pressure mercury horizontal lamp

Energy Technology Data Exchange (ETDEWEB)

Ben Hamida, M. B.; Charrada, K. [Unite d' Etude des Milieux Ionises et Reactifs, IPEIM, 5019 route de Kairouan Monastir (Tunisia)

2012-06-15

This paper is devoted to study the dynamics of a discharge lamp with high intensity in a horizontal position. As an example of application, we chose the high-pressure mercury lamp. For this, we realized a three-dimensional model, a stable and powered DC. After the validation of this model, we used it to reproduce the influence of some parameters that have appeared on major transport phenomena of mass and energy in studying the lamp operating in a horizontal position. Indeed, the mass of mercury and the electric current are modified and the effect of convective transport is studied.

Electroporation-delivered transdermal neostigmine in rats: equivalent action to intravenous administration

Directory of Open Access Journals (Sweden)

Berkó S

2016-05-01

Full Text Available Szilvia Berkó,1,* Kálmán F Szűcs,2,* Boglárka Balázs,1,3 Erzsébet Csányi,1 Gábor Varju,4 Anita Sztojkov-Ivanov,2 Mária Budai-Szűcs,1 Judit Bóta,2 Róbert Gáspár2 1Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Szeged, Szeged, Hungary; 2Department of Pharmacodynamics and Biopharmacy, Faculty of Pharmacy, University of Szeged, Szeged, Hungary; 3Gedeon Richter Plc., Budapest, 4Dr Derm Clinic of Anti-Aging Dermatology, Aesthetic Laser and Plastic Surgery, Budapest, Hungary *These authors contributed equally to this work Purpose: Transdermal electroporation has become one of the most promising noninvasive methods for drug administration, with greatly increased transport of macromolecules through the skin. The cecal-contracting effects of repeated transdermal electroporation delivery and intravenous administration of neostigmine were compared in anesthetized rats. Methods: The cecal contractions were detected with implantable strain gauge sensors, and the plasma levels of neostigmine were followed by high-performance liquid chromatography. Results: Both intravenously and EP-administered neostigmine (0.2–66.7 µg/kg increased the cecal contractions in a dose-dependent manner. For both the low doses and the highest dose, the neostigmine plasma concentrations were the same after the two modes of administration, while an insignificantly higher level was observed at a dose of 20 µg/kg after intravenous administration as compared with the electroporation route. The contractile responses did not differ significantly after the two administration routes. Conclusion: The results suggest that electroporation-delivered neostigmine elicits action equivalent to that observed after intravenous administration as concerning both time and intensity. Electroporation permits the delivery of even lower doses of water-soluble compounds through the skin, which is very promising for clinical practice. Keywords: transdermal

Antigen Cross-Presentation of Immune Complexes

Science.gov (United States)

Platzer, Barbara; Stout, Madeleine; Fiebiger, Edda

2014-01-01

The ability of dendritic cells (DCs) to cross-present tumor antigens has long been a focus of interest to physicians, as well as basic scientists, that aim to establish efficient cell-based cancer immune therapy. A prerequisite for exploiting this pathway for therapeutic purposes is a better understanding of the mechanisms that underlie the induction of tumor-specific cytotoxic T-lymphocyte (CTL) responses when initiated by DCs via cross-presentation. The ability of humans DC to perform cross-presentation is of utmost interest, as this cell type is a main target for cell-based immunotherapy in humans. The outcome of a cross-presentation event is guided by the nature of the antigen, the form of antigen uptake, and the subpopulation of DCs that performs presentation. Generally, CD8α+ DCs are considered to be the most potent cross-presenting DCs. This paradigm, however, only applies to soluble antigens. During adaptive immune responses, immune complexes form when antibodies interact with their specific epitopes on soluble antigens. Immunoglobulin G (IgG) immune complexes target Fc-gamma receptors on DCs to shuttle exogenous antigens efficiently into the cross-presentation pathway. This receptor-mediated cross-presentation pathway is a well-described route for the induction of strong CD8+ T cell responses. IgG-mediated cross-presentation is intriguing because it permits the CD8− DCs, which are commonly considered to be weak cross-presenters, to efficiently cross-present. Engaging multiple DC subtypes for cross-presentation might be a superior strategy to boost CTL responses in vivo. We here summarize our current understanding of how DCs use IgG-complexed antigens for the efficient induction of CTL responses. Because of its importance for human cell therapy, we also review the recent advances in the characterization of cross-presentation properties of human DC subsets. PMID:24744762

Transfection of small numbers of human endothelial cells by electroporation and synthetic amphiphiles

NARCIS (Netherlands)

van Leeuwen, E B; van der Veen, A Y; Hoekstra, D; Engberts, J B; Halie, M R; van der Meer, J; Ruiters, M H

OBJECTIVES: This study compared the efficiency of electroporation and synthetic amphiphiles. (SAINT-2pp/DOPE) in transfecting small numbers of human endothelial cells. METHODS AND RESULTS: Optimal transfection conditions were tested and appeared to be 400 V and 960 microF for electroporation and a

Immunohistochemical identification of messenger RNA-related proteins in basophilic inclusions of adult-onset atypical motor neuron disease.

Science.gov (United States)

Fujita, Kengo; Ito, Hidefumi; Nakano, Satoshi; Kinoshita, Yoshimi; Wate, Reika; Kusaka, Hirofumi

2008-10-01

This report concerns an immunohistochemical investigation on RNA-related proteins in the basophilic inclusions (BIs) from patients with adult-onset atypical motor neuron disease. Formalin-fixed, paraffin-embedded sections of the motor cortex and the lumbar spinal cord were examined. The BIs appeared blue in color with H&E and Nissl stain, and pink with methylgreen-pyronin stain. Ribonuclease pretreatment abolished the methylgreen-pyronin staining, suggesting that the BIs contained RNA. Immunohistochemically, the BIs were distinctly labeled with the antibodies against poly(A)-binding protein 1, T cell intracellular antigen 1, and ribosomal protein S6. These proteins are essential constituents of stress granules. In contrast, the BIs were not immunoreactive for ribosomal protein L28 and decapping enzyme 1, which are core components of transport ribonucleoprotein particles and processing bodies, respectively. Moreover, the BIs were not immunopositive for TDP-43. Our results imply that translation attenuation could be involved in the processes of BI formation in this disorder.

Effects of deformability and thermal motion of lipid membrane on electroporation: By molecular dynamics simulations

International Nuclear Information System (INIS)

Sun, Sheng; Yin, Guangyao; Lee, Yi-Kuen; Wong, Joseph T.Y.; Zhang, Tong-Yi

2011-01-01

Research highlights: → MD simulations show that deformability and thermal motion of membrane affect electroporation. → Stiffer membrane inhibits electroporation and makes water penetrate from both sides. → Higher temperature accelerates electroporation. -- Abstract: Effects of mechanical properties and thermal motion of POPE lipid membrane on electroporation were studied by molecular dynamics simulations. Among simulations in which specific atoms of lipids were artificially constrained at their equilibrium positions using a spring with force constant of 2.0 kcal/(mol A 2 ) in the external electric field of 1.4 kcal/(mol A e), only constraint on lateral motions of lipid tails prohibited electroporation while non-tail parts had little effects. When force constant decreased to 0.2 kcal/(mol A 2 ) in the position constraints on lipid tails in the external electric field of 2.0 kcal/(mol A e), water molecules began to enter the membrane. Position constraints of lipid tails allow water to penetrate from both sides of membrane. Thermal motion of lipids can induce initial defects in the hydrophobic core of membrane, which are favorable nucleation sites for electroporation. Simulations at different temperatures revealed that as the temperature increases, the time taken to the initial pore formation will decrease.

In vivo electroporation enhances vaccine-mediated therapeutic control of human papilloma virus-associated tumors by the activation of multifunctional and effector memory CD8+ T cells.

Science.gov (United States)

Sales, Natiely S; Silva, Jamile R; Aps, Luana R M M; Silva, Mariângela O; Porchia, Bruna F M M; Ferreira, Luís Carlos S; Diniz, Mariana O

2017-12-19

In vivo electroporation (EP) has reignited the clinical interest on DNA vaccines as immunotherapeutic approaches to control different types of cancer. EP has been associated with increased immune response potency, but its capacity in influencing immunomodulation remains unclear. Here we evaluated the impact of in vivo EP on the induction of cellular immune responses and therapeutic effects of a DNA vaccine targeting human papillomavirus-induced tumors. Our results demonstrate that association of EP with the conventional intramuscular administration route promoted a more efficient activation of multifunctional and effector memory CD8 + T cells with enhanced cytotoxic activity. Furthermore, EP increased tumor infiltration of CD8 + T cells and avoided tumor recurrences. Finally, our results demonstrated that EP promotes local migration of antigen presenting cells that enhances with vaccine co-delivery. Altogether the present evidences shed further light on the in vivo electroporation action and its impact on the immunogenicity of DNA vaccines. Copyright © 2017 Elsevier Ltd. All rights reserved.

Regulatory RNAs derived from transfer RNA?

Science.gov (United States)

Pederson, Thoru

2010-10-01

Four recent studies suggest that cleavages of transfer RNAs generate products with microRNA-like features, with some evidence of function. If their regulatory functions were to be confirmed, these newly revealed RNAs would add to the expanding repertoire of small noncoding RNAs and would also provide new perspectives on the coevolution of transfer RNA and messenger RNA.

Scalable Device for Automated Microbial Electroporation in a Digital Microfluidic Platform.

Science.gov (United States)

Madison, Andrew C; Royal, Matthew W; Vigneault, Frederic; Chen, Liji; Griffin, Peter B; Horowitz, Mark; Church, George M; Fair, Richard B

2017-09-15

Electrowetting-on-dielectric (EWD) digital microfluidic laboratory-on-a-chip platforms demonstrate excellent performance in automating labor-intensive protocols. When coupled with an on-chip electroporation capability, these systems hold promise for streamlining cumbersome processes such as multiplex automated genome engineering (MAGE). We integrated a single Ti:Au electroporation electrode into an otherwise standard parallel-plate EWD geometry to enable high-efficiency transformation of Escherichia coli with reporter plasmid DNA in a 200 nL droplet. Test devices exhibited robust operation with more than 10 transformation experiments performed per device without cross-contamination or failure. Despite intrinsic electric-field nonuniformity present in the EP/EWD device, the peak on-chip transformation efficiency was measured to be 8.6 ± 1.0 × 10 8 cfu·μg -1 for an average applied electric field strength of 2.25 ± 0.50 kV·mm -1 . Cell survival and transformation fractions at this electroporation pulse strength were found to be 1.5 ± 0.3 and 2.3 ± 0.1%, respectively. Our work expands the EWD toolkit to include on-chip microbial electroporation and opens the possibility of scaling advanced genome engineering methods, like MAGE, into the submicroliter regime.

Electroporation of Postimplantation Mouse Embryos In Utero.

Science.gov (United States)

Huang, Cheng-Chiu; Carcagno, Abel

2018-02-01

Gene transfer by electroporation is possible in mouse fetuses within the uterus. As described in this protocol, the pregnant female is anesthetized, the abdominal cavity is opened, and the uterus with the fetuses is exteriorized. A solution of plasmid DNA is injected through the uterine wall directly into the fetus, typically into a cavity like the brain ventricle, guided by fiber optic illumination. Electrodes are positioned on the uterus around the region of the fetus that was injected, and electrical pulses are delivered. The uterus is returned to the abdominal cavity, the body wall is sutured closed, and the female is allowed to recover. The manipulated fetuses can then be collected and analyzed at various times after the electroporation. This method allows experimental access to later-stage developing mouse embryos. © 2018 Cold Spring Harbor Laboratory Press.

The Messenger Mission to Mercury

CERN Document Server

Domingue, D. L

2007-01-01

NASA’s MESSENGER mission, launched on 3 August, 2004 is the seventh mission in the Discovery series. MESSENGER encounters the planet Mercury four times, culminating with an insertion into orbit on 18 March 2011. It carries a comprehensive package of geophysical, geological, geochemical, and space environment experiments to complete the complex investigations of this solar-system end member, which begun with Mariner 10. The articles in this book, written by the experts in each area of the MESSENGER mission, describe the mission, spacecraft, scientific objectives, and payload. The book is of interest to all potential users of the data returned by the MESSENGER mission, to those studying the nature of the planet Mercury, and by all those interested in the design and implementation of planetary exploration missions.

Multivalent human papillomavirus l1 DNA vaccination utilizing electroporation.

Directory of Open Access Journals (Sweden)

Kihyuck Kwak

Full Text Available Naked DNA vaccines can be manufactured simply and are stable at ambient temperature, but require improved delivery technologies to boost immunogenicity. Here we explore in vivo electroporation for multivalent codon-optimized human papillomavirus (HPV L1 and L2 DNA vaccination.Balb/c mice were vaccinated three times at two week intervals with a fusion protein comprising L2 residues ∼11-88 of 8 different HPV types (11-88×8 or its DNA expression vector, DNA constructs expressing L1 only or L1+L2 of a single HPV type, or as a mixture of several high-risk HPV types and administered utilizing electroporation, i.m. injection or gene gun. Serum was collected two weeks and 3 months after the last vaccination. Sera from immunized mice were tested for in-vitro neutralization titer, and protective efficacy upon passive transfer to naive mice and vaginal HPV challenge. Heterotypic interactions between L1 proteins of HPV6, HPV16 and HPV18 in 293TT cells were tested by co-precipitation using type-specific monoclonal antibodies.Electroporation with L2 multimer DNA did not elicit detectable antibody titer, whereas DNA expressing L1 or L1+L2 induced L1-specific, type-restricted neutralizing antibodies, with titers approaching those induced by Gardasil. Co-expression of L2 neither augmented L1-specific responses nor induced L2-specific antibodies. Delivery of HPV L1 DNA via in vivo electroporation produces a stronger antibody response compared to i.m. injection or i.d. ballistic delivery via gene gun. Reduced neutralizing antibody titers were observed for certain types when vaccinating with a mixture of L1 (or L1+L2 vectors of multiple HPV types, likely resulting from heterotypic L1 interactions observed in co-immunoprecipitation studies. High titers were restored by vaccinating with individual constructs at different sites, or partially recovered by co-expression of L2, such that durable protective antibody titers were achieved for each type

Structure, dynamics and RNA binding of the multi-domain splicing factor TIA-1

Science.gov (United States)

Wang, Iren; Hennig, Janosch; Jagtap, Pravin Kumar Ankush; Sonntag, Miriam; Valcárcel, Juan; Sattler, Michael

2014-01-01

Alternative pre-messenger ribonucleic acid (pre-mRNA) splicing is an essential process in eukaryotic gene regulation. The T-cell intracellular antigen-1 (TIA-1) is an apoptosis-promoting factor that modulates alternative splicing of transcripts, including the pre-mRNA encoding the membrane receptor Fas. TIA-1 is a multi-domain ribonucleic acid (RNA) binding protein that recognizes poly-uridine tract RNA sequences to facilitate 5′ splice site recognition by the U1 small nuclear ribonucleoprotein (snRNP). Here, we characterize the RNA interaction and conformational dynamics of TIA-1 by nuclear magnetic resonance (NMR), isothermal titration calorimetry (ITC) and small angle X-ray scattering (SAXS). Our NMR-derived solution structure of TIA-1 RRM2–RRM3 (RRM2,3) reveals that RRM2 adopts a canonical RNA recognition motif (RRM) fold, while RRM3 is preceded by an non-canonical helix α0. NMR and SAXS data show that all three RRMs are largely independent structural modules in the absence of RNA, while RNA binding induces a compact arrangement. RRM2,3 binds to pyrimidine-rich FAS pre-mRNA or poly-uridine (U9) RNA with nanomolar affinities. RRM1 has little intrinsic RNA binding affinity and does not strongly contribute to RNA binding in the context of RRM1,2,3. Our data unravel the role of binding avidity and the contributions of the TIA-1 RRMs for recognition of pyrimidine-rich RNAs. PMID:24682828

NADPH oxidase-2 derived ROS dictates murine DC cytokine-mediated cell fate decisions during CD4 T helper-cell commitment.

Directory of Open Access Journals (Sweden)

Meghan A Jendrysik

Full Text Available NADPH oxidase-2 (Nox2/gp91(phox and p47(phox deficient mice are prone to hyper-inflammatory responses suggesting a paradoxical role for Nox2-derived reactive oxygen species (ROS as anti-inflammatory mediators. The molecular basis for this mode of control remains unclear. Here we demonstrate that IFNγ/LPS matured p47(phox-/--ROS deficient mouse dendritic cells (DC secrete more IL-12p70 than similarly treated wild type DC, and in an in vitro co-culture model IFNγ/LPS matured p47(phox-/- DC bias more ovalbumin-specific CD4(+ T lymphocytes toward a Th1 phenotype than wild type (WT DC through a ROS-dependent mechanism linking IL-12p70 expression to regulation of p38-MAPK activation. The Nox2-dependent ROS production in DC negatively regulates proinflammatory IL-12 expression in DC by constraining p38-MAPK activity. Increasing endogenous H(2O(2 attenuates p38-MAPK activity in IFNγ/LPS stimulated WT and p47(phox-/- DC, which suggests that endogenous Nox 2-derived ROS functions as a secondary messenger in the activated p38-MAPK signaling pathway during IL-12 expression. These findings indicate that ROS, generated endogenously by innate and adaptive immune cells, can function as important secondary messengers that can regulate cytokine production and immune cell cross-talk to control during the inflammatory response.

Lower glutamic acid decarboxylase 65-kDa isoform messenger RNA and protein levels in the prefrontal cortex in schizoaffective disorder but not schizophrenia.

Science.gov (United States)

Glausier, Jill R; Kimoto, Sohei; Fish, Kenneth N; Lewis, David A

2015-01-15

Altered gamma-aminobutyric acid (GABA) signaling in the prefrontal cortex (PFC) has been associated with cognitive dysfunction in patients with schizophrenia and schizoaffective disorder. Levels of the GABA-synthesizing enzyme glutamic acid decarboxylase 67-kDa isoform (GAD67) in the PFC have been consistently reported to be lower in patients with these disorders, but the status of the second GABA-synthesizing enzyme, glutamic acid decarboxylase 65-kDa isoform (GAD65), remains unclear. GAD65 messenger RNA (mRNA) levels were quantified in PFC area 9 by quantitative polymerase chain reaction from 62 subjects with schizophrenia or schizoaffective disorder and 62 matched healthy comparison subjects. In a subset of subject pairs, GAD65 relative protein levels were quantified by confocal immunofluorescence microscopy. Mean GAD65 mRNA levels were 13.6% lower in subjects with schizoaffective disorder but did not differ in subjects with schizophrenia relative to their matched healthy comparison subjects. In the subjects with schizoaffective disorder, mean GAD65 protein levels were 19.4% lower and were correlated with GAD65 mRNA levels. Lower GAD65 mRNA and protein levels within subjects with schizoaffective disorder were not attributable to factors commonly comorbid with the diagnosis. In concert with previous studies, these findings suggest that schizoaffective disorder is associated with lower levels of both GAD65 and GAD67 mRNA and protein in the PFC, whereas subjects with schizophrenia have lower mean levels of only GAD67 mRNA and protein. Because cognitive function is generally better preserved in patients with schizoaffective disorder relative to patients with schizophrenia, these findings may support an interpretation that GAD65 downregulation provides a homeostatic response complementary to GAD67 downregulation that serves to reduce inhibition in the face of lower PFC network activity. Copyright © 2015 Society of Biological Psychiatry. Published by Elsevier Inc

CT-guided Irreversible Electroporation in an Acute Porcine Liver Model: Effect of Previous Transarterial Iodized Oil Tissue Marking on Technical Parameters, 3D Computed Tomographic Rendering of the Electroporation Zone, and Histopathology

International Nuclear Information System (INIS)

Sommer, C. M.; Fritz, S.; Vollherbst, D.; Zelzer, S.; Wachter, M. F.; Bellemann, N.; Gockner, T.; Mokry, T.; Schmitz, A.; Aulmann, S.; Stampfl, U.; Pereira, P.; Kauczor, H. U.; Werner, J.; Radeleff, B. A.

2015-01-01

PurposeTo evaluate the effect of previous transarterial iodized oil tissue marking (ITM) on technical parameters, three-dimensional (3D) computed tomographic (CT) rendering of the electroporation zone, and histopathology after CT-guided irreversible electroporation (IRE) in an acute porcine liver model as a potential strategy to improve IRE performance.MethodsAfter Ethics Committee approval was obtained, in five landrace pigs, two IREs of the right and left liver (RL and LL) were performed under CT guidance with identical electroporation parameters. Before IRE, transarterial marking of the LL was performed with iodized oil. Nonenhanced and contrast-enhanced CT examinations followed. One hour after IRE, animals were killed and livers collected. Mean resulting voltage and amperage during IRE were assessed. For 3D CT rendering of the electroporation zone, parameters for size and shape were analyzed. Quantitative data were compared by the Mann–Whitney test. Histopathological differences were assessed.ResultsMean resulting voltage and amperage were 2,545.3 ± 66.0 V and 26.1 ± 1.8 A for RL, and 2,537.3 ± 69.0 V and 27.7 ± 1.8 A for LL without significant differences. Short axis, volume, and sphericity index were 16.5 ± 4.4 mm, 8.6 ± 3.2 cm 3 , and 1.7 ± 0.3 for RL, and 18.2 ± 3.4 mm, 9.8 ± 3.8 cm 3 , and 1.7 ± 0.3 for LL without significant differences. For RL and LL, the electroporation zone consisted of severely widened hepatic sinusoids containing erythrocytes and showed homogeneous apoptosis. For LL, iodized oil could be detected in the center and at the rim of the electroporation zone.ConclusionThere is no adverse effect of previous ITM on technical parameters, 3D CT rendering of the electroporation zone, and histopathology after CT-guided IRE of the liver

CT-guided Irreversible Electroporation in an Acute Porcine Liver Model: Effect of Previous Transarterial Iodized Oil Tissue Marking on Technical Parameters, 3D Computed Tomographic Rendering of the Electroporation Zone, and Histopathology

Energy Technology Data Exchange (ETDEWEB)

Sommer, C. M., E-mail: christof.sommer@med.uni-heidelberg.de [University Hospital Heidelberg, Department of Diagnostic and Interventional Radiology (Germany); Fritz, S., E-mail: stefan.fritz@med.uni-heidelberg.de [University Hospital Heidelberg, Department of General Visceral and Transplantation Surgery (Germany); Vollherbst, D., E-mail: dominikvollherbst@web.de [University Hospital Heidelberg, Department of Diagnostic and Interventional Radiology (Germany); Zelzer, S., E-mail: s.zelzer@dkfz-heidelberg.de [German Cancer Research Center (dkfz), Medical and Biological Informatics (Germany); Wachter, M. F., E-mail: fredericwachter@googlemail.com; Bellemann, N., E-mail: nadine.bellemann@med.uni-heidelberg.de; Gockner, T., E-mail: theresa.gockner@med.uni-heidelberg.de; Mokry, T., E-mail: theresa.mokry@med.uni-heidelberg.de; Schmitz, A., E-mail: anne.schmitz@med.uni-heidelberg.de [University Hospital Heidelberg, Department of Diagnostic and Interventional Radiology (Germany); Aulmann, S., E-mail: sebastian.aulmann@mail.com [University Hospital Heidelberg, Department of General Pathology (Germany); Stampfl, U., E-mail: ulrike.stampfl@med.uni-heidelberg.de [University Hospital Heidelberg, Department of Diagnostic and Interventional Radiology (Germany); Pereira, P., E-mail: philippe.pereira@slk-kliniken.de [SLK Kliniken Heilbronn GmbH, Clinic for Radiology, Minimally-invasive Therapies and Nuclear Medicine (Germany); Kauczor, H. U., E-mail: hu.kauczor@med.uni-heidelberg.de [University Hospital Heidelberg, Department of Diagnostic and Interventional Radiology (Germany); Werner, J., E-mail: jens.werner@med.uni-heidelberg.de [University Hospital Heidelberg, Department of General Visceral and Transplantation Surgery (Germany); Radeleff, B. A., E-mail: boris.radeleff@med.uni-heidelberg.de [University Hospital Heidelberg, Department of Diagnostic and Interventional Radiology (Germany)

2015-02-15

PurposeTo evaluate the effect of previous transarterial iodized oil tissue marking (ITM) on technical parameters, three-dimensional (3D) computed tomographic (CT) rendering of the electroporation zone, and histopathology after CT-guided irreversible electroporation (IRE) in an acute porcine liver model as a potential strategy to improve IRE performance.MethodsAfter Ethics Committee approval was obtained, in five landrace pigs, two IREs of the right and left liver (RL and LL) were performed under CT guidance with identical electroporation parameters. Before IRE, transarterial marking of the LL was performed with iodized oil. Nonenhanced and contrast-enhanced CT examinations followed. One hour after IRE, animals were killed and livers collected. Mean resulting voltage and amperage during IRE were assessed. For 3D CT rendering of the electroporation zone, parameters for size and shape were analyzed. Quantitative data were compared by the Mann–Whitney test. Histopathological differences were assessed.ResultsMean resulting voltage and amperage were 2,545.3 ± 66.0 V and 26.1 ± 1.8 A for RL, and 2,537.3 ± 69.0 V and 27.7 ± 1.8 A for LL without significant differences. Short axis, volume, and sphericity index were 16.5 ± 4.4 mm, 8.6 ± 3.2 cm{sup 3}, and 1.7 ± 0.3 for RL, and 18.2 ± 3.4 mm, 9.8 ± 3.8 cm{sup 3}, and 1.7 ± 0.3 for LL without significant differences. For RL and LL, the electroporation zone consisted of severely widened hepatic sinusoids containing erythrocytes and showed homogeneous apoptosis. For LL, iodized oil could be detected in the center and at the rim of the electroporation zone.ConclusionThere is no adverse effect of previous ITM on technical parameters, 3D CT rendering of the electroporation zone, and histopathology after CT-guided IRE of the liver.

Enhancing Irreversible Electroporation by Manipulating Cellular Biophysics with a Molecular Adjuvant.

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Ivey, Jill W; Latouche, Eduardo L; Richards, Megan L; Lesser, Glenn J; Debinski, Waldemar; Davalos, Rafael V; Verbridge, Scott S

2017-07-25

Pulsed electric fields applied to cells have been used as an invaluable research tool to enhance delivery of genes or other intracellular cargo, as well as for tumor treatment via electrochemotherapy or tissue ablation. These processes involve the buildup of charge across the cell membrane, with subsequent alteration of transmembrane potential that is a function of cell biophysics and geometry. For traditional electroporation parameters, larger cells experience a greater degree of membrane potential alteration. However, we have recently demonstrated that the nuclear/cytoplasm ratio (NCR), rather than cell size, is a key predictor of response for cells treated with high-frequency irreversible electroporation (IRE). In this study, we leverage a targeted molecular therapy, ephrinA1, known to markedly collapse the cytoplasm of cells expressing the EphA2 receptor, to investigate how biophysical cellular changes resulting from NCR manipulation affect the response to IRE at varying frequencies. We present evidence that the increase in the NCR mitigates the cell death response to conventional electroporation pulsed-electric fields (∼100 μs), consistent with the previously noted size dependence. However, this same molecular treatment enhanced the cell death response to high-frequency electric fields (∼1 μs). This finding demonstrates the importance of considering cellular biophysics and frequency-dependent effects in developing electroporation protocols, and our approach provides, to our knowledge, a novel and direct experimental methodology to quantify the relationship between cell morphology, pulse frequency, and electroporation response. Finally, this novel, to our knowledge, combinatorial approach may provide a paradigm to enhance in vivo tumor ablation through a molecular manipulation of cellular morphology before IRE application. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Design and testing of a separate-type lighting system using solar energy and cold-cathode fluorescent lamps

Energy Technology Data Exchange (ETDEWEB)

Yang, J.-P. [Department of Electrical Engineering, China Institute of Technology, Taipei, Taiwan 115, Taiwan (China)]. E-mail: april4120@tp.edu.tw; Hsiao, H.-C. [Department of Electrical Engineering, National Taiwan University of Science and Technology, Taipei, Taiwan 106, Taiwan (China)]. E-mail: hsiao@mouse.ee.ntust.edu.tw

2007-01-15

This paper presents a solar-powered lighting system, using cold-cathode fluorescent-lamps (CCFLs), with its battery-charging circuit and lamp-ignition circuit being separated so that its solar panels can be installed at any distance deemed necessary away from the lighting site in order to receive the maximum solar energy available. This system adopts the maximum-power point tracking (MPPT) method to control the power output of the solar panels and uses the zero-voltage switching (ZVS) DC-DC converter, as the charging circuit, to increase the panels' power generation efficiency and the charging circuit's conversion efficiency. The electronic ballast circuit for the CCFL is constructed with a half-bridge inverter, a resonant inductor, and a Rosen-type piezoelectric transformer, which forms a piezoelectric resonant-type inverter: to simplify the circuitry and to improve the power conversion efficiency, the ballast circuit is designed to directly step up the battery voltage in igniting the lamp. We also establish the transmission-parameter model for the piezoelectric resonant-type inverter to provide the base for the electric-power circuit design. Our experimental results indicate that the proposed system possesses some advantages, such as greater energy efficiency, circuitry simplicity, and so on, and is suitable for night lighting in house yards, parks and advertising panels.

Design and testing of a separate-type lighting system using solar energy and cold-cathode fluorescent lamps

International Nuclear Information System (INIS)

Yang, J.-P.; Hsiao, H.-C.

2007-01-01

This paper presents a solar-powered lighting system, using cold-cathode fluorescent-lamps (CCFLs), with its battery-charging circuit and lamp-ignition circuit being separated so that its solar panels can be installed at any distance deemed necessary away from the lighting site in order to receive the maximum solar energy available. This system adopts the maximum-power point tracking (MPPT) method to control the power output of the solar panels and uses the zero-voltage switching (ZVS) DC-DC converter, as the charging circuit, to increase the panels' power generation efficiency and the charging circuit's conversion efficiency. The electronic ballast circuit for the CCFL is constructed with a half-bridge inverter, a resonant inductor, and a Rosen-type piezoelectric transformer, which forms a piezoelectric resonant-type inverter: to simplify the circuitry and to improve the power conversion efficiency, the ballast circuit is designed to directly step up the battery voltage in igniting the lamp. We also establish the transmission-parameter model for the piezoelectric resonant-type inverter to provide the base for the electric-power circuit design. Our experimental results indicate that the proposed system possesses some advantages, such as greater energy efficiency, circuitry simplicity, and so on, and is suitable for night lighting in house yards, parks and advertising panels

Lightning-triggered electroporation and electrofusion as possible contributors to natural horizontal gene transfer.

Science.gov (United States)

Kotnik, Tadej

2013-09-01

Phylogenetic studies show that horizontal gene transfer (HGT) is a significant contributor to genetic variability of prokaryotes, and was perhaps even more abundant during the early evolution. Hitherto, research of natural HGT has mainly focused on three mechanisms of DNA transfer: conjugation, natural competence, and viral transduction. This paper discusses the feasibility of a fourth such mechanism--cell electroporation and/or electrofusion triggered by atmospheric electrostatic discharges (lightnings). A description of electroporation as a phenomenon is followed by a review of experimental evidence that electroporation of prokaryotes in aqueous environments can result in release of non-denatured DNA, as well as uptake of DNA from the surroundings and transformation. Similarly, a description of electrofusion is followed by a review of experiments showing that prokaryotes devoid of cell wall can electrofuse into hybrids expressing the genes of their both precursors. Under sufficiently fine-tuned conditions, electroporation and electrofusion are efficient tools for artificial transformation and hybridization, respectively, but the quantitative analysis developed here shows that conditions for electroporation-based DNA release, DNA uptake and transformation, as well as for electrofusion are also present in many natural aqueous environments exposed to lightnings. Electroporation is thus a plausible contributor to natural HGT among prokaryotes, and could have been particularly important during the early evolution, when the other mechanisms might have been scarcer or nonexistent. In modern prokaryotes, natural absence of the cell wall is rare, but it is reasonable to assume that the wall has formed during a certain stage of evolution, and at least prior to this, electrofusion could also have contributed to natural HGT. The concluding section outlines several guidelines for assessment of the feasibility of lightning-triggered HGT. © 2013 Elsevier B.V. All rights

1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies

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Kramar, Peter

2016-01-01

This volume presents the proceedings of the 1st World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies (WC2015). The congress took place in Portorož, Slovenia, during the week of September 6th to 10th, 2015. The scientific part of the Congress covered different aspects of electroporation and related technologies and included the following main topics:   ·         Application of pulsed electric fields technology in food: challenges and opportunities ·         Electrical impedance measurement for assessment of electroporation yield ·         Electrochemistry and electroporation ·         Electroporation meets electrostimulation ·         Electrotechnologies for food and biomass treatment ·         Food and biotechnology applications ·         In vitro electroporation - basic mechanisms ·         Interfacial behaviour of lipid-assemblies, membranes and cells in electric f...

LAMP-1-chimeric DNA vaccines enhance the antibody response in Japanese flounder, Paralichthys olivaceus.

Science.gov (United States)

Rondón-Barragán, Iang; Nozaki, Reiko; Hirono, Ikuo; Kondo, Hidehiro

2017-08-01

DNA vaccination is one method to protect farmed fish from viral and bacterial diseases. Chimeric antigens encoded by DNA vaccines have been shown to increase the resistance to viral diseases. Here, we sequenced the gene encoding lysosome-associated membrane protein-1 from Japanese flounder, Paralichthys olivaceus, (JfLAMP-1) and assessed its use in a chimeric DNA vaccine fused with the major capsule protein (MCP) from red seabream iridovirus (RSIV). JfLAMP-1 cDNA has a length of 1248 bp encoding 415 aa, which contains transmembrane and cytoplasmic domains. JfLAMP-1 is constitutively expressed in several tissues and its expression in spleen was upregulated following injection of formalin-killed cells (FKC) of Edwardsiella tarda. Immunofluorescence analysis showed that JfLAMP-1 is distributed in the small and large granules in the cytoplasm and groups close to the nucleus. The DNA encoding the luminal domain of JfLAMP-1 was replaced with the gene for the RSIV MCP, and the construct was cloned in an expression vector (pCIneo). Fish vaccinated with pCLAMP-MCP had significantly higher antibody levels than fish vaccinated with pCIneo vector harboring the MCP gene (p day 30 post-vaccination. Copyright © 2017 Elsevier Ltd. All rights reserved.

Effects of insulin on messenger RNA activities in rat liver

International Nuclear Information System (INIS)

Hill, R.E.; Lee, K.L.; Kenney, F.T.

1981-01-01

Liver poly(A) RNA, isolated from adrenalectomized rats after insulin treatment, was translated in a nuclease-treated lysate of rabbit reticulocytes and quantitated for both total activity and the capacity to synthesize the insulin-inducible enzyme tyrosine amino-transferase. Analysis of the translated products from poly(A) RNA isolated 1 h after insulin treatment showed a 2.7-fold increase in activity of tyrosine aminotransferase mRNA. During the same interval, the capacity of poly(A) RNA to direct the synthesis of total protein in lysates also changed, showing a 30 to 40% increase in translational activity/unit of RNA. Increased translatability was apparent in all fractions of poly(A) RNA separated by centrifugation on sucrose gradients. Insulin thus appears to mediated a generalized changed in mRNAs leading to increased capacity for translation; induction of tyrosine aminotransferase may reflect unusual sensitivity to this effect of the hormone

Lamp-Ballast Compatibility Index for Efficient Ceramic Metal Halide Lamp Operation

OpenAIRE

Sourish Chatterjee

2013-01-01

Development of energy efficient products and exploration of energy saving potential are major challenges for present day’s technology. Ceramic Metal Halide lamp is the latest improved version of metal halide lamp that finds its wide applications in indoor commercial lighting especially in retail shop lighting. This lamp shows better performance in terms of higher lumen per watt and colour constancy in comparison to conventional metal halide lamp. The inherent negative incremental impedance of...

Irreversible electroporation ablation area enhanced by synergistic high- and low-voltage pulses.

Directory of Open Access Journals (Sweden)

Chenguo Yao

Full Text Available Irreversible electroporation (IRE produced by a pulsed electric field can ablate tissue. In this study, we achieved an enhancement in ablation area by using a combination of short high-voltage pulses (HVPs to create a large electroporated area and long low-voltage pulses (LVPs to ablate the electroporated area. The experiments were conducted in potato tuber slices. Slices were ablated with an array of four pairs of parallel steel electrodes using one of the following four electric pulse protocols: HVP, LVP, synergistic HVP+LVP (SHLVP or LVP+HVP. Our results showed that the SHLVPs more effectively necrotized tissue than either the HVPs or LVPs, even when the SHLVP dose was the same as or lower than the HVP or LVP doses. The HVP and LVP order mattered and only HVPs+LVPs (SHLVPs treatments increased the size of the ablation zone because the HVPs created a large electroporated area that was more susceptible to the subsequent LVPs. Real-time temperature change monitoring confirmed that the tissue was non-thermally ablated by the electric pulses. Theoretical calculations of the synergistic effects of the SHLVPs on tissue ablation were performed. Our proposed SHLVP protocol provides options for tissue ablation and may be applied to optimize the current clinical IRE protocols.

Irreversible electroporation ablation area enhanced by synergistic high- and low-voltage pulses.

Science.gov (United States)

Yao, Chenguo; Lv, Yanpeng; Dong, Shoulong; Zhao, Yajun; Liu, Hongmei

2017-01-01

Irreversible electroporation (IRE) produced by a pulsed electric field can ablate tissue. In this study, we achieved an enhancement in ablation area by using a combination of short high-voltage pulses (HVPs) to create a large electroporated area and long low-voltage pulses (LVPs) to ablate the electroporated area. The experiments were conducted in potato tuber slices. Slices were ablated with an array of four pairs of parallel steel electrodes using one of the following four electric pulse protocols: HVP, LVP, synergistic HVP+LVP (SHLVP) or LVP+HVP. Our results showed that the SHLVPs more effectively necrotized tissue than either the HVPs or LVPs, even when the SHLVP dose was the same as or lower than the HVP or LVP doses. The HVP and LVP order mattered and only HVPs+LVPs (SHLVPs) treatments increased the size of the ablation zone because the HVPs created a large electroporated area that was more susceptible to the subsequent LVPs. Real-time temperature change monitoring confirmed that the tissue was non-thermally ablated by the electric pulses. Theoretical calculations of the synergistic effects of the SHLVPs on tissue ablation were performed. Our proposed SHLVP protocol provides options for tissue ablation and may be applied to optimize the current clinical IRE protocols.

mRNA Cancer Vaccines-Messages that Prevail.

Science.gov (United States)

Grunwitz, Christian; Kranz, Lena M

2017-01-01

During the last decade, mRNA became increasingly recognized as a versatile tool for the development of new innovative therapeutics. Especially for vaccine development, mRNA is of outstanding interest and numerous clinical trials have been initiated. Strikingly, all of these studies have proven that large-scale GMP production of mRNA is feasible and concordantly report a favorable safety profile of mRNA vaccines. Induction of T-cell immunity is a multi-faceted process comprising antigen acquisition, antigen processing and presentation, as well as immune stimulation. The effectiveness of mRNA vaccines is critically dependent on making the antigen(s) of interest available to professional antigen-presenting cells, especially DCs. Efficient delivery of mRNA into DCs in vivo remains a major challenge in the mRNA vaccine field. This review summarizes the principles of mRNA vaccines and highlights the importance of in vivo mRNA delivery and recent advances in harnessing their therapeutic potential.

Electroporation of Mammalian Cells by Nanosecond Electric Field Oscillations and its Inhibition by the Electric Field Reversal

Science.gov (United States)

2015-09-08

Report 3. DATES COVERED (From – To) March 2013 to July 2015 4. TITLE AND SUBTITLE Electroporation of mammalian cells by nanosecond electric field...Prescribed by ANSI Std. Z39.18 1Scientific RepoRts | 5:13818 | DOi: 10.1038/srep13818 www.nature.com/scientificreports Electroporation of mammalian cells...first to demonstrate that mammalian cells can be electroporated by damped sine wave electric stimuli of nanosecond duration. By comparing the

Dominant role of antigen dose in CD4+Foxp3+ regulatory T cell induction and expansion1

Science.gov (United States)

Turner, Michael S.; Kane, Lawrence P.; Morel, Penelope A.

2009-01-01

The definitions of tolerogenic vs. immunogenic dendritic cells (DC) remain controversial. Immature DC have been shown to induce T regulatory cells (Treg) specific for foreign and allo-antigens. However, we have previously reported that mature DC (G4DC) prevented the onset of autoimmune diabetes whereas immature DC (GMDC) were therapeutically ineffective. In this study, islet-specific CD4+ T cells from BDC2.5 TCR Tg mice were stimulated, in the absence of exogenous cytokine, with GMDC or G4DC pulsed with high- or low-affinity antigenic peptides and examined for Treg induction. Both GMDC and G4DC presenting low peptide doses induced weak TCR signaling via the Akt/mTOR pathway, resulting in significant expansion of Foxp3+ Treg. Furthermore, unpulsed G4DC, but not GMDC, also induced Treg. High peptide doses induced strong Akt/mTOR signaling and favored the expansion of Foxp3neg Th cells. The inverse correlation of Foxp3 and Akt/mTOR signaling was also observed in DO11.10 and OT-II TCR-Tg T cells and was recapitulated with anti-CD3/CD28 stimulation in the absence of DC. IL-6 production in these cultures correlated positively with antigen dose and inversely with Treg expansion. Studies with T cells or DC from IL-6−/− mice revealed that IL-6 production by T cells was more important in the inhibition of Treg induction at low antigen doses. These studies indicate that strength of Akt/mTOR signaling, a critical T cell intrinsic determinant for Treg vs Th induction, can be controlled by adjusting the dose of antigenic peptide. Furthermore, this operates in a dominant fashion over DC phenotype and cytokine production. PMID:19801514

Antibody-antigen-adjuvant conjugates enable co-delivery of antigen and adjuvant to dendritic cells in cis but only have partial targeting specificity.

Directory of Open Access Journals (Sweden)

Martin Kreutz

Full Text Available Antibody-antigen conjugates, which promote antigen-presentation by dendritic cells (DC by means of targeted delivery of antigen to particular DC subsets, represent a powerful vaccination approach. To ensure immunity rather than tolerance induction the co-administration of a suitable adjuvant is paramount. However, co-administration of unlinked adjuvant cannot ensure that all cells targeted by the antibody conjugates are appropriately activated. Furthermore, antigen-presenting cells (APC that do not present the desired antigen are equally strongly activated and could prime undesired responses against self-antigens. We, therefore, were interested in exploring targeted co-delivery of antigen and adjuvant in cis in form of antibody-antigen-adjuvant conjugates for the induction of anti-tumour immunity. In this study, we report on the assembly and characterization of conjugates consisting of DEC205-specific antibody, the model antigen ovalbumin (OVA and CpG oligodeoxynucleotides (ODN. We show that such conjugates are more potent at inducing cytotoxic T lymphocyte (CTL responses than control conjugates mixed with soluble CpG. However, our study also reveals that the nucleic acid moiety of such antibody-antigen-adjuvant conjugates alters their binding and uptake and allows delivery of the antigen and the adjuvant to cells partially independently of DEC205. Nevertheless, antibody-antigen-adjuvant conjugates are superior to antibody-free antigen-adjuvant conjugates in priming CTL responses and efficiently induce anti-tumour immunity in the murine B16 pseudo-metastasis model. A better understanding of the role of the antibody moiety is required to inform future conjugate vaccination strategies for efficient induction of anti-tumour responses.

MESSENGER'S First Flyby of Mercury

Science.gov (United States)

Slavin, James A.

2008-01-01

The MESSENGER mission to Mercury offers our first opportunity to explore this planet's miniature magnetosphere since Mariner 10's brief fly-bys in 1974-5. The magnetosphere of Mercury is the smallest in the solar system with its magnetic field typically standing off the solar wind only - 1000 to 2000 km above the surface. An overview of the MESSENGER mission and its January 14th close flyby of Mercury will be provided. Primary science objectives and the science instrumentation will be described. Initial results from MESSENGER'S first flyby on January 14th, 2008 will be discussed with an emphasis on the magnetic field and charged particle measurements.

A leukocyte antigen, Leu-13, is involved in induction of resistance of human cells to x-ray cell killing by interferon-α

International Nuclear Information System (INIS)

Kita, Kazuko; Zhai, Ling; Sugaya, Shigeru; Suzuki, Nobuo

2003-01-01

We previously reported on human interferon (HuIFN)-induced resistance of human cells to X-ray and UV cell killing. In this study, we searched for the genes whose expression is responsible for the resistance, using a PCR-based mRNA differential display method and Northern blotting analysis. RSa cells were used for this analysis, because they show increased resistance to X-ray- and UV-caused cell killing by HuIFN-α treatment prior to irradiation. Messenger RNA expression levels for Leu-13, a leukocyte antigen, were markedly up-regulated in RSa cells after HuIFN-α treatment. Furthermore, pretreatment of RSa cells with antisense oligonucleotides for Leu-13 mRNA resulted in the suppression of the HuIFN-α-induced resistance of the cells to X-ray cell killing, but did not modulate HuIFN-α-induced resistance to UV cell killing. These results suggest that Leu-13 is involved in HuIFN-α-induced resistance of human cells to X-ray cell killing, but not to UV cell killing. (author)

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

Science.gov (United States)

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 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 transfection of Sertoli cells.

siRNAs targeted to certain polyadenylation sites promote specific, RISC-independent degradation of messenger RNAs.

Science.gov (United States)

Vickers, Timothy A; Crooke, Stanley T

2012-07-01

While most siRNAs induce sequence-specific target mRNA cleavage and degradation in a process mediated by Ago2/RNA-induced silencing complex (RISC), certain siRNAs have also been demonstrated to direct target RNA reduction through deadenylation and subsequent degradation of target transcripts in a process which involves Ago1/RISC and P-bodies. In the current study, we present data suggesting that a third class of siRNA exist, which are capable of promoting target RNA reduction that is independent of both Ago and RISC. These siRNAs bind the target messenger RNA at the polyA signal and are capable of redirecting a small amount of polyadenylation to downstream polyA sites when present, however, the majority of the activity appears to be due to inhibition of polyadenylation or deadenylation of the transcript, followed by exosomal degradation of the immature mRNA.

Translational Influence on Messenger Stability

DEFF Research Database (Denmark)

Eriksen, Mette

-termination to be a global phenomena in gene regulation. The influence of codon usage in the early coding region on messenger stability was examined, in order to establish how fast or slow the ribosome has to decode the sequence for it to protect the messenger from degradation. The experiments demonstrated that very fast...

Transformation of group A streptococci by electroporation

NARCIS (Netherlands)

Suvorov, Alexander; Kok, Jan; Venema, Gerhardus

1988-01-01

The introduction, via electroporation, of free plasmid DNA into three strains of Streptococcus pyogenes is described. The method is very simple and rapid and efficiencies vary from 1 × 10^3 to 4 × 10^4 per µg of DNA. The method was also used to introduce an integrative plasmid and transformants were

Hollow-Core Fiber Lamp

Science.gov (United States)

Yi, Lin (Inventor); Tjoelker, Robert L. (Inventor); Burt, Eric A. (Inventor); Huang, Shouhua (Inventor)

2016-01-01

Hollow-core capillary discharge lamps on the millimeter or sub-millimeter scale are provided. The hollow-core capillary discharge lamps achieve an increased light intensity ratio between 194 millimeters (useful) and 254 millimeters (useless) light than conventional lamps. The capillary discharge lamps may include a cone to increase light output. Hollow-core photonic crystal fiber (HCPCF) may also be used.

49 CFR 393.25 - Requirements for lamps other than head lamps.

Science.gov (United States)

2010-10-01

... listed in paragraph (c) of this section. If motor vehicle equipment (e.g., mirrors, snow plows, wrecker...: J586—Stop Lamps for Use on Motor Vehicles Less Than 2032 mm in Overall Width, March 2000; J2261 Stop Lamps and Front- and Rear-Turn Signal Lamps for Use on Motor Vehicles 2032 mm or More in Overall Width...

Controlled release of optimized electroporation enhances the transdermal efficiency of sinomenine hydrochloride for treating arthritis in vitro and in clinic

Science.gov (United States)

Feng, Shun; Zhu, Lijun; Huang, Zhisheng; Wang, Haojia; Li, Hong; Zhou, Hua; Lu, Linlin; Wang, Ying; Liu, Zhongqiu; Liu, Liang

2017-01-01

Sinomenine hydrochloride (SH) is an ideal drug for the treatment of rheumatoid arthritis and osteoarthritis. However, high plasma concentration of systemically administered SH can release histamine, which can cause rash and gastrointestinal side effects. Topical delivery can increase SH concentration in the synovial fluid without high plasma level, thus minimizing systemic side effects. However, passive diffusion of SH was found to be inefficient because of the presence of the stratum corneum layer. Therefore, an effective method is required to compensate for the low efficiency of SH passive diffusion. In this study, transdermal experiments in vitro and clinical tests were utilized to explore the optimized parameters for electroporation of topical delivery for SH. Fluorescence experiment and hematoxylin and eosin staining analysis were performed to reveal the mechanism by which electroporation promoted permeation. In vitro, optimized electroporation parameters were 3 KHz, exponential waveform, and intensity 10. Using these parameters, transdermal permeation of SH was increased by 1.9–10.1 fold in mice skin and by 1.6–47.1 fold in miniature pig skin compared with passive diffusion. After the electroporation stimulation, the intercellular intervals and epidermal cracks in the skin increased. In clinical tests, SH concentration in synovial fluid was 20.84 ng/mL after treatment with electroporation. Therefore, electroporation with optimized parameters could significantly enhance transdermal permeation of SH. The mechanism by which electroporation promoted permeation was that the electronic pulses made the skin structure looser. To summarize, electroporation may be an effective complementary method for transdermal permeation of SH. The controlled release of electroporation may be a promising clinical method for transdermal drug administration. PMID:28670109

RNA-based, transient modulation of gene expression in human haematopoietic stem and progenitor cells

Science.gov (United States)

Diener, Yvonne; Jurk, Marion; Kandil, Britta; Choi, Yeong-Hoon; Wild, Stefan; Bissels, Ute; Bosio, Andreas

2015-01-01

Modulation of gene expression is a useful tool to study the biology of haematopoietic stem and progenitor cells (HSPCs) and might also be instrumental to expand these cells for therapeutic approaches. Most of the studies so far have employed stable gene modification by viral vectors that are burdensome when translating protocols into clinical settings. Our study aimed at exploring new ways to transiently modify HSPC gene expression using non-integrating, RNA-based molecules. First, we tested different methods to deliver these molecules into HSPCs. The delivery of siRNAs with chemical transfection methods such as lipofection or cationic polymers did not lead to target knockdown, although we observed more than 90% fluorescent cells using a fluorochrome-coupled siRNA. Confocal microscopic analysis revealed that despite extensive washing, siRNA stuck to or in the cell surface, thereby mimicking a transfection event. In contrast, electroporation resulted in efficient, siRNA-mediated protein knockdown. For transient overexpression of proteins, we used optimised mRNA molecules with modified 5′- and 3′-UTRs. Electroporation of mRNA encoding GFP resulted in fast, efficient and persistent protein expression for at least seven days. Our data provide a broad-ranging comparison of transfection methods for hard-to-transfect cells and offer new opportunities for DNA-free, non-integrating gene modulation in HSPCs. PMID:26599627

Lamp-2 deficiency prevents high-fat diet-induced obese diabetes via enhancing energy expenditure

International Nuclear Information System (INIS)

Yasuda-Yamahara, Mako; Kume, Shinji; Yamahara, Kosuke; Nakazawa, Jun; Chin-Kanasaki, Masami; Araki, Hisazumi; Araki, Shin-ichi; Koya, Daisuke; Haneda, Masakzu; Ugi, Satoshi; Maegawa, Hiroshi; Uzu, Takashi

2015-01-01

Autophagy process is essential for maintaining intracellular homeostasis and consists of autophagosome formation and subsequent fusion with lysosome for degradation. Although the role of autophagosome formation in the pathogenesis of diabetes has been recently documented, the role of the latter process remains unclear. This study analyzed high-fat diet (HFD)-fed mice lacking lysosome-associated membrane protein-2 (lamp-2), which is essential for the fusion with lysosome and subsequent degradation of autophagosomes. Although lamp-2 deficient mice showed little alteration in glucose metabolism under normal diet feeding, they showed a resistance against high-fat diet (HFD)-induced obesity, hyperinsulinemic hyperglycemia and tissues lipid accumulation, accompanied with higher energy expenditure. The expression levels of thermogenic genes in brown adipose tissue were significantly increased in HFD-fed lamp-2-deficient mice. Of some serum factors related to energy expenditure, the serum level of fibroblast growth factor (FGF) 21 and its mRNA expression level in the liver were significantly higher in HFD-fed lamp-2-deficient mice in an ER stress-, but not PPARα-, dependent manner. In conclusion, a lamp-2-depenedent fusion and degradation process of autophagosomes is involved in the pathogenesis of obese diabetes, providing a novel insight into autophagy and diabetes. - Highlights: • Lamp-2 is essential for autophagosome fusion with lysosome and its degradation. • Lamp-2 deficiency lead to a resistance to diet-induced obese diabetes in mice. • Lamp-2 deficiency increased whole body energy expenditure under HFD-feeding. • Lamp-2 deficiency elevated the serum level of FGF21 under HFD-feeding

Lamp-2 deficiency prevents high-fat diet-induced obese diabetes via enhancing energy expenditure

Energy Technology Data Exchange (ETDEWEB)

Yasuda-Yamahara, Mako [Department of Medicine, Shiga University of Medical Science, Otsu, Shiga (Japan); Kume, Shinji, E-mail: skume@belle.shiga-med.ac.jp [Department of Medicine, Shiga University of Medical Science, Otsu, Shiga (Japan); Yamahara, Kosuke; Nakazawa, Jun; Chin-Kanasaki, Masami; Araki, Hisazumi; Araki, Shin-ichi [Department of Medicine, Shiga University of Medical Science, Otsu, Shiga (Japan); Koya, Daisuke [Department of Diabetology and Endocrinology, Kanazawa Medical University, Kahoku-Gun, Ishikawa (Japan); Haneda, Masakzu [Division of Metabolism and Biosystemic Science, Asahikawa Medical University, Asahikawa, Hokkaido (Japan); Ugi, Satoshi; Maegawa, Hiroshi; Uzu, Takashi [Department of Medicine, Shiga University of Medical Science, Otsu, Shiga (Japan)

2015-09-18

Autophagy process is essential for maintaining intracellular homeostasis and consists of autophagosome formation and subsequent fusion with lysosome for degradation. Although the role of autophagosome formation in the pathogenesis of diabetes has been recently documented, the role of the latter process remains unclear. This study analyzed high-fat diet (HFD)-fed mice lacking lysosome-associated membrane protein-2 (lamp-2), which is essential for the fusion with lysosome and subsequent degradation of autophagosomes. Although lamp-2 deficient mice showed little alteration in glucose metabolism under normal diet feeding, they showed a resistance against high-fat diet (HFD)-induced obesity, hyperinsulinemic hyperglycemia and tissues lipid accumulation, accompanied with higher energy expenditure. The expression levels of thermogenic genes in brown adipose tissue were significantly increased in HFD-fed lamp-2-deficient mice. Of some serum factors related to energy expenditure, the serum level of fibroblast growth factor (FGF) 21 and its mRNA expression level in the liver were significantly higher in HFD-fed lamp-2-deficient mice in an ER stress-, but not PPARα-, dependent manner. In conclusion, a lamp-2-depenedent fusion and degradation process of autophagosomes is involved in the pathogenesis of obese diabetes, providing a novel insight into autophagy and diabetes. - Highlights: • Lamp-2 is essential for autophagosome fusion with lysosome and its degradation. • Lamp-2 deficiency lead to a resistance to diet-induced obese diabetes in mice. • Lamp-2 deficiency increased whole body energy expenditure under HFD-feeding. • Lamp-2 deficiency elevated the serum level of FGF21 under HFD-feeding.

Effects of transgenic sterilization constructs and their repressor compounds on hatch, developmental rate and early survival of electroporated channel catfish embryos and fry.

Science.gov (United States)

Su, Baofeng; Shang, Mei; Li, Chao; Perera, Dayan A; Pinkert, Carl A; Irwin, Michael H; Peatman, Eric; Grewe, Peter; Patil, Jawahar G; Dunham, Rex A

2015-04-01

Channel catfish (Ictalurus punctatus) embryos were electroporated with sterilization constructs targeting primordial germ cell proteins or with buffer. Some embryos then were treated with repressor compounds, cadmium chloride, copper sulfate, sodium chloride or doxycycline, to prevent expression of the transgene constructs. Promoters included channel catfish nanos and vasa, salmon transferrin (TF), modified yeast Saccharomyces cerevisiae copper transport protein (MCTR) and zebrafish racemase (RM). Knock-down systems were the Tet-off (nanos and vasa constructs), MCTR, RM and TF systems. Knock-down genes included shRNAi targeting 5' nanos (N1), 3' nanos (N2) or dead end (DND), or double-stranded nanos RNA (dsRNA) for overexpression of nanos mRNA. These constructs previously were demonstrated to knock down nanos, vasa and dead end, with the repressors having variable success. Exogenous DNA affected percentage hatch (% hatch), as all 14 constructs, except for the TF dsRNA, TF N1 (T), RM DND (C), vasa DND (C), vasa N1 (C) and vasa N2 (C), had lower % hatch than the control electroporated with buffer. The MCTR and RM DND (T) constructs resulted in delayed hatch, and the vasa and nanos constructs had minimal effects on time of hatch (P nanos constructs, doxycycline greatly delayed hatch (P < 0.05). Adverse effects of the transgenes and repressors continued for several treatments for the first 6 days after hatch, but only in a few treatments during the next 10 days. Repressors and gene expression impacted the yield of putative transgenic channel catfish fry, and need to be considered and accounted for in the hatchery phase of producing transgenically sterilized catfish fry and their fertile counterparts. This fry output should be considered to ensure that sufficient numbers of transgenic fish are produced for future applications and for defining repressor systems that are the most successful.

Strong and multi-antigen specific immunity by hepatitis B core antigen (HBcAg)-based vaccines in a murine model of chronic hepatitis B: HBcAg is a candidate for a therapeutic vaccine against hepatitis B virus.

Science.gov (United States)

Akbar, Sheikh Mohammad Fazle; Chen, Shiyi; Al-Mahtab, Mamun; Abe, Masanori; Hiasa, Yoichi; Onji, Morikazu

2012-10-01

Experimental evidence suggests that hepatitis B core antigen (HBcAg)-specific cytotoxic T lymphocytes (CTL) are essential for the control of hepatitis B virus (HBV) replication and prevention of liver damage in patients with chronic hepatitis B (CHB). However, most immune therapeutic approaches in CHB patients have been accomplished with hepatitis B surface antigen (HBsAg)-based prophylactic vaccines with unsatisfactory clinical outcomes. In this study, we prepared HBsAg-pulsed dendritic cells (DC) and HBcAg-pulsed DC by culturing spleen DC from HBV transgenic mice (HBV TM) and evaluated the immunomodulatory capabilities of these antigens, which may serve as a better therapy for CHB. The kinetics of HBsAg, antibody levels against HBsAg (anti-HBs), proliferation of HBsAg- and HBcAg-specific lymphocytes, production of antigen-specific CTL, and activation of endogenous DC were compared between HBV TM vaccinated with either HBsAg- or HBcAg-pulsed DC. Vaccination with HBsAg-pulsed DC induced HBsAg-specific immunity, but failed to induce HBcAg-specific immunity in HBV TM. However, immunization of HBV TM with HBcAg-pulsed DC resulted in: (1) HBsAg negativity, (2) production of anti-HBs, and (3) development of HBsAg- and HBcAg-specific T cells and CTL in the spleen and the liver. Additionally, significantly higher levels of activated endogenous DC were detected in HBV TM immunized with HBcAg-pulsed DC compared to HBsAg-pulsed DC (pdamage suggests that HBcAg should be an integral component of the therapeutic vaccine against CHB. Copyright © 2012 Elsevier B.V. All rights reserved.

Effect of electroporation on radiosensitization with cisplatin in two cell lines with different chemo- and radiosensitivity

International Nuclear Information System (INIS)

Kranjc, S.; Cemazar, M.; Grosel, A.; Pipan, Z.; Sersa, G.

2003-01-01

Aim. Radiosensitization with cisplatin can be enhanced by electroporation of cells and tumours. The aim of this study was to extend our previous studies on two carcinoma tumour models with different chemo- and radiosensitivity in order to evaluate whether this treatment is effective also on less chemo- and radiosensitive tumour cells. Materials and methods. This in vitro study was performed on carcinoma SCK and EAT-E cells. The cytotoxicity of three-modality treatment consisting of cisplatin, electroporation and irradiation was determined by the clonogenic assay. Results. The radiosensitizing effect of cisplatin on the two cell lines was greatly enhanced by electroporation. By this combined treatment, less chemo and radiosensitive EAT-E cells were rendered as sensitive as more chemo and radiosensitive SCK cells. Conclusion. The enhancement of cisplatin-induced radiosensitization of cells by electroporation could be beneficially used in the treatment of intrinsically less chemo- and radiosensitive tumours. (author)

Discharge lamp technologies

Science.gov (United States)

Dakin, James

1994-01-01

This talk is an overview of discharge lamp technology commonly employed in general lighting, with emphasis on issues pertinent to lighting for plant growth. Since the audience is primarily from the plant growth community, and this begins the light source part of the program, we will start with a brief description of the discharge lamps. Challenges of economics and of thermal management make lamp efficiency a prime concern in controlled environment agriculture, so we will emphasize science considerations relating to discharge lamp efficiency. We will then look at the spectra and ratings of some representative lighting products, and conclude with a discussion of technological advances.

A generic RNA-pulsed dendritic cell vaccine strategy for renal cell carcinoma

Science.gov (United States)

Geiger, Christiane; Regn, Sybille; Weinzierl, Andreas; Noessner, Elfriede; Schendel, Dolores J

2005-01-01

We present a generic dendritic cell (DC) vaccine strategy for patients with renal cell carcinoma (RCC) based on the use of RNA as a source of multiplex tumor-associated antigens (TAAs). Instead of preparing RNA from tumor tissue of each individual RCC patient, we propose to substitute RNA prepared from a well characterized highly immunogenic RCC cell line (RCC-26 tumor cells) as a generic source of TAAs for loading of DCs. We demonstrate here that efficient RNA transfer can be achieved using lipofection of immature DCs, which are subsequently matured with a cytokine cocktail to express high levels of MHC and costimulatory molecules as well as the chemokine receptor CCR7. Neither RNA itself nor the lipid component impacted on the phenotype or the cytokine secretion of mature DCs. Following RNA loading, DCs derived from HLA-A2-positive donors were able to activate effector-memory cytotoxic T lymphocytes (CTLs) specific for a TAA ligand expressed by the RCC-26 cell line. CTL responses to RNA-loaded DCs reached levels comparable to those stimulated directly by the RCC-26 tumor cells. Furthermore, DCs expressing tumor cell RNA primed naïve T cells, yielding T cell lines with cytotoxicity and cytokine secretion after contact with RCC tumor cells. RCC-26 cell lines are available as good manufacturing practice (GMP)-certified reagents enabling this source of RNA to be easily standardized and adapted for clinical testing. In addition, well defined immune monitoring tools, including the use of RNA expressing B cell lines, are available. Thus, this DC vaccine strategy can be directly compared with an ongoing gene therapy trial using genetically-engineered variants of the RCC-26 cell line as vaccines for RCC patients with metastatic disease. PMID:16045799

A generic RNA-pulsed dendritic cell vaccine strategy for renal cell carcinoma

Directory of Open Access Journals (Sweden)

Noessner Elfriede

2005-07-01

Full Text Available Abstract We present a generic dendritic cell (DC vaccine strategy for patients with renal cell carcinoma (RCC based on the use of RNA as a source of multiplex tumor-associated antigens (TAAs. Instead of preparing RNA from tumor tissue of each individual RCC patient, we propose to substitute RNA prepared from a well characterized highly immunogenic RCC cell line (RCC-26 tumor cells as a generic source of TAAs for loading of DCs. We demonstrate here that efficient RNA transfer can be achieved using lipofection of immature DCs, which are subsequently matured with a cytokine cocktail to express high levels of MHC and costimulatory molecules as well as the chemokine receptor CCR7. Neither RNA itself nor the lipid component impacted on the phenotype or the cytokine secretion of mature DCs. Following RNA loading, DCs derived from HLA-A2-positive donors were able to activate effector-memory cytotoxic T lymphocytes (CTLs specific for a TAA ligand expressed by the RCC-26 cell line. CTL responses to RNA-loaded DCs reached levels comparable to those stimulated directly by the RCC-26 tumor cells. Furthermore, DCs expressing tumor cell RNA primed naïve T cells, yielding T cell lines with cytotoxicity and cytokine secretion after contact with RCC tumor cells. RCC-26 cell lines are available as good manufacturing practice (GMP-certified reagents enabling this source of RNA to be easily standardized and adapted for clinical testing. In addition, well defined immune monitoring tools, including the use of RNA expressing B cell lines, are available. Thus, this DC vaccine strategy can be directly compared with an ongoing gene therapy trial using genetically-engineered variants of the RCC-26 cell line as vaccines for RCC patients with metastatic disease.

Higgs mass from neutrino-messenger mixing

International Nuclear Information System (INIS)

Byakti, Pritibhajan; Khosa, Charanjit K.; Mummidi, V.S.; Vempati, Sudhir K.

2017-01-01

The discovery of the Higgs particle at 125 GeV has put strong constraints on minimal messenger models of gauge mediation, pushing the stop masses into the multi-TeV regime. Extensions of these models with matter-messenger mixing terms have been proposed to generate a large trilinear parameter, A t , relaxing these constraints. The detailed survey of these models (DOI: 10.1007/JHEP05(2013)055; 10.1007/JHEP08(2013)093 ) so far considered messenger mixings with only MSSM superfields. In the present work, we extend the survey to MSSM with inverse-seesaw mechanism. The neutrino-sneutrino corrections to the Higgs mass in the inverse seesaw model are not significant in the minimal gauge mediation model, unless one considers messenger-matter interaction terms. We classify all possible models with messenger-matter interactions and perform thorough numerical analysis to find out the promising models. We found that out of the 17 possible models 9 of them can lead to Higgs mass within the observed value without raising the sfermion masses significantly. The successful models have stop masses ∼1.5 TeV with small or negligible mixing and yet a light CP even Higgs at 125 GeV.

Nonlinear electro-mechanobiological behavior of cell membrane during electroporation

KAUST Repository

Deng, Peigang; Lee, Yi-Kuen; Lin, Ran; Zhang, Tong-Yi

2012-01-01

A nonlinear electroporation (EP) model is proposed to study the electro-mechanobiological behavior of cell membrane during EP, by taking the nonlinear large deformation of the membrane into account. The proposed model predicts the critical

Martini Coarse-Grained Force Field : Extension to RNA

NARCIS (Netherlands)

Uusitalo, Jaakko J.; Ingolfsson, Helgi I.; Marrink, Siewert J.; Faustino, Ignacio

2017-01-01

RNA has an important role not only as the messenger of genetic information but also as a regulator of gene expression. Given its central role in cell biology, there is significant interest in studying the structural and dynamic behavior of RNA in relation to other biomolecules. Coarse-grain

Identification of a novel dendritic cell surface antigen defined by carbohydrate specific CD24 antibody cross-reactivity.

Science.gov (United States)

Williams, L A; McLellan, A D; Summers, K L; Sorg, R V; Fearnley, D B; Hart, D N

1996-01-01

Dendritic cells (DC) are characterized as leucocytes that lack mature lineage specific markers and stimulate naive T-lymphocyte proliferation in vitro and in vivo. The mouse heat stable antigen (HSA) participates in T lymphocyte co-stimulation and is expressed by DC isolated from thymus, skin and spleen. The human HSA homologue, CD24, is predominantly expressed by B lymphocytes and granulocytes, but its expression on DC has not been studied in detail. CD24 clearly participates in B-lymphocyte signalling but co-stimulatory activity for T lymphocytes has not yet been described. We have examined the expression of CD24 on human peripheral blood DC populations isolated directly or following in vitro culture. The CD24 antigen was absent from blood DC however, cross-reactive sialylated carbohydrate epitopes were detected on DC with some CD24 monoclonal antibodies (mAb). These CD24 mAb define a protein surface antigen, which is expressed by an immature or resting subpopulation of peripheral blood DC and is down-regulated following activation differentiation in vitro. PMID:8911149

Predicting electroporation of cells in an inhomogeneous electric field based on mathematical modeling and experimental CHO-cell permeabilization to propidium iodide determination.

Science.gov (United States)

Dermol, Janja; Miklavčič, Damijan

2014-12-01

High voltage electric pulses cause electroporation of the cell membrane. Consequently, flow of the molecules across the membrane increases. In our study we investigated possibility to predict the percentage of the electroporated cells in an inhomogeneous electric field on the basis of the experimental results obtained when cells were exposed to a homogeneous electric field. We compared and evaluated different mathematical models previously suggested by other authors for interpolation of the results (symmetric sigmoid, asymmetric sigmoid, hyperbolic tangent and Gompertz curve). We investigated the density of the cells and observed that it has the most significant effect on the electroporation of the cells while all four of the mathematical models yielded similar results. We were able to predict electroporation of cells exposed to an inhomogeneous electric field based on mathematical modeling and using mathematical formulations of electroporation probability obtained experimentally using exposure to the homogeneous field of the same density of cells. Models describing cell electroporation probability can be useful for development and presentation of treatment planning for electrochemotherapy and non-thermal irreversible electroporation. Copyright © 2014 Elsevier B.V. All rights reserved.

Messengers of the universe

International Nuclear Information System (INIS)

Becker, J.K.; Spurio, M.

2011-01-01

The observation of the solar neutrinos and of a neutrino burst from the supernova explosion 1987A opened a new observation field which in the next years could be complemented with the detection of astrophysical highenergy neutrinos. Neutrino astronomy is a young discipline derived from the fundamental necessity of extending conventional astronomy beyond the usual electro-magnetic messengers. This is a summary of recent results on those new 'messengers of the universe', based on the presentations in Branch IV of the Neutrino Oscillation Workshop 2010 (NOW2010).

Enhanced Dendritic Cell-Mediated Antigen-Specific CD4+ T Cell Responses: IFN-Gamma Aids TLR Stimulation

Directory of Open Access Journals (Sweden)

Kuo-Ching Sheng

2013-01-01

Full Text Available Phenotypic maturation and T cell stimulation are two functional attributes of DCs critical for immune induction. The combination of antigens, including those from cancer, with Toll-like receptor (TLR ligands induces far superior cellular immune responses compared to antigen alone. In this study, IFN-gamma treatment of bone marrow-derived DC, followed by incubation with the TLR2, TLR4, or TLR9 agonists, enhanced DC activation compared to TLR ligation alone. Most notably, the upregulation of CD40 with LPS stimulation and CD86 with CpG stimulation was observed in in vitro cultures. Similarly, IFN-gamma coinjected with TLR ligands was able to promote DC activation in vivo, with DCs migrating from the site of immunization to the popliteal lymph nodes demonstrating increased expression of CD80 and CD86. The heightened DC activation translated to a drastic increase in T cell stimulatory capacity in both antigen independent and antigen dependent fashions. This is the first time that IFN-gamma has been shown to have a combined effect with TLR ligation to enhance DC activation and function. The results demonstrate the novel use of IFN-gamma together with TLR agonists to enhance antigen-specific T cell responses, for applications in the development of enhanced vaccines and drug targets against diseases including cancer.

Growth differentiation factor 3 is induced by bone morphogenetic protein 6 (BMP-6) and BMP-7 and increases luteinizing hormone receptor messenger RNA expression in human granulosa cells.

Science.gov (United States)

Shi, Jia; Yoshino, Osamu; Osuga, Yutaka; Akiyama, Ikumi; Harada, Miyuki; Koga, Kaori; Fujimoto, Akihisa; Yano, Tetsu; Taketani, Yuji

2012-04-01

To examine the relevance of growth differentiation factor 3 (GDF-3) and bone morphogenetic protein (BMP) cytokines in human ovary. Molecular studies. Research laboratory. Eight women undergoing salpingo-oophorectomy and 30 women undergoing ovarian stimulation for in vitro fertilization. Localizing GDF-3 protein in human ovaries; granulosa cells (GC) cultured with GDF-3, BMP-6, or BMP-7 followed by RNA extraction. The localization of GDF-3 protein in normal human ovaries via immunohistochemical analysis, GDF-3 messenger RNA (mRNA) expression evaluation via quantitative real-time reverse transcription and polymerase chain reaction (RT-PCR), and evaluation of the effect of GDF-3 on leuteinizing hormone (LH) receptor mRNA expression via quantitative real-time RT-PCR. In the ovary, BMP cytokines, of the transforming growth factor beta (TGF-β) superfamily, are known as a luteinization inhibitor by suppressing LH receptor expression in GC. Growth differentiation factor 3, a TGF-β superfamily cytokine, is recognized as an inhibitor of BMP cytokines in other cells. Immunohistochemical analysis showed that GDF-3 was strongly detected in the GC of antral follicles. An in vitro assay revealed that BMP-6 or BMP-7 induced GDF-3 mRNA in GC. Also, GDF-3 increased LH receptor mRNA expression and inhibited the effect of BMP-7, which suppressed the LH receptor mRNA expression in GC. GDF-3, induced with BMP-6 and BMP-7, might play a role in folliculogenesis by inhibiting the effect of BMP cytokines. Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

A new equivalent circuit model for micro electroporation systems

KAUST Repository

Shagoshtasbi, Hooman; Lee, Yi-Kuen

2011-01-01

Electroporation (EP) is a unique biotechnique in which intense electric pulses are applied on the cell membrane to temporarily generate nanoscale electropores and to increase the membrane permeability for the delivery of exogenous biomolecules or drugs. We propose a new equivalent circuit model with 8 electric components to predict the electrodynamic response of a micro EP system. As the permeability of the cell membrane increases, the membrane resistance decreases. The numerical simulations of the transmembrane current responses to different applied voltages (1∼6V) are consistent with the experimental results using HeLa cells. Besides, the transmembrane voltage as a function of applied voltages is determined as well. These transmembrane current and voltage responses can be extremely useful for the design of new generation of micro EP systems for transfection of large DNA molecules in the future. © 2011 IEEE.

A new equivalent circuit model for micro electroporation systems

KAUST Repository

Shagoshtasbi, Hooman

2011-02-01

Electroporation (EP) is a unique biotechnique in which intense electric pulses are applied on the cell membrane to temporarily generate nanoscale electropores and to increase the membrane permeability for the delivery of exogenous biomolecules or drugs. We propose a new equivalent circuit model with 8 electric components to predict the electrodynamic response of a micro EP system. As the permeability of the cell membrane increases, the membrane resistance decreases. The numerical simulations of the transmembrane current responses to different applied voltages (1∼6V) are consistent with the experimental results using HeLa cells. Besides, the transmembrane voltage as a function of applied voltages is determined as well. These transmembrane current and voltage responses can be extremely useful for the design of new generation of micro EP systems for transfection of large DNA molecules in the future. © 2011 IEEE.

Bystander Effect Induced by Electroporation is Possibly Mediated by Microvesicles and Dependent on Pulse Amplitude, Repetition Frequency and Cell Type.

Science.gov (United States)

Prevc, Ajda; Bedina Zavec, Apolonija; Cemazar, Maja; Kloboves-Prevodnik, Veronika; Stimac, Monika; Todorovic, Vesna; Strojan, Primoz; Sersa, Gregor

2016-10-01

Bystander effect, a known phenomenon in radiation biology, where irradiated cells release signals which cause damage to nearby, unirradiated cells, has not been explored in electroporated cells yet. Therefore, our aim was to determine whether bystander effect is present in electroporated melanoma cells in vitro, by determining viability of non-electroporated cells exposed to medium from electroporated cells and by the release of microvesicles as potential indicators of the bystander effect. Here, we demonstrated that electroporation of cells induces bystander effect: Cells exposed to electric pulses mediated their damage to the non-electroporated cells, thus decreasing cell viability. We have shown that shedding microvesicles may be one of the ways used by the cells to mediate the death signals to the neighboring cells. The murine melanoma B16F1 cell line was found to be more electrosensitive and thus more prone to bystander effect than the canine melanoma CMeC-1 cell line. In B16F1 cell line, bystander effect was present above the level of electropermeabilization of the cells, with the threshold at 800 V/cm. Furthermore, with increasing electric field intensities and the number of pulses, the bystander effect also increased. In conclusion, electroporation can induce bystander effect which may be mediated by microvesicles, and depends on pulse amplitude, repetition frequency and cell type.

Higgs mass from neutrino-messenger mixing

Energy Technology Data Exchange (ETDEWEB)

Byakti, Pritibhajan [Center for High Energy Physics, Indian Institute of Science,C.V. Raman Ave, Bangalore 560012 (India); Department of Theoretical Physics, Indian Association for the Cultivation of Science,2A & 2B Raja S.C. Mullick Road, Kolkata 700 032 (India); Khosa, Charanjit K. [Center for High Energy Physics, Indian Institute of Science,C.V. Raman Ave, Bangalore 560012 (India); Mummidi, V.S. [Harish-Chandra Research Institute,Chhatnag Road, Jhusi, Allahabad 211019 (India); Vempati, Sudhir K. [Center for High Energy Physics, Indian Institute of Science,C.V. Raman Ave, Bangalore 560012 (India)

2017-03-06

The discovery of the Higgs particle at 125 GeV has put strong constraints on minimal messenger models of gauge mediation, pushing the stop masses into the multi-TeV regime. Extensions of these models with matter-messenger mixing terms have been proposed to generate a large trilinear parameter, A{sub t}, relaxing these constraints. The detailed survey of these models (DOI: 10.1007/JHEP05(2013)055; 10.1007/JHEP08(2013)093 ) so far considered messenger mixings with only MSSM superfields. In the present work, we extend the survey to MSSM with inverse-seesaw mechanism. The neutrino-sneutrino corrections to the Higgs mass in the inverse seesaw model are not significant in the minimal gauge mediation model, unless one considers messenger-matter interaction terms. We classify all possible models with messenger-matter interactions and perform thorough numerical analysis to find out the promising models. We found that out of the 17 possible models 9 of them can lead to Higgs mass within the observed value without raising the sfermion masses significantly. The successful models have stop masses ∼1.5 TeV with small or negligible mixing and yet a light CP even Higgs at 125 GeV.

Expression of somatotropin receptor messenger ribonucleic acid in bovine tissues

International Nuclear Information System (INIS)

Lucy, M.C.; Boyd, C.K.; Koenigsfeld, A.T.; Okamura, C.S.

1998-01-01

The somatotropin receptor mRNA is controlled by at least two different gene promoters that generate 2 two variants with different exon 1 sequences (1A and 1B). The location of 1A and 1B somatotropin receptor mRNA within cattle tissues and, hence, the tissue specificity of the 1A and 1B promoters are unknown. In addition, the cDNA sequence of the 1B somatotropin receptor has not been determined. Our objective, therefore, was to sequence a cDNA for the 1B somatotropin receptor and to analyze bovine tissues for expression of 1A and 1B somatotropin receptor mRNA. Twenty adult tissues and six fetal tissues were collected at slaughter from each of four cows and two fetuses. Messenger RNA was analyzed using ribonuclease protection assays. The adult liver expressed both 1A and 1B mRNA. All other adult tissues expressed 1B mRNA but not 1A mRNA. The greatest amount of 1B mRNA was detected in liver and adipose (abdominal and subcutaneous) tissues. Other tissues had approximately one-half to one-tenth of the amount of 1B mRNA in the liver or adipose tissue. Fetal tissues (including fetal liver) expressed 1B mRNA and not 1A mRNA. Based on cDNA sequencing, the protein encoded by the 1A and 1B mRNA was nearly identical. We concluded that 1A somatotropin receptor mRNA is specific to adult bovine liver. Other adult and fetal bovine tissues expressed 1B somatotropin receptor mRNA with a predicted protein sequence that was similar to the 1A somatotropin receptor

Regulatory network controlling extracellular proteins in Erwinia carotovora subsp. carotovora: FlhDC, the master regulator of flagellar genes, activates rsmB regulatory RNA production by affecting gacA and hexA (lrhA) expression.

Science.gov (United States)

Cui, Yaya; Chatterjee, Asita; Yang, Hailian; Chatterjee, Arun K

2008-07-01

Erwinia carotovora subsp. carotovora produces an array of extracellular proteins (i.e., exoproteins), including plant cell wall-degrading enzymes and Harpin, an effector responsible for eliciting hypersensitive reaction. Exoprotein genes are coregulated by the quorum-sensing signal, N-acyl homoserine lactone, plant signals, an assortment of transcriptional factors/regulators (GacS/A, ExpR1, ExpR2, KdgR, RpoS, HexA, and RsmC) and posttranscriptional regulators (RsmA, rsmB RNA). rsmB RNA production is positively regulated by GacS/A, a two-component system, and negatively regulated by HexA (PecT in Erwinia chrysanthemi; LrhA [LysR homolog A] in Escherichia coli) and RsmC, a putative transcriptional adaptor. While free RsmA, an RNA-binding protein, promotes decay of mRNAs of exoprotein genes, binding of RsmA with rsmB RNA neutralizes the RsmA effect. In the course of studies of GacA regulation, we discovered that a locus bearing strong homology to the flhDC operon of E. coli also controls extracellular enzyme production. A transposon insertion FlhDC(-) mutant produces very low levels of pectate lyase, polygalacturonase, cellulase, protease, and E. carotovora subsp. carotovora Harpin (Harpin(Ecc)) and is severely attenuated in its plant virulence. The production of these exoproteins is restored in the mutant carrying an FlhDC(+) plasmid. Sequence analysis and transcript assays disclosed that the flhD operon of E. carotovora subsp. carotovora, like those of other enterobacteria, consists of flhD and flhC. Complementation analysis revealed that the regulatory effect requires functions of both flhD and flhC products. The data presented here show that FlhDC positively regulates gacA, rsmC, and fliA and negatively regulates hexA (lrhA). Evidence shows that FlhDC controls extracellular protein production through cumulative effects on hexA and gacA. Reduced levels of GacA and elevated levels of HexA in the FlhDC(-) mutant are responsible for the inhibition of rsmB RNA

The content of DNA and RNA in microparticles released by Jurkat and HL-60 cells undergoing in vitro apoptosis

International Nuclear Information System (INIS)

Reich, Charles F.; Pisetsky, David S.

2009-01-01

Microparticles are small membrane-bound vesicles that are released from apoptotic cells during blebbing. These particles contain DNA and RNA and display important functional activities, including immune system activation. Furthermore, nucleic acids inside the particle can be analyzed as biomarkers in a variety of disease states. To elucidate the nature of microparticle nucleic acids, DNA and RNA released in microparticles from the Jurkat T and HL-60 promyelocytic cell lines undergoing apoptosis in vitro were studied. Microparticles were isolated from culture media by differential centrifugation and characterized by flow cytometry and molecular approaches. In these particles, DNA showed laddering by gel electrophoresis and was present in a form that allowed direct binding by a monoclonal anti-DNA antibody, suggesting antigen accessibility even without fixation. Analysis of RNA by gel electrophoresis showed intact 18s and 28s ribosomal RNA bands, although lower molecular bands consistent with 28s ribosomal RNA degradation products were also present. Particles also contained messenger RNA as shown by RT-PCR amplification of sequences for β-actin and GAPDH. In addition, gel electrophoresis showed the presence of low molecular weight RNA in the size range of microRNA. Together, these results indicate that microparticles from apoptotic Jurkat and HL-60 cells contain diverse nucleic acid species, indicating translocation of both nuclear and cytoplasmic DNA and RNA as particle release occurs during death

Bleomycin--electrical pulse delivery: electroporation therapy-bleomycin--Genetronics; MedPulser-bleomycin--Genetronics.

Science.gov (United States)

2004-01-01

Genetronics Biomedical is using its electroporation therapy technology to deliver bleomycin to tumour cells for the treatment of cancer. Genetronics have developed the MedPulser Electroporation Therapy System, which consists of an electrical pulse generator and disposable electrode applicators. The MedPulser system enables the delivery of large molecules into cells by briefly applying an electric field to the cell. This causes a transient permeability in the cell's outer membrane characterised by the appearance of pores across the membrane. After the field is discontinued, the pores close, trapping the therapeutic molecules inside the target cells. Genetronics is using the MedPulser System in conjunction with bleomycin, an antineoplastic antibiotic that binds to DNA causing strand scissions. Genetronics is seeking a licensing partner for the use of electroporation for the delivery of drugs in chemotherapy. In 1998, Genetronics entered a licensing and development agreement with Ethicon for electroporation and electrofusion. Under the terms of this agreement, Ethicon was to develop and clinically test the Genetronics electroporation delivery system and conduct all regulatory activities throughout the world except Canada. Ethicon would also market the products once regulatory approval has been obtained and Genetronics was to receive a percentage of the net sales and as license fees. However, in July 2000, Ethicon exercised its rights to terminate the agreement without cause. All rights were returned to Genetronics in January 2001. In 1997, Genetronics entered an agreement with Abbott Laboratories for the manufacture of bleomycin for use in the US in its MedPulsar system after regulatory approval had been granted for its use in the treatment of solid tumours. In a separate supply agreement, Faulding Inc. has agreed to manufacture bleomycin for Genetronic for use in Canada after regulatory approval had been granted. The MedPulsar Electroporation Therapy System with

Semi-allogeneic dendritic cells can induce antigen-specific T-cell activation, which is not enhanced by concurrent alloreactivity.

Science.gov (United States)

Wells, James W; Cowled, Chris J; Darling, David; Guinn, Barbara-Ann; Farzaneh, Farzin; Noble, Alistair; Galea-Lauri, Joanna

2007-12-01

Alloreactive T-cell responses are known to result in the production of large amounts of proinflammatory cytokines capable of activating and maturing dendritic cells (DC). However, it is unclear whether these allogeneic responses could also act as an adjuvant for concurrent antigen-specific responses. To examine effects of simultaneous alloreactive and antigen-specific T-cell responses induced by semi-allogeneic DC. Semi-allogeneic DC were generated from the F(1) progeny of inbred strains of mice (C57BL/6 and C3H, or C57BL/6 and DBA). We directly primed antigen-specific CD8(+) and CD4(+) T-cells from OT-I and OT-II mice, respectively, in the absence of allogeneic responses, in vitro, and in the presence or absence of alloreactivity in vivo. In vitro, semi-allogeneic DC cross-presented ovalbumin (OVA) to naïve CD8(+) OT-I transgenic T-cells, primed naïve CD4(+) OT-II transgenic T-cells and could stimulate strong alloreactive T-cell proliferation in a primary mixed lymphocyte reaction (MLR). In vivo, semi-allogeneic DC migrated efficiently to regional lymph nodes but did not survive there as long as autologous DC. In addition, they were not able to induce cytotoxic T-lymphocyte (CTL) activity to a target peptide, and only weakly stimulated adoptively transferred OT-II cells. The CD4(+) response was unchanged in allo-tolerized mice, indicating that alloreactive T-cell responses could not provide help for concurrently activated antigen-specific responses. In an EL4 tumour-treatment model, vaccination with semi-allogeneic DC/EL4 fusion hybrids, but not allogeneic DC/EL4 hybrids, significantly increased mouse survival. Expression of self-Major histocompatibility complex (MHC) by semi-allogeneic DC can cause the induction of antigen-specific immunity, however, concurrently activated allogeneic bystander responses do not provide helper or adjuvant effects.

Genomic Characterization of Variable Surface Antigens Reveals a Telomere Position Effect as a Prerequisite for RNA Interference-Mediated Silencing in Paramecium tetraurelia

Science.gov (United States)

Baranasic, Damir; Oppermann, Timo; Cheaib, Miriam; Cullum, John; Schmidt, Helmut

2014-01-01

ABSTRACT Antigenic or phenotypic variation is a widespread phenomenon of expression of variable surface protein coats on eukaryotic microbes. To clarify the mechanism behind mutually exclusive gene expression, we characterized the genetic properties of the surface antigen multigene family in the ciliate Paramecium tetraurelia and the epigenetic factors controlling expression and silencing. Genome analysis indicated that the multigene family consists of intrachromosomal and subtelomeric genes; both classes apparently derive from different gene duplication events: whole-genome and intrachromosomal duplication. Expression analysis provides evidence for telomere position effects, because only subtelomeric genes follow mutually exclusive transcription. Microarray analysis of cultures deficient in Rdr3, an RNA-dependent RNA polymerase, in comparison to serotype-pure wild-type cultures, shows cotranscription of a subset of subtelomeric genes, indicating that the telomere position effect is due to a selective occurrence of Rdr3-mediated silencing in subtelomeric regions. We present a model of surface antigen evolution by intrachromosomal gene duplication involving the maintenance of positive selection of structurally relevant regions. Further analysis of chromosome heterogeneity shows that alternative telomere addition regions clearly affect transcription of closely related genes. Consequently, chromosome fragmentation appears to be of crucial importance for surface antigen expression and evolution. Our data suggest that RNAi-mediated control of this genetic network by trans-acting RNAs allows rapid epigenetic adaptation by phenotypic variation in combination with long-term genetic adaptation by Darwinian evolution of antigen genes. PMID:25389173

Electroporation-based treatment planning for deep-seated tumors based on automatic liver segmentation of MRI images.

Science.gov (United States)

Pavliha, Denis; Mušič, Maja M; Serša, Gregor; Miklavčič, Damijan

2013-01-01

Electroporation is the phenomenon that occurs when a cell is exposed to a high electric field, which causes transient cell membrane permeabilization. A paramount electroporation-based application is electrochemotherapy, which is performed by delivering high-voltage electric pulses that enable the chemotherapeutic drug to more effectively destroy the tumor cells. Electrochemotherapy can be used for treating deep-seated metastases (e.g. in the liver, bone, brain, soft tissue) using variable-geometry long-needle electrodes. To treat deep-seated tumors, patient-specific treatment planning of the electroporation-based treatment is required. Treatment planning is based on generating a 3D model of the organ and target tissue subject to electroporation (i.e. tumor nodules). The generation of the 3D model is done by segmentation algorithms. We implemented and evaluated three automatic liver segmentation algorithms: region growing, adaptive threshold, and active contours (snakes). The algorithms were optimized using a seven-case dataset manually segmented by the radiologist as a training set, and finally validated using an additional four-case dataset that was previously not included in the optimization dataset. The presented results demonstrate that patient's medical images that were not included in the training set can be successfully segmented using our three algorithms. Besides electroporation-based treatments, these algorithms can be used in applications where automatic liver segmentation is required.

The optimization of voltage parameter for tissue electroporation in ...

African Journals Online (AJOL)

USER

2010-07-19

Jul 19, 2010 ... bodies, peptides or pharmaceuticals into the cell. Electro- ... acetic acid; MS, Murashige and Skoog; 2,4-D, 2,4-dichloro- ... sterile water and transferred to each ice-cold 0.4 cm electroporation ... (X-gluc) and 20% methanol.

Alternative RNA splicing and cancer

Science.gov (United States)

Liu, Sali; Cheng, Chonghui

2015-01-01

Alternative splicing of pre-messenger RNA (mRNA) is a fundamental mechanism by which a gene can give rise to multiple distinct mRNA transcripts, yielding protein isoforms with different, even opposing, functions. With the recognition that alternative splicing occurs in nearly all human genes, its relationship with cancer-associated pathways has emerged as a rapidly growing field. In this review, we summarize recent findings that have implicated the critical role of alternative splicing in cancer and discuss current understandings of the mechanisms underlying dysregulated alternative splicing in cancer cells. PMID:23765697

Wood's lamp examination

Science.gov (United States)

... this page: //medlineplus.gov/ency/article/003386.htm Wood lamp examination To use the sharing features on this page, please enable JavaScript. A Wood lamp examination is a test that uses ultraviolet ( ...

Detection of phytoplasma by loop-mediated isothermal amplification of DNA (LAMP).

Science.gov (United States)

Obura, E; Masiga, D; Wachira, F; Gurja, B; Khan, Z R

2011-02-01

Napier stunt phytoplasma (16SrXI and 16SrIII) in eastern Africa is a serious threat to the expansion of Napier grass (Pennisetum purpureum) farming in the region, where it is widely cultivated as fodder in zero grazing livestock systems. The grass has high potential for bio-fuel production, and has been adopted by farmers as a countermeasure to cereal stem borer Lepidoptera, since it attracts and traps the insect. Diagnosis of stunt phytoplasma have been largely by nested polymerase chain reaction (nPCR) targeting the 16S rRNA gene. However, the method is laborious, costly and technically demanding. This investigation has developed a simpler but effective phytoplasma diagnostic tool, called; loop-mediated isothermal amplification of DNA (LAMP). The assay was tested on 8 symptomatic and 8 asymptomatic plants, while its detection limit was compared to nested PCR using samples serially diluted from 3 ng/μl to 0.38 pg/μl. Molecular typing of LAMP products was determined by BsrI restriction digestion and Southern blot analysis. The assay sensitivity, positive and negative predictive values were estimated, while the specificity was tested on 11 phytoplasma groups. LAMP was specific to 5 phytoplasma groups: 16SrVI, X, XI and XVI. BsrI restriction digestion produced two predicted fragments, and there was specific binding of probe DNA to the LAMP amplicons in Southern blot analysis. The assay sensitivity was 100%, while the positive and negative predictive values were 63 and 100% respectively. LAMP was 20-fold more sensitive than nested PCR. This study validates LAMP for routine diagnosis of Napier stunt and other closely related phytoplasmas. Copyright © 2010 Elsevier B.V. All rights reserved.

Spontaneous reverse movement of mRNA-bound tRNA through the ribosome.

Science.gov (United States)

Konevega, Andrey L; Fischer, Niels; Semenkov, Yuri P; Stark, Holger; Wintermeyer, Wolfgang; Rodnina, Marina V

2007-04-01

During the translocation step of protein synthesis, a complex of two transfer RNAs bound to messenger RNA (tRNA-mRNA) moves through the ribosome. The reaction is promoted by an elongation factor, called EF-G in bacteria, which, powered by GTP hydrolysis, induces an open, unlocked conformation of the ribosome that allows for spontaneous tRNA-mRNA movement. Here we show that, in the absence of EF-G, there is spontaneous backward movement, or retrotranslocation, of two tRNAs bound to mRNA. Retrotranslocation is driven by the gain in affinity when a cognate E-site tRNA moves into the P site, which compensates the affinity loss accompanying the movement of peptidyl-tRNA from the P to the A site. These results lend support to the diffusion model of tRNA movement during translocation. In the cell, tRNA movement is biased in the forward direction by EF-G, which acts as a Brownian ratchet and prevents backward movement.

Ultrasensitive direct competitive FLISA using highly luminescent quantum dot beads for tuning affinity of competing antigens to antibodies

Energy Technology Data Exchange (ETDEWEB)

Xiong, Sicheng; Zhou, Yaofeng; Huang, Xiaolin [State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047 (China); Yu, Ruijin [College of Science, Northwest A& F University, Yangling, Shaanxi 712100 (China); Lai, Weihua [State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047 (China); Xiong, Yonghua, E-mail: yhxiongchen@163.com [State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047 (China)

2017-06-15

Herein, for the first time we report a novel direct competitive fluorescence-linked immunosorbent assay (dcFLISA) for the ultrasensitive detection of ochratoxin A (OTA) by introducing a large size polymer beads loaded with quantum dots (QBs) as carrier of competing antigen for decreasing binding affinity to antibody and enhancing the fluorescent signal intensity. When using 255 nm QBs as carrier of competing antigen, the equilibrium dissociation constant of QB based competing antigen to antibodies can be tuned to 100 times higher than that of the horseradish peroxidase (HRP) based competing antigen by controlling labeled amounts of antigen on the surface of QBs. Various parameters that influenced the sensitivity of dcFLISA were investigated and optimized. Under optimum detection parameters, the dynamic linear range of developed dcFLISA for detecting OTA was established at 0.05 pg/mL to 1.56 pg/mL with a half maximal inhibitory concentration at 0.14 ± 0.04 pg/mL (n = 5), which is three orders of magnitude lower than that of conventional HRP-based dcELISA (0.24 ng/mL). The developed FLISA is also highly accurate, reliable, and shows no cross reaction to other mycotoxins. In summary, the proposed method offers a straightforward approach to improve the sensitivity of direct competitive immunoassay for trace small chemical molecule detection in food quality control, environmental monitoring, and clinical diagnosis. - Highlights: • Highly luminescent QBs were used as a carrier of competing antigen for ultrasensitive detection of OTA. • It is the first time to use a large size QBs as a carrier for tuning affinity of competing antigen to antibodies. • IC{sub 50} value of QB-based dcFLISA is three orders of magnitude lower than that of HRP-based dcELISA.

Ultrasensitive direct competitive FLISA using highly luminescent quantum dot beads for tuning affinity of competing antigens to antibodies

International Nuclear Information System (INIS)

Xiong, Sicheng; Zhou, Yaofeng; Huang, Xiaolin; Yu, Ruijin; Lai, Weihua; Xiong, Yonghua

2017-01-01

Herein, for the first time we report a novel direct competitive fluorescence-linked immunosorbent assay (dcFLISA) for the ultrasensitive detection of ochratoxin A (OTA) by introducing a large size polymer beads loaded with quantum dots (QBs) as carrier of competing antigen for decreasing binding affinity to antibody and enhancing the fluorescent signal intensity. When using 255 nm QBs as carrier of competing antigen, the equilibrium dissociation constant of QB based competing antigen to antibodies can be tuned to 100 times higher than that of the horseradish peroxidase (HRP) based competing antigen by controlling labeled amounts of antigen on the surface of QBs. Various parameters that influenced the sensitivity of dcFLISA were investigated and optimized. Under optimum detection parameters, the dynamic linear range of developed dcFLISA for detecting OTA was established at 0.05 pg/mL to 1.56 pg/mL with a half maximal inhibitory concentration at 0.14 ± 0.04 pg/mL (n = 5), which is three orders of magnitude lower than that of conventional HRP-based dcELISA (0.24 ng/mL). The developed FLISA is also highly accurate, reliable, and shows no cross reaction to other mycotoxins. In summary, the proposed method offers a straightforward approach to improve the sensitivity of direct competitive immunoassay for trace small chemical molecule detection in food quality control, environmental monitoring, and clinical diagnosis. - Highlights: • Highly luminescent QBs were used as a carrier of competing antigen for ultrasensitive detection of OTA. • It is the first time to use a large size QBs as a carrier for tuning affinity of competing antigen to antibodies. • IC_5_0 value of QB-based dcFLISA is three orders of magnitude lower than that of HRP-based dcELISA.

Sweet Spot Supersymmetry and Composite Messengers

International Nuclear Information System (INIS)

Ibe, Masahiro; Kitano, Ryuichiro

2007-01-01

Sweet spot supersymmetry is a phenomenologically and cosmologically perfect framework to realize a supersymmetric world at short distance. We discuss a class of dynamical models of supersymmetry breaking and its mediation whose low-energy effective description falls into this framework. Hadron fields in the dynamical models play a role of the messengers of the supersymmetry breaking. As is always true in the models of the sweet spot supersymmetry, the messenger scale is predicted to be 10 5 GeV ∼ mess ∼ 10 GeV. Various values of the effective number of messenger fields N mess are possible depending on the choice of the gauge group

Theoretical and experimental study of electroporation of red blood cells using MEMS technology

KAUST Repository

Deng, Peigang; Yin, Guangyao; Zhang, Tong Yi; Chang, Donald C.; Lee, Yi Kuen

2010-01-01

A theoretical and experimental study of electroporation (EP) of red blood cells (RBCs) was presented in this paper. With additional strain energy, an energy-based model of an electropore induced on a RBC's membrane at different electric fields was proposed to predict the critical EP electric field strength. In addition, EP experiments with red blood cells at single-cell level was carried out on a micro EP chip. The measured critical EP electric field strengths are in agreement with the numerical predictions. ©2010 IEEE.

Theoretical and experimental study of electroporation of red blood cells using MEMS technology

KAUST Repository

Deng, Peigang

2010-01-01

A theoretical and experimental study of electroporation (EP) of red blood cells (RBCs) was presented in this paper. With additional strain energy, an energy-based model of an electropore induced on a RBC\\'s membrane at different electric fields was proposed to predict the critical EP electric field strength. In addition, EP experiments with red blood cells at single-cell level was carried out on a micro EP chip. The measured critical EP electric field strengths are in agreement with the numerical predictions. ©2010 IEEE.

RF Noise Generation in High-Pressure Short-Arc DC Xenon Lamps

Science.gov (United States)

Minayeva, Olga; Doughty, Douglas

2007-10-01

Continuous direct current xenon arcs will generate RF noise under certain circumstance, which can lead to excessive electro- magnetic interference in systems that use these arcs as light sources. Phenomenological observations are presented for xenon arcs having arc gaps ˜1 mm, cold fill pressures of ˜2.5 MPa, and currents up to 30 amps. Using a loop antenna in the vicinity of an operating lamp, it is observed that as the current to the arc is lowered there is a reproducible threshold at which the RF noise generation begins. This threshold is accompanied by a small abrupt drop in voltage (˜0.2 volts). The RF emission appears in pulses ˜150 nsec wide separated by ˜300 nec - the pulse interval decreases with decreasing current. The properties of the RF emission as a function of arc parameters (such as pressure, arc gap, electrode design) will be discussed and a semi-quantitative model presented.

Keratinocyte Growth Factor Gene Electroporation into Skeletal Muscle as a Novel Gene Therapeutic Approach for Elastase-Induced Pulmonary Emphysema in Mice

International Nuclear Information System (INIS)

Tobinaga, Shuichi; Matsumoto, Keitaro; Nagayasu, Takeshi; Furukawa, Katsuro; Abo, Takafumi; Yamasaki, Naoya; Tsuchiya, Tomoshi; Miyazaki, Takuro; Koji, Takehiko

2015-01-01

Pulmonary emphysema is a progressive disease with airspace destruction and an effective therapy is needed. Keratinocyte growth factor (KGF) promotes pulmonary epithelial proliferation and has the potential to induce lung regeneration. The aim of this study was to determine the possibility of using KGF gene therapy for treatment of a mouse emphysema model induced by porcine pancreatic elastase (PPE). Eight-week-old BALB/c male mice treated with intra-tracheal PPE administration were transfected with 80 μg of a recombinant human KGF (rhKGF)-expressing FLAG-CMV14 plasmid (pKGF-FLAG gene), or with the pFLAG gene expressing plasmid as a control, into the quadriceps muscle by electroporation. In the lung, the expression of proliferating cell nuclear antigen (PCNA) was augmented, and surfactant protein A (SP-A) and KGF receptor (KGFR) were co-expressed in PCNA-positive cells. Moreover, endogenous KGF and KGFR gene expression increased significantly by pKGF-FLAG gene transfection. Arterial blood gas analysis revealed that the PaO 2 level was not significantly reduced on day 14 after PPE instillation with pKGF-FLAG gene transfection compared to that of normal mice. These results indicated that KGF gene therapy with electroporation stimulated lung epithelial proliferation and protected depression of pulmonary function in a mouse emphysema model, suggesting a possible method of treating pulmonary emphysema

Mercury's Reference Frames After the MESSENGER Mission

Science.gov (United States)

Stark, A.; Oberst, J.; Preusker, F.; Burmeister, S.; Steinbrügge, G.; Hussmann, H.

2018-05-01

We provide an overview of Mercury's reference frames based on MESSENGER observations. We discuss the dynamical, the principal-axes, the ellipsoid, as well as the cartographic frame, which was adopted for MESSENGER data products.

Illuminating Messengers: An Update and Outlook on RNA Visualization in Bacteria

Directory of Open Access Journals (Sweden)

Lieke A. van Gijtenbeek

2017-06-01

Full Text Available To be able to visualize the abundance and spatiotemporal features of RNAs in bacterial cells would permit obtaining a pivotal understanding of many mechanisms underlying bacterial cell biology. The first methods that allowed observing single mRNA molecules in individual cells were introduced by Bertrand et al. (1998 and Femino et al. (1998. Since then, a plethora of techniques to image RNA molecules with the aid of fluorescence microscopy has emerged. Many of these approaches are useful for the large eukaryotic cells but their adaptation to study RNA, specifically mRNA molecules, in bacterial cells progressed relatively slow. Here, an overview will be given of fluorescent techniques that can be used to reveal specific RNA molecules inside fixed and living single bacterial cells. It includes a critical evaluation of their caveats as well as potential solutions.

Retail Lamps Study 3.1: Dimming, Flicker, and Power Quality Characteristics of LED A Lamps

Energy Technology Data Exchange (ETDEWEB)

Royer, Michael P. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Poplawski, Michael E. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Brown, Charles C. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

2014-12-01

To date, all three reports in the retail lamps series have focused on basic performance parameters, such as lumen output, efficacy, and color quality. This report goes a step further, examining the photoelectric characteristics (i.e., dimming and flicker) of a subset of lamps from CALiPER Retails Lamps Study 3. Specifically, this report focuses on the dimming, power quality, and flicker characteristics of 14 LED A lamps, as controlled by four different retail-available dimmers. The results demonstrate notable variation across the various lamps, but little variation between the four dimmers. Overall, the LED lamps: ~tended to have higher relative light output compared to the incandescent and halogen benchmark at the same dimmer output signal (RMS voltage). The lamps’ dimming curves (i.e., the relationship between control signal and relative light output) ranged from linear to very similar to the square-law curve typical of an incandescent lamp. ~generally exhibited symmetrical behavior—the same dimming curve—when measured proceeding from maximum to minimum or minimum to maximum control signal. ~mostly dimmed below 10% of full light output, with some exceptions for specific lamp and dimmer combinations ~exhibited a range of flicker characteristics, with many comparing favorably to the level typical of a magnetically-ballasted fluorescent lamp through at least a majority of the dimming range. ~ always exceeded the relative (normalized) efficacy over the dimming range of the benchmark lamps, which rapidly decline in efficacy when they are dimmed. This report generally does not attempt to rank the performance of one product compared to another, but instead focuses on the collective performance of the group versus conventional incandescent or halogen lamps, the performance of which is likely to be the baseline for a majority of consumers. Undoubtedly, some LED lamps perform better—or more similar to conventional lamps—than others. Some perform desirably for one

Anistropically varying conductivity in irreversible electroporation simulations.

Science.gov (United States)

Labarbera, Nicholas; Drapaca, Corina

2017-11-01

One recent area of cancer research is irreversible electroporation (IRE). Irreversible electroporation is a minimally invasive procedure where needle electrodes are inserted into the body to ablate tumor cells with electricity. The aim of this paper is to propose a mathematical model that incorporates a tissue's conductivity increasing more in the direction of the electrical field as this has been shown to occur in experiments. It was necessary to mathematically derive a valid form of the conductivity tensor such that it is dependent on the electrical field direction and can be easily implemented into numerical software. The derivation of a conductivity tensor that can take arbitrary functions for the conductivity in the directions tangent and normal to the electrical field is the main contribution of this paper. Numerical simulations were performed for isotropic-varying and anisotropic-varying conductivities to evaluate the importance of including the electrical field's direction in the formulation for conductivity. By starting from previously published experimental results, this paper derived a general formulation for an anistropic-varying tensor for implementation into irreversible electroporation modeling software. The anistropic-varying tensor formulation allows the conductivity to take into consideration both electrical field direction and magnitude, as opposed to previous published works that only took into account electrical field magnitude. The anisotropic formulation predicts roughly a five percent decrease in ablation size for the monopolar simulation and approximately a ten percent decrease in ablation size for the bipolar simulations. This is a positive result as previously reported results found the isotropic formulation to overpredict ablation size for both monopolar and bipolar simulations. Furthermore, it was also reported that the isotropic formulation overpredicts the ablation size more for the bipolar case than the monopolar case. Thus, our

Impact of aging on antigen presentation cell function of dendritic cells.

Science.gov (United States)

Wong, Christine; Goldstein, Daniel R

2013-08-01

Older people exhibit increased mortality to infections and cancer as compared to younger people, indicating that aging impairs immunity. Dendritic cells (DCs) are key for bridging the innate and adaptive arms of the immune system by priming antigen specific T cells. Discerning how aging impacts DC function to initiate adaptive immune responses is of great biomedical importance as this could lead to the development of novel therapeutics to enhance immunity with aging. This review details reports indicating that aging impairs the antigen presenting function of DCs but highlights other studies indicating preserved DC function with aging. How aging impacts antigen presentation by DCs is complex and without a clear unifying biological underpinning. Copyright © 2013 Elsevier Ltd. All rights reserved.

Discharge lamp technologies

Energy Technology Data Exchange (ETDEWEB)

Dakin, J. [GE Lighting, Cleveland, OH (United States)

1994-12-31

This talk is an overview of discharge lamp technology commonly employed in general lighting, with emphasis on issues pertinent to lighting for plant growth. Since the audience is primarily from the plant growth community, and this begins the light source part of the program, we will start with a brief description of the discharge lamps. Challenges of economics and of thermal management make lamp efficiency a prime concern in controlled environment agriculture, so we will emphasize science considerations relating to discharge lamp efficiency. We will then look at the spectra and ratings of some representative lighting products, and conclude with a discussion of technological advance. A general overview of discharge lighting technology can be found in the book of Waymouth (1971). A recent review of low pressure lighting discharge science is found in Dakin (1991). The pioneering paper of Reiling (1964) provides a good introduction to metal halide discharges. Particularly relevant to lighting for plant growth, a recent and thorough treatment of high pressure Na lamps is found in the book by deGroot and vanVliet (1986). Broad practical aspects of lighting application are thoroughly covered in the IES Lighting Handbook edited by Kaufman (1984).

Identification and molecular profiling of DC-SIGN-like from big belly seahorse (Hippocampus abdominalis) inferring its potential relevancy in host immunity.

Science.gov (United States)

Jo, Eunyoung; Elvitigala, Don Anushka Sandaruwan; Wan, Qiang; Oh, Minyoung; Oh, Chulhong; Lee, Jehee

2017-12-01

Dendritic-cell-specific ICAM-3-grabbing non-integrin (DC-SIGN) is a C-type lectin that functions as a pattern recognition receptor by recognizing pathogen-associated molecular patterns (PAMPs). It is also involved in various events of the dendritic cell (DC) life cycle, such as DC migration, antigen capture and presentation, and T cell priming. In this study, a DC-SIGN-like gene from the big belly seahorse Hippocampus abdominalis (designated as ShDCS-like) was identified and molecularly characterized. The putative, complete ORF was found to be 1368 bp in length, encoding a protein of 462 amino acids with a molecular mass of 52.6 kDa and a theoretical isoelectric point of 8.26. The deduced amino acid sequence contains a single carbohydrate recognition domain (CRD), in which six conserved cysteine residues and two Ca 2+ -binding site motifs (QPN, WND) were identified. Based on pairwise sequence analysis, ShDCS-like exhibits the highest amino acid identity (94.6%) and similarity (97.4%) with DC-SIGN-like counterpart from tiger tail seahorse Hippocampus comes. Quantitative real-time PCR revealed that ShDCS-like mRNA is transcribed universally in all tissues examined, but with abundance in kidney and gill tissues. The basal mRNA expression of ShDCS-like was modulated in blood cell, kidney, gill and liver tissues in response to the stimulation of healthy fish with lipopolysaccharides (LPS), Edwardsiella tarda, or Streptococcus iniae. Moreover, recombinant ShDCS-like-CRD domain exhibited detectable agglutination activity against different bacteria. Collectively, these results suggest that ShDCS-like may potentially involve in immune function in big belly seahorses. Copyright © 2017 Elsevier Ltd. All rights reserved.

Electroporation Enhanced Effect of Dystrophin Splice Switching PNA Oligomers in Normal and Dystrophic Muscle

DEFF Research Database (Denmark)

Hjortkjær, Camilla Brolin; Shiraishi, Takehiko; Hojman, Pernille

2015-01-01

for improvement of in vivo cellular availability, we have investigated the effect of electrotransfer upon intramuscular (i.m.) PNA administration in vivo. Antisense PNA targeting exon 23 of the murine dystrophin gene was administered by i.m. injection to the tibialis anterior (TA) muscle of normal NMRI......Peptide nucleic acid (PNA) is a synthetic DNA mimic that has shown potential for discovery of novel splice switching antisense drugs. However, in vivo cellular delivery has been a limiting factor for development, and only few successful studies have been reported. As a possible modality...... switching was detected at the RNA level up to 4 weeks after a single-dose treatment. In dystrophic muscles of the MDX mouse, electroporation increased the number of dystrophin-positive fibers about 2.5-fold at 2 weeks after a single PNA administration compared to injection only. In conclusion, we find...

Genetic recombination in plant-infecting messenger-sense RNA viruses: overview and research perspectives.

Science.gov (United States)

Bujarski, Jozef J

2013-01-01

RNA recombination is one of the driving forces of genetic variability in (+)-strand RNA viruses. Various types of RNA-RNA crossovers were described including crosses between the same or different viral RNAs or between viral and cellular RNAs. Likewise, a variety of molecular mechanisms are known to support RNA recombination, such as replicative events (based on internal or end-to-end replicase switchings) along with non-replicative joining among RNA fragments of viral and/or cellular origin. Such mechanisms as RNA decay or RNA interference are responsible for RNA fragmentation and trans-esterification reactions which are likely accountable for ligation of RNA fragments. Numerous host factors were found to affect the profiles of viral RNA recombinants and significant differences in recombination frequency were observed among various RNA viruses. Comparative analyses of viral sequences allowed for the development of evolutionary models in order to explain adaptive phenotypic changes and co-evolving sites. Many questions remain to be answered by forthcoming RNA recombination research. (1) How various factors modulate the ability of viral replicase to switch templates, (2) What is the intracellular location of RNA-RNA template switchings, (3) Mechanisms and factors responsible for non-replicative RNA recombination, (4) Mechanisms of integration of RNA viral sequences with cellular genomic DNA, and (5) What is the role of RNA splicing and ribozyme activity. From an evolutionary stand point, it is not known how RNA viruses parasitize new host species via recombination, nor is it obvious what the contribution of RNA recombination is among other RNA modification pathways. We do not understand why the frequency of RNA recombination varies so much among RNA viruses and the status of RNA recombination as a form of sex is not well documented.

Genetic recombination in plant-infecting messenger-sense RNA viruses: overview and research perspectives

Directory of Open Access Journals (Sweden)

Jozef Julian Bujarski

2013-03-01

Full Text Available RNA recombination is one of the driving forces of genetic variability in (+-strand RNA viruses. Various types of RNA-RNA crossovers were described including crosses between the same or different viral RNAs or between viral and cellular RNAs. Likewise, a variety of molecular mechanisms are known to support RNA recombination, such as replicative events (based on internal or end-to-end replicase switchings along with nonreplicative joining among RNA fragments of viral and/or cellular origin. Such mechanisms as RNA decay or RNA interference are responsible for RNA fragmentation and trans-esterification reactions which are likely accountable for ligation of RNA fragments. Numerous host factors were found to affect the profiles of viral RNA recombinants and significant differences in recombination frequency were observed among various RNA viruses. Comparative analyses of viral sequences allowed for the development of evolutionary models in order to explain adaptive phenotypic changes and co-evolving sites. Many questions remain to be answered by forthcoming RNA recombination research. (i How various factors modulate the ability of viral replicase to switch templates, (ii What is the intracellular location of RNA-RNA template switchings, (iii Mechanisms and factors responsible for non-replicative RNA recombination, (iv Mechanisms of integration of RNA viral sequences with cellular genomic DNA, and (v What is the role of RNA splicing and ribozyme activity. From an evolutionary stand point, it is not known how RNA viruses parasitize new host species via recombination, nor is it obvious what the contribution of RNA recombination is among other RNA modification pathways. We do not understand why the frequency of RNA recombination varies so much among RNA viruses and the status of RNA recombination as a form of sex is not well documented.

Targeting of macrophage galactose-type C-type lectin (MGL) induces DC signaling and activation

DEFF Research Database (Denmark)

Napoletano, Chiara; Zizzari, Ilaria G; Rughetti, Aurelia

2012-01-01

NAc or Tn)-carrying tumor-associated antigens to improve DC performance. MGL expressed by ex vivo-generated iDCs from healthy donors was engaged by a 60-mer MUC1(9Tn) -glycopeptide as a Tn-carrying tumor-associated antigen, and an anti-MGL antibody, as a specific MGL binder. We demonstrated that MGL......Dendritic cells (DCs) sense the microenvironment through several types of receptors recognizing pathogen-associated molecular patterns. In particular, C-type lectins, expressed by distinct subsets of DCs, recognize and internalize specific carbohydrate antigen in a Ca(2+) -dependent manner....... Targeting of these receptors is becoming an efficient strategy of delivering antigens in DC-based anticancer immunotherapy. Here we investigated the role of the macrophage galactose type C-lectin receptor (MGL), expressed by immature DCs (iDCs), as a molecular target for a-N-acetylgalactosamine (Gal...

Lamp for sunshine simulation

DEFF Research Database (Denmark)

2016-01-01

A lamp system is provided, comprising a lamp with a lamp housing accommodating a plurality of light sources for emission of visible light, including blue light, a time keeping unit, a light sensor for sensing intensity of light incident upon it, and a light controller configured for controlling...... the plurality of light sources in response to the intensity of light sensed by the light sensor and the time provided by the time keeping unit, characterized in that the lamp emits blue light for a selected time period, wherein the blue light has a luminous flux ranging from 50 lux to 200 lux and, preferably......, an irradiance that is larger than 5 mW/nm/m2 in a selected wavelength range, such as in the wavelength range from 440 nm to 500 nm, as measured at a distance of 3 metres from the lamp....

T-lymphocyte cytokine mRNA expression in cystic echinococcosis.

Science.gov (United States)

Fauser, S; Kern, P

1997-04-01

In the present study we investigated cytokine mRNA expression by peripheral blood mononuclear cells (PBMC) from patients with cystic echinococcosis (CE) after stimulation with different antigens. By using reverse transcriptase polymerase chain reaction (RT-PCR) we could demonstrate that restimulation with crude Echinococcus granulosus antigen (Eg-Ag) induced or enhanced Th2 cytokine mRNA expression, especially IL-5 (by using antigen from sheep cyst fluid) in 23 out of 26 investigated CE patients and IL-10 (by using antigen from camel cyst fluid) in 10 out of 10 investigated CE patients. In contrast, IL-5 mRNA expression was absent in PBMC of healthy controls after Eg-Ag stimulation. To determine the specificity of this reaction we stimulated PBMC from 11 CE patients with crude Echinococcus multilocularis antigen (Em-Ag) and PBMC from 8 CE patients with Toxocara canis antigen (Tc-Ag). We found that the PBMC of patients showed a similar mRNA cytokine pattern on stimulation with Em-Ag when compared with Eg-Ag stimulation. The cytokine mRNA pattern on stimulation with Tc-Ag, however, resembled the cytokine mRNA pattern of unstimulated PBMC. Furthermore, the stimulation of PBMC with crude Mycobacterium tuberculosis antigen (H37Ra) and purified protein derivative (PPD) of M. tuberculosis revealed distinct IL-5 mRNA expression in all investigated CE patients, whereas in healthy controls IL-5 mRNA expression was very weak or totally absent. Thus, our results indicate an induction of Th2 cytokine mRNA expression in CE patients, which is frequently observed in parasite infections. Interestingly, this response persists after stimulation with tuberculosis antigens, which normally induce Th1 response.

Semiautomated improvement of RNA alignments

DEFF Research Database (Denmark)

Andersen, Ebbe Sloth; Lind-Thomsen, Allan; Knudsen, Bjarne

2007-01-01

connects to external tools to provide a flexible semiautomatic editing environment. A new method, Pcluster, is introduced for dividing the sequences of an RNA alignment into subgroups with secondary structure differences. Pcluster was used to evaluate 574 seed alignments obtained from the Rfam database...... and we identified 71 alignments with significant prediction of inconsistent base pairs and 102 alignments with significant prediction of novel base pairs. Four RNA families were used to illustrate how SARSE can be used to manually or automatically correct the inconsistent base pairs detected by Pcluster......: the mir-399 RNA, vertebrate telomase RNA (vert-TR), bacterial transfer-messenger RNA (tmRNA), and the signal recognition particle (SRP) RNA. The general use of the method is illustrated by the ability to accommodate pseudoknots and handle even large and divergent RNA families. The open architecture...

Application of RNA interference in treating human diseases

Indian Academy of Sciences (India)

ference than either strand individually. After injection into ... antisense strand to messenger RNAs (mRNAs) that bear ... processing of longer dsRNA and stem loop precursors (Nov- ... RNAi has several applications in biomedical research,.

Harmonics Monitoring Survey on LED Lamps

Directory of Open Access Journals (Sweden)

Abdelrahman Ahmed Akila

2017-03-01

Full Text Available Light Emitting Diode (LED lamps are being increasingly used in many applications. These LED lamps operate using a driver, which is a switching device. Hence, LED lamps will be a source of harmonics in the power system. These harmonics if not well treated, may cause severe performance and operational problems. In this paper, harmonics (amplitude and phase angles generated by both LED lamps and conventional fluorescent lamps will be studied practically. Then they will be analyzed and evaluated. Compared to each other harmonics generated by both LED and conventional florescent lamps, self mitigation may occur based on the phase angle of these harmonics. All data will be measured using power analyzer and will be done on a sample of actual lamps.

Finite-element modelling and preliminary validation of microneedle-based electrodes for enhanced tissue electroporation.

Science.gov (United States)

Houlihan, Ruth; Grygoryev, Konstantin; Zhenfei Ning; Williams, John; Moore, Tom; O'Mahony, Conor

2017-07-01

This paper investigates the use of microneedle-based electrodes for enhanced testis electroporation, with specific application to the production of transgenic mice. During the design phase, finite-element software has been used to construct a tissue model and to compare the relative performance of electrodes employing a) conventional flat plates, b) microneedle arrays, and c) invasive needles. Results indicate that microneedle-based electrodes can achieve internal tissue field strengths which are an order of magnitude higher than those generated using conventional flat electrodes, and which are comparable to fields produced using invasive needles. Using a double-sided etching process, conductive microneedle arrays were then fabricated and used in prototype electrodes. In a series of mouse model experiments involving injection of a DNA vector expressing Green Fluorescent Protein (GFP), the performance of flat and microneedle electrodes was compared by measuring GFP expression after electroporation. The main finding, supported by experimental and simulated data, is that use of microneedle-based electrodes significantly enhanced electroporation of testis.

Mercury's Messenger

Science.gov (United States)

Chapman, Clark R.

2004-01-01

Forty years after Mariner 2, planetary exploration has still only just begun, and many more missions are on drawing boards, nearing the launch pad, or even en route across interplanetary space to their targets. One of the most challenging missions that will be conducted this decade is sending the MESSENGER spacecraft to orbit the planet Mercury.…

Evaluation of the reverse transcription loop-mediated isothermal amplification (RT-LAMP) as a screening method for the detection of influenza viruses in the fecal materials of water birds.

Science.gov (United States)

Yoshida, Hiromi; Sakoda, Yoshihiro; Endo, Mayumi; Motoshima, Masayuki; Yoshino, Fumi; Yamamoto, Naoki; Okamatsu, Masatoshi; Soejima, Takahiro; Senba, Syouhei; Kanda, Hidetoshi; Kida, Hiroshi

2011-06-01

Migratory water birds are a natural reservoir for influenza A viruses. Viruses replicate in the intestines of ducks and are shed with the fecal materials. Virus isolation from collected fecal materials, therefore, is an integral part of the surveillance of avian influenza in water birds. In the present study, reverse transcription loop-mediated isothermal amplification (RT-LAMP) was assessed for its usefulness in detecting the RNA of influenza A viruses in fecal materials. It was found that, RT-LAMP specifically and sensitively detects the matrix gene of influenza A viruses. Influenza A viruses were isolated from the fecal materials in which viral RNA were detected by RT-LAMP in 35 min. The present findings indicate that RT-LAMP is useful as a high throughput screening method for field samples prior to virus isolation, allowing the processing of hundreds of samples per day.

Marker lamps

International Nuclear Information System (INIS)

Watkins, D.V.

1980-01-01

A marker lamp is described which consists of a block of transparent plastics material encapsulated in which is a radioactive light source. These lights comprise a small sealed glass capsule, the hollow inside surface of which is coated with phosphor and which contains tritium or similar radioactive gas. The use of such lamps for identification marking of routes, for example roads, and for identification of underwater oil pipelines is envisaged. (U.K.)

A thermostable messenger RNA based vaccine against rabies.

Science.gov (United States)

Stitz, Lothar; Vogel, Annette; Schnee, Margit; Voss, Daniel; Rauch, Susanne; Mutzke, Thorsten; Ketterer, Thomas; Kramps, Thomas; Petsch, Benjamin

2017-12-01

Although effective rabies virus vaccines have been existing for decades, each year, rabies virus infections still cause around 50.000 fatalities worldwide. Most of these cases occur in developing countries, where these vaccines are not available. The reasons for this are the prohibitive high costs of cell culture or egg grown rabies virus vaccines and the lack of a functional cold chain in many regions in which rabies virus is endemic. Here, we describe the excellent temperature resistance of a non-replicating mRNA based rabies virus vaccine encoding the rabies virus glycoprotein (RABV-G). Prolonged storage of the vaccine from -80°C to up to +70°C for several months did not impact the protective capacity of the mRNA vaccine. Efficacy after storage was demonstrated by the induction of rabies specific virus neutralizing antibodies and protection in mice against lethal rabies infection. Moreover, storing the vaccine at oscillating temperatures between +4° and +56°C for 20 cycles in order to simulate interruptions of the cold chain during vaccine transport, did not affect the vaccine's immunogenicity and protective characteristics, indicating that maintenance of a cold chain is not essential for this vaccine.

Mobile MSN Messenger: Still a Complement?

Directory of Open Access Journals (Sweden)

Marcus Nyberg

2008-10-01

Full Text Available In order to understand how mobile instant messaging services can fit into the users’ current communication behavior, Ericsson Research performed a qualitative user study in Sweden in May 2007. The results showed that the respondents were positive towards (free of charge mobile MSN Messenger and perceived it as an ex¬tension of the computer-based version that could be used anywhere. However, although MSN Messenger on the com¬puter definitely was considered as a ‘must-have’ application, the mobile version was only perceived as a ‘nice-to-have’ application and a complement to text mes¬saging (SMS. Almost one year later, in April 2008, Ericsson Research performed a short qualita¬tive follow-up study with the same set of respondents to un¬derstand if and how the mobile MSN Messenger usage had changed. The results actually revealed that none of the re¬spondents used mobile MSN Messenger anymore as the application no longer was free of charge. On a general level, the study highlights important considera¬tions when intro¬ducing computer-based concepts and Internet services in a mo¬bile environment.

A Critical Analysis of the Role of SNARE Protein SEC22B in Antigen Cross-Presentation

Directory of Open Access Journals (Sweden)

S. Julia Wu

2017-06-01

Full Text Available Cross-presentation initiates immune responses against tumors and viral infections by presenting extracellular antigen on MHC I to activate CD8+ T cell-mediated cytotoxicity. In vitro studies in dendritic cells (DCs established SNARE protein SEC22B as a specific regulator of cross-presentation. However, the in vivo contribution of SEC22B to cross-presentation has not been tested. To address this, we generated DC-specific Sec22b knockout (CD11c-Cre Sec22bfl/fl mice. Contrary to the paradigm, SEC22B-deficient DCs efficiently cross-present both in vivo and in vitro. Although in vitro small hairpin RNA (shRNA-mediated Sec22b silencing in bone-marrow-derived dendritic cells (BMDCs reduced cross-presentation, treatment of SEC22B-deficient BMDCs with the same shRNA produced a similar defect, suggesting the Sec22b shRNA modulates cross-presentation through off-target effects. RNA sequencing of Sec22b shRNA-treated SEC22B-deficient BMDCs demonstrated several changes in the transcriptome. Our data demonstrate that contrary to the accepted model, SEC22B is not necessary for cross-presentation, cautioning against extrapolating phenotypes from knockdown studies alone.

Transparent ceramic lamp envelope materials

Energy Technology Data Exchange (ETDEWEB)

Wei, G C [OSRAM SYLVANIA, 71 Cherry Hill Drive, Beverly, MA 01915 (United States)

2005-09-07

Transparent ceramic materials with optical qualities comparable to single crystals of similar compositions have been developed in recent years, as a result of the improved understanding of powder-processing-fabrication- sintering-property inter-relationships. These high-temperature materials with a range of thermal and mechanical properties are candidate envelopes for focused-beam, short-arc lamps containing various fills operating at temperatures higher than quartz. This paper reviews the composition, structure and properties of transparent ceramic lamp envelope materials including sapphire, small-grained polycrystalline alumina, aluminium oxynitride, yttrium aluminate garnet, magnesium aluminate spinel and yttria-lanthana. A satisfactory thermal shock resistance is required for the ceramic tube to withstand the rapid heating and cooling cycles encountered in lamps. Thermophysical properties, along with the geometry, size and thickness of a transparent ceramic tube, are important parameters in the assessment of its resistance to fracture arising from thermal stresses in lamps during service. The corrosive nature of lamp-fill liquid and vapour at high temperatures requires that all lamp components be carefully chosen to meet the target life. The wide range of new transparent ceramics represents flexibility in pushing the limit of envelope materials for improved beamer lamps.

miRNA-target chimeras reveal miRNA 3'-end pairing as a major determinant of Argonaute target specificity

DEFF Research Database (Denmark)

Moore, Michael J; Scheel, Troels K H; Luna, Joseph M

2015-01-01

microRNAs (miRNAs) act as sequence-specific guides for Argonaute (AGO) proteins, which mediate posttranscriptional silencing of target messenger RNAs. Despite their importance in many biological processes, rules governing AGO-miRNA targeting are only partially understood. Here we report a modifie...

RNA-binding proteins ZFP36L1 and ZFP36L2 promote cell quiescence.

Science.gov (United States)

Galloway, Alison; Saveliev, Alexander; Łukasiak, Sebastian; Hodson, Daniel J; Bolland, Daniel; Balmanno, Kathryn; Ahlfors, Helena; Monzón-Casanova, Elisa; Mannurita, Sara Ciullini; Bell, Lewis S; Andrews, Simon; Díaz-Muñoz, Manuel D; Cook, Simon J; Corcoran, Anne; Turner, Martin

2016-04-22

Progression through the stages of lymphocyte development requires coordination of the cell cycle. Such coordination ensures genomic integrity while cells somatically rearrange their antigen receptor genes [in a process called variable-diversity-joining (VDJ) recombination] and, upon successful rearrangement, expands the pools of progenitor lymphocytes. Here we show that in developing B lymphocytes, the RNA-binding proteins (RBPs) ZFP36L1 and ZFP36L2 are critical for maintaining quiescence before precursor B cell receptor (pre-BCR) expression and for reestablishing quiescence after pre-BCR-induced expansion. These RBPs suppress an evolutionarily conserved posttranscriptional regulon consisting of messenger RNAs whose protein products cooperatively promote transition into the S phase of the cell cycle. This mechanism promotes VDJ recombination and effective selection of cells expressing immunoglobulin-μ at the pre-BCR checkpoint. Copyright © 2016, American Association for the Advancement of Science.

The gas phase emitter effect of lanthanum within ceramic metal halide lamps and its dependence on the La vapor pressure and operating frequency

Energy Technology Data Exchange (ETDEWEB)

Ruhrmann, C.; Hoebing, T.; Bergner, A.; Groeger, S.; Awakowicz, P.; Mentel, J. [Electrical Engineering and Plasma Technology, Ruhr University Bochum, D-44780 Bochum (Germany); Denissen, C.; Suijker, J. [Philips Lighting, Category Professional Lamps, P.O. Box 80020, NL-5600JM Eindhoven (Netherlands)

2015-08-07

The gas phase emitter effect increases the lamp lifetime by lowering the work function and, with it, the temperature of the tungsten electrodes of metal halide lamps especially for lamps in ceramic vessels due to their high rare earth pressures. It is generated by a monolayer on the electrode surface of electropositive atoms of certain emitter elements, which are inserted into the lamp bulb by metal iodide salts. They are vaporized, dissociated, ionized, and deposited by an emitter ion current onto the electrode surface within the cathodic phase of lamp operation with a switched-dc or ac-current. The gas phase emitter effect of La and the influence of Na on the emitter effect of La are studied by spatially and phase-resolved pyrometric measurements of the electrode tip temperature, La atom, and ion densities by optical emission spectroscopy as well as optical broadband absorption spectroscopy and arc attachment images by short time photography. An addition of Na to the lamp filling increases the La vapor pressure within the lamp considerably, resulting in an improved gas phase emitter effect of La. Furthermore, the La vapor pressure is raised by a heating of the cold spot. In this way, conditions depending on the La vapor pressure and operating frequency are identified, at which the temperature of the electrodes becomes a minimum.

Accurate microRNA target prediction correlates with protein repression levels

Directory of Open Access Journals (Sweden)

Simossis Victor A

2009-09-01

Full Text Available Abstract Background MicroRNAs are small endogenously expressed non-coding RNA molecules that regulate target gene expression through translation repression or messenger RNA degradation. MicroRNA regulation is performed through pairing of the microRNA to sites in the messenger RNA of protein coding genes. Since experimental identification of miRNA target genes poses difficulties, computational microRNA target prediction is one of the key means in deciphering the role of microRNAs in development and disease. Results DIANA-microT 3.0 is an algorithm for microRNA target prediction which is based on several parameters calculated individually for each microRNA and combines conserved and non-conserved microRNA recognition elements into a final prediction score, which correlates with protein production fold change. Specifically, for each predicted interaction the program reports a signal to noise ratio and a precision score which can be used as an indication of the false positive rate of the prediction. Conclusion Recently, several computational target prediction programs were benchmarked based on a set of microRNA target genes identified by the pSILAC method. In this assessment DIANA-microT 3.0 was found to achieve the highest precision among the most widely used microRNA target prediction programs reaching approximately 66%. The DIANA-microT 3.0 prediction results are available online in a user friendly web server at http://www.microrna.gr/microT

Efficacy of irreversible electroporation in human pancreatic adenocarcinoma: advanced murine model

Directory of Open Access Journals (Sweden)

Prejesh Philips

Full Text Available Irreversible electroporation (IRE is a promising cell membrane ablative modality for pancreatic cancer. There have been recent concerns regarding local recurrence and the potential use of IRE as a debulking (partial ablation modality. We hypothesize that incomplete ablation leads to early recurrence and a more aggressive biology. We created the first ever heterotopic murine model by inoculating BALB/c nude mice in the hindlimb with a subcutaneous injection of Panc-1 cells, an immortalized human pancreatic adenocarcinoma cell line. Tumors were allowed to grow from 0.75 to 1.5 cm and then treated with the goal of complete ablation or partial ablation using standard IRE settings. Animals were recovered and survived for 2 days (n = 6, 7 (n = 6, 14 (n = 6, 21 (n = 6, 30 (n = 8, and 60 (n = 8 days. All 40 animals/tumors underwent successful IRE under general anesthesia with muscle paralysis. The mean tumor volume of the animals undergoing ablation was 1,447.6 mm3 ± 884. Histologically, in the 14-, 21-, 30-, and 60-day survival groups the entire tumor was nonviable, with a persistent tumor nodule completely replaced fibrosis. In the group treated with partial ablation, incomplete electroporation/recurrences (N = 10 animals were seen, of which 66% had confluent tumors and this was a significant predictor of recurrence (P < 0.001. Recurrent tumors were also significantly larger (mean 4,578 mm3 ± SD 877 versus completed electroporated tumors 925.8 ± 277, P < 0.001. Recurrent tumors had a steeper growth curve (slope = 0.73 compared with primary tumors (0.60, P = 0.02. Recurrent tumors also had a significantly higher percentage of EpCAM expression, suggestive of stem cell activation. Tumors that recur after incomplete electroporation demonstrate a biologically aggressive tumor that could be more resistant to standard of care chemotherapy. Clinical correlation of this data is limited, but should be considered when IRE of pancreatic cancer is being

Peroxisome proliferator-activated receptors, estrogenic responses and biotransformation system in the liver of salmon exposed to tributyltin and second messenger activator

Energy Technology Data Exchange (ETDEWEB)

Pavlikova, Nela [Department of Biology, Norwegian University of Science and Technology (NTNU), Hogskoleringen 5, 7491 Trondheim (Norway); RECETOX Research Centre for Environmental Chemistry and Ecotoxicology, Masaryk University, Kamenice 3, CZ62500 Brno (Czech Republic); Kortner, Trond M. [Department of Biology, Norwegian University of Science and Technology (NTNU), Hogskoleringen 5, 7491 Trondheim (Norway); Arukwe, Augustine, E-mail: arukwe@bio.ntnu.no [Department of Biology, Norwegian University of Science and Technology (NTNU), Hogskoleringen 5, 7491 Trondheim (Norway)

2010-08-15

The mechanisms by which organotin compounds produce modulations of the endocrine systems and other biological responses are not fully understood. In this study, juvenile salmon were force-fed diet containing TBT (0: solvent control, 0.1, 1 and 10 mg/kg fish) for 72 h. Subsequently, fish exposed to solvent control and 10 mg TBT were exposed to waterborne concentration (200 {mu}g/l) of the adenylate cyclase (AC) stimulator, forskolin for 2 and 4 h. The overall aim of the study was to explore whether TBT endocrine disruptive effects involve second messenger activation. Liver was sampled from individual fish (n = 8) at the end of the exposures. The transcription patterns of peroxisome proliferator-activated receptor (PPAR) isotype and acyl-coenzyme A oxidase 1 (ACOX1), aromatase isoform, estrogen receptor-{alpha} (ER{alpha}), pregnane X receptor (PXR), CYP3A and glutathione S-transferase (GST) genes were measured by quantitative polymerase chain reaction (qPCR). Our data showed a consistent increase in PPAR{alpha}, PPAR{beta} and PPAR{gamma} mRNA and protein expression after TBT exposure that were inversely correlated with ACOX1 mRNA levels. Forskolin produced PPAR isotype-specific mRNA and protein effects that were modulated by TBT. ACOX1 expression was decreased (at 2 h) and increased (at 4 h) by forskolin and the presence of TBT potentiated these effects. TBT apparently increased mRNA and protein levels of cyp19a, compared to the solvent control, whereas cyp19b mRNA levels were unaffected by TBT treatment. Combined TBT and forskolin exposure produced respective decrease and increase of mRNA levels of cyp19a and cyp19b, compared with control. TBT decreased ER{alpha} mRNA at low dose (1 mg/kg) and forskolin exposure alone produced a consistent decrease of ER{alpha} mRNA levels that were not affected by the presence of TBT. Interestingly, PXR and CYP3A mRNA levels were differentially affected, either decreased or increased, after exposure to TBT and forskolin, singly

Peroxisome proliferator-activated receptors, estrogenic responses and biotransformation system in the liver of salmon exposed to tributyltin and second messenger activator

International Nuclear Information System (INIS)

Pavlikova, Nela; Kortner, Trond M.; Arukwe, Augustine

2010-01-01

The mechanisms by which organotin compounds produce modulations of the endocrine systems and other biological responses are not fully understood. In this study, juvenile salmon were force-fed diet containing TBT (0: solvent control, 0.1, 1 and 10 mg/kg fish) for 72 h. Subsequently, fish exposed to solvent control and 10 mg TBT were exposed to waterborne concentration (200 μg/l) of the adenylate cyclase (AC) stimulator, forskolin for 2 and 4 h. The overall aim of the study was to explore whether TBT endocrine disruptive effects involve second messenger activation. Liver was sampled from individual fish (n = 8) at the end of the exposures. The transcription patterns of peroxisome proliferator-activated receptor (PPAR) isotype and acyl-coenzyme A oxidase 1 (ACOX1), aromatase isoform, estrogen receptor-α (ERα), pregnane X receptor (PXR), CYP3A and glutathione S-transferase (GST) genes were measured by quantitative polymerase chain reaction (qPCR). Our data showed a consistent increase in PPARα, PPARβ and PPARγ mRNA and protein expression after TBT exposure that were inversely correlated with ACOX1 mRNA levels. Forskolin produced PPAR isotype-specific mRNA and protein effects that were modulated by TBT. ACOX1 expression was decreased (at 2 h) and increased (at 4 h) by forskolin and the presence of TBT potentiated these effects. TBT apparently increased mRNA and protein levels of cyp19a, compared to the solvent control, whereas cyp19b mRNA levels were unaffected by TBT treatment. Combined TBT and forskolin exposure produced respective decrease and increase of mRNA levels of cyp19a and cyp19b, compared with control. TBT decreased ERα mRNA at low dose (1 mg/kg) and forskolin exposure alone produced a consistent decrease of ERα mRNA levels that were not affected by the presence of TBT. Interestingly, PXR and CYP3A mRNA levels were differentially affected, either decreased or increased, after exposure to TBT and forskolin, singly and also in combination. GST mRNA was

Transformation of undomesticated strains of Bacillus subtilis by protoplast electroporation

NARCIS (Netherlands)

Romero, Diego; Perez-Garcia, Alejandro; Veening, Jan-Willem; de Vicente, Antonio; Kuipers, Oscar P.; de, Vicente A.

A rapid method combining the use of protoplasts and electroporation was developed to transform recalcitrant wild strains of Bacillus subtilis. The method described here allows transformation with both replicative and integrative plasmids, as well as with chromosomal DNA, and provides a valuable tool

Low vulnerability of the right phrenic nerve to electroporation ablation

NARCIS (Netherlands)

van Driel, Vincent J. H. M.; Neven, KGEJ; van Wessel, Harri; Vink, Aryan; Doevendans, Pieter A. F. M.; Wittkampf, Fred H. M.

BACKGROUND Circular electroporation ablation is a novel ablation modality for electrical pulmonary vein isolation. With a single 200-3 application, deep circular myocardial lesions can be created. However, the acute and chronic effects of this energy source on phrenic nerve (PN) function are

RNA Localization in Astrocytes

DEFF Research Database (Denmark)

Thomsen, Rune

2012-01-01

, regulation of the blood brain barrier and glial scar tissue formation. Despite the involvement in various CNS functions only a limited number of studies have addressed mRNA localization in astrocytes. This PhD project was initially focused on developing and implementing methods that could be used to asses mRNA......Messenger RNA (mRNA) localization is a mechanism by which polarized cells can regulate protein synthesis to specific subcellular compartments in a spatial and temporal manner, and plays a pivotal role in multiple physiological processes from embryonic development to cell differentiation...... localization in astrocyte protrusions, and following look into the subcellular localization pattern of specific mRNA species of both primary astrocytes isolated from cortical hemispheres of newborn mice, and the mouse astrocyte cell line, C8S. The Boyden chamber cell fractionation assay was optimized, in a way...

Detection of Zika virus using reverse-transcription LAMP coupled with reverse dot blot analysis in saliva.

Directory of Open Access Journals (Sweden)

Maite Sabalza

Full Text Available In recent years, there have been increasing numbers of infectious disease outbreaks that spread rapidly to population centers resulting from global travel, population vulnerabilities, environmental factors, and ecological disasters such as floods and earthquakes. Some examples of the recent outbreaks are the Ebola epidemic in West Africa, Middle East respiratory syndrome coronavirus (MERS-Co in the Middle East, and the Zika outbreak through the Americas. We have created a generic protocol for detection of pathogen RNA and/or DNA using loop-mediated isothermal amplification (LAMP and reverse dot-blot for detection (RDB and processed automatically in a microfluidic device. In particular, we describe how a microfluidic assay to detect HIV viral RNA was converted to detect Zika virus (ZIKV RNA. We first optimized the RT-LAMP assay to detect ZIKV RNA using a benchtop isothermal amplification device. Then we implemented the assay in a microfluidic device that will allow analyzing 24 samples simultaneously and automatically from sample introduction to detection by RDB technique. Preliminary data using saliva samples spiked with ZIKV showed that our diagnostic system detects ZIKV RNA in saliva. These results will be validated in further experiments with well-characterized ZIKV human specimens of saliva. The described strategy and methodology to convert the HIV diagnostic assay and platform to a ZIKV RNA detection assay provides a model that can be readily utilized for detection of the next emerging or re-emerging infectious disease.

Geodesy at Mercury with MESSENGER

Science.gov (United States)

Smith, David E.; Zuber, Maria t.; Peale, Stanley J.; Phillips, Roger J.; Solomon, Sean C.

2006-01-01

In 2011 the MESSENGER (MErcury Surface, Space ENvironment, GEochemistry, and Ranging) spacecraft will enter Mercury orbit and begin the mapping phase of the mission. As part of its science objectives the MESSENGER mission will determine the shape and gravity field of Mercury. These observations will enable the topography and the crustal thickness to be derived for the planet and will determine the small libration of the planet about its axis, the latter critical to constraining the state of the core. These measurements require very precise positioning of the MESSENGER spacecraft in its eccentric orbit, which has a periapsis altitude as low as 200 km, an apoapsis altitude near 15,000 km, and a closest approach to the surface varying from latitude 60 to about 70 N. The X-band tracking of MESSENGER and the laser altimetry are the primary data that will be used to measure the planetary shape and gravity field. The laser altimeter, which has an expected range of 1000 to 1200 km, is expected to provide significant data only over the northern hemisphere because of MESSENGER's eccentric orbit. For the southern hemisphere, radio occultation measurements obtained as the spacecraft passes behind the planet as seen from Earth and images obtained with the imaging system will be used to provide the long-wavelength shape of the planet. Gravity, derived from the tracking data, will also have greater resolution in the northern hemisphere, but full global models for both topography and gravity will be obtained at low harmonic order and degree. The limiting factor for both gravity and topography is expected to be knowledge of the spacecraft location. Present estimations are that in a combined tracking, altimetry, and occultation solution the spacecraft position uncertainty is likely to be of order 10 m. This accuracy should be adequate for establishing an initial geodetic coordinate system for Mercury that will enable positioning of imaged features on the surface, determination of

MESSENGER'S First and Second Flybys of Mercury

Science.gov (United States)

Slavin, James A.

2009-01-01

The MESSENGER mission to Mercury offers our first opportunity to explore this planet's miniature magnetosphere since Mariner 10's brief fly-bys in 1974-5. The magnetosphere of Mercury is the smallest in the solar system with its magnetic field typically standing off the solar wind only approximately 1000 km above the surface. An overview of the MESSENGER mission and its January 14th and October 6th, 2008 close flybys of Mercury will be provided. Primary science objectives and the science instrumentation will be described. Initial results from MESSENGER will be discussed with an emphasis on the magnetic field and charged particle measurements.

Harnessing Dendritic Cells for Tumor Antigen Presentation

Energy Technology Data Exchange (ETDEWEB)

Nierkens, Stefan [Department of Tumor Immunology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Geert Grooteplein 28, Nijmegen 6525 GA (Netherlands); Janssen, Edith M., E-mail: edith.janssen@cchmc.org [Division of Molecular Immunology, Cincinnati Children' s Hospital Research Foundation, University of Cincinnati College of Medicine, 3333 Burnet Avenue, Cincinnati, OH 45229 (United States)

2011-04-26

Dendritic cells (DC) are professional antigen presenting cells that are crucial for the induction of anti-tumor T cell responses. As a consequence, research has focused on the harnessing of DCs for therapeutic interventions. Although current strategies employing ex vivo-generated and tumor-antigen loaded DCs have been proven feasible, there are still many obstacles to overcome in order to improve clinical trial successes and offset the cost and complexity of customized cell therapy. This review focuses on one of these obstacles and a pivotal step for the priming of tumor-specific CD8{sup +} and CD4{sup +} T cells; the in vitro loading of DCs with tumor antigens.

Materials for incandescent and fluorescent lamps

DEFF Research Database (Denmark)

Thorsen, Knud Aage

1996-01-01

The article gives an overview of the materials systems used for incandescent lamps as well as a brief introduction to the systems used for fluorescent lamps. The materials used for incandescent lamps are doped tungsten used for the filaments, metals and alloys used for terminal and support posts......, lead wires and internal reflectors and screens as well as glasses for the envelope. The physics of bulbs and changes in bulbs during use are elucidated. The cost and energy savings and environmental benefits by replacement of incandescent lamps by fluorescent lamps are presented....

Delineating the cell death mechanisms associated with skin electroporation.

Science.gov (United States)

Schultheis, Katherine; Smith, Trevor R F; Kiosses, William B; Kraynyak, Kimberly A; Wong, Amelia; Oh, Janet; Broderick, Kate Elizabeth

2018-06-28

The immune responses elicited following delivery of DNA vaccines to the skin has previously been shown to be significantly enhanced by the addition of electroporation (EP) to the treatment protocol. Principally, EP increases the transfection of pDNA into the resident skin cells. In addition to increasing the levels of in vivo transfection, the physical insult induced by EP is associated with activation of innate pathways which are believed to mediate an adjuvant effect, further enhancing DNA vaccine responses. Here, we have investigated the possible mechanisms associated with this adjuvant effect, primarily focusing on the cell death pathways associated with the skin EP procedure independent of pDNA delivery. Using the minimally invasive CELLECTRA®-3P intradermal electroporation device that penetrates the epidermal and dermal layers of the skin, we have investigated apoptotic and necrotic cell death in relation to the vicinity of the electrode needles and electric field generated. Employing the well-established TUNEL assay, we detected apoptosis beginning as early as one hour after EP and peaking at the 4 hour time point. The majority of the apoptotic events were detected in the epidermal region directly adjacent to the electrode needle. Using a novel propidium iodide in vivo necrotic cell death assay, we detected necrotic events concentrated in the epidermal region adjacent to the electrode. Furthermore, we detected up-regulation of calreticulin expression on skin cells after EP, thus labeling these cells for uptake by dendritic cells and macrophages. These results allow us to delineate the cell death mechanisms occurring in the skin following intradermal EP independently of pDNA delivery. We believe these events contribute to the adjuvant effect observed following electroporation at the skin treatment site.

The Effect of Electroporation of a Lyotroic Liquid Crystal Genistein-Based Formulation in the Recovery of Murine Melanoma Lesions.

Science.gov (United States)

Danciu, Corina; Berkó, Szilvia; Varju, Gábor; Balázs, Boglárka; Kemény, Lajos; Németh, István Balázs; Cioca, Andreea; Petruș, Alexandra; Dehelean, Cristina; Cosmin, Citu Ioan; Amaricai, Elena; Toma, Claudia Crina

2015-07-08

A lamellar lyotropic liquid crystal genistein-based formulation (LLC-Gen) was prepared in order to increase the aqueous solubility of the lipophilic phytocompound genistein. The formulation was applied locally, in a murine model of melanoma, with or without electroporation. The results demonstrated that, when the formulation was applied by electroporation, the tumors appeared later. During the 21 days of the experiment, the LLC-Gen formulation decreased the tumor volume, the amount of melanin and the degree of erythema, but when electroporation was applied, all these parameters indicated a better prognosis even (lower tumor volume, amount of melanin and degree of erythema). Although hematoxylin-eosin (HE) staining confirmed the above events, application of the LLC-Gen formulation by electroporation did not lead to a significant effect in terms of the serum concentrations of the protein S100B and serum neuron specific enolase (NSE), or the tissue expression of the platelet-derived growth factor receptor β (PDGFRβ) antibody.

The Effect of Electroporation of a Lyotroic Liquid Crystal Genistein-Based Formulation in the Recovery of Murine Melanoma Lesions

Directory of Open Access Journals (Sweden)

Corina Danciu

2015-07-01

Full Text Available A lamellar lyotropic liquid crystal genistein-based formulation (LLC-Gen was prepared in order to increase the aqueous solubility of the lipophilic phytocompound genistein. The formulation was applied locally, in a murine model of melanoma, with or without electroporation. The results demonstrated that, when the formulation was applied by electroporation, the tumors appeared later. During the 21 days of the experiment, the LLC-Gen formulation decreased the tumor volume, the amount of melanin and the degree of erythema, but when electroporation was applied, all these parameters indicated a better prognosis even (lower tumor volume, amount of melanin and degree of erythema. Although hematoxylin–eosin (HE staining confirmed the above events, application of the LLC-Gen formulation by electroporation did not lead to a significant effect in terms of the serum concentrations of the protein S100B and serum neuron specific enolase (NSE, or the tissue expression of the platelet-derived growth factor receptor β (PDGFRβ antibody.

Wood's lamp illumination (image)

Science.gov (United States)

A Wood's lamp emits ultraviolet light and can be a diagnostic aid in determining if someone has a fungal ... is an infection on the area where the Wood's lamp is illuminating, the area will fluoresce. Normally ...

Cytoplasmic Control of Sense-Antisense mRNA Pairs

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Flore Sinturel

2015-09-01

Full Text Available Transcriptome analyses have revealed that convergent gene transcription can produce many 3′-overlapping mRNAs in diverse organisms. Few studies have examined the fate of 3′-complementary mRNAs in double-stranded RNA-dependent nuclear phenomena, and nothing is known about the cytoplasmic destiny of 3′-overlapping messengers or their impact on gene expression. Here, we demonstrate that the complementary tails of 3′-overlapping mRNAs can interact in the cytoplasm and promote post-transcriptional regulatory events including no-go decay (NGD in Saccharomyces cerevisiae. Genome-wide experiments confirm that these messenger-interacting mRNAs (mimRNAs form RNA duplexes in wild-type cells and thus have potential roles in modulating the mRNA levels of their convergent gene pattern under different growth conditions. We show that the post-transcriptional fate of hundreds of mimRNAs is controlled by Xrn1, revealing the extent to which this conserved 5′-3′ cytoplasmic exoribonuclease plays an unexpected but key role in the post-transcriptional control of convergent gene expression.

Cytoplasmic Control of Sense-Antisense mRNA Pairs.

Science.gov (United States)

Sinturel, Flore; Navickas, Albertas; Wery, Maxime; Descrimes, Marc; Morillon, Antonin; Torchet, Claire; Benard, Lionel

2015-09-22

Transcriptome analyses have revealed that convergent gene transcription can produce many 3'-overlapping mRNAs in diverse organisms. Few studies have examined the fate of 3'-complementary mRNAs in double-stranded RNA-dependent nuclear phenomena, and nothing is known about the cytoplasmic destiny of 3'-overlapping messengers or their impact on gene expression. Here, we demonstrate that the complementary tails of 3'-overlapping mRNAs can interact in the cytoplasm and promote post-transcriptional regulatory events including no-go decay (NGD) in Saccharomyces cerevisiae. Genome-wide experiments confirm that these messenger-interacting mRNAs (mimRNAs) form RNA duplexes in wild-type cells and thus have potential roles in modulating the mRNA levels of their convergent gene pattern under different growth conditions. We show that the post-transcriptional fate of hundreds of mimRNAs is controlled by Xrn1, revealing the extent to which this conserved 5'-3' cytoplasmic exoribonuclease plays an unexpected but key role in the post-transcriptional control of convergent gene expression. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

MESSENGER at Mercury: Early Orbital Operations

Science.gov (United States)

McNutt, Ralph L., Jr; Solomon, Sean C.; Bedini, Peter D.; Anderson, Brian J.; Blewett, David T.; Evans, Larry G.; Gold, Robert E.; Krimigis, Stamatios M.; Murchie, Scott L.; Nittler, Larry R.;

Guide totheNomenclatureofKinetoplastidRNA Editing: AProposal

Czech Academy of Sciences Publication Activity Database

Simpson, L.; Aphasizhev, R.; Lukeš, Julius; Cruz-Reyes, J.

2010-01-01

Roč. 161, č. 1 (2010), s. 2-6 ISSN 1434-4610 Institutional research plan: CEZ:AV0Z60220518 Keywords : TRYPANOSOMA-BRUCEI MITOCHONDRIA * BINDING COMPLEX * EDITOSOME INTEGRITY * MESSENGER-RNA * U-DELETION * LEISHMANIA Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.329, year: 2010

A Single-Stage LED Tube Lamp Driver with Power-Factor Corrections and Soft Switching for Energy-Saving Indoor Lighting Applications

Directory of Open Access Journals (Sweden)

Chun-An Cheng

2017-01-01

Full Text Available This paper presents a single-stage alternating current (AC/direct current (DC light-emitting diode (LED tube lamp driver for energy-saving indoor lighting applications; this driver features power-factor corrections and soft switching, and also integrates a dual buck-boost converter with coupled inductors and a half-bridge series resonant converter cascaded with a bridge rectifier into a single-stage power-conversion topology. The features of the presented driver are high efficiency (>91%, satisfying power factor (PF > 0.96, low input-current total-harmonic distortion (THD < 10%, low output voltage ripple factor (<7.5%, low output current ripple factor (<8%, and zero-voltage switching (ZVS obtained on both power switches. Operational principles are described in detail, and experimental results obtained from an 18 W-rated LED tube lamp for T8/T10 fluorescent lamp replacements with input utility-line voltages ranging from 100 V to 120 V have demonstrated the functionality of the presented driver suitable for indoor lighting applications.

MicroRNA and Cancer: Tiny Molecules with Major Implications

OpenAIRE

VandenBoom II, Timothy G; Li, Yiwei; Philip, Philip A; Sarkar, Fazlul H

2008-01-01

Cancer is currently a major public health problem and, as such, emerging research is making significant progress in identifying major players in its biology. One recent topic of interest involves microRNAs (miRNAs) which are small, non-coding RNA molecules that inhibit gene expression post-transcriptionally. They accomplish this by binding to the 3? untranslated region (3?UTR) of target messengerRNA (mRNA), resulting in either their degradation or inhibition of translation, depending on the d...

Appendix: a solution hybridization assay to detect radioactive globin messenger RNA nucleotide sequences

Energy Technology Data Exchange (ETDEWEB)

Ross, J

1976-09-15

In view of the sensitivity and specificity of the solution hybridization assay for unlabeled globin mRNA a similar technique has been devised to detect radioactive globin mRNA sequences with unlabeled globin cDNA. Several properties of the hybridization reaction are presented since RNA kinetic experiments reported recently depend on the validity of this assay. Data on hybridization analysis of (/sup 3/H)RNA from mouse fetal liver or erythroleukemia cell cytoplasm are presented. These data indicate that the excess cDNA solution assay for radioactive globin mRNA detection is specific for globin mRNA sequences. It can be performed rapidly and is highly reproducible from experiment. It is at least 500-fold less sensitive than the assay for unlabeled globin mRNA, due to the RNAase backgrounds of 0.05 to 0.15 %. However, this limitation has not affected kinetic experiments with non-dividing fetal liver erythroid cells, which synthesize relatively large quantities of globin mRNA.

Regulatory Network Controlling Extracellular Proteins in Erwinia carotovora subsp. carotovora: FlhDC, the Master Regulator of Flagellar Genes, Activates rsmB Regulatory RNA Production by Affecting gacA and hexA (lrhA) Expression▿

Science.gov (United States)

Cui, Yaya; Chatterjee, Asita; Yang, Hailian; Chatterjee, Arun K.

2008-01-01

Erwinia carotovora subsp. carotovora produces an array of extracellular proteins (i.e., exoproteins), including plant cell wall-degrading enzymes and Harpin, an effector responsible for eliciting hypersensitive reaction. Exoprotein genes are coregulated by the quorum-sensing signal, N-acyl homoserine lactone, plant signals, an assortment of transcriptional factors/regulators (GacS/A, ExpR1, ExpR2, KdgR, RpoS, HexA, and RsmC) and posttranscriptional regulators (RsmA, rsmB RNA). rsmB RNA production is positively regulated by GacS/A, a two-component system, and negatively regulated by HexA (PecT in Erwinia chrysanthemi; LrhA [LysR homolog A] in Escherichia coli) and RsmC, a putative transcriptional adaptor. While free RsmA, an RNA-binding protein, promotes decay of mRNAs of exoprotein genes, binding of RsmA with rsmB RNA neutralizes the RsmA effect. In the course of studies of GacA regulation, we discovered that a locus bearing strong homology to the flhDC operon of E. coli also controls extracellular enzyme production. A transposon insertion FlhDC− mutant produces very low levels of pectate lyase, polygalacturonase, cellulase, protease, and E. carotovora subsp. carotovora Harpin (HarpinEcc) and is severely attenuated in its plant virulence. The production of these exoproteins is restored in the mutant carrying an FlhDC+ plasmid. Sequence analysis and transcript assays disclosed that the flhD operon of E. carotovora subsp. carotovora, like those of other enterobacteria, consists of flhD and flhC. Complementation analysis revealed that the regulatory effect requires functions of both flhD and flhC products. The data presented here show that FlhDC positively regulates gacA, rsmC, and fliA and negatively regulates hexA (lrhA). Evidence shows that FlhDC controls extracellular protein production through cumulative effects on hexA and gacA. Reduced levels of GacA and elevated levels of HexA in the FlhDC− mutant are responsible for the inhibition of rsmB RNA production

EVIR: chimeric receptors that enhance dendritic cell cross-dressing with tumor antigens.

Science.gov (United States)

Squadrito, Mario Leonardo; Cianciaruso, Chiara; Hansen, Sarah K; De Palma, Michele

2018-03-01

We describe a lentivirus-encoded chimeric receptor, termed extracellular vesicle (EV)-internalizing receptor (EVIR), which enables the selective uptake of cancer-cell-derived EVs by dendritic cells (DCs). The EVIR enhances DC presentation of EV-associated tumor antigens to CD8 + T cells primarily through MHCI recycling and cross-dressing. EVIRs should facilitate exploring the mechanisms and implications of horizontal transfer of tumor antigens to antigen-presenting cells.

Lysosome-Associated Membrane Proteins (LAMP Maintain Pancreatic Acinar Cell Homeostasis: LAMP-2–Deficient Mice Develop PancreatitisSummary

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Olga A. Mareninova

2015-11-01

Full Text Available Background & Aims: The pathogenic mechanism of pancreatitis is poorly understood. Recent evidence implicates defective autophagy in pancreatitis responses; however, the pathways mediating impaired autophagy in pancreas remain largely unknown. Here, we investigate the role of lysosome associated membrane proteins (LAMPs in pancreatitis. Methods: We analyzed changes in LAMPs in experimental models and human pancreatitis, and the underlying mechanisms: LAMP deglycosylation and degradation. LAMP cleavage by cathepsin B (CatB was analyzed by mass spectrometry. We used mice deficient in LAMP-2 to assess its role in pancreatitis. Results: Pancreatic levels of LAMP-1 and LAMP-2 greatly decrease across various pancreatitis models and in human disease. Pancreatitis does not trigger the LAMPs’ bulk deglycosylation but induces their degradation via CatB-mediated cleavage of the LAMP molecule close to the boundary between luminal and transmembrane domains. LAMP-2 null mice spontaneously develop pancreatitis that begins with acinar cell vacuolization due to impaired autophagic flux, and progresses to severe pancreas damage characterized by trypsinogen activation, macrophage-driven inflammation, and acinar cell death. LAMP-2 deficiency causes a decrease in pancreatic digestive enzymes content, and stimulates the basal and inhibits cholecystokinin-induced amylase secretion by acinar cells. The effects of LAMP-2 knockout and acute cerulein pancreatitis overlap, which corroborates the pathogenic role of LAMP decrease in experimental pancreatitis models. Conclusions: The results indicate a critical role for LAMPs, particularly LAMP-2, in maintaining pancreatic acinar cell homeostasis and provide evidence that defective lysosomal function, resulting in impaired autophagy, leads to pancreatitis. Mice with LAMP-2 deficiency present a novel genetic model of human pancreatitis caused by lysosomal/autophagic dysfunction. Keywords: Amylase Secretion, Autophagy

Diode-laser-illuminated automotive lamp systems

Science.gov (United States)

Marinelli, Michael A.; Remillard, Jeffrey T.

1998-05-01

We have utilized the high brightness of state-of-the-art diode laser sources, and a variety of emerging optical technologies to develop a new class of thin, uniquely styled automotive brake and signal lamps. Using optics based on thin (5 mm) plastic sheets, these lamps provide appearance and functional advantages not attainable with traditional automotive lighting systems. The light is coupled into the sheets using a 1 mm diameter glass fiber, and manipulated using refraction and reflection from edges, surfaces, and shaped cut-outs. Light can be extracted with an efficiency of approximately 50% and formed into a luminance distribution that meets the Society of Automotive Engineers (SAE) photometric requirements. Prototype lamps using these optics have been constructed and are less than one inch in thickness. Thin lamps reduce sheet metal costs, complexity, material usage, weight, and allow for increased trunk volume. In addition, these optics enhance lamp design flexibility. When the lamps are not energized, they can appear body colored, and when lighted, the brightness distribution across the lamp can be uniform or structured. A diode laser based brake lamp consumes seven times less electrical power than one using an incandescent source and has instant on capability. Also, diode lasers have the potential to be 10-year/150,000 mile light sources.

Model of discharge lamps with magnetic ballast

OpenAIRE

Molina, Julio; Sainz Sapera, Luis; Mesas García, Juan José; Bergas Jané, Joan Gabriel

2013-01-01

Magnetic ballast discharge lamp modeling has been extensively studied because these lamps can be an important source of harmonics. Discharge lamp models usually represent the arc voltage by a square waveform. However, this waveform can be far from actual arc voltages, which affects the accuracy of the lamp models. This paper investigates the actual arc voltage behavior of discharge lamps from laboratory measurements and proposes a novel characterization of these voltages to reformulate the co...

Capturing microRNA targets using an RNA-induced silencing complex (RISC)-trap approach.

Science.gov (United States)

Cambronne, Xiaolu A; Shen, Rongkun; Auer, Paul L; Goodman, Richard H

2012-12-11

Identifying targets is critical for understanding the biological effects of microRNA (miRNA) expression. The challenge lies in characterizing the cohort of targets for a specific miRNA, especially when targets are being actively down-regulated in miRNA- RNA-induced silencing complex (RISC)-messengerRNA (mRNA) complexes. We have developed a robust and versatile strategy called RISCtrap to stabilize and purify targets from this transient interaction. Its utility was demonstrated by determining specific high-confidence target datasets for miR-124, miR-132, and miR-181 that contained known and previously unknown transcripts. Two previously unknown miR-132 targets identified with RISCtrap, adaptor protein CT10 regulator of kinase 1 (CRK1) and tight junction-associated protein 1 (TJAP1), were shown to be endogenously regulated by miR-132 in adult mouse forebrain. The datasets, moreover, differed in the number of targets and in the types and frequency of microRNA recognition element (MRE) motifs, thus revealing a previously underappreciated level of specificity in the target sets regulated by individual miRNAs.

Messenger Observations of Mercury's Bow Shock and Magnetopause

Science.gov (United States)

Slavin J. A.; Acuna, M. H.; Anderson, B. J.; Benna, M.; Gloeckler, G.; Krimigis, S. M.; Raines, M.; Schriver, D.; Travnicek, P.; Zurbuchen, T. H.

2008-01-01

The MESSENGER spacecraft made the first of three flybys of Mercury on January 14.2008 (1). New observations of solar wind interaction with Mercury were made with MESSENGER'S Magnetometer (MAG) (2.3) and Energetic Particle and Plasma Spectrometer (EPPS) - composed of the Energetic Particle Spectrometer (EPS) and Fast Imaging Plasma Spectrometer (FIPS) (3,4). These MESSENGER observations show that Mercury's magnetosphere has a large-scale structure that is distinctly Earth-like, but it is immersed in a comet-like cloud of planetary ions [5]. Fig. 1 provides a schematic view of the coupled solar wind - magnetosphere - neutral atmosphere - solid planet system at Mercury.

Mechanistic evaluation of the pros and cons of digital RT-LAMP for HIV-1 viral load quantification on a microfluidic device and improved efficiency via a two-step digital protocol.

Science.gov (United States)

Sun, Bing; Shen, Feng; McCalla, Stephanie E; Kreutz, Jason E; Karymov, Mikhail A; Ismagilov, Rustem F

2013-02-05

Here we used a SlipChip microfluidic device to evaluate the performance of digital reverse transcription-loop-mediated isothermal amplification (dRT-LAMP) for quantification of HIV viral RNA. Tests are needed for monitoring HIV viral load to control the emergence of drug resistance and to diagnose acute HIV infections. In resource-limited settings, in vitro measurement of HIV viral load in a simple format is especially needed, and single-molecule counting using a digital format could provide a potential solution. We showed here that when one-step dRT-LAMP is used for quantification of HIV RNA, the digital count is lower than expected and is limited by the yield of desired cDNA. We were able to overcome the limitations by developing a microfluidic protocol to manipulate many single molecules in parallel through a two-step digital process. In the first step we compartmentalize the individual RNA molecules (based on Poisson statistics) and perform reverse transcription on each RNA molecule independently to produce DNA. In the second step, we perform the LAMP amplification on all individual DNA molecules in parallel. Using this new protocol, we increased the absolute efficiency (the ratio between the concentration calculated from the actual count and the expected concentration) of dRT-LAMP 10-fold, from ∼2% to ∼23%, by (i) using a more efficient reverse transcriptase, (ii) introducing RNase H to break up the DNA:RNA hybrid, and (iii) adding only the BIP primer during the RT step. We also used this two-step method to quantify HIV RNA purified from four patient samples and found that in some cases, the quantification results were highly sensitive to the sequence of the patient's HIV RNA. We learned the following three lessons from this work: (i) digital amplification technologies, including dLAMP and dPCR, may give adequate dilution curves and yet have low efficiency, thereby providing quantification values that underestimate the true concentration. Careful

A new LED lamp for the collection of nocturnal Lepidoptera and a spectral comparison of light-trapping lamps

Directory of Open Access Journals (Sweden)

Gunnar Brehm

2017-04-01

Full Text Available Most nocturnal Lepidoptera can be attracted to artificial light sources, particularly to those that emit a high proportion of ultraviolet radiation. Here, I describe a newly developed LED lamp set for the use in the field that is lightweight, handy, robust, and energy efficient. The emitted electromagnetic spectrum corresponds to the peak sensitivity in most Lepidoptera eye receptors (ultraviolet, blue and green. Power LEDs with peaks at 368 nm (ultraviolet, 450 nm (blue, 530 nm (green, and 550 nm (cool white are used. I compared the irradiance (Ee of many commonly used light-trapping lamps at a distance of 50 cm. Between wavelengths of 300 and 1000 nm, irradiance from the new lamp was 1.43 W m-2. The new lamp proved to be the most energy efficient, and it emitted more radiation in the range between 300 and 400 nm than any other lamp tested. Cold cathodes are the second most energy-efficient lamps. Irradiation from fluorescent actinic tubes is higher than from fluorescent blacklight-blue tubes. High-wattage incandescent lamps and self-ballasted mercury vapour lamps have highest irradiance, but they mainly emit in the long wave spectrum. The use of gauze and sheets decreases the proportion of UV radiation and increases the share of blue light, probably due to optical brighteners. Compared with sunlight, UV irradiance is low at a distance of 50 cm from the lamp, but (safety glasses as well as keeping sufficient distance from the lamp are recommended. In field tests, the new LED lamp attracted large numbers of Lepidoptera in both the Italian Alps and in the Peruvian Andes.

Bacterial nucleotide-based second messengers.

Science.gov (United States)

Pesavento, Christina; Hengge, Regine

2009-04-01

In all domains of life nucleotide-based second messengers transduce signals originating from changes in the environment or in intracellular conditions into appropriate cellular responses. In prokaryotes cyclic di-GMP has emerged as an important and ubiquitous second messenger regulating bacterial life-style transitions relevant for biofilm formation, virulence, and many other bacterial functions. This review describes similarities and differences in the architecture of the cAMP, (p)ppGpp, and c-di-GMP signaling systems and their underlying signaling principles. Moreover, recent advances in c-di-GMP-mediated signaling will be presented and the integration of c-di-GMP signaling with other nucleotide-based signaling systems will be discussed.

Capacitive discharge exciplex lamps

Energy Technology Data Exchange (ETDEWEB)

Sosnin, E A; Erofeev, M V; Tarasenko, V F [High Current Electronics Institute, 2/3, Akademichesky Ave., Tomsk 634055 (Russian Federation)

2005-09-07

Simple-geometry exciplex lamps of a novel type excited by a capacitive discharge (CD-excilamps) have been investigated. An efficient radiation has been obtained on KrBr*, KrCl*, XeBr*, XeCl* molecules and I* atom. The highest values of efficiency of various working molecules are approximately 10-18%. The lifetime of the operating gas mixture in KrCl*, XeCl*, I* and XeBr* exciplex lamps excited by a CD is above 1000 h. Owing to the above-mentioned characteristics, the exciplex lamps excited by a CD are supposed to be very promising for various applications.

Capacitive discharge exciplex lamps

International Nuclear Information System (INIS)

Sosnin, E A; Erofeev, M V; Tarasenko, V F

2005-01-01

Simple-geometry exciplex lamps of a novel type excited by a capacitive discharge (CD-excilamps) have been investigated. An efficient radiation has been obtained on KrBr*, KrCl*, XeBr*, XeCl* molecules and I* atom. The highest values of efficiency of various working molecules are approximately 10-18%. The lifetime of the operating gas mixture in KrCl*, XeCl*, I* and XeBr* exciplex lamps excited by a CD is above 1000 h. Owing to the above-mentioned characteristics, the exciplex lamps excited by a CD are supposed to be very promising for various applications

49 CFR 234.221 - Lamp voltage.

Science.gov (United States)

2010-10-01

... 49 Transportation 4 2010-10-01 2010-10-01 false Lamp voltage. 234.221 Section 234.221 Transportation Other Regulations Relating to Transportation (Continued) FEDERAL RAILROAD ADMINISTRATION..., Inspection, and Testing Maintenance Standards § 234.221 Lamp voltage. The voltage at each lamp shall be...

Max Tech and Beyond: Fluorescent Lamps

Energy Technology Data Exchange (ETDEWEB)

Scholand, Michael

2012-04-01

Fluorescent lamps are the most widely used artificial light source today, responsible for approximately 70% of the lumens delivered to our living spaces globally. The technology was originally commercialized in the 1930's, and manufacturers have been steadily improving the efficacy of these lamps over the years through modifications to the phosphors, cathodes, fill-gas, operating frequency, tube diameter and other design attributes. The most efficient commercially available fluorescent lamp is the 25 Watt T5 lamp. This lamp operates at 114-116 lumens per watt while also providing good color rendering and more than 20,000 hours of operating life. Industry experts interviewed indicated that while this lamp is the most efficient in the market today, there is still a further 10 to 14% of potential improvements that may be introduced to the market over the next 2 to 5 years. These improvements include further developments in phosphors, fill-gas, cathode coatings and ultraviolet (UV) reflective glass coatings. The commercialization of these technology improvements will combine to bring about efficacy improvements that will push the technology up to a maximum 125 to 130 lumens per watt. One critical issue raised by researchers that may present a barrier to the realization of these improvements is the fact that technology investment in fluorescent lamps is being reduced in order to prioritize research into light emitting diodes (LEDs) and ceramic metal halide high intensity discharge (HID) lamps. Thus, it is uncertain whether these potential efficacy improvements will be developed, patented and commercialized. The emphasis for premium efficacy will continue to focus on T5 lamps, which are expected to continue to be marketed along with the T8 lamp. Industry experts highlighted the fact that an advantage of the T5 lamp is the fact that it is 40% smaller and yet provides an equivalent lumen output to that of a T8 or T12 lamp. Due to its smaller form factor, the T5 lamp

Nanosecond pulsed electric fields (nsPEFs) low cost generator design using power MOSFET and Cockcroft-Walton multiplier circuit as high voltage DC source

International Nuclear Information System (INIS)

Sulaeman, M. Y.; Widita, R.

2014-01-01

Purpose: Non-ionizing radiation therapy for cancer using pulsed electric field with high intensity field has become an interesting field new research topic. A new method using nanosecond pulsed electric fields (nsPEFs) offers a novel means to treat cancer. Not like the conventional electroporation, nsPEFs able to create nanopores in all membranes of the cell, including membrane in cell organelles, like mitochondria and nucleus. NsPEFs will promote cell death in several cell types, including cancer cell by apoptosis mechanism. NsPEFs will use pulse with intensity of electric field higher than conventional electroporation, between 20–100 kV/cm and with shorter duration of pulse than conventional electroporation. NsPEFs requires a generator to produce high voltage pulse and to achieve high intensity electric field with proper pulse width. However, manufacturing cost for creating generator that generates a high voltage with short duration for nsPEFs purposes is highly expensive. Hence, the aim of this research is to obtain the low cost generator design that is able to produce a high voltage pulse with nanosecond width and will be used for nsPEFs purposes. Method: Cockcroft-Walton multiplier circuit will boost the input of 220 volt AC into high voltage DC around 1500 volt and it will be combined by a series of power MOSFET as a fast switch to obtain a high voltage with nanosecond pulse width. The motivation using Cockcroft-Walton multiplier is to acquire a low-cost high voltage DC generator; it will use capacitors and diodes arranged like a step. Power MOSFET connected in series is used as voltage divider to share the high voltage in order not to damage them. Results: This design is expected to acquire a low-cost generator that can achieve the high voltage pulse in amount of −1.5 kV with falltime 3 ns and risetime 15 ns into a 50Ω load that will be used for nsPEFs purposes. Further detailed on the circuit design will be explained at presentation

Cosmic Microwave Background Mapmaking with a Messenger Field

Science.gov (United States)

Huffenberger, Kevin M.; Næss, Sigurd K.

2018-01-01

We apply a messenger field method to solve the linear minimum-variance mapmaking equation in the context of Cosmic Microwave Background (CMB) observations. In simulations, the method produces sky maps that converge significantly faster than those from a conjugate gradient descent algorithm with a diagonal preconditioner, even though the computational cost per iteration is similar. The messenger method recovers large scales in the map better than conjugate gradient descent, and yields a lower overall χ2. In the single, pencil beam approximation, each iteration of the messenger mapmaking procedure produces an unbiased map, and the iterations become more optimal as they proceed. A variant of the method can handle differential data or perform deconvolution mapmaking. The messenger method requires no preconditioner, but a high-quality solution needs a cooling parameter to control the convergence. We study the convergence properties of this new method and discuss how the algorithm is feasible for the large data sets of current and future CMB experiments.

Fragile X Mental Retardation Protein and Dendritic Local Translation of the Alpha Subunit of the Calcium/Calmodulin-Dependent Kinase II Messenger RNA Are Required for the Structural Plasticity Underlying Olfactory Learning.

Science.gov (United States)

Daroles, Laura; Gribaudo, Simona; Doulazmi, Mohamed; Scotto-Lomassese, Sophie; Dubacq, Caroline; Mandairon, Nathalie; Greer, Charles August; Didier, Anne; Trembleau, Alain; Caillé, Isabelle

2016-07-15

In the adult brain, structural plasticity allowing gain or loss of synapses remodels circuits to support learning. In fragile X syndrome, the absence of fragile X mental retardation protein (FMRP) leads to defects in plasticity and learning deficits. FMRP is a master regulator of local translation but its implication in learning-induced structural plasticity is unknown. Using an olfactory learning task requiring adult-born olfactory bulb neurons and cell-specific ablation of FMRP, we investigated whether learning shapes adult-born neuron morphology during their synaptic integration and its dependence on FMRP. We used alpha subunit of the calcium/calmodulin-dependent kinase II (αCaMKII) mutant mice with altered dendritic localization of αCaMKII messenger RNA, as well as a reporter of αCaMKII local translation to investigate the role of this FMRP messenger RNA target in learning-dependent structural plasticity. Learning induces profound changes in dendritic architecture and spine morphology of adult-born neurons that are prevented by ablation of FMRP in adult-born neurons and rescued by an metabotropic glutamate receptor 5 antagonist. Moreover, dendritically translated αCaMKII is necessary for learning and associated structural modifications and learning triggers an FMRP-dependent increase of αCaMKII dendritic translation in adult-born neurons. Our results strongly suggest that FMRP mediates structural plasticity of olfactory bulb adult-born neurons to support olfactory learning through αCaMKII local translation. This reveals a new role for FMRP-regulated dendritic local translation in learning-induced structural plasticity. This might be of clinical relevance for the understanding of critical periods disruption in autism spectrum disorder patients, among which fragile X syndrome is the primary monogenic cause. Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

30 CFR 57.17010 - Electric lamps.

Science.gov (United States)

2010-07-01

... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Electric lamps. 57.17010 Section 57.17010 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR METAL AND NONMETAL MINE....17010 Electric lamps. Individual electric lamps shall be carried for illumination by all persons...

A non-canonical landscape of the microRNA system

Directory of Open Access Journals (Sweden)

Gabriel Adelman Cipolla

2014-09-01

Full Text Available Microribonucleic acids, best known as microRNAs or miRNAs, are small, non-coding RNAs with important regulatory roles in eukaryotic cells. Here, I present a broad review about highly relevant but generally non-depicted features of miRNAs, among which stand out the non-conventional miRNA seed sites, the unusual messenger RNA (mRNA target regions, the non-canonical miRNA-guided mechanisms of gene expression regulation and the recently identified new class of miRNA ligands. Furthermore, I address the miRNA uncommon genomic location, transcription, and subcellular localization. Altogether, these unusual features and roles place the miRNA system as a very diverse, complex and intriguing biological mechanism.

Effects of deformability and thermal motion of lipid membrane on electroporation: By molecular dynamics simulations

KAUST Repository

Sun, Sheng

2011-01-01

Effects of mechanical properties and thermal motion of POPE lipid membrane on electroporation were studied by molecular dynamics simulations. Among simulations in which specific atoms of lipids were artificially constrained at their equilibrium positions using a spring with force constant of 2.0kcal/(molÅ2) in the external electric field of 1.4kcal/(molÅe), only constraint on lateral motions of lipid tails prohibited electroporation while non-tail parts had little effects. When force constant decreased to 0.2kcal/(molÅ2) in the position constraints on lipid tails in the external electric field of 2.0kcal/(molÅe), water molecules began to enter the membrane. Position constraints of lipid tails allow water to penetrate from both sides of membrane. Thermal motion of lipids can induce initial defects in the hydrophobic core of membrane, which are favorable nucleation sites for electroporation. Simulations at different temperatures revealed that as the temperature increases, the time taken to the initial pore formation will decrease. © 2010 Elsevier Inc.

Lamps recycling aiming at the environment preservation

International Nuclear Information System (INIS)

Yamachita, Roberto Akira; Gama, Paulo Henrique R. Pereira; Haddad, Jamil; Santos, Afonso H. Moreira; Guardia, Eduardo C.

1999-01-01

The article discusses the following issues of lamps recycling in Brazil: mercury lamps recycling, recycling potential, energy conservation and environmental impacts, enterprises lamps recycling, and incentives policy

CALiPER Retail Lamps Study 3.1: Dimming, Flicker, and Power Quality Characteristics of LED A Lamps

Energy Technology Data Exchange (ETDEWEB)

none,

2014-12-31

This CALiPER report examines the characteristics of a subset of lamps from CALiPER Retail Lamps Study 3 in more detail. Specifically, it focuses on the dimming, power quality, and flicker characteristics of 14 LED A lamps, as controlled by four different retail-available dimmers.

Comparison of LAMP and PCR for molecular mass screening of sand flies for Leishmania martiniquensis infection.

Science.gov (United States)

Tiwananthagorn, Saruda; Kato, Hirotomo; Yeewa, Ranchana; Muengpan, Amontip; Polseela, Raxsina; Leelayoova, Saovanee

2017-02-01

Leishmaniasis caused by Leishmania martiniquensis infection has been reported in human and domestic animals of Martinique Island, Germany, Switzerland, USA, Myanmar and Thailand. The peculiar clinical features of disseminated cutaneous and visceral forms co-existence render the urgent need of specific diagnostic tool to identify the natural sand fly vectors for effective prevention and control strategies. Loop-mediated isothermal amplification (LAMP) of 18S rRNA gene as well as polymerase chain reaction (PCR) of minicircle kinetoplast DNA gene (PCR-mkDNA) have never been applied to detect L. martiniquensis and L. siamensis in sand fly vectors. The present study was aimed to validate malachite green-LAMP (MG-LAMP) and PCR-mkDNA techniques to detect L. martiniquensis in sand fly vectors, compared with the conventional PCR of internal transcribed spacer 1 (PCR-ITS1). We compared the validity of LAMP of 18S rRNA gene and PCR-mkDNA, to PCR-ITS1 in simulation model of L. martiniquensis infection in Sergentomyia gemmea sand flies. Attributable to the sensitivity and specificity, PCR-mkDNA was consecutively applied to detect L. martiniquensis in 380 female sand fly individuals captured in the newly identified affected region of Lamphun Province, Thailand. Results showed that PCR-mkDNA could detect at least one promastigote per sand fly, which was 10-time superior to LAMP and PCR-ITS1. In addition, PCR-mkDNA was more specific, able to differentiate L. martiniquensis from other viscerotropic Leishmania species, such as L. siamensis, L. (L.) donovani, and L. (L.) infantum. Consecutively, mass screening of L. martiniquensis in 380 female sand fly individuals by PCR-mkDNA was implemented in a new affected area of Thailand where a patient with leishmaniasis/HIV co-infection resides; however Leishmania DNA was undetected. In conclusion, PCR-mkDNA is a promising tool for molecular mass screening of L. martiniquensis infection in outbreak areas where several species of Leishmania

Comparison of LAMP and PCR for molecular mass screening of sand flies for Leishmania martiniquensis infection

Science.gov (United States)

Tiwananthagorn, Saruda; Kato, Hirotomo; Yeewa, Ranchana; Muengpan, Amontip; Polseela, Raxsina; Leelayoova, Saovanee

2017-01-01

BACKGROUND Leishmaniasis caused by Leishmania martiniquensis infection has been reported in human and domestic animals of Martinique Island, Germany, Switzerland, USA, Myanmar and Thailand. The peculiar clinical features of disseminated cutaneous and visceral forms co-existence render the urgent need of specific diagnostic tool to identify the natural sand fly vectors for effective prevention and control strategies. Loop-mediated isothermal amplification (LAMP) of 18S rRNA gene as well as polymerase chain reaction (PCR) of minicircle kinetoplast DNA gene (PCR-mkDNA) have never been applied to detect L. martiniquensis and L. siamensis in sand fly vectors. OBJECTIVE The present study was aimed to validate malachite green-LAMP (MG-LAMP) and PCR-mkDNA techniques to detect L. martiniquensis in sand fly vectors, compared with the conventional PCR of internal transcribed spacer 1 (PCR-ITS1). METHODS We compared the validity of LAMP of 18S rRNA gene and PCR-mkDNA, to PCR-ITS1 in simulation model of L. martiniquensis infection in Sergentomyia gemmea sand flies. Attributable to the sensitivity and specificity, PCR-mkDNA was consecutively applied to detect L. martiniquensis in 380 female sand fly individuals captured in the newly identified affected region of Lamphun Province, Thailand. FINDINGS AND MAIN CONCLUSIONS Results showed that PCR-mkDNA could detect at least one promastigote per sand fly, which was 10-time superior to LAMP and PCR-ITS1. In addition, PCR-mkDNA was more specific, able to differentiate L. martiniquensis from other viscerotropic Leishmania species, such as L. siamensis, L. (L.) donovani, and L. (L.) infantum. Consecutively, mass screening of L. martiniquensis in 380 female sand fly individuals by PCR-mkDNA was implemented in a new affected area of Thailand where a patient with leishmaniasis/HIV co-infection resides; however Leishmania DNA was undetected. In conclusion, PCR-mkDNA is a promising tool for molecular mass screening of L. martiniquensis

Efficacy of In Vivo Electroporation-Mediated IL-10 Gene Delivery on Survival of Skin Flaps.

Science.gov (United States)

Seyed Jafari, S Morteza; Shafighi, Maziar; Beltraminelli, Helmut; Weber, Benedikt; Schmid, Ralph A; Geiser, Thomas; Gazdhar, Amiq; Hunger, Robert E

2018-04-01

Despite advances in understanding the underlying mechanisms of flap necrosis and improvement in surgical techniques, skin flap necrosis after reconstructive surgery remains a crucial issue. We investigated the efficacy of electroporation-mediated IL-10 gene transfer to random skin flap with an aim to accelerate wound healing and improve skin flap survival. Nine male Wistar rats (300-330 g) were divided in two groups (a) control group (n = 5), only surgery no gene transfer, and (b) experimental group, received electroporation-mediated IL-10 gene transfer 24 h before the surgery as prophylaxis (n = 4). Random skin flap (McFarlane) was performed in both groups. Planimetry, Laser Doppler imaging, and immunohistochemistry were used to evaluate the effect of IL-10 gene transfer between study groups at day 7. Electroporation-mediated IL-10 gene transfer decreased percentage of flap necrosis (p value = 0.0159) and increased cutaneous perfusion compared to the control group (p value = 0.0159). In addition, Spearman's rank correlation showed a significant negative correlation between percentage of flap necrosis and Laser Index (p value = 0.0083, r -0.83, respectively). Furthermore, significantly higher mean CD31 + vessel density was detected in the experimental group compared to the control group (p value = 0.0159). Additionally, semi-quantitative image analysis showed lower inflammatory cell count in experimental group compared to control group (p value = 0.0317). In vivo electroporation-mediated IL-10 gene transfer reduced necrosis, enhanced survival and vascularity in the ischemic skin flap.

tRNA--the golden standard in molecular biology.

Science.gov (United States)

Barciszewska, Mirosława Z; Perrigue, Patrick M; Barciszewski, Jan

2016-01-01

Transfer RNAs (tRNAs) represent a major class of RNA molecules. Their primary function is to help decode a messenger RNA (mRNA) sequence in order to synthesize protein and thus ensures the precise translation of genetic information that is imprinted in DNA. The discovery of tRNA in the late 1950's provided critical insight into a genetic machinery when little was known about the central dogma of molecular biology. In 1965, Robert Holley determined the first nucleotide sequence of alanine transfer RNA (tRNA(Ala)) which earned him the 1968 Nobel Prize in Physiology or Medicine. Today, tRNA is one of the best described and characterized biological molecules. Here we review some of the key historical events in tRNA research which led to breakthrough discoveries and new developments in molecular biology.

Spectroscopy on metal-halide lamps under varying gravity conditions

NARCIS (Netherlands)

Flikweert, A.J.

2008-01-01

Worldwide, 20% of all electricity is used for lighting. For this reason, efficient lamps are economically and ecologically important. High intensity discharge (HID) lamps are efficient lamps. The most common HID lamp these days is the metal-halide (MH) lamp. MH lamps have a good colour rendering

A method of combined single-cell electrophysiology and electroporation.

Science.gov (United States)

Graham, Lyle J; Del Abajo, Ricardo; Gener, Thomas; Fernandez, Eduardo

2007-02-15

This paper describes a method of extracellular recording and subsequent electroporation with the same electrode in single retinal ganglion cells in vitro. We demonstrate anatomical identification of neurons whose receptive fields were measured quantitatively. We discuss how this simple method should also be applicable for the delivery of a variety of intracellular agents, including gene delivery, to physiologically characterized neurons, both in vitro and in vivo.

CALiPER Retail Lamps Study 3

Energy Technology Data Exchange (ETDEWEB)

Royer, Michael P. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Beeson, Tracy A. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

2014-02-01

The CALiPER program first began investigating LED lamps sold at retail stores in 2010, purchasing 33 products from eight retailers and covering six product categories. The findings revealed a fragmented marketplace, with large disparities in performance of different products, accuracy of manufacturer claims, and offerings from different retail outlets. Although there were some good products, looking back many would not be considered viable competitors to other available options, with too little lumen output, not high enough efficacy, or poor color quality. CALiPER took another look in late 2011purchasing 38 products of five different types from nine retailers and the improvement was marked. Performance was up; retailer claims were more accurate; and the price per lumen and price per unit efficacy were down, although the price per product had not changed much. Nonetheless, there was still plenty of room for improvement, with the performance of LED lamps not yet reaching that of well-established classes of conventional lamps (e.g., 75 W incandescent A19 lamps). Since the second retail lamp study was published in early 2012, there has been substantial progress in all aspects of LED lamps available from retailers. To document this progress, CALiPER again purchased a sample of lamps from retail stores 46 products in total, focusing on A19, PAR30, and MR16 lamps but instead of a random sample, sought to select products to answer specific hypotheses about performance. These hypotheses focused on expanding ranges of LED equivalency, the accuracy of lifetime claims, efficacy and price trends, as well as changes to product designs. Among other results, key findings include: There are now very good LED options to compete with 60 W, 75 W, and 100 W incandescent A19 lamps, and 75 W halogen PAR30 lamps. MR16 lamps have shown less progress, but there are now acceptable alternatives to 35 W, 12 V halogen MR16 lamps and 50 W, 120 V halogen MR16 lamps for some applications. Other

Trypanosome RNA editing: the complexity of getting U in and taking U out

Czech Academy of Sciences Publication Activity Database

Read, L. K.; Lukeš, Julius; Hashimi, Hassan

2016-01-01

Roč. 7, č. 1 (2016), s. 33-51 ISSN 1757-7004 R&D Projects: GA ČR GA15-21974S EU Projects: European Commission(XE) 289007 Institutional support: RVO:60077344 Keywords : messenger RNA * guide RNA * mitochondria Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.838, year: 2016

Electroporation - a biophysical method for transferring nano-sized systems and drugs in vitro and in vivo

International Nuclear Information System (INIS)

Nikolova, B.; Atanasova, S.; Pehlivanova, V.; Jelev, J.; Tsoneva, Y.; Bakalova, R.; Peycheva, E.

2017-01-01

The aim of this study was to investigate the electrostatic internalisation of quinatum dots (QDs) and QDs containing nano-hydrogels in the Colon 26 cell line and their effect on cell viability as well as their passive and electrically mediated delivery in solid murine tumor models. Materials and methods: Colon 26 cancer cell line was used for in vitro experiments, survival was followed by a MTS test, and images were obtained by confocal microscopy. For in vivo experiments, mouse models with implanted Colon 26 cells were used. All in vivo measurements are carried out ~ 9-10 days after the inoculation, when the tumor size is ~ 100 mm 3 . Results: Electroporation facilitates the delivery of nanoparticles - both in vivo and in vitro. We demonstrate that increasing the applied tension leads to increased nanoparticle penetration into the cells without significantly reducing cell survival. The penetration of nano-hydrogels into tumor tissue is visualized by fluorescence imaging and MRI. The highest intensity of the tumor signal was recorded 30 minutes after the combined treatment (electroporation and QDs loaded nano-hydrogels), even 48 hours post electroporation. The data show a more efficient penetration and long retention of nanoparticles in the tumor after electroporation, due to the increased permeability of the cell membranes and local cleavage of the blood vessels. Conclusion: The internalization and retention of nano-hydrogels is a promising tool both in future strategies for the treatment of cancer and nano medicine. [bg

UHP lamp systems for projection applications

International Nuclear Information System (INIS)

Derra, Guenther; Moench, Holger; Fischer, Ernst; Giese, Hermann; Hechtfischer, Ulrich; Heusler, Gero; Koerber, Achim; Niemann, Ulrich; Noertemann, Folke-Charlotte; Pekarski, Pavel; Pollmann-Retsch, Jens; Ritz, Arnd; Weichmann, Ulrich

2005-01-01

Projection systems have found widespread use in conference rooms and other professional applications during the last decade and are now entering the home TV market at a considerable pace. Projectors as small as about one litre are able to deliver several thousand screen lumens and are, with a system efficacy of over 10 lm W -1 , the most efficient display systems realized today. Short arc lamps are a key component for projection systems of the highest efficiency for small-size projection displays. The introduction of the ultra high performance (UHP) lamp system by Philips in 1995 can be identified as one of the key enablers of the commercial success of projection systems. The UHP lamp concept features outstanding arc luminance, a well suited spectrum, long life and excellent lumen maintenance. For the first time it combines a very high pressure mercury discharge lamp with extremely short and stable arc gap with a regenerative chemical cycle keeping the discharge walls free from blackening, leading to lifetimes of over 10 000 h. Since the introduction of the UHP lamp system, many important new technology improvements have been realized: burner designs for higher lamp power, advanced ignition systems, miniaturized electronic drivers and innovative reflector concepts. These achievements enabled the impressive increase of projector light output, a remarkable reduction in projector size and even higher optical efficiency in projection systems during the last years. In this paper the concept of the UHP lamp system is described, followed by a discussion of the technological evolution the UHP lamp has undergone so far. Last, but not least, the important improvements of the UHP lamp system including the electronic driver and the reflector are discussed. (review article)

Reproducible pattern of microRNA in normal human skin

DEFF Research Database (Denmark)

Holst, Line; Kaczkowski, Bogumil; Gniadecki, Robert

2010-01-01

RNA expression pattern in normal human skin. Here we investigated miRNA expression profiles from skin biopsies of 8 healthy volunteers taken from sun protected and mildly photo damaged skin using the modified protocol for miRNA extraction. We were able to show a constant pattern of miRNA expression between......MicroRNAs (miRNAs) regulate cell growth, differentiation and apoptosis via specific targeting of messenger RNA (mRNA). Aberrant mRNA expression contributes to pathological processes such as carcinogenesis. To take advantage of miRNA profiling in skin disease it is essential to investigate mi...... different individuals. We did not find any significant differences in miRNA expression between sun protected and mildly photodamaged skin. These results may be valuable for future design of studies on miRNA expression in skin disease....

Reproducible pattern of microRNA in normal human skin

DEFF Research Database (Denmark)

Holst, Line; Kaczkowski, Bogumil; Gniadecki, Robert

2010-01-01

RNA expression pattern in normal human skin. Here we investigated miRNA expression profiles from skin biopsies of 8 healthy volunteers taken from sun protected and mildly photo damaged skin using the modified protocol for miRNA extraction. We were able to show a constant pattern of miRNA expression between...... different individuals. We did not find any significant differences in miRNA expression between sun protected and mildly photodamaged skin. These results may be valuable for future design of studies on miRNA expression in skin disease.......MicroRNAs (miRNAs) regulate cell growth, differentiation and apoptosis via specific targeting of messenger RNA (mRNA). Aberrant mRNA expression contributes to pathological processes such as carcinogenesis. To take advantage of miRNA profiling in skin disease it is essential to investigate mi...

Analysis of the performance of domestic lighting lamps

International Nuclear Information System (INIS)

Aman, M.M.; Jasmon, G.B.; Mokhlis, H.; Bakar, A.H.A.

2013-01-01

The power crisis problem is getting worse in the developing countries. Measures are being taken to overcome the power shortage problem by efficiently utilizing the available power. Replacement of high-power consumption lamps with energy efficient lamps is also among these steps. This paper presents a detailed comparative analysis between domestic lighting lamps (DLLs) use for producing artificial light. DLLs include incandescent lamp (IL), fluorescent lamp (FL) and compact fluorescent lamp (CFL). Light emitting diodes (LED) based lamp technology is relatively new in comparison with conventional incandescent and discharge lamps. However, the present study will also cover the LED lamps. Power quality based experiments have been conducted on DLLs in Power System Laboratory and power consumption based calculations are carried out using the lighting design software DIALux. The result shows that with the current technology, the use of FL and LED lamp is beneficial for utility as well as for consumer. However, with the current pace in the development of LED technology, it is possible LED lamps will lead the lighting market in the near future. The paper has also presented the uncertainties that exist in lighting market and proposed the guidelines that will help in making future energy policy. - Highlights: ► Performances of domestic lighting lamps are compared. ► Power quality and power consumption based case study results are presented. ► For future energy policies, recommendations are also given.

Solid-state semiconductors are better alternatives to arc-lamps for efficient and uniform illumination in minimal access surgery.

Science.gov (United States)

Lee, Alex C H; Elson, Daniel S; Neil, Mark A; Kumar, Sunil; Ling, Bingo W; Bello, Fernando; Hanna, George B

2009-03-01

Current arc-lamp illumination systems have a number of technical and ergonomic limitations. White light-emitting diodes (LEDs) are energy-efficient solid-state lighting devices which are small, durable and inexpensive. Their use as an alternative to arc-lamp light sources in minimal access surgery has not been explored. This study aims to develop an LED-based endo-illuminator and to determine its lighting characteristics for use in minimal access surgery. We developed an LED endo-illuminator using a white LED mounted at the tip of a steel rod. Offline image analysis was carried out to compare the illuminated field using the LED endo-illuminator or an arc-lamp based endoscope in terms of uniformity, shadow sharpness and overall image intensity. Direct radiometric power measurements in light intensity and stability were obtained. Visual perception of fine details at the peripheral endoscopic field was assessed by 13 subjects using the different illumination systems. Illumination from the LED endo-illuminator was more uniform compared to illumination from an arc-lamp source, especially at the closer distance of 4 cm (0.0006 versus 0.0028 arbitrary units--lower value indicates more uniform illumination). The shadows were also sharper (edge widths of 16 versus 44 pixels for the first edge and 15 versus 61 pixels for the second edge). The overall mean image intensity was higher (127 versus 100 arbitrary units) when using the autoshutter mode despite the lower direct radiometric power, about one tenth of the arc-lamp endoscopic system. The illumination was also more stable with less flickering (0.02% versus 5% of total power in non-DC components). Higher median scores on visual perception was also obtained (237 versus 157, p arc-lamp-based system currently used.

Inductive tuners for microwave driven discharge lamps

Science.gov (United States)

Simpson, James E.

1999-01-01

An RF powered electrodeless lamp utilizing an inductive tuner in the waveguide which couples the RF power to the lamp cavity, for reducing reflected RF power and causing the lamp to operate efficiently.

A comparison of cell-free placental messenger ribonucleic acid and color Doppler ultrasound for the prediction of placental invasion in patients with placenta accreta

OpenAIRE

Naghshineh, Elham; Khorvash, Elahe; Kamali, Sara

2015-01-01

Background: The aim of the present study was to comparison between cell-free placental messenger ribonucleic acid (mRNA) and Doppler ultrasound for the prediction of placental invasion in women with placenta accreta. Materials and Methods: In this cross-sectional study, 50 pregnant women at risk for placenta accreta underwent color Doppler and assessment of cell-free placental mRNA. Real-time reverse-transcription polymerase chain reaction was used for measurement of cell-free placental m...

Skin-Resident T Cells Drive Dermal Dendritic Cell Migration in Response to Tissue Self-Antigen.

Science.gov (United States)

Ali, Niwa; Zirak, Bahar; Truong, Hong-An; Maurano, Megan M; Gratz, Iris K; Abbas, Abul K; Rosenblum, Michael D

2018-05-01

Migratory dendritic cell (DC) subsets deliver tissue Ags to draining lymph nodes (DLNs) to either initiate or inhibit T cell-mediated immune responses. The signals mediating DC migration in response to tissue self-antigen are largely unknown. Using a mouse model of inducible skin-specific self-antigen expression, we demonstrate that CD103 + dermal DCs (DDCs) rapidly migrate from skin to skin DLN (SDLNs) within the first 48 h after Ag expression. This window of time was characterized by the preferential activation of tissue-resident Ag-specific effector T cells (Teffs), with no concurrent activation of Ag-specific Teffs in SDLNs. Using genetic deletion and adoptive transfer approaches, we show that activation of skin-resident Teffs is required to drive CD103 + DDC migration in response to tissue self-antigen and this Batf3-dependent DC population is necessary to mount a fulminant autoimmune response in skin. Conversely, activation of Ag-specific Teffs in SDLNs played no role in DDC migration. Our studies reveal a crucial role for skin-resident T cell-derived signals, originating at the site of self-antigen expression, to drive DDC migration during the elicitation phase of an autoimmune response. Copyright © 2018 by The American Association of Immunologists, Inc.

A small RNA activates CFA synthase by isoform-specific mRNA stabilization.

Science.gov (United States)

Fröhlich, Kathrin Sophie; Papenfort, Kai; Fekete, Agnes; Vogel, Jörg

2013-11-13

Small RNAs use a diversity of well-characterized mechanisms to repress mRNAs, but how they activate gene expression at the mRNA level remains not well understood. The predominant activation mechanism of Hfq-associated small RNAs has been translational control whereby base pairing with the target prevents the formation of an intrinsic inhibitory structure in the mRNA and promotes translation initiation. Here, we report a translation-independent mechanism whereby the small RNA RydC selectively activates the longer of two isoforms of cfa mRNA (encoding cyclopropane fatty acid synthase) in Salmonella enterica. Target activation is achieved through seed pairing of the pseudoknot-exposed, conserved 5' end of RydC to an upstream region of the cfa mRNA. The seed pairing stabilizes the messenger, likely by interfering directly with RNase E-mediated decay in the 5' untranslated region. Intriguingly, this mechanism is generic such that the activation is equally achieved by seed pairing of unrelated small RNAs, suggesting that this mechanism may be utilized in the design of RNA-controlled synthetic circuits. Physiologically, RydC is the first small RNA known to regulate membrane stability.

A review of electroporation-based antitumor skin therapies and investigation of betulinic acid-loaded ointment.

Science.gov (United States)

Bakonyi, Monika; Berko, Szilvia; Eros, Gabor; Varju, Gabor; Dehelean, Cristina; Szucs, Maria Budai; Csanyi, Erzsebet

2017-11-13

Electrochemotherapy is a novel treatment for cutaneous and subcutaneous tumors utilizing the combination of electroporation and chemotherapeutic agents. Since tumors have an increasing incidence nowadays as a result of environmental and genetic factors, electrochemotherapy could be a promising treatment for cancer patients. The aim of this article is to summarize the novel knowledge about the use of electroporation for antitumor treatments and to present a new application of electrochemotherapy with a well-known plant derived antitumor drug betulinic acid. For the review we have searched the databases of scientific and medical research to collect the available publications about the use of electrochemotherapy in the treatment of various types of cancer. By the utilization of the available knowledge, we investigated the effect of electroporation on the penetration of a topically applied betulinic acid formulation into the skin by ex vivo Raman spectroscopy on hairless mouse skin Results: Raman measurements have demonstrated that the penetration depth of betulinic acid can be remarkably ameliorated by the use of electroporation, so this protocol can be a possibility for the treatment of deeper localized cancer nodules. Furthermore, it proved the influence of various treatment times, since they caused different spatial distributions of the drug in the skin. The review demonstrates that electrochemotherapy is a promising tool to treat different kinds of tumors with high efficiency and with only a few moderate adverse effects. Moreover, it presents a non-invasive method to enhance the penetration of antitumor agents, which can offer novel prospects for antitumor therapies. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Influence of DMPS on the water retention capacity of electroporated stratum corneum: ATR-FTIR study.

Science.gov (United States)

Sckolnick, Maria; Hui, Sek-Wen; Sen, Arindam

2008-02-28

Anionic lipids like phosphatidylserine are known to significantly enhance electroporation mediated transepidermal transport of polar solutes of molecular weights up to 10kDa. The underlying mechanism of the effect of anionic lipids on transdermal transport is not fully understood. The main barrier to transdermal transport lies within the intercellular lipid matrix (ILM) of the stratum corneum (SC) and our previous studies indicate that dimyristoyl phosphatidylserine (DMPS) can perturb the packing of this lipid matrix. Here we report on our investigation on water retention in the SC following electroporation in the presence and the absence of DMPS. The water content in the outer most layers of the SC of full thickness porcine skin was determined using ATR-FTIR-spectroscopy. The results show that in the presence of DMPS, the SC remains in a state of enhanced hydration for longer periods after electroporation. This increase in water retention in the SC by DMPS is likely to play an important role in trans-epidermal transport, since improved hydration of the skin barrier can be expected to increase the partitioning of polar solutes and possibly the permeability.

Visualization through Magnetic Resonance Imaging of DNA Internalized Following “In Vivo” Electroporation

Directory of Open Access Journals (Sweden)

Simonetta Geninatti Crich

2005-01-01

Full Text Available The ability to visualize plasmid DNA entrapment in muscle cells undergoing an “in vivo” electroporation treatment was investigated on BALB/c mice using a 7-T magnetic resonance imaging (MRI scanner using the paramagnetic Gd–DOTA–spd complex as imaging reporter. Gd–DOTA–spd bears a tripositively charged spermidine residue that yields a strong binding affinity toward the negatively charged DNA chain (6.4 kb, Ka = 2.2 × 103 M−1 for approximately 2500 ± 500 binding sites. Cellular colocalization of Gd-DOTA-spd and plasmid DNA has been validated by histological analysis of excised treated muscle. In vivo MRI visualization of Gd-DOTA-spd distribution provides an excellent route to access the cellular entrapment of plasmid DNA upon applying an electroporation pulse.

DC-SIGN, a C-type lectin on dendritic cells that unveils many aspects of dendritic cell biology

NARCIS (Netherlands)

Geijtenbeek, Teunis B. H.; Engering, Anneke; van Kooyk, Yvette

2002-01-01

Dendritic cells (DC) are present in essentially every tissue where they operate at the interface of innate and acquired immunity by recognizing pathogens and presenting pathogen-derived peptides to T cells. It is becoming clear that not all C-type lectins on DC serve as antigen receptors recognizing

Injection molded chips with integrated conducting polymer electrodes for electroporation of cells

DEFF Research Database (Denmark)

Andresen, Kristian; Hansen, Morten; Matschuk, Maria

2010-01-01

We present the design-concept for an all polymer injection molded single use microfluidic device. The fabricated devices comprise integrated conducting polymer electrodes and Luer fitting ports to allow for liquid and electrical access. A case study of low voltage electroporation of biological...

A method of genetically engineering acidophilic, heterotrophic, bacteria by electroporation and conjugation

Energy Technology Data Exchange (ETDEWEB)

Roberto, F.F.; Glenn, A.W.; Ward, T.E.

1990-08-07

A method of genetically manipulating an acidophilic bacteria is provided by two different procedures. Using electroporation, chimeric and broad-host range plasmids are introduced into Acidiphilium. Conjugation is also employed to introduce broad-host range plasmids into Acidiphilium at neutral pH.

Efficient ex vivo delivery of chemically modified messenger RNA using lipofection and magnetofection.

Science.gov (United States)

Badieyan, Zohreh Sadat; Pasewald, Tamara; Mykhaylyk, Olga; Rudolph, Carsten; Plank, Christian

2017-01-22

Recently, chemically modified mRNA (cmRNA) therapeutics have been the subject of extensive application-oriented research in both academia and industry as a safer alternative for gene and recombinant protein therapies. However, the lack of an efficient delivery system hinders widespread application. Here we used ∼100-nm lipoplexes and magnetic lipoplexes that can protect cmRNA from RNases and efficiently deliver it into muscle and fat tissues as well as to the endothelium of the carotid artery. Establishing magnetofection for ex vivo cmRNA delivery for the first time, we suggest this method for potential enhanced and targeted delivery of cmRNA. This study introduces optimal cmRNA complexes with high ex vivo efficiency as good candidates for further in vivo cmRNA delivery. Copyright © 2016 Elsevier Inc. All rights reserved.

Pre-mRNA Splicing in Plants: In Vivo Functions of RNA-Binding Proteins Implicated in the Splicing Process

Directory of Open Access Journals (Sweden)

Katja Meyer

2015-07-01

Full Text Available Alternative pre-messenger RNA splicing in higher plants emerges as an important layer of regulation upon exposure to exogenous and endogenous cues. Accordingly, mutants defective in RNA-binding proteins predicted to function in the splicing process show severe phenotypic alterations. Among those are developmental defects, impaired responses to pathogen threat or abiotic stress factors, and misregulation of the circadian timing system. A suite of splicing factors has been identified in the model plant Arabidopsis thaliana. Here we summarize recent insights on how defects in these splicing factors impair plant performance.

Conformational Selection and Induced Fit for RNA Polymerase and RNA/DNA Hybrid Backtracked Recognition

Directory of Open Access Journals (Sweden)

Haifeng eChen

2015-11-01

Full Text Available RNA polymerase catalyzes transcription with a high fidelity. If DNA/RNA mismatch or DNA damage occurs downstream, a backtracked RNA polymerase can proofread this situation. However, the backtracked mechanism is still poorly understood. Here we have performed multiple explicit-solvent molecular dynamics (MD simulations on bound and apo DNA/RNA hybrid to study backtracked recognition. MD simulations at room temperature suggest that specific electrostatic interactions play key roles in the backtracked recognition between the polymerase and DNA/RNA hybrid. Kinetics analysis at high temperature shows that bound and apo DNA/RNA hybrid unfold via a two-state process. Both kinetics and free energy landscape analyses indicate that bound DNA/RNA hybrid folds in the order of DNA/RNA contracting, the tertiary folding and polymerase binding. The predicted Φ-values suggest that C7, G9, dC12, dC15 and dT16 are key bases for the backtracked recognition of DNA/RNA hybrid. The average RMSD values between the bound structures and the corresponding apo ones and Kolmogorov-Smirnov (KS P test analyses indicate that the recognition between DNA/RNA hybrid and polymerase might follow an induced fit mechanism for DNA/RNA hybrid and conformation selection for polymerase. Furthermore, this method could be used to relative studies of specific recognition between nucleic acid and protein.

Translation affects YoeB and MazF messenger RNA interferase activities by different mechanisms

DEFF Research Database (Denmark)

Christensen-Dalsgaard, Mikkel; Gerdes, Kenn

2008-01-01

Prokaryotic toxin-antitoxin loci encode mRNA cleaving enzymes that inhibit translation. Two types are known: those that cleave mRNA codons at the ribosomal A site and those that cleave any RNA site specifically. RelE of Escherichia coli cleaves mRNA at the ribosomal A site in vivo and in vitro bu...

Propagation of ionization waves during ignition of fluorescent lamps

International Nuclear Information System (INIS)

Langer, R; Tidecks, R; Horn, S; Garner, R; Hilscher, A

2008-01-01

The propagation of the first ionization wave in a compact fluorescent lamp (T4 tube with standard electrodes) during ignition was investigated for various initial dc-voltages (both polarities measured against ground) and gas compositions (with and without mercury). In addition the effect of the presence of a fluorescent powder coating was studied. The propagation velocity of the initial wave was measured by an assembly of photomultipliers installed along the tube, which detected the light emitted by the wave head. The propagation was found to be faster for positive than for negative polarity. This effect is explained involving processes in the electrode region as well as in the wave head. Waves propagate faster in the presence of a fluorescent powder coating than without it and gases of lighter mass show a faster propagation than gases with higher mass

Transforming growth factor-beta messenger RNA and protein in murine colitis

DEFF Research Database (Denmark)

Whiting, C V; Williams, A M; Claesson, Mogens Helweg

2001-01-01

Using a CD4+ T-cell-transplanted SCID mouse model of colitis, we have analyzed TGF-beta transcription and translation in advanced disease. By in situ hybridization, the epithelium of both control and inflamed tissues transcribed TGF-beta1 and TGF-beta3 mRNAs, but both were expressed significantly...... farther along the crypt axis in disease. Control lamina propria cells transcribed little TGF-beta1 or TGF-beta3 mRNA, but in inflamed tissues many cells expressed mRNA for both isoforms. No TGF-beta2 message was detected in either control or inflamed tissues. Immunohistochemistry for latent and active TGF...

Geometric Modelling of Octagonal Lamp Poles

Science.gov (United States)

Chan, T. O.; Lichti, D. D.

2014-06-01

Lamp poles are one of the most abundant highway and community components in modern cities. Their supporting parts are primarily tapered octagonal cones specifically designed for wind resistance. The geometry and the positions of the lamp poles are important information for various applications. For example, they are important to monitoring deformation of aged lamp poles, maintaining an efficient highway GIS system, and also facilitating possible feature-based calibration of mobile LiDAR systems. In this paper, we present a novel geometric model for octagonal lamp poles. The model consists of seven parameters in which a rotation about the z-axis is included, and points are constrained by the trigonometric property of 2D octagons after applying the rotations. For the geometric fitting of the lamp pole point cloud captured by a terrestrial LiDAR, accurate initial parameter values are essential. They can be estimated by first fitting the points to a circular cone model and this is followed by some basic point cloud processing techniques. The model was verified by fitting both simulated and real data. The real data includes several lamp pole point clouds captured by: (1) Faro Focus 3D and (2) Velodyne HDL-32E. The fitting results using the proposed model are promising, and up to 2.9 mm improvement in fitting accuracy was realized for the real lamp pole point clouds compared to using the conventional circular cone model. The overall result suggests that the proposed model is appropriate and rigorous.

Performance of ARCHITECT HCV core antigen test with specimens from US plasma donors and injecting drug users.

Science.gov (United States)

Mixson-Hayden, Tonya; Dawson, George J; Teshale, Eyasu; Le, Thao; Cheng, Kevin; Drobeniuc, Jan; Ward, John; Kamili, Saleem

2015-05-01

Hepatitis C virus (HCV) core antigen is a serological marker of current HCV infection. The aim of this study was mainly to evaluate the performance characteristics of the ARCHITECT HCV core antigen assay with specimens from US plasma donors and injecting drug users. A total of 551 serum and plasma samples with known anti-HCV and HCV RNA status were tested for HCV core antigen using the Abbott ARCHITECT HCV core antigen test. HCV core antigen was detectable in 100% of US plasma donor samples collected during the pre-seroconversion phase of infection (anti-HCV negative/HCV RNA positive). Overall sensitivity of the HCV core antigen assay was 88.9-94.3% in samples collected after seroconversion. The correlation between HCV core antigen and HCV RNA titers was 0.959. HCV core antigen testing may be reliably used to identify current HCV infection. Published by Elsevier B.V.

Increased efficiency of exogenous messenger RNA translation in a Krebs ascites cell lysate.

Science.gov (United States)

Metafora, S; Terada, M; Dow, L W; Marks, P A; Bank, A

1972-05-01

Addition of a 0.5 M KCl wash fraction from rabbit reticulocyte ribosomes causes a 3- to 10-fold increase in the extent of translation of natural mRNAs by Krebs-cell lysates. In the presence of the wash fraction, 1 pmol of rabbit or mouse 10S RNA directs the incorporation of 80 pmol of leucine into rabbit globin. The addition of human 10S RNA results in the synthesis of equal amounts of human alpha and beta chains, identified by column chromatography. The stimulation by the wash fraction is almost completely dependent on added mammalian tRNA. In contrast to the wash fraction from rabbit reticulocytes, the wash fraction isolated from Krebs-cell ribosomes is inhibitory to both endogenous and exogenous mRNA translation. The stimulation by the wash fraction from rabbit ribosomes is not specific for globin mRNAs, but also increases endogenous, phage Qbeta, and viral RNA-directed protein synthesis.

Careful treatment planning enables safe ablation of liver tumors adjacent to major blood vessels by percutaneous irreversible electroporation (IRE

Directory of Open Access Journals (Sweden)

Kos Bor

2015-09-01

Full Text Available Background. Irreversible electroporation (IRE is a tissue ablation method, which relies on the phenomenon of electroporation. When cells are exposed to a sufficiently electric field, the plasma membrane is disrupted and cells undergo an apoptotic or necrotic cell death. Although heating effects are known IRE is considered as non-thermal ablation technique and is currently applied to treat tumors in locations where thermal ablation techniques are contraindicated.

30 CFR 75.1703 - Portable electric lamps.

Science.gov (United States)

2010-07-01

... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Portable electric lamps. 75.1703 Section 75... HEALTH MANDATORY SAFETY STANDARDS-UNDERGROUND COAL MINES Miscellaneous § 75.1703 Portable electric lamps. [Statutory Provisions] Persons underground shall use only permissible electric lamps approved by the...

Single Cell Electroporation Method for Mammalian CNS Neurons in Organotypic Slice Cultures

Science.gov (United States)

Uesaka, Naofumi; Hayano, Yasufumi; Yamada, Akito; Yamamoto, Nobuhiko

Axon tracing is an essential technique to study the projection pattern of neurons in the CNS. Horse radish peroxidase and lectins have contributed to revealing many neural connection patterns in the CNS (Itaya and van Hoesen, 1982; Fabian and Coulter, 1985; Yoshihara, 2002). Moreover, a tracing method with fluorescent dye has enabled the observation of growing axons in living conditions, and demon strated a lot of developmental aspects in axon growth and guidance (Harris et al., 1987; O'Rourke and Fraser, 1990; Kaethner and Stuermer, 1992; Halloran and Kalil, 1994; Yamamoto et al., 1997). More recently, genetically encoded fluores cent proteins can be used as a powerful tool to observe various biological events. Several gene transfer techniques such as microinjection, biolistic gene gun, viral infection, lipofection and transgenic technology have been developed (Feng et al., 2000; Ehrengruber et al., 2001; O'Brien et al., 2001; Ma et al., 2002; Sahly et al., 2003). In particular, the electroporation technique was proved as a valuable tool, since it can be applied to a wide range of tissues and cell types with little toxicity and can be performed with relative technical easiness. Most methods, including a stand ard electroporation technique, are suitable for gene transfer to a large number of cells. However, this is not ideal for axonal tracing, because observation of individ ual axons is occasionally required. To overcome this problem, we have developed an electroporation method using glass micropipettes containing plasmid solutions and small current injection. Here we introduce the method in detail and exemplified results with some example applications and discuss its usefulness.

Modulation of antigen presenting cell functions during chronic HPV infection

Directory of Open Access Journals (Sweden)

Abate Assefa Bashaw

2017-12-01

Full Text Available High-risk human papillomaviruses (HR-HPV infect basal keratinocytes, where in some individuals they evade host immune responses and persist. Persistent HR-HPV infection of the cervix causes precancerous neoplasia that can eventuate in cervical cancer. Dendritic cells (DCs are efficient in priming/cross-priming antigen-specific T cells and generating antiviral and antitumor cytotoxic CD8+ T cells. However, HR-HPV have adopted various immunosuppressive strategies, with modulation of DC function crucial to escape from the host adaptive immune response. HPV E6 and E7 oncoproteins alter recruitment and localization of epidermal DCs, while soluble regulatory factors derived from HPV-induced hyperplastic epithelium change DC development and influence initiation of specific cellular immune responses. This review focuses on current evidence for HR-HPV manipulation of antigen presentation in dendritic cells and escape from host immunity.

SV40 T antigen alone drives karyotype instability that precedes neoplastic transformation of human diploid fibroblasts.

Science.gov (United States)

Ray, F A; Peabody, D S; Cooper, J L; Cram, L S; Kraemer, P M

1990-01-01

To define the role of SV40 large T antigen in the transformation and immortalization of human cells, we have constructed a plasmid lacking most of the unique coding sequences of small t antigen as well as the SV40 origin of replication. The promoter for T antigen, which lies within the origin of replication, was deleted and replaced by the Rous sarcoma virus promoter. This minimal construct was co-electroporated into normal human fibroblasts of neonatal origin along with a plasmid containing the neomycin resistance gene (neo). Three G418-resistant, T antigen-positive clones were expanded and compared to three T antigen-positive clones that received the pSV3neo plasmid (capable of expressing large and small T proteins and having two origins of replication). Autonomous replication of plasmid DNA was observed in all three clones that received pSV3neo but not in any of the three origin minus clones. Immediately after clonal expansion, several parameters of neoplastic transformation were assayed. Low percentages of cells in T antigen-positive populations were anchorage independent or capable of forming colonies in 1% fetal bovine serum. The T antigen-positive clones generally exhibited an extended lifespan in culture but rarely became immortalized. Large numbers of dead cells were continually generated in all T antigen-positive, pre-crisis populations. Ninety-nine percent of all T antigen-positive cells had numerical or structural chromosome aberrations. Control cells that received the neo gene did not have an extended life span, did not have noticeable numbers of dead cells, and did not exhibit karyotype instability. We suggest that the role of T antigen protein in the transformation process is to generate genetic hypervariability, leading to various consequences including neoplastic transformation and cell death.

MicroRNA-22 impairs anti-tumor ability of dendritic cells by targeting p38.

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Xue Liang

Full Text Available Dendritic cells (DCs play a critical role in triggering anti-tumor immune responses. Their intracellular p38 signaling is of great importance in controlling DC activity. In this study, we identified microRNA-22 (miR-22 as a microRNA inhibiting p38 protein expression by directly binding to the 3' untranslated region (3'UTR of its mRNA. The p38 down-regulation further interfered with the synthesis of DC-derived IL-6 and the differentiation of DC-driven Th17 cells. Moreover, overexpression of miR-22 in DCs impaired their tumor-suppressing ability while miR-22 inhibitor could reverse this phenomenon and improve the curative effect of DC-based immunotherapy. Thus, our results highlight a suppressive role for miR-22 in the process of DC-invoked anti-tumor immunity and that blocking this microRNA provides a new strategy for generating potent DC vaccines for patients with cancer.

Holographic gauge mediation via strongly coupled messengers

International Nuclear Information System (INIS)

McGuirk, Paul; Shiu, Gary; Sumitomo, Yoske

2010-01-01

We consider a relative of semidirect gauge mediation where the hidden sector exists at large 't Hooft coupling. Such scenarios can be difficult to describe using perturbative field theory methods but may fall into the class of holographic gauge mediation scenarios, meaning that they are amenable to the techniques of gauge/gravity duality. We use a recently found gravity solution to examine one such case, where the hidden sector is a cascading gauge theory resulting in a confinement scale not much smaller than the messenger mass. In the original construction of holographic gauge mediation, as in other examples of semidirect gauge mediation at strong coupling, the primary contributions to visible sector soft terms come from weakly coupled messenger mesons. In contrast to these examples, we describe the dual of a gauge theory where there are significant contributions from scales in which the strongly coupled messenger quarks are the effective degrees of freedom. In this regime, the visible sector gaugino mass can be calculated entirely from holography.

Fluorescent discharge lamp

Science.gov (United States)

Mukai, E.; Otsuka, H.; Nomi, K.; Honmo, I.

1982-01-01

A rapidly illuminating fluorescent lamp 1,200 mm long and 32.5 mm in diameter with an interior conducting strip which is compatible with conventional fixtures and ballasts is described. The fluorescent lamp is composed of a linear glass tube, electrodes sealed at both ends, mercury and raregas sealed in the glass tube, a fluorescent substance clad on the inner walls of the glass tube, and a clad conducting strip extending the entire length of the glass tube in the axial direction on the inner surface of the tube.

Detection of AR-V7 mRNA in whole blood may not predict the effectiveness of novel endocrine drugs for castration-resistant prostate cancer.

Science.gov (United States)

Takeuchi, Takumi; Okuno, Yumiko; Hattori-Kato, Mami; Zaitsu, Masayoshi; Mikami, Koji

2016-01-01

A splice variant of androgen receptor (AR), AR-V7, lacks in androgen-binding portion and leads to aggressive cancer characteristics. Reverse transcription-polymerase chain reactions (PCRs) and subsequent nested PCRs for the amplification of AR-V7 and prostate-specific antigen (PSA) transcripts were done for whole blood of patients with prostate cancer and male controls. With primary reverse transcription PCRs, AR-V7 and PSA were detected in 4.5% and 4.7% of prostate cancer, respectively. With nested PCRs, AR-V7 messenger RNA (mRNA) was positive in 43.8% of castration-sensitive prostate cancer and 48.1% of castration-resistant prostate cancer (CRPC), while PSA mRNA was positive in 6.3% of castration-sensitive prostate cancer and 18.5% of CRPC. Whole-blood samples of controls showed AR-V7 mRNA expression by nested PCR. Based on multivariate analysis, expression of AR-V7 mRNA in whole blood was not significantly correlated with clinical parameters and PSA mRNA in blood, while univariate analysis showed a correlation between AR-V7 mRNA and metastasis at initial diagnosis. Detection of AR-V7 mRNA did not predict the reduction of serum PSA in patients with CRPC following abiraterone and enzalutamide administration. In conclusion, AR-V7 mRNA expression in normal hematopoietic cells may have annihilated the manifestation of aggressiveness of prostate cancer and the prediction of the effectiveness of abiraterone and enzalutamide by the assessment of AR-V7 mRNA in blood.

Collisional and radiative processes in fluorescent lamps

International Nuclear Information System (INIS)

Lister, Graeme G.

2003-01-01

Since electrode life is the major limiting factor in operating fluorescent lamps, many lighting companies have introduced 'electrodeless' fluorescent lamps, using inductively coupled discharges. These lamps often operate at much higher power loadings than standard lamps and numerical models have not been successful in reproducing experimental measurements in the parameter ranges of interest. A comprehensive research program was undertaken to study the fundamental physical processes of these discharges, co-funded by the Electric Power Research Institute (EPRI) and OSRAM SYLVANIA under the name of ALITE. The program included experiments and modeling of radiation transport, computations of electron-atom and atom-atom cross sections and the first comprehensive power balance studies of a highly loaded fluorescent lamp. Results from the program and their importance to the understanding of the physics of fluorescent lamps are discussed, with particular emphasis on the important collisional and radiative processes. Comparisons between results of experimental measurements and numerical models are presented

DC electric springs with DC/DC converters

DEFF Research Database (Denmark)

Wang, Qingsong; Cheng, Ming; Jiang, Yunlei

2016-01-01

The concept of DC electric spring (DCES) was recently proposed to solve the stability issue caused by integrating intermittent renewable energy sources (RESs) to DC microgrids. In this paper, a new topology of DCES is proposed based on DC/DC converters. The proposed DCES consists of a bi-directio......The concept of DC electric spring (DCES) was recently proposed to solve the stability issue caused by integrating intermittent renewable energy sources (RESs) to DC microgrids. In this paper, a new topology of DCES is proposed based on DC/DC converters. The proposed DCES consists of a bi...... and/or constant discharging for batteries is adopted and four operating modes are analyzed as charging-positive, charging-negative, discharging-positive and discharging-negative modes. An additional mechanism for fast charging or fast discharging is also designed to secure normal operation...... of batteries. With the proposed DCES, the power fluctuations due to intermittent RESs can be passed to non-critical loads (NCLs) and batteries while power on critical loads (CLs) is kept stable. This is possibly the first attempt to design a DCES with only DC/DC converters. The performances of the proposed...

Phase out of incandescent lamps

Energy Technology Data Exchange (ETDEWEB)

NONE

2010-07-01

Since early 2007 almost all OECD and many non-OECD governments have announced policies aimed at phasing-out incandescent lighting within their jurisdictions. This study considers the implications of these policy developments in terms of demand for regulatory compliant lamps and the capacity and motivation of the lamp industry to produce efficient lighting products in sufficient volume to meet future demand. To assess these issues, it reviews the historic international screw-based lamp market, describes the status of international phase-out policies and presents projections of anticipated market responses to regulatory requirements to determine future demand for CFLs.

30 CFR 57.12035 - Weatherproof lamp sockets.

Science.gov (United States)

2010-07-01

... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Weatherproof lamp sockets. 57.12035 Section 57.12035 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR METAL AND NONMETAL... Surface and Underground § 57.12035 Weatherproof lamp sockets. Lamp sockets shall be of a weatherproof type...

30 CFR 56.12035 - Weatherproof lamp sockets.

Science.gov (United States)

2010-07-01

... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Weatherproof lamp sockets. 56.12035 Section 56.12035 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR METAL AND NONMETAL....12035 Weatherproof lamp sockets. Lamp sockets shall be of a weatherproof type where they are exposed to...

Lamp with a truncated reflector cup

Science.gov (United States)

Li, Ming; Allen, Steven C.; Bazydola, Sarah; Ghiu, Camil-Daniel

2013-10-15

A lamp assembly, and method for making same. The lamp assembly includes first and second truncated reflector cups. The lamp assembly also includes at least one base plate disposed between the first and second truncated reflector cups, and a light engine disposed on a top surface of the at least one base plate. The light engine is configured to emit light to be reflected by one of the first and second truncated reflector cups.

47 CFR 17.54 - Rated lamp voltage.

Science.gov (United States)

2010-10-01

... 47 Telecommunication 1 2010-10-01 2010-10-01 false Rated lamp voltage. 17.54 Section 17.54... voltage. To insure the necessary lumen output by obstruction lights, the rated voltage of incandescent lamps used shall correspond to be within 3 percent higher than the voltage across the lamp socket during...

Brugia malayi Antigen (BmA Inhibits HIV-1 Trans-Infection but Neither BmA nor ES-62 Alter HIV-1 Infectivity of DC Induced CD4+ Th-Cells.

Directory of Open Access Journals (Sweden)

Emily E I M Mouser

Full Text Available One of the hallmarks of HIV-1 disease is the association of heightened CD4+ T-cell activation with HIV-1 replication. Parasitic helminths including filarial nematodes have evolved numerous and complex mechanisms to skew, dampen and evade human immune responses suggesting that HIV-1 infection may be modulated in co-infected individuals. Here we studied the effects of two filarial nematode products, adult worm antigen from Brugia malayi (BmA and excretory-secretory product 62 (ES-62 from Acanthocheilonema viteae on HIV-1 infection in vitro. Neither BmA nor ES-62 influenced HIV-1 replication in CD4+ enriched T-cells, with either a CCR5- or CXCR4-using virus. BmA, but not ES-62, had the capacity to bind the C-type lectin dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN thereby inhibiting HIV-1 trans-infection of CD4+ enriched T-cells. As for their effect on DCs, neither BmA nor ES-62 could enhance or inhibit DC maturation as determined by CD83, CD86 and HLA-DR expression, or the production of IL-6, IL-10, IL-12 and TNF-α. As expected, due to the unaltered DC phenotype, no differences were found in CD4+ T helper (Th cell phenotypes induced by DCs treated with either BmA or ES-62. Moreover, the HIV-1 susceptibility of the Th-cell populations induced by BmA or ES-62 exposed DCs was unaffected for both CCR5- and CXCR4-using HIV-1 viruses. In conclusion, although BmA has the potential capacity to interfere with HIV-1 transmission or initial viral dissemination through preventing the virus from interacting with DCs, no differences in the Th-cell polarizing capacity of DCs exposed to BmA or ES-62 were observed. Neither antigenic source demonstrated beneficial or detrimental effects on the HIV-1 susceptibility of CD4+ Th-cells induced by exposed DCs.

Dissociation of SERPINE1 mRNA from the translational repressor proteins Ago2 and TIA-1 upon platelet activation.

Science.gov (United States)

Corduan, Aurélie; Plé, Hélène; Laffont, Benoit; Wallon, Thérèse; Plante, Isabelle; Landry, Patricia; Provost, Patrick

2015-05-01

Platelets play an important role in haemostasis, as well as in thrombosis and coagulation processes. They harbour a wide variety of messenger RNAs (mRNAs), that can template de novo protein synthesis, and an abundant array of microRNAs, which are known to mediate mRNA translational repression through proteins of the Argonaute (Ago) family. The relationship between platelet microRNAs and proteins capable of mediating translational repression, however, remains unclear. Here, we report that half of platelet microRNAs is associated to mRNA-regulatory Ago2 protein complexes, in various proportions. Associated to these Ago2 complexes are platelet mRNAs known to support de novo protein synthesis. Reporter gene activity assays confirmed the capacity of the platelet microRNAs, found to be associated to Ago2 complexes, to regulate translation of these platelet mRNAs through their 3'UTR. Neither the microRNA repertoire nor the microRNA composition of Ago2 complexes of human platelets changed upon activation with thrombin. However, under conditions favoring de novo synthesis of Plasminogen Activator Inhibitor-1 (PAI-1) protein, we documented a rapid dissociation of the encoding platelet SERPINE1 mRNA from Ago2 protein complexes as well as from the translational repressor protein T-cell-restricted intracellular antigen-1 (TIA-1). These findings are consistent with a scenario by which lifting of the repressive effects of Ago2 and TIA-1 protein complexes, involving a rearrangement of proteinmRNA complexes rather than disassembly of Ago2microRNA complexes, would allow translation of SERPINE1 mRNA into PAI-1 in response to platelet activation.