Sigma Receptor 1 activation attenuates release of inflammatory cytokines MIP1γ, MIP2, MIP3α and IL12 (p40/p70) by retinal Müller glial cells

PubMed Central

Shanmugam, A.; Wang, J.; Markand, S.; Perry, R.L.; Tawfik, A.; Zorrilla, E.; Ganapathy, V.; Smith, S.B.

2015-01-01

The high affinity Sigma Receptor 1 (σR1) ligand (+)-pentazocine ((+)-PTZ) affords profound retinal neuroprotection in vitro and in vivo by a yet-unknown mechanism. A common feature of retinal disease is Müller cell reactive gliosis, which includes cytokine release. Here we investigated whether LPS stimulates cytokine release by primary mouse Müller cells and whether (+)-PTZ alters release. Using a highly sensitive inflammatory antibody array we observed significant release of macrophage inflammatory proteins (MIP1γ, MIP2, MIP3α) and interleukin-12 (IL12 (p40/p70)) in LPS-treated cells compared to controls, and a significant decrease in secretion upon (+)-PTZ treatment. Müller cells from σR1 knockout mice demonstrated increased MIP1γ, MIP2, MIP3α and IL12 (p40/p70) secretion when exposed to LPS compared to LPS-stimulated WT cells. We investigated whether cytokine secretion was accompanied by cytosolic-to-nuclear NFκB translocation and whether endothelial cell adhesion/migration was altered by released cytokines. Cells exposed to LPS demonstrated increased NFκB nuclear location, which was reduced significantly in (+)-PTZ-treated cells. Media conditioned by LPS-stimulated-Müller cells induced leukocyte-endothelial cell adhesion and endothelial cell migration, which was attenuated by (+)-PTZ treatment. The findings suggest that release of certain inflammatory cytokines by Müller cells can be attenuated by σR1 ligands providing insights into the retinal neuroprotective role of this receptor. PMID:25439327

Bacteriophage-based vectors for site-specific insertion of DNA in the chromosome of Corynebacteria.

PubMed

Oram, Mark; Woolston, Joelle E; Jacobson, Andrew D; Holmes, Randall K; Oram, Diana M

2007-04-15

In Corynebacterium diphtheriae, diphtheria toxin is encoded by the tox gene of some temperate corynephages such as beta. beta-like corynephages are capable of inserting into the C. diphtheriae chromosome at two specific sites, attB1 and attB2. Transcription of the phage-encoded tox gene, and many chromosomally encoded genes, is regulated by the DtxR protein in response to Fe(2+) levels. Characterizing DtxR-dependent gene regulation is pivotal in understanding diphtheria pathogenesis and mechanisms of iron-dependent gene expression; although this has been hampered by a lack of molecular genetic tools in C. diphtheriae and related Coryneform species. To expand the systems for genetic manipulation of C. diphtheriae, we constructed plasmid vectors capable of integrating into the chromosome. These plasmids contain the beta-encoded attP site and the DIP0182 integrase gene of C. diphtheriae NCTC13129. When these vectors were delivered to the cytoplasm of non-lysogenic C. diphtheriae, they integrated into either the attB1 or attB2 sites with comparable frequency. Lysogens were also transformed with these vectors, by virtue of the second attB site. An integrated vector carrying an intact dtxR gene complemented the mutant phenotypes of a C. diphtheriae DeltadtxR strain. Additionally, strains of beta-susceptible C. ulcerans, and C. glutamicum, a species non-permissive for beta, were each transformed with these vectors. This work significantly extends the tools available for targeted transformation of both pathogenic and non-pathogenic Corynebacterium species.

Potentiometric determination of pantoprazole using an ion-selective sensor based on polypyrrole doped films.

PubMed

Noronha, Bárbara V; Bindewald, Eduardo H; de Oliveira, Michelle C; Papi, Maurício A P; Bergamini, Márcio F; Marcolino, Luiz H

2014-10-01

The present work reports for the first time the use of polypyrrole (PPy) doped film for development of a potentiometric disposable sensor for determination of pantoprazole (PTZ), a drug used for ulcer treatment. Selective potentiometric response has been found by using a membrane of PPy doped with PTZ anions prepared under galvanostatic conditions at graphite pencil electrode (GPEM/PPy-PTZ) surface. Potentiometric response has been influenced for conditions adopted in polymerization and measurement step. After optimization of experimental (e.g. pH and time of conditioning) and instrumental parameters (e.g. current density and electrical charge) a linear analytical curve from 1.0 × 10(-5) to 1.1 × 10(-2) mol L(-1) with a slope of calibration of the 57.6 mV dec(-1) and limit of detection (LOD) of 6.9 × 10(-6) mol L(-1) was obtained. The determination of the PTZ content in pharmaceutical samples using the proposed methodology and official method recommended by Brazilian Pharmacopeia are in agreement at the 95% confidence level and within an acceptable range of error. Copyright © 2014 Elsevier B.V. All rights reserved.

Hyaluronic acid synthase-2 gene transfer into the joints of Beagles by use of recombinant adeno-associated viral vectors.

PubMed

Kyostio-Moore, Sirkka; Berthelette, Patricia; Cornell, Cathleen Sookdeo; Nambiar, Bindu; Figueiredo, Monica Dias

2018-05-01

OBJECTIVE To evaluate gene transfer of recombinant adeno-associated viral (rAAV) vectors with AAV2 or AAV5 capsid and encoding hyaluronic acid (HA) synthase-2 (HAS2) into joints of healthy dogs. ANIMALS 22 purpose-bred Beagles. PROCEDURES Plasmid expression cassettes encoding canine HAS2 (cHAS2) were assessed in vitro for concentration and molecular size of secreted HA. Thereafter, rAAV2-cHAS2 vectors at 3 concentrations and rAAV5-cHAS2 vectors at 1 concentration were each administered intra-articularly into the left stifle joint of 5 dogs; 2 dogs received PBS solution instead. Synovial fluid HA concentration and serum and synovial fluid titers of neutralizing antibodies against AAV capsids were measured at various points. Dogs were euthanized 28 days after treatment, and cartilage and synovium samples were collected for vector DNA and mRNA quantification and histologic examination. RESULTS Cell transfection with plasmids encoding cHAS2 resulted in an increase in production and secretion of HA in vitro. In vivo, the rAAV5-cHAS2 vector yielded uniform genome transfer and cHAS2 expression in collected synovium and cartilage samples. In contrast, rAAV2-cHAS2 vectors were detected inconsistently in synovium and cartilage samples and failed to produce clear dose-related responses. Histologic examination revealed minimal synovial inflammation in joints injected with rAAV vectors. Neutralizing antibodies against AAV capsids were detected in serum and synovial fluid samples from all vector-treated dogs. CONCLUSIONS AND CLINICAL RELEVANCE rAAV5-mediated transfer of the gene for cHAS2 into healthy joints of dogs by intra-articular injection appeared safe and resulted in vector-derived cHAS2 production by synoviocytes and chondrocytes. Whether this treatment may increase HA production by synoviocytes and chondrocytes in osteoarthritic joints remains to be determined.

Induction of immunity to antigens expressed by recombinant adeno-associated virus depends on the route of administration.

PubMed

Brockstedt, D G; Podsakoff, G M; Fong, L; Kurtzman, G; Mueller-Ruchholtz, W; Engleman, E G

1999-07-01

Recombinant adeno-associated virus (rAAV) is a replication-defective parvovirus which is being explored as a vector for gene therapy because of its broad host range, excellent safety profile, and durable transgene expression in infected hosts. rAAV has also been reported by several groups to induce little or no immune response to its encoded transgene products. In this study we examined the immunogenicity of rAAV by studying the immune response of C57BL/6 mice to a single dose of rAAV-encoding ovalbumin (AAV-Ova) administered by a variety of routes. Mice injected with AAV-Ova intraperitoneally (ip), intravenously, or subcutaneously developed potent ovalbumin-specific cytotoxic T lymphocytes (CTL) as well as anti-ovalbumin antibodies and antibodies to AAV. In contrast, mice injected with AAV-Ova intramuscularly developed a humoral response to the virus and the transgene but minimal ovalbumin-specific CTLs. The induced CTL response after ip administration of AAV-Ova protected mice against a subsequent tumor challenge with an ovalbumin-transfected B16 melanoma cell line. Studies of the mechanism by which AAV-Ova induces CTL confirmed that the virus delivers the transgene product into the classical MHC class I pathway of antigen processing. Mice that previously had been exposed to rAAV vectors failed to develop ovalbumin-specific CTL following administration of AAV-Ova. Analysis of these mice revealed the presence of circulating anti-AAV antibodies that blocked rAAV transduction in vitro and inhibited CTL induction in vivo. These results suggest a possible role for rAAV in the immunotherapy of malignancies and viral infections, although induced antibody responses to AAV may limit its ability to be administered for repeated vaccinations. Copyright 1999 Academic Press.

Bacteriophage-based Vectors for Site-specific Insertion of DNA in the Chromosome of Corynebacteria

PubMed Central

Oram, Mark; Woolston, Joelle E.; Jacobson, Andrew D.; Holmes, Randall K.; Oram, Diana M.

2007-01-01

In Corynebacterium diphtheriae, diphtheria toxin is encoded by the tox gene of some temperate corynephages such as β. β-like corynephages are capable of inserting into the C. diphtheriae chromosome at two specific sites, attB1 and attB2. Transcription of the phage-encoded tox gene, and many chromosomally-encoded genes, is regulated by the DtxR protein in response to Fe2+ levels. Characterizing DtxR-dependent gene regulation is pivotal in understanding diphtheria pathogenesis and mechanisms of iron-dependent gene expression; although this has been hampered by a lack of molecular genetic tools in C. diphtheriae and related Coryneform species. To expand the systems for genetic manipulation of C. diphtheriae, we constructed plasmid vectors capable of integrating into the chromosome. These plasmids contain the β-encoded attP site and the DIP0182 integrase gene of C. diphtheriae NCTC13129. When these vectors were delivered to the cytoplasm of non-lysogenic C. diphtheriae, they integrated into either the attB1 or attB2 sites with comparable frequency. Lysogens were also transformed with these vectors, by virtue of the second attB site. An integrated vector carrying an intact dtxR gene complemented the mutant phenotypes of a C. diphtheriae ΔdtxR strain. Additionally, strains of β-susceptible C. ulcerans, and C. glutamicum, a species non-permissive for β, were each transformed with these vectors. This work significantly extends the tools available for targeted transformation of both pathogenic and non-pathogenic Corynebacterium species. PMID:17275217

Characteristics of Minimally Oversized Adeno-Associated Virus Vectors Encoding Human Factor VIII Generated Using Producer Cell Lines and Triple Transfection.

PubMed

Nambiar, Bindu; Cornell Sookdeo, Cathleen; Berthelette, Patricia; Jackson, Robert; Piraino, Susan; Burnham, Brenda; Nass, Shelley; Souza, David; O'Riordan, Catherine R; Vincent, Karen A; Cheng, Seng H; Armentano, Donna; Kyostio-Moore, Sirkka

2017-02-01

Several ongoing clinical studies are evaluating recombinant adeno-associated virus (rAAV) vectors as gene delivery vehicles for a variety of diseases. However, the production of vectors with genomes >4.7 kb is challenging, with vector preparations frequently containing truncated genomes. To determine whether the generation of oversized rAAVs can be improved using a producer cell-line (PCL) process, HeLaS3-cell lines harboring either a 5.1 or 5.4 kb rAAV vector genome encoding codon-optimized cDNA for human B-domain deleted Factor VIII (FVIII) were isolated. High-producing "masterwells" (MWs), defined as producing >50,000 vg/cell, were identified for each oversized vector. These MWs provided stable vector production for >20 passages. The quality and potency of the AAVrh8R/FVIII-5.1 and AAVrh8R/FVIII-5.4 vectors generated by the PCL method were then compared to those prepared via transient transfection (TXN). Southern and dot blot analyses demonstrated that both production methods resulted in packaging of heterogeneously sized genomes. However, the PCL-derived rAAV vector preparations contained some genomes >4.7 kb, whereas the majority of genomes generated by the TXN method were ≤4.7 kb. The PCL process reduced packaging of non-vector DNA for both the AAVrh8R/FVIII-5.1 and the AAVrh8R/FVIII-5.4 kb vector preparations. Furthermore, more DNA-containing viral particles were obtained for the AAVrh8R/FVIII-5.1 vector. In a mouse model of hemophilia A, animals administered a PCL-derived rAAV vector exhibited twofold higher plasma FVIII activity and increased levels of vector genomes in the liver than mice treated with vector produced via TXN did. Hence, the quality of oversized vectors prepared using the PCL method is greater than that of vectors generated using the TXN process, and importantly this improvement translates to enhanced performance in vivo.

Evaluation of Pharmacodynamic Interactions Between Telavancin and Aztreonam or Piperacillin/Tazobactam Against Pseudomonas aeruginosa, Escherichia coli and Methicillin-Resistant Staphylococcus aureus.

PubMed

Yim, Juwon; Smith, Jordan R; Barber, Katie E; Hallesy, Jessica A; Rybak, Michael J

2016-09-01

In clinical trials comparing telavancin (TLV) with vancomycin for treatment of hospital-acquired pneumonia, TLV demonstrated lower clinical cure rates than vancomycin in patients who had mixed gram-positive and -negative infections and were concomitantly treated with either aztreonam (ATM) or piperacillin/tazobactam (PTZ). Here, we investigated therapeutic interactions between TLV and ATM or PTZ in an in vitro pharmacokinetic/pharmacodynamic (PK/PD) model under simulated reduced renal function conditions. In vitro one-compartment PK/PD models were run over 96 h simulating TLV 10 mg/kg every 48 h, ATM 500 mg every 8 h and PTZ continuous infusion 13.5 g over 24 h alone and in combination against P. aeruginosa, E. coli and methicillin-resistant S. aureus (MRSA). The efficacy of antimicrobials was evaluated by plotting time-kill curves and calculating the reduction in log10 cfu/ml over 96 h. Against both MRSA strains, TLV was rapidly bactericidal at 4 h and maintained its activity over 96 h with no observed antagonism by either ATM or PTZ. PTZ maintained bacteriostatic and bactericidal activities against E. coli ATCC 25922 and clinical strain R1022 at 96 h, whereas both strains regrew as soon as 24 h in ATM models. Against P. aeruginosa ATCC 27853, regrowth was noted at 24 h in models simulating ATM and PTZ. The addition of TLV to ATM or PTZ had no appreciable impact on activity against the two E. coli strains and P. aeruginosa strain. The combinations of TLV and either ATM or PTZ did not demonstrate any antagonistic activity. Clinical variables and patient characteristics should be further explored to determine possible reasons for discrepancies in outcomes. Theravance Biopharma Antibiotics, Inc.

Methods of treating Parkinson's disease using viral vectors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bankiewicz, Krystof; Cunningham, Janet

Methods of delivering viral vectors, particularly recombinant adeno-associated virus (rAAV) virions, to the central nervous system (CNS) using convection enhanced delivery (CED) are provided. The rAAV virions include a nucleic acid sequence encoding a therapeutic polypeptide. The methods can be used for treating CNS disorders such as for treating Parkinson's Disease.

Novel Visual Sensor Coverage and Deployment in Time Aware PTZ Wireless Visual Sensor Networks.

PubMed

Yap, Florence G H; Yen, Hong-Hsu

2016-12-30

In this paper, we consider the visual sensor deployment algorithm in Pan-Tilt-Zoom (PTZ) Wireless Visual Sensor Networks (WVSNs). With PTZ capability, a sensor's visual coverage can be extended to reduce the number of visual sensors that need to be deployed. The coverage zone of a visual sensor in PTZ WVSN is composed of two regions, a Direct Coverage Region (DCR) and a PTZ Coverage Region (PTZCR). In the PTZCR, a visual sensor needs a mechanical pan-tilt-zoom operation to cover an object. This mechanical operation can take seconds, so the sensor might not be able to adjust the camera in time to capture the visual data. In this paper, for the first time, we study this PTZ time-aware PTZ WVSN deployment problem. We formulate this PTZ time-aware PTZ WVSN deployment problem as an optimization problem where the objective is to minimize the total visual sensor deployment cost so that each area is either covered in the DCR or in the PTZCR while considering the PTZ time constraint. The proposed Time Aware Coverage Zone (TACZ) model successfully captures the PTZ visual sensor coverage in terms of camera focal range, angle span zone coverage and camera PTZ time. Then a novel heuristic, called Time Aware Deployment with PTZ camera (TADPTZ) algorithm, is proposed to solve the problem. From our computational experiments, we found out that TACZ model outperforms the existing M coverage model under all network scenarios. In addition, as compared to the optimal solutions, the TACZ model is scalable and adaptable to the different PTZ time requirements when deploying large PTZ WVSNs.

Novel Visual Sensor Coverage and Deployment in Time Aware PTZ Wireless Visual Sensor Networks

PubMed Central

Yap, Florence G. H.; Yen, Hong-Hsu

2016-01-01

In this paper, we consider the visual sensor deployment algorithm in Pan-Tilt-Zoom (PTZ) Wireless Visual Sensor Networks (WVSNs). With PTZ capability, a sensor’s visual coverage can be extended to reduce the number of visual sensors that need to be deployed. The coverage zone of a visual sensor in PTZ WVSN is composed of two regions, a Direct Coverage Region (DCR) and a PTZ Coverage Region (PTZCR). In the PTZCR, a visual sensor needs a mechanical pan-tilt-zoom operation to cover an object. This mechanical operation can take seconds, so the sensor might not be able to adjust the camera in time to capture the visual data. In this paper, for the first time, we study this PTZ time-aware PTZ WVSN deployment problem. We formulate this PTZ time-aware PTZ WVSN deployment problem as an optimization problem where the objective is to minimize the total visual sensor deployment cost so that each area is either covered in the DCR or in the PTZCR while considering the PTZ time constraint. The proposed Time Aware Coverage Zone (TACZ) model successfully captures the PTZ visual sensor coverage in terms of camera focal range, angle span zone coverage and camera PTZ time. Then a novel heuristic, called Time Aware Deployment with PTZ camera (TADPTZ) algorithm, is proposed to solve the problem. From our computational experiments, we found out that TACZ model outperforms the existing M coverage model under all network scenarios. In addition, as compared to the optimal solutions, the TACZ model is scalable and adaptable to the different PTZ time requirements when deploying large PTZ WVSNs. PMID:28042829

Combination recombinant simian or chimpanzee adenoviral vectors for vaccine development.

PubMed

Cheng, Cheng; Wang, Lingshu; Ko, Sung-Youl; Kong, Wing-Pui; Schmidt, Stephen D; Gall, Jason G D; Colloca, Stefano; Seder, Robert A; Mascola, John R; Nabel, Gary J

2015-12-16

Recombinant adenoviral vector (rAd)-based vaccines are currently being developed for several infectious diseases and cancer therapy, but pre-existing seroprevalence to such vectors may prevent their use in broad human populations. In this study, we investigated the potential of low seroprevalence non-human primate rAd vectors to stimulate cellular and humoral responses using HIV/SIV Env glycoprotein (gp) as the representative antigen. Mice were immunized with novel simian or chimpanzee rAd (rSAV or rChAd) vectors encoding HIV gp or SIV gp by single immunization or in heterologous prime/boost combinations (DNA/rAd; rAd/rAd; rAd/NYVAC or rAd/rLCM), and adaptive immunity was assessed. Among the rSAV and rChAd tested, rSAV16 or rChAd3 vector alone generated the most potent immune responses. The DNA/rSAV regimen also generated immune responses similar to the DNA/rAd5 regimen. rChAd63/rChAd3 and rChAd3 /NYVAC induced similar or even higher levels of CD4+ and CD8+ T-cell and IgG responses as compared to rAd28/rAd5, one of the most potent combinations of human rAds. The optimized vaccine regimen stimulated improved cellular immune responses and neutralizing antibodies against HIV compared to the DNA/rAd5 regimen. Based on these results, this type of novel rAd vector and its prime/boost combination regimens represent promising candidates for vaccine development. Published by Elsevier Ltd.

Glutamate receptor antibodies directed against AMPA receptors subunit 3 peptide B (GluR3B) can be produced in DBA/2J mice, lower seizure threshold and induce abnormal behavior.

PubMed

Ganor, Yonatan; Goldberg-Stern, Hadassa; Cohen, Ran; Teichberg, Vivian; Levite, Mia

2014-04-01

Anti-GluR3B antibodies (GluR3B Ab's), directed against peptide B/aa372-395 of GluR3 subunit of glutamate/AMPA receptors, are found in ∼35% of epilepsy patients, activate glutamate/AMPA receptors, evoke ion currents, kill neurons and damage the brain. We recently found that GluR3B Ab's also associate with neurological/psychiatric/behavioral abnormalities in epilepsy patients. Here we asked if GluR3B Ab's could be produced in DBA/2J mice, and also modulate seizure threshold and/or cause behavioral/motor impairments in these mice. DBA/2J mice were immunized with the GluR3B peptide in Complete Freund's Adjuvant (CFA), or with controls: ovalbumin (OVA), CFA, or phosphate-buffer saline (PBS). GluR3B Ab's and OVA Ab's were tested. Seizures were induced in all mice by the chemoconvulsant pentylenetetrazole (PTZ) at three time points, each time with less PTZ to avoid non-specific death. Behavior was examined in Open-Field, RotaRod and Grip tests. GluR3B Ab's were produced only in GluR3B-immunized mice, while OVA Ab's were produced only in OVA-immunized mice, showing high Ab's specificity. In GluR3B Ab's negative mice, seizure severity scores and percentages of animals developing generalized seizures declined in response to decreasing PTZ doses. In contrast, both parameters remained unchanged/high in the GluR3B Ab's positive mice, showing that these mice were more susceptible to seizures. The seizure scores associated significantly with the GluR3B Ab's levels. GluR3B Ab's positive mice were also more anxious in Open-Field test, fell faster in RotaRod test, and fell more in Grip test, compared to all the control mice. GluR3B Ab's are produced in DBA/2J mice, facilitate seizures and induce behavioral/motor impairments. This animal model can therefore serve for studying autoimmune epilepsy and abnormal behavior mediated by pathogenic anti-GluR3B Ab's. Copyright © 2014 Elsevier Ltd. All rights reserved.

Lentiviral vectors encoding shRNAs efficiently transduce and knockdown LINGO-1 but induce an interferon response and cytotoxicity in CNS neurons

PubMed Central

Hutson, Thomas H.; Foster, Edmund; Dawes, John M.; Hindges, Robert; Yáñez-Muñoz, Rafael J.; Moon, Lawrence D.F.

2017-01-01

Background Knocking down neuronal LINGO-1 using short hairpin RNAs (shRNAs) might enhance axon regeneration in the CNS. Integration-deficient lentiviral vectors have great potential as a therapeutic delivery system for CNS injuries. However, recent studies have revealed that shRNAs can induce an interferon response resulting in off-target effects and cytotoxicity. Methods CNS neurons were transduced with integration-deficient lentiviral vectors in vitro. The transcriptional effect of shRNA expression was analysed using qRT-PCR and northern blots were used to assess shRNA production. Results Integration-deficient lentiviral vectors efficiently transduced CNS neurons and knocked down LINGO-1 mRNA in vitro. However, an increase in cell death was observed when lentiviral vectors encoding an shRNA were applied or when high vector concentrations were used. We demonstrate that high doses of vector or the use of vectors encoding shRNAs can induce an up-regulation of interferon stimulated genes (OAS1 and PKR) and a down-regulation of off- target genes (including p75NTR and NgR1). Furthermore, the northern blot demonstrated that these negative consequences occur even when lentiviral vectors express low levels of shRNAs. Together, these results may explain why neurite outgrowth was not enhanced on an inhibitory substrate after transduction with lentiviral vectors encoding an shRNA targeting LINGO-1. Conclusions These findings highlight the importance of including appropriate controls to verify silencing specificity and the requirement to check for an interferon response when conducting RNA interference experiments. However, the potential benefits that RNA interference and viral vectors offer to gene-based therapies to CNS injuries cannot be overlooked and demand further investigation. PMID:22499506

Synthesis and biological evaluation of novel 2,3-disubstituted benzofuran analogues of GABA as neurotropic agents.

PubMed

Coaviche-Yoval, Arturo; Luna, Hector; Tovar-Miranda, Ricardo; Soriano-Ursua, Marvin Antonio; Trujillo-Ferrara, Jose G

2018-05-23

Benzofurans are heterocyclic compounds with neurotropic activity. Some have been developed for the treatment of acute and degenerative neuronal injuries. To evaluate the in silico binding of some promising benzofurans on the GABA receptors, and the in vivo neurotropic activity of benzofuran analogues (BZF 6-10) of gamma-aminobutyric acid (GABA) on a seizure model. The ligands with the best physicochemical attributes were docked on two GABA receptors (the alpha-1 subunit of GABAA-R and GBR1 subunit of GABAB-R). Selected benzofuran derivatives were synthesized by a multistep procedure and characterized. To examine the neurotropic effects, mice were pretreated with different concentrations of the compounds prior to PTZ- or 4-AP-induced seizures. We assessed acute toxicity, motor behavior, and the effects on seizures. The tested ligands that complied with Lipinski's rule of five were tested in silico with GABAA-R (ΔG = -5.51 to -5.84 kcal/mol) at the allosteric site for benzodiazepines. They bound to a similar cluster of residues as the reference compound (gaboxadol, ΔG = -5.51 kcal/mol). Synthesis was achieved with good overall yields (42-9.7%). Two compounds were selected for biological tests (BZF-7 and rac-BZF-10) on a mouse model of seizures, induced by pentylenetetrazol (PTZ) or 4-aminopyridine (4-AP). PTZ-induced seizures are associated with GABA receptors, and those 4-AP-induced with the blockage of the delayed rectifier-type potassium channel, which promotes the release of the NMDA-sensitive glutamatergic ionotropic receptor and other neurotransmitters. The biological assays demonstrated that BZF-7 and rac-BZF-10 do not protect against seizures. Indeed, BZF-7 increased the number of PTZ-induced seizures and decreased latency time. The 4-AP model apparently showed a potentiation of seizure effects after administration of the BZF-analogues, evidenced by the incidence and severity of the seizures and reduced latency time. The results suggest that the test compounds are GABAergic antagonists with stimulatory activity on the CNS. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Anticonvulsant and Antioxidant Effects of Pitavastatin Against Pentylenetetrazol-Induced Kindling in Mice.

PubMed

Faghihi, Nastaran; Mohammadi, Mohammad Taghi

2017-06-01

Purpose: The pleiotropic effects of statins (antioxidant and anti-inflammation) have been reported by previous studies. Therefore, we aimed to determine whether pitavastatin has protective effects against pentylenetetrazol (PTZ)-induced kindling in mice and also whether pitavastatin improves the brain antioxidant capacity and attenuates the oxidative injuries in kindled mice. Methods: Twenty-four mice were randomly divided into four groups (each group n=6); control, PTZ-kindling and PTZ-kindled rats treated with pitavastatin (1&4 mg/kg). PTZ kindling seizures were induced by repetitive intraperitoneal injections of PTZ (65 mg/kg) every 48 hours till day twenty-one. Animals received daily oral pitavastatin for twenty-one days. Latency, score and duration of the seizures were recorded. The activities of catalase (CAT) ad superoxide dismutase (SOD), and likewise the contents of malondialdehyde (MDA) and nitrate were assessed in the brains of all rats. Results: There was a progressive reduction in latency of the kindled rats in the next injections of PTZ. Pitavastatin reduced this value (latency) particularly at higher dose. Seizures duration and score also decreased in treatment groups. SOD and CAT activities significantly decreased in PTZ-kindling group by 62% and 64%, respectively, but pitavastatin did not significantly change the SOD and CAT activities. Brain MDA and nitrate significantly increased in PTZ-kindling group by 53% and 30%, respectively. Pitavastatin at higher dose significantly decreased the MDA and nitrate contents of PTZ-kindling rats by 45% and 32%, respectively. Conclusion: Our findings revealed that pitavastatin can improve the behavioral expression of the PTZ-kindling rats and attenuate the seizure-induced oxidative/nitrosative damage.

Protective effect of naringin on pentylenetetrazole (PTZ)-induced kindling; possible mechanisms of antikindling, memory improvement, and neuroprotection.

PubMed

Kola, Phani Kumar; Akula, Annapurna; NissankaraRao, Lakshmi Sudeepthi; Danduga, R Ch Sekhara Reddy

2017-10-01

The present study investigated the effects of Naringin on seizure severity, progress of kindling, memory impairment, oxidative stress, neurochemicals, and neural damage in Pentylenetetrazole (PTZ)-induced kindling. Alternate intra-peritoneal injections of PTZ induced kindling at 22 injections of PTZ. In comparison with the PTZ group, pretreatment with Naringin 30 min prior to PTZ administration and on a PTZ-free day was found to lead to a decreased seizure score, a mitigated progress of kindling, decreased transfer latency, and increased total number of arm entries, % alternation behavior in Y maze, and % conditioned avoidance response in a pole climbing apparatus. Biochemical analysis of the frontal and temporal cortexes and the hippocampus of the brain showed that Naringin attenuated the level of lipid peroxidation (MDA) and augmented the reduced glutathione, superoxide dismutase, catalase, and total thiol results in decreased oxidative stress compared with the PTZ group and control group. Investigation of neurochemicals revealed a minute change in gamma amino butyric acid (GABA), glutamate and dopamine, and decreased AChE in the three regions. Increased CA1 neuronal density in the hippocampus and increased cell density in the frontal and temporal regions indicate the potential of naringin to act against PTZ-induced kindling, memory impairment, oxidative stress, neurochemical changes, and histological aberrations. Copyright © 2017 Elsevier Inc. All rights reserved.

Prophylaxis and Treatment of Alzheimer's Disease by Delivery of an Adeno-Associated Virus Encoding a Monoclonal Antibody Targeting the Amyloid Beta Protein

PubMed Central

Shimada, Masaru; Abe, Shinya; Takahashi, Toru; Shiozaki, Kazumasa; Okuda, Mitsue; Mizukami, Hiroaki; Klinman, Dennis M.; Ozawa, Keiya; Okuda, Kenji

2013-01-01

We previously reported on a monoclonal antibody (mAb) that targeted amyloid beta (Aß) protein. Repeated injection of that mAb reduced the accumulation of Aß protein in the brain of human Aß transgenic mice (Tg2576). In the present study, cDNA encoding the heavy and light chains of this mAb were subcloned into an adeno-associated virus type 1 (AAV) vector with a 2A/furin adapter. A single intramuscular injection of 3.0×1010 viral genome of these AAV vectors into C57BL/6 mice generated serum anti-Aß Ab levels up to 0.3 mg/ml. Anti-Aß Ab levels in excess of 0.1 mg/ml were maintained for up to 64 weeks. The effect of AAV administration on Aß levels in vivo was examined. A significant decrease in Aß levels in the brain of Tg2576 mice treated at 5 months (prophylactic) or 10 months (therapeutic) of age was observed. These results support the use of AAV vector encoding anti-Aß Ab for the prevention and treatment of Alzheimer's disease. PMID:23555563

The thiostrepton-resistance-encoding gene in Streptomyces laurentii is located within a cluster of ribosomal protein operons.

PubMed

Smith, T M; Jiang, Y F; Shipley, P; Floss, H G

1995-10-16

A common approach to identify and clone biosynthetic gene from an antibiotic-producing streptomycete is to clone the resistance gene for the antibiotic of interest and then use that gene to clone DNA that is linked to it. As a first step toward cloning the genes responsible for the biosynthesis of thiostrepton (Th) in Streptomyces laurentii (Sl), the Th resistance-encoding gene (tsnR) was cloned as a 1.5-kb BamHI-PvuII fragment in Escherichia coli (Ec), and shown to confer Th resistance when introduced into S. lividans TK24. The tsnR-containing DNA fragment was used as a probe to isolate clones from cosmid libraries of DNA in the Ec cosmid vector SuperCos, and pOJ446 (an Ec/streptomycete) cosmid vector. Sequence and genetic analysis of the DNA flanking the tsnR indicates that the Sl tsnR is not closely linked to biosynthetic genes. Instead it is located within a cluster of ribosomal protein operons.

Degradable polyethylenimine derivate coupled to a bifunctional peptide R13 as a new gene-delivery vector

PubMed Central

Liu, Kehai; Wang, Xiaoyu; Fan, Wei; Zhu, Qing; Yang, Jingya; Gao, Jing; Gao, Shen

2012-01-01

Background To solve the efficiency versus cytotoxicity and tumor-targeting problems of polyethylenimine (PEI) used as a nonviral gene delivery vector, a degradable PEI derivate coupled to a bifunctional peptide R13 was developed. Methods First, we synthesized a degradable PEI derivate by crosslinking low-molecular-weight PEI with pluronic P123, then used tumor-targeting peptide arginine-glycine-aspartate-cysteine (RGDC), in conjunction with the cell-penetrating peptide Tat (49–57), to yield a bifunctional peptide RGDC-Tat (49–57) named R13, which can improve cell selection and increase cellular uptake, and, lastly, adopted R13 to modify the PEI derivates so as to prepare a new polymeric gene vector (P123-PEI-R13). The new gene vector was characterized in terms of its chemical structure and biophysical parameters. We also investigated the specificity, cytotoxicity, and gene transfection efficiency of this vector in αvβ3-positive human cervical carcinoma Hela cells and murine melanoma B16 cells in vitro. Results The vector showed controlled degradation, strong targeting specificity to αvβ3 receptor, and noncytotoxicity in Hela cells and B16 cells at higher doses, in contrast to PEI 25 KDa. The particle size of P123-PEI-R13/DNA complexes was around 100–250 nm, with proper zeta potential. The nanoparticles can protect plasmid DNA from being digested by DNase I at a concentration of 6 U DNase I/μg DNA. The nanoparticles were resistant to dissociation induced by 50% fetal bovine serum and 600 μg/mL sodium heparin. P123-PEI-R13 also revealed higher transfection efficiency in two cell lines as compared with PEI 25 KDa. Conclusion P123-PEI-R13 is a potential candidate as a safe and efficient gene-delivery carrier for gene therapy. PMID:22412301

Electron transfer triggered by optical excitation of phenothiazine-tris(meta-phenylene-ethynylene)-(tricarbonyl)(bpy)(py)rhenium(I).

PubMed

Bingöl, Bahar; Durrell, Alec C; Keller, Gretchen E; Palmer, Joshua H; Grubbs, Robert H; Gray, Harry B

2013-04-25

We have investigated excited-state electron transfer in a donor-bridge-acceptor complex containing phenothiazine (PTZ) linked via tris(meta-phenylene-ethynylene) to a tricarbonyl(bipyridine)(pyridine)Re(I) unit. Time-resolved luminescence experiments reveal two excited-state (*Re) decay regimes, a multiexponential component with a mean lifetime of 2.7 ns and a longer monoexponential component of 530 ns in dichloromethane solution. The faster decay is attributed to PTZ → *Re electron transfer in a C-shaped PTZ-bridge-Re conformer (PTZ-Re ≈ 7.5 Å). We assign the longer lifetime, which is virtually identical to that of free *Re, to an extended conformer (PTZ-Re > 20 Å). The observed biexponential *Re decay requires that interconversion of PTZ-bridge-Re conformers be slower than 10(6) s(-1).

Effects of pentylenetetrazole and glutamate on metabolism of [U-(13)C]glucose in cultured cerebellar granule neurons.

PubMed

Eloqayli, Haytham; Qu, Hong; Unsgård, Geirmund; Sletvold, Olav; Hadidi, Hakam; Sonnewald, Ursula

2002-02-01

This study was performed to analyze the effects of glutamate and the epileptogenic agent pentylenetetrazole (PTZ) on neuronal glucose metabolism. Cerebellar granule neurons were incubated for 2 h in medium containing 3 mM [U-(13)C]glucose, with and without 0.25 mM glutamate and/or 10 mM PTZ. In the presence of PTZ, decreased glucose consumption with unchanged lactate release was observed, indicating decreased glucose oxidation. PTZ also slowed down tricarboxylic acid (TCA) cycle activity as evidenced by the decreased amounts of labeled aspartate and [1,2-(13)C]glutamate. When glutamate was present, glucose consumption was also decreased. However, the amount of glutamate, derived from [U-(13)C]glucose via the first turn of the TCA cycle, was increased. The decreased amount of [1,2-(13)C]glutamate, derived from the second turn in the TCA cycle, and increased amount of aspartate indicated the dilution of label due to the entrance of unlabeled glutamate into TCA cycle. In the presence of glutamate plus PTZ, the effect of PTZ was enhanced by glutamate. Labeled alanine was detected only in the presence of glutamate plus PTZ, which indicated that oxaloacetate was a better amino acid acceptor than pyruvate. Furthermore, there was also evidence for intracellular compartmentation of oxaloacetate metabolism. Glutamate and PTZ caused similar metabolic changes, however, via different mechanisms. Glutamate substituted for glucose as energy substrate in the TCA cycle, whereas, PTZ appeared to decrease mitochondrial activity.

Targeted Modifications in Adeno-Associated Virus Serotype 8 Capsid Improves Its Hepatic Gene Transfer Efficiency In Vivo

PubMed Central

Sen, Dwaipayan; Gadkari, Rupali A; Sudha, Govindarajan; Gabriel, Nishanth; Kumar, Yesupatham Sathish; Selot, Ruchita; Samuel, Rekha; Rajalingam, Sumathi; Ramya, V.; Nair, Sukesh C.; Srinivasan, Narayanaswamy; Srivastava, Alok

2013-01-01

Abstract Recombinant adeno-associated virus vectors based on serotype 8 (AAV8) have shown significant promise for liver-directed gene therapy. However, to overcome the vector dose dependent immunotoxicity seen with AAV8 vectors, it is important to develop better AAV8 vectors that provide enhanced gene expression at significantly low vector doses. Since it is known that AAV vectors during intracellular trafficking are targeted for destruction in the cytoplasm by the host–cellular kinase/ubiquitination/proteasomal machinery, we modified specific serine/threonine kinase or ubiquitination targets on the AAV8 capsid to augment its transduction efficiency. Point mutations at specific serine (S)/threonine (T)/lysine (K) residues were introduced in the AAV8 capsid at the positions equivalent to that of the effective AAV2 mutants, generated successfully earlier. Extensive structure analysis was carried out subsequently to evaluate the structural equivalence between the two serotypes. scAAV8 vectors with the wild-type (WT) and each one of the S/T→Alanine (A) or K-Arginine (R) mutant capsids were evaluated for their liver transduction efficiency in C57BL/6 mice in vivo. Two of the AAV8-S→A mutants (S279A and S671A), and a K137R mutant vector, demonstrated significantly higher enhanced green fluorescent protein (EGFP) transcript levels (∼9- to 46-fold) in the liver compared to animals that received WT-AAV8 vectors alone. The best performing AAV8 mutant (K137R) vector also had significantly reduced ubiquitination of the viral capsid, reduced activation of markers of innate immune response, and a concomitant two-fold reduction in the levels of neutralizing antibody formation in comparison to WT-AAV8 vectors. Vector biodistribution studies revealed that the K137R mutant had a significantly higher and preferential transduction of the liver (106 vs. 7.7 vector copies/mouse diploid genome) when compared to WT-AAV8 vectors. To further study the utility of the K137R-AAV8 mutant in therapeutic gene transfer, we delivered human coagulation factor IX (h.FIX) under the control of liver-specific promoters (LP1 or hAAT) into C57BL/6 mice. The circulating levels of h.FIX:Ag were higher in all the K137R-AAV8 treated groups up to 8 weeks post-hepatic gene transfer. These studies demonstrate the feasibility of the use of this novel AAV8 vectors for potential gene therapy of hemophilia B. PMID:23442071

Lack of Humoral Immune Response to the Tetracycline (Tet) Activator in Rats Injected Intracranially with Tet-off rAAV Vectors

PubMed Central

Han, Ye; Chang, Qin A.; Virag, Tamas; West, Neva C.; George, David; Castro, Maria G.; Bohn, Martha C.

2010-01-01

The ability to safely control transgene expression from viral vectors is a long-term goal in the gene therapy field. We have previously reported tight regulation of GFP expression in rat brain using a self-regulating tet-off rAAV vector. The immune responses against tet regulatory elements observed by other groups in nonhuman primates after intramuscular injection of tet-on encoding vectors raise concerns about the clinical value of tet-regulated vectors. However, previous studies have not examined immune responses following injection of AAV vectors into brain. Therefore, rat striatum was injected with tet-off rAAV harboring a therapeutic gene for Parkinson's disease, either hAADC or hGDNF. The expression of each gene was tightly controlled by the tet-off regulatory system. Using an ELISA developed with purified GST-tTA protein, no detectable immunogenicity against tTA was observed in sera of rats that received an intrastriatal injection of either vector. In contrast, sera from rats intradermally injected with an adenovirus containing either tTA or rtTA, as positive controls, had readily detectable antibodies. These observations suggest that tet-off rAAV vectors do not elicit an immune response when injected into rat brain and that these may offer safer vectors for Parkinson's disease than vectors with constitutive expression. PMID:20164859

Methods of treating Parkinson's disease using viral vectors

DOEpatents

Bankiewicz, Krys; Cunningham, Janet

2012-11-13

Methods of delivering viral vectors, particularly recombinant AAV virions, to the central nervous system (CNS) are provided for the treatment of CNS disorders, particularly those disorders which involve the neurotransmitter dopamine. The methods entail providing rAAV virions that comprise a transgene encoding aromatic amino acid decarboxylase (AADC) and administering the virions to the brain of a mammal using a non-manual pump.

Improving effect of mild foot electrical stimulation on pentylenetetrazole-induced impairment of learning and memory.

PubMed

Abasi-Moghadam, Monir; Ghasemi-Dehno, Arefe; Sadegh, Mehdi; Palizvan, Mohammad Reza

2018-05-10

Epilepsy is a common neurological disorder that affects learning and memory. Recently it has been shown that mild foot electrical stimulation (MFES) can increase learning and memory in normal rats. Pentylenetetrazole (PTZ) kindling is a model of human epilepsy. As with human epilepsy, PTZ kindling impairs learning and memory in rats. The purpose of this study was to investigate the effect MFES on kindling-induced learning and memory deficits in rats. Forty-nine male Wistar rats weighting 200 to 250 g were divided into the following seven groups: PTZ only, phenytoin only, MFES only, PTZ plus phenytoin, PTZ plus MFES, phenytoin plus MFES, and saline (control), with the treatments administered for 26 days. Forty-eight hours after the last injection, the animals performed the Morris water maze (MWM) task, and spatial learning and memory were measured. The results indicated that although chronic administration of phenytoin inhibited the development of PTZ kindling, it did not exert a protective effect against kindling-induced spatial learning and memory impairment in rats. On the other hand, pretreatment of PTZ-kindled animals with MFES significantly improved spatial working and reference memory. The results point to potential novel beneficial effects of MFES on learning and memory impairment induced by PTZ kindling in rats. Copyright © 2018 Elsevier Inc. All rights reserved.

Piperacillin-tazobactam vs. imipenem-cilastatin as empirical therapy in hematopoietic stem cell transplantation recipients with febrile neutropenia.

PubMed

Jing, Yu; Li, Jian; Yuan, Lei; Zhao, Xiaoli; Wang, Quanshun; Yu, Li; Zhou, Daobin; Huang, Wenrong

2016-03-01

This randomized, dual-center study compared the efficacy and safety of piperacillin-tazobactam (PTZ) and imipenem-cilastatin (IMP) in hematopoietic stem cell transplantation (HSCT) recipients with febrile neutropenia. HSCT recipients with febrile neutropenia were randomized into two groups receiving either PTZ or IMP as initial empiric antibiotic. Endpoints were defervescence rate after empiric antibiotic for 48 h, success at end of therapy, and side effects. Defervescence within 48 h after empiric antibiotic was observed in 46 patients with PTZ (75.4%) and 59 patients with IMP (95.2%) (p = 0.002). Ten patients (10/46) in the PTZ group and two patients (2/59) in the IMP group switched empiric antibiotics due to recurrent fever (p = 0.005). Success of initial antibiotic with modification was achieved in 34 patients with PTZ (55.7%) and 53 patients with IMP (85.5%) at the end of therapy (p = 0.001). To treat the bacteremia, seven of 10 patients in the PTZ group and one of eight patients in the IMP group needed to switch the empiric antibiotic (p = 0.025). Compared with PTZ, IMP had more gastrointestinal adverse events (p = 0.045). This study demonstrates that IMP had better efficacy than PTZ as an empiric antibiotic for febrile neutropenia in the HSCT setting, but with more gastrointestinal side reactions. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Subpicosecond Photoinduced Hole Transfer from a CdS Quantum Dot to a Molecular Acceptor Bound Through an Exciton-Delocalizing Ligand.

PubMed

Lian, Shichen; Weinberg, David J; Harris, Rachel D; Kodaimati, Mohamad S; Weiss, Emily A

2016-06-28

This paper describes the enhancement of the rate of hole transfer from a photoexcited CdS quantum dot (QD), with radius R = 2.0 nm, to a molecular acceptor, phenothiazine (PTZ), by linking the donor and acceptor through a phenyldithiocarbamate (PTC) linker, which is known to lower the confinement energy of the excitonic hole. Upon adsorption of PTC, the bandgap of the QD decreases due to delocalization of the exciton, primarily the excitonic hole, into interfacial states of mixed QD/PTC character. This delocalization enables hole transfer from the QD to PTZ in <300 fs (within the instrument response of the laser system) when linked by PTC, but not when linked by a benzoate group, which has a similar length and conjugation as PTC but does not delocalize the excitonic hole. Comparison of the two systems was aided by quantification of the surface coverage of benzoate and PTC-linked PTZ by (1)H NMR. This work provides direct spectroscopic evidence of the enhancement of the rate of hole extraction from a colloidal QD through covalent linkage of a hole acceptor through an exciton-delocalizing ligand.

Ultrafast photoinduced charge transport in Pt(II) donor-acceptor assembly bearing naphthalimide electron acceptor and phenothiazine electron donor.

PubMed

Sazanovich, Igor V; Best, Jonathan; Scattergood, Paul A; Towrie, Michael; Tikhomirov, Sergei A; Bouganov, Oleg V; Meijer, Anthony J H M; Weinstein, Julia A

2014-12-21

Visible light-induced charge transfer dynamics were investigated in a novel transition metal triad acceptor-chromophore-donor, (NDI-phen)Pt(II)(-C≡C-Ph-CH2-PTZ)2 (1), designed for photoinduced charge separation using a combination of time-resolved infrared (TRIR) and femtosecond electronic transient absorption (TA) spectroscopy. In 1, the electron acceptor is 1,4,5,8-naphthalene diimide (NDI), and the electron donor is phenothiazine (PTZ), and [(phen)Pt(-C≡C-Ph-)], where phen is 1,10-phenanthroline, represents the chromophoric core. The first excited state observed in 1 is a (3)MLCT/LL'CT, with {Pt(II)-acetylide}-to-phen character. Following that, charge transfer from the phen-anion onto the NDI subunit to form NDI(-)-phen-[Pt-(C≡C)2](+)-PTZ2 occurs with a time constant of 2.3 ps. This transition is characterised by appearance of the prominent NDI-anion features in both TRIR and TA spectra. The final step of the charge separation in 1 proceeds with a time constant of ∼15 ps during which the hole migrates from the [Pt-(C≡C)2] subunit to one of the PTZ groups. Charge recombination in 1 then occurs with two distinct time constants of 36 ns and 107 ns, corresponding to the back electron transfer to each of the two donor groups; a rather rare occurrence which manifests that the hole in the final charge-separated state is localised on one of the two donor PTZ groups. The assignment of the nature of the excited states and dynamics in 1 was assisted by TRIR investigations of the analogous previously reported ((COOEt)2bpy)Pt(C≡C-Ph-CH2-PTZ)2 (2), (J. E. McGarrah and R. Eisenberg, Inorg. Chem., 2003, 42, 4355; J. E. McGarrah, J. T. Hupp and S. N. Smirnov, J. Phys. Chem. A, 2009, 113, 6430) as well as (bpy)Pt(C≡C-Ph-C7H15)2, which represent the acceptor-free dyad, and the chromophoric core, respectively. Thus, the step-wise formation of the full charge-separated state on the picosecond time scale and charge recombination via tunnelling have been established; and the presence of two distinct charge recombination pathways has been observed.

Brain redox imaging in the pentylenetetrazole (PTZ)-induced kindling model of epilepsy by using in vivo electron paramagnetic resonance and a nitroxide imaging probe.

PubMed

Emoto, Miho C; Yamato, Mayumi; Sato-Akaba, Hideo; Yamada, Ken-ichi; Fujii, Hirotada G

2015-11-03

Much evidence supports the idea that oxidative stress is involved in the pathogenesis of epilepsy, and therapeutic interventions with antioxidants are expected as adjunct antiepileptic therapy. The aims of this study were to non-invasively obtain spatially resolved redox data from control and pentylenetetrazole (PTZ)-induced kindled mouse brains by electron paramagnetic resonance (EPR) imaging and to visualize the brain regions that are sensitive to oxidative damage. After infusion of the redox-sensitive imaging probe 3-methoxycarbonyl-2,2,5,5-tetramethyl-piperidine-1-oxyl (MCP), a series of EPR images of PTZ-induced mouse heads were measured. Based on the pharmacokinetics of the reduction reaction of MCP in the mouse heads, the pixel-based rate constant of its reduction reaction was calculated as an index of redox status in vivo and mapped as a redox map. The obtained redox map showed heterogeneity in the redox status in PTZ-induced mouse brains compared with control. The co-registered image of the redox map and magnetic resonance imaging (MRI) for both control and PTZ-induced mice showed a clear change in the redox status around the hippocampus after PTZ. To examine the role of antioxidants on the brain redox status, the levels of antioxidants were measured in brain tissues of control and PTZ-induced mice. Significantly lower concentrations of glutathione in the hippocampus of PTZ-kindled mice were detected compared with control. From the results of both EPR imaging and the biochemical assay, the hippocampus was found to be susceptible to oxidative damage in the PTZ-induced animal model of epilepsy. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Anticonvulsant effects of isomeric nonimidazole histamine H3 receptor antagonists

PubMed Central

Sadek, Bassem; Saad, Ali; Schwed, Johannes Stephan; Weizel, Lilia; Walter, Miriam; Stark, Holger

2016-01-01

Phenytoin (PHT), valproic acid, and modern antiepileptic drugs (AEDs), eg, remacemide, loreclezole, and safinamide, are only effective within a maximum of 70%–80% of epileptic patients, and in many cases the clinical use of AEDs is restricted by their side effects. Therefore, a continuous need remains to discover innovative chemical entities for the development of active and safer AEDs. Ligands targeting central histamine H3 receptors (H3Rs) for epilepsy might be a promising therapeutic approach. To determine the potential of H3Rs ligands as new AEDs, we recently reported that no anticonvulsant effects were observed for the (S)-2-(4-(3-(piperidin-1-yl)propoxy)benzylamino)propanamide (1). In continuation of our research, we asked whether anticonvulsant differences in activities will be observed for its R-enantiomer, namely, (R)-2-(4-(3-(piperidin-1-yl)propoxy)benzylamino)propaneamide (2) and analogs thereof, in maximum electroshock (MES)-, pentylenetetrazole (PTZ)-, and strychnine (STR)-induced convulsion models in rats having PHT and valproic acid (VPA) as reference AEDs. Unlike the S-enantiomer (1), the results show that animals pretreated intraperitoneally (ip) with the R-enantiomer 2 (10 mg/kg) were moderately protected in MES and STR induced models, whereas proconvulsant effect was observed for the same ligand in PTZ-induced convulsion models. However, animals pretreated with intraperitoneal doses of 5, 10, or 15 mg/kg of structurally bulkier (R)-enantiomer (3), in which 3-piperidinopropan-1-ol in ligand 2 was replaced by (4-(3-(piperidin-1-yl)propoxy)phenyl)methanol, and its (S)-enantiomer (4) significantly and in a dose-dependent manner reduced convulsions or exhibited full protection in MES and PTZ convulsions model, respectively. Interestingly, the protective effects observed for the (R)-enantiomer (3) in MES model were significantly greater than those of the standard H3R inverse agonist/antagonist pitolisant, comparable with those observed for PHT, and reversed when rats were pretreated with the selective H3R agonist R-(α)-methyl-histamine. Comparisons of the observed antagonistic in vitro affinities among the ligands 1–6 revealed profound stereoselectivity at human H3Rs with varying preferences for this receptor subtype. Moreover, the in vivo anticonvulsant effects observed in this study for ligands 1–6 showed stereoselectivity in different convulsion models in male adult rats. PMID:27853355

Anticonvulsant effects of isomeric nonimidazole histamine H3 receptor antagonists.

PubMed

Sadek, Bassem; Saad, Ali; Schwed, Johannes Stephan; Weizel, Lilia; Walter, Miriam; Stark, Holger

2016-01-01

Phenytoin (PHT), valproic acid, and modern antiepileptic drugs (AEDs), eg, remacemide, loreclezole, and safinamide, are only effective within a maximum of 70%-80% of epileptic patients, and in many cases the clinical use of AEDs is restricted by their side effects. Therefore, a continuous need remains to discover innovative chemical entities for the development of active and safer AEDs. Ligands targeting central histamine H 3 receptors (H 3 Rs) for epilepsy might be a promising therapeutic approach. To determine the potential of H 3 Rs ligands as new AEDs, we recently reported that no anticonvulsant effects were observed for the ( S )-2-(4-(3-(piperidin-1-yl)propoxy)benzylamino)propanamide ( 1 ). In continuation of our research, we asked whether anticonvulsant differences in activities will be observed for its R -enantiomer, namely, ( R )-2-(4-(3-(piperidin-1-yl)propoxy)benzylamino)propaneamide ( 2 ) and analogs thereof, in maximum electroshock (MES)-, pentylenetetrazole (PTZ)-, and strychnine (STR)-induced convulsion models in rats having PHT and valproic acid (VPA) as reference AEDs. Unlike the S -enantiomer ( 1 ), the results show that animals pretreated intraperitoneally (ip) with the R -enantiomer 2 (10 mg/kg) were moderately protected in MES and STR induced models, whereas proconvulsant effect was observed for the same ligand in PTZ-induced convulsion models. However, animals pretreated with intraperitoneal doses of 5, 10, or 15 mg/kg of structurally bulkier ( R )-enantiomer ( 3 ), in which 3-piperidinopropan-1-ol in ligand 2 was replaced by (4-(3-(piperidin-1-yl)propoxy)phenyl)methanol, and its ( S )-enantiomer ( 4 ) significantly and in a dose-dependent manner reduced convulsions or exhibited full protection in MES and PTZ convulsions model, respectively. Interestingly, the protective effects observed for the ( R )-enantiomer ( 3 ) in MES model were significantly greater than those of the standard H 3 R inverse agonist/antagonist pitolisant, comparable with those observed for PHT, and reversed when rats were pretreated with the selective H 3 R agonist R -(α)-methyl-histamine. Comparisons of the observed antagonistic in vitro affinities among the ligands 1 - 6 revealed profound stereoselectivity at human H 3 Rs with varying preferences for this receptor subtype. Moreover, the in vivo anticonvulsant effects observed in this study for ligands 1 - 6 showed stereoselectivity in different convulsion models in male adult rats.

Construction of New Campylobacter Cloning Vectors and a New Mutational Cat Cassette

DTIC Science & Technology

1993-01-01

mutational cat cassette PE - 61102A PR - 3M161102 6. AUTHOR(S) TA - BS13AK Yao R, Aim RA, Trust TJ, Guerry P WU- 1291 7. PERFORMING ORGANIZATION NAME(S) AND...mutational cat cassette %~ccesion For (Site-specific mutagenesis; recombinant DNA; multiple cloning site; PCR; shuttle vectors) NTIS CRA&I OTIC TAB E...campylobacter portion of these vectors, only three CAT , Cm acetyllraaseriase; car, gene encoding CAT , Cm, restriction sites in the IacZ MCS remain unique

Isolation, cloning and expression mapping of a gene encoding an anti-diuretic hormone and other CAPA-related peptides in the disease vector, Rhodnius prolixus

USDA-ARS?s Scientific Manuscript database

Following a blood meal, Rhodnius prolixus undergoes a rapid diuresis in order to eliminate excess water and salts. During the voiding of this primary urine, R. prolixus acts as a vector of Chagas’ disease, with the causative agent, Trypanosoma cruzi, infecting the human host via the urine. Diuresi...

Mechanism of protection against alcoholism by an alcohol dehydrogenase polymorphism: development of an animal model.

PubMed

Rivera-Meza, Mario; Quintanilla, María Elena; Tampier, Lutske; Mura, Casilda V; Sapag, Amalia; Israel, Yedy

2010-01-01

Humans who carry a point mutation in the gene coding for alcohol dehydrogenase-1B (ADH1B*2; Arg47His) are markedly protected against alcoholism. Although this mutation results in a 100-fold increase in enzyme activity, it has not been reported to cause higher levels of acetaldehyde, a metabolite of ethanol known to deter alcohol intake. Hence, the mechanism by which this mutation confers protection against alcoholism is unknown. To study this protective effect, the wild-type rat cDNA encoding rADH-47Arg was mutated to encode rADH-47His, mimicking the human mutation. The mutated cDNA was incorporated into an adenoviral vector and administered to genetically selected alcohol-preferring rats. The V(max) of rADH-47His was 6-fold higher (P<0.001) than that of the wild-type rADH-47Arg. Animals transduced with rAdh-47His showed a 90% (P<0.01) increase in liver ADH activity and a 50% reduction (P<0.001) in voluntary ethanol intake. In animals transduced with rAdh-47His, administration of ethanol (1g/kg) produced a short-lived increase of arterial blood acetaldehyde concentration to levels that were 3.5- to 5-fold greater than those in animals transduced with the wild-type rAdh-47Arg vector or with a noncoding vector. This brief increase (burst) in arterial acetaldehyde concentration after ethanol ingestion may constitute the mechanism by which humans carrying the ADH1B*2 allele are protected against alcoholism.

Effect of harmane on the convulsive threshold in epilepsy models in mice.

PubMed

Aricioglu, Feyza; Yillar, Okan; Korcegez, Eylem; Berkman, Kemal

2003-12-01

The study investigated the activity of harmane on maximal electroshock seizures (MES) and seizures induced by pentilentetrazole (PTZ) in mice. Initial studies established convulsive current 50 (CC(50)) values or MES and effective dose 50 (ED(50)) for PTZ to produce seizures. Harmane (2.5, 5.0, or 10 mg/kg intraperitoneally) increased the threshold of seizures in MES dose-dependently. The convulsions produced by PTZ were decreased by the low dose of harmane (2.5 mg/kg), but the high dose of harmane (10 mg/kg) resulted in worse grade V convulsions followed by more lethality compared with PTZ alone. Therefore, harmane seems to be protective against grand mal seizures in the MES model but not against a petit mal seizure model (PTZ) in mice.

Treatment with pentylenetetrazole (PTZ) and 4-aminopyridine (4-AP) differently affects survival, locomotor activity, and biochemical markers in Drosophila melanogaster.

PubMed

Soares, Deividi C S; Portela, José L R; Roos, Daniel H; Rodrigues, Nathane R; Gomes, Karen K; Macedo, Giulianna E; Posser, Thais; Franco, Jeferson L; Hassan, Waseem; Puntel, Robson L

2018-05-01

PTZ is a convulsive agent that acts via selective blockage of GABA A receptor channels, whereas 4-AP leads to a convulsive episode via blockage of K + channels. However, the mechanism(s) by which pentylenetetrazole (PTZ) and 4-aminopyridine (4-AP) cause toxicity to Drosophila melanogaster needs to be properly explored, once it will help in establishing an alternative model for development of proper therapeutic strategies and also to counteract the changes associated with exposure to both epileptic drugs. For the purpose, we investigated the effects of exposure (48 h) to PTZ (60 mM) and/or 4-AP (20 mM) on survival, locomotor performance, and biochemical markers in the body and/or head of flies. 4-AP-fed flies presented a higher incidence of mortality and a worse performance in the open field test as compared to non-treated flies. 4-AP also caused a significant increase in the reactive species (RS) and protein carbonyl (PC) content in the body and head. Also a significant increase in catalase and acetylcholinesterase (AChE) activities was observed in the body. In the same vein, PTZ exposure resulted in a significant increase in RS, thiobarbituric acid reactive substances (TBARS), PC content, and catalase activity in the body. PTZ exposure also caused a significant increase in AChE activity both in body and head. It is important to note that PTZ-treated flies also down-regulated the NRF 2 expression. Moreover, both 4AP- and PTZ-fed flies presented a significant decrease in MTT reduction, down-regulation, and inhibition of SOD in body. However, SOD was significantly more active in the head of both 4-AP and PTZ-treated flies. Our findings provide evidence regarding the toxicological potential of both PTZ and/or 4-AP to flies. This model will help in decoding the underlying toxicological mechanisms of the stated drugs. It will also help to properly investigate the therapeutic strategies and to counteract the drastic changes associated with both epileptogenic drugs.

Generation of Recombinant Modified Vaccinia Virus Ankara Encoding VP2, NS1, and VP7 Proteins of Bluetongue Virus.

PubMed

Marín-López, Alejandro; Ortego, Javier

2016-01-01

Modified Vaccinia Virus Ankara (MVA) is employed widely as an experimental vaccine vector for its lack of replication in mammalian cells and high expression level of foreign/heterologous genes. Recombinant MVAs (rMVAs) are used as platforms for protein production as well as vectors to generate vaccines against a high number of infectious diseases and other pathologies. The portrait of the virus combines desirable elements such as high-level biological safety, the ability to activate appropriate innate immune mediators upon vaccination, and the capacity to deliver substantial amounts of heterologous antigens. Recombinant MVAs encoding proteins of bluetongue virus (BTV), an Orbivirus that infects domestic and wild ruminants transmitted by biting midges of the Culicoides species, are excellent vaccine candidates against this virus. In this chapter we describe the methods for the generation of rMVAs encoding VP2, NS1, and VP7 proteins of bluetongue virus as a model example for orbiviruses. The protocols included cover the cloning of VP2, NS1, and VP7 BTV-4 genes in a transfer plasmid, the construction of recombinant MVAs, the titration of virus working stocks and the protein expression analysis by immunofluorescence and radiolabeling of rMVA infected cells as well as virus purification.

A Single-Vector, Single-Injection Trivalent Filovirus Vaccine: Proof of Concept Study in Outbred Guinea Pigs.

PubMed

Mire, Chad E; Geisbert, Joan B; Versteeg, Krista M; Mamaeva, Natalia; Agans, Krystle N; Geisbert, Thomas W; Connor, John H

2015-10-01

The filoviruses, Marburg marburgvirus (MARV), Zaire ebolavirus (ZEBOV), and Sudan ebolavirus (SEBOV), cause severe and often fatal hemorrhagic fever in humans and nonhuman primates (NHPs). Monovalent recombinant vesicular stomatitis virus (rVSV)-based vaccine vectors, which encode a filovirus glycoprotein (GP) in place of the VSV glycoprotein, have shown 100% efficacy against homologous filovirus challenge in rodent and NHP studies. Here, we examined the utility of a single-vector, single-injection trivalent rVSV vector expressing MARV, ZEBOV, and SEBOV GPs to protect against MARV-, ZEBOV-, and SEBOV-induced disease in outbred Hartley guinea pigs where we observed protection from effects of all 3 filoviruses. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Whole-genome sequence of “Candidatus Liberibacter solanacearum” strain R1 from California

USDA-ARS?s Scientific Manuscript database

The draft whole-genome sequence of “Candidatus Liberibacter solanacearum” strain R1, isolated from a tomato plant in California, United States, is reported. The R1 strain genome is 1,204,257 bp in size (G+C content of 35.3%), encoding 1,101 open reading frames and 57 RNA genes....

Structural and Functional Elucidation of Yeast Lanosterol 14α-Demethylase in Complex with Agrochemical Antifungals

PubMed Central

Sagatova, Alia A.; Keniya, Mikhail V.; Negroni, Jacopo; Wilson, Rajni K.; Woods, Matthew A.; Monk, Brian C.

2016-01-01

Azole antifungals, known as demethylase inhibitors (DMIs), target sterol 14α-demethylase (CYP51) in the ergosterol biosynthetic pathway of fungal pathogens of both plants and humans. DMIs remain the treatment of choice in crop protection against a wide range of fungal phytopathogens that have the potential to reduce crop yields and threaten food security. We used a yeast membrane protein expression system to overexpress recombinant hexahistidine-tagged S. cerevisiae lanosterol 14α-demethylase and the Y140F or Y140H mutants of this enzyme as surrogates in order characterize interactions with DMIs. The whole-cell antifungal activity (MIC50 values) of both the R- and S-enantiomers of tebuconazole, prothioconazole (PTZ), prothioconazole-desthio, and oxo-prothioconazole (oxo-PTZ) as well as for fluquinconazole, prochloraz and a racemic mixture of difenoconazole were determined. In vitro binding studies with the affinity purified enzyme were used to show tight type II binding to the yeast enzyme for all compounds tested except PTZ and oxo-PTZ. High resolution X-ray crystal structures of ScErg11p6×His in complex with seven DMIs, including four enantiomers, reveal triazole-mediated coordination of all compounds and the specific orientation of compounds within the relatively hydrophobic binding site. Comparison with CYP51 structures from fungal pathogens including Candida albicans, Candida glabrata and Aspergillus fumigatus provides strong evidence for a highly conserved CYP51 structure including the drug binding site. The structures obtained using S. cerevisiae lanosterol 14α-demethylase in complex with these agrochemicals provide the basis for understanding the impact of mutations on azole susceptibility and a platform for the structure-directed design of the next-generation of DMIs. PMID:27907120

Intranasal adenovirus-vectored vaccine for induction of long-lasting humoral immunity-mediated broad protection against influenza in mice.

PubMed

Kim, Eun Hye; Park, Hae-Jung; Han, Gye-Yeong; Song, Man-Ki; Pereboev, Alexander; Hong, Jeong S; Chang, Jun; Byun, Young-Ho; Seong, Baik Lin; Nguyen, Huan H

2014-09-01

Influenza vaccines aimed at inducing antibody (Ab) responses against viral surface hemagglutinin (HA) and neuraminidase (NA) provide sterile immunity to infection with the same subtypes. Vaccines targeting viral conserved determinants shared by the influenza A viruses (IAV) offer heterosubtypic immunity (HSI), a broad protection against different subtypes. We proposed that vaccines targeting both HA and the conserved ectodomain of matrix protein 2 (M2e) would provide protection against infection with the same subtype and also HSI against other subtypes. We report here that single intranasal immunization with a recombinant adenovirus (rAd) vector encoding both HA of H5 virus and M2e (rAdH5/M2e) induced significant HA- and M2e-specific Ab responses, along with protection against heterosubtypic challenge in mice. The protection is superior compared to that induced by rAd vector encoding either HA (rAdH5), or M2e (rAdM2e). While protection against homotypic H5 virus is primarily mediated by virus-neutralizing Abs, the cross-protection is associated with Abs directed to conserved stalk HA and M2e that seem to have an additive effect. Consistently, adoptive transfer of antisera induced by rAdH5/M2e provided the best protection against heterosubtypic challenge compared to that provided by antisera derived from mice immunized with rAdH5 or rAdM2e. These results support the development of rAd-vectored vaccines encoding both H5 and M2e as universal vaccines against different IAV subtypes. Current licensed influenza vaccines provide protection limited to the infection with same virus strains; therefore, the composition of influenza vaccines has to be revised every year. We have developed a new universal influenza vaccine that is highly efficient in induction of long-lasting cross-protection against different influenza virus strains. The cross-protection is associated with a high level of vaccine-induced antibodies against the conserved stalk domain of influenza virus hemagglutinin and the ectodomain of matrix protein. The vaccine could be used to stimulate cross-protective antibodies for the prevention and treatment of influenza with immediate effect for individuals who fail to respond to or receive the vaccine in due time. The vaccine offers a new tool to control influenza outbreaks, including pandemics. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

The involvement of neuronal nitric oxide synthase in antiepileptic action of alpha-asarone on pentylenetetrazol molding rats.

PubMed

Su, Jing; Zhu, Wenting; Liu, Jing; Yin, Jian; Qin, Wei; Jiang, Changbin

2014-01-01

The aim of the present study was to research the role of nitric oxide (NO) as a mediator of alpha (α)-asarone effect at the pentylenetetrazol (PTZ)-induced epileptiform discharge in rat. α-Asarone that was injected intraperitoneally twenty minutes before PTZ injection suppressed the clonic discharge effectively and the significant actions lasted for 30 min with no change of clonic amplitude. Administration of α-asarone did not influence interictal discharge. Four kinds of NO regulators were administered, including non-selective NG-nitro-L-arginine methyl ester (L-NAME), selective neuronal nitric oxide synthase (nNOS) inhibitor, 7-nitroindazole (7-NI), inducible nitric oxide synthase (iNOS) inhibitor, aminoguanidine (AG) and NO substrate, L-arginine (ARG) and their influence on the actions of α-asarone were studied, and all of the regulators were administered fifteen minutes before α-asarone injection. L-NAME and 7-NI reversed the anticlonic activity of α-asarone, and a significant increase of clonic activity was induced by L-NAME later in L-NAME +.α-asarone + PTZ group. There were no significant differences between AG + α-asarone + PTZ and α-asarone + PTZ group. L-ARG played a dual role in this study. It aggravated clonic discharge in the early stage but relieved interictal discharge in the late stage compared with PTZ group alone, and the beneficial effect of α-asarone was also reversed. All the above results suggest that nNOS/NO pathway mediates the anticonvulsant effect of α-asarone, and NO played a biphasic role in PTZ modeling process, while iNOS was unrelated to the inhibition effect of α-asarone on PTZ induced epileptiform activity.

Curcumin attenuates inflammatory response and cognitive deficits in experimental model of chronic epilepsy.

PubMed

Kaur, Harpreet; Patro, Ishan; Tikoo, Kulbhushan; Sandhir, Rajat

2015-10-01

Evidence suggests that glial cells play a critical role in inflammation in chronic epilepsy, contributing to perpetuation of seizures and cognitive dysfunctions. The present study was designed to evaluate the beneficial effect of curcumin, a polyphenol with pleiotropic properties, on cognitive deficits and inflammation in chronic epilepsy. Kindled model of epilepsy was induced by administering sub-convulsive dose of pentylenetetrazole (PTZ) at 40 mg/kg, i.p. every alternative day for 30 days to Wistar rats. The animals were assessed for cognitive deficits by Morris water maze and inflammatory response in terms of microglial and astrocyte activation. PTZ treated animals had increased escape latency suggesting impaired cognitive functions. Further, an increased expression of astrocyte (GFAP) and microglial (Iba-1) activation markers were observed in terms of mRNA and protein levels in the PTZ treated animals. Concomitantly, mRNA and protein levels of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) and chemokine (MCP-1) were increased in hippocampus and cortex. Immunoreactivity to anti-GFAP and anti-Iba-1 antibodies was also enhanced in hippocampus and cortex suggesting gliosis in PTZ treated animals. However, curcumin administration at a dose of 100 mg/kg to PTZ animals prevented cognitive deficits. A significant decrease in pro-inflammatory cytokines and chemokine expression was observed in hippocampus and cortex of PTZ treated rats supplemented with curcumin. In addition, curcumin also attenuated increased expression of GFAP and Iba-1 in animals with PTZ induced chronic epilepsy. Moreover, immunohistochemical analysis also showed significant reduction in number of activated glial cells on curcumin administration to PTZ treated animals. Taken together, these findings suggest that curcumin is effective in attenuating glial activation and ameliorates cognitive deficits in chronic epilepsy. Copyright © 2015 Elsevier Ltd. All rights reserved.

Anti-epileptogenic and antioxidant effect of Lavandula officinalis aerial part extract against pentylenetetrazol-induced kindling in male mice.

PubMed

Rahmati, Batool; Khalili, Mohsen; Roghani, Mehrdad; Ahghari, Parisa

2013-06-21

Repeated application of Lavandula officinalis (L. officinalis) has been recommended for a long time in Iranian traditional medicine for some of nervous disorders like epilepsy and dementia. However, there is no available report for the effect of chronic administration of Lavandula extract in development (acquisition) of epilepsy. Therefore, this study was designed to investigate the anti-epileptogenic and antioxidant activity of repeated administration of Lavandula officinalis extract on pentylenetetrazol (PTZ) kindling seizures in mice model. Lavandula officinalis was tested for its ability (i) to suppress the seizure intensity and lethal effects of PTZ in kindled mice (anti-epileptogenic effect), (ii) to attenuate the PTZ-induced oxidative injury in the brain tissue (antioxidant effect) when given as a pretreatment prior to each PTZ injection during kindling development. Valproate (Val), a major antiepileptic drug, was also tested for comparison. Val and Lavandula officinalis extract showed anti-epileptogenic properties as they reduced seizure score of kindled mice and PTZ-induced mortality. In this regard, Lavandula officinalis was more effective than Val. Both Lavandula officinalis and Val suppressed brain nitric oxide (NO) level of kindled mice in comparison with the control and PTZ group. Meanwhile, Lavandula officinalis suppressed NO level more than Val and Lavandula officinalis also decreased brain MDA level relative to PTZ group. This is the first report to demonstrate NO suppressing and anti-epileptogenic effect of chronic administration of Lavandula officinalis extract on acquisition of epilepsy in PTZ kindling mice model. In this regard, Lavandula officinalis extract was more effective than Val, possibly and in part via brain NO suppression. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Neuronal zinc-α2-glycoprotein is decreased in temporal lobe epilepsy in patients and rats.

PubMed

Liu, Ying; Wang, Teng; Liu, Xi; Wei, Xin; Xu, Tao; Yin, Maojia; Ding, Xueying; Mo, Lijuan; Chen, Lifen

2017-08-15

Zinc-α2-glycoprotein (ZAG) is a 42-kDa protein encoded by the AZGP1 gene that is known as a lipid mobilizing factor and is highly homologous to major histocompatibility complex class I family molecules. Recently, transcriptomic research has shown that AZGP1 expression is reduced in the brain tissue of epilepsy patients. However, the cellular distribution and biological role of ZAG in the brain and epilepsy are unclear. Patients with refractory temporal lobe epilepsy (TLE) and brain trauma were included in this study, and pentylenetetrazole (PTZ)-kindled rats were also used. The existence and level of ZAG in the brain were identified using immunohistochemistry, double-labeled immunofluorescence and western blot, and the expression level of AZGP1 mRNA was determined with quantitative real-time polymerase chain reaction (qrt-PCR). To explore the potential biological role of ZAG in the brain, co-immunoprecipitation (Co-IP) of phosphorylated ERK (p-ERK), TGF-β1 and ZAG was also performed. ZAG was found in the cytoplasm of neurons in brain tissue from both patients and rats. The levels of AZGP1 mRNA and ZAG were lower in refractory TLE patients and PTZ-kindled rats than in controls. In addition, the ZAG level decreased as PTZ kindling continued. Co-IP identified direct binding between p-ERK, TGF-β1 and ZAG. ZAG was found to be synthesized in neurons, and both the AZGP1 mRNA and ZAG protein levels were decreased in epilepsy patients and rat models. The reduction in ZAG may participate in the pathogenesis and pathophysiology of epilepsy by interacting with p-ERK and TGF-β1, promoting inflammation, regulating the metabolism of ketone bodies, or affecting other epilepsy-related molecules. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

New phenotypes generated by the G57R mutation of BUD23 in Saccharomyces cerevisiae.

PubMed

Lin, Jyun-Liang; Yu, Hui-Chia; Chao, Ju-Lan; Wang, Chung; Cheng, Ming-Yuan

2012-12-01

BUD23 in Saccharomyces cerevisiae encodes for a class I methyltransferase, and deletion of the gene results in slow growth and random budding phenotypes. Herein, two BUD23 mutants defective in methyltransferase activity were generated to investigate whether the phenotypes of the null mutant might be correlated with a loss in enzymatic activity. Expression at the physiological level of both D77A and G57R mutants was able to rescue the phenotypes of the bud23-null mutant. The result implied that the methyltransferase activity of the protein was not necessary for supporting normal growth and bud site selection of the cells. High-level expression of Bud23 (G57R), but not Bud23 or Bud23 (D77A), in BUD23 deletion cells failed to complement these phenotypes. However, just like Bud23, Bud23 (G57R) was localized in a DAPI-poor region in the nucleus. Distinct behaviour in Bud23 (G57R) could not be originated from a mislocalization of the protein. Over-expression of Bud23 (G57R) in null cells also produced changes in actin organization and additional septin mutant-like phenotypes. Therefore, the absence of Bud23, Bud23 (G57R) at a high level might affect the cell division of yeast cells through an as yet unidentified mechanism. Copyright © 2012 John Wiley & Sons, Ltd.

Safety and Immunogenicity of a Recombinant Adenovirus Serotype 35-Vectored HIV-1 Vaccine in Adenovirus Serotype 5 Seronegative and Seropositive Individuals.

PubMed

Fuchs, Jonathan D; Bart, Pierre-Alexandre; Frahm, Nicole; Morgan, Cecilia; Gilbert, Peter B; Kochar, Nidhi; DeRosa, Stephen C; Tomaras, Georgia D; Wagner, Theresa M; Baden, Lindsey R; Koblin, Beryl A; Rouphael, Nadine G; Kalams, Spyros A; Keefer, Michael C; Goepfert, Paul A; Sobieszczyk, Magdalena E; Mayer, Kenneth H; Swann, Edith; Liao, Hua-Xin; Haynes, Barton F; Graham, Barney S; McElrath, M Juliana

2015-05-01

Recombinant adenovirus serotype 5 (rAd5)-vectored HIV-1 vaccines have not prevented HIV-1 infection or disease and pre-existing Ad5 neutralizing antibodies may limit the clinical utility of Ad5 vectors globally. Using a rare Ad serotype vector, such as Ad35, may circumvent these issues, but there are few data on the safety and immunogenicity of rAd35 directly compared to rAd5 following human vaccination. HVTN 077 randomized 192 healthy, HIV-uninfected participants into one of four HIV-1 vaccine/placebo groups: rAd35/rAd5, DNA/rAd5, and DNA/rAd35 in Ad5-seronegative persons; and DNA/rAd35 in Ad5-seropositive persons. All vaccines encoded the HIV-1 EnvA antigen. Antibody and T-cell responses were measured 4 weeks post boost immunization. All vaccines were generally well tolerated and similarly immunogenic. As compared to rAd5, rAd35 was equally potent in boosting HIV-1-specific humoral and cellular immunity and responses were not significantly attenuated in those with baseline Ad5 seropositivity. Like DNA, rAd35 efficiently primed rAd5 boosting. All vaccine regimens tested elicited cross-clade antibody responses, including Env V1/V2-specific IgG responses. Vaccine antigen delivery by rAd35 is well-tolerated and immunogenic as a prime to rAd5 immunization and as a boost to prior DNA immunization with the homologous insert. Further development of rAd35-vectored prime-boost vaccine regimens is warranted.

ACT-Vision: active collaborative tracking for multiple PTZ cameras

NASA Astrophysics Data System (ADS)

Broaddus, Christopher; Germano, Thomas; Vandervalk, Nicholas; Divakaran, Ajay; Wu, Shunguang; Sawhney, Harpreet

2009-04-01

We describe a novel scalable approach for the management of a large number of Pan-Tilt-Zoom (PTZ) cameras deployed outdoors for persistent tracking of humans and vehicles, without resorting to the large fields of view of associated static cameras. Our system, Active Collaborative Tracking - Vision (ACT-Vision), is essentially a real-time operating system that can control hundreds of PTZ cameras to ensure uninterrupted tracking of target objects while maintaining image quality and coverage of all targets using a minimal number of sensors. The system ensures the visibility of targets between PTZ cameras by using criteria such as distance from sensor and occlusion.

Zinc-finger nuclease-mediated gene correction using single AAV vector transduction and enhancement by Food and Drug Administration-approved drugs

PubMed Central

Ellis, BL; Hirsch, ML; Porter, SN; Samulski, RJ; Porteus, MH

2016-01-01

An emerging strategy for the treatment of monogenic diseases uses genetic engineering to precisely correct the mutation(s) at the genome level. Recent advancements in this technology have demonstrated therapeutic levels of gene correction using a zinc-finger nuclease (ZFN)-induced DNA double-strand break in conjunction with an exogenous DNA donor substrate. This strategy requires efficient nucleic acid delivery and among viral vectors, recombinant adeno-associated virus (rAAV) has demonstrated clinical success without pathology. However, a major limitation of rAAV is the small DNA packaging capacity and to date, the use of rAAV for ZFN gene delivery has yet to be reported. Theoretically, an ideal situation is to deliver both ZFNs and the repair substrate in a single vector to avoid inefficient gene targeting and unwanted mutagenesis, both complications of a rAAV co-transduction strategy. Therefore, a rAAV format was generated in which a single polypeptide encodes the ZFN monomers connected by a ribosome skipping 2A peptide and furin cleavage sequence. On the basis of this arrangement, a DNA repair substrate of 750 nucleotides was also included in this vector. Efficient polypeptide processing to discrete ZFNs is demonstrated, as well as the ability of this single vector format to stimulate efficient gene targeting in a human cell line and mouse model derived fibroblasts. Additionally, we increased rAAV-mediated gene correction up to sixfold using a combination of Food and Drug Administration-approved drugs, which act at the level of AAV vector transduction. Collectively, these experiments demonstrate the ability to deliver ZFNs and a repair substrate by a single AAV vector and offer insights for the optimization of rAAV-mediated gene correction using drug therapy. PMID:22257934

Site-specific integration of Streptomyces PhiC31 integrase-based vectors in the chromosome of Rhodococcus equi.

PubMed

Hong, Yang; Hondalus, Mary K

2008-10-01

Streptomyces PhiC31-based site-specific integration was used to transform the facultative intracellular pathogen Rhodococcus equi. The transformation efficiency of vectors incorporating the PhiC31 integrase and attP sites was comparable to that of replication plasmids using the same electroporation procedure. A single attB integration site was identified within an ORF encoding a pirin-like protein, which deviates slightly from the consensus sequence of Streptomyces attB sites. Vector integration was stably maintained in the R. equi chromosome for as many as 100 generations during unselected passage in vitro. In addition, integration does not appear to affect the replication of bacteria inside macrophages. Finally, this integration system was also used to successfully complement an R. equi mutant.

Characterization of the cryptic plasmid pOfk55 from Legionella pneumophila and construction of a pOfk55-derived shuttle vector.

PubMed

Nishida, Takashi; Watanabe, Kenta; Tachibana, Masato; Shimizu, Takashi; Watarai, Masahisa

2017-03-01

In this study, a cryptic plasmid pOfk55 from Legionella pneumophila was isolated and characterized. pOfk55 comprised 2584bp with a GC content of 37.3% and contained three putative open reading frames (ORFs). orf1 encoded a protein of 195 amino acids and the putative protein shared 39% sequence identity with a putative plasmid replication protein RepL. ORF1 was needed for replication in L. pneumophila but pOfk55 did not replicate in Escherichia coli. orf2 and orf3 encoded putative hypothetical proteins of 114 amino acids and 78 amino acids, respectively, but the functions of the putative proteins ORF2 and OFR3 are not clear. The transfer mechanism for pOfk55 was independent on the type IVB secretion system in the original host. A L. pneumophila-E. coli shuttle vector, pNT562 (5058bp, Km R ), was constructed by In-Fusion Cloning of pOfk55 with a kanamycin-resistance gene from pUTmini-Tn5Km and the origin of replication from pBluescript SK(+) (pNT561). Multiple cloning sites from pBluescript SK(+) as well as the tac promoter region and lacI gene from pAM239-GFP were inserted into pNT561 to construct pNT562. The transformation efficiency of pNT562 in L. pneumophila strains ranged from 1.6×10 1 to 1.0×10 5 CFU/ng. The relative number of pNT562 was estimated at 5.7±1.0 copies and 73.6% of cells maintained the plasmid after 1week in liquid culture without kanamycin. A green fluorescent protein (GFP) expression vector, pNT563, was constructed by ligating pNT562 with the gfpmut3 gene from pAM239-GFP. pNT563 was introduced into L. pneumophila Lp02 and E. coli DH5α, and both strains expressed GFP successfully. These results suggest that the shuttle vector is useful for genetic studies in L. pneumophila. Copyright © 2017 Elsevier Inc. All rights reserved.

Glutamate receptor 1 phosphorylation at Serine 831 and 845 modulates seizure susceptibility and hippocampal hyperexcitability following early life seizures

PubMed Central

Rakhade, S.N.; Fitzgerald, E.F.; Klein, P.M.; Zhou, C.; Sun, H; Huganir, R.L.; Jensen, F.E.

2012-01-01

Neonatal seizures can lead to later life epilepsy and neurobehavioral deficits, and there are no treatments to prevent these sequelae. We previously showed that hypoxia-induced seizures in a neonatal rat model induce rapid phosphorylation of S831 and S845 sites of the AMPA receptor GluR1 subunit and later neuronal hyperexcitability and epilepsy, suggesting that seizure-induced post-translational modifications may represent a novel therapeutic target. To unambiguously assess the contribution of these sites, we examined seizure susceptibility in wild type mice versus transgenic knock-in mice with deficits in GluR1 S831 and S845 phosphorylation (GluR1 double phosphomutant (GluR1DPM) mice). Phosphorylation of the GluR1 S831 and S845 sites was significantly increased in the hippocampus and cortex following a single episode of pentyleneterazol (PTZ) induced seizures in postnatal day 9 (P9) wild type mouse pups, and that transgenic knock-in mice have a higher threshold and longer latencies to seizures. Like the rat, hypoxic seizures in P9 C57BL/6N wild type mice resulted in transient increases in GluR1 S831 and GluR1 S845 phosphorylation in cortex, and were associated with enhanced seizure susceptibility to later-life kainic acid induced seizures. In contrast, later-life seizure susceptibility following hypoxia-induced seizures was attenuated in GluR1 DPM mice, supporting a role for post-translational modifications in seizure-induced network excitability. Finally, human hippocampal samples from neonatal seizure autopsy cases also showed an increase in GluR1 S831 and S845, supporting the validation of this potential therapeutic target in human tissue. PMID:23223299

Preventive effect of Coriandrum sativum on neuronal damages in pentylentetrazole-induced seizure in rats

PubMed Central

Pourzaki, Mojtaba; Homayoun, Mansour; Sadeghi, Saeed; Seghatoleslam, Masoumeh; Hosseini, Mahmoud; Ebrahimzadeh Bideskan, Alireza

2017-01-01

Objective: Coriandrum sativum (C. sativum) as a medicinal plant has been pointed to have analgesic, hypnotic and anti-oxidant effects. In the current study, a possible preventive effect of the hydro-alcoholic extract of the plant on neuronal damages was examined in pentylenetetrazole (PTZ) rat model of seizure. Materials and Methods: Forty male rats were divided into five main groups and treated by (1) saline, (2) PTZ: 100 mg/kg PTZ (i.p) and (3-5) 50, 100 and 200 mg/kg of hydro-alcoholic extract of C. sativum during seven consecutive days before PTZ injection. After electrocorticography (ECoG), the brains were removed to use for histological examination. Results: All doses of the extract reduced duration, frequency and amplitude of the burst discharges while prolonged the latency of the seizure attacks (p<0.05, p<0.01, and p<0.001). Administration of all 3 doses of the extract significantly prevented from production of dark neurons (p<0.01, and p<0.001) and apoptotic cells (p<0.05, p<0.01, and p<0.001) in different areas of the hippocampus compared to PTZ group. Conclusion: The results of this study allow us to conclude that C. sativum, because of its antioxidant properties, prevents from neuronal damages in PTZ rat model of seizure. PMID:28348967

Ultraviolet A photosensitivity profile of dexchlorpheniramine maleate and promethazine-based creams: Anti-inflammatory, antihistaminic, and skin barrier protection properties.

PubMed

Facchini, Gustavo; Eberlin, Samara; Clerici, Stefano Piatto; Alves Pinheiro, Ana Lucia Tabarini; Costa, Adilson

2017-12-01

Unwanted side effects such as dryness, hypersensitivity, and cutaneous photosensitivity are challenge for adherence and therapeutical success for patients using treatments for inflammatory and allergic skin response. In this study, we compared the effects of two dermatological formulations, which are used in inflammatory and/or allergic skin conditions: dexchlorpheniramine maleate (DCP; 10 mg/g) and promethazine (PTZ; 20 mg/g). We evaluated both formulations for phototoxicity potential, skin irritation, anti-inflammatory and antihistaminic abilities, and skin barrier repair in vitro and ex vivo using the standard OECD test guideline n° 432, the ECVAM protocol n° 78, and cultured skin explants from a healthy patient. Ultraviolet A was chosen as exogenous agent to induce allergic and inflammatory response. Both PTZ and DCP promoted increases in interleukin-1 (IL-1) synthesis in response to ultraviolet A (UVA) radiation compared to control. However, the increase observed with PTZ was significantly greater than the DCP, indicating that the latter has a lower irritant potential. DCP also demonstrated a protective effect on UVA-induced leukotriene B4 and nuclear factor kappa B (NF-κB) synthesis. Conversely, PTZ demonstrates more robust UVA antihistaminic activity. Likewise, PTZ promoted a significantly greater increase in the production of involucrin and keratin 14, both associated with protective skin barrier property. In conclusion, these data suggest possible diverging UVA response mechanisms of DCP and PTZ, which gives greater insight into the contrasting photosensitizing potential between DCP and PTZ observed in the patients. © 2017 Wiley Periodicals, Inc.

Development of SYBR Green and TaqMan quantitative real-time PCR assays for hepatopancreatic parvovirus (HPV) infecting Penaeus monodon in India.

PubMed

Yadav, Reena; Paria, Anutosh; Mankame, Smruti; Makesh, M; Chaudhari, Aparna; Rajendran, K V

2015-12-01

Hepatopancreatic parvovirus (HPV) infects Penaeus monodon and causes mortality in the larval stages. Further, it has been implicated in the growth retardation in cultured P. monodon. Though different geographical isolates of HPV show large sequence variations, a sensitive PCR assay specific to Indian isolate has not yet been reported. Here, we developed a sensitive SYBR Green-based and TaqMan real-time PCR for the detection and quantification of the virus. A 441-bp PCR amplicon was cloned in pTZ57 R/T vector and the plasmid copy number was estimated. A 10-fold serial dilution of the plasmid DNA from 1 × 10(9) copies to 1 copy was prepared and used as the standard. The primers were tested initially using the standard on a conventional PCR format to determine the linearity of detection. The standards were further tested on real-time PCR format using SYBR Green and TaqMan chemistry and standard curves were generated based on the Ct values from three well replicates for each dilution. The assays were found to be sensitive, specific and reproducible with a wide dynamic range (1 × 10(9) to 10 copies) with coefficient of regression (R(2)) > 0.99, calculated average slope -3.196 for SYBR Green assay whereas, for TaqMan assay it was >0.99 and -3.367, respectively. The intra- and inter-assay variance of the Ct values ranged from 0.26% to 0.94% and 0.12% to 0.81%, respectively, for SYBR Green assay, and the inter-assay variance of the Ct values for TaqMan assay ranged from 0.07% to 1.93%. The specificity of the assays was proved by testing other DNA viruses of shrimp such as WSSV, IHHNV and MBV. Standardized assays were further tested to detect and quantify HPV in the post-larvae of P. monodon. The result was further compared with conventional PCR to test the reproducibility of the test. The assay was also used to screen Litopeneaus vannamei, Macrobrachium rosenbergii and Scylla serrata for HPV. Copyright © 2015 Elsevier Ltd. All rights reserved.

Electromagnetic radiation (Wi-Fi) and epilepsy induce calcium entry and apoptosis through activation of TRPV1 channel in hippocampus and dorsal root ganglion of rats.

PubMed

Ghazizadeh, Vahid; Nazıroğlu, Mustafa

2014-09-01

Incidence rates of epilepsy and use of Wi-Fi worldwide have been increasing. TRPV1 is a Ca(2+) permeable and non-selective channel, gated by noxious heat, oxidative stress and capsaicin (CAP). The hyperthermia and oxidant effects of Wi-Fi may induce apoptosis and Ca(2+) entry through activation of TRPV1 channel in epilepsy. Therefore, we tested the effects of Wi-Fi (2.45 GHz) exposure on Ca(2+) influx, oxidative stress and apoptosis through TRPV1 channel in the murine dorsal root ganglion (DRG) and hippocampus of pentylentetrazol (PTZ)-induced epileptic rats. Rats in the present study were divided into two groups as controls and PTZ. The PTZ groups were divided into two subgroups namely PTZ + Wi-Fi and PTZ + Wi-Fi + capsazepine (CPZ). The hippocampal and DRG neurons were freshly isolated from the rats. The DRG and hippocampus in PTZ + Wi-Fi and PTZ + Wi-Fi + CPZ groups were exposed to Wi-Fi for 1 hour before CAP stimulation. The cytosolic free Ca(2+), reactive oxygen species production, apoptosis, mitochondrial membrane depolarization, caspase-3 and -9 values in hippocampus were higher in the PTZ group than in the control although cell viability values decreased. The Wi-Fi exposure induced additional effects on the cytosolic Ca(2+) increase. However, pretreatment of the neurons with CPZ, results in a protection against epilepsy-induced Ca(2+) influx, apoptosis and oxidative damages. In results of whole cell patch-clamp experiments, treatment of DRG with Ca(2+) channel antagonists [thapsigargin, verapamil + diltiazem, 2-APB, MK-801] indicated that Wi-Fi exposure induced Ca(2+) influx via the TRPV1 channels. In conclusion, epilepsy and Wi-Fi in our experimental model is involved in Ca(2+) influx and oxidative stress-induced hippocampal and DRG death through activation of TRPV1 channels, and negative modulation of this channel activity by CPZ pretreatment may account for the neuroprotective activity against oxidative stress.

Experimental re-evaluation of flunarizine as add-on antiepileptic therapy.

PubMed

Thakur, Anamika; Sahai, A K; Thakur, J S

2011-04-01

Experimental studies have found several calcium channel blockers with anticonvulsant property. Flunarizine is one of the most potent calcium channel blockers, which has shown anticonvulsant effect against pentylenetetrazole (PTZ) and maximal electroshock (MES)-induced seizures. However, further experimental and clinical trials have shown varied results. We conducted a PTZ model experimental study to re-evaluate the potential of flunarizine for add-on therapy in the management of refractory epilepsy. Experiments were conducted in PTZ model involving Swiss strain mice. Doses producing seizures in 50% and 99% mice, i.e. CD(50) and CD(99) values of PTZ were obtained from the dose-response study. Animals received graded, single dose of sodium valproate (100-300 mg/kg), lamotrigine (3-12 mg/kg) and flunarizine (5-20 mg/kg), and then each group of mice was injected with CD(99) dose of PTZ (65mg/kg i.p.). Another group of mice received single ED(50) dose (dose producing seizure protection in 50% mice) of sodium valproate and flunarizine separately in left and right side of abdomen. Results were analysed by Kruskal-Wallis ANOVA on Ranks test. As compared to control, sodium valproate at 250 mg/kg and 300 mg/kg produced statistical significant seizure protection. At none of the pre-treatment dose levels of lamotrigine, the seizure score with PTZ differed significantly from that observed in the vehicle-treated group. Pre-treatment with flunarizine demonstrated dose-dependent decrease in the seizure score to PTZ administration. As compared to control group, flunarizine at 20 mg/kg produced statistical significant seizure protection. As combined use of sodium valproate and flunarizine has shown significant seizure protection in PTZ model, flunarizine has a potential for add-on therapy in refractory cases of partial seizures. It is therefore, we conclude that further experimental studies and multicenter clinical trials involving large sample size are needed to establish flunarizine as add-on therapy in refractory epilepsy.

Intracellularly applied anti-P70 antibody blocks the induction of abnormal membrane properties by pentylenetetrazole in identified Euhadra neurons.

PubMed

Onozuka, M; Watanabe, K

1996-04-15

Using the voltage-clamp technique combined with pressure injection, we have studied the action of pentylenetetrazole (PTZ) on identified Euhadra neurons by examining how the PTZ-induced changes in membrane properties are affected by an antibody against P70, a protein found in the experimentally-induced epileptogenic cortex of rats. Intracellular injection of anti-P70 antibody blocked the induction by PTZ; bursting activity with both of development of negative slope resistance region in the steady state 1-V curve and a reduction in the delayed outward potassium current. These results suggest a novel mechanism of action for PTZ, involving intracellular protein(s) which react with anti-P70 antibody.

Expressing Transgenes That Exceed the Packaging Capacity of Adeno-Associated Virus Capsids

PubMed Central

Chamberlain, Kyle; Riyad, Jalish Mahmud; Weber, Thomas

2016-01-01

Recombinant adeno-associated virus vectors (rAAV) are being explored as gene delivery vehicles for the treatment of various inherited and acquired disorders. rAAVs are attractive vectors for several reasons: wild-type AAVs are nonpathogenic, and rAAVs can trigger long-term transgene expression even in the absence of genome integration—at least in postmitotic tissues. Moreover, rAAVs have a low immunogenic profile, and the various AAV serotypes and variants display broad but distinct tropisms. One limitation of rAAVs is that their genome-packaging capacity is only ∼5 kb. For most applications this is not of major concern because the median human protein size is 375 amino acids. Excluding the ITRs, for a protein of typical length, this allows the incorporation of ∼3.5 kb of DNA for the promoter, polyadenylation sequence, and other regulatory elements into a single AAV vector. Nonetheless, for certain diseases the packaging limit of AAV does not allow the delivery of a full-length therapeutic protein by a single AAV vector. Hence, approaches to overcome this limitation have become an important area of research for AAV gene therapy. Among the most promising approaches to overcome the limitation imposed by the packaging capacity of AAV is the use of dual-vector approaches, whereby a transgene is split across two separate AAV vectors. Coinfection of a cell with these two rAAVs will then—through a variety of mechanisms—result in the transcription of an assembled mRNA that could not be encoded by a single AAV vector because of the DNA packaging limits of AAV. The main purpose of this review is to assess the current literature with respect to dual-AAV-vector design, to highlight the effectiveness of the different methodologies and to briefly discuss future areas of research to improve the efficiency of dual-AAV-vector transduction. PMID:26757051

Thioredoxin System from Deinococcus radiodurans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Obiero, Josiah; Pittet, Vanessa; Bonderoff, Sara A.

2010-05-03

This paper describes the cloning, purification, and characterization of thioredoxin (Trx) and thioredoxin reductase (TrxR) and the structure determination of TrxR from the ionizing radiation-tolerant bacterium Deinococcus radiodurans strain R1. The genes from D. radiodurans encoding Trx and TrxR were amplified by PCR, inserted into a pET expression vector, and overexpressed in Escherichia coli. The overexpressed proteins were purified by metal affinity chromatography, and their activity was demonstrated using well-established assays of insulin precipitation (for Trx), 5,5{prime}-dithiobis(2-nitrobenzoic acid) (DTNB) reduction, and insulin reduction (for TrxR). In addition, the crystal structure of oxidized TrxR was determined at 1.9-{angstrom} resolution. The overallmore » structure was found to be very similar to that of E. coli TrxR and homodimeric with both NADPH- and flavin adenine dinucleotide (FAD)-binding domains containing variants of the canonical nucleotide binding fold, the Rossmann fold. The K{sub m} (5.7 {micro}M) of D. radiodurans TrxR for D. radiodurans Trx was determined and is about twofold higher than that of the E. coli thioredoxin system. However, D. radiodurans TrxR has a much lower affinity for E. coli Trx (K{sub m}, 44.4 {micro}M). Subtle differences in the surface charge and shape of the Trx binding site on TrxR may account for the differences in recognition. Because it has been suggested that TrxR from D. radiodurans may have dual cofactor specificity (can utilize both NADH and NADPH), D. radiodurans TrxR was tested for its ability to utilize NADH as well. Our results show that D. radiodurans TrxR can utilize only NADPH for activity.« less

Transformation of Rhodococcus fascians by High-Voltage Electroporation and Development of R. fascians Cloning Vectors

PubMed Central

Desomer, Jan; Dhaese, Patrick; Montagu, Marc Van

1990-01-01

The analysis of the virulence determinants of phytopathogenic Rhodococcus fascians has been hampered by the lack of a system for introducing exogenous DNA. We investigated the possibility of genetic transformation of R. fascians by high-voltage electroporation of intact bacterial cells in the presence of plasmid DNA. Electrotransformation in R. fascians D188 resulted in transformation frequencies ranging from 105/μg of DNA to 107/μg of DNA, depending on the DNA concentration. The effects of different electrical parameters and composition of electroporation medium on transformation efficiency are presented. By this transformation method, a cloning vector (pRF28) for R. fascians based on an indigenous 160-kilobase (chloramphenicol and cadmium resistance-encoding) plasmid pRF2 from strain NCPPB 1675 was developed. The origin of replication and the chloramphenicol resistance gene on pRF28 were used to construct cloning vectors that are capable of replication in R. fascians and Escherichia coli. The electroporation method presented was efficient enough to allow detection of the rare integration of replication-deficient pRF28 derivatives in the R. fascians D188 genome via either homologous or illegitimate recombination. Images PMID:16348290

Anti-kindling Effect of Bezafibrate, a Peroxisome Proliferator-activated Receptors Alpha Agonist, in Pentylenetetrazole Induced Kindling Seizure Model

PubMed Central

Saha, Lekha; Bhandari, Swati; Bhatia, Alka; Banerjee, Dibyajyoti; Chakrabarti, Amitava

2014-01-01

Background and Purpose: Studies in the animals suggested that Peroxisome proliferators activated receptors (PPARs) may be involved in seizure control and selective agonists of PPAR α or PPAR γ raise seizure thresholds. The present study was contemplated with the aim of evaluating the anti kindling effects and the mechanism of bezafibrate, a Peroxisome proliferator-activated receptors α (PPAR-α) agonist in pentylenetetrazole (PTZ) induced kindling model of seizures in rats. Methods: In a PTZ kindled Wistar rat model, different doses of bezafibrate (100 mg/kg, 200 mg/kg and 300 mg/kg) were administered intraperitoneally 30 minutes before the PTZ injection. The PTZ injection was given on alternate day till the animal became fully kindled or till 10 weeks. The parameters measured were the latency to develop kindling and incidence of kindling, histopathological study of hippocampus, hippocampal lipid peroxidation studies, serum neuron specific enolase, and hippocampal DNA fragmentation study. Results: In this study, bezafibrate significantly reduced the incidence of kindling in PTZ treated rats and exhibited a marked prolongation in the latencies to seizures. In the present study bezafibrate decreased the thiobarbituric acid-reactive substance i.e. Malondialdehyde levels, increased the reduced glutathione levels, catalase and superoxide dismutase activity in the brain. This added to its additional neuroprotective effects. Bezafibrate also reduced the neuronal damage and apoptosis in hippocampal area of the brain. Therefore bezafibrate exerted anticonvulsant properties in PTZ induced kindling model in rats. Conclusions: These findings may provide insights into the understanding of the mechanism of bezafibrate as an anti kindling agent and could offer a useful support to the basic antiepileptic therapy in preventing the development of PTZ induced seizures, suggesting its potential for therapeutic applications in temporal lobe epilepsy. PMID:25625088

[(18)F]FDG PET Neuroimaging Predicts Pentylenetetrazole (PTZ) Kindling Outcome in Rats.

PubMed

Bascuñana, Pablo; Javela, Julián; Delgado, Mercedes; Fernández de la Rosa, Rubén; Shiha, Ahmed Anis; García-García, Luis; Pozo, Miguel Ángel

2016-10-01

Epileptogenesis, i.e., development of epilepsy, involves a number of processes that alter the brain function in the way that triggers spontaneous seizures. Kindling is one of the most used animal models of temporal lobe epilepsy (TLE) and epileptogenesis, although chemical kindling suffers from high inter-assay success unpredictability. This study was aimed to analyze the eventual regional brain metabolic changes during epileptogenesis in the pentylenetetrazole (PTZ) kindling model in order to obtain a predictive kindling outcome parameter. In vivo longitudinal positron emission tomography (PET) scans with 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) along the PTZ kindling protocol (35 mg/kg intraperitoneally (i.p.), 18 sessions) in adult male rats were performed in order to evaluate the regional brain metabolism. The half of the PTZ-injected rats reached the kindled state. In addition, a significant decrease of [(18)F]FDG uptake at the end of the protocol in most of the brain structures of kindled animals was found, reflecting the characteristic epilepsy-associated hypometabolism. However, PTZ-injected animals but not reaching the kindled state did not show this widespread brain hypometabolism. Retrospective analysis of the data revealed that hippocampal [(18)F]FDG uptake normalized to pons turned out to be a predictive index of the kindling outcome. Thus, a 19.06 % reduction (p = 0.008) of the above parameter was found in positively kindled rats compared to non-kindled ones just after the fifth PTZ session. Non-invasive PET neuroimaging was a useful tool for discerning epileptogenesis progression in this animal model. Particularly, the [(18)F]FDG uptake of the hippocampus proved to be an early predictive parameter to differentiate resistant and non-resistant animals to the PTZ kindling.

Acute seizure suppression by transcranial direct current stimulation in rats

PubMed Central

Dhamne, Sameer C; Ekstein, Dana; Zhuo, Zhihong; Gersner, Roman; Zurakowski, David; Loddenkemper, Tobias; Pascual-Leone, Alvaro; Jensen, Frances E; Rotenberg, Alexander

2015-01-01

Objective Cathodal transcranial direct current stimulation (tDCS) is a focal neuromodulation technique that suppresses cortical excitability by low-amplitude constant electrical current, and may have an antiepileptic effect. Yet, tDCS has not been tested in status epilepticus (SE). Furthermore, a combined tDCS and pharmacotherapy antiseizure approach is unexplored. We therefore examined in the rat pentylenetetrazol (PTZ) SE model whether cathodal tDCS (1) suppresses seizures, (2) augments lorazepam (LZP) efficacy, and (3) enhances GABAergic cortical inhibition. Methods Experiment 1 aimed to identify an effective cathodal tDCS intensity. Rats received intraperitoneal PTZ followed by tDCS (sham, cathodal 1 mA, or cathodal 0.1 mA; for 20 min), and then a second PTZ challenge. In Experiment 2, two additional animal groups received a subtherapeutic LZP dose after PTZ, and then verum or sham tDCS. Clinical and electroencephalography (EEG) epileptic activity were compared between all groups. In Experiment 3, we measured GABA-mediated paired-pulse inhibition of the motor evoked potential by paired-pulse transcranial magnetic stimulation (ppTMS) in rats that received PTZ or saline, and either verum or sham tDCS. Results Cathodal 1 mA tDCS (1) reduced EEG spike bursts, and suppressed clinical seizures after the second PTZ challenge, (2) in combination with LZP was more effective in seizure suppression and improved the clinical seizure outcomes compared to either tDCS or LZP alone, and (3) prevented the loss of ppTMS motor cortex inhibition that accompanied PTZ injection. Interpretation These results suggest that cathodal 1 mA tDCS alone and in combination with LZP can suppress seizures by augmenting GABAergic cortical inhibition. PMID:26339678

Immune Protection of Nonhuman Primates against Ebola Virus with Single Low-Dose Adenovirus Vectors Encoding Modified GPs

PubMed Central

Geisbert, Joan B; Shedlock, Devon J; Xu, Ling; Lamoreaux, Laurie; Custers, Jerome H. H. V; Popernack, Paul M; Yang, Zhi-Yong; Pau, Maria G; Roederer, Mario; Koup, Richard A; Goudsmit, Jaap; Jahrling, Peter B; Nabel, Gary J

2006-01-01

Background Ebola virus causes a hemorrhagic fever syndrome that is associated with high mortality in humans. In the absence of effective therapies for Ebola virus infection, the development of a vaccine becomes an important strategy to contain outbreaks. Immunization with DNA and/or replication-defective adenoviral vectors (rAd) encoding the Ebola glycoprotein (GP) and nucleoprotein (NP) has been previously shown to confer specific protective immunity in nonhuman primates. GP can exert cytopathic effects on transfected cells in vitro, and multiple GP forms have been identified in nature, raising the question of which would be optimal for a human vaccine. Methods and Findings To address this question, we have explored the efficacy of mutant GPs from multiple Ebola virus strains with reduced in vitro cytopathicity and analyzed their protective effects in the primate challenge model, with or without NP. Deletion of the GP transmembrane domain eliminated in vitro cytopathicity but reduced its protective efficacy by at least one order of magnitude. In contrast, a point mutation was identified that abolished this cytopathicity but retained immunogenicity and conferred immune protection in the absence of NP. The minimal effective rAd dose was established at 1010 particles, two logs lower than that used previously. Conclusions Expression of specific GPs alone vectored by rAd are sufficient to confer protection against lethal challenge in a relevant nonhuman primate model. Elimination of NP from the vaccine and dose reductions to 1010 rAd particles do not diminish protection and simplify the vaccine, providing the basis for selection of a human vaccine candidate. PMID:16683867

An adenovirus prime/plasmid boost strategy for induction of equipotent immune responses to two dengue virus serotypes.

PubMed

Khanam, Saima; Rajendra, Pilankatta; Khanna, Navin; Swaminathan, Sathyamangalam

2007-02-15

Dengue is a public health problem of global significance for which there is neither an effective antiviral therapy nor a preventive vaccine. It is a mosquito-borne viral disease, caused by dengue (DEN) viruses, which are members of the Flaviviridae family. There are four closely related serotypes, DEN-1, DEN-2, DEN-3 and DEN-4, each of which is capable of causing disease. As immunity to any one serotype can potentially sensitize an individual to severe disease during exposure to a heterologous serotype, the general consensus is that an effective vaccine should be tetravalent, that is, it must be capable of affording protection against all four serotypes. The current strategy of creating tetravalent vaccine formulations by mixing together four monovalent live attenuated vaccine viruses has revealed the phenomenon of viral interference leading to the manifestation of immune responses biased towards a single serotype. This work stems from the emergence of (i) the DEN virus envelope (E) domain III (EDIII) as the most important region of the molecule from a vaccine perspective and (ii) the adenovirus (Ad) as a promising vaccine vector platform. We describe the construction of a recombinant, replication-defective Ad (rAd) vector encoding a chimeric antigen made of in-frame linked EDIIIs of DEN virus serotypes 2 and 4. Using this rAd vector, in conjunction with a plasmid vector encoding the same chimeric bivalent antigen, in a prime-boost strategy, we show that it is possible to elicit equipotent neutralizing and T cell responses specific to both DEN serotypes 2 and 4. Our data support the hypothesis that a DEN vaccine targeting more than one serotype may be based on a single DNA-based vector to circumvent viral interference. This work lays the foundation for developing a single Ad vector encoding EDIIIs of all four DEN serotypes to evoke a balanced immune response against each one of them. Thus, this work has implications for the development of safe and effective tetravalent dengue vaccines.

Antiapoptotic and neuroprotective role of Curcumin in Pentylenetetrazole (PTZ) induced kindling model in rat.

PubMed

Saha, Lekha; Chakrabarti, Amitava; Kumari, Sweta; Bhatia, Alka; Banerjee, Dibyojyoti

2016-02-01

Kindling, a sub threshold chemical or electrical stimulation, increases seizure duration and enhances accompanied behavior until it reaches a sort of equilibrium state. The present study aimed to explore the effect of curcumin on the development of kindling in PTZ kindled rats and its role in apoptosis and neuronal damage. In a PTZ kindled Wistar rat model, different doses of curcumin (100, 200 and 300 mg/kg) were administrated orally one hour before the PTZ injections on alternate day during the whole kindling days. The following parameters were compared between control and experimental groups: the course of kindling, stages of seizures, Histopathological scoring of hippocampus, antioxidant parameters in the hippocampus, DNA fragmentation and caspase-3 expression in hippocampus, and neuron-specific enolase in the blood. One way ANOVA followed by Bonferroni post hoc analysis and Fischer's Exact test were used for statistical analyses. PTZ, 30 mg/kg, induced kindling in rats after 32.0 ± 1.4 days. Curcumin showed dose-dependent anti-seizure effect. Curcumin (300 mg/kg) significantly increased the latency to myoclonic jerks, clonic seizures as well as generalized tonic-clonic seizures, improved the seizure score and decreased the number of myoclonic jerks. PTZ kindling induced a significant neuronal injury, oxidative stress and apoptosis which were reversed by pretreatment with curcumin in a dose-dependent manner. Our study suggests that curcumin has a potential antiepileptogenic effect on kindling-induced epileptogenesis.

Sub-Inhibitory Concentration of Piperacillin-Tazobactam May be Related to Virulence Properties of Filamentous Escherichia coli.

PubMed

de Andrade, João Paulo Lopes; de Macêdo Farias, Luiz; Ferreira, João Fernando Gonçalves; Bruna-Romero, Oscar; da Glória de Souza, Daniele; de Carvalho, Maria Auxiliadora Roque; dos Santos, Kênia Valéria

2016-01-01

Sub-inhibitory concentrations of antibiotics are always generated as a consequence of antimicrobial therapy and the effects of such residual products in bacterial morphology are well documented, especially the filamentation generated by beta-lactams. The aim of this study was to investigate some morphological and pathological aspects (virulence factors) of Escherichia coli cultivated under half-minimum inhibitory concentration (1.0 µg/mL) of piperacillin-tazobactam (PTZ sub-MIC). PTZ sub-MIC promoted noticeable changes in the bacterial cells which reach the peak of morphological alterations (filamentation) and complexity at 16 h of antimicrobial exposure. Thereafter the filamentous cells and a control one, not treated with PTZ, were comparatively tested for growth curve; biochemical profile; oxidative stress tolerance; biofilm production and cell hydrophobicity; motility and pathogenicity in vivo. PTZ sub-MIC attenuated the E. coli growth rate, but without changes in carbohydrate fermentation or in traditional biochemical tests. Overall, the treatment of E. coli with sub-MIC of PTZ generated filamentous forms which were accompanied by the inhibition of virulence factors such as the oxidative stress response, biofilm formation, cell surface hydrophobicity, and motility. These results are consistent with the reduced pathogenicity observed for the filamentous E. coli in the murine model of intra-abdominal infection. In other words, the treatment of E. coli with sub-MIC of PTZ suggests a decrease in their virulence.

Novel sull binary vectors enable an inexpensive foliar selection method in Arabidopsis

USDA-ARS?s Scientific Manuscript database

Sulfonamide resistance is conferred by the sulI gene found on many Enterobacteriaceae R plasmids and Tn21 type transposons. The sulI gene encodes a sulfonamide insensitive dihydropteroate synthase enzyme required for folate biosynthesis. Transformation of tobacco, potato or Arabidopsis using sulI as...

Evaluation of the anticonvulsant effect of Centella asiatica (gotu kola) in pentylenetetrazol-induced seizures with respect to cholinergic neurotransmission.

PubMed

Visweswari, Gopalreddygari; Prasad, Kanchi Siva; Chetan, Pandanaboina Sahitya; Lokanatha, Valluru; Rajendra, Wudayagiri

2010-03-01

The study described here was carried out to investigate the anticonvulsant effect of different extracts of Centella asiatica with respect to cholinergic activity on pentylenetetrazol (PTZ)-induced seizures. Rats were randomly divided into eight groups of six rats each: nonepileptic rats treated with saline; PTZ (60 mg/kg, IP)-induced seizure rats treated with saline; PTZ-induced seizure rats pretreated with n-hexane, chloroform, ethyl acetate, n-butanol, and water extracts of C. asiatica; and PTZ-induced seizure rats pretreated with diazepam (2mg/kg body wt). The seized rats pretreated with different extracts were administered a dose of 200mg/kg body wt orally for 1 week before induction of epilepsy. Increased acetylcholine content and decreased acetylcholinesterase activity were recorded in different brain regions during PTZ-induced seizures. Pretreatment with C. asiatica extracts caused recovery of the levels of acetylcholine and acetylcholinesterase. These findings suggest that C. asiatica causes perceptible changes in the cholinergic system as one of the facets of its anticonvulsant activity. (c) 2010 Elsevier Inc. All rights reserved.

Efficacy assessment of an MVA vectored Rift Valley Fever vaccine in lambs.

PubMed

Busquets, Núria; Lorenzo, Gema; López-Gil, Elena; Rivas, Raquel; Solanes, David; Galindo-Cardiel, Iván; Abad, F Xavier; Rodríguez, Fernando; Bensaid, Albert; Warimwe, George; Gilbert, Sarah C; Domingo, Mariano; Brun, Alejandro

2014-08-01

The present study has evaluated the protection conferred by a single subcutaneous dose of a modified vaccinia virus Ankara (MVA) vectored vaccine encoding the Rift Valley Fever virus (RVFV) glycoproteins Gn and Gc in lambs. Three groups of six to seven lambs were immunized as follows: one group received the vaccine (termed rMVA-GnGc), a second group received an MVA vector (vector control) and a third group received saline solution (non-vaccinated control). Fourteen days later, all animals were subcutaneously challenged with 10(5) TCID50 of the virulent RVFV isolate 56/74 and vaccine efficacy assessed using standard endpoints. Two lambs (one from the vaccine group and one from the vector control group) succumbed to RVFV challenge, showing characteristic liver lesions. Lambs from both the vector control and non-vaccinated groups were febrile from days 2 to 5 post challenge (pc) while those in the rMVA-GnGc group showed a single peak of pyrexia at day 3 pc. RVFV RNA was detected in both nasal and oral swabs from days 3 to 7 pc in some lambs from the vector control and non-vaccinated groups, but no viral shedding could be detected in the surviving lambs vaccinated with rMVA-GnGc. Together, the data suggest that a single dose of the rMVA-GnGc vaccine may be sufficient to reduce RVFV shedding and duration of viremia but does not provide sterile immunity nor protection from disease. Further optimization of this vaccine approach in lambs is warranted. Copyright © 2014 Elsevier B.V. All rights reserved.

Epileptogenic effects of G protein-coupled estrogen receptor 1 in the rat pentylenetetrazole kindling model of epilepsy.

PubMed

Kurt, Akif Hakan; Bosnak, Mehmet; Inan, Salim Yalcın; Celik, Ahmet; Uremis, Muhammed Mehdi

2016-02-01

G protein-coupled estrogen receptor 1 (GPER-1) has been demonstrated in several parts of the brain and may play an important role in estrogen downstream signaling pathway. However, the effects of this receptor on epileptic seizure are not clearly known. Therefore, the effects of GPER-1 agonist, G-1, GPER-1 antagonist, G-15 and the main estrogenic hormone, 17β-estradiol were investigated on seizures and brain tissue oxidative damages induced by pentylenetetrazole (PTZ) in rats. In this study, 30 adult male Wistar albino rats were used. Due to intraperitoneal (ip) injections of a subconvulsant dose of PTZ (35mg/kg) which was repeated 12 times every 48h, chemical kindling occurred and kindling seizure was recorded for 30min. The rats were injected with 17β-estradiol (5μg/kg, ip) or G-1 (5μg/kg, ip), G-15 (5μg/kg, ip), Saline, Ethanol and Dimethyl sulfoxide (DMSO) 30min before each dose of PTZ. Observed seizures were classified between the phase 0-5. Thirty minutes later when the last 12. PTZ administration, all rats were sacrificed and the brain cortex, hippocampus sections were removed and the tissue superoxide dismutase (SOD), malondialdehyde (MDA) and nitric oxide (NO) levels on these tissues were studied. GPER1 agonist, G-1 and estrogenic hormone, 17β-estradiol significantly increased the development of PTZ kindling the seizures. However, GPER1 antagonist, G-15 did not change the development of PTZ kindling the seizures. In the cortex and hippocampus homogenates, the NO levels after G-1 administration had significantly increased (p<0.05) compared to the PTZ groups but SOD activities and MDA levels demonstrated no difference between the groups. This is the first study that explores that GPER-1 receptors have epileptogenic effect on PTZ-induced kindling rat. GPER1 may mediate the epileptogenic effect of estrogens by changing the oxidative or anti-oxidative parameters in the brain. Copyright © 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

[The ability of NMDA glutamate receptor blockers to prevent a pentylenetetrazole kindling in mice and morphological changes in the hippocampus].

PubMed

Vasil'ev, D S; Tumanova, N L; Lavrent'eva, V V; Starshinova, L A; Zhabko, E P; Lukomskaia, N Ia; Zhuravin, I A; Magazanik, L G

2013-09-01

We investigated in mice the relationship between convulsions and morphological changes of hippocampal neurons that arise in the development of pentylentetrazol (PTZ)-induced kindling. The kindling was caused by of 35 mg/kg PTZ i.p. 3 times a week for a month. By the end of this period, 70% of the mice responded to the injections of PTZ with pronounced clonic or tonic-clonic seizures. The hippocampal slices (layer stratum pyramidale, CA1, Nissl's stain) obtained from mice exhibiting seizures revealed a large number of modified cells (24.7 +/- 2.1%). These hyperchromic neurons have been characterized by a decrease of the size cell body, there was a loss of turgor, the body cells shrink, and dendritic spines curl. Part of the cells took the shape of elongated neck. Such modified the dark type of neurons contained only 2.3 +/- 2.3% in the hippocampus of intact mice, and 30% of the mice resistant to the convulsive action ofPTZ during the period of observation. The expression of protein NeuN (Fox3) in hippocamal neuron including the hyperchromic once suggests that neurons on the whole did not die and were relatively viable. Preventive administration of NMDA receptor blockers (0.5 mg/kg, memantine 0.1 mg/kg or IEM-1958 1 mg/kg, s.c.) 30 minutes prior to PTZ reduced the proportion of mice which exhibited PTZ kindling from 70% to 40%. The modified neurons were observed in which the PTZ kindling due to the blocker presence did not develop, i.e., the same as in intact mice. Contrary, 24.0 +/- 5.6% of hyperchromic neurons were found in the hippocampal slices from mice manifested seizures, despite the co-administration of NMDA blockers. The data obtained indicate that modified neurons are the result of seizures suffered by the animals in the course of PTZ kindling, and that the blockade of NMDA glutamate receptors can suppress manifestations of seizures and the accompanying morphological changes of hippocampal neurons.

Comparative Analysis of the Magnitude, Quality, Phenotype and Protective Capacity of SIV Gag-Specific CD8+ T Cells Following Human-, Simian- and Chimpanzee-Derived Recombinant Adenoviral Vector Immunisation

PubMed Central

Quinn, Kylie M.; Costa, Andreia Da; Yamamoto, Ayako; Berry, Dana; Lindsay, Ross W.B.; Darrah, Patricia A.; Wang, Lingshu; Cheng, Cheng; Kong, Wing-Pui; Gall, Jason G.D.; Nicosia, Alfredo; Folgori, Antonella; Colloca, Stefano; Cortese, Riccardo; Gostick, Emma; Price, David A.; Gomez, Carmen E.; Esteban, Mariano; Wyatt, Linda S.; Moss, Bernard; Morgan, Cecilia; Roederer, Mario; Bailer, Robert T.; Nabel, Gary J.; Koup, Richard A.; Seder, Robert A.

2013-01-01

Recombinant adenoviral vectors (rAds) are the most potent recombinant vaccines for eliciting CD8+ T cell-mediated immunity in humans; however, prior exposure from natural adenoviral infection can decrease such responses. Here we show low seroreactivity in humans against simian- (sAd11, sAd16), or chimpanzee-derived (chAd3, chAd63) compared to human-derived (rAd5, rAd28, rAd35) vectors across multiple geographic regions. We then compared the magnitude, quality, phenotype and protective capacity of CD8+ T cell responses in mice vaccinated with rAds encoding SIV Gag. Using a dose range (1 × 107 to 109 PU), we defined a hierarchy among rAd vectors based on the magnitude and protective capacity of CD8+ T cell responses, from most to least as: rAd5 and chAd3, rAd28 and sAd11, chAd63, sAd16, and rAd35. Selection of rAd vector or dose could modulate the proportion and/or frequency of IFNγ+TNFα+IL-2+ and KLRG1+CD127- CD8+ T cells, but strikingly ~30–80% of memory CD8+ T cells co-expressed CD127 and KLRG1. To further optimise CD8+ T cell responses, we assessed rAds as part of prime-boost regimens. Mice primed with rAds and boosted with NYVAC generated Gag-specific responses that approached ~60% of total CD8+ T cells at peak. Alternatively, priming with DNA or rAd28 and boosting with rAd5 or chAd3 induced robust and equivalent CD8+ T cell responses compared to prime or boost alone. Collectively, these data provide the immunologic basis for using specific rAd vectors alone or as part of prime-boost regimens to induce CD8+ T cells for rapid effector function or robust long-term memory, respectively. PMID:23390298

A recursive technique for adaptive vector quantization

NASA Technical Reports Server (NTRS)

Lindsay, Robert A.

1989-01-01

Vector Quantization (VQ) is fast becoming an accepted, if not preferred method for image compression. The VQ performs well when compressing all types of imagery including Video, Electro-Optical (EO), Infrared (IR), Synthetic Aperture Radar (SAR), Multi-Spectral (MS), and digital map data. The only requirement is to change the codebook to switch the compressor from one image sensor to another. There are several approaches for designing codebooks for a vector quantizer. Adaptive Vector Quantization is a procedure that simultaneously designs codebooks as the data is being encoded or quantized. This is done by computing the centroid as a recursive moving average where the centroids move after every vector is encoded. When computing the centroid of a fixed set of vectors the resultant centroid is identical to the previous centroid calculation. This method of centroid calculation can be easily combined with VQ encoding techniques. The defined quantizer changes after every encoded vector by recursively updating the centroid of minimum distance which is the selected by the encoder. Since the quantizer is changing definition or states after every encoded vector, the decoder must now receive updates to the codebook. This is done as side information by multiplexing bits into the compressed source data.

Method and system for efficient video compression with low-complexity encoder

NASA Technical Reports Server (NTRS)

Chen, Jun (Inventor); He, Dake (Inventor); Sheinin, Vadim (Inventor); Jagmohan, Ashish (Inventor); Lu, Ligang (Inventor)

2012-01-01

Disclosed are a method and system for video compression, wherein the video encoder has low computational complexity and high compression efficiency. The disclosed system comprises a video encoder and a video decoder, wherein the method for encoding includes the steps of converting a source frame into a space-frequency representation; estimating conditional statistics of at least one vector of space-frequency coefficients; estimating encoding rates based on the said conditional statistics; and applying Slepian-Wolf codes with the said computed encoding rates. The preferred method for decoding includes the steps of; generating a side-information vector of frequency coefficients based on previously decoded source data, encoder statistics, and previous reconstructions of the source frequency vector; and performing Slepian-Wolf decoding of at least one source frequency vector based on the generated side-information, the Slepian-Wolf code bits and the encoder statistics.

Acute exposure to caffeine decreases the anticonvulsant action of ethosuximide, but not that of clonazepam, phenobarbital and valproate against pentetrazole-induced seizures in mice.

PubMed

Luszczki, Jarogniew J; Zuchora, Marek; Sawicka, Katarzyna M; Kozińska, Justyna; Czuczwar, Stanisław J

2006-01-01

This study examines the effect of acute administration of caffeine sodium benzoate (CAF) on the anticonvulsant action of four conventional antiepileptic drugs (AEDs: clonazepam - CZP, ethosuximide - ETS, phenobarbital - PB and valproate - VPA) against pentetrazole (PTZ)-induced clonic seizures in mice. The results indicate that CAF at a dose of 92.4 mg/kg significantly reduced the threshold for PTZ-induced clonic seizures in mice from 69.5 to 51.7 mg/kg (p<0.05), being ineffective at lower doses of 69.3 and 46.2 mg/kg. Moreover, CAF at doses of and 92.4 mg/kg attenuated the protective action of ETS against PTZ-induced seizures, by increasing its median effective dose (ED50) from 127.7 to 182.3 (p<0.05), and 198.3 mg/kg (p<0.01), respectively. In this case, no pharmacokinetic changes in total brain ETS concentrations after systemic ip administration of CAF (at 92.4 mg/kg) were observed, indicating a pharmacodynamic nature of interaction between ETS and CAF in the PTZ-test in mice. In contrast, CAF (at a dose of 92.4 mg/kg reducing the threshold for PTZ-induced seizures) combined with other AEDs (CZP, PB and VPA) did not affect their anticonvulsant action in the PTZ test in mice. Moreover, CAF (92.4 mg/kg) did not alter significantly total brain concentrations of the remaining AEDs (CZP, PB and VPA). The evaluation of potential acute adverse effects produced by AEDs in combination with CAF revealed that neither CAF (up to 92.4 mg/kg) administered alone nor combined with the studied drugs (at doses corresponding to their ED(50) values in the PTZ-test) affected motor performance of animals in the chimney test. In conclusion, the acute exposure to CAF may diminish the antiseizure protection offered by ETS in epileptic patients. Therefore, patients treated with ETS should avoid CAF.

Integration of multispectral face recognition and multi-PTZ camera automated surveillance for security applications

NASA Astrophysics Data System (ADS)

Chen, Chung-Hao; Yao, Yi; Chang, Hong; Koschan, Andreas; Abidi, Mongi

2013-06-01

Due to increasing security concerns, a complete security system should consist of two major components, a computer-based face-recognition system and a real-time automated video surveillance system. A computerbased face-recognition system can be used in gate access control for identity authentication. In recent studies, multispectral imaging and fusion of multispectral narrow-band images in the visible spectrum have been employed and proven to enhance the recognition performance over conventional broad-band images, especially when the illumination changes. Thus, we present an automated method that specifies the optimal spectral ranges under the given illumination. Experimental results verify the consistent performance of our algorithm via the observation that an identical set of spectral band images is selected under all tested conditions. Our discovery can be practically used for a new customized sensor design associated with given illuminations for an improved face recognition performance over conventional broad-band images. In addition, once a person is authorized to enter a restricted area, we still need to continuously monitor his/her activities for the sake of security. Because pantilt-zoom (PTZ) cameras are capable of covering a panoramic area and maintaining high resolution imagery for real-time behavior understanding, researches in automated surveillance systems with multiple PTZ cameras have become increasingly important. Most existing algorithms require the prior knowledge of intrinsic parameters of the PTZ camera to infer the relative positioning and orientation among multiple PTZ cameras. To overcome this limitation, we propose a novel mapping algorithm that derives the relative positioning and orientation between two PTZ cameras based on a unified polynomial model. This reduces the dependence on the knowledge of intrinsic parameters of PTZ camera and relative positions. Experimental results demonstrate that our proposed algorithm presents substantially reduced computational complexity and improved flexibility at the cost of slightly decreased pixel accuracy as compared to Chen and Wang's method [18].

Cell Cycle Dependent Regulation of Human Progesterone Receptor in Breast Cancer

DTIC Science & Technology

2004-10-01

wt PR or S400A PR (0.01-1.0 [tg each), CDK2 (ljig), and PRE-2x-TATA-luc reporter plasmid (lig) along with Renilla plasmid (10ng) as a control for...8 hours prior to treatment. Cells were collected following treatment with 10 nM R5020 or ETOH vehicle control for 18 hrs. Luciferase and renilla ...reporter, a renilla reporter construct as a transfection control, either wt PR-B or S400A mutant PR-B, and control parental vector or a vector encoding an

Phenomenal enhancement of optical nonlinearity in PTZ-I based ZnS/ZnSe nanocomposites

NASA Astrophysics Data System (ADS)

Divyasree, M. C.; Shiju, E.; Vijisha, M. V.; Ramesan, M. T.; Chandrasekharan, K.

2018-05-01

The enhanced nonlinear optical properties of phenothiazine-iodine (PTZ-I) charge transfer complex (CTC) on composite formation with ZnS/ZnSe nanostructures are reported. The interaction between the components was confirmed by the FTIR spectra. Structural and morphological changes on nanocomposite formation were analyzed by scanning electron microscopy and X-ray diffraction spectra. The absorption and emission features of both the nanocomposites and their constituent components were studied. Nonlinear optical properties of all the samples in nanosecond regime were investigated by the Z-scan technique using Nd: YAG laser with 532 nm wavelength and 7 ns pulse width. The optical nonlinearity of PTZ-I CTC was found to be improved considerably on composite formation and the new systems can be proposed as excellent candidates for photonic devices. Enhanced optical nonlinearity of the composites could be attributed to charge/energy transfer mechanism between PTZ-I CTC and the nanostructures.

Transduction of skeletal muscles with common reporter genes can promote muscle fiber degeneration and inflammation.

PubMed

Winbanks, Catherine E; Beyer, Claudia; Qian, Hongwei; Gregorevic, Paul

2012-01-01

Recombinant adeno-associated viral vectors (rAAV vectors) are promising tools for delivering transgenes to skeletal muscle, in order to study the mechanisms that control the muscle phenotype, and to ameliorate diseases that perturb muscle homeostasis. Many studies have employed rAAV vectors carrying reporter genes encoding for β-galactosidase (β-gal), human placental alkaline phosphatase (hPLAP), and green fluorescent protein (GFP) as experimental controls when studying the effects of manipulating other genes. However, it is not clear to what extent these reporter genes can influence signaling and gene expression signatures in skeletal muscle, which may confound the interpretation of results obtained in experimentally manipulated muscles. Herein, we report a strong pro-inflammatory effect of expressing reporter genes in skeletal muscle. Specifically, we show that the administration of rAAV6:hPLAP vectors to the hind limb muscles of mice is associated with dose- and time-dependent macrophage recruitment, and skeletal muscle damage. Dose-dependent expression of hPLAP also led to marked activity of established pro-inflammatory IL-6/Stat3, TNFα, IKKβ and JNK signaling in lysates obtained from homogenized muscles. These effects were independent of promoter type, as expression cassettes featuring hPLAP under the control of constitutive CMV and muscle-specific CK6 promoters both drove cellular responses when matched for vector dose. Importantly, the administration of rAAV6:GFP vectors did not induce muscle damage or inflammation except at the highest doses we examined, and administration of a transgene-null vector (rAAV6:MCS) did not cause damage or inflammation at any of the doses tested, demonstrating that GFP-expressing, or transgene-null vectors may be more suitable as experimental controls. The studies highlight the importance of considering the potential effects of reporter genes when designing experiments that examine gene manipulation in vivo.

Construction of a New Phage Integration Vector pFIV-Val for Use in Different Francisella Species

PubMed Central

Tlapák, Hana; Köppen, Kristin; Rydzewski, Kerstin; Grunow, Roland; Heuner, Klaus

2018-01-01

We recently identified and described a putative prophage on the genomic island FhaGI-1 located within the genome of Francisella hispaniensis AS02-814 (F. tularensis subsp. novicida-like 3523). In this study, we constructed two variants of a Francisella phage integration vector, called pFIV1-Val and pFIV2-Val (Francisella Integration Vector-tRNAVal-specific), using the attL/R-sites and the site-specific integrase (FN3523_1033) of FhaGI-1, a chloramphenicol resistance cassette and a sacB gene for counter selection of transformants against the vector backbone. We inserted the respective sites and genes into vector pUC57-Kana to allow for propagation in Escherichia coli. The constructs generated a circular episomal form in E. coli which could be used to transform Francisella spp. where FIV-Val stably integrated site specifically into the tRNAVal gene of the genome, whereas pUC57-Kana is lost due to counter selection. Functionality of the new vector was demonstrated by the successfully complementation of a Francisella mutant strain. The vectors were stable in vitro and during host-cell infection without selective pressure. Thus, the vectors can be applied as a further genetic tool in Francisella research, expanding the present genetic tools by an integrative element. This new element is suitable to perform long-term experiments with different Francisella species. PMID:29594068

Construction of a New Phage Integration Vector pFIV-Val for Use in Different Francisella Species.

PubMed

Tlapák, Hana; Köppen, Kristin; Rydzewski, Kerstin; Grunow, Roland; Heuner, Klaus

2018-01-01

We recently identified and described a putative prophage on the genomic island FhaGI-1 located within the genome of Francisella hispaniensis AS02-814 ( F. tularensis subsp. novicida -like 3523). In this study, we constructed two variants of a Francisella phage integration vector, called pFIV1-Val and pFIV2-Val ( Francisella Integration Vector-tRNA Val -specific), using the attL/R- sites and the site-specific integrase (FN3523_1033) of FhaGI-1, a chloramphenicol resistance cassette and a sacB gene for counter selection of transformants against the vector backbone. We inserted the respective sites and genes into vector pUC57-Kana to allow for propagation in Escherichia coli . The constructs generated a circular episomal form in E. coli which could be used to transform Francisella spp . where FIV-Val stably integrated site specifically into the tRNA Val gene of the genome, whereas pUC57-Kana is lost due to counter selection. Functionality of the new vector was demonstrated by the successfully complementation of a Francisella mutant strain. The vectors were stable in vitro and during host-cell infection without selective pressure. Thus, the vectors can be applied as a further genetic tool in Francisella research, expanding the present genetic tools by an integrative element. This new element is suitable to perform long-term experiments with different Francisella species.

Study of Jet Quenching with Z+jet Correlations in Pb-Pb and pp Collisions at sqrt[s]_{NN}=5.02  TeV.

PubMed

Sirunyan, A M; Tumasyan, A; Adam, W; Asilar, E; Bergauer, T; Brandstetter, J; Brondolin, E; Dragicevic, M; Erö, J; Flechl, M; Friedl, M; Frühwirth, R; Ghete, V M; Hartl, C; Hörmann, N; Hrubec, J; Jeitler, M; König, A; Krätschmer, I; Liko, D; Matsushita, T; Mikulec, I; Rabady, D; Rad, N; Rahbaran, B; Rohringer, H; Schieck, J; Strauss, J; Waltenberger, W; Wulz, C-E; Chekhovsky, V; Dvornikov, O; Dydyshka, Y; Emeliantchik, I; Litomin, A; Makarenko, V; Mossolov, V; Stefanovitch, R; Suarez Gonzalez, J; Zykunov, V; Shumeiko, N; Alderweireldt, S; De Wolf, E A; Janssen, X; Lauwers, J; Van De Klundert, M; Van Haevermaet, H; Van Mechelen, P; Van Remortel, N; Van Spilbeeck, A; Abu Zeid, S; Blekman, F; D'Hondt, J; Daci, N; De Bruyn, I; Deroover, K; Lowette, S; Moortgat, S; Moreels, L; Olbrechts, A; Python, Q; Skovpen, K; Tavernier, S; Van Doninck, W; Van Mulders, P; Van Parijs, I; Brun, H; Clerbaux, B; De Lentdecker, G; Delannoy, H; Fasanella, G; Favart, L; Goldouzian, R; Grebenyuk, A; Karapostoli, G; Lenzi, T; Léonard, A; Luetic, J; Maerschalk, T; Marinov, A; Randle-Conde, A; Seva, T; Vander Velde, C; Vanlaer, P; Vannerom, D; Yonamine, R; Zenoni, F; Zhang, F; Cimmino, A; Cornelis, T; Dobur, D; Fagot, A; Gul, M; Khvastunov, I; Poyraz, D; Salva, S; Schöfbeck, R; Tytgat, M; Van Driessche, W; Yazgan, E; Zaganidis, N; Bakhshiansohi, H; Beluffi, C; Bondu, O; Brochet, S; Bruno, G; Caudron, A; De Visscher, S; Delaere, C; Delcourt, M; Francois, B; Giammanco, A; Jafari, A; Komm, M; Krintiras, G; Lemaitre, V; Magitteri, A; Mertens, A; Musich, M; Nuttens, C; Piotrzkowski, K; Quertenmont, L; Selvaggi, M; Vidal Marono, M; Wertz, S; Beliy, N; Aldá Júnior, W L; Alves, F L; Alves, G A; Brito, L; Hensel, C; Moraes, A; Pol, M E; Rebello Teles, P; Belchior Batista Das Chagas, E; Carvalho, W; Chinellato, J; Custódio, A; Da Costa, E M; Da Silveira, G G; De Jesus Damiao, D; De Oliveira Martins, C; Fonseca De Souza, S; Huertas Guativa, L M; Malbouisson, H; Matos Figueiredo, D; Mora Herrera, C; Mundim, L; Nogima, H; Prado Da Silva, W L; Santoro, A; Sznajder, A; Tonelli Manganote, E J; Vilela Pereira, A; Ahuja, S; Bernardes, C A; Dogra, S; Fernandez Perez Tomei, T R; Gregores, E M; Mercadante, P G; Moon, C S; Novaes, S F; Padula, Sandra S; Romero Abad, D; Ruiz Vargas, J C; Aleksandrov, A; Hadjiiska, R; Iaydjiev, P; Rodozov, M; Stoykova, S; Sultanov, G; Vutova, M; Dimitrov, A; Glushkov, I; Litov, L; Pavlov, B; Petkov, P; Fang, W; Ahmad, M; Bian, J G; Chen, G M; Chen, H S; Chen, M; Chen, Y; Cheng, T; Jiang, C H; Leggat, D; Liu, Z; Romeo, F; Ruan, M; Shaheen, S M; Spiezia, A; Tao, J; Wang, C; Wang, Z; Zhang, H; Zhao, J; Ban, Y; Chen, G; Li, Q; Liu, S; Mao, Y; Qian, S J; Wang, D; Xu, Z; Avila, C; Cabrera, A; Chaparro Sierra, L F; Florez, C; Gomez, J P; González Hernández, C F; Ruiz Alvarez, J D; Sanabria, J C; Godinovic, N; Lelas, D; Puljak, I; Ribeiro Cipriano, P M; Sculac, T; Antunovic, Z; Kovac, M; Brigljevic, V; Ferencek, D; Kadija, K; Mesic, B; Susa, T; Attikis, A; Mavromanolakis, G; Mousa, J; Nicolaou, C; Ptochos, F; Razis, P A; Rykaczewski, H; Tsiakkouri, D; Finger, M; Finger, M; Carrera Jarrin, E; Assran, Y; Elkafrawy, T; Mahrous, A; Kadastik, M; Perrini, L; Raidal, M; Tiko, A; Veelken, C; Eerola, P; Pekkanen, J; Voutilainen, M; Härkönen, J; Järvinen, T; Karimäki, V; Kinnunen, R; Lampén, T; Lassila-Perini, K; Lehti, S; Lindén, T; Luukka, P; Tuominiemi, J; Tuovinen, E; Wendland, L; Talvitie, J; Tuuva, T; Besancon, M; Couderc, F; Dejardin, M; Denegri, D; Fabbro, B; Faure, J L; Favaro, C; Ferri, F; Ganjour, S; Ghosh, S; Givernaud, A; Gras, P; Hamel de Monchenault, G; Jarry, P; Kucher, I; Locci, E; Machet, M; Malcles, J; Rander, J; Rosowsky, A; Titov, M; Zghiche, A; Abdulsalam, A; Antropov, I; Baffioni, S; Beaudette, F; Blanco, J M; Busson, P; Cadamuro, L; Chapon, E; Charlot, C; Davignon, O; Granier de Cassagnac, R; Jo, M; Lisniak, S; Miné, P; Nguyen, M; Ochando, C; Ortona, G; Paganini, P; Pigard, P; Regnard, S; Salerno, R; Sirois, Y; Strebler, T; Yilmaz, Y; Zabi, A; Agram, J-L; Andrea, J; Aubin, A; Bloch, D; Brom, J-M; Buttignol, M; Chabert, E C; Chanon, N; Collard, C; Conte, E; Coubez, X; Fontaine, J-C; Gelé, D; Goerlach, U; Le Bihan, A-C; Van Hove, P; Gadrat, S; Beauceron, S; Bernet, C; Boudoul, G; Carrillo Montoya, C A; Chierici, R; Contardo, D; Courbon, B; Depasse, P; El Mamouni, H; Fan, J; Fay, J; Gascon, S; Gouzevitch, M; Grenier, G; Ille, B; Lagarde, F; Laktineh, I B; Lethuillier, M; Mirabito, L; Pequegnot, A L; Perries, S; Popov, A; Sabes, D; Sordini, V; Vander Donckt, M; Verdier, P; Viret, S; Toriashvili, T; Bagaturia, I; Autermann, C; Beranek, S; Feld, L; Kiesel, M K; Klein, K; Lipinski, M; Preuten, M; Schomakers, C; Schulz, J; Verlage, T; Albert, A; Brodski, M; Dietz-Laursonn, E; Duchardt, D; Endres, M; Erdmann, M; Erdweg, S; Esch, T; Fischer, R; Güth, A; Hamer, M; Hebbeker, T; Heidemann, C; Hoepfner, K; Knutzen, S; Merschmeyer, M; Meyer, A; Millet, P; Mukherjee, S; Olschewski, M; Padeken, K; Pook, T; Radziej, M; Reithler, H; Rieger, M; Scheuch, F; Sonnenschein, L; Teyssier, D; Thüer, S; Cherepanov, V; Flügge, G; Kargoll, B; Kress, T; Künsken, A; Lingemann, J; Müller, T; Nehrkorn, A; Nowack, A; Pistone, C; Pooth, O; Stahl, A; Aldaya Martin, M; Arndt, T; Asawatangtrakuldee, C; Beernaert, K; Behnke, O; Behrens, U; Bin Anuar, A A; Borras, K; Campbell, A; Connor, P; Contreras-Campana, C; Costanza, F; Diez Pardos, C; Dolinska, G; Eckerlin, G; Eckstein, D; Eichhorn, T; Eren, E; Gallo, E; Garay Garcia, J; Geiser, A; Gizhko, A; Grados Luyando, J M; Grohsjean, A; Gunnellini, P; Harb, A; Hauk, J; Hempel, M; Jung, H; Kalogeropoulos, A; Karacheban, O; Kasemann, M; Keaveney, J; Kleinwort, C; Korol, I; Krücker, D; Lange, W; Lelek, A; Leonard, J; Lipka, K; Lobanov, A; Lohmann, W; Mankel, R; Melzer-Pellmann, I-A; Meyer, A B; Mittag, G; Mnich, J; Mussgiller, A; Ntomari, E; Pitzl, D; Placakyte, R; Raspereza, A; Roland, B; Sahin, M Ö; Saxena, P; Schoerner-Sadenius, T; Seitz, C; Spannagel, S; Stefaniuk, N; Van Onsem, G P; Walsh, R; Wissing, C; Blobel, V; Centis Vignali, M; Draeger, A R; Dreyer, T; Garutti, E; Gonzalez, D; Haller, J; Hoffmann, M; Junkes, A; Klanner, R; Kogler, R; Kovalchuk, N; Lapsien, T; Lenz, T; Marchesini, I; Marconi, D; Meyer, M; Niedziela, M; Nowatschin, D; Pantaleo, F; Peiffer, T; Perieanu, A; Poehlsen, J; Sander, C; Scharf, C; Schleper, P; Schmidt, A; Schumann, S; Schwandt, J; Stadie, H; Steinbrück, G; Stober, F M; Stöver, M; Tholen, H; Troendle, D; Usai, E; Vanelderen, L; Vanhoefer, A; Vormwald, B; Akbiyik, M; Barth, C; Baur, S; Baus, C; Berger, J; Butz, E; Caspart, R; Chwalek, T; Colombo, F; De Boer, W; Dierlamm, A; Fink, S; Freund, B; Friese, R; Giffels, M; Gilbert, A; Goldenzweig, P; Haitz, D; Hartmann, F; Heindl, S M; Husemann, U; Katkov, I; Kudella, S; Mildner, H; Mozer, M U; Müller, Th; Plagge, M; Quast, G; Rabbertz, K; Röcker, S; Roscher, F; Schröder, M; Shvetsov, I; Sieber, G; Simonis, H J; Ulrich, R; Wayand, S; Weber, M; Weiler, T; Williamson, S; Wöhrmann, C; Wolf, R; Anagnostou, G; Daskalakis, G; Geralis, T; Giakoumopoulou, V A; Kyriakis, A; Loukas, D; Topsis-Giotis, I; Kesisoglou, S; Panagiotou, A; Saoulidou, N; Tziaferi, E; Evangelou, I; Flouris, G; Foudas, C; Kokkas, P; Loukas, N; Manthos, N; Papadopoulos, I; Paradas, E; Filipovic, N; Bencze, G; Hajdu, C; Horvath, D; Sikler, F; Veszpremi, V; Vesztergombi, G; Zsigmond, A J; Beni, N; Czellar, S; Karancsi, J; Makovec, A; Molnar, J; Szillasi, Z; Bartók, M; Raics, P; Trocsanyi, Z L; Ujvari, B; Bahinipati, S; Choudhury, S; Mal, P; Mandal, K; Nayak, A; Sahoo, D K; Sahoo, N; Swain, S K; Bansal, S; Beri, S B; Bhatnagar, V; Chawla, R; Bhawandeep, U; Kalsi, A K; Kaur, A; Kaur, M; Kumar, R; Kumari, P; Mehta, A; Mittal, M; Singh, J B; Walia, G; Kumar, Ashok; Bhardwaj, A; Choudhary, B C; Garg, R B; Keshri, S; Malhotra, S; Naimuddin, M; Nishu, N; Ranjan, K; Sharma, R; Sharma, V; Bhattacharya, R; Bhattacharya, S; Chatterjee, K; Dey, S; Dutt, S; Dutta, S; Ghosh, S; Majumdar, N; Modak, A; Mondal, K; Mukhopadhyay, S; Nandan, S; Purohit, A; Roy, A; Roy, D; Roy Chowdhury, S; Sarkar, S; Sharan, M; Thakur, S; Behera, P K; Chudasama, R; Dutta, D; Jha, V; Kumar, V; Mohanty, A K; Netrakanti, P K; Pant, L M; Shukla, P; Topkar, A; Aziz, T; Dugad, S; Kole, G; Mahakud, B; Mitra, S; Mohanty, G B; Parida, B; Sur, N; Sutar, B; Banerjee, S; Bhowmik, S; Dewanjee, R K; Ganguly, S; Guchait, M; Jain, Sa; Kumar, S; Maity, M; Majumder, G; Mazumdar, K; Sarkar, T; Wickramage, N; Chauhan, S; Dube, S; Hegde, V; Kapoor, A; Kothekar, K; Pandey, S; Rane, A; Sharma, S; Chenarani, S; Eskandari Tadavani, E; Etesami, S M; Khakzad, M; Mohammadi Najafabadi, M; Naseri, M; Paktinat Mehdiabadi, S; Rezaei Hosseinabadi, F; Safarzadeh, B; Zeinali, M; Felcini, M; Grunewald, M; Abbrescia, M; Calabria, C; Caputo, C; Colaleo, A; Creanza, D; Cristella, L; De Filippis, N; De Palma, M; Fiore, L; Iaselli, G; Maggi, G; Maggi, M; Miniello, G; My, S; Nuzzo, S; Pompili, A; Pugliese, G; Radogna, R; Ranieri, A; Selvaggi, G; Sharma, A; Silvestris, L; Venditti, R; Verwilligen, P; Abbiendi, G; Battilana, C; Bonacorsi, D; Braibant-Giacomelli, S; Brigliadori, L; Campanini, R; Capiluppi, P; Castro, A; Cavallo, F R; Chhibra, S S; Codispoti, G; Cuffiani, M; Dallavalle, G M; Fabbri, F; Fanfani, A; Fasanella, D; Giacomelli, P; Grandi, C; Guiducci, L; Marcellini, S; Masetti, G; Montanari, A; Navarria, F L; Perrotta, A; Rossi, A M; Rovelli, T; Siroli, G P; Tosi, N; Albergo, S; Costa, S; Di Mattia, A; Giordano, F; Potenza, R; Tricomi, A; Tuve, C; Barbagli, G; Ciulli, V; Civinini, C; D'Alessandro, R; Focardi, E; Lenzi, P; Meschini, M; Paoletti, S; Russo, L; Sguazzoni, G; Strom, D; Viliani, L; Benussi, L; Bianco, S; Fabbri, F; Piccolo, D; Primavera, F; Calvelli, V; Ferro, F; Monge, M R; Robutti, E; Tosi, S; Brianza, L; Brivio, F; Ciriolo, V; Dinardo, M E; Fiorendi, S; Gennai, S; Ghezzi, A; Govoni, P; Malberti, M; Malvezzi, S; Manzoni, R A; Menasce, D; Moroni, L; Paganoni, M; Pedrini, D; Pigazzini, S; Ragazzi, S; Tabarelli de Fatis, T; Buontempo, S; Cavallo, N; De Nardo, G; Di Guida, S; Esposito, M; Fabozzi, F; Fienga, F; Iorio, A O M; Lanza, G; Lista, L; Meola, S; Paolucci, P; Sciacca, C; Thyssen, F; Bacchetta, N; Benato, L; Bisello, D; Boletti, A; Carlin, R; Checchia, P; Dall'Osso, M; De Castro Manzano, P; Dorigo, T; Dosselli, U; Gasparini, F; Gasparini, U; Gozzelino, A; Lacaprara, S; Margoni, M; Meneguzzo, A T; Pazzini, J; Pegoraro, M; Pozzobon, N; Sgaravatto, M; Simonetto, F; Torassa, E; Ventura, S; Zanetti, M; Zotto, P; Zumerle, G; Braghieri, A; Fallavollita, F; Magnani, A; Montagna, P; Ratti, S P; Re, V; Riccardi, C; Salvini, P; Vai, I; Vitulo, P; Alunni Solestizi, L; Bilei, G M; Ciangottini, D; Fanò, L; Lariccia, P; Leonardi, R; Mantovani, G; Menichelli, M; Saha, A; Santocchia, A; Androsov, K; Azzurri, P; Bagliesi, G; Bernardini, J; Boccali, T; Castaldi, R; Ciocci, M A; Dell'Orso, R; Donato, S; Fedi, G; Giassi, A; Grippo, M T; Ligabue, F; Lomtadze, T; Martini, L; Messineo, A; Palla, F; Rizzi, A; Savoy-Navarro, A; Spagnolo, P; Tenchini, R; Tonelli, G; Venturi, A; Verdini, P G; Barone, L; Cavallari, F; Cipriani, M; Del Re, D; Diemoz, M; Gelli, S; Longo, E; Margaroli, F; Marzocchi, B; Meridiani, P; Organtini, G; Paramatti, R; Preiato, F; Rahatlou, S; Rovelli, C; Santanastasio, F; Amapane, N; Arcidiacono, R; Argiro, S; Arneodo, M; Bartosik, N; Bellan, R; Biino, C; Cartiglia, N; Cenna, F; Costa, M; Covarelli, R; Degano, A; Demaria, N; Finco, L; Kiani, B; Mariotti, C; Maselli, S; Migliore, E; Monaco, V; Monteil, E; Monteno, M; Obertino, M M; Pacher, L; Pastrone, N; Pelliccioni, M; Pinna Angioni, G L; Ravera, F; Romero, A; Ruspa, M; Sacchi, R; Shchelina, K; Sola, V; Solano, A; Staiano, A; Traczyk, P; Belforte, S; Casarsa, M; Cossutti, F; Della Ricca, G; Zanetti, A; Kim, D H; Kim, G N; Kim, M S; Lee, S; Lee, S W; Oh, Y D; Sekmen, S; Son, D C; Yang, Y C; Lee, A; Kim, H; Brochero Cifuentes, J A; Kim, T J; Cho, S; Choi, S; Go, Y; Gyun, D; Ha, S; Hong, B; Jo, Y; Kim, Y; Lee, K; Lee, K S; Lee, S; Lim, J; Park, S K; Roh, Y; Almond, J; Kim, J; Lee, H; Oh, S B; Radburn-Smith, B C; Seo, S H; Yang, U K; Yoo, H D; Yu, G B; Choi, M; Kim, H; Kim, J H; Lee, J S H; Park, I C; Ryu, G; Ryu, M S; Choi, Y; Goh, J; Hwang, C; Lee, J; Yu, I; Dudenas, V; Juodagalvis, A; Vaitkus, J; Ahmed, I; Ibrahim, Z A; Komaragiri, J R; Md Ali, M A B; Mohamad Idris, F; Wan Abdullah, W A T; Yusli, M N; Zolkapli, Z; Castilla-Valdez, H; De La Cruz-Burelo, E; Heredia-De La Cruz, I; Hernandez-Almada, A; Lopez-Fernandez, R; Magaña Villalba, R; Mejia Guisao, J; Sanchez-Hernandez, A; Carrillo Moreno, S; Oropeza Barrera, C; Vazquez Valencia, F; Carpinteyro, S; Pedraza, I; Salazar Ibarguen, H A; Uribe Estrada, C; Morelos Pineda, A; Krofcheck, D; Butler, P H; Ahmad, A; Ahmad, M; Hassan, Q; Hoorani, H R; Khan, W A; Saddique, A; Shah, M A; Shoaib, M; Waqas, M; Bialkowska, H; Bluj, M; Boimska, B; Frueboes, T; Górski, M; Kazana, M; Nawrocki, K; Romanowska-Rybinska, K; Szleper, M; Zalewski, P; Bunkowski, K; Byszuk, A; Doroba, K; Kalinowski, A; Konecki, M; Krolikowski, J; Misiura, M; Olszewski, M; Walczak, M; Bargassa, P; Beirão Da Cruz E Silva, C; Calpas, B; Di Francesco, A; Faccioli, P; Ferreira Parracho, P G; Gallinaro, M; Hollar, J; Leonardo, N; Lloret Iglesias, L; Nemallapudi, M V; Rodrigues Antunes, J; Seixas, J; Toldaiev, O; Vadruccio, D; Varela, J; Vischia, P; Afanasiev, S; Bunin, P; Gavrilenko, M; Golutvin, I; Gorbunov, I; Kamenev, A; Karjavin, V; Lanev, A; Malakhov, A; Matveev, V; Palichik, V; Perelygin, V; Shmatov, S; Shulha, S; Skatchkov, N; Smirnov, V; Voytishin, N; Zarubin, A; Chtchipounov, L; Golovtsov, V; Ivanov, Y; Kim, V; Kuznetsova, E; Murzin, V; Oreshkin, V; Sulimov, V; Vorobyev, A; Andreev, Yu; Dermenev, A; Gninenko, S; Golubev, N; Karneyeu, A; Kirsanov, M; Krasnikov, N; Pashenkov, A; Tlisov, D; Toropin, A; Epshteyn, V; Gavrilov, V; Lychkovskaya, N; Popov, V; Pozdnyakov, I; Safronov, G; Spiridonov, A; Toms, M; Vlasov, E; Zhokin, A; Bylinkin, A; Chadeeva, M; Danilov, M; Rusinov, V; Andreev, V; Azarkin, M; Dremin, I; Kirakosyan, M; Leonidov, A; Terkulov, A; Baskakov, A; Belyaev, A; Boos, E; Demiyanov, A; Ershov, A; Gribushin, A; Kodolova, O; Korotkikh, V; Lokhtin, I; Miagkov, I; Obraztsov, S; Petrushanko, S; Savrin, V; Snigirev, A; Vardanyan, I; Blinov, V; Skovpen, Y; Shtol, D; Azhgirey, I; Bayshev, I; Bitioukov, S; Elumakhov, D; Kachanov, V; Kalinin, A; Konstantinov, D; Krychkine, V; Petrov, V; Ryutin, R; Sobol, A; Troshin, S; Tyurin, N; Uzunian, A; Volkov, A; Adzic, P; Cirkovic, P; Devetak, D; Dordevic, M; Milosevic, J; Rekovic, V; Alcaraz Maestre, J; Barrio Luna, M; Calvo, E; Cerrada, M; Chamizo Llatas, M; Colino, N; De La Cruz, B; Delgado Peris, A; Escalante Del Valle, A; Fernandez Bedoya, C; Fernández Ramos, J P; Flix, J; Fouz, M C; Garcia-Abia, P; Gonzalez Lopez, O; Goy Lopez, S; Hernandez, J M; Josa, M I; Navarro De Martino, E; Pérez-Calero Yzquierdo, A; Puerta Pelayo, J; Quintario Olmeda, A; Redondo, I; Romero, L; Soares, M S; de Trocóniz, J F; Missiroli, M; Moran, D; Cuevas, J; Fernandez Menendez, J; Gonzalez Caballero, I; González Fernández, J R; Palencia Cortezon, E; Sanchez Cruz, S; Suárez Andrés, I; Vizan Garcia, J M; Cabrillo, I J; Calderon, A; Curras, E; Fernandez, M; Garcia-Ferrero, J; Gomez, G; Lopez Virto, A; Marco, J; Martinez Rivero, C; Matorras, F; Piedra Gomez, J; Rodrigo, T; Ruiz-Jimeno, A; Scodellaro, L; Trevisani, N; Vila, I; Vilar Cortabitarte, R; Abbaneo, D; Auffray, E; Auzinger, G; Bachtis, M; Baillon, P; Ball, A H; Barney, D; Bloch, P; Bocci, A; Botta, C; Camporesi, T; Castello, R; Cepeda, M; Cerminara, G; Chen, Y; d'Enterria, D; Dabrowski, A; Daponte, V; David, A; De Gruttola, M; De Roeck, A; Di Marco, E; Dobson, M; Dorney, B; du Pree, T; Duggan, D; Dünser, M; Dupont, N; Elliott-Peisert, A; Everaerts, P; Fartoukh, S; Franzoni, G; Fulcher, J; Funk, W; Gigi, D; Gill, K; Girone, M; Glege, F; Gulhan, D; Gundacker, S; Guthoff, M; Harris, P; Hegeman, J; Innocente, V; Janot, P; Kieseler, J; Kirschenmann, H; Knünz, V; Kornmayer, A; Kortelainen, M J; Kousouris, K; Krammer, M; Lange, C; Lecoq, P; Lourenço, C; Lucchini, M T; Malgeri, L; Mannelli, M; Martelli, A; Meijers, F; Merlin, J A; Mersi, S; Meschi, E; Milenovic, P; Moortgat, F; Morovic, S; Mulders, M; Neugebauer, H; Orfanelli, S; Orsini, L; Pape, L; Perez, E; Peruzzi, M; Petrilli, A; Petrucciani, G; Pfeiffer, A; Pierini, M; Racz, A; Reis, T; Rolandi, G; Rovere, M; Sakulin, H; Sauvan, J B; Schäfer, C; Schwick, C; Seidel, M; Sharma, A; Silva, P; Sphicas, P; Steggemann, J; Stoye, M; Takahashi, Y; Tosi, M; Treille, D; Triossi, A; Tsirou, A; Veckalns, V; Veres, G I; Verweij, M; Wardle, N; Wöhri, H K; Zagozdzinska, A; Zeuner, W D; Bertl, W; Deiters, K; Erdmann, W; Horisberger, R; Ingram, Q; Kaestli, H C; Kotlinski, D; Langenegger, U; Rohe, T; Bachmair, F; Bäni, L; Bianchini, L; Casal, B; Dissertori, G; Dittmar, M; Donegà, M; Grab, C; Heidegger, C; Hits, D; Hoss, J; Kasieczka, G; Lustermann, W; Mangano, B; Marionneau, M; Martinez Ruiz Del Arbol, P; Masciovecchio, M; Meinhard, M T; Meister, D; Micheli, F; Musella, P; Nessi-Tedaldi, F; Pandolfi, F; Pata, J; Pauss, F; Perrin, G; Perrozzi, L; Quittnat, M; Rossini, M; Schönenberger, M; Starodumov, A; Tavolaro, V R; Theofilatos, K; Wallny, R; Aarrestad, T K; Amsler, C; Caminada, L; Canelli, M F; De Cosa, A; Galloni, C; Hinzmann, A; Hreus, T; Kilminster, B; Ngadiuba, J; Pinna, D; Rauco, G; Robmann, P; Salerno, D; Yang, Y; Zucchetta, A; Candelise, V; Doan, T H; Jain, Sh; Khurana, R; Konyushikhin, M; Kuo, C M; Lin, W; Lu, Y J; Pozdnyakov, A; Yu, S S; Kumar, Arun; Chang, P; Chang, Y H; Chao, Y; Chen, K F; Chen, P H; Fiori, F; Hou, W-S; Hsiung, Y; Liu, Y F; Lu, R-S; Miñano Moya, M; Paganis, E; Psallidas, A; Tsai, J F; Asavapibhop, B; Singh, G; Srimanobhas, N; Suwonjandee, N; Adiguzel, A; Damarseckin, S; Demiroglu, Z S; Dozen, C; Eskut, E; Girgis, S; Gokbulut, G; Guler, Y; Hos, I; Kangal, E E; Kara, O; Kayis Topaksu, A; Kiminsu, U; Oglakci, M; Onengut, G; Ozdemir, K; Ozturk, S; Polatoz, A; Tali, B; Turkcapar, S; Zorbakir, I S; Zorbilmez, C; Bilin, B; Bilmis, S; Isildak, B; Karapinar, G; Yalvac, M; Zeyrek, M; Gülmez, E; Kaya, M; Kaya, O; Yetkin, E A; Yetkin, T; Cakir, A; Cankocak, K; Sen, S; Grynyov, B; Levchuk, L; Sorokin, P; Aggleton, R; Ball, F; Beck, L; Brooke, J J; Burns, D; Clement, E; Cussans, D; Flacher, H; Goldstein, J; Grimes, M; Heath, G P; Heath, H F; Jacob, J; Kreczko, L; Lucas, C; Newbold, D M; Paramesvaran, S; Poll, A; Sakuma, T; Seif El Nasr-Storey, S; Smith, D; Smith, V J; Belyaev, A; Brew, C; Brown, R M; Calligaris, L; Cieri, D; Cockerill, D J A; Coughlan, J A; Harder, K; Harper, S; Olaiya, E; Petyt, D; Shepherd-Themistocleous, C H; Thea, A; Tomalin, I R; Williams, T; Baber, M; Bainbridge, R; Buchmuller, O; Bundock, A; Burton, D; Casasso, S; Citron, M; Colling, D; Corpe, L; Dauncey, P; Davies, G; De Wit, A; Della Negra, M; Di Maria, R; Dunne, P; Elwood, A; Futyan, D; Haddad, Y; Hall, G; Iles, G; James, T; Lane, R; Laner, C; Lucas, R; Lyons, L; Magnan, A-M; Malik, S; Mastrolorenzo, L; Nash, J; Nikitenko, A; Pela, J; Penning, B; Pesaresi, M; Raymond, D M; Richards, A; Rose, A; Seez, C; Summers, S; Tapper, A; Uchida, K; Vazquez Acosta, M; Virdee, T; Wright, J; Zenz, S C; Cole, J E; Hobson, P R; Khan, A; Kyberd, P; Reid, I D; Symonds, P; Teodorescu, L; Turner, M; Borzou, A; Call, K; Dittmann, J; Hatakeyama, K; Liu, H; Pastika, N; Bartek, R; Dominguez, A; Cooper, S I; Henderson, C; Rumerio, P; West, C; Arcaro, D; Avetisyan, A; Bose, T; Gastler, D; Rankin, D; Richardson, C; Rohlf, J; Sulak, L; Zou, D; Benelli, G; Cutts, D; Garabedian, A; Hakala, J; Heintz, U; Hogan, J M; Jesus, O; Kwok, K H M; Laird, E; Landsberg, G; Mao, Z; Narain, M; Piperov, S; Sagir, S; Spencer, E; Syarif, R; Breedon, R; Burns, D; Calderon De La Barca Sanchez, M; Chauhan, S; Chertok, M; Conway, J; Conway, R; Cox, P T; Erbacher, R; Flores, C; Funk, G; Gardner, M; Ko, W; Lander, R; Mclean, C; Mulhearn, M; Pellett, D; Pilot, J; Shalhout, S; Shi, M; Smith, J; Squires, M; Stolp, D; Tos, K; Tripathi, M; Bravo, C; Cousins, R; Dasgupta, A; Florent, A; Hauser, J; Ignatenko, M; Mccoll, N; Saltzberg, D; Schnaible, C; Valuev, V; Weber, M; Bouvier, E; Burt, K; Clare, R; Ellison, J; Gary, J W; Ghiasi Shirazi, S M A; Hanson, G; Heilman, J; Jandir, P; Kennedy, E; Lacroix, F; Long, O R; Olmedo Negrete, M; Paneva, M I; Shrinivas, A; Si, W; Wei, H; Wimpenny, S; Yates, B R; Branson, J G; Cerati, G B; Cittolin, S; Derdzinski, M; Gerosa, R; Holzner, A; Klein, D; Krutelyov, V; Letts, J; Macneill, I; Olivito, D; Padhi, S; Pieri, M; Sani, M; Sharma, V; Simon, S; Tadel, M; Vartak, A; Wasserbaech, S; Welke, C; Wood, J; Würthwein, F; Yagil, A; Zevi Della Porta, G; Amin, N; Bhandari, R; Bradmiller-Feld, J; Campagnari, C; Dishaw, A; Dutta, V; Franco Sevilla, M; George, C; Golf, F; Gouskos, L; Gran, J; Heller, R; Incandela, J; Mullin, S D; Ovcharova, A; Qu, H; Richman, J; Stuart, D; Suarez, I; Yoo, J; Anderson, D; Bendavid, J; Bornheim, A; Bunn, J; Duarte, J; Lawhorn, J M; Mott, A; Newman, H B; Pena, C; Spiropulu, M; Vlimant, J R; Xie, S; Zhu, R Y; Andrews, M B; Ferguson, T; Paulini, M; Russ, J; Sun, M; Vogel, H; Vorobiev, I; Weinberg, M; Cumalat, J P; Ford, W T; Jensen, F; Johnson, A; Krohn, M; Mulholland, T; Stenson, K; Wagner, S R; Alexander, J; Chaves, J; Chu, J; Dittmer, S; Mcdermott, K; Mirman, N; Nicolas Kaufman, G; Patterson, J R; Rinkevicius, A; Ryd, A; Skinnari, L; Soffi, L; Tan, S M; Tao, Z; Thom, J; Tucker, J; Wittich, P; Zientek, M; Winn, D; Abdullin, S; Albrow, M; Apollinari, G; Apresyan, A; Banerjee, S; Bauerdick, L A T; Beretvas, A; Berryhill, J; Bhat, P C; Bolla, G; Burkett, K; Butler, J N; Cheung, H W K; Chlebana, F; Cihangir, S; Cremonesi, M; Elvira, V D; Fisk, I; Freeman, J; Gottschalk, E; Gray, L; Green, D; Grünendahl, S; Gutsche, O; Hare, D; Harris, R M; Hasegawa, S; Hirschauer, J; Hu, Z; Jayatilaka, B; Jindariani, S; Johnson, M; Joshi, U; Klima, B; Kreis, B; Lammel, S; Linacre, J; Lincoln, D; Lipton, R; Liu, M; Liu, T; Lopes De Sá, R; Lykken, J; Maeshima, K; Magini, N; Marraffino, J M; Maruyama, S; Mason, D; McBride, P; Merkel, P; Mrenna, S; Nahn, S; O'Dell, V; Pedro, K; Prokofyev, O; Rakness, G; Ristori, L; Sexton-Kennedy, E; Soha, A; Spalding, W J; Spiegel, L; Stoynev, S; Strait, J; Strobbe, N; Taylor, L; Tkaczyk, S; Tran, N V; Uplegger, L; Vaandering, E W; Vernieri, C; Verzocchi, M; Vidal, R; Wang, M; Weber, H A; Whitbeck, A; Wu, Y; Acosta, D; Avery, P; Bortignon, P; Bourilkov, D; Brinkerhoff, A; Carnes, A; Carver, M; Curry, D; Das, S; Field, R D; Furic, I K; Konigsberg, J; Korytov, A; Low, J F; Ma, P; Matchev, K; Mei, H; Mitselmakher, G; Rank, D; Shchutska, L; Sperka, D; Thomas, L; Wang, J; Wang, S; Yelton, J; Linn, S; Markowitz, P; Martinez, G; Rodriguez, J L; Ackert, A; Adams, T; Askew, A; Bein, S; Hagopian, S; Hagopian, V; Johnson, K F; Prosper, H; Santra, A; Yohay, R; Baarmand, M M; Bhopatkar, V; Colafranceschi, S; Hohlmann, M; Noonan, D; Roy, T; Yumiceva, F; Adams, M R; Apanasevich, L; Berry, D; Betts, R R; Bucinskaite, I; Cavanaugh, R; Evdokimov, O; Gauthier, L; Gerber, C E; Hofman, D J; Jung, K; Kamin, J; Sandoval Gonzalez, I D; Varelas, N; Wang, H; Wu, Z; Zakaria, M; Zhang, J; Bilki, B; Clarida, W; Dilsiz, K; Durgut, S; Gandrajula, R P; Haytmyradov, M; Khristenko, V; Merlo, J-P; Mermerkaya, H; Mestvirishvili, A; Moeller, A; Nachtman, J; Ogul, H; Onel, Y; Ozok, F; Penzo, A; Snyder, C; Tiras, E; Wetzel, J; Yi, K; Anderson, I; Blumenfeld, B; Cocoros, A; Eminizer, N; Fehling, D; Feng, L; Gritsan, A V; Maksimovic, P; Osherson, M; Roskes, J; Sarica, U; Swartz, M; Xiao, M; Xin, Y; You, C; Al-Bataineh, A; Baringer, P; Bean, A; Boren, S; Bowen, J; Castle, J; Forthomme, L; Kenny, R P; Khalil, S; Kropivnitskaya, A; Majumder, D; Mcbrayer, W; Murray, M; Sanders, S; Stringer, R; Tapia Takaki, J D; Wang, Q; Ivanov, A; Kaadze, K; Maravin, Y; Mohammadi, A; Saini, L K; Skhirtladze, N; Toda, S; Rebassoo, F; Wright, D; Anelli, C; Baden, A; Baron, O; Belloni, A; Calvert, B; Eno, S C; Ferraioli, C; Gomez, J A; Hadley, N J; Jabeen, S; Kellogg, R G; Kolberg, T; Kunkle, J; Lu, Y; Mignerey, A C; Ricci-Tam, F; Shin, Y H; Skuja, A; Tonjes, M B; Tonwar, S C; Abercrombie, D; Allen, B; Apyan, A; Azzolini, V; Barbieri, R; Baty, A; Bi, R; Bierwagen, K; Brandt, S; Busza, W; Cali, I A; D'Alfonso, M; Demiragli, Z; Di Matteo, L; Gomez Ceballos, G; Goncharov, M; Hsu, D; Iiyama, Y; Innocenti, G M; Klute, M; Kovalskyi, D; Krajczar, K; Lai, Y S; Lee, Y-J; Levin, A; Luckey, P D; Maier, B; Marini, A C; Mcginn, C; Mironov, C; Narayanan, S; Niu, X; Paus, C; Roland, C; Roland, G; Salfeld-Nebgen, J; Stephans, G S F; Tatar, K; Varma, M; Velicanu, D; Veverka, J; Wang, J; Wang, T W; Wyslouch, B; Yang, M; Benvenuti, A C; Chatterjee, R M; Evans, A; Hansen, P; Kalafut, S; Kao, S C; Kubota, Y; Lesko, Z; Mans, J; Nourbakhsh, S; Ruckstuhl, N; Rusack, R; Tambe, N; Turkewitz, J; Acosta, J G; Oliveros, S; Avdeeva, E; Bloom, K; Claes, D R; Fangmeier, C; Gonzalez Suarez, R; Kamalieddin, R; Kravchenko, I; Malta Rodrigues, A; Meier, F; Monroy, J; Siado, J E; Snow, G R; Stieger, B; Alyari, M; Dolen, J; Godshalk, A; Harrington, C; Iashvili, I; Kaisen, J; Kharchilava, A; Parker, A; Rappoccio, S; Roozbahani, B; Alverson, G; Barberis, E; Hortiangtham, A; Massironi, A; Morse, D M; Nash, D; Orimoto, T; Teixeira De Lima, R; Trocino, D; Wang, R-J; Wood, D; Bhattacharya, S; Charaf, O; Hahn, K A; Kumar, A; Mucia, N; Odell, N; Pollack, B; Schmitt, M H; Sung, K; Trovato, M; Velasco, M; Dev, N; Hildreth, M; Hurtado Anampa, K; Jessop, C; Karmgard, D J; Kellams, N; Lannon, K; Marinelli, N; Meng, F; Mueller, C; Musienko, Y; Planer, M; Reinsvold, A; Ruchti, R; Smith, G; Taroni, S; Wayne, M; Wolf, M; Woodard, A; Alimena, J; Antonelli, L; Bylsma, B; Durkin, L S; Flowers, S; Francis, B; Hart, A; Hill, C; Hughes, R; Ji, W; Liu, B; Luo, W; Puigh, D; Winer, B L; Wulsin, H W; Cooperstein, S; Driga, O; Elmer, P; Hardenbrook, J; Hebda, P; Lange, D; Luo, J; Marlow, D; Medvedeva, T; Mei, K; Olsen, J; Palmer, C; Piroué, P; Stickland, D; Svyatkovskiy, A; Tully, C; Malik, S; Barker, A; Barnes, V E; Folgueras, S; Gutay, L; Jha, M K; Jones, M; Jung, A W; Khatiwada, A; Miller, D H; Neumeister, N; Schulte, J F; Shi, X; Sun, J; Wang, F; Xie, W; Parashar, N; Stupak, J; Adair, A; Akgun, B; Chen, Z; Ecklund, K M; Geurts, F J M; Guilbaud, M; Li, W; Michlin, B; Northup, M; Padley, B P; Roberts, J; Rorie, J; Tu, Z; Zabel, J; Betchart, B; Bodek, A; de Barbaro, P; Demina, R; Duh, Y T; Ferbel, T; Galanti, M; Garcia-Bellido, A; Han, J; Hindrichs, O; Khukhunaishvili, A; Lo, K H; Tan, P; Verzetti, M; Agapitos, A; Chou, J P; Gershtein, Y; Gómez Espinosa, T A; Halkiadakis, E; Heindl, M; Hughes, E; Kaplan, S; Kunnawalkam Elayavalli, R; Kyriacou, S; Lath, A; Nash, K; Saka, H; Salur, S; Schnetzer, S; Sheffield, D; Somalwar, S; Stone, R; Thomas, S; Thomassen, P; Walker, M; Delannoy, A G; Foerster, M; Heideman, J; Riley, G; Rose, K; Spanier, S; Thapa, K; Bouhali, O; Celik, A; Dalchenko, M; De Mattia, M; Delgado, A; Dildick, S; Eusebi, R; Gilmore, J; Huang, T; Juska, E; Kamon, T; Mueller, R; Pakhotin, Y; Patel, R; Perloff, A; Perniè, L; Rathjens, D; Safonov, A; Tatarinov, A; Ulmer, K A; Akchurin, N; Cowden, C; Damgov, J; De Guio, F; Dragoiu, C; Dudero, P R; Faulkner, J; Gurpinar, E; Kunori, S; Lamichhane, K; Lee, S W; Libeiro, T; Peltola, T; Undleeb, S; Volobouev, I; Wang, Z; Greene, S; Gurrola, A; Janjam, R; Johns, W; Maguire, C; Melo, A; Ni, H; Sheldon, P; Tuo, S; Velkovska, J; Xu, Q; Arenton, M W; Barria, P; Cox, B; Goodell, J; Hirosky, R; Ledovskoy, A; Li, H; Neu, C; Sinthuprasith, T; Sun, X; Wang, Y; Wolfe, E; Xia, F; Clarke, C; Harr, R; Karchin, P E; Sturdy, J; Belknap, D A; Buchanan, J; Caillol, C; Dasu, S; Dodd, L; Duric, S; Gomber, B; Grothe, M; Herndon, M; Hervé, A; Klabbers, P; Lanaro, A; Levine, A; Long, K; Loveless, R; Ojalvo, I; Perry, T; Pierro, G A; Polese, G; Ruggles, T; Savin, A; Smith, N; Smith, W H; Taylor, D; Woods, N

2017-08-25

The production of jets in association with Z bosons, reconstructed via the μ^{+}μ^{-} and e^{+}e^{-} decay channels, is studied in pp and, for the first time, in Pb-Pb collisions. Both data samples were collected by the CMS experiment at the LHC, at a nucleon-nucleon center-of-mass energy of 5.02 TeV. The Pb-Pb collisions were analyzed in the 0%-30% centrality range. The back-to-back azimuthal alignment was studied in both pp and Pb-Pb collisions for Z bosons with transverse momentum p_{T}^{Z}>60  GeV/c and a recoiling jet with p_{T}^{jet}>30  GeV/c. The p_{T} imbalance x_{jZ}=p_{T}^{jet}/p_{T}^{Z}, as well as the average number of jet partners per Z, R_{jZ}, was studied in intervals of p_{T}^{Z}. The R_{jZ} is found to be smaller in Pb-Pb than in pp collisions, which suggests that in Pb-Pb collisions a larger fraction of partons associated with the Z bosons fall below the 30  GeV/c p_{T}^{jet} threshold because they lose energy.

Increased seizure latency and decreased severity of pentylenetetrazol-induced seizures in mice after essential oil administration.

PubMed

Koutroumanidou, Eleni; Kimbaris, Athanasios; Kortsaris, Alexandros; Bezirtzoglou, Eugenia; Polissiou, Moschos; Charalabopoulos, Konstantinos; Pagonopoulou, Olga

2013-01-01

The effect of pretreatment with essential oils (EOs) from eight aromatic plants on the seizure latency and severity of pentylenetetrazol- (PTZ-) induced seizures in mice was evaluated. Weight-dependent doses of Rosmarinus officinalis, Ocimum basilicum, Mentha spicata, Mentha pulegium, Lavandula angustifolia, Mentha piperita, Origanum dictamnus, and Origanum vulgare, isolated from the respective aromatic plants from NE Greece, were administered 60 minutes prior to intraperitoneal (i.p.) injection of a lethal dose of PTZ to eight respective groups of Balb-c mice. Control group received only one i.p. PTZ injection. Motor and behavioral activity of the animals after EOs administration, development of tonic-clonic seizures, seizure latency and severity, and percentage of survival after PTZ administration were determined for each group. All groups of mice treated with the EOs showed reduced activity and stability after the administration of the oil, except for those treated with O. vulgare (100% mortality after the administration of the oil). After PTZ administration, mice from the different groups showed increased latency and reduced severity of seizures (ranging from simple twitches to complete seizures). Mice who had received M. piperita demonstrated no seizures and 100% survival. The different drastic component and its concentration could account for the diversity of anticonvulsant effects.

Pulse Vector-Excitation Speech Encoder

NASA Technical Reports Server (NTRS)

Davidson, Grant; Gersho, Allen

1989-01-01

Proposed pulse vector-excitation speech encoder (PVXC) encodes analog speech signals into digital representation for transmission or storage at rates below 5 kilobits per second. Produces high quality of reconstructed speech, but with less computation than required by comparable speech-encoding systems. Has some characteristics of multipulse linear predictive coding (MPLPC) and of code-excited linear prediction (CELP). System uses mathematical model of vocal tract in conjunction with set of excitation vectors and perceptually-based error criterion to synthesize natural-sounding speech.

Involvement of over-expressed BMP4 in pentylenetetrazol kindling-induced cell proliferation in the dentate gyrus of adult rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yin Jinbo; Ma Yuxin; Yin Qing

2007-03-30

The dentate gyrus (DG) of the hippocampus is one of a few regions in the adult mammalian brain characterized by ongoing neurogenesis. Proliferation of neural precursors in the granule cell layer of the DG has been identified in pentylenetetrazol (PTZ) kindling epilepsy model, however, little is known about the molecular mechanism. We previously reported that the expression pattern of bone morphogenetic proteins-4 (BMP4) mRNA in the hippocampus was developmentally regulated and mainly localized in the DG of the adult. To explore the role of BMP4 in epileptic activity, we detected BMP4 expression in the DG during PTZ kindling process andmore » explore its correlation with cell proliferation combined with bromodeoxyuridine (BrdU) labeling technique. We found that dynamic changes in BMP4 level and BrdU labeled cells dependent on the kindling stage of PTZ induced seizure-prone state. The number of BMP4 mRNA-positive cells and BrdU labeled cells reached the top level 1 day after PTZ kindled, then declined to base level 2 months later. Furthermore, there was a significant correlation between increased BMP4 mRNA expression and increased number of BrdU labeled cells. After effectively blocked expression of BMP4 with antisense oligodeoxynucleotides(ASODN), the BrdU labeled cells in the dentate gyrus subgranular zone(DG-SGZ) and hilus were significantly decreased 16d after First PTZ injection and 1, 3, 7, 14d after kindled respectively. These findings suggest that increased proliferation in the DG of the hippocampus resulted from kindling epilepsy elicited by PTZ maybe be modulated by BMP4 over-expression.« less

l-Carnitine Modulates Epileptic Seizures in Pentylenetetrazole-Kindled Rats via Suppression of Apoptosis and Autophagy and Upregulation of Hsp70.

PubMed

Hussein, Abdelaziz M; Adel, Mohamed; El-Mesery, Mohamed; Abbas, Khaled M; Ali, Amr N; Abulseoud, Osama A

2018-03-14

l-Carnitine is a unique nutritional supplement for athletes that has been recently studied as a potential treatment for certain neuropsychiatric disorders. However, its efficacy in seizure control has not been investigated. Sprague Dawley rats were randomly assigned to receive either saline (Sal) (negative control) or pentylenetetrazole (PTZ) 40 mg/kg i.p. × 3 times/week × 3 weeks. The PTZ group was further subdivided into two groups, the first received oral l-carnitine (l-Car) (100 mg/kg/day × 4 weeks) (PTZ + l-Car), while the second group received saline (PTZ + Sal). Daily identification and quantification of seizure scores, time to the first seizure and the duration of seizures were performed in each animal. Molecular oxidative markers were examined in the animal brains. l-Car treatment was associated with marked reduction in seizure score ( p = 0.0002) that was indicated as early as Day 2 of treatment and continued throughout treatment duration. Furthermore, l-Car significantly prolonged the time to the first seizure ( p < 0.0001) and shortened seizure duration ( p = 0.028). In addition, l-Car administration for four weeks attenuated PTZ-induced increase in the level of oxidative stress marker malondialdehyde (MDA) ( p < 0.0001) and reduced the activity of catalase enzyme ( p = 0.0006) and increased antioxidant GSH activity ( p < 0.0001). Moreover, l-Car significantly reduced PTZ-induced elevation in protein expression of caspase-3 ( p < 0.0001) and β-catenin ( p < 0.0001). Overall, our results suggest a potential therapeutic role of l-Car in seizure control and call for testing these preclinical results in a proof of concept pilot clinical study.

Evaluation of possible antioxidant and anticonvulsant effects of the ethyl acetate fraction from Platonia insignis Mart. (Bacuri) on epilepsy models.

PubMed

Júnior, Joaquim Soares da Costa; de Almeida, Antonia Amanda C; Tomé, Adriana da Rocha; Citó, Antonia Maria das Graças Lopes; Saffi, Jenifer; de Freitas, Rivelilson Mendes

2011-12-01

The aim of present study was to examine the effects of the ethyl acetate fraction (EAF) from Platonia insignis on lipid peroxidation level, nitrite formation, and superoxide dismutase and catalase activities in rat striatum prior to pilocarpine-induced seizures as well as to explore its anticonvulsant activity in adult rats prior to pentylenetetrazole (PTZ)- and picrotoxin (PIC)-induced seizures. Wistar rats were treated with vehicle, atropine (25mg/kg), EAF (0.1, 1, and 10mg/kg), pilocarpine (400mg/kg, P400 group), PTZ (60 mg/kg, PTZ group), PIC (8 mg/kg, PIC group), atropine+P400, EAF+P400, EAF+PTZ, or EAF+PIC. Significant decreases in number of crossings and rearings were observed in the P400 group. The EAF 10+P400 group also had significant increases in these parameters. In addition, in rats treated with P400, there were significant increases in lipid peroxidation and nitrite levels; however, there were no alterations in SOD and catalase activities. In the EAF 10+P400 group, lipid peroxidation and nitrite levels significantly decreased and SOD and catalase activities significantly increased after pilocarpine-induced seizures. Additionally, effects of the EAF were evaluated in PTZ and PIC models. EAF did not increase the latency to development of convulsions induced with PTZ and PIC at the doses tested. Our findings strongly support the hypothesis that EAF does not have anticonvulsant activity in the different models of epilepsy studied. Our results indicate that in the in vivo model of pilocarpine-induced seizures, EAF has antioxidant activity, but not anticonvulsant properties at the doses tested. Copyright © 2011. Published by Elsevier Inc.

Effects of hydroalcoholic extract of Coriandrum sativum on oxidative damage in pentylenetetrazole-induced seizures in rats

PubMed Central

Karami, Reza; Hosseini, Mahmoud; Mohammadpour, Toktam; Ghorbani, Ahmad; Sadeghnia, Hamid Reza; Rakhshandeh, Hassan; Vafaee, Farzaneh; Esmaeilizadeh, Mahdi

2015-01-01

Background: An important role for oxidative stress, as a consequence of epileptic seizures, has been suggested. Coriandrum sativum has been shown that have antioxidant effects. Central nervous system depressant effects of C. sativum have also been reported. In this study, the effects of hydroalcoholic extract of aerial parts of the plants on brain tissues oxidative damages following seizures induced by pentylenetetrazole (PTZ) was investigated in rats. Methods: The rats were divided into five groups and treated: (1) Control (saline), (2) PTZ (90 mg/kg, i.p.), (3-5) three doses (100, 500 and 1000 mg/kg of C. sativum extract (CSE) before PTZ. Latencies to the first minimal clonic seizures (MCS) and the first generalized tonic-clonic seizures (GTCS) were recorded. The cortical and hippocampal tissues were then removed for biochemical measurements. Results: The extract significantly increased the MCS and GTCS latencies (P < 0.01, P < 0.001) following PTZ-induced seizures. The malondialdehyde (MDA) levels in both cortical and hippocampal tissues of PTZ group were significantly higher than those of the control animals (P < 0.001). Pretreatment with the extract prevented elevation of the MDA levels (P < 0.010–P < 0.001). Following PTZ administration, a significant reduction in total thiol groups was observed in both cortical and hippocampal tissues (P < 0.050). Pre-treatment with the 500 mg/kg of the extract caused a significant prevention of decreased in total thiol concentration in the cortical tissues (P < 0.010). Conclusion: The present study showed that the hydroalcoholic extract of the aerial parts of C. sativum possess significant antioxidant and anticonvulsant activities. PMID:26056549

Vaccination with an adenoviral vector that encodes and displays a retroviral antigen induces improved neutralizing antibody and CD4+ T-cell responses and confers enhanced protection.

PubMed

Bayer, Wibke; Tenbusch, Matthias; Lietz, Ruth; Johrden, Lena; Schimmer, Simone; Uberla, Klaus; Dittmer, Ulf; Wildner, Oliver

2010-02-01

We present a new type of adenoviral vector that both encodes and displays a vaccine antigen on the capsid, thus combining in itself gene-based and protein vaccination; this vector resulted in an improved vaccination outcome in the Friend virus (FV) model. For presentation of the envelope protein gp70 of Friend murine leukemia virus on the adenoviral capsid, gp70 was fused to the adenovirus capsid protein IX. When compared to vaccination with conventional FV Env- and Gag-encoding adenoviral vectors, vaccination with the adenoviral vector that encodes and displays pIX-gp70 combined with an FV Gag-encoding vector resulted in significantly improved protection against systemic FV challenge infection, with highly controlled viral loads in plasma and spleen. This improved protection correlated with improved neutralizing antibody titers and stronger CD4(+) T-cell responses. Using a vector that displays gp70 without encoding it, we found that while the antigen display on the capsid alone was sufficient to induce high levels of binding antibodies, in vivo expression was necessary for the induction of neutralizing antibodies. This new type of adenovirus-based vaccine could be a valuable tool for vaccination.

Solid lipid nanoparticle-based vectors intended for the treatment of X-linked juvenile retinoschisis by gene therapy: In vivo approaches in Rs1h-deficient mouse model.

PubMed

Apaolaza, P S; Del Pozo-Rodríguez, A; Torrecilla, J; Rodríguez-Gascón, A; Rodríguez, J M; Friedrich, U; Weber, B H F; Solinís, M A

2015-11-10

X-linked juvenile retinoschisis (XLRS), which results from mutations in the gene RS1 that encodes the protein retinoschisin, is a retinal degenerative disease affecting between 1/5000 and 1/25,000 people worldwide. Currently, there is no cure for this disease and the treatment is based on the application of low-vision aids. The aim of the present work was the in vitro and in vivo evaluation of two different non-viral vectors based on solid lipid nanoparticles (SLNs), protamine and two anionic polysaccharides, hyaluronic acid (HA) or dextran (DX), for the treatment of XLRS. First, the vectors containing a plasmid which encodes both the reporter green fluorescent protein (GFP) and the therapeutic protein retinoschisin, under the control of CMV promoters, were characterized in vitro. Then, the vectors were subretinally or intravitreally administrated to C57BL/6 wild type mice. One week later, GFP was detected in all treated mice and in all retinal layers except in the Outer Nuclear Layer (ONL) and the Inner Nuclear Layer (INL), regardless of the administration route and the vector employed. Finally, two weeks after subretinal or intravitreal injection to Rs1h-deficient mice, GFP and retinoschisin expression was detected in all retinal layers, except in the ONL, which was maintained for at least two months after subretinal administration. The structural analysis of the treated Rs1h-deficient eyes showed a partial recovery of the retina related to the production of retinoschisin. This work shows for the first time a successful RS1 gene transfer to Rs1h-deficient animals using non-viral nanocarriers, with promising results that point to non-viral gene therapy as a feasible future therapeutic tool for retinal disorders.

Seizure-mediated neuronal activation induces DREAM gene expression in the mouse brain.

PubMed

Matsu-ura, Toru; Konishi, Yoshiyuki; Aoki, Tsutomu; Naranjo, Jose R; Mikoshiba, Katsuhiko; Tamura, Taka-aki

2002-12-30

Various transcriptional activators are induced in neurons concomitantly with long-lasting neural activity, whereas only a few transcription factors are known to act as neural activity-inducible transcription repressors. In this study, mRNA of DREAM (DRE-antagonizing modulator), a Ca(2+)-modulated transcriptional repressor, was demonstrated to accumulate in the mouse brain after pentylenetetrazol (PTZ)-induced seizures. Accumulation in the mouse hippocampus reached maximal level in the late phase (at 7-8 h) after PTZ injection. Kainic acid induced the same response. Interestingly, the late induction of DREAM expression required new protein synthesis and was blocked by MK801 suggesting that Ca(2+)-influx via NMDA receptors is necessary for the PTZ-mediated DREAM expression. In situ hybridization revealed that PTZ-induced DREAM mRNA accumulation was observed particularly in the dentate gyrus, cerebral cortex, and piriform cortex. The results of the present study demonstrate that DREAM is a neural activity-stimulated late gene and suggest its involvement in adaptation to long-lasting neuronal activity.

Identification, Characterization and Rescue of a Novel Vasopressin-2 Receptor Mutation Causing Nephrogenic Diabetes Insipidus

PubMed Central

Ranadive, Sayali A.; Ersoy, Baran; Favre, Helene; Cheung, Clement C.; Rosenthal, Stephen M.; Miller, Walter L.; Vaisse, Christian

2018-01-01

SUMMARY Objective X-linked nephrogenic diabetes insipidus (XNDI), caused by mutations in the V2 vasopressin receptor (V2R), is clinically distinguished from central diabetes insipidus (CDI) by elevated serum vasopressin (AVP) levels and unresponsiveness to 1-desamino-8-D-arginine vasopressin (DDAVP). We report two infants with XNDI, and present the characterization and functional rescue of a novel V2R mutation. Patients Two male infants presented with poor growth and hypernatremia. Both patients had measurable pre-treatment serum AVP and polyuria that did not respond to DDAVP, suggesting NDI. However, both also had absent posterior pituitary bright spot on MRI, which is a finding more typical of CDI. Methods The AVPR2 gene encoding V2R was sequenced. The identified novel missense mutation was re-created by site-directed mutagenesis and expressed in HEK293 cells. V2R activity was assessed by the ability of transfected cells to produce cAMP in response to stimulation with DDAVP. Membrane localization of V2R was assessed by fluorescence microscopy. Results Patient 1 had a deletion of AVPR2; patient 2 had the novel mutation L57R. In transiently transfected HEK293 cells, DDAVP induced detectable but severely impaired L57R V2R activity compared to cells expressing wild-type V2R. Fluorescence microscopy showed that myc-tagged wild-type V2R localized to the cell membrane while L57R V2R remained intracellular. A non-peptide V2R chaperone, SR121463, partially rescued L57R V2R function by allowing it to reach the cell membrane. Conclusions L57R V2R has impaired in vitro activity that can be partially improved by treatment with a V2R chaperone. The posterior pituitary hyperintensity may be absent in infants with XNDI. PMID:19170711

Method and system for efficiently searching an encoded vector index

DOEpatents

Bui, Thuan Quang; Egan, Randy Lynn; Kathmann, Kevin James

2001-09-04

Method and system aspects for efficiently searching an encoded vector index are provided. The aspects include the translation of a search query into a candidate bitmap, and the mapping of data from the candidate bitmap into a search result bitmap according to entry values in the encoded vector index. Further, the translation includes the setting of a bit in the candidate bitmap for each entry in a symbol table that corresponds to candidate of the search query. Also included in the mapping is the identification of a bit value in the candidate bitmap pointed to by an entry in an encoded vector.

Adeno-associated virus type 8 vector–mediated expression of siRNA targeting vascular endothelial growth factor efficiently inhibits neovascularization in a murine choroidal neovascularization model

PubMed Central

Igarashi, Tsutomu; Miyake, Noriko; Fujimoto, Chiaki; Yaguchi, Chiemi; Iijima, Osamu; Shimada, Takashi; Takahashi, Hiroshi

2014-01-01

Purpose To assess the feasibility of a gene therapeutic approach to treating choroidal neovascularization (CNV), we generated an adeno-associated virus type 8 vector (AAV2/8) encoding an siRNA targeting vascular endothelial growth factor (VEGF), and determined the AAV2/8 vector’s ability to inhibit angiogenesis. Methods We initially transfected 3T3 cells expressing VEGF with the AAV2/8 plasmid vector psiRNA-VEGF using the H1 promoter and found that VEGF expression was significantly diminished in the transfectants. We next injected 1 μl (3 × 1014 vg/ml) of AAV2/8 vector encoding siRNA targeting VEGF (AAV2/8/SmVEGF-2; n = 12) or control vector encoding green fluorescent protein (GFP) (AAV2/8/GFP; n = 14) into the subretinal space in C57BL/6 mice. One week later, CNV was induced by using a diode laser to make four separate choroidal burns around the optic nerve in each eye. After an additional 2 weeks, the eyes were removed for flat mount analysis of the CNV surface area. Results Subretinal delivery of AAV2/8/SmVEGF-2 significantly diminished CNV at the laser lesions, compared to AAV8/GFP (1597.3±2077.2 versus 5039.5±4055.9 µm2; p<0.05). Using an enzyme-linked immunosorbent assay, we found that VEGF levels were reduced by approximately half in the AAV2/8/SmVEGF-2 treated eyes. Conclusions These results suggest that siRNA-VEGF can be expressed across the retina and that long-term suppression of CNV is possible through the use of stable AAV2/8-mediated siRNA-VEGF expression. In vivo gene therapy may thus be a feasible approach to the clinical management of CNV in conditions such as age-related macular degeneration. PMID:24744609

Behavioral and genotoxic evaluation of rosmarinic and caffeic acid in acute seizure models induced by pentylenetetrazole and pilocarpine in mice.

PubMed

Coelho, Vanessa Rodrigues; Vieira, Caroline Gonçalves; de Souza, Luana Pereira; da Silva, Lucas Lima; Pflüger, Pricila; Regner, Gabriela Gregory; Papke, Débora Kuck Mausolff; Picada, Jaqueline Nascimento; Pereira, Patrícia

2016-11-01

The goal of this study was to investigate the effects of rosmarinic acid (RA) and caffeic acid (CA) in the acute pentylenetetrazole (PTZ) and pilocarpine (PIL) seizure models. We also evaluated the effect of RA and CA on the diazepam (DZP)-induced sleeping time test and its possible neuroprotective effect against the genotoxic damage induced by PTZ and PIL. Mice were treated intraperitoneally (i.p.) with saline, RA (2 or 4 mg/kg), or CA (4 or 8 mg/kg) alone or associated to low-dose DZP. After, mice received a single dose of PTZ (88 mg/kg) or PIL (250 mg/kg) and were monitored for the percentage of seizures and the latency to first seizure (LFS) >3 s. Vigabatrin and DZP were used as positive controls. In the DZP-induced sleeping time test, mice were treated with RA and CA and 30 min after receiving DZP (25 mg/kg, i.p.). The alkaline comet assay was performed after acute seizure tests to evaluate the antigenotoxic profiles of RA and CA. The doses of RA and CA tested alone did not reduce the occurrence of seizures induced by PTZ or PIL. The association of 4 mg/kg RA + low-dose DZP was shown to increase LFS in the PTZ model, compared to the group that received only the DZP. In the DZP-induced sleeping time test, the latency to sleep was reduced by 4 mg/kg RA and 8 mg/kg CA. The PTZ-induced genotoxic damage was not prevented by RA or CA, but the PIL-induced genotoxic damage was decreased by pretreatment with 4 mg/kg RA (in cortex) and 4 mg/kg CA (in hippocampus). In conclusion, RA and CA presented neuroprotective effect against PIL-induced genotoxic damage and reduced the latency to DZP-induced sleep. Of the rosmarinic acid, 4 mg/kg enhanced the DZP effect in the increase of latency to clonic PTZ-induced seizures.

Adeno-associated virus vectors can be efficiently produced without helper virus.

PubMed

Matsushita, T; Elliger, S; Elliger, C; Podsakoff, G; Villarreal, L; Kurtzman, G J; Iwaki, Y; Colosi, P

1998-07-01

The purpose of this work was to develop an efficient method for the production of adeno-associated virus (AAV) vectors in the absence of helper virus. The adenovirus regions that mediate AAV vector replication were identified and assembled into a helper plasmid. These included the VA, E2A and E4 regions. When this helper plasmid was cotransfected into 293 cells, along with plasmids encoding the AAV vector, and rep and cap genes, AAV vector was produced as efficiently as when using adenovirus infection as a source of help. CMV-driven constructs expressing the E4orf6 and the 72-M(r), E2A proteins were able to functionally replace the E4 and E2A regions, respectively. Therefore the minimum set of genes required to produce AAV helper activity equivalent to that provided by adenovirus infection consists of, or is a subset of, the following genes: the E4orf6 gene, the 72-M(r), E2A protein gene, the VA RNA genes and the E1 region. AAV vector preparations made with adenovirus and by the helper virus-free method were essentially indistinguishable with respect to particle density, particle to infectivity ratio, capsimer ratio and efficiency of muscle transduction in vivo. Only AAV vector preparations made by the helper virus-free method were not reactive with anti-adenovirus sera.

Creatine Revealed Anticonvulsant Properties on Chemically and Electrically Induced Seizures in Mice.

PubMed

Shafaroodi, Hamed; Shahbek, Farnaz; Faizi, Mehrdad; Ebrahimi, Farzad; Moezi, Leila

2016-01-01

Creatine exerts beneficial effects on a variety of pathologies in which energy metabolism and oxidative stress play an etiological role. Creatine supplements have shown beneficial effects on neurological disorders including Parkinson׳s disease, Huntington›s disease, amyotrophic lateral sclerosis, as well as Alzheimer›s disease and stroke. However, the potential benefits of creatine for patients with convulsive disorders remain poorly defined. While some authors did not suggest any anti- or pro-convulsant roles for creatine treatment, others suggest that creatine may be an anticonvulsant agent. In this study, we investigated the effects of creatine on seizures in mice. Three models were used to explore the role of creatine on seizures in mice including intravenous pentylenetetrazole (PTZ), intraperitoneal PTZ, and electroshock models. Acute creatine treatment (10, 20, 40 and 80 mg/Kg) significantly increased the clonic seizure threshold in the intravenous PTZ model. Sub-chronic administration of creatine (10 and 20 mg/Kg) revealed a significant anticonvulsant effect in intravenous PTZ model. Acute creatine administration (10, 20 and 40 mg/Kg) significantly decreased the frequency of clonic seizures in the intraperitoneal PTZ model. Besides, acute creatine (40 and 80 mg/Kg) decreased the incidence of tonic seizures after electroshock. In conclusion, creatine exerts anticonvulsant effects in three seizure models; therefore, it may act as a potential drug to help patients with convulsions. However, further investigations should be done to clarify these results more.

Creatine Revealed Anticonvulsant Properties on Chemically and Electrically Induced Seizures in Mice

PubMed Central

Shafaroodi, Hamed; Shahbek, Farnaz; Faizi, Mehrdad; Ebrahimi, Farzad; Moezi, Leila

2016-01-01

Creatine exerts beneficial effects on a variety of pathologies in which energy metabolism and oxidative stress play an etiological role. Creatine supplements have shown beneficial effects on neurological disorders including Parkinson׳s disease, Huntington›s disease, amyotrophic lateral sclerosis, as well as Alzheimer›s disease and stroke. However, the potential benefits of creatine for patients with convulsive disorders remain poorly defined. While some authors did not suggest any anti- or pro-convulsant roles for creatine treatment, others suggest that creatine may be an anticonvulsant agent. In this study, we investigated the effects of creatine on seizures in mice. Three models were used to explore the role of creatine on seizures in mice including intravenous pentylenetetrazole (PTZ), intraperitoneal PTZ, and electroshock models. Acute creatine treatment (10, 20, 40 and 80 mg/Kg) significantly increased the clonic seizure threshold in the intravenous PTZ model. Sub-chronic administration of creatine (10 and 20 mg/Kg) revealed a significant anticonvulsant effect in intravenous PTZ model. Acute creatine administration (10, 20 and 40 mg/Kg) significantly decreased the frequency of clonic seizures in the intraperitoneal PTZ model. Besides, acute creatine (40 and 80 mg/Kg) decreased the incidence of tonic seizures after electroshock. In conclusion, creatine exerts anticonvulsant effects in three seizure models; therefore, it may act as a potential drug to help patients with convulsions. However, further investigations should be done to clarify these results more. PMID:28243281

Cloning and Characterization of the Genes Encoding the Murine Homologues of the Human Melanoma Antigens MART1 and gp100

PubMed Central

Zhai, Yifan; Yang, James C.; Spiess, Paul; Nishimura, Michael I.; Overwijk, Willem W.; Roberts, Bruce; Restifo, Nicholas P.; Rosenberg, Steven A.

2008-01-01

The recent identification of genes encoding melanoma-associated antigens has opened new possibilities for the development of cancer vaccines designed to cause the rejection of established tumors. To develop a syngeneic animal model for evaluating antigen-specific vaccines in cancer therapy, the murine homologues of the human melanoma antigens MART1 and gp 100, which were specifically recognized by tumor-infiltrating lymphocytes from patients with melanoma, were cloned and sequenced from a murine B16 melanoma cDNA library. The open reading frames of murine MART1 and gp 100 encode proteins of 113- and 626-amino acids with 68.8 and 77% identity to the respective human proteins. Comparison of the DNA sequences of the murine MART1 genes, derived from normal melanocytes, the immortalized nontumorgenic melanocyte line Melan-a and the B16 melanoma, showed all to be identical. Northern and Western blot analyses confirmed that both genes encoded products that were melanocyte lineage proteins. Mice immunized with murine MART1 or gp 100 using recombinant vaccinia virus failed to produce any detectable T-cell responses or protective immunity against B16 melanoma. In contrast, immunization of mice with human gp 100 using recombinant adenoviruses elicited T cells specific for hgp100, but these T cells also cross reacted with B16 tumor in vitro and induced significant but weak protection against B16 challenge. Immunization with human and mouse gp100 together [adenovirus type 2 (Ad2)-hep100 plus recombinant vaccinia virus (rVV)-mgp100], or immunization with human gp100 (Ad2-hgp100) and boosting with heterologous vector (rVV-hgp100 or rVV-mgp100) or homologous vector (Ad2-hgp100), did not significantly enhance the protective response against B16 melanoma. These results may suggest that immunization with heterologous tumor antigen, rather than self, may be more effective as an immunotherapeutic reagent in designing antigen-specific cancer vaccines. PMID:9101410

The convulsive and electroencephalographic changes produced by nonpeptidic delta-opioid agonists in rats: comparison with pentylenetetrazol.

PubMed

Jutkiewicz, Emily M; Baladi, Michelle G; Folk, John E; Rice, Kenner C; Woods, James H

2006-06-01

delta-Opioid agonists produce convulsions and antidepressant-like effects in rats. It has been suggested that the antidepressant-like effects are produced through a convulsant mechanism of action either through overt convulsions or nonconvulsive seizures. This study evaluated the convulsive and seizurogenic effects of nonpeptidic delta-opioid agonists at doses that previously were reported to produce antidepressant-like effects. In addition, delta-opioid agonist-induced electroencephalographic (EEG) and behavioral changes were compared with those produced by the chemical convulsant pentylenetetrazol (PTZ). For these studies, EEG changes were recorded using a telemetry system before and after injections of the delta-opioid agonists [(+)-4-[(alphaR)-alpha-[(2S,5R)-2,5-dimethyl-4-(2-propenyl)-1-piperazinyl]-(3-methoxyphenyl)methyl]-N,N-diethylbenz (SNC80) and [(+)-4-[alpha(R)-alpha-[(2S,5R)-2,5-dimethyl-4-(2-propenyl)-1-piperazinyl]-(3-hydroxyphenyl)methyl]-N,N-diethylbenzamide [(+)-BW373U86]. Acute administration of nonpeptidic delta-opioid agonists produced bilateral ictal and paroxysmal spike and/or sharp wave discharges. delta-Opioid agonists produced brief changes in EEG recordings, and tolerance rapidly developed to these effects; however, PTZ produced longer-lasting EEG changes that were exacerbated after repeated administration. Studies with antiepileptic drugs demonstrated that compounds used to treat absence epilepsy blocked the convulsive effects of nonpeptidic delta-opioid agonists. Overall, these data suggest that delta-opioid agonist-induced EEG changes are not required for the antidepressant-like effects of these compounds and that neural circuitry involved in absence epilepsy may be related to delta-opioid agonist-induced convulsions. In terms of therapeutic development, these data suggest that it may be possible to develop delta-opioid agonists devoid of convulsive properties.

Firewalls Prevent Systemic Dissemination of Vectors Derived from Human Adenovirus Type 5 and Suppress Production of Transgene-Encoded Antigen in a Murine Model of Oral Vaccination

PubMed Central

Revaud, Julien; Unterfinger, Yves; Rol, Nicolas; Suleman, Muhammad; Shaw, Julia; Galea, Sandra; Gavard, Françoise; Lacour, Sandrine A.; Coulpier, Muriel; Versillé, Nicolas; Havenga, Menzo; Klonjkowski, Bernard; Zanella, Gina; Biacchesi, Stéphane; Cordonnier, Nathalie; Corthésy, Blaise; Ben Arous, Juliette; Richardson, Jennifer P.

2018-01-01

To define the bottlenecks that restrict antigen expression after oral administration of viral-vectored vaccines, we tracked vectors derived from the human adenovirus type 5 at whole body, tissue, and cellular scales throughout the digestive tract in a murine model of oral delivery. After intragastric administration of vectors encoding firefly luciferase or a model antigen, detectable levels of transgene-encoded protein or mRNA were confined to the intestine, and restricted to delimited anatomical zones. Expression of luciferase in the form of multiple small bioluminescent foci in the distal ileum, cecum, and proximal colon suggested multiple crossing points. Many foci were unassociated with visible Peyer's patches, implying that transduced cells lay in proximity to villous rather than follicle-associated epithelium, as supported by detection of transgene-encoded antigen in villous epithelial cells. Transgene-encoded mRNA but not protein was readily detected in Peyer's patches, suggesting that post-transcriptional regulation of viral gene expression might limit expression of transgene-encoded antigen in this tissue. To characterize the pathways by which the vector crossed the intestinal epithelium and encountered sentinel cells, a fluorescent-labeled vector was administered to mice by the intragastric route or inoculated into ligated intestinal loops comprising a Peyer's patch. The vector adhered selectively to microfold cells in the follicle-associated epithelium, and, after translocation to the subepithelial dome region, was captured by phagocytes that expressed CD11c and lysozyme. In conclusion, although a large number of crossing events took place throughout the intestine within and without Peyer's patches, multiple firewalls prevented systemic dissemination of vector and suppressed production of transgene-encoded protein in Peyer's patches. PMID:29423380

An alternative approach in regulation of expression of a transgene by endogenous miR-145 in carcinoma and normal breast cell lines.

PubMed

Ghanbari Safari, Maryam; Baesi, Kazem; Hosseinkhani, Saman

2017-03-01

MicroRNAs are small noncoding RNAs that regulate gene expression by repressing translation of target cellular transcripts. Increasing evidences indicate that miRNAs have different expression profiles and play crucial roles in numerous cellular processes. Delivery and expression of transgenes for cancer therapy must be specific for tumors to avoid killing of healthy tissues. Many investigators have shown that transgene expression can be suppressed in normal cells using vectors that are responsive to microRNA regulation. To overcome this problem, miR-145 that exhibits downregulation in many types of cancer cells was chosen for posttranscriptional regulatory systems mediated by microRNAs. In this study, a psiCHECK-145T vector carrying four tandem copies of target sequences of miR-145 into 3'-UTR of the Renilla luciferase gene was constructed. Renilla luciferase activity from the psiCHECK-145T vector was 57% lower in MCF10A cells with high miR-145 expression as compared to a control condition. Additionally, overexpression of miR-145 in MCF-7 cells with low expression level of miR-145 showed more than 76% reduction in the Renilla luciferase activity from the psiCHECK-145T vector. Inclusion of miR-145 target sequences into the 3'-UTR of the Renilla luciferase gene is a feasible strategy for restricting transgene expression in a breast cancer cell line while sparing a breast normal cell line. © 2015 International Union of Biochemistry and Molecular Biology, Inc.

Anatomical Reconstruction and Functional Imaging Reveal an Ordered Array of Skylight Polarization Detectors in Drosophila

PubMed Central

Bleul, Christiane; Baumann-Klausener, Franziska; Labhart, Thomas; Dickinson, Michael H.

2016-01-01

Many insects exploit skylight polarization as a compass cue for orientation and navigation. In the fruit fly, Drosophila melanogaster, photoreceptors R7 and R8 in the dorsal rim area (DRA) of the compound eye are specialized to detect the electric vector (e-vector) of linearly polarized light. These photoreceptors are arranged in stacked pairs with identical fields of view and spectral sensitivities, but mutually orthogonal microvillar orientations. As in larger flies, we found that the microvillar orientation of the distal photoreceptor R7 changes in a fan-like fashion along the DRA. This anatomical arrangement suggests that the DRA constitutes a detector for skylight polarization, in which different e-vectors maximally excite different positions in the array. To test our hypothesis, we measured responses to polarized light of varying e-vector angles in the terminals of R7/8 cells using genetically encoded calcium indicators. Our data confirm a progression of preferred e-vector angles from anterior to posterior in the DRA, and a strict orthogonality between the e-vector preferences of paired R7/8 cells. We observed decreased activity in photoreceptors in response to flashes of light polarized orthogonally to their preferred e-vector angle, suggesting reciprocal inhibition between photoreceptors in the same medullar column, which may serve to increase polarization contrast. Together, our results indicate that the polarization-vision system relies on a spatial map of preferred e-vector angles at the earliest stage of sensory processing. SIGNIFICANCE STATEMENT The fly's visual system is an influential model system for studying neural computation, and much is known about its anatomy, physiology, and development. The circuits underlying motion processing have received the most attention, but researchers are increasingly investigating other functions, such as color perception and object recognition. In this work, we investigate the early neural processing of a somewhat exotic sense, called polarization vision. Because skylight is polarized in an orientation that is rigidly determined by the position of the sun, this cue provides compass information. Behavioral experiments have shown that many species use the polarization pattern in the sky to direct locomotion. Here we describe the input stage of the fly's polarization-vision system. PMID:27170135

Prime-boost vaccination with heterologous live vectors encoding SIV gag and multimeric HIV-1 gp160 protein: efficacy against repeated mucosal R5 clade C SHIV challenges

PubMed Central

Lakhashe, Samir K.; Velu, Vijayakumar; Sciaranghella, Gaia; Siddappa, Nagadenahalli B.; DiPasquale, Janet M.; Hemashettar, Girish; Yoon, John K.; Rasmussen, Robert A.; Yang, Feng; Lee, Sandra J.; Montefiori, David C.; Novembre, Francis J.; Villinger, François; Amara, Rama Rao; Kahn, Maria; Hu, Shiu-Lok; Li, Sufen; Li, Zhongxia; Frankel, Fred R.; Robert-Guroff, Marjorie; Johnson, Welkin E.; Lieberman, Judy; Ruprecht, Ruth M.

2011-01-01

We sought to induce primate immunodeficiency virus-specific cellular and neutralizing antibody (nAb) responses in rhesus macaques (RM) through a bimodal vaccine approach. RM were immunized intragastrically (i.g.) with the live-attenuated Listeria monocytogenes (Lm) vector Lmdd-BdopSIVgag encoding SIVmac239 gag. SIV Gag-specific cellular responses were boosted by intranasal and intratracheal administration of replication-competent adenovirus (Ad5hr-SIVgag) encoding the same gag. To broaden antiviral immunity, the RM were immunized with multimeric HIV clade C (HIV-C) gp160 and HIV Tat. SIV Gag-specific cellular immune responses and HIV-1 nAb developed in some RM. The animals were challenged intrarectally with five low doses of R5 SHIV-1157ipEL-p, encoding a heterologous HIV-C Env (22.1% divergent to the Env immunogen). All five controls became viremic. One out of ten vaccinees was completely protected and another had low peak viremia. Sera from the completely and partially protected RM neutralized the challenge virus >90%; these RM also had strong SIV Gag-specific proliferation of CD8+ T cells. Peak and area under the curve of plasma viremia (during acute phase) among vaccinees was lower than for controls, but did not attain significance. The completely protected RM showed persistently low numbers of the α4β7-expressing CD4+ T cells; the latter have been implicated as preferential virus targets in-vivo. Thus, vaccine-induced immune responses and relatively lower numbers of potential target cells were associated with protection. PMID:21693155

Prime-boost vaccination with heterologous live vectors encoding SIV gag and multimeric HIV-1 gp160 protein: efficacy against repeated mucosal R5 clade C SHIV challenges.

PubMed

Lakhashe, Samir K; Velu, Vijayakumar; Sciaranghella, Gaia; Siddappa, Nagadenahalli B; Dipasquale, Janet M; Hemashettar, Girish; Yoon, John K; Rasmussen, Robert A; Yang, Feng; Lee, Sandra J; Montefiori, David C; Novembre, Francis J; Villinger, François; Amara, Rama Rao; Kahn, Maria; Hu, Shiu-Lok; Li, Sufen; Li, Zhongxia; Frankel, Fred R; Robert-Guroff, Marjorie; Johnson, Welkin E; Lieberman, Judy; Ruprecht, Ruth M

2011-08-05

We sought to induce primate immunodeficiency virus-specific cellular and neutralizing antibody (nAb) responses in rhesus macaques (RM) through a bimodal vaccine approach. RM were immunized intragastrically (i.g.) with the live-attenuated Listeria monocytogenes (Lm) vector Lmdd-BdopSIVgag encoding SIVmac239 gag. SIV Gag-specific cellular responses were boosted by intranasal and intratracheal administration of replication-competent adenovirus (Ad5hr-SIVgag) encoding the same gag. To broaden antiviral immunity, the RM were immunized with multimeric HIV clade C (HIV-C) gp160 and HIV Tat. SIV Gag-specific cellular immune responses and HIV-1 nAb developed in some RM. The animals were challenged intrarectally with five low doses of R5 SHIV-1157ipEL-p, encoding a heterologous HIV-C Env (22.1% divergent to the Env immunogen). All five controls became viremic. One out of ten vaccinees was completely protected and another had low peak viremia. Sera from the completely and partially protected RM neutralized the challenge virus > 90%; these RM also had strong SIV Gag-specific proliferation of CD8⁺ T cells. Peak and area under the curve of plasma viremia (during acute phase) among vaccinees was lower than for controls, but did not attain significance. The completely protected RM showed persistently low numbers of the α4β7-expressing CD4⁺ T cells; the latter have been implicated as preferential virus targets in vivo. Thus, vaccine-induced immune responses and relatively lower numbers of potential target cells were associated with protection. Copyright © 2011 Elsevier Ltd. All rights reserved.

Diminished pheromone-induced sexual behavior in neurokinin-1 receptor deficient (TACR1(-/-)) mice.

PubMed

Berger, A; Tran, A H; Dida, J; Minkin, S; Gerard, N P; Yeomans, J; Paige, C J

2012-07-01

Studies in mice with targeted deletions of tachykinin genes suggest that tachykinins and their receptors influence emotional behaviors such as aggression, depression and anxiety. Here, we investigated whether TAC1- and TAC4-encoded peptides (substance P and hemokinin-1, respectively) and the neurokinin-1 receptor (NK-1R) are involved in the modulation of sexual behaviors. Male mice deficient for the NK-1R (TACR1 (-/-)) exhibited decreased exploration of female urine in contrast to C57BL/6 control mice and mice deficient for NK-1R ligands such as TAC1 (-/-), TAC4 (-/-) and the newly generated TAC1 (-/-) /TAC4 (-/-) mice. In comparison to C57BL/6 mice, mounting frequency and duration were decreased in male TACR1 (-/-) mice, while mounting latency was increased. Decreased preference for sexual pheromones was also seen in female TACR1 (-/-) mice. Furthermore, administration of the NK-1R-antagonist L-703,606 decreased investigation of female urine by male C57BL/6 mice, suggesting an involvement of NK-1R in urine sniffing behavior. Our results provide evidence for the NK-1R in facilitating sexual approach behavior, as male TACR1 (-/-) mice exhibited blunted approach behavior toward females following the initial interaction compared with C57BL/6 mice. NK-1R signaling may therefore play an important role in pheromone-induced sexual behavior. © 2012 The Authors. Genes, Brain and Behavior © 2012 Blackwell Publishing Ltd and International Behavioural and Neural Genetics Society.

Calibration of a dual-PTZ camera system for stereo vision

NASA Astrophysics Data System (ADS)

Chang, Yau-Zen; Hou, Jung-Fu; Tsao, Yi Hsiang; Lee, Shih-Tseng

2010-08-01

In this paper, we propose a calibration process for the intrinsic and extrinsic parameters of dual-PTZ camera systems. The calibration is based on a complete definition of six coordinate systems fixed at the image planes, and the pan and tilt rotation axes of the cameras. Misalignments between estimated and ideal coordinates of image corners are formed into cost values to be solved by the Nelder-Mead simplex optimization method. Experimental results show that the system is able to obtain 3D coordinates of objects with a consistent accuracy of 1 mm when the distance between the dual-PTZ camera set and the objects are from 0.9 to 1.1 meters.

Hydrothermal syntheses and anion-induced structural transformation of three Cadmium phosphonates

NASA Astrophysics Data System (ADS)

Hu, Han; Zhai, Fupeng; Liu, Xiaofeng; Ling, Yun; Chen, Zhenxia; Zhou, Yaming

2018-05-01

Three cadmium phosphonate coordinated polymers, namely as [Cd5(ptz)3(SO4)2(5H2O)]·6H2O (Cdptz-1), [Cd3(ptz)2(Cl)2(4H2O)]·2H2O (Cdptz-2) and [Cd4(ptz)2(SO4)(Cl)(OH)H2O]·H2O (Cdptz-3) have been hydrothermally synthesized using 4-(1,2,4-triazol-4-yl)phenylphosphonic acid (H2ptz) as ligand. Single crystal X-ray analyses revealed Cdptz-2 as layered structure and Cdptz-1,3 as pillar-layered structures with Cl- or SO42- as bridging anions. Due to the weak bonding between metal and anions, Cdptz-1 and 2 can reversibly convert into each other by simple immersing in the corresponding solution at room temperature. While the transformations between Cdptz-1,2 and Cdptz-3 can only happen under hydrothermal condition. The causes for the transformation involve the metal-ligand bond breaking/formation, replacement of anions and enhancement/decrement of the network dimensionality.

Naringin ameliorates pentylenetetrazol-induced seizures and associated oxidative stress, inflammation, and cognitive impairment in rats: possible mechanisms of neuroprotection.

PubMed

Golechha, Mahaveer; Sarangal, Vikas; Bhatia, Jagriti; Chaudhry, Uma; Saluja, Daman; Arya, Dharmveer Singh

2014-12-01

Oxidative stress and cognitive impairment are associated with PTZ-induced convulsions. Naringin is a bioflavonoid present in the grapefruit. It is a potent antioxidant, and we evaluated its effect on PTZ-induced convulsions. Rats were pretreated with normal saline, naringin (20, 40, and 80 mg/kg, i.p.), or diazepam (5mg/kg, i.p.) 30 min prior to the administration of PTZ. The administration of PTZ induced myoclonic jerks and generalized tonic-clonic seizures (GTSs). We observed that naringin significantly prolonged the induction of myoclonic jerks dose-dependently. Naringin (80 mg/kg, i.p.) pretreatment protected all rats, and this protective effect was annulled by the GABAA receptor antagonist, flumazenil. In addition, naringin reduced brain MDA and TNF-α levels and conserved GSH. The pretreatment also enhanced the performance of rats in the passive avoidance task. Our observations highlight the antioxidant, antiinflammatory, and anticonvulsant potential of naringin. Also, naringin modulates the GABAA receptor to produce anticonvulsant effects and to ameliorate cognitive impairment. Copyright © 2014 Elsevier Inc. All rights reserved.

vGLUT2 heterozygous mice show more susceptibility to clonic seizures induced by pentylenetetrazol.

PubMed

Schallier, Anneleen; Massie, Ann; Loyens, Ellen; Moechars, Diederik; Drinkenburg, Wilhelmus; Michotte, Yvette; Smolders, Ilse

2009-01-01

Glutamate, the most abundant excitatory neurotransmitter in the central nervous system, is well known to be implicated in epileptic seizures. Therefore, impairments in glutamate transport could have an involvement in the mechanism of epileptogenesis. The uptake of glutamate into synaptic vesicles is mediated by vesicular glutamate transporters (vGLUTs). There are three known vGLUT isoforms, vGLUT1-3. In this study, we are particularly interested in the vGLUT2 isoform. We investigated the possible role of vGLUT2 in pentylenetetrazol (PTZ)-induced seizure generation. Seizure threshold of PTZ was compared in vGLUT2 heterozygous knock out (HET) and wild type (WT) mice. In comparison with their WT littermates a lower dose of PTZ was needed in the vGLUT2 HET mice until the onset of the first myoclonic jerk. The threshold for PTZ-induced clonic seizure activity was also lower in the vGLUT2 HET mice. These results indicate, for the first time, that vGLUT2 is likely involved in the epileptogenesis of generalized seizures.

β-Glucuronidase as a Sensitive and Versatile Reporter in Actinomycetes ▿

PubMed Central

Myronovskyi, Maksym; Welle, Elisabeth; Fedorenko, Viktor; Luzhetskyy, Andriy

2011-01-01

Here we describe a versatile and sensitive reporter system for actinomycetes that is based on gusA, which encodes the β-glucuronidase enzyme. A series of gusA-containing transcriptional and translational fusion vectors were constructed and utilized to study the regulatory cascade of the phenalinolactone biosynthetic gene cluster. Furthermore, these vectors were used to study the efficiency of translation initiation at the ATG, GTG, TTG, and CTG start codons. Surprisingly, constructs using a TTG start codon showed the best activity, whereas those using ATG or GTG were approximately one-half or one-third as active, respectively. The CTG fusion showed only 5% of the activity of the TTG fusion. A suicide vector, pKGLP2, carrying gusA in its backbone was used to visually detect merodiploid formation and resolution, making gene targeting in actinomycetes much faster and easier. Three regulatory genes, plaR1, plaR2, and plaR3, involved in phenalinolactone biosynthesis were efficiently replaced with an apramycin resistance marker using this system. Finally, we expanded the genetic code of actinomycetes by introducing the nonproteinogenic amino acid N-epsilon-cyclopentyloxycarbonyl-l-lysine with the GusA protein as a reporter. PMID:21685164

Effects of bee products on pentylenetetrazole-induced seizures in the rat.

PubMed

Zárraga-Galindo, N; Vergara-Aragón, P; Rosales-Meléndez, S; Ibarra-Guerrero, P; Domínguez-Marrufo, L E; Oviedo-García, R E; Hernández-Ramírez, H; Hernández-Téllez, B; López-Martínez, I E; Sánchez-Cervantes, I; Vázquez-García, M; Santiago, J

2011-01-01

Bee products (BP) have been used for centuries as a diet complement with claimed curative properties. The aim of this study was to determine whether oral administration of BP prevented behavioral, histological, and biochemical alterations, caused by pentylenetetrazole (PTZ)-induced kindling in rats. Male Wistar rats were employed to evaluate seizure latency, number and duration, performance in the open field test, histological alterations and mortality following BP administration. Oral administration of BP at two doses, 30 and 60 mg/kg/day, significantly lengthened latency of both clonic and tonic PTZ-induced seizures, decreased the duration and frequency of seizures and reduced mortality. In the Open Field test, BP treated groups showed increases in the number of crossed squares and rearing counts, and on optimal dose, decreases in fecal boli. Histological analysis showed in PTZ (50 and 80 mg/kg) kindling rats, lungs with inflammatory peribronchiolar, and perialveolar infiltrates. In the liver, mild losses of trabeculae, multi-vesiculated hepatocytes (steatosis) and inflammatory infiltrates in hepatic parenchyma were observed. Interestingly, in the heart, fibers were markedly separated. In testis, stratified epithelium of seminal tubules lost its normal structure, tubules had epithelium loss, spermatids were absent, and spermatogonia and Leydig cells diminished. In PTZ kindling rats treated with BP, the lungs had no inflammatory infiltrates, although the heart showed some inflammatory infiltrates. Remaining structures had normal characteristics. These results, suggest that BP can protect rats from effects of PTZ-induced kindling.

The effects of different fractions of Coriandrum sativum on pentylenetetrazole-induced seizures and brain tissues oxidative damage in rats.

PubMed

Anaeigoudari, Akbar; Hosseini, Mahmoud; Karami, Reza; Vafaee, Farzaneh; Mohammadpour, Toktam; Ghorbani, Ahmad; Sadeghnia, Hamid Reza

2016-01-01

In the present work, the effects of different fractions of Coriandrum sativum (C. sativum), on pentylenetetrazole (PTZ)-induced seizures and brain tissues oxidative damage were investigated in rats. The rats were divided into the following groups: (1) vehicle, (2) PTZ (90 mg/kg), (3) water fraction (WF) of C. sativum (25 and 100 mg/kg), (4) n-butanol fraction (NBF) of C. sativum (25 and 100 mg/kg), and (5) ethyl acetate fraction (EAF) of C. sativum (25 and 100 mg/kg). The first generalized tonic-clonic seizures (GTCS) latency in groups treated with 100 mg /kg of WF or EAF was significantly higher than that of PTZ group (p<0.01). In contrast to WF, the EAF and NBF were not effective in increasing the first minimal clonic seizure (MCS) latency. Malondialdehyde (MDA) levels in both cortical and hippocampal tissues of PTZ group were significantly higher than those of control animals (p<0.001). Pretreatment with WF, NBF, or EAF resulted in a significant reduction in the MDA levels of hippocampi (p<0.01 - p<0.001). Following PTZ administration, a significant reduction in total thiol groups was observed in the brain tissues (p<0.05). Pretreatment with WF and NBF significantly elevated thiol concentrations in cortical and hippocampal tissues, respectively (p<0.05). The present study showed that different fractions of C. sativum possess antioxidant activity in the brain and WF and EAF of this plant have anticonvulsant effects.

Non-Additive Voltametric Currents From a Mixture of Two, Three and Four Redox-Active Compounds and Electroanalytical Implications

NASA Technical Reports Server (NTRS)

Dass, Amala; Oh, Woon Su; Gao, Xue-Rong; Rawashdeh, Abdel M.; Leventis, Nicholas

2004-01-01

We have published recently the effect of dissimilar diffusion coefficients on the size of the voltammetric waves from a mixture of two redox-active compounds. Similarly, at the potential range where three redox-active species, decamethylferrocene (dMeFc), ferrocene (Fc) and N-methylphenothiazine (MePTZ), are oxidized simultaneously with rates controlled by linear diffusion, electrogenerated radicals diffusing outwards from the electrode react with the original species diffusing towards the electrode from the bulk; thus, Fc(+) reacts with dMeFc producing Fc and dMeFc(+), while MePTZ(+) reacts both with dMeFc producing MePTZ and dMeFc(+), and with Fc producing MePTZ and Fc(+). These reactions replace dMeFc with Fc at the second plateau, and both dMeFc and Fc with MePTZ at the third plateau. Since the diffusion coefficients of the three species are not equal, the mass-transfer limited currents of the second and the third oxidation wave plateaus change by approx. 10%. Numerical simulations of the experimental voltamograms support this mechanism. Similar results were also obtained for a mixture of four redoxactive compounds. The implications of this non-additive nature of currents on: (a) the use of internal voltammetric standards for quantitative analysis of a mixture of redox-active compounds; and, (b) the half wave potentials (E1/2) of the 2nd, 3rd and 4th waves for qualitative analysis, will be discussed.

Curcumin-loaded chitosan-alginate-STPP nanoparticles ameliorate memory deficits and reduce glial activation in pentylenetetrazol-induced kindling model of epilepsy.

PubMed

Hashemian, Mona; Anissian, Diana; Ghasemi-Kasman, Maryam; Akbari, Atefeh; Khalili-Fomeshi, Mohsen; Ghasemi, Shahram; Ahmadi, Fatemeh; Moghadamnia, Ali Akbar; Ebrahimpour, Anahita

2017-10-03

Despite several beneficial effects of curcumin, its medical application has been hampered due to low water solubility. To improve the aqueous solubility of curcumin, it has been loaded on chitosan (CS)-alginate (ALG) - sodium tripolyphosphate (STPP) nanoparticles (NPs). Then, the effect of curcumin NPs on memory improvement and glial activation was investigated in pentylenetetrazol (PTZ)-induced kindling model. Male NMRI mice have received the daily injection of curcumin NPs at dose of 12.5 or 25mg/kg. All interventions were injected intraperitoneally (i.p), 10days before PTZ administration and the injections were continued until 1h before each PTZ injection. Spatial learning and memory was evaluated using Morris water maze test after the 7th PTZ injection. Animals have received 10 injections of PTZ and then, brain tissues were removed for histological evaluation. Nissl staining was used to determine the level of cell death in hippocampus and immunostaining method was performed against NeuN and GFAP/Iba1 for assessment of neuronal density and glial activation respectively. Behavioral results showed that curcumin NPs exhibit anticonvulsant activity and prevent cognitive impairment in fully kindled animals. The level of cell death and glial activation reduced in animals which have received curcumin NPs compared to those received free curcumin. To conclude, these findings suggest that curcumin NPs effectively ameliorate memory impairment and attenuate the level of activated glial cells in a mice model of chronic epilepsy. Copyright © 2017. Published by Elsevier Inc.

Randomized controlled trial of piperacillin-tazobactam, cefepime and ertapenem for the treatment of urinary tract infection caused by extended-spectrum beta-lactamase-producing Escherichia coli.

PubMed

Seo, Yu Bin; Lee, Jacob; Kim, Young Keun; Lee, Seung Soon; Lee, Jeong-A; Kim, Hyo Youl; Uh, Young; Kim, Han-Sung; Song, Wonkeun

2017-06-07

Due to limited therapeutic options, the spread of extended-spectrum beta-lactamases (ESBLs) have become a major public health concern. We conducted a prospective, randomized, open-label comparison of the therapeutic efficacy of piperacillin-tazobactam (PTZ), cefepime, and ertapenem in febrile nosocomial urinary tract infection with ESBL-producing Escherichia coli (ESBL-EC). This study was conducted at three university hospitals between January 2013 and August 2015. Hospitalized adult patients presenting with fever were screened for healthcare-associated urinary tract infection (HA-UTI). When ESBL-EC was solely detected and susceptible to a randomized antibiotic in vitro, the case was included in the final analysis. Participants were treated for 10-14 days with PTZ, cefepime, or ertapenem. A total of 66 participants were evenly assigned to the PTZ and ertapenem treatment groups. After the recruitment of six participants, assignment to the cefepime treatment group was stopped because of an unexpectedly high treatment failure rate. The baseline characteristics of these participants did not differ from participants in other treatment groups. The clinical and microbiological response to PTZ treatment was estimated to be 94% and was similar to the response to ertapenem treatment. The efficacy of cefepime was 33.3%. In the cefepime group, age, Charlson comorbidity index, genotype, and minimal inhibitory concentration (MIC) did not significantly affect the success of treatment. Similarly, genotype seemed to be irrelevant with respect to clinical outcome in the PTZ group. Expired cases tended to involve septic shock with a high Charlson comorbidity index and high MIC. Results from this study suggest that PTZ is effective in the treatment of urinary tract infection caused by ESBL-EC when the in vitro test indicates susceptibility. In addition, cefepime should not be used as an alternative treatment for urinary tract infection caused by ESBL-EC. The trial was registered with the Clinical Research Information Service of Korea Centers for Disease Control and Prevention. (KCT0001895).

"Triplet" polycistronic vectors encoding Gata4, Mef2c, and Tbx5 enhances postinfarct ventricular functional improvement compared with singlet vectors.

PubMed

Mathison, Megumi; Singh, Vivek P; Gersch, Robert P; Ramirez, Maricela O; Cooney, Austin; Kaminsky, Stephen M; Chiuchiolo, Maria J; Nasser, Ahmed; Yang, Jianchang; Crystal, Ronald G; Rosengart, Todd K

2014-10-01

The in situ reprogramming of cardiac fibroblasts into induced cardiomyocytes by the administration of gene transfer vectors encoding Gata4 (G), Mef2c (M), and Tbx5 (T) has been shown to improve ventricular function in myocardial infarction models. The efficacy of this strategy could, however, be limited by the need for fibroblast targets to be infected 3 times--once by each of the 3 transgene vectors. We hypothesized that a polycistronic "triplet" vector encoding all 3 transgenes would enhance postinfarct ventricular function compared with use of "singlet" vectors. After validation of the polycistronic vector expression in vitro, adult male Fischer 344 rats (n=6) underwent coronary ligation with or without intramyocardial administration of an adenovirus encoding all 3 major vascular endothelial growth factor (VEGF) isoforms (AdVEGF-All6A positive), followed 3 weeks later by the administration to AdVEGF-All6A-positive treated rats of singlet lentivirus encoding G, M, or T (1×10(5) transducing units each) or the same total dose of a GMT "triplet" lentivirus vector. Western blots demonstrated that triplet and singlet vectors yielded equivalent GMT transgene expression, and fluorescence activated cell sorting demonstrated that triplet vectors were nearly twice as potent as singlet vectors in generating induced cardiomyocytes from cardiac fibroblasts. Echocardiography demonstrated that GMT triplet vectors were more effective than the 3 combined singlet vectors in enhancing ventricular function from postinfarct baselines (triplet, 37%±10%; singlet, 13%±7%; negative control, 9%±5%; P<.05). These data have confirmed that the in situ administration of G, M, and T induces postinfarct ventricular functional improvement and that GMT polycistronic vectors enhance the efficacy of this strategy. Copyright © 2014 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.

The restriction-modification genes of Escherichia coli K-12 may not be selfish: they do not resist loss and are readily replaced by alleles conferring different specificities.

PubMed

O'Neill, M; Chen, A; Murray, N E

1997-12-23

Type II restriction and modification (R-M) genes have been described as selfish because they have been shown to impose selection for the maintenance of the plasmid that encodes them. In our experiments, the type I R-M system EcoKI does not behave in the same way. The genes specifying EcoKI are, however, normally residents of the chromosome and therefore our analyses were extended to monitor the deletion of chromosomal genes rather than loss of plasmid vector. If EcoKI were to behave in the same way as the plasmid-encoded type II R-M systems, the loss of the relevant chromosomal genes by mutation or recombination should lead to cell death because the cell would become deficient in modification enzyme and the bacterial chromosome would be vulnerable to the restriction endonuclease. Our data contradict this prediction; they reveal that functional type I R-M genes in the chromosome are readily replaced by mutant alleles and by alleles encoding a type I R-M system of different specificity. The acquisition of allelic genes conferring a new sequence specificity, but not the loss of the resident genes, is dependent on the product of an unlinked gene, one predicted [Prakash-Cheng, A., Chung, S. S. & Ryu, J. (1993) Mol. Gen. Genet. 241, 491-496] to be relevant to control of expression of the genes that encode EcoKI. Our evidence suggests that not all R-M systems are evolving as "selfish" units; rather, the diversity and distribution of the family of type I enzymes we have investigated require an alternative selective pressure.

Viral Vector-Based Targeting of miR-21 in Cardiac Nonmyocyte Cells Reduces Pathologic Remodeling of the Heart

PubMed Central

Ramanujam, Deepak; Sassi, Yassine; Laggerbauer, Bernhard; Engelhardt, Stefan

2016-01-01

Systemic inhibition of miR-21 has proven effective against myocardial fibrosis and dysfunction, while studies in cardiac myocytes suggested a protective role in this cell type. Considering potential implications for therapy, we aimed to determine the cell fraction where miR-21 exerts its pathological activity. We developed a viral vector-based strategy for gene targeting of nonmyocyte cardiac cells in vivo and compared global to cardiac myocyte-specific and nonmyocyte-specific deletion of miR-21 in chronic left ventricular pressure overload. Murine moloney virus and serotype 9 of adeno-associated virus were engineered to encode improved Cre recombinase for genetic deletion in miR-21fl/fl mice. Pericardial injection of murine moloney virus-improved Cre recombinase to neonates achieved highly selective genetic ablation of miR-21 in nonmyocyte cardiac cells, identified as cardiac fibroblasts and endothelial cells. Upon left ventricular pressure overload, cardiac function was only preserved in mice with miR-21 deficiency in nonmyocyte cardiac cells, but not in mice with global or cardiac myocyte-specific ablation. Our data demonstrate that miR-21 exerts its pathologic activity directly in cardiac nonmyocytes and encourage further development of antimiR-21 therapy toward cellular tropism. PMID:27545313

Decoding the Ubiquitin-Mediated Pathway of Arthropod Disease Vectors

PubMed Central

Choy, Anthony; Severo, Maiara S.; Sun, Ruobai; Girke, Thomas; Gillespie, Joseph J.; Pedra, Joao H. F.

2013-01-01

Protein regulation by ubiquitin has been extensively described in model organisms. However, characterization of the ubiquitin machinery in disease vectors remains mostly unknown. This fundamental gap in knowledge presents a concern because new therapeutics are needed to control vector-borne diseases, and targeting the ubiquitin machinery as a means for disease intervention has been already adopted in the clinic. In this study, we employed a bioinformatics approach to uncover the ubiquitin-mediated pathway in the genomes of Anopheles gambiae, Aedes aegypti, Culex quinquefasciatus, Ixodes scapularis, Pediculus humanus and Rhodnius prolixus. We observed that (1) disease vectors encode a lower percentage of ubiquitin-related genes when compared to Drosophila melanogaster, Mus musculus and Homo sapiens but not Saccharomyces cerevisiae; (2) overall, there are more proteins categorized as E3 ubiquitin ligases when compared to E2-conjugating or E1-activating enzymes; (3) the ubiquitin machinery within the three mosquito genomes is highly similar; (4) ubiquitin genes are more than doubled in the Chagas disease vector (R. prolixus) when compared to other arthropod vectors; (5) the deer tick I. scapularis and the body louse (P. humanus) genomes carry low numbers of E1-activating enzymes and HECT-type E3 ubiquitin ligases; (6) R. prolixus have low numbers of RING-type E3 ubiquitin ligases; and (7) C. quinquefasciatus present elevated numbers of predicted F-box E3 ubiquitin ligases, JAB and UCH deubiquitinases. Taken together, these findings provide novel opportunities to study the interaction between a pathogen and an arthropod vector. PMID:24205097

Rational plasmid design and bioprocess optimization to enhance recombinant adeno-associated virus (AAV) productivity in mammalian cells.

PubMed

Emmerling, Verena V; Pegel, Antje; Milian, Ernest G; Venereo-Sanchez, Alina; Kunz, Marion; Wegele, Jessica; Kamen, Amine A; Kochanek, Stefan; Hoerer, Markus

2016-02-01

Viral vectors used for gene and oncolytic therapy belong to the most promising biological products for future therapeutics. Clinical success of recombinant adeno-associated virus (rAAV) based therapies raises considerable demand for viral vectors, which cannot be met by current manufacturing strategies. Addressing existing bottlenecks, we improved a plasmid system termed rep/cap split packaging and designed a minimal plasmid encoding adenoviral helper function. Plasmid modifications led to a 12-fold increase in rAAV vector titers compared to the widely used pDG standard system. Evaluation of different production approaches revealed superiority of processes based on anchorage- and serum-dependent HEK293T cells, exhibiting about 15-fold higher specific and volumetric productivity compared to well-established suspension cells cultivated in serum-free medium. As for most other viral vectors, classical stirred-tank bioreactor production is thus still not capable of providing drug product of sufficient amount. We show that manufacturing strategies employing classical surface-providing culture systems can be successfully transferred to the new fully-controlled, single-use bioreactor system Integrity(TM) iCELLis(TM) . In summary, we demonstrate substantial bioprocess optimizations leading to more efficient and scalable production processes suggesting a promising way for flexible large-scale rAAV manufacturing. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Recombinant vaccines against T. gondii: comparison between homologous and heterologous vaccination protocols using two viral vectors expressing SAG1.

PubMed

Mendes, Érica Araújo; Fonseca, Flavio G; Casério, Bárbara M; Colina, Janaína P; Gazzinelli, Ricardo Tostes; Caetano, Braulia C

2013-01-01

The use of recombinant viral vectors expressing T. gondii antigens is a safe and efficient approach to induce immune response against the parasite and a valuable tool for vaccine development. We have previously protected mice from toxoplasmosis by immunizing the animals with an adenovirus expressing the protein SAG1 (AdSAG1) of T. gondii. We are now looking for ways to improve the vaccination strategy and enhance protection. One limitation of homologous vaccinations (sequential doses of the same vector) is induction of anti-vector immune response that blocks cell transduction, restricts transgene expression and, consequently, compromises the overall outcome of vaccination. One way to avert the effects of anti-vector response is to use different viruses in prime and boost (heterologous vaccination). Bearing this in mind, we generated a modified Vaccinia Virus Ankara encoding SAG1 (MVASAG1), to be tested as boost agent after prime with AdSAG1. Although minor differences were observed in the magnitude of the anti-SAG1 immune response induced by each vaccination protocol, the heterologous immunization with AdSAG1 followed by MVASAG1 resulted in improved capacity to control brain cyst formation in a model of chronic toxoplasmosis in C57BL/6 mice.

A Geometric Framework for the Kinematics of Crystals With Defects

DTIC Science & Technology

2006-02-01

which parallel transport preserves dot products of vectors, i.e. r G G ¼ 0. It is called the Levi - Civita connection [57] or the Riemannian connection...yielding a null covariant derivative of the metric tensor is called a metric connection. The Levi – Civita connection of (8) is metric. Note that in...tensor formed by inserting the Levi – Civita con- nection (8) into (10). A geometric space B0 with metric G having R G ¼ 0 is called flat. One may show

Isolation of a novel plasmid from Couchioplanes caeruleus and construction of two plasmid vectors for gene expression in Actinoplanes missouriensis.

PubMed

Jang, Moon-Sun; Fujita, Azusa; Ikawa, Satomi; Hanawa, Keitaro; Yamamura, Hideki; Tamura, Tomohiko; Hayakawa, Masayuki; Tezuka, Takeaki; Ohnishi, Yasuo

2015-01-01

To date, no plasmid vector has been developed for the rare actinomycete Actinoplanes missouriensis. Moreover, no small circular plasmid has been reported to exist in the genus Actinoplanes. Here, a novel plasmid, designated pCAZ1, was isolated from Couchioplanes caeruleus subsp. azureus via screening for small circular plasmids in Actinoplanes (57 strains) and Couchioplanes (2 strains). Nucleotide sequencing revealed that pCAZ1 is a 5845-bp circular molecule with a G + C content of 67.5%. The pCAZ1 copy number was estimated at 30 per chromosome. pCAZ1 contains seven putative open reading frames, one of which encodes a protein containing three motifs conserved among plasmid-encoded replication proteins that are involved in the rolling-circle mechanism of replication. Detection of single-stranded DNA intermediates in C. caeruleus confirmed that pCAZ1 replicates by this mechanism. The ColE1 origin from pBluescript SK(+) and the oriT sequence with the apramycin resistance gene aac(3)IV from pIJ773 were inserted together into pCAZ1, to construct the Escherichia coli-A. missouriensis shuttle vectors, pCAM1 and pCAM2, in which the foreign DNA fragment was inserted into pCAZ1 in opposite directions. pCAM1 and pCAM2 were successfully transferred to A. missouriensis through the E. coli-mediated conjugative transfer system. The copy numbers of pCAM1 and pCAM2 in A. missouriensis were estimated to be one and four per chromosome, respectively. Thus, these vectors can be used as effective genetic tools for homologous and heterologous gene expression studies in A. missouriensis. Copyright © 2014 Elsevier Inc. All rights reserved.

Herpes simplex virus vector-mediated gene delivery of glutamic acid decarboxylase reduces detrusor overactivity in spinal cord injured rats

PubMed Central

Miyazato, Minoru; Sugaya, Kimio; Goins, William F.; Goss, James R.; Chancellor, Michael B.; de Groat, William C.; Glorioso, Joseph C.; Yoshimura, Naoki

2010-01-01

We examined whether replication-defective herpes simplex virus (HSV) vectors encoding the 67 Kd form of the glutamic acid decarboxylase (GAD67) gene product, the gamma-aminobutyric acid (GABA) synthesis enzyme, can suppress detrusor overactivity (DO) in spinal cord injury (SCI) rats. One week after spinalization, HSV vectors expressing GAD and green fluorescent protein (GFP) (HSV-GAD) were injected into the bladder wall. SCI rats without HSV injection (HSV-untreated) and those injected with lacZ-encoding reporter gene HSV vectors (HSV-LacZ) were used as controls. Three weeks after viral injection, continuous cystometry was performed under awake conditions in all three groups. In the HSV-GAD group, the number and amplitude of non-voiding contractions (NVCs) were significantly decreased (40–45% and 38–40%, respectively) along with an increase in voiding efficiency, compared with HSV-untreated and HSV-LacZ groups, but micturition pressure was not different among the three groups. Intrathecal application of bicuculline partly reversed the decreased number and amplitude of NVCs, and decreased voiding efficiency in the HSV-GAD group. In the HSV-GAD group, GAD67 mRNA and protein levels were significantly increased in L6-S1 dorsal root ganglia (DRG) compared with the HSV-LacZ group while 57% of DRG cells were GFP-positive, and these neurons showed increased GAD67-like immunoreactivity compared with the HSV-LacZ group. These results indicate that GAD gene therapy effectively suppresses DO following SCI predominantly via activation of spinal GABAA receptors. Thus, HSV-based GAD gene transfer to bladder afferent pathways may represent a novel approach for the treatment of neurogenic DO. PMID:19225548

Reduced estradiol synthesis by letrozole, an aromatase inhibitor, is protective against development of pentylenetetrazole-induced kindling in mice.

PubMed

Rashid, Davood; Panda, B P; Vohora, Divya

2015-11-01

Neurosteroids, such as testosterone and their metabolites, are known to modulate neuronal excitability. The enzymes regulating the metabolism of these neurosteroids, thus, may be targeted as a noval strategy for the development of new antiepileptic drugs. The present work targeted two such enzymes i,e aromatase and 5α-reductase in order to explore the potential of letrozole (an aromatase inhibitor) on pentylenetetrazole (PTZ)-induced kindling in mice and the ability of finasteride (a 5α-reductase inhibitor) to modulate any such effects. PTZ (30 mg/kg, i.p.), when administered once every two days (for a total of 24 doses) induced kindling in Swiss albino mice. Letrozole (1 mg/kg, p.o.), administered prior to PTZ, significantly reduced the % incidence of kindling, delayed mean onset time of seizures and reduced seizure severity score. Letrozole reduced the levels of plasma 17β-estradiol after induction of kindling. The concurrent administration of finasteride and letrozole produced effects similar to letrozole on PTZ-kindling and on estradiol levels. This implies that the ability of letrozole to redirect the synthesis of dihydrotestosterone (DHT) and 5α-androstanediol from testosterone doesn't appear to play a significant role in the protective effects of letrozole against PTZ kindling. Letrozole, however, increased the levels of 5α-DHT in mice plasma. The aromatase inhibitors, thus, may be exploited for inhibiting the synthesis of proconvulsant (17β-estradiol) and/or redirecting the synthesis of anticonvulsant (DHT and 5α-androstanediol) neurosteroids. Copyright © 2015 Elsevier Ltd. All rights reserved.

Reversal of pentylenetetrazole-altered swimming and neural activity-regulated gene expression in zebrafish larvae by valproic acid and valerian extract.

PubMed

Torres-Hernández, Bianca A; Colón, Luis R; Rosa-Falero, Coral; Torrado, Aranza; Miscalichi, Nahira; Ortíz, José G; González-Sepúlveda, Lorena; Pérez-Ríos, Naydi; Suárez-Pérez, Erick; Bradsher, John N; Behra, Martine

2016-07-01

Ethnopharmacology has documented hundreds of psychoactive plants awaiting exploitation for drug discovery. A robust and inexpensive in vivo system allowing systematic screening would be critical to exploiting this knowledge. The objective of this study was to establish a cheap and accurate screening method which can be used for testing psychoactive efficacy of complex mixtures of unknown composition, like plant crude extracts. We used automated recording of zebrafish larval swimming behavior during light vs. dark periods which we reproducibly altered with an anxiogenic compound, pentylenetetrazole (PTZ). First, we reversed this PTZ-altered swimming by co-treatment with a well-defined synthetic anxiolytic drug, valproic acid (VPA). Next, we aimed at reversing it by adding crude root extracts of Valeriana officinalis (Val) from which VPA was originally derived. Finally, we assessed how expression of neural activity-regulated genes (c-fos, npas4a, and bdnf) known to be upregulated by PTZ treatment was affected in the presence of Val. Both VPA and Val significantly reversed the PTZ-altered swimming behaviors. Noticeably, Val at higher doses was affecting swimming independently of the presence of PTZ. A strong regulation of all three neural-activity genes was observed in Val-treated larvae which fully supported the behavioral results. We demonstrated in a combined behavioral-molecular approach the strong psychoactivity of a natural extract of unknown composition made from V. officinalis. Our results highlight the efficacy and sensitivity of such an approach, therefore offering a novel in vivo screening system amenable to high-throughput testing of promising ethnobotanical candidates.

The effects of different fractions of Coriandrum sativum on pentylenetetrazole-induced seizures and brain tissues oxidative damage in rats

PubMed Central

Anaeigoudari, Akbar; Hosseini, Mahmoud; Karami, Reza; Vafaee, Farzaneh; Mohammadpour, Toktam; Ghorbani, Ahmad; Sadeghnia, Hamid Reza

2016-01-01

Objective: In the present work, the effects of different fractions of Coriandrum sativum (C. sativum), on pentylenetetrazole (PTZ)-induced seizures and brain tissues oxidative damage were investigated in rats. Materials and Methods: The rats were divided into the following groups: (1) vehicle, (2) PTZ (90 mg/kg), (3) water fraction (WF) of C. sativum (25 and 100 mg/kg), (4) n-butanol fraction (NBF) of C. sativum (25 and 100 mg/kg), and (5) ethyl acetate fraction (EAF) of C. sativum (25 and 100 mg/kg). Results: The first generalized tonic-clonic seizures (GTCS) latency in groups treated with 100 mg /kg of WF or EAF was significantly higher than that of PTZ group (p<0.01). In contrast to WF, the EAF and NBF were not effective in increasing the first minimal clonic seizure (MCS) latency. Malondialdehyde (MDA) levels in both cortical and hippocampal tissues of PTZ group were significantly higher than those of control animals (p<0.001). Pretreatment with WF, NBF, or EAF resulted in a significant reduction in the MDA levels of hippocampi (p<0.01 - p<0.001). Following PTZ administration, a significant reduction in total thiol groups was observed in the brain tissues (p<0.05). Pretreatment with WF and NBF significantly elevated thiol concentrations in cortical and hippocampal tissues, respectively (p<0.05). Conclusion: The present study showed that different fractions of C. sativum possess antioxidant activity in the brain and WF and EAF of this plant have anticonvulsant effects. PMID:27222836

Does tachyphylaxis occur in long-term management of scalp seborrheic dermatitis with pyrithione zinc-based treatments?

PubMed

Schwartz, James R; Rocchetta, Heather; Asawanonda, Pravit; Luo, Fangyi; Thomas, Jennifer H

2009-01-01

Scalp seborrheic dermatitis and dandruff (SD/D) are chronic conditions requiring long-term treatment. There is a common belief that patients frequently experience decreasing benefits over time when using a single product. This physiologic accommodation is termed tachyphylaxis. To systematically investigate the anecdotal belief that tachyphylaxis occurs in long-term treatment of SD/D using quantitative clinical assessments. An international questionnaire completed by 722 dermatologists assessed the belief of tachyphylaxis incidence with pyrithione zinc (PTZ)-based shampoos, time course, occurrence relative to active ingredients, and effect of switching products. Two double-blind, randomized, clinical evaluations were conducted, 24- and 48-week studies, whereby a 1% PTZ shampoo, a 2% PTZ shampoo, or a matched placebo control shampoo was used by each subject for the duration of the study. Dermatologists assessed the adherent scalp flaking (scale of 0-10) at baseline and at specified intervals. Sixty-four per cent of responding dermatologists believed tachyphylaxis occurred with PTZ products, and most felt that tachyphylaxis occurred within 3 months of use. Evaluation of mean treatment responses vs. placebo and individual responses as a function of study duration showed a consistent benefit for all products at all time points; therefore, no evidence for tachyphylaxis was found (within 48 weeks of treatment). No evidence for tachyphylaxis in SD/D treatment by PTZ-based shampoos was found. Compliance could explain the decreasing response rate seen over time; the solution is to choose an affordable therapeutic product that is effective long term without cosmetic trade-offs.

Development of oral CTL vaccine using a CTP-integrated Sabin 1 poliovirus-based vector system.

PubMed

Han, Seung-Soo; Lee, Jinjoo; Jung, Yideul; Kang, Myeong-Ho; Hong, Jung-Hyub; Cha, Min-Suk; Park, Yu-Jin; Lee, Ezra; Yoon, Cheol-Hee; Bae, Yong-Soo

2015-09-11

We developed a CTL vaccine vector by modification of the RPS-Vax system, a mucosal vaccine vector derived from a poliovirus Sabin 1 strain, and generated an oral CTL vaccine against HIV-1. A DNA fragment encoding a cytoplasmic transduction peptide (CTP) was integrated into the RPS-Vax system to generate RPS-CTP, a CTL vaccine vector. An HIV-1 p24 cDNA fragment was introduced into the RPS-CTP vector system and a recombinant poliovirus (rec-PV) named vRPS-CTP/p24 was produced. vRPS-CTP/p24 was genetically stable and efficiently induced Th1 immunity and p24-specific CTLs in immunized poliovirus receptor-transgenic (PVR-Tg) mice. In challenge experiments, PVR-Tg mice that were pre-immunized orally with vRPS-CTP/p24 were resistant to challenge with a lethal dose of p24-expressing recombinant vaccinia virus (rMVA-p24). These results suggested that the RPS-CTP vector system had potential for developing oral CTL vaccines against infectious diseases. Copyright © 2015 Elsevier Ltd. All rights reserved.

A single Alal 39-to-Glu substitution in the Renibacterium salmoninarum virulence-associated protein p57 results in antigenic variation and is associated with enhanced p57 binding to Chinook salmon leukocytes

USGS Publications Warehouse

Wiens, Gregory D.; Pascho, Ron; Winton, James R.

2002-01-01

The gram-positive bacterium Renibacterium salmoninarum produces relatively large amounts of a 57-kDa protein (p57) implicated in the pathogenesis of salmonid bacterial kidney disease. Antigenic variation in p57 was identified by using monoclonal antibody 4C11, which exhibited severely decreased binding to R. salmoninarum strain 684 p57 and bound robustly to the p57 proteins of seven other R. salmoninarum strains. This difference in binding was not due to alterations in p57 synthesis, secretion, or bacterial cell association. The molecular basis of the 4C11 epitope loss was determined by amplifying and sequencing the two identical genes encoding p57, msa1 and msa2. The 5′ and coding sequences of the 684 msa1 and msa2 genes were identical to those of the ATCC 33209 msa1and msa2 genes except for a single C-to-A nucleotide mutation. This mutation was identified in both the msa1 and msa2 genes of strain 684 and resulted in an Ala139-to-Glu substitution in the amino-terminal region of p57. We examined whether this mutation in p57 altered salmonid leukocyte and rabbit erythrocyte binding activities. R. salmoninarum strain 684 extracellular protein exhibited a twofold increase in agglutinating activity for chinook salmon leukocytes and rabbit erythrocytes compared to the activity of the ATCC 33209 extracellular protein. A specific and quantitative p57 binding assay confirmed the increased binding activity of 684 p57. Monoclonal antibody 4C11 blocked the agglutinating activity of the ATCC 33209 extracellular protein but not the agglutinating activity of the 684 extracellular protein. These results indicate that the Ala139-to-Glu substitution altered immune recognition and was associated with enhanced biological activity of R. salmoninarum 684 p57.

Interleukin-Encoding Adenoviral Vectors as Genetic Adjuvant for Vaccination against Retroviral Infection

PubMed Central

Ohs, Inga; Windmann, Sonja; Wildner, Oliver; Dittmer, Ulf; Bayer, Wibke

2013-01-01

Interleukins (IL) are cytokines with stimulatory and modulatory functions in the immune system. In this study, we have chosen interleukins which are involved in the enhancement of TH2 responses and B cell functions to analyze their potential to improve a prophylactic adenovirus-based anti-retroviral vaccine with regard to antibody and virus-specific CD4+ T cell responses. Mice were vaccinated with an adenoviral vector which encodes and displays the Friend Virus (FV) surface envelope protein gp70 (Ad.pIXgp70) in combination with adenoviral vectors encoding the interleukins IL4, IL5, IL6, IL7 or IL23. Co-application of Ad.pIXgp70 with Ad.IL5, Ad.IL6 or Ad.IL23 resulted in improved protection with high control over FV-induced splenomegaly and reduced viral loads. Mice co-immunized with adenoviral vectors encoding IL5 or IL23 showed increased neutralizing antibody responses while mice co-immunized with Ad.IL6 or Ad.IL23 showed improved FV-specific CD4+ T cell responses compared to mice immunized with Ad.pIXgp70 alone. We show that the co-application of adenoviral vectors encoding specific interleukins is suitable to improve the vaccination efficacy of an anti-retroviral vaccine. Improved protection correlated with improved CD4+ T cell responses and especially with higher neutralizing antibody titers. The co-application of selected interleukin-encoding adenoviral vectors is a valuable tool for vaccination with regard to enhancement of antibody mediated immunity. PMID:24349306

Novel Genes Encoding Hexadecanoic Acid Δ6-Desaturase Activity in a Rhodococcus sp.

PubMed

Araki, Hiroyuki; Hagihara, Hiroshi; Takigawa, Hirofumi; Tsujino, Yukiharu; Ozaki, Katsuya

2016-11-01

cis-6-Hexadecenoic acid, a major component of human sebaceous lipids, is involved in the defense mechanism against Staphylococcus aureus infection in healthy skin and closely related to atopic dermatitis. Previously, Koike et al. (Biosci Biotechnol Biochem 64:1064-1066, 2000) reported that a mutant strain of Rhodococcus sp. produced cis-6-hexadecenoate derivatives from palmitate alkyl esters. From the mutant Rhodococcus strain, we identified and sequenced two open reading frames present in an amplified 5.7-kb region; these open reading frames encoded tandemly repeated Δ6-desaturase-like genes, Rdes1 and Rdes2. A phylogenetic tree indicated that Rdes1 and Rdes2 were different from previously known Δ6-desaturase genes, and that they formed a new cluster. Rdes1 and Rdes2 were each introduced into vectors and then expressed separately in Escherichia coli, and the fatty acid composition of the transformed cells was analyzed by gas chromatography and mass spectrometry. The amount of cis-6-hexadecenoic acid was significantly higher in Rdes1- or Rdes2-transformed E. coli cells (twofold and threefold, respectively) than in vector-only control cells. These results showed that cis-6-hexadecenoic acid was produced in E. coli cells by the rhodococcal Δ6-desaturase-like proteins.

Paradigms for pharmacological characterization of C. elegans synaptic transmission mutants.

PubMed

Locke, Cody; Berry, Kalen; Kautu, Bwarenaba; Lee, Kyle; Caldwell, Kim; Caldwell, Guy

2008-08-18

The nematode, Caenorhabditis elegans, has become an expedient model for studying neurotransmission. C. elegans is unique among animal models, as the anatomy and connectivity of its nervous system has been determined from electron micrographs and refined by pharmacological assays. In this video, we describe how two complementary neural stimulants, an acetylcholinesterase inhibitor, called aldicarb, and a gamma-aminobutyric acid (GABA) receptor antagonist, called pentylenetetrazole (PTZ), may be employed to specifically characterize signaling at C. elegans neuromuscular junctions (NMJs) and facilitate our understanding of antagonistic neural circuits. Of 302 C. elegans neurons, nineteen GABAergic D-type motor neurons innervate body wall muscles (BWMs), while four GABAergic neurons, called RMEs, innervate head muscles. Conversely, thirty-nine motor neurons express the excitatory neurotransmitter, acetylcholine (ACh), and antagonize GABA transmission at BWMs to coordinate locomotion. The antagonistic nature of GABAergic and cholinergic motor neurons at body wall NMJs was initially determined by laser ablation and later buttressed by aldicarb exposure. Acute aldicarb exposure results in a time-course or dose-responsive paralysis in wild-type worms. Yet, loss of excitatory ACh transmission confers resistance to aldicarb, as less ACh accumulates at worm NMJs, leading to less stimulation of BWMs. Resistance to aldicarb may be observed with ACh-specific or general synaptic function mutants. Consistent with antagonistic GABA and ACh transmission, loss of GABA transmission, or a failure to negatively regulate ACh release, confers hypersensitivity to aldicarb. Although aldicarb exposure has led to the isolation of numerous worm homologs of neurotransmission genes, aldicarb exposure alone cannot efficiently determine prevailing roles for genes and pathways in specific C. elegans motor neurons. For this purpose, we have introduced a complementary experimental approach, which uses PTZ. Neurotransmission mutants display clear phenotypes, distinct from aldicarb-induced paralysis, in response to PTZ. Wild-type worms, as well as mutants with specific inabilities to release or receive ACh, do not show apparent sensitivity to PTZ. However, GABA mutants, as well as general synaptic function mutants, display anterior convulsions in a time-course or dose-responsive manner. Mutants that cannot negatively regulate general neurotransmitter release and, thus, secrete excessive amounts of ACh onto BWMs, become paralyzed on PTZ. The PTZ-induced phenotypes of discrete mutant classes indicate that a complementary approach with aldicarb and PTZ exposure paradigms in C. elegans may accelerate our understanding of neurotransmission. Moreover, videos demonstrating how we perform pharmacological assays should establish consistent methods for C. elegans research.

Anatomical Reconstruction and Functional Imaging Reveal an Ordered Array of Skylight Polarization Detectors in Drosophila.

PubMed

Weir, Peter T; Henze, Miriam J; Bleul, Christiane; Baumann-Klausener, Franziska; Labhart, Thomas; Dickinson, Michael H

2016-05-11

Many insects exploit skylight polarization as a compass cue for orientation and navigation. In the fruit fly, Drosophila melanogaster, photoreceptors R7 and R8 in the dorsal rim area (DRA) of the compound eye are specialized to detect the electric vector (e-vector) of linearly polarized light. These photoreceptors are arranged in stacked pairs with identical fields of view and spectral sensitivities, but mutually orthogonal microvillar orientations. As in larger flies, we found that the microvillar orientation of the distal photoreceptor R7 changes in a fan-like fashion along the DRA. This anatomical arrangement suggests that the DRA constitutes a detector for skylight polarization, in which different e-vectors maximally excite different positions in the array. To test our hypothesis, we measured responses to polarized light of varying e-vector angles in the terminals of R7/8 cells using genetically encoded calcium indicators. Our data confirm a progression of preferred e-vector angles from anterior to posterior in the DRA, and a strict orthogonality between the e-vector preferences of paired R7/8 cells. We observed decreased activity in photoreceptors in response to flashes of light polarized orthogonally to their preferred e-vector angle, suggesting reciprocal inhibition between photoreceptors in the same medullar column, which may serve to increase polarization contrast. Together, our results indicate that the polarization-vision system relies on a spatial map of preferred e-vector angles at the earliest stage of sensory processing. The fly's visual system is an influential model system for studying neural computation, and much is known about its anatomy, physiology, and development. The circuits underlying motion processing have received the most attention, but researchers are increasingly investigating other functions, such as color perception and object recognition. In this work, we investigate the early neural processing of a somewhat exotic sense, called polarization vision. Because skylight is polarized in an orientation that is rigidly determined by the position of the sun, this cue provides compass information. Behavioral experiments have shown that many species use the polarization pattern in the sky to direct locomotion. Here we describe the input stage of the fly's polarization-vision system. Copyright © 2016 the authors 0270-6474/16/365397-08$15.00/0.

Negative kinetic temperature effect on the hydride transfer from NADH analogue BNAH to the radical cation of N-benzylphenothiazine in acetonitrile.

PubMed

Zhu, Xiao-Qing; Zhang, Jian-Yu; Cheng, Jin-Pei

2006-09-01

The reaction rates of 1-(p-substituted benzyl)-1,4-dihydronicotinamide (G-BNAH) with N-benzylphenothiazine radical cation (PTZ(*+)) in acetonitrile were determined. The results show that the reaction rates (k(obs)) decreased from 2.80 x 10(7) to 2.16 x 10(7) M(-1) s(-1) for G = H as the reaction temperature increased from 298 to 318 K. The activation enthalpies of the reactions were estimated according to Eyring equation to give negative values (-3.4 to -2.9 kcal/mol). Investigation of the reaction intermediate shows that the charge-transfer complex (CT-complex) between G-BNAH and PTZ(*+) was formed in front of the hydride transfer from G-BNAH to PTZ(*+). The formation enthalpy of the CT-complex was estimated by using the Benesi-Hildebrand equation to give the values from -6.4 to -6.0 kcal/mol when the substituent G in G-BNAH changes from CH(3)O to Br. Detailed thermodynamic analyses on each elementary step in the possible reaction pathways suggest that the hydride transfer from G-BNAH to PTZ(*+) occurs by a concerted hydride transfer via a CT-complex. The effective charge distribution on the pyridine ring in G-BNAH at the various stages-the reactant G-BNAH, the charge-transfer complex, the transition-state, and the product G-BNA(+)-was estimated by using the method of Hammett-type linear free energy analysis, and the results show that the pyridine ring carries relative effective positive charges of 0.35 in the CT-complex and 0.45 in the transition state, respectively, which indicates that the concerted hydride transfer from G-BNAH to PTZ(*+) was practically performed by the initial charge (-0.35) transfer from G-BNAH to PTZ(*+) and then followed by the transfer of hydrogen atom with partial negative charge (-0.65). It is evident that the present work would be helpful in understanding the nature of the negative temperature effect, especially on the reaction of NADH coenzyme with the drug phenothiazine in vivo.

Enhancement of Recombinant Protein Production in Transgenic Nicotiana benthamiana Plant Cell Suspension Cultures with Co-Cultivation of Agrobacterium Containing Silencing Suppressors.

PubMed

Huang, Ting-Kuo; Falk, Bryce W; Dandekar, Abhaya M; McDonald, Karen A

2018-05-24

We have previously demonstrated that the inducible plant viral vector (CMViva) in transgenic plant cell cultures can significantly improve the productivity of extracellular functional recombinant human alpha-1-antiryspin (rAAT) compared with either a common plant constitutive promoter ( Cauliflower mosaic virus (CaMV) 35S) or a chemically inducible promoter (estrogen receptor-based XVE) system. For a transgenic plant host system, however, viral or transgene-induced post-transcriptional gene silencing (PTGS) has been identified as a host response mechanism that may dramatically reduce the expression of a foreign gene. Previous studies have suggested that viral gene silencing suppressors encoded by a virus can block or interfere with the pathways of transgene-induced PTGS in plant cells. In this study, the capability of nine different viral gene silencing suppressors were evaluated for improving the production of rAAT protein in transgenic plant cell cultures (CMViva, XVE or 35S system) using an Agrobacterium -mediated transient expression co-cultivation process in which transgenic plant cells and recombinant Agrobacterium carrying the viral gene silencing suppressor were grown together in suspension cultures. Through the co-cultivation process, the impacts of gene silencing suppressors on the rAAT production were elucidated, and promising gene silencing suppressors were identified. Furthermore, the combinations of gene silencing suppressors were optimized using design of experiments methodology. The results have shown that in transgenic CMViva cell cultures, the functional rAAT as a percentage of total soluble protein is increased 5.7 fold with the expression of P19, and 17.2 fold with the co-expression of CP, P19 and P24.

Vesicular stomatitis virus-based Ebola vaccines with improved cross-protective efficacy.

PubMed

Marzi, Andrea; Ebihara, Hideki; Callison, Julie; Groseth, Allison; Williams, Kinola J; Geisbert, Thomas W; Feldmann, Heinz

2011-11-01

For Ebola virus (EBOV), 4 different species are known: Zaire, Sudan, Côte d'Ivoire, and Reston ebolavirus. The newly discovered Bundibugyo ebolavirus has been proposed as a 5th species. So far, no cross-neutralization among EBOV species has been described, aggravating progress toward cross-species protective vaccines. With the use of recombinant vesicular stomatitis virus (rVSV)-based vaccines, guinea pigs could be protected against Zaire ebolavirus (ZEBOV) infection only when immunized with a vector expressing the homologous, but not a heterologous, EBOV glycoprotein (GP). However, infection of guinea pigs with nonadapted wild-type strains of the different species resulted in full protection of all animals against subsequent challenge with guinea pig-adapted ZEBOV, showing that cross-species protection is possible. New vectors were generated that contain EBOV viral protein 40 (VP40) or EBOV nucleoprotein (NP) as a second antigen expressed by the same rVSV vector that encodes the heterologous GP. After applying a 2-dose immunization approach, we observed an improved cross-protection rate, with 5 of 6 guinea pigs surviving the lethal ZEBOV challenge if vaccinated with rVSV-expressing SEBOV-GP and -VP40. Our data demonstrate that cross-protection between the EBOV species can be achieved, although EBOV-GP alone cannot induce the required immune response.

An exploration of diffusion tensor eigenvector variability within human calf muscles.

PubMed

Rockel, Conrad; Noseworthy, Michael D

2016-01-01

To explore the effect of diffusion tensor imaging (DTI) acquisition parameters on principal and minor eigenvector stability within human lower leg skeletal muscles. Lower leg muscles were evaluated in seven healthy subjects at 3T using an 8-channel transmit/receive coil. Diffusion-encoding was performed with nine signal averages (NSA) using 6, 15, and 25 directions (NDD). Individual DTI volumes were combined into aggregate volumes of 3, 2, and 1 NSA according to number of directions. Tensor eigenvalues (λ1 , λ2 , λ3 ), eigenvectors (ε1 , ε2 , ε3 ), and DTI metrics (fractional anisotropy [FA] and mean diffusivity [MD]) were calculated for each combination of NSA and NDD. Spatial maps of signal-to-noise ratio (SNR), λ3 :λ2 ratio, and zenith angle were also calculated for region of interest (ROI) analysis of vector orientation consistency. ε1 variability was only moderately related to ε2 variability (r = 0.4045). Variation of ε1 was affected by NDD, not NSA (P < 0.0002), while variation of ε2 was affected by NSA, not NDD (P < 0.0003). In terms of tensor shape, vector variability was weakly related to FA (ε1 :r = -0.1854, ε2 : ns), but had a stronger relation to the λ3 :λ2 ratio (ε1 :r = -0.5221, ε2 :r = -0.1771). Vector variability was also weakly related to SNR (ε1 :r = -0.2873, ε2 :r = -0.3483). Zenith angle was found to be strongly associated with variability of ε1 (r = 0.8048) but only weakly with that of ε2 (r = 0.2135). The second eigenvector (ε2 ) displayed higher directional variability relative to ε1 , and was only marginally affected by experimental conditions that impacted ε1 variability. © 2015 Wiley Periodicals, Inc.

Comparative studies on the effects of clinically used anticonvulsants on the oxidative stress biomarkers in pentylenetetrazole-induced kindling model of epileptogenesis in mice.

PubMed

Mazhar, Faizan; Malhi, Saima M; Simjee, Shabana U

2017-01-01

Oxidative stress plays a key role in the pathogenesis of epilepsy and contributes in underlying epileptogenesis process. Anticonvulsant drugs targeting the oxidative stress domain of epileptogenesis may provide better control of seizure. The present study was carried out to investigate the effect of clinically used anti-epileptic drugs (AEDs) on the course of pentylenetetrazole (PTZ)-induced kindling and oxidative stress markers in mice. Six mechanistically heterogeneous anticonvulsants: phenobarbital, phenytoin, levetiracetam, pregabalin, topiramate, and felbamate were selected and their redox profiles were determined. Diazepam was used as a drug control for comparison. Kindling was induced by repeated injections of a sub-convulsive dose of PTZ (50 mg/kg, s.c.) on alternate days until seizure score 5 was evoked in the control kindled group. Anticonvulsants were administered daily. Following PTZ kindling, oxidative stress biomarkers were assessed in homogenized whole brain samples and estimated for the levels of nitric oxide, peroxide, malondialdehyde, protein carbonyl, reduced glutathione, and activities of nitric oxide synthase and superoxide dismutase. Biochemical analysis revealed a significant increase in the levels of reactive oxygen species with a parallel decrease in endogenous anti-oxidants in PTZ-kindled control animals. Daily treatment with levetiracetam and felbamate significantly decreased the PTZ-induced seizure score as well as the levels of nitric oxide (p<0.001), nitric oxide synthase activity (p<0.05), peroxide levels (p<0.05), and malondialdehyde (p<0.05). Levetiracetam and felbamate significantly decreased lipid and protein peroxidation whereas topiramate was found to reduce lipid peroxidation only. An AED that produces anticonvulsant effect by the diversified mechanism of action such as levetiracetam, felbamate, and topiramate exhibited superior anti-oxidative stress activity in addition to their anticonvulsant activity.

Antiepileptic and antipsychotic activities of standardized Śilājatu (Shilajit) in experimental animals.

PubMed

Durg, Sharanbasappa; Veerapur, Veeresh P; Thippeswamy, B S; Ahamed, Syed Mansoor

2015-01-01

Śilājatu (Shilajit; SJ) is claimed in traditional Indian medical practice to be useful in the treatment of nervous disorders, epilepsy and as antistress. To investigate whether SJ possesses antiepileptic and antipsychotic activities in rodents. Isonicotinyl hydrazine (INH), pentylenetetrazole (PTZ), apomorphine, phenytoin, diazepam, haloperidol and other chemicals of analytical grade were procured from standard companies. The antiepileptic activity of SJ was assessed using maximal electro shock (MES)-induced seizures in rats, INH and PTZ-induced seizures in mice. The antipsychotic effect of SJ was evaluated using apomorphine-induced climbing and stereotyped behaviours respectively, in mice and rats. SJ (25 and 50 mg/kg, p.o.) was given orally once daily for 15 days in all the rodent models. On the test day, SJ was administered 1 h prior to electric shock or chemical inducers (INH/PTZ/apomorphine) in experimental animals; the animals were then observed for different phases of seizures and psychotic behaviours. In addition, gamma-aminobutyric acid (GABA) content in the brain of rats and mice was estimated in seizure models. The data were expressed as mean ± standard error of mean. Statistical comparisons were performed by one-way ANOVA followed by Tukey's post-test using Graph Pad Prism version 5.0, USA. A P < 0.05 was considered significant. SJ pretreatment significantly inhibited the seizures induced by MES, INH and PTZ in a dose dependent manner. Further, SJ augmented brain GABA levels to normal, decreased by INH and PTZ in mice brain. SJ pretreatment also significantly inhibited the climbing and stereotyped behaviours induced by apomorphine. The present data seems to confirm the antiepileptic activity of SJ which may be because of enhancing the GABAergic system. The antipsychotic activity observed may be due to anti-dopaminergic and/or GABA-mimetic actions.

Dual Regulation of Bacillus subtilis kinB Gene Encoding a Sporulation Trigger by SinR through Transcription Repression and Positive Stringent Transcription Control.

PubMed

Fujita, Yasutaro; Ogura, Mitsuo; Nii, Satomi; Hirooka, Kazutake

2017-01-01

It is known that transcription of kinB encoding a trigger for Bacillus subtilis sporulation is under repression by SinR, a master repressor of biofilm formation, and under positive stringent transcription control depending on the adenine species at the transcription initiation nucleotide (nt). Deletion and base substitution analyses of the kinB promoter (P kinB ) region using lacZ fusions indicated that either a 5-nt deletion (Δ5, nt -61/-57, +1 is the transcription initiation nt) or the substitution of G at nt -45 with A (G-45A) relieved kinB repression. Thus, we found a pair of SinR-binding consensus sequences (GTTCTYT; Y is T or C) in an inverted orientation (SinR-1) between nt -57/-42, which is most likely a SinR-binding site for kinB repression. This relief from SinR repression likely requires SinI, an antagonist of SinR. Surprisingly, we found that SinR is essential for positive stringent transcription control of P kinB . Electrophoretic mobility shift assay (EMSA) analysis indicated that SinR bound not only to SinR-1 but also to SinR-2 (nt -29/-8) consisting of another pair of SinR consensus sequences in a tandem repeat arrangement; the two sequences partially overlap the '-35' and '-10' regions of P kinB . Introduction of base substitutions (T-27C C-26T) in the upstream consensus sequence of SinR-2 affected positive stringent transcription control of P kinB , suggesting that SinR binding to SinR-2 likely causes this positive control. EMSA also implied that RNA polymerase and SinR are possibly bound together to SinR-2 to form a transcription initiation complex for kinB transcription. Thus, it was suggested in this work that derepression of kinB from SinR repression by SinI induced by Spo0A∼P and occurrence of SinR-dependent positive stringent transcription control of kinB might induce effective sporulation cooperatively, implying an intimate interplay by stringent response, sporulation, and biofilm formation.

Integrative proteomics to understand the transmission mechanism of Barley yellow dwarf virus-GPV by its insect vector Rhopalosiphum padi

PubMed Central

Wang, Hui; Wu, Keke; Liu, Yan; Wu, Yunfeng; Wang, Xifeng

2015-01-01

Barley yellow dwarf virus-GPV (BYDV-GPV) is transmitted by Rhopalosiphum padi and Schizaphis graminum in a persistent nonpropagative manner. To improve our understanding of its transmission mechanism by aphid vectors, we used two approaches, isobaric tags for relative and absolute quantitation (iTRAQ) and yeast two-hybrid (YTH) system, to identify proteins in R. padi that may interact with or direct the spread of BYDV-GPV along the circulative transmission pathway. Thirty-three differential aphid proteins in viruliferous and nonviruliferous insects were identified using iTRAQ coupled to 2DLC-MS/MS. With the yeast two-hybrid system, 25 prey proteins were identified as interacting with the readthrough protein (RTP) and eight with the coat protein (CP), which are encoded by BYDV-GPV. Among the aphid proteins identified, most were involved in primary energy metabolism, synaptic vesicle cycle, the proteasome pathway and the cell cytoskeleton organization pathway. In a systematic comparison of the two methods, we found that the information generated by the two methods was complementary. Taken together, our findings provide useful information on the interactions between BYDV-GPV and its vector R. padi to further our understanding of the mechanisms regulating circulative transmission in aphid vectors. PMID:26161807

miR-Sens--a retroviral dual-luciferase reporter to detect microRNA activity in primary cells.

PubMed

Beillard, Emmanuel; Ong, Siau Chi; Giannakakis, Antonis; Guccione, Ernesto; Vardy, Leah A; Voorhoeve, P Mathijs

2012-05-01

MicroRNA-mRNA interactions are commonly validated and deconstructed in cell lines transfected with luciferase reporters. However, due to cell type-specific variations in microRNA or RNA-binding protein abundance, such assays may not reliably reflect microRNA activity in other cell types that are less easily transfected. In order to measure miRNA activity in primary cells, we constructed miR-Sens, a MSCV-based retroviral vector that encodes both a Renilla luciferase reporter gene controlled by microRNA binding sites in its 3' UTR and a Firefly luciferase normalization gene. miR-Sens sensors can be efficiently transduced in primary cells such as human fibroblasts and mammary epithelial cells, and allow the detection of overexpressed and, more importantly, endogenous microRNAs. Notably, we find that the relative luciferase activity is correlated to the miRNA expression, allowing quantitative measurement of microRNA activity. We have subsequently validated the miR-Sens 3' UTR vectors with known human miRNA-372, miRNA-373, and miRNA-31 targets (LATS2 and TXNIP). Overall, we observe that miR-Sens-based assays are highly reproducible, allowing detection of the independent contribution of multiple microRNAs to 3' UTR-mediated translational control of LATS2. In conclusion, miR-Sens is a new tool for the efficient study of microRNA activity in primary cells or panels of cell lines. This vector will not only be useful for studies on microRNA biology, but also more broadly on other factors influencing the translation of mRNAs.

miR-Sens—a retroviral dual-luciferase reporter to detect microRNA activity in primary cells

PubMed Central

Beillard, Emmanuel; Ong, Siau Chi; Giannakakis, Antonis; Guccione, Ernesto; Vardy, Leah A.; Voorhoeve, P. Mathijs

2012-01-01

MicroRNA–mRNA interactions are commonly validated and deconstructed in cell lines transfected with luciferase reporters. However, due to cell type-specific variations in microRNA or RNA-binding protein abundance, such assays may not reliably reflect microRNA activity in other cell types that are less easily transfected. In order to measure miRNA activity in primary cells, we constructed miR-Sens, a MSCV-based retroviral vector that encodes both a Renilla luciferase reporter gene controlled by microRNA binding sites in its 3′ UTR and a Firefly luciferase normalization gene. miR-Sens sensors can be efficiently transduced in primary cells such as human fibroblasts and mammary epithelial cells, and allow the detection of overexpressed and, more importantly, endogenous microRNAs. Notably, we find that the relative luciferase activity is correlated to the miRNA expression, allowing quantitative measurement of microRNA activity. We have subsequently validated the miR-Sens 3′ UTR vectors with known human miRNA-372, miRNA-373, and miRNA-31 targets (LATS2 and TXNIP). Overall, we observe that miR-Sens-based assays are highly reproducible, allowing detection of the independent contribution of multiple microRNAs to 3′ UTR–mediated translational control of LATS2. In conclusion, miR-Sens is a new tool for the efficient study of microRNA activity in primary cells or panels of cell lines. This vector will not only be useful for studies on microRNA biology, but also more broadly on other factors influencing the translation of mRNAs. PMID:22417692

Collaborative real-time scheduling of multiple PTZ cameras for multiple object tracking in video surveillance

NASA Astrophysics Data System (ADS)

Liu, Yu-Che; Huang, Chung-Lin

2013-03-01

This paper proposes a multi-PTZ-camera control mechanism to acquire close-up imagery of human objects in a surveillance system. The control algorithm is based on the output of multi-camera, multi-target tracking. Three main concerns of the algorithm are (1) the imagery of human object's face for biometric purposes, (2) the optimal video quality of the human objects, and (3) minimum hand-off time. Here, we define an objective function based on the expected capture conditions such as the camera-subject distance, pan tile angles of capture, face visibility and others. Such objective function serves to effectively balance the number of captures per subject and quality of captures. In the experiments, we demonstrate the performance of the system which operates in real-time under real world conditions on three PTZ cameras.

Potentiation of anthrax vaccines using protective antigen-expressing viral replicon vectors.

PubMed

Wang, Hai-Chao; An, Huai-Jie; Yu, Yun-Zhou; Xu, Qing

2015-02-01

DNA vaccines require improvement for human use because they are generally weak stimulators of the immune system in humans. The efficacy of DNA vaccines can be improved using a viral replicon as vector to administer antigen of pathogen. In this study, we comprehensively evaluated the conventional non-viral DNA, viral replicon DNA or viral replicon particles (VRP) vaccines encoding different forms of anthrax protective antigen (PA) for specific immunity and protective potency against anthrax. Our current results clearly suggested that these viral replicon DNA or VRP vaccines derived from Semliki Forest virus (SFV) induced stronger PA-specific immune responses than the conventional non-viral DNA vaccines when encoding the same antigen forms, which resulted in potent protection against challenge with the Bacillus anthracis strain A16R. Additionally, the naked PA-expressing SFV replicon DNA or VRP vaccines without the need for high doses or demanding particular delivery regimens elicited robust immune responses and afforded completely protective potencies, which indicated the potential of the SFV replicon as vector of anthrax vaccines for use in clinical application. Therefore, our results suggest that these PA-expressing SFV replicon DNA or VRP vaccines may be suitable as candidate vaccines against anthrax. Copyright © 2015 Elsevier B.V. All rights reserved.

Genome sequences of the human body louse and its primary endosymbiont provide insights into the permanent parasitic lifestyle.

PubMed

Kirkness, Ewen F; Haas, Brian J; Sun, Weilin; Braig, Henk R; Perotti, M Alejandra; Clark, John M; Lee, Si Hyeock; Robertson, Hugh M; Kennedy, Ryan C; Elhaik, Eran; Gerlach, Daniel; Kriventseva, Evgenia V; Elsik, Christine G; Graur, Dan; Hill, Catherine A; Veenstra, Jan A; Walenz, Brian; Tubío, José Manuel C; Ribeiro, José M C; Rozas, Julio; Johnston, J Spencer; Reese, Justin T; Popadic, Aleksandar; Tojo, Marta; Raoult, Didier; Reed, David L; Tomoyasu, Yoshinori; Kraus, Emily; Krause, Emily; Mittapalli, Omprakash; Margam, Venu M; Li, Hong-Mei; Meyer, Jason M; Johnson, Reed M; Romero-Severson, Jeanne; Vanzee, Janice Pagel; Alvarez-Ponce, David; Vieira, Filipe G; Aguadé, Montserrat; Guirao-Rico, Sara; Anzola, Juan M; Yoon, Kyong S; Strycharz, Joseph P; Unger, Maria F; Christley, Scott; Lobo, Neil F; Seufferheld, Manfredo J; Wang, Naikuan; Dasch, Gregory A; Struchiner, Claudio J; Madey, Greg; Hannick, Linda I; Bidwell, Shelby; Joardar, Vinita; Caler, Elisabet; Shao, Renfu; Barker, Stephen C; Cameron, Stephen; Bruggner, Robert V; Regier, Allison; Johnson, Justin; Viswanathan, Lakshmi; Utterback, Terry R; Sutton, Granger G; Lawson, Daniel; Waterhouse, Robert M; Venter, J Craig; Strausberg, Robert L; Berenbaum, May R; Collins, Frank H; Zdobnov, Evgeny M; Pittendrigh, Barry R

2010-07-06

As an obligatory parasite of humans, the body louse (Pediculus humanus humanus) is an important vector for human diseases, including epidemic typhus, relapsing fever, and trench fever. Here, we present genome sequences of the body louse and its primary bacterial endosymbiont Candidatus Riesia pediculicola. The body louse has the smallest known insect genome, spanning 108 Mb. Despite its status as an obligate parasite, it retains a remarkably complete basal insect repertoire of 10,773 protein-coding genes and 57 microRNAs. Representing hemimetabolous insects, the genome of the body louse thus provides a reference for studies of holometabolous insects. Compared with other insect genomes, the body louse genome contains significantly fewer genes associated with environmental sensing and response, including odorant and gustatory receptors and detoxifying enzymes. The unique architecture of the 18 minicircular mitochondrial chromosomes of the body louse may be linked to the loss of the gene encoding the mitochondrial single-stranded DNA binding protein. The genome of the obligatory louse endosymbiont Candidatus Riesia pediculicola encodes less than 600 genes on a short, linear chromosome and a circular plasmid. The plasmid harbors a unique arrangement of genes required for the synthesis of pantothenate, an essential vitamin deficient in the louse diet. The human body louse, its primary endosymbiont, and the bacterial pathogens that it vectors all possess genomes reduced in size compared with their free-living close relatives. Thus, the body louse genome project offers unique information and tools to use in advancing understanding of coevolution among vectors, symbionts, and pathogens.

Genome sequences of the human body louse and its primary endosymbiont provide insights into the permanent parasitic lifestyle

PubMed Central

Kirkness, Ewen F.; Haas, Brian J.; Sun, Weilin; Braig, Henk R.; Perotti, M. Alejandra; Clark, John M.; Lee, Si Hyeock; Robertson, Hugh M.; Kennedy, Ryan C.; Elhaik, Eran; Gerlach, Daniel; Kriventseva, Evgenia V.; Elsik, Christine G.; Graur, Dan; Hill, Catherine A.; Veenstra, Jan A.; Walenz, Brian; Tubío, José Manuel C.; Ribeiro, José M. C.; Rozas, Julio; Johnston, J. Spencer; Reese, Justin T.; Popadic, Aleksandar; Tojo, Marta; Raoult, Didier; Reed, David L.; Tomoyasu, Yoshinori; Kraus, Emily; Mittapalli, Omprakash; Margam, Venu M.; Li, Hong-Mei; Meyer, Jason M.; Johnson, Reed M.; Romero-Severson, Jeanne; VanZee, Janice Pagel; Alvarez-Ponce, David; Vieira, Filipe G.; Aguadé, Montserrat; Guirao-Rico, Sara; Anzola, Juan M.; Yoon, Kyong S.; Strycharz, Joseph P.; Unger, Maria F.; Christley, Scott; Lobo, Neil F.; Seufferheld, Manfredo J.; Wang, NaiKuan; Dasch, Gregory A.; Struchiner, Claudio J.; Madey, Greg; Hannick, Linda I.; Bidwell, Shelby; Joardar, Vinita; Caler, Elisabet; Shao, Renfu; Barker, Stephen C.; Cameron, Stephen; Bruggner, Robert V.; Regier, Allison; Johnson, Justin; Viswanathan, Lakshmi; Utterback, Terry R.; Sutton, Granger G.; Lawson, Daniel; Waterhouse, Robert M.; Venter, J. Craig; Strausberg, Robert L.; Collins, Frank H.; Zdobnov, Evgeny M.; Pittendrigh, Barry R.

2010-01-01

As an obligatory parasite of humans, the body louse (Pediculus humanus humanus) is an important vector for human diseases, including epidemic typhus, relapsing fever, and trench fever. Here, we present genome sequences of the body louse and its primary bacterial endosymbiont Candidatus Riesia pediculicola. The body louse has the smallest known insect genome, spanning 108 Mb. Despite its status as an obligate parasite, it retains a remarkably complete basal insect repertoire of 10,773 protein-coding genes and 57 microRNAs. Representing hemimetabolous insects, the genome of the body louse thus provides a reference for studies of holometabolous insects. Compared with other insect genomes, the body louse genome contains significantly fewer genes associated with environmental sensing and response, including odorant and gustatory receptors and detoxifying enzymes. The unique architecture of the 18 minicircular mitochondrial chromosomes of the body louse may be linked to the loss of the gene encoding the mitochondrial single-stranded DNA binding protein. The genome of the obligatory louse endosymbiont Candidatus Riesia pediculicola encodes less than 600 genes on a short, linear chromosome and a circular plasmid. The plasmid harbors a unique arrangement of genes required for the synthesis of pantothenate, an essential vitamin deficient in the louse diet. The human body louse, its primary endosymbiont, and the bacterial pathogens that it vectors all possess genomes reduced in size compared with their free-living close relatives. Thus, the body louse genome project offers unique information and tools to use in advancing understanding of coevolution among vectors, symbionts, and pathogens. PMID:20566863

Identification of compounds with anti-convulsant properties in a zebrafish model of epileptic seizures

PubMed Central

Baxendale, Sarah; Holdsworth, Celia J.; Meza Santoscoy, Paola L.; Harrison, Michael R. M.; Fox, James; Parkin, Caroline A.; Ingham, Philip W.; Cunliffe, Vincent T.

2012-01-01

SUMMARY The availability of animal models of epileptic seizures provides opportunities to identify novel anticonvulsants for the treatment of people with epilepsy. We found that exposure of 2-day-old zebrafish embryos to the convulsant agent pentylenetetrazole (PTZ) rapidly induces the expression of synaptic-activity-regulated genes in the CNS, and elicited vigorous episodes of calcium (Ca2+) flux in muscle cells as well as intense locomotor activity. We then screened a library of ∼2000 known bioactive small molecules and identified 46 compounds that suppressed PTZ-inducedtranscription of the synaptic-activity-regulated gene fos in 2-day-old (2 dpf) zebrafish embryos. Further analysis of a subset of these compounds, which included compounds with known and newly identified anticonvulsant properties, revealed that they exhibited concentration-dependent inhibition of both locomotor activity and PTZ-induced fos transcription, confirming their anticonvulsant characteristics. We conclude that this in situ hybridisation assay for fos transcription in the zebrafish embryonic CNS is a robust, high-throughput in vivo indicator of the neural response to convulsant treatment and lends itself well to chemical screening applications. Moreover, our results demonstrate that suppression of PTZ-induced fos expression provides a sensitive means of identifying compounds with anticonvulsant activities. PMID:22730455

Molecular cloning, expression and characterization of a functional GSTmu class from the cattle tick Boophilus annulatus.

PubMed

Shahein, Yasser Ezzat; El Sayed El-Hakim, Amr; Abouelella, Amira Mohamed Kamal; Hamed, Ragaa Reda; Allam, Shaimaa Abdul-Moez; Farid, Nevin Mahmoud

2008-03-25

A full-length cDNA of a glutathione S-transferase (GST) was cloned from a cDNA library of the local Egyptian cattle tick Boophilus annulatus. The 672 bp cloned fragment was sequenced and showed an open reading frame encoding a protein of 223 amino acids. Comparison of the deduced amino acid sequence with GSTs from other species revealed that the sequence is closely related to the mammalian mu-class GST. The cloned gene was expressed in E. coli under T7 promotor of pET-30b vector, and purified under native conditions. The purified enzyme appeared as a single band on 12% SDS-PAGE and has a molecular weight of 30.8 kDa including the histidine tag of the vector. The purified enzyme was assayed upon the chromogenic substrate 1-chloro-2,4-dinitrobenzene (CDNB) and the recombinant enzyme showed high level of activity even in the presence of the beta-galactosidase region on its 5' end and showed maximum activity at pH 7.5. The Km values for CDNB and GSH were 0.57 and 0.79 mM, respectively. The over expressed rBaGST showed high activity toward CDNB (121 units/mg protein) and less toward DCNB (29.3 units/mg protein). rBaGST exhibited peroxidatic activity on cumene hydroperoxide sharing this property with GSTs belonging to the GST alpha class. I50 values for cibacron blue and bromosulfophthalein were 0.22 and 8.45 microM, respectively, sharing this property with the mammalian GSTmu class. Immunoblotting revealed the presence of the GST molecule in B. annulatus protein extracts; whole tick, larvae, gut, salivary gland and ovary. Homologues to the GSTmu were also detected in other tick species as Hyalomma dromedarii and Rhipicephalus sp. while in Ornithodoros moubata, GSTmu homologue could not be detected.

Vector Adaptive/Predictive Encoding Of Speech

NASA Technical Reports Server (NTRS)

Chen, Juin-Hwey; Gersho, Allen

1989-01-01

Vector adaptive/predictive technique for digital encoding of speech signals yields decoded speech of very good quality after transmission at coding rate of 9.6 kb/s and of reasonably good quality at 4.8 kb/s. Requires 3 to 4 million multiplications and additions per second. Combines advantages of adaptive/predictive coding, and code-excited linear prediction, yielding speech of high quality but requires 600 million multiplications and additions per second at encoding rate of 4.8 kb/s. Vector adaptive/predictive coding technique bridges gaps in performance and complexity between adaptive/predictive coding and code-excited linear prediction.

Chemical Communications

DTIC Science & Technology

2012-04-27

TOTAL: Patents Submitted Submitted: "Systems and Methods for Amplification and Phage Display", Derda, R., Tang, S.K.Y., Whitesides, G.M. PCT/US11...College 60 Oxford Street Cambridge MA 02138 5a: 5f-1a: 5f-c: Systems and Methods for Amplification and Phage Display Patent Filed in US? (5d-1) Y NPatent...the bacteriophage- T7 promoter (See S.I. for details). This series of FP encoding vectors contain the ampicillin-resistant gene as a selective marker

Method and System for Temporal Filtering in Video Compression Systems

NASA Technical Reports Server (NTRS)

Lu, Ligang; He, Drake; Jagmohan, Ashish; Sheinin, Vadim

2011-01-01

Three related innovations combine improved non-linear motion estimation, video coding, and video compression. The first system comprises a method in which side information is generated using an adaptive, non-linear motion model. This method enables extrapolating and interpolating a visual signal, including determining the first motion vector between the first pixel position in a first image to a second pixel position in a second image; determining a second motion vector between the second pixel position in the second image and a third pixel position in a third image; determining a third motion vector between the first pixel position in the first image and the second pixel position in the second image, the second pixel position in the second image, and the third pixel position in the third image using a non-linear model; and determining a position of the fourth pixel in a fourth image based upon the third motion vector. For the video compression element, the video encoder has low computational complexity and high compression efficiency. The disclosed system comprises a video encoder and a decoder. The encoder converts the source frame into a space-frequency representation, estimates the conditional statistics of at least one vector of space-frequency coefficients with similar frequencies, and is conditioned on previously encoded data. It estimates an encoding rate based on the conditional statistics and applies a Slepian-Wolf code with the computed encoding rate. The method for decoding includes generating a side-information vector of frequency coefficients based on previously decoded source data and encoder statistics and previous reconstructions of the source frequency vector. It also performs Slepian-Wolf decoding of a source frequency vector based on the generated side-information and the Slepian-Wolf code bits. The video coding element includes receiving a first reference frame having a first pixel value at a first pixel position, a second reference frame having a second pixel value at a second pixel position, and a third reference frame having a third pixel value at a third pixel position. It determines a first motion vector between the first pixel position and the second pixel position, a second motion vector between the second pixel position and the third pixel position, and a fourth pixel value for a fourth frame based upon a linear or nonlinear combination of the first pixel value, the second pixel value, and the third pixel value. A stationary filtering process determines the estimated pixel values. The parameters of the filter may be predetermined constants.

Fusion of Huntingtin interacting protein 1 to platelet-derived growth factor beta receptor (PDGFbetaR) in chronic myelomonocytic leukemia with t(5;7)(q33;q11.2).

PubMed

Ross, T S; Bernard, O A; Berger, R; Gilliland, D G

1998-06-15

We report the fusion of the Huntingtin interactin protein 1 (HIP1) gene to the platelet-derived growth factor betareceptor (PDGFbetaR) gene in a patient with chronic myelomonocytic leukemia (CMML) with a t(5;7)(q33;q11.2) translocation. Southern blot analysis of patient bone marrow cells with a PDGFbetaR gene probe demonstrated rearrangement of the PDGFbetaR gene. Anchored polymerase chain reaction using PDGFbetaR primers identified a chimeric transcript containing the HIP1 gene located at 7q11.2 fused to the PDGFbetaR gene on 5q33. HIP1 is a 116-kD protein recently cloned by yeast two-hybrid screening for proteins that interact with Huntingtin, the mutated protein in Huntington's disease. The consequence of t(5;7)(q33;q11.2) is an HIP1/PDGFbetaR fusion gene that encodes amino acids 1 to 950 of HIP1 joined in-frame to the transmembrane and tyrosine kinase domains of the PDGFbetaR. The reciprocal PDGFbetaR/HIP1 transcript is not expressed. HIP1/PDGFbetaR is a 180-kD protein when expressed in the murine hematopoietic cell line, Ba/F3, and is constitutively tyrosine phosphorylated. Furthermore, HIP1/PDGFbetaR transforms the Ba/F3 cells to interleukin-3-independent growth. These data are consistent with an alternative mechanism for activation of PDGFbetaR tyrosine kinase activity by fusion with HIP1, leading to transformation of hematopoietic cells, and may implicate Huntingtin or HIP1 in the pathogenesis of hematopoietic malignancies.

Development of a nested polymerase chain reaction for amplification of a sequence of the p57 gene of Renibacterium salmoninarum that provides a highly sensitive method for detection of the bacterium in salmonid kidney

USGS Publications Warehouse

Chase, D.M.; Pascho, R.J.

1998-01-01

Nucleic acid-based assays have shown promise for diagnosing Renibacterium salmoninarum in tissues and body fluids of salmonids. DeVelopment of a nested polymerase chain reaction (PCR) method to detect a 320 bp DNA segment of the gene encoding the p57 protein of R. salmoninarum is described. Whereas a conventional PCR for a 383 bp segment of the p57 gene reliably detected 1000 R. salmoninarum cells per reaction in kidney tissue, the nested PCR detected as few as 10 R. salmoninarum per reaction in kidney tissue. Two DNA extraction methods for the nested PCR were compared and the correlation between replicate samples was generally higher in samples extracted by the QIAamp system compared with those extracted by the phenol/chloroform method. The specificity of the nested PCR was confirmed by testing DNA extracts of common bacterial fish pathogens and a panel of bacterial species reported to cause false-positive reactions in the enzyme-linked immunosorbent assay (ELISA) and the fluorescent antibody test (FAT) for R. salmoninarum. Kidney samples from 74 naturally infected chinook Salmon were examined by the nested PCR, the ELISA, and the FAT, and the detected prevalences of R. salmoninarum were 61, 47, and 43%, respectively.

Single-Dose Intracardiac Injection of Pro-Regenerative MicroRNAs Improves Cardiac Function After Myocardial Infarction.

PubMed

Lesizza, Pierluigi; Prosdocimo, Giulia; Martinelli, Valentina; Sinagra, Gianfranco; Zacchigna, Serena; Giacca, Mauro

2017-04-14

Recent evidence indicates that a few human microRNAs (miRNAs), in particular hsa-miR-199a-3p and hsa-miR-590-3p, stimulate proliferation of cardiomyocytes and, once expressed in the mouse heart using viral vectors, induce cardiac regeneration after myocardial infarction. Viral vectors, however, are not devoid of safety issues and, more notably, drive expression of the encoded miRNAs for indefinite periods of time, which might not be desirable in light of human therapeutic application. As an alternative to the use of viral vectors, we wanted to assess the efficacy of synthetic miRNA mimics in inducing myocardial repair after single intracardiac injection using synthetic lipid formulations. We comparatively analyzed the efficacy of different lipid formulations in delivering hsa-miR-199a-3p and hsa-miR-590-3p both in primary neonatal mouse cardiomyocytes and in vivo. We established a transfection protocol allowing persistence of these 2 mimics for at least 12 days after a single intracardiac injection, with minimal dispersion to other organs and long-term preservation of miRNA functional activity, as assessed by monitoring the expression of 2 mRNA targets. Administration of this synthetic formulation immediately after myocardial infarction in mice resulted in marked reduction of infarct size and persistent recovery of cardiac function. A single administration of synthetic miRNA-lipid formulations is sufficient to stimulate cardiac repair and restoration of cardiac function. © 2017 American Heart Association, Inc.

An equine herpesvirus type 1 (EHV-1) vector expressing Rift Valley fever virus (RVFV) Gn and Gc induces neutralizing antibodies in sheep.

PubMed

Said, Abdelrahman; Elmanzalawy, Mona; Ma, Guanggang; Damiani, Armando Mario; Osterrieder, Nikolaus

2017-08-14

Rift Valley fever virus (RVFV) is an arthropod-borne bunyavirus that can cause serious and fatal disease in humans and animals. RVFV is a negative-sense RNA virus of the Phlebovirus genus in the Bunyaviridae family. The main envelope RVFV glycoproteins, Gn and Gc, are encoded on the M segment of RVFV and known inducers of protective immunity. In an attempt to develop a safe and efficacious RVF vaccine, we constructed and tested a vectored equine herpesvirus type 1 (EHV-1) vaccine that expresses RVFV Gn and Gc. The Gn and Gc genes were custom-synthesized after codon optimization and inserted into EHV-1 strain RacH genome. The rH_Gn-Gc recombinant virus grew in cultured cells with kinetics that were comparable to those of the parental virus and stably expressed Gn and Gc. Upon immunization of sheep, the natural host, neutralizing antibodies against RVFV were elicited by rH_Gn-Gc and protective titers reached to 1:320 at day 49 post immunization but not by parental EHV-1, indicating that EHV-1 is a promising vector alternative in the development of a safe marker RVFV vaccine.

Correction of murine hemophilia A following nonmyeloablative transplantation of hematopoietic stem cells engineered to encode an enhanced human factor VIII variant using a safety-augmented retroviral vector

PubMed Central

Ramezani, Ali

2009-01-01

Insertional mutagenesis by retroviral vectors is a major impediment to the clinical application of hematopoietic stem cell gene transfer for the treatment of hematologic disorders. We recently developed an insulated self-inactivating gammaretroviral vector, RMSinOFB, which uses a novel enhancer-blocking element that significantly decreases genotoxicity of retroviral integration. In this study, we used the RMSinOFB vector to evaluate the efficacy of a newly bioengineered factor VIII (fVIII) variant (efVIII)—containing a combination of A1 domain point mutations (L303E/F309S) and an extended partial B domain for improved secretion plus A2 domain mutations (R484A/R489A/P492A) for reduced immunogenicity—toward successful treatment of murine hemophilia A. In cell lines, efVIII was secreted at up to 6-fold higher levels than an L303E/F309S A1 domain–only fVIII variant (sfVIIIΔB). Most important, when compared with a conventional gammaretroviral vector expressing sfVIIIΔB, lower doses of RMSin-efVIII-OFB–transduced hematopoietic stem cells were needed to generate comparable curative fVIII levels in hemophilia A BALB/c mice after reduced-intensity total body irradiation or nonmyeloablative chemotherapy conditioning regimens. These data suggest that the safety-augmented RMSin-efVIII-OFB platform represents an encouraging step in the development of a clinically appropriate gene addition therapy for hemophilia A. PMID:19470695

Indomethacin treatment prior to pentylenetetrazole-induced seizures downregulates the expression of il1b and cox2 and decreases seizure-like behavior in zebrafish larvae.

PubMed

Barbalho, Patrícia Gonçalves; Lopes-Cendes, Iscia; Maurer-Morelli, Claudia Vianna

2016-03-09

It has been demonstrated that the zebrafish model of pentylenetetrazole (PTZ)-evoked seizures and the well-established rodent models of epilepsy are similar pertaining to behavior, electrographic features, and c-fos expression. Although this zebrafish model is suitable for studying seizures, to date, inflammatory response after seizures has not been investigated using this model. Because a relationship between epilepsy and inflammation has been established, in the present study we investigated the transcript levels of the proinflammatory cytokines interleukin-1 beta (il1b) and cyclooxygenase-2 (cox2a and cox2b) after PTZ-induced seizures in the brain of zebrafish 7 days post fertilization. Furthermore, we exposed the fish to the nonsteroidal anti-inflammatory drug indomethacin prior to PTZ, and we measured its effect on seizure latency, number of seizure behaviors, and mRNA expression of il1b, cox2b, and c-fos. We used quantitative real-time PCR to assess the mRNA expression of il1b, cox2a, cox2b, and c-fos, and visual inspection was used to monitor seizure latency and the number of seizure-like behaviors. We found a short-term upregulation of il1b, and we revealed that cox2b, but not cox2a, was induced after seizures. Indomethacin treatment prior to PTZ-induced seizures downregulated the mRNA expression of il1b, cox2b, and c-fos. Moreover, we observed that in larvae exposed to indomethacin, seizure latency increased and the number of seizure-like behaviors decreased. This is the first study showing that il1b and cox-2 transcripts are upregulated following PTZ-induced seizures in zebrafish. In addition, we demonstrated the anticonvulsant effect of indomethacin based on (1) the inhibition of PTZ-induced c-fos transcription, (2) increase in seizure latency, and (3) decrease in the number of seizure-like behaviors. Furthermore, anti-inflammatory effect of indomethacin is clearly demonstrated by the downregulation of the mRNA expression of il1b and cox2b. Our results are supported by previous evidences suggesting that zebrafish is a suitable alternative for studying inflammation, seizures, and the effect of anti-inflammatory compounds on seizure suppression.

Production of SV40-derived vectors.

PubMed

Strayer, David S; Mitchell, Christine; Maier, Dawn A; Nichols, Carmen N

2010-06-01

Recombinant simian virus 40 (rSV40)-derived vectors are particularly useful for gene delivery to bone marrow progenitor cells and their differentiated derivatives, certain types of epithelial cells (e.g., hepatocytes), and central nervous system neurons and microglia. They integrate rapidly into cellular DNA to provide long-term gene expression in vitro and in vivo in both resting and dividing cells. Here we describe a protocol for production and purification of these vectors. These procedures require only packaging cells (e.g., COS-7) and circular vector genome DNA. Amplification involves repeated infection of packaging cells with vector produced by transfection. Cotransfection is not required in any step. Viruses are purified by centrifugation using discontinuous sucrose or cesium chloride (CsCl) gradients and resulting vectors are replication-incompetent and contain no detectable wild-type SV40 revertants. These approaches are simple, give reproducible results, and may be used to generate vectors that are deleted only for large T antigen (Tag), or for all SV40-coding sequences capable of carrying up to 5 kb of foreign DNA. These vectors are best applied to long-term expression of proteins normally encoded by mammalian cells or by viruses that infect mammalian cells, or of untranslated RNAs (e.g., RNA interference). The preparative approaches described facilitate application of these vectors and allow almost any laboratory to exploit their strengths for diverse gene delivery applications.

Effects of cell phone radiation on lipid peroxidation, glutathione and nitric oxide levels in mouse brain during epileptic seizure.

PubMed

Esmekaya, Meric Arda; Tuysuz, Mehmet Zahid; Tomruk, Arın; Canseven, Ayse G; Yücel, Engin; Aktuna, Zuhal; Keskil, Semih; Seyhan, Nesrin

2016-09-01

The objective of the this study was to evaluate the effects of cellular phone radiation on oxidative stress parameters and oxide levels in mouse brain during pentylenetetrazole (PTZ) induced epileptic seizure. Eight weeks old mice were used in the study. Animals were distributed in the following groups: Group I: Control group treated with PTZ, Group II: 15min cellular phone radiation+PTZ treatment+30min cellular phone radiation, Group III: 30min cellular phone radiation+PTZ treatment+30min cellular phone radiation. The RF radiation was produced by a 900MHz cellular phone. Lipid peroxidation, which is the indicator of oxidative stress was quantified by measuring the formation of thiobarbituric acid reactive substances (TBARS). The glutathione (GSH) levels were determined by the Ellman method. Tissue total nitric oxide (NOx) levels were obtained using the Griess assay. Lipid peroxidation and NOx levels of brain tissue increased significantly in group II and III compared to group I. On the contrary, GSH levels were significantly lower in group II and III than group I. However, no statistically significant alterations in any of the endpoints were noted between group II and Group III. Overall, the experimental findings demonstrated that cellular phone radiation may increase the oxidative damage and NOx level during epileptic activity in mouse brain. Copyright © 2016 Elsevier B.V. All rights reserved.

Protracted ethanol withdrawal in rats: Tolerance to the anxiolytic effects of diazepam and pentobarbital but not phenobarbital

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lai, H.; Prather, P.L.

1990-02-26

Anxiety is a common symptom during ethanol withdrawal contributing to its continuous abuse and alcoholism. Ethanol withdrawal in rats produces an interoceptive discriminative stimulus (IDS) similar to that produced by the anxiogenic drug pentylenetetrazol (PTZ). This stimulus peaks at 12 hours after last dose of ethanol and thereafter the IDS is detected for several days (protracted withdrawal) by sensitization to a probe drug. previously, the authors have shown that during the protracted withdrawal, the IDS is enhanced by GABA receptor antagonists suggesting alteration of brain GABA systems. This report provides further evidence that chronic ethanol alters GABAergic systems. Rats weremore » trained to discriminate PTZ (20 mg/kg, ip) from saline. Diazepam, pentobarbital and phenobarbital blocked the PTZ-IDS dose dependently. Ethanol, 4.5% w/v, was then given in a nutritionally complete diet for a week. On termination of the ethanol diet, rats exhibited signs and symptoms of withdrawal which returned to baseline within 3 days. During the protracted withdrawal period, the authors then redetermined the blockade of the PTZ-IDS. Significant tolerance was observed to the effectiveness of diazepam and pentobarbital, but not to phenobarbital. Since diazepam and pentobarbital produce significantly more enhancement of GABAergic activity than does phenobarbital, these data further suggest alteration of brain GABAergic systems during protracted withdrawal from ethanol.« less

Efficacy of a Combined Intracerebral and Systemic Gene Delivery Approach for the Treatment of a Severe Lysosomal Storage Disorder

PubMed Central

Spampanato, Carmine; De Leonibus, Elvira; Dama, Paola; Gargiulo, Annagiusi; Fraldi, Alessandro; Sorrentino, Nicolina Cristina; Russo, Fabio; Nusco, Edoardo; Auricchio, Alberto; Surace, Enrico M; Ballabio, Andrea

2011-01-01

Multiple sulfatase deficiency (MSD), a severe autosomal recessive disease is caused by mutations in the sulfatase modifying factor 1 gene (Sumf1). We have previously shown that in the Sumf1 knockout mouse model (Sumf1−/−) sulfatase activities are completely absent and, similarly to MSD patients, this mouse model displays growth retardation and early mortality. The severity of the phenotype makes MSD unsuitable to be treated by enzyme replacement or bone marrow transplantation, hence the importance of testing the efficacy of novel treatment strategies. Here we show that recombinant adeno-associated virus serotype 9 (rAAV9) vector injected into the cerebral ventricles of neonatal mice resulted in efficient and widespread transduction of the brain parenchyma. In addition, we compared a combined, intracerebral ventricles and systemic, administration of an rAAV9 vector encoding SUMF1 gene to the single administrations—either directly in brain, or systemic alone —in MSD mice. The combined treatment resulted in the global activation of sulfatases, near-complete clearance of glycosaminoglycans (GAGs) and decrease of inflammation in both the central nervous system (CNS) and visceral organs. Furthermore, behavioral abilities were improved by the combined treatment. These results underscore that the “combined” mode of rAAV9 vector administration is an efficient option for the treatment of severe whole-body disorders. PMID:21326216

Preclinical Potency and Biodistribution Studies of an AAV 5 Vector Expressing Human Interferon-β (ART-I02) for Local Treatment of Patients with Rheumatoid Arthritis

PubMed Central

Aalbers, Caroline J.; Bevaart, Lisette; Loiler, Scott; de Cortie, Karin; Wright, J. Fraser; Mingozzi, Federico; Tak, Paul P.; Vervoordeldonk, Margriet J.

2015-01-01

Introduction Proof of concept for local gene therapy for the treatment of arthritis with immunomodulatory cytokine interferon beta (IFN-β) has shown promising results in animal models of rheumatoid arthritis (RA). For the treatment of RA patients, we engineered a recombinant adeno-associated serotype 5 vector (rAAV5) encoding human (h)IFN-β under control of a nuclear factor κB promoter (ART-I02). Methods The potency of ART-I02 in vitro as well as biodistribution in vivo in arthritic animals was evaluated to characterize the vector prior to clinical application. ART-I02 expression and bioactivity after transduction was evaluated in fibroblast-like synoviocytes (FLS) from different species. Biodistribution of the vector after local injection was assessed in a rat adjuvant arthritis model through qPCR analysis of vector DNA. In vivo imaging was used to investigate transgene expression and kinetics in a mouse collagen induced arthritis model. Results Transduction of RA FLS in vitro with ART-I02 resulted in high expression levels of bioactive hIFN-β. Transduction of FLS from rhesus monkeys, rodents and rabbits with ART-I02 showed high transgene expression, and hIFN-β proved bioactive in FLS from rhesus monkeys. Transgene expression and bioactivity in RA FLS were unaltered in the presence of methotrexate. In vivo, vector biodistribution analysis in rats after intra-articular injection of ART-I02 demonstrated that the majority of vector DNA remained in the joint (>93%). In vivo imaging in mice confirmed local expression of rAAV5 in the knee joint region and demonstrated rapid detectable and sustained expression up until 7 weeks. Conclusions These data show that hIFN-β produced by RA FLS transduced with ART-I02 is bioactive and that intra-articular delivery of rAAV5 drives expression of a therapeutic transgene in the joint, with only limited biodistribution of vector DNA to other tissues, supporting progress towards a phase 1 clinical trial for the local treatment of arthritis in patients with RA. PMID:26107769

Preclinical Potency and Biodistribution Studies of an AAV 5 Vector Expressing Human Interferon-β (ART-I02) for Local Treatment of Patients with Rheumatoid Arthritis.

PubMed

Aalbers, Caroline J; Bevaart, Lisette; Loiler, Scott; de Cortie, Karin; Wright, J Fraser; Mingozzi, Federico; Tak, Paul P; Vervoordeldonk, Margriet J

2015-01-01

Proof of concept for local gene therapy for the treatment of arthritis with immunomodulatory cytokine interferon beta (IFN-β) has shown promising results in animal models of rheumatoid arthritis (RA). For the treatment of RA patients, we engineered a recombinant adeno-associated serotype 5 vector (rAAV5) encoding human (h)IFN-β under control of a nuclear factor κB promoter (ART-I02). The potency of ART-I02 in vitro as well as biodistribution in vivo in arthritic animals was evaluated to characterize the vector prior to clinical application. ART-I02 expression and bioactivity after transduction was evaluated in fibroblast-like synoviocytes (FLS) from different species. Biodistribution of the vector after local injection was assessed in a rat adjuvant arthritis model through qPCR analysis of vector DNA. In vivo imaging was used to investigate transgene expression and kinetics in a mouse collagen induced arthritis model. Transduction of RA FLS in vitro with ART-I02 resulted in high expression levels of bioactive hIFN-β. Transduction of FLS from rhesus monkeys, rodents and rabbits with ART-I02 showed high transgene expression, and hIFN-β proved bioactive in FLS from rhesus monkeys. Transgene expression and bioactivity in RA FLS were unaltered in the presence of methotrexate. In vivo, vector biodistribution analysis in rats after intra-articular injection of ART-I02 demonstrated that the majority of vector DNA remained in the joint (>93%). In vivo imaging in mice confirmed local expression of rAAV5 in the knee joint region and demonstrated rapid detectable and sustained expression up until 7 weeks. These data show that hIFN-β produced by RA FLS transduced with ART-I02 is bioactive and that intra-articular delivery of rAAV5 drives expression of a therapeutic transgene in the joint, with only limited biodistribution of vector DNA to other tissues, supporting progress towards a phase 1 clinical trial for the local treatment of arthritis in patients with RA.

Tyrosine triple mutated AAV2-BDNF gene therapy in a rat model of transient IOP elevation

PubMed Central

Igarashi, Tsutomu; Kobayashi, Maika; Kameya, Shuhei; Fujimoto, Chiaki; Nakamoto, Kenji; Takahashi, Hisatomo; Igarashi, Toru; Miyake, Noriko; Iijima, Osamu; Hirai, Yukihiko; Shimada, Takashi; Okada, Takashi; Takahashi, Hiroshi

2016-01-01

Purpose We examined the neuroprotective effects of exogenous brain-derived neurotrophic factor (BDNF), which provides protection to retinal ganglion cells (RGCs) in rodents, in a model of transient intraocular pressure (IOP) elevation using a mutant (triple Y-F) self-complementary adeno-associated virus type 2 vector encoding BDNF (tm-scAAV2-BDNF). Methods The tm-scAAV2-BDNF or control vector encoding green fluorescent protein (GFP; tm-scAAV2-GFP) was intravitreally administered to rats, which were then divided into four groups: control, ischemia/reperfusion (I/R) injury only, I/R injury with tm-scAAV2-GFP, and tm-scAAV2-BDNF. I/R injury was then induced by transiently increasing IOP, after which the rats were euthanized to measure the inner retinal thickness and cell counts in the RGC layer. Results Intravitreous injection of tm-scAAV2-BDNF resulted in high levels of BDNF expression in the neural retina. Histological analysis showed that the inner retinal thickness and cell numbers in the RGC layer were preserved after transient IOP elevation in eyes treated with tm-scAAV2-BDNF but not in the other I/R groups. Significantly reduced glial fibrillary acidic protein (GFAP) immunostaining after I/R injury in the rats that received tm-scAAV2-BDNF indicated reduced retinal stress, and electroretinogram (ERG) analysis confirmed preservation of retinal function in the tm-scAAV2-BDNF group. Conclusions These results demonstrate the feasibility and effectiveness of neuroprotective gene therapy using tm-scAAV2-BDNF to protect the inner retina from transiently high intraocular pressure. An in vivo gene therapeutic approach to the clinical management of retinal diseases in conditions such as glaucoma, retinal artery occlusion, hypertensive retinopathy, and diabetic retinopathy thus appears feasible. PMID:27440998

Highly selective SGLT2 inhibitor dapagliflozin reduces seizure activity in pentylenetetrazol-induced murine model of epilepsy.

PubMed

Erdogan, Mumin Alper; Yusuf, Dimas; Christy, Joanna; Solmaz, Volkan; Erdogan, Arife; Taskiran, Emin; Erbas, Oytun

2018-06-07

Worldwide, over 10 million individuals suffer from drug-resistant epilepsy. New therapeutic strategies are needed to address this debilitating disease. Inhibition of sodium-glucose linked transporters (SGLTs), which are variably expressed in the brain, has been demonstrated to reduce seizure activity in murine models of epilepsy. Here we investigated the effects of dapagliflozin, a highly competitive SGLT2 inhibitor currently used as a drug for diabetes mellitus, on seizure activity in rats with pentylenetetrazol (PTZ) induced seizures. Laboratory rats (n = 48) were evenly randomized into two experiments, each with four study arms: (1) a vehicle-treated (placebo) arm infused with saline; (2) a control arm infused with PTZ; (3) a treatment arm with PTZ and dapagliflozin at 75 mg/kg, and (4) another treatment arm with PTZ and dapagliflozin at 150 mg/kg. Study subjects were assessed for seizures either via EEG as measured by spike wave percentage (SWP), or clinically via Racine's scales scores (RSS) and time to first myoclonic jerk (TFMJ). Rats treated with dapagliflozin had lower mean SWP on EEG (20.4% versus 75.3% for untreated rats). Behaviorally, treatment with dapagliflozin improved means RSS (2.33 versus 5.5) and mean TFMJ (68.3 versus 196.7 s). All of these findings were statistically significant with p-values of < 0.0001. There was a trend towards even better seizure control with the higher dose of dapagliflozin at 150 mg/kg, however this was not consistently statistically significant. Dapagliflozin decreased seizure activity in rats with PTZ-induced seizures. This may be explained by the anti-seizure effects of decreased glucose availability and a reduction in sodium transport across neuronal membranes which can confer a stabilizing effect against excitability and unwanted depolarization. The potential clinical role of dapagliflozin and other SGLT2 inhibitors as anti-seizure medications should be further explored.

Transcriptomic Analysis in a Drosophila Model Identifies Previously Implicated and Novel Pathways in the Therapeutic Mechanism in Neuropsychiatric Disorders

PubMed Central

Singh, Priyanka; Mohammad, Farhan; Sharma, Abhay

2011-01-01

We have taken advantage of a newly described Drosophila model to gain insights into the potential mechanism of antiepileptic drugs (AEDs), a group of drugs that are widely used in the treatment of several neurological and psychiatric conditions besides epilepsy. In the recently described Drosophila model that is inspired by pentylenetetrazole (PTZ) induced kindling epileptogenesis in rodents, chronic PTZ treatment for 7 days causes a decreased climbing speed and an altered CNS transcriptome, with the latter mimicking gene expression alterations reported in epileptogenesis. In the model, an increased climbing speed is further observed 7 days after withdrawal from chronic PTZ. We used this post-PTZ withdrawal regime to identify potential AED mechanism. In this regime, treatment with each of the five AEDs tested, namely, ethosuximide, gabapentin, vigabatrin, sodium valproate, and levetiracetam, resulted in rescuing of the altered climbing behavior. The AEDs also normalized PTZ withdrawal induced transcriptomic perturbation in fly heads; whereas AED untreated flies showed a large number of up- and down-regulated genes which were enriched in several processes including gene expression and cell communication, the AED treated flies showed differential expression of only a small number of genes that did not enrich gene expression and cell communication processes. Gene expression and cell communication related upregulated genes in AED untreated flies overrepresented several pathways – spliceosome, RNA degradation, and ribosome in the former category, and inositol phosphate metabolism, phosphatidylinositol signaling, endocytosis, and hedgehog signaling in the latter. Transcriptome remodeling effect of AEDs was overall confirmed by microarray clustering that clearly separated the profiles of AED treated and untreated flies. Besides being consistent with previously implicated pathways, our results provide evidence for a role of other pathways in psychiatric drug mechanism. Overall, we provide an amenable model to understand neuropsychiatric mechanism in cellular and molecular terms. PMID:21503142

Antiepileptic and antipsychotic activities of standardized Śilājatu (Shilajit) in experimental animals

PubMed Central

Durg, Sharanbasappa; Veerapur, Veeresh P.; Thippeswamy, B. S.; Ahamed, Syed Mansoor

2015-01-01

Background: Śilājatu (Shilajit; SJ) is claimed in traditional Indian medical practice to be useful in the treatment of nervous disorders, epilepsy and as antistress. Aim: To investigate whether SJ possesses antiepileptic and antipsychotic activities in rodents. Materials and Methods: Isonicotinyl hydrazine (INH), pentylenetetrazole (PTZ), apomorphine, phenytoin, diazepam, haloperidol and other chemicals of analytical grade were procured from standard companies. The antiepileptic activity of SJ was assessed using maximal electro shock (MES)-induced seizures in rats, INH and PTZ-induced seizures in mice. The antipsychotic effect of SJ was evaluated using apomorphine-induced climbing and stereotyped behaviours respectively, in mice and rats. Settings and Designs: SJ (25 and 50 mg/kg, p.o.) was given orally once daily for 15 days in all the rodent models. On the test day, SJ was administered 1 h prior to electric shock or chemical inducers (INH/PTZ/apomorphine) in experimental animals; the animals were then observed for different phases of seizures and psychotic behaviours. In addition, gamma-aminobutyric acid (GABA) content in the brain of rats and mice was estimated in seizure models. Statistical Analysis: The data were expressed as mean ± standard error of mean. Statistical comparisons were performed by one-way ANOVA followed by Tukey's post-test using Graph Pad Prism version 5.0, USA. A P < 0.05 was considered significant. Results and Conclusions: SJ pretreatment significantly inhibited the seizures induced by MES, INH and PTZ in a dose dependent manner. Further, SJ augmented brain GABA levels to normal, decreased by INH and PTZ in mice brain. SJ pretreatment also significantly inhibited the climbing and stereotyped behaviours induced by apomorphine. The present data seems to confirm the antiepileptic activity of SJ which may be because of enhancing the GABAergic system. The antipsychotic activity observed may be due to anti-dopaminergic and/or GABA-mimetic actions. PMID:26865744

Harnessing endogenous miR-181a to segregate transgenic antigen receptor expression in developing versus post-thymic T cells in murine hematopoietic chimeras.

PubMed

Papapetrou, Eirini P; Kovalovsky, Damian; Beloeil, Laurent; Sant'angelo, Derek; Sadelain, Michel

2009-01-01

MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression by targeting complementary sequences, referred to as miRNA recognition elements (MREs), typically located in the 3' untranslated region of mRNAs. miR-181a is highly expressed in developing thymocytes and markedly downregulated in post-thymic T cells. We investigated whether endogenous miR-181a can be harnessed to segregate expression of chimeric antigen receptors (CARs) and TCRs between developing and mature T cells. Lentiviral-encoded antigen receptors were tagged with a miR-181a-specific MRE and transduced into mouse BM cells that were used to generate hematopoietic chimeras. Expression of a CAR specific for human CD19 (hCD19) was selectively suppressed in late double-negative and double-positive thymocytes, coinciding with the peak in endogenous miR-181a expression. Receptor expression was fully restored in post-thymic resting and activated T cells, affording protection against a subsequent challenge with hCD19+ tumors. Hematopoietic mouse chimeras engrafted with a conalbumin-specific TCR prone to thymic clonal deletion acquired peptide-specific T cell responsiveness only when the vector-encoded TCR transcript was similarly engineered to be subject to regulation by miR-181a. These results demonstrate the potential of miRNA-regulated transgene expression in stem cell-based therapies, including cancer immunotherapy.

Vector adaptive predictive coder for speech and audio

NASA Technical Reports Server (NTRS)

Chen, Juin-Hwey (Inventor); Gersho, Allen (Inventor)

1990-01-01

A real-time vector adaptive predictive coder which approximates each vector of K speech samples by using each of M fixed vectors in a first codebook to excite a time-varying synthesis filter and picking the vector that minimizes distortion. Predictive analysis for each frame determines parameters used for computing from vectors in the first codebook zero-state response vectors that are stored at the same address (index) in a second codebook. Encoding of input speech vectors s.sub.n is then carried out using the second codebook. When the vector that minimizes distortion is found, its index is transmitted to a decoder which has a codebook identical to the first codebook of the decoder. There the index is used to read out a vector that is used to synthesize an output speech vector s.sub.n. The parameters used in the encoder are quantized, for example by using a table, and the indices are transmitted to the decoder where they are decoded to specify transfer characteristics of filters used in producing the vector s.sub.n from the receiver codebook vector selected by the vector index transmitted.

Variants of glycoside hydrolases

DOEpatents

Teter, Sarah; Ward, Connie; Cherry, Joel; Jones, Aubrey; Harris, Paul; Yi, Jung

2013-02-26

The present invention relates to variants of a parent glycoside hydrolase, comprising a substitution at one or more positions corresponding to positions 21, 94, 157, 205, 206, 247, 337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513 of SEQ ID NO: 2, and optionally further comprising a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2 a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2, wherein the variants have glycoside hydrolase activity. The present invention also relates to nucleotide sequences encoding the variant glycoside hydrolases and to nucleic acid constructs, vectors, and host cells comprising the nucleotide sequences.

Variants of glycoside hydrolases

DOEpatents

Teter, Sarah [Davis, CA; Ward, Connie [Hamilton, MT; Cherry, Joel [Davis, CA; Jones, Aubrey [Davis, CA; Harris, Paul [Carnation, WA; Yi, Jung [Sacramento, CA

2011-04-26

The present invention relates to variants of a parent glycoside hydrolase, comprising a substitution at one or more positions corresponding to positions 21, 94, 157, 205, 206, 247, 337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513 of SEQ ID NO: 2, and optionally further comprising a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2 a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2, wherein the variants have glycoside hydrolase activity. The present invention also relates to nucleotide sequences encoding the variant glycoside hydrolases and to nucleic acid constructs, vectors, and host cells comprising the nucleotide sequences.

Variants of glycoside hydrolases

DOEpatents

Teter, Sarah; Ward, Connie; Cherry, Joel; Jones, Aubrey; Harris, Paul; Yi, Jung

2017-07-11

The present invention relates to variants of a parent glycoside hydrolase, comprising a substitution at one or more positions corresponding to positions 21, 94, 157, 205, 206, 247, 337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513 of SEQ ID NO: 2, and optionally further comprising a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2 a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2, wherein the variants have glycoside hydrolase activity. The present invention also relates to nucleotide sequences encoding the variant glycoside hydrolases and to nucleic acid constructs, vectors, and host cells comprising the nucleotide sequences.

Comparative seroprevalence and immunogenicity of six rare serotype recombinant adenovirus vaccine vectors from subgroups B and D.

PubMed

Abbink, Peter; Lemckert, Angelique A C; Ewald, Bonnie A; Lynch, Diana M; Denholtz, Matthew; Smits, Shirley; Holterman, Lennart; Damen, Irma; Vogels, Ronald; Thorner, Anna R; O'Brien, Kara L; Carville, Angela; Mansfield, Keith G; Goudsmit, Jaap; Havenga, Menzo J E; Barouch, Dan H

2007-05-01

Recombinant adenovirus serotype 5 (rAd5) vector-based vaccines are currently being developed for both human immunodeficiency virus type 1 and other pathogens. The potential limitations associated with rAd5 vectors, however, have led to the construction of novel rAd vectors derived from rare Ad serotypes. Several rare serotype rAd vectors have already been described, but a detailed comparison of multiple rAd vectors from subgroups B and D has not previously been reported. Such a comparison is critical for selecting optimal rAd vectors for advancement into clinical trials. Here we describe the construction of three novel rAd vector systems from Ad26, Ad48, and Ad50. We report comparative seroprevalence and immunogenicity studies involving rAd11, rAd35, and rAd50 vectors from subgroup B; rAd26, rAd48, and rAd49 vectors from subgroup D; and rAd5 vectors from subgroup C. All six rAd vectors from subgroups B and D exhibited low seroprevalence in a cohort of 200 individuals from sub-Saharan Africa, and they elicited Gag-specific cellular immune responses in mice both with and without preexisting anti-Ad5 immunity. The rAd vectors from subgroup D were also evaluated using rhesus monkeys and were shown to be immunogenic after a single injection. The rAd26 vectors proved the most immunogenic among the rare serotype rAd vectors studied, although all rare serotype rAd vectors were still less potent than rAd5 vectors in the absence of anti-Ad5 immunity. These studies substantially expand the portfolio of rare serotype rAd vectors that may prove useful as vaccine vectors for the developing world.

Progress on adenovirus-vectored universal influenza vaccines.

PubMed

Xiang, Kui; Ying, Guan; Yan, Zhou; Shanshan, Yan; Lei, Zhang; Hongjun, Li; Maosheng, Sun

2015-01-01

Influenza virus (IFV) infection causes serious health problems and heavy financial burdens each year worldwide. The classical inactivated influenza virus vaccine (IIVV) and live attenuated influenza vaccine (LAIV) must be updated regularly to match the new strains that evolve due to antigenic drift and antigenic shift. However, with the discovery of broadly neutralizing antibodies that recognize conserved antigens, and the CD8(+) T cell responses targeting viral internal proteins nucleoprotein (NP), matrix protein 1 (M1) and polymerase basic 1 (PB1), it is possible to develop a universal influenza vaccine based on the conserved hemagglutinin (HA) stem, NP, and matrix proteins. Recombinant adenovirus (rAd) is an ideal influenza vaccine vector because it has an ideal stability and safety profile, induces balanced humoral and cell-mediated immune responses due to activation of innate immunity, provides 'self-adjuvanting' activity, can mimic natural IFV infection, and confers seamless protection against mucosal pathogens. Moreover, this vector can be developed as a low-cost, rapid-response vaccine that can be quickly manufactured. Therefore, an adenovirus vector encoding conserved influenza antigens holds promise in the development of a universal influenza vaccine. This review will summarize the progress in adenovirus-vectored universal flu vaccines and discuss future novel approaches.

Mild overexpression of Mecp2 in mice causes a higher susceptibility toward seizures.

PubMed

Bodda, Chiranjeevi; Tantra, Martesa; Mollajew, Rustam; Arunachalam, Jayamuruga P; Laccone, Franco A; Can, Karolina; Rosenberger, Albert; Mironov, Sergej L; Ehrenreich, Hannelore; Mannan, Ashraf U

2013-07-01

An intriguing finding about the gene encoding methyl-CpG binding protein 2 (MeCP2) is that the loss-of-function mutations cause Rett syndrome and duplication (gain-of-function) of MECP2 leads to another neurological disorder termed MECP2 duplication syndrome. To ensure proper neurodevelopment, a precise regulation of MeCP2 expression is critical, and any gain or loss of MeCP2 over a narrow threshold level may lead to postnatal neurological impairment. To evaluate MeCP2 dosage effects, we generated Mecp2(WT_EGFP) transgenic (TG) mouse in which MeCP2 (endogenous plus TG) is mildly overexpressed (approximately 1.5×). The TG MeCP2(WT_EGFP) fusion protein is functionally active, as cross breeding of these mice with Mecp2 knockout mice led to alleviation of major phenotypes in the null mutant mice, including premature lethality. To characterize the Mecp2(WT_EGFP) mouse model, we performed an extensive battery of behavioral tests, which revealed that these mice manifest increased aggressiveness and higher pentylenetetrazole (PTZ)-induced seizure propensity. Evaluation of neuronal parameters revealed a reduction in the number of tertiary branching sites and increased spine density in Mecp2(WT_EGFP) transgenic (TG) neurons. Treatment of TG neurons with epileptogenic compound-PTZ led to a marked increase in amplitude and frequency of calcium spikes. Based on our ex vivo and in vivo data, we conclude that epileptic seizures are manifested as the first symptom when MeCP2 is mildly overexpressed in mice. Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Reconstruction of an 8-lead surface ECG from two subcutaneous ICD vectors.

PubMed

Wilson, David G; Cronbach, Peter L; Panfilo, D; Greenhut, Saul E; Stegemann, Berthold P; Morgan, John M

2017-06-01

Techniques exist which allow surface ECGs to be reconstructed from reduced lead sets. We aimed to reconstruct an 8-lead ECG from two independent S-ICD sensing electrodes vectors as proof of this principle. Participants with ICDs (N=61) underwent 3minute ECGs using a TMSi Porti7 multi-channel signal recorder (TMS international, The Netherlands) with electrodes in the standard S-ICD and 12-lead positions. Participants were randomised to either a training (N=31) or validation (N=30) group. The transformation used was a linear combination of the 2 independent S-ICD vectors to each of the 8 independent leads of the 12-lead ECG, with coefficients selected that minimized the root mean square error (RMSE) between recorded and derived ECGs when applied to the training group. The transformation was then applied to the validation group and agreement between the recorded and derived lead pairs was measured by Pearson correlation coefficient (r) and normalised RMSE (NRMSE). In total, 27 patients with complete data sets were included in the validation set consisting of 57,888 data points from 216 full lead sets. The distribution of the r and NRMSE were skewed. Mean r=0.770 (SE 0.024), median r=0.925. NRMSE mean=0.233 (SE 0.015) median=0.171. We have demonstrated that the reconstruction of an 8-lead ECG from two S-ICD vectors is possible. If perfected, the ability to generate accurate multi-lead surface ECG data from an S-ICD would potentially allow recording and review of clinical arrhythmias at follow-up. Copyright © 2017 Elsevier B.V. All rights reserved.

Dynamic calibration of pan-tilt-zoom cameras for traffic monitoring.

PubMed

Song, Kai-Tai; Tai, Jen-Chao

2006-10-01

Pan-tilt-zoom (PTZ) cameras have been widely used in recent years for monitoring and surveillance applications. These cameras provide flexible view selection as well as a wider observation range. This makes them suitable for vision-based traffic monitoring and enforcement systems. To employ PTZ cameras for image measurement applications, one first needs to calibrate the camera to obtain meaningful results. For instance, the accuracy of estimating vehicle speed depends on the accuracy of camera calibration and that of vehicle tracking results. This paper presents a novel calibration method for a PTZ camera overlooking a traffic scene. The proposed approach requires no manual operation to select the positions of special features. It automatically uses a set of parallel lane markings and the lane width to compute the camera parameters, namely, focal length, tilt angle, and pan angle. Image processing procedures have been developed for automatically finding parallel lane markings. Interesting experimental results are presented to validate the robustness and accuracy of the proposed method.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sirunyan, Albert M; et al.

The production of jets in association with Z bosons, reconstructed via the mu+mu- and e+e- decay channels, is studied in pp and, for the first time, in PbPb collisions. Both data samples were collected by the CMS experiment at the LHC, at a center-of-mass energy of 5.02 TeV. The PbPb collisions were analyzed in the 0-30% centrality range. The back-to-back azimuthal alignment was studied in both pp and PbPb collisions for Z bosons with transverse momentum ptz > 60 GeV/c and a recoiling jet with ptj > 30 GeV/c. The pt imbalance, xjz= ptj/ptz, as well as the average numbermore » of jet partners per Z, rjz, were studied in intervals of ptz, in both pp and PbPb collisions. The rjz is found to be smaller in PbPb than in pp collisions, which suggests that in PbPb collisions a larger fraction of partons, associated with the Z bosons, lose energy and fall below the 30 GeV/c ptj threshold.« less

Adeno-associated-virus-mediated transduction of the mammary gland enables sustained production of recombinant proteins in milk

PubMed Central

Wagner, Stefan; Thresher, Rosemary; Bland, Ross; Laible, Götz

2015-01-01

Biopharming for the production of recombinant pharmaceutical proteins in the mammary gland of transgenic animals is an attractive but laborious alternative compared to mammalian cell fermentation. The disadvantage of the lengthy process of genetically modifying an entire animal could be circumvented with somatic transduction of only the mammary epithelium with recombinant, replication-defective viruses. While other viral vectors offer very limited scope for this approach, vectors based on adeno-associated virus (AAV) appear to be ideal candidates because AAV is helper-dependent, does not induce a strong immune response and has no association with disease. Here, we sought to test the suitability of recombinant AAV (rAAV) for biopharming. Using reporter genes, we showed that injected rAAV efficiently transduced mouse mammary cells. When rAAV encoding human myelin basic protein (hMBP) was injected into the mammary glands of mice and rabbits, this resulted in the expression of readily detectable protein levels of up to 0.5 g/L in the milk. Furthermore we demonstrated that production of hMBP persisted over extended periods and that protein expression could be renewed in a subsequent lactation by re-injection of rAAV into a previously injected mouse gland. PMID:26463440

Error control techniques for satellite and space communications

NASA Technical Reports Server (NTRS)

Costello, Daniel J., Jr.

1994-01-01

The unequal error protection capabilities of convolutional and trellis codes are studied. In certain environments, a discrepancy in the amount of error protection placed on different information bits is desirable. Examples of environments which have data of varying importance are a number of speech coding algorithms, packet switched networks, multi-user systems, embedded coding systems, and high definition television. Encoders which provide more than one level of error protection to information bits are called unequal error protection (UEP) codes. In this work, the effective free distance vector, d, is defined as an alternative to the free distance as a primary performance parameter for UEP convolutional and trellis encoders. For a given (n, k), convolutional encoder, G, the effective free distance vector is defined as the k-dimensional vector d = (d(sub 0), d(sub 1), ..., d(sub k-1)), where d(sub j), the j(exp th) effective free distance, is the lowest Hamming weight among all code sequences that are generated by input sequences with at least one '1' in the j(exp th) position. It is shown that, although the free distance for a code is unique to the code and independent of the encoder realization, the effective distance vector is dependent on the encoder realization.

Effects of transcranial focal electrical stimulation via tripolar concentric ring electrodes on pentylenetetrazole-induced seizures in rats

PubMed Central

Besio, W.G.; Makeyev, O.; Medvedev, A.; Gale, K.

2013-01-01

Purpose To study the effects of noninvasive transcranial focal electrical stimulation (TFS) via tripolar concentric ring electrodes (TCRE) on the electrographic and behavioral activity from pentylenetetrazole (PTZ)-induced seizures in rats. Methods The TCREs were attached to the rat scalp. PTZ was administered and, after the first myoclonic jerk was observed, TFS was applied to the TFS treated group. The electroencephalogram (EEG) and behavioral activity were recorded and studied. Results In the case of the TFS treated group, after TFS, there was a significant (p = 0.001) decrease in power compared to the control group in delta, theta, and alpha frequency bands. The number of myoclonic jerks was significantly different (p = 0.002) with median of 22 and 4.5 for the control group and the TFS treated groups, respectively. The duration of myoclonic activity was also significantly different (p= 0.031) with median of 17.56 min for the control group versus 8.63 min for the TFS treated group. At the same time there was no significant difference in seizure onset latency and maximal behavioral seizure activity score between control and TFS treated groups. Conclusions TFS via TCREs interrupted PTZ-induced seizures and electrographic activity was reduced towards the “baseline.” The significantly reduced electrographic power, number of myoclonic jerks, and duration of myoclonic activity of PTZ-induced seizures suggests that TFS may have an anticonvulsant effect. PMID:23290195

Effect of lamotrigine, oxcarbazepine and topiramate on cognitive functions and oxidative stress in PTZ-kindled mice.

PubMed

Agarwal, Nidhi Bharal; Agarwal, Nitin Kumar; Mediratta, Pramod Kumari; Sharma, Krishna Kishore

2011-04-01

Cognitive impairment is frequently observed in epileptic patients. It has been seen that not only epilepsy but antiepileptic drugs also impair cognitive functions. The present study was undertaken to assess the effect of three anticonvulsants viz. lamotrigine (5mg/kg, p.o.), oxcarbazepine (15mg/kg, p.o.) and topiramate (10mg/kg, p.o.) on cognitive function and oxidative stress during pentylenetetrazole (PTZ)-kindling in mice. Kindling was induced by the administration of PTZ (25mg/kg, i.p.) on every alternate day till 5 weeks. Cognition was assessed after the development of kindling. Elevated plus maze (EPM) and passive avoidance response (PAR) tests were carried out after 24h and 48h of the last PTZ administration. After completion of behavioural tests malondialdehyde (MDA), glutathione levels, superoxide dismutase and catalase activity were measured as an indicator of oxidative stress. The results of the present study indicate that topiramate (10mg/kg) administration to kindled animals increased transfer latency and decreased step-down latency in EPM and PAR tests, respectively. However, lamotrigine and oxcarbazepine did not alter the two parameters. Topiramate administration to kindled as well as non-kindled animals has shown increase in MDA and decrease in glutathione levels. Lamotrigine and oxcarbazepine did not show significant alteration in oxidative stress parameters. To conclude, long term administration of topiramate impairs cognitive functions during experimental epilepsy while lamotrigine and oxcarbazepine are safer. Copyright © 2010 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.

Ferulic acid exhibits antiepileptogenic effect and prevents oxidative stress and cognitive impairment in the kindling model of epilepsy.

PubMed

Hassanzadeh, Parichehr; Arbabi, Elham; Atyabi, Fatemeh; Dinarvand, Rassoul

2017-06-15

Some conventional antiepileptic drugs induce oxidative stress and cognitive impairment which may limit their clinical applications. Ferulic acid is a phenolic phytochemical with antioxidant and neuroprotective properties that prompted us to evaluate its therapeutic potential in epilepsy which is usually associated with oxidative stress and cognitive decline. Male Wistar rats received 30mg/kg of pentylenetetrazole (PTZ) intraperitoneally (i.p.) once every alternate day until the development of kindling. The locomotor activity, elevated plus maze, and passive avoidance tests were performed. Oxidative stress was evaluated by the determination of brain malondialdehyde and reduced glutathione. The effects of pre-treatment with ferulic acid (25, 50, 75, and 100mg/kg, i.p.) against PTZ-kindled seizures, cognitive impairment, and oxidative stress were investigated. Kindling was developed 34.18±1.54days after PTZ treatment which was associated with generalized tonic-clonic seizures (GTCS), myoclonic jerks, cognitive deficit, and oxidative stress. Ferulic acid at doses of 75 and 100mg/kg significantly reduced the seizure score, number of myoclonic jerks, cognitive decline and oxidative stress. Spontaneous locomotor activity did not significantly differ between the groups. Ferulic acid exhibits antiepileptogenic effect and prevents oxidative stress and cognitive impairment induced by PTZ kindling. Therefore, this phenolic phytochemical appears as a promising adjuvant for antiepileptic drugs. Meanwhile, further experimental and clinical studies are required to provide insights into the cellular/molecular mechanism(s) underlying the action of ferulic acid. Copyright © 2016 Elsevier Inc. All rights reserved.

Interference of TRPV1 function altered the susceptibility of PTZ-induced seizures.

PubMed

Jia, Yun-Fang; Li, Ying-Chao; Tang, Yan-Ping; Cao, Jun; Wang, Li-Ping; Yang, Yue-Xiong; Xu, Lin; Mao, Rong-Rong

2015-01-01

Transient receptor potential vanilloid 1 (TRPV1) is widely distributed in the central nervous system (CNS) including hippocampus, and regulates the balance of excitation and inhibition in CNS, which imply its important role in epilepsy. We used both pharmacological manipulations and transgenic mice to disturb the function of TRPV1 and then studied the effects of these alterations on the susceptibility of pentylenetetrazol (PTZ)-induced seizures. Our results showed that systemic administration of TRPV1 agonist capsaicin (CAP, 40 mg/kg) directly induced tonic-clonic seizures (TCS) without PTZ induction. The severity of seizure was increased in lower doses of CAP groups (5 and 10 mg/kg), although the latency to TCS was delayed. On the other hand, systemic administration of TRPV1 antagonist capsazepine (CPZ, 0.05 and 0.5 mg/kg) and TRPV1 knockout mice exhibited delayed latency to TCS and reduced mortality. Furthermore, hippocampal administration of CPZ (10 and 33 nmol/μL/side) was firstly reported to increase the latency to TCS, decrease the maximal grade of seizure and mortality. It is worth noting that decreased susceptibility of PTZ-induced seizures was observed in hippocampal TRPV1 overexpression mice and hippocampal CAP administration (33 nmol/μL/side), which is opposite from results of systemic agonist CAP. Our findings suggest that the systemic administration of TRPV1 antagonist may be a novel therapeutic target for epilepsy, and alteration of hippocampal TRPV1 function exerts a critical role in seizure susceptibility.

Effects of transcranial focal electrical stimulation via tripolar concentric ring electrodes on pentylenetetrazole-induced seizures in rats.

PubMed

Besio, W G; Makeyev, O; Medvedev, A; Gale, K

2013-07-01

To study the effects of noninvasive transcranial focal electrical stimulation (TFS) via tripolar concentric ring electrodes (TCRE) on the electrographic and behavioral activity from pentylenetetrazole (PTZ)-induced seizures in rats. The TCREs were attached to the rat scalp. PTZ was administered and, after the first myoclonic jerk was observed, TFS was applied to the TFS treated group. The electroencephalogram (EEG) and behavioral activity were recorded and studied. In the case of the TFS treated group, after TFS, there was a significant (p=0.001) decrease in power compared to the control group in delta, theta, and alpha frequency bands. The number of myoclonic jerks was significantly different (p=0.002) with median of 22 and 4.5 for the control group and the TFS treated groups, respectively. The duration of myoclonic activity was also significantly different (p=0.031) with median of 17.56 min for the control group versus 8.63 min for the TFS treated group. At the same time there was no significant difference in seizure onset latency and maximal behavioral seizure activity score between control and TFS treated groups. TFS via TCREs interrupted PTZ-induced seizures and electrographic activity was reduced toward the "baseline." The significantly reduced electrographic power, number of myoclonic jerks, and duration of myoclonic activity of PTZ-induced seizures suggests that TFS may have an anticonvulsant effect. Copyright © 2012 Elsevier B.V. All rights reserved.

Involvement of ATP-sensitive potassium channels and the opioid system in the anticonvulsive effect of zolpidem in mice.

PubMed

Sheikhi, Mehdi; Shirzadian, Armin; Dehdashtian, Amir; Amiri, Shayan; Ostadhadi, Sattar; Ghasemi, Mehdi; Dehpour, Ahmad Reza

2016-09-01

Zolpidem is a hypnotic medication that mainly exerts its function through activating γ-aminobutyric acid (GABA)A receptors. There is some evidence that zolpidem may have anticonvulsive effects. However, the mechanisms underlying this effect have not been elucidated yet. In the present study, we used the pentylentetrazole (PTZ)-induced generalized seizure model in mice to investigate whether zolpidem can affect seizure threshold. We also further evaluated the roles of ATP-sensitive potassium (KATP) channels as well as μ-opioid receptors in the effects of zolpidem on seizure threshold. Our data showed that zolpidem in a dose-dependent manner increased the PTZ-induced seizure threshold. The noneffective (i.e., did not significantly alter the PTZ-induced seizure threshold by itself) doses of KATP channel blocker (glibenclamide) and nonselective opioid receptor antagonist (naloxone) were able to inhibit the anticonvulsive effect of zolpidem. Additionally, noneffective doses of either KATP channel opener (cromakalim) or nonselective μ-opioid receptor agonist (morphine) in combination with a noneffective dose of zolpidem exerted a significant anticonvulsive effect on PTZ-induced seizures in mice. A combination of noneffective doses of naloxone and glibenclamide, which separately did not affect zolpidem effect on seizure threshold, inhibited the anticonvulsive effects of zolpidem. These results suggest a role for KATP channels and the opioid system, alone or in combination, in the anticonvulsive effects of zolpidem. Copyright © 2016 Elsevier Inc. All rights reserved.

A larval zebrafish model of bipolar disorder as a screening platform for neuro-therapeutics.

PubMed

Ellis, Lee David; Soanes, Kelly Howard

2012-08-01

Modelling neurological diseases has proven extraordinarily difficult due to the phenotypic complexity of each disorder. The zebrafish has become a useful model system with which to study abnormal neurological and behavioural activity and holds promise as a model of human disease. While most of the disease modelling using zebrafish has made use of adults, larvae hold tremendous promise for the high-throughput screening of potential therapeutics. The further development of larval disease models will strengthen their ability to contribute to the drug screening process. Here we have used zebrafish larvae to model the symptoms of bipolar disorder by treating larvae with sub-convulsive concentrations of the GABA antagonist pentylenetetrazol (PTZ). A number of therapeutics that act on different targets, in addition to those that have been used to treat bipolar disorder, were tested against this model to assess its predictive value. Carbamazepine, valproic acid, baclofen and honokiol, were found to oppose various aspects of the PTZ-induced changes in activity. Lidocaine and haloperidol exacerbated the PTZ-induced activity changes and sulpiride had no effect. By comparing the degree of phenotypic rescue with the mechanism of action of each therapeutic we have shown that the low-concentration PTZ model can produce a number of intermediate phenotypes that model symptoms of bipolar disorder, may be useful in modelling other disease states, and will help predict the efficacy of novel therapeutics. Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.

Rhizomucor miehei triglyceride lipase is processed and secreted from transformed Aspergillus oryzae.

PubMed

Huge-Jensen, B; Andreasen, F; Christensen, T; Christensen, M; Thim, L; Boel, E

1989-09-01

The cDNA encoding the precursor of the Rhizomucor miehei triglyceride lipase was inserted in an Aspergillus oryzae expression vector. In this vector the expression of the lipase cDNA is under control of the Aspergillus oryzae alpha-amylase gene promoter and the Aspergillus niger glucoamylase gene terminator. The recombinant plasmid was introduced into Aspergillus oryzae, and transformed colonies were selected and screened for lipase expression. Lipase-positive transformants were grown in a small fermentor, and recombinant triglyceride lipase was purified from the culture broth. The purified enzymatically active recombinant lipase (rRML) secreted from A. oryzae was shown to have the same characteristics with respect to mobility on reducing SDS-gels and amino acid composition as the native enzyme. N-terminal amino acid sequencing indicated that approximately 70% of the secreted rRML had the same N-terminal sequence as the native Rhizomucor miehei enzyme, whereas 30% of the secreted rRML was one amino acid residue shorter in the N-terminal. The recombinant lipase precursor, which has a 70 amino acid propeptide, is thus processed in and secreted from Aspergillus oryzae. We have hereby demonstrated the utility of this organism as a host for the production of recombinant triglyceride lipases.

Isolation and characterization of a cDNA clone for the complete protein coding region of the delta subunit of the mouse acetylcholine receptor.

PubMed Central

LaPolla, R J; Mayne, K M; Davidson, N

1984-01-01

A mouse cDNA clone has been isolated that contains the complete coding region of a protein highly homologous to the delta subunit of the Torpedo acetylcholine receptor (AcChoR). The cDNA library was constructed in the vector lambda 10 from membrane-associated poly(A)+ RNA from BC3H-1 mouse cells. Surprisingly, the delta clone was selected by hybridization with cDNA encoding the gamma subunit of the Torpedo AcChoR. The nucleotide sequence of the mouse cDNA clone contains an open reading frame of 520 amino acids. This amino acid sequence exhibits 59% and 50% sequence homology to the Torpedo AcChoR delta and gamma subunits, respectively. However, the mouse nucleotide sequence has several stretches of high homology with the Torpedo gamma subunit cDNA, but not with delta. The mouse protein has the same general structural features as do the Torpedo subunits. It is encoded by a 3.3-kilobase mRNA. There is probably only one, but at most two, chromosomal genes coding for this or closely related sequences. Images PMID:6096870

Red-shifted fluorescent proteins mPlum and mRaspberry and polynucleotides encoding the same

DOEpatents

Tsien, Roger Y [La Jolla, CA; Wang, Lei [San Diego, CA

2008-07-01

Methods using somatic hypermutation (SHM) for producing polypeptide and nucleic acid variants, and nucleic acids encoding such polypeptide variants are disclosed. Such variants may have desired properties. Also disclosed are novel polypeptides, such as improved fluorescent proteins, produced by the novel methods, and nucleic acids, vectors, and host cells comprising such vectors.

DNA encoding for plant digalactosyldiacylglycerol galactosyltransferase and methods of use

DOEpatents

Benning, Christoph; Doermann, Peter

2003-11-04

The cDNA encoding digalactosyldiacylglycerol galactosyltransferase (DGD1) is provided. The deduced amino acid sequence is also provided. Methods of making and using DGD1 to screen for new herbicides and alter a plant's leaf lipid composition are also provided, as well as expression vectors, transgenic plants or other organisms transfected with said vectors.

Vector assembly of colloids on monolayer substrates

NASA Astrophysics Data System (ADS)

Jiang, Lingxiang; Yang, Shenyu; Tsang, Boyce; Tu, Mei; Granick, Steve

2017-06-01

The key to spontaneous and directed assembly is to encode the desired assembly information to building blocks in a programmable and efficient way. In computer graphics, raster graphics encodes images on a single-pixel level, conferring fine details at the expense of large file sizes, whereas vector graphics encrypts shape information into vectors that allow small file sizes and operational transformations. Here, we adapt this raster/vector concept to a 2D colloidal system and realize `vector assembly' by manipulating particles on a colloidal monolayer substrate with optical tweezers. In contrast to raster assembly that assigns optical tweezers to each particle, vector assembly requires a minimal number of optical tweezers that allow operations like chain elongation and shortening. This vector approach enables simple uniform particles to form a vast collection of colloidal arenes and colloidenes, the spontaneous dissociation of which is achieved with precision and stage-by-stage complexity by simply removing the optical tweezers.

Fusagene vectors: a novel strategy for the expression of multiple genes from a single cistron.

PubMed

Gäken, J; Jiang, J; Daniel, K; van Berkel, E; Hughes, C; Kuiper, M; Darling, D; Tavassoli, M; Galea-Lauri, J; Ford, K; Kemeny, M; Russell, S; Farzaneh, F

2000-12-01

Transduction of cells with multiple genes, allowing their stable and co-ordinated expression, is difficult with the available methodologies. A method has been developed for expression of multiple gene products, as fusion proteins, from a single cistron. The encoded proteins are post-synthetically cleaved and processed into each of their constituent proteins as individual, biologically active factors. Specifically, linkers encoding cleavage sites for the Golgi expressed endoprotease, furin, have been incorporated between in-frame cDNA sequences encoding different secreted or membrane bound proteins. With this strategy we have developed expression vectors encoding multiple proteins (IL-2 and B7.1, IL-4 and B7.1, IL-4 and IL-2, IL-12 p40 and p35, and IL-12 p40, p35 and IL-2 ). Transduction and analysis of over 100 individual clones, derived from murine and human tumour cell lines, demonstrate the efficient expression and biological activity of each of the encoded proteins. Fusagene vectors enable the co-ordinated expression of multiple gene products from a single, monocistronic, expression cassette.

Csk Homologous Kinase, a Potential Regulator of CXCR4-Medicated Breast Cancer Cell Metastasis

DTIC Science & Technology

2011-08-01

is a non-receptor tyrosine kinase and a second member of the Csk family. Like Csk, CHK has Src homology 2 ( SH2 ) and SH3 domains and lacks the...MSCV-retroviral vectors encoding either wild-type CHK or kinase -dead CHK or wild type SH2 domain or SH2 -R147A or SH2 -G129A. All these constructs were... Kinase , a Potential Regulator of CXCR4-Medicated Breast Cancer Cell Metastasis Byeong-Chel Lee The University of Pittsburgh Pittsburgh, PA 15213

Vesicular stomatitis virus-based vaccines protect nonhuman primates against Bundibugyo ebolavirus.

PubMed

Mire, Chad E; Geisbert, Joan B; Marzi, Andrea; Agans, Krystle N; Feldmann, Heinz; Geisbert, Thomas W

2013-01-01

Ebola virus (EBOV) causes severe and often fatal hemorrhagic fever in humans and nonhuman primates (NHPs). Currently, there are no licensed vaccines or therapeutics for human use. Recombinant vesicular stomatitis virus (rVSV)-based vaccine vectors, which encode an EBOV glycoprotein in place of the VSV glycoprotein, have shown 100% efficacy against homologous Sudan ebolavirus (SEBOV) or Zaire ebolavirus (ZEBOV) challenge in NHPs. In addition, a single injection of a blend of three rVSV vectors completely protected NHPs against challenge with SEBOV, ZEBOV, the former Côte d'Ivoire ebolavirus, and Marburg virus. However, recent studies suggest that complete protection against the newly discovered Bundibugyo ebolavirus (BEBOV) using several different heterologous filovirus vaccines is more difficult and presents a new challenge. As BEBOV caused nearly 50% mortality in a recent outbreak any filovirus vaccine advanced for human use must be able to protect against this new species. Here, we evaluated several different strategies against BEBOV using rVSV-based vaccines. Groups of cynomolgus macaques were vaccinated with a single injection of a homologous BEBOV vaccine, a single injection of a blended heterologous vaccine (SEBOV/ZEBOV), or a prime-boost using heterologous SEBOV and ZEBOV vectors. Animals were challenged with BEBOV 29-36 days after initial vaccination. Macaques vaccinated with the homologous BEBOV vaccine or the prime-boost showed no overt signs of illness and survived challenge. In contrast, animals vaccinated with the heterologous blended vaccine and unvaccinated control animals developed severe clinical symptoms consistent with BEBOV infection with 2 of 3 animals in each group succumbing. These data show that complete protection against BEBOV will likely require incorporation of BEBOV glycoprotein into the vaccine or employment of a prime-boost regimen. Fortunately, our results demonstrate that heterologous rVSV-based filovirus vaccine vectors employed in the prime-boost approach can provide protection against BEBOV using an abbreviated regimen, which may have utility in outbreak settings.

The effect of leptin, ghrelin, and neuropeptide-Y on serum Tnf-Α, Il-1β, Il-6, Fgf-2, galanin levels and oxidative stress in an experimental generalized convulsive seizure model.

PubMed

Oztas, Berrin; Sahin, Deniz; Kir, Hale; Eraldemir, Fatma Ceyla; Musul, Mert; Kuskay, Sevinç; Ates, Nurbay

2017-02-01

The objective of this study is to examine the effects of the endogenous ligands leptin, ghrelin, and neuropeptide Y (NPY) on seizure generation, the oxidant/antioxidant balance, and cytokine levels, which are a result of immune response in a convulsive seizure model. With this goal, Wistar rats were divided into 5 groups-Group 1: Saline, Group 2: Saline+PTZ (65mg/kg), Group 3: leptin (4mg/kg)+PTZ, Group 4: ghrelin (80μg/kg)+PTZ, and Group 5: NPY (60μg/kg)+PTZ. All injections were delivered intraperitoneally, and simultaneous electroencephalography (EEG) records were obtained. Seizure activity was scored by observing seizure behavior, and the onset time, latency, and seizure duration were determined according to the EEG records. At the end of the experiments, blood samples were obtained in all groups to assess the serum TNF-α, IL-1β, IL-6, FGF-2, galanin, nitric oxide (NOֹ), malondialdehyde (MDA), and glutathione (GSH) levels. The electrophysiological and biochemical findings (p<0.05) of this study show that all three peptides have anticonvulsant effects in the pentylenetetrazol (PTZ)-induced generalized tonic-clonic convulsive seizure model. The reduction of the levels of the pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 caused by leptin, ghrelin, and NPY shows that these peptides may have anti-inflammatory effects in epileptic seizures. Also, leptin significantly increases the serum levels of the endogenous anticonvulsive agent galanin. The fact that each one of these endogenous peptides reduces the levels of MDA and increases the serum levels of GSH leads to the belief that they may have protective effects against oxidative damage that is thought to play a role in the pathogenesis of epilepsy. Our study contributes to the clarification of the role of these peptides in the brain in seizure-induced oxidative stress and immune system physiology and also presents new approaches to the etiology and treatment of tendency to epileptic seizures. Copyright © 2016 Elsevier Ltd. All rights reserved.

Differential expression of the virulence-associated protein p57 and characterization of its duplicated gene rosa in virulent and attenuated strains of Renibacterium salmoninarum

USGS Publications Warehouse

O'Farrell, C. L.; Strom, M.S.

1999-01-01

Virulence mechanisms utilized by the salmonid fish pathogen Renibacterium salmoninarum are poorly understood. One potential virulence factor is p57 (also designated MSA for major soluble antigen), an abundant 57 kDa soluble protein that is predominately localized on the bacterial cell surface with significant levels released into the extracellular milieu. Previous studies of an attenuated strain, MT 239, indicated that it differs from virulent strains in the amount of surface-associated p57. In this report, we show overall expression of p57 in R. salmoninarum MT 239 is considerably reduced as compared to a virulent strain, ATCC 33209. The amount of cell-associated p57 is decreased while the level of p57 in the culture supernatant is nearly equivalent between the strains. To determine if lowered amount of cell-associated p57 was due to a sequence defect in p57, a genetic comparison was performed. Two copies of the gene encoding p57 (msa1 and msa2) were found in 33209 and MT 239, as well as in several other virulent isolates. Both copies from 33209 and MT 239 were cloned and sequenced and found to be identical to each other, and identical between the 2 strains. A comparison of msa1 and msa2 within each strain showed that their sequences diverge 40 base pairs 5, to the open reading frame, while sequences 3' to the open reading frame are essentially identical for at least 225 base pairs. Northern blot analysis showed no difference in steady state levels of rosa mRNA between the 2 strains. These data suggest that while cell-surface localization of p57 may be important for R. salmoninarum virulence, the differences in localization, and total p57 expression between 33209 anti MT 239 are not due to differences in rosa sequence or differences in steady state transcript levels.

Evaluation of anticonvulsant and nootropic effect of ondansetron in mice.

PubMed

Jain, S; Agarwal, N B; Mediratta, P K; Sharma, K K

2012-09-01

The role of serotonin receptors have been implicated in various types of experimentally induced seizures. Ondansetron is a highly selective 5-hydroxytryptamine 3 (5-HT(3)) receptor antagonist used as antiemetic agent for chemotherapy-, and radiotherapy-induced nausea and vomiting. The present study was carried out to examine the effect of ondansetron on electroshock, pentylenetetrazole (PTZ)-induced seizures and cognitive functions in mice. Ondansetron was administered intraperitoneally (i.p.) at doses of 0.5, 1.0 and 2.0 mg/kg (single dose) to observe its effect on the increasing current electroshock seizure (ICES) test and PTZ-induced seizure test. In addition, a chronic study (21 days) was also performed to assess the effects of ondansetron on electroshock-induced convulsions and cognitive functions. The effect on cognition was assessed by elevated plus maze and passive avoidance paradigms. Phenytoin (25 mg/kg, i.p.) was used as a standard anticonvulsant drug and piracetam (200 mg/kg) was administered as a standard nootropic drug. The results were compared with an acute study, wherein it was found that the administration of ondansetron (1.0 and 2.0 mg/kg) significantly raised the seizure-threshold current as compared to control group in the ICES test. Similar results were observed after chronic administration of ondansetron. In PTZ test, ondansetron in all the three tested doses failed to show protective effect against PTZ-induced seizure test. Administration of ondansetron for 21 days significantly decreased the transfer latency (TL) and prolonged the step-down latency (SDL). The results of present study suggest the anticonvulsant and memory-enhancing effect of ondansetron in mice.

Аnticonvulsant effects of citicoline and diazepam at their combined application on model of the acute generalized convulsions induced by pentylenetetrazole in Wistar rats.

PubMed

Kuznetsova, L V; Karpova, M N; Zinkovski, K A; Klishina, N Yu

2016-01-01

Studying of efficiency of the combined application of the citicoline possessing nootropic and anticonvulsive action and antiepileptic drug of diazepam on the acute generalized convulsions (AGC) caused by a convulsant pentylentetrazole (PTZ). Experiments are executed on the male Wistar rats (n = 68) weighing 160-190 g on the AGС model caused by of PTZ in a dose of 80 mg/kg, intraperitoneally (i.p.). For studying of efficiency of the combined use of drugs determined the minimum anticonvulsive action of a citicoline (Tserakson, «Nicomed Ferrer Internacional, S.A.») and diazepam (Relanium, Warsaw pharmaceutical plant of Polf AO, Warsaw, Poland). For this citicoline were administered i.p. in doses 500 and 300 mg/kg 1 hour before the PTZ and diazepam - in doses of 0,5 and 0,25 mg/kg 30 min before administration of PTZ. Control animals were injected with saline to the same extent and under the same experimental conditions. It is shown that the combined administration of a citicoline and diazepam in minimum active doses (300 and 0.25 mg/kg respectively), increases anticonvulsive properties of both drugs. The combined administration of citicoline with diazepam in minimally active doses enhances anticonvulsant properties of both drugs, thereby reducing the risk of development of side effects. In addition, the research may serve as experimental justification for the use of drugs in case of convulsions for the purpose beneficial effect on cognitive function and delays of progressing of neurodegenerative processes.

Acute administration of ginger (Zingiber officinale rhizomes) extract on timed intravenous pentylenetetrazol infusion seizure model in mice.

PubMed

Hosseini, Abdolkarim; Mirazi, Naser

2014-03-01

Zingiber officinale (Zingiberaceae) or ginger, which is used in traditional medicine has antioxidant activity and neuroprotective effects. The effects of this plant on clonic seizure have not yet been studied. The present study evaluated the anticonvulsant effect of ginger in a model of clonic seizures induced with pentylenetetrazole (PTZ) in male mice. The anticonvulsant effect of Z. officinale was investigated using i.v. PTZ-induced seizure models in mice. Different doses of the hydroethanolic extract of Z. officinale (25, 50, and 100mg/kg) were administered intraperitonal (i.p.), 2 and 24h before induction of PTZ. Phenobarbital sodium (30mg/kg), a reference standard, was also tested for comparison. The effect of ginger on to the appearance of three separate seizure endpoints (myoclonic, generalized clonus and forelimb tonic extension phase) was recorded. The results showed that the ginger extract has anticonvulsant effects in all the experimental treatment groups of seizure tested as it significantly increased the seizure threshold. Hydroethanolic extract of Z. officinale significantly increased the onset time of myoclonic seizure at doses of 25-100mg/kg (p<0.001) and significantly prevented generalized clonic (p<0.001) and increased the threshold for the forelimb tonic extension (p<0.01) seizure 2 and 24h before induction of PTZ compared with control group. Based on the results the hydroethanolic extract of ginger has anticonvulsant effects, possibly through an interaction with inhibitory and excitatory system, antioxidant mechanisms, oxidative stress and calcium channel inhibition. Copyright © 2014 Elsevier B.V. All rights reserved.

Alteration of pentylenetetrazole-induced seizure threshold by chronic administration of ginger (Zingiber officinale) extract in male mice.

PubMed

Hosseini, Abdolkarim; Mirazi, Naser

2015-05-01

Zingiber officinale Roscoe (Zingiberaceae), or ginger, used in traditional Chinese medicine, has antioxidant activity and neuroprotective effects. The effects of this plant on clonic seizure have not yet been studied. The present study evaluated the anticonvulsant effect of ginger in a model of clonic seizures induced with pentylenetetrazole (PTZ) in male mice. The anticonvulsant effect of Z. officinale was investigated using i.v. PTZ-induced seizure models in mice. Different doses of the hydroethanolic extract of Z. officinale (25, 50, and 100 mg/kg) were administered intraperitonal (i.p.), daily for 1 week before induction of PTZ. Phenobarbital sodium (30 mg/kg), a reference standard, was also tested for comparison. The effect of ginger on to the appearance of three separate seizure endpoints, e.g., myoclonic, generalized clonic, and tonic extension phase, was recorded. Hydroethanolic extract of Z. officinale significantly increased the onset time of myoclonic seizure at doses of 25-100 mg/kg (55.33 ± 1.91 versus 24.47 ± 1.33 mg/kg, p < 0.001) and significantly prevented generalized clonic (74.64 ± 3.52 versus 47.72 ± 2.31 mg/kg, p < 0.001) and increased the threshold for the forelimb tonic extension (102.6 ± 5.39 versus 71.82 ± 7.82 mg/kg, p < 0.01) seizure induced by PTZ compared with the control group. Based on the results, the hydroethanolic extract of ginger has anticonvulsant effects, possibly through an interaction with inhibitory and excitatory systems, antioxidant mechanisms, and oxidative stress inhibition.

Transgenic fertile Scoparia dulcis L., a folk medicinal plant, conferred with a herbicide-resistant trait using an Ri binary vector.

PubMed

Yamazaki, M; Son, L; Hayashi, T; Morita, N; Asamizu, T; Mourakoshi, I; Saito, K

1996-01-01

Transgenic herbicide-resistant Scoparia dulcis plants were obtained by using an Ri binary vector system. The chimeric bar gene encoding phosphinothricin acetyltransferase flanked by the promoter for cauliflower mosaic virus 35S RNA and the terminal sequence for nopaline synthase was introduced in the plant genome by Agrobacterium-mediated transformation by means of scratching young plants. Hairy roots resistant to bialaphos were selected and plantlets (R0) were regenerated. Progenies (S1) were obtained by self-fertilization. The transgenic state was confirmed by DNA-blot hybridization and assaying of neomycin phosphotransferase II. Expression of the bar gene in the transgenic R0 and S1 progenies was indicated by the activity of phosphinothricin acetyltransferase. Transgenic plants accumulated scopadulcic acid B, a specific secondary metabolite of S. dulcis, in amounts of 15-60% compared with that in normal plants. The transgenic plants and progenies showed resistant trait towards bialaphos and phosphinothricin. These results suggest that an Ri binary system is one of the useful tools for the transformation of medicinal plants for which a regeneration protocol has not been established.

Optoporation of impermeable molecules and genes for visualization and activation of cells

NASA Astrophysics Data System (ADS)

Dhakal, Kamal; Batbyal, Subrata; Kim, Young-Tae; Mohanty, Samarendra

2015-03-01

Visualization, activation, and detection of the cell(s) and their electrical activity require delivery of exogenous impermeable molecules and targeted expression of genes encoding labeling proteins, ion-channels and voltage indicators. While genes can be delivered by viral vector to cells, delivery of other impermeable molecules into the cytoplasm of targeted cells requires microinjection by mechanical needle or microelectrodes, which pose significant challenge to the viability of the cells. Further, it will be useful to localize the expression of the targeted molecules not only in specific cell types, but to specific cells in restricted spatial regions. Here, we report use of focused near-infrared (NIR) femtosecond laser beam to transiently perforate targeted cell membrane to insert genes encoding blue light activatable channelrhodopsin-2 (ChR2) and red-shifted opsin (ReachR). Optoporation of nanomolar concentrations of rhodamine phalloidin (an impermeable dye molecule for staining filamentous actin) into targeted living mammalian cells (both HEK and primary cortical neurons) is also achieved allowing imaging of dynamics and intact morphology of cellular structures without requiring fixation.

Predication-based semantic indexing: permutations as a means to encode predications in semantic space.

PubMed

Cohen, Trevor; Schvaneveldt, Roger W; Rindflesch, Thomas C

2009-11-14

Corpus-derived distributional models of semantic distance between terms have proved useful in a number of applications. For both theoretical and practical reasons, it is desirable to extend these models to encode discrete concepts and the ways in which they are related to one another. In this paper, we present a novel vector space model that encodes semantic predications derived from MEDLINE by the SemRep system into a compact spatial representation. The associations captured by this method are of a different and complementary nature to those derived by traditional vector space models, and the encoding of predication types presents new possibilities for knowledge discovery and information retrieval.

The effect of dorsal hippocampal administration of nicotinic and muscarinic cholinergic ligands on pentylenetetrazol-induced generalized seizures in rats.

PubMed

Gholami, Morteza; Saboory, Ehsan; Zare, Samad; Roshan-Milani, Shiva; Hajizadeh-Moghaddam, Akbar

2012-10-01

In the present study, the effects of intrahippocampal injections of cholinergic ligands on pentylenetetrazol (PTZ)-induced seizures were investigated in rats. The rats were assigned to 1 of the following 9 groups: saline, nicotine (0.5 or 1 μg), atropine (0.25 or 1 μg), oxotremorine-M (0.1 or 1 μg), or mecamylamine (2 or 8 μg). Cholinergic ligands were administered via intrahippocampal infusion 30 min before seizure induction (intraperitoneal injection of 80 mg/kg PTZ). Results show that antagonists caused nonsignificant increases in the latency of tonic-clonic seizures, significant decreases in the duration of tonic-clonic seizures, significant decreases in the latency of death, and increases in mortality rate. Agonists led to increases in the duration of tonic-clonic seizures, decreases in the latency of death, and decreases in mortality rate. These results provide compelling evidence that cholinergic ligands show modulatory effects on a PTZ model of acute seizure in the rat hippocampus. Copyright © 2012 Elsevier Inc. All rights reserved.

Rebound increase in seizure susceptibility but not isolation-induced calls after single administration of clonazepam and Ro 19-8022 in infant rats.

PubMed

Mikulecká, A; Mareš, P; Kubová, H

2011-01-01

The purpose of our study was to determine whether a single administration of anticonvulsant doses of two ligands of benzodiazepine receptors, clonazepam and Ro 19-8022, leads to development of rebound phenomena in immature 12-day-old rats. Three tests were used: pentylenetetrazole (PTZ)-induced seizures, isolation-induced ultrasonic vocalizations, and motor performance. Susceptibility to the convulsant effects of PTZ decreased 24 hours, but increased 48 hours, after clonazepam administration. Ultrasonic vocalizations were completely suppressed 30 minutes and 3 hours after clonazepam; a moderate inhibitory effect persisted even at 48 hours. Motor abilities were slightly compromised up to 3 hours. Similar effects of Ro 19-8022 on PTZ-induced seizures and ultrasonic vocalizations were observed 24 and 48 hours after administration; motor performance was not affected. Rebound proconvulsant effects followed different time courses after administration of the two benzodiazepine receptor ligands in developing animals. Anxiolytic-like effects of these drugs were still present at the time when animals exhibited rebound proconvulsant effects. Copyright © 2010 Elsevier Inc. All rights reserved.

Identification of marine neuroactive molecules in behaviour-based screens in the larval zebrafish.

PubMed

Long, Si-Mei; Liang, Feng-Yin; Wu, Qi; Lu, Xi-Lin; Yao, Xiao-Li; Li, Shi-Chang; Li, Jing; Su, Huanxing; Pang, Ji-Yan; Pei, Zhong

2014-05-30

High-throughput behavior-based screen in zebrafish is a powerful approach for the discovery of novel neuroactive small molecules for treatment of nervous system diseases such as epilepsy. To identify neuroactive small molecules, we first screened 36 compounds (1-36) derived from marine natural products xyloketals and marine isoprenyl phenyl ether obtained from the mangrove fungus. Compound 1 demonstrated the most potent inhibition on the locomotor activity in larval zebrafish. Compounds 37-42 were further synthesized and their potential anti-epilepsy action was then examined in a PTZ-induced epilepsy model in zebrafish. Compound 1 and compounds 39, 40 and 41 could significantly attenuate PTZ-induced locomotor hyperactivity and elevation of c-fos mRNA in larval zebrafish. Compound 40 showed the most potent inhibitory action against PTZ-induced hyperactivity. The structure-activity analysis showed that the OH group at 12-position played a critical role and the substituents at the 13-position were well tolerated in the inhibitory activity of xyloketal derivatives. Thus, these derivatives may provide some novel drug candidates for the treatment of epilepsy.

Critical design criteria for minimal antibiotic-free plasmid vectors necessary to combine robust RNA Pol II and Pol III-mediated eukaryotic expression with high bacterial production yields

PubMed Central

Carnes, Aaron E.; Luke, Jeremy M.; Vincent, Justin M.; Anderson, Sheryl; Schukar, Angela; Hodgson, Clague P.; Williams, James A.

2010-01-01

Background For safety considerations, regulatory agencies recommend elimination of antibiotic resistance markers and nonessential sequences from plasmid DNA-based gene medicines. In the present study we analyzed antibiotic-free (AF) vector design criteria impacting bacterial production and mammalian transgene expression. Methods Both CMV-HTLV-I R RNA Pol II promoter (protein transgene) and murine U6 RNA Pol III promoter (RNA transgene) vector designs were studied. Plasmid production yield was assessed through inducible fed-batch fermentation. RNA Pol II-directed EGFP and RNA Pol III-directed RNA expression were quantified by fluorometry and quantitative real-time polymerase chain reaction (RT-PCR), respectively, after transfection of human HEK293 cells. Results Sucrose-selectable minimalized protein and therapeutic RNA expression vector designs that combined an RNA-based AF selection with highly productive fermentation manufacturing (>1,000 mg/L plasmid DNA) and high level in vivo expression of encoded products were identified. The AF selectable marker was also successfully applied to convert existing kanamycin-resistant DNA vaccine plasmids gWIZ and pVAX1 into AF vectors, demonstrating a general utility for retrofitting existing vectors. A minimum vector size for high yield plasmid fermentation was identified. A strategy for stable fermentation of plasmid dimers with improved vector potency and fermentation yields up to 1,740 mg/L was developed. Conclusions We report the development of potent high yield AF gene medicine expression vectors for protein or RNA (e.g. short hairpin RNA or microRNA) products. These AF expression vectors were optimized to exceed a newly identified size threshold for high copy plasmid replication and direct higher transgene expression levels than alternative vectors. PMID:20806425

Preventing vaccinia virus class-I epitopes presentation by HSV-ICP47 enhances the immunogenicity of a TAP-independent cancer vaccine epitope.

PubMed

Raafat, Nermin; Sadowski-Cron, Charlotte; Mengus, Chantal; Heberer, Michael; Spagnoli, Giulio C; Zajac, Paul

2012-09-01

Herpes simplex virus protein ICP47, encoded by US12 gene, strongly downregulates major histocompatibility complex (MHC) class-I antigen restricted presentation by blocking transporter associated with antigen processing (TAP) protein. To decrease viral vector antigenic immunodominance and MHC class-I driven clearance, we engineered recombinant vaccinia viruses (rVV) expressing ICP47 alone (rVV-US12) or together with endoplasmic reticulum (ER)-targeted Melan-A/MART-1(27-35) model tumor epitope (rVV-MUS12). In this study, we show that antigen presenting cells (APC), infected with rVV-US12, display a decreased ability to present TAP dependent MHC class-I restricted viral antigens to CD8+ T-cells. While HLA class-I cell surface expression is strongly downregulated, other important immune related molecules such as CD80, CD44 and, most importantly, MHC class-II are unaffected. Characterization of rVV-MUS12 infected cells demonstrates that over-expression of a TAP-independent peptide, partially compensates for ICP47 induced surface MHC class-I downregulation (30% vs. 70% respectively). Most importantly, in conditions where clearance of infected APC by virus-specific CTL represents a limiting factor, a significant enhancement of CTL responses to the tumor epitope can be detected in cultures stimulated with rVV-MUS12, as compared to those stimulated by rVV-MART alone. Such reagents could become of high relevance in multiple boost protocols required for cancer immunotherapy, to limit vector-specific responsiveness. Copyright © 2011 UICC.

Horizontal Transfer of Plasmid-Mediated Cephalosporin Resistance Genes in the Intestine of Houseflies (Musca domestica).

PubMed

Fukuda, Akira; Usui, Masaru; Okubo, Torahiko; Tamura, Yutaka

2016-06-01

Houseflies are a mechanical vector for various types of bacteria, including antimicrobial-resistant bacteria (ARB). If the intestine of houseflies is a suitable site for the transfer of antimicrobial resistance genes (ARGs), houseflies could also serve as a biological vector for ARB. To clarify whether cephalosporin resistance genes are transferred efficiently in the housefly intestine, we compared with conjugation experiments in vivo (in the intestine) and in vitro by using Escherichia coli with eight combinations of four donor and two recipient strains harboring plasmid-mediated cephalosporin resistance genes and chromosomal-encoded rifampicin resistance genes, respectively. In the in vivo conjugation experiment, houseflies ingested donor strains for 6 hr and then recipient strains for 3 hr, and 24 hr later, the houseflies were surface sterilized and analyzed. In vitro conjugation experiments were conducted using the broth-mating method. In 3/8 combinations, the in vitro transfer frequency (Transconjugants/Donor) was ≥1.3 × 10(-4); the in vivo transfer rates of cephalosporin resistance genes ranged from 2.0 × 10(-4) to 5.7 × 10(-5). Moreover, cephalosporin resistance genes were transferred to other species of enteric bacteria of houseflies such as Achromobacter sp. and Pseudomonas fluorescens. These results suggest that houseflies are not only a mechanical vector for ARB but also a biological vector for the occurrence of new ARB through the horizontal transfer of ARGs in their intestine.

Prediction of blood-brain partitioning: a model based on molecular electronegativity distance vector descriptors.

PubMed

Zhang, Yong-Hong; Xia, Zhi-Ning; Qin, Li-Tang; Liu, Shu-Shen

2010-09-01

The objective of this paper is to build a reliable model based on the molecular electronegativity distance vector (MEDV) descriptors for predicting the blood-brain barrier (BBB) permeability and to reveal the effects of the molecular structural segments on the BBB permeability. Using 70 structurally diverse compounds, the partial least squares regression (PLSR) models between the BBB permeability and the MEDV descriptors were developed and validated by the variable selection and modeling based on prediction (VSMP) technique. The estimation ability, stability, and predictive power of a model are evaluated by the estimated correlation coefficient (r), leave-one-out (LOO) cross-validation correlation coefficient (q), and predictive correlation coefficient (R(p)). It has been found that PLSR model has good quality, r=0.9202, q=0.7956, and R(p)=0.6649 for M1 model based on the training set of 57 samples. To search the most important structural factors affecting the BBB permeability of compounds, we performed the values of the variable importance in projection (VIP) analysis for MEDV descriptors. It was found that some structural fragments in compounds, such as -CH(3), -CH(2)-, =CH-, =C, triple bond C-, -CH<, =C<, =N-, -NH-, =O, and -OH, are the most important factors affecting the BBB permeability. (c) 2010. Published by Elsevier Inc.

Identification of candidate transmission-blocking antigen genes in Theileria annulata and related vector-borne apicomplexan parasites.

PubMed

Lempereur, Laetitia; Larcombe, Stephen D; Durrani, Zeeshan; Karagenc, Tulin; Bilgic, Huseyin Bilgin; Bakirci, Serkan; Hacilarlioglu, Selin; Kinnaird, Jane; Thompson, Joanne; Weir, William; Shiels, Brian

2017-06-05

Vector-borne apicomplexan parasites are a major cause of mortality and morbidity to humans and livestock globally. The most important disease syndromes caused by these parasites are malaria, babesiosis and theileriosis. Strategies for control often target parasite stages in the mammalian host that cause disease, but this can result in reservoir infections that promote pathogen transmission and generate economic loss. Optimal control strategies should protect against clinical disease, block transmission and be applicable across related genera of parasites. We have used bioinformatics and transcriptomics to screen for transmission-blocking candidate antigens in the tick-borne apicomplexan parasite, Theileria annulata. A number of candidate antigen genes were identified which encoded amino acid domains that are conserved across vector-borne Apicomplexa (Babesia, Plasmodium and Theileria), including the Pfs48/45 6-cys domain and a novel cysteine-rich domain. Expression profiling confirmed that selected candidate genes are expressed by life cycle stages within infected ticks. Additionally, putative B cell epitopes were identified in the T. annulata gene sequences encoding the 6-cys and cysteine rich domains, in a gene encoding a putative papain-family cysteine peptidase, with similarity to the Plasmodium SERA family, and the gene encoding the T. annulata major merozoite/piroplasm surface antigen, Tams1. Candidate genes were identified that encode proteins with similarity to known transmission blocking candidates in related parasites, while one is a novel candidate conserved across vector-borne apicomplexans and has a potential role in the sexual phase of the life cycle. The results indicate that a 'One Health' approach could be utilised to develop a transmission-blocking strategy effective against vector-borne apicomplexan parasites of animals and humans.

Inhibition of KLF7-Targeting MicroRNA 146b Promotes Sciatic Nerve Regeneration.

PubMed

Li, Wen-Yuan; Zhang, Wei-Ting; Cheng, Yong-Xia; Liu, Yan-Cui; Zhai, Feng-Guo; Sun, Ping; Li, Hui-Ting; Deng, Ling-Xiao; Zhu, Xiao-Feng; Wang, Ying

2018-06-01

A previous study has indicated that Krüppel-like factor 7 (KLF7), a transcription factor that stimulates Schwann cell (SC) proliferation and axonal regeneration after peripheral nerve injury, is a promising therapeutic transcription factor in nerve injury. We aimed to identify whether inhibition of microRNA-146b (miR-146b) affected SC proliferation, migration, and myelinated axon regeneration following sciatic nerve injury by regulating its direct target KLF7. SCs were transfected with miRNA lentivirus, miRNA inhibitor lentivirus, or KLF7 siRNA lentivirus in vitro. The expression of miR146b and KLF7, as well as SC proliferation and migration, were subsequently evaluated. In vivo, an acellular nerve allograft (ANA) followed by injection of GFP control vector or a lentiviral vector encoding an miR-146b inhibitor was used to assess the repair potential in a model of sciatic nerve gap. miR-146b directly targeted KLF7 by binding to the 3'-UTR, suppressing KLF7. Up-regulation of miR-146b and KLF7 knockdown significantly reduced the proliferation and migration of SCs, whereas silencing miR-146b resulted in increased proliferation and migration. KLF7 protein was localized in SCs in which miR-146b was expressed in vivo. Similarly, 4 weeks after the ANA, anti-miR-146b increased KLF7 and its target gene nerve growth factor cascade, promoting axonal outgrowth. Closer analysis revealed improved nerve conduction and sciatic function index score, and enhanced expression of neurofilaments, P0 (anti-peripheral myelin), and myelinated axon regeneration. Our findings provide new insight into the regulation of KLF7 by miR-146b during peripheral nerve regeneration and suggest a potential therapeutic strategy for peripheral nerve injury.

Gene Transfer of ZMapp Antibodies Mediated by Recombinant Adeno-Associated Virus Protects Against Ebola Infections.

PubMed

Robert, Marc-André; Nassoury, Nasha; Chahal, Parminder S; Venne, Marie-Hélène; Racine, Trina; Qiu, Xiangguo; Kobinger, Gary; Kamen, Amine; Gilbert, Rénald; Gaillet, Bruno

2018-04-01

Vectored delivery of the ZMapp antibody cocktail (c2G4, c4G7, and c13C6) by using recombinant adeno-associated viruses (rAAVs) could be useful for preventive immunization against Ebola virus infections because rAAVs can generate long-term antibody expression. Three rAAVs (serotype 9) encoding chimeric ZMapp antibodies were produced by triple-plasmid transfection up to 10 L-scale in WAVE bioreactors using HEK293 cells grown in suspension/serum-free conditions. Efficacy of AAV-c2G4 via intravenous (i.v.), intramuscular (i.m.), and intranasal (i.n.) routes of administration was evaluated in mice with two different doses of 2.7 × 10 10 and 13.0 × 10 10 vector genomes (vg). The best protective efficacies after Ebola challenge were obtained with the i.v. and i.m. routes. Serum concentrations of ZMapp antibodies positively correlated with survivability. Efficacy of the rAAV-ZMapp cocktail was then evaluated at a higher dose of 30.0 × 10 10 vg. It conferred a more robust protection (90% i.v. and 60% i.m.) than rAAV-c4G7 (30%) and rAAV-c13C6 (70%), both administered separately at the same dose. Delivery of rAAV-c2G4 alone achieved up to 100% protection (100% i.v. and 90% i.m.) at the same dose. In conclusion, the preventive treatment was effective in mice. However, no advantage was observed for using the rAAV-ZMapp cocktail in comparison to the utilization of the single rAAV-c2G4.

Weaving Knotted Vector Fields with Tunable Helicity.

PubMed

Kedia, Hridesh; Foster, David; Dennis, Mark R; Irvine, William T M

2016-12-30

We present a general construction of divergence-free knotted vector fields from complex scalar fields, whose closed field lines encode many kinds of knots and links, including torus knots, their cables, the figure-8 knot, and its generalizations. As finite-energy physical fields, they represent initial states for fields such as the magnetic field in a plasma, or the vorticity field in a fluid. We give a systematic procedure for calculating the vector potential, starting from complex scalar functions with knotted zero filaments, thus enabling an explicit computation of the helicity of these knotted fields. The construction can be used to generate isolated knotted flux tubes, filled by knots encoded in the lines of the vector field. Lastly, we give examples of manifestly knotted vector fields with vanishing helicity. Our results provide building blocks for analytical models and simulations alike.

Unrestricted Hepatocyte Transduction with Adeno-Associated Virus Serotype 8 Vectors in Mice

PubMed Central

Nakai, Hiroyuki; Fuess, Sally; Storm, Theresa A.; Muramatsu, Shin-ichi; Nara, Yuko; Kay, Mark A.

2005-01-01

Recombinant adeno-associated virus (rAAV) vectors can mediate long-term stable transduction in various target tissues. However, with rAAV serotype 2 (rAAV2) vectors, liver transduction is confined to only a small portion of hepatocytes even after administration of extremely high vector doses. In order to investigate whether rAAV vectors of other serotypes exhibit similar restricted liver transduction, we performed a dose-response study by injecting mice with β-galactosidase-expressing rAAV1 and rAAV8 vectors via the portal vein. The rAAV1 vector showed a blunted dose-response similar to that of rAAV2 at high doses, while the rAAV8 vector dose-response remained unchanged at any dose and ultimately could transduce all the hepatocytes at a dose of 7.2 × 1012 vector genomes/mouse without toxicity. This indicates that all hepatocytes have the ability to process incoming single-stranded vector genomes into duplex DNA. A single tail vein injection of the rAAV8 vector was as efficient as portal vein injection at any dose. In addition, intravascular administration of the rAAV8 vector at a high dose transduced all the skeletal muscles throughout the body, including the diaphragm, the entire cardiac muscle, and substantial numbers of cells in the pancreas, smooth muscles, and brain. Thus, rAAV8 is a robust vector for gene transfer to the liver and provides a promising research tool for delivering genes to various target organs. In addition, the rAAV8 vector may offer a potential therapeutic agent for various diseases affecting nonhepatic tissues, but great caution is required for vector spillover and tight control of tissue-specific gene expression. PMID:15596817

HR38, an ortholog of NR4A family nuclear receptors, mediates 20-hydroxyecdysone regulation of carbohydrate metabolism during mosquito reproduction.

PubMed

Dong, Dujuan; Zhang, Yang; Smykal, Vlastimil; Ling, Lin; Raikhel, Alexander S

2018-05-01

The Aedes aegypti mosquito is the principal vector for many dangerous human viral diseases. Carbohydrate metabolism (CM) is essential for supplying the energy necessary for host seeking, blood digestion and rapid egg development of this vector insect. The steroid hormone 20-hydroxyecdysone (20E) and the ecdysone receptor (EcR) are important regulators of CM, coordinating it with female reproductive events. We report here that the NR4A nuclear receptor AHR38 plays a critical role in mediating these actions of 20E and EcR. AHR38 RNA interference (RNAi) depletion in female mosquitoes blocked the transcriptional activation of CM genes encoding phosphoglucomutase (PGM) and trehalose-6-phophate synthase (TPS); it caused an increase of glycogen accumulation and a decrease of the circulating sugar trehalose. This treatment also resulted in a dramatic reduction in fecundity. Considering that these phenotypes resulting from AHR38 RNAi depletion are similar to those of EcR RNAi, we investigated a possible connection between these transcription factors in CM regulation. EcR RNAi inhibits the AHR38 gene expression. Moreover, the 20E-induced EcR complex directly activates AHR38 by binding to the ecdysone response element (EcRE) in the upstream regulatory region of this gene. The present work has implicated AHR38 in the 20E-mediated control of CM and provided new insight into mechanisms of 20E regulation of metabolism during female mosquito reproduction. © 2018 Published by Elsevier Ltd.

Functional Characterization of the Mannitol Promoter of Pseudomonas fluorescens DSM 50106 and Its Application for a Mannitol-Inducible Expression System for Pseudomonas putida KT2440

PubMed Central

Hoffmann, Jana; Altenbuchner, Josef

2015-01-01

A new pBBR1MCS-2-derived vector containing the Pseudomonas fluorescens DSM10506 mannitol promoter PmtlE and mtlR encoding its AraC/XylS type transcriptional activator was constructed and optimized for low basal expression. Mannitol, arabitol, and glucitol-inducible gene expression was demonstrated with Pseudomonas putida and eGFP as reporter gene. The new vector was applied for functional characterization of PmtlE. Identification of the DNA binding site of MtlR was achieved by in vivo eGFP measurement with PmtlE wild type and mutants thereof. Moreover, purified MtlR was applied for detailed in vitro investigations using electrophoretic mobility shift assays and DNaseI footprinting experiments. The obtained data suggest that MtlR binds to PmtlE as a dimer. The proposed DNA binding site of MtlR is AGTGC-N5-AGTAT-N7-AGTGC-N5-AGGAT. The transcription activation mechanism includes two binding sites with different binding affinities, a strong upstream binding site and a weaker downstream binding site. The presence of the weak downstream binding site was shown to be necessary to sustain mannitol-inducibility of PmtlE. Two possible functions of mannitol are discussed; the effector might stabilize binding of the second monomer to the downstream half site or promote transcription activation by inducing a conformational change of the regulator that influences the contact to the RNA polymerase. PMID:26207762

Duration of treatment and activation of α1-containing GABAA receptors variably affect the level of anxiety and seizure susceptibility after diazepam withdrawal in rats

PubMed Central

Kovačević, Jovana; Timić, Tamara; Tiruveedhula, Veera V.; Batinić, Bojan; Namjoshi, Ojas A.; Milić, Marija; Joksimović, Srđan; Cook, James M.; Savić, Miroslav M.

2014-01-01

Long-term use of benzodiazepine-type drugs may lead to physical dependence, manifested by withdrawal syndrome after abrupt cessation of treatment. The aim of the present study was to investigate the influence of duration of treatment, as well as the role of α1-containing GABAA receptors, in development of physical dependence to diazepam, assessed through the level of anxiety and susceptibility to pentylenetetrazole (PTZ)-induced seizures, 24 h after withdrawal from protracted treatment in rats. Withdrawal of 2 mg/kg diazepam after 28, but not after 14 or 21 days of administration led to an anxiety-like behavior in the elevated plus maze. Antagonism of the diazepam effects at α1-containing GABAA receptors, achieved by daily administration of the neutral modulator βCCt (5 mg/kg), did not affect the anxiety level during withdrawal. An increased susceptibility to PTZ-induced seizures was observed during diazepam withdrawal after 21 and 28 days of treatment. Daily co-administration of βCCt further decreased the PTZ-seizure threshold after 21 days of treatment, whilst it prevented the diazepam withdrawal-elicited decrease of the PTZ threshold after 28 days of treatment. In conclusion, the current study suggests that the role of α1-containing GABAA receptors in mediating the development of physical dependence may vary based on the effect being studied and duration of protracted treatment. Moreover, the present data supports previous findings that the lack of activity at α1-containing GABAA receptors is not sufficient to eliminate physical dependence liability of ligands of the benzodiazepine type. PMID:24695241

MicroRNA-137 represses FBI-1 to inhibit proliferation and in vitro invasion and migration of hepatocellular carcinoma cells.

PubMed

Zhu, Min; Li, Mingyang; Wang, Tao; Linghu, Enqiang; Wu, Benyan

2016-10-01

The pro-oncogene factor that binds to inducer of short transcripts-1 (FBI-1), which is encoded by ZBTB7A gene and belongs to POK (POZ/BTB and KrÜppel) protein family, has been shown to enhance hepatocellular carcinoma (HCC) cells proliferation and multi-drug resistance (MDR) process. However, the possibility that FBI-1 is a therapeutic target for further HCC treatment remains poorly determined. In the current study, two microRNA (miRNA) target prediction programs (TargetScan and MiRanda) were used to identify miR-137 as a potential regulator of FBI-1. Our results showed that expression of miR-137 was downregulated, while FBI-1 was upregulated in clinical HCC specimens, compared with paired non-tumor specimens. Overexpression of miR-137 via adenoviral vector inhibited the proliferation and anchorage-independent growth of HCC cells, HepG2 and MHCC-97H. Our data also showed that miR-137 repressed endogenous expression level of FBI-1, as well as Notch-1 and Survivin. MiR-137 also inhibited in vitro invasion and migration of HCC cells and attenuated their epithelial-mesenchymal transition (EMT) process. Moreover, miR-137 suppressed the growth rate of HepG2 cells in nude mice model. Overexpression of miR-137 via its adenoviral vector enhanced the sensitivity of HepG2 cells to anti-tumor drugs and attenuated the MDR process of a resistance cell line HepG2/adriamycin (ADR). Thus, FBI-1 downregulation mediated by miR-137 overexpression may be a potential strategy for HCC treatment.

Silencing of HaAce1 gene by host-delivered artificial microRNA disrupts growth and development of Helicoverpa armigera.

PubMed

Saini, Ravi Prakash; Raman, Venkat; Dhandapani, Gurusamy; Malhotra, Era Vaidya; Sreevathsa, Rohini; Kumar, Polumetla Ananda; Sharma, Tilak R; Pattanayak, Debasis

2018-01-01

The polyphagous insect-pest, Helicoverpa armigera, is a serious threat to a number of economically important crops. Chemical application and/or cultivation of Bt transgenic crops are the two strategies available now for insect-pest management. However, environmental pollution and long-term sustainability are major concerns against these two options. RNAi is now considered as a promising technology to complement Bt to tackle insect-pests menace. In this study, we report host-delivered silencing of HaAce1 gene, encoding the predominant isoform of H. armigera acetylcholinesterase, by an artificial microRNA, HaAce1-amiR1. Arabidopsis pre-miRNA164b was modified by replacing miR164b/miR164b* sequences with HaAce1-amiR1/HaAce1-amiR1* sequences. The recombinant HaAce1-preamiRNA1 was put under the control of CaMV 35S promoter and NOS terminator of plant binary vector pBI121, and the resultant vector cassette was used for tobacco transformation. Two transgenic tobacco lines expressing HaAce1-amiR1 was used for detached leaf insect feeding bioassays. Larval mortality of 25% and adult deformity of 20% were observed in transgenic treated insect group over that control tobacco treated insect group. The reduction in the steady-state level of HaAce1 mRNA was 70-80% in the defective adults compared to control. Our results demonstrate promise for host-delivered amiRNA-mediated silencing of HaAce1 gene for H. armigera management.

Practical auxiliary basis implementation of Rung 3.5 functionals

DOE Office of Scientific and Technical Information (OSTI.GOV)

Janesko, Benjamin G., E-mail: b.janesko@tcu.edu; Scalmani, Giovanni; Frisch, Michael J.

2014-07-21

Approximate exchange-correlation functionals for Kohn-Sham density functional theory often benefit from incorporating exact exchange. Exact exchange is constructed from the noninteracting reference system's nonlocal one-particle density matrix γ(r{sup -vector},r{sup -vector}′). Rung 3.5 functionals attempt to balance the strengths and limitations of exact exchange using a new ingredient, a projection of γ(r{sup -vector},r{sup -vector} ′) onto a semilocal model density matrix γ{sub SL}(ρ(r{sup -vector}),∇ρ(r{sup -vector}),r{sup -vector}−r{sup -vector} ′). γ{sub SL} depends on the electron density ρ(r{sup -vector}) at reference point r{sup -vector}, and is closely related to semilocal model exchange holes. We present a practical implementation of Rung 3.5 functionals, expandingmore » the r{sup -vector}−r{sup -vector} ′ dependence of γ{sub SL} in an auxiliary basis set. Energies and energy derivatives are obtained from 3D numerical integration as in standard semilocal functionals. We also present numerical tests of a range of properties, including molecular thermochemistry and kinetics, geometries and vibrational frequencies, and bandgaps and excitation energies. Rung 3.5 functionals typically provide accuracy intermediate between semilocal and hybrid approximations. Nonlocal potential contributions from γ{sub SL} yield interesting successes and failures for band structures and excitation energies. The results enable and motivate continued exploration of Rung 3.5 functional forms.« less

Isolation of Onchocerca lupi in Dogs and Black Flies, California, USA

PubMed Central

Hassan, Hassan K.; Bolcen, Shanna; Kubofcik, Joseph; Nutman, Thomas B.; Eberhard, Mark L.; Middleton, Kelly; Wekesa, Joseph Wakoli; Ruedas, Gimena; Nelson, Kimberly J.; Dubielzig, Richard; De Lombaert, Melissa; Silverman, Bruce; Schorling, Jamie J.; Adler, Peter H.; Beeler, Emily S.

2015-01-01

In southern California, ocular infections caused by Onchocerca lupi were diagnosed in 3 dogs (1 in 2006, 2 in 2012). The infectious agent was confirmed through morphologic analysis of fixed parasites in tissues and by PCR and sequencing of amplicons derived from 2 mitochondrially encoded genes and 1 nuclear-encoded gene. A nested PCR based on the sequence of the cytochrome oxidase subunit 1 gene of the parasite was developed and used to screen Simulium black flies collected from southern California for O. lupi DNA. Six (2.8%; 95% CI 0.6%–5.0%) of 213 black flies contained O. lupi DNA. Partial mitochondrial16S rRNA gene sequences from the infected flies matched sequences derived from black fly larvae cytotaxonomically identified as Simulium tribulatum. These data implicate S. tribulatum flies as a putative vector for O. lupi in southern California. PMID:25897954

Isolation and characterization of a cDNA clone coding for a glutathione S-transferase class delta enzyme from the biting midge Culicoides variipennis sonorensis Wirth and Jones.

PubMed

Abdallah, M A; Pollenz, R S; Droog, F N; Nunamaker, R A; Tabachnick, W J; Murphy, K E

2000-12-01

Culicoides variipennis sonorensis is the primary vector of bluetongue viruses in North America. Glutathione S-transferases (GSTs) are enzymes that catalyze nucleophilic substitutions, converting reactive lipophilic molecules into soluble conjugates. Increased GST activity is associated with development of insecticide resistance. Described here is the isolation of the first cDNA encoding a C. variipennis GST. The clone consists of 720 translated bases encoding a protein with a M(r) of approximately 24,800 composed of 219 amino acids. The deduced amino acid sequence is similar (64%-74%) to class Delta (previously named Theta) GSTs from the dipteran genera Musca, Drosophila, Lucilia and Anopheles. The cDNA was subcloned into pET-11b, expressed in Epicurian coli BL21 (DE3) and has a specific activity of approximately 28,000 units/mg for the substrate 1-chloro-2,4-dinitrobenzene.

Survey of Navy Funded Marine Mammal Research and Studies FY 00-01

DTIC Science & Technology

2001-05-10

protein of canine distemper virus as a reporter system in order to evaluate 103 the humoral response to DNA-mediated vaccination in cetaceans. If...PCR/ RT PCR, DNA cloning and sequencing, etc. Efforts are ongoing to design and clone a vector encoding Canine Distemper Virus, a virus closely...alternative plasmid as our reporter gene delivery vector. This alternate plasmid will encode for Canine Distemper virus genes, closely related to

Polypeptides having laccase activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Ye; Tang, Lan; Duan, Junxin

The present invention relates to isolated polypeptides having laccase activity and polynucleotides encoding the polypeptides and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

OmniBird: a miniature PTZ NIR sensor system for UCAV day/night autonomous operations

NASA Astrophysics Data System (ADS)

Yi, Steven; Li, Hui

2007-04-01

Through a SBIR funding from NAVAIR, we have successfully developed an innovative, miniaturized, and lightweight PTZ UCAV imager called OmniBird for UCAV taxiing. The proposed OmniBird will be able to fit in a small space. The designed zoom capability allows it to acquire focused images for targets ranging from 10 to 250 feet. The innovative panning mechanism also allows the system to have a field of view of +/- 100 degrees within the provided limited spacing (6 cubic inches). The integrated optics, camera sensor, and mechanics solution will allow the OmniBird to stay optically aligned and shock-proof under harsh environments.

Design, synthesis, and anticonvulsant activity of new hybrid compounds derived from 2-(2,5-dioxopyrrolidin-1-yl)propanamides and 2-(2,5-dioxopyrrolidin-1-yl)butanamides.

PubMed

Kamiński, Krzysztof; Zagaja, Mirosław; Łuszczki, Jarogniew J; Rapacz, Anna; Andres-Mach, Marta; Latacz, Gniewomir; Kieć-Kononowicz, Katarzyna

2015-07-09

The library of 27 new 1-(4-phenylpiperazin-1-yl)- or 1-(morpholin-4-yl)-(2,5-dioxopyrrolidin-1-yl)propanamides and (2,5-dioxopyrrolidin-1-yl)butanamides as potential new hybrid anticonvulsant agents was synthesized. These hybrid molecules join the chemical fragments of well-known antiepileptic drugs (AEDs) such as ethosuximide, levetiracetam, and lacosamide. Compounds 5, 10, 11, and 24 displayed the broad spectra of activity across the preclinical seizure models, namely, the maximal electroshock (MES) test, the subcutaneous pentylenetetrazole (scPTZ) test, and the six-hertz (6 Hz) model of pharmacoresistant limbic seizures. The highest protection was demonstrated by 11 (ED50 MES = 88.4 mg/kg, ED50 scPTZ = 59.9 mg/kg, ED50 6 Hz = 21.0 mg/kg). This molecule did not impair the motor coordination of animals in the chimney test even at high doses (TD50 > 1500 mg/kg), yielding superb protective indexes (PI MES > 16.97, PI PTZ > 25.04, PI 6 Hz > 71.43). As a result, 11 displayed distinctly better safety profile than clinically relevant AEDs ethosuximide, lacosamide, or valproic acid.

Design, synthesis and biological evaluation of new hybrid anticonvulsants derived from N-benzyl-2-(2,5-dioxopyrrolidin-1-yl)propanamide and 2-(2,5-dioxopyrrolidin-1-yl)butanamide derivatives.

PubMed

Kamiński, Krzysztof; Rapacz, Anna; Łuszczki, Jarogniew J; Latacz, Gniewomir; Obniska, Jolanta; Kieć-Kononowicz, Katarzyna; Filipek, Barbara

2015-05-15

The purpose of this study was to synthesize the library of 33 new N-benzyl-2-(2,5-dioxopyrrolidin-1-yl)propanamides, 2-(3-methyl-2,5-dioxopyrrolidin-1-yl)propanamides, and 2-(2,5-dioxopyrrolidin-1-yl)butanamides as potential new hybrid anticonvulsant agents. These hybrid molecules join the chemical fragments of well-known antiepileptic drugs (AEDs) such as ethosuximide, levetiracetam, and lacosamide. The coupling reaction of the 2-(2,5-dioxopyrrolidin-1-yl)propanoic acid, 2-(3-methyl-2,5-dioxopyrrolidin-1-yl)propanoic acid, or 2-(2,5-dioxopyrrolidin-1-yl)butanoic acid with the appropriately substituted benzylamines in the presence of the coupling reagent, N,N-carbonyldiimidazole (CDI) generated the final compounds 4-36. Spectral data acquired via (1)H NMR, (13)C NMR, and LC-MS confirmed the chemical structures of the newly prepared compounds. The initial anticonvulsant screening was performed in mice intraperitoneally (ip), using the maximal electroshock seizure (MES) and subcutaneous pentylenetetrazole (scPTZ) seizure tests. The rotarod test determined the acute neurological toxicity (NT). The results of preliminary pharmacological screening revealed that 25 compounds showed protection in half or more of the animals tested in the MES and/or scPTZ seizure models at the fixed dose of 100mg/kg. The broad spectra of activity across the preclinical seizure models displayed compounds 4, 7, 8, 13, 15-18, 24, and 26. The quantitative pharmacological studies in mice demonstrated the highest protection for compounds 4 (ED50 MES=67.65 mg/kg, ED50scPTZ=42.83 mg/kg); 8 (ED50 MES=54.90 mg/kg, ED50scPTZ=50.29 mg/kg); and 20 (ED50scPTZ=47.39 mg/kg). These compounds were distinctly more potent and provided better safety profiles in the rotarod test compared to valproic acid or ethosuximide, which were used as model AEDs. Compound 8 underwent only a slight metabolic change by the human liver microsomes (HLMs), and also did not affect the activity of human cytochrome P450 isoform, CYP3A4, in the in vitro assays. Copyright © 2015 Elsevier Ltd. All rights reserved.

Ribosomal DNA Integrating rAAV-rDNA Vectors Allow for Stable Transgene Expression

PubMed Central

Lisowski, Leszek; Lau, Ashley; Wang, Zhongya; Zhang, Yue; Zhang, Feijie; Grompe, Markus; Kay, Mark A

2012-01-01

Although recombinant adeno-associated virus (rAAV) vectors are proving to be efficacious in clinical trials, the episomal character of the delivered transgene restricts their effectiveness to use in quiescent tissues, and may not provide lifelong expression. In contrast, integrating vectors enhance the risk of insertional mutagenesis. In an attempt to overcome both of these limitations, we created new rAAV-rDNA vectors, with an expression cassette flanked by ribosomal DNA (rDNA) sequences capable of homologous recombination into genomic rDNA. We show that after in vivo delivery the rAAV-rDNA vectors integrated into the genomic rDNA locus 8–13 times more frequently than control vectors, providing an estimate that 23–39% of the integrations were specific to the rDNA locus. Moreover, a rAAV-rDNA vector containing a human factor IX (hFIX) expression cassette resulted in sustained therapeutic levels of serum hFIX even after repeated manipulations to induce liver regeneration. Because of the relative safety of integration in the rDNA locus, these vectors expand the usage of rAAV for therapeutics requiring long-term gene transfer into dividing cells. PMID:22990671

More About Vector Adaptive/Predictive Coding Of Speech

NASA Technical Reports Server (NTRS)

Jedrey, Thomas C.; Gersho, Allen

1992-01-01

Report presents additional information about digital speech-encoding and -decoding system described in "Vector Adaptive/Predictive Encoding of Speech" (NPO-17230). Summarizes development of vector adaptive/predictive coding (VAPC) system and describes basic functions of algorithm. Describes refinements introduced enabling receiver to cope with errors. VAPC algorithm implemented in integrated-circuit coding/decoding processors (codecs). VAPC and other codecs tested under variety of operating conditions. Tests designed to reveal effects of various background quiet and noisy environments and of poor telephone equipment. VAPC found competitive with and, in some respects, superior to other 4.8-kb/s codecs and other codecs of similar complexity.

Functional characterization of adenoviral/retroviral chimeric vectors and their use for efficient screening of retroviral producer cell lines.

PubMed

Duisit, G; Salvetti, A; Moullier, P; Cosset, F L

1999-01-20

We have generated three different E1-deleted replication-defective adenoviral vectors expressing either Moloney murine leukemia virus (Mo-MuLV) Gag-Pol core particle proteins, gibbon ape leukemia virus (GALV) envelope glycoproteins, or an MuLV-derived retroviral vector genome encoding mCD2 antigen, a murine cell surface marker easily detectable by flow cytometry. Each of the three vectors was first characterized individually by infection of cells providing the complementary retroviral function(s) and able to induce the production of retroviral vectors with an efficiency similar to or higher than that of FLY stable retroviral packaging cells [Cosset, F.-L., Takeuchi, Y., Battini, J.-L., Weiss, R.A., and Collins, M.K.L., (1995). J. Virol. 69, 7430-7436]. In small-scale pilot experiments, TE671 cells simultaneously coinfected with the three adenoviral vectors efficiently released helper-free retroviral vectors in their supernatant, with titers greater than 10(6) infectious particles per milliliter by end-point titrations. Our results also indicated that in contrast to retroviral vector-packageable RNAs, the adenovirus-mediated overexpression of both Gag-Pol and Env packaging functions had limited impact on retroviral titers. The primary mechanism suspected is the premature intracellular cleavage of the Pr65gag precursor that we found in gag-pol-expressing cells, which in turn may impair the normal incorporation of high loads of functional Env. Last, the characterization of the adenoviral/retroviral chimeric vectors allowed the screening of various primate cells for retroviral production and we found that three hepatocyte-derived cell lines were highly efficient in the assembly and release of infectious retroviral particles.

Encoding the local connectivity patterns of fMRI for cognitive task and state classification.

PubMed

Onal Ertugrul, Itir; Ozay, Mete; Yarman Vural, Fatos T

2018-06-15

In this work, we propose a novel framework to encode the local connectivity patterns of brain, using Fisher vectors (FV), vector of locally aggregated descriptors (VLAD) and bag-of-words (BoW) methods. We first obtain local descriptors, called mesh arc descriptors (MADs) from fMRI data, by forming local meshes around anatomical regions, and estimating their relationship within a neighborhood. Then, we extract a dictionary of relationships, called brain connectivity dictionary by fitting a generative Gaussian mixture model (GMM) to a set of MADs, and selecting codewords at the mean of each component of the mixture. Codewords represent connectivity patterns among anatomical regions. We also encode MADs by VLAD and BoW methods using k-Means clustering. We classify cognitive tasks using the Human Connectome Project (HCP) task fMRI dataset and cognitive states using the Emotional Memory Retrieval (EMR). We train support vector machines (SVMs) using the encoded MADs. Results demonstrate that, FV encoding of MADs can be successfully employed for classification of cognitive tasks, and outperform VLAD and BoW representations. Moreover, we identify the significant Gaussians in mixture models by computing energy of their corresponding FV parts, and analyze their effect on classification accuracy. Finally, we suggest a new method to visualize the codewords of the learned brain connectivity dictionary.

The Vector-Ballot Approach for Online Voting Procedures

NASA Astrophysics Data System (ADS)

Kiayias, Aggelos; Yung, Moti

Looking at current cryptographic-based e-voting protocols, one can distinguish three basic design paradigms (or approaches): (a) Mix-Networks based, (b) Homomorphic Encryption based, and (c) Blind Signatures based. Each of the three possesses different advantages and disadvantages w.r.t. the basic properties of (i) efficient tallying, (ii) universal verifiability, and (iii) allowing write-in ballot capability (in addition to predetermined candidates). In fact, none of the approaches results in a scheme that simultaneously achieves all three. This is unfortunate, since the three basic properties are crucial for efficiency, integrity and versatility (flexibility), respectively. Further, one can argue that a serious business offering of voting technology should offer a flexible technology that achieves various election goals with a single user interface. This motivates our goal, which is to suggest a new "vector-ballot" based approach for secret-ballot e-voting that is based on three new notions: Provably Consistent Vector Ballot Encodings, Shrink-and-Mix Networks and Punch-Hole-Vector-Ballots. At the heart of our approach is the combination of mix networks and homomorphic encryption under a single user interface; given this, it is rather surprising that it achieves much more than any of the previous approaches for e-voting achieved in terms of the basic properties. Our approach is presented in two generic designs called "homomorphic vector-ballots with write-in votes" and "multi-candidate punch-hole vector-ballots"; both of our designs can be instantiated over any homomorphic encryption function.

Characterization of intravitreally delivered capsid mutant AAV2-Cre vector to induce tissue-specific mutations in murine retinal ganglion cells.

PubMed

Langouet-Astrie, Christophe J; Yang, Zhiyong; Polisetti, Sraavya M; Welsbie, Derek S; Hauswirth, William W; Zack, Donald J; Merbs, Shannath L; Enke, Raymond A

2016-10-01

Targeted expression of Cre recombinase in murine retinal ganglion cells (RGCs) by viral vector is an effective strategy for creating tissue-specific gene knockouts for investigation of genetic contribution to RGC degeneration associated with optic neuropathies. Here we characterize dosage, efficacy and toxicity for sufficient intravitreal delivery of a capsid mutant Adeno-associated virus 2 (AAV2) vector encoding Cre recombinase. Wild type and Rosa26 (R26) LacZ mice were intravitreally injected with capsid mutant AAV2 viral vectors. Murine eyes were harvested at intervals ranging from 2 weeks to 15 weeks post-injection and were assayed for viral transduction, transgene expression and RGC survival. 10(9) vector genomes (vg) were sufficient for effective in vivo targeting of murine ganglion cell layer (GCL) retinal neurons. Transgene expression was observed as early as 2 weeks post-injection of viral vectors and persisted to 11 weeks. Early expression of Cre had no significant effect on RGC survival, while significant RGC loss was detected beginning 5 weeks post-injection. Early expression of viral Cre recombinase was robust, well-tolerated and predominantly found in GCL neurons suggesting this strategy can be effective in short-term RGC-specific mutation studies in experimental glaucoma models such as optic nerve crush and transection experiments. RGC degeneration with Cre expression for more than 4 weeks suggests that Cre toxicity is a limiting factor for targeted mutation strategies in RGCs. Copyright © 2016 Elsevier Ltd. All rights reserved.

Deletion of the Vaccinia Virus Gene A46R, Encoding for an Inhibitor of TLR Signalling, Is an Effective Approach to Enhance the Immunogenicity in Mice of the HIV/AIDS Vaccine Candidate NYVAC-C

PubMed Central

Perdiguero, Beatriz; Gómez, Carmen Elena; Di Pilato, Mauro; Sorzano, Carlos Oscar S.; Delaloye, Julie; Roger, Thierry; Calandra, Thierry; Pantaleo, Giuseppe; Esteban, Mariano

2013-01-01

Viruses have developed strategies to counteract signalling through Toll-like receptors (TLRs) that are involved in the detection of viruses and induction of proinflammatory cytokines and IFNs. Vaccinia virus (VACV) encodes A46 protein which disrupts TLR signalling by interfering with TLR: adaptor interactions. Since the innate immune response to viruses is critical to induce protective immunity, we studied whether deletion of A46R gene in a NYVAC vector expressing HIV-1 Env, Gag, Pol and Nef antigens (NYVAC-C) improves immune responses against HIV-1 antigens. This question was examined in human macrophages and in mice infected with a single A46R deletion mutant of the vaccine candidate NYVAC-C (NYVAC-C-ΔA46R). The viral gene A46R is not required for virus replication in primary chicken embryo fibroblast (CEF) cells and its deletion in NYVAC-C markedly increases TNF, IL-6 and IL-8 secretion by human macrophages. Analysis of the immune responses elicited in BALB/c mice after DNA prime/NYVAC boost immunization shows that deletion of A46R improves the magnitude of the HIV-1-specific CD4 and CD8 T cell immune responses during adaptive and memory phases, maintains the functional profile observed with the parental NYVAC-C and enhances anti-gp120 humoral response during the memory phase. These findings establish the immunological role of VACV A46R on innate immune responses of macrophages in vitro and antigen-specific T and B cell immune responses in vivo and suggest that deletion of viral inhibitors of TLR signalling is a useful approach for the improvement of poxvirus-based vaccine candidates. PMID:24069354

Does ITV vaginal procedure ensure dosimetric coverage during IMRT of post-operative gynaecological tumours without instructions concerning rectal filling?

PubMed

Verges, Ramona; Giraldo, Alexandra; Seoane, Alejandro; Toral, Elisabet; Ruiz, M Carmen; Pons, Ariadna; Giralt, Jordi

2018-01-01

To find out whether the internal target volume (ITV) vaginal procedure ensures dosimetric coverage during intensity-modulated radiation therapy (IMRT) of post-operative gynaecological tumours without instructions on rectal filling. The ITV vaginal procedure does not necessarily include all movements of the bladder, and does not include changes in the rectal volume. We should know if the vaginal ITV is a useful tool in maintaining CTV coverage during treatment. A retrospective analysis of 24 patients treated between July 2012 and July 2014 with adjuvant IMRT for gynaecological cancer. All patients underwent empty and full bladder CT on simulation (CT-planning) and three weeks later (CT-control). ITV displacement was measured and the 3D vector was calculated. ITV coverage was then evaluated by comparing the volume covered by the prescription isodose on both CT's. Patients were asked to have full bladder but they did not follow recommendations for the rectum. The mean 3D vector was 0.64 ± 0.32 cm (0.09-1.30). The mean ITV coverage loss was 5.8 ± 5.7% (0-20.2). We found a significant positive correlation between the 3D vector and the loss of coverage (Pearson correlation, r  = 0.493, 95% CI: 0.111-0.748, p  = 0.0144). We did not find any significant correlation between the bladder and rectal parameters with the 3D vector and loss of dosimetric coverage. We found a trend between the maximum rectal diameter in CT-planning and 3D vector ( r  = 0.400, 95% CI: -0.004 to 0.692, p  = 0.0529). ITV vaginal procedure contributed to ensuring a good dose coverage without instructions on rectal filling.

Immune Protection of Nonhuman Primates Against Ebola Virus with Single Low-Dose Adenovirus Vectors Encoding Modified GPs

DTIC Science & Technology

2006-06-01

21. Geisbert TW, Hensley LE , Larsen T, Young HA, Reed DS, et al. (2003) Pathogenesis of Ebola hemorrhagic fever in cynomolgus macaques: Evidence that...Shedlock DJ, Xu L, et al. (2006) Immune protection of nonhuman primates against Ebola virus with single low-dose adenovirus vectors encoding modified...CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT SAR 18. NUMBER OF PAGES 9 19a. NAME OF RESPONSIBLE PERSON a. REPORT unclassified b. ABSTRACT

Immunogenicity and protective efficacy of a replication-defective infectious bronchitis virus vaccine using an adenovirus vector and administered in ovo.

PubMed

Zeshan, Basit; Zhang, Lili; Bai, Juan; Wang, Xinglong; Xu, Jiarong; Jiang, Ping

2010-06-01

In ovo vaccination remains an attractive option for a cost effective, uniform and mass application of vaccines for commercial poultry. However, the vaccines which can be delivered safely by this method are limited and there is no currently licensed embryo-safe vaccine against infectious bronchitis virus (IBV). In this study, a recombinant adenovirus expressing the S1 gene of nephropathogenic IBV (rAd-S1) was constructed and the immune responses and protective efficacy against homologous challenge were evaluated after in ovo vaccination. The results showed that the rAd-S1 led to dramatic augmentation of humoral and cellular responses in birds vaccinated in ovo followed by an intramuscular inoculation. Both IFN-gamma and IL-4 in chicken's lymphocytes were produced by this strategy. Following challenge with IBV, the chickens vaccinated with recombinant adenovirus showed fewer nephropathic lesions and less severe clinical signs as compared to those receiving wild-type adenovirus or PBS. The construction of non-replicating human adenovirus vector encoding S1 gene of IBV and its in ovo delivery demonstrated the potential of an alternative vaccination strategy against IBV. Copyright 2010 Elsevier B.V. All rights reserved.

Vaccination with Replication Deficient Adenovectors Encoding YF-17D Antigens Induces Long-Lasting Protection from Severe Yellow Fever Virus Infection in Mice.

PubMed

Bassi, Maria R; Larsen, Mads A B; Kongsgaard, Michael; Rasmussen, Michael; Buus, Søren; Stryhn, Anette; Thomsen, Allan R; Christensen, Jan P

2016-02-01

The live attenuated yellow fever vaccine (YF-17D) has been successfully used for more than 70 years. It is generally considered a safe vaccine, however, recent reports of serious adverse events following vaccination have raised concerns and led to suggestions that even safer YF vaccines should be developed. Replication deficient adenoviruses (Ad) have been widely evaluated as recombinant vectors, particularly in the context of prophylactic vaccination against viral infections in which induction of CD8+ T-cell mediated immunity is crucial, but potent antibody responses may also be elicited using these vectors. In this study, we present two adenobased vectors targeting non-structural and structural YF antigens and characterize their immunological properties. We report that a single immunization with an Ad-vector encoding the non-structural protein 3 from YF-17D could elicit a strong CD8+ T-cell response, which afforded a high degree of protection from subsequent intracranial challenge of vaccinated mice. However, full protection was only observed using a vector encoding the structural proteins from YF-17D. This vector elicited virus-specific CD8+ T cells as well as neutralizing antibodies, and both components were shown to be important for protection thus mimicking the situation recently uncovered in YF-17D vaccinated mice. Considering that Ad-vectors are very safe, easy to produce and highly immunogenic in humans, our data indicate that a replication deficient adenovector-based YF vaccine may represent a safe and efficient alternative to the classical live attenuated YF vaccine and should be further tested.

Tick Salivary Cholinesterase: A Probable Immunomodulator of Host-parasite Interactions.

PubMed

Temeyer, Kevin B; Tuckow, Alexander P

2016-05-01

The southern cattle tick, Rhipicephalus (Boophilus) microplus (Canestrini), is the most economically important cattle ectoparasite in the world. Rhipicephalus microplus and Rhipicephalus annulatus (Say) continue to threaten U.S. cattle producers despite eradication and an importation barrier based on inspection, dipping of imported cattle in organophosphate (OP) acaricide, and quarantine of infested premises. OP acaricides inhibit acetylcholinesterase (AChE), essential to tick central nervous system function. Unlike vertebrates, ticks possess at least three genes encoding AChEs, differing in amino acid sequence and biochemical properties. Genomic analyses of R. microplus and the related tick, Ixodes scapularis, suggest that ticks contain many genes encoding different AChEs. This work is the first report of a salivary cholinesterase (ChE) activity in R. microplus, and discusses complexity of the cholinergic system in ticks and significance of tick salivary ChE at the tick-host interface. It further provides three hypotheses that the salivary ChE plausibly functions 1) to reduce presence of potentially toxic acetylcholine present in the large bloodmeal imbibed during rapid engorgement, 2) to modulate the immune response (innate and/or acquired) of the host to tick antigens, and 3) to influence transmission and establishment of pathogens within the host animal. Ticks are vectors for a greater number and variety of pathogens than any other parasite, and are second only to mosquitoes (owing to malaria) as vectors of serious human disease. Saliva-assisted transmission (SAT) of pathogens is well-known; however, the salivary components participating in the SAT process remain to be elucidated. Published by Oxford University Press on behalf of Entomological Society of America 2016. This work is written by US Government employees and is in the public domain in the US.

High-level expression of recombinant beta-galactosidases in Lactobacillus plantarum and Lactobacillus sakei using a Sakacin P-based expression system.

PubMed

Halbmayr, Elisabeth; Mathiesen, Geir; Nguyen, Thu-Ha; Maischberger, Thomas; Peterbauer, Clemens K; Eijsink, Vincent G H; Haltrich, Dietmar

2008-06-25

This work presents the cloning and expression of the genes encoding heterodimeric beta-galactosidases from Lactobacillus reuteri L103, Lactobacillus acidophilus R22, Lactobacillus plantarum WCFS1, and Lactobacillus sakei Lb790. These enzymes consist of two subunits of approximately 73 and 35 kDa, which are encoded by two overlapping genes, lacL and lacM, respectively. We have cloned these genes into the lactobacillal expression vectors pSIP403 and pSIP409, which are based on the sakacin P operon of L. sakei ( Sørvig et al. Microbiology 2005, 151, 2439- 2449 ), and expressed them in the host strains L. plantarum WCFS1 and L. sakei Lb790. Results varied considerably, ranging from 2.23 to 61.1 U/mg of beta-galactosidase activity, depending on the origin of the lacLM genes, the host strain, and the expression vector used. Highest expression levels were obtained in a laboratory cultivation of L. plantarum WCFS1 harboring the plasmid pEH3R containing the lacLM gene from L. reuteri L103. These cultivations yielded approximately 23 000 U of beta-galactosidase activity per liter, corresponding to the formation of roughly 100 mg of recombinant protein per liter of fermentation medium, and beta-galactosidase levels amounted to 55% of the total intracellular protein of the host organism. To further verify the suitability of this expression system, recombinant beta-galactosidase from L. reuteri was purified to apparent homogeneity. The properties of the purified enzyme were essentially identical with the properties of purified native beta-galactosidase from L. reuteri L103. The presented results lead the way to efficient overproduction of beta-galactosidase in a food-grade expression system, which is of high interest for applications in food industry.

In situ reprogramming to transdifferentiate fibroblasts into cardiomyocytes using adenoviral vectors: Implications for clinical myocardial regeneration.

PubMed

Mathison, Megumi; Singh, Vivek P; Chiuchiolo, Maria J; Sanagasetti, Deepthi; Mao, Yun; Patel, Vivekkumar B; Yang, Jianchang; Kaminsky, Stephen M; Crystal, Ronald G; Rosengart, Todd K

2017-02-01

The reprogramming of cardiac fibroblasts into induced cardiomyocyte-like cells improves ventricular function in myocardial infarction models. Only integrating persistent expression vectors have thus far been used to induce reprogramming, potentially limiting its clinical applicability. We therefore tested the reprogramming potential of nonintegrating, acute expression adenoviral (Ad) vectors. Ad or lentivirus vectors encoding Gata4 (G), Mef2c (M), and Tbx5 (T) were validated in vitro. Sprague-Dawley rats then underwent coronary ligation and Ad-mediated administration of vascular endothelial growth factor to generate infarct prevascularization. Three weeks later, animals received Ad or lentivirus encoding G, M, or T (AdGMT or LentiGMT) or an equivalent dose of a null vector (n = 11, 10, and 10, respectively). Outcomes were analyzed by echocardiography, magnetic resonance imaging, and histology. Ad and lentivirus vectors provided equivalent G, M, and T expression in vitro. AdGMT and LentiGMT both likewise induced expression of the cardiomyocyte marker cardiac troponin T in approximately 6% of cardiac fibroblasts versus <1% cardiac troponin T expression in AdNull (adenoviral vector that does not encode a transgene)-treated cells. Infarcted myocardium that had been treated with AdGMT likewise demonstrated greater density of cells expressing the cardiomyocyte marker beta myosin heavy chain 7 compared with AdNull-treated animals. Echocardiography demonstrated that AdGMT and LentiGMT both increased ejection fraction compared with AdNull (AdGMT: 21% ± 3%, LentiGMT: 14% ± 5%, AdNull: -0.4% ± 2%; P < .05). Ad vectors are at least as effective as lentiviral vectors in inducing cardiac fibroblast transdifferentiation into induced cardiomyocyte-like cells and improving cardiac function in postinfarct rat hearts. Short-term expression Ad vectors may represent an important means to induce cardiac cellular reprogramming in humans. Copyright © 2016 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.

A plasmid toolkit for cloning chimeric cDNAs encoding customized fusion proteins into any Gateway destination expression vector

PubMed Central

2013-01-01

Background Valuable clone collections encoding the complete ORFeomes for some model organisms have been constructed following the completion of their genome sequencing projects. These libraries are based on Gateway cloning technology, which facilitates the study of protein function by simplifying the subcloning of open reading frames (ORF) into any suitable destination vector. The expression of proteins of interest as fusions with functional modules is a frequent approach in their initial functional characterization. A limited number of Gateway destination expression vectors allow the construction of fusion proteins from ORFeome-derived sequences, but they are restricted to the possibilities offered by their inbuilt functional modules and their pre-defined model organism-specificity. Thus, the availability of cloning systems that overcome these limitations would be highly advantageous. Results We present a versatile cloning toolkit for constructing fully-customizable three-part fusion proteins based on the MultiSite Gateway cloning system. The fusion protein components are encoded in the three plasmids integral to the kit. These can recombine with any purposely-engineered destination vector that uses a heterologous promoter external to the Gateway cassette, leading to the in-frame cloning of an ORF of interest flanked by two functional modules. In contrast to previous systems, a third part becomes available for peptide-encoding as it no longer needs to contain a promoter, resulting in an increased number of possible fusion combinations. We have constructed the kit’s component plasmids and demonstrate its functionality by providing proof-of-principle data on the expression of prototype fluorescent fusions in transiently-transfected cells. Conclusions We have developed a toolkit for creating fusion proteins with customized N- and C-term modules from Gateway entry clones encoding ORFs of interest. Importantly, our method allows entry clones obtained from ORFeome collections to be used without prior modifications. Using this technology, any existing Gateway destination expression vector with its model-specific properties could be easily adapted for expressing fusion proteins. PMID:23957834

A plasmid toolkit for cloning chimeric cDNAs encoding customized fusion proteins into any Gateway destination expression vector.

PubMed

Buj, Raquel; Iglesias, Noa; Planas, Anna M; Santalucía, Tomàs

2013-08-20

Valuable clone collections encoding the complete ORFeomes for some model organisms have been constructed following the completion of their genome sequencing projects. These libraries are based on Gateway cloning technology, which facilitates the study of protein function by simplifying the subcloning of open reading frames (ORF) into any suitable destination vector. The expression of proteins of interest as fusions with functional modules is a frequent approach in their initial functional characterization. A limited number of Gateway destination expression vectors allow the construction of fusion proteins from ORFeome-derived sequences, but they are restricted to the possibilities offered by their inbuilt functional modules and their pre-defined model organism-specificity. Thus, the availability of cloning systems that overcome these limitations would be highly advantageous. We present a versatile cloning toolkit for constructing fully-customizable three-part fusion proteins based on the MultiSite Gateway cloning system. The fusion protein components are encoded in the three plasmids integral to the kit. These can recombine with any purposely-engineered destination vector that uses a heterologous promoter external to the Gateway cassette, leading to the in-frame cloning of an ORF of interest flanked by two functional modules. In contrast to previous systems, a third part becomes available for peptide-encoding as it no longer needs to contain a promoter, resulting in an increased number of possible fusion combinations. We have constructed the kit's component plasmids and demonstrate its functionality by providing proof-of-principle data on the expression of prototype fluorescent fusions in transiently-transfected cells. We have developed a toolkit for creating fusion proteins with customized N- and C-term modules from Gateway entry clones encoding ORFs of interest. Importantly, our method allows entry clones obtained from ORFeome collections to be used without prior modifications. Using this technology, any existing Gateway destination expression vector with its model-specific properties could be easily adapted for expressing fusion proteins.

Cloning and characterization of an inulinase gene from the marine yeast Candida membranifaciens subsp. flavinogenie W14-3 and its expression in Saccharomyces sp. W0 for ethanol production.

PubMed

Zhang, Lin-Lin; Tan, Mei-Juan; Liu, Guang-Lei; Chi, Zhe; Wang, Guang-Yuan; Chi, Zhen-Ming

2015-04-01

The INU1 gene encoding an exo-inulinase from the marine-derived yeast Candida membranifaciens subsp. flavinogenie W14-3 was cloned and characterized. It had an open reading frame of 1,536 bp long encoding an inulinase. The coding region of it was not interrupted by any intron. The cloned gene encoded 512 amino acid residues of a protein with a putative signal peptide of 23 amino acids and a calculated molecular mass of 57.8 kDa. The protein sequence deduced from the inulinase gene contained the inulinase consensus sequences (WMNDPNGL), (RDP), ECP FS and Q. The protein also had six conserved putative N-glycosylation sites. The deduced inulinase from the yeast strain W14-3 was found to be closely related to that from Candida kutaonensis sp. nov. KRF1, Kluyveromyces marxianus, and Cryptococcus aureus G7a. The inulinase gene with its signal peptide encoding sequence was subcloned into the pMIRSC11 expression vector and expressed in Saccharomyces sp. W0. The recombinant yeast strain W14-3-INU-112 obtained could produce 16.8 U/ml of inulinase activity and 12.5 % (v/v) ethanol from 250 g/l of inulin within 168 h. The monosaccharides were detected after the hydrolysis of inulin with the crude inulinase (the yeast culture). All the results indicated that the cloned gene and the recombinant yeast strain W14-3-INU-112 had potential applications in biotechnology.

Rift Valley fever virus MP-12 vaccine encoding Toscana virus NSs retains neuroinvasiveness in mice.

PubMed

Indran, Sabarish V; Lihoradova, Olga A; Phoenix, Inaia; Lokugamage, Nandadeva; Kalveram, Birte; Head, Jennifer A; Tigabu, Bersabeh; Smith, Jennifer K; Zhang, Lihong; Juelich, Terry L; Gong, Bin; Freiberg, Alexander N; Ikegami, Tetsuro

2013-07-01

Rift Valley fever is a mosquito-borne zoonotic disease endemic to sub-Saharan Africa. Rift Valley fever virus (RVFV; genus Phlebovirus, family Bunyaviridae) causes high rates of abortion and fetal malformation in pregnant ruminants, and haemorrhagic fever, neurological disorders or blindness in humans. The MP-12 strain is a highly efficacious and safe live-attenuated vaccine candidate for both humans and ruminants. However, MP-12 lacks a marker to differentiate infected from vaccinated animals. In this study, we originally aimed to characterize the efficacy of a recombinant RVFV MP-12 strain encoding Toscana virus (TOSV) NSs gene in place of MP-12 NSs (rMP12-TOSNSs). TOSV NSs promotes the degradation of dsRNA-dependent protein kinase (PKR) and inhibits interferon-β gene up-regulation without suppressing host general transcription. Unexpectedly, rMP12-TOSNSs increased death in vaccinated outbred mice and inbred BALB/c or C57BL/6 mice. Immunohistochemistry showed diffusely positive viral antigens in the thalamus, hypothalamus and brainstem, including the medulla. No viral antigens were detected in spleen or liver, which is similar to the antigen distribution of moribund mice infected with MP-12. These results suggest that rMP12-TOSNSs retains neuroinvasiveness in mice. Our findings demonstrate that rMP12-TOSNSs causes neuroinvasion without any hepatic disease and will be useful for studying the neuroinvasion mechanism of RVFV and TOSV.

Rift Valley fever virus MP-12 vaccine encoding Toscana virus NSs retains neuroinvasiveness in mice

PubMed Central

Indran, Sabarish V.; Lihoradova, Olga A.; Phoenix, Inaia; Lokugamage, Nandadeva; Kalveram, Birte; Head, Jennifer A.; Tigabu, Bersabeh; Smith, Jennifer K.; Zhang, Lihong; Juelich, Terry L.; Gong, Bin; Freiberg, Alexander N.

2013-01-01

Rift Valley fever is a mosquito-borne zoonotic disease endemic to sub-Saharan Africa. Rift Valley fever virus (RVFV; genus Phlebovirus, family Bunyaviridae) causes high rates of abortion and fetal malformation in pregnant ruminants, and haemorrhagic fever, neurological disorders or blindness in humans. The MP-12 strain is a highly efficacious and safe live-attenuated vaccine candidate for both humans and ruminants. However, MP-12 lacks a marker to differentiate infected from vaccinated animals. In this study, we originally aimed to characterize the efficacy of a recombinant RVFV MP-12 strain encoding Toscana virus (TOSV) NSs gene in place of MP-12 NSs (rMP12-TOSNSs). TOSV NSs promotes the degradation of dsRNA-dependent protein kinase (PKR) and inhibits interferon-β gene up-regulation without suppressing host general transcription. Unexpectedly, rMP12-TOSNSs increased death in vaccinated outbred mice and inbred BALB/c or C57BL/6 mice. Immunohistochemistry showed diffusely positive viral antigens in the thalamus, hypothalamus and brainstem, including the medulla. No viral antigens were detected in spleen or liver, which is similar to the antigen distribution of moribund mice infected with MP-12. These results suggest that rMP12-TOSNSs retains neuroinvasiveness in mice. Our findings demonstrate that rMP12-TOSNSs causes neuroinvasion without any hepatic disease and will be useful for studying the neuroinvasion mechanism of RVFV and TOSV. PMID:23515022

Compositional Verification with Abstraction, Learning, and SAT Solving

DTIC Science & Technology

2015-05-01

arithmetic, and bit-vectors (currently, via bit-blasting). The front-end is based on an existing tool called UFO [8] which converts C programs to the Horn...supports propositional logic, linear arithmetic, and bit-vectors (via bit-blasting). The front-end is based on the tool UFO [8]. It encodes safety of...tool UFO [8]. The encoding in Horn-SMT only uses the theory of Linear Rational Arithmetic. All experiments were carried out on an Intel R© CoreTM2 Quad

Gene encoding herbicide safener binding protein

DOEpatents

Walton, Jonathan D.; Scott-Craig, John S.

1999-01-01

The cDNA encoding safener binding protein (SafBP), also referred to as SBP1, is set forth in FIG. 5 and SEQ ID No. 1. The deduced amino acid sequence is provided in FIG. 5 and SEQ ID No. 2. Methods of making and using SBP1 and SafBP to alter a plant's sensitivity to certain herbicides or a plant's responsiveness to certain safeners are also provided, as well as expression vectors, transgenic plants or other organisms transfected with said vectors and seeds from said plants.

Pulse Code Modulation (PCM) encoder handbook for Aydin Vector MMP-900 series system

NASA Technical Reports Server (NTRS)

Raphael, David

1995-01-01

This handbook explicates the hardware and software properties of a time division multiplex system. This system is used to sample analog and digital data. The data is then merged with frame synchronization information to produce a serial pulse coded modulation (PCM) bit stream. Information in this handbook is required by users to design congruous interface and attest effective utilization of this encoder system. Aydin Vector provides all of the components for these systems to Goddard Space Flight Center/Wallops Flight Facility.

Allelic Variation of the Tas1r3 Taste Receptor Gene Selectively Affects Behavioral and Neural Taste Responses to Sweeteners in the F2 Hybrids between C57BL/6ByJ and 129P3/J Mice

PubMed Central

Inoue, Masashi; Reed, Danielle R.; Li, Xia; Tordoff, Michael G.; Beauchamp, Gary K.; Bachmanov, Alexander A.

2006-01-01

Recent studies have shown that the T1R3 receptor protein encoded by the Tas1r3 gene is involved in transduction of sweet taste. To assess ligand specificity of the T1R3 receptor, we analyzed the association of Tas1r3 allelic variants with taste responses in mice. In the F2 hybrids between the C57BL/6ByJ (B6) and 129P3/J (129) inbred mouse strains, we determined genotypes of markers on chromosome 4, where Tas1r3 resides, measured consumption of taste solutions presented in two-bottle preference tests, and recorded integrated responses of the chorda tympani gustatory nerve to lingual application of taste stimuli. For intakes and preferences, significant linkages to Tas1r3 were found for the sweeteners sucrose, saccharin, and d-phenylalanine but not glycine. For chorda tympani responses, significant linkages to Tas1r3 were found for the sweeteners sucrose, saccharin, d-phenylalanine, d-tryptophan, and SC-45647 but not glycine, l-proline, l-alanine, or l-glutamine. No linkages to distal chromosome 4 were detected for behavioral or neural responses to non-sweet quinine, citric acid, HCl, NaCl, KCl, monosodium glutamate, inosine 5′-monophosphate, or ammonium glutamate. These results demonstrate that allelic variation of the Tas1r3 gene affects gustatory neural and behavioral responses to some, but not all, sweeteners. This study describes the range of ligand sensitivity of the T1R3 receptor using an in vivo approach and, to our knowledge, is the first genetic mapping study of activity in gustatory nerves. PMID:14999080

Langerin negative dendritic cells promote potent CD8+ T-cell priming by skin delivery of live adenovirus vaccine microneedle arrays

PubMed Central

Bachy, Veronique; Hervouet, Catherine; Becker, Pablo D.; Chorro, Laurent; Carlin, Leo M.; Herath, Shanthi; Papagatsias, Timos; Barbaroux, Jean-Baptiste; Oh, Sea-Jin; Benlahrech, Adel; Athanasopoulos, Takis; Dickson, George; Patterson, Steven; Kwon, Sung-Yun; Geissmann, Frederic; Klavinskis, Linda S.

2013-01-01

Stabilization of virus protein structure and nucleic acid integrity is challenging yet essential to preserve the transcriptional competence of live recombinant viral vaccine vectors in the absence of a cold chain. When coupled with needle-free skin delivery, such a platform would address an unmet need in global vaccine coverage against HIV and other global pathogens. Herein, we show that a simple dissolvable microneedle array (MA) delivery system preserves the immunogenicity of vaccines encoded by live recombinant human adenovirus type 5 (rAdHu5). Specifically, dried rAdHu5 MA immunization induced CD8+ T-cell expansion and multifunctional cytokine responses equipotent with conventional injectable routes of immunization. Intravital imaging demonstrated MA cargo distributed both in the epidermis and dermis, with acquisition by CD11c+ dendritic cells (DCs) in the dermis. The MA immunizing properties were attributable to CD11c+ MHCIIhi CD8αneg epithelial cell adhesion molecule (EpCAMneg) CD11b+ langerin (Lang; CD207)neg DCs, but neither Langerhans cells nor Lang+ DCs were required for CD8+ T-cell priming. This study demonstrates an important technical advance for viral vaccine vectors progressing to the clinic and provides insights into the mechanism of CD8+ T-cell priming by live rAdHu5 MAs. PMID:23386724

Langerin negative dendritic cells promote potent CD8+ T-cell priming by skin delivery of live adenovirus vaccine microneedle arrays.

PubMed

Bachy, Veronique; Hervouet, Catherine; Becker, Pablo D; Chorro, Laurent; Carlin, Leo M; Herath, Shanthi; Papagatsias, Timos; Barbaroux, Jean-Baptiste; Oh, Sea-Jin; Benlahrech, Adel; Athanasopoulos, Takis; Dickson, George; Patterson, Steven; Kwon, Sung-Yun; Geissmann, Frederic; Klavinskis, Linda S

2013-02-19

Stabilization of virus protein structure and nucleic acid integrity is challenging yet essential to preserve the transcriptional competence of live recombinant viral vaccine vectors in the absence of a cold chain. When coupled with needle-free skin delivery, such a platform would address an unmet need in global vaccine coverage against HIV and other global pathogens. Herein, we show that a simple dissolvable microneedle array (MA) delivery system preserves the immunogenicity of vaccines encoded by live recombinant human adenovirus type 5 (rAdHu5). Specifically, dried rAdHu5 MA immunization induced CD8(+) T-cell expansion and multifunctional cytokine responses equipotent with conventional injectable routes of immunization. Intravital imaging demonstrated MA cargo distributed both in the epidermis and dermis, with acquisition by CD11c(+) dendritic cells (DCs) in the dermis. The MA immunizing properties were attributable to CD11c(+) MHCII(hi) CD8α(neg) epithelial cell adhesion molecule (EpCAM(neg)) CD11b(+) langerin (Lang; CD207)(neg) DCs, but neither Langerhans cells nor Lang(+) DCs were required for CD8(+) T-cell priming. This study demonstrates an important technical advance for viral vaccine vectors progressing to the clinic and provides insights into the mechanism of CD8(+) T-cell priming by live rAdHu5 MAs.

Amino acid "little Big Bang": representing amino acid substitution matrices as dot products of Euclidian vectors.

PubMed

Zimmermann, Karel; Gibrat, Jean-François

2010-01-04

Sequence comparisons make use of a one-letter representation for amino acids, the necessary quantitative information being supplied by the substitution matrices. This paper deals with the problem of finding a representation that provides a comprehensive description of amino acid intrinsic properties consistent with the substitution matrices. We present a Euclidian vector representation of the amino acids, obtained by the singular value decomposition of the substitution matrices. The substitution matrix entries correspond to the dot product of amino acid vectors. We apply this vector encoding to the study of the relative importance of various amino acid physicochemical properties upon the substitution matrices. We also characterize and compare the PAM and BLOSUM series substitution matrices. This vector encoding introduces a Euclidian metric in the amino acid space, consistent with substitution matrices. Such a numerical description of the amino acid is useful when intrinsic properties of amino acids are necessary, for instance, building sequence profiles or finding consensus sequences, using machine learning algorithms such as Support Vector Machine and Neural Networks algorithms.

Life cycle, feeding and defecation patterns of Rhodnius ecuadoriensis (Lent & León 1958) (Hemiptera: Reduviidae: Triatominae) under laboratory conditions.

PubMed

Villacís, Anita G; Arcos-Terán, Laura; Grijalva, Mario J

2008-11-01

Rhodnius ecuadoriensis is the second most important vector of Chagas Disease (CD) in Ecuador. The objective of this study was to describe (and compare) the life cycle, the feeding and defecation patterns under laboratory conditions of two populations of this specie [from the provinces of Manabí (Coastal region) and Loja (Andean region)]. Egg-to-adult (n = 57) development took an average of 189.9 +/- 20 (Manabí) and 181.3 +/- 6.4 days (Loja). Mortality rates were high among Lojan nymphs. Pre-feeding time (from contact with host to feeding initiation) ranged from 4 min 42 s [nymph I (NI)] to 8 min 30 s (male); feeding time ranged from 14 min 45 s (NI)-28 min 25 s (male) (Manabí) and from 15 min 25 s (NI)-28 min 57 s (nymph V) (Loja). The amount of blood ingested increased significantly with instar and was larger for Manabí specimens (p < 0.001). Defecation while feeding was observed in Manabí specimens from stage nymph III and in Lojan bugs from stage nymph IV. There was a gradual, age-related increase in the frequency of this behaviour in both populations. Our results suggest that R. ecuadoriensis has the bionomic traits of an efficient vector of Trypanosoma cruzi. Together with previous data on the capacity of this species to infest rural households, these results indicate that control of synanthropic R. ecuadoriensis populations in the coastal and Andean regions may have a significant impact for CD control in Ecuador and Northern Peru.

Protection of Chickens against Avian Influenza with Non-Replicating Adenovirus-Vectored Vaccine

PubMed Central

Toro, Haroldo; Tang, De-chu C.; Suarez, David L.; Shi, Z.

2009-01-01

Protective immunity against avian influenza (AI) virus was elicited in chickens by single-dose vaccination with a replication competent adenovirus (RCA) -free human adenovirus (Ad) vector encoding an H7 AI hemagglutinin (AdChNY94.H7). Chickens vaccinated in ovo with an Ad vector encoding an AI H5 (AdTW68.H5) previously described, which were subsequently vaccinated intramuscularly with AdChNY94.H7 post-hatch, responded with robust antibody titers against both the H5 and H7 AI proteins. Antibody responses to Ad vector in ovo vaccination follow a dose-response kinetic. The use of a synthetic AI H5 gene codon optimized to match the chicken cell tRNA pool was more potent than the cognate H5 gene. The use of Ad-vectored vaccines to increase resistance of chicken populations against multiple AI strains could reduce the risk of an avian-originating influenza pandemic in humans. PMID:18384919

Subpial Adeno-associated Virus 9 (AAV9) Vector Delivery in Adult Mice.

PubMed

Tadokoro, Takahiro; Miyanohara, Atsushi; Navarro, Michael; Kamizato, Kota; Juhas, Stefan; Juhasova, Jana; Marsala, Silvia; Platoshyn, Oleksandr; Curtis, Erik; Gabel, Brandon; Ciacci, Joseph; Lukacova, Nada; Bimbova, Katarina; Marsala, Martin

2017-07-13

The successful development of a subpial adeno-associated virus 9 (AAV9) vector delivery technique in adult rats and pigs has been reported on previously. Using subpially-placed polyethylene catheters (PE-10 or PE-5) for AAV9 delivery, potent transgene expression through the spinal parenchyma (white and gray matter) in subpially-injected spinal segments has been demonstrated. Because of the wide range of transgenic mouse models of neurodegenerative diseases, there is a strong desire for the development of a potent central nervous system (CNS)-targeted vector delivery technique in adult mice. Accordingly, the present study describes the development of a spinal subpial vector delivery device and technique to permit safe and effective spinal AAV9 delivery in adult C57BL/6J mice. In spinally immobilized and anesthetized mice, the pia mater (cervical 1 and lumbar 1-2 spinal segmental level) was incised with a sharp 34 G needle using an XYZ manipulator. A second XYZ manipulator was then used to advance a blunt 36G needle into the lumbar and/or cervical subpial space. The AAV9 vector (3-5 µL; 1.2 x 10 13 genome copies (gc)) encoding green fluorescent protein (GFP) was then injected subpially. After injections, neurological function (motor and sensory) was assessed periodically, and animals were perfusion-fixed 14 days after AAV9 delivery with 4% paraformaldehyde. Analysis of horizontal or transverse spinal cord sections showed transgene expression throughout the entire spinal cord, in both gray and white matter. In addition, intense retrogradely-mediated GFP expression was seen in the descending motor axons and neurons in the motor cortex, nucleus ruber, and formatio reticularis. No neurological dysfunction was noted in any animals. These data show that the subpial vector delivery technique can successfully be used in adult mice, without causing procedure-related spinal cord injury, and is associated with highly potent transgene expression throughout the spinal neuraxis.

Cellobiohydrolase variants and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wogulis, Mark

The present invention relates to variants of a parent cellobiohydrolase II. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

Polypeptides having catalase activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Ye; Duan, Junxin; Zhang, Yu

Provided are isolated polypeptides having catalase activity and polynucleotides encoding the polypeptides. Also provided are nucleic acid constructs, vectors and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

A novel, broad-range, CTXΦ-derived stable integrative expression vector for functional studies.

PubMed

Das, Bhabatosh; Kumari, Reena; Pant, Archana; Sen Gupta, Sourav; Saxena, Shruti; Mehta, Ojasvi; Nair, Gopinath Balakrish

2014-12-01

CTXΦ, a filamentous vibriophage encoding cholera toxin, uses a unique strategy for its lysogeny. The single-stranded phage genome forms intramolecular base-pairing interactions between two inversely oriented XerC and XerD binding sites (XBS) and generates a functional phage attachment site, attP(+), for integration. The attP(+) structure is recognized by the host-encoded tyrosine recombinases XerC and XerD (XerCD), which enables irreversible integration of CTXΦ into the chromosome dimer resolution site (dif) of Vibrio cholerae. The dif site and the XerCD recombinases are widely conserved in bacteria. We took advantage of these conserved attributes to develop a broad-host-range integrative expression vector that could irreversibly integrate into the host chromosome using XerCD recombinases without altering the function of any known open reading frame (ORF). In this study, we engineered two different arabinose-inducible expression vectors, pBD62 and pBD66, using XBS of CTXΦ. pBD62 replicates conditionally and integrates efficiently into the dif of the bacterial chromosome by site-specific recombination using host-encoded XerCD recombinases. The expression level of the gene of interest could be controlled through the PBAD promoter by modulating the functions of the vector-encoded transcriptional factor AraC. We validated the irreversible integration of pBD62 into a wide range of pathogenic and nonpathogenic bacteria, such as V. cholerae, Vibrio fluvialis, Vibrio parahaemolyticus, Escherichia coli, Salmonella enterica, and Klebsiella pneumoniae. Gene expression from the PBAD promoter of integrated vectors was confirmed in V. cholerae using the well-studied reporter genes mCherry, eGFP, and lacZ. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

vanC Cluster of Vancomycin-Resistant Enterococcus gallinarum BM4174

PubMed Central

Arias, Cesar A.; Courvalin, Patrice; Reynolds, Peter E.

2000-01-01

Glycopeptide-resistant enterococci of the VanC type synthesize UDP-muramyl-pentapeptide[d-Ser] for cell wall assembly and prevent synthesis of peptidoglycan precursors ending in d-Ala. The vanC cluster of Enterococcus gallinarum BM4174 consists of five genes: vanC-1, vanXYC, vanT, vanRC, and vanSC. Three genes are sufficient for resistance: vanC-1 encodes a ligase that synthesizes the dipeptide d-Ala-d-Ser for addition to UDP-MurNAc-tripeptide, vanXYC encodes a d,d-dipeptidase–carboxypeptidase that hydrolyzes d-Ala-d-Ala and removes d-Ala from UDP-MurNAc-pentapeptide[d-Ala], and vanT encodes a membrane-bound serine racemase that provides d-Ser for the synthetic pathway. The three genes are clustered: the start codons of vanXYC and vanT overlap the termination codons of vanC-1 and vanXYC, respectively. Two genes which encode proteins with homology to the VanS-VanR two-component regulatory system were present downstream from the resistance genes. The predicted amino acid sequence of VanRC exhibited 50% identity to VanR and 33% identity to VanRB. VanSC had 40% identity to VanS over a region of 308 amino acids and 24% identity to VanSB over a region of 285 amino acids. All residues with important functions in response regulators and histidine kinases were conserved in VanRC and VanSC, respectively. Induction experiments based on the determination of d,d-carboxypeptidase activity in cytoplasmic extracts confirmed that the genes were expressed constitutively. Using a promoter-probing vector, regions upstream from the resistance and regulatory genes were identified that have promoter activity. PMID:10817725

Modular protein expression by RNA trans-splicing enables flexible expression of antibody formats in mammalian cells from a dual-host phage display vector.

PubMed

Shang, Yonglei; Tesar, Devin; Hötzel, Isidro

2015-10-01

A recently described dual-host phage display vector that allows expression of immunoglobulin G (IgG) in mammalian cells bypasses the need for subcloning of phage display clone inserts to mammalian vectors for IgG expression in large antibody discovery and optimization campaigns. However, antibody discovery and optimization campaigns usually need different antibody formats for screening, requiring reformatting of the clones in the dual-host phage display vector to an alternative vector. We developed a modular protein expression system mediated by RNA trans-splicing to enable the expression of different antibody formats from the same phage display vector. The heavy-chain region encoded by the phage display vector is directly and precisely fused to different downstream heavy-chain sequences encoded by complementing plasmids simply by joining exons in different pre-mRNAs by trans-splicing. The modular expression system can be used to efficiently express structurally correct IgG and Fab fragments or other antibody formats from the same phage display clone in mammalian cells without clone reformatting. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Simple extraction method for quantification of phenothiazine residues in pork muscle using liquid chromatography-triple quadrupole tandem mass spectrometry.

PubMed

Zhang, Dan; Park, Jin-A; Kim, Seong-Kwan; Cho, Sang-Hyun; Cho, Soo-Min; Shim, Jae-Han; Kim, Jin-Suk; Abd El-Aty, A M; Shin, Ho-Chul

2017-06-01

In this study, an analytical method was developed for quantification of residues of the anthelmintic drug phenothiazine (PTZ) in pork muscle using liquid chromatography-tandem mass spectrometry. Muscles were extracted using 0.2% formic acid and 10 mm ammonium formate in acetonitrile, defatted and purified using n-hexane. The drug was well separated on a Waters XBridge™ C 18 analytical column using a binary solvent system consisting of 0.2% formic acid and 10 mm ammonium formate in ultrapure water (A) and acetonitrile (B). Good linearity was achieved over a six-point concentration range in matrix-matched calibration with determination coefficient =0.9846. Fortified pork muscle having concentrations equivalent to and double the limit of quantification (1 ng/g) yielded recovery ranges between 100.82 and 104.03% and relative standard deviations <12%. Samples (n = 5) collected from large markets located in Seoul City tested negative for PTZ residue. In conclusion, 0.2% formic acid and ammonium formate in acetonitrile can effectively extract PTZ from pork muscle without solid-phase extraction, a step normally required for cleanup before analysis and the validated method can be used for routine analysis to ensure the quality of animal products. Copyright © 2016 John Wiley & Sons, Ltd.

2, 5-Disubstituted phthalimides: design, synthesis and anticonvulsant activity in scPTZ and MES models.

PubMed

Saadabadi, Atefeh; Kohen, Babak; Irandoust, Maryam; Shafaroudi, Hamed; Mohammadpour, Tara; Rezayat, Mahdi; Davood, Asghar

2018-05-15

In this study, fifteenth new 2,5-disubstituted analgouges of phthalimide were designed and synthesized using the appropriate synthetic route to evaluate anticonvulsant activity against the maximal electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ) compare to phenytoin as a positive control. The structures of the synthesized compounds were confirmed by FT-IR, H-NMR, C-NMR and MASS spectroscopy. All the tested compounds were found to be effective in the PTZ model at the dose of 60 mg/kg and most of the compounds showed protection against MES test indicative of their ability to inhibit the seizure spread at the all dose ranges. Compound 3 has illustrated the best efficacy among all compounds and showed more potency than phenytoin in clonic seizure and was potent as phenytoin in tonic seizure. Using a model of the Na channel, these derivatives were docked in the active site. Docking studies displayed that all synthesized compounds have more negative binding energy compare to reference drug and inhibition-constant less than phenytoin that means they can block the receptor more efficiently and usually form hydrophobic interactions or hydrogen binding interaction frequently with the domains I, II, III and rarely with domain IV. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Neurotoxic lesions of the pedunculopontine tegmental nucleus impair the elaboration of postictal antinociception.

PubMed

de Oliveira, Rithiele Cristina; de Oliveira, Ricardo; Falconi-Sobrinho, Luiz Luciano; Biagioni, Audrey Franceschi; Almada, Rafael Carvalho; Dos Anjos-Garcia, Tayllon; Bazaglia-de-Sousa, Guilherme; Khan, Asmat Ullah; Coimbra, Norberto Cysne

2018-05-12

Generalised tonic-clonic seizures, generated by abnormal neuronal hyper-activity, cause a significant and long-lasting increase in the nociceptive threshold. The pedunculopontine tegmental nucleus (PPTN) plays a crucial role in the regulation of seizures as well as the modulation of pain, but its role in postictal antinociceptive processes remains unclear. In the present study, we aimed to investigate the involvement of PPTN neurons in the postictal antinociception. Wistar rats had their tail-flick baseline recorded and were injected with ibotenic acid (1.0 μg/0.2 μL) into the PPTN, aiming to promote a local neurotoxic lesion. Five days after the neuronal damage, pentylenetetrazole (PTZ; 64 mg/kg) was intraperitoneally administered to induce tonic-clonic seizures. The tail-withdrawal latency was measured immediately after the seizures (0 min) and subsequently at 10-min intervals until 130 min after the seizures were induced pharmacologically. Ibotenic acid microinjected into the PPTN did not reduce the PTZ-induced seizure duration and severity, but it diminished the postictal antinociception from 0 to 130 min after the end of the PTZ-induced tonic-clonic seizures. These results suggest that the postictal antinociception depends on the PPTN neuronal cells integrity. Copyright © 2018 Elsevier Inc. All rights reserved.

Construction of improved temperature-sensitive and mobilizable vectors and their use for constructing mutations in the adhesin-encoding acm gene of poorly transformable clinical Enterococcus faecium strains.

PubMed

Nallapareddy, Sreedhar R; Singh, Kavindra V; Murray, Barbara E

2006-01-01

Inactivation by allelic exchange in clinical isolates of the emerging nosocomial pathogen Enterococcus faecium has been hindered by lack of efficient tools, and, in this study, transformation of clinical isolates was found to be particularly problematic. For this reason, a vector for allelic replacement (pTEX5500ts) was constructed that includes (i) the pWV01-based gram-positive repAts replication region, which is known to confer a high degree of temperature intolerance, (ii) Escherichia coli oriR from pUC18, (iii) two extended multiple-cloning sites located upstream and downstream of one of the marker genes for efficient cloning of flanking regions for double-crossover mutagenesis, (iv) transcriptional terminator sites to terminate undesired readthrough, and (v) a synthetic extended promoter region containing the cat gene for allelic exchange and a high-level gentamicin resistance gene, aph(2'')-Id, to distinguish double-crossover recombination, both of which are functional in gram-positive and gram-negative backgrounds. To demonstrate the functionality of this vector, the vector was used to construct an acm (encoding an adhesin to collagen from E. faecium) deletion mutant of a poorly transformable multidrug-resistant E. faecium endocarditis isolate, TX0082. The acm-deleted strain, TX6051 (TX0082Deltaacm), was shown to lack Acm on its surface, which resulted in the abolishment of the collagen adherence phenotype observed in TX0082. A mobilizable derivative (pTEX5501ts) that contains oriT of Tn916 to facilitate conjugative transfer from the transformable E. faecalis strain JH2Sm::Tn916 to E. faecium was also constructed. Using this vector, the acm gene of a nonelectroporable E. faecium wound isolate was successfully interrupted. Thus, pTEX5500ts and its mobilizable derivative demonstrated their roles as important tools by helping to create the first reported allelic replacement in E. faecium; the constructed this acm deletion mutant will be useful for assessing the role of acm in E. faecium pathogenesis using animal models.

Preparation and infrared/raman classification of 630 spectroscopically encoded styrene copolymers.

PubMed

Fenniri, Hicham; Chun, Sangki; Terreau, Owen; Bravo-Vasquez, Juan-Pablo

2008-01-01

The barcoded resins (BCRs) were introduced recently as a platform for encoded combinatorial chemistry. One of the main challenges yet to be overcome is the demonstration that a large number of BCRs could be generated and classified with high confidence. Here, we describe the synthesis and classification of 630 polystyrene-based copolymers prepared from the combinatorial association of 15 spectroscopically active styrene monomers. Each of the 630 copolymers displayed a unique vibrational fingerprint (infrared and Raman), which was converted into a spectral vector. To each of the 630 copolymers, a vector of the known (reference) composition was assigned. Unknown (prediction) vectors were decoded using multivariate data analysis. From the inner product of the reference and prediction vectors, a correlation map comparing 396 900 copolymer pairs (630 x 630) was generated. In 100% of the cases, the highest correlation was obtained for polymer pairs in which the reference and prediction vectors correspond to copolymers prepared from identical styrene monomers, thus demonstrating the high reliability of this encoding strategy. We have also established that the spectroscopic barcodes generated from the Raman and infrared spectra are independent of the copolymers' morphology (beaded versus bulk polymers). Besides the demonstration of the generality of the polymer barcoding strategy, the analytical methods developed here could in principle be extended to the investigation of the composition and purity of any other synthetic polymer and biopolymer library, or even scaffold-based combinatorial libraries.

An Oral DNA Vaccine Encoding Endoglin Eradicates Breast Tumors by Blocking Their Blood Supply

DTIC Science & Technology

2007-05-01

Marcantonio , Ph.D. Research Associate Dorothy...SB *pɘ.05 0 2 0 4 0 6 0 8 0 5 :1 1 0 :1 20 :1 E:T Ratio % S p e c i f i c L y s i s 100 101 102 103 104 FL2-Height Human Endoglin C o u n t s A B...Fig3. Cytotoxicity assay of breast cancer specific CTL. 0 5000 10000 15000 F l u o r e s c e i n l e v e l s / g HuEGEmpty Vector SB * *pɘ.005 B

Image Coding Based on Address Vector Quantization.

NASA Astrophysics Data System (ADS)

Feng, Yushu

Image coding is finding increased application in teleconferencing, archiving, and remote sensing. This thesis investigates the potential of Vector Quantization (VQ), a relatively new source coding technique, for compression of monochromatic and color images. Extensions of the Vector Quantization technique to the Address Vector Quantization method have been investigated. In Vector Quantization, the image data to be encoded are first processed to yield a set of vectors. A codeword from the codebook which best matches the input image vector is then selected. Compression is achieved by replacing the image vector with the index of the code-word which produced the best match, the index is sent to the channel. Reconstruction of the image is done by using a table lookup technique, where the label is simply used as an address for a table containing the representative vectors. A code-book of representative vectors (codewords) is generated using an iterative clustering algorithm such as K-means, or the generalized Lloyd algorithm. A review of different Vector Quantization techniques are given in chapter 1. Chapter 2 gives an overview of codebook design methods including the Kohonen neural network to design codebook. During the encoding process, the correlation of the address is considered and Address Vector Quantization is developed for color image and monochrome image coding. Address VQ which includes static and dynamic processes is introduced in chapter 3. In order to overcome the problems in Hierarchical VQ, Multi-layer Address Vector Quantization is proposed in chapter 4. This approach gives the same performance as that of the normal VQ scheme but the bit rate is about 1/2 to 1/3 as that of the normal VQ method. In chapter 5, a Dynamic Finite State VQ based on a probability transition matrix to select the best subcodebook to encode the image is developed. In chapter 6, a new adaptive vector quantization scheme, suitable for color video coding, called "A Self -Organizing Adaptive VQ Technique" is presented. In addition to chapters 2 through 6 which report on new work, this dissertation includes one chapter (chapter 1) and part of chapter 2 which review previous work on VQ and image coding, respectively. Finally, a short discussion of directions for further research is presented in conclusion.

Induction of complex immune responses and strong protection against retrovirus challenge by adenovirus-based immunization depends on the order of vaccine delivery.

PubMed

Kaulfuß, Meike; Wensing, Ina; Windmann, Sonja; Hrycak, Camilla Patrizia; Bayer, Wibke

2017-02-06

In the Friend retrovirus mouse model we developed potent adenovirus-based vaccines that were designed to induce either strong Friend virus GagL 85-93 -specific CD8 + T cell or antibody responses, respectively. To optimize the immunization outcome we evaluated vaccination strategies using combinations of these vaccines. While the vaccines on their own confer strong protection from a subsequent Friend virus challenge, the simple combination of the vaccines for the establishment of an optimized immunization protocol did not result in a further improvement of vaccine effectivity. We demonstrate that the co-immunization with GagL 85-93 /leader-gag encoding vectors together with envelope-encoding vectors abrogates the induction of GagL 85-93 -specific CD8 + T cells, and in successive immunization protocols the immunization with the GagL 85-93 /leader-gag encoding vector had to precede the immunization with an envelope encoding vector for the efficient induction of GagL 85-93 -specific CD8 + T cells. Importantly, the antibody response to envelope was in fact enhanced when the mice were adenovirus-experienced from a prior immunization, highlighting the expedience of this approach. To circumvent the immunosuppressive effect of envelope on immune responses to simultaneously or subsequently administered immunogens, we developed a two immunizations-based vaccination protocol that induces strong immune responses and confers robust protection of highly Friend virus-susceptible mice from a lethal Friend virus challenge.

Antiseizure Effects of Ketogenic Diet on Seizures Induced with Pentylenetetrazole, 4-Aminopyridine and Strychnine in Wistar Rats.

PubMed

Sanya, E O; Soladoye, A O; Desalu, O O; Kolo, P M; Olatunji, L A; Olarinoye, J K

2017-03-06

The ketogenic diet (KD) is a cheap and effective alternative therapy for most epilepsy. There are paucity of experimental data in Nigeria on the usefulness of KD in epilepsy models. This is likely to be responsible for the poor clinical acceptability of the diet in the country. This study therefore aimed at providing experimental data on usefulness of KD on seizure models.  The study used 64 Wistar rats that were divided into two dietary groups [normal diet (ND) and ketogenic diet (KD)]. Animal in each group were fed for 35days. Medium chain triglyceride ketogenic diet (MCT-KD) was used and it consisted of 15% carbohydrate in normal rat chow long with 5ml sunflower oil (25% (v/w). The normal diet was the usual rat chow. Seizures were induced with one of Pentelyntetrazole (PTZ), 4-Aminopyridine (AP) and Strychnine (STR). Fasting glucose, ketosis level and serum chemistry were determined and seizure parameters recorded. Serum ketosis was significantly higher in MCT-KD-fed rats (12.7 ±2.6) than ND-fed (5.17±0.86) rats. Fasting blood glucose was higher in ND-fed rats (5.3±0.9mMol/l) than in MCT-KD fed rats (5.1±0.5mMol/l) with p=0.9. Seizure latency was significantly prolonged in ND-fed compared with MCT-KD fed rats after PTZ-induced seizures (61±9sec vs 570±34sec) and AP-induced seizures (49±11sec vs 483±41sec). The difference after Str-induced seizure (51±7 vs 62±8 sec) was not significan. The differences in seizure duration between ND-fed and MCT-KD fed rats with PTZ (4296±77sec vs 366±46sec) and with AP (5238±102sec vs 480±67sec) were significant (p<0.05), but not with STR (3841±94sec vs 3510±89sec) respectively. The mean serum Na+ was significantly higher in MCT-KD fed (141.7±2.1mMol/l) than ND-fed rats (137±2.3mMol/l). There was no significant difference in mean values of other serum electrolytes between the MCT-KD fed and ND-fed animals. MCT-KD caused increase resistance to PTZ-and AP-induced seizures, but has no effect on STR-induced seizures. This antiseizure property is probably mediated through GABAergic receptors (PTZ effect) and blockade of membrane bound KATP channels (AP effect) with some enhancement by serum ketosis.

Polypeptides having beta-glucosidase activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Ye; Duan, Junxin; Zhang, Yu

Provided are isolated polypeptides having beta-glucosidase activity and polynucleotides encoding the polypeptides. Also provided are nucleic acid constructs, vectors and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having endoglucanase activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Yu; Liu, Ye; Duan, Junxin

Provided are isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. Also provided are nucleic acid constructs, vectors and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having beta-xylosidase activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Ye; Tang, Lan; Zhang, Yu

Provided are isolated polypeptides having beta-xylosidase activity and polynucleotides encoding the polypeptides. Also provided are nucleic acid constructs, vectors and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Cellobiohydrolase variants and polynucleotides encoding the same

DOEpatents

Wogulis, Mark

2014-09-09

The present invention relates to variants of a parent cellobiohydrolase. The present invention also relates to polynucleotides encoding the cellobiohydrolase variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the cellobiohydrolase variants.

Hybrid polypeptides having cellobiohydrolase activity and polynucleotides encoding same

DOEpatents

Liu, Ye; Shaghasi, Tarana

2016-11-01

The present invention provides hybrid polypeptides having cellobiohydrolase activity. The present invention also provides polynucleotides encoding the hybrid polypeptides; nucleic acid constructs, vectors and host cells comprising the polynucleotides; and processes of using the hybrid polypeptides.

Adenovirus-based genetic vaccines for biodefense.

PubMed

Boyer, Julie L; Kobinger, Gary; Wilson, James M; Crystal, Ronald G

2005-02-01

The robust host responses elicited against transgenes encoded by (E1-)(E3-) adenovirus (Ad) gene transfer vectors can be used to develop Ad-based vectors as platform technologies for vaccines against potential bioterror pathogens. This review focuses on pathogens of major concern as bioterror agents and why Ad vectors are ideal as anti-bioterror vaccine platforms, providing examples from our laboratories of using Ad vectors as vaccines against potential bioterror pathogens and how Ad vectors can be developed to enhance vaccine efficacy in the bioterror war.

A Family of LIC Vectors for High-Throughput Cloning and Purification of Proteins1

PubMed Central

Eschenfeldt, William H.; Stols, Lucy; Millard, Cynthia Sanville; Joachimiak, Andrzej; Donnelly, Mark I.

2009-01-01

Summary Fifteen related ligation-independent cloning vectors were constructed for high-throughput cloning and purification of proteins. The vectors encode a TEV protease site for removal of tags that facilitate protein purification (his-tag) or improve solubility (MBP, GST). Specialized vectors allow coexpression and copurification of interacting proteins, or in vivo removal of MBP by TVMV protease to improve screening and purification. All target genes and vectors are processed by the same protocols, which we describe here. PMID:18988021

Food-grade host/vector expression system for Lactobacillus casei based on complementation of plasmid-associated phospho-beta-galactosidase gene lacG.

PubMed

Takala, T M; Saris, P E J; Tynkkynen, S S H

2003-01-01

A new food-grade host/vector system for Lactobacillus casei based on lactose selection was constructed. The wild-type non-starter host Lb. casei strain E utilizes lactose via a plasmid-encoded phosphotransferase system. For food-grade cloning, a stable lactose-deficient mutant was constructed by deleting a 141-bp fragment from the phospho-beta-galactosidase gene lacG via gene replacement. The deletion resulted in an inactive phospho-beta-galactosidase enzyme with an internal in-frame deletion of 47 amino acids. A complementation plasmid was constructed containing a replicon from Lactococcus lactis, the lacG gene from Lb. casei, and the constitutive promoter of pepR for lacG expression from Lb. rhamnosus. The expression of the lacG gene from the resulting food-grade plasmid pLEB600 restored the ability of the lactose-negative mutant strain to grow on lactose to the wild-type level. The vector pLEB600 was used for expression of the proline iminopeptidase gene pepI from Lb. helveticus in Lb. casei. The results show that the food-grade expression system reported in this paper can be used for expression of foreign genes in Lb. casei.

Nucleus accumbens lentiviral-mediated gain of function of the oxytocin receptor regulates anxiety- and ethanol-related behaviors in adult mice.

PubMed

Bahi, Amine; Al Mansouri, Shamma; Al Maamari, Elyazia

2016-10-01

Anxiety is believed to influence ethanol use human in alcoholics. Studies using laboratory animals suggested an interaction between oxytocin and the behavioral effects of ethanol. Our previous study implicated a potential role for the oxytocin receptor (OxtR) in regulating ethanol-conditioned place preference. Here, we examined anxiety and the behavioral responses to ethanol in C57BL/6 mice stereotaxically injected in the nucleus accumbens (NAcc) with lentiviral vectors expressing an empty vector (Mock) or the OxtR cDNA. For anxiety we used the elevated-plus maze, the open-field and the marble-burying tests and for ethanol we used the two-bottle choice paradigm, the wire-hanging and ethanol-induced loss-of-righting-reflex tests. We found that, compared to Mock, OxtR overexpression led to anxiolytic-like behavior without altering spontaneous locomotor activity. Most importantly, we found that, relative to Mock controls, increased expression of the OxtR in the NAcc led to decreased ethanol consumption and preference in the two-bottle choice protocol and increased resistance to ethanol-induced sedation. We also compared the consequence of OxtR modulation on the consumption and preference of saccharin and quinine and found that the two experimental groups did not differ for any tastant. These results provide further evidence that the oxytocin system contributes to the regulation of ethanol drinking and sensitivity and position OxtR as a central molecular mediator of ethanol's effects within the mesolimbic system. Taken together, the current findings suggest that OxtR manipulation may be a relevant strategy to address ethanol use disorders. Copyright © 2016 Elsevier Inc. All rights reserved.

Hybrid polypeptides having cellobiohydrolase activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Ye; Shaghasi, Tarana

The present invention relates to hybrid polypeptides having cellobiohydrolase activity. The present invention also relates to polynucleotides encoding the hybrid polypeptides; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and processes of using the hybrid polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Zhang, Yu; Duan, Junxin; Tang, Lan; Wu, Wenping

2015-06-09

Provided are isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. Also provided are nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having endoglucanase activity and polynucleotides encoding same

DOEpatents

Liu, Ye; Duan, Junxin; Tang, Lan

2015-09-22

The present invention provides isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also provides nucleic acid constructs, vectors, and host cell comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellobiohydrolase activitiy and polynucleotides encoding same

DOEpatents

Liu, Ye; Tang, Lan; Duan, Junxin

2015-12-15

The present invention provides isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also provides nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

DOEpatents

Liu, Ye; Tang, Lan

2015-07-14

The present invention provides isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also provides nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Pulse Code Modulation (PCM) encoder handbook for Aydin Vector MMP-600 series system

NASA Technical Reports Server (NTRS)

Currier, S. F.; Powell, W. R.

1986-01-01

The hardware and software characteristics of a time division multiplex system are described. The system is used to sample analog and digital data. The data is merged with synchronization information to produce a serial pulse coded modulation (PCM) bit stream. Information presented herein is required by users to design compatible interfaces and assure effective utilization of this encoder system. GSFC/Wallops Flight Facility has flown approximately 50 of these systems through 1984 on sounding rockets with no inflight failures. Aydin Vector manufactures all of the components for these systems.

Trastuzumab-binding peptide display by Tobacco mosaic virus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Frolova, Olga Y.; Petrunia, Igor V.; Komarova, Tatiana V.

2010-11-10

Human epidermal growth factor receptor-2 (HER2/neu) is a target for the humanized monoclonal antibody trastuzumab. Recently, trastuzumab-binding peptides (TBP) of HER2/neu that inhibit proliferation of breast cancer cells were identified. We have now studied conditions of efficient assembly in vivo of Tobacco mosaic virus (TMV)-based particles displaying TBP on its surface. The system is based on an Agrobacterium-mediated co-delivery of binary vectors encoding TMV RNA and coat protein (CP) with TBP in its C-terminal extension into plant leaves. We show how the fusion of amino acid substituted TBP (sTBP) to CP via a flexible peptide linker can improve the manufacturabilitymore » of recombinant TMV (rTMV). We also reveal that rTMV particles with exposed sTBP retained trastuzumab-binding capacity but lost an anti-HER2/neu immunogenic scaffold function. Mouse antibodies against rTMV did not recognize HER2/neu on surface of human SK-BR-3 cells.« less

CrpP Is a Novel Ciprofloxacin-Modifying Enzyme Encoded by the Pseudomonas aeruginosa pUM505 Plasmid.

PubMed

Chávez-Jacobo, Víctor M; Hernández-Ramírez, Karen C; Romo-Rodríguez, Pamela; Pérez-Gallardo, Rocío Viridiana; Campos-García, Jesús; Gutiérrez-Corona, J Félix; García-Merinos, Juan Pablo; Meza-Carmen, Víctor; Silva-Sánchez, Jesús; Ramírez-Díaz, Martha I

2018-06-01

The pUM505 plasmid, isolated from a clinical Pseudomonas aeruginosa isolate, confers resistance to ciprofloxacin (CIP) when transferred into the standard P. aeruginosa strain PAO1. CIP is an antibiotic of the quinolone family that is used to treat P. aeruginosa infections. In silico analysis, performed to identify CIP resistance genes, revealed that the 65-amino-acid product encoded by the orf131 gene in pUM505 displays 40% amino acid identity to the Mycobacterium smegmatis aminoglycoside phosphotransferase (an enzyme that phosphorylates and inactivates aminoglycoside antibiotics). We cloned orf131 (renamed crpP , for c iprofloxacin r esistance p rotein, p lasmid encoded) into the pUCP20 shuttle vector. The resulting recombinant plasmid, pUC- crpP , conferred resistance to CIP on Escherichia coli strain J53-3, suggesting that this gene encodes a protein involved in CIP resistance. Using coupled enzymatic analysis, we determined that the activity of CrpP on CIP is ATP dependent, while little activity against norfloxacin was detected, suggesting that CIP may undergo phosphorylation. Using a recombinant His-tagged CrpP protein and liquid chromatography-tandem mass spectrometry, we also showed that CIP was phosphorylated prior to its degradation. Thus, our findings demonstrate that CrpP, encoded on the pUM505 plasmid, represents a new mechanism of CIP resistance in P. aeruginosa , which involves phosphorylation of the antibiotic. Copyright © 2018 American Society for Microbiology.

Expression and activity analysis of a new fusion protein targeting ovarian cancer cells.

PubMed

Su, Manman; Chang, Weiqin; Wang, Dingding; Cui, Manhua; Lin, Yang; Wu, Shuying; Xu, Tianmin

2015-09-01

The aim of the present study was to develop a new therapeutic drug to improve the prognosis of ovarian cancer patients. Human urokinase-type plasminogen activator (uPA)17-34-kunitz-type protease inhibitor (KPI) eukaryotic expression vector was constructed and recombinant human uPA17-34-KPI (rhuPA17-34-KPI) in P. pastoris was expressed. In the present study, the DNA sequences that encode uPA 17-34 amino acids were created according to the native amino acids sequence and inserted into the KPI-pPICZαC vector, which was constructed. Then, uPA17‑34-KPI-pPICZαC was transformed into P. pastoris X-33, and rhuPA17-34-KPI was expressed by induction of methanol. The bioactivities of a recombinant fusion protein were detected with trypsin inhibition analysis, and the inhibitory effects on the growth of ovarian cancer cells were identified using the TUNEL assay, in vitro wound‑healing assay and Matrigel model analysis. The results of the DNA sequence analysis of the recombinant vector uPA17-34-KPI‑pPICZα demonstrated that the DNA‑encoding human uPA 17-34 amino acids, 285-288 amino acids of amyloid precursor protein (APP) and 1-57 amino acids of KPI were correctly inserted into the pPICZαC vector. Following induction by methonal, the fusion protein with a molecular weight of 8.8 kDa was observed using SDS-PAGE and western blot analysis. RhuPA17-34-KPI was expressed in P. pastoris with a yield of 50 mg/l in a 50-ml tube. The recombinant fusion protein was able to inhibit the activity of trypsin, inhibit growth and induce apoptosis of SKOV3 cells, and inhibit the invasion and metastasis of ovarian cancer cells. By considering uPA17-34 amino acid specific binding uPAR as the targeted part of fusion protein and utilizing the serine protease inhibitor activity of KPI, it was found that the recombinant fusion protein uPA17-34-KPI inhibited the invasion and metastasis of ovarian tumors, and may therefore be regarded as effective in targeted treatment.

The upstream enhancer elements of the G6PC promoter are critical for optimal G6PC expression in murine glycogen storage disease type Ia.

PubMed

Lee, Young Mok; Pan, Chi-Jiunn; Koeberl, Dwight D; Mansfield, Brian C; Chou, Janice Y

2013-11-01

Glycogen storage disease type-Ia (GSD-Ia) patients deficient in glucose-6-phosphatase-α (G6Pase-α or G6PC) manifest impaired glucose homeostasis characterized by fasting hypoglycemia, growth retardation, hepatomegaly, nephromegaly, hyperlipidemia, hyperuricemia, and lactic acidemia. Two efficacious recombinant adeno-associated virus pseudotype 2/8 (rAAV8) vectors expressing human G6Pase-α have been independently developed. One is a single-stranded vector containing a 2864-bp of the G6PC promoter/enhancer (rAAV8-GPE) and the other is a double-stranded vector containing a shorter 382-bp minimal G6PC promoter/enhancer (rAAV8-miGPE). To identify the best construct, a direct comparison of the rAAV8-GPE and the rAAV8-miGPE vectors was initiated to determine the best vector to take forward into clinical trials. We show that the rAAV8-GPE vector directed significantly higher levels of hepatic G6Pase-α expression, achieved greater reduction in hepatic glycogen accumulation, and led to a better toleration of fasting in GSD-Ia mice than the rAAV8-miGPE vector. Our results indicated that additional control elements in the rAAV8-GPE vector outweigh the gains from the double-stranded rAAV8-miGPE transduction efficiency, and that the rAAV8-GPE vector is the current choice for clinical translation in human GSD-Ia. © 2013.

Molecular characterization of enolase gene from Taenia multiceps.

PubMed

Li, W H; Qu, Z G; Zhang, N Z; Yue, L; Jia, W Z; Luo, J X; Yin, H; Fu, B Q

2015-10-01

Taenia multiceps is a cestode parasite with its larval stage, known as Coenurus cerebralis, mainly encysts in the central nervous system of sheep and other livestocks. Enolase is a key glycolytic enzyme and represents multifunction in most organisms. In the present study, a 1617bp full-length cDNA encoding enolase was cloned from T. multiceps and designated as TmENO. A putative encoded protein of 433 amino acid residues that exhibited high similarity to helminth parasites. The recombinant TmENO protein (rTmENO) showed the catalytic and plasminogen-binding characteristics after the TmENO was subcloned and expressed in the pET30a(+) vector. The TmENO gene was transcribed during the adult and larval stages and was also identified in both cyst fluid and as a component of the adult worms and the metacestode by western blot analysis. Taken together, our results will facilitate further structural characterization for TmENO and new potential control strategies for T. multiceps. Copyright © 2015 Elsevier Ltd. All rights reserved.

An improved Red/ET recombineering system and mouse ES cells culture conditions for the generation of targeted mutant mice.

PubMed

Kumagai, Katsuyoshi; Takanashi, Masakatsu; Ohno, Shin-Ichiro; Kuroda, Masahiko; Sudo, Katsuko

2017-05-03

Targeted mutant mice generated on a C57BL/6 background are powerful tools for analysis of the biological functions of genes, and gene targeting technologies using mouse embryonic stem (ES) cells have been used to generate such mice. Recently, a bacterial artificial chromosome (BAC) recombineering system was established for the construction of targeting vectors. However, gene retrieval from BACs for the generation of gene targeting vectors using this system remains difficult. Even when construction of a gene targeting vector is successful, the efficiency of production of targeted mutant mice from ES cells derived from C57BL/6 mice are poor. Therefore, in this study, we first improved the strategy for the retrieval of genes from BACs and their transfer into a DT-A plasmid, for the generation of gene targeting vectors using the BAC recombineering system. Then, we attempted to generate targeted mutant mice from ES cell lines derived from C57BL/6 mice, by culturing in serum-free medium. In conclusion, we established an improved strategy for the efficient generation of targeted mutant mice on a C57BL/6 background, which are useful for the in vivo analysis of gene functions and regulation.

Gene Transfer of Brain-derived Neurotrophic Factor (BDNF) Prevents Neurodegeneration Triggered by FXN Deficiency.

PubMed

Katsu-Jiménez, Yurika; Loría, Frida; Corona, Juan Carlos; Díaz-Nido, Javier

2016-05-01

Friedreich's ataxia is a predominantly neurodegenerative disease caused by recessive mutations that produce a deficiency of frataxin (FXN). Here, we have used a herpesviral amplicon vector carrying a gene encoding for brain-derived neurotrophic factor (BDNF) to drive its overexpression in neuronal cells and test for its effect on FXN-deficient neurons both in culture and in the mouse cerebellum in vivo. Gene transfer of BDNF to primary cultures of mouse neurons prevents the apoptosis which is triggered by the knockdown of FXN gene expression. This neuroprotective effect of BDNF is also observed in vivo in a viral vector-based knockdown mouse cerebellar model. The injection of a lentiviral vector carrying a minigene encoding for a FXN-specific short hairpin ribonucleic acid (shRNA) into the mouse cerebellar cortex triggers a FXN deficit which is accompanied by significant apoptosis of granule neurons as well as loss of calbindin in Purkinje cells. These pathological changes are accompanied by a loss of motor coordination of mice as assayed by the rota-rod test. Coinjection of a herpesviral vector encoding for BDNF efficiently prevents both the development of cerebellar neuropathology and the ataxic phenotype. These data demonstrate the potential therapeutic usefulness of neurotrophins like BDNF to protect FXN-deficient neurons from degeneration.

Fast higher-order MR image reconstruction using singular-vector separation.

PubMed

Wilm, Bertram J; Barmet, Christoph; Pruessmann, Klaas P

2012-07-01

Medical resonance imaging (MRI) conventionally relies on spatially linear gradient fields for image encoding. However, in practice various sources of nonlinear fields can perturb the encoding process and give rise to artifacts unless they are suitably addressed at the reconstruction level. Accounting for field perturbations that are neither linear in space nor constant over time, i.e., dynamic higher-order fields, is particularly challenging. It was previously shown to be feasible with conjugate-gradient iteration. However, so far this approach has been relatively slow due to the need to carry out explicit matrix-vector multiplications in each cycle. In this work, it is proposed to accelerate higher-order reconstruction by expanding the encoding matrix such that fast Fourier transform can be employed for more efficient matrix-vector computation. The underlying principle is to represent the perturbing terms as sums of separable functions of space and time. Compact representations with this property are found by singular-vector analysis of the perturbing matrix. Guidelines for balancing the accuracy and speed of the resulting algorithm are derived by error propagation analysis. The proposed technique is demonstrated for the case of higher-order field perturbations due to eddy currents caused by diffusion weighting. In this example, image reconstruction was accelerated by two orders of magnitude.

Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Ye; Tang, Lan; Duan, Junxin

The present invention relates to isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having xylanase activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spodsberg, Nikolaj

The present invention relates to isolated polypeptides having xylanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having xylanase activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lopez de Leon, Alfredo; Rey, Michael

The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Variants of polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sweeney, Matt; Wogulis, Mark

The present invention relates to polypeptide having cellulolytic enhancing activity variants. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spodsberg, Nikolaj

The present invention relates to isolated polypeptides having cellobiohydrolase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having endoglucanase activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Yu; Liu, Ye; Duan, Junxin

The present invention relates to isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having endoglucanase activity and polynucleotides encoding same

DOEpatents

Lopez de Leon, Alfredo; Rey, Michael

2012-09-18

The present invention relates to isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having xylanase activity and polynucleotides encoding same

DOEpatents

Lopez de Leon, Alfredo; Rey, Michael

2010-12-14

The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having endoglucanase activity and polynucleotides encoding same

DOEpatents

Harris, Paul [Carnation, WA; Lopez de Leon, Alfredo [Davis, CA; Rey, Micheal [Davis, CA; Ding, Hanshu [Davis, CA; Vlasenko, Elena [Davis, CA

2012-02-21

The present invention relates to isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

DOEpatents

Spodsberg, Nikolaj

2016-06-28

The present invention relates to isolated polypeptides having cellobiohydrolase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having xylanase activity and polynucleotides encoding same

DOEpatents

Lopez de Leon, Alfredo; Rey, Michael

2016-05-31

The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having endoglucanase activity and polynucleotides encoding same

DOEpatents

Spodsberg, Nikolaj

2015-02-10

The present invention relates to isolated polypeptides having endoglucanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having endoglucanase activity and polynucleotides encoding same

DOEpatents

Spodsberg, Nikolaj

2016-02-23

The present invention relates to isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having xylanase activity and polynucleotides encoding same

DOEpatents

Tang, Lan; Liu, Ye; Duan, Junxin; Ding, Hanshu

2013-04-30

The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having xylanase activity and polynucleotides encoding same

DOEpatents

Tang, Lan; Liu, Ye; Duan, Junxin; Hanshu, Ding

2012-10-30

The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

DOEpatents

Liu, Ye; Tang, Lan

2015-11-20

The present invention relates to isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having xylanase activity and polynucleotides encoding same

DOEpatents

Lopez de Leon, Alfredo; Rey, Michael

2015-01-27

The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having xylanase activity and polynucleotides encoding same

DOEpatents

Spodsberg, Nikolaj

2014-10-21

The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having endoglucanase activity and polynucleotides encoding same

DOEpatents

Lopez de Leon, Alfredo; Rey, Michael

2015-03-10

The present invention relates to isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having xylanase activity and polynucleotides encoding same

DOEpatents

Spodsberg, Nikolaj

2017-05-02

The present invention relates to isolated polypeptides having xylanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

DOEpatents

Spodsberg, Nikolaj

2015-03-31

The present invention relates to isolated polypeptides having cellobiohydrolase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

DOEpatents

Spodsberg, Nikolaj

2015-07-14

The present invention relates to isolated polypeptides having cellobiohydrolase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

DOEpatents

Brown, Kimberly [Elk Grove, CA; Harris, Paul [Carnation, WA; Lopez De Leon, Alfredo [Davis, CA; Merino, Sandra [West Sacremento, CA

2007-05-22

The present invention relates to isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

DOEpatents

Liu, Ye; Harris, Paul; Tang, Lan; Wu, Wenping

2013-11-19

The present invention relates to isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

DOEpatents

Morant, Marc D.; Harris, Paul

2015-10-13

The present invention relates to isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

DOEpatents

Liu, Ye; Tang, Lan; Harris, Paul; Wu, Wenping

2012-10-02

The present invention relates to isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polynucleotides encoding polypeptides having beta-glucosidase activity

DOEpatents

Harris, Paul; Golightly, Elizabeth

2010-03-02

The present invention relates to isolated polypeptides having beta-glucosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

Polypeptides having endoglucanase activity and polynucleotides encoding same

DOEpatents

Lopez de Leon, Alfredo; Rey, Michael

2013-06-18

The present invention relates to isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spodsberg, Nikolaj

2016-12-13

The present invention relates to isolated polypeptides having cellobiohydrolase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having xylanase activity and polynucleotides encoding same

DOEpatents

Spodsberg, Nikolaj

2014-10-14

The present invention relates to isolated polypeptides having xylanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Zhang, Yu; Tang, Lan; Henriksen, Svend Hostgaard Bang

2016-05-17

The present invention provides isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also provides nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Analytic calculation of finite-population reproductive numbers for direct- and vector-transmitted diseases with homogeneous mixing.

PubMed

Keegan, Lindsay; Dushoff, Jonathan

2014-05-01

The basic reproductive number, R0, provides a foundation for evaluating how various factors affect the incidence of infectious diseases. Recently, it has been suggested that, particularly for vector-transmitted diseases, R0 should be modified to account for the effects of finite host population within a single disease transmission generation. Here, we use a transmission factor approach to calculate such "finite-population reproductive numbers," under the assumption of homogeneous mixing, for both vector-borne and directly transmitted diseases. In the case of vector-borne diseases, we estimate finite-population reproductive numbers for both host-to-host and vector-to-vector generations, assuming that the vector population is effectively infinite. We find simple, interpretable formulas for all three of these quantities. In the direct case, we find that finite-population reproductive numbers diverge from R0 before R0 reaches half of the population size. In the vector-transmitted case, we find that the host-to-host number diverges at even lower values of R0, while the vector-to-vector number diverges very little over realistic parameter ranges.

Quantitative modeling of peptide binding to TAP using support vector machine.

PubMed

Diez-Rivero, Carmen M; Chenlo, Bernardo; Zuluaga, Pilar; Reche, Pedro A

2010-01-01

The transport of peptides to the endoplasmic reticulum by the transporter associated with antigen processing (TAP) is a necessary step towards determining CD8 T cell epitopes. In this work, we have studied the predictive performance of support vector machine models trained on single residue positions and residue combinations drawn from a large dataset consisting of 613 nonamer peptides of known affinity to TAP. Predictive performance of these TAP affinity models was evaluated under 10-fold cross-validation experiments and measured using Pearson's correlation coefficients (R(p)). Our results show that every peptide position (P1-P9) contributes to TAP binding (minimum R(p) of 0.26 +/- 0.11 was achieved by a model trained on the P6 residue), although the largest contributions to binding correspond to the C-terminal end (R(p) = 0.68 +/- 0.06) and the P1 (R(p) = 0.51 +/- 0.09) and P2 (0.57 +/- 0.08) residues of the peptide. Training the models on additional peptide residues generally improved their predictive performance and a maximum correlation (R(p) = 0.89 +/- 0.03) was achieved by a model trained on the full-length sequences or a residue selection consisting of the first 5 N- and last 3 C-terminal residues of the peptides included in the training set. A system for predicting the binding affinity of peptides to TAP using the methods described here is readily available for free public use at http://imed.med.ucm.es/Tools/tapreg/. (c) 2009 Wiley-Liss, Inc.

Practical somewhat-secure quantum somewhat-homomorphic encryption with coherent states

NASA Astrophysics Data System (ADS)

Tan, Si-Hui; Ouyang, Yingkai; Rohde, Peter P.

2018-04-01

We present a scheme for implementing homomorphic encryption on coherent states encoded using phase-shift keys. The encryption operations require only rotations in phase space, which commute with computations in the code space performed via passive linear optics, and with generalized nonlinear phase operations that are polynomials of the photon-number operator in the code space. This encoding scheme can thus be applied to any computation with coherent-state inputs, and the computation proceeds via a combination of passive linear optics and generalized nonlinear phase operations. An example of such a computation is matrix multiplication, whereby a vector representing coherent-state amplitudes is multiplied by a matrix representing a linear optics network, yielding a new vector of coherent-state amplitudes. By finding an orthogonal partitioning of the support of our encoded states, we quantify the security of our scheme via the indistinguishability of the encrypted code words. While we focus on coherent-state encodings, we expect that this phase-key encoding technique could apply to any continuous-variable computation scheme where the phase-shift operator commutes with the computation.

Ichnocarpus frutescens Ameliorates Experimentally Induced Convulsion in Rats

PubMed Central

Singh, Narendra Kumar; Laloo, Damiki; Garabadu, Debapriya; Singh, Tryambak Deo; Singh, Virendra Pratap

2014-01-01

The present study was carried out to evaluate the anticonvulsant activity and probable mechanism of action of the methanol root extract from I. frutescens (MEIF) using different experimental animal models. Anticonvulsant activity of the single dose of MEIF (100, 200, and 400 mg/kg, p.o.) was evaluated in maximal electroshock- (MES-), pentylenetetrazole- (PTZ-), and isoniazid- (INH-) induced convulsions models in rats. The levels of γ-amino butyric acid (GABA), glutamate, GABA-transaminase (GABA-T) activity and oxidative stress markers were measured in pretreated rat's brain homogenate to corroborate the mechanism of observed anticonvulsant activity. MEIF (200–400 mg/kg, p.o.) protected the animals in all the behavioral models used. Pretreatment of MEIF (200–400 mg/kg, p.o.) and diazepam (1.0 mg/kg, i.p.) to the animals in INH-induced convulsion model showed 100% and 80% protection, respectively, as well as significant restoration of GABA and glutamate level in the rat's brain. MEIF and vigabatrin (50 mg/kg, i.p.) reduced the PTZ-induced increase in the activity of GABA-T (46%) in the brain. Further, MEIF reversed the PTZ-induced increase in lipid peroxidase (LPO) and decrease in reduced glutathione (GSH), catalase (CAT), and superoxide dismutase (SOD) activities. The findings of this study validate the anticonvulsant activity of I. frutescens. PMID:27379268

Effects of Dorema ammoniacum Gum on Neuronal Epileptiform Activity-Induced by Pentylenetetrazole

PubMed Central

Ghasemi, Fatemeh; Tamadon, Hanieh; Hosseinmardi, Narges; Janahmadi, Mahyar

2018-01-01

Epilepsy is a chronic neurological disease which disrupts the neuronal electrical activity. One-third of patients are resistant to treatment with available antiepileptic agents. The use of herbal medicine for treating several diseases including epilepsy is on the rise. Therefore, further investigation is required to verify the safety and effectiveness of Phytomedicine in treating diseases. The current study is an attempt to elucidate the electrophysiological mechanism of the effect of Dorema ammoniacum gum on a cellular model of epilepsy, using intracellular recording method. The gum was applied either after or before pentylenetetrazole, as an epileptic drug, in order to explore the possible therapeutic and preventive effects of gum. Treatment with D. ammoniacum gum alone increased the neuronal excitability and when applied before or after treatment with PTZ not only did not prevent or change the electrophysiological changes induced by PTZ but also re-enhanced the induction of hyperexcitability and epileptiform activity through depolarizing membrane potential, increasing the firing frequency and decreasing the AHP amplitude. However, phenobarbital, as a standard anti-epileptic agent, almost reversed the effect of PTZ and preserved the normal firing properties of F1 neurons. The possible candidate mechanism of the effect of gum on neuronal excitability could be suppressive effects of gum on voltage and/or Ca2+ dependent K+ channels currents underlying AHP. PMID:29881430

Detection of nonlinear interactions of EEG alpha waves in the brain by a new coherence measure and its application to epilepsy and anti-epileptic drug therapy.

PubMed

Sherman, David; Zhang, Ning; Garg, Shikha; Thakor, Nitish V; Mirski, Marek A; White, Mirinda Anderson; Hinich, Melvin J

2011-04-01

EEG and field potential rhythms established in the cortex and thalamus may accommodate the propagation of seizures. This article describes the interaction between thalamus and cortex during pentylenetetrazol (PTZ) seizures in rats with and without prior treatment with ethosuximide (ESM), a well-known antiepileptic drug (AED) that raises the threshold for seizures, was given before PTZ. The AED was given before PTZ convulsant administration. We track this thalamo-cortical association with a novel measure we have called the cross-bicoherence gain, or BISCOH. This quantity allows us to measure the spectral coherence in a purely higher order spectralmethodology. BISCOH is able to track the formation of nonlinearities at specific frequencies in the recorded EEG. BISCOH showed a strong increase in low alpha wave harmonic generationat 10 and 12.5 Hz after ESM treatment (p < 0.02 and p < 0.007, respectively). Conventional coherence failed to show distinctive and significant changes in thalamo-cortical coupling after ESM treatment at those frequencies and instead showed changes at 5 Hz. This rise in cortical rhythms is evidence of harmonic generation or new frequency formation in the thalamo-cortical system withAED therapy. BISCOH could become a powerful tool in unraveling changes in coherence due to neuroelectric modulation resulting from drug treatment or electrical stimulation.

Agmatine reduces extracellular glutamate during pentylenetetrazole-induced seizures in rat brain: A potential mechanism for the anticonvulsive effects

PubMed Central

Feng, Yangzheng; LeBlanc, Michael H.; Regunathan, Soundar

2010-01-01

Glutamate has been implicated in the initiation and spread of seizure activity. Agmatine, an endogenous neuromodulator, is an antagonist of NMDA receptors and has anticonvulsive effects. Whether agmatine regulate glutamate release, as measured by in vivo microdialysis, is not known. In this study, we used pentylenetetrazole (PTZ)-induced seizure model to determine the effect of agmatine on extracellular glutamate in rat brain. We also determined the time course and the amount of agmatine that reached brain after peripheral injection. After i.p. injection of agmatine (50 mg/kg), increase of agmatine in rat cortex and hippocampus was observed in 15 min with levels returning to baseline in one hour. Rats, naïve and implanted with microdialysis cannula into the cortex, were administered PTZ (60 mg/kg, i.p.) with prior injection of agmatine (100 mg/kg, i.p.) or saline. Seizure grades were recorded and microdialysis samples were collected every 15 min for 75 min. Agmatine pre-treatment significantly reduced the seizure grade and increased the onset time. The levels of extracellular glutamate in frontal cortex rose two- to three-fold after PTZ injection and agmatine significantly inhibited this increase. In conclusion, the present data suggest that the anticonvulsant activity of agmatine, in part, could be related to the inhibition glutamate release. PMID:16125317

Anticonvulsant activity of Aloe vera leaf extract in acute and chronic models of epilepsy in mice.

PubMed

Rathor, Naveen; Arora, Tarun; Manocha, Sachin; Patil, Amol N; Mediratta, Pramod K; Sharma, Krishna K

2014-03-01

The effect of Aloe vera in epilepsy has not yet been explored. This study was done to explore the effect of aqueous extract of Aloe vera leaf powder on three acute and one chronic model of epilepsy. In acute study, aqueous extract of Aloe vera leaf (extract) powder was administered in doses 100, 200 and 400 mg/kg p.o. Dose of 400 mg/kg of Aloe vera leaf extract was chosen for chronic administration. Oxidative stress parameters viz. malondialdehyde (MDA) and reduced glutathione (GSH) were also estimated in brain of kindled animals. In acute study, Aloe vera leaf (extract) powder in a dose-dependent manner significantly decreased duration of tonic hind limb extension in maximal electroshock seizure model, increased seizure threshold current in increasing current electroshock seizure model, and increased latency to onset and decreased duration of clonic convulsion in pentylenetetrazole (PTZ) model as compared with control group. In chronic study, Aloe vera leaf (extract) powder prevented progression of kindling in PTZ-kindled mice. Aloe vera leaf (extract) powder 400 mg/kg p.o. also reduced brain levels of MDA and increased GSH levels as compared to the PTZ-kindled non-treated group. The results of study showed that Aloe vera leaf (extract) powder possessed significant anticonvulsant and anti-oxidant activity. © 2013 Royal Pharmaceutical Society.

7-Nitroindazole, a nitric oxide synthase inhibitor, enhances the anticonvulsive action of ethosuximide and clonazepam against pentylenetetrazol-induced convulsions.

PubMed

Borowicz, K K; Luszczki, J; Kleinrok, Z; Czuczwar, S J

2000-01-01

The interaction of 7-nitroindazole (7-NI), a nitric oxide synthase (NOS) inhibitor, with the protective activity of conventional antiepileptics against pentylenetetrazol (PTZ)-induced seizures was tested in mice. Alone, 7-nitroindazole (up to 50mg/kg) was ineffective in this model of experimental epilepsy. However, it potentiated the anticonvulsive activity of ethosuximide and clonazepam, significantly reducing their ED50S against PTZ-induced convulsions (from 144 to 76 mg/kg, and from 0.05 to 0.016 mg/kg, respectively). Conversely, the protective actions of valproate and phenobarbital were not affected by the NOS inhibitor. Since the nitric oxide precursor, L-arginine, did not reverse the action of 7-NI on ethosuximide or clonazepam, an involvement of central NO does not seem probable. Neither ethosuximide nor clonazepam, administered at their ED50S (144 and 0.05 mg/kg, respectively), produced significant adverse effects as regards motor coordination (chimney test) and long-term memory (passive avoidance task). Also 7-NI (50 mg/kg) and its combinations with ethosuximide and clonazepam (providing a 50% protection against PTZ-evoked seizures) did not disturb motor and mnemonic performance in mice. The interaction at the pharmacokinetic level does not seem probable, at least in the case of ethosuximide, because the NOS inhibitor did not interfere with its plasma or brain concentrations.

Characterization of the Sorbitol Utilization Cluster of the Probiotic Pediococcus parvulus 2.6: Genetic, Functional and Complementation Studies in Heterologous Hosts

PubMed Central

Pérez-Ramos, Adrian; Werning, Maria L.; Prieto, Alicia; Russo, Pasquale; Spano, Giuseppe; Mohedano, Mari L.; López, Paloma

2017-01-01

Pediococcus parvulus 2.6 secretes a 2-substituted (1,3)-β-D-glucan with prebiotic and immunomodulatory properties. It is synthesized by the GTF glycosyltransferase using UDP-glucose as substrate. Analysis of the P. parvulus 2.6 draft genome revealed the existence of a sorbitol utilization cluster of six genes (gutFRMCBA), whose products should be involved in sorbitol utilization and could generate substrates for UDP-glucose synthesis. Southern blot hybridization analysis showed that the cluster is located in a plasmid. Analysis of metabolic fluxes and production of the exopolysaccharide revealed that: (i) P. parvulus 2.6 is able to metabolize sorbitol, (ii) sorbitol utilization is repressed in the presence of glucose and (iii) sorbitol supports the synthesis of 2-substituted (1,3)-β-D-glucan. The sorbitol cluster encodes two putative regulators, GutR and GutM, in addition to a phosphoenolpyruvate-dependent phosphotransferase transport system and sorbitol-6-phosphate dehydrogenase. Therefore, we investigated the involvement of GutR and GutM in the expression of gutFRMCBA. The promoter-probe vector pRCR based on the mrfp gene, which encodes the fluorescence protein mCherry, was used to test the potential promoter of the cluster (Pgut) and the genes encoding the regulators. This was performed by transferring by electrotransformation the recombinant plasmids into two hosts, which metabolize sorbitol: Lactobacillus plantarum and Lactobacillus casei. Upon growth in the presence of sorbitol, but not of glucose, only the presence of Pgut was required to support expression of mrfp in L. plantarum. In L. casei the presence of sorbitol in the growth medium and the pediococcal gutR or gutR plus gutM in the genome was required for Pgut functionality. This demonstrates that: (i) Pgut is required for expression of the gut cluster, (ii) Pgut is subjected to catabolic repression in lactobacilli, (iii) GutR is an activator, and (iv) in the presence of sorbitol, trans-complementation for activation of Pgut exists in L. plantarum but not in L. casei. PMID:29259592

An adaptive vector quantization scheme

NASA Technical Reports Server (NTRS)

Cheung, K.-M.

1990-01-01

Vector quantization is known to be an effective compression scheme to achieve a low bit rate so as to minimize communication channel bandwidth and also to reduce digital memory storage while maintaining the necessary fidelity of the data. However, the large number of computations required in vector quantizers has been a handicap in using vector quantization for low-rate source coding. An adaptive vector quantization algorithm is introduced that is inherently suitable for simple hardware implementation because it has a simple architecture. It allows fast encoding and decoding because it requires only addition and subtraction operations.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schnorr, Kirk; Kramer, Randall

2017-08-08

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tang, Lan; Liu, Ye; Duan, Junxin

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having beta-xylosidase activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Yu; Liu, Ye; Duan, Junxin

The present invention relates to isolated polypeptides having beta-xylosidase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Lopez de Leon, Alfredo [Davis, CA; Ding, Hanshu [Davis, CA; Brown, Kimberly [Elk Grove, CA

2011-10-25

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having beta-glucosidase activity and polynucleotides encoding same

DOEpatents

Harris, Paul; Golightly, Elizabeth

2012-11-27

The present invention relates to isolated polypeptides having beta-glucosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Zhang, Yu; Duan, Junxin; Tang, Lan; Wu, Wenping

2016-06-14

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Zhang, Yu; Duan, Junxin; Tang, Lan; Wu, Wenping

2016-11-22

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Tang, Lan [Beijing, CN; Liu, Ye [Beijing, CN; Duan, Junxin [Beijing, CN; Zhang, Yu [Beijing, CN; Jorgensen, Christian Isak [Bagsvaerd, DK; Kramer, Randall [Lincoln, CA

2012-04-03

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Duan, Junxin [Beijing, CN; Liu, Ye [Beijing, CN; Tang, Lan [Beijing, CN; Wu, Wenping [Beijing, CN; Quinlan, Jason [Albany, CA; Kramer, Randall [Lincoln, CA

2012-03-27

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Tang, Lan; Liu, Ye; Duan, Junxin; Zhang, Yu; Joergensen, Christian; Kramer, Randall

2016-11-29

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Tang, Lan; Liu, Ye; Duan, Junxin; Zhang, Yu; Joergensen, Christian; Kramer, Randall

2014-09-16

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having xylanase activity and polynucleotides encoding the same

DOEpatents

Spodsberg, Nikolaj [Bagsvaed, DK

2014-01-07

The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The inventino also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Tang, Lan; Liu, Ye; Duan, Junxin; Wu, Wenping; Kramer, Randall

2014-10-21

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Schnorr, Kirk; Kramer, Randall

2016-04-05

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having beta-glucosidase activity and polynucleotides encoding same

DOEpatents

Harris, Paul [Carnation, WA; Golightly, Elizabeth [Reno, NV

2007-07-17

The present invention relates to isolated polypeptides having beta-glucosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Maiyuran, Suchindra; Kramer, Randall; Harris, Paul

2013-10-29

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having beta-glucosidase activity and polynucleotides encoding same

DOEpatents

Harris, Paul [Carnation, WA; Golightly, Elizabeth [Reno, NV

2011-06-14

The present invention relates to isolated polypeptides having beta-glucosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Tang, Lan; Liu, Ye; Duan, Junxin; Zhang, Yu; Jorgensen, Christian Isak; Kramer, Randall

2013-04-16

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Duan, Junxin; Tang, Lan; Liu, Ye; Wu, Wenping; Quinlan, Jason; Kramer, Randall

2013-06-18

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Schnorr, Kirk; Kramer, Randall

2016-08-09

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Elevated expression of activins promotes muscle wasting and cachexia.

PubMed

Chen, Justin L; Walton, Kelly L; Winbanks, Catherine E; Murphy, Kate T; Thomson, Rachel E; Makanji, Yogeshwar; Qian, Hongwei; Lynch, Gordon S; Harrison, Craig A; Gregorevic, Paul

2014-04-01

In models of cancer cachexia, inhibiting type IIB activin receptors (ActRIIBs) reverse muscle wasting and prolongs survival, even with continued tumor growth. ActRIIB mediates signaling of numerous TGF-β proteins; of these, we demonstrate that activins are the most potent negative regulators of muscle mass. To determine whether activin signaling in the absence of tumor-derived factors induces cachexia, we used recombinant serotype 6 adeno-associated virus (rAAV6) vectors to increase circulating activin A levels in C57BL/6 mice. While mice injected with control vector gained ~10% of their starting body mass (3.8±0.4 g) over 10 wk, mice injected with increasing doses of rAAV6:activin A exhibited weight loss in a dose-dependent manner, to a maximum of -12.4% (-4.2±1.1 g). These reductions in body mass in rAAV6:activin-injected mice correlated inversely with elevated serum activin A levels (7- to 24-fold). Mechanistically, we show that activin A reduces muscle mass and function by stimulating the ActRIIB pathway, leading to deleterious consequences, including increased transcription of atrophy-related ubiquitin ligases, decreased Akt/mTOR-mediated protein synthesis, and a profibrotic response. Critically, we demonstrate that the muscle wasting and fibrosis that ensues in response to excessive activin levels is fully reversible. These findings highlight the therapeutic potential of targeting activins in cachexia.

AAV vector encoding human VEGF165-transduced pectineus muscular flaps increase the formation of new tissue through induction of angiogenesis in an in vivo chamber for tissue engineering: A technique to enhance tissue and vessels in microsurgically engineered tissue.

PubMed

Moimas, Silvia; Manasseri, Benedetto; Cuccia, Giuseppe; Stagno d'Alcontres, Francesco; Geuna, Stefano; Pattarini, Lucia; Zentilin, Lorena; Giacca, Mauro; Colonna, Michele R

2015-01-01

In regenerative medicine, new approaches are required for the creation of tissue substitutes, and the interplay between different research areas, such as tissue engineering, microsurgery and gene therapy, is mandatory. In this article, we report a modification of a published model of tissue engineering, based on an arterio-venous loop enveloped in a cross-linked collagen-glycosaminoglycan template, which acts as an isolated chamber for angiogenesis and new tissue formation. In order to foster tissue formation within the chamber, which entails on the development of new vessels, we wondered whether we might combine tissue engineering with a gene therapy approach. Based on the well-described tropism of adeno-associated viral vectors for post-mitotic tissues, a muscular flap was harvested from the pectineus muscle, inserted into the chamber and transduced by either AAV vector encoding human VEGF165 or AAV vector expressing the reporter gene β-galactosidase, as a control. Histological analysis of the specimens showed that muscle transduction by AAV vector encoding human VEGF165 resulted in enhanced tissue formation, with a significant increase in the number of arterioles within the chamber in comparison with the previously published model. Pectineus muscular flap, transduced by adeno-associated viral vectors, acted as a source of the proangiogenic factor vascular endothelial growth factor, thus inducing a consistent enhancement of vessel growth into the newly formed tissue within the chamber. In conclusion, our present findings combine three different research fields such as microsurgery, tissue engineering and gene therapy, suggesting and showing the feasibility of a mixed approach for regenerative medicine.

Chimpanzee adenovirus vaccine generates acute and durable protective immunity against ebolavirus challenge.

PubMed

Stanley, Daphne A; Honko, Anna N; Asiedu, Clement; Trefry, John C; Lau-Kilby, Annie W; Johnson, Joshua C; Hensley, Lisa; Ammendola, Virginia; Abbate, Adele; Grazioli, Fabiana; Foulds, Kathryn E; Cheng, Cheng; Wang, Lingshu; Donaldson, Mitzi M; Colloca, Stefano; Folgori, Antonella; Roederer, Mario; Nabel, Gary J; Mascola, John; Nicosia, Alfredo; Cortese, Riccardo; Koup, Richard A; Sullivan, Nancy J

2014-10-01

Ebolavirus disease causes high mortality, and the current outbreak has spread unabated through West Africa. Human adenovirus type 5 vectors (rAd5) encoding ebolavirus glycoprotein (GP) generate protective immunity against acute lethal Zaire ebolavirus (EBOV) challenge in macaques, but fail to protect animals immune to Ad5, suggesting natural Ad5 exposure may limit vaccine efficacy in humans. Here we show that a chimpanzee-derived replication-defective adenovirus (ChAd) vaccine also rapidly induced uniform protection against acute lethal EBOV challenge in macaques. Because protection waned over several months, we boosted ChAd3 with modified vaccinia Ankara (MVA) and generated, for the first time, durable protection against lethal EBOV challenge.

Electronic and structural aspects of spin transitions observed by optical microscopy. The case of [Fe(ptz)6](BF4)2.

PubMed

Chong, Christian; Mishra, Haritosh; Boukheddaden, Kamel; Denise, Stéphane; Bouchez, Guillaume; Collet, Eric; Ameline, Jean-Claude; Naik, Anil D; Garcia, Yann; Varret, François

2010-02-11

The colorimetric analysis of images recorded with an optical microscope during the onset of the spin crossover transformation allows monitoring separately the involved electronic and structural aspects, through the separation of resonant absorption and scattering effects. Complementary information can also be obtained by using the polarized modes of the microscope. These potentialities are illustrated by the observation of [Fe(ptz)(6)](BF(4))(2) single crystals during the onset of the thermal transitions in the 110-140 K range. We characterized the interplay between the electronic (HS <--> LS) and structural (order <--> disorder) transformations. Elastic stresses and mechanical effects (hopping, self-cleavage) generated by the volume change upon electronic transition are also illustrated, with their impact on the photoswitching properties of the crystals.

EGVII endoglucanase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

2014-02-25

The present invention provides a novel endoglucanase nucleic acid sequence, designated egl7, and the corresponding EGVII amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVII, recombinant EGVII proteins and methods for producing the same.

Polypeptides having cellulolytic enhancing activity and nucleic acids encoding same

DOEpatents

Brown, Kimberly; Harris, Paul; Zaretsky, Elizabeth; Re, Edward; Vlasenko, Elena; McFarland, Keith; Lopez de Leon, Alfredo

2012-10-16

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

EGVII endoglucanase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

2006-05-16

The present invention provides a novel endoglucanase nucleic acid sequence, designated egl7, and the corresponding EGVII amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVII, recombinant EGVII proteins and methods for producing the same.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOEpatents

Dotson, William D.; Greenier, Jennifer; Ding, Hanshu

2007-09-18

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated nucleic acids encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acids as well as methods for producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding the same

DOEpatents

Tang, Lan; Liu, Ye; Duan, Junxin; Wu, Wenping; Kramer, Randall

2013-11-19

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

EGVI endoglucanase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel [Los Gatos, CA; Goedegebuur, Frits [Vlaardingen, NL; Ward, Michael [San Francisco, CA; Yao, Jian [Sunnyvale, CA

2008-04-01

The present invention provides a novel endoglucanase nucleic acid sequence, designated egl6, and the corresponding EGVI amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVI, recombinant EGVI proteins and methods for producing the same.

EGVI endoglucanase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

2010-10-12

The present invention provides a novel endoglucanase nucleic acid sequence, designated egl6, and the corresponding EGVI amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVI, recombinant EGVI proteins and methods for producing the same.

EGVIII endoglucanase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

2006-05-23

The present invention provides a novel endoglucanase nucleic acid sequence, designated egl8, and the corresponding EGVIII amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVIII, recombinant EGVIII proteins and methods for producing the same.

EGVI endoglucanase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

2010-10-05

The present invention provides a novel endoglucanase nucleic acid sequence, designated egl6, and the corresponding EGVI amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVI, recombinant EGVI proteins and methods for producing the same.

EGVI endoglucanase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

2006-06-06

The present invention provides a novel endoglucanase nucleic acid sequence, designated egl6, and the corresponding EGVI amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVI, recombinant EGVI proteins and methods for producing the same.

EGVII endoglucanase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel [Los Gatos, CA; Goedegebuur, Frits [Vlaardingen, NL; Ward, Michael [San Francisco, CA; Yao, Jian [Sunnyvale, CA

2009-05-05

The present invention provides an endoglucanase nucleic acid sequence, designated egl7, and the corresponding EGVII amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVII, recombinant EGVII proteins and methods for producing the same.

EGVII endoglucanase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

2013-07-16

The present invention provides a novel endoglucanase nucleic acid sequence, designated egl7, and the corresponding EGVII amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVII, recombinant EGVII proteins and methods for producing the same.

EGVII endoglucanase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel [Los Gatos, CA; Goedegebuur, Frits [Vlaardingen, NL; Ward, Michael [San Francisco, CA; Yao, Jian [Sunnyvale, CA

2012-02-14

The present invention provides a novel endoglucanase nucleic acid sequence, designated egl7, and the corresponding EGVII amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVII, recombinant EGVII proteins and methods for producing the same.

EGVII endoglucanase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

2015-04-14

The present invention provides a novel endoglucanase nucleic acid sequence, designated egl7, and the corresponding EGVII amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVII, recombinant EGVII proteins and methods for producing the same.

Polypeptides having cellulolytic enhancing activity and nucleic acids encoding same

DOEpatents

Brown, Kimberly; Harris, Paul; Zaretsky, Elizabeth; Re, Edward; Vlasenko, Elena; McFarland, Keith; Lopez de Leon, Alfredo

2014-09-30

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and nucleic acids encoding same

DOEpatents

Brown, Kimberly; Harris, Paul; Zaretsky, Elizabeth; Re, Edward; Vlasenko, Elena; McFarland, Keith; Lopez de Leon, Alfredo

2017-09-05

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and nucleic acids encoding same

DOEpatents

Brown, Kimberly; Harris, Paul; Zaretsky, Elizabeth; Re, Edward; Vlasenko, Elena; McFarland, Keith; Lopez de Leon, Alfredo

2010-06-22

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

Polypeptides having beta-glucosidase activity and polynucleotides encoding the same

DOEpatents

Brown, Kimberly; Harris, Paul

2013-12-17

The present invention relates to isolated polypeptides having beta-glucosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and nucleic acids encoding same

DOEpatents

Brown, Kimberly; Harris, Paul; Zaretsky, Elizabeth; Re, Edward; Vlasenko, Elena; McFarland, Keith; Lopez de Leon, Alfredo

2016-08-09

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

Polypeptides having cellulolytic enhancing activity and polynucleotides encoding the same

DOEpatents

Tang, Lan; Liu, Ye; Duan, Junxin; Zhang, Yu; Jorgensen, Christian Isak; Kramer, Randall

2013-12-24

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

A defective retroviral vector encoding human interferon-alpha2 can transduce human leukemic cell lines.

PubMed

Austruy, E; Bagnis, C; Carbuccia, N; Maroc, C; Birg, F; Dubreuil, P; Mannoni, P; Chabannon, C

1998-01-01

Using the LXSN backbone, a defective retroviral vector (LISN) was constructed that encodes the human interferon (IFN)-alpha2 (hIFN-alpha2) gene and the neomycin resistance gene; the hIFN-alpha2 gene was cloned from human placental genomic DNA. High titers of the LISN retrovirus were produced by the amphotropic packaging cell line GP+envAM12. LISN is able to infect three human hematopoietic and leukemic cell lines: K562, LAMA-84, and TF-1. G418-resistant cells were detected in a similar proportion after infection with either the LISN retroviral vector or the LnLSN retroviral vector (encoding the nlsLacZ gene instead of hIFN-alpha2), suggesting that hIFN-alpha2 does not inhibit (or only partially inhibits) the production of retroviral particles by the packaging cell line and the infection of human cells. LISN-infected cells express and secrete hIFN-alpha2 as demonstrated by Northern blot analysis of poly(A)+ RNA, detection of the intracellular protein by fluorescence-activated cell sorter analysis, and detection of secreted hIFN-alpha in cell supernatants using an enzyme-linked immunosorbent assay. Retrovirally produced hIFN-alpha2 is biologically active, as demonstrated by the partial inhibition of the growth of K562 and TF-1, the modulation of the expression of cell surface antigens, the induction of the (2'-5') oligoadenylate synthetase, and, for LAMA-84, the down-modulation of the BCR-ABL protein. We conclude that the infection of human leukemic cell lines with a retroviral vector encoding hIFN-alpha2 is feasible and induces the expected biological effects. This experimental model will be useful in investigating the possibility of transducing normal and leukemic cells and hematopoietic progenitors and in determining the consequences of the autocrine production of hIFN-alpha2 on the behavior of these cells.

Overexpression of cystatin C in synovium does not reduce synovitis or cartilage degradation in established osteoarthritis.

PubMed

Kyostio-Moore, Sirkka; Piraino, Susan; Berthelette, Patricia; Moran, Nance; Serriello, Joseph; Bendele, Alison; Sookdeo, Cathleen; Nambiar, Bindu; Ewing, Patty; Armentano, Donna; Matthews, Gloria L

2015-01-16

Cathepsin K (catK) expression is increased in cartilage, bone and synovium during osteoarthritis (OA). To study the role of catK expression and elevated cathepsin activity in the synovium on cartilage destruction in established OA, we overexpressed cystatin C (cysC), a natural cysteine protease inhibitor, in the synovium of rabbit OA joints. The ability of cysC to inhibit activity of cathepsins in rabbit OA synovium lysates was tested in vitro using protease activity assay. In vivo, the tissue localization of recombinant adeno-associated virus (rAAV) with LacZ gene after intra-articular injection was determined by β-galactosidase staining of rabbit joints 4 weeks later. To inhibit cathepsin activity in the synovium, a rAAV2-encoding cysC was delivered intra-articularly into rabbit joints 4 weeks after OA was induced by anterior cruciate ligament transection (ACLT). Seven weeks postinjection, endogenous catK and cysC levels as well as the vector-derived cysC expression in the synovium of normal and OA joints were examined by RNA quantification. Synovial cathepsin activity and catK, catB and catL protein levels were determined by activity and Western blot analyses, respectively. Synovitis and cartilage degradation were evaluated by histopathological scoring. In vitro, the ability of cysC to efficiently inhibit activity of purified catK and OA-induced cathepsins in rabbit synovial lysates was demonstrated. In vivo, the intra-articular delivery of rAAV2/LacZ showed transduction of mostly synovium. Induction of OA in rabbit joints resulted in fourfold increase in catK mRNA compared to sham controls while no change was detected in endogenous cysC mRNA levels in the synovium. Protein levels for catK, catB and catL were also increased in the synovium with a concomitant fourfold increase in cathepsin activity. Joints treated with rAAV2/cysC showed both detection of vector genomes and vector-derived cysC transcripts in the synovium. Production of functional cysC by the vector was demonstrated by complete block of cathepsin activity in the synovium. However, this did not decrease synovitis, bone sclerosis or progression of cartilage degradation. Increased production of natural cathepsin inhibitor, cysC, in OA synovium does not alleviate synovitis or cartilage pathology during a preexisting OA.

[Ubiquitination of recombinant adeno-associated viral vector and its application].

PubMed

Wang, Qi-zhao; Lu, Ying-hui; Diao, Yong; Xu, Rui-an

2012-09-01

Recombinant adeno-associated virus (rAAV) has been widely used as vector for gene therapy. However, the effectiveness of gene therapy based on rAAV needs to be further improved. Enhancement of the transduction efficiency is one of the most important fields for rAAV-based gene therapy. Recent results have showed that the ubiquitin-proteasome system plays an important role in the trafficking of rAAV vector in cytoplasm, and regulation of its function may significantly improve the transduction efficiency of rAAV vector in various types of cells and tissues.

Density-dependent host choice by disease vectors: epidemiological implications of the ideal free distribution.

PubMed

Basáñez, María-Gloria; Razali, Karina; Renz, Alfons; Kelly, David

2007-03-01

The proportion of vector blood meals taken on humans (the human blood index, h) appears as a squared term in classical expressions of the basic reproduction ratio (R(0)) for vector-borne infections. Consequently, R(0) varies non-linearly with h. Estimates of h, however, constitute mere snapshots of a parameter that is predicted, from evolutionary theory, to vary with vector and host abundance. We test this prediction using a population dynamics model of river blindness assuming that, before initiation of vector control or chemotherapy, recorded measures of vector density and human infection accurately represent endemic equilibrium. We obtain values of h that satisfy the condition that the effective reproduction ratio (R(e)) must equal 1 at equilibrium. Values of h thus obtained decrease with vector density, decrease with the vector:human ratio and make R(0) respond non-linearly rather than increase linearly with vector density. We conclude that if vectors are less able to obtain human blood meals as their density increases, antivectorial measures may not lead to proportional reductions in R(0) until very low vector levels are achieved. Density dependence in the contact rate of infectious diseases transmitted by insects may be an important non-linear process with implications for their epidemiology and control.

Immunization of Chickens with Newcastle Disease Virus Expressing H5 Hemagglutinin Protects against Highly Pathogenic H5N1 Avian Influenza Viruses

PubMed Central

Nayak, Baibaswata; Rout, Subrat N.; Kumar, Sachin; Khalil, Mohammed S.; Fouda, Moustafa M.; Ahmed, Luay E.; Earhart, Kenneth C.; Perez, Daniel R.; Collins, Peter L.; Samal, Siba K.

2009-01-01

Background Highly-pathogenic avian influenza virus (HPAIV) and Newcastle disease virus (NDV) are the two most important poultry viruses in the world. Natural low-virulence NDV strains have been used as vaccines over the past 70 years with proven track records. We have previously developed a reverse genetics system to produce low-virulent NDV vaccine strain LaSota from cloned cDNA. This system allows us to use NDV as a vaccine vector for other avian pathogens. Methodology/Principal Finding Here, we constructed two recombinant NDVs (rNDVs) each of which expresses the hemagglutinin (HA) gene of HPAIV H5N1strain A/Vietnam/1203/2004 from an added gene. In one, rNDV (rNDV-HA), the open reading frame (ORF) of HA gene was expressed without modification. In the second, rNDV (rNDV-HAF), the ORF was modified so that the transmembrane and cytoplasmic domains of the encoded HA gene were replaced with those of the NDV F protein. The insertion of either version of the HA ORF did not increase the virulence of the rNDV vector. The HA protein was found to be incorporated into the envelopes of both rNDV-HA and rNDV-HAF. However, there was an enhanced incorporation of the HA protein in rNDV-HAF. Chickens immunized with a single dose of either rNDV-HA or rNDV-HAF induced a high titer of HPAIV H5-specific antibodies and were completely protected against challenge with NDV as well as lethal challenges of both homologous and heterologous HPAIV H5N1. Conclusion and Significance Our results suggest that these chimeric viruses have potential as safe and effective bivalent vaccines against NDV and. HPAIV. These vaccines will be convenient and affordable, which will be highly beneficial to the poultry industry. Furthermore, immunization with these vaccines will permit serological differentiation of vaccinated and avian influenza field virus infected animals. PMID:19654873

Impaired Glucose Metabolism in Mice Lacking the Tas1r3 Taste Receptor Gene.

PubMed

Murovets, Vladimir O; Bachmanov, Alexander A; Zolotarev, Vasiliy A

2015-01-01

The G-protein-coupled sweet taste receptor dimer T1R2/T1R3 is expressed in taste bud cells in the oral cavity. In recent years, its involvement in membrane glucose sensing was discovered in endocrine cells regulating glucose homeostasis. We investigated importance of extraorally expressed T1R3 taste receptor protein in age-dependent control of blood glucose homeostasis in vivo, using nonfasted mice with a targeted mutation of the Tas1r3 gene that encodes the T1R3 protein. Glucose and insulin tolerance tests, as well as behavioral tests measuring taste responses to sucrose solutions, were performed with C57BL/6ByJ (Tas1r3+/+) inbred mice bearing the wild-type allele and C57BL/6J-Tas1r3tm1Rfm mice lacking the entire Tas1r3 coding region and devoid of the T1R3 protein (Tas1r3-/-). Compared with Tas1r3+/+ mice, Tas1r3-/- mice lacked attraction to sucrose in brief-access licking tests, had diminished taste preferences for sucrose solutions in the two-bottle tests, and had reduced insulin sensitivity and tolerance to glucose administered intraperitoneally or intragastrically, which suggests that these effects are due to absence of T1R3. Impairment of glucose clearance in Tas1r3-/- mice was exacerbated with age after intraperitoneal but not intragastric administration of glucose, pointing to a compensatory role of extraoral T1R3-dependent mechanisms in offsetting age-dependent decline in regulation of glucose homeostasis. Incretin effects were similar in Tas1r3+/+ and Tas1r3-/- mice, which suggests that control of blood glucose clearance is associated with effects of extraoral T1R3 in tissues other than the gastrointestinal tract. Collectively, the obtained data demonstrate that the T1R3 receptor protein plays an important role in control of glucose homeostasis not only by regulating sugar intake but also via its extraoral function, probably in the pancreas and brain.

Genomic characterization of an extensively-drug resistance Salmonella enterica serotype Indiana strain harboring blaNDM-1 gene isolated from a chicken carcass in China.

PubMed

Wang, Wei; Peng, Zixin; Baloch, Zulqarnain; Hu, Yujie; Xu, Jin; Zhang, Wenhui; Fanning, Séamus; Li, Fengqin

2017-11-01

The objective of this study was to genetically characterize the antimicrobial resistance mechanisms of Salmonella enterica serotype Indiana C629 isolated from a chicken carcass in China in 2014. Antimicrobial susceptibility against a panel of 23 antimicrobial agents was carried out on Salmonella enterica serotype Indiana C629 and assessed according to CLSI standards. Whole-genome sequencing of this isolate was conducted to obtain the complete genome of S. Indiana. Salmonella Indiana C629 expressed an XDR phenotype being resistant to more than 20 antimicrobial agents, including imipenem and meropenem. From the analysis of the resistance mechanisms, two mutations were identified in subunit A of DNA gyrase within the quinolone resistance determining region, in addition to the acquisition of mobile efflux pumps encoding oqxA/B/R. Additionally, four beta-lactamases resistance genes (bla CTX-M-65 , bla TEM-1 , bla OXA-1 , and bla NDM-1 ), five aminoglycosides resistance genes (aac(3)-IV, aac(6')-Ib-cr, aadA2, aadA5, and aph(4)-Ia), two phenicol resistance genes (catB3 and floR), and five trimethoprim/sulfamethoxazole resistance genes (sul1/2/3 and dfrA12/17) were also identified. A total of 191 virulence genes were identified. Among them, 57 belonged to type-three secretion system (T3SS) encoding genes, 55 belonged to fimbrial adherence encoding genes, and 39 belonged to flagella-encoding genes CONCLUSIONS: This study demonstrated that multi-resistance mechanisms consistent with an XDR-phenotype, along with various virulence encoding genes of a S. Indiana strain in China These findings highlight the importance of cooperation among different sectors in order to monitor the spread of resistant pathogens among food animal, foods of animal origin and human beings that might further take measures to protect consumers' health. Copyright © 2017 Elsevier GmbH. All rights reserved.

Alphavirus vector-based replicon particles expressing multivalent cross-protective Lassa virus glycoproteins

PubMed Central

Wang, Min; Jokinen, Jenny; Tretyakova, Irina; Pushko, Peter; Lukashevich, Igor S.

2018-01-01

Lassa virus (LASV) is the most prevalent rodent-borne arenavirus circulated in West Africa. With population at risk from Senegal to Nigeria, LASV causes Lassa fever and is responsible for thousands of deaths annually. High genetic diversity of LASV is one of the challenges for vaccine R&D. We developed multivalent virus-like particle vectors (VLPVs) derived from the human Venezuelan equine encephalitis TC-83 IND vaccine (VEEV) as the next generation of alphavirus-based bicistronic RNA replicon particles. The genes encoding VEEV structural proteins were replaced with LASV glycoproteins (GPC) from distantly related clades I and IV with individual 26S promoters. Bicistronic RNA replicons encoding wild-type LASV GPC (GPCwt) and C-terminally deleted, non-cleavable modified glycoprotein (ΔGPfib), were encapsidated into VLPV particles using VEEV capsid and glycoproteins provided in trans. In transduced cells, VLPVs induced simultaneous expression of LASV GPCwt and ΔGPfib from 26S alphavirus promoters. LASV ΔGPfib was predominantly expressed as trimers, accumulated in the endoplasmic reticulum, induced ER stress and apoptosis promoting antigen cross-priming. VLPV vaccines were immunogenic and protective in mice and upregulated CD11c+/CD8+ dendritic cells playing the major role in cross-presentation. Notably, VLPV vaccination resulted in induction of cross-reactive multifunctional T cell responses after stimulation of immune splenocytes with peptide cocktails derived from LASV from clades I-IV. Multivalent RNA replicon-based LASV vaccines can be applicable for first responders, international travelers visiting endemic areas, military and lab personnel. PMID:29287681

UMG Lenti: novel lentiviral vectors for efficient transgene- and reporter gene expression in human early hematopoietic progenitors.

PubMed

Chiarella, Emanuela; Carrà, Giovanna; Scicchitano, Stefania; Codispoti, Bruna; Mega, Tiziana; Lupia, Michela; Pelaggi, Daniela; Marafioti, Maria G; Aloisio, Annamaria; Giordano, Marco; Nappo, Giovanna; Spoleti, Cristina B; Grillone, Teresa; Giovannone, Emilia D; Spina, Raffaella; Bernaudo, Francesca; Moore, Malcolm A S; Bond, Heather M; Mesuraca, Maria; Morrone, Giovanni

2014-01-01

Lentiviral vectors are widely used to investigate the biological properties of regulatory proteins and/or of leukaemia-associated oncogenes by stably enforcing their expression in hematopoietic stem and progenitor cells. In these studies it is critical to be able to monitor and/or sort the infected cells, typically via fluorescent proteins encoded by the modified viral genome. The most popular strategy to ensure co-expression of transgene and reporter gene is to insert between these cDNAs an IRES element, thus generating bi-cistronic mRNAs whose transcription is driven by a single promoter. However, while the product of the gene located upstream of the IRES is generally abundantly expressed, the translation of the downstream cDNA (typically encoding the reporter protein) is often inconsistent, which hinders the detection and the isolation of transduced cells. To overcome these limitations, we developed novel lentiviral dual-promoter vectors (named UMG-LV5 and -LV6) where transgene expression is driven by the potent UBC promoter and that of the reporter protein, EGFP, by the minimal regulatory element of the WASP gene. These vectors, harboring two distinct transgenes, were tested in a variety of human haematopoietic cell lines as well as in primary human CD34+ cells in comparison with the FUIGW vector that contains the expression cassette UBC-transgene-IRES-EGFP. In these experiments both UMG-LV5 and UMG-LV6 yielded moderately lower transgene expression than FUIGW, but dramatically higher levels of EGFP, thereby allowing the easy distinction between transduced and non-transduced cells. An additional construct was produced, in which the cDNA encoding the reporter protein is upstream, and the transgene downstream of the IRES sequence. This vector, named UMG-LV11, proved able to promote abundant expression of both transgene product and EGFP in all cells tested. The UMG-LVs represent therefore useful vectors for gene transfer-based studies in hematopoietic stem and progenitor cells, as well as in non-hematopoietic cells.

The Role of Proto-Thrusts in Frontal Accretion and Accommodation of Plate Convergence, Hikurangi Subduction Margin, New Zealand

NASA Astrophysics Data System (ADS)

Barnes, P.; Ghisetti, F.; Ellis, S. M.; Morgan, J.

2016-12-01

Proto-thrusts are an enigmatic structural feature at the toe of many subduction accretionary wedges. They are commonly recognised in seismic reflection sections as relatively small-displacement (tens of metres) faults seaward of the primary deformation front. Although widely assumed to reflect incipient accretionary deformation and to mark the location of future thrusts, proto-thrusts have received relatively little attention. Few studies have attempted to characterise their displacement properties, evolution, and kinematic role in frontal accretion processes associated with propagation of the interface décollement. In this study, we make use of excellent quality geophysical and bathymetric imaging of the spectacular 25 km-wide Hikurangi margin proto-thrust zone (PTZ), the structure of which varies significantly along strike. From a detailed structural analysis, we provide the first substantial quantitative dataset on proto-thrust geometry, displacement profiles, fault scaling relationships, and fault population characteristics. These analyses provide new insights into the role of inferred stratigraphic inhomogeneity in proto-thrust development, and the role of proto-thrust arrays in frontal accretion. Our observations, combined with our own recently published reconstructions of the wedge, and ongoing numerical simulations, indicate a migrating wave of proto-thrust activity in association with forward-advancement of the décollement. Calculation of tectonic shortening accommodated by the active PTZ east of the present deformation front, from measurements of seismically-imaged fault displacements and estimates of sub-seismic faulting derived from power law relationships, reveal their surprisingly significant role in accommodating regional plate convergence. South of the colliding Bennett Knoll Seamount, the predominantly seaward-vergent PTZ has accommodated 3.3 km of tectonic shortening, of which 70% is at sub-seismic scale. In comparison, north of Bennett Knoll Seamount, the predominantly landward-vergent PTZ has accommodated 4 km of shortening, of which 87% is at sub-seismic scale. These data combined with estimates of stratigraphic ages and deformation duration, indicate that proto-thrusts potentially accommodate up 30-50% of the total convergence rate.

A novel model for NSAID induced gastroenteropathy in rats.

PubMed

Singh, Devendra Pratap; Borse, Swapnil P; Nivsarkar, Manish

2016-01-01

Progress in management of Nonsteroidal anti-inflammatory drug (NSAID) induced gastrointestinal toxicity requires the availability of appropriate experimental animal models that are as close to humans as feasible. Our objective was to develop a rat model for NSAID-induced gastroenteropathy and also to simulate the common clinical scenario of co-administration of NSAID and proton pump inhibitor (PPI) to explore if PPI contribute to exacerbation of NSAID-enteropathy. Rats were treated twice daily with pantoprazole sodium (PTZ; 10mg/kg peroral) or vehicle for a total of 10days. In some experiments, Diclofenac sodium (DCF; 9mg/kg) or vehicle was administered orally twice daily for the final 5days of PTZ/vehicle administration. After the last dose on 9th day, rats in all the groups were fasted but water was provided ad libitum. 12h after the last dose on 10th day, rats in all the groups were euthanized and their gastrointestinal tracts were assessed for haemorrhagic lesions, lipid peroxidation, intestinal permeability and gastrointestinal luminal pH alterations. Changes in haemoglobin, haematocrit and serum levels of albumin, total protein, ALT and bilirubin were calculated. The macroscopic and histological evidence suggested that administration of DCF resulted in significant gastroenteropathic damage and co-administration of PTZ resulted in significant exacerbation of NSAID enteropathy, while attenuation of NSAID induced gastropathy was observed. Our results were further supported by the significant decrease in haemoglobin and haematocrit levels and serum levels of albumin and total proteins, an increase in oxidative stress and intestinal permeability with the use of DCF either alone or in combination with PTZ. This model was developed to simulate the human clinical situation during NSAID therapy and indeed the present DCF regimen caused both gastric and small bowel alterations, such as multiple erosive lesions, together with a decrease in haemoglobin, haematocrit, serum albumin, serum total protein levels and IP alteration, known to occur in patients receiving NSAIDs. Additionally, this paper provides yet another evidence for PPI induced exacerbation of NSAID enteropathy. Copyright © 2015 Elsevier Inc. All rights reserved.

Huperzine A prophylaxis against pentylenetetrazole-induced seizures in rats is associated with increased cortical inhibition.

PubMed

Gersner, R; Ekstein, D; Dhamne, S C; Schachter, S C; Rotenberg, A

2015-11-01

Huperzine A (HupA) is a naturally occurring compound found in the firmoss Huperzia serrata. While HupA is a potent acetylcholinesterase inhibitor, its full pharmacologic profile is incompletely described. Since previous works suggested a capacity for HupA to prophylax against seizures, we tested the HupA antiepileptic potential in pentylenetetrazole (PTZ) rat epilepsy model and explored its mechanism of action by spectral EEG analysis and by paired-pulse transcranial magnetic stimulation (ppTMS), a measure of GABA-mediated intracortical inhibition. We tested whether HupA suppresses seizures in the rat PTZ acute seizure model, and quantified latency to first myoclonus and to generalized tonic-clonic seizure, and spike frequency on EEG. Additionally, we measured power in the EEG gamma frequency band which is associated with GABAergic cortical interneuron activation. Then, as a step toward further examining the HupA antiepileptic mechanism of action, we tested long-interval intracortical inhibition (LICI) using ppTMS coupled with electromyography to assess whether HupA augments GABA-mediated paired-pulse inhibition of the motor evoked potential. We also tested whether the HupA effect on paired-pulse inhibition was central or peripheral by comparison of outcomes following administration of HupA or the peripheral acetylcholinesterase inhibitor pyridostigmine. We also tested whether the HupA effect was dependent on central muscarinic or GABAA receptors by co-administration of HupA and atropine or PTZ, respectively. In tests of antiepileptic potential, HupA suppressed seizures and epileptic spikes on EEG. Spectral EEG analysis also revealed enhanced gamma frequency band power with HupA treatment. By ppTMS we found that HupA increases intracortical inhibition and blocks PTZ-induced cortical excitation. Atropine co-administration with HupA did not alter HupA-induced intracortical inhibition suggesting independent of muscarinic acetylcholine receptors mechanism in this model. Last, pyridostigmine did not affect the ppTMS-measured cortical inhibition suggesting that HupA-induced effect is centrally-mediated. Our data support antiepileptic HupA applications, and suggest that such activity may be via enhancement of GABAergic intracortical inhibition. Copyright © 2015 Elsevier B.V. All rights reserved.

Chimeric polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wogulis, Mark; Sweeney, Matthew; Heu, Tia

The present invention relates to chimeric GH61 polypeptides having cellulolytic enhancing activity. The present invention also relates to polynucleotides encoding the chimeric GH61 polypeptides; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the chimeric GH61 polypeptides.

Recombinant host cells and nucleic acid constructs encoding polypeptides having cellulolytic enhancing activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schnorr, Kirk; Kramer, Randall

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Ex Vivo Adenoviral Vector Gene Delivery Results in Decreased Vector-associated Inflammation Pre- and Post–lung Transplantation in the Pig

PubMed Central

Yeung, Jonathan C; Wagnetz, Dirk; Cypel, Marcelo; Rubacha, Matthew; Koike, Terumoto; Chun, Yi-Min; Hu, Jim; Waddell, Thomas K; Hwang, David M; Liu, Mingyao; Keshavjee, Shaf

2012-01-01

Acellular normothermic ex vivo lung perfusion (EVLP) is a novel method of donor lung preservation for transplantation. As cellular metabolism is preserved during perfusion, it represents a potential platform for effective gene transduction in donor lungs. We hypothesized that vector-associated inflammation would be reduced during ex vivo delivery due to isolation from the host immune system response. We compared ex vivo with in vivo intratracheal delivery of an E1-, E3-deleted adenoviral vector encoding either green fluorescent protein (GFP) or interleukin-10 (IL-10) to porcine lungs. Twelve hours after delivery, the lung was transplanted and the post-transplant function assessed. We identified significant transgene expression by 12 hours in both in vivo and ex vivo delivered groups. Lung function remained excellent in all ex vivo groups after viral vector delivery; however, as expected, lung function decreased in the in vivo delivered adenovirus vector encoding GFP (AdGFP) group with corresponding increases in IL-1β levels. Transplanted lung function was excellent in the ex vivo transduced lungs and inferior lung function was seen in the in vivo group after transplantation. In summary, ex vivo delivery of adenoviral gene therapy to the donor lung is superior to in vivo delivery in that it leads to less vector-associated inflammation and provides superior post-transplant lung function. PMID:22453765

Joint capsule treatment with enkephalin-encoding HSV-1 recombinant vector reduces inflammatory damage and behavioural sequelae in rat CFA monoarthritis.

PubMed

Lu, Ying; McNearney, Terry A; Wilson, Steven P; Yeomans, David C; Westlund, Karin N

2008-03-01

This study assessed enkephalin expression induced by intra-articular application of recombinant, enkephalin-encoding herpes virus (HSV-1) and the impact of expression on nociceptive behaviours and synovial lining inflammation in arthritic rats. Replication-conditional HSV-1 recombinant vectors with cDNA encoding preproenkephalin (HSV-ENK), or control transgene beta-galactosidase cDNA (HSV-beta-gal; control) were injected into knee joints with complete Freund's adjuvant (CFA). Joint temperatures, circumferences and nociceptive behaviours were monitored on days 0, 7, 14 and 21 post CFA and vector treatments. Lumbar (L4-6) dorsal root ganglia (DRG) and spinal cords were immunostained for met-enkephalin (met-ENK), beta-gal, HSV-1 proteins and Fos. Joint tissues were immunostained for met-ENK, HSV-1 proteins, and inflammatory mediators Regulated on Activation, Normal T-cell Expressed and Secreted (RANTES) and cyclo-oxygenase-2, or stained with haematoxylin and eosin for histopathology. Compared to exuberant synovial hypertrophy and inflammatory cell infiltration seen in arthritic rats treated with CFA only or CFA and HSV-beta-gal, the CFA- and HSV-ENK-treated arthritic rats had: (i) striking preservation of synovial membrane cytoarchitecture with minimal inflammatory cell infiltrates; (ii) significantly improved nociceptive behavioural responses to mechanical and thermal stimuli; (iii) normalized Fos staining in lumbar dorsal horn; and (iv) significantly increased met-ENK staining in ipsilateral synovial tissue, lumbar DRG and spinal cord. The HSV-1 and transgene product expression were confined to ipsilateral lumbar DRG (HSV-1, met-ENK, beta-gal). Only transgene product (met-ENK and beta-gal) was seen in lumbar spinal cord sections. Targeted delivery of enkephalin-encoding HSV-1 vector generated safe, sustained opioid-induced analgesia with protective anti-inflammatory blunting in rat inflammatory arthritis.

Performance Improvements of the Phoneme Recognition Algorithm.

DTIC Science & Technology

1984-06-01

VECTOR NUNDIRt"tIVECTj Beginning vector ; of speech to be extracted INDEX * MOD(IVECT,4) ICheck for vector being ; last vector in block 63 ILE u ((JUICT...1fIER0) ; Read header block. CALL CHECK(IERO) IHEADER(57) : ICOfP ; Change a of freq. components ICHECK : (IHEADER(56) - ([LENGTH - 1)) * ILENGTH...ICONT .Al. ICHECK ) GO TO 55 ; Check to make sure correct ;amount of vectors have been created# INCR u INCR + 32 ;Jump over last set of components

Effects of Homology Length in the Repeat Region on Minus-Strand DNA Transfer and Retroviral Replication

PubMed Central

Dang, Que; Hu, Wei-Shau

2001-01-01

Homology between the two repeat (R) regions in the retroviral genome mediates minus-strand DNA transfer during reverse transcription. We sought to define the effects of R homology lengths on minus-strand DNA transfer. We generated five murine leukemia virus (MLV)-based vectors that contained identical sequences but different lengths of the 3′ R (3, 6, 12, 24 and 69 nucleotides [nt]); 69 nt is the full-length MLV R. After one round of replication, viral titers from the vector with a full-length downstream R were compared with viral titers generated from the other four vectors with reduced R lengths. Viral titers generated from vectors with R lengths reduced to one-third (24 nt) or one-sixth (12 nt) that of the wild type were not significantly affected; however, viral titers generated from vectors with only 3- or 6-nt homology in the R region were significantly lower. Because expression and packaging of the RNA were similar among all the vectors, the differences in the viral titers most likely reflected the impact of the homology lengths on the efficiency of minus-strand DNA transfer. The molecular nature of minus-strand DNA transfer was characterized in 63 proviruses. Precise R-to-R transfer was observed in most proviruses generated from vectors with 12-, 24-, or 69-nt homology in R, whereas aberrant transfers were predominantly used to generate proviruses from vectors with 3- or 6-nt homology. Reverse transcription using RNA transcribed from an upstream promoter, termed read-in RNA transcripts, resulted in most of the aberrant transfers. These data demonstrate that minus-strand DNA transfer is homology driven and a minimum homology length is required for accurate and efficient minus-strand DNA transfer. PMID:11134294

Modification to the Capsid of the Adenovirus Vector That Enhances Dendritic Cell Infection and Transgene-Specific Cellular Immune Responses

PubMed Central

Worgall, Stefan; Busch, Annette; Rivara, Michael; Bonnyay, David; Leopold, Philip L.; Merritt, Robert; Hackett, Neil R.; Rovelink, Peter W.; Bruder, Joseph T.; Wickham, Thomas J.; Kovesdi, Imi; Crystal, Ronald G.

2004-01-01

Adenovirus (Ad) gene transfer vectors can be used to transfer and express antigens and function as strong adjuvants and thus are useful platforms for the development of genetic vaccines. Based on the hypothesis that Ad vectors with enhanced infectibility of dendritic cells (DC) may be able to evoke enhanced immune responses against antigens encoded by the vector in vivo, the present study analyzes the vaccine potential of an Ad vector expressing β-galactosidase as a model antigen and genetically modified with RGD on the fiber knob [AdZ.F(RGD)] to more selectively infect DC and consequently enhance immunity against the β-galactosidase antigen. Infection of murine DC in vitro with AdZ.F(RGD) showed an eightfold-increased transgene expression following infection compared to AdZ (also expressing β-galactosidase, but with a wild-type capsid). Binding, cellular uptake, and trafficking in DC were also increased with AdZ.F(RGD) compared to AdZ. To determine whether AdZ.F(RGD) could evoke enhanced immune responses to β-galactosidase in vivo, C57BL/6 mice were immunized with AdZ.F(RGD) or AdZ subcutaneously via the footpad. Humoral responses with both vectors were comparable, with similar anti-β-galactosidase antibody levels following vector administration. However, cellular responses to β-galactosidase were significantly enhanced, with the frequency of CD4+ as well as the CD8+ β-galactosidase-specific gamma interferon response in cells isolated from the draining lymph nodes increased following immunization with AdZ.F(RGD) compared to Ad.Z (P < 0.01). Importantly, this enhanced cellular immune response of the AdZ.F(RGD) vector was sufficient to evoke enhanced inhibition of the growth of preexisting tumors expressing β-galactosidase: BALB/c mice implanted with the CT26 syngeneic β-galactosidase-expressing colon carcinoma cell line and subsequently immunized with AdZ.F(RGD) showed decreased tumor growth and improved survival compared to mice immunized with AdZ. These data demonstrate that addition of an RGD motif to the Ad fiber knob increases the infectibility of DC and leads to enhanced cellular immune responses to the Ad-transferred transgene, suggesting that the RGD capsid modification may be useful in developing Ad-based vaccines. PMID:14963160

Security authentication using phase-encoded nanoparticle structures and polarized light.

PubMed

Carnicer, Artur; Hassanfiroozi, Amir; Latorre-Carmona, Pedro; Huang, Yi-Pai; Javidi, Bahram

2015-01-15

Phase-encoded nanostructures such as quick response (QR) codes made of metallic nanoparticles are suggested to be used in security and authentication applications. We present a polarimetric optical method able to authenticate random phase-encoded QR codes. The system is illuminated using polarized light, and the QR code is encoded using a phase-only random mask. Using classification algorithms, it is possible to validate the QR code from the examination of the polarimetric signature of the speckle pattern. We used Kolmogorov-Smirnov statistical test and Support Vector Machine algorithms to authenticate the phase-encoded QR codes using polarimetric signatures.

Vector-mediated chromosomal integration of the glutamate decarboxylase gene in streptococcus thermophilus

USDA-ARS?s Scientific Manuscript database

The integrative vector pINTRS was used to transfer glutamate decarboxylase (GAD) activity to Streptococcus thermophilus ST128, thus allowing for the production of '-aminobutyric acid (GABA). In pINTRS, the gene encoding glutamate decarboxylase, gadB, was flanked by DNA fragments homologous to a S. ...

Murine Cytomegalovirus m02 Gene Family Protects against Natural Killer Cell-Mediated Immune Surveillance

PubMed Central

Oliveira, Sofia A.; Park, Se-Ho; Lee, Peter; Bendelac, Albert; Shenk, Thomas E.

2002-01-01

The murine cytomegalovirus m02 gene family encodes putative type I membrane glycoproteins named m02 through m16. A subset of these genes were fused to an epitope tag and cloned into an expression vector. In transfected and murine cytomegalovirus-infected cells, m02, m04, m05, m06, m07, m09, m10, and m12 localized to cytoplasmic structures near the nucleus, whereas m08 and m13 localized to a filamentous structure surrounding the nucleus. Substitution mutants lacking the m02 gene (SMsubm02) or the entire m02 gene family (SMsubm02-16) grew like their wild-type parent in cultured cells. However, whereas SMsubm02 was as pathogenic as the wild-type virus, SMsubm02-16 was markedly less virulent. SMsubm02-16 produced less infectious virus in most organs compared to wild-type virus in BALB/c and C57BL/6J mice, but it replicated to wild-type levels in the organs of immunodeficient γc/Rag2 mice, lacking multiple cell types including natural killer cells, and in C57BL/6J mice depleted of natural killer cells. These results argue that one or more members of the m02 gene family antagonize natural killer cell-mediated immune surveillance. PMID:11752177

Antipathogen genes and the replacement of disease-vectoring mosquito populations: a model-based evaluation

PubMed Central

Robert, Michael A; Okamoto, Kenichi W; Gould, Fred; Lloyd, Alun L

2014-01-01

Recently, genetic strategies aimed at controlling populations of disease-vectoring mosquitoes have received considerable attention as alternatives to traditional measures. Theoretical studies have shown that female-killing (FK), antipathogen (AP), and reduce and replace (R&R) strategies can each decrease the number competent vectors. In this study, we utilize a mathematical model to evaluate impacts on competent Aedes aegypti populations of FK, AP, and R&R releases as well as hybrid strategies that result from combinations of these three approaches. We show that while the ordering of efficacy of these strategies depends upon population life history parameters, sex ratio of releases, and switch time in combination strategies, AP-only and R&R/AP releases typically lead to the greatest long-term reduction in competent vectors. R&R-only releases are often less effective at long-term reduction of competent vectors than AP-only releases or R&R/AP releases. Furthermore, the reduction in competent vectors caused by AP-only releases is easier to maintain than that caused by FK-only or R&R-only releases even when the AP gene confers a fitness cost. We discuss the roles that density dependence and inclusion of females play in the order of efficacy of the strategies. We anticipate that our results will provide added impetus to continue developing AP strategies. PMID:25558284

BGL7 beta-glucosidase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel; Ward, Michael

2013-01-29

The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl7, and the corresponding BGL7 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL7, recombinant BGL7 proteins and methods for producing the same.

BGL6 .beta.-glucosidase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel; Ward, Michael

2012-10-02

The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl6, and the corresponding BGL6 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL6, recombinant BGL6 proteins and methods for producing the same.

BGL5 .beta.-glucosidase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

2006-02-28

The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl5, and the corresponding BGL5 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL5, recombinant BGL5 proteins and methods for producing the same.

BGL5 .beta.-glucosidase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel [Los Gatos, CA; Goedegebuur, Frits [Vlaardingen, NL; Ward, Michael [San Francisco, CA; Yao, Jian [Sunnyvale, CA

2008-03-18

The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl5, and the corresponding BGL5 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL5, recombinant BGL5 proteins and methods for producing the same.

Polypeptides having beta-glucosidase activity and polynucleotides encoding same

DOEpatents

Morant, Marc

2014-01-14

The present invention relates to isolated polypeptides having beta-glucosidase activity, beta-xylosidase, or beta-glucosidase activity and isolated polynucleotides encoding polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

BGL6 beta-glucosidase and nucleic acids encoding the same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dunn-Coleman, Nigel; Ward, Michael

The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl6, and the corresponding BGL6 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL6, recombinant BGL6 proteins and methods for producing the same.

BGL6 beta-glucosidase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel; Ward, Michael

2014-03-04

The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl6, and the corresponding BGL6 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL6, recombinant BGL6 proteins and methods for producing the same.

BGL7 beta-glucosidase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel; Ward, Michael

2015-04-14

The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl7, and the corresponding BGL7 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL7, recombinant BGL7 proteins and methods for producing the same.

BGL7 beta-glucosidase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel; Ward, Michael

2014-03-25

The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl7, and the corresponding BGL7 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL7, recombinant BGL7 proteins and methods for producing the same.

BGL6 beta-glucosidase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel; Ward, Michael

2015-08-11

The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl6, and the corresponding BGL6 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL6, recombinant BGL6 proteins and methods for producing the same.

Nucleic acids encoding mosaic clade M human immunodeficiency virus type 1 (HIV-1) envelope immunogens

DOEpatents

Korber, Bette T; Fischer, William; Liao, Hua-Xin; Haynes, Barton F; Letvin, Norman; Hahn, Beatrice H

2015-04-21

The present invention relates to nucleic acids encoding mosaic clade M HIV-1 Env polypeptides and to compositions and vectors comprising same. The nucleic acids of the invention are suitable for use in inducing an immune response to HIV-1 in a human.

BGL3 beta-glucosidase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

2007-09-25

The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl3, and the corresponding BGL3 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL3, recombinant BGL3 proteins and methods for producing the same.

BGL3 beta-glucosidase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel [Los Gatos, CA; Goedegebuur, Frits [Vlaardingen, NL; Ward, Michael [San Francisco, CA; Yao, Jian [Sunnyvale, CA

2008-04-01

The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl3, and the corresponding BGL3 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL3, recombinant BGL3 proteins and methods for producing the same.

BGL4 beta-glucosidase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel [Los Gatos, CA; Goedegebuur, Frits [Vlaardingen, NL; Ward, Michael [San Francisco, CA; Yao, Jian [Sunnyvale, CA

2011-12-06

The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl4, and the corresponding BGL4 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL4, recombinant BGL4 proteins and methods for producing the same.

BGL4 .beta.-glucosidase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

2006-05-16

The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl4, and the corresponding BGL4 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL4, recombinant BGL4 proteins and methods for producing the same.

BGL3 beta-glucosidase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel [Los Gatos, CA; Goedegebuur, Frits [Vlaardingen, NL; Ward, Michael [San Francisco, CA; Yao, Jian [Sunnyvale, CA

2011-06-14

The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl3, and the corresponding BGL3 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL3, recombinant BGL3 proteins and methods for producing the same.

BGL6 beta-glucosidase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel [Los Gatos, CA; Ward, Michael [San Francisco, CA

2009-09-01

The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl6, and the corresponding BGL6 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL6, recombinant BGL6 proteins and methods for producing the same.

BGL3 beta-glucosidase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

2012-10-30

The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl3, and the corresponding BGL3 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL3, recombinant BGL3 proteins and methods for producing the same.

BGL4 beta-glucosidase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel [Los Gatos, CA; Goedegebuur, Frits [Vlaardingen, NL; Ward, Michael [San Francisco, CA; Yao, Jian [Sunnyvale, CA

2008-01-22

The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl4, and the corresponding BGL4 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL4, recombinant BGL4 proteins and methods for producing the same.

Cre-lox Univector acceptor vectors for functional screening in protoplasts: analysis of Arabidopsis donor cDNAs encoding ABSCISIC ACID INSENSITIVE1-Like protein phosphatases

PubMed Central

Jia, Fan; Gampala, Srinivas S.L.; Mittal, Amandeep; Luo, Qingjun; Rock, Christopher D.

2009-01-01

The 14,200 available full length Arabidopsis thaliana cDNAs in the Universal Plasmid System (UPS) donor vector pUNI51 should be applied broadly and efficiently to leverage a “functional map-space” of homologous plant genes. We have engineered Cre-lox UPS host acceptor vectors (pCR701- 705) with N-terminal epitope tags in frame with the loxH site and downstream from the maize Ubiquitin promoter for use in transient protoplast expression assays and particle bombardment transformation of monocots. As an example of the utility of these vectors, we recombined them with several Arabidopsis cDNAs encoding Ser/Thr protein phosphatase type 2C (PP2Cs) known from genetic studies or predicted by hierarchical clustering meta-analysis to be involved in ABA and stress responses. Our functional results in Zea mays mesophyll protoplasts on ABA-inducible expression effects on the Late Embryogenesis Abundant promoter ProEm:GUS reporter were consistent with predictions and resulted in identification of novel activities of some PP2Cs. Deployment of these vectors can facilitate functional genomics and proteomics and identification of novel gene activities. PMID:19499346

Expansion of the Lyme Disease Vector Ixodes Scapularis in Canada Inferred from CMIP5 Climate Projections

PubMed Central

McPherson, Michelle; García-García, Almudena; Cuesta-Valero, Francisco José; Hansen-Ketchum, Patti; MacDougall, Donna; Ogden, Nicholas Hume

2017-01-01

Background: A number of studies have assessed possible climate change impacts on the Lyme disease vector, Ixodes scapularis. However, most have used surface air temperature from only one climate model simulation and/or one emission scenario, representing only one possible climate future. Objectives: We quantified effects of different Representative Concentration Pathway (RCP) and climate model outputs on the projected future changes in the basic reproduction number (R0) of I. scapularis to explore uncertainties in future R0 estimates. Methods: We used surface air temperature generated by a complete set of General Circulation Models from the Coupled Model Intercomparison Project Phase 5 (CMIP5) to hindcast historical (1971–2000), and to forecast future effects of climate change on the R0 of I. scapularis for the periods 2011–2040 and 2041–2070. Results: Increases in the multimodel mean R0 values estimated for both future periods, relative to 1971–2000, were statistically significant under all RCP scenarios for all of Nova Scotia, areas of New Brunswick and Quebec, Ontario south of 47°N, and Manitoba south of 52°N. When comparing RCP scenarios, only the estimated R0 mean values between RCP6.0 and RCP8.5 showed statistically significant differences for any future time period. Conclusion: Our results highlight the potential for climate change to have an effect on future Lyme disease risk in Canada even if the Paris Agreement’s goal to keep global warming below 2°C is achieved, although mitigation reducing emissions from RCP8.5 levels to those of RCP6.0 or less would be expected to slow tick invasion after the 2030s. https://doi.org/10.1289/EHP57 PMID:28599266

Treatment of collagenase-induced osteoarthritis with a viral vector encoding TSG-6 results in ectopic bone formation.

PubMed

Broeren, Mathijs G A; Di Ceglie, Irene; Bennink, Miranda B; van Lent, Peter L E M; van den Berg, Wim B; Koenders, Marije I; Blaney Davidson, Esmeralda N; van der Kraan, Peter M; van de Loo, Fons A J

2018-01-01

Tumor necrosis factor-inducible gene 6 (TSG-6) has anti-inflammatory and chondroprotective effects in mouse models of inflammatory arthritis. Because cartilage damage and inflammation are also observed in osteoarthritis (OA), we determined the effect of viral overexpression of TSG-6 in experimental osteoarthritis. Bone marrow-derived cells were differentiated to multinucleated osteoclasts in the presence of recombinant TSG-6 or after transduction with a lentiviral TSG-6 expression vector. Multi-nucleated osteoclasts were analyzed after tartrate resistant acid phosphatase staining and resorption activity was determined on dentin slices. Collagenase-induced osteoarthritis (CIOA) was induced in C57BL/6 mice after intra-articular injection of an adenoviral TSG-6 or control luciferase expression vector. Inflammation-related protease activity was measured using bioluminescent Prosense probes. After a second adenovirus injection, cartilage damage was assessed in histological sections stained with Safranin-O. Ectopic bone formation was scored in X-ray images of the affected knees. TSG-6 did not inhibit the formation of multi-nucleated osteoclasts, but caused a significant reduction in the resorption activity on dentin slices. Adenoviral TSG-6 gene therapy in CIOA could not reduce the cartilage damage compared to the luciferase control virus and no significant difference in inflammation-related protease activity was noted between the TSG-6 and control treated group. Instead, X-ray analysis and histological analysis revealed the presence of ectopic bone formation in the TSG-6 treated group. Gene therapy based on the expression of TSG-6 could not provide cartilage protection in experimental osteoarthritis, but instead resulted in increased ectopic bone formation.

What Limits the Encoding Effect of Note-Taking? A Meta-Analytic Examination

ERIC Educational Resources Information Center

Kobayashi, K.

2005-01-01

Previous meta-analyses indicate that the overall encoding effect of note-taking is positive but modest. This meta-analysis of 57 note-taking versus no note-taking comparison studies explored what limits the encoding effect by examining the moderating influence of seven variables: intervention, schooling level, presentation mode and length, test…

Screening a wide host-range, waste-water metagenomic library in tryptophan auxotrophs of Rhizobium leguminosarum and of Escherichia coli reveals different classes of cloned trp genes.

PubMed

Li, Youguo; Wexler, Margaret; Richardson, David J; Bond, Philip L; Johnston, Andrew W B

2005-12-01

A metagenomic cosmid library was constructed, in which the insert DNA was derived from bacteria in a waste-water treatment plant and the vector was the wide host-range cosmid pLAFR3. The library was screened for clones that could correct defined tryptophan auxotrophs of the alpha-proteobacterium Rhizobium leguminosarum and of Escherichia coli. A total of 26 different cosmids that corrected at least one trp mutant in one or both of these species were obtained. Several cosmids corrected the auxotrophy of one or more R. leguminosarum trp mutants, but not the corresponding mutants in E. coli. Conversely, one cosmid corrected trpA, B, C, D and E mutants of E. coli but none of the trp mutants of R. leguminosarum. Two of the Trp+ cosmids were examined in more detail. One contained a trp operon that resembled that of the pathogen Chlamydophila caviae, containing the unusual kynU gene, which specifies kynureninase. The other, whose trp genes functioned in R. leguminosarum but not in E. coli, contained trpDCFBA in an operon that is likely co-transcribed with five other genes, most of which had no known link with tryptophan synthesis. The sequences of these TRP proteins, and the products of nine other genes encoded by this cosmid, failed to affiliate them with any known bacterial lineage. For one metagenomic cosmid, lac reporter fusions confirmed that its cloned trp genes were transcribed in R. leguminosarum, but not in E. coli. Thus, rhizobia, with their many sigma-factors, may be well-suited hosts for metagenomic libraries, cloned in wide host-range vectors.

In Vitro Antimicrobial Activity of Razupenem (SMP-601, PTZ601) against Anaerobic Bacteria▿

PubMed Central

Tran, Chau Minh; Tanaka, Kaori; Yamagishi, Yuka; Goto, Takatsugu; Mikamo, Hiroshige; Watanabe, Kunitomo

2011-01-01

We evaluated the in vitro antianaerobic activity of razupenem (SMP-601, PTZ601), a new parenterally administered carbapenem, against 70 reference strains and 323 clinical isolates. Razupenem exhibited broad-spectrum activity against anaerobes, inhibiting most of the reference strains when used at a concentration of ≤1 μg/ml. Furthermore, it exhibited strong activity, comparable to those of other carbapenems (meropenem and doripenem), against clinically isolated non-fragilis Bacteroides spp. (MIC90s of 2 μg/ml), with MIC90 values of 0.06, 0.03, and 0.5 μg/ml against Prevotella spp., Porphyromonas spp., and Fusobacterium spp., respectively. Clinical isolates of anaerobic Gram-positive cocci, Eggerthella spp., and Clostridium spp. were highly susceptible to razupenem (MIC90s, 0.03 to 1 μg/ml). PMID:21343447

Anti-convulsant action and amelioration of oxidative stress by Glycyrrhiza glabra root extract in pentylenetetrazole- induced seizure in albino rats

PubMed Central

Chowdhury, Bimalendu; Bhattamisra, Subrat K.; Das, Mangala C.

2013-01-01

Objectives: The aim of the present study was to evaluate the anti-convulsant potential of aqueous and ethanol e xtract of Glycyrrhiza glabra (AEGG and EEGG) and its action on markers of oxidant stress in albino rats. Materials and Methods: The aqueous and ethanol extract of Glycyrrhiza glabra was tested at three doses viz. 100, 200, and 400 mg/kg i.p. for its anti-convulsant activity using pentylenetetrazole (PTZ)-induced seizure in rat. The effect of EEGG (400 mg/kg, i.p.) on oxidative stress markers like malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) of rat brain tissue homogenate was tested. Results: The onset of seizure was delayed (P < 0.01) by all the three doses of EEGG, but the duration of convulsion was reduced (P < 0.01) only in higher dose level (200 and 400 mg/ kg), whereas AEGG up to 400 mg/kg did not alter any of the parameters significantly. Biochemical analysis of rat brain tissue revealed that MDA was increased (P < 0.01), whereas SOD and CAT were decreased (P < 0.01) in PTZ-induced seizure rat, whereas pre-treatment with EEGG (400 mg/kg) decreased (P < 0.01) the MDA and increased (P < 0.01) both SOD and CAT, indicating attenuation of lipid peroxidation due to increase in antioxidant enzymes. Conclusion: The results demonstrated that EEGG poses anti-convulsant potential and ameliorates ROS induced neuronal damage in PTZ-induced seizure. PMID:23543836

Surface Engineering of ITO Substrates to Improve the Memory Performance of an Asymmetric Conjugated Molecule with a Side Chain.

PubMed

Hou, Xiang; Cheng, Xue-Feng; Xiao, Xin; He, Jing-Hui; Xu, Qing-Feng; Li, Hua; Li, Na-Jun; Chen, Dong-Yun; Lu, Jian-Mei

2017-09-05

Organic multilevel random resistive access memory (RRAM) devices with an electrode/organic layer/electrode sandwich-like structure suffer from poor reproducibility, such as low effective ternary device yields and a wide threshold voltage distribution, and improvements through organic material renovation are rather limited. In contrast, engineering of the electrode surfaces rather than molecule design has been demonstrated to boost the performance of organic electronics effectively. Herein, we introduce surface engineering into organic multilevel RRAMs to enhance their ternary memory performance. A new asymmetric conjugated molecule composed of phenothiazine and malononitrile with a side chain (PTZ-PTZO-CN) was fabricated in an indium tin oxide (ITO)/PTZ-PTZO-CN/Al sandwich-like memory device. Modification of the ITO substrate with a phosphonic acid (PA) prior to device fabrication increased the ternary device yield (the ratio of effective ternary device) and narrowed the threshold voltage distribution. The crystallinity analysis revealed that PTZ-PTZO-CN grown on untreated ITO crystallized into two phases. After the surface engineering of ITO, this crystalline ambiguity was eliminated and a sole crystal phase was obtained that was the same as in the powder state. The unified crystal structure and improved grain mosaicity resulted in a lower threshold voltage and, therefore, a higher ternary device yield. Our result demonstrated that PA modification also improved the memory performance of an asymmetric conjugated molecule with a side chain. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

A species dependent response to the pro-epileptic drug pentylentetrazole in birds.

PubMed

Amin, Faiq; Dar, Asim H; Osama, Khan; Khan, Faezah; Mitha, Rida; Tharwani, Arsal; Haider, Ghulam; Chand, Prem; Arain, Fazal M

2017-09-01

Epilepsy is common disorder that affects over 50 million people worldwide. Birds remain a promising yet largely under-explored model of epilepsy. This study reports the comparison of the response of two species of birds, Australian Parrots (APs) and Sparrows (SPs), to a pro-epileptic drug, Pentylenetetrazole (PTZ). PTZ injections caused myoclonic jerks (MCJs) and tonic clonic seizures (TCSs) in both species. The frequency of MCJs in APs was greater at the dose of 75mg/kg compared to both 50mg/kg and 25mg/kg while it was not significantly different in SPs. The comparison of APs and SPs showed that the frequency of MCJs was greater in APs compared to SPs at 25mg/kg and 75mg/kg while its latency was reduced at 25mg/kg and 50mg/kg. Interestingly SPs had a reduced latency of TCSs compared to APs at 75mg/kg. Glutamatergic and Gabaergic cell count was conducted to determine an association with the epileptic response to PTZ. The Glutamatergic cell counts for SPs was significantly greater than APs and conversely the Gabaergic cell counts in APs was higher compared to SPs. The reason for this difference in findings needs to be further investigated. This study shows that birds, and APs and SPs in particular, are a valid, interesting and under-explored model of epilepsy that should be further explored in order to understand the mysteries of epilepsy. Copyright © 2017 Elsevier Inc. All rights reserved.

Generation and purification of recombinant fimbrillin from Porphyromonas (Bacteroides) gingivalis 381.

PubMed Central

Washington, O R; Deslauriers, M; Stevens, D P; Lyford, L K; Haque, S; Yan, Y; Flood, P M

1993-01-01

Fimbrillin is the major subunit protein of fimbriae from the human periodontal pathogen Porphyromonas (Bacteroides) gingivalis. We describe here the generation and initial characterization of recombinant fimbrillin (r-fimbrillin) isolated from P. gingivalis 381. A fragment of DNA encoding the gene for fimbrillin was generated by polymerase chain reaction and cloned into the expression vector pET11b. Plasmids containing the recombinant gene were transfected into Escherichia coli. Clones were selected on plates for ampicillin resistance and individually screened by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for protein production after activation with IPTG (isopropyl-beta-D- thiogalactopyranoside). One clone, OW0.2, produced significant amounts of a 42-kDa protein after induction with IPTG. This clone contained the pET11b plasmid with a 1-kb insert that had sequence homology to the gene encoding fimbrillin. The majority of recombinant protein from clone OW0.2 was found in the cytoplasm within inclusion bodies. Protein aggregates were solubilized in 8 M urea, and SDS-PAGE analysis showed two major protein bands, one at 42 kDa and the other at 17 kDa. These two proteins coeluted from a DEAE-Sepharose column at 0.15 M NaCl and were reactive to rabbit antiserum to fimbrillin in a Western blot (immunoblot). A preparation giving a single protein band at 42 kDa in SDS-PAGE was obtained by size fractionation by using continuous-elution electrophoresis. Lymph node cells from animals immunized with either fimbrillin from P. gingivalis or r-fimbrillin showed antigen-specific proliferation to both P. gingivalis fimbrillin and r-fimbrillin in an in vitro recall assay. Therefore, it appears that r-fimbrillin is chemically, antigenically, and serologically identical to fimbrillin isolated from P. gingivalis 381. Images PMID:8094377

Cloning of a methanol-inducible moxF promoter and its analysis in moxB mutants of Methylobacterium extorquens AM1rif.

PubMed Central

Morris, C J; Lidstrom, M E

1992-01-01

In Methylobacterium extorquens AM1, gene encoding methanol dehydrogenase polypeptides are transcriptionally regulated in response to C1 compounds, including methanol (M. E. Lidstrom and D. I. Stirling, Annu. Rev. Microbiol. 44:27-57, 1990). In order to study this regulation, a transcriptional fusion has been constructed between a beta-galactosidase reporter gene and a 1.55-kb XhoI-SalI fragment of M. extorquens AM1rif DNA encoding the N terminus of the methanol dehydrogenase large subunit (moxF) and 1,289 bp of upstream DNA. The fusion exhibited orientation-specific promoter activity in M. extorquens AM1rif but was expressed constitutively when the transcriptional fusion was located on the plasmid. However, correct regulation was restored when the construction was inserted in the M. extorquens AM1rif chromosome. This DNA fragment was shown to contain both the moxFJGI promoter and the sequences necessary in cis for its transcriptional regulation by methanol. Transcription from this promoter was studied in the M. extorquens AM1rif moxB mutant strains UV4rif and UV25rif, which have a pleiotropic phenotype with regard to the components of methanol oxidation. In these mutants, beta-galactosidase activity from the fusion was reduced to a level equal to that of the vector background when the fusion was present in both plasmid and chromosomal locations. Since both constitutive and methanol-inducible promoter activities were lost in the mutants, moxB appears to be required for transcription of the genes encoding the methanol dehydrogenase polypeptides. Images PMID:1624436

Secure multiparty computation of a comparison problem.

PubMed

Liu, Xin; Li, Shundong; Liu, Jian; Chen, Xiubo; Xu, Gang

2016-01-01

Private comparison is fundamental to secure multiparty computation. In this study, we propose novel protocols to privately determine [Formula: see text], or [Formula: see text] in one execution. First, a 0-1-vector encoding method is introduced to encode a number into a vector, and the Goldwasser-Micali encryption scheme is used to compare integers privately. Then, we propose a protocol by using a geometric method to compare rational numbers privately, and the protocol is information-theoretical secure. Using the simulation paradigm, we prove the privacy-preserving property of our protocols in the semi-honest model. The complexity analysis shows that our protocols are more efficient than previous solutions.

Peptide Processing Is Critical for T-Cell Memory Inflation and May Be Optimized to Improve Immune Protection by CMV-Based Vaccine Vectors.

PubMed

Dekhtiarenko, Iryna; Ratts, Robert B; Blatnik, Renata; Lee, Lian N; Fischer, Sonja; Borkner, Lisa; Oduro, Jennifer D; Marandu, Thomas F; Hoppe, Stephanie; Ruzsics, Zsolt; Sonnemann, Julia K; Mansouri, Mandana; Meyer, Christine; Lemmermann, Niels A W; Holtappels, Rafaela; Arens, Ramon; Klenerman, Paul; Früh, Klaus; Reddehase, Matthias J; Riemer, Angelika B; Cicin-Sain, Luka

2016-12-01

Cytomegalovirus (CMV) elicits long-term T-cell immunity of unparalleled strength, which has allowed the development of highly protective CMV-based vaccine vectors. Counterintuitively, experimental vaccines encoding a single MHC-I restricted epitope offered better immune protection than those expressing entire proteins, including the same epitope. To clarify this conundrum, we generated recombinant murine CMVs (MCMVs) encoding well-characterized MHC-I epitopes at different positions within viral genes and observed strong immune responses and protection against viruses and tumor growth when the epitopes were expressed at the protein C-terminus. We used the M45-encoded conventional epitope HGIRNASFI to dissect this phenomenon at the molecular level. A recombinant MCMV expressing HGIRNASFI on the C-terminus of M45, in contrast to wild-type MCMV, enabled peptide processing by the constitutive proteasome, direct antigen presentation, and an inflation of antigen-specific effector memory cells. Consequently, our results indicate that constitutive proteasome processing of antigenic epitopes in latently infected cells is required for robust inflationary responses. This insight allows utilizing the epitope positioning in the design of CMV-based vectors as a novel strategy for enhancing their efficacy.

Peptide Processing Is Critical for T-Cell Memory Inflation and May Be Optimized to Improve Immune Protection by CMV-Based Vaccine Vectors

PubMed Central

Blatnik, Renata; Lee, Lian N.; Fischer, Sonja; Borkner, Lisa; Oduro, Jennifer D.; Marandu, Thomas F.; Hoppe, Stephanie; Ruzsics, Zsolt; Sonnemann, Julia K.; Meyer, Christine; Holtappels, Rafaela; Arens, Ramon; Früh, Klaus; Reddehase, Matthias J.; Riemer, Angelika B.; Cicin-Sain, Luka

2016-01-01

Cytomegalovirus (CMV) elicits long-term T-cell immunity of unparalleled strength, which has allowed the development of highly protective CMV-based vaccine vectors. Counterintuitively, experimental vaccines encoding a single MHC-I restricted epitope offered better immune protection than those expressing entire proteins, including the same epitope. To clarify this conundrum, we generated recombinant murine CMVs (MCMVs) encoding well-characterized MHC-I epitopes at different positions within viral genes and observed strong immune responses and protection against viruses and tumor growth when the epitopes were expressed at the protein C-terminus. We used the M45-encoded conventional epitope HGIRNASFI to dissect this phenomenon at the molecular level. A recombinant MCMV expressing HGIRNASFI on the C-terminus of M45, in contrast to wild-type MCMV, enabled peptide processing by the constitutive proteasome, direct antigen presentation, and an inflation of antigen-specific effector memory cells. Consequently, our results indicate that constitutive proteasome processing of antigenic epitopes in latently infected cells is required for robust inflationary responses. This insight allows utilizing the epitope positioning in the design of CMV-based vectors as a novel strategy for enhancing their efficacy. PMID:27977791

Gene therapy-mediated delivery of targeted cytotoxins for glioma therapeutics

PubMed Central

Candolfi, Marianela; Xiong, Weidong; Yagiz, Kader; Liu, Chunyan; Muhammad, A. K. M. G.; Puntel, Mariana; Foulad, David; Zadmehr, Ali; Ahlzadeh, Gabrielle E.; Kroeger, Kurt M.; Tesarfreund, Matthew; Lee, Sharon; Debinski, Waldemar; Sareen, Dhruv; Svendsen, Clive N.; Rodriguez, Ron; Lowenstein, Pedro R.; Castro, Maria G.

2010-01-01

Restricting the cytotoxicity of anticancer agents by targeting receptors exclusively expressed on tumor cells is critical when treating infiltrative brain tumors such as glioblastoma multiforme (GBM). GBMs express an IL-13 receptor (IL13Rα2) that differs from the physiological IL4R/IL13R receptor. We developed a regulatable adenoviral vector (Ad.mhIL-4.TRE.mhIL-13-PE) encoding a mutated human IL-13 fused to Pseudomonas exotoxin (mhIL-13-PE) that specifically binds to IL13Rα2 to provide sustained expression, effective anti-GBM cytotoxicity, and minimal neurotoxicity. The therapeutic Ad also encodes mutated human IL-4 that binds to the physiological IL4R/IL13R without interacting with IL13Rα2, thus inhibiting potential binding of mhIL-13-PE to normal brain cells. Using intracranial GBM xenografts and syngeneic mouse models, we tested the Ad.mhIL-4.TRE.mhIL-13-PE and two protein formulations, hIL-13-PE used in clinical trials (Cintredekin Besudotox) and a second-generation mhIL-13-PE. Cintredekin Besudotox doubled median survival without eliciting long-term survival and caused severe neurotoxicity; mhIL-13-PE led to ∼40% long-term survival, eliciting severe neurological toxicity at the high dose tested. In contrast, Ad-mediated delivery of mhIL-13-PE led to tumor regression and long-term survival in over 70% of the animals, without causing apparent neurotoxicity. Although Cintredekin Besudotox was originally developed to target GBM, when tested in a phase III trial it failed to achieve clinical endpoints and revealed neurotoxicity. Limitations of Cintredekin Besudotox include its short half-life, which demanded frequent or continued administration, and binding to IL4R/IL13R, present in normal brain cells. These shortcomings were overcome by our therapeutic Ad, thus representing a significant advance in the development of targeted therapeutics for GBM. PMID:21030678

Development of the gateway recycling cloning system for multiple linking of expression cassettes in a defined order, and direction on gateway compatible binary vectors.

PubMed

Kimura, Tetsuya; Nakao, Akihide; Murata, Sachiko; Kobayashi, Yasuyuki; Tanaka, Yuji; Shibahara, Kenta; Kawazu, Tetsu; Nakagawa, Tsuyoshi

2013-01-01

We developed the Gateway recycling cloning system, which allows multiple linking of expression cassettes by multiple rounds of the Gateway LR reaction. Employing this system, the recycling donor vector pRED419 was subjected to the first LR reaction with an attR1-attR2 type destination vector. Then conversion vector pCON was subjected to an LR reaction to restore the attR1-attR2 site on the destination vector for the next cloning cycle. By repetition of these two simple steps, we linked four expression cassettes of a reporter gene in Gateway binary vector pGWB1, introduced the constructs into tobacco BY-2 cells, and observed the expression of transgenes.

The ribosome as a missing link in prebiotic evolution II: Ribosomes encode ribosomal proteins that bind to common regions of their own mRNAs and rRNAs.

PubMed

Root-Bernstein, Robert; Root-Bernstein, Meredith

2016-05-21

We have proposed that the ribosome may represent a missing link between prebiotic chemistries and the first cells. One of the predictions that follows from this hypothesis, which we test here, is that ribosomal RNA (rRNA) must have encoded the proteins necessary for ribosomal function. In other words, the rRNA also functioned pre-biotically as mRNA. Since these ribosome-binding proteins (rb-proteins) must bind to the rRNA, but the rRNA also functioned as mRNA, it follows that rb-proteins should bind to their own mRNA as well. This hypothesis can be contrasted to a "null" hypothesis in which rb-proteins evolved independently of the rRNA sequences and therefore there should be no necessary similarity between the rRNA to which rb-proteins bind and the mRNA that encodes the rb-protein. Five types of evidence reported here support the plausibility of the hypothesis that the mRNA encoding rb-proteins evolved from rRNA: (1) the ubiquity of rb-protein binding to their own mRNAs and autogenous control of their own translation; (2) the higher-than-expected incidence of Arginine-rich modules associated with RNA binding that occurs in rRNA-encoded proteins; (3) the fact that rRNA-binding regions of rb-proteins are homologous to their mRNA binding regions; (4) the higher than expected incidence of rb-protein sequences encoded in rRNA that are of a high degree of homology to their mRNA as compared with a random selection of other proteins; and (5) rRNA in modern prokaryotes and eukaryotes encodes functional proteins. None of these results can be explained by the null hypothesis that assumes independent evolution of rRNA and the mRNAs encoding ribosomal proteins. Also noteworthy is that very few proteins bind their own mRNAs that are not associated with ribosome function. Further tests of the hypothesis are suggested: (1) experimental testing of whether rRNA-encoded proteins bind to rRNA at their coding sites; (2) whether tRNA synthetases, which are also known to bind to their own mRNAs, are encoded by the tRNA sequences themselves; (3) and the prediction that archaeal and prokaryotic (DNA-based) genomes were built around rRNA "genes" so that rRNA-related sequences will be found to make up an unexpectedly high proportion of these genomes. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Real-time optical laboratory solution of parabolic differential equations

NASA Technical Reports Server (NTRS)

Casasent, David; Jackson, James

1988-01-01

An optical laboratory matrix-vector processor is used to solve parabolic differential equations (the transient diffusion equation with two space variables and time) by an explicit algorithm. This includes optical matrix-vector nonbase-2 encoded laboratory data, the combination of nonbase-2 and frequency-multiplexed data on such processors, a high-accuracy optical laboratory solution of a partial differential equation, new data partitioning techniques, and a discussion of a multiprocessor optical matrix-vector architecture.

Cell culture compositions

DOEpatents

Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yiao, Jian

2014-03-18

The present invention provides a novel endoglucanase nucleic acid sequence, designated egl6 (SEQ ID NO:1 encodes the full length endoglucanase; SEQ ID NO:4 encodes the mature form), and the corresponding endoglucanase VI amino acid sequence ("EGVI"; SEQ ID NO:3 is the signal sequence; SEQ ID NO:2 is the mature sequence). The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVI, recombinant EGVI proteins and methods for producing the same.

A recombinant novirhabdovirus presenting at the surface the E Glycoprotein from West Nile Virus (WNV) is immunogenic and provides partial protection against lethal WNV challenge in BALB/c mice.

PubMed

Nzonza, Angella; Lecollinet, Sylvie; Chat, Sophie; Lowenski, Steeve; Mérour, Emilie; Biacchesi, Stéphane; Brémont, Michel

2014-01-01

West Nile Virus (WNV) is a zoonotic mosquito-transmitted flavivirus that can infect and cause disease in mammals including humans. Our study aimed at developing a WNV vectored vaccine based on a fish Novirhabdovirus, the Viral Hemorrhagic Septicemia virus (VHSV). VHSV replicates at temperatures lower than 20°C and is naturally inactivated at higher temperatures. A reverse genetics system has recently been developed in our laboratory for VHSV allowing the addition of genes in the viral genome and the recovery of the respective recombinant viruses (rVHSV). In this study, we have generated rVHSV vectors bearing the complete WNV envelope gene (EWNV) (rVHSV-EWNV) or fragments encoding E subdomains (either domain III alone or domain III fused to domain II) (rVHSV-DIIIWNV and rVHSV-DII-DIIIWNV, respectively) in the VHSV genome between the N and P cistrons. With the objective to enhance the targeting of the EWNV protein or EWNV-derived domains to the surface of VHSV virions, Novirhadovirus G-derived signal peptide and transmembrane domain (SPG and TMG) were fused to EWNV at its amino and carboxy termini, respectively. By Western-blot analysis, electron microscopy observations or inoculation experiments in mice, we demonstrated that both the EWNV and the DIIIWNV could be expressed at the viral surface of rVHSV upon addition of SPG. Every constructs expressing EWNV fused to SPG protected 40 to 50% of BALB/cJ mice against WNV lethal challenge and specifically rVHSV-SPGEWNV induced a neutralizing antibody response that correlated with protection. Surprisingly, rVHSV expressing EWNV-derived domain III or II and III were unable to protect mice against WNV challenge, although these domains were highly incorporated in the virion and expressed at the viral surface. In this study we demonstrated that a heterologous glycoprotein and non membrane-anchored protein, can be efficiently expressed at the surface of rVHSV making this approach attractive to develop new vaccines against various pathogens.

Biocatalytic Conversion of Avermectin to 4"-Oxo-Avermectin: Heterologous Expression of the ema1 Cytochrome P450 Monooxygenase

PubMed Central

Molnár, István; Hill, D. Steven; Zirkle, Ross; Hammer, Philip E.; Gross, Frank; Buckel, Thomas G.; Jungmann, Volker; Pachlatko, Johannes Paul; Ligon, James M.

2005-01-01

The cytochrome P450 monooxygenase Ema1 from Streptomyces tubercidicus R-922 and its homologs from closely related Streptomyces strains are able to catalyze the regioselective oxidation of avermectin into 4"-oxo-avermectin, a key intermediate in the manufacture of the agriculturally important insecticide emamectin benzoate (V. Jungmann, I. Molnár, P. E. Hammer, D. S. Hill, R. Zirkle, T. G. Buckel, D. Buckel, J. M. Ligon, and J. P. Pachlatko, Appl. Environ. Microbiol. 71:6968-6976, 2005). The gene for Ema1 has been expressed in Streptomyces lividans, Streptomyces avermitilis, and solvent-tolerant Pseudomonas putida strains using different promoters and vectors to provide biocatalytically competent cells. Replacing the extremely rare TTA codon with the more frequent CTG codon to encode Leu4 in Ema1 increased the biocatalytic activities of S. lividans strains producing this enzyme. Ferredoxins and ferredoxin reductases were also cloned from Streptomyces coelicolor and biocatalytic Streptomyces strains and tested in ema1 coexpression systems to optimize the electron transport towards Ema1. PMID:16269733

An adenovirus-vectored nasal vaccine confers rapid and sustained protection against anthrax in a single-dose regimen.

PubMed

Zhang, Jianfeng; Jex, Edward; Feng, Tsungwei; Sivko, Gloria S; Baillie, Leslie W; Goldman, Stanley; Van Kampen, Kent R; Tang, De-chu C

2013-01-01

Bacillus anthracis is the causative agent of anthrax, and its spores have been developed into lethal bioweapons. To mitigate an onslaught from airborne anthrax spores that are maliciously disseminated, it is of paramount importance to develop a rapid-response anthrax vaccine that can be mass administered by nonmedical personnel during a crisis. We report here that intranasal instillation of a nonreplicating adenovirus vector encoding B. anthracis protective antigen could confer rapid and sustained protection against inhalation anthrax in mice in a single-dose regimen in the presence of preexisting adenovirus immunity. The potency of the vaccine was greatly enhanced when codons of the antigen gene were optimized to match the tRNA pool found in human cells. In addition, an adenovirus vector encoding lethal factor can confer partial protection against inhalation anthrax and might be coadministered with a protective antigen-based vaccine.

An Adenovirus-Vectored Nasal Vaccine Confers Rapid and Sustained Protection against Anthrax in a Single-Dose Regimen

PubMed Central

Jex, Edward; Feng, Tsungwei; Sivko, Gloria S.; Baillie, Leslie W.; Goldman, Stanley; Van Kampen, Kent R.; Tang, De-chu C.

2013-01-01

Bacillus anthracis is the causative agent of anthrax, and its spores have been developed into lethal bioweapons. To mitigate an onslaught from airborne anthrax spores that are maliciously disseminated, it is of paramount importance to develop a rapid-response anthrax vaccine that can be mass administered by nonmedical personnel during a crisis. We report here that intranasal instillation of a nonreplicating adenovirus vector encoding B. anthracis protective antigen could confer rapid and sustained protection against inhalation anthrax in mice in a single-dose regimen in the presence of preexisting adenovirus immunity. The potency of the vaccine was greatly enhanced when codons of the antigen gene were optimized to match the tRNA pool found in human cells. In addition, an adenovirus vector encoding lethal factor can confer partial protection against inhalation anthrax and might be coadministered with a protective antigen-based vaccine. PMID:23100479

CYTOMEGALOVIRUS VECTORS VIOLATE CD8+ T CELL EPITOPE RECOGNITION PARADIGMS

PubMed Central

Hansen, Scott G.; Sacha, Jonah B.; Hughes, Colette M.; Ford, Julia C.; Burwitz, Benjamin J.; Scholz, Isabel; Gilbride, Roxanne M.; Lewis, Matthew S.; Gilliam, Awbrey N.; Ventura, Abigail B.; Malouli, Daniel; Xu, Guangwu; Richards, Rebecca; Whizin, Nathan; Reed, Jason S.; Hammond, Katherine B.; Fischer, Miranda; Turner, John M.; Legasse, Alfred W.; Axthelm, Michael K.; Edlefsen, Paul T.; Nelson, Jay A.; Lifson, Jeffrey D.; Früh, Klaus; Picker, Louis J.

2013-01-01

CD8+ T cell responses focus on a small fraction of pathogen- or vaccine-encoded peptides, and for some pathogens, these restricted recognition hierarchies limit the effectiveness of anti-pathogen immunity. We found that simian immunodeficiency virus (SIV) protein-expressing Rhesus Cytomegalovirus (RhCMV) vectors elicit SIV-specific CD8+ T cells that recognize unusual, diverse and highly promiscuous epitopes, including dominant responses to epitopes restricted by class II major histocompatibility complex (MHC) molecules. Induction of canonical SIV epitope-specific CD8+ T cell responses is suppressed by the RhCMV-encoded Rh189 (US11) gene, and the promiscuous MHC class I- and class II-restricted CD8+ T cell responses only occur in the absence of the Rh157.4-.6 (UL128-131) genes. Thus, CMV vectors can be genetically programmed to achieve distinct patterns of CD8+ T cell epitope recognition. PMID:23704576

High-quality animation of 2D steady vector fields.

PubMed

Lefer, Wilfrid; Jobard, Bruno; Leduc, Claire

2004-01-01

Simulators for dynamic systems are now widely used in various application areas and raise the need for effective and accurate flow visualization techniques. Animation allows us to depict direction, orientation, and velocity of a vector field accurately. This paper extends a former proposal for a new approach to produce perfectly cyclic and variable-speed animations for 2D steady vector fields (see [1] and [2]). A complete animation of an arbitrary number of frames is encoded in a single image. The animation can be played using the color table animation technique, which is very effective even on low-end workstations. A cyclic set of textures can be produced as well and then encoded in a common animation format or used for texture mapping on 3D objects. As compared to other approaches, the method presented in this paper produces smoother animations and is more effective, both in memory requirements to store the animation, and in computation time.

Investigation of a Third Order Baro-Damped Vertical Channel of INS.

DTIC Science & Technology

1982-12-01

SI%(TINCL3.S:*4SA-") SALP -IA = CQS(TI4CLj/Ch.Ar IF(GA"-GI! )24.25,25 25 Z-F GAM-GI�.924 26 CALP..,A:SGRt4 -SA PMA.SALPHAI 32 G) T’) 2? 24...55 39 CALPHA:-iQR?(1-!ALPmA& SALP "A) C SEGUENT IAEE 24 VZ=0. 135 iS.OU?!%E TPAJ ?4/F4 0PTZ1 F4.*.564 IO/2 1..3 147 1031. V40R ZZ GAwoor.(EQ-HT) CTLAT...TSORT( COS(GA.4l.C:3(GAM8.( (ocrNL- 7#(AI-2 STLAr = S.Nc.%cJS(GI SALP ,4* = CoS(TINCL)/CTLAT 5334 :F(GA"-G1213!.3S,33 35 ChLP’m*:SOfT(1-SALPA.SALPMIAJ

Suppression of cotton leaf curl disease symptoms in Gossypium hirsutum through over expression of host-encoded miRNAs.

PubMed

Akmal, Mohd; Baig, Mirza S; Khan, Jawaid A

2017-12-10

Cotton leaf curl disease (CLCuD), a major factor resulting in the enormous yield losses in cotton crop, is caused by a distinct monopartite begomovirus in association with Cotton leaf curl Multan betasatellite (CLCuMB). Micro(mi)RNAs are known to regulate gene expression in eukaryotes, including antiviral defense in plants. In a previous study, we had computationally identified a set of cotton miRNAs, which were shown to have potential targets in the genomes of Cotton leaf curl Multan virus (CLCuMuV) and CLCuMB at multiple loci. In the current study, effect of Gossypium arboreum-encoded miRNAs on the genome of CLCuMuV and CLCuMB was investigated in planta. Two computationally predicted cotton-encoded miRNAs (miR398 and miR2950) that showed potential to bind multiple Open Reading Frames (ORFs; C1, C4, V1, and non- coding intergenic region) of CLCuMuV, and (βC1) of CLCuMB were selected. Functional validation of miR398 and miR2950 was done by overexpression approach in G. hirsutum var. HS6. A total of ten in vitro cotton plants were generated from independent events and subjected to biological and molecular analyses. Presence of the respective Precursor (pre)-miRNA was confirmed through PCR and Southern blotting, and their expression level was assessed by semi quantitative RT-PCR, Real Time quantitative PCR and northern hybridization in the PCR-positive lines. Southern hybridization revealed 2-4 copy integration of T-DNA in the genome of the transformed lines. Remarkably, expression of pre-miRNAs was shown up to 5.8-fold higher in the transgenic (T 0 ) lines as revealed by Real Time PCR. The virus resistance was monitored following inoculation of the transgenic cotton lines with viruliferous whitefly (Bemisia tabaci) insect vector. After inoculation, four of the transgenic lines remained apparently symptom free. While a very low titre of viral DNA could be detected by Rolling circle amplification, betasatellite responsible for symptom induction could not be detected in any of the healthy looking transgenic lines. In this study for the first time, efficacy of the host (G. arboreum)-encoded miRNAs against CLCuD symptoms was experimentally demonstrated through overexpression of miR398 and miR2950 in G. hirsutum var. HS6 plants. Computational prediction of miRNAs targeting virus genome and their subsequent implication in translational inhibition or cleavage based suppression of viral mRNA via overexpression could help in generating virus resistant plants. Copyright © 2017 Elsevier B.V. All rights reserved.

Adaptive Hybrid Picture Coding. Volume 2.

DTIC Science & Technology

1985-02-01

ooo5 V.a Measurement Vector ..eho..............57 V.b Size Variable o .entroi* Vector .......... .- 59 V * c Shape Vector .Ř 0-60o oe 6 I V~d...the Program for the Adaptive Line of Sight Method .i.. 18.. o ... .... .... 1 B Details of the Feature Vector FormationProgram .. o ...oo..-....- .122 C ...shape recognition is analogous to recognition of curves in space. Therefore, well known concepts and theorems from differential geometry can be 34 . o

Systems and methods for the secretion of recombinant proteins in gram negative bacteria

DOE Office of Scientific and Technical Information (OSTI.GOV)

Withers, III, Sydnor T.; Dominguez, Miguel A.; DeLisa, Matthew P.

Disclosed herein are systems and methods for producing recombinant proteins utilizing mutant E. coli strains containing expression vectors carrying nucleic acids encoding the proteins, and secretory signal sequences to direct the secretion of the proteins to the culture medium. Host cells transformed with the expression vectors are also provided.

Systems and methods for the secretion of recombinant proteins in gram negative bacteria

DOEpatents

Withers, III, Sydnor T.; Dominguez, Miguel A; DeLisa, Matthew P.; Haitjema, Charles H.

2016-08-09

Disclosed herein are systems and methods for producing recombinant proteins utilizing mutant E. coli strains containing expression vectors carrying nucleic acids encoding the proteins, and secretory signal sequences to direct the secretion of the proteins to the culture medium. Host cells transformed with the expression vectors are also provided.

Adenoviral production of interleukin-2 at the tumor site removes the need for systemic postconditioning in adoptive cell therapy.

PubMed

Santos, Joao Manuel; Havunen, Riikka; Siurala, Mikko; Cervera-Carrascon, Víctor; Tähtinen, Siri; Sorsa, Suvi; Anttila, Marjukka; Karell, Pauliina; Kanerva, Anna; Hemminki, Akseli

2017-10-01

Systemic high dose interleukin-2 (IL-2) postconditioning has long been utilized in boosting the efficacy of T cells in adoptive cell therapy (ACT) of solid tumors. The resulting severe off-target toxicity of these regimens renders local production at the tumor an attractive concept with possible safety gains. We evaluated the efficacy and safety of intratumorally administered IL-2-coding adenoviruses in combination with tumor-infiltrating lymphocyte therapy in syngeneic Syrian hamsters bearing HapT1 pancreatic tumors and with T cell receptor transgenic ACT in B16.OVA melanoma bearing C57BL/6 mice. The models are complementary: hamsters are semi-permissive for human oncolytic adenovirus, whereas detailed immunological analyses are possible in mice. In both models, local production of IL-2 successfully replaced the need for systemic recombinant IL-2 (rIL-2) administration and increased the efficacy of the cell therapy. Furthermore, vectored delivery of IL-2 significantly enhanced the infiltration of CD8+ T cells, M1-like macrophages, and B-cells while systemic rIL-2 increased CD25 + FoxP3+ T cells at the tumor. In contrast with vectored delivery, histopathological analysis of systemic rIL-2-treated animals revealed significant changes in lungs, livers, hearts, spleens, and kidneys. In summary, local IL-2 production results in efficacy and safety gains in the context of ACT. These preclinical assessments provide the rationale for ongoing clinical translation. © 2017 UICC.

Immunogenicity of ORFV-based vectors expressing the rabies virus glycoprotein in livestock species.

PubMed

Martins, Mathias; Joshi, Lok R; Rodrigues, Fernando S; Anziliero, Deniz; Frandoloso, Rafael; Kutish, Gerald F; Rock, Daniel L; Weiblen, Rudi; Flores, Eduardo F; Diel, Diego G

2017-11-01

The parapoxvirus Orf virus (ORFV) encodes several immunomodulatory proteins (IMPs) that modulate host-innate and pro-inflammatory responses and has been proposed as a vaccine delivery vector for use in animal species. Here we describe the construction and characterization of two recombinant ORFV vectors expressing the rabies virus (RABV) glycoprotein (G). The RABV-G gene was inserted in the ORFV024 or ORFV121 gene loci, which encode for IMPs that are unique to parapoxviruses and inhibit activation of the NF-κB signaling pathway. The immunogenicity of the resultant recombinant viruses (ORFV ∆024 RABV-G or ORFV ∆121 RABV-G, respectively) was evaluated in pigs and cattle. Immunization of the target species with ORFV ∆024 RABV-G and ORFV ∆121 RABV-G elicited robust neutralizing antibody responses against RABV. Notably, neutralizing antibody titers induced in ORFV ∆121 RABV-G-immunized pigs and cattle were significantly higher than those detected in ORFV ∆024 RABV-G-immunized animals, indicating a higher immunogenicity of ORFV Δ121 -based vectors in these animal species. Copyright © 2017 Elsevier Inc. All rights reserved.

Beta-glucosidase variants and polynucleotides encoding same

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wogulis, Mark; Harris, Paul; Osborn, David

The present invention relates to beta-glucosidase variants, e.g. beta-glucosidase variants of a parent Family GH3A beta-glucosidase from Aspergillus fumigatus. The present invention also relates to polynucleotides encoding the beta-glucosidase variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the beta-glucosidase variants.

Polypeptides having beta-glucosidase activity and polynucleotides encoding same

DOEpatents

Morant, Marc Dominique

2014-10-14

The present invention relates to isolated polypeptides having beta-glucosidase activity, beta-xylosidase activity, or beta-glucosidase and beta-xylosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

One-Step Bioprocess of Inulin to Product Inulo-Oligosaccharides Using Bacillus subtilis Secreting an Extracellular Endo-Inulinase.

PubMed

Jiang, Ruifan; Qiu, Yibin; Huang, Weiwei; Zhang, Li; Xue, Feng; Ni, Hao; Mei, Difen; Gao, Jian; Xu, Hong

2018-06-18

Inulo-oligosaccharides (IOSs), a novel food additive and health product, represent a promising alternative to antibiotics. As prebiotics, IOSs can be obtained from inulin by endo-inulinase-mediated hydrolysis. Nonetheless, enzymatic catalysis is not feasible industrially because of the required catalytic conditions and cost. In this study, a 2331-bp optimized gene inuQ (from Pseudomonas mucidolens) encoding endo-inulinase was cloned into shuttle vector PHY300PLK and transfected into Bacillus subtilis WB800-R, with the simultaneous deletion of gene sacC encoding levanase. The maximal IOS yield after hydrolysis of the crude extract of inulin was 67.84 ± 0.72 g/L for a recombinant strain with the signal peptide nprB from alkaline protease and promoter P 43 . The conversion rate reached 75.38%. For the major IOSs, the degree of polymerization was between 3 and 5. This study offers a simple and efficient one-step bioprocess for IOS production from inulin through secretion of an extracellular heterologous endo-inulinase by B. subtilis.

Partial Gene Cloning and Enzyme Structure Modeling of Exolevanase Fragment from Bacillus subtilis

NASA Astrophysics Data System (ADS)

Azhar, M.; Natalia, D.; Syukur, S.; Andriani, N.; Jamsari, J.

2018-04-01

Inulin hydrolysis thermophilic and thermotolerant bacteria are potential sources of inulin hydrolysis enzymes. Partial gene that encodes inulin hydrolysis enzymes had been isolated from Bacillus subtilis using polymerase chain reaction (PCR) method with the DPE.slFandDPE.eR degenerative primers. The partial gene was cloned into pGEM-T Easy vector with E. coli as host cells and analyzed using BLASTx, CrustalW2, and Phyre2 programs. Size of thepartial gene had been found539 bp that encoded 179aminoacid residues of protein fragment. The sequences of protein fragment was more similar to exolevanase than exoinulinase. The protein fragment had conserved motif FSGS, and specific hits GH32 β-fructosidase. It had three residues of active site and five residues of substrate binding. The active site on the protein fragment were D (1-WLNDP-5), D (125-FRDPK-129) and E (177-WEC-179). Substrate binding on the protein fragment were ND (1-WLNDP-5), Q (18-FYQY-21), FS (60-FSGS-63) RD (125-FRDPK-129) and E (177-WEC-179).

A fosmid cloning strategy for detecting the widest possible spectrum of microbes from the international space station drinking water system.

PubMed

Choi, Sangdun; Chang, Mi Sook; Stuecker, Tara; Chung, Christine; Newcombe, David A; Venkateswaran, Kasthuri

2012-12-01

In this study, fosmid cloning strategies were used to assess the microbial populations in water from the International Space Station (ISS) drinking water system (henceforth referred to as Prebiocide and Tank A water samples). The goals of this study were: to compare the sensitivity of the fosmid cloning strategy with that of traditional culture-based and 16S rRNA-based approaches and to detect the widest possible spectrum of microbial populations during the water purification process. Initially, microbes could not be cultivated, and conventional PCR failed to amplify 16S rDNA fragments from these low biomass samples. Therefore, randomly primed rolling-circle amplification was used to amplify any DNA that might be present in the samples, followed by size selection by using pulsed-field gel electrophoresis. The amplified high-molecular-weight DNA from both samples was cloned into fosmid vectors. Several hundred clones were randomly selected for sequencing, followed by Blastn/Blastx searches. Sequences encoding specific genes from Burkholderia, a species abundant in the soil and groundwater, were found in both samples. Bradyrhizobium and Mesorhizobium, which belong to rhizobia, a large community of nitrogen fixers often found in association with plant roots, were present in the Prebiocide samples. Ralstonia, which is prevalent in soils with a high heavy metal content, was detected in the Tank A samples. The detection of many unidentified sequences suggests the presence of potentially novel microbial fingerprints. The bacterial diversity detected in this pilot study using a fosmid vector approach was higher than that detected by conventional 16S rRNA gene sequencing.

Molecular Diagnostic Tools for Detection and Differentiation of Phytoplasmas Based on Chaperonin-60 Reveal Differences in Host Plant Infection Patterns

PubMed Central

Dumonceaux, Tim J.; Green, Margaret; Hammond, Christine; Perez, Edel; Olivier, Chrystel

2014-01-01

Phytoplasmas (‘Candidatus Phytoplasma’ spp.) are insect-vectored bacteria that infect a wide variety of plants, including many agriculturally important species. The infections can cause devastating yield losses by inducing morphological changes that dramatically alter inflorescence development. Detection of phytoplasma infection typically utilizes sequences located within the 16S–23S rRNA-encoding locus, and these sequences are necessary for strain identification by currently accepted standards for phytoplasma classification. However, these methods can generate PCR products >1400 bp that are less divergent in sequence than protein-encoding genes, limiting strain resolution in certain cases. We describe a method for accessing the chaperonin-60 (cpn60) gene sequence from a diverse array of ‘Ca.Phytoplasma’ spp. Two degenerate primer sets were designed based on the known sequence diversity of cpn60 from ‘Ca.Phytoplasma’ spp. and used to amplify cpn60 gene fragments from various reference samples and infected plant tissues. Forty three cpn60 sequences were thereby determined. The cpn60 PCR-gel electrophoresis method was highly sensitive compared to 16S-23S-targeted PCR-gel electrophoresis. The topology of a phylogenetic tree generated using cpn60 sequences was congruent with that reported for 16S rRNA-encoding genes. The cpn60 sequences were used to design a hybridization array using oligonucleotide-coupled fluorescent microspheres, providing rapid diagnosis and typing of phytoplasma infections. The oligonucleotide-coupled fluorescent microsphere assay revealed samples that were infected simultaneously with two subtypes of phytoplasma. These tools were applied to show that two host plants, Brassica napus and Camelina sativa, displayed different phytoplasma infection patterns. PMID:25551224

Targeted Knock-Down of miR21 Primary Transcripts Using snoMEN Vectors Induces Apoptosis in Human Cancer Cell Lines.

PubMed

Ono, Motoharu; Yamada, Kayo; Avolio, Fabio; Afzal, Vackar; Bensaddek, Dalila; Lamond, Angus I

2015-01-01

We have previously reported an antisense technology, 'snoMEN vectors', for targeted knock-down of protein coding mRNAs using human snoRNAs manipulated to contain short regions of sequence complementarity with the mRNA target. Here we characterise the use of snoMEN vectors to target the knock-down of micro RNA primary transcripts. We document the specific knock-down of miR21 in HeLa cells using plasmid vectors expressing miR21-targeted snoMEN RNAs and show this induces apoptosis. Knock-down is dependent on the presence of complementary sequences in the snoMEN vector and the induction of apoptosis can be suppressed by over-expression of miR21. Furthermore, we have also developed lentiviral vectors for delivery of snoMEN RNAs and show this increases the efficiency of vector transduction in many human cell lines that are difficult to transfect with plasmid vectors. Transduction of lentiviral vectors expressing snoMEN targeted to pri-miR21 induces apoptosis in human lung adenocarcinoma cells, which express high levels of miR21, but not in human primary cells. We show that snoMEN-mediated suppression of miRNA expression is prevented by siRNA knock-down of Ago2, but not by knock-down of Ago1 or Upf1. snoMEN RNAs colocalise with Ago2 in cell nuclei and nucleoli and can be co-immunoprecipitated from nuclear extracts by antibodies specific for Ago2.

Broad-spectrum anti-tumor and anti-metastatic DNA vaccine based on p62-encoding vector

PubMed Central

Sherman, Michael Y.; Gabai, Vladimir; Kiselev, Oleg; Komissarov, Andrey; Grudinin, Mikhail; Shartukova, Maria; Romanovskaya-Romanko, Ekaterina A.; Kudryavets, Yuri; Bezdenezhnykh, Natalya; Lykhova, Oleksandra; Semesyuk, Nadiia; Concetti, Antonio; Tsyb, Anatoly; Filimonova, Marina; Makarchuk, Victoria; Yakubovsky, Raisa; Chursov, Andrey; Shcherbinina, Vita; Shneider, Alexander

2013-01-01

Autophagy plays an important role in neoplastic transformation of cells and in resistance of cancer cells to radio- and chemotherapy. p62 (SQSTM1) is a key component of autophagic machinery which is also involved in signal transduction. Although recent empirical observations demonstrated that p62 is overexpressed in variety of human tumors, a mechanism of p62 overexpression is not known. Here we report that the transformation of normal human mammary epithelial cells with diverse oncogenes (RAS, PIK3CA and Her2) causes marked accumulation of p62. Based on this result, we hypothesized that p62 may be a feasible candidate to be an anti-cancer DNA vaccine. Here we performed a preclinical study of a novel DNA vaccine encoding p62. Intramuscularly administered p62-encoding plasmid induced anti-p62 antibodies and exhibited strong antitumor activity in four models of allogeneic mouse tumors – B16 melanoma, Lewis lung carcinoma (LLC), S37 sarcoma, and Ca755 breast carcinoma. In mice challenged with Ca755 cells, p62 treatment had dual effect: inhibited tumor growth in some mice and prolonged life in those mice which developed tumor size similar to control. P62-encoding plasmid has demonstrated its potency both as a preventive and therapeutic vaccine. Importantly, p62 vaccination drastically suppressed metastasis formation: in B16 melanoma where tumor cells where injected intravenously, and in LLC and S37 sarcoma with spontaneous metastasis. Overall, we conclude that a p62-encoding vector(s) constitute(s) a novel, effective broad-spectrum antitumor and anti-metastatic vaccine feasible for further development and clinical trials. PMID:24121124

Low-complexity video encoding method for wireless image transmission in capsule endoscope.

PubMed

Takizawa, Kenichi; Hamaguchi, Kiyoshi

2010-01-01

This paper presents a low-complexity video encoding method applicable for wireless image transmission in capsule endoscopes. This encoding method is based on Wyner-Ziv theory, in which side information available at a transmitter is treated as side information at its receiver. Therefore complex processes in video encoding, such as estimation of the motion vector, are moved to the receiver side, which has a larger-capacity battery. As a result, the encoding process is only to decimate coded original data through channel coding. We provide a performance evaluation for a low-density parity check (LDPC) coding method in the AWGN channel.

Neutralized adenovirus-immune complexes can mediate effective gene transfer via an Fc receptor-dependent infection pathway.

PubMed

Leopold, Philip L; Wendland, Rebecca L; Vincent, Theresa; Crystal, Ronald G

2006-10-01

Neutralization of adenovirus (Ad) by anti-Ad neutralizing antibodies in serum involves formation of Ad-immune complexes that prevent the virus from interacting with target cells. We hypothesized that Ad-immune complexes likely contain viable Ad vectors which, although no longer capable of gaining access to receptors on target cells, may be able to express transgenes in cells bearing Fc receptors for immunoglobulins, i.e., that antibody-based "neutralization" of Ad vectors may be circumvented by the Fc receptor pathway. To test this hypothesis, we expressed the Fcgamma receptor IIA (FcgammaR) in A549 lung epithelial cells or human dermal fibroblasts and evaluated gene transfer in the presence of human neutralizing anti-Ad serum. FcgammaR-expressing cells bound and internalized copious amounts of Ad, with a distinct population of internalized Ad trafficking to the nucleus. The dose-response curves for inhibition of gene transfer revealed that FcgammaR-expressing cells required a more-than-10-fold higher concentration of anti-Ad serum to achieve 50% inhibition of Ad-encoded beta-galactosidase expression compared with non-FcgammaR-expressing cells. The discrepancy between neutralization of Ad during infection of FcgammaR-expressing cells and neutralization of Ad during infection of non-FcgammaR-expressing cells occurred with either heat-inactivated or non-heat-inactivated sera, was blocked by addition of purified Fc domain protein, and did not require the cytoplasmic domain of FcgammaR, suggesting that immune complex internalization proceeded via endocytosis rather than phagocytosis. FcgammaR-mediated infection by Ad-immune complexes did not require expression of the coxsackie virus-Ad receptor (CAR) since similar data were obtained when CAR-deficient human dermal fibroblasts were engineered to express FcgammaR. However, interaction of the Ad penton base with cell surface integrins contributed to the difference in neutralization between FcgammaR-expressing and non-FcgammaR-expressing cells. The data indicate that complexes formed from Ad and anti-Ad neutralizing antibodies, while compromised with respect to infection of non-FcgammaR-expressing target cells, maintain the potential to transfer genes to FcgammaR-expressing cells, with consequent expression of the transgene. The formation of Ad-immune complexes that can target viable virus to antigen-presenting cells may account for the success of Ad-based vaccines administered in the presence of low levels of neutralizing anti-Ad antibody.

Vaccination With a Highly Attenuated Recombinant Vesicular Stomatitis Virus Vector Protects Against Challenge With a Lethal Dose of Ebola Virus

PubMed Central

Matassov, Demetrius; Marzi, Andrea; Latham, Terri; Xu, Rong; Ota-Setlik, Ayuko; Feldmann, Friederike; Geisbert, Joan B.; Mire, Chad E.; Hamm, Stefan; Nowak, Becky; Egan, Michael A.; Geisbert, Thomas W.; Eldridge, John H.; Feldmann, Heinz; Clarke, David K.

2015-01-01

Previously, recombinant vesicular stomatitis virus (rVSV) pseudotypes expressing Ebolavirus glycoproteins (GPs) in place of the VSV G protein demonstrated protection of nonhuman primates from lethal homologous Ebolavirus challenge. Those pseudotype vectors contained no additional attenuating mutations in the rVSV genome. Here we describe rVSV vectors containing a full complement of VSV genes and expressing the Ebola virus (EBOV) GP from an additional transcription unit. These rVSV vectors contain the same combination of attenuating mutations used previously in the clinical development pathway of an rVSV/human immunodeficiency virus type 1 vaccine. One of these rVSV vectors (N4CT1-EBOVGP1), which expresses membrane-anchored EBOV GP from the first position in the genome (GP1), elicited a balanced cellular and humoral GP-specific immune response in mice. Guinea pigs immunized with a single dose of this vector were protected from any signs of disease following lethal EBOV challenge, while control animals died in 7–9 days. Subsequently, N4CT1-EBOVGP1 demonstrated complete, single-dose protection of 2 macaques following lethal EBOV challenge. A single sham-vaccinated macaque died from disease due to EBOV infection. These results demonstrate that highly attenuated rVSV vectors expressing EBOV GP may provide safer alternatives to current EBOV vaccines. PMID:26109675

Effect of the Leaf Essential Oil from Cinnamosma madagascariensis Danguy on Pentylenetetrazol-induced Seizure in Rats.

PubMed

Rakotosaona, Rianasoambolanoro; Randrianarivo, Emmanuel; Rasoanaivo, Philippe; Nicoletti, Marcello; Benelli, Giovanni; Maggi, Filippo

2017-10-01

In the Malagasy traditional practices, the smoke from burning leaves of Cinnamosma madagascariensis Danguy is inhaled to treat brain disorders such as dementia, epilepsy, and headache. In the present work, we have evaluated the in vivo anticonvulsant effects of the essential oil from leaves of C. madagascariensis (CMEO). CMEO was isolated by steam distillation. The anticonvulsant activity of CMEO (0.4 and 0.8 ml/kg bw) administered subcutaneously was evaluated on pentylenetetrazol (PTZ)-induced seizures in Wistar rats; diazepam was used as positive control. Linalool, limonene, and myrcene were the major CMEO constituents. At the dose of 0.8 ml/kg, CMEO completely arrested the PTZ-induced convulsions with moderate sedative effects. The traditional anticonvulsant use of C. madagascariensis was confirmed allowing us to candidate molecules from CMEO as potential drugs to treat convulsions associated with strong agitation. © 2017 Wiley-VHCA AG, Zurich, Switzerland.

Measurement of the transverse momentum distribution of Z /γ* bosons in proton-proton collisions at √{ s} = 7 TeV with the ATLAS detector

NASA Astrophysics Data System (ADS)

Aad, G.; Abbott, B.; Abdallah, J.; Abdelalim, A. A.; Abdesselam, A.; Abdinov, O.; Abi, B.; Abolins, M.; Abramowicz, H.; Abreu, H.; Acerbi, E.; Acharya, B. S.; Adams, D. L.; Addy, T. N.; Adelman, J.; Aderholz, M.; Adomeit, S.; Adragna, P.; Adye, T.; Aefsky, S.; Aguilar-Saavedra, J. A.; Aharrouche, M.; Ahlen, S. P.; Ahles, F.; Ahmad, A.; Ahsan, M.; Aielli, G.; Akdogan, T.; Åkesson, T. P. A.; Akimoto, G.; Akimov, A. V.; Akiyama, A.; Alam, M. S.; Alam, M. A.; Albert, J.; Albrand, S.; Aleksa, M.; Aleksandrov, I. N.; Alessandria, F.; Alexa, C.; Alexander, G.; Alexandre, G.; Alexopoulos, T.; Alhroob, M.; Aliev, M.; Alimonti, G.; Alison, J.; Aliyev, M.; Allport, P. P.; Allwood-Spiers, S. E.; Almond, J.; Aloisio, A.; Alon, R.; Alonso, A.; Alviggi, M. G.; Amako, K.; Amaral, P.; Amelung, C.; Ammosov, V. V.; Amorim, A.; Amorós, G.; Amram, N.; Anastopoulos, C.; Andari, N.; Andeen, T.; Anders, C. F.; Anderson, K. J.; Andreazza, A.; Andrei, V.; Andrieux, M.-L.; Anduaga, X. S.; Angerami, A.; Anghinolfi, F.; Anjos, N.; Annovi, A.; Antonaki, A.; Antonelli, M.; Antonov, A.; Antos, J.; Anulli, F.; Aoun, S.; Aperio Bella, L.; Apolle, R.; Arabidze, G.; Aracena, I.; Arai, Y.; Arce, A. T. H.; Archambault, J. P.; Arfaoui, S.; Arguin, J.-F.; Arik, E.; Arik, M.; Armbruster, A. J.; Arnaez, O.; Arnault, C.; Artamonov, A.; Artoni, G.; Arutinov, D.; Asai, S.; Asfandiyarov, R.; Ask, S.; Åsman, B.; Asquith, L.; Assamagan, K.; Astbury, A.; Astvatsatourov, A.; Atoian, G.; Aubert, B.; Auerbach, B.; Auge, E.; Augsten, K.; Aurousseau, M.; Austin, N.; Avolio, G.; Avramidou, R.; Axen, D.; Ay, C.; Azuelos, G.; Azuma, Y.; Baak, M. A.; Baccaglioni, G.; Bacci, C.; Bach, A. M.; Bachacou, H.; Bachas, K.; Bachy, G.; Backes, M.; Backhaus, M.; Badescu, E.; Bagnaia, P.; Bahinipati, S.; Bai, Y.; Bailey, D. C.; Bain, T.; Baines, J. T.; Baker, O. K.; Baker, M. D.; Baker, S.; Baltasar Dos Santos Pedrosa, F.; Banas, E.; Banerjee, P.; Banerjee, Sw.; Banfi, D.; Bangert, A.; Bansal, V.; Bansil, H. S.; Barak, L.; Baranov, S. P.; Barashkou, A.; Barbaro Galtieri, A.; Barber, T.; Barberio, E. L.; Barberis, D.; Barbero, M.; Bardin, D. Y.; Barillari, T.; Barisonzi, M.; Barklow, T.; Barlow, N.; Barnett, B. M.; Barnett, R. M.; Baroncelli, A.; Barone, G.; Barr, A. J.; Barreiro, F.; Barreiro Guimarães da Costa, J.; Barrillon, P.; Bartoldus, R.; Barton, A. E.; Bartsch, D.; Bartsch, V.; Bates, R. L.; Batkova, L.; Batley, J. R.; Battaglia, A.; Battistin, M.; Battistoni, G.; Bauer, F.; Bawa, H. S.; Beare, B.; Beau, T.; Beauchemin, P. H.; Beccherle, R.; Bechtle, P.; Beck, H. P.; Beckingham, M.; Becks, K. H.; Beddall, A. J.; Beddall, A.; Bedikian, S.; Bednyakov, V. A.; Bee, C. P.; Begel, M.; Behar Harpaz, S.; Behera, P. K.; Beimforde, M.; Belanger-Champagne, C.; Bell, P. J.; Bell, W. H.; Bella, G.; Bellagamba, L.; Bellina, F.; Bellomo, M.; Belloni, A.; Beloborodova, O.; Belotskiy, K.; Beltramello, O.; Ben Ami, S.; Benary, O.; Benchekroun, D.; Benchouk, C.; Bendel, M.; Benedict, B. H.; Benekos, N.; Benhammou, Y.; Benjamin, D. P.; Benoit, M.; Bensinger, J. R.; Benslama, K.; Bentvelsen, S.; Berge, D.; Bergeaas Kuutmann, E.; Berger, N.; Berghaus, F.; Berglund, E.; Beringer, J.; Bernardet, K.; Bernat, P.; Bernhard, R.; Bernius, C.; Berry, T.; Bertin, A.; Bertinelli, F.; Bertolucci, F.; Besana, M. I.; Besson, N.; Bethke, S.; Bhimji, W.; Bianchi, R. M.; Bianco, M.; Biebel, O.; Bieniek, S. P.; Biesiada, J.; Biglietti, M.; Bilokon, H.; Bindi, M.; Binet, S.; Bingul, A.; Bini, C.; Biscarat, C.; Bitenc, U.; Black, K. M.; Blair, R. E.; Blanchard, J.-B.; Blanchot, G.; Blazek, T.; Blocker, C.; Blocki, J.; Blondel, A.; Blum, W.; Blumenschein, U.; Bobbink, G. J.; Bobrovnikov, V. B.; Bocchetta, S. S.; Bocci, A.; Boddy, C. R.; Boehler, M.; Boek, J.; Boelaert, N.; Böser, S.; Bogaerts, J. A.; Bogdanchikov, A.; Bogouch, A.; Bohm, C.; Boisvert, V.; Bold, T.; Boldea, V.; Bolnet, N. M.; Bona, M.; Bondarenko, V. G.; Boonekamp, M.; Boorman, G.; Booth, C. N.; Bordoni, S.; Borer, C.; Borisov, A.; Borissov, G.; Borjanovic, I.; Borroni, S.; Bos, K.; Boscherini, D.; Bosman, M.; Boterenbrood, H.; Botterill, D.; Bouchami, J.; Boudreau, J.; Bouhova-Thacker, E. V.; Boulahouache, C.; Bourdarios, C.; Bousson, N.; Boveia, A.; Boyd, J.; Boyko, I. R.; Bozhko, N. I.; Bozovic-Jelisavcic, I.; Bracinik, J.; Braem, A.; Branchini, P.; Brandenburg, G. W.; Brandt, A.; Brandt, G.; Brandt, O.; Bratzler, U.; Brau, B.; Brau, J. E.; Braun, H. M.; Brelier, B.; Bremer, J.; Brenner, R.; Bressler, S.; Breton, D.; Britton, D.; Brochu, F. M.; Brock, I.; Brock, R.; Brodbeck, T. J.; Brodet, E.; Broggi, F.; Bromberg, C.; Brooijmans, G.; Brooks, W. K.; Brown, G.; Brown, H.; Bruckman de Renstrom, P. A.; Bruncko, D.; Bruneliere, R.; Brunet, S.; Bruni, A.; Bruni, G.; Bruschi, M.; Buanes, T.; Bucci, F.; Buchanan, J.; Buchanan, N. J.; Buchholz, P.; Buckingham, R. M.; Buckley, A. G.; Buda, S. I.; Budagov, I. A.; Budick, B.; Büscher, V.; Bugge, L.; Buira-Clark, D.; Bulekov, O.; Bunse, M.; Buran, T.; Burckhart, H.; Burdin, S.; Burgess, T.; Burke, S.; Busato, E.; Bussey, P.; Buszello, C. P.; Butin, F.; Butler, B.; Butler, J. M.; Buttar, C. M.; Butterworth, J. M.; Buttinger, W.; Byatt, T.; Cabrera Urbán, S.; Caforio, D.; Cakir, O.; Calafiura, P.; Calderini, G.; Calfayan, P.; Calkins, R.; Caloba, L. P.; Caloi, R.; Calvet, D.; Calvet, S.; Camacho Toro, R.; Camarri, P.; Cambiaghi, M.; Cameron, D.; Campana, S.; Campanelli, M.; Canale, V.; Canelli, F.; Canepa, A.; Cantero, J.; Capasso, L.; Capeans Garrido, M. D. M.; Caprini, I.; Caprini, M.; Capriotti, D.; Capua, M.; Caputo, R.; Caramarcu, C.; Cardarelli, R.; Carli, T.; Carlino, G.; Carminati, L.; Caron, B.; Caron, S.; Carrillo Montoya, G. D.; Carter, A. A.; Carter, J. R.; Carvalho, J.; Casadei, D.; Casado, M. P.; Cascella, M.; Caso, C.; Castaneda Hernandez, A. M.; Castaneda-Miranda, E.; Castillo Gimenez, V.; Castro, N. F.; Cataldi, G.; Cataneo, F.; Catinaccio, A.; Catmore, J. R.; Cattai, A.; Cattani, G.; Caughron, S.; Cauz, D.; Cavalleri, P.; Cavalli, D.; Cavalli-Sforza, M.; Cavasinni, V.; Ceradini, F.; Cerqueira, A. S.; Cerri, A.; Cerrito, L.; Cerutti, F.; Cetin, S. A.; Cevenini, F.; Chafaq, A.; Chakraborty, D.; Chan, K.; Chapleau, B.; Chapman, J. D.; Chapman, J. W.; Chareyre, E.; Charlton, D. G.; Chavda, V.; Chavez Barajas, C. A.; Cheatham, S.; Chekanov, S.; Chekulaev, S. V.; Chelkov, G. A.; Chelstowska, M. A.; Chen, C.; Chen, H.; Chen, S.; Chen, T.; Chen, X.; Cheng, S.; Cheplakov, A.; Chepurnov, V. F.; Cherkaoui El Moursli, R.; Chernyatin, V.; Cheu, E.; Cheung, S. L.; Chevalier, L.; Chiefari, G.; Chikovani, L.; Childers, J. T.; Chilingarov, A.; Chiodini, G.; Chizhov, M. V.; Choudalakis, G.; Chouridou, S.; Christidi, I. A.; Christov, A.; Chromek-Burckhart, D.; Chu, M. L.; Chudoba, J.; Ciapetti, G.; Ciba, K.; Ciftci, A. K.; Ciftci, R.; Cinca, D.; Cindro, V.; Ciobotaru, M. D.; Ciocca, C.; Ciocio, A.; Cirilli, M.; Ciubancan, M.; Clark, A.; Clark, P. J.; Cleland, W.; Clemens, J. C.; Clement, B.; Clement, C.; Clifft, R. W.; Coadou, Y.; Cobal, M.; Coccaro, A.; Cochran, J.; Coe, P.; Cogan, J. G.; Coggeshall, J.; Cogneras, E.; Cojocaru, C. D.; Colas, J.; Colijn, A. P.; Collard, C.; Collins, N. J.; Collins-Tooth, C.; Collot, J.; Colon, G.; Conde Muiño, P.; Coniavitis, E.; Conidi, M. C.; Consonni, M.; Consorti, V.; Constantinescu, S.; Conta, C.; Conventi, F.; Cook, J.; Cooke, M.; Cooper, B. D.; Cooper-Sarkar, A. M.; Cooper-Smith, N. J.; Copic, K.; Cornelissen, T.; Corradi, M.; Corriveau, F.; Cortes-Gonzalez, A.; Cortiana, G.; Costa, G.; Costa, M. J.; Costanzo, D.; Costin, T.; Côté, D.; Coura Torres, R.; Courneyea, L.; Cowan, G.; Cowden, C.; Cox, B. E.; Cranmer, K.; Crescioli, F.; Cristinziani, M.; Crosetti, G.; Crupi, R.; Crépé-Renaudin, S.; Cuciuc, C.-M.; Cuenca Almenar, C.; Cuhadar Donszelmann, T.; Cuneo, S.; Curatolo, M.; Curtis, C. J.; Cwetanski, P.; Czirr, H.; Czyczula, Z.; D'Auria, S.; D'Onofrio, M.; D'Orazio, A.; da Silva, P. V. M.; da Via, C.; Dabrowski, W.; Dai, T.; Dallapiccola, C.; Dam, M.; Dameri, M.; Damiani, D. S.; Danielsson, H. O.; Dannheim, D.; Dao, V.; Darbo, G.; Darlea, G. L.; Daum, C.; Dauvergne, J. P.; Davey, W.; Davidek, T.; Davidson, N.; Davidson, R.; Davies, E.; Davies, M.; Davison, A. R.; Davygora, Y.; Dawe, E.; Dawson, I.; Dawson, J. W.; Daya, R. K.; de, K.; de Asmundis, R.; de Castro, S.; de Castro Faria Salgado, P. E.; de Cecco, S.; de Graat, J.; de Groot, N.; de Jong, P.; de La Taille, C.; de la Torre, H.; de Lotto, B.; de Mora, L.; de Nooij, L.; de Oliveira Branco, M.; de Pedis, D.; de Saintignon, P.; de Salvo, A.; de Sanctis, U.; de Santo, A.; de Vivie de Regie, J. B.; Dean, S.; Dedovich, D. V.; Degenhardt, J.; Dehchar, M.; Deile, M.; Del Papa, C.; Del Peso, J.; Del Prete, T.; Deliyergiyev, M.; Dell'Acqua, A.; Dell'Asta, L.; Della Pietra, M.; Della Volpe, D.; Delmastro, M.; Delpierre, P.; Delruelle, N.; Delsart, P. A.; Deluca, C.; Demers, S.; Demichev, M.; Demirkoz, B.; Deng, J.; Denisov, S. P.; Derendarz, D.; Derkaoui, J. E.; Derue, F.; Dervan, P.; Desch, K.; Devetak, E.; Deviveiros, P. O.; Dewhurst, A.; Dewilde, B.; Dhaliwal, S.; Dhullipudi, R.; di Ciaccio, A.; di Ciaccio, L.; di Girolamo, A.; di Girolamo, B.; di Luise, S.; di Mattia, A.; di Micco, B.; di Nardo, R.; di Simone, A.; di Sipio, R.; Diaz, M. A.; Diblen, F.; Diehl, E. B.; Dietrich, J.; Dietzsch, T. A.; Diglio, S.; Dindar Yagci, K.; Dingfelder, J.; Dionisi, C.; Dita, P.; Dita, S.; Dittus, F.; Djama, F.; Djobava, T.; Do Vale, M. A. B.; Do Valle Wemans, A.; Doan, T. K. O.; Dobbs, M.; Dobinson, R.; Dobos, D.; Dobson, E.; Dobson, M.; Dodd, J.; Doglioni, C.; Doherty, T.; Doi, Y.; Dolejsi, J.; Dolenc, I.; Dolezal, Z.; Dolgoshein, B. A.; Dohmae, T.; Donadelli, M.; Donega, M.; Donini, J.; Dopke, J.; Doria, A.; Dos Anjos, A.; Dosil, M.; Dotti, A.; Dova, M. T.; Dowell, J. D.; Doxiadis, A. D.; Doyle, A. T.; Drasal, Z.; Drees, J.; Dressnandt, N.; Drevermann, H.; Driouichi, C.; Dris, M.; Dubbert, J.; Dubbs, T.; Dube, S.; Duchovni, E.; Duckeck, G.; Dudarev, A.; Dudziak, F.; Dührssen, M.; Duerdoth, I. P.; Duflot, L.; Dufour, M.-A.; Dunford, M.; Duran Yildiz, H.; Duxfield, R.; Dwuznik, M.; Dydak, F.; Dzahini, D.; Düren, M.; Ebenstein, W. L.; Ebke, J.; Eckert, S.; Eckweiler, S.; Edmonds, K.; Edwards, C. A.; Edwards, N. C.; Ehrenfeld, W.; Ehrich, T.; Eifert, T.; Eigen, G.; Einsweiler, K.; Eisenhandler, E.; Ekelof, T.; El Kacimi, M.; Ellert, M.; Elles, S.; Ellinghaus, F.; Ellis, K.; Ellis, N.; Elmsheuser, J.; Elsing, M.; Ely, R.; Emeliyanov, D.; Engelmann, R.; Engl, A.; Epp, B.; Eppig, A.; Erdmann, J.; Ereditato, A.; Eriksson, D.; Ernst, J.; Ernst, M.; Ernwein, J.; Errede, D.; Errede, S.; Ertel, E.; Escalier, M.; Escobar, C.; Espinal Curull, X.; Esposito, B.; Etienne, F.; Etienvre, A. I.; Etzion, E.; Evangelakou, D.; Evans, H.; Fabbri, L.; Fabre, C.; Fakhrutdinov, R. M.; Falciano, S.; Fang, Y.; Fanti, M.; Farbin, A.; Farilla, A.; Farley, J.; Farooque, T.; Farrington, S. M.; Farthouat, P.; Fassnacht, P.; Fassouliotis, D.; Fatholahzadeh, B.; Favareto, A.; Fayard, L.; Fazio, S.; Febbraro, R.; Federic, P.; Fedin, O. L.; Fedorko, W.; Fehling-Kaschek, M.; Feligioni, L.; Fellmann, D.; Felzmann, C. U.; Feng, C.; Feng, E. J.; Fenyuk, A. B.; Ferencei, J.; Ferland, J.; Fernando, W.; Ferrag, S.; Ferrando, J.; Ferrara, V.; Ferrari, A.; Ferrari, P.; Ferrari, R.; Ferrer, A.; Ferrer, M. L.; Ferrere, D.; Ferretti, C.; Ferretto Parodi, A.; Fiascaris, M.; Fiedler, F.; Filipčič, A.; Filippas, A.; Filthaut, F.; Fincke-Keeler, M.; Fiolhais, M. C. N.; Fiorini, L.; Firan, A.; Fischer, G.; Fischer, P.; Fisher, M. J.; Fisher, S. M.; Flechl, M.; Fleck, I.; Fleckner, J.; Fleischmann, P.; Fleischmann, S.; Flick, T.; Flores Castillo, L. R.; Flowerdew, M. J.; Föhlisch, F.; Fokitis, M.; Fonseca Martin, T.; Forbush, D. A.; Formica, A.; Forti, A.; Fortin, D.; Foster, J. M.; Fournier, D.; Foussat, A.; Fowler, A. J.; Fowler, K.; Fox, H.; Francavilla, P.; Franchino, S.; Francis, D.; Frank, T.; Franklin, M.; Franz, S.; Fraternali, M.; Fratina, S.; French, S. T.; Froeschl, R.; Froidevaux, D.; Frost, J. A.; Fukunaga, C.; Fullana Torregrosa, E.; Fuster, J.; Gabaldon, C.; Gabizon, O.; Gadfort, T.; Gadomski, S.; Gagliardi, G.; Gagnon, P.; Galea, C.; Gallas, E. J.; Gallas, M. V.; Gallo, V.; Gallop, B. J.; Gallus, P.; Galyaev, E.; Gan, K. K.; Gao, Y. S.; Gapienko, V. A.; Gaponenko, A.; Garberson, F.; Garcia-Sciveres, M.; García, C.; García Navarro, J. E.; Gardner, R. W.; Garelli, N.; Garitaonandia, H.; Garonne, V.; Garvey, J.; Gatti, C.; Gaudio, G.; Gaumer, O.; Gaur, B.; Gauthier, L.; Gavrilenko, I. L.; Gay, C.; Gaycken, G.; Gayde, J.-C.; Gazis, E. N.; Ge, P.; Gee, C. N. P.; Geerts, D. A. A.; Geich-Gimbel, Ch.; Gellerstedt, K.; Gemme, C.; Gemmell, A.; Genest, M. H.; Gentile, S.; George, M.; George, S.; Gerlach, P.; Gershon, A.; Geweniger, C.; Ghazlane, H.; Ghez, P.; Ghodbane, N.; Giacobbe, B.; Giagu, S.; Giakoumopoulou, V.; Giangiobbe, V.; Gianotti, F.; Gibbard, B.; Gibson, A.; Gibson, S. M.; Gilbert, L. M.; Gilchriese, M.; Gilewsky, V.; Gillberg, D.; Gillman, A. R.; Gingrich, D. M.; Ginzburg, J.; Giokaris, N.; Giordano, R.; Giorgi, F. M.; Giovannini, P.; Giraud, P. F.; Giugni, D.; Giunta, M.; Giusti, P.; Gjelsten, B. K.; Gladilin, L. K.; Glasman, C.; Glatzer, J.; Glazov, A.; Glitza, K. W.; Glonti, G. L.; Godfrey, J.; Godlewski, J.; Goebel, M.; Göpfert, T.; Goeringer, C.; Gössling, C.; Göttfert, T.; Goldfarb, S.; Goldin, D.; Golling, T.; Golovnia, S. N.; Gomes, A.; Gomez Fajardo, L. S.; Gonçalo, R.; Goncalves Pinto Firmino da Costa, J.; Gonella, L.; Gonidec, A.; Gonzalez, S.; González de La Hoz, S.; Gonzalez Silva, M. L.; Gonzalez-Sevilla, S.; Goodson, J. J.; Goossens, L.; Gorbounov, P. A.; Gordon, H. A.; Gorelov, I.; Gorfine, G.; Gorini, B.; Gorini, E.; Gorišek, A.; Gornicki, E.; Gorokhov, S. A.; Goryachev, V. N.; Gosdzik, B.; Gosselink, M.; Gostkin, M. I.; Gouanère, M.; Gough Eschrich, I.; Gouighri, M.; Goujdami, D.; Goulette, M. P.; Goussiou, A. G.; Goy, C.; Grabowska-Bold, I.; Grabski, V.; Grafström, P.; Grah, C.; Grahn, K.-J.; Grancagnolo, F.; Grancagnolo, S.; Grassi, V.; Gratchev, V.; Grau, N.; Gray, H. M.; Gray, J. A.; Graziani, E.; Grebenyuk, O. G.; Greenfield, D.; Greenshaw, T.; Greenwood, Z. D.; Gregor, I. M.; Grenier, P.; Griffiths, J.; Grigalashvili, N.; Grillo, A. A.; Grinstein, S.; Grishkevich, Y. V.; Grivaz, J.-F.; Grognuz, J.; Groh, M.; Gross, E.; Grosse-Knetter, J.; Groth-Jensen, J.; Grybel, K.; Guarino, V. J.; Guest, D.; Guicheney, C.; Guida, A.; Guillemin, T.; Guindon, S.; Guler, H.; Gunther, J.; Guo, B.; Guo, J.; Gupta, A.; Gusakov, Y.; Gushchin, V. N.; Gutierrez, A.; Gutierrez, P.; Guttman, N.; Gutzwiller, O.; Guyot, C.; Gwenlan, C.; Gwilliam, C. B.; Haas, A.; Haas, S.; Haber, C.; Hackenburg, R.; Hadavand, H. K.; Hadley, D. R.; Haefner, P.; Hahn, F.; Haider, S.; Hajduk, Z.; Hakobyan, H.; Haller, J.; Hamacher, K.; Hamal, P.; Hamilton, A.; Hamilton, S.; Han, H.; Han, L.; Hanagaki, K.; Hance, M.; Handel, C.; Hanke, P.; Hansen, J. R.; Hansen, J. B.; Hansen, J. D.; Hansen, P. H.; Hansson, P.; Hara, K.; Hare, G. A.; Harenberg, T.; Harkusha, S.; Harper, D.; Harrington, R. D.; Harris, O. M.; Harrison, K.; Hartert, J.; Hartjes, F.; Haruyama, T.; Harvey, A.; Hasegawa, S.; Hasegawa, Y.; Hassani, S.; Hatch, M.; Hauff, D.; Haug, S.; Hauschild, M.; Hauser, R.; Havranek, M.; Hawes, B. M.; Hawkes, C. M.; Hawkings, R. J.; Hawkins, D.; Hayakawa, T.; Hayden, D.; Hayward, H. S.; Haywood, S. J.; Hazen, E.; He, M.; Head, S. J.; Hedberg, V.; Heelan, L.; Heim, S.; Heinemann, B.; Heisterkamp, S.; Helary, L.; Heller, M.; Hellman, S.; Hellmich, D.; Helsens, C.; Henderson, R. C. W.; Henke, M.; Henrichs, A.; Henriques Correia, A. M.; Henrot-Versille, S.; Henry-Couannier, F.; Hensel, C.; Henß, T.; Hernandez, C. M.; Hernández Jiménez, Y.; Herrberg, R.; Hershenhorn, A. D.; Herten, G.; Hertenberger, R.; Hervas, L.; Hessey, N. P.; Hidvegi, A.; Higón-Rodriguez, E.; Hill, D.; Hill, J. C.; Hill, N.; Hiller, K. H.; Hillert, S.; Hillier, S. J.; Hinchliffe, I.; Hines, E.; Hirose, M.; Hirsch, F.; Hirschbuehl, D.; Hobbs, J.; Hod, N.; Hodgkinson, M. C.; Hodgson, P.; Hoecker, A.; Hoeferkamp, M. R.; Hoffman, J.; Hoffmann, D.; Hohlfeld, M.; Holder, M.; Holmes, A.; Holmgren, S. O.; Holy, T.; Holzbauer, J. L.; Homma, Y.; Hong, T. M.; Hooft van Huysduynen, L.; Horazdovsky, T.; Horn, C.; Horner, S.; Horton, K.; Hostachy, J.-Y.; Hou, S.; Houlden, M. A.; Hoummada, A.; Howarth, J.; Howell, D. F.; Hristova, I.; Hrivnac, J.; Hruska, I.; Hryn'ova, T.; Hsu, P. J.; Hsu, S.-C.; Huang, G. S.; Hubacek, Z.; Hubaut, F.; Huegging, F.; Huffman, T. B.; Hughes, E. W.; Hughes, G.; Hughes-Jones, R. E.; Huhtinen, M.; Hurst, P.; Hurwitz, M.; Husemann, U.; Huseynov, N.; Huston, J.; Huth, J.; Iacobucci, G.; Iakovidis, G.; Ibbotson, M.; Ibragimov, I.; Ichimiya, R.; Iconomidou-Fayard, L.; Idarraga, J.; Idzik, M.; Iengo, P.; Igonkina, O.; Ikegami, Y.; Ikeno, M.; Ilchenko, Y.; Iliadis, D.; Imbault, D.; Imhaeuser, M.; Imori, M.; Ince, T.; Inigo-Golfin, J.; Ioannou, P.; Iodice, M.; Ionescu, G.; Irles Quiles, A.; Ishii, K.; Ishikawa, A.; Ishino, M.; Ishmukhametov, R.; Issever, C.; Istin, S.; Itoh, Y.; Ivashin, A. V.; Iwanski, W.; Iwasaki, H.; Izen, J. M.; Izzo, V.; Jackson, B.; Jackson, J. N.; Jackson, P.; Jaekel, M. R.; Jain, V.; Jakobs, K.; Jakobsen, S.; Jakubek, J.; Jana, D. K.; Jankowski, E.; Jansen, E.; Jantsch, A.; Janus, M.; Jarlskog, G.; Jeanty, L.; Jelen, K.; Jen-La Plante, I.; Jenni, P.; Jeremie, A.; Jež, P.; Jézéquel, S.; Jha, M. K.; Ji, H.; Ji, W.; Jia, J.; Jiang, Y.; Jimenez Belenguer, M.; Jin, G.; Jin, S.; Jinnouchi, O.; Joergensen, M. D.; Joffe, D.; Johansen, L. G.; Johansen, M.; Johansson, K. E.; Johansson, P.; Johnert, S.; Johns, K. A.; Jon-And, K.; Jones, G.; Jones, R. W. L.; Jones, T. W.; Jones, T. J.; Jonsson, O.; Joram, C.; Jorge, P. M.; Joseph, J.; Jovin, T.; Ju, X.; Juranek, V.; Jussel, P.; Kabachenko, V. V.; Kabana, S.; Kaci, M.; Kaczmarska, A.; Kadlecik, P.; Kado, M.; Kagan, H.; Kagan, M.; Kaiser, S.; Kajomovitz, E.; Kalinin, S.; Kalinovskaya, L. V.; Kama, S.; Kanaya, N.; Kaneda, M.; Kanno, T.; Kantserov, V. A.; Kanzaki, J.; Kaplan, B.; Kapliy, A.; Kaplon, J.; Kar, D.; Karagoz, M.; Karnevskiy, M.; Karr, K.; Kartvelishvili, V.; Karyukhin, A. N.; Kashif, L.; Kasmi, A.; Kass, R. D.; Kastanas, A.; Kataoka, M.; Kataoka, Y.; Katsoufis, E.; Katzy, J.; Kaushik, V.; Kawagoe, K.; Kawamoto, T.; Kawamura, G.; Kayl, M. S.; Kazanin, V. A.; Kazarinov, M. Y.; Keates, J. R.; Keeler, R.; Kehoe, R.; Keil, M.; Kekelidze, G. D.; Kelly, M.; Kennedy, J.; Kenney, C. J.; Kenyon, M.; Kepka, O.; Kerschen, N.; Kerševan, B. P.; Kersten, S.; Kessoku, K.; Ketterer, C.; Keung, J.; Khakzad, M.; Khalil-Zada, F.; Khandanyan, H.; Khanov, A.; Kharchenko, D.; Khodinov, A.; Kholodenko, A. G.; Khomich, A.; Khoo, T. J.; Khoriauli, G.; Khoroshilov, A.; Khovanskiy, N.; Khovanskiy, V.; Khramov, E.; Khubua, J.; Kim, H.; Kim, M. S.; Kim, P. C.; Kim, S. H.; Kimura, N.; Kind, O.; King, B. T.; King, M.; King, R. S. B.; Kirk, J.; Kirsch, G. P.; Kirsch, L. E.; Kiryunin, A. E.; Kisielewska, D.; Kittelmann, T.; Kiver, A. M.; Kiyamura, H.; Kladiva, E.; Klaiber-Lodewigs, J.; Klein, M.; Klein, U.; Kleinknecht, K.; Klemetti, M.; Klier, A.; Klimentov, A.; Klingenberg, R.; Klinkby, E. B.; Klioutchnikova, T.; Klok, P. F.; Klous, S.; Kluge, E.-E.; Kluge, T.; Kluit, P.; Kluth, S.; Kneringer, E.; Knobloch, J.; Knoops, E. B. F. G.; Knue, A.; Ko, B. R.; Kobayashi, T.; Kobel, M.; Kocian, M.; Kocnar, A.; Kodys, P.; Köneke, K.; König, A. C.; Koenig, S.; Köpke, L.; Koetsveld, F.; Koevesarki, P.; Koffas, T.; Koffeman, E.; Kohn, F.; Kohout, Z.; Kohriki, T.; Koi, T.; Kokott, T.; Kolachev, G. M.; Kolanoski, H.; Kolesnikov, V.; Koletsou, I.; Koll, J.; Kollar, D.; Kollefrath, M.; Kolya, S. D.; Komar, A. A.; Komaragiri, J. R.; Komori, Y.; Kondo, T.; Kono, T.; Kononov, A. I.; Konoplich, R.; Konstantinidis, N.; Kootz, A.; Koperny, S.; Kopikov, S. V.; Korcyl, K.; Kordas, K.; Koreshev, V.; Korn, A.; Korol, A.; Korolkov, I.; Korolkova, E. V.; Korotkov, V. A.; Kortner, O.; Kortner, S.; Kostyukhin, V. V.; Kotamäki, M. J.; Kotov, S.; Kotov, V. M.; Kotwal, A.; Kourkoumelis, C.; Kouskoura, V.; Koutsman, A.; Kowalewski, R.; Kowalski, T. Z.; Kozanecki, W.; Kozhin, A. S.; Kral, V.; Kramarenko, V. A.; Kramberger, G.; Krasel, O.; Krasny, M. W.; Krasznahorkay, A.; Kraus, J.; Kreisel, A.; Krejci, F.; Kretzschmar, J.; Krieger, N.; Krieger, P.; Kroeninger, K.; Kroha, H.; Kroll, J.; Kroseberg, J.; Krstic, J.; Kruchonak, U.; Krüger, H.; Kruker, T.; Krumshteyn, Z. V.; Kruth, A.; Kubota, T.; Kuehn, S.; Kugel, A.; Kuhl, T.; Kuhn, D.; Kukhtin, V.; Kulchitsky, Y.; Kuleshov, S.; Kummer, C.; Kuna, M.; Kundu, N.; Kunkle, J.; Kupco, A.; Kurashige, H.; Kurata, M.; Kurochkin, Y. A.; Kus, V.; Kuykendall, W.; Kuze, M.; Kuzhir, P.; Kvasnicka, O.; Kvita, J.; Kwee, R.; La Rosa, A.; La Rotonda, L.; Labarga, L.; Labbe, J.; Lablak, S.; Lacasta, C.; Lacava, F.; Lacker, H.; Lacour, D.; Lacuesta, V. R.; Ladygin, E.; Lafaye, R.; Laforge, B.; Lagouri, T.; Lai, S.; Laisne, E.; Lamanna, M.; Lampen, C. L.; Lampl, W.; Lancon, E.; Landgraf, U.; Landon, M. P. J.; Landsman, H.; Lane, J. L.; Lange, C.; Lankford, A. J.; Lanni, F.; Lantzsch, K.; Laplace, S.; Lapoire, C.; Laporte, J. F.; Lari, T.; Larionov, A. V.; Larner, A.; Lasseur, C.; Lassnig, M.; Lau, W.; Laurelli, P.; Lavorato, A.; Lavrijsen, W.; Laycock, P.; Lazarev, A. B.; Lazzaro, A.; Le Dortz, O.; Le Guirriec, E.; Le Maner, C.; Le Menedeu, E.; Lebel, C.; Lecompte, T.; Ledroit-Guillon, F.; Lee, H.; Lee, J. S. H.; Lee, S. C.; Lee, L.; Lefebvre, M.; Legendre, M.; Leger, A.; Legeyt, B. C.; Legger, F.; Leggett, C.; Lehmacher, M.; Lehmann Miotto, G.; Lei, X.; Leite, M. A. L.; Leitner, R.; Lellouch, D.; Lellouch, J.; Leltchouk, M.; Lendermann, V.; Leney, K. J. C.; Lenz, T.; Lenzen, G.; Lenzi, B.; Leonhardt, K.; Leontsinis, S.; Leroy, C.; Lessard, J.-R.; Lesser, J.; Lester, C. G.; Leung Fook Cheong, A.; Levêque, J.; Levin, D.; Levinson, L. J.; Levitski, M. S.; Lewandowska, M.; Lewis, A.; Lewis, G. H.; Leyko, A. M.; Leyton, M.; Li, B.; Li, H.; Li, S.; Li, X.; Liang, Z.; Liang, Z.; Liberti, B.; Lichard, P.; Lichtnecker, M.; Lie, K.; Liebig, W.; Lifshitz, R.; Lilley, J. N.; Limbach, C.; Limosani, A.; Limper, M.; Lin, S. C.; Linde, F.; Linnemann, J. T.; Lipeles, E.; Lipinsky, L.; Lipniacka, A.; Liss, T. M.; Lissauer, D.; Lister, A.; Litke, A. M.; Liu, C.; Liu, D.; Liu, H.; Liu, J. B.; Liu, M.; Liu, S.; Liu, Y.; Livan, M.; Livermore, S. S. A.; Lleres, A.; Llorente Merino, J.; Lloyd, S. L.; Lobodzinska, E.; Loch, P.; Lockman, W. S.; Lockwitz, S.; Loddenkoetter, T.; Loebinger, F. K.; Loginov, A.; Loh, C. W.; Lohse, T.; Lohwasser, K.; Lokajicek, M.; Loken, J.; Lombardo, V. P.; Long, R. E.; Lopes, L.; Lopez Mateos, D.; Losada, M.; Loscutoff, P.; Lo Sterzo, F.; Losty, M. J.; Lou, X.; Lounis, A.; Loureiro, K. F.; Love, J.; Love, P. A.; Lowe, A. J.; Lu, F.; Lubatti, H. J.; Luci, C.; Lucotte, A.; Ludwig, A.; Ludwig, D.; Ludwig, I.; Ludwig, J.; Luehring, F.; Luijckx, G.; Lumb, D.; Luminari, L.; Lund, E.; Lund-Jensen, B.; Lundberg, B.; Lundberg, J.; Lundquist, J.; Lungwitz, M.; Lupi, A.; Lutz, G.; Lynn, D.; Lys, J.; Lytken, E.; Ma, H.; Ma, L. L.; Macana Goia, J. A.; Maccarrone, G.; Macchiolo, A.; Maček, B.; Machado Miguens, J.; Macina, D.; Mackeprang, R.; Madaras, R. J.; Mader, W. F.; Maenner, R.; Maeno, T.; Mättig, P.; Mättig, S.; Magalhaes Martins, P. J.; Magnoni, L.; Magradze, E.; Mahalalel, Y.; Mahboubi, K.; Mahout, G.; Maiani, C.; Maidantchik, C.; Maio, A.; Majewski, S.; Makida, Y.; Makovec, N.; Mal, P.; Malecki, Pa.; Malecki, P.; Maleev, V. P.; Malek, F.; Mallik, U.; Malon, D.; Maltezos, S.; Malyshev, V.; Malyukov, S.; Mameghani, R.; Mamuzic, J.; Manabe, A.; Mandelli, L.; Mandić, I.; Mandrysch, R.; Maneira, J.; Mangeard, P. S.; Manjavidze, I. D.; Mann, A.; Manning, P. M.; Manousakis-Katsikakis, A.; Mansoulie, B.; Manz, A.; Mapelli, A.; Mapelli, L.; March, L.; Marchand, J. F.; Marchese, F.; Marchiori, G.; Marcisovsky, M.; Marin, A.; Marino, C. P.; Marroquim, F.; Marshall, R.; Marshall, Z.; Martens, F. K.; Marti-Garcia, S.; Martin, A. J.; Martin, B.; Martin, B.; Martin, F. F.; Martin, J. P.; Martin, Ph.; Martin, T. A.; Martin Dit Latour, B.; Martinez, M.; Martinez Outschoorn, V.; Martyniuk, A. C.; Marx, M.; Marzano, F.; Marzin, A.; Masetti, L.; Mashimo, T.; Mashinistov, R.; Masik, J.; Maslennikov, A. L.; Maß, M.; Massa, I.; Massaro, G.; Massol, N.; Mastrandrea, P.; Mastroberardino, A.; Masubuchi, T.; Mathes, M.; Matricon, P.; Matsumoto, H.; Matsunaga, H.; Matsushita, T.; Mattravers, C.; Maugain, J. M.; Maxfield, S. J.; Maximov, D. A.; May, E. N.; Mayne, A.; Mazini, R.; Mazur, M.; Mazzanti, M.; Mazzoni, E.; Mc Kee, S. P.; McCarn, A.; McCarthy, R. L.; McCarthy, T. G.; McCubbin, N. A.; McFarlane, K. W.; McFayden, J. A.; McGlone, H.; McHedlidze, G.; McLaren, R. A.; McLaughlan, T.; McMahon, S. J.; McPherson, R. A.; Meade, A.; Mechnich, J.; Mechtel, M.; Medinnis, M.; Meera-Lebbai, R.; Meguro, T.; Mehdiyev, R.; Mehlhase, S.; Mehta, A.; Meier, K.; Meinhardt, J.; Meirose, B.; Melachrinos, C.; Mellado Garcia, B. R.; Mendoza Navas, L.; Meng, Z.; Mengarelli, A.; Menke, S.; Menot, C.; Meoni, E.; Mercurio, K. M.; Mermod, P.; Merola, L.; Meroni, C.; Merritt, F. S.; Messina, A.; Metcalfe, J.; Mete, A. S.; Meuser, S.; Meyer, C.; Meyer, J.-P.; Meyer, J.; Meyer, J.; Meyer, T. C.; Meyer, W. T.; Miao, J.; Michal, S.; Micu, L.; Middleton, R. P.; Miele, P.; Migas, S.; Mijović, L.; Mikenberg, G.; Mikestikova, M.; Mikuž, M.; Miller, D. W.; Miller, R. J.; Mills, W. J.; Mills, C.; Milov, A.; Milstead, D. A.; Milstein, D.; Minaenko, A. A.; Miñano, M.; Minashvili, I. A.; Mincer, A. I.; Mindur, B.; Mineev, M.; Ming, Y.; Mir, L. M.; Mirabelli, G.; Miralles Verge, L.; Misiejuk, A.; Mitrevski, J.; Mitrofanov, G. Y.; Mitsou, V. A.; Mitsui, S.; Miyagawa, P. S.; Miyazaki, K.; Mjörnmark, J. U.; Moa, T.; Mockett, P.; Moed, S.; Moeller, V.; Mönig, K.; Möser, N.; Mohapatra, S.; Mohn, B.; Mohr, W.; Mohrdieck-Möck, S.; Moisseev, A. M.; Moles-Valls, R.; Molina-Perez, J.; Monk, J.; Monnier, E.; Montesano, S.; Monticelli, F.; Monzani, S.; Moore, R. W.; Moorhead, G. F.; Mora Herrera, C.; Moraes, A.; Morais, A.; Morange, N.; Morel, J.; Morello, G.; Moreno, D.; Moreno Llácer, M.; Morettini, P.; Morii, M.; Morin, J.; Morita, Y.; Morley, A. K.; Mornacchi, G.; Morone, M.-C.; Morozov, S. V.; Morris, J. D.; Morvaj, L.; Moser, H. G.; Mosidze, M.; Moss, J.; Mount, R.; Mountricha, E.; Mouraviev, S. V.; Moyse, E. J. W.; Mudrinic, M.; Mueller, F.; Mueller, J.; Mueller, K.; Müller, T. A.; Muenstermann, D.; Muijs, A.; Muir, A.; Munwes, Y.; Murakami, K.; Murray, W. J.; Mussche, I.; Musto, E.; Myagkov, A. G.; Myska, M.; Nadal, J.; Nagai, K.; Nagano, K.; Nagasaka, Y.; Nairz, A. M.; Nakahama, Y.; Nakamura, K.; Nakano, I.; Nanava, G.; Napier, A.; Nash, M.; Nation, N. R.; Nattermann, T.; Naumann, T.; Navarro, G.; Neal, H. A.; Nebot, E.; Nechaeva, P. Yu.; Negri, A.; Negri, G.; Nektarijevic, S.; Nelson, S.; Nelson, T. K.; Nemecek, S.; Nemethy, P.; Nepomuceno, A. A.; Nessi, M.; Nesterov, S. Y.; Neubauer, M. S.; Neusiedl, A.; Neves, R. M.; Nevski, P.; Newman, P. R.; Nguyen Thi Hong, V.; Nickerson, R. B.; Nicolaidou, R.; Nicolas, L.; Nicquevert, B.; Niedercorn, F.; Nielsen, J.; Niinikoski, T.; Nikiforov, A.; Nikolaenko, V.; Nikolaev, K.; Nikolic-Audit, I.; Nikolics, K.; Nikolopoulos, K.; Nilsen, H.; Nilsson, P.; Ninomiya, Y.; Nisati, A.; Nishiyama, T.; Nisius, R.; Nodulman, L.; Nomachi, M.; Nomidis, I.; Nordberg, M.; Nordkvist, B.; Norton, P. R.; Novakova, J.; Nozaki, M.; Nožička, M.; Nozka, L.; Nugent, I. M.; Nuncio-Quiroz, A.-E.; Nunes Hanninger, G.; Nunnemann, T.; Nurse, E.; Nyman, T.; O'Brien, B. J.; O'Neale, S. W.; O'Neil, D. C.; O'Shea, V.; Oakham, F. G.; Oberlack, H.; Ocariz, J.; Ochi, A.; Oda, S.; Odaka, S.; Odier, J.; Ogren, H.; Oh, A.; Oh, S. H.; Ohm, C. C.; Ohshima, T.; Ohshita, H.; Ohska, T. K.; Ohsugi, T.; Okada, S.; Okawa, H.; Okumura, Y.; Okuyama, T.; Olcese, M.; Olchevski, A. G.; Oliveira, M.; Oliveira Damazio, D.; Oliver Garcia, E.; Olivito, D.; Olszewski, A.; Olszowska, J.; Omachi, C.; Onofre, A.; Onyisi, P. U. E.; Oram, C. J.; Oreglia, M. J.; Oren, Y.; Orestano, D.; Orlov, I.; Oropeza Barrera, C.; Orr, R. S.; Osculati, B.; Ospanov, R.; Osuna, C.; Otero Y Garzon, G.; P Ottersbach, J.; Ouchrif, M.; Ould-Saada, F.; Ouraou, A.; Ouyang, Q.; Owen, M.; Owen, S.; Øye, O. K.; Ozcan, V. E.; Ozturk, N.; Pacheco Pages, A.; Padilla Aranda, C.; Pagan Griso, S.; Paganis, E.; Paige, F.; Pajchel, K.; Palestini, S.; Pallin, D.; Palma, A.; Palmer, J. D.; Pan, Y. B.; Panagiotopoulou, E.; Panes, B.; Panikashvili, N.; Panitkin, S.; Pantea, D.; Panuskova, M.; Paolone, V.; Papadelis, A.; Papadopoulou, Th. D.; Paramonov, A.; Park, W.; Parker, M. A.; Parodi, F.; Parsons, J. A.; Parzefall, U.; Pasqualucci, E.; Passeri, A.; Pastore, F.; Pastore, Fr.; Pásztor, G.; Pataraia, S.; Patel, N.; Pater, J. R.; Patricelli, S.; Pauly, T.; Pecsy, M.; Pedraza Morales, M. I.; Peleganchuk, S. V.; Peng, H.; Pengo, R.; Penson, A.; Penwell, J.; Perantoni, M.; Perez, K.; Perez Cavalcanti, T.; Perez Codina, E.; Pérez García-Estañ, M. T.; Perez Reale, V.; Perini, L.; Pernegger, H.; Perrino, R.; Perrodo, P.; Persembe, S.; Peshekhonov, V. D.; Peters, O.; Petersen, B. A.; Petersen, J.; Petersen, T. C.; Petit, E.; Petridis, A.; Petridou, C.; Petrolo, E.; Petrucci, F.; Petschull, D.; Petteni, M.; Pezoa, R.; Phan, A.; Phillips, A. W.; Phillips, P. W.; Piacquadio, G.; Piccaro, E.; Piccinini, M.; Pickford, A.; Piec, S. M.; Piegaia, R.; Pilcher, J. E.; Pilkington, A. D.; Pina, J.; Pinamonti, M.; Pinder, A.; Pinfold, J. L.; Ping, J.; Pinto, B.; Pirotte, O.; Pizio, C.; Placakyte, R.; Plamondon, M.; Plano, W. G.; Pleier, M.-A.; Pleskach, A. V.; Poblaguev, A.; Poddar, S.; Podlyski, F.; Poggioli, L.; Poghosyan, T.; Pohl, M.; Polci, F.; Polesello, G.; Policicchio, A.; Polini, A.; Poll, J.; Polychronakos, V.; Pomarede, D. M.; Pomeroy, D.; Pommès, K.; Pontecorvo, L.; Pope, B. G.; Popeneciu, G. A.; Popovic, D. S.; Poppleton, A.; Portell Bueso, X.; Porter, R.; Posch, C.; Pospelov, G. E.; Pospisil, S.; Potrap, I. N.; Potter, C. J.; Potter, C. T.; Poulard, G.; Poveda, J.; Prabhu, R.; Pralavorio, P.; Prasad, S.; Pravahan, R.; Prell, S.; Pretzl, K.; Pribyl, L.; Price, D.; Price, L. E.; Price, M. J.; Prichard, P. M.; Prieur, D.; Primavera, M.; Prokofiev, K.; Prokoshin, F.; Protopopescu, S.; Proudfoot, J.; Prudent, X.; Przysiezniak, H.; Psoroulas, S.; Ptacek, E.; Purdham, J.; Purohit, M.; Puzo, P.; Pylypchenko, Y.; Qian, J.; Qian, Z.; Qin, Z.; Quadt, A.; Quarrie, D. R.; Quayle, W. B.; Quinonez, F.; Raas, M.; Radescu, V.; Radics, B.; Rador, T.; Ragusa, F.; Rahal, G.; Rahimi, A. M.; Rahm, D.; Rajagopalan, S.; Rammensee, M.; Rammes, M.; Ramstedt, M.; Randrianarivony, K.; Ratoff, P. N.; Rauscher, F.; Rauter, E.; Raymond, M.; Read, A. L.; Rebuzzi, D. M.; Redelbach, A.; Redlinger, G.; Reece, R.; Reeves, K.; Reichold, A.; Reinherz-Aronis, E.; Reinsch, A.; Reisinger, I.; Reljic, D.; Rembser, C.; Ren, Z. L.; Renaud, A.; Renkel, P.; Rescigno, M.; Resconi, S.; Resende, B.; Reznicek, P.; Rezvani, R.; Richards, A.; Richter, R.; Richter-Was, E.; Ridel, M.; Rieke, S.; Rijpstra, M.; Rijssenbeek, M.; Rimoldi, A.; Rinaldi, L.; Rios, R. R.; Riu, I.; Rivoltella, G.; Rizatdinova, F.; Rizvi, E.; Robertson, S. H.; Robichaud-Veronneau, A.; Robinson, D.; Robinson, J. E. M.; Robinson, M.; Robson, A.; Rocha de Lima, J. G.; Roda, C.; Roda Dos Santos, D.; Rodier, S.; Rodriguez, D.; Roe, A.; Roe, S.; Røhne, O.; Rojo, V.; Rolli, S.; Romaniouk, A.; Romanov, V. M.; Romeo, G.; Romero Maltrana, D.; Roos, L.; Ros, E.; Rosati, S.; Rosbach, K.; Rose, M.; Rosenbaum, G. A.; Rosenberg, E. I.; Rosendahl, P. L.; Rosselet, L.; Rossetti, V.; Rossi, E.; Rossi, L. P.; Rossi, L.; Rotaru, M.; Roth, I.; Rothberg, J.; Rousseau, D.; Royon, C. R.; Rozanov, A.; Rozen, Y.; Ruan, X.; Rubinskiy, I.; Ruckert, B.; Ruckstuhl, N.; Rud, V. I.; Rudolph, C.; Rudolph, G.; Rühr, F.; Ruggieri, F.; Ruiz-Martinez, A.; Rulikowska-Zarebska, E.; Rumiantsev, V.; Rumyantsev, L.; Runge, K.; Runolfsson, O.; Rurikova, Z.; Rusakovich, N. A.; Rust, D. R.; Rutherfoord, J. P.; Ruwiedel, C.; Ruzicka, P.; Ryabov, Y. F.; Ryadovikov, V.; Ryan, P.; Rybar, M.; Rybkin, G.; Ryder, N. C.; Rzaeva, S.; Saavedra, A. F.; Sadeh, I.; Sadrozinski, H. F.-W.; Sadykov, R.; Safai Tehrani, F.; Sakamoto, H.; Salamanna, G.; Salamon, A.; Saleem, M.; Salihagic, D.; Salnikov, A.; Salt, J.; Salvachua Ferrando, B. M.; Salvatore, D.; Salvatore, F.; Salvucci, A.; Salzburger, A.; Sampsonidis, D.; Samset, B. H.; Sanchez, A.; Sandaker, H.; Sander, H. G.; Sanders, M. P.; Sandhoff, M.; Sandoval, T.; Sandstroem, R.; Sandvoss, S.; Sankey, D. P. C.; Sansoni, A.; Santamarina Rios, C.; Santoni, C.; Santonico, R.; Santos, H.; Saraiva, J. G.; Sarangi, T.; Sarkisyan-Grinbaum, E.; Sarri, F.; Sartisohn, G.; Sasaki, O.; Sasaki, T.; Sasao, N.; Satsounkevitch, I.; Sauvage, G.; Sauvan, E.; Sauvan, J. B.; Savard, P.; Savinov, V.; Savu, D. O.; Savva, P.; Sawyer, L.; Saxon, D. H.; Says, L. P.; Sbarra, C.; Sbrizzi, A.; Scallon, O.; Scannicchio, D. A.; Schaarschmidt, J.; Schacht, P.; Schäfer, U.; Schaepe, S.; Schaetzel, S.; Schaffer, A. C.; Schaile, D.; Schamberger, R. D.; Schamov, A. G.; Scharf, V.; Schegelsky, V. A.; Scheirich, D.; Schernau, M.; Scherzer, M. I.; Schiavi, C.; Schieck, J.; Schioppa, M.; Schlenker, S.; Schlereth, J. L.; Schmidt, E.; Schmieden, K.; Schmitt, C.; Schmitt, S.; Schmitz, M.; Schöning, A.; Schott, M.; Schouten, D.; Schovancova, J.; Schram, M.; Schroeder, C.; Schroer, N.; Schuh, S.; Schuler, G.; Schultes, J.; Schultz-Coulon, H.-C.; Schulz, H.; Schumacher, J. W.; Schumacher, M.; Schumm, B. A.; Schune, Ph.; Schwanenberger, C.; Schwartzman, A.; Schwemling, Ph.; Schwienhorst, R.; Schwierz, R.; Schwindling, J.; Scott, W. G.; Searcy, J.; Sedykh, E.; Segura, E.; Seidel, S. C.; Seiden, A.; Seifert, F.; Seixas, J. M.; Sekhniaidze, G.; Seliverstov, D. M.; Sellden, B.; Sellers, G.; Seman, M.; Semprini-Cesari, N.; Serfon, C.; Serin, L.; Seuster, R.; Severini, H.; Sevior, M. E.; Sfyrla, A.; Shabalina, E.; Shamim, M.; Shan, L. Y.; Shank, J. T.; Shao, Q. T.; Shapiro, M.; Shatalov, P. B.; Shaver, L.; Shaw, C.; Shaw, K.; Sherman, D.; Sherwood, P.; Shibata, A.; Shichi, H.; Shimizu, S.; Shimojima, M.; Shin, T.; Shmeleva, A.; Shochet, M. J.; Short, D.; Shupe, M. A.; Sicho, P.; Sidoti, A.; Siebel, A.; Siegert, F.; Siegrist, J.; Sijacki, Dj.; Silbert, O.; Silva, J.; Silver, Y.; Silverstein, D.; Silverstein, S. B.; Simak, V.; Simard, O.; Simic, Lj.; Simion, S.; Simmons, B.; Simonyan, M.; Sinervo, P.; Sinev, N. B.; Sipica, V.; Siragusa, G.; Sisakyan, A. N.; Sivoklokov, S. Yu.; Sjölin, J.; Sjursen, T. B.; Skinnari, L. A.; Skovpen, K.; Skubic, P.; Skvorodnev, N.; Slater, M.; Slavicek, T.; Sliwa, K.; Sloan, T. J.; Sloper, J.; Smakhtin, V.; Smirnov, S. Yu.; Smirnova, L. N.; Smirnova, O.; Smith, B. C.; Smith, D.; Smith, K. M.; Smizanska, M.; Smolek, K.; Snesarev, A. A.; Snow, S. W.; Snow, J.; Snuverink, J.; Snyder, S.; Soares, M.; Sobie, R.; Sodomka, J.; Soffer, A.; Solans, C. A.; Solar, M.; Solc, J.; Soldatov, E.; Soldevila, U.; Solfaroli Camillocci, E.; Solodkov, A. A.; Solovyanov, O. V.; Sondericker, J.; Soni, N.; Sopko, V.; Sopko, B.; Sorbi, M.; Sosebee, M.; Soukharev, A.; Spagnolo, S.; Spanò, F.; Spighi, R.; Spigo, G.; Spila, F.; Spiriti, E.; Spiwoks, R.; Spousta, M.; Spreitzer, T.; Spurlock, B.; St. Denis, R. D.; Stahl, T.; Stahlman, J.; Stamen, R.; Stanecka, E.; Stanek, R. W.; Stanescu, C.; Stapnes, S.; Starchenko, E. A.; Stark, J.; Staroba, P.; Starovoitov, P.; Staude, A.; Stavina, P.; Stavropoulos, G.; Steele, G.; Steinbach, P.; Steinberg, P.; Stekl, I.; Stelzer, B.; Stelzer, H. J.; Stelzer-Chilton, O.; Stenzel, H.; Stevenson, K.; Stewart, G. A.; Stillings, J. A.; Stockmanns, T.; Stockton, M. C.; Stoerig, K.; Stoicea, G.; Stonjek, S.; Strachota, P.; Stradling, A. R.; Straessner, A.; Strandberg, J.; Strandberg, S.; Strandlie, A.; Strang, M.; Strauss, E.; Strauss, M.; Strizenec, P.; Ströhmer, R.; Strom, D. M.; Strong, J. A.; Stroynowski, R.; Strube, J.; Stugu, B.; Stumer, I.; Stupak, J.; Sturm, P.; Soh, D. A.; Su, D.; Subramania, H. S.; Succurro, A.; Sugaya, Y.; Sugimoto, T.; Suhr, C.; Suita, K.; Suk, M.; Sulin, V. V.; Sultansoy, S.; Sumida, T.; Sun, X.; Sundermann, J. E.; Suruliz, K.; Sushkov, S.; Susinno, G.; Sutton, M. R.; Suzuki, Y.; Svatos, M.; Sviridov, Yu. M.; Swedish, S.; Sykora, I.; Sykora, T.; Szeless, B.; Sánchez, J.; Ta, D.; Tackmann, K.; Taffard, A.; Tafirout, R.; Taga, A.; Taiblum, N.; Takahashi, Y.; Takai, H.; Takashima, R.; Takeda, H.; Takeshita, T.; Talby, M.; Talyshev, A.; Tamsett, M. C.; Tanaka, J.; Tanaka, R.; Tanaka, S.; Tanaka, S.; Tanaka, Y.; Tani, K.; Tannoury, N.; Tappern, G. P.; Tapprogge, S.; Tardif, D.; Tarem, S.; Tarrade, F.; Tartarelli, G. F.; Tas, P.; Tasevsky, M.; Tassi, E.; Tatarkhanov, M.; Taylor, C.; Taylor, F. E.; Taylor, G. N.; Taylor, W.; Teixeira Dias Castanheira, M.; Teixeira-Dias, P.; Temming, K. K.; Ten Kate, H.; Teng, P. K.; Terada, S.; Terashi, K.; Terron, J.; Terwort, M.; Testa, M.; Teuscher, R. J.; Thadome, J.; Therhaag, J.; Theveneaux-Pelzer, T.; Thioye, M.; Thoma, S.; Thomas, J. P.; Thompson, E. N.; Thompson, P. D.; Thompson, P. D.; Thompson, A. S.; Thomson, E.; Thomson, M.; Thun, R. P.; Tic, T.; Tikhomirov, V. O.; Tikhonov, Y. A.; Timmermans, C. J. W. P.; Tipton, P.; Tique Aires Viegas, F. J.; Tisserant, S.; Tobias, J.; Toczek, B.; Todorov, T.; Todorova-Nova, S.; Toggerson, B.; Tojo, J.; Tokár, S.; Tokunaga, K.; Tokushuku, K.; Tollefson, K.; Tomoto, M.; Tompkins, L.; Toms, K.; Tong, G.; Tonoyan, A.; Topfel, C.; Topilin, N. D.; Torchiani, I.; Torrence, E.; Torres, H.; Torró Pastor, E.; Toth, J.; Touchard, F.; Tovey, D. R.; Traynor, D.; Trefzger, T.; Tremblet, L.; Tricoli, A.; Trigger, I. M.; Trincaz-Duvoid, S.; Trinh, T. N.; Tripiana, M. F.; Trischuk, W.; Trivedi, A.; Trocmé, B.; Troncon, C.; Trottier-McDonald, M.; Trzupek, A.; Tsarouchas, C.; Tseng, J. C.-L.; Tsiakiris, M.; Tsiareshka, P. V.; Tsionou, D.; Tsipolitis, G.; Tsiskaridze, V.; Tskhadadze, E. G.; Tsukerman, I. I.; Tsulaia, V.; Tsung, J.-W.; Tsuno, S.; Tsybychev, D.; Tua, A.; Tuggle, J. M.; Turala, M.; Turecek, D.; Turk Cakir, I.; Turlay, E.; Turra, R.; Tuts, P. M.; Tykhonov, A.; Tylmad, M.; Tyndel, M.; Tyrvainen, H.; Tzanakos, G.; Uchida, K.; Ueda, I.; Ueno, R.; Ugland, M.; Uhlenbrock, M.; Uhrmacher, M.; Ukegawa, F.; Unal, G.; Underwood, D. G.; Undrus, A.; Unel, G.; Unno, Y.; Urbaniec, D.; Urkovsky, E.; Urrejola, P.; Usai, G.; Uslenghi, M.; Vacavant, L.; Vacek, V.; Vachon, B.; Vahsen, S.; Valenta, J.; Valente, P.; Valentinetti, S.; Valkar, S.; Valladolid Gallego, E.; Vallecorsa, S.; Valls Ferrer, J. A.; van der Graaf, H.; van der Kraaij, E.; van der Leeuw, R.; van der Poel, E.; van der Ster, D.; van Eijk, B.; van Eldik, N.; van Gemmeren, P.; van Kesteren, Z.; van Vulpen, I.; Vandelli, W.; Vandoni, G.; Vaniachine, A.; Vankov, P.; Vannucci, F.; Varela Rodriguez, F.; Vari, R.; Varnes, E. W.; Varouchas, D.; Vartapetian, A.; Varvell, K. E.; Vassilakopoulos, V. I.; Vazeille, F.; Vegni, G.; Veillet, J. J.; Vellidis, C.; Veloso, F.; Veness, R.; Veneziano, S.; Ventura, A.; Ventura, D.; Venturi, M.; Venturi, N.; Vercesi, V.; Verducci, M.; Verkerke, W.; Vermeulen, J. C.; Vest, A.; Vetterli, M. C.; Vichou, I.; Vickey, T.; Viehhauser, G. H. A.; Viel, S.; Villa, M.; Villaplana Perez, M.; Vilucchi, E.; Vincter, M. G.; Vinek, E.; Vinogradov, V. B.; Virchaux, M.; Virzi, J.; Vitells, O.; Viti, M.; Vivarelli, I.; Vives Vaque, F.; Vlachos, S.; Vlasak, M.; Vlasov, N.; Vogel, A.; Vokac, P.; Volpi, G.; Volpi, M.; Volpini, G.; von der Schmitt, H.; von Loeben, J.; von Radziewski, H.; von Toerne, E.; Vorobel, V.; Vorobiev, A. P.; Vorwerk, V.; Vos, M.; Voss, R.; Voss, T. T.; Vossebeld, J. H.; Vranjes, N.; Vranjes Milosavljevic, M.; Vrba, V.; Vreeswijk, M.; Vu Anh, T.; Vuillermet, R.; Vukotic, I.; Wagner, W.; Wagner, P.; Wahlen, H.; Wakabayashi, J.; Walbersloh, J.; Walch, S.; Walder, J.; Walker, R.; Walkowiak, W.; Wall, R.; Waller, P.; Wang, C.; Wang, H.; Wang, H.; Wang, J.; Wang, J.; Wang, J. C.; Wang, R.; Wang, S. M.; Warburton, A.; Ward, C. P.; Warsinsky, M.; Watkins, P. M.; Watson, A. T.; Watson, M. F.; Watts, G.; Watts, S.; Waugh, A. T.; Waugh, B. M.; Weber, J.; Weber, M.; Weber, M. S.; Weber, P.; Weidberg, A. R.; Weigell, P.; Weingarten, J.; Weiser, C.; Wellenstein, H.; Wells, P. S.; Wen, M.; Wenaus, T.; Wendler, S.; Weng, Z.; Wengler, T.; Wenig, S.; Wermes, N.; Werner, M.; Werner, P.; Werth, M.; Wessels, M.; Weydert, C.; Whalen, K.; Wheeler-Ellis, S. J.; Whitaker, S. P.; White, A.; White, M. J.; White, S.; Whitehead, S. R.; Whiteson, D.; Whittington, D.; Wicek, F.; Wicke, D.; Wickens, F. J.; Wiedenmann, W.; Wielers, M.; Wienemann, P.; Wiglesworth, C.; Wiik, L. A. M.; Wijeratne, P. A.; Wildauer, A.; Wildt, M. A.; Wilhelm, I.; Wilkens, H. G.; Will, J. Z.; Williams, E.; Williams, H. H.; Willis, W.; Willocq, S.; Wilson, J. A.; Wilson, M. G.; Wilson, A.; Wingerter-Seez, I.; Winkelmann, S.; Winklmeier, F.; Wittgen, M.; Wolter, M. W.; Wolters, H.; Wooden, G.; Wosiek, B. K.; Wotschack, J.; Woudstra, M. J.; Wraight, K.; Wright, C.; Wrona, B.; Wu, S. L.; Wu, X.; Wu, Y.; Wulf, E.; Wunstorf, R.; Wynne, B. M.; Xaplanteris, L.; Xella, S.; Xie, S.; Xie, Y.; Xu, C.; Xu, D.; Xu, G.; Yabsley, B.; Yamada, M.; Yamamoto, A.; Yamamoto, K.; Yamamoto, S.; Yamamura, T.; Yamaoka, J.; Yamazaki, T.; Yamazaki, Y.; Yan, Z.; Yang, H.; Yang, U. K.; Yang, Y.; Yang, Y.; Yang, Z.; Yanush, S.; Yao, W.-M.; Yao, Y.; Yasu, Y.; Ybeles Smit, G. V.; Ye, J.; Ye, S.; Yilmaz, M.; Yoosoofmiya, R.; Yorita, K.; Yoshida, R.; Young, C.; Youssef, S.; Yu, D.; Yu, J.; Yu, J.; Yuan, L.; Yurkewicz, A.; Zaets, V. G.; Zaidan, R.; Zaitsev, A. M.; Zajacova, Z.; Zalite, Yo. K.; Zanello, L.; Zarzhitsky, P.; Zaytsev, A.; Zeitnitz, C.; Zeller, M.; Zemla, A.; Zendler, C.; Zenin, A. V.; Zenin, O.; Ženiš, T.; Zenonos, Z.; Zenz, S.; Zerwas, D.; Zevi Della Porta, G.; Zhan, Z.; Zhang, D.; Zhang, H.; Zhang, J.; Zhang, X.; Zhang, Z.; Zhao, L.; Zhao, T.; Zhao, Z.; Zhemchugov, A.; Zheng, S.; Zhong, J.; Zhou, B.; Zhou, N.; Zhou, Y.; Zhu, C. G.; Zhu, H.; Zhu, J.; Zhu, Y.; Zhuang, X.; Zhuravlov, V.; Zieminska, D.; Zimmermann, R.; Zimmermann, S.; Zimmermann, S.; Ziolkowski, M.; Zitoun, R.; Živković, L.; Zmouchko, V. V.; Zobernig, G.; Zoccoli, A.; Zolnierowski, Y.; Zsenei, A.; Zur Nedden, M.; Zutshi, V.; Zwalinski, L.; Atlas Collaboration

2011-11-01

A measurement of the Z /γ* transverse momentum (pTZ) distribution in proton-proton collisions at √{ s} = 7 TeV is presented using Z /γ* →e+e- and Z /γ* →μ+μ- decays collected with the ATLAS detector in data sets with integrated luminosities of 35 pb-1 and 40 pb-1, respectively. The normalized differential cross sections are measured separately for electron and muon decay channels as well as for their combination up to pTZ of 350 GeV for invariant dilepton masses 66 GeV

Characterization of leptospiral proteins that afford partial protection in hamsters against lethal challenge with Leptospira interrogans.

PubMed

Atzingen, Marina V; Gonçales, Amane P; de Morais, Zenaide M; Araújo, Eduardo R; De Brito, Thales; Vasconcellos, Silvio A; Nascimento, Ana L T O

2010-09-01

Leptospirosis is a worldwide zoonosis caused by pathogenic Leptospira. The whole-genome sequence of Leptospira interrogans serovar Copenhageni together with bioinformatic tools allow us to search for novel antigen candidates suitable for improved vaccines against leptospirosis. This study focused on three genes encoding conserved hypothetical proteins predicted to be exported to the outer membrane. The genes were amplified by PCR from six predominant pathogenic serovars in Brazil. The genes were cloned and expressed in Escherichia coli strain BL21-SI using the expression vector pDEST17. The recombinant proteins tagged with N-terminal 6xHis were purified by metal-charged chromatography. The proteins were recognized by antibodies present in sera from hamsters that were experimentally infected. Immunization of hamsters followed by challenge with a lethal dose of a virulent strain of Leptospira showed that the recombinant protein rLIC12730 afforded statistically significant protection to animals (44 %), followed by rLIC10494 (40 %) and rLIC12922 (30 %). Immunization with these proteins produced an increase in antibody titres during subsequent boosters, suggesting the involvement of a T-helper 2 response. Although more studies are needed, these data suggest that rLIC12730 and rLIC10494 are promising candidates for a multivalent vaccine for the prevention of leptospirosis.

sal Genes Determining the Catabolism of Salicylate Esters Are Part of a Supraoperonic Cluster of Catabolic Genes in Acinetobacter sp. Strain ADP1

PubMed Central

Jones, Rheinallt M.; Pagmantidis, Vassilis; Williams, Peter A.

2000-01-01

A 5-kbp region upstream of the are-ben-cat genes was cloned from Acinetobacter sp. strain ADP1, extending the supraoperonic cluster of catabolic genes to 30 kbp. Four open reading frames, salA, salR, salE, and salD, were identified from the nucleotide sequence. Reverse transcription-PCR studies suggested that these open reading frames are organized into two convergent transcription units, salAR and salDE. The salE gene, encoding a protein of 239 residues, was ligated into expression vector pET5a. Its product, SalE, was shown to have esterase activity against short-chain alkyl esters of 4-nitrophenol but was also able to hydrolyze ethyl salicylate to ethanol and salicylic acid. A mutant of ADP1 with a Kmr cassette introduced into salE had lost the ability to utilize only ethyl and methyl salicylates of the esters tested as sole carbon sources, and no esterase activity against ethyl salicylate could be detected in cell extracts. SalE was induced during growth on ethyl salicylate but not during growth on salicylate itself. salD encoded a protein of undetermined function with homologies to the Escherichia coli FadL membrane protein, which is involved in facilitating fatty acid transport, and a number of other proteins detected during aromatic catabolism, which may also function in hydrocarbon transport or uptake processes. A Kmr cassette insertion in salD deleteriously affected cell growth and viability. The salA and salR gene products closely resemble two Pseudomonas proteins, NahG and NahR, respectively encoding salicylate hydroxylase and the LysR family regulator of both salicylate and naphthalene catabolism. salA was cloned into pUC18 together with salR and salE, and its gene product showed salicylate-inducible hydroxylase activity against a range of substituted salicylates, with the same relative specific activities as found in wild-type ADP1 grown on salicylate. Mutations involving insertion of Kmr cassettes into salA and salR eliminated expression of salicylate hydroxylase activity and the ability to grow on either salicylate or ethyl salicylate. Studies of mutants with disruptions of genes of the β-ketoadipate pathway with or without an additional salE mutation confirmed that ethyl salicylate and salicylate were channeled into the β-ketoadipate pathway at the level of catechol and thence dissimilated by the cat gene products. SalR appeared to regulate expression of salA but not salE. PMID:10715011

A scalable method for the production of high-titer and high-quality adeno-associated type 9 vectors using the HSV platform

PubMed Central

Adamson-Small, Laura; Potter, Mark; Falk, Darin J; Cleaver, Brian; Byrne, Barry J; Clément, Nathalie

2016-01-01

Recombinant adeno-associated vectors based on serotype 9 (rAAV9) have demonstrated highly effective gene transfer in multiple animal models of muscular dystrophies and other neurological indications. Current limitations in vector production and purification have hampered widespread implementation of clinical candidate vectors, particularly when systemic administration is considered. In this study, we describe a complete herpes simplex virus (HSV)-based production and purification process capable of generating greater than 1 × 1014 rAAV9 vector genomes per 10-layer CellSTACK of HEK 293 producer cells, or greater than 1 × 105 vector genome per cell, in a final, fully purified product. This represents a 5- to 10-fold increase over transfection-based methods. In addition, rAAV vectors produced by this method demonstrated improved biological characteristics when compared to transfection-based production, including increased infectivity as shown by higher transducing unit-to-vector genome ratios and decreased total capsid protein amounts, shown by lower empty-to-full ratios. Together, this data establishes a significant improvement in both rAAV9 yields and vector quality. Further, the method can be readily adapted to large-scale good laboratory practice (GLP) and good manufacturing practice (GMP) production of rAAV9 vectors to enable preclinical and clinical studies and provide a platform to build on toward late-phases and commercial production. PMID:27222839

Cloning, Expression and Characteristics of a Novel Alkalistable and Thermostable Xylanase Encoding Gene (Mxyl) Retrieved from Compost-Soil Metagenome

PubMed Central

Verma, Digvijay; Kawarabayasi, Yutaka; Miyazaki, Kentaro; Satyanarayana, Tulasi

2013-01-01

Background The alkalistable and thermostable xylanases are in high demand for pulp bleaching in paper industry and generating xylooligosaccharides by hydrolyzing xylan component of agro-residues. The compost-soil samples, one of the hot environments, are expected to be a rich source of microbes with thermostable enzymes. Methodology/Principal Findings Metagenomic DNA from hot environmental samples could be a rich source of novel biocatalysts. While screening metagenomic library constructed from DNA extracted from the compost-soil in the p18GFP vector, a clone (TSDV-MX1) was detected that exhibited clear zone of xylan hydrolysis on RBB xylan plate. The sequencing of 6.321 kb DNA insert and its BLAST analysis detected the presence of xylanase gene that comprised 1077 bp. The deduced protein sequence (358 amino acids) displayed homology with glycosyl hydrolase (GH) family 11 xylanases. The gene was subcloned into pET28a vector and expressed in E. coli BL21 (DE3). The recombinant xylanase (rMxyl) exhibited activity over a broad range of pH and temperature with optima at pH 9.0 and 80°C. The recombinant xylanase is highly thermostable having T1/2 of 2 h at 80°C and 15 min at 90°C. Conclusion/Significance This is the first report on the retrieval of xylanase gene through metagenomic approach that encodes an enzyme with alkalistability and thermostability. The recombinant xylanase has a potential application in paper and pulp industry in pulp bleaching and generating xylooligosaccharides from the abundantly available agro-residues. PMID:23382818

UMG Lenti: Novel Lentiviral Vectors for Efficient Transgene- and Reporter Gene Expression in Human Early Hematopoietic Progenitors

PubMed Central

Chiarella, Emanuela; Carrà, Giovanna; Scicchitano, Stefania; Codispoti, Bruna; Mega, Tiziana; Lupia, Michela; Pelaggi, Daniela; Marafioti, Maria G.; Aloisio, Annamaria; Giordano, Marco; Nappo, Giovanna; Spoleti, Cristina B.; Grillone, Teresa; Giovannone, Emilia D.; Spina, Raffaella; Bernaudo, Francesca; Moore, Malcolm A. S.; Bond, Heather M.; Mesuraca, Maria; Morrone, Giovanni

2014-01-01

Lentiviral vectors are widely used to investigate the biological properties of regulatory proteins and/or of leukaemia-associated oncogenes by stably enforcing their expression in hematopoietic stem and progenitor cells. In these studies it is critical to be able to monitor and/or sort the infected cells, typically via fluorescent proteins encoded by the modified viral genome. The most popular strategy to ensure co-expression of transgene and reporter gene is to insert between these cDNAs an IRES element, thus generating bi-cistronic mRNAs whose transcription is driven by a single promoter. However, while the product of the gene located upstream of the IRES is generally abundantly expressed, the translation of the downstream cDNA (typically encoding the reporter protein) is often inconsistent, which hinders the detection and the isolation of transduced cells. To overcome these limitations, we developed novel lentiviral dual-promoter vectors (named UMG-LV5 and –LV6) where transgene expression is driven by the potent UBC promoter and that of the reporter protein, EGFP, by the minimal regulatory element of the WASP gene. These vectors, harboring two distinct transgenes, were tested in a variety of human haematopoietic cell lines as well as in primary human CD34+ cells in comparison with the FUIGW vector that contains the expression cassette UBC-transgene-IRES-EGFP. In these experiments both UMG-LV5 and UMG–LV6 yielded moderately lower transgene expression than FUIGW, but dramatically higher levels of EGFP, thereby allowing the easy distinction between transduced and non-transduced cells. An additional construct was produced, in which the cDNA encoding the reporter protein is upstream, and the transgene downstream of the IRES sequence. This vector, named UMG-LV11, proved able to promote abundant expression of both transgene product and EGFP in all cells tested. The UMG-LVs represent therefore useful vectors for gene transfer-based studies in hematopoietic stem and progenitor cells, as well as in non-hematopoietic cells. PMID:25502183

Evaluation of fiber-modified adenovirus vector-vaccine against foot-and-mouth diseaes in cattle

USDA-ARS?s Scientific Manuscript database

Novel vaccination approaches against foot-and-mouth-disease (FMD) include the use of a replication-defective human adenovirus type 5 vector (Ad5) that contains the capsid encoding regions of FMD virus (FMDV). An Ad5.A24 has proven effective as a vaccine against FMD in swine and cattle. However, ther...