Gene transfer into genomes of human cells by the sleeping beauty transposon system

The Sleeping Beauty (SB) transposon system, derived from teleost fish sequences, is extremely effective at delivering DNA to vertebrate genomes, including those of humans. We have examined several parameters of the SB system to improve it as a potential, nonviral vector for gene therapy. Our investigation centered on three features: the carrying capacity of the transposon for efficient integration into chromosomes of HeLa cells, the effects of overexpression of the SB transposase gene on transposition rates, and improvements in the activity of SB transposase to increase insertion rates of transgenes into cellular chromosomes. We found that SB transposons of about 6 kb retained 50% of the maximal efficiency of transposition, which is sufficient to deliver 70-80% of identified human cDNAs with appropriate transcriptional regulatory sequences. Overexpression inhibition studies revealed that there are optimal ratios of SB transposase to transposon for maximal rates of transposition, suggesting that conditions of delivery of the two-part transposon system are important for the best gene-transfer efficiencies. We further refined the SB transposase to incorporate several amino acid substitutions, the result of which led to an improved transposase called SB11. With SB11 we are able to achieve transposition rates that are about 100-fold above those achieved with plasmids that insert into chromosomes by random recombination. With the recently described improvements to the transposon itself, the SB system appears to be a potential gene-transfer tool for human gene therapy.

Construction and evaluation of DNA vaccines encoding Edwardsiella tarda antigens

Edwardsiella tarda is an opportunistic pathogen that can infect humans, animal, and fish. Two E. tarda antigens, Eta6 and FliC, which are homologues to an ecotin precursor and the FliC flagellin, respectively, were identified by in vivo-induced antigen technology from a pathogenic E. tarda strain isolated from diseased fish. When used as a subunit vaccine, purified recombinant Eta6 was moderately protective against lethal challenge of E. tarda in a Japanese flounder model, whereas purified recombinant FliC showed no apparent immunciprotectivity. Similarly, DNA vaccines based on eta6 and fliC in the form of plasmids pEta6 and pFliC induced, respectively, moderate and marginal protection against E. tarda infection. To improve the vaccine efficacy of eta6, a chimeric DNA vaccine, pCE6, was constructed, which encodes Eta6 fused in-frame to FliC. pCE6 was found to induce significantly higher level of protection than pEta6. Likewise, another chimeric DNA vaccine, pCE18, which expresses FliC fused to a previously identified E. tarda antigen Et18, elicited significantly stronger protective immunity than the DNA vaccine based on et18 alone. Fish immunized with pEta6 and pCE6 produced specific serum antibodies and exhibited significantly enhanced expression of the genes encoding elements that are involved in both innate and adaptive immune responses. Furthermore, the induction magnitudes of most of these genes were significantly higher in pCE6-vaccinated fish than in pEta6-vaccinated fish. (C) 2009 Elsevier Ltd. All rights reserved.

Immunoprotective analysis of VhhP2, a Vibrio harveyi vaccine candidate

VhhP2 is an Outer membrane protein identified in a pathogenic Vibrio harveyi strain, T4, isolated from diseased fish. When used as a Subunit Vaccine, purified recombinant VhhP2 affords high level of protection upon Japanese flounder against V harveyi challenge. Vaccination with VhhP2 induced the expression of a number of immune-related genes, especially those encoding immunoglobulin M (IgM) and major histocompatibility complex (MHC) II alpha. A VhhP2 surface display system, in the form of the fish commensal strain FIR harboring the vhhP2-expressing plasmid pJVP, was constructed. PF3/pJVP is able to produce and present recombinant VhhP2 on cell surface. Vaccination of fish with live PF3/pJVP via intraperitoneal injection elicited Strong immunoprotection. Vaccination of fish orally with live PF3/pJVP embedded in alginate microspheres also induced effective immunoprotection. In addition, a VhhP2-based surface display system was created, in which VhhP2 serves as a carrier for the Surface delivery of a heterologous Edwardsiella tarda immunogen, Et18, that is fused in-frame to VhhP2. DH5 alpha/pJVP18, which expresses and surface-displays the VhhP2-Et18 chimera, proved to be an effective vaccine that call protect fish against infections by V. harveyi and E. tarda to the extents comparable to those produced by vaccination with purified recombinant VhhP2 and Et18, respectively. These data suggest that VhhP2 may be applied as a vaccine and a vaccine carrier against infections by V. harveyi and other pathogens such as F. tarda. (C) 2009 Elsevier Ltd. All rights reserved.

Construction of an attenuated Pseudomonas fluorescens strain and evaluation of its potential as a cross-protective vaccine

Ferric uptake regulator (Fur) is a global transcription regulator that is ubiquitous to Gram-negative bacteria and regulates diverse biological processes, including iron uptake, cellular metabolism, stress response, and production of virulence determinants. As a result, for many pathogenic bacteria, Fur plays a crucial role in the course of infection and disease development. In this study, the fur gene was cloned from a pathogenic Pseudomonas fluorescens strain, TSS, isolated from diseased Japanese flounder cultured in a local farm. TSS Fur can partially complement the defective phenotype of an Escherichia coli fur mutant. A TSS fur null mutant, TFM, was constructed. Compared to TSS, TFM exhibits reduced growth ability, aberrant production of outer membrane proteins, decreased resistance against host serum bactericidal activity, impaired ability to disseminate in host blood and tissues, and drastic attenuation in overall bacterial virulence in a Japanese flounder infection model. When used as a live vaccine administered via the injection, immersion, and oral routes, TFM affords high levels of protection upon Japanese flounder against not only P.fluorescens infection but also Aeromonas hydrophila infection. Furthermore, a plasmid, pJAQ, was constructed, which expresses the coding element of the Vibrio harveyi antigen AgaV-DegQ. TFM harboring pJAQ can secret AgaV-DegQ into the extracellular milieu. Vaccination of Japanese flounder with live TFM/pJAQ elicited strong immunoprotection against both V. harveyi and A. hydrophila infections. (C) 2009 Elsevier Ltd. All rights reserved.

Dissection and localization of the immunostimulating domain of Edwardsiella tarda FliC

Bacterial flagellin is known to induce potent immune response in vertebrate systems via the toll-like receptor (TLR) 5. As a result, flagellin has been studied extensively as a vaccine adjuvant. In a previous study, we examined the vaccine and adjuvant potentials of the flagellin (FliC) of the fish pathogen Edwardsiella tarda. We found that E. tarda FliC induced low protective immunity by itself but could function as a molecular adjuvant and potentiate the specific immune response induced by the E. tarda antigen Eta6. Since FliC is a large protein and organized into distinct structural domains, we wondered whether the immunostimulating effect observed with the full-length protein could be localized to a certain region. To investigate this question, we in the present study dissected the FliC protein into several segments according to its structural features: (i) N163, which consists of the conserved N-terminal 163 residues of FliC; (ii) M160, which consists of the variable middle 160 residues; (iii) C94, which consists of the conserved C-terminal 94 residues; (iv) NC257, which is an artificial fusion of N163 and C94. To examine the adjuvanticity of the FliC fragments, DNA vaccine plasmids expressing FliC fragments in fusion with Eta6 were constructed and used to immunize Japanese flounder. The results showed that N163 produced the best adjuvant effect, which, in respect to improvement in the relative percent survival of the vaccinated fish, was comparable to that of the full-length FliC. None of the other FliC fragments exhibited apparent immunopotentiating effect. Further analysis showed that N163 enhanced the production of serum specific antibodies and, like full-length FliC, significantly upregulated the expression of the genes that are possibly involved in innate and adaptive immunity. These results indicate that N163 is the immunodominant region of FliC and suggest that E. tarda FliC may induce immune responses in Japanese flounder via mechanisms alternative to that involving TLR5. (C) 2010 Elsevier Ltd. All rights reserved.

Identification of an Edwardsiella tarda surface antigen and analysis of its immunoprotective potential as a purified recombinant subunit vaccine and a surface-anchored subunit vaccine expressed by a fish commensal strain

Edwardsiella tarda is the etiological agent of edwardsiellosis, a systematic disease that affects a wide range of marine and freshwater fish cultured worldwide. In order to identify E. tarda antigens with vaccine potential, we in this study conducted a systematic search for E. tarda proteins with secretion capacity. One of the proteins thus identified was Esa1, which contains 795 amino acid residues and shares extensive overall sequence identities with the D15-like surface antigens of several bacterial species. In silico analyses indicated that Esa1 localizes to outer membrane and possesses domain structures that are conserved among bacterial surface antigens. The vaccine potential of purified recombinant Esa1 was examined in a Japanese flounder (Paralichthys olivaceus) model, which showed that fish vaccinated with Esa1 exhibited a high level of survival and produced specific serum antibodies. Passive immunization of naive fish with antisera raised against Esa1 resulted in significant protection against E. tarda challenge. Taking advantage of the secretion capacity of Esa1 and the natural gut-colonization ability of a fish commensal strain, we constructed an Esa1-expressing recombinant strain, FP3/pJsa1. Western immunoblot and agglutination analyses showed that FP3/pJsa1 produces outer membrane-localized Esa1 and forms aggregates in the presence of anti-Esa1 antibodies. Vaccination analyses showed that FP3/pJsa1 as an intraperitoneal injection vaccine and an oral vaccine embedded in alginate microspheres produced relative percent survival rates of 79% and 52%, respectively, under severe challenging conditions that resulted in 92-96% mortality in control fish. Further analyses showed that following oral vaccination, FP3/pJsa1 was able to colonize in the gut but unable to disseminate into other tissues. Together these results indicate that Esa1 is a protective immunogen and an effective oral vaccine when delivered by FP3/pJsa1 as a surface-anchored antigen. (c) 2010 Elsevier Ltd. All rights reserved.

Isolation and analysis of the vaccine potential of an attenuated Edwardsiella tarda strain

Edwardsiella tarda is an important aquaculture pathogen that can infect a wide range of marine and freshwater fish worldwide. In this study, a modified E. tarda strain, TX5RM, was selected by multiple passages of the pathogenic E. tarda strain TX5 on growth medium containing the antibiotic rifampicin. Compared to the wild type strain, the rifampicin-resistant mutant TX5RM (i) shows drastically increased median lethal dose and reduced capacity to disseminate in and colonize fish tissues and blood; (ii) exhibits slower growth rates when cultured in rich medium or under conditions of iron depletion; and (iii) differs in the production profile of whole-cell proteins. The immunoprotective potential of TX5RM was examined in a Japanese flounder (Paralichthys olivaceus) model as a vaccine delivered via intraperitoneal injection, oral feeding, bath immersion, and oral feeding plus immersion. All the vaccination trials, except those of injection, were performed with a booster at 3-week after the first vaccination. The results showed that TX5RM administered via all four approaches produced significant protection, with the highest protection levels observed with TX5RM administered via oral feeding plus immersion, which were, in terms of relative percent of survival (RPS), 80.6% and 69.4% at 5- and 8-week post-vaccination, respectively. Comparable levels of specific serum antibody production were induced by TX5RM-vaccinated via different routes. Microbiological analyses showed that TX5RM was recovered from the gut, liver, and spleen of the fish at 1-10 days post-oral vaccination and from the spleen, liver, kidney, and blood of the fish at 1-14 days post-immersion vaccination. Taken together, these results indicate that TX5RM is an attenuated E. tarda strain with good vaccine potential and that a combination of oral and immersion vaccinations may be a good choice for the administration of live attenuated vaccines. (C) 2010 Elsevier Ltd. All rights reserved.

Identification and analysis of a CpG motif that protects turbot (Scophthalmus maximus) against bacterial challenge and enhances vaccine-induced specific immunity

Oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs in certain contexts are known to be immunostimulatory in vertebrate systems. CpG ODNs with immune effects have been identified for many fish species but, to our knowledge, not for turbot. In this study, a turbot-effective CpG ODN, ODN 205, was identified and a plasmid, pCN5, was constructed which contains the CpG motif of ODN 205. When administered into turbot via intraperitoneal (i.p.) injection, both ODN 205 and pCN5 could (i) inhibit bacterial dissemination in blood in dose and time dependent manners, and (ii) protect against lethal bacterial challenge. Immunological analyses showed that in vitro treatment with ODN 205 stimulated peripheral blood leukocyte proliferation, while i.p. injection with ODN 205 enhanced the respiratory burst activity, chemiluminescence response, and acid phosphatase activity of turbot head kidney macrophages. pCN5 treatment-induced immune responses similar to those induced by ODN 205 treatment except that pCN5 could also enhance serum bactericidal activity in a calcium-independent manner. To examine whether ODN 205 and pCN5 had any effect on specific immunity, ODN 205 and pCN5 were co-administered into turbot with a Vibrio harveyi subunit vaccine, DegQ. The results showed that pCN5, but not ODN 205, significantly increased the immunoprotective efficacy of DegQ and enhanced the production of specific serum antibodies in the vaccinated fish. Further analysis indicated that vaccination with DegQ in the presence of pCN5 upregulated the expression of the genes encoding MHC class II alpha, IgM, Mx, and IL-8 receptor. Taken together, these results demonstrate that ODN 205 and pCN5 can stimulate the immune system of turbot and induce protection against bacterial challenge. In addition, pCN5 also possesses adjuvant property and can potentiate vaccine-induced specific immunity. (C) 2010 Elsevier Ltd. All rights reserved.

Construction and analysis of an experimental Streptococcus iniae DNA vaccine

Streptococcus iniae is a severe aquaculture pathogen that can also infect humans and animal. A putative secretory antigen, Slat 0, was identified from a pathogenic S. iniae strain by in vivo-induced antigen technology. Using turbot as an animal model, the immunoprotective effect of Sia10 was examined as a DNA vaccine in the form of plasmid pSia10, which expresses sia10 under the cytomegalovirus immediate-early promoter. In fish vaccinated with pSia10, transcription of sia10 was detected in muscle, liver, spleen, and kidney at 7, 14, 21, 28, 35, 42, and 49 days post-vaccination. In addition, production of Sia10 protein was also detected in the muscle tissues of pSia10-vaccinated fish. Fish vaccinated with pSia10 exhibited a relative percent survival (RPS) of 73.9% and 92.3%, respectively, when challenged with high and low doses (producing a cumulative mortality of 92% and 52%, respectively, in the control groups) of S. iniae. Immunological and transcriptional analyses showed that vaccination with pSia10(i) induced much stronger chemiluminescence response and significantly higher levels of nitric oxide production and acid phosphatase activity in head kidney macrophages; (ii) caused the production of specific serum antibodies, which afforded apparent immunoprotection when transferred passively into naive fish; and (iii) upregulated the expression of the genes encoding proteins that are possibly involved in both innate and adaptive immune responses. Taken together, these results indicated that pSia10 is an effective vaccine candidate and may be used in the control of S. iniae infection in aquaculture. (C) 2010 Elsevier Ltd. All rights reserved.

Standardized review of atrial anatomy for cardiac electrophysiologists

2.691 JCR (2013) Q2, 50/125 Cardiac & cardiovascular systems, 51/122 Medicine, research & experimental

Corrigendum to "Short-term changes in light distortion in orthokeratology subjects"

2.134 JCR (2015) Q3, 74/124 Medicine, research & experimental, 81/161 Biotechnology & applied microbiology

Hospital clinicians information behaviour and attitudes towards the 'Clinical Informationist': an Irish survey

Background: Hospital clinicians are increasingly expected to practice evidence-based medicine (EBM) in order to minimize medical errors and ensure quality patient care, but experience obstacles to information-seeking. The introduction of a Clinical Informationist (CI) is explored as a possible solution. Aims:  This paper investigates the self-perceived information needs, behaviour and skill levels of clinicians in two Irish public hospitals. It also explores clinicians perceptions and attitudes to the introduction of a CI into their clinical teams. Methods: A questionnaire survey approach was utilised for this study, with 22 clinicians in two hospitals. Data analysis was conducted using descriptive statistics. Results: Analysis showed that clinicians experience diverse information needs for patient care, and that barriers such as time constraints and insufficient access to resources hinder their information-seeking. Findings also showed that clinicians struggle to fit information-seeking into their working day, regularly seeking to answer patient-related queries outside of working hours. Attitudes towards the concept of a CI were predominantly positive. Conclusion: This paper highlights the factors that characterise and limit hospital clinicians information-seeking, and suggests the CI as a potentially useful addition to the clinical team, to help them to resolve their information needs for patient care.

Combining environmental and medical datasets to explore potential associations between environmental factors and health: Policy implications for human health risk assessments

This review paper discusses the use of Tellus and Tellus Border soil and stream geochemistry data to investigate the relationship between medical data and naturally occurring background levels of potentially toxic elements (PTEs) such as heavy metals in soils and water. The research hypothesis is that long-term low level oral exposure of PTEs via soil and water may result in cumulative exposures that may act as risk factors for progressive diseases including cancer and chronic kidney disease. A number of public policy implications for regional human health risk assessments, public health policy and education are also explored alongside the argument for better integration of multiple data sets to enhance ongoing medical and social research. This work presents a partnership between the School of Geography, Archaeology and Palaeoecology, Northern Ireland Cancer Registry, Queen’s University Belfast, and the nephrology (kidney medicine) research group.

Plaster body wrap: effects on abdominal fat

Background Abdominal fat is associated with metabolic disorders, leading to cardiovascular risk factors and numerous diseases. This study aimed to analyze the effect of plaster body wrap in combination with aerobic exercise on abdominal fat. Methods Nineteen female volunteers were randomly divided into intervention group (IG; n = 10) performing aerobic exercise with plaster body wrap, and control group (CG; n = 9) performing only exercise. Subcutaneous and visceral fat were measured using ultrasound; subcutaneous fat was also estimated on analysis of skinfolds and abdominal perimeters. Results At the end of the 10-sessions protocol, the IG demonstrated a significant decrease (p ≤ 0.05) in subcutaneous fat at the left anterior superior iliac spine (ASIS) level and in iliac crest perimeter measurements. A large intervention effect size strength (0.80) was found in subcutaneous fat below the navel and a moderate effect size strength on the vertical abdominal skinfold (0.62) and the perimeter of the most prominent abdominal point (0.57). Comparing the initial and final data of each group, the IG showed a significant decrease in numerous variables including visceral and subcutaneous fat above and below the navel measured by ultrasound (p ≤ 0.05). Conclusion Plaster body wrap in combination with aerobic exercise seems to be effective for abdominal fat reduction.

Le discours de l'innovation technologique en médecine régénératrice

Le discours sur l’innovation oriente la recherche scientifique médicale publique vers un développement technologique et économique à court terme. À ce titre, la médecine régénératrice est une thérapie innovatrice marquée par une logique d’accumulation spéculative qui porte à la fois sur les cellules humaines et sur la façon de mener la recherche. Or, une réorganisation de la recherche scientifique liée à une nouvelle conception économique de la science et de la technologie ainsi qu’un rôle différent attribué à l’État constituent le cadre institutionnel contemporain qui émerge à la fin des années 1970. Le changement induit par cette idée d’innovation et sur lequel s’attarde ce mémoire porte non pas sur l’usage ou la destination de la science, mais sur l’extension du raisonnement économique. Celui-ci ne survient pas à l’étape du développement, après que la recherche ait été effectuée en vertu du modèle de la « Big Science ». Au contraire, il remonte du marché pour s’installer très tôt au stade de la compréhension des mécanismes biologiques et dans un espace qui relève de la propriété collective : le laboratoire public. Le passage du caractère « exogène » à « endogène » de la recherche scientifique publique vis-à-vis de l’économie est au cœur d’une discussion sur l’hégémonie de la logique de marché.