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Validation of RNAi Silencing Specificity Using Synthetic Genes: Salicylic Acid-Binding Protein 2 Is Required for Innate Immunity in PlantsKumar, Dhirendra, Gustafsson, Claes, Klessig, Daniel F. 01 March 2006 (has links)
RNA interference (RNAi) is widely used to specifically silence the expression of any gene to study its function and to identify and validate therapeutic targets. Despite the popularity of this technology, recent studies have shown that RNAi may also silence non-targeted genes. Here we demonstrate the utility of a quick, efficient and robust approach to directly validate the specificity of RNAi as an alternative to indirect validation of RNAi through gene expression profiling. Our approach involves reversing (complementing) the RNAi-induced phenotype by introducing a synthetic version of the target gene that is designed to escape silencing. This synthetic gene complementation approach can also be used for mutational analysis of the target gene, or to provide a functional version of a defective protein after silencing the defective gene by RNAi. Using this approach we demonstrate that the loss of systemic acquired resistance, a form of innate immunity in plants, is indeed due to the silencing of salicylic acid-binding protein 2 rather than to off-target effects.
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Prey-mediated effects of imidacloprid on Laricobius nigrinus (Coleoptera: Derodontidae) and Sasajiscymnus tsugae (Coleoptera: Coccinellidae), two predators of hemlock woolly adelgidEisenback, Brian Matthew 31 July 2008 (has links)
Prey-mediated effects of imidacloprid were evaluated for Laricobius nigrinus Fender and Sasajiscymnus tsugae Sasaji and McClure after feeding on hemlock woolly adelgid (HWA), Adelges tsugae Annand (Hemiptera: Adelgidae). Two methods were evaluated for detecting imidacloprid in hemlock tissues: a commercially available enzyme linked immunoassay (ELISA) kit and a high performance thin-layer chromatography technique for detecting and quantifying imidacloprid residues in hemlock wood and needle tissues. ELISA is advantageous because of its cost, sensitivity, and ease of use. However, matrix effects in the form of false positives and overestimated imidacloprid concentrations were evident in hemlock wood and needle tissue extracts. Matrix effects could be reduced by dilution with water, effectively raising the lower detection range of the kit from 0.2 to 200 ppb. High performance thin-layer chromatography was accurate, quick, easy to use, and matrix effects were not evident. However, the technique is sensitive in the lower ppm range and tissue samples from field-treated hemlocks are often in the ppb range, making this technique less desirable than more sensitive analytical methods.
Lethal and sublethal effects on both predators were evident after eastern hemlock branches infested with HWA were spiked with imidacloprid in the laboratory. HWA mortality increased with dosage and time, and its 30 d LC50 was determined to be 242 ppb. Both predator species exhibited reduced survivorship and fitness parameters after feeding on HWA from the treated branches. In a topical application bio-assay, 6 d imidacloprid LD50 values for L. nigrinus and S. tsugae were 2.43 and 1.82 µg/g, respectively. Imidacloprid and its major metabolites in hemlock tissues were analyzed by liquid chromatography-tandem mass spectrometry. Imidacloprid recovery from beetle cadavers was correlated with beetle mortality from feeding on treated hemlock branches. Olefin was the primary imidacloprid metabolite recovered from hemlock wood tissues.
When predators fed on HWA from field-treated trees, impacts on survivorship and fitness were variable. In 2005, significantly higher proportions of both species of beetles were affected by feeding on control branches compared with treated branches. In 2006, beetles feeding on HWA from some of the trees treated in the field exhibited longer fliptimes compared with beetles feeding on controls, although beetle mortality was not significant among treatements. In the field, imidacloprid controlled HWA populations 1-3 years post-treatment. Hemlock health improved in the highest dosage group, with significantly greater lengths of new shoots compared with shoots from control trees. Eastern hemlock trees primarily metabolized imidacloprid into the olefin metabolite, which can have increased insecticidal toxicity compared with imidacloprid. Imidacloprid was detected in beetle cadavers after feeding on HWA from treated branches, suggesting that prey-mediated impacts of systemic imidacloprid are possible on nontarget predators. However, because of HWA's sensitivity to imidacloprid, in field situations predators are more likely to be affected by reduced adelgid density and quality. / Ph. D.
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A toolkit for analysis of gene editing and off-target effects of engineered nucleasesFine, Eli Jacob 27 May 2016 (has links)
Several tools were developed to help researchers facilitate clinical translation of the use of engineered nucleases towards their disease gene of interest. Two major issues addressed were the inability to accurately predict nuclease off-target sites by user-friendly \textit{in silico} methods and the lack of a high-throughput, sensitive measurement of gene editing activity at endogenous loci. These objectives were accomplished by the completion of the following specific aims. An online search interface to allow exhaustive searching of a genome for potential nuclease off-target sites was implemented. Previously discovered off-target sites were collated and ranking algorithms developed that preferentially score validated off-target sites higher than other predictions. HEK-293T cells transfected with newly developed TALENs and ZFNs targeting the beta-globin gene were analyzed at the off-target sites predicted by the tool. Many samples of genomic DNA from cells treated with different ZFNs and TALENs were analyzed for off-target effects to generate a greatly expanded training set of bona fide off-target sites. Modifications to the off-target prediction algorithm parameters were evaluated for improvement through Precision-Recall analysis and several other metrics. An analysis pipeline was developed to process SMRT reads to simultaneously measure the rates of different DNA repair mechanisms by directly examining the DNA sequences. K562 cells were transfected with different types of nucleases and donor repair templates in order to optimize conditions for repairing the beta-globin gene. This work will have significant impact on future studies as the methods developed herein allow other laboratories to optimize nuclease-based therapies for single gene disorders.
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The spread of the harlequin ladybird Harmonia axyridis (Coleoptera: coccinellidae) in Europe and its effects on native ladybirdsBrown, Peter M. J. January 2010 (has links)
Native to Asia, Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae) is an invasive non-native ladybird in Europe and North America, where it was widely introduced as a biological control agent of aphids and coccids. There were three main objectives of this study: firstly, to investigate the spread and distribution of H. axyridis in Europe from the start of the invasion process; secondly, to study its ecology in Great Britain; and thirdly, to examine its effects on native ladybirds in Britain. In Europe, collaborations with a network of scientists allowed the collation of H. axyridis occurrence data from across the continent. In Great Britain, a web-based public survey (www.harlequin-survey.org) was used, leading to the receipt of over 10,000 verified records of H. axyridis between 2004 and 2008. National land cover data enabled the habitats used by H. axyridis to be identified across Great Britain. These datasets were analysed in order to study the spread and ecology of H. axyridis in Europe and Britain. Fieldwork over a three year period (2006 to 2008) was carried out to examine changes in ladybird species assemblages during the invasion phase of H. axyridis in eastern England. Laboratory work was conducted to detect intraguild predation by H. axyridis, through PCR analyses of gut contents of field-collected ladybirds. In Europe, H. axyridis has spread since 2001 at the rate of approximately 200km yr-1. It has become established in at least 23 European countries. The established range extends from Norway in the north to southern France in the south, and from Ukraine in the east to Great Britain in the west. In the first five years of establishment H. axyridis spread north through Britain at the rate of 105km yr-1 and west at the rate of 145km yr-1. Evidence of the production of two generations per year was found, giving H. axyridis an advantage over most native ladybirds in Britain. Although H. axyridis was very common in urban habitats, it increasingly used semi-natural habitats. In addition, whilst the species was most common on deciduous trees, it was increasingly found on herbaceous plants. Aceraceae, Rosaceae and Malvaceae were the dominant plant families used by H. axyridis, especially for breeding. In eastern England H. axyridis increased from 0.1% to 40% of total ladybirds in three years, whilst native aphidophagous ladybirds declined from 84% to 41% of total ladybirds in the same period. Three species in particular experienced declines: Adalia 2-punctata, Coccinella 7-punctata and Propylea 14-punctata. Harmonia axyridis was the most abundant species by the end of the study. Detection of intraguild predation by one coccinellid on another, in the field in Europe, was shown for the first time using PCR techniques: A. 2-punctata DNA was detected in the gut of one of 112 field-collected H. axyridis. Harmonia axyridis has spread very quickly since 2001 and has become one of the most widely distributed coccinellids in Europe. Populations of native aphidophagous ladybirds were negatively affected by the arrival of H. axyridis, partly through intraguild predation.
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Non-target Effects of Genetically Modified TreesBlomberg, Patrik January 2007 (has links)
To date, few studies have focused on the effects of genetically modified trees (GM trees) on the environment. One concern with GM trees is that they may have unanticipated effects on non-target organisms, i.e. effects on organisms that are not direct targets of the genetically modified trait. The main objective of this thesis was to study potential non-target effects from the interaction between GM trees and natural enemies, including phytopathogens and herbivorous insects. To study this I used a system consisting of GM trees featuring changes in growth-related characteristics, and naturally occurring enemies. The GM trees used were the aspen hybrids Populus tremula x tremuloides: one unmodified wild type clone T89 (control) and transgenic lines with altered expression of gibberellin (GA 20-oxidase), sucrose (SPS) or pectin (PME); and Populus tremula x alba: one unmodified wild type clone INRA 717-1-B4 (control) and lines modified to suppress the activity of the enzymes in the lignin biosynthetic pathway, i.e. CAD, COMT, CCR or CCoAOMT. The natural enemies used were the parasitic phytopathogens Melampsora pinitorqua, M. populnea and Venturia tremulae, and the herbivorous leaf-beetle Phratora vitellinae. To address this question inoculation experiments, feeding preference experiments, analyses of secondary chemistry and field inventories were performed. The results of the studies showed that the GM trees significantly affected the interaction with the natural enemies, both in the laboratory as well as in the field. For instance, both M. pinitorqua and V. tremulae showed an altered disease incidence on the GM trees of P. tremula x tremuloides compared to the unmodified wild type T89, where all tested transgenic lines exhibited altered susceptibility to the pathogens. However, there were also differences in aggressiveness to the aspens depending on pathogen population. The results from the field inventory showed that lines within all tested transgenic construct, COMT, CAD, CCoAOMT and CCR of P. tremula x alba differed significantly from the wild type INRA 717-1-B4 in susceptibility to M. populnea. In addition, the susceptibility to the rust also differed significantly between lines carrying the same transgenic constructs. Furthermore, we found that overexpression of SPS in P. tremula x tremuloides, unintentionally induced changes in plant secondary chemistry, where the GM-line SPS33A exhibited the largest deviation from the wild type T89 in contents of plant phenolics and nitrogen, and that these changes coincide with a concurrent decrease in herbivory by P. vitellinae on this line. I argue that the altered interactions are the result of physiological changes in the trees. They can originate from direct effects i.e. altered expression of the modified trait, indirect effects of the genetic modification process e.g. pleiotropy, or effects from the transformation process e.g. position effects, to which the tested natural enemies respond. The result stresses the importance of further research on the causes and mechanisms responsible for the altered interaction between GM trees and non-target organisms, as well as evaluating the potential environmental effects of cultivation of GM trees in the field. Such research will require collaboration between researchers from different disciplines, such as plant ecology and physiology, functional genomics, proteomics and metabolomics.
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Mort cellulaire immunogène induite par le crizotinib dans le cancer poumon non à petites cellules. / Crizotinib-Induced Immunogenic Cell Death in Non-Small Cell Lung CancerLiu, Peng 20 June 2018 (has links)
De nombreuses données suggèrent que le succès thérapeutique de certaines chimiothérapies conventionnelles, radiothérapies, ainsi que des thérapies ciblées est dû à leur capacité a induire la mort cellulaire immunogène (ICD), ce qui stimule la libération ou l'exposition des motifs moléculaires associés à un dommage (DAMPs) conduisant à leur reconnaissance par le système immunitaire, rétablissant ainsi l'immunosurveillance. En utilisant un criblage non polarisé, le crizotinib a été identifié en tant qu'inhibiteur de tyrosine-kinase ayant la capacité de stimuler la libération de caractéristiques distinctives de l’ICD. Des expériences faites par la suite ont montréque le crizotinib induit l'exposition de la calreticulin, la sécrétion d'ATP et la libération d’HMGB1, ainsi que le stress du réticulum endoplasmique dans les lignées cellulaires cancéreuses murines et humaines, notamment en combinaison avec les agents non immunogènes tel que le cisplatine. L’ICD causée par la combinaison du crizotinib avec la chimiothérapie a aussi été observée dans les cellules du cancer bronchique non à petites cellules (NSCLC), cellules ne possédant pas de mutations activatrices d’ALK ou ROS1 ; cela suggère un mode d'action hors-cible. Des études comparatives ont montré que seule la conformation utilisée en clinique, l’isoforme (R)-crizotinib, a la capacité de stimuler l’ICD ; le (S)-énantiomère ne possède pas ces caractéristiques. Combinées au cisplatine, les cellules fibrosarcome MCA205 et les cellules cancéreuses du poumon TC-1 traitées avec le crizotinib ont vacciné efficacement les souris immunocompétentes syngéniques contre la croissance des cellules vivantes de même type. Le crizotinib a amélioré l'efficacité de la chimiothérapie dans trois modèles de cancer du poumon orthotopiques : transplantable, induit par des carcinogènes et induites par les oncogènes. De façon remarquable, l’effet du crizotinib est aboli si un des signaux de l’ICD est bloqué. L'efficacité anticancéreuse dans chaque modèle s’est révélé être lié à l'infiltration de lymphocytes T, montrant l’implication d’une réaction immunitaire. Cela a été confirmé par des expériences chez des souris immunodéficientes (nu/nu, déficientes en thymodépendantes lymphocytes T) et dans des souris immunocompétentes dans lesquelles l’interféron gamma a été neutralisé à l’aide d’un anticorps ; l’effet du crizotinib était aboli dans les deux modèles. La combinaison du crizotinib avec le cisplatine a entraîné un accroissement de l'expression de PD-1, PDL-1 et CTLA-4 dans la tumeur, s’accompagnant par conséquent d'une sensibilisation importante des NSCLC à l'immunothérapie avec des anticorps anti-PD-1 et CTLA-4. Ainsi, la combinaison du crizotinib avec la chimiothérapie conventionnelle et les inhibiteurs des points de contrôle immunitaire peuvent être actifs contre le NSCLC. Les données présentées dans cette thèse pourraient faciliter la conception d’essais cliniques afin d’établir de nouvelles stratégies combinatoires pour le traitement des NSCLC. / Accumulating evidence suggests that certain conventional chemotherapies, radiotherapies, as well as targeted therapies mediate their long-term therapeutic success by inducing immunogenic cell death (ICD), which stimulate the release or exposure of danger-associated molecular patterns from or on cancer cells, causing their recognition by the immune system, thus reinstating immunosurveillance. An unbiased screen identified crizotinib as a tyrosine kinase inhibitor that is potent in provoking hallmarks of ICD. In subsequent low-throughput validation experiments, crizotinib promoted Calreticulin exposure, ATP secretion, HMGB1 release, as well as ER stress in both human and murine cancer cells, especially if it is combined with normally non-ICD inducing chemotherapeutics such as cisplatin. ICD induced by the combination of chemotherapy and crizotinib was also observed in non-small cell lung carcinoma (NSCLC) cells lacking activating mutations of the crizotinib targets ALK and ROS1, suggesting an off-target-mediated mode of action. Comparative studies indicated that exclusively the clinically used (R) isoform of crizotinib was efficient in inducing cell death and stimulating ICD hallmarks whereas the (S) enantiomer lacked those characteristics. When combined with cisplatin, crizotinib-killed fibrosarcoma MCA205 cells as well as lung cancer TC-1 cells efficiently vaccinated syngeneic immunocompetent mice against a re-challenge with live cancer cells of the same types. Crizotinib improved the efficacy of chemotherapy with non-ICD inducers (such as cisplatin and mitomycin C) on three distinct (transplantable, carcinogen- or oncogene induced) orthotopic NSCLC models, none of which relied on the activation of ALK or ROS1. Of note these anticancer effects were completely lost if any of the ICD signals was blocked. These anticancer efficacies in different models were linked to an increased T lymphocyte infiltration as a sign of an immune response and were lost if such tumors grew on immunodeficient (nu/nu) mice that are athymic and hence lack thymus-dependent T lymphocytes, or on immunocompetent mice with a neutralization of interferon-. The combination of cisplatin and crizotinib led to an increase in the expression of CTLA-4, PD-1 and PD-L1 in tumors, coupled to a strong sensitization of NSCLC to immunotherapy with antibodies blocking CTLA-4 and PD-1. Hence, a combination of crizotinib, conventional chemotherapy and immune checkpoint blockade may be active against NSCLC, and these data might facilitate the design of clinical trials to evaluated novel combination regiments for the treatment of NSCLC.
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Population ecology of the red admiral butterfly (Bassaris gonerilla) and the effects of non-target parasitism by Pteromalus puparumBarron, M. C. January 2004 (has links)
There is anecdotal evidence that populations of the New Zealand endemic red admiral butterfly Bassaris gonerilla (F.) have declined since the early 1900s. This decline has been associated with the introduction of the generalist pupal parasitoids Pteromalus puparum (L.) and Echthromorpha intricatoria (F.). The former was deliberately introduced for the biological control of the cabbage white butterfly (Pieris rapae (L.)); the latter is an adventitious arrival from Australia. The objective of this thesis was to quantify, using population models, the effect that P. puparum is having on B. gonerilla abundance. Population monitoring and a phenology model (based on temperature-related development rates) indicated that B. gonerilla has two full generations and one partial generation per summer in the Banks Peninsula region of New Zealand. B. gonerilla abundance was greatly reduced in drought summers, which was probably due to the negative effects of drought on the quality and quantity of the larval host plant Urtica ferox Forst. A life table study showed that egg parasitism by the unidentified scelionid Telenomus sp. was the largest mortality factor for the pre-imaginal stages of B. gonerilla, followed by "disappearance" mortality (predation and dispersal) in the larval stages. Pupal mortality due to P. puparum was lower compared with that caused by E. intricatoria, with 1-19% and 20-30% of pupae being parasitised by P. puparum and E. intricatoria, respectively. Collection of B. gonerilla pupae from the Christchurch, Dunedin and Wellington areas confirmed higher rates of percentage parasitism by E. intricatoria. B. gonerilla collected from the Banks Peninsula had a 50: 50 sex ratio and lifetime fecundity was estimated in the laboratory as 312 eggs per female. There was no evidence of density-dependent parasitism of B. gonerilla pupae by P. puparum in the field, although there was a significant positive relationship between life table estimates of E. intricatoria parasitism and B. gonerilla pupal abundance. Larval dispersal from the host plant showed a positive relationship with larval instar but no relationship with larval density. Rates of change in B. gonerilla adult abundance between generations within a year showed evidence of density dependence, and this negative feedback was stronger in a drought year. A discrete-time model for B. gonerilla population dynamics was constructed which had two summer generations per year and a partial overwintering generation. The model showed that the presence of this overwintering generation provides a temporal refuge from high levels of E. intricatoria parasitism. Removal of parasitoid mortality from the model suggested that P. puparum was suppressing B. Gonerilla populations on the Banks Peninsula by 5% and E. intricatoria by 30%. An important assumption of the model was that parasitism rates were independent of B. gonerilla density. This assumption appears valid for P. puparum parasitism, but may not be valid for E. intricatoria; therefore the estimated suppression levels due to this adventive parasitoid should be viewed with some caution. It is too soon to generalise on what determines the magnitude of non-target effects by arthropod biocontrol agents, this being only the second study to quantify effects at a population level. However, in this case retrospective analysis has shown that the impact of non-target parasitism by P. puparum on B. gonerilla abundance has been small. There is anecdotal evidence that populations of the New Zealand endemic red admiral butterfly Bassaris gonerilla (F.) have declined since the early 1900s. This decline has been associated with the introduction of the generalist pupal parasitoids Pteromalus puparum (L.) and Echthromorpha intricatoria (F.). The former was deliberately introduced for the biological control of the cabbage white butterfly (Pieris rapae (L.)); the latter is an adventitious arrival from Australia. The objective of this thesis was to quantify, using population models, the effect that P. puparum is having on B. gonerilla abundance. Population monitoring and a phenology model (based on temperature-related development rates) indicated that B. gonerilla has two full generations and one partial generation per summer in the Banks Peninsula region of New Zealand. B. gonerilla abundance was greatly reduced in drought summers, which was probably due to the negative effects of drought on the quality and quantity of the larval host plant Urtica ferox Forst.. A life table study showed that egg parasitism by the unidentified scelionid Telenomus sp. was the largest mortality factor for the pre-imaginal stages of B. gonerilla, followed by "disappearance" mortality (predation and dispersal) in the larval stages. Pupal mortality due to P. puparum was lower compared with that caused by E. intricatoria, with 1-19% and 20-30% of pupae being parasitised by P. puparum and E. intricatoria, respectively. Collection of B. gonerilla pupae from the Christchurch, Dunedin and Wellington areas confirmed higher rates of percentage parasitism by E. intricatoria. B. gonerilla collected from the Banks Peninsula had a 50: 50 sex ratio and lifetime fecundity was estimated in the laboratory as 312 eggs per female. There was no evidence of density-dependent parasitism of B. gonerilla pupae by P. puparum in the field, although there was a significant positive relationship between life table estimates of E. intricatoria parasitism and B. gonerilla pupal abundance. Larval dispersal from the host plant showed a positive relationship with larval instar but no relationship with larval density. Rates of change in B. gonerilla adult abundance between generations within a year showed evidence of density dependence, and this negative feedback was stronger in a drought year. A discrete-time model for B. gonerilla population dynamics was constructed which had two summer generations per year and a partial overwintering generation. The model showed that the presence of this overwintering generation provides a temporal refuge from high levels of E. intricatoria parasitism. Removal of parasitoid mortality from the model suggested that P. puparum was suppressing B. Gonerilla populations on the Banks Peninsula by 5% and E. intricatoria by 30%. An important assumption of the model was that parasitism rates were independent of B. gonerilla density. This assumption appears valid for P. puparum parasitism, but may not be valid for E. intricatoria; therefore the estimated suppression levels due to this adventive parasitoid should be viewed with some caution. It is too soon to generalise on what determines the magnitude of non-target effects by arthropod biocontrol agents, this being only the second study to quantify effects at a population level. However, in this case retrospective analysis has shown that the impact of non-target parasitism by P. puparum on B. gonerilla abundance has been small.
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