scholarly journals Danio Rerio as Model Organism for Adenoviral Vector Evaluation

Genes ◽  
2019 ◽  
Vol 10 (12) ◽  
pp. 1053
Author(s):  
Paola Gulías ◽  
Jorge Guerra-Varela ◽  
Manuela Gonzalez-Aparicio ◽  
Ana Ricobaraza ◽  
Africa Vales ◽  
...  
Keyword(s):  
Danio Rerio ◽  
Viral Vector ◽  
Early Stage ◽  
High Capacity ◽  
Model Organism ◽  
Adenoviral Vectors ◽  
Regulatory Agencies ◽  
In Vivo Model ◽  
Efficient Alternative

Viral vector use is wide-spread in the field of gene therapy, with new clinical trials starting every year for different human pathologies and a growing number of agents being approved by regulatory agencies. However, preclinical testing is long and expensive, especially during the early stages of development. Nowadays, the model organism par excellence is the mouse (Mus musculus), and there are few investigations in which alternative models are used. Here, we assess the possibility of using zebrafish (Danio rerio) as an in vivo model for adenoviral vectors. We describe how E1/E3-deleted adenoviral vectors achieve efficient transduction when they are administered to zebrafish embryos via intracranial injection. In addition, helper-dependent (high-capacity) adenoviral vectors allow sustained transgene expression in this organism. Taking into account the wide repertoire of genetically modified zebrafish lines, the ethical aspects, and the affordability of this model, we conclude that zebrafish could be an efficient alternative for the early-stage preclinical evaluation of adenoviral vectors.

scholarly journals Simultaneous Assessment of the Efficacy and Toxicity of Marine Mollusc–Derived Brominated Indoles in an In Vivo Model for Early Stage Colon Cancer

2017 ◽  
Vol 17 (2) ◽  
pp. 248-262 ◽  
Author(s):  
Babak Esmaeelian ◽  
Kirsten Benkendorff ◽  
Richard K. Le Leu ◽  
Catherine A. Abbott
Keyword(s):  
Colon Cancer ◽  
Natural Products ◽  
Liver Toxicity ◽  
Early Stage ◽  
Blood Biochemistry ◽  
In Vivo Model ◽  
Liver Histopathology ◽  
Genotoxic Carcinogens ◽  
Early Stage Colon Cancer

The acute apoptotic response to genotoxic carcinogens animal model has been extensively used to assess the ability of drugs and natural products like dietary components to promote apoptosis in the colon and protect against colorectal cancer (CRC). This work aimed to use this model to identify the main chemopreventative agent in extracts from an Australian mollusc Dicathais orbita, while simultaneously providing information on their potential in vivo toxicity. After 2 weeks of daily oral gavage with bioactive extracts and purified brominated indoles, mice were injected with the chemical carcinogen azoxymethane (AOM; 10 mg/kg) and then killed 6 hours later. Efficacy was evaluated using immunohistochemical and hematoxylin staining, and toxicity was assessed via hematology, blood biochemistry, and liver histopathology. Comparison of saline- and AOM-injected controls revealed that potential toxic side effects can be interpreted from blood biochemistry and hematology using this short-term model, although AOM negatively affected the ability to detect histopathological effects in the liver. Purified 6-bromoisatin was identified as the main cancer preventive agent in the Muricidae extract, significantly enhancing apoptosis and reducing cell proliferation in the colonic crypts at 0.05 mg/g. There was no evidence of liver toxicity associated with 6-bromoisatin, whereas 0.1 mg/g of the brominated indole tyrindoleninone led to elevated aspartate aminotransferase levels and a reduction in red blood cells. As tyrindoleninone is converted to 6-bromoisatin by oxidation, this information will assist in the optimization and quality control of a chemopreventative nutraceutical from Muricidae. In conclusion, preliminary data on in vivo safety can be simultaneously collected when testing the efficacy of new natural products, such as 6-bromoisatin from Muricidae molluscs for early stage prevention of colon cancer.

scholarly journals Herpes Simplex Virus Type 1 Thymidine Kinase Sequence Fused to the lacZ Gene Increases Levels of β-Galactosidase Activity per Genome of High-Capacity but Not First-Generation Adenoviral Vectors In Vitro and In Vivo

2008 ◽  
Vol 83 (4) ◽  
pp. 2004-2010 ◽  
Author(s):  
M. Puntel ◽  
R. J. Barrett ◽  
S. Mondkar ◽  
V. Saxena ◽  
K. M. Kroeger ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Transgene Expression ◽  
First Generation ◽  
High Capacity ◽  
Adenoviral Vectors ◽  
Simplex Virus

ABSTRACT Increased transgene expression per vector genome is an important goal in the optimization of viral vectors for gene therapy. Herein we demonstrate that herpes simplex virus type 1 (HSV1) thymidine kinase (TK) gene sequences (1,131 bp) fused to the 3′ end of lacZ increase transgene expression from high-capacity adenoviral vectors (HCAd), but not from first-generation (Ad) vectors. The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), in contrast, increased transgene expression levels from Ad but not HCAd vectors. The differential activity of the HSV1 TK gene and WPRE sequences was detected both in vitro and in vivo and suggests potentially different mechanisms of action or the interaction of these elements with vector genomic sequences.

scholarly journals Optimization of Non-Thermal Plasma Treatment in an In Vivo Model Organism

PLoS ONE ◽  
2016 ◽  
Vol 11 (8) ◽  
pp. e0160676 ◽  
Author(s):  
Amanda Lee ◽  
Abraham Lin ◽  
Kajol Shah ◽  
Harpreet Singh ◽  
Vandana Miller ◽  
...  
Keyword(s):  
Plasma Treatment ◽  
Thermal Plasma ◽  
Model Organism ◽  
In Vivo Model ◽  
Non Thermal Plasma

scholarly journals Development and Validation of Endovascular Chemotherapy Filter Device for Removing High-Dose Doxorubicin: Preclinical Study

2014 ◽  
Vol 8 (4) ◽  
Author(s):  
Anand S. Patel ◽  
Maythem Saeed ◽  
Erin J. Yee ◽  
Jeffrey Yang ◽  
Gregory J. Lam ◽  
...  
Keyword(s):  
Flow Model ◽  
Vena Cava ◽  
High Capacity ◽  
Ion Exchange Resin ◽  
In Vivo Model ◽  
High Dose ◽  
Single Pass ◽  
Porcine Serum

To develop a novel endovascular chemotherapy filter (CF) able to remove excess drug from the blood during intra-arterial chemotherapy delivery (IAC), thus preventing systemic toxicities and thereby enabling higher dose IAC. A flow circuit containing 2.5 mL of ion-exchange resin was constructed. Phosphate-buffered saline (PBS) containing 50 mg doxorubicin (Dox) was placed in the flow model with the hypothesis that doxorubicin would bind rapidly to resin. To simulate IAC, 50 mg of doxorubicin was infused over 10 min into the flow model containing resin. Similar testing was repeated with porcine serum. Doxorubicin concentrations were measured over 60 min and compared to controls (without resin). Single-pass experiments were also performed. Based on these experiments, an 18F CF was constructed with resin in its tip. In a pilot porcine study, the device was deployed under fluoroscopy. A control hepatic doxorubicin IAC model (no CF placed) was developed in another animal. A second CF device was created with a resin membrane and tested in the infrarenal inferior vena cava (IVC) of a swine. In the PBS model, resin bound 76% of doxorubicin in 10 min, and 92% in 30 min (P < 0.001). During IAC simulation, 64% of doxorubicin bound in 10 min and 96% in 60 min (P < 0.001). On average, 51% of doxorubicin concentration was reduced during each pass in single pass studies. In porcine serum, 52% of doxorubicin bound in 10 min, and 80% in 30 min (P < 0.05). CF device placement and administration of IAC were successful in three animals. No clot was present on the resin within the CF following the in vivo study. The infrarenal IVC swine study demonstrated promising results with up to 85% reduction in peak concentration by the CF device. An endovascular CF device was developed and shown feasible in vitro. An in vivo model was established with promising results supporting high-capacity rapid doxorubicin filtration from the blood that can be further evaluated in future studies.

scholarly journals Expression of glucose-dependent insulinotropic polypeptide in the zebrafish

2009 ◽  
Vol 297 (6) ◽  
pp. R1803-R1812 ◽  
Author(s):  
Michelle C. Musson ◽  
Lisa I. Jepeal ◽  
Patrick D. Mabray ◽  
Irina V. Zhdanova ◽  
Wellington V. Cardoso ◽  
...  
Keyword(s):  
Early Stage ◽  
Model Organism ◽  
Receptor Activation ◽  
Evolutionary Development ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Glucagon Receptor ◽  
Nutrient Deposition ◽  
Zebrafish Larvae

In mammals, glucose-dependent insulinotropic polypeptide (GIP) is synthesized predominately in the small intestine and functions in conjunction with insulin to promote nutrient deposition. However, little is known regarding GIP expression and function in early vertebrates like the zebrafish, a model organism representing an early stage in the evolutionary development of the compound vertebrate pancreas. Analysis of GIP and insulin ( insa) expression in zebrafish larvae by RT-PCR demonstrated that although insa was detected as early as 24 h postfertilization (hpf), GIP expression was not demonstrated until 72 hpf, shortly after the completion of endocrine pancreatic development but prior to the commencement of independent feeding. Furthermore, whole mount in situ hybridization of zebrafish larvae showed expression of GIP and insa in the same tissues, and in adult zebrafish, RT-PCR and immunohistochemistry demonstrated GIP expression in both the intestine and the pancreas. Receptor activation studies showed that zebrafish GIP was capable of activating the rat GIP receptor. Although previous studies have identified four receptors with glucagon receptor-like sequences in the zebrafish, one of which possesses the capacity to bind GIP, a functional analysis of these receptors has not been performed. This study demonstrates interactions between the latter receptor and zebrafish GIP, identifying it as a potential in vivo target for the ligand. Finally, food deprivation studies in larvae demonstrated an increase in GIP and proglucagon II mRNA levels in response to fasting. In conclusion, the results of these studies suggest that although the zebrafish appears to be a model of an early stage of evolutionary development of GIP expression, the peptide may not possess incretin properties in this species.

scholarly journals Evaluation of Cosmetic Properties of Natural Ingredients in the Trás-os-Montes area: a PhD Project

2021 ◽  
Author(s):  
Sara Gonçalves ◽  
Isabel Gaivão
Keyword(s):  
Molecular Mechanisms ◽  
Developmental Stages ◽  
Human Lymphocytes ◽  
Ecological Impact ◽  
Model Organism ◽  
The Body ◽  
In Vivo Model ◽  
Dna Integrity ◽  
The European Union

The term cosmetics refers to a product applied to the body for the purpose of beautifying, cleansing or improving appearance and enhancing attractive features. The natural cosmetics market has grown since the consumer took consciousness of the concept of natural-based ingredients. A great number of cosmetics have noxious and chemically-potent substances and have an ecological impact on the environment. A study performed by the Danish Council THINK Chemicalsfound that in total 65 chemicals of concern were found in 39 products. This means consumers are exposed to these chemicals, perhaps in a daily basis. They also found that three products contained illegal ingredients in the European Union. Thus, the use of natural and organic cosmetics becomes increasingly important. This requires a strong investigation into the benefits that fruits and plants can bring to health. The PhD project will focus on four natural ingredients common in the Trás-os-Montes area: almond (Prunus dulcis), elderberry (Sambucus nigra), olive (Olea europaea) and grapes (Vitis vinifera). The general purpose of this PhD project is to evaluate the cosmetic properties of the natural ingredients towards the DNA integrity promotion. Additionally, it is intended to evaluate genoprotection, longevity and prolificacy of the natural ingredients in Drosophila melanogaster. The short life cycle, the distinct developmental stages, the availability of various tools and reagents, known genome sequence and the physiological similarity of Drosophila with humans make them an excellent in vivo model organism to rapidly test toxicity in whole organism and elucidate the molecular mechanisms underlying the toxicity. The natural product with the best result will be used to evaluate genoprotection in human lymphocytes. These are used as a surrogate tissue, as they are easily obtained, in large numbers, do not require cell culture, are diploids and are almost all in the same phase of the cell cycle. This project is in an initial phase and lacks results, which will be available along this year.

Thrombin Generation in Zebrafish

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4218-4218
Author(s):  
Evelien Schurgers ◽  
Martijn Moorlag ◽  
Hilde Kelchtermans ◽  
Coenraad Hemker ◽  
Bas De Laat
Keyword(s):  
Whole Blood ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Genetic Research ◽  
Model Organism ◽  
Lag Time ◽  
In Vivo Model ◽  
Time To Peak ◽  
Paper Disk

Abstract Introduction Recently, the whole blood calibrated automated thrombogram (CAT) was miniaturized enabling the measurement of thrombin generation (TG) in a limited volume (5 µl) of whole blood. Consequently, this approach may be used to determine TG in small lab animals. Zebrafish are readily available test animals for genetic research. However, their small size has been a hurdle in thrombosis and hemostasis research since most assays require large amounts of plasma. In this study we verified the possibility to measure TG in zebrafish using our newly developed miniaturized whole blood assay. Methods For TG, 5 µl of whole blood was mixed with 5 µl of buffer containing a rhodamin-based thrombin-sensitive P2Rho substrate (final concentration (fc) 300 µM). 5 µl of this mixture was put on a paper disk and covered with mineral oil to prevent evaporation. Calibration was done as described previously (Ninivaggi et al. Clin Chem 2012) by adding 5 µl of whole blood to 5 µl of a mixture containing P2Rho (fc 300 µM), a2M-thrombin calibrator (fc 100 nM) and citrate (fc 9,8 mM). Fluorescence was detected with a fluorometer (485/538 nm). Results Due to their limited blood volume, it is impossible to perform both a TG and calibrator measurement on the same fish. Since calibrator measurements performed on blood from different fish demonstrated acceptable variation (CV calibrator slopes < 15%), the average calibrator slope was used for calculations. The calculated TG parameters from 2 independent experiments are depicted in Table 1. TG measured in individual fish showed the same amount of inter-individual variation as in humans. Striking differences with human TG parameters were the short lag time, high peak and high velocity index. Moreover a further analysis of the fibrin network of the clot, by means of scanning electron microscopy (SEM), showed a much denser network composed of thinner fibers compared to humans. Table 1. Thrombin generation parameters and their coefficient of variance (CV) in zebrafish in two independent experiments. Experiment 1(n=4) Experiment 2(n=9) Mean CV Mean CV Peak ETP (nM.min) 575.68 19.69 689.71 24.91 Peak (nM) 1573.16 29.58 1668.05 14.46 Lag time (min) 0.27 0.00 0.49 10.92 Time to peak (min) 1.08 5.30 1.37 11.49 Velocity (nM/min) 4002.33 38.21 3826.35 17.04 Conclusion These results demonstrate the feasibility of measuring TG in whole blood collected from zebrafish. Consequently, zebrafish may be used as a in vivo model to test the effect of (novel) anticoagulant therapeutics on thrombin generation and serve as a model organism for mechanistical research in thrombosis and haemostasis. Disclosures No relevant conflicts of interest to declare.

Characterization and subcellular localization of a bacterial flotillin homologue

Microbiology ◽  
2009 ◽  
Vol 155 (6) ◽  
pp. 1786-1799 ◽  
Author(s):  
Catriona Donovan ◽  
Marc Bramkamp
Keyword(s):  
Early Stage ◽  
Model Organism ◽  
Cell Types ◽  
Specific Gene ◽  
Cell Type ◽  
Distinct Cell ◽  
Sporulation Efficiency ◽  
Cell Type Specific ◽  
Detergent Resistant Membranes

The process of endospore formation in Bacillus subtilis is complex, requiring the generation of two distinct cell types, a forespore and larger mother cell. The development of these cell types is controlled and regulated by cell type-specific gene expression, activated by a σ-factor cascade. Activation of these cell type-specific sigma factors is coupled with the completion of polar septation. Here, we describe a novel protein, YuaG, a eukaryotic reggie/flotillin homologue that is involved in the early stages of sporulation of the Gram-positive model organism B. subtilis. YuaG localizes in discrete foci in the membrane and is highly dynamic. Purification of detergent-resistant membranes revealed that YuaG is associated with negatively charged phospholipids, e.g. phosphatidylglycerol (PG) or cardiolipin (CL). However, localization of YuaG is not always dependent on PG/CL in vivo. A yuaG disruption strain shows a delay in the onset of sporulation along with reduced sporulation efficiency, where the spores develop to a certain stage and then appear to be trapped at this stage. Our results indicate that YuaG is involved in the early stage of spore development, probably playing a role in the signalling cascade at the onset of sporulation.

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