Development of a non-viral gene therapy strategy for choroideremia

Non-viral plasmids harbouring scaffold matrix attachment regions (S/MARs) provide stable and functional episomal maintenance, resistance to epigenetic silencing and have been shown to be capable of sustained expression in murine tissues. S/MARs are an attractive alternative to conventional viral gene delivery vectors due to their low toxicity and reduced immunogenicity. This thesis documents the development of this novel non-viral episomal plasmid DNA (pDNA) expression system for persistently expressing vectors for Choroideremia (CHM) gene therapy. To this end, our goal was to analyse the ability of S/MAR plasmids to generate efficient and stable transgene expression in the mouse retina following subretinal injections. We generated and analysed a series of S/MAR constructs expressing either REP1 cDNA or the reporter enhanced green fluorescent protein (EGFP) or Luciferase (Luc) genes driven by the elongation factor-1 short (EFS) or cytomegalovirus (CMV) early enhancer/chicken beta actin (CAG) promoter. We demonstrate using the AtT20 cell line, the principle that S/MAR containing plasmids can provide long-term episomal EGFP and REP1 transgene expression in vitro. Furthermore, long-term REP1 expression was also analysed in CHM fibroblasts, where we showed in preliminary experiments that DNA constructs expressing REP1 were able to rescue CHM derived cells by rescue of their Rab27a prenylation defect and provide long-term expression of hREP1 after transfection of human and mouse CHM fibroblasts. We also developed an optimised method for delivering S/MAR containing plasmids in the eye via subretinal injections of complexed pDNAs, and provide evidence for the utility and versatility of S/MAR plasmids in ocular tissue. We show for the first time the longitudinal transgene expression of Luc in the retina as measured using bioluminescent imaging. Long- term maintenance and expression of EGFP and REP1 transgenes were also observed by PCR and Western blot analysis. Further analysis provided evidence for the long term episomal maintenance of these vectors in the eye as shown by Southern analysis. We provide evidence for the superiority of an S/MAR containing plasmid in providing long-term expression in the eye. All control pDNA constructs were shown to be incapable of sustaining significant transgene expression beyond one-month following pDNA delivery. We further demonstrate the lack of toxicity within the eye and show that fundus examinations as well as detailed histological examinations of retinal sections do not elicit an inflammatory response to our plasmids once subretinally injected in the eye. Finally, we explored the therapeutic potential of the REP1-S/MAR episome, by subretinally injecting mouse models of CHM. Further repeat experiments in CHM mouse models would add confidence to the overall experiments; however initial findings were encouraging where a partial correction of the REP1 protein and functional rescue in the eye was shown.Open Acces

Antipseudomonal activity of Artemisia quettensis Podlech essential oil and its synergy with imipenem

Context: The problems associated with hospital infections caused by Pseudomonas aeruginosa, and the emergence of new and the re-emergence of old infectious diseases have become increasingly evident. Therefore, medicinal plants take precedence over the development of new antibacterial agents. The combination effects of antibiotics and plant compounds might be an appropriate solution for microbial resistance and useful method for assessment of synergistic interactions for inhibition of bacterial growth. This study is an experimental design for the discovery and finding of natural and harmless compounds for the treatment of infectious diseases. Aim: To determine the antibacterial potency of Artemisia quettensis essential oil, and in combination with imipenem, to inhibit the growth of Pseudomonas aeruginosa. Methods: The essential oil was obtained through hydrodistillation from aerial parts of the plant and analysis using GC and GC-MS. To demonstrate the in vitro antibacterial activity of the essential oil against Pseudomonas aeruginosa (ATCC 27853) disc diffusion assay was used, either alone or in combination with a standard antibiotic. Results: The most dominant components were homoadamantane (9.38%), Camphor (7.91%) and Eugenol (10.46%). The oil and antibiotic showed high antibacterial activity against Pseudomonas aeruginosa with minimal inhibitory concentration (MIC) 0.5 µL/mL and 16 µg/mL and minimal bactericidal concentration (MBC) 4 µL/mL and 32 µL/mL, respectively. The synergistic effect of the oil and antibiotic showed MIC 0.2 µL/mL and 4 µg/mL and MBC 2 µL/mL and 8 µL/mL, respectively. This study showed that Artemisia quettensis oil has significant antibacterial activity against Pseudomonas aeruginosa infections. Conclusions: The essential oil exhibited synergism with imipenem displaying the ability to enhance the activity of this compound and it may be useful in the fight against emerging microbial drug resistance

5,10-methylene tetrahydrofolate reductase C677T gene polymorphism, homocysteine concentration and the extent of premature coronary artery disease in Southern Iran

Elevated level of plasma homocysteine (Hcy) has been identified as an independent risk factor for coronary artery disease (CAD). Furthermore, numerous studies have documented the influences of a common polymorphism (C677T) of methylenetetrahydrofolate reductase (MTHFR) on homocysteine levels. However the relationship between this mutation and cardiovascular diseases (CVD) has remained as a controversial issue. The present study was undertaken to investigate the relationship between C677T polymorphism of MTHFR gene, plasma total Hcy levels and the number of affected vessels as a criterion for the extent of CAD. MTHFR genotypes and plasma homocysteine (HCY) concentrations were examined in 231 patients and 300 healthy subjects who underwent diagnostic coronary angiography. A multiple linear regression analysis was performed to identify the predictors of Hcy levels whereas logistic regression model was built to determine the association of Hcy quartiles with the risk of CAD adjusted for risk factors. The prevalence of MTHFR genotypes was similar between CAD patients and non-CAD individuals while the geometric mean of Hcy values was significantly higher in patient group (14.13 ± 4.11 μmol/l) than in control group (10.19 ± 3.52 μmol/l) (P < 0.001). Moreover, unlike the MTHFR polymorphism, Hcy concentration increased with increasing number of stenosed vessels and the CAD risk increased about 2 folds in the top two Hcy quartiles (≥ 17.03 and 13.20-17.02 μmol/l) compared with the lowest quartile (≤ 9.92 μmol/l) after controlling for conventional risk factors (P<0.001 for both). Our data suggest that hyperhomocysteinaemia (HHcy) is significantly associated to CAD risk increase as well as to the extent of coronary atherosclerosis

Effect of quercetin on methotrexate-induced hepatic and renal damages in male rats

Abstract Background and purpose: Methotrexate as a chemotherapy drug causes chronic liver damage, infiltration of neutrophils, oxidative stress, and direct renal tubular damage. Quercetin is a flavonoid with antioxidant and anti-inflammatory properties. Therefore, the aim of this study was to investigate the effect of quercetin on eliminating the liver and kidney toxicity of methotrexate. Materials and methods: In this experimental study, 32 rats were divided into 4 groups. Group I (control) was given regular diet. Group II received single-dose methotrexate. Group III received methotrexate + a single dose quercetin and the last group (positive control) received methotrexate + a single dose silymarin. After five days, blood samples were taken and the serum GOT, GPT, ALP, Cr, urea and antioxidant capacity of plasma were measured. Some parts of liver and kidney were removed to measure the liver and kidney SOD, MDA, catalase activity and histopathological studies. Results: Serum GOT, GPT, ALP, Cr, and liver and kidney MDA were significantly higher (P<0.05) in group II, compared with those of the control group. These parameters significantly decreased (P<0.05) in group III. Compared to the control group, antioxidant capacity of plasma, activity of the liver and kidney SOD, catalase and serum urea decreased significantly in group II (P<0.05). Administration of quercetin significantly increased these parameters (P<0.05) and decreased hepatic and renal lymphocyte infiltration. Conclusion: According to the results, administration of quercetin could have a protective role in preventing liver and renal toxicity induced by methotrexate which could be due to its antioxidant property

The clinical and genetic spectrum of autosomal-recessive TOR1A-related disorders.

In the field of rare diseases, progress in molecular diagnostics led to the recognition that variants linked to autosomal-dominant neurodegenerative diseases of later onset can, in the context of biallelic inheritance, cause devastating neurodevelopmental disorders and infantile or childhood-onset neurodegeneration. TOR1A-associated arthrogryposis multiplex congenita 5 (AMC5) is a rare neurodevelopmental disorder arising from biallelic variants in TOR1A, a gene that in the heterozygous state is associated to torsion dystonia-1 (DYT1 or DYT-TOR1A), an early-onset dystonia with reduced penetrance. While 15 individuals with TOR1A-AMC5 have been reported (less than 10 in detail), a systematic investigation of the full disease-associated spectrum has not been conducted. Here, we assess the clinical, radiological and molecular characteristics of 57 individuals from 40 families with biallelic variants in TOR1A. Median age at last follow-up was 3 years (0-24 years). Most individuals presented with severe congenital flexion contractures (95%) and variable developmental delay (79%). Motor symptoms were reported in 79% and included lower limb spasticity and pyramidal signs, as well as gait disturbances. Facial dysmorphism was an integral part of the phenotype, with key features being a broad/full nasal tip, narrowing of the forehead and full cheeks. Analysis of disease-associated manifestations delineated a phenotypic spectrum ranging from normal cognition and mild gait disturbance to congenital arthrogryposis, global developmental delay, intellectual disability, absent speech and inability to walk. In a subset, the presentation was consistent with fetal akinesia deformation sequence with severe intrauterine abnormalities. Survival was 71% with higher mortality in males. Death occurred at a median age of 1.2 months (1 week - 9 years) due to respiratory failure, cardiac arrest, or sepsis. Analysis of brain MRI studies identified non-specific neuroimaging features, including a hypoplastic corpus callosum (72%), foci of signal abnormality in the subcortical and periventricular white matter (55%), diffuse white matter volume loss (45%), mega cisterna magna (36%) and arachnoid cysts (27%). The molecular spectrum included 22 distinct variants, defining a mutational hotspot in the C-terminal domain of the Torsin-1A protein. Genotype-phenotype analysis revealed an association of missense variants in the 3-helix bundle domain to an attenuated phenotype, while missense variants near the Walker A/B motif as well as biallelic truncating variants were linked to early death. In summary, this systematic cross-sectional analysis of a large cohort of individuals with biallelic TOR1A variants across a wide age-range delineates the clinical and genetic spectrum of TOR1A-related autosomal-recessive disease and highlights potential predictors for disease severity and survival

A methodology for identifying critical links and estimating macroscopic fundamental diagram in large-scale urban networks

The Macroscopic Fundamental Diagram (MFD), which exhibits the relationship between average flow and average density of an urban network, is a promising framework for monitoring and controlling urban traffic networks. Given that monitoring resources (e.g. loop detectors, probe vehicle data, etc.) are limited in real-world networks, acquiring adequate data to estimate an MFD is of crucial importance. This study presents a novel, network-wide approach to identifying critical links and estimating average traffic flow and density. The proposed model estimates the MFD using flow and density measurements from those critical links, which constitute only a small subset of all the links in the network. To find the critical links, we rely on historical probe vehicle data, and propose a model that builds on Principal Component Analysis (PCA), a dimensionality reduction and a feature selection method. Essentially, using PCA, a large number of possibly interrelated variables in a dataset can be reduced to a set of smaller uncorrelated variables, while maintaining as much information as possible in the dataset. The resulting uncorrelated variables, or the principal components, indicate the major patterns or the dominating features of the original dataset. Additionally, PCA enables the (approximate) reconstruction of the full-scale dataset from the selected features (or principal components). In this work, we apply PCA in order to identify the main traffic features from a probe vehicle dataset; then, we find the links that are associated with these features (i.e., critical links); then, we locate loop detectors on those links to collect flow and density data; and finally, we reconstruct the full-scale data, building on the PCA mechanism. This gives us the flow and density of all links, from which we can effectively estimate the MFD

Management Science Letters A new memetic algorithm for solving split delivery vehicle routing problem

Split delivery vehicle routing problem is one of the traditional types of routing problems in which the demand of different points can be divided among vehicles and the objective is to minimize the path length, which vehicles travel. In this paper, fuel cost of vehicles which is assumed to be dependent on their traveled path and load is considered as the objective functions. Namely, the cost of the consumed fuel is proportionate to the unit of load carried per unit of distance. In order to solve the proposed model a new memetic algorithm is developed which has two rows. The performance of the proposed algorithm for 21 standard problems is compared with the optimum solutions obtained from mathematical programming standard solver and the solutions of the same algorithm with single row solution representation. The results express the efficiency of developed algorithm. Growing Science Ltd. All rights reserved.

A new memetic algorithm for solving split delivery vehicle routing problem

Split delivery vehicle routing problem is one of the traditional types of routing problems in which the demand of different points can be divided among vehicles and the objective is to minimize the path length, which vehicles travel. In this paper, fuel cost of vehicles which is assumed to be dependent on their traveled path and load is considered as the objective functions. Namely, the cost of the consumed fuel is proportionate to the unit of load carried per unit of distance. In order to solve the proposed model a new memetic algorithm is developed which has two rows. The performance of the proposed algorithm for 21 standard problems is compared with the optimum solutions obtained from mathematical programming standard solver and the solutions of the same algorithm with single row solution representation. The results express the efficiency of developed algorithm