Emerging Gene Correction Strategies for Muscular Dystrophies: Scientific Progress and Regulatory Impact

Muscular dystrophies comprise a heterogeneous cluster of inherited muscle degenerative disorders with the common feature of progressive muscle weakness. These represent good candidates for treatment with gene-based therapies. Progress in gene transfer technologies has raised hopes for successful therapeutic restoration of mutated genes such as dystrophin in Duchenne muscular dystrophy. Delivery to enough muscle cells, however, remains a challenge for a successful gene replacement therapy. Other approaches based on exon skipping to correct mutant dystrophin’s pre-mRNA splicing patterns have been tried, and partial restoration of dystrophin expression was reported in late-stage clinical trials, but full therapeutic efficacy is yet to be confirmed. The emergence of gene editing and its recent success in AIDS have opened a new therapeutic era for muscular dystrophies. This chapter will cover new gene correction strategies for muscular dystrophies and their regulatory challenges before they can become routine treatment modalities in the clinic

A High-level Petri Net Based Approach for Modeling and Composition of Web Services

AbstractWeb services are modular, self-describing, self-contained and loosely coupled applications, which intercommuni-cate via messages exchanging. The evolution of the internet and the emergence of new technologies like e-business have influenced the use of these last ones, which have become popular. The composition of web services is a topic that attracts the interest of researchers. It offers complex problems process ability even with simple existing web services while cooperating with each other. However, modeling tools and formal techniques for the completion of this task are required.In this paper, we show how simple existing web services can be composed, in order to create a composite service, which offers new features. In this context, we propose an expressive object-oriented Petri net based algebra that succeeds in the complex composition of Web services

MOLECULAR CHARACTERIZATION OF METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS ISOLATED FROM CLINICAL CASES IN EAST ALGERIA

Objective: The purpose of this study is to investigate the methicillin resistance gene, and some virulence factors in methicillin-resistant Staphylococcus aureus (MRSA) isolates by polymerase chain reaction (PCR).Methods: This study has included 12 MRSA isolates. All isolates were previously identified as S. aureus by a standard microbiological procedure, and a detection of methicillin resistance was realized by phenotypic methods. Following genomic DNA extraction, the presence of gyrA, mecA, lukPV, and tst genes was analyzed by duplex PCR. All retained S. aureus species have been found to contain gyrA gene.Results: Ten stains have been found to harbor mecA gene indicating it's responsibility for methicillin resistance in those strains. Among the 12 strains, six of which were found to be Panton-Valentine leukocidine positive while none of which has tst gene encoding the toxic shock syndrome toxin.Conclusion: The pathogenesis of MRSA infections is related to the expression of a wide variety of virulence factors.Keywords: Methicillin-resistant Staphylococcus aureus, Panton-Valentine leukocidine, Toxic shock syndrome toxin 1, Multidrug resistance, mecA

Pyrosequencing identification of Mycobacterium tuberculosis W-Beijing

<p>Abstract</p> <p>Background</p> <p>The worldwide expanding <it>Mycobacterium tuberculosis </it>W-Beijing family is associated with treatment failure and relapse. Its identification currently relies on spoligotyping and conventional sequencing. We developed pyrosequencing as an alternative method for its identification.</p> <p>Findings</p> <p>Pyrosequencing found a G/A substitution in the Rv0927c-pstS3 intergenic spacer and a RD105 deletion, identifying 8/104 <it>M. tuberculosis </it>isolates as W-Beijing isolates. In addition, pyrosequencing found a previously unreported TGC deletion in the Rv0927c gene of W-Beijing isolates. Total concordance was found between the pyrosequencing data and conventional sequencing, as well as reference molecular identification. Multispacer Sequence Typing assigned the W-Beijing isolates to the Asian lineage and the 96 non-W-Beijing isolates to the Euro-American lineage (P < 10^-5). The W-Beijing isolates were all susceptible to streptomycin, rifampin, isoniazid, ethambutol, and pyrazinamide; no resistance-associated mutations were detected in these eight W-Beijing isolates. There were no statistically significant differences in the antibiotic susceptibility of W-Beijing and non-W-Beijing isolates (<it>p </it>= 0.2, X²test). Pyrosequencing correctly identified <it>M. tuberculosis </it>organisms in 26/26 sputum specimens exhibiting acid-fast bacilli. Pyrosequencing results were obtained within four hours, incurring an estimated cost of 1.86 €/test.</p> <p>Conclusion</p> <p>Pyrosequencing of the Rv0927c gene and adjacent intergenic spacer is an efficient, low-cost technique for the rapid identification of W-Beijing isolates.</p

SAFETY ASSESSMENT OF LINUM USITATISSIMUM (LINN.) INGESTION IN NEW ZEALAND RABBITS

Background: The therapeutic safety of herbal medicine is a major concern for consumers and users. After studying the effects of linseed on hair growth in rabbits, the turn is to evaluate its safety by the observation of some clinical, biological and anatomo-pathological aspects. Materials and Methods: A study was conducted during a period of three months on two groups of rabbits (control and test). Test group daily received feed supplemented with 3g of ground linseed while the control animals received the same feed without any additives. Weekly, rabbits were weighed and monthly blood samples were taken. By the end of the trial, liver and kidneys biopsies were analyzed for histological and cytological lesions. Results: There was no significant improvement in weight gain in the test group rabbits, in which biochemical parameters had differentially evolved with a decrease in their Glycemia and cholesterolemia. There were also no modifications in their serum hepatic and renal marker enzymes and their liver and kidneys exhibited noticeably normal histology without any anatomically detectable anomalies. Conclusion: These findings confirm that prolonged linseed ingestion in rabbits is safe

Quantum dot labelling of adenovirus allows for highly sensitive single cell flow and imaging cytometry

A quantum dot method for highly efficient labelling of single adenoviral particles is developed. The technique has no impact on viral fitness and allows the imaging and tracking of virus binding and internalisation events using a variety of techniques including imaging cytometry and confocal microscopy. The method is applied to characterise the tropism of different adenoviral vectors

Caractérisation des bactéries lactiques autochtones productrices d¿exopolysaccharides et étude de leurs effets intrinsèques sur une matrice laitière

Tesis llevada a cabo para conseguir el grado de Doctor por la Université des Frères Mentouri Constantine 1.--2020-11-02[FR] Les produits laitiers naturellement fermentés constituent une source intéressante de nouvelles souches de bactéries lactiques (BL) au potentiel technologique avéré. Certaines d¿entre elles, sont susceptibles de synthétiser des exopolysaccharides (EPS) au cours de la fermentation, qui améliorent les propriétés rhéologiques des matrices laitières. Ce travail de thèse porte sur la sélection de BL productrices d¿EPS, à partir de produits laitiers fermentés locaux. Au total, 30 échantillons ont été collectés dans l¿Est de l¿Algérie, à partir desquels 584 isolats présumés des BL ont été testés pour la production des EPS. Seuls 18 isolats ont présenté un phénotype producteur, dont la caractérisation polyphasique a confirmé leur appartenance aux deux espèces : Leuconostoc mesenteroides et Lactobacillus plantarum. Une présélection basée sur la stabilité du caractère de production d¿EPS a permis de retenir 6 souches en plus d¿une souche témoin de phénotype non producteur, toutes affiliées à l¿espèce Lactobacillus plantarum. La caractérisation macroscopique de leurs gels lactiques, a permis ensuite de sélectionner deux souches LBIO1 et LBIO28 produisant des EPS viscosifiants en milieu laitier. L¿étude de leurs productions par les méthodes gravimétriques et chromatographiques a révélé que les polymères produits sont des métabolites primaires, de haut poids moléculaire. En parallèle, une caractérisation de la macro et microstructure au microscope confocale à balayage laser des deux gels lactiques SMCLBIO1 et SMCLBIO28, comparativement au gel témoin de SMCLBIO14, a conclu que les EPS des deux souches filantes ont contribué significativement à l¿amélioration des attributs physiques des matrices laitières. D¿autre part, les micrographies réalisées par cryo-microscopie électronique ont montré l¿existence d¿un web-like EPS dense attaché aux cellules des deux souches filantes contrairement à celle de LBIO14. Enfin, un séquençage complet des trois génomes suivi d¿une analyse...[EN] The naturally fermented dairy products are an interesting source of new strains of lactic acid bacteria (LAB) with proven technological potential. Some of them are capable of synthesizing exopolysaccharides (EPS) during fermentation, which improve the rheological properties of dairy matrices. This thesis work focuses on the selection of LAB producing EPS from local fermented dairy products. A total of 30 samples were collected in Eastern Algeria, from which 584 presumptive isolates of LAB were tested for the production of EPS. Only 18 isolates presented a productive phenotype, their polyphasic characterization confirmed that they belong to two species: Leuconostoc mesenteroides and Lactobacillus plantarum. A preselection based on the stability of EPS production character made it possible to retain 6 strains in addition to a control strain, all affiliated to the Lactobacillus plantarum species. Then macroscopic characterization of their lactic gels allowed to select two strains LBIO1 and LBIO28 producing viscosifying EPS in a dairy matrix. The study of their production revealed that the polymers produced are primary metabolites, with a high molecular weight. In parallel, a characterization of the macro and microstructure under confocal laser scanning microscopy of the two gels SMCLBIO1 and SMCLBIO28, compared to the control gel SMCLBIO14, concluded that the EPS of the two ropy strains contribute significantly to the improvement of the physical attributes of lactic gels. Furthermore, micrographs made by cryo-electron microscopy showed the existence of a dense web-like EPS attached to the cells for both ropy strains, which is not observed for LBIO14. Finally, a complete sequencing of the three genomes followed by an in silico genomic analysis confirmed the safety of the strains and the existence of genes coding for bacteriocins and vitamins. The study of the biosynthetic machinery of EPS in comparison with the reference strain Lactobacillus plantarum WCF1 revealed the presence of 2 complete eps clusters cps3 and cps4 in the three genomes with 100% similarity between them, all chromosomal. However, the two strains LBIO1 and LBIO28 have in addition genes belonging to cps2, which would therefore be involved in the synthesis of the ropy polymer with high molecular weight. The two ropy strains LBIO1 and LBIO28 can therefore be exploited in dairy fermentation, based on the techno-functional properties of their polymers, which contribute to the improvement of the rheological characteristics of the matrices generated

Targeting polymer coated adenovirus to tumour-associated vasculature

Tumour-associated vasculature provides an accessible target for systemic gene therapy using targeted adenoviruses. The aim of this thesis is to develop strategies for targeting adenovirus infection to tumour-associated endothelium.Adenovirus expressing luciferase (Adluc) was coated with an amino-reactive polymer based on poly [N-(2-hydroxypropyl) methacrylamide] [pHPMA] to ablate normal infection pathways¬. This was a pre-requisite to redirecting virus tropism to infect endothelial cells via specific receptors.Direct attachment to the pHPMA-adenovirus (pcAdluc) of ligands including vascular endothelial growth factor (VEGF165) and a monoclonal antibody (RAFL) recognising VEGF receptor 2 (VEGFR-2) retargeted infectivity to VEGFR-2-positive endothelial cells and not to receptor-negative cells. Specificity of transduction in vitro was shown by competition with excess antibody. In vivo however, the VEGF165-retargeted virus failed to transduce tumour-associated endothelia following systemic administration. Similarly, direct linkage of a monoclonal antibody against E-Selectin (MHES) demonstrated E-Selectin-specific transduction of tumour necrosis factor-α (TNF-α)-activated endothelial cells, although overall levels of infection were not increased compared to unmodified Adluc.A two-component targeting system using protein A or protein G as ‘bridging’ agents was developed to ensure the required orientation of targeting antibodies. Using this system MHES mediated greater transduction of TNF-α-activated endothelial cells than Adluc. Conjugation using protein A also gave non-specific effects which were not seen with protein G. Whereas the unmodified Adluc virus failed to transduce TNF-α-activated endothelium in an umbilical vein model ex vivo, the MHES-protein G-pHPMA-adenovirus (MHES-StrepGpcAdluc) mediated good transduction. Similarly, StrepGpcAdluc retargeted with a chimeric P-Selectin Glycoprotein Ligand-1 (PSGL-1)-Fc fusion protein, showed good circulation kinetics and significant uptake into HepG2 xenografts following intravenous administration. Histological studies suggested selective targeting to tumour-associated endothelial cells.Overall these findings support the assertion that tumour-associated vasculature is an accessible target for systemic gene delivery, and the use of protein G as bridging agent facilitates rapid screening of Fc-bearing ligands for retargeting pcAd infection to tumour-associated endothelium.</p