Resistance to Direct-Acting Antiviral Agents in Treatment of Hepatitis C Virus Infections

Compounds targeting nonstructural (NS) proteins of hepatitis C virus (HCV) demonstrate clinical promise, suggesting that NS3/NS4a, NS5A, or NS5B inhibitors are potential components in direct-acting antiviral (DAA) combination therapies. In vitro studies revealed dramatic inhibition of viral replication or alteration in subcellular localization of NS proteins. DAAs bind either to catalytic sites (NS3 and NS5B) or to domain-1 of NS5A. Although >90% of the patients clear HCV RNA from their sera, a significant portion of cirrhotic patients suffer from resistance or virological relapse. Mutations in specific residues (Q80K) in NS3 (M28, A30, L31, and Y93 in genotypes 1a and 1b or L28, L30, M31, and Y93 in genotype 4) in NS5A and A282T in NS5B are associated with resistance to DAA [resistance-associated variants (RAVs)]. Current knowledge on the NS functions, mode of action of DAAs, and impacts of RAVs on treatment response are discussed. Not only mutations affecting the binding of DAAs to target proteins but also substitutions affecting the replication fitness of mutant quasispecies are major determinants of treatment failures. These resistance-associated substitutions (RASs) are now considered the major viral mutants that influence the virological outcome after DAA treatment

Evaluation of the genetic effects of the in vitro antimicrobial activities of Rhazya stricta leaf extract using molecular techniques and scanning electron microscope

Rhazya stricta plants have always played a major role in the treatment of human and animal diseases and it has main role in the folk medicine. The aim of this study was to explore the potential antimicrobial activities of the aqueous leaves extract of R. stricta on Gram-negative and Gram-positive food-borne bacteria and evaluate the antimicrobial effect at the molecular level. The results indicate that the aqueous leaves extract of R. stricta exhibited the antimicrobial activity against tested microorganisms. A clear, but significantly smaller, inhibition zones were formed after the treatment of two Gram-negative bacteria (Escherichia coli and Aeromonas hydrophila) and one Gram-positive bacteria (Staphylococcus aureus) with the aqueous leaves extract of R. stricta (50 mg) comparing with those formed after the treatment with streptomycin (15 mg). Moreover, the results obtained after the treatments of bacterial strains with elevated concentrations of aqueous extracts of the wild plant of R. stricta leaves reveled that the extract has potent lethal activities as the growth turbidity decreased as the concentration or time of exposure increased. In addition, the observation by the scanning electron microscope showed that cells of the bacterial strains were damaged after the treatment with plant extracts. The noticed antimicrobial effect was explored at the molecular level, using restriction fragment length polymorphism (RFLP) analysis of the plasmid DNA and random amplification of polymorphic DNA (RAPD) analysis of the genomic DNA extracted from the control (untreated) and R. stricta leaf extract-treated bacterial strains. The results demonstrate polymorphic band pattern for most treated microbes compared with the wild type (untreated) strain. Concerning gene expression under the same conditions, total protein contents of the three treated bacteria showed significantly gradual increase in all of the treatment doses compared to control. In addition, the SDS-PAGE of the bacterial cellular proteins resulted in the induction of some protein bands under the treatment conditions. All these results strongly point out the mutagenicity, lethal and antimicrobial effect of the leaves extract of R. stricta. The results indicate the possibility of using the leaves extract of R. stricta as a source of antibacterial compounds for treatment of infections caused by multi-drug resistant (MDR) bacterial pathogens.Keywords: Medicinal plants, Rhazya stricta, antimicrobial, mutagenicity, RAPD, RFLP, SEM, E. coli, S. aureus, A. hydrophilaAfrican Journal of Biotechnology Vol. 12(21), pp. 3171-318

Genetic diversity and DNA fingerprint study in tomato (Solanum lycopersicum L) cultivars grown in Egypt using simple sequence repeats (SSR) markers

A collection of ten cultivars of tomato grown in Egypt were screened with 20 simple sequence repeat (SSR) primers in order to determine genetic identities, genetic diversity and genetic relationships among these  cultivars. On an average, 38 alleles were amplified using SSR primers with scorable fragment sizes ranging  from approximately 75 to 275 bp. 23 alleles were polymorphic thus revealing 60.5% of polymorphism. The  genetic similarity estimated according to SSR data was scaled between 17.6 and 93.2%, suggesting the  potential of SSR markers in discriminating among plants of close or distant genetic backgrounds. Unweighted pair group method with arithmetic mean (UPGMA) clustering grouped the cultivars into two groups where the  two Egyptian cultivars Edkawy and Giza 80 were clustered in different group. In addition, clustering was found  consistent with the known information regarding growth habit. The genetic distance information obtained in  this study might be useful to breeder for planning crosses among these cultivars.Key words: Tomato cultivars, diversity, Simple sequence repeats (SSR), Egypt

Increased expression of T- cell- surface CXCR4 in asthmatic children

Background: Signals delivered through the chemokine receptor CXCR4 upon interaction with its ligand, SDF-1 α/β result in the most efficacious chemoattraction of T lymphocytes to the asthmatic airways with the resultant lung inflammation and airway hyperresponsiveness. Objective: The extensive pharmacological and physiological evidence that CXCR4 chemokine receptor influences the allergic airway disease has stimulated us to study the relation between its expression in peripheral blood T lymphocytes and the exacerbation of asthmatic attacks of varying severity. Methods: The chemokine receptor CXCR4 was assayed by flow cytometry in peripheral blood T lymphocytes from 25 asthmatic children, during asthma exacerbation and after complete remission of symptoms and physical signs. The results were compared to those of 30 healthy children. Results: The CXCR4 expression in peripheral blood T lymphocytes was significantly increased in children with acute exacerbations of bronchial asthma as compared to controls (mean ± SD = 62.27 ± 17.57% versus 24.76 ± 6.88%; p < 0.001). After remission of acute attacks, the CXCR4 expression decreased significantly as compared to the values during attacks (mean ± SD = 40.90 ± 13.25%), however, the level of expression during quiescence was still significantly higher than the values of the controls (mean ± SD = 40.90 ± 13.25%; p < 0.001). The CXCR4 expression was significantly higher in children with acute severe asthma as compared to those with either mild or moderate attacks. During remission, patients with mild intermittent asthma had less expression of CXCR4 when compared to any grade of persistent asthma, while the results were comparable between all groups of persistent asthma of varying severity. A significant positive correlation could link the CXCR4% to the absolute eosinophilic count during acute asthma attacks. Conclusion: CXCR4 is over-expressed in T lymphocytes of asthmatic children. It was found to be related to disease activity and seems to be involved in the establishment and maintenance of chronic inflammation of the airways.Keywords: Asthma, chemokine receptors, CXCR4, children, T lymphocytesEgypt J Pediatr Allergy Immunol 2003; 1(2): 80-

Clinical significance of anti-Scl-70 antibody estimation in pediatric patients with systemic lupus erythematosus

Background: Some patients with scleroderma have overlap features with systemic lupus erythematosus (SLE). Anti-Scl-70 antibody was reported in as many as 35% of patients with scleroderma and signifies an increased risk for the development of pulmonary fibrosis and pulmonary hypertension. This antibody was detected in 25% of adult patients with SLE. Objective: We hypothesized that a particular subset of lupus patients might be at an increased risk of certain complications if anti-Scl-70 antibody was elevated in their sera. Methods: Serum anti-Scl-70 antibody levels were assayed by ELISA from 34 pediatric patients with SLE and from 24 healthy controls. Patients were also subjected to clinical evaluation for system involvement and for disease activity by systemic lupus erythematosus disease activity index (SLEDAI). The ESR, serum anti DNA antibody, serum complement 3, creatinine clearance and 24 hours urinary protein excretion were assessed and renal biopsy for histopathology was performed in selected cases. Results: Anti Scl-70 antibody was elevated (> 25 U/ml) in 6 SLE patients (18%), borderline (15-25 U/ml) in 17 patients (50%) and normal (< 15 U/ml) in 11 of them (32%). The patient group had significant elevation of serum anti-Scl-70 antibody levels (mean [SD] = 32.5 [8.8] U/ml) as compared to the controls, (mean [SD]= 9.3 [4.1] U/ml; p < 0.001). Patients with clinical lupus nephritis had significant higher levels of this autoantibody as compared to those without clinically evident renal involvement. Also, pulmonary hypertension was found significantly related to the high serum levels of anti-Scl-70 antibody. A significant positive correlation could link anti-Scl-70 levels to the ESR values and SLEDAI scores. Anti-Scl-70 levels were neither affected by the presence of neuropsychiatric involvement nor by intake of cytotoxic drugs. Conclusion: Anti-Scl-70 antibody is present in a significant subset of patients with SLE. For this subset, it offers a good correlate of disease activity and suggests an increased risk for pulmonary hypertension and renal involvement.Keywords: SLE; anti-Scl-70; pulmonary hypertension; lupus nephritis; SLEDAIEgypt J Pediatr Allergy Immunol 2006; 4(2): 53-60

A reproducible protocol for regeneration and transformation in canola (Brassica napus L.)

The objective of the present study is to develop an efficient protocol for shoot and plant regeneration using five commercial canola cultivars grown under the Egyptian agricultural conditions. The regeneration efficiency from hypocotyl explants was examined. The data indicated that embryonic calli were formed within two weeks in the presence of 1 mgl-1 2,4-D. Adventitious shoots emerged from the embryonic callus in the presence of 4.5 mgl-1 BA. The cultivars showed a varied response to shoot regeneration. Regeneration frequency was high in the cultivar Sarow-4 (68%) followed by Masrri L-16 (64%) compared with the other cultivars tested. Hypocotyl explants from the cultivars Sarow-4 and Semu-249 were inoculated and co-cultivated with Agrobacterium tumefaciens strain LBA4404 harboring a binary vector pBI-121 containing the neomycin phosphotransferase-II gene (NPT-II). The resulted putative transgenic plantlets were able to grow under knanamycin containing medium. The stable integration of the NPT-II gene into the plant genomes was tested by PCR using NPT-II -specific primers. The GUS gene expression can be detected only in the transgenic plants. The reported protocol in the present study is repeatable and can be used to regenerate transgenic canola plants expressing the genes present in A. tumifaciens binary vectors.Keywords: Agrobacterium, canola, GUS assay, regeneration, fransformation, NPT II gen

SOIL EROSION BY TILLAGE IN RAINFED N-W. EGYPTION COAST

Tillage erosion is one of the main causes of land degradation. The objective of the present study is to evaluate the effect of tillage systems (up and downslope and contour tillage), soil conditions (consolidated and loosened soils), tillage depth, speed and slope on tillage erosion rate. Field experiments of variable slopes (3 - 16%) were established on sandy soil of Wadi El Ramala, west Mersa Matruh City. Soil translocation as affected by tillage systems and soil conditions were examined. In addition, soil losses by tillage erosion and water erosion were measured and evaluated. The experimental results showed that the average displacement distance is a function of gradient slopes, soil condition, tillage system, depth and speed. The validation of the soil translocation model developed by (Van Muysen et al 2000), under different gradient slopes, tillage depth, speed and soil condition were evaluated. This validation showed that variations in tracer displacement distance can be successfully predicted. Consequently, such model can be used under sandy soil. Finally, experiment results showed that tillage operations with a chisel plow under present agricultural practices are responsible for the major field redistribution of soil. Furthermore, it is clear that tillage of a loosened soil is far more erosive than tillage of a consolidated soil, where the tillage transport coefficient (K) was 105 kg.m-1 per tillage operation for consolidated soil and 179 kg.m-1 for loosened soil under contour tillage. However, 256 kg.m-1 per tillage prevailed for consolidated soil and 454 kg.m-1 for loosened soil under up and downslope tillage treatments