Rapid and direct spectrophotometric method for kinetics studies and routine assay of peroxidase based on aniline diazo substrates

Peroxidases are ubiquitous enzymes that play an important role in living organisms. Current spectrophotometrically based peroxidase assay methods are based on the production of chromophoric substances at the end of the enzymatic reaction. The ambiguity regarding the formation and identity of the final chromophoric product and its possible reactions with other molecules have raised concerns about the accuracy of these methods. This can be of serious concern in inhibition studies. A novel spectrophotometric assay for peroxidase, based on direct measurement of a soluble aniline diazo substrate, is introduced. In addition to the routine assays, this method can be used in comprehensive kinetics studies. 4-[(4-Sulfophenyl)azo]aniline (λ_max?=?390?nm, ??=?32 880 M^?1 cm^?1 at pH 4.5 to 9) was introduced for routine assay of peroxidase. This compound is commercially available and is indexed as a food dye. Using this method, a detection limit of 0.05?nmol mL^?1 was achieved for peroxidase.     

<Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis> Increases the Anti-apoptotic Micro RNA-21 and Decreases the Pro-inflammatory Micro RNA-155 in the LPS-Treated Human Endothelial Cells

Given the anti-inflammatory and protective role of probiotics in atherosclerosis and the regulatory role of micro RNA (miRNA) in endothelial cell (dys) functions, this study aimed to investigate the effect of Lactobacillus acidophilus (La) on cellular death and the expression of miRNA-21, 92a, 155, and 663 in human umbilical vein endothelial cell (HUVEC) induced by Escherichia coli lipopolysaccharide (Ec-LPS). LPS-treated and untreated HUVECs were cultured in the presence of different La conditions such as La-conditioned media (LaCM), La water extract (LaWE), La culture-filtered (LaFS) and unfiltered supernatants (LaUFS). After 24 h, apoptosis, necrosis and the levels of the mentioned miRNAs were measured using flow cytometry and real-time PCR methods, respectively. LaCM decreased apoptosis, necrosis and inflammatory miR-155 and conversely increased anti-apoptotic miR-21 in Ec-LPS-treated HUVECs. Association analysis revealed negative correlations between necrosis and the levels of miR-21, miR-92a, and miR-155. The beneficial effects of L. acidophilus on the ECs death and expression of atherosclerosis related miRNAs in these cells imply a new aspect of its regulation in cardiovascular diseases rather than previously described ones and suggest this probiotic bacterium as a candidate in the preventative therapy of atherosclerosis.     

Increased Serum Levels of IL-17A and IL-23 Are Associated with Decreased Vitamin D3 and Increased Pain in Osteoarthritis

IntroductionOsteoarthritis (OA) is the most common type of arthritis and proinflammatory cytokines have been considered as the main etiologic factor in the pathogenesis of the disease. Serum levels of cytokines, that are associated with innate immunity and TH1 cells, have been analyzed in OA patients, however, there is limited research that profiles cytokines associated with Th17 cells and their relation to vitamin D3 and pain.ResultsSerum levels of IL-17A, and IL-23 were statistically higher in OA patients than in healthy controls, while IL-21 and vitamin D3 were significantly lower in OA patients when compared to controls. A significant positive correlation was found between the serum levels of IL-17A and IL-23 using WOMAC pain scores and vitamin D3 serum levels.DiscussionThe results suggest that IL-17A plays a significant role in OA pathogenesis and the induction of pain. Decreased serum levels of vitamin D3 may reflect a positive role played by the factor in the regulation of immune responses in OA patients.     

In vitro knock-out of miR-155 suppresses leukemic and HCV virus loads in pediatric HCV-4–associated acute lymphoid leukemia: A promising target therapy

Hepatitis C virus (HCV) infection is a major public health problem, having a high prevalence in Egypt. Leukemia and lymphoma have been associated with HCV infection. MicroRNA-155 (miR-155) has been reported to play a regulatory role in cancer, inflammation, and immune response to infection. The expression level of miR-155 in HCV viremic patients is controversial; although high miR-155 levels were demonstrated in HCV genotypes 1,2, and 3, low levels of miR-155 were detected in Egyptian patients with HCV genotype 4. Several studies have investigated the correlation between the levels of miRNA-155 and the replication of HCV, others have evaluated miRNA-155 as a prognostic biomarker in different types of cancer. No studies have investigated the impact of miRNA-155 knockdown on HCV pediatric patients associated with childhood acute lymphoblastic leukemia (ALL). We knocked-out the miR_155a in cultured polymorphonuclear cells (PBMCs) obtained from 60 children with ALL; 30 were associated with HCV-4 infection and 30 were HCV negative. The miR_155a, HCV viral load, and cell proliferation werre assessed in treated and untreated cells using TaqMan assay quantitative polymerase chain reaction. We found that miRNA-155 was significantly upregulated by seven folds in the HCV-4 associated ALL group; while being linked to high HCV viral load and leukemic burden, miR_155a knock-out can improve the disease outcome. We conclude that miR-155 is a critical miRNA that is considered a therapeutic target in pediatric HCV leukemic patients.     

Optimization of immersion frequency and medium substitution on microtuberization of Chlorophytum borivilianum in RITA system on production of saponins

In this study, different immersion times were compared for optimization of Chlorophytum borivilianum microtuber production in a RITA (Récipient à Immersion Temporaire Automatique) system. Significantly higher mean number and length of microtubers and growth index were obtained (16, 52 mm and 16, respectively) with 15 min immersion in microtuberization medium (MM) for every 60 min rest period, and could eliminate the occurrence of hyperhydricity. The substitution of MM with liquid hormone-free MS (MSO) medium on week 6 of culture differed significantly in terms of mean number and length of microtubers and growth index compared to earlier substitution at week 3 with MSO but did not differ significantly in comparison to cultures maintained throughout the 9 weeks in MM. Comparison of total saponin of TIS-derived microtubers with tubers of field grown mother plant showed a 1.3-fold increase of saponin (≈21% diosgenin) in the microtubers. The above results hold a promising application of TIS for large scale production of microtubers and accumulated saponin in C. borivilianum.     

Characterization of uropathogenic E. coli O25b-B2-ST131, O15:K52:H1, and CGA: Neutrophils apoptosis,serum bactericidal assay,biofilm formation,and virulence typing

Pathogenic and drug-resistant strains of Escherichia coli (E. coli) O25b-B2-ST131, O15:H1-D-ST393, and CGA (clonal group A) clonal groups have spread worldwide. This study aimed at determining E. coli epidemic clonal groups, their virulence factors, biofilm formation, neutrophils apoptosis, and antimicrobial resistance pattern of uropathogenic E. coli. A total of 95 CTX-M-1-producing E. coli clinical isolates were enrolled. E. coli O25b-B2-ST131, CGA, and O15:K52:H1 were identified by serotyping and phylogrouping and allele-specific polymerase chain reaction-based assay. Antibiotic susceptibility, biofilm formation, hemolysis, and human serum bactericidal assay were performed. Neutrophil apoptosis was assayed by flow cytometry. Nine E. coli clonal groups including six O25b-B2-ST131 strains, two CGA, and one O15:K52:H1-D-ST393 strains were detected. One O25b-B2-ST131 isolate was a strong biofilm-producer. Three ST131 isolates had type I fimbriae. Furthermore, all the CGA and O15:K52:H1 and three of ST131 isolates harbored the P fimbriae. The virulence genes ompT, fimH, and traT were detected among all the clonal groups. The apoptosis was induced by O25b-B2-ST131, CGA, and O15:K52:H1 E. coli. There was no significant difference regarding apoptosis induction among clonal groups. Furthermore, the presence of the cdt, usp, and vat genes was significantly associated with the apoptosis of neutrophils by O25b-B2-ST131, CGA, and O15:K52:H1-D-ST393 clonal groups.