Reversion of the gal3 mutation of Escherichia coli: Partial deletion of the insertion sequence

Summary The gal3 mutation of E. coli is an insertion of a DNA sequence, 1,100 base pairs in length, into the operator-promoter region of the galactose operon. This mutation reverts spontaneously to gal⁺ by excision of the insertion to produce stable, inducible revertants, or by tandem duplications of the gal operon to produce unstable, constitutive revertants. The nature of a third class of revertants, which are stable and constitutive, is the subject of the present study.The stable, constitutive class of revertants included approximately 30% of all gal⁺ revertants obtained from a gal3() strain. Although the constitutive reversions could be transduced by , the efficiency was found to be extremely poor and the rare transductants which did appear seemed to originate from abnormal transducing particles. It was concluded that these reversions were not normally packaged by .In order to facilitate the packaging of these reversions, the chlD-pgl region was deleted from the parent gal3() strain. Unexpectedly, the gal3 mutation in the majority of these deletions reverted to produce stable, constitutive reversions exclusively. The explanation proposed was that the chlD-pgl deletions had also removed part of the gal operator-promoter up to the gal3 insertion, so that simple excisions of the insertion yielded stable, constitutive revertants by connecting the gal structural genes to a different promoter. These revertants were not considered to be true representatives of the stable, constitutive class. The specificity of deletion end-points at the insertion was found only in the gal3() strain, and not in gal ⁺, gal ⁺(), or gal3 strains. Moreover, the frequency of spontaneous chlD-pgl deletions increased 10- to 15-fold in presence of the gal3 insertion.A gal phage bearing a true stable, constitutive reversion (gal ^c 200) was isolated from the revertant strain by subsequent deletion of the chlD-pgl segment (31). Electron micrographs of gal ⁺ and gal ^c 200 31(chlD pgl) DNA heteroduplexes were interpreted to indicate that the stable, constitutive reversion had arisen by a deletion of 3/4 of the gal3 insertion sequence.The main conclusions are: (i) the stable, constitutive reversions of gal3 can arise by partial deletions of the insertion sequence, apparently by elimination of the nucleotide sequence which causes polarity; (ii) the chlD-pgl deletions may exhibit preferential termination at the right extremity of the gal3 insertion in presence of prophage ; and (iii) the gal3 insertion appears to inhibit the production of gal particles by providing a nucleotide sequence which is recognized and degraded by a specific endonuclease. It is suggested that inhibition of transducing particle formation by gal3 and the preferred termination of deletions at gal3 might represent related phenomena.     

Discovery of new nicotinamides as apoptotic VEGFR-2 inhibitors: virtual screening,synthesis, anti-proliferative,immunomodulatory, ADMET,toxicity, and molecular dynamic simulation studies

A library of modified VEGFR-2 inhibitors was designed as VEGFR-2 inhibitors. Virtual screening was conducted for the hypothetical library using in silico docking, ADMET, and toxicity studies. Four compounds exhibited high in silico affinity against VEGFR-2 and an acceptable range of the drug-likeness. These compounds were synthesised and subjected to in vitro cytotoxicity assay against two cancer cell lines besides VEGFR-2 inhibitory determination. Compound D-1 showed cytotoxic activity against HCT-116 cells almost double that of sorafenib. Compounds A-1, C-6, and D-1 showed good IC₅₀ values against VEGFR-2. Compound D-1 markedly increased the levels of caspase-8 and BAX expression and decreased the anti-apoptotic Bcl-2 level. Additionally, compound D-1 caused cell cycle arrest at pre-G1 and G2-M phases in HCT-116 cells and induced apoptosis at both early and late apoptotic stages. Compound D-1 decreased the level of TNF-α and IL6 and inhibited TNF-α and IL6. MD simulations studies were performed over 100 ns.     

Association of long non-coding RNAs NEAT1, and MALAT1 expression and pathogenesis of Behçet's disease among Egyptian patients

Behçet''s disease (BD) is a chronic inflammatory disease. Immunological defects have been shown to play a significant role in the progression of BD. The serum levels of two long non-coding RNAs (lncRNAs), NEAT1 and MALAT1, were examined in patients with BD to identify their role in the disease pathogenesis. Both lncRNAs were mentioned as essential regulators of innate immune responses and have a crucial role in inflammatory diseases. Fifty patients with BD and a similar number of control individuals were involved in our study. At enrollment, data was collected from patients and controls, and the disease severity in active cases was determined using the Behçet''s Disease Current Activity Form (BDCAF). Levels of the two studied biomarkers in the serum, NEAT1 and MALAT1, were investigated by quantitative RT-PCR (qRT-PCR). NEAT1 levels were significantly turned down in BD patients (fold changes = 0.77, p = 0.0001) and correlated negatively with the BDCAF (r = −0.41; p = 0.003). On the other hand, the MALAT1 levels were significantly up-regulated in BD patients (fold changes = 2.65, p = 0.003). Serum levels of NEAT1 were significantly decreased in patients with active states than in stationary cases (0.387 versus 1.99, respectively; p = 0.01) and compared with controls (p = 0.001). Also, NEAT1 levels were significantly increased in patients with stationary states compared to controls (p = 0.03). There was a positive association between NEAT1 and MALAT1 levels among BD patients (r = 0.29, p = 0.04). Our findings demonstrate a possible role of NEAT1 and MALAT1 in the pathogenesis of BD.     

Design and synthesis of thiazolidine-2,4-diones hybrids with 1,2-dihydroquinolones and 2-oxindoles as potential VEGFR-2 inhibitors: in-vitro anticancer evaluation and in-silico studies

A thiazolidine-2,4-dione nucleus was molecularly hybridised with the effective antitumor moieties; 2-oxo-1,2-dihydroquinoline and 2-oxoindoline to obtain new hybrids with potential activity against VEGFR-2. The cytotoxic effects of the synthesised derivatives against Caco-2, HepG-2, and MDA-MB-231 cell lines were investigated. Compound 12a was found to be the most potent candidate against the investigated cell lines with IC₅₀ values of 2, 10, and 40 µM, respectively. Furthermore, the synthesised derivatives were tested in vitro for their VEGFR-2 inhibitory activity showing strong inhibition. Moreover, an in vitro viability study against Vero non-cancerous cell line was investigated and the results reflected a high safety profile of all tested compounds. Compound 12a was further investigated for its apoptotic behaviour by assessing the gene expression of four genes (Bcl2, Bcl-xl, TGF, and Survivin). Molecular dynamic simulations authenticated the high affinity, accurate binding, and perfect dynamics of compound 12a against VEGFR-2.     

Rational design of biodegradable sulphonamide candidates treating septicaemia by synergistic dual inhibition of COX-2/PGE2 axis and DHPS enzyme

A new series of co-drugs was designed based on hybridising the dihydropteroate synthase (DHPS) inhibitor sulphonamide scaffold with the COX-2 inhibitor salicylamide pharmacophore through biodegradable linkage to achieve compounds with synergistic dual inhibition of COX-2/PGE2 axis and DHPS enzyme to enhance antibacterial activity for treatment of septicaemia. Compounds 5 b, 5j, 5n and 5o demonstrated potent in vitro COX-2 inhibitory activity comparable to celecoxib. 5j and 5o exhibited ED₅₀ lower than celecoxib in carrageenan-induced paw edoema test with % PGE2 inhibition higher than celecoxib. Furthermore, 5 b, 5j and 5n showed gastric safety profile like celecoxib. Moreover, in vivo antibacterial screening revealed that, 5j showed activity against S.aureus and E.coli higher than sulfasalazine. While, 5o revealed activity against E.coli higher than sulfasalazine and against S.aureus comparable to sulfasalazine. Compound 5j achieved the target goal as potent inhibitor of COX-2/PGE2 axis and in vivo broad-spectrum antibacterial activity against induced septicaemia in mice.     

Prediction and expression analysis of deleterious nonsynonymous SNPs of Arabidopsis ACD11 gene by combining computational algorithms and molecular docking approach

Accelerated cell death 11 (ACD11) is an autoimmune gene that suppresses pathogen infection in plants by preventing plant cells from becoming infected by any pathogen. This gene is widely known for growth inhibition, premature leaf chlorosis, and defense-related programmed cell death (PCD) in seedlings before flowering in Arabidopsis plant. Specific amino acid changes in the ACD11 protein’s highly conserved domains are linked to autoimmune symptoms including constitutive defensive responses and necrosis without pathogen awareness. The molecular aspect of the aberrant activity of the ACD11 protein is difficult to ascertain. The purpose of our study was to find the most deleterious mutation position in the ACD11 protein and correlate them with their abnormal expression pattern. Using several computational methods, we discovered PCD vulnerable single nucleotide polymorphisms (SNPs) in ACD11. We analysed the RNA-Seq data, identified the detrimental nonsynonymous SNPs (nsSNP), built genetically mutated protein structures and used molecular docking to assess the impact of mutation. Our results demonstrated that the A15T and A39D mutations in the GLTP domain were likely to be extremely detrimental mutations that inhibit the expression of the ACD11 protein domain by destabilizing its composition, as well as disrupt its catalytic effectiveness. When compared to the A15T mutant, the A39D mutant was more likely to destabilize the protein structure. In conclusion, these mutants can aid in the better understanding of the vast pool of PCD susceptibilities connected to ACD11 gene GLTP domain activation.