Discovery of potent polyphosphate kinase 1 (PPK1) inhibitors using structure‐based exploration of PPK1Pharmacophoric space coupled with docking analyses

Inorganic polyphosphate (polyP) is present in all living forms of life. Studied mainly in prokaryotes, polyP and its associated enzymes are vital in diverse metabolic activities, in some structural functions, and most importantly in stress responses. Bacterial species, including many pathogens, encode a homolog of a major polyP synthesis enzyme, Poly Phosphate Kinase (PPK) with 2 different genes coding for PPK1 and PPK2. Genetic deletion of the ppk1 gene leads to reduced polyP levels and the consequent loss of virulence and stress adaptation responses. This far, no PPK1 homolog has been identified in higher‐order eukaryotes, and, therefore, PPK1 represents a novel target for chemotherapy. The aim of the current study is to investigate PPK1 from Escherichia coli with comprehensive understanding of the enzyme's structure and binding sites, which were used to design pharmacophores and screen a library of compounds for potential discovery of selective PPK1 inhibitors. Verification of the resultant inhibitors activities was conducted using a combination of mutagenic and chemical biological approaches. The metabolic phenotypic maps of the wild type E. coli (WT) and ppk1 knockout mutant were generated and compared with the metabolic map of the chemically inhibited WT. In addition, biofilm formation ability was measured in WT, ppk1 knockout mutant, and the chemically inhibited WT. The results demonstrated that chemical inhibition of PPK1, with the designed inhibitors, was equivalent to gene deletion in altering specific metabolic pathways, changing the metabolic fingerprint, and suppressing the ability of E. coli to form a biofilm.     

Human C-reactive protein gene polymorphism and metabolic syndrome are associated with premature coronary artery disease

The aim of this study was to investigate the association between C-reactive protein (CRP) gene polymorphism and metabolic syndrome (MetS) with premature coronary artery disease (PCAD). 116 patients with PCAD (58 with MetS and 58 without MetS) and 119 controls were included in the study. CRP gene + 1059 G>C polymorphism was analyzed by polymerase chain reaction. Serum hs-CRP was measured using high-sensitivity enzyme-linked immunosorbent assay. Carriers of C allele of the CRP + 1059 G>C polymorphism had 3.37 fold increased risk to develop MetS in patients with PCAD. In addition CRP gene and hs-CRP levels were independent risk factors for PCAD and MetS. The present study provides new evidence that the presence of CRP + 1059 G>C polymorphism and hs-CRP levels are independent determinants of PCAD and MetS in Egyptians. The results of our study suggest a synergistic effect of CRP C allele with classical risk factors such as hypertension, obesity, dyslipidemia and MetS.     

Design of HIV-1 integrase inhibitors targeting the catalytic domain as well as its interaction with LEDGF/p75: a scaffold hopping approach using salicylate and catechol groups

HIV-1 integrase (IN) is a validated therapeutic target for antiviral drug design. However, the emergence of viral strains resistant to clinically studied IN inhibitors demands the discovery of novel inhibitors that are structurally as well mechanistically different. Herein, we describe the design and discovery of novel IN inhibitors targeting the catalytic domain as well as its interaction with LEDGF/p75, which is essential for the HIV-1 integration as an IN cofactor. By merging the pharmacophores of salicylate and catechol, the 2,3-dihydroxybenzamide (5a) was identified as a new scaffold to inhibit the strand transfer reaction efficiently. Further structural modifications on the 2,3-dihydroxybenzamide scaffold revealed that the heteroaromatic functionality attached on the carboxamide portion and the piperidin-1-ylsulfonyl substituted at the phenyl ring are beneficial for the activity, resulting in a low micromolar IN inhibitor (5p, IC(50)=5 μM) with more than 40-fold selectivity for the strand transfer over the 3'-processing reaction. More significantly, this active scaffold remarkably inhibited the interaction between IN and LEDGF/p75 cofactor. The prototype example, N-(cyclohexylmethyl)-2,3-dihydroxy-5-(piperidin-1-ylsulfonyl) benzamide (5u) inhibited the IN-LEDGF/p75 interaction with an IC(50) value of 8 μM. Using molecular modeling, the mechanism of action was hypothesized to involve the chelation of the divalent metal ions inside the IN active site. Furthermore, the inhibitor of IN-LEDGF/p75 interaction was properly bound to the LEDGF/p75 binding site on IN. This work provides a new and efficient approach to evolve novel HIV-1 IN inhibitors from rational integration and optimization of previously reported inhibitors.     

In Vitro Selection and Identification of Potential Probiotics Isolated from the Gastrointestinal Tract of Nile Tilapia, <Emphasis Type="Italic">Oreochromis niloticus</Emphasis>

Fish gut bacteria can be used as probiotics for aquaculture. The aim of this study is to screen and identify beneficial probiotic bacteria from the gut of Nile tilapia, Oreochromis niloticus. Nine out of one hundred thirty-five isolates were non-pathogenic through intraperitoneal injection and had antibacterial activities with at least a strain from the five isolated fish pathogens, Aeromonas sobria, Aeromonas hydrophila, Pseudomonas aeruginosa, Pseudomonas putida, and Staphylococcus aureus. Further tests showed that such isolates can survive in the presence of high bile concentration (10%) and at different acidic pH values. A strains (14HT) was sensitive to all selected antibiotics, two strains were (9HT and 11HT) resistant to streptomycin and three strains (9HT, 11HT and 38HT) had resistance to two antibiotics. Four isolates (11HT, 33HT, 38HT and 41HT) had an amylase and a protease activities and one strain (47HT) showed only amylase activity. Based on 16S rRNA gene analysis, the isolated strains were identified as follows: Lactococcus lactis (8HT, 9HT, 11HT and 33HT); Enterococcus faecalis (14HT), Lysinibacillus sp. (38HT) and Citrobacter freundii (39HT, 41HT and 47HT).     

<Emphasis Type="Italic">Braf</Emphasis>, <Emphasis Type="Italic">Kras</Emphasis> and <Emphasis Type="Italic">Helicobacter pylori</Emphasis> epigenetic changes-associated chronic gastritis in Egyptian patients with and without gastric cancer

We aimed to study MLH1 and MGMT methylation status in Helicobacter pylori-associated chronic gastritis in Egyptian patients with and without gastric cancer. 39 patients were included in our study. They were divided into 2 groups; patients without (group I) and with gastric adenocarcinoma (group II). Patients were subjected to clinical examination, abdominal ultrasound and upper endoscopy for gastric biopsy. Biopsies were subjected to urease test, histological examination, and DNA purification. H. pylori, Braf, Kras, MLH1 and MGMT methylation were assessed by quantitative PCR. DNA sequencing was performed to assess Braf and Kras genes mutation. qPCR of H. pylori was significantly higher in patients with adenocarcinoma (group II) than those without adenocarcinoma (group I); with a p < 0.001 as well as in patients with age above 50 years with a p value = 0.008. By applying logistic regression analysis it was reported that the H. pylori qPCR is a significant predictor to the adenocarcinoma with OR = 1.025 (95 % CI: 1. 002–1.048), with sensitivity of 90 % and specificity of 100 %. Adenocarcinoma patients had a significantly higher mean age and levels of H. Pylori, Braf, K-ras, methylated MGMT and methylated MLH1 than those of gastritis patients. DNA sequence analysis of Braf (codon 12) and Kras (codon 600) had genes mutation in gastric adenocarcinoma versus chronic gastritis. Conclusion: H. pylori may cause epigenetic changes predisposing the patients to cancer stomach. Estimation of H. pylori by qPCR can be a good predictor to adenocarcinoma. Braf and Kras genes mutation were reveled in gastritis and adenocarcinoma patients.     

The role of calcium,silicon and salicylic acid treatment in protection of canola plants against boron toxicity stress

Boron (B) toxicity often limits crop yield and the quality of production in agricultural areas. Here, we investigated the effects of calcium (Ca), silicon (Si) and salicylic acid (SA) on development of B toxicity, B allocation in canola (Brassica napus cultivar Sarw 4) and its role in non-enzymatic antioxidants in relation to yield of this cultivar under B toxicity. Canola seedlings were subjected to four B levels induced by boric acid in the absence or presence of Ca, Si and SA. The results showed that Ca, Si and SA addition ameliorated the inhibition in canola growth, water content (WC), and improved siliqua number, siliqua weight and seed index. The B content in shoots and roots and total B accumulation in the whole plant were increased in control plants under B-toxicity-stress, and these parameters were significantly decreased by addition of Ca, Si and SA. The shoot ascorbate pool (ascorbate, AsA, and dehydroascorbate, DHA), α-tocopherol and phenolics (free and bound) were increased under B toxicity, and were significantly decreased in most cases by addition of Ca, Si and SA, except α-tocopherol, which increased at low B levels (0, 25 and 50 mg kg soil^?1). The glutathione content did not obviously change by B stress, while added Ca, Si and SA inhibited its accumulation under B stress. In addition, B toxicity reduced the shoot flavonoids content; however, this reduction was not alleviated by the use of Ca, Si and SA treatments. It could be concluded that growth and yield of canola plants grown under high B concentration improved after external application of Ca, Si or SA.     

The beneficial effects of a multistrain potential probiotic,formic, and lactic acids with different vaccination regimens on broiler chickens challenged with multidrug-resistant Escherichia coli and Salmonella

The effects of a multistrain potential probiotic (Protexin®), acids, and a bacterin from multidrug-resistant E. coli O26, O78, S. Enteritidis (1,9,12 g.m1,7), and S. Typhimurium (1,4,5,12.i.1,2) on the immune response, haematological parameters, cytokines, and growth parameters of broiler chickens challenged with bacterin live serotypes were investigated. Two experiments were designed using 300 one-day-old chicks (Arbor Acres) randomly assigned to 15 groups. The first experiment comprised 9 groups, including positive and negative control groups and other groups received Protexin®, acids, and the bacterin (0.2 ml/SC), either alone or in combination, on the 1st day. The second experiment contained 6 groups, including positive and negative control groups and other groups received a combination of Protexin®, acids, and the bacterin (0.5 ml/SC) on the 8th day. All the groups except the negative control groups were challenged on the 8th and 16th days in both experiments, respectively, with mixed live bacterin serotypes. The groups that received Protexin®, acids, and the bacterin either alone or in combination revealed significant improvements in the immune response to the bacterin (p ≤ 0.05). The groups in the 1st experiment and most the 2nd experiment groups showed a reduced mortality rate and decreased levels IFN-γ, IL-4, and IL-12 cytokines (p ≤ 0.05). Moreover, these groups demonstrated increases in haematological parameters and reduced rates of infection-caused anaemia. These groups showed significant increases in growth performance parameters, such as body weight, weight gain, and the feed conversion ratio (FCR) (p ≤ 0.05). There was a beneficial effect on 1-day-old chickens produced by combining Protexin®, acids, and the bacterin (0.2 ml/SC).