Iraqi propolis increases degradation of IL-1β and NLRC4 by autophagy following Pseudomonas aeruginosa infection

Autophagy is a cellular process for maintaining cellular homeostasis. This process can be induced by different factors, such as immune stimuli and pathogen-associated molecules. Autophagy has an important role in the control of IL-1β secretion by macrophages and other cell types. In present study, we describe a novel role for Iraqi propolis affecting autophagy in controlling the secretion of IL-1β in bone-marrow macrophages (BMDMs). After infection with Pseudomonas aeruginosa in the presence of propolis, the degradation of IL-1β was induced, and the activity of inflammasome was reduced. Iraqi propolis-induced autophagy in in vitro and in vivo models decreased the levels of IL-1β and caspase-1. Results indicated that IL-1β pathway production is regulated by autophagy via two different novel mechanisms, namely, regulation of the activation of NLRC4 inflammasome and IL-1β targeting for lysosomal degradation.     

The phage P.E1 isolated from hospital sewage reduces the growth of Escherichia coli

This study involves partial characterisation of a lytic bacteriophage P.E₁ against a multi drug-resistant clinical isolate of Escherichia coli, isolated from hospital sewage supply. The phage P.E₁ has showed a narrow host range suitable for its use in phage therapy. Phage showed lytic activity up to 70°C and at alkaline conditions, but at higher acidic conditions its activity decreased. Latent period and burst size of P.E₁ estimated from single-step growth curve was 40 min and 185 plaque-forming units per cell, respectively. The phage P.E₁ reduced the growth of host bacteria during the initial 12?h of infection; however, the host bacteria developed resistance afterwards. During the 24-hour observation period, the bacteriophage could still reduce the growth of its host bacteria evident by lower optical density in the phage-treated samples compared with control. The phage genome was double-stranded DNA and larger than 12?kb in size. Further manipulations of genome and proteins may help to unveil the unique aspects of this phage, to use it in phage therapy against E. coli.     

Functional analysis of the group A streptococcal luxS/AI-2 system in metabolism, adaptation to stress and interaction with host cells

Background

The luxS/AI-2 signaling pathway has been reported to interfere with important physiological and pathogenic functions in a variety of bacteria. In the present study, we investigated the functional role of the streptococcal luxS/AI-2 system in metabolism and diverse aspects of pathogenicity including the adaptation of the organism to stress conditions using two serotypes of Streptococcus pyogenes, M1 and M19.

Results

Exposing wild-type and isogenic luxS-deficient strains to sulfur-limited media suggested a limited role for luxS in streptococcal activated methyl cycle metabolism. Interestingly, loss of luxS led to an increased acid tolerance in both serotypes. Accordingly, luxS expression and AI-2 production were reduced at lower pH, thus linking the luxS/AI-2 system to stress adaptation in S. pyogenes. luxS expression and AI-2 production also decreased when cells were grown in RPMI medium supplemented with 10% serum, considered to be a host environment-mimicking medium. Furthermore, interaction analysis with epithelial cells and macrophages showed a clear advantage of the luxS-deficient mutants to be internalized and survive intracellularly in the host cells compared to the wild-type parents. In addition, our data revealed that luxS influences the expression of two virulence-associated factors, the fasX regulatory RNA and the virulence gene sibA (psp).

Conclusion

Here, we suggest that the group A streptococcal luxS/AI-2 system is not only involved in the regulation of virulence factor expression but in addition low level of luxS expression seems to provide an advantage for bacterial survival in conditions that can be encountered during infections.     

Spectrofluorometric determination of clonazepam in dosage forms: Application to content uniformity testing and human plasma

The present paper describes a developed and validated simple, highly sensitive and cost‐effective spectrofluorometric method for determination of clonazepam (CNP). The proposed method depends on forming a highly fluorescent product through the reduction of CNP with Zn/HCl. The produced fluorophore exhibits a strong fluorescence at λ_em 350 nm after excitation at λ_ex 250 nm. The use of carboxymethylcellulose (CMC) greatly enhanced the fluorescence intensity of the produced fluorophore to the extent of about 100%. Calibration curve showed good linear regression (r ² > 0.9998) within test ranges of 20–400 ng ml^?1 with a lower detection limit of 0.67 ng ml^?1 and lower quantification limit of 2.22 ng ml^?1 upon using CMC. The method was successfully applied to the analysis of CNP in its pharmaceutical formulations and the results were in agreement with those obtained using a reference method. Furthermore, the content uniformity testing of the tablets was also performed. The application of the proposed method was extended to determine CNP in spiked human plasma sample as a preliminary investigation and the results were satisfactory.     

Structural Characterization of a Gcn5-Related N-Acetyltransferase from Staphylococcus aureus

The Gcn5-related N-acetyltransferases (GNATs) are ubiquitously expressed in nature and perform a diverse range of cellular functions through the acetylation of small molecules and protein substrates. Using activated acetyl coenzyme A as a common acetyl donor, GNATs catalyse the transfer of an acetyl group to acceptor molecules including aminoglycoside antibiotics, glucosamine-6-phosphate, histones, serotonin and spermidine. There is often only very limited sequence conservation between members of the GNAT superfamily, in part, reflecting their capacity to bind a diverse array of substrates. In contrast, the secondary and tertiary structures are highly conserved, but then at the quaternary level there is further diversity, with GNATs shown to exist in monomeric, dimeric, or tetrameric states. Here we describe the X-ray crystallographic structure of a GNAT enzyme from Staphyloccocus aureus with only low sequence identity to previously solved GNAT proteins. It contains many of the classical GNAT motifs, but lacks other hallmarks of the GNAT fold including the classic β-bulge splayed at the β-sheet interface. The protein is likely to be a dimer in solution based on analysis of the asymmetric unit within the crystal structure, homology with related GNAT family members, and size exclusion chromatography. The study provides the first high resolution structure of this enzyme, providing a strong platform for substrate and cofactor modelling, and structural/functional comparisons within this diverse enzyme superfamily.     

Vangl2/RhoA Signaling Pathway Regulates Stem Cell Self-Renewal Programs and Growth in Rhabdomyosarcoma

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A lethal phenotype associated with tissue plasminogen deficiency in humans

The role of plasminogen in preventing thrombosis requires activation by tissue plasminogen activator (t-PA) encoded by PLAT. While case–control associations have been pursued for common variants in PLAT, no disease-causing mutations have been reported. We describe a consanguineous family with two children who died shortly after birth due to complications related to severe hydranencephaly and diaphragmatic hernia. A combined exome/autozygome analysis was carried out with informed consent. We identified a homozygous null mutation in PLAT that abrogated t-PA level in patient cells. This is the first reported human knockout mutation of PLAT. The apparent association with hydranencephaly, diaphragmatic hernia and postnatal lethality requires further validation.