Overproduction and purification of Mycobacterium tuberculosis WhiB3 in Escherichia coli is enhanced by co-expression with trigger factor chaperone.

Members of the WhiB-like (Wbl) family of proteins are found in Acintomycetes and are somewhat recalcitrant to overproduction as soluble proteins in the laboratory protein expression workhorse Esherichia coli. The aim of this study was to evaluate the effects of culture conditions and co-expression of the chaperone protein, trigger factor (TF), on the soluble production of recombinant Mycobacterium tuberculosis (Mtb) WhiB3. A pET28a derived expression plasmid coding for His6-WhiB3 was created and the effects of varying the concentration of inducer (IPTG), the timing of induction, the nature of the inducer (auto-induction medium) and the temperature of the cultivation on the production of soluble His6-WhiB3 were tested. Whilst His6-WhiB3 protein was readily detected, the overwhelming majority of the protein was present in the insoluble fraction of cell-free extracts. However, co-expression of the tig from pTf16, coding for TF, increased His6-WhiB3 solubility dramatically, facilitating its isolation by affinity chromatography. Purified His6-WhiB3 was shown to be monomeric, and UV-visible spectra suggested that ∼10% of the isolated protein possessed a [4Fe-4S] cluster. The secondary structural properties of His6-WhiB3 were altered by acquisition of an iron-sulfur cluster. By developing a protocol to readily overproduce and purify WhiB3, this study paves the way for future structure-function experiments.

Correlation of GSTP1 gene variants of male Iraqi waterpipe (Hookah) tobacco smokers and the risk of lung cancer.

Glutathione-S-transferases (GSTs) play a role in the detoxification of environmental chemicals and mutagens, such as those inhaled during tobacco smoking. There have been conflicting reports concerning GST polymorphisms as risk factors in the development of lung cancer. No studies focused on Arab populations exposed to Waterpipe (WP) tobacco smoke have been undertaken. Here Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) and gene sequencing were applied to analyze allelic variations in GSTP1-rs1695 and -rs1138272 amongst 123 lung cancer patients and 129 controls. The data suggest that WP smoking raised the risk of lung cancer more than three-fold (OR 3.6; 95% CI 2.1-6.0; p < 0.0001). However, there was no significant association between individual GSTP1 polymorphisms and the risk of lung cancer. In contrast, analysis of the rs1695 and rs1138272 combination suggested that the risk of lung cancer was raised more than two-fold for carriers of the GSTP1-rs1695 (G) allele (OR 2.5; 95% CI 1.0-6.4; p < 0.05), however, the presence of the GSTP1-rs1138272 (T) allele, in addition to GSTP1-rs1695, did not significantly change the risk ratio (OR 2.8; 95% CI 1.4-5.7; p < 0.004). WP tobacco smokers who carried the GSTP1-rs1695, but not GSTP1-rs1138272, allele were similarly susceptible to lung cancer (OR 2.4; 95% CI 1.1-5.3; p < 0.03). Hence, the results suggest that smoking WP tobacco and carrying GSTP1-rs1695 polymorphisms are risk factors for lung cancer in Arab Iraqi males.

The correlation of combined OGG1, CYP1A1 and GSTP1 gene variants and risk of lung cancer of male Iraqi waterpipe tobacco smokers.

Genetic polymorphisms of genes whose products are responsible for activities, such as xenobiotic metabolism, mutagen detoxification and DNA-repair, have been predicted to be associated with the risk of developing lung cancer (LC). The association of LC with tobacco smoking has been extensively investigated, but no studies have focused on the Arab ethnicity. Previously, we examined the association between genetic polymorphisms among Phase I and Phase II metabolism genes and the risk of LC. Here, we extend the data by examining the correlation of OGG1 Ser326Cys combined with CYP1A1 (Ile462Val and MspI) and GSTP1 (Ile105Val and Ala103Val) polymorphisms with the risk of LC. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and gene sequencing were carried out for genotyping the OGG1 polymorphisms of 123 LC patients and 129 controls. No significant differences in the frequencies of the OGG1 mutant allele between patients and controls were found. The distributions of heterozygous Ser/Cys or Cys/Cys genotypes of OGG1 were not associated with the risk of LC either according to the histological types of LC or based on waterpipe tobacco (WP) smoking status. In contrast, the combined effect of OGG1 variants with CYP1A1 and GSTP1 variants revealed a significant correlation with the OGG1 Ser326Cys-CYP1A1 MspI variants pairing. This association was significant (p = 0.001) in individuals who carried homozygous or heterozygous variant type genotypes of both genes in a reference with carriers of both wild-type genotypes (wt/wt - wt/wt). The odds ratios were 2.99 (95% CI 1.67-5.36), 2.68 (95% CI 1.08-6.62), and 2.80 (95% CI 1.18-6.69) for those who carried (wt/wt - wt/vt + vt/vt), (wt/vt + vt/vt - wt/wt), and (wt/vt + vt/vt - wt/vt + vt/vt), respectively. The study suggests a limited correlation is present between carrying OGG1 Ser326Cys polymorphism and the risk of developing LC in Arab populations.

Analysis of Kytococcus sedentarius Strain Isolated from a Dehumidifier Operating in a University Lecture Theatre: Systems for Aerobic Respiration, Resisting Osmotic Stress, and Sensing Nitric Oxide.

A strain of Kytococcus sedentarius was isolated from a dehumidifier operating in a university lecture theatre. Genome analysis and phenotypic characterisation showed that this strain, K. sedentarius MBB13, was a moderately halotolerant aerobe with a branched aerobic electron transport chain and genes that could contribute to erythromycin resistance. The major compatible solute was glycine betaine, with ectoine and proline being deployed at higher osmolarities. Actinobacteria possess multiple WhiB-like (Wbl) regulatory proteins, and K. sedentarius MBB13 has four (WhiB1, WhiB2, WhiB3, and WhiB7). Wbls are iron-sulfur proteins that regulate gene expression through interactions with RNA polymerase sigma factors and/or other regulatory proteins. Bacterial two-hybrid analyses suggested that WhiB1 and WhiB2, but not WhiB3 and WhiB7, interact with the C-terminal domain of the major sigma factor, σA; no interaction was detected between any of the Wbl proteins and the only alternative sigma factors, σB, σH, or σJ. The interaction between σA and WhiB1 or WhiB2 was disrupted in a heterologous system under growth conditions that produce nitric oxide and the iron-sulfur clusters of the isolated WhiB1 and WhiB2 proteins reacted with nitric oxide. Thus, K. sedentarius strain exhibits the major phenotypic characteristics of the type strain and a comprehensive examination of the interactions between its four Wbl proteins and four sigma factors suggested that the Wbl proteins all operate through interaction with σA.

Functionalized graphene oxide/Fe3O4 nanocomposite: A biocompatible and robust nanocarrier for targeted delivery and release of anticancer agents.

The development of efficient drug nanocarriers has remained an important challenge in advanced drug delivery in human body. Combination of graphene-based nanomaterials and cyanuric chloride (CC), as a linker, may improve the success of drug delivery. Herein, a simple approach was used for the synthesis of superparamagnetic graphene oxide (SPMGO) nanocomposite through a chemical precipitation method. The nanocomposite was readily functionalized with cyanuric chloride as a linker for loading the drug. The FTIR spectroscopy confirmed the efficient synthesis of nanocarriers. So did the transmission electron microscopy, atomic force microscopy, and thermo-gravimetric analysis, X-ray diffraction and X-ray photoelectron spectroscopy. Subsequently, the synthesized nanocarriers were studied in terms of their potential for biomedical applications. Immobilization of methotrexate (MTX), as a drug for treatment of cancer was taken into action on the SPMGO and SPMGO/CC. The in vitro assays indicated that the drug nanocarrier systems, SPMGO/MTX and SPMGO/CC/MTX, are hemo-compatible and increase the efficiency of MTX against Caov-4, HeLa and MCF-7 cell lines. The MTX nanocarriers represented a considerably high drug loading and controlled drug release. The overall results indicated the great potential of SPMGO/CC/MTX nanocarrier for targeted drug delivery, particularly in MTX chemotherapy.

Structure of a Wbl protein and implications for NO sensing by M. tuberculosis.

Mycobacterium tuberculosis causes pulmonary tuberculosis (TB) and claims ~1.8 million human lives per annum. Host nitric oxide (NO) is important in controlling TB infection. M. tuberculosis WhiB1 is a NO-responsive Wbl protein (actinobacterial iron-sulfur proteins first identified in the 1970s). Until now, the structure of a Wbl protein has not been available. Here a NMR structural model of WhiB1 reveals that Wbl proteins are four-helix bundles with a core of three α-helices held together by a [4Fe-4S] cluster. The iron-sulfur cluster is required for formation of a complex with the major sigma factor (σA) and reaction with NO disassembles this complex. The WhiB1 structure suggests that loss of the iron-sulfur cluster (by nitrosylation) permits positively charged residues in the C-terminal helix to engage in DNA binding, triggering a major reprogramming of gene expression that includes components of the virulence-critical ESX-1 secretion system.