Bassam K. Kudhair Shairoza

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.

STAG proteins, which are part of the cohesin complex and encoded by the STAG genes, are known as Irr1/Scc3 in yeast and as SA/STAG/stromalin in mammals. There are more variants as there are alternate splice sites, maybe three open reading frames (ORFs) code for three main proteins, including: SA1 (STAG1), SA2 (STAG2) and SA3 (STAG3). The cohesin protein complex has various essential roles in eukaryotic cell biology. This study compared the expression of the STAG1 gene in four different breast cancer cell lines, including: MCF-7, T-47D, MDA-MB-468, and MDA-MB-231 and normal breast tissue. RNA was extracted from these cell lines and mRNA was converted to cDNA, and then expression of the STAG1 gene was quantified by three sets of specific primer pairs using Real Time-quantitative PCR (RT-qPCR). The findings show significantly different over-expression of STAG1 in these cancer cell lines in comparison with the normal tissue, and the cell lines were different in their expression levels. In conclusion, the STAG1 gene can be postulated as a candidate breast cancer biomarker that needs to be further evaluated in breast tumor biopsies.

Background The gene responsible for encoding the protein of cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) has been found to be associated with rheumatoid arthritis (RA) in different ethnic populations. But the association of +49A/G CTLA-4 polymorphism with susceptibility of RA among Iraqi Arab populations has not yet been determined. Methods One hundred and seventy-eight patients were examined, 67 of them were males (mean age 54.71 ± 10.4 years), while 167 were examined for the control group, of whom 64 were males and the rest were females. CTLA-4 DNA genotyping was carried on to determine the +49 A/G (rs231775) polymorphism using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Enzyme-linked immunosorbent assay (ELISA) was also applied here to measure the antibodies level for cyclic citrullinated peptides (anti-CCP) and Rheumatoid factor (RF). Results The frequency of AG and GG genotypes in CTLA-4 + 49 were significantly higher among RA patients in comparing with controls (55.61% vs 42.51%, OR = 2.18, 95% CI = 1.62–3.79, P = 0.003) and (20.22% vs 10.77%, OR = 2.61, 95% CI = 1.31–6.46, P = 0.002) respectively. G allele frequency was also significantly higher among RA cases (52.24% vs. 31.73%, OR = 3.02; 95% CI = 1.61–7.39, P = 0.001). The frequencies of the AA genotype and A allele, however, were significantly lower in cases than controls (24.15% vs 46.70%, P = 0.001) and (47.75% vs 68.26%, P = 0.001) respectively. Moreover, the levels of Anti-CCP and RF were raised significantly among RA patients than controls (P = 0.0001), but none of these parameters were correlated with genotypes of CTLA-4. Conclusions Carries of CTLA-4 + 49 AG and GG alleles were at a high risk of developing functional disability of RA, unlike the AA allele carriers.

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.

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.

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.