A Detailed Chemical and Biological Investigation of Twelve Allium Species from Eastern Anatolia with Chemometric Studies

Allium species are widely consumed as food all over the world. The phenolic profile of ethanol extracts of aerial parts and roots of 12 Allium species, collected from five different Eastern Anatolia regions, were studied using LC-MS/MS. In vitro antioxidant, anticholinesterase, cytotoxic and antimicrobial activities were also tested. The multivariate analyses were performed using principal component and hierarchical cluster analyses. Seventeen of 27 standard compounds were detected in all Allium species. The major components were mainly identified as quinic acid, malic acid, vanillin, and p-coumaric acid. The aerial parts possessed better antioxidant activity than roots. Aerial parts of A. atroviolaceum, A. chrysantherum, A. kharputense, and A. shirnakiense exhibited high cytotoxic activity against DLD-1 colon cancer cell lines (IC₅₀ 12.5 μg/mL). A. shatakiense and A. vineale demonstrated good antimicrobial activity against S. aureus and E. coli (MIC 75 μg/mL). According to chemometric analysis, differences were detected between aerial parts and the roots. The aerial parts of A. atroviolaceum, A. chrysantherum, A. kharputense, and A. shirnakiense could be potent in the pharmaceutical industry while A. shatakiense and A. vineale in the food industry after further investigations.     

Mechanistic and structural insights into the in vitro inhibitory action of hypericin on glutathione reductase purified from baker's yeast

This work aims at studying the interaction between glutathione reductase (GR) and hypericin. The type of inhibition was determined by measuring changes in GR activity at increasing concentrations of hypericin as well as at varying concentrations of glutathione disulfide (GSSG) and nicotinamide adenine dinucleotide phosphate (NADPH), and the binding pose of hypericin was predicted by molecular docking. Accordingly, hypericin emerges as an effective inhibitor of GR. When the variable substrate is GSSG, the type of inhibition is competitive. When the variable substrate is NADPH, however, the type of inhibition appears to be linear mixed‐type competitive. Our computational analyses suggest that hypericin binds in the large intermonomer cavity of GR, and that it may interfere with the normal positioning/functioning of the redox‐active disulfide center at the enzyme's active site. Overall, besides its contributory role in promoting oxidative stress via the formation of reactive oxygen species in photodynamic therapy, hypericin can also weaken cancer cells through inhibiting GR.     

Identification QTLs Controlling Genes for Se Uptake in Lentil Seeds

Lentil (Lens culinaris Medik.) is an excellent source of protein and carbohydrates and is also rich in essential trace elements for the human diet. Selenium (Se) is an essential micronutrient for human health and nutrition, providing protection against several diseases and regulating important biological systems. Dietary intake of 55 μg of Se per day is recommended for adults, with inadequate Se intake causing significant health problems. The objective of this study was to identify and map quantitative trait loci (QTL) of genes controlling Se accumulation in lentil seeds using a population of 96 recombinant inbred lines (RILs) developed from the cross “PI 320937” × “Eston” grown in three different environments for two years (2012 and 2013). Se concentration in seed varied between 119 and 883 μg/kg. A linkage map consisting of 1,784 markers (4 SSRs, and 1,780 SNPs) was developed. The map spanned a total length of 4,060.6 cM, consisting of 7 linkage groups (LGs) with an average distance of 2.3 cM between adjacent markers. Four QTL regions and 36 putative QTL markers, with LOD scores ranging from 3.00 to 4.97, distributed across two linkage groups (LG2 and LG5) were associated with seed Se concentration, explaining 6.3–16.9% of the phenotypic variation.     

Expression and Polymorphism of Toll-Like Receptor 4 and Effect on NF-κB Mediated Inflammation in Colon Cancer Patients

Our aim was to evaluate the association between the expression and the polymorphism of TLR4/NF-κB pathways and colon cancer. TLR4 (rs4986790, rs10759932, rs10759931 and rs2770150) were genotyped in blood samples from Colorectal patients and healthy controls. TLR4 and cytokines inflammatory expression were evaluated by real time PCR on 40 matching normal and colon tissues and the protein level by Immunohistochemistry. The high level of TLR4 expression in colon cancer tissues is mainly due to infections by bacteria in the human colon and leads to induction of an acute secretion of inflammatory cytokines mediated by NF-κB. Also, we report here a clear evidence for an association between TLR4 rs10759931 polymorphism (OR = 0.086, CI: 0.04–0.18, P = <0.00001). This polymorphism affects the entire population without being specific to either gender or to any age group. In contrast, the rs2770150 is associated with colon cancer in women aged over 50 years and is closely linked with the decreased levels of female sex hormones during the post-menopausal period (OR = 0.188, CI: 0.074–0.48, P = <0.00084). rs10759932 and rs4986790 appear to have any association with colon cancer. Our data suggest that TLR4 SNPs could possibly serve as biomarkers for decision making in colon cancer treatment.     

Probing Native Protein Structures by Chemical Cross-linking, Mass Spectrometry, and Bioinformatics

Chemical cross-linking of reactive groups in native proteins and protein complexes in combination with the identification of cross-linked sites by mass spectrometry has been in use for more than a decade. Recent advances in instrumentation, cross-linking protocols, and analysis software have led to a renewed interest in this technique, which promises to provide important information about native protein structure and the topology of protein complexes. In this article, we discuss the critical steps of chemical cross-linking and its implications for (structural) biology: reagent design and cross-linking protocols, separation and mass spectrometric analysis of cross-linked samples, dedicated software for data analysis, and the use of cross-linking data for computational modeling. Finally, the impact of protein cross-linking on various biological disciplines is highlighted.The concept of protein cross-linking as a (bio)chemical tool to infer structural information about protein conformations and protein-protein interactions in combination with mass spectrometry was introduced at the end of the 1990s (1). In a seminal paper, Young et al. (1) used chemical cross-linking of lysine residues in bovine basic fibroblast growth factor FGF-2 (heparin-binding growth factor 2) to provide distance constraints for the computational derivation of the fold of this small (17-kDa) protein. FGF-2 was cross-linked with bis(sulfosuccinimidyl) suberate, purified by size exclusion chromatography, and digested with trypsin. Cross-linked peptides were separated by HPLC and analyzed on line by ESI-TOF and off line by MALDI-TOF mass spectrometry. Putative cross-links were then assigned based on their precursor masses, and some of them were verified by MALDI postsource decay. The authors could identify 15 cross-links that did not bridge directly adjacent lysines and therefore provided information on the three-dimensional structure of the protein. These data were used to assign FGF-2 to the β-trefoil family by excluding calculated models that did not fit the distance constraints.In the last decade, the application of protein cross-linking has expanded, first and foremost driven by developments in mass spectrometry as the method of choice for the high throughput identification of proteins and their modifications. Reviews by Back et al. (2), Sinz (3), and most recently Lee (4) give an overview on the evolution of the field. However, despite the progress that has undoubtedly been made, cross-linking is still considered a “niche” technique that has not (yet) lived up to its promises. High throughput generation of data supporting protein fold prediction and the determination of protein-protein interactions have not been realized routinely. There may be several reasons for that such as the necessity of access to high end mass spectrometers, the requirement of specialized reagents, and the need for tailored software. However, recent years have seen an increased interest in this technique, which is reflected in the literature and by the emergence of new reagents and software tools.Here, we present an overview of recent developments in methodology, instrumentation, and bioinformatics related to chemical cross-linking of proteins and the analysis of cross-linked peptides by mass spectrometry. Other cross-linking areas such as protein-DNA cross-linking, photoinduced cross-linking, or the characterization of disulfide bonds will not be covered in detail in this paper. We critically discuss advantages and limitations of different concepts and look beyond the immediate outcome of cross-linking experiments (putative interactions and/or distance constraints) and examine the potential role of chemical cross-linking in the analysis of protein interaction networks and, more generally, for structural and systems biology.     

Identification of novel large genomic rearrangements at the BRCA1 locus in Malaysian women with breast cancer

Background: The incidence of breast cancer has been on the rise in Malaysia. It is suggested that a subset of breast cancer cases were associated with germline mutation in breast cancer susceptibility (BRCA) genes. Most of the BRCA mutations reported in Malaysia were point mutations, small deletions and insertions. Here we report the first study of BRCA large genomic rearrangements (LGRs) in Malaysia. We aimed to detect the presence of LGRs in the BRCA genes of Malaysian patients with breast cancer. Methods: Multiplex ligation-dependent probe amplification (MLPA) for BRCA LGRs was carried out on 100 patients (60 were high-risk breast cancer patients previously tested negative/positive for BRCA1 and BRCA2 mutations, and 40 were sporadic breast cancer patients), recruited from three major referral centres, Universiti Kebangsaan Malaysia Medical Centre (UKMMC), Hospital Kuala Lumpur (HKL) and Hospital Putrajaya (HPJ). Results: Two novel BRCA1 rearrangements were detected in patients with sporadic breast cancer; both results were confirmed by quantitative PCR. No LGRs were found in patients with high-risk breast cancer. The two large genomic rearrangements detected were genomic amplifications of exon 3 and exon 10. No BRCA2 genomic rearrangement was found in both high-risk and sporadic breast cancer patients. Conclusion: These results will be helpful to understand the mutation spectrum of BRCA1 and BRCA2 genes in Malaysian patients with breast cancer. Further studies involving larger samples are required to establish a genetic screening strategy for both high-risk and sporadic breast cancer patients.