Heterogeneity of KRAS Mutation Status in Rectal Cancer

IntroductionAnti-EGFR targeted therapy is of increasing importance in advanced colorectal cancer and prior KRAS mutation testing is mandatory for therapy. However, at which occasions this should be performed is still under debate. We aimed to assess in patients with locally advanced rectal cancer whether there is intra-specimen KRAS heterogeneity prior to and upon preoperative chemoradiotherapy (CRT), and if there are any changes in KRAS mutation status due to this intervention.ResultsFor 20 (43%) out of the 47 patients, a KRAS mutation was detected. With 12 out of 20, the majority of these mutations affected codon 35. We did not obtained evidence that CRT results in changes of the KRAS mutation pattern. In addition, no intratumoral heterogeneity in the KRAS mutational status could be proven. This was true for both the biopsies prior to CRT and the resection specimens thereafter. The discrepancy observed in some samples when using the SNaPshot™ assay was due to insufficient sensitivity of this technique upon massive tumor regression by CRT as application of the therascreen® KRAS test revealed concordant results.ConclusionOur results indicate that the KRAS mutation status at the primary tumor site of rectal cancer is homogenous. Its assessment for therapeutic decisions is feasible in pre-therapeutic biopsies as well as in post-therapeutic resected specimens. The amount of viable tumor cells seems to be an important determinant for assay sensitivity and should thus be considered for selection of the analytical method.     

A new species of Myrmozercon Berlese (Acari,Mesostigmata, Laelapidae) associated?with?ant?from?Iran

This paper report on a new species of mites of the genus Myrmozercon associated with ant in Iran – Myrmozercon cyrusi Ghafarian and Joharchi sp. n. was collected associated of the Monomorium sp. in Kenevist Rural District in the Central District of Mashhad County, Khorasan Razavi Province, Iran. This new species is described and illustrations provided. Myrmozercon ovatum Karawajew, 1909 is suspected to be a junior synonym of Myrmozercon brevipes Berlese, 1902 and host-specificity and host range of Myrmozercon are also reviewed.     

Aptamer-based electrochemical biosensor by using Au-Pt nanoparticles,carbon nanotubes and acriflavine platform

Herein, an ultrasensitive electrochemical aptasensor for quantitative detection of bisphenol A (BPA) was fabricated based on a novel signal amplification strategy. This aptasensor was developed by electrodeposition of gold-platinum nanoparticles (Au-PtNPs) on glassy carbon (GC) electrode modified with acid-oxidized carbon nanotubes (CNTs-COOH). In this protocol, acriflavine (ACF) was covalently immobilized at the surface of glassy carbon electrode modified with Au-PtNPs/CNTs-COOH nanocomposite. Attachment of BPA-aptamer at the surface of modified electrode was performed through the formation of phosphoramidate bonds between the amino group of ACF and phosphate group of the aptamer at 5′end. By interaction of BPA with the aptamer, the conformational of aptamer was changed which lead to retarding the interfacial electron transfer of ACF as a probe. Sensitive quantitative detection of BPA was carried out by monitoring the decrease of differential pulse voltammetric (DPV) responses of ACF peak current with increasing the BPA concentration. The resultant aptasensor exhibited good specificity, stability and reproducibility, indicating that the present strategy was promising for broad potential application.     

Incorporation and activation of a membrane-bound enzyme in bilayers of liposomes

Summary The effects of lipid composition and fluidity of lipid bilayers on incorporation and activation of membrane-bound d-fructose dehydrogenase are described in this study. The incorporation of the enzyme into bilayers of small unilamellar vesicles (SUV) made of several phospholipids resulted in enzyme activation with magnitudes higher than that observed in the presence of Triton X-100, indicating that this higher activation is due to lipid-protein interaction. The activity was highest in the presence of SUV formed by the addition of 10% dl--dipalmitoylphosphatidylethanolamine to l--dimyristoylphosphatidylcholine, which resulted in eightfold higher activation compared with that of the enzyme in its free state. This activation did not appear to be due to the degree of incorporation of the enzyme, indicating that incorporation is distinct from the activation event. Thus, it is probably the lipid environment that leads to higher activation of the enzyme. A break in the Arrhenius plot of the activity of the membrane-bound enzyme at temperatures close to the phase transition of the phospholipid implies that changes in the physical state of the lipid bilayer influence the enzyme activity. Furthermore, immobilization of d-fructose dehydrogenase, previously adsorbed to SUV, on urethane prepolymer also resulted in about eightfold higher activation than that of the free enzyme. Offprint requests to: S. Katoh     

Prevention of thermal aggregation of an allosteric protein by small molecules: some mechanistic insights

Detailed knowledge of conformation and dynamics of native, intermediate and unfolded states of a protein is essential in searching for effective small molecules to prevent its aggregation. In a recent study we have demonstrated how allosteric effectors may influence protein-protein interactions at high temperatures using glutamate dehydrogenase (GDH) as a model allosteric protein. In the present study, thermal aggregation of this well-characterized enzyme was investigated in the presence of a number of amino acids (including Gly, Glu, Trp, Pro, Lys, Arg), polyamines (putrescine and spermidine) and chaperone-like molecules (cyclodextrins and caseins) as non-specific effectors. It was shown that some amino acids and polyamines may suppress aggregation via interaction with native species and may preserve the activity of the enzyme while cyclodextrins and caseins may exert their anti-aggregation potential via binding to aggregation-prone intermediates, without having any capacity to protect its native structure from unfolding. Observations describing the similarities and differences between the specific ligands and non-specific small molecules related to their interaction with native and aggregation-prone states of GDH are presented and discussed. It is argued that the type of studies described in the present communication is useful for the development of effective strategies for prevention of aggregation by small molecules.     

Role of electrostatic interactions in 2,2,2-trifluoroethanol-induced structural changes and aggregation of alpha-chymotrypsin

It has been recently demonstrated that alpha-chymotrypsin (CT) can be driven toward amyloid aggregation by addition of 2,2,2-trifluoroethanol (TFE), at intermediate concentrations. In the present article, the process of TFE-induced CT aggregation was investigated in more detailed kinetic terms where the effects of medium conditions, such as temperature, presence of kosmotropic and chaotropic salts, pH and chemical modification of lysine residues were examined. Various techniques, including light scattering, fluorescence and circular dichroism spectroscopy, were used to follow and characterize this process. The kinetics of aggregation was found to obey a second-order reaction with respect to protein concentration. The aggregation-prone A-state and aggregation-deficient TFE- or T-state of CT were found to be induced at lower TFE concentrations in the presence of salts. Use of acidic and alkaline conditions and lysine modification also promoted the formation of the T-state. Results presented suggest a role for electrostatic interactions in the aggregation process.     

Hepatic mechanisms of the Walnut antidiabetic effect in mice

The present study was designed to explore the mechanism of action of walnut (the seed of Juglans regia) leaf and ridge on hepatic glucose metabolism in diabetic mice. Experimental diabetes was induced by intravenous administration of streptozotocin (60 mg/kg)and confirmed with an increase of blood glucose, 90–100% of the control, 72 hours later. Isolated extracts from walnut leaf and ridges were administered in a single effective dose of 400 mg/kg orally. Firstly, blood glucose was determined every 1 hour until 5 hours post administration of extracts. In the second experiment, the liver was surgically removed, 2 hours post treatment of diabetic animals with extracts, homogenized and used for measurement of key enzymes of glycogenolysis (glycogen phosphorylase, GP) and gluconeogenesis (phosphoenolpyruvate carboxykinase, PEPCK). Treatment by both leaf and ridge extracts decreased blood glucose and liver PEPCK activity and increased blood insulin and liver GP activity. It is concluded that walnut is able to lower blood glucose through inhibition of hepatic gluconeogenesis and secretion of pancreatic insulin.     

Association of the human aryl hydrocarbon receptor repressor (AhRR)-c.565C>G transversion with male infertility: A case-control study from Iran

Dioxins (eg, 2,3,7,8-tetrachlorodibenzo-p-dioxin/TCDD), as environmental endocrine disruptors and toxic carcinogens, can affect male reproductive health. The influence of dioxins is mediated via the aryl hydrocarbon receptor (AhR) pathway and its repressor (AhRR). In this study, we investigated the association of AhRR-c.565C>G transversion polymorphism with male infertility. In a hospital-based case-control study, 221 semen samples (111 infertile and 110 healthy controls) based on World Health Organization guidelines were collected from in vitro fertilization centers of Babol, Iran. The AhRR-c.565C>G (rs2292596) polymorphism was genotyped using a polymerase chain reaction-restriction fragment length polymorphism analysis. The difference in the allele frequency of AhRR-c.565C>G transversion polymorphism did not reach a significant level. The genotype frequency was statistically significantly different between fertile and infertile men. We found that polymorphism rs2292596 (Pro185Ala) was statistically s ignificantly associated with the risk of male infertility. In addition, the statistical difference became more significant when the frequency was compared between the Pro/Pro genotype and the Pro/Ala plus Ala/Ala genotype. The 185 Pro wild-type alleles of AhRR may be associated with the risk of male infertility. The proallele also may diminish inhibition of AhR-mediated signaling of exposure to environmental pollutants.     

Vitamin D3 affects mitochondrial biogenesis through mitogen-activated protein kinase in polycystic ovary syndrome mouse model

Polycystic ovarian syndrome (PCOS) is a disorder characterized by oligomenorrhea, anovulation, and hyperandrogenism. Altered mitochondrial biogenesis can result in hyperandrogenism. The goal of this study was to examine the effect of vitamin D3 on mitochondrial biogenesis of the granulosa cells in the PCOS-induced mouse model. Vitamin D3 applies its effect via the mitogen-activated pathway kinase-extracellular signal-regulated kinases (MAPK-ERK1/2) pathway. The PCOS mouse model was induced by the injection of dehydroepiandrosterone (DHEA). Isolated granulosa cells were subsequently treated with vitamin D3, MAPK activator, and MAPK inhibitor. Gene expression levels were measured using real-time polymerase chain reaction. MAPK proteins were investigated by western blot analysis. We also determined reactive oxygen species (ROS) levels with 2′, 7′-dichlorofluorescein diacetate. Mitochondrial membrane potential (mtMP) was also measured by TMJC1. Mitochondrial biogenesis (peroxisome proliferator-activated receptor gamma coactivator 1-α and nuclear respiratory factor), antioxidant (superoxide dismutase, glutathione peroxidase, and catalase), and antiapoptotic (B-cell lymphoma-2) genes were upregulated in the PCOS mice that treated with vitamin D3 compared with the PCOS mice without any treatment. Vitamin D3 and MAPK activator-treated groups also reduced ROS levels compared with the nontreated PCOS group. In summary, vitamin D3 and MAPK activator increased the levels of mitochondrial biogenesis, MAPK pathway, and mtMP markers, while concomitantly decreased ROS levels in granulosa cells of the PCOS-induced mice. This study suggests that vitamin D3 may improve mitochondrial biogenesis through stimulation of the MAPK pathway in cultured granulosa cells of DHEA-induced PCOS mice which yet to be investigated.