GHSR DNA hypermethylation is a new epigenetic biomarker for gastric adenocarcinoma and beyond

Aberrations of DNA methylation are early events in the development of tumors. In this study, we investigated the DNA methylation status of growth hormone secretagogue receptor (GHSR), a promising pan-cancer biomarker, in gastric cancer (GC). Initially, data sets from DNA methylation and gene expression studies available at Gene Expression Omnibus (GEO) were analyzed. Confirmation was done on primary tumor specimens and adjacent normal stomach tissue samples. Both analyses showed significant hypermethylation of GHSR. For further validation, The Cancer Genome Atlas data on stomach cancer was used. A receiver operating characteristic curve analysis yielded an area under the curve value of 0.85, corroborating its usefulness as a diagnostic marker. A genome-wide comethylation analysis revealed several correlated genes. CREB1 was found to act as an upstream regulator of this gene network. Furthermore, GHSR methylation was found to be a biomarker in several other tumor entities, namely cancers of the bladder, endometrium, esophagus, head and neck, liver, thyroid, kidney, and ovary. Our findings along with previous reports on other types of cancer suggest a high potential of GHSR gene methylation as a pan-cancer biomarker, which could be considered for liquid biopsy applications.     

A Neuron-Glial Trans-Signaling Cascade Mediates LRRK2-Induced Neurodegeneration

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Ethanol-induced impairment of polyamine homeostasis – A potential cause of neural tube defect and intrauterine growth restriction in fetal alcohol syndrome

Introduction

Polyamines play a fundamental role during embryogenesis by regulating cell growth and proliferation and by interacting with RNA, DNA and protein. The polyamine pools are regulated by metabolism and uptake from exogenous sources. The use of certain inhibitors of polyamine synthesis causes similar defects to those seen in alcohol exposure e.g. retarded embryo growth and endothelial cell sprouting.

Methods

CD-1 mice received two intraperitoneal injections of 3 g/kg ethanol at 4 h intervals 8.75 days post coitum (dpc). The fetal head, trunk, yolk sac and placenta were collected at 9.5 and 12.5 dpc and polyamine concentrations were determined.

Results

No measurable quantity of polyamines could be detected in the embryo head at 9.5 dpc, 12 h after ethanol exposure. Putrescine was not detectable in the trunk of the embryo at that time, whereas polyamines in yolk sac and placenta were at control level. Polyamine deficiency was associated with slow cell growth, reduction in endothelial cell sprouting, an altered pattern of blood vessel network formation and consequently retarded migration of neural crest cells and growth restriction.

Discussion

Our results indicate that the polyamine pools in embryonic and extraembryonic tissues are developmentally regulated. Alcohol administration, at the critical stage, perturbs polyamine levels with various patterns, depending on the tissue and its developmental stage. The total absence of polyamines in the embryo head at 9.5 dpc may explain why this stage is so vulnerable to the development of neural tube defect, and growth restriction, the findings previously observed in fetal alcohol syndrome.     

Development of an enhanced chemiluminescence immunoassay (CLIA) for detecting urinary albumin

In the present study, a sensitive and competitive chemiluminescence immunoassay (CLIA) was developed in order to detect human serum albumin (HSA) in urine specimen. The method utilizes a home-made monoclonal anti-albumin antibody conjugated to horseradish peroxidase enzyme (mAb-HRP). Sensitivity, specificity and linearity of the assay were evaluated. According to the results, the proper concentration of HSA and mAb-HRP conjugates was 800?ng/100?μl and 1:200 respectively. In optimal conditions, this method could detect HSA in a high linear range of 10–200?μg?ml^?1 with the low detection limit of 0.025?μg?ml^?1. No evidence of interference with presence of probable substances in the urine samples indicated its high specificity and selectivity. Moreover, high reproducibility as well as high sensitivity and specificity of the test were confirmed using diabetic and non-diabetic samples. Significant concordance was observed between CLIA and immunoturbidimetry assay regarding detection of HSA. The results of the present study can be considered in accordance with the current demands such as reliability, accuracy, convenience and high speed of performance for a precise protein detection method. Furthermore, it may be regarded as a more rapid, simpler and cheaper alternative compared to other sophisticated assays.     

Stability improvement of immobilized lactoperoxidase using polyaniline polymer

Enzyme engineering via immobilization techniques is perfectly compatible against the other chemical or biological approximate to improve enzyme functions and stability. In this study lactoperoxidase was immobilized onto polyaniline polymer activated with glutaraldehyde as a bifunctional agent, to improve enzyme properties. Polyaniline polymer was used due its unique physical and chemical properties to immobilize lactoperoxidase (LPO). The optimum activity of immobilized LPO was observed at pH 6 and 55?°C, which has been increased about 10?°C for the immobilized enzyme. The immobilized enzyme maintained absolutely active for 60?days whereas the native enzyme lost 80?% of its initial activity within this period of time. Moreover, the immobilized enzyme can be reused for several times without loss of activity. The kinetic parameter studies showed slight differences between free and immobilized enzymes. The K_m and K_m.app were calculated to be 0.6 and 0.4; also V_max and V_max.app were 1.3 and 0.9 respectively.     

DNA-binding studies of fluoxetine antidepressant

Fluoxetine is a selective serotonin reuptake inhibitor (SSRI) antidepressant that is widely prescribed. The DNA-binding behavior of fluoxetine antidepressant and calf thymus DNA was investigated in Tris-HCl buffer at physiological pH 7.4 with a series of techniques, including UV-Vis and circular dichroism spectroscopies, competitive study with Hoechst 33258, viscometry, and cyclic voltammetry. Fluoxetine molecules bind to DNA via groove mode as illustrated by hypochromism with no red shift in the UV absorption band of fluoxetine, decrease in Hoechst-DNA solution fluorescence, and no significant changes in viscosity of DNA. The CD spectra of DNA molecules show a little change in stacking mode of base pair but no modification changes in DNA conformation, for example, from B-DNA to A or C-DNA. The binding constant (K(b)) of DNA with fluoxetine was calculated to be 6.7 × 10(4) M(-1), which is in the range of reported and known groove binders, such as distamycin. All results showed the groove-binding mode of interaction of fluoxetine with DNA.     

In vitro study of DNA interaction with clodinafop-propargyl herbicide

The interaction of native calf thymus DNA with clodinafop-propargyl (CP), in 10 mM HEPES aqueous solutions at neutral pH 7.2, has been investigated by spectrophotometric, circular dichroism (CD), spectrofluorometric, melting temperature (Tm), and viscosimetric techniques. It was found that CP molecules could intercalate between base pairs of DNA as evidenced by hyperchromism in UV absorption band of DNA, an increase in melting temperature, a sharp increase in specific viscosity of DNA, induced CD spectral changes, and increase in the fluorescence of methylene blue (MB)-DNA solutions in the presence of increasing amounts of CP, which indicates that it is able to release the intercalated MB completely. All results suggest that the CP interacts with calf thymus DNA by an intercalative mode of binding.     

Single domain antibodies: a new concept for epidermal growth factor receptor and EGFRvIII targeting

Epidermal growth factor receptor (EGFR) is one of the major molecular targets for cancer diagnosis and therapy. EGFR and EGFRvIII, mutated form of EGFR, have been identified as participating in pathogenesis of some forms of human cancers. Monoclonal antibodies (mAbs) targeting EGFR/EGFRvIII have been shown to suppress the signal transduction pathways controlling tumor cell growth, proliferation, and apoptosis. Until now, different types of mAbs or antibody fragments against EGFR family have been established. Some of these antibodies have been used clinically for treating various forms of human malignancies. More recently, a single domain antibody (sdAb) targeting this family of receptors has been introduced. The heavy chain antibodies (HCAbs) that made up variable regions of heavy chain, CH2, and CH3 domains are shown in camelids. SdAbs derived from camel HCAbs are the smallest known natural building parts for binding to antigen. They also possess a longer antigen recognizing region, which increases their capability for being more specific in target antigen enhancement. Camelid antibodies are highly valuable for their special characteristics, including heat resistance, small size, high solubility in an aqueous environment, and non-immunogenicity in a human environment. Due to these abilities, research on biotechnological production and treatment applications of recombinant smaller fragments of these only HCAbs is widely in progress. In this article, we will discuss the challenges and successes of different types of mAbs targeting EGFR/EGFRvIII in human cancer.     

Inhibition of the Jun kinase pathway blocks DNA repair, enhances p53-mediated apoptosis and promotes gene amplification.

We have previously shown, by expression of a nonphosphorylatable dominant inhibitor mutant of c-Jun [cJun(S63A,S73A)], that activation of the NH2-terminal Jun kinase/stress-activated protein kinase by genotoxic damage is required for DNA repair. Here, we examine the consequences of inhibition of DNA repair on p53-induced apoptosis in T98G cells, which are devoid of endogenous wild-type p53. Relative to parental or wild-type c-Jun-expressing control cells, mutant Jun-expressing T98G clones show similar growth rates and plating efficiencies. However, these cells are unable to repair DNA (PCR-stop assays) and exhibit up to an 80-fold increased methotrexate-induced colony formation due to amplification of the dihydrofolate reductase gene. Moreover, the mutant c-Jun clones exhibit increased apoptosis and elevated bax:bcl2 ratios on expression of wild-type p53. These results indicate that inhibition of DNA repair leads to accumulation of DNA damage in tumor cells with unstable genomes and this, in turn, enhances p53mediated apoptosis.