Analysis of TP53 gene and particular infrastructural alterations in invasive ductal mammary carcinoma.

This study was conducted in order to determine the mutational status of TP53 gene and to determine some particular aspects from ultrastructural level in invasive mammary ductal carcinoma. The cellular signaling pathway involving the TP53 gene acts in biological deoxyribonucleic acid (DNA) repair processes and cell cycle arrest following a signal transmitted to the p53 protein when posttranslational changes occur in the cell due to stress induced in the cell by both intrinsic and extrinsic factors. Cellular stress activates the transcription factor function of the protein that initiates, as the case may be, either DNA repair or programmed cell death (apoptosis). The TP53 gene is commonly mutated in many human cancers and also has a highly polymorphic grade. To determine the mutational status of the exons 4-9 of the TP53 gene, we used extracted DNA from fresh breast tissue, and we analyzed it through direct sequencing. In mammary carcinoma, the mutation frequency of TP53 is running between 20-40% and, in regards the polymorphism, at least 14 different forms were identified, that are associated with cancer risk. The mutation type distribution showed a predominance of deletions and a reduced frequency of substitutions comparing with International Agency for Research on Cancer (IARC) database. Taken in consideration the importance of the tumor associated stroma in tumor development, we have also investigated some particular aspects at the infrastructural level of invasive mammary ductal carcinoma, notably concerning telocytes as tumor stroma interstitial cells by transmission electron microscopy analysis.

Molecular analysis of BRCA1 and BRCA2 genes by next generation sequencing and ultrastructural aspects of breast tumor tissue.

In this paper, we focus our interest on the dynamics alterations of the tumor-stroma interface at the ultrastructural level and to detect BRCA1 and BRCA2 mutations using next generation sequencing (NGS) of breast tumor tissue. Electron microscopic investigation revealed some peculiar infrastructural alterations of the tumor cells per se as well as of the tumor-stroma interface: invadopodia, shedding microvesicles, altered morphology and reduced number of telocytes, different abnormalities of the microvasculature. Tumor suppressor genes BRCA1 and BRCA2 are the genes with most hereditary predisposition to breast and ovarian cancer. An early identification of mutation within these genes is essential for determining classification and therapeutic approach to patients. Genetic tests used to determine mutations in BRCA1 and BRCA2 genes are laborious analysis methods which include, among others, NGS. We analyzed a total of eight samples, in which genomic DNA was amplified using Ion AmpliSeq panel BRCA1 and BRCA2. DNA libraries were created, amplified and sequenced with Ion Torrent Personal Genome Machine. The bio-information data obtained allow us to detect all known pathogenic mutation and uncertain polymorphisms.

Particular molecular and ultrastructural aspects in invasive mammary carcinoma.

Electron microscopic investigations of invasive mammary carcinoma tumors revealed that intercellular junctions, namely desmosomes are severely altered; some desmosomes became internalized. Tumor cells, especially by their invadopodia, generate and disseminate membrane vesicles, including exosomes, inside of peritumoral stroma. Telocytes, a new described interstitial/stromal cell phenotype, considered to play important roles in cell signaling, exhibited a reduced number of hetero-cellular contacts, which suggests a possible perturbation of tissular homeostasis modulation. Signaling PIK3/Akt pathway plays an important role both in carcinogenesis and in proliferation, differentiation, and cell survival. Alteration of this pathway has been observed in many human cancers, often involving an increase in the activity of PIK3CA, p110α catalytic subunit of PI3K. Our study confirms the high prevalence of PIK3CA mutations in breast cancer. In accordance with the results of the largest previous studies, 87.5% of mutations detected by DNA direct sequencing were hot spot mutations, most of them located in the kinase domain. High percentage of mutations detected by high-resolution melting makes the assay an attractive choice for mutation scanning, especially, in samples with low percentage of tumor cell.

Prognostic value of melanoma inhibitory activity protein in localized cutaneous malignant melanoma.

Background. Cutaneous malignant melanoma (CMM) is a heterogeneous disease, acknowledged for its lack of predictability regarding clinical evolution. In order to appreciate a patient's individual prognosis, an attempt is made to find new tumor markers that parallel the disease progression. Objective. To identify if melanoma inhibitory activity (MIA) protein could represent a tool for selecting high risk early stages melanoma patients. Method. Between 2008 and 2013, 155 patients with CMM were treated in our clinic. 84 of them were classified into stages I and II, according to TNM 2009. MIA serum concentration was measured in all patients and 50 healthy donors. A cut-off value of 9.4 ng/ml was established using the ROC curve. Results. All patients were followed up by periodic investigations every 6 months. We have noticed that 66% of patients with MIA serum values at diagnosis greater than 9.4 ng/mL have relapsed, while only 5% of patients with MIA serum concentration below the estimated threshold, recurred during the follow-up period (P = 0.000). The death risk was 12 times higher in pathological MIA group of patients (P = 0.0001). Conclusions. Our data suggest that MIA is an independent prognostic factor for patients with localized CMM.

Green light effects on biological systems: a new biophysical phenomenon.

This paper reports a new phenomenon connected with the influence of green light (GL) on biological systems. Our experiments have revealed an antioxidant effect of GL on cells subjected to lethal doses of UV at the cellular level and a protective effect of GL on DNA denatured by UV, coupled with a structural modification of DNA macromolecules under GL irradiation, at the molecular level. Mouse melanocyte cultures are subjected to UV irradiations with L(50) fluxes of 16.0 J m(-2) s(-1). GL is obtained from a strontium aluminate pigment, which emits GL under UV activation. Cells grown in GL, prior to UV irradiation, present a clear surprising protective effect with surviving values close to the controls. A GL antioxidant effect is suggested to be mediated through GL influence on cellular water cluster dynamics. To test this hypothesis, reactive oxygen species (ROS) are determined in cell cultures. The results revealed a decrease of cellular ROS generation in the UV-irradiated samples protected by a previous 24 h of GL irradiation. At the DNA level, the same type of GL protection against UV damage is recorded by gel electrophoresis and by UV spectroscopy of the irradiated DNA molecules. Two physical methods, impedance spectroscopy and chronoamperometry, have revealed at the level of GL-irradiated DNA molecules spectral modifications that correlate with the UV spectroscopy results. The interaction between the chargeless photons and the field of water molecules from the cellular compartments is discussed in relation with the new field of macroscopic quantum coherence phenomena.

Combined microwave and electron beam exposure facilities for medical studies and applications.

The paper presents two radiation exposure facilities (REFs) which permit separate and simultaneous irradiation with microwaves (MW) of 2.45 GHz and electron beams (EB) of 6.23 MeV for malignant melanoma (MM) cell investigations, in vitro (MW+EB-REF-vitro) and in vivo (MW+EB-REF-vivo). The REFs are specifically designed for the following medical studies: 1) The effects of separate and combined (successive and simultaneous) MW and EB irradiation on the B16F10 mouse--MM cell cultures without/with drugs incubation, 2) The effects of separate and combined MW and EB irradiation on human blood components irradiated in samples of integral blood from healthy donors and from donors with MM; 3) The effects of separate and combined MW and EB whole body irradiation on the C57 BL/6 mice bearing MM without/with drugs administration. Several representative results obtained by experiments with REFs in vitro and in vivo are discussed. The most important conclusion of the experimental results is that low dose-total body MW+EB irradiation combined with drugs administration could present a valuable potential for an advanced study in malignant melanoma therapy.

Cell investigations simultaneously with exposure to 2.45 GHz microwaves.

The paper presents two microwave (MW) exposure systems (MWESs) that permit observations and measurements on cell cultures during their exposure to MW of 2.45 GHz: MWES-1 and MWES-2. MWES-1 is designed for the measurement of the cell membrane fluorescence anisotropies (MFA) simultaneously with MW exposure. MWES-2 is designed for the cells culture exploration under an inverted microscope before, during and after MW exposure. MWES-1 consists mainly of a 2.45 GHz microwave generator (MWG-2.45 GHz-SAIREM) of 0-25 W, equipped with forward power and reflected power displaying, and an adjustable coaxial antenna immersed directly into the cuvette with the cells-suspension of a Spex type spectrofluorometer. The MW effect on membrane fluidity of B16F10 malignant melanoma (B16F10-MM) cells in suspension were investigated with MWES-1, by MFA measurements. We observed a MW induced transition temperature (ITT) rising strongly during the MW exposure as compared with ITT obtained by classical heating (CH). The MWES-2 consists of the MWG-2.45 GHz-SAIREM generator and a rectangular waveguide applicator with traveling wave placed between the condenser and the objective of a Zeiss Axiovert 200 microscope, equipped with a fluorescence device and image acquisition. The MW effects on shape and apoptosis of the B16F10-MM cells were investigate with MWES-2. The B16F10-MM cells exhibited visible shape changes during MW exposure up to 37 degrees C. The MW exposure induced cells apoptosis/necrosis in several seconds after that MW are applied, beginning with SAR = 1.5 W/sample, compared to CH controls exposed at the same temperature dynamics.