Live-cell biosensor for assessment of adhesion qualities of biomaterials.

The present study describes a live-cell biosensor, suitable for general evaluation of adhesion qualities of different substrates. It is based on NIH/3T3 fibroblast cell line stably expressing fusion fluorescently tagged proteins mCherry-vinculin and GFP-tensin as quantifiable markers for assessment not only of focal but also of fibrillar contacts. Four measurable parameters - spreading, polarization and development of focal and fibrillar adhesions were used to standardize the adhesion of biosensor cells after plating on five substrates of natural origin - fibronectin, vitronectin, laminin-111, laminin-521 and collagen type I. The obtained set of values (adhesion quality map) were utilized to describe the default biosensor behavior and as a standard for evaluation of surface biocompatibility of materials with unknown adhesive properties. To demonstrate the applicability of the biosensor we studied two PDMS-based artificial materials. The results demonstrated the superior adhesive properties of the poly(acrylic acid)-containing polymer (PDMS-PAA) over that of poly(vinyl pyrrolidone) copolymer (PDMS-PVP), and pointed out the formation of focal adhesions as a parameter for possible further improvements.

Frequency of the common promoter polymorphism MMP2 -1306 C>T in a population from central Bulgaria.

Matrix metalloproteinases (MMPs) are a family of highly homologous extracellular Zn2+-dependent endopeptidases, also known as matrixins. MMP-2 (gelatinase A) and MMP-9 (gelatinase B) are considered to play a key role in a variety of physiological processes as well as in the development and progression of a vast majority of pathological conditions. Most of the genes encoding MMPs, including MMP-2, are highly polymorphic. One of the single nucleotide polymorphisms with functional activity in the promoter region of MMP2 is the transition MMP2 -1306C>T (rs243865). The aim of the present study was to evaluate the genotype and allele frequencies of the common promoter polymorphism -1306C>T in MMP2 in 75 individuals from central Bulgaria and to compare our results with those of other population studies. We found that 76.0% of the randomly enrolled individuals are carriers of the CC genotype, 17.3% of CT, and 6.7% of the TT genotype. The minor allele frequency (MAF) was 15.3%. Interestingly, the obtained genotype frequencies appeared to differ from those of some other Caucasian populations (USA - 55/38/7, MAF 26%; The Netherlands - 52.8/40.5/6.7, MAF 26.9%; Austria - 55.6/35.5/8.9, MAF 27.2%), but were closer to the values of the reported global genotype distribution (75.3/21.3/3.4, MAF 14%).

Current state of the opportunities for derivation of germ-like cells from pluripotent stem cells: are you a man, or a mouse?

The concept of pluripotency as a prerogative of cells of early mammal embryos and cultured embryonic stem cells (ESC) has been invalidated with the advent of induced pluripotent stem cells. Later, it became clear that the ability to generate all cell types of the adult organism is also a questionable aspect of pluripotency, as there are cell types, such as germ cells, which are difficult to produce from pluripotent stem cells. Recently it has been proposed that there are at least two different states of pluripotency; namely, the naïve, or ground state, and the primed state, which may differ radically in terms of timeline of existence, signalling mechanisms, cell properties, capacity for differentiation into different cell types, etc. Germ-like male and female rodent cells have been successfully produced in vitro from ESC and induced pluripotent stem cells. The attempts to derive primate primordial germ cells (PGC) and germ cells in vitro from pluripotent stem cells, however, still have a low success rate, especially with the female germline. The paper reviews the properties of rodent and primate ESC with regard to their capacity for differentiation in vitro to germ-like cells, outlining the possible caveats to derivation of PGC and germ cells from primate and human pluripotent cells.

Microbial community development of biofilm in Amaranth decolourization technology analysed by FISH.

The aim of this study was to elucidate the role, the space distribution and the relationships of the bacteria from the genus Pseudomonas in a biofilm community during semi-continuous Amaranth decolourization process in model sand biofilters. The examined parameters of the process were as follows: technological parameters; key enzyme activities (azoreductase, succinate dehydrogenase, catechol-1,2-dioxygenase, catechol-2,3-dioxygenase); the number of azo-degrading bacteria and the bacteria from genus Pseudomonas (plate count technique); the amount and the location of Pseudomonas sp. using fluorescent in situ hybridization (FISH). The results showed that the increase of the Amaranth removal rate with 120% was accompanied with increase of the enzyme activities of the biofilm (azoreductase activity - with 25.90% and succinate dehydrogenase - with 10.61%). The enzyme assays showed absence of activity for сatechol-1,2-dioxygenase and catechol-2,3-dioxygenase at the early phase and high activities of the same oxygenases at the late phase (2.76 and 1.74 µmol/min mg protein, respectively). In the beginning of the process (0-191 h), the number of the culturable microorganisms from genus Pseudomonas was increased with 48.76% but at the late phase (191-455 h) they were decreased with 15.25% while the quantity of the non-culturable bacteria from this genus with synergetic relationships was increased with 23.26%. The dominant microbial factors were identified in the structure of the biofilm during the azo-degradation process by using FISH analysis. Furthermore, the inner mechanisms for increase of the rate and the range of the detoxification were revealed during the complex wastewater treatment processes.

Investigation of the role of MMP3 -1171insA polymorphism in cutaneous malignant melanoma - a preliminary study.

Coetaneous malignant melanoma is the most aggressive cancer of the skin with a high rate of mortality worldwide. Degradation of basement membranes and extracellular matrix is an essential step in cancer invasion and metastasis. Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) play key roles in this step. MMP-3 also called stromelysin-1 was one of the first proteinases found to be associated with cancer. In the gene of MMP-3 (MMP3), an insertion/deletion of an A nucleotide at position -1171 in promoter region has been identified and shown to effect the expression activity of the gene. The present study was conducted to investigate the relation of MMP3 -1171insA polymorphism with skin malignant melanoma risk in a pilot case-control study of Bulgarian patients (n = 26) and unaffected controls (n = 172). The genotypes of controls and melanoma patients were in Hardy-Weinberg equilibrium. The results showed no statistically significant difference both in genotype and allele frequencies of MMP3 -1171insA polymorphism between melanoma patients and healthy controls either in crude analyses (p = 0.360 and 0.790, c2-test) or after adjustment for age and sex. The comparison of some clinical characteristics between the patients with different genotypes showed a trend for longer survival of patients with 6A/6A genotype compared to the carriers of 5A allele (5A/5A+5A/6A genotypes, p = 0.118, Log rank test). The results of our current preliminary study do not provide evidence for the role of the promoter polymorphism -1171insA in MMP3 as a risk factor for development of coetaneous melanoma, but suggest its implication in progression of the diseases.

Disoxaril mutants of Coxsackievirus B1: phenotypic characteristics and analysis of the target VP1 gene.

Disoxaril inhibits enterovirus replication by binding to the hydrophobic pocket within the VP1 coat protein, thus stabilizing the virion and blocking its uncoating. Disoxaril-resistant (RES) mutants of the Coxsackievirus B1 (CVB1/RES) were derived from the wild disoxaril-sensitive (SOF) strain (CVB1/SOF) using a selection approach. A disoxaril-dependent (DEP) mutant (CVB1/DEP) was obtained following nine consecutive passages of the disoxaril-resistant mutant in the presence of disoxaril. Phenotypic characteristics of the disoxaril mutants were investigated. A timing-of-addition study of the CVB1/DEP replication demonstrated that in the absence of disoxaril the virus particle assembly stopped. VP1 RNA sequences of disoxaril mutants were compared with the existing Gen Bank CVB1 reference structure. The amino acid sequence of a large VP1 196-258 peptide (disoxaril-binding region) of CVB1/RES was significantly different from that of the CVB1/SOF. Crucially important changes in CVB1/RES were two point mutations, M213H and F237L, both in the ligand-binding pocket. The sequence analysis of the CVB1/DEP showed some reversion to CVB1/SOF. The amino acid sequences of the three VP1 proteins are presented.