Development of Innovative Biocomposites with Collagen, Keratin and Hydroxyapatite for Bone Tissue Engineering.

This study follows the process for the development of an innovative biomimetic composite derived from bovine collagen with keratin, with hydroxyapatite being hybridized into its architecture, and it builds a comprehensive evaluation of the composite's characteristics. The novel biomimetic materials are tailored with special traits to be achieved for the repair of osteochondral defects (OCDs). The purpose of the present research is to create a reliable effective alternative to existing bone graft materials while leveraging the intrinsic properties of the components for enhanced osteoinduction and integration. The composites were characterized based on their morphological properties, including water absorption, through scanning electron microscopy (SEM), and their structural properties were characterized by Fourier-Transform Infrared Spectroscopy (FTIR). Biological performance was assessed in vitro using human bone marrow mesenchymal stem cells (BMSCs), focusing on cytotoxicity, cell viability, and the ability to support cell colonization with forthcoming results. This in vivo study illustrates the real potential that this class of novel composites exhibits in regard to bone and cartilage tissue engineering and encourages further exploration and development for future clinical applications.

Circulating Biomarkers for Laboratory Diagnostics of Atherosclerosis-Literature Review.

Atherosclerosis is still considered a disease burden with long-term damaging processes towards the cardiovascular system. Evaluation of atherosclerotic stages requires the use of independent markers such as those already considered traditional, that remain the main therapeutic target for patients with atherosclerosis, together with emerging biomarkers. The challenge is finding models of predictive markers that are particularly tailored to detect and evaluate the evolution of incipient vascular lesions. Important advances have been made in this field, resulting in a more comprehensible and stronger linkage between the lipidic profile and the continuous inflammatory process. In this paper, we analysed the most recent data from the literature studying the molecular mechanisms of biomarkers and their involvement in the cascade of events that occur in the pathophysiology of atherosclerosis.

Selection of Optimal Operating Conditions for Extraction of Myrtus Communis L. Essential Oil by the Steam Distillation Method.

Myrtus communis L. is one of the important aromatic and medicinal species from the Mediterranean area. It is used in various fields such as culinary, cosmetic, pharmaceutical, therapeutic, and industrial applications. Thus, a Box-Wilson experimental plan was used in this study to select the optimal operating conditions in order to obtain high volumes of essential oils. The factorial design method was applied to evaluate at an industrial scale the effect of major process variables on the essential oil extraction from Myrtus communis L. herbs by the steam distillation method. The input variables considered as significant operating conditions were: X1-boiler occupancy rate (boilers were filled to 50%, 75%, and 100%), X2-distillation duration (distillation was continued 60, 75, and 90 min), and X3-particle size (herbs were cut in sizes of 10, 20, and 30 mm via guillotine). The dependent variable selected, coded as Y, was the essential oil volume obtained (mL). The steps of the classical statistical experimental design technique were complemented with the Taguchi method to improve the extraction efficacy of essential oil from Myrtus communis L., and the optimum parameter conditions were selected: boiler occupancy rate 100%, distillation duration 75 min, and particle size 20 mm. Following the optimum parameters, the GC-MS assay revealed for the Myrtus communis L. essential oil two predominant components, α-pinene-33.14% and eucalyptol-55.09%.

Efficiency of Multiparticulate Delivery Systems Loaded with Flufenamic Acid Designed for Burn Wound Healing Applications.

Burns are soft tissue injuries that require particular care for wound healing. Current tissue engineering approaches are aimed at identifying the most efficient treatment combinations to restore the tissue properties and function by using adapted scaffolds or delivery platforms for tissue repair and regeneration by triggering molecules. To reduce the inflammation associated with skin burns, the addition of an anti-inflammatory factor in these scaffolds would greatly increase the quality of the therapy. Therefore, this study is aimed at obtaining and validating a novel multiparticulate system based on a collagen matrix with controlled delivery of flufenamic acid anti-inflammatory drug for burn wound healing applications. In this work, we have characterized the properties and biocompatibility of these multiparticulate drug delivery systems (MDDS) and we have demonstrated their efficiency against burns and soft tissue lesions, particularly when the drug was microencapsulated, and thus with a controlled release. This study contributes to the advancement in therapy of burns and burn wound healing applications.

Rad51 overexpression and resistance to genotoxic agents. A study in the fission yeast Schizosaccharomyces pombe.

BACKGROUND: Many cancer cell lines have been found to overexpress the recombinase Rad51. The overexpression is associated with increased invasive potential and resistance to DNA-damaging therapeutic agents. This has been attributed to an increased capacity of cells overexpressing Rad51 to repair DNA lesions or to a genetic stabilization of the genome. AIM: As the explanations are somewhat controversial, we attempted to reproduce overexpression in the unicellular eukaryote Schizosaccharomyces pombe to have a simpler tool to study the problem of Rad51 overexpression and its induced resistance to DNA-damaging agents. METHODS: We used the nmt1 promoter inserted upstream of rad51 gene to induce its overexpression and studied the phenotype of the transformed strain, especially its sensitivity to camptothecin and hydroxyurea. RESULTS: We found that overexpression induced sensitivity to the two drugs even when it was associated with the deletion of a recombination mediator rad22/rad52 gene. However, when overexpression was associated with the deletion of the helicase-encoding fbh1 gene, the sensitivity to camptothecin was diminished.

Rad52 protein function study by fluorescence tagging.

Rad52 protein plays a significant role in DNA lesions repair by homologous recombination in eukariotic cells. Human Rad52 function somewhat overlaps with BRCA2 and has a role in cell survival in the absence of BRCA1-BRCA2 mediated recombination. Additional Rad52 function analysis and intracellular localization studies are probably necessary. We present a method for Rad22 protein tagging, a Schizosaccharomyces pombe Rad52 homologue, by Crerecombinase-mediated cassette exchange (RMCE) using the versatile pAW8 plasmid. Rad22 protein was C-termini yEGFP tagged; the resulting strain was analyzed by fluorescence microscopy. The yEGFP signal was observed (Rad22 foci) for 7.5 microM camptothecin, 0.005% methyl methanesulfonate, and 4 mM hydroxyurea treated cells. The RMCE method was efficient, and the presence of tagged Rad22 protein was confirmed by Western-Blot and fluorescence microscopy.

Hereditary hemochromatosis in north-eastern Romania.

UNLABELLED: Hereditary hemochromatosis is a genetic disturbance of iron metabolism resulting in iron overload in several organs and their functional failure. Early diagnosis is necessary to start a simple and effective therapy: bleeding. It is the most frequent genetic disease in some populations. Our objectives were 1. An estimate of the expectancy of the disease in the population of our geographic region, and 2. To diagnose the disease by applying the established methods and estimate the efficiency of our diagnosis. METHODS: 1.To estimate the expectancy, we genotyped 200 persons for the most frequent mutations causing the disease: HFE-C282Y and HFE-H63D by PCR-RFLP. 2. To diagnose the disease phenotypically we determined plasma iron level, ferritin level and transferrin saturation index in 549 patients previously diagnosed as chronic hepatitis or cirrhosis and genotyped those with hemochromatosis phenotype. RESULTS: 1. We found allelic frequencies of 1.75% and 13.25% for the HFE-C282Y and H63D mutant alleles respectively. From these frequencies we calculated that a severe case caused by a C282Y/C282Y homozygote can arise in 816 people and a mild case caused by a C282Y/H63D compound heterozygote can arise in 100 people 2. Among 549 patients we found 10 to have the phenotype of hemochromatosis and 3 out of the 10 were found to carry mutations: two in the HFE gene (one homozygous C282Y and one compound heterozygous C282Y/H63D) and one in the hemojuvelin (HJV) gene (a G320V).

In Schizosaccharomyces pombe the 14-3-3 protein Rad24p is involved in negative control of pho1 gene expression.

Expression of Schizosaccharomyces pombe pho1-encoded acid phosphatase is transcriptionally regulated by adenine and phosphate. Four genes, anr1-3 and anr5, encode negative regulators of pho1 expression. Apart from being designated as loci, the anr genes have not been further characterized. In this study we provide evidence that a strain carrying the deletion of rad24, a 14-3-3 protein-encoding gene, exhibits an anr mutant like the phenotype (higher phosphatase activity, higher transcript levels of pho1, lower sensitivity to adenine of pho1 expression) and that rad24 is closely linked, probably allelic, to anr5. By sequencing the two exons of the rad24 gene in a strain carrying the mutant allele anr5-13, we found a T/A-to-C/G transition in the 225th codon of its ORF, causing a leucine-to-serine substitution in a highly conserved region of all proteins of the 14-3-3 family. anr2 and anr3 are not allelic to rad24. The mutant alleles of anr2 and anr3 are recessive to their wild-type alleles and do not belong to the same epistasis group as rad24.

Method for protein tagging in Schizosaccharomyces pombe.

Tagging is a useful method for the investigation of proteins. It allows the localization of the proteins in the cell, their purification in order to investigate their function and the determination of their expression. The aim of the present study was to tag the Rad32 protein of fission yeast (which is the homologue of Mre11 protein from humans) at its N-terminus. Rad32p as well as Mre11p are involved in the repair of DNA double strand breaks and in the DNA damage checkpoint. We carried out this tagging using the Cre-loxp recombination system. In a first step, a 2 kb DNA fragment was integrated upstream of the initiating codon of rad32 gene. This fragment encoded the TAP-tag (tandem affinity purification), a loxp site, a selectable marker (sup3-5), an exogenous promoter (nmt1) and a second loxp site, in this sequence. Following transformation of this DNA fragment into S. pombe cells, rad32 was under the control of the artificial promotor, which allows a controlled expression of the gene by thiamine. In a second step, the cells were transformed with a plasmid coding for Cre recombinase, which catalyses the excision of the DNA sequence between the two loxp sites, removing the marker and the artificial promotor. Thus the tag became attached to the rad32 gene upstream of the ATG, placing the gene under the control of its native promotor. The strain thus obtained will be subsequently used for evidencing the tagged protein by Western blotting and then for its purification in order to investigate its function.

[Mutagenic aspects of the lipoprotein lipase gene]. / Aspecte mutagenice la nivelul genei lipoprotein lipazei.

Lipoprotein lipase (LPL) is an enzyme involved in the metabolism of triglyceride-rich lipoproteins (chylomicrons and VLDL), participating also in the remodeling of HDL particles. It is encoded by a gene located on chromosome 8 (8p22), containing 10 exons. Abnormalities in this gene lead to the development of LPL enzyme deficiency. About 100 mutations have been described in the LPL gene, the most frequent being Asp9Asn, Gly188Glu and Asn291Ser. Mutations in the homozygous form are associated with type I hyperlipoproteinemia (familial chylomicronemia). Mutations in the heterozygous state have a significant incidence in population (3-7%) and lead to a decrease in the LPL activity with up to 50%, which causes the modification of the plasma lipid profile, meaning an increase in triglycerides and a decrease in HDL cholesterol. Both modifications represent cardiovascular risk factors, so that the carriers of LPL gene mutations have an increased predisposition to develop coronary heart disease. Moreover, the association of heterozygous mutations in the LPL gene in individuals with genetic-type hyperlipoproteinemias may aggravate the perturbations of the lipid profile and, consequently, they can increase the cardiovascular risk in these patients. The accumulated data may be considered an argument for the importance of investigating LPL gene mutations in the population, in order to detect precociously the individuals with an increased atherogenic predisposition and to decide on the appropriate therapy.

[Method for the molecular diagnosis of lipoprotein lipase genetic deficiency]. / Metoda de diagnostic molecular al deficitului genetic al lipoprotein lipazei.

Modifications of plasma lipid profile is one of the major causes of a high cardiovascular risk. They can be the consequences of mutations in the gene encoding lipoprotein lipase (LPL), an enzyme that has an important role in the metabolism of plasma lipoproteins. The aim of the present study was to put into practice a method for detecting the Gly188Glu mutation in the LPL gene. The search was performed on a group of 107 patients with cardiovascular diseases and/or dyslipidemias. DNA investigation consisted, in a first stage, in the enzymatic digestion of exon 5 of the LPL gene, previously amplified by the PCR reaction, with the AvaII restriction endonuclease. Three of the subjects were further investigated by the sequencing of exon 5, in order to search for the presence of other mutations. We didn't detect the Gly188Glu mutation in none of the cases, and no other mutation in exon 5 was found in the three patients tested by DNA sequencing. We conclude that the amplification-restriction method can be used for the detection of known mutations in the LPL gene, allowing an early identification of the subjects with a high cardiovascular risk and the onset of the appropriate therapy. In order to detect mutations which don't affect the recognition sequence of a restriction enzyme and eventually new mutations, the sequencing of that gene is recommended.