Marieta Costache

Evolution of Fucosyltransferase Genes in Vertebrates

Cloning and expression of chimpanzee FUT3, FUT5, and FUT6 genes confirmed the hypothesis that the gene duplications at the origin of the present human cluster of genes occurred between: (i) the great mammalian radiation 80 million years ago and (ii) the separation of man and chimpanzee 10 million years ago. The phylogeny of fucosyltransferase genes was completed by the addition of the FUT8 family of α(1,6)fucosyltransferase genes, which are the oldest genes of the fucosyltransferase family. By analysis of data banks, a newFUT8 alternative splice expressed in human retina was identified, which allowed mapping the human FUT8 gene to 14q23. The results suggest that the fucosyltransferase genes have evolved by successive duplications, followed by translocations, and divergent evolution from a single ancestral gene.

Synthesis, characterization, and in vitro studies of graphene oxide/chitosan-polyvinyl alcohol films.

Nanocomposites based on chitosan-polyvinyl alcohol (CS-PVA) and graphene oxide (GO) were prepared by casting the stable aqueous mixture of the components. SEM, TEM and X-ray diffraction showed that graphene oxide is largely dispersed on molecular scale within CS-PVA matrix. FTIR investigation indicated the occurrence of some interaction between graphene oxide nanosheets and CS-PVA. The obtained composites are mechanically strong and exhibit improved thermal stability. By addition of 6 wt.% GO within CS-PVA blend, the elastic modulus increased over 200%. The cell viability and proliferation results showed that MC3T3-E1 mouse osteoblastic cells can adhere and developed on the CS-PVA/GO composite films. A significant proliferation potential was displayed by the cells in contact with CS-PVA/GO 6 wt.%. Graphene oxide reinforced CS-PVA with high mechanical and bioactive properties are potential candidates for tissue engineering.

Reduced inflammatory activity of RAW 264.7 macrophages on titania nanotube modified Ti surface.

Macrophages play a pivotal role in the hosts response to biomaterials being considered as an essential cell type during both optimal tissue-implant integration and pathologic process of implant failure. Hence, understanding of their cellular activity on biomaterials is important for improving evaluation and design of biomaterials for biomedical applications. In the present study, we have comparatively investigated the interactions of titania nanotubes (78 nm diameter) and commercial pure Ti with RAW 264.7 macrophages in both standard and pro-inflammatory (stimulation with lipopolysaccharide, LPS) culture conditions. In vitro tests showed that TiO2 nanotubes exhibited significantly decreased inflammatory activity of macrophages with respect to cytokine and chemokine gene expression/protein secretion, induction of foreign body giant cells (FBGCs) and nitric oxide (NO) release thereby mitigating the inflammatory response induced by LPS as compared to flat Ti surface. Therefore, our results suggest a novel role of TiO2 nanotubes in modulating macrophage response in biomaterial-associated bacterial infections. Overall, the current study provides new insight into how TiO2 nanotubes can be involved in macrophage activation and supports the great promise of such surface modifications for biomedical applications.

Zoonotic Cryptosporidium parvum in Romanian newborn lambs (Ovis aries).

This study was undertaken to investigate the occurrence and public health significance of Cryptosporidium species/genotypes and subtypes in a newborn lambs. A total of 175 diarrheic fecal samples from lambs (younger than 21 days) were collected in seven sheep flocks located in western Romania, and were microscopically examined for the presence of Cryptosporidium oocysts after staining with modified Ziehl-Neelsen technique. Twenty-four (13.7%) fecal samples were tested Cryptosporidium positive by microscopy and were subjected for molecular characterization. All positive samples were successfully amplified through a nested polymerase chain reaction (PCR) of the small subunit (SSU) rRNA gene (18S). Cryptosporidium species were determined by restriction fragment length polymorphism (RFLP) analysis of the secondary PCR products using the conventional SspI and VspI restriction enzymes. The identified species were: Cryptosporidium parvum (20/24), C. ubiquitum (2/24) and C. xiaoi (2/24), respectively. PCR-RFLP results for C. ubiquitum and C. xiaoi isolates were confirmed by DNA sequencing. Subsequently, subtyping of seven randomly selected C. parvum isolates, based on sequence analysis of the GP60 gene, revealed the presence of five different subtypes (IIaA17G1R1, IIaA16G1R1, IIdA20G1, IIdA24G1 and IIdA22G2R1) belonging in two zoonotic subtype families (IIa and IId). These findings may suggest the potential role of the newborn lambs as a source for human cryptosporidiosis. This is the first published report about the presence of C. ubiquitum and C. xiaoi in lambs from Romania.

In vitro cytocompatibility evaluation of chitosan/graphene oxide 3D scaffold composites designed for bone tissue engineering

Extensively studied nowadays, graphene oxide (GO) has a benefic effect on cell proliferation and differentiation, thus holding promise for bone tissue engineering (BTE) approaches. The aim of this study was not only to design a chitosan 3D scaffold improved with GO for optimal BTE, but also to analyze its physicochemical properties and to evaluate its cytocompatibility and ability to support cell metabolic activity and proliferation. Overall results show that the addition of GO in the scaffolds composition improved mechanical properties and pore formation and enhanced the bioactivity of the scaffold material for tissue engineering. The new developed CHT/GO 3 wt% scaffold could be a potential candidate for further in vitro and in vivo osteogenesis studies and BTE approaches.

The activity of some digestive enzymes in domestic rabbits before and after weaning

The activity ofamylase, maitase, lipase, pepsin, trypsin and chymotrypsin in suckling (15 days of age) and in 1-day weaned (43 days of age) domestic rabbits was assayed and compared with older (90 and 180 days) rabbits. It was found that amylase was active in the pancreas at 15 days (11 580 amylase units (AU) per mg protein) and increased during growth, reaching a maximum level (58 960 AU per mg protein) at 90 days of age. Specific activity (SA) of maitase from the small intestine mucosa varied depending on the intestinal segment and the age of the rabbits: activity in the duodenal mucosa decreased, while in the jejunal and Heal mucosa activity increased, during growth. Lipase SA reached a maximum level in suckling rabbits at the age of 15 days (in gastric mucosa 242 and in pancreas 608 mequiv. liberated oleic acid per mg protein per h, 37°C) and decreased sharply at weaning: in gastric mucosa down to 86 and in pancreas down to 89 mequiv. oleic acid per mg protein per h, 37°C. SA of pepsin remained relatively constant for all the studied categories of rabbits: 38 to 39 nmol tyrosine per mg protein per min, 25°C. Trypsin and chymotrypsin SA reached a peak at about weaning: 1·83 nmol benzoyl-arginyl-ethyl-ester per mg protein per min, 25°C and 40·1 nmol benzoyl-phenyl-naphtyl-ester per mg protein per min, 35·5°C respectively.

Histopatological alterations and oxidative stress in liver and kidney of Leuciscus cephalus following exposure to heavy metals in the Tur River, North Western Romania

Pollution of the aquatic environment by heavy metals is a great concern worldwide. Freshwater fish ingests various metals through gills, skin or diet. Our aim was to investigate the oxidative stress and histopathological injuries induced by Fe, Cu, Zn, Pb, Cd in the liver and kidney of Leuciscus cephalus. Fish samples were collected from two sites in the Tur River, NW Romania, in upstream and downstream of a pollution source. Metals were differently distributed in the liver and kidney of fish. The highest concentrations of Fe, Cu and Pb were found in liver, whereas Zn and Cd concentrations were the highest in kidney in specimens collected from the downstream site. The histopathological changes were associated with metal bioaccumulation, being more severe in kidney than liver. Malondialdehyde (MDA) and advanced oxidation protein products (AOPP) increased significantly in the liver and kidney of fish from downstream site compared to upstream one, whereas reduced glutathione (GSH) decreased. The activities of superoxide dismutase (SOD), catalase (CAT) and glutathione-S-transferase (GST) increased significantly in livers, whereas SOD increased in kidney. Our study revealed that liver has a higher capacity and adaptability to counteract ROS compared to kidney. The more pronounced increase of hepatic SOD, CAT and GST activities is related milder structural changes observed in liver compared to kidney, where lesions were not reduced by antioxidant defense system.

Layer-shaped alginate hydrogels enhance the biological performance of human adipose-derived stem cells

BackgroundThe reconstruction of adipose tissue defects is often challenged by the complications that may occur following plastic and reconstructive surgery, including donor-site morbidity, implant migration and foreign body reaction. To overcome these problems, adipose tissue engineering (ATE) using stem cell-based regeneration strategies has been widely explored in the last years. Mounting evidence has shown that adipose-derived stem cells (ADSCs) represent a promising cell source for ATE. In the context of a small number of reports concerning adipose tissue regeneration using three-dimensional (3-D) systems, the present study was designed to evaluate the biological performance of a novel alginate matrix that incorporates human ADSCs (hADSCs).ResultsCulture-expanded cells isolated from the stromal vascular fraction (SVF), corresponding to the third passage which showed the expression of mesenchymal stem cell (MSC) markers, were used in the 3-D culture systems. The latter represented a calcium alginate hydrogel, obtained by the diffusion of calcium gluconate (CGH matrix), and shaped as discoid-thin layer. For comparative purposes, a similar hADSC-laden alginate hydrogel cross-linked with calcium chloride was considered as reference hydrogel (RH matrix). Both hydrogels showed a porous structure under scanning electron microscopy (SEM) and the hADSCs embedded displayed normal spherical morphologies, some of them showing signs of mitosis. More than 85% of the entrapped cells survived throughout the incubation period of 7 days. The percentage of viable cells was significantly higher within CGH matrix at 2 days post-seeding, and approximately similar within both hydrogels after 7 days of culture. Moreover, both alginate-based hydrogels stimulated cell proliferation. The number of hADSC within hydrogels has increased during the incubation period of 7 days and was higher in the case of CGH matrix. Cells grown under adipogenic conditions for 21 days showed that both analyzed 3-D culture systems support adipogenic differentiation in terms of neutral lipid accumulation and perillipin expression. Furthermore, the cells encapsulated in CGH matrix displayed a more differentiated phenotype.ConclusionsThe results of this study suggest that both CGH and RH matrices successfully support the survival and adipogenesis of hADSC. An enhancement of biological performance was detected in the case of CGH matrix, suggesting its promising application in ATE.

Adapted response of the antioxidant defense system to oxidative stress induced by deoxynivalenol in Hek-293 cells

The mycotoxin deoxynivalenol (DON), a contaminant of certain foods and feeds, is cytotoxic and genotoxic to mammalians cells. Exposure of human embryonic kidney (Hek-293) cells to DON led to a dose- and time-dependent decrease in cell viability, with an IC(50) about 7.6 μM. The DON effects on Hek-293 morphology, reactive oxygen species, lipid peroxidation and antioxidative system and caspase 3 and bcl-2 expression were studied. Cells became round and in some are progressive loss of cell attachment appeared. These biochemical parameters were assessed after 6, 12 and 24 h of treatment with 2.5 and 5 μM DON. An increase in superoxide dismutase activity within the interval 6-12 h and almost complete recovery by the end of experiment for both concentrations was observed, whereas the profile of catalase activity was the same with the superoxide dismutase one for 2.5 μM and decreased in a time-dependent manner for 5 μM. A temporary activation of glutathione peroxidase and glutathione reductase was recorded at 12 h post-exposure, while the glutathione-S-transferase activity was unchanged for both concentrations. The NADP(+)-dependent isocitrate dehydrogenase activity showed a transient increase at the 12 h post-exposure. The caspase 3 expression remained unchanged and the bcl-2 one decreased after 24 h of exposure for the two concentrations. Our results showed the dose- and time specific changes in the antioxidants system of Hek-293 cells, which could not counteract efficiently the effects DON exposure. The different types of cell death which could be activated by this DON induced changes are mentioned.

Nuclear Markers of Danube Sturgeons Hybridization

Acipenseriformes are composed of 25 sturgeon species and two paddlefish species distributed exclusively in the northern hemisphere. The Danube River and the Black Sea were originally inhabited by six sturgeon species but two are extinct and only four are still reproducing currently in the Lower Danube: Huso huso, Acipenser stellatus, A. gueldenstaedtii and A. ruthenus. Sturgeon species hybridize more easily than other fish and the determination of pure species or hybrid status is important for conservation and for breeding in fish farms. This survey demonstrated that morphological determination of this status is not reliable and a molecular tool, based on eight microsatellites genotypes is proposed. This method, based on three successive statistical analyses including Factorial Correspondence Analysis (FCA), Structure assignation and NewHybrids status determination, showed a high efficiency in discriminating pure species specimens from F1, F2 and two kinds of backcross individuals involving three of the four reproducing Lower Danube sturgeon species.