Modulation of cell adhesion, proliferation and differentiation on materials designed for body implants

The interaction of cells and tissues with artificial materials designed for applications in biotechnologies and in medicine is governed by the physical and chemical properties of the material surface. There is optimal cell adhesion to moderately hydrophilic and positively charged substrates, due to the adsorption of cell adhesion-mediating molecules (e.g. vitronectin, fibronectin) in an advantageous geometrical conformation, which makes specific sites on these molecules (e.g. specific amino acid sequences) accessible to cell adhesion receptors (e.g. integrins). Highly hydrophilic surfaces prevent the adsorption of proteins, or these molecules are bound very weakly. On highly hydrophobic materials, however, proteins are adsorbed in rigid and denatured forms, hampering cell adhesion. The wettability of the material surface, particularly in synthetic polymers, can be effectively regulated by physical treatments, e.g. by irradiation with ions, plasma or UV light. The irradiation-activated material surface can be functionalized by various biomolecules and nanoparticles, and this further enhances its attractiveness for cells and its effectiveness in regulating cell functions. Another important factor for cell-material interaction is surface roughness and surface topography. Nanostructured substrates (i.e. substrates with irregularities smaller than 100nm), are generally considered to be beneficial for cell adhesion and growth, while microstructured substrates behave more controversially (e.g. they can hamper cell spreading and proliferation but they enhance cell differentiation, particularly in osteogenic cells). A factor which has been relatively less investigated, but which is essential for cell-material interaction, is material deformability. Highly soft and deformable substrates cannot resist the tractional forces generated by cells during cell adhesion, and cells are not able to attach, spread and survive on such materials. Local variation in the physical and chemical properties of the material surface can be advantageously used for constructing patterned surfaces. Micropatterned surfaces enable regionally selective cell adhesion and directed growth, which can be utilized in tissue engineering, in constructing microarrays and in biosensorics. Nanopatterned surfaces are an effective tool for manipulating the type, number, spacing and distribution of ligands for cell adhesion receptors on the material surface. As a consequence, these surfaces are able to control the size, shape, distribution and maturity of focal adhesion plaques on cells, and thus cell adhesion, proliferation, differentiation and other cell functions.     

Role of duodenal iron transporters and hepcidin in patients with alcoholic liver disease

Patients with alcoholic liver disease (ALD) often display disturbed iron indices. Hepcidin, a key regulator of iron metabolism, has been shown to be down‐regulated by alcohol in cell lines and animal models. This down‐regulation led to increased duodenal iron transport and absorption in animals. In this study, we investigated gene expression of duodenal iron transport molecules and hepcidin in three groups of patients with ALD (with anaemia, with iron overload and without iron overload) and controls. Expression of DMT1, FPN1, DCYTB, HEPH, HFE and TFR1 was measured in duodenal biopsies by using real‐time PCR and Western blot. Serum hepcidin levels were measured by using ELISA. Serum hepcidin was decreased in patients with ALD. At the mRNA level, expressions of DMT1, FPN1 and TFR1 genes were significantly increased in ALD. This pattern was even more pronounced in the subgroups of patients without iron overload and with anaemia. Protein expression of FPN1 paralleled the increase at the mRNA level in the group of patients with ALD. Serum ferritin was negatively correlated with DMT1 mRNA. The down‐regulation of hepcidin expression leading to up‐regulation of iron transporters expression in the duodenum seems to explain iron metabolism disturbances in ALD. Alcohol consumption very probably causes suppression of hepcidin expression in patients with ALD.     

NMR Assignment of the mTOR Domain Responsible for Rapamycin Binding

A fully automated, NOE-based NMR structure determination of a uniformly ¹³C,¹⁵N-labeled protein was achieved in crude cell-extract, without purification of the overexpressed protein. Essentially complete sequence-specific assignments were obtained using triple resonance experiments, based on the high intensity of the resonances from the overexpressed protein relative to those of the background. For the collection of NOE distance constraints, efficient discrimination between NOE cross peaks from the target protein and background signals was achieved using the programs ATNOS and CANDID. In the iterative ATNOS/CANDID procedure, the identification of the desired protein NOEs is initially guided by the self-consistency of the protein NOE-network. Although the intensities of the signals in this network vary over a wide range, and are in many instances comparable to or smaller than those of the background, the first cycle of calculations resulted in the correct global polypeptide fold, and the structure was then refined in six subsequent cycles using the intermediate NMR structures for additional guidance. The experience gained with this work demonstrates that the ATNOS/CANDID procedure for automatic protein structure determination is highly robust and reliable in the presence of intense background signals, and might thus also represent a platform for future protein structure determinations in physiological fluids.     

Determination of content of metallothionein and low molecular mass stress peptides in transgenic tobacco plants

Phytoremediation is a process that utilizes plants to remove, transfer, stabilize, or destroy pollutants in soil, sediment, and groundwater. Plants used for such purposes have several requirements. Genetic engineering these plants could be an effective tool used to acquire features needed for such purposes within a substantial amount of time. This paper aims to utilize electrochemical techniques to analyze transgenic tobacco and, thus, to reveal their heavy metals phytoremediation potential. Total thiol and metallothionein (MT) quantities were determined in the control and transgenic tobacco plants. The total content of thiols in transgenic plants varied within the range of 561 to 1,671 μg g⁻¹. Furthermore, the determination of MT was done on transgenic tobacco plants. The level of human MT in transgenic tobacco plants varied between 25 and 95 μg g⁻¹. However, a plant cell protects itself by synthesizing low molecular mass thiols such as reduced glutathione and phytochelatins to protect itself against heavy metals toxicity. The most important thiols, cysteine (Cys), glutathione (GSH), oxidised glutathione (GSSG) and phytochelatin 2 (PC2), were determined in the non-transgenic and transgenic tobacco plants by high performance liquid chromatography with electrochemical detection. Tobacco plants synthesizing the highest amount of metallothionein have the highest basal level of phytochelatin 2 as well as reduced glutathione and free cysteine. It clearly follows from the results obtained that the biosynthesis of particular thiols is mutually linked, which contributes to a better protection of a transgenic plant against heavy metals effects.     

Deep,subsurface microflora after excavation respiration and biomass and its potential role in degradation of fossil organic matter

Three types of Miocene claystones (amorphous, lamellar, and transitional) were aseptically sampled from depths of 30 m and 150 m below the soil surface. Respiration of these sediments was measured under conditions that prevented inoculation by other microorganisms not indigenous to the claystones in situ. Microbial respiration was higher in lamellar than amorphous claystones and was not affected by sampling depth. During cultivation, microbial biomass (as indicated by PLFA) significantly increased. Microbial biomass after cultivation was significantly higher in sediments from 30 m than from 150 m depth. Both microbial respiration and biomass increased after glucose addition.     

New 4-deoxy-(1→3)-β-D-glucan-based oligosaccharides and their immunostimulating potential

(1→3)-β-D-Glucans are well-established natural biological immunomodulators. However, problems inherited with the natural origin of these polysaccharides bring about significant setbacks, including batch-to-batch heterogeneity and significant differences based on the source and isolation techniques. In this study, we tried to overcome these problems by preparation of a quantitatively new set of oligo-(1→3)-β-D-glucan-based synthetic immunomodulators. Some of these non-natural oligosaccharides showed biological activities, such as stimulation of phagocytosis, modulation of gene expression, and anti-cancer activity, which were superior to natural glucans.     

Solution structure of the Mycobacterium tuberculosis EsxG·EsxH complex: functional implications and comparisons with other M. tuberculosis Esx family complexes

Mycobacterium tuberculosis encodes five type VII secretion systems that are responsible for exporting a number of proteins, including members of the Esx family, which have been linked to tuberculosis pathogenesis and survival within host cells. The gene cluster encoding ESX-3 is regulated by the availability of iron and zinc, and secreted protein products such as the EsxG·EsxH complex have been associated with metal ion acquisition. EsxG and EsxH have previously been shown to form a stable 1:1 heterodimeric complex, and here we report the solution structure of the complex, which features a core four-helix bundle decorated at both ends by long, highly flexible, N- and C-terminal arms that contain a number of highly conserved residues. Despite clear similarities in the overall backbone fold to the EsxA·EsxB complex, the structure reveals some striking differences in surface features, including a potential protein interaction site on the surface of the EsxG·EsxH complex. EsxG·EsxH was also found to contain a specific Zn(2+) binding site formed from a cluster of histidine residues on EsxH, which are conserved across obligate mycobacterial pathogens including M. tuberculosis and Mycobacterium leprae. This site may reflect an essential role in zinc ion acquisition or point to Zn(2+)-dependent regulation of its interaction with functional partner proteins. Overall, the surface features of both the EsxG·EsxH and the EsxA·EsxB complexes suggest functions mediated via interactions with one or more target protein partners.     

MicroRNA Editing Facilitates Immune Elimination of HCMV Infected Cells

The human cytomegalovirus (HCMV) is extremely prevalent in the human population. Infection by HCMV is life threatening in immune compromised individuals and in immune competent individuals it can cause severe birth defects, developmental retardation and is even associated with tumor development. While numerous mechanisms were developed by HCMV to interfere with immune cell activity, much less is known about cellular mechanisms that operate in response to HCMV infection. Here we demonstrate that in response to HCMV infection, the expression of the short form of the RNA editing enzyme ADAR1 (ADAR1-p110) is induced. We identified the specific promoter region responsible for this induction and we show that ADAR1-p110 can edit miR-376a. Accordingly, we demonstrate that the levels of the edited-miR-376a (miR-376a(e)) increase during HCMV infection. Importantly, we show that miR-376a(e) downregulates the immune modulating molecule HLA-E and that this consequently renders HCMV infected cells susceptible to elimination by NK cells.     

Estimating a Logistic Discrimination Functions When One of the Training Samples Is Subject to Misclassification: A Maximum Likelihood Approach

The problem of discrimination and classification is central to much of epidemiology. Here we consider the estimation of a logistic regression/discrimination function from training samples, when one of the training samples is subject to misclassification or mislabeling, e.g. diseased individuals are incorrectly classified/labeled as healthy controls. We show that this leads to zero-inflated binomial model with a defective logistic regression or discrimination function, whose parameters can be estimated using standard statistical methods such as maximum likelihood. These parameters can be used to estimate the probability of true group membership among those, possibly erroneously, classified as controls. Two examples are analyzed and discussed. A simulation study explores properties of the maximum likelihood parameter estimates and the estimates of the number of mislabeled observations.