Study of apoptotic deletion mediated by Bifidobacterium longum with construction of recombinant strains for Serpin encoding gene and phenotypes comparison in a pig cell model

The first part of the research project of the Co-Advisorship Ph.D Thesis was aimed to select the best Bifidobacterium longum strains suitable to set the basis of our study. We were looking for strains with the abilities to colonize the intestinal mucosa and with good adhesion capacities, so that we can test these strains to investigate their ability to induce apoptosis in “damaged” intestinal cells. Adhesion and apoptosis are the two process that we want to study to better understand the role of an adhesion protein that we have previously identified and that have top scores homologies with the recent serpin encoding gene identified in B. longum by Nestlè researchers. Bifidobacterium longum is a probiotic, known for its beneficial effects to the human gut and even for its immunomodulatory and antitumor activities. Recently, many studies have stressed out the intimate relation between probiotic bacteria and the GIT mucosa and their influence on human cellular homeostasis. We focused on the apoptotic deletion of cancer cells induced by B. longum. This has been valued in vitro, performing the incubation of three B.longum strains with enterocyte-like Caco- 2 cells, to evidence DNA fragmentation, a cornerstone of apoptosis. The three strains tested were defined for their adhesion properties using adhesion and autoaggregation assays. These features are considered necessary to select a probiotic strain. The three strains named B12, B18 and B2990 resulted respectively: “strong adherent”, “adherent” and “non adherent”. Then, bacteria were incubated with Caco-2 cells to investigate apoptotic deletion. Cocultures of Caco-2 cells with B. longum resulted positive in DNA fragmentation test, only when adherent strains were used (B12 and B18). These results indicate that the interaction with adherent B. longum can induce apoptotic deletion of Caco-2 cells, suggesting a role in cellular homeostasis of the gastrointestinal tract and in restoring the ecology of damaged colon tissues. These results were used to keep on researching and the strains tested were used as recipient of recombinant techniques aimed to originate new B.longum strains with enhanced capacity of apoptotic induction in “damaged” intestinal cells. To achieve this new goal it was decided to clone the serpin encoding gene of B. longum, so that we can understand its role in adhesion and apoptosis induction. Bifidobacterium longum has immunostimulant activity that in vitro can lead to apoptotic response of Caco-2 cell line. It secretes a hypothetical eukaryotic type serpin protein, which could be involved in this kind of deletion of damaged cells. We had previously characterised a protein that has homologies with the hypothetical serpin of B. longum (DD087853). In order to create Bifidobacterium serpin transformants, a B. longum cosmid library was screened with a PCR protocol using specific primers for serpin gene. After fragment extraction, the insert named S1 was sub-cloned into pRM2, an Escherichia coli - Bifidobacterium shuttle vector, to construct pRM3. Several protocols for B. longum transformation were performed and the best efficiency was obtained using MRS medium and raffinose. Finally bacterial cell supernatants were tested in a dotblot assay to detect antigens presence against anti-antitrypsin polyclonal antibody. The best signal was produced by one starin that has been renamed B. longum BLKS 7. Our research study was aimed to generate transformants able to over express serpin encoding gene, so that we can have the tools for a further study on bacterial apoptotic induction of Caco-2 cell line. After that we have originated new trasformants the next step to do was to test transformants abilities when exposed to an intestinal cell model. In fact, this part of the project was achieved in the Department of Biochemistry of the Medical Faculty of the University of Maribor, guest of the abroad supervisor of the Co-Advisorship Doctoral Thesis: Prof. Avrelija Cencic. In this study we examined the probiotic ability of some bacterial strains using intestinal cells from a 6 years old pig. The use of intestinal mammalian cells is essential to study this symbiosis and a functional cell model mimics a polarised epithelium in which enterocytes are separated by tight junctions. In this list of strains we have included the Bifidobacterium longum BKS7 transformant strain that we have previously originated; in order to compare its abilities. B. longum B12 wild type and B. longum BKS7 transformant and eight Lactobacillus strains of different sources were co-cultured with porcine small intestine epithelial cells (PSI C1) and porcine blood monocytes (PoM2) in Transwell filter inserts. The strains, including Lb. gasseri, Lb. fermentum, Lb. reuterii, Lb. plantarum and unidentified Lactobacillus from kenyan maasai milk and tanzanian coffee, were assayed for activation of cell lines, measuring nitric oxide by Griess reaction, H202 by tetramethylbenzidine reaction and O2 - by cytochrome C reduction. Cytotoxic effect by crystal violet staining and induction on metabolic activity by MTT cell proliferation assay were tested too. Transepithelial electrical resistance (TER) of polarised PSI C1 was measured during 48 hours co-culture. TER, used to observe epithelium permeability, decrease during pathogenesis and tissue becomes permeable to ion passive flow lowering epithelial barrier function. Probiotics can prevent or restore increased permeability. Lastly, dot-blot was achieved against Interleukin-6 of treated cells supernatants. The metabolic activity of PoM2 and PSI C1 increased slightly after co-culture not affecting mitochondrial functions. No strain was cytotoxic over PSI C1 and PoM2 and no cell activation was observed, as measured by the release of NO2, H202 and O2 - by PoM2 and PSI C1. During coculture TER of polarised PSI C1 was two-fold higher comparing with constant TER (~3000 ) of untreated cells. TER raise generated by bacteria maintains a low permeability of the epithelium. During treatment Interleukin-6 was detected in cell supernatants at several time points, confirming immunostimulant activity. All results were obtained using Lactobacillus paracasei Shirota e Carnobacterium divergens as controls. In conclusion we can state that both the list of putative probiotic bacteria and our new transformant strain of B. longum are not harmful when exposed to intestinal cells and could be selected as probiotics, because can strengthen epithelial barrier function and stimulate nonspecific immunity of intestinal cells on a pig cell model. Indeed, we have found out that none of the strains tested that have good adhesion abilities presents citotoxicity to the intestinal cells and that non of the strains tested can induce cell lines to produce high level of ROS, neither NO2. Moreover we have assayed even the capacity of producing certain citokynes that are correlated with immune response. The detection of Interleukin-6 was assayed in all our samples, including B.longum transformant BKS 7 strain, this result indicates that these bacteria can induce a non specific immune response in the intestinal cells. In fact, when we assayed the presence of Interferon-gamma in cells supernatant after bacterial exposure, we have no positive signals, that means that there is no activation of a specific immune response, thus confirming that these bacteria are not recognize as pathogen by the intestinal cells and are certainly not harmful for intestinal cells. The most important result is the measure of Trans Epithelial Electric Resistance that have shown how the intestinal barrier function get strengthen when cells are exposed to bacteria, due to a reduction of the epithelium permeability. We have now a new strain of B. longum that will be used for further studies above the mechanism of apoptotic induction to “damaged cells” and above the process of “restoring ecology”. This strain will be the basis to originate new transformant strains for Serpin encoding gene that must have better performance and shall be used one day even in clinical cases as in “gene therapy” for cancer treatment and prevention.

Engineering data;

Mode of access: Internet.

Computer simulation for tube-making by the cold roll-forming process

The conventional design of forming rolls depends heavily on the individual skill of roll designers which is based on intuition and knowledge gained from previous work. Roll design is normally a trial an error procedure, however with the progress of computer technology, CAD/CAM systems for the cold roll-forming industry have been developed. Generally, however, these CAD systems can only provide a flower pattern based on the knowledge obtained from previously successful flower patterns. In the production of ERW (Electric Resistance Welded) tube and pipe, the need for a theoretical simulation of the roll-forming process, which can not only predict the occurrence of the edge buckling but also obtain the optimum forming condition, has been recognised. A new simulation system named "CADFORM" has been devised that can carry out the consistent forming simulation for this tube-making process. The CADFORM system applied an elastic-plastic stress-strain analysis and evaluate edge buckling by using a simplified model of the forming process. The results can also be visualised graphically. The calculated longitudinal strain is obtained by considering the deformation of lateral elements and takes into account the reduction in strains due to the fin-pass roll. These calculated strains correspond quite well with the experimental results. Using the calculated strains, the stresses in the strip can be estimated. The addition of the fin-pass roll reduction significantly reduces the longitudinal compressive stress and therefore effectively suppresses edge buckling. If the calculated longitudinal stress is controlled, by altering the forming flower pattern so it does not exceed the buckling stress within the material, then the occurrence of edge buckling can be avoided. CADFORM predicts the occurrence of edge buckling of the strip in tube-making and uses this information to suggest an appropriate flower pattern and forming conditions which will suppress the occurrence of the edge buckling.

A novel regulatory role for tissue transglutaminase in epithelial-mesenchymal transition in cystic fibrosis

Cystic fibrosis (CF) is a genetic disorder caused by mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) for which there is no overall effective treatment. Recent work indicates tissue transglutaminase (TG2) plays a pivotal intracellular role in proteostasis in CF epithelia and that the pan TG inhibitor cysteamine improves CFTR stability. Here we show TG2 has another role in CF pathology linked with TGFβ1 activation and signalling, induction of epithelial-mesenchymal transition (EMT), CFTR stability and induction of matrix deposition. We show that increased TG2 expression in normal and CF bronchial epithelial cells increases TGFβ1 levels, promoting EMT progression, and impairs tight junctions as measured by Transepithelial Electric Resistance (TEER) which can be reversed by selective inhibition of TG2 with an observed increase in CFTR stability. Our data indicate that selective inhibition of TG2 provides a potential therapeutic avenue for reducing fibrosis and increasing CFTR stability in CF.

Electric-field-dependent average resistance and the resistance fluctuation in one-dimensional disordered systems

Following an invariant-imbedding approach, we obtain analytical expressions for the ensemble-averaged resistance (ρ) and its Sinai’s fluctuations for a one-dimensional disordered conductor in the presence of a finite electric field F. The mean resistance shows a crossover from the exponential to the power-law length dependence with increasing field strength in agreement with known numerical results. More importantly, unlike the zero-field case the resistance distribution saturates to a Poissonian-limiting form proportional to A‖F‖exp(-A‖F‖ρ) for large sample lengths, where A is constant.

Electric-tunable magneto resistance effect in a two-dimensional electron gas modulated by hybrid magnetic-electric barrier nanostructure

The electric-tunable spin-independent magneto resistance effect has been theoretically investigated in ballistic regime within a two-dimensional electron gas modulated by magnetic-electric barrier nanostructure. By including the omitted stray field in previous investigations oil analogous structures, it is demonstrated based on this improved approximation that the magnetoresistance ratio for the considered structure can be efficiently enhanced by a proper electric barrier up to the maximum value depending on the specific magnetic suppression. Besides, it is also shown the introduction of positive electrostatic modulation can effectively overcome the degradation of magnetoresistance ratio for asymmetric configuration and enhance the visibility of periodic pattern induced by the size effect, while for an opposite modulation the system magnetoresistance ratio concerned may change its sign. (C) 2009 Elsevier B.V. All rights reserved.

Quantum-Ohmic Resistance Fluctuation In Disordered Conductors - An Invariant Imbedding Approach

It is now well known that in extreme quantum limit, dominated by the elastic impurity scattering and the concomitant quantum interference, the zero-temperature d.c. resistance of a strictly one-dimensional disordered system is non-additive and non-self-averaging. While these statistical fluctuations may persist in the case of a physically thin wire, they are implicitly and questionably ignored in higher dimensions. In this work, we have re-examined this question. Following an invariant imbedding formulation, we first derive a stochastic differential equation for the complex amplitude reflection coefficient and hence obtain a Fokker-Planck equation for the full probability distribution of resistance for a one-dimensional continuum with a Gaussian white-noise random potential. We then employ the Migdal-Kadanoff type bond moving procedure and derive the d-dimensional generalization of the above probability distribution, or rather the associated cumulant function –‘the free energy’. For d=3, our analysis shows that the dispersion dominates the mobilitly edge phenomena in that (i) a one-parameter B-function depending on the mean conductance only does not exist, (ii) an approximate treatment gives a diffusion-correction involving the second cumulant. It is, however, not clear whether the fluctuations can render the transition at the mobility edge ‘first-order’. We also report some analytical results for the case of the one dimensional system in the presence of a finite electric fiekl. We find a cross-over from the exponential to the power-low length dependence of resistance as the field increases from zero. Also, the distribution of resistance saturates asymptotically to a poissonian form. Most of our analytical results are supported by the recent numerical simulation work reported by some authors.

Negative differential resistance in Gd0.5Sr0.5MnO3: A consequence of Joule heating

Negative differential resistance (NDR) in current-voltage (I-V) characteristics and apparent colossal electroresistance were observed in Gd0.5Sr0.5MnO3 single crystals at low temperatures. The continuous dc I-V measurements showed a marked thermal drift. In addition, temperature of the sample surface was found to be significantly higher than that of the base at high applied currents. Two different strategies namely estimation and diminution of the Joule heating (pulsed I-V measurements) were employed to investigate its role in the electric transport properties. Our experiments reveal that the NDR in Gd0.5Sr0.5MnO3 is a consequence of Joule heating rather than the melting of charge order. (C) 2010 American Institute of Physics. doi:10.1063/1.3486221]

Influence of Bi 3+ ions in enhancing the magnitude of positive temperature coefficients of resistance in n-BaTiO3 ceramics

Bi3+ ions substituting at Ba-sites in a limited concentration range with another donor dopant occupying the Ti-sites in polycrystalline BaTiO3 enhanced the positive temperature coefficient of resistance (PTCR) by over seven orders of magnitude. These ceramics did not require normal post sinter annealing or a change to an oxygen atmosphere during annealing. These ceramics had low porosities coupled with better stabilities to large applied electric fields and chemically reducing atmospheres. Bi3+ ions limited the grain growth to less than 8 mum in size, they enhanced the concentration of acceptor-type trap centres at the grain-boundary-layer regions and maintained complete tetragonality at low grain sizes in BaTiO3 ceramics.

Electric Field Directed Growth of Molecular Wires of Charge Transfer Molecules on Prefabricated Metal Electrodes

Molecular wires of charge transfer molecules were formed by co-evaporating the 7 7 8 8-Tetracyanoquinodimethane [TCNQ] (acceptor) and Tetrathiafulvalene [TTF] (donor) molecules across prefabricated metal electrodes. Molecular wires of TTF TCNQ were also formed by evaporating single complex of TTF:TCNQ across prefabricated metal electrodes The prefabricated metal electrodes were made using electron beam lithography on SiO2 and glass cover slip substrates. Even though TTF: TCNQ wires grown from both co-evaporation and evaporation techniques show semiconductor like behavior in temperature dependence of resistance they show different activation energies due the difference in stoichiometry of TTF and TCNQ.

Potential and Electric Field Profiles for Transmission line Insulators

Transmission of bulk power at high voltages over very long distances has become very imperative. At present, throughout the globe, this task has been mostly performed by overhead transmission lines. The dual task of mechanically supporting and electrically isolating the live phase conductors from the support tower is performed by string insulators. Whether in clean condition or under polluted conditions, the electrical stress distribution along the insulators governs the possible flashover, which is quite detrimental to the system. However, a reliable data on stress distribution in commonly employed string insulators are rather scarce. Considering this, the present work has made an attempt to study accurately, the field distribution in 220 kV strings for six different types of porcelain/ceramic insulators (Normal and Antifog discs) used for high voltage transmission. The surface charge simulation method is employed for the required field computation. Voltage and electric stress distribution is deduced and compared across different types of discs. A comparison on normalised surface resistance, which is an indicator for the stress concentration under polluted condition, is also attempted.

Electrical and magneto-resistance of Co/CNT/epoxy thin film for strain and magnetic field sensing

Cobalt and iron nanoparticles are doped in carbon nanotube (CNT)/polymer matrix composites and studied for strain and magnetic field sensing properties. Characterization of these samples is done for various volume fractions of each constituent (Co and Fe nanoparticles and CNTs) and also for cases when only either of the metallic components is present. The relation between the magnetic field and polarization-induced strain are exploited. The electronic bandgap change in the CNTs is obtained by a simplified tight-binding formulation in terms of strain and magnetic field. A nonlinear constitutive model of glassy polymer is employed to account for (1) electric bias field dependent softening/hardening (2) CNT orientations as a statistical ensemble and (3) CNT volume fraction. An effective medium theory is then employed where the CNTs and nanoparticles are treated as inclusions. The intensity of the applied magnetic field is read indirectly as the change in resistance of the sample. Very small magnetic fields can be detected using this technique since the resistance is highly sensitive to strain. Its sensitivity due to the CNT volume fraction is also discussed. The advantage of this sensor lies in the fact that it can be molded into desirable shape and can be used in fabrication of embedded sensors where the material can detect external magnetic fields on its own. Besides, the stress-controlled hysteresis of the sample can be used in designing memory devices. These composites have potential for use in magnetic encoders, which are made of a magnetic field sensor and a barcode.

Simulation of Potential and Electric Field Profiles for transmission line Insulators

Since the end of second world war, extra high voltage ac transmission has seen its development. The distances between generating and load centres as well as the amount of power to be handled increased tremendously for last 50 years. The highest commercial voltage has increased to 765 kV in India and 1,200 kV in many other countries. The bulk power transmission has been mostly performed by overhead transmission lines. The dual task of mechanically supporting and electrically isolating the live phase conductors from the support tower is performed by string insulators. Whether in clean condition or under polluted conditions, the electrical stress distribution along the insulators governs the possible flashover, which is quite detrimental to the system. Hence the present investigation aims to study accurately, the field distribution for various types of porcelain/ceramic insulators (Normal and Antifog discs) used for high-voltage transmission. The surface charge simulation method is employed for the field computation. A comparison on normalised surface resistance, which is an indicator for the stress concentration under polluted condition, is also attempted.

Electrical Resistance and Seebeck Coefficient in PbTe Nanowires

We address a physics-based simplified analytical formulation of the diffusive electrical resistance ( (Omega)) and Seebeck coefficient () in a PbTe nanowire dominated by acoustic phonon scattering under the presence of a low static longitudinal electric field. The use of a second-order nonparabolic electron energy band structure involving a geometry-dependent band gap has been selected in principle to demonstrate that the electron mean free path (MFP) in such a system can reach as low as about 8 nm at room temperature for a 10-nm-wide PbTe nanowire. This is followed by the formulation of the carrier back-scattering coefficient for determination of (Omega) and as functions of wire dimensions, temperature, and the field, respectively. The present analytical formulation agrees well with the available experimental data and may find extensive use in determination of various electrothermal transport phenomena in PbTe-based one-dimensional electron devices.

Radiation from a short electric dipole antenna in a hot uniaxial plasma

The effects of electron temperature on the radiation fields and the resistance of a short dipole antenna embedded in a uniaxial plasma have been studied. It is found that for ω < ω_p the antenna excites two waves, a slow wave and a fast wave. These waves propagate only within a cone whose axis is parallel to the biasing magnetostatic field B_o and whose semicone angle is slightly less than sin ^(-1) (ω/ω_p). In the case of ω > ω_p the antenna excites two separate modes of radiation. One of the modes is the electromagnetic mode, while the other mode is of hot plasma origin. A characteristic interference structure is noted in the angular distribution of the field. The far fields are evaluated by asymptotic methods, while the near fields are calculated numerically. The effects of antenna length ℓ, electron thermal speed, collisional and Landau damping on the near field patterns have been studied.

The input and the radiation resistances are calculated and are shown to remain finite for nonzero electron thermal velocities. The effect of Landau damping and the antenna length on the input and radiation resistances has been considered.

The radiation condition for solving Maxwell's equations is discussed and the phase and group velocities for propagation given. It is found that for ω < ω_p in the radial direction (cylindrical coordinates) the power flow is in the opposite direction to that of the phase propagation. For ω > ω_p the hot plasma mode has similar characteristics.