Inactive endosialidase-based detection of bacterial and oncofetal polysialic acid

Polysialic acid is a carbohydrate polymer which consist of N-acetylneuraminic acid units joined by alpha2,8-linkages. It is developmentally regulated and has an important role during normal neuronal development. In adults, it participates in complex neurological processes, such as memory, neural plasticity, tumor cell growth and metastasis. Polysialic acid also constitutes the capsule of some meningitis and sepsis-causing bacteria, such as Escherichia coli K1, group B meningococci, Mannheimia haemolytica A2 and Moraxella nonliquefaciens. Polysialic acid is poorly immunogenic; therefore high affinity antibodies against it are difficult to prepare, thus specific and fast detection methods are needed. Endosialidase is an enzyme derived from the E. coli K1 bacteriophage, which specifically recognizes and degrades polysialic acid. In this study, a novel detection method for polysialic acid was developed based on a fusion protein of inactive endosialidase and the green fluorescent protein. It utilizes the ability of the mutant, inactive endosialidase to bind but not cleave polysialic acid. Sequencing of the endosialidase gene revealed that amino acid substitutions near the active site of the enzyme differentiate the active and inactive forms of the enzyme. The fusion protein was applied for the detection of polysialic acid in bacteria and neuroblastoma. The results indicate that the fusion protein is a fast, sensitive and specific reagent for the detection of polysialic acid. The use of an inactive enzyme as a specific molecular tool for the detection of its substrate represents an approach which could potentially find wide applicability in the specific detection of diverse macromolecules.

Effect of alcohol concentration and electrode composition on the ethanol electrochemical oxidation

The electrochemical oxidation on platinum and platinum rhodium bimetallic electrodes was studied by Differential Electrochemical Mass Spectrometry for several ethanol concentrations in solution. It is found that increasing the ethanol concentration the production of the partially oxidized products (acetaldehyde) increases as the concentration increases. On the other hand, addition of 25% at. of rhodium increases the full oxidation to CO2. Another interesting result observed is a correlation between the intensity of the dehydrogenations peak at 0.3 V vs. RHE and the CO2 yield for the different ethanol concentration studied.

Spectrophotometric study of iron oxidation in the iron(II)/thiocyanate/acetone system and some analytical applications

The possibility of using thiocyanate to determine iron(II) and/or iron(III) in water-acetone mixture has been re-examined as part of a systematic and comparative study involving metallic complexes of pseudohalide ligands. Some parameters that affect the complete oxidation of the ferrous cations, their subsequent complexation and the system stability have been studied to optimize the experimental conditions. Our results show the viability and potentiality of this simply methodology as an alternative analytical procedure to determine iron cations with high sensitivity, precision and accuracy. Studies on the calibration, stability, precision, and effect of various different ions have been carried out by using absorbance values measured at 480 nm. The analytical curve for the total iron determination obeys Beer's law (r = 0.9993), showing a higher sensitivity (molar absorptivity of 2.10x10(4) L cm-1 mol-1) when compared with other traditional systems (ligands) or even with the "similar" azide ion [1.53x10(4) L cm-1 mol-1, for iron-III/azide complexes, in 70% (v/v) tetrahydrofuran/water, at 396 nm]. Under such optimized experimental conditions, it is possible to determine iron in the concentration range from 0.5 to 2 ppm (15-65% T for older equipments, quartz cells of 1.00 cm). Analytical applications have been tested for some different materials (iron ores), also including pharmaceutical products for anemia, and results were compared with atomic absorption determinations. Very good agreement was obtained with these two different techniques, showing the potential of the present experimental conditions for the total iron spectrophotometric determinations (errors < 5%). The possibility of iron speciation was made evident by using another specific and auxiliary method for iron(II) or (III).

Role of oxidation-reduction cycle of iron in direct leaching of zinc concentrate

Direct leaching is an alternative to conventional roast-leach-electrowin (RLE) zinc production method. The basic reaction of direct leach method is the oxidation of sphalerite concentrate in acidic liquid by ferric iron. The reaction mechanism and kinetics, mass transfer and current modifications of zinc concentrate direct leaching process are considered. Particular attention is paid to the oxidation-reduction cycle of iron and its role in direct leaching of zinc concentrate, since it can be one of the limiting factors of the leaching process under certain conditions. The oxidation-reduction cycle of iron was experimentally studied with goal of gaining new knowledge for developing the direct leaching of zinc concentrate. In order to obtain this aim, ferrous iron oxidation experiments were carried out. Affect of such parameters as temperature, pressure, sulfuric acid concentration, ferrous iron and copper concentrations was studied. Based on the experimental results, mathematical model of the ferrous iron oxidation rate was developed. According to results obtained during the study, the reaction rate orders for ferrous iron concentration, oxygen concentration and copper concentration are 0.777, 0.652 and 0.0951 respectively. Values predicted by model were in good concordance with the experimental results. The reliability of estimated parameters was evaluated by MCMC analysis which showed good parameters reliability.

Control of tomato bacterial wilt through the incorporation of aerial part of pigeon pea and crotalaria to soil

The use of organic matter that improves the physical, chemical and biological soil properties has been studied as an inducer of suppressiveness to soilborne plant pathogens. The objective of this work was to evaluate the effect of different sources and concentrations of organic matter on tomato bacterial wilt control. Two commercially available organic composts and freshly cut aerial parts of pigeon pea (Cajanus cajan) and crotalaria (Crotalaria juncea) were incorporated, in concentrations of 10, 20 and 30 % (v/v), into soil infested with Ralstonia solanacearum. The soil with the fresh organic matter of pigeon pea and crotalaria was incubated for 30 and 60 days before planting. Tomato seedlings of cv. Santa Clara were transplanted into polyethylene bags with 3 kg of the planting substrate (infested soil + organic matter). The wilting symptoms and percentage of flowering plants were evaluated for 45 days. All evaluated concentrations with incorporation and incubation for 30 days of aerial parts of pigeon pea and crotalaria controlled 100% tomato bacterial wilt. With 60 days of incubation, only the 10 % concentration of pigeon pea and crotalaria did not control the disease. These results suggest that soil incorporation of fresh aerial parts of pigeon pea and crotalaria is an effective method for bacterial wilt control.

Functional studies on bacterial nucleotide-regulated inorganic pyrophosphatases

CBS domains are ~60 amino acid tandemly repeated regulatory modules forming a widely distributed domain superfamily. Found in thousands of proteins from all kingdoms of life, CBS domains have adopted a variety of functions during evolution, one of which is regulation of enzyme activity through binding of adenylate-containing compounds in a hydrophobic cavity. Mutations in human CBS domain-containing proteins cause hereditary diseases. Inorganic pyrophosphatases (PPases) are ubiquitous enzymes, which pull pyrophosphate (PPi) producing reactions forward by hydrolyzing PPi into phosphate. Of the two nonhomologous soluble PPases, dimeric family II PPases, belonging to the DHH family of phosphoesterases, require a transition metal and magnesium for maximal activity. A quarter of the almost 500 family II PPases, found in bacteria and archaea, contain a 120-250 amino acid N-terminal insertion, comprised of two CBS domains separated in sequence by a DRTGG domain. These enzymes are thus named CBS-PPases. The function of the DRTGG domain in proteins is unknown. The aim of this PhD thesis was to elucidate the structural and functional differences of CBS-PPases in comparison to family II PPases lacking the regulatory insert. To this end, we expressed, purified and characterized the CBS-PPases from Clostridium perfringens (cpCBS-PPase) and Moorella thermoacetica (mtCBS-PPase), the latter lacking a DRTGG domain. Both enzymes are homodimers in solution and display maximal activity against PPi in the presence of Co2+ and Mg2+. Uniquely, the DRTGG domain was found to enable tripolyphosphate hydrolysis at rates similar to that of PPi. Additionally, we found that AMP and ADP inhibit, while ATP and AP4A activate CBSPPases, thus enabling regulation in response to changes in cellular energy status. We then observed substrate- and nucleotide-induced conformational transitions in mtCBS-PPase and found that the enzyme exists in two differentially active conformations, interconverted through substrate binding and resulting in a 2.5-fold enzyme activation. AMP binding was shown to produce an alternate conformation, which is reached through a different pathway than the substrate-induced conformation. We solved the structure of the regulatory insert from cpCBS-PPase in complex with AMP and AP4A and proposed that conformational changes in the loops connecting the catalytic and regulatory domains enable activity regulation. We examined the effects of mutations in the CBS domains of mtCBS-PPase on catalytic activity, as well as, nucleotide binding and inhibition.

DPS-Like Peroxide Resistance Protein: Structural and Functional Studies on a Versatile Nanocontainer

Oxidative stress is a constant threat to almost all organisms. It damages a number of biomolecules and leads to the disruption of many crucial cellular functions. It is caused by reactive oxygen species (ROS), such as hydrogen peroxide (H2O₂), superoxide (•O₂ -), and hydroxyl radical (•OH). The most harmful of these compounds is •OH, which is only formed in cells in the presence of redox-cycling transition metals, such as iron and copper. Bacteria have developed a number of mechanisms to cope with ROS. One of the most widespread means employed by bacteria is the DNA-binding proteins from starved cells (Dps). Dps proteins protect the cells by binding and oxidizing Fe2+, thus greatly reducing the production of •OH. The oxidized iron is stored inside the protein as an iron core. In addition, Dps proteins bind directly to DNA forming a protective coating that shields DNA from harmful agents. Moreover, Dps proteins have been found to elicit other protective functions in cells and to participate in bacterial virulence. Dps proteins are of special importance to Streptococci owing to the lack of catalase in this genus of bacteria.This study was focused on structural and functional characterization of streptococcal Dpslike peroxide resistance (Dpr) proteins. Initially, crystal structures of Streptococcus pyogenes Dpr were determined. The data confirmed the presence of a di-metal ferroxidase center (FOC) in Dpr proteins and revealed the presence of a novel N-terminal helix as well as a surface metal-binding site. The crystal structures of Streptococcus suis Dpr complexed with transition metals demonstrated the metal specificity of the FOC. Solution binding studies also indicated the presence of a di-metal FOC. These results suggested a possible role for Dpr in the detoxification of various metals. Iron was found to mineralize inside the protein as ferrihydrite based on X-ray absorption spectroscopy data. The iron core was found to exhibit clear superparamagnetic behaviour using magnetic and Mössbauer measurements. The results from this study are expected to further increase our understanding on the binding, oxidation, and mineralization of iron and other metals in Dpr proteins. In particular, the structural and magnetic properties of the iron core can form a basis for potential new applications in nanotechnology. From the streptococcal viewpoint, the results would help in understanding better the complicated picture of bacterial pathogenesis. Dpr proteins may also provide a novel target for drug design due to their tight involvement in bacterial virulence.

Effects of low intensity laser in in vitro bacterial culture and in vivo infected wounds

OBJECTIVE: to compare the effects of low intensity laser therapy on in vitro bacterial growth and in vivo in infected wounds, and to analyze the effectiveness of the AsGa Laser technology in in vivo wound infections. METHODS: in vitro: Staphylococcus aureus were incubated on blood agar plates, half of them being irradiated with 904 nm wavelength laser and dose of 3J/cm2 daily for seven days. In vivo: 32 male Wistar rats were divided into control group (uninfected) and Experimental Group (Infected). Half of the animals had their wounds irradiated. RESULTS: in vitro: there was no statistically significant variation between the experimental groups as for the source plates and the derived ones (p>0.05). In vivo: there was a significant increase in the deposition of type I and III collagen in the wounds of the infected and irradiated animals when assessed on the fourth day of the experiment (p=0.034). CONCLUSION: low-intensity Laser Therapy applied with a wavelength of 904nm and dose 3J/cm2 did not alter the in vitro growth of S. aureus in experimental groups; in vivo, however, it showed significant increase in the deposition of type I and III collagen in the wound of infected and irradiated animals on the fourth day of the experiment.

Identification and characterisation of a novel adhesin of Streptococcus suis and its use as a target of adhesion inhibition and bacterial detection

Streptococcus suis is an important pig pathogen but it is also zoonotic, i.e. capable of causing diseases in humans. Human S. suis infections are quite uncommon but potentially life-threatening and the pathogen is an emerging public health concern. This Gram-positive bacterium possesses a galabiose-specific (Galalpha1−4Gal) adhesion activity, which has been studied for over 20 years. P-fimbriated Escherichia coli−bacteria also possess a similar adhesin activity targeting the same disaccharide. The galabiose-specific adhesin of S. suis was identified by an affinity proteomics method. No function of the protein identified was formerly known and it was designated streptococcal adhesin P (SadP). The peptide sequence of SadP contains an LPXTG-motif and the protein was proven to be cell wall−anchored. SadP may be multimeric since in SDS-PAGE gel it formed a protein ladder starting from about 200 kDa. The identification was confirmed by producing knockout strains lacking functional adhesin, which had lost their ability to bind to galabiose. The adhesin gene was cloned in a bacterial expression host and properties of the recombinant adhesin were studied. The galabiose-binding properties of the recombinant protein were found to be consistent with previous results obtained studying whole bacterial cells. A live-bacteria application of surface plasmon resonance was set up, and various carbohydrate inhibitors of the galabiose-specific adhesins were studied with this assay. The potencies of the inhibitors were highly dependent on multivalency. Compared with P-fimbriated E. coli, lower concentrations of galabiose derivatives were needed to inhibit the adhesion of S. suis. Multivalent inhibitors of S. suis adhesion were found to be effective at low nanomolar concentrations. To specifically detect galabiose adhesin−expressing S. suis bacteria, a technique utilising magnetic glycoparticles and an ATP bioluminescence bacterial detection system was also developed. The identification and characterisation of the SadP adhesin give valuable information on the adhesion mechanisms of S. suis, and the results of this study may be helpful for the development of novel inhibitors and specific detection methods of this pathogen.

Development of an environmentally friendly method of starch oxidation by hydrogen peroxide and a complex water-soluble Iron catalyst

Oxidized starch is a key component in the paper industry, where it is used as both surfacing sizer and filler. Large quantities are annually used for this purpose; however, the methods for the oxidation are not environmentally friendly. In our research, we have studied the possibility to replace the harmful oxidation agents, such as hypochlorite or iodates and transition metal catalysts, with a more environmentally friendly oxidant, hydrogen peroxide (H2O2), and a special metal complex catalyst (FePcS), of which only a small amount is needed. The work comprised batch and semi-batch studies by H2O2, ultrasound studies of starch particles, determination of low-molecular by-products and determination of the decomposition kinetics of H2O2 in the presence of starch and the catalyst. This resulted in a waste-free oxidation method, which only produces water and oxygen as side products. The starch oxidation was studied in both semi-batch and batch modes in respective to the oxidant (H2O2) addition. The semi-batch mode proved to yield a sufficient degree of substitution (COOH groups) for industrial purposes. Treatment of starch granules by ultrasound was found to improve the reactivity of starch. The kinetic results were found out to have a rather complex pattern – several oxidation phases were observed, apparently due to the fact that the oxidation reaction in the beginning only took place on the surface, whereas after a prolonged reaction time, partial degradation of the solid starch granules allowed further reaction in the interior parts. Batch-mode experiments enabled a more detailed study of the mechanisms of starch in the presence of H2O2 and the catalyst, but yielded less oxidized starch due to rapid decomposition of H2O2 due to its high concentrations. The effect of the solid-liquid (S/L) ratio in the reaction system was studied in batch experiments. These studies revealed that the presence of the catalyst and the starch enhance the H2O2 decomposition.

Vaccination protocol and bacterial strain affect the serological response of beef calves against blackleg

The serological response of beef calves was evaluated with different vaccination regimens against blackleg, using an official strain (MT) and a field-collected strain of Clostridium chauvoei as antigens. Sixty calves were randomly allocated to four different groups and were submitted to distinct vaccination protocols with a commercial polyvalent vaccine. Group G1 was first vaccinated at four months of age and a booster shot was given after weaning, at eight months. Group G2 was given the first dose at eight months and a booster shot 30 days later. Group G3 was vaccinated only once at eight months and the control group was not vaccinated. These alternative vaccination regimens were proposed in an effort to adequately protect cattle under open-field farming conditions. Serological evaluations were made by Elisa at 4, 8, 9 and 10 months of age. Both groups receiving booster shots had a significantly increased serological response 30 days later. However, the serum IgG levels against C. chauvoei were significantly higher in the calves that were first vaccinated at four months. At 10 months, the two booster shot groups (G1 and G2) had similar serological responses, while the calves that were treated with a single dose of vaccine at weaning (G3) had a response that was similar to that of the control group. The serological response of the calves was significantly inferior at several of the evaluation times when the field strain of the bacteria was used as a challenge antigen instead of the official MT strain. The serological response of calves that are vaccinated twice was found to be satisfactory, independent of the first injection being made at four or eight months of age. It was also concluded that it would be useful to include local bacterial strains in commercial vaccine production.

Oxidation rates of carbon and nitrogen in char residues from solid fuels

Computational fluid dynamics (CFD) modeling is an important tool in designing new combustion systems. By using CFD modeling, entire combustion systems can be modeled and the emissions and the performance can be predicted. CFD modeling can also be used to develop new and better combustion systems from an economical and environmental point of view. In CFD modeling of solid fuel combustion, the combustible fuel is generally treated as single fuel particles. One of the limitations with the CFD modeling concerns the sub-models describing the combustion of single fuel particles. Available models in the scientific literature are in many cases not suitable as submodels for CFD modeling since they depend on a large number of input parameters and are computationally heavy. In this thesis CFD-applicable models are developed for the combustion of single fuel particles. The single particle models can be used to improve the combustion performance in various combustion devices or develop completely new technologies. The investigated fields are oxidation of carbon (C) and nitrogen (N) in char residues from solid fuels. Modeled char-C oxidation rates are compared to experimental oxidation rates for a large number of pulverized solid fuel chars under relevant combustion conditions. The experiments have been performed in an isothermal plug flow reactor operating at 1123-1673 K and 3-15 vol.% O2. In the single particle model, the char oxidation is based on apparent kinetics and depends on three fuel specific parameters: apparent pre-exponential factor, apparent activation energy, and apparent reaction order. The single particle model can be incorporated as a sub-model into a CFD code. The results show that the modeled char oxidation rates are in good agreement with experimental char oxidation rates up to around 70% of burnout. Moreover, the results show that the activation energy and the reaction order can be assumed to be constant for a large number of bituminous coal chars under conditions limited by the combined effects of chemical kinetics and pore diffusion. Based on this, a new model based on only one fuel specific parameter is developed (Paper III). The results also show that reaction orders of bituminous coal chars and anthracite chars differ under similar conditions (Paper I and Paper II); reaction orders of bituminous coal chars were found to be one, while reaction orders of anthracite chars were determined to be zero. This difference in reaction orders has not previously been observed in the literature and should be considered in future char oxidation models. One of the most frequently used comprehensive char oxidation models could not explain the difference in the reaction orders. In the thesis (Paper II), a modification to the model is suggested in order to explain the difference in reaction orders between anthracite chars and bituminous coal chars. Two single particle models are also developed for the NO formation and reduction during the oxidation of single biomass char particles. In the models the char-N is assumed to be oxidized to NO and the NO is partly reduced inside the particle. The first model (Paper IV) is based on the concentration gradients of NO inside and outside the particle and the second model is simplified to such an extent that it is based on apparent kinetics and can be incorporated as a sub-model into a CFD code (Paper V). Modeled NO release rates from both models were in good agreement with experimental measurements from a single particle reactor of quartz glass operating at 1173-1323 K and 3-19 vol.% O2. In the future, the models can be used to reduce NO emissions in new combustion systems.