Mechanical Properties and Microstructure of Dissimilar S355/AA6061-T6 FSW Butt Joints.

The aim of this paper is to analyse the mechanical properties of butt joints between S355 steel and 6061-T6 aluminium alloy, as well as their relationship to changes in the structure of the material caused by welding. The effect of the tool offset was analysed in particular. For the analysis, tensile tests were carried out using macro- and mini-specimens taken from S355/AA6061-T6 joints and base materials. In addition, the macro- and microstructure of the joints was determined, the hardness profiles in the joints were analysed, and fractographic analysis of the fractures of the specimens was carried out. Based on the results of the macro- and microstructure examinations, typical friction stir welding (FSW) joint zones were characterised. The microstructure was observed in the interface line of the materials on the root side, the negative effect of which on the quality of the joint was confirmed by digital image correlation (DIC) strain analysis during the monotonic tensile test. The highest average value of su = 141 MPa for the entire joint was obtained for a 0.4 mm tool offset. The highest average value of su = 185 MPa for the selected joint layer was obtained for a 0.3 mm tool offset. Fracturing of the joint in the selected layer for the tool offset values of 0.3 mm and 0.4 mm occurred in the weld nugget zone (WNZ) where the lowest hardness was recorded.

A Functionalized Membrane Layer as Part of a Dressing to Aid Wound Healing.

PURPOSE: This study is an approach to a dressing platform based on support functionalized with oxygenating factors within an alginate layer, constituting a safe and even contact surface for interface with a wound. METHODS: An alginate layer with incorporated oxygenating elements deposited on the support patch was assessed. As an oxygenating factor, perfluorooctyl was applied, and the layer coatings in two options, cross-linked and not, were evaluated. The function of human dermal fibroblast cells cultured in the presence of these constructs was analyzed, as well as their morphology using flow cytometry, fluorescence microscopy, and scanning electron microscopy. In addition, the membrane coating material was assessed using FTIR, AFM, and SEM-EDX characterization. RESULTS: The applied membrane coatings adsorbed on the patch ensured the viability of the human fibroblasts cultured on the membranes during 10 days of culture. However, on the sixth day of culture, the percentage of live cells grown in the presence of cross-linked alginate with oxygenating factor ((ALG-PFC)net) was significantly higher than that of the cells cultured in the presence of the alginate coatings alone. SEM-EDX analysis of the (ALG-PFC)net confirmed the presence of oxygenating and cross-linking factors. In addition, the regular granular branched structure of the layer coating material involving the oxygenating and cross-linking factors was observed using the AFM technique. CONCLUSION: The topography of the layer coating material involving the oxygenating and cross-linking factors ensures an even contact surface for interface with the wound. Considering 5-day intervals between dressing replacements, the platform with an oxygenating configuration ensuring the growth and morphology of the human fibroblasts can be recommended at this time as an element of a dressing system.

Dissecting transmembrane bicarbonate transport by 1,8-di(thio)amidocarbazoles.

Synthetic ionophores able to transport bicarbonate and chloride anions across lipid bilayers are appealing for their wide range of potential biological applications. We have studied the bicarbonate and chloride transport by carbazoles with two amido/thioamido groups using a bicarbonate-sensitive europium(III) probe in liposomes and found a highly remarkable transporter concentration dependence. This can be explained by a combination of two distinct transport mechanisms: HCO3-/Cl- exchange and a combination of unassisted CO2 diffusion and HCl transport, of which the respective contributions were quantified. The compounds studied were found to be highly potent HCl transporters. Based on the mechanistic insights on anion transport, we have tested the antimicrobial activity of these compounds and found a good correlation with their ion transport properties and a high activity against Gram-positive bacteria.

Composite Membrane Dressings System with Metallic Nanoparticles as an Antibacterial Factor in Wound Healing.

Wound management is the burning problem of modern medicine, significantly burdening developed countries' healthcare systems. In recent years, it has become clear that the achievements of nanotechnology have introduced a new quality in wound healing. The application of nanomaterials in wound dressing significantly improves their properties and promotes the healing of injuries. Therefore, this review paper presents the subjectively selected nanomaterials used in wound dressings, including the metallic nanoparticles (NPs), and refers to the aspects of their application as antimicrobial factors. The literature review was supplemented with the results of our team's research on the elements of multifunctional new-generation dressings containing nanoparticles. The wound healing multiple molecular pathways, mediating cell types, and affecting agents are discussed herein. Moreover, the categorization of wound dressings is presented. Additionally, some materials and membrane constructs applied in wound dressings are described. Finally, bacterial participation in wound healing and the mechanism of the antibacterial function of nanoparticles are considered. Membranes involving NPs as the bacteriostatic factors for improving wound healing of skin and bones, including our experimental findings, are discussed in the paper. In addition, some studies of our team concerning the selected bacterial strains' interaction with material involving different metallic NPs, such as AuNPs, AgNPs, Fe3O4NPs, and CuNPs, are presented. Furthermore, nanoparticles' influence on selected eukaryotic cells is mentioned. The ideal, universal wound dressing still has not been obtained; thus, a new generation of products have been developed, represented by the nanocomposite materials with antibacterial, anti-inflammatory properties that can influence the wound-healing process.

Polyelectrolyte Membrane with Hydroxyapatite and Silver Nanoparticles as a Material for Modern Wound Dressings.

Modern wound dressings not only play a covering role but also facilitate the function of the wound, contributing to a faster healing process. In this paper, we present a polyelectrolyte system with nanosized elements that could stimulate the growth of eukaryotic cells while providing antimicrobial properties, which may be recommended as a potential dressing material. The proposed platform consisted of polyethyleneimine, hydroxyapatite, and silver nanoparticles and was characterized using various macroscopic techniques. The constructed membrane scaffold was evaluated with immobilized WEHI 164 cells as a model system for cells sustained at the interface of bone and skin. Moreover, the bacteriostatic function of the designed membrane material was evaluated using different bacterial strains.

Characterization of Mutations Conferring Resistance to Rifampin in Mycobacterium tuberculosis Clinical Strains.

Resistance of Mycobacterium tuberculosis to rifampin (RMP), mediated by mutations in the rpoB gene coding for the beta-subunit of RNA polymerase, poses a serious threat to the efficacy of clinical management and, thus, control programs for tuberculosis (TB). The contribution of many individual rpoB mutations to the development and level of RMP resistance remains elusive. In this study, the incidence of mutations throughout the rpoB gene among 115 Mycobacterium tuberculosis clinical isolates, both resistant and susceptible to RMP, was determined. Of the newly discovered rpoB mutations, the role of three substitutions in the causation of RMP resistance was empirically tested. The results from in vitro mutagenesis experiments were combined with the assessment of the prevalence of rpoB mutations, and their reciprocal co-occurrences, across global M. tuberculosis populations. Twenty-two different types of mutations in the rpoB gene were identified and distributed among 58 (89.2%) RMP-resistant strains. The MICs of RMP were within the range of 40 to 800 mg/liter, with MIC50 and MIC90 values of 400 and 800 mg/liter, respectively. None of the mutations (Gln429His, Met434Ile, and Arg827Cys) inspected for their role in the development of RMP resistance produced an RMP-resistant phenotype in isogenic M. tuberculosis H37Rv strain-derived mutants. These mutations are supposed to compensate for fitness impairment incurred by other mutations directly associated with drug resistance.

Gold Nanoparticle-Modified Poly(vinyl chloride) Surface with Improved Antimicrobial Properties for Medical Devices.

Despite the significant technological progress achieved in the past decades in the medical field, device-related infections carry a heavy social and economic burden. Surface modification of medical equipment is one of the most interesting approaches employed to improve the antibacterial activity of a material. Herein, we developed a process for the gold nanoparticle modification of a poly(vinyl chloride) laryngeal tube, which typically serves as an airway management device. In our study, we focused specifically on increasing the antimicrobial properties of the material while maintaining its biocompatibility. We applied two different modification methods to the poly(vinyl chloride) laryngeal tube. An increase in the antimicrobial activity of the surface was observed for both methods. In addition, the adsorption of bacterial cells on the material surface was assessed. We determined that the number of colonies cultured in the presence of the gold nanoparticle-modified samples or absorbed to the material surface decreased significantly compared with the control group. The trend was observed for both Gram-positive and Gram-negative strains. Moreover, it was established that the designed material did not exhibit a lethal impact on a control cell line. Finally, we noted discrepancies in the growth of bacteria cultured in the presence of modified or unmodified PVC material as well as differences in cell adherence to its surface. The proposed poly(vinyl chloride) modifications are most effective against Gram-positive bacteria, especially L. monocytogenes. Nevertheless, it ought to be emphasized that due to their different properties, each strain requires an individual approach.

Delivery of Chicken Egg Ovalbumin to Dendritic Cells by Listeriolysin O-Secreting Vegetative Bacillus subtilis.

Listeriolysin O (LLO), one of the most immunogenic proteins of Listeria monocytogenes and its main virulence factor, mediates bacterial escape from the phagosome of the infected cell. Thus, its expression in a nonpathogenic bacterial host may enable effective delivery of heterologous antigens to the host cell cytosol and lead to their processing predominantly through the cytosolic MHC class I presentation pathway. The aim of this project was to characterize the delivery of a model antigen, chicken egg ovalbumin (OVA), to the cytosol of dendritic cells by recombinant Bacillus subtilis vegetative cells expressing LLO. Our work indicated that LLO produced by non-sporulating vegetative bacteria was able to support OVA epitope presentation by MHC I molecules on the surface of antigen presenting cells and consequently influence OVA-specific cytotoxic T cell activation. Additionally, it was proven that the genetic context of the epitope sequence is of great importance, as only the native full-sequence OVA fused to the N-terminal fragment of LLO was sufficient for effective epitope delivery and activation of CD8⁺ lymphocytes. These results demonstrate the necessity for further verification of the fusion antigen potency of enhancing the MHC I presentation, and they prove that LLO-producing B. subtilis may represent a novel and attractive candidate for a vaccine vector.

Stabilized nanosystem of nanocarriers with an immobilized biological factor for anti-tumor therapy.

OBJECTIVE: The inadequate efficiency of existing therapeutic anti-cancer regiments and the increase in the multidrug resistance of cancer cells underscore the need to investigate novel anticancer strategies. The induction of apoptosis in tumors by cytotoxic agents produced by pathogenic microorganisms is an example of such an approach. Nevertheless, even the most effective drug should be delivered directly to targeted sites to reduce any negative impact on other cells. Accordingly, the stabilized nanosystem (SNS) for active agent delivery to cancer cells was designed for further application in local anti-tumor therapy. A product of genetically modified Escherichia coli, listeriolysin O (LLO), was immobilized within the polyelectrolyte membrane (poly(ethylenimine)|hyaluronic acid) shells of 'LLO nanocarriers' coupled with the stabilizing element of natural origin. METHODS AND RESULTS: The impact of LLO was evaluated in human leukemia cell lines in vitro. Correspondingly, the influence of the SNS and its elements was assessed in vitro. The viability of targeted cells was evaluated by flow cytometry. Visualization of the system structure was performed using confocal microscopy. The membrane shell applied to the nanocarriers was analyzed using atomic force microscopy and Fourier transform infrared spectroscopy techniques. Furthermore, the presence of a polyelectrolyte layer on the nanocarrier surface and/or in the cell was confirmed by flow cytometry. Finally, the structural integrity of the SNS and the corresponding release of the fluorescent solute listeriolysin were investigated. CONCLUSION: The construction of a stabilized system offers LLO release with a lethal impact on model eukaryotic cells. The applied platform design may be recommended for local anti-tumor treatment purposes.

Lmo0171, a novel internalin-like protein, determines cell morphology of Listeria monocytogenes and its ability to invade human cell lines.

Internalins comprise a class of Listeria monocytogenes proteins responsible for activation of signalling pathways leading to phagocytic uptake of the bacterium by the host cell. In this paper, a possible role of Lmo0171-a new member of the internalin family was investigated. Disruption of the lmo0171 gene resulted in important cell morphology alterations along with a decrease in the ability to invade three eukaryotic cell lines, that is Int407, Hep-2 and HeLa and diminished adhesion efficiency to int407, thereby suggesting bifunctionality of the newly characterised Lmo0171 internalin.

Cytotoxicity of purified listeriolysin O on mouse and human leukocytes and leukaemia cells.

BACKGROUND: Listeriolysin O (LLO) is the main virulence factor of Listeria monocytogenes and facilitates the intracellular survival of the pathogen. Some of its characteristics endorse the growing popularity of LLO for use in biotechnology, particularly in the development of novel vaccines. Here, we evaluate the use of LLO to eradicate leukaemia cells. RESULTS: A purified LLO preparation was obtained by affinity chromatography. The LLO preparation procedure was optimized and purified LLO was tested for optimal conditions of storage including temperature, application of proteinase inhibitors and serum components. We demonstrated the possibility of regulating LLO activity by adjusting cell membrane cholesterol content. The LLO preparation had haemolytic activity and had a cytotoxic effect on the human T-leukaemia Jurkat cell line as well as mouse and human peripheral blood mononuclear cells. CONCLUSIONS: LLO has a very potent cytotoxic activity towards human leukocytes. Importantly, the cytotoxic activity was easily regulated in vitro and could be restricted to areas containing malignant cells, raising the possibility of future clinical application of LLO for leukaemia treatment.

Identification and analysis of mutations in the katG gene in multidrug-resistant Mycobacterium tuberculosis clinical isolates.

INTRODUCTION: A major role in the development of resistance of Mycobacterium tuberculosis to isoniazid (INH) is attributed to mutations in the katG gene coding for the catalase/peroxidase, an enzyme required for obtaining a pharmacologically active form of the drug. Analysis of mutations in the katG gene in M. tuberculosis strains may contribute to the development of reliable and rapid tests for detection of INH resistance. The aim of the study was to identify and characterize mutations in the katG gene in multidrug-resistant M. tuberculosis clinical isolates. MATERIAL AND METHODS: The study included 46 strains of M. tuberculosis, recovered from MDR-TB patients in Poland in 2004. Mutations in the katG gene were detected by comparing DNA sequences with the corresponding sequence of a wild-type reference laboratory strain (M. tuberculosis H37Rv). The obtained results were interpreted in the context of MIC values of INH and catalase activity of the strains tested. RESULTS: A total of 43 (93%) strains contained mutations in the katG gene. The most frequently observed were mutations at codon 315, found in 34 (74%) strains. Mutations at other codons were rare: 4 strains contained mutations at codon 463, 2 at codon 131 and another 2 at codon 234. Mutations at codons 68, 91, 101, 126, 128 and 194 were found in single strains only. Two strains, for which no mutations at codon 315 of the katG gene were identified, had a unique translation termination mutation, which would invariably result in polypeptide truncation leading to the generation of dysfunctional catalase polypeptides. Both these strains presented the highest MIC values for INH (80 and 100 µg/mL) and showed a complete loss of catalase activity. For the remaining 41 strains with katG mutations, the MICs of INH were within the range 0.2-10 µg/mL. Thirty-six (88%) of those strains retained their catalase activity. CONCLUSIONS: Mutations at codon 315 within the katG gene, depending on their type might be useful for the prediction of INH resistance. Whereas the missense mutations do not affect the catalase activity or the level of INH resistance, the nonsense mutations result in high-level resistance to INH and a total loss of catalase activity.

Multicentre Etest evaluation of in vitro activity of conventional antifungal drugs against European bovine mastitis Prototheca spp. isolates.

OBJECTIVES: Bovine mammary protothecosis is a serious pathology that entails high economic losses in the dairy industry. The disease, the frequency of which has recently been increasing worldwide, is caused by unicellular, achlorophyllous, yeast-like algae of two species: Prototheca zopfii and Prototheca blaschkeae. The objective of this study was to investigate the in vitro activity of a panel of conventional antifungal drugs against Prototheca spp. isolates. METHODS: A total of 144 P. zopfii genotype 2 and P. blaschkeae strains isolated from milk of mastitic cows were subjected to drug susceptibility testing by Etest methodology. RESULTS: Five out of ten antifungal drugs tested exhibited no activity against Prototheca spp. isolates. The best activity against Prototheca spp. was demonstrated by amphotericin B (MIC90 of 1.5 mg/L). The MICs differed significantly (P < 0.01) between P. zopfii genotype 2 and P. blaschkeae, with the latter species being more susceptible to amphotericin B and azoles. Marked differences (P < 0.05) in azole and amphotericin B activities were noted among Prototheca spp. isolates originating from different European countries. Based on the correlation coefficients, a considerable cross-interaction was found among MICs of azoles and between MICs of azoles and amphotericin B for Prototheca spp. (P < 0.03). CONCLUSIONS: This study represents the largest, cross-European evaluation of antifungal activity against Prototheca spp. to date. The activity of amphotericin B against Prototheca spp. validates its potential use as a therapeutic agent against bovine protothecosis. For laboratory testing of drug activity against Prototheca spp., the Etest method is encouraged, due to its technical simplicity, rapidity and high intra- and inter-laboratory reproducibility.

The use of hollow fiber membranes combined with cytometry in analysis of bacteriological samples.

To avoid destruction of the implanted biological material it may be separated from host immunological system by enclosure within a permiselective membrane. Two-directional diffusion through the membrane of nutrients, metabolic products, as well as bioactive products of encapsulated cells is required to ensure their survival and functional activities. The system of cells encapsulated within the membrane releasing the biologically active substance may be applied either locally to give an opportunity of therapeutic agent activity in the specified place and/or at some convenient site (tissue) for a prolonged period of time.The novel system of bacteria bio-encapsulation using modified membranes, and its assessment by flow cytometry is described and discussed. The encapsulated in membrane bacteria, functioning and releasing their products were evaluated in the systems in vitro and in vivo. The bacteria cells products impact on Eukariotic cells was evaluated. The cytometric evaluation demonstrates the membrane ability to avoid the release of bacteria enclosed within the membrane wall. In experiments with treatment of the bacteria with antibiotic to release products from damaged bacteria it was possible to distinguish stages of the applied antibiotic impact on encapsulated bacteria cells. In E. coli following stages were distinguished: induction of membrane permeability to PI, activation of proteases targeting GFP (protein) and subsequent nucleic acids degradation. In the another experiment the evidence was presented of the cytotoxic activity of live Bacillus subtilis encapsulated within the membrane system. The Bacilus products mediated by secreted listeriolysin O (LLO) on the chosen eukaryotic cells was evaluated. Similar systems releasing bacterial products locally and continuously may selectively affect different types of cells and may have possible application in the anticancer treatment at localized sites.

Contribution of cysteine residue to the properties of Listeria monocytogenes listeriolysin O.

Listeriolysin (LLO) is the key virulence factor critical for Listeria monocytogenes pathogenesis. Listerial cytolysin belongs to the family of cholesterol-dependent cytolysins (CDCs), a group of pore-forming toxins produced by related gram-positive bacteria. Most CDCs contain a cysteine residue in the conserved undecapeptide - a sequence that is highly preserved among this group of proteins. Substitutions of cysteine do not always lead to loss of hemolytic activity, questioning the purpose of such strong conservation of this amino acid in the sequence of CDC. The properties of 3 L. monocytogenes strains, a wild type and 2 mutants expressing modified LLO within the cysteine residue, were analyzed in this work. The first of these mutants producing a toxin with cysteine to alanine substitution showed similar features to the wild type except that a thiol-reducing agent was not necessary for hemolytic activity. Another strain secreting LLO containing serine instead of cysteine exhibited strikingly different properties than the wild type. Modified toxin is independent of the reducing reagents, less stable, and shows accelerated kinetics of cytolysis in comparison with the unchanged protein. However, both mutant strains are less invasive in the cell culture model showing the important role of cysteine in L. monocytogenes virulence.