Porcine parvovirus VP1/VP2 on a time series epitope mapping: exploring the effects of high hydrostatic pressure on the immune recognition of antigens.

Porcine parvovirus (PPV) is a DNA virus that causes reproductive failure in gilts and sows, resulting in embryonic and fetal losses worldwide. Epitope mapping of PPV is important for developing new vaccines. In this study, we used spot synthesis analysis for epitope mapping of the capsid proteins of PPV (NADL-2 strain) and correlated the findings with predictive data from immunoinformatics. The virus was exposed to three conditions prior to inoculation in pigs: native (untreated), high hydrostatic pressure (350 MPa for 1 h) at room temperature and high hydrostatic pressure (350 MPa for 1 h) at - 18 °C, and was compared with a commercial vaccine produced using inactivated PPV. The screening of serum samples detected 44 positive spots corresponding to 20 antigenic sites. Each type of inoculated antigen elicited a distinct epitope set. In silico prediction located linear and discontinuous epitopes in B cells that coincided with several epitopes detected in spot synthesis of sera from pigs that received different preparations of inoculum. The conditions tested elicited antibodies against the VP1/VP2 antigen that differed in relation to the response time and the profile of structurally available regions that were recognized.

Synergism between high hydrostatic pressure and glutaraldehyde for the inactivation of Staphylococcus aureus at moderate temperature.

The sterilization of transplant and medical devices should be effective but not detrimental to the structural properties of the materials used. In this study, we examined the effectiveness of chemical and physical agents for inactivating Staphylococcus aureus, a gram-positive bacterium and important cause of infections and biofilm production. The treatment conditions in this work were chosen to facilitate their subsequent use with sensitive materials. The effects of temperature, high hydrostatic pressure, and glutaraldehyde disinfectant on the growth of two strains of S. aureus (ATCC 25923 and BEC 9393) were investigated individually and/or in combinations. A low concentration of glutaraldehyde (0.5 mM), high hydrostatic pressure (300 MPa for 10 min), and moderate temperature (50 °C), when used in combination, significantly potentiated the inactivation of both bacterial strains by > 8 orders of magnitude. Transmission electron microscopy revealed structural damage and changes in area that correlated with the use of pressure in the presence of glutaraldehyde at room temperature in both strains. Biofilm from strain ATCC 25923 was particularly susceptible to inactivation. The conditions used here provided effective sterilization that can be applied to sensitive surgical devices and biomaterials, with negligible damage. The use of this experimental approach to investigate other pathogens could lead to the adoption of this procedure for sterilizing sensitive materials.

The polygonal model: A simple representation of biomolecules as a tool for teaching metabolism.

Metabolism involves numerous reactions and organic compounds that the student must master to understand adequately the processes involved. Part of biochemical learning should include some knowledge of the structure of biomolecules, although the acquisition of such knowledge can be time-consuming and may require significant effort from the student. In this report, we describe the "polygonal model" as a new means of graphically representing biomolecules. This model is based on the use of geometric figures such as open triangles, squares, and circles to represent hydroxyl, carbonyl, and carboxyl groups, respectively. The usefulness of the polygonal model was assessed by undergraduate students in a classroom activity that consisted of "transforming" molecules from Fischer models to polygonal models and vice and versa. The survey was applied to 135 undergraduate Biology and Nursing students. Students found the model easy to use and we noted that it allowed identification of students' misconceptions in basic concepts of organic chemistry, such as in stereochemistry and organic groups that could then be corrected. The students considered the polygonal model easier and faster for representing molecules than Fischer representations, without loss of information. These findings indicate that the polygonal model can facilitate the teaching of metabolism when the structures of biomolecules are discussed. Overall, the polygonal model promoted contact with chemical structures, e.g. through drawing activities, and encouraged student-student dialog, thereby facilitating biochemical learning. © 2017 by The International Union of Biochemistry and Molecular Biology, 46(1):66-75, 2018.

Influence of high hydrostatic pressure on epitope mapping of tobacco mosaic virus coat protein.

In this study, we investigated the effect of high hydrostatic pressure (HHP) on tobacco mosaic virus (TMV), a model virus in immunology and one of the most studied viruses to date. Exposure to HHP significantly altered the recognition epitopes when compared to sera from mice immunized with native virus. These alterations were studied further by combining HHP with urea or low temperature and then inoculating the altered virions into Balb-C mice. The antibody titers and cross-reactivity of the resulting sera were determined by ELISA. The antigenicity of the viral particles was maintained, as assessed by using polyclonal antibodies against native virus. The antigenicity of canonical epitopes was maintained, although binding intensities varied among the treatments. The patterns of recognition determined by epitope mapping were cross checked with the prediction algorithms for the TMVcp amino acid sequence to infer which alterations had occurred. These findings suggest that different cleavage sites were exposed after the treatments and this was confirmed by epitope mapping using sera from mice immunized with virus previously exposed to HHP.

Potentiation of high hydrostatic pressure inactivation of Mycobacterium by combination with physical and chemical conditions.

Mycobacterium abscessus is an important hospital-acquired pathogen involved in infections associated with medical, surgical, and biopharmaceutical materials. In this work, we investigated the pressure-induced inactivation of two strains [2544 and American Type Culture Collection (ATCC) 19977] of M. abscessus in combination with different temperatures and pH conditions. For strain 2544, exposure to 250 MPa for 90 min did not significantly inactivate the bacteria at 20 °C, whereas at -15 °C, there was complete inactivation. Exposure to 250 MPa at ≥60 °C caused rapid inactivation, with no viable bacteria after 45 min. With 45 min of exposure, there were no viable bacteria at any temperature when a higher pressure (350 MPa) was used. Extremes of pH (4 or 9) also markedly enhanced the pressure-induced inactivation of bacteria at 250 MPa, with complete inactivation after 45 min. In comparison, exposure of this strain to the disinfecting agent glutaraldehyde (0.5 %) resulted in total inactivation within 5 min. Strain 19977 was more sensitive to high pressure but less sensitive to glutaraldehyde than strain 2544. These results indicate that high hydrostatic pressure in combination with other physical parameters may be useful in reducing the mycobacterial contamination of medical materials and pharmaceuticals that are sensitive to autoclaving.

Tendency for oxidation of annelid hemoglobin at alkaline pH and dissociated states probed by redox titration.

The redox titration of extracellular hemoglobin of Glossoscolex paulistus (Annelidea) was investigated in different pH conditions and after dissociation induced by pressure. Oxidation increased with increasing pH, as shown by the reduced amount of ferricyanide necessary for the oxidation of hemoglobin. This behavior was the opposite of that of vertebrate hemoglobins. The potential of half oxidation (E1/2) changed from -65.3 to +146.8 mV when the pH increased from 4.50 to 8.75. The functional properties indicated a reduction in the log P50 from 1.28 to 0.28 in this pH range. The dissociation at alkaline pH or induced by high pressure, confirmed by HPLC gel filtration, suggested that disassembly of the hemoglobin could be involved in the increased potential for oxidation. These results suggest that the high stability and prolonged lifetime common to invertebrate hemoglobins is related to their low tendency to oxidize at acidic pH, in contrast to vertebrate hemoglobins.