Degradation of Toxins Derived from Foodborne Pathogens by Atmospheric-Pressure Dielectric-Barrier Discharge.

Foodborne diseases can be attributed not only to contamination with bacterial or fungal pathogens but also their associated toxins. Thus, to maintain food safety, innovative decontamination techniques for toxins are required. We previously demonstrated that an atmospheric-pressure dielectric-barrier discharge (APDBD) plasma generated by a roller conveyer plasma device is effective at inactivating bacteria and fungi in foods. Here, we have further examined whether the roller conveyer plasma device can be used to degrade toxins produced by foodborne bacterial pathogens, including aflatoxin, Shiga toxins (Stx1 and Stx2), enterotoxin B and cereulide. Each toxin was spotted onto an aluminum plate, allowed to dry, and then treated with APDBD plasma applied by the roller conveyer plasma device for different time periods. Assessments were conducted using a competitive enzyme-linked immunosorbent assay (ELISA) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results demonstrate a significant time-dependent decrease in the levels of these toxins. ELISA showed that aflatoxin B1 concentrations were reduced from 308.6 µg/mL to 74.4 µg/mL within 1 min. For Shiga toxins, Stx1 decreased from 913.8 µg/mL to 65.1 µg/mL, and Stx2 from 2309.0 µg/mL to 187.6 µg/mL within the same time frame (1 min). Enterotoxin B levels dropped from 62.67 µg/mL to 1.74 µg/mL at 15 min, and 1.43 µg/mL at 30 min, but did not display a significant decrease within 5 min. LC-MS/MS analysis verified that cereulide was reduced to below the detection limit following 30 min of APDBD plasma treatment. Taken together, these findings highlight that a range of foodborne toxins can be degraded by a relatively short exposure to plasma generated by an APDBD using a roller conveyer device. This technology offers promising advancements in food safety, providing a novel method to alleviate toxin contamination in the food processing industry.

Plasma Biology 2.0.

Plasma biology is a cutting-edge research field that involves plasma technology [...].

Recent Advances in Prion Inactivation by Plasma Sterilizer.

Prions, which cause transmissible spongiform encephalopathies (TSEs), are a notorious group of infectious agents with possibly the highest resistance to complete inactivation. Although various gas plasma instruments have been developed, studies on prion inactivation using gas plasma instruments are limited. Among them, the hydrogen peroxide gas plasma instrument, STERRAD® (Advanced Sterilization Products; ASP, Johnson & Johnson, Irvine, CA, USA), is recommended for prion inactivation of heat-sensitive medical devices. However, STERRAD® is not a plasma sterilizer but a hydrogen peroxide gas sterilizer. In STERRAD®, plasma generated by radio frequency (RF) discharge removes excess hydrogen peroxide gas and does not contribute to sterilization. This is also supported by evidence that the instrument was not affected by the presence or absence of RF gas plasma. However, recent studies have shown that other gas plasma instruments derived from air, nitrogen, oxygen, Ar, and a mixture of gases using corona, dielectric barrier, microwave, and pulse discharges can inactivate scrapie prions. As inactivation studies on prions other than scrapie are limited, further accumulation of evidence on the effectiveness of gas plasma using human-derived prion samples is warranted for practical purposes.

Plasma Biology.

It is now more than 90 years since Irving Langmuir used the technical term "plasma" to describe an ionized gas [...].

Inactivation Methods for Prions.

Incidences of iatrogenic Creutzfeldt-Jakob disease (iCJD) are caused by transplantation of prion-contaminated hormones, cornea and dura mater as well as contact with prion- contaminated medical devices, such as stereotactic electrodes, used in neurosurgery. Because prions are highly resistant and difficult to inactivate, prion contamination is a severe risk when medical instruments are reused after surgical procedures involving suspicious and confirmed cases of patients with prion diseases. Therefore, when high-risk procedures such as cerebral surgery, craniotomy surgery, orthopaedic spinal surgery and ophthalmic surgery are performed for high-risk patients or individuals with prion diseases, it is neces- sary to appropriately treat the medical devices using scientifically proven prion inactivation methods. In this chapter, we introduce fundamental aspects of prion inactivation methods, looking specifically at the practical issues involved in their implementation.

Chronic Wasting Disease: Current Assessment of Transmissibility.

Chronic wasting disease (CWD) is a prion disease of cervids characterized by clini- cal symptoms of progressive weight loss, abnormal behaviour and excessive salivation. Incidents have been reported in North America and Korea as well as in Europe. Current knowledge, based on in vitro and in vivo experiments, suggests direct CWD transmis- sion to humans is unlikely. Nonetheless, humans may consume CWD-infected materials, which presents a potential risk. Studies indicate that transmission by horizontal infection of cervids probably occurs via saliva, faeces, and urine as well as from environmental res- ervoirs of prions found in soil and water. In addition, infectivity in the skeletal muscle of infected deer has been observed. These findings suggest that direct contact with infected animals and indirect contact with prion-contaminated materials are potential sources of infection. However, recent studies on the detection of pregnancy-related prion infectivity imply the potential transmission of CWD from mother to offspring. In this review, fundamental aspects of CWD are reviewed.

Introduction to Current Progress in Advanced Research on Prions.

Prion diseases or transmissible spongiform encephalopathies (TSEs) are fatal neurological diseases that include Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep and goats, bovine spongiform encephalopathy (BSE) in cattle, camel spongiform encephalopathy (CSE) in camels and chronic wasting disease (CWD) in cervids. A key event in prion diseases is the conversion of the cellular, host-encoded prion protein (PrPC) to its abnormal isoform (PrPSc) predominantly in the central nervous system of the infected host (Aguzzi et al., 2004). These diseases are transmissible under some circumstances, but unlike other transmissible disorders, prion diseases can also be caused by mutations in the host gene. The mechanism of prion spread among sheep and goats that develop natural scrapie is unknown. CWD, transmissible mink encephalopathy (TME), BSE, feline spongiform encephalopathy (FSE), and exotic ungulate encephalopathy (EUE) are all thought to occur after the consumption of prion-infected material. Most cases of human prion disease occur from unknown reasons, and greater than 20 mutations in the prion protein (PrP) gene may lead to inherited prion disease. In other instances, prion diseases are contracted by exposure to prion infectivity. These considerations raise the question of how a mere protein aggregate can bypass mucosal barriers, circumvent innate and adoptive immunity, and traverse the blood-brain barrier to give rise to brain disease. Here, we will briefly introduce a few topics in current prion studies.

Function of Prion Protein and the Family Member, Shadoo.

Lowering cellular prion protein (PrPC) levels in the brain is predicted to be a powerful therapeutic strategy for the prion disease. PrPC may act as an antiapoptotic agent by blocking some of the internal environmental factors that initiate apoptosis. Prion protein (PrP)-knockout methods provide powerful indications on the neuroprotective function of PrPC. Using PrPC-knockout cell lines, the inhibition of apoptosis through stress inducible protein1 (STI1) is mediated by PrPC-dependent superoxide dismutase (SOD) activation. Besides, PrP-knockout exhibited wide spread alterations of oscillatory activity in the olfactory bulb as well as altered paired-pulse plasticity at the dendrodendric synapse. Both the behavioural and electro-physiological phenotypes could be rescued by neuronal PrPC expression. Neuprotein Shadoo (Sho), similarly to PrPC, can prevent neuronal cell death induced by the expression of PrP△HD mutants, an artificial PrP mutant devoid of internal hydrophobic domain. Sho can efficiently protect cells against exito-toxin-induced cell death by glutamates. Sho and PrP seem to be dependent on similar domains, in particular N-terminal (N), and their internal hydrophobic domain. Sho△N and Sho△HD displayed a reduced stress-protective activity but are complex glycosylated and attached to the outer leaflet of the plasma membrane via glycosylphosphatidylinositol (GPI) anchor indicating that impaired activity is not due to incorrect cellular trafficking. In Sho, over-expressed mice showed large amyloid plaques not seen in wild-type mice. However, Shadoo is not a major modulator of abnormal prion protein (PrPSc) accumulation. Sho and PrP share a stress-protective activity. The ability to adopt a toxic conformation of PrPSc seems to be specific for PrP.

Effect of Microglial Inflammation in Prion Disease.

Prion diseases are a group of transmissible fatal neurodegenerative disorders. Neuropatho- logical features of prion diseases include neuroinflammation featuring the infiltration of activated microglia in affected brain areas as well as the accumulation of an abnormal isoform of the cellular prion protein and neuronal loss. Recent studies have elucidated that inflammation in the brain induced by microglia plays an important role in the pathogenesis of neurodegenerative disorders including prion disease. Thus, the regulation of neuroin- flammation is key in terms of therapeutic and preventative approaches. The functions of neuroinflammation and microglia in this disease are discussed in this article.

Antibiotic-Resistant and Non-Resistant Bacteria Display Similar Susceptibility to Dielectric Barrier Discharge Plasma.

Here, we examined whether antibiotic-resistant and non-resistant bacteria show a differential susceptibility to plasma treatment. Escherichia coli DH5α were transformed with pPRO-EX-HT-CAT, which encodes an ampicillin resistance gene and chloramphenicol acetyltransferase (CAT) gene, and then treated with a dielectric barrier discharge (DBD) plasma torch. Plasma treatment reduced the viable cell count of E. coli after transformation/selection and further cultured in ampicillin-containing and ampicillin-free medium. However, there was no significant difference in viable cell count between the transformed and untransformed E. coli after 1 min- and 2 min-plasma treatment. Furthermore, the enzyme-linked immunosorbent assay (ELISA) and acetyltransferase activity assay showed that the CAT activity was reduced after plasma treatment in both transformed and selected E. coli grown in ampicillin-containing or ampicillin-free medium. Loss of lipopolysaccharide and DNA damage caused by plasma treatment were confirmed by a Limulus test and polymerase chain reaction, respectively. Taken together, these findings suggest the plasma acts to degrade components of the bacteria and is therefore unlikely to display a differential affect against antibiotic-resistant and non-resistant bacteria. Therefore, the plasma method may be useful in eliminating bacteria that are recalcitrant to conventional antibiotic therapy.

Disinfection and Sterilization Using Plasma Technology: Fundamentals and Future Perspectives for Biological Applications.

Recent studies have shown that plasma can efficiently inactivate microbial pathogens such as bacteria, fungi, and viruses in addition to degrading toxins. Moreover, this technology is effective at inactivating pathogens on the surface of medical and dental devices, as well as agricultural products. The current practical applications of plasma technology range from sterilizing therapeutic medical devices to improving crop yields, as well as the area of food preservation. This review introduces recent advances and future perspectives in plasma technology, especially in applications related to disinfection and sterilization. We also introduce the latest studies, mainly focusing on the potential applications of plasma technology for the inactivation of microorganisms and the degradation of toxins.

PrP Knockout Cells Expressing Transmembrane PrP Resist Prion Infection.

Glycosylphosphatidylinositol (GPI) anchoring of the prion protein (PrPC) influences PrPC misfolding into the disease-associated isoform, PrPres, as well as prion propagation and infectivity. GPI proteins are found in cholesterol- and sphingolipid-rich membrane regions called rafts. Exchanging the GPI anchor for a nonraft transmembrane sequence redirects PrPC away from rafts. Previous studies showed that nonraft transmembrane PrPC variants resist conversion to PrPres when transfected into scrapie-infected N2a neuroblastoma cells, likely due to segregation of transmembrane PrPC and GPI-anchored PrPres in distinct membrane environments. Thus, it remained unclear whether transmembrane PrPC might convert to PrPres if seeded by an exogenous source of PrPres not associated with host cell rafts and without the potential influence of endogenous expression of GPI-anchored PrPC To further explore these questions, constructs containing either a C-terminal wild-type GPI anchor signal sequence or a nonraft transmembrane sequence containing a flexible linker were expressed in a cell line derived from PrP knockout hippocampal neurons, NpL2. NpL2 cells have physiological similarities to primary neurons, representing a novel and advantageous model for studying transmissible spongiform encephalopathy (TSE) infection. Cells were infected with inocula from multiple prion strains and in different biochemical states (i.e., membrane bound as in brain microsomes from wild-type mice or purified GPI-anchorless amyloid fibrils). Only GPI-anchored PrPC supported persistent PrPres propagation. Our data provide strong evidence that in cell culture GPI anchor-directed membrane association of PrPC is required for persistent PrPres propagation, implicating raft microdomains as a location for conversion. IMPORTANCE: Mechanisms of prion propagation, and what makes them transmissible, are poorly understood. Glycosylphosphatidylinositol (GPI) membrane anchoring of the prion protein (PrPC) directs it to specific regions of cell membranes called rafts. In order to test the importance of the raft environment on prion propagation, we developed a novel model for prion infection where cells expressing either GPI-anchored PrPC or transmembrane-anchored PrPC, which partitions it to a different location, were treated with infectious, misfolded forms of the prion protein, PrPres We show that only GPI-anchored PrPC was able to convert to PrPres and able to serially propagate. The results strongly suggest that GPI anchoring and the localization of PrPC to rafts are crucial to the ability of PrPC to propagate as a prion.

Antibody-integrated and functionalized graphite-encapsulated magnetic beads, produced using ammonia gas plasma technology, for capturing Salmonella.

Salmonella spp. is the single and most important causative agent of foodborne infections, especially involving foods such as eggs, milk and meat. To prevent infection, a reliable surveillance system is required that can quickly and sensitively detect Salmonella. Here, we describe the development of antibody-integrated magnetic beads that are functionalized by a novel strategy using ammonia gas plasma. Ammonia plasma, produced by a radio frequency (RF) power supply, was allowed to react with the surface of graphite-encapsulated magnetic beads, resulting in the introduction of amino groups. An anti-Salmonella antibody was then anchored by sulfide groups present on the protein surface to the amino groups of the magnetic beads via N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). The potential usefulness of these magnetic beads for capturing Salmonella was examined as follows. The beads were incubated with Salmonella in liquid medium and then separated from the supernatant by applying a magnetic field. After thorough washing, adsorption of Salmonella to the beads was confirmed by immunochromatography, polymerase chain reaction and a direct culture assay. Our findings indicate that the capture and concentration of Salmonella using the antibody-integrated magnetic beads was more efficient than commercial Dynabeads® anti-Salmonella, which are conventionally used for concentrating Salmonella from liquid cultures. We believe this novel bead technology will contribute to the enhanced detection of Salmonella.

Integration of antibody by surface functionalization of graphite-encapsulated magnetic beads using ammonia gas plasma technology for capturing influenza A virus.

Antibody-integrated magnetic beads have been functionalized for influenza A virus capture. First, ammonia plasma produced by a radio frequency power source was reacted with the surface of graphite-encapsulated magnetic beads to introduce amino groups. Anti-influenza A virus hemagglutinin antibody was then anchored by its surface sulfide groups to the amino groups on the beads via N-succinimidyl 3-(2-pyridyldithio) propionate. After incubation with influenza A virus, adsorption of the virus to the beads was confirmed by immunochromatography, polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and inoculation of chicken embryonated eggs, indicating that virus infectivity is maintained and that the proposed method is useful for the enhanced detection and isolation of influenza A virus.

15-Deoxy-Δ12,14-prostaglandin J2 induces PPARγ- and p53-independent apoptosis in rabbit synovial cells.

A ligand of peroxisome proliferator-activated receptor γ (PPARγ), 15-deoxy-Δ(12,14)-prostaglandin J2 (15d-PGJ2) induces apoptosis in various cells. However, the mechanism appears to be complex and cell-type specific. We investigated the mechanism of 15d-PGJ2-induced apoptosis of rabbit synovial cells. Exposure to 15d-PGJ2 resulted in DNA fragmentation accompanied by caspase-3 and -9 activations in the cells, suggesting occurrence of mitochondria-mediated apoptosis. Although the exposure also induced remarkable increase in p53 protein, its transcriptional activity was rather reduced, suggesting non-necessity of p53 in 15d-PGJ2-induced apoptosis. Covalent binding of 15d-PGJ2 to cellular proteins including p53 resulted in their insolubilization. N-acetylcysteine inhibited not only the 15d-PGJ2-induced apoptotic events but also the protein insolubilizations via its interaction with 15d-PGJ2. The studies using a PPARγ-agonist and -antagonist showed noninvolvement of PPARγ in 15d-PGJ2-induced apoptosis. The pre-exposure to pro-inflammatory cytokines did not affect the cytotoxicity of 15d-PGJ2 in synovial cells. Taken together, these results show that 15d-PGJ2 induces a mitochondria-mediated apoptotic pathway in p53- and PPARγ-independent manners.

Review of studies that have used knockout mice to assess normal function of prion protein under immunological or pathophysiological stress.

Deletion of cellular isoform of prion protein (PrP(C)) increases neuronal predisposition to damage by modulating apoptosis and the negative consequences of oxidative stress. In vivo studies have demonstrated that PrP(C)-deficient mice are more prone to seizure, depression, and induction of epilepsy and experience extensive cerebral damage following ischemic challenge or viral infection. In addition, adenovirus-mediated overexpression of PrP(C) reduces brain damage in rat models of cerebral ischemia. In experimental autoimmune encephalomyelitis, PrP(C)-deficient mice reportedly have a more aggressive disease onset and less clinical improvement during the chronic phase than wild-type mice mice. In mice given oral dextran sulfate, PrP(C) has a potential protective role against inflammatory bowel disease. PrP(C)-deficient mice demonstrate significantly greater increases in blood glucose concentrations after intraperitoneal injection of glucose than wild-type mice. Further in vivo challenges to PrP gene-deficient models and conditional knockout models with siRNA and in vivo administration of PrP-ligating agents may assist in refining knowledge of the lymphoid function of PrP(C) and predicting the effects of anti-PrP treatment on the immune system. Together, these findings indicate that PrP(C) may have multiple neuroprotective and anti-inflammatory roles, which explains why this protein is so widely expressed.

Proteolysis of abnormal prion protein with a thermostable protease from Thermococcus kodakarensis KOD1.

The abnormal prion protein (scrapie-associated prion protein, PrP(Sc)) is considered to be included in the group of infectious agents of transmissible spongiform encephalopathies. Since PrP(Sc) is highly resistant to normal sterilization procedures, the decontamination of PrP(Sc) is a significant public health issue. In the present study, a hyperthermostable protease, Tk-subtilisin, was used to degrade PrP(Sc). Although PrP(Sc) is known to be resistant toward proteolytic enzymes, Tk-subtilisin was able to degrade PrP(Sc) under extreme conditions. The level of PrP(Sc) in brain homogenates was found to decrease significantly in vitro following Tk-subtilisin treatment at 100 °C, whereas some protease-resistant fractions remain after proteinase K treatment. Rather small amounts of Tk-subtilisin (0.3 U) were required to degrade PrP(Sc) at 100 °C and pH 8.0. In addition, Tk-subtilisin was observed to degrade PrP(Sc) in the presence of sodium dodecyl sulfate or other industrial surfactants. Although several proteases degrading PrP(Sc) have been reported, practical decontamination procedures using enzymes are not available. This report aims to provide basic information for the practical use of a proteolytic enzyme for PrP(Sc) degradation.

Effect of combined infection with Salmonella and influenza virus on their respective proliferation in chicken embryonated eggs.

Background: Salmonella is a major food-borne bacterial pathogen that causes food poisoning related to the consumption of eggs, milk, and meat. Food safety in relation to Salmonella is particularly important for eggs because their shells as well as their contents can be a source of contamination. Chicken can also be infected with influenza virus, but it remains unclear how co-infection of Salmonella and influenza virus affect each other. Aim: The potential influence of co-infection of Salmonella and influenza virus was examined. Methods: Salmonella Abony and influenza virus were injected into chicken embryonated eggs. After incubation, proliferation of Salmonella and influenza virus was measured using a direct culture assay for bacteria and an enzyme-linked immunosorbent assay for influenza virus, respectively. Results: Our findings indicate that the number of colony-forming units (CFUs) of Salmonella did not vary between chicken embryonated eggs co-infected with influenza A virus and Salmonella-only infected eggs. Furthermore, we found the proliferation of influenza A or B virus was not significantly influenced by co-infection of the eggs with Salmonella. Conclusion: These results suggest that combined infection of Salmonella with influenza virus does not affect each other, at least in terms of their proliferation.

Enzymatic activity of a subtilisin homolog, Tk-SP, from Thermococcus kodakarensis in detergents and its ability to degrade the abnormal prion protein.

BACKGROUND: Tk-SP is a member of subtilisin-like serine proteases from a hyperthermophilic archaeon Thermococcus kodakarensis. It has been known that the hyper-stable protease, Tk-SP, could exhibit enzymatic activity even at high temperature and in the presence of chemical denaturants. In this work, the enzymatic activity of Tk-SP was measured in the presence of detergents and EDTA. In addition, we focused to demonstrate that Tk-SP could degrade the abnormal prion protein (PrPSc), a protease-resistant isoform of normal prion protein (PrPC). RESULTS: Tk-SP was observed to maintain its proteolytic activity with nonionic surfactants and EDTA at 80°C. We optimized the condition in which Tk-SP functions efficiently, and demonstrated that the enzyme is highly stable in the presence of 0.05% (w/v) nonionic surfactants and 0.01% (w/v) EDTA, retaining up to 80% of its activity. Additionally, we also found that Tk-SP can degrade PrPSc to a level undetectable by western-blot analysis. CONCLUSIONS: Our results indicate that Tk-SP has a great potential for technological applications, such as thermo-stable detergent additives. In addition, it is also suggested that Tk-SP-containing detergents can be developed to decrease the secondary infection risks of transmissible spongiform encephalopathies (TSE).

Sterilization mechanism of nitrogen gas plasma: induction of secondary structural change in protein.

The mechanism of action on biomolecules of N2 gas plasma, a novel sterilization technique, remains unclear. Here, the effect of N2 gas plasma on protein structure was investigated. BSA, which was used as the model protein, was exposed to N2 gas plasma generated by short-time high voltage pulses from a static induction thyristor power supply. N2 gas plasma-treated BSA at 1.5 kilo pulses per second showed evidence of degradation and modification when assessed by Coomassie brilliant blue staining and ultraviolet spectroscopy at 280 nm. Fourier transform infrared spectroscopy analysis was used to determine the protein's secondary structure. When the amide I region was analyzed in the infrared spectra according to curve fitting and Fourier self-deconvolution, N2 gas plasma-treated BSA showed increased α-helix and decreased ß-turn content. Because heating decreased α-helix and increased ß-sheet content, the structural changes induced by N2 gas plasma-treatment of BSA were not caused by high temperatures. Thus, the present results suggest that conformational changes induced by N2 gas plasma are mediated by mechanisms distinct from heat denaturation.