High-pressure CO 2 treatment has been studied as a promising method for inactivating bacterial spores. In the present study, we compared this method with other sterilization techniques, including heat and pressure treatment. Spores of Bacillus coagulans, Bacillus subtilis, Bacillus cereus, Bacillus licheniformis, and Geobacillus stearothermophilus were subjected to CO 2 treatment at 30 MPa and 35°C, to high-hydrostatic-pressure treatment at 200 MPa and 65°C, or to heat treatment at 0.1 MPa and 85°C. All of the bacterial spores except the G. stearothermophilus spores were easily inactivated by the heat treatment. The highly heat-and pressureresistant spores of G. stearothermophilus were not the most resistant to CO 2 treatment. We also investigated the influence of temperature on CO 2 inactivation of G. stearothermophilus. Treatment with CO 2 and 30 MPa of pressure at 95°C for 120 min resulted in 5-log-order spore inactivation, whereas heat treatment at 95°C for 120 min and high-hydrostatic-pressure treatment at 30 MPa and 95°C for 120 min had little effect. The activation energy required for CO 2 treatment of G. stearothermophilus spores was lower than the activation energy for heat or pressure treatment. Although heat was not necessary for inactivation by CO 2 treatment of G. stearothermophilus spores, CO 2 treatment at 95°C was more effective than treatment at 95°C alone.
In this study, we investigated the effects of proteinase gene disruption on heterologous protein production by Aspergillus oryzae. The human lysozyme (HLY) was selected for recombinant production as a model for the heterologous protein. A tandem HLY construct fused with alpha-amylase (AmyB) was expressed by A. oryzae in which the Kex2 cleavage site was inserted at the upstream of HLY. HLY was successfully processed from AmyB and produced in the medium. We performed a systematic disruption analysis of five proteinase genes (pepA, pepE, alpA, tppA, and palB) in the HLY-producing strain with the adeA selectable marker. Comparative analysis indicated that disruption of the tppA gene encoding a tripeptidyl peptidase resulted in the highest increase (36%) in the HLY production. We further deleted the tppA gene in the pepE or palB disruptant with another selectable marker, argB. Consequently, a double disruption of the tppA and pepE genes led to a 63% increase in the HLY production compared to the control strain. This is the first study to report that the double disruption of the tppA and pepE genes improved the production level of a heterologous protein by filamentous fungi.
Platelet-rich fibrin (PRF) therapy has been widely applied in regenerative dentistry, and PRF preparation has been optimized to efficiently form fibrin clots using plain glass tubes. Currently, a shortage of commercially available glass tubes has forced PRF users to utilize silica-coated plastic tubes. However, most plastic tubes are approved by regulatory authorities only for diagnostic use and remain to be approved for PRF therapy. To clarify this issue, we quantified silica microparticles incorporated into the PRF matrix. Blood samples were collected into three different brands of silica-containing plastic tubes and were immediately centrifuged following the protocol for advanced-PRF (A-PRF). Advanced-PRF-like matrices were examined using a scanning electron microscope (SEM), and silica microparticles were quantified using a spectrophotometer. Each brand used silica microparticles of specific size and appearance. Regardless of tube brands and individual donors, significant, but not accidental, levels of silica microparticles were found to be incorporated into the A-PRF-like matrix, which will be consequently incorporated into the implantation sites. Presently, from the increasing data for cytotoxicity of amorphous silica, we cannot exclude the possibility that such A-PRF-like matrices negatively influence tissue regeneration through induction of inflammation. Further investigation should be performed to clarify such potential risks.
BackgroundBased on the notion that full activation of platelets is required for a growth factor release, in regenerative dentistry, platelet-rich plasma (PRP) in liquid form is usually clotted by addition of CaCl2 in glassware before topical implantation. However, there has been no evidence as to which is better, full or partial activation of platelets, for minimizing the loss of growth factors and improving the controlled release of growth factors from coagulated PRP. To address this matter, here, we primarily examined direct effects of CaCl2 on platelets in PBS and on coagulation in citrated PRP.MethodsPRP was prepared from healthy volunteers’ blood. Platelets’ actions were monitored by scanning electron microscopy, flow cytometry, digital holographic microscopy, and immunofluorescent staining. Clot formation was examined in plasma.ResultsIn plasma-free PBS, 0.1% CaCl2 immediately upregulated CD62P and CD63, causing a release of microparticles and fibrinogen/fibrin; consequently, platelets aggregated and adhered to polystyrene culture dishes with enlargement of their attachment area. In a clot formation assay in plasma, CaCl2 initially induced platelet aggregation, which triggered loop-like matrix formation and subsequently induced coagulation on a watch glass. Such changes were not clearly observed either with PRP in a plastic dish or in platelet-poor plasma on a watch glass: coagulation was delayed in both conditions.ConclusionsThese findings indicate that besides the well-known coagulation pathway, which activates platelets via thrombin conversion in a coagulation cascade, CaCl2 directly activates platelets, which then facilitate clot formation independently and in cooperation with the coagulation pathway.
Because of its simple operation, platelet-rich fibrin (PRF) is becoming more popular than the original form, platelet-rich plasma (PRP), in regenerative dentistry. PRF preparation requires plain glass blood-collection tubes, but not either anticoagulants or coagulation factors. However, such glass tubes designed for laboratory testing are no longer commercially available. Although several glass tubes specifically designed for PRF preparation are available, many clinicians prefer to obtain stably supplied substitutes, such as silica-coated plastic tubes produced by major medical device companies. The quality of PRF prepared by silica-coated tubes has not been assessed and we previously reported significant contamination of silica microparticles in the resulting PRF matrix and alerted clinicians against the use for PRF preparation. To further assess the biosafety of the silica microparticles, we presently examined their effects on human normal periosteal cells derived from alveolar bone. The periosteal cells were obtained from explant cultures of small periosteal tissues obtained from healthy donors. Silica microparticles were obtained from silica-coated tubes and added to cell cultures. Cellular responses were monitored using a tetrazolium assay, phase-contract inverted microscopy, an immunofluorescence method, and scanning electron microscopy. Silica microparticles adsorbed onto the cell surface with seemingly high affinity and induced apoptosis, resulting in significant reduction of cell proliferation and viability. These findings suggest that silica microparticles contained in plastic tubes for the purpose of blood coagulation are hazardous for various cell types around sites where silicacontaminated PRF matrices are implanted.Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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