Osteogenesis of 3D-Printed PCL/TCP/bdECM Scaffold Using Adipose-Derived Stem Cells Aggregates; An Experimental Study in the Canine Mandible.

Three-dimensional (3D) printing is perceived as an innovative tool for change in tissue engineering and regenerative medicine based on research outcomes on the development of artificial organs and tissues. With advances in such technology, research is underway into 3D-printed artificial scaffolds for tissue recovery and regeneration. In this study, we fabricated artificial scaffolds by coating bone demineralized and decellularized extracellular matrix (bdECM) onto existing 3D-printed polycaprolactone/tricalcium phosphate (PCL/TCP) to enhance osteoconductivity and osteoinductivity. After injecting adipose-derived stem cells (ADSCs) in an aggregate form found to be effective in previous studies, we examined the effects of the scaffold on ossification during mandibular reconstruction in beagle dogs. Ten beagles were divided into two groups: group A (PCL/TCP/bdECM + ADSC injection; n = 5) and group B (PCL/TCP/bdECM; n = 5). The results were analyzed four and eight weeks after intervention. Computed tomography (CT) findings showed that group A had more diffuse osteoblast tissue than group B. Evidence of infection or immune rejection was not detected following histological examination. Goldner trichrome (G/T) staining revealed rich ossification in scaffold pores. ColI, Osteocalcin, and Runx2 gene expressions were determined using real-time polymerase chain reaction. Group A showed greater expression of these genes. Through Western blotting, group A showed a greater expression of genes that encode ColI, Osteocalcin, and Runx2 proteins. In conclusion, intervention group A, in which the beagles received the additional ADSC injection together with the 3D-printed PCL/TCP coated with bdECM, showed improved mandibular ossification in and around the pores of the scaffold.

Inhalation toxicity of benzalkonium chloride and triethylene glycol mixture in rats.

Benzalkonium chloride (BAC), a disinfectant, and triethylene glycol (TEG), an organic solvent/sanitizer, are frequently combined in commercially available household sprays. To assess the respiratory effect of this combination, Sprague-Dawley rats were exposed to an aerosol containing BAC (0.5%, w/v) and TEG (10%, w/v) for up to 2 weeks in a whole-body inhalation chamber. BAC (4.1-4.5 mg/m3, sprayed from 0.5% solution) promoted pulmonary cell damage and inflammation as depicted by the increase in total protein, lactate dehydrogenase, polymorphonuclear leukocytes, and macrophage inflammatory protein-2 in the bronchoalveolar lavage fluid, whereas TEG (85.3-94.5 mg/m3, sprayed from 10% solution) did not affect the lung. Rats exposed to the BAC/TEG mixture for 2 weeks showed severe respiratory symptoms (sneezing, wheezing, breath shortness, and chest tightness), but no lung damage or inflammation was observed. However, significant ulceration and degenerative necrosis were observed in the nasal cavities of rats repeatedly exposed to the BAC/TEG mixture. The mass median aerodynamic diameters of the aqueous, BAC, TEG and BAC/TEG aerosols were 1.24, 1.27, 3.11 and 3.24 µm, respectively, indicating that TEG-containing aerosols have larger particles than those of the aqueous and BAC alone aerosols. These results suggest that the toxic effects of BAC and BAC/TEG aerosols on the different respiratory organs may be associated with the difference in particle diameter, since particle size is important in determining the deposition site of inhaled materials.

DNA translocation through a nanopore in an ultrathin self-assembled peptide membrane.

Here, we explore the possibility of using peptide-based materials as a membrane in solid-state nanopore devices in an effort to develop a sequence-specific, programmable biological membrane platform. We use a recently developed tyrosine-mediated self-assembly peptide sheet. At the air/water interface, the 5mer peptide YFCFY self-assembles into a uniform and robust two-dimensional (2D) structure, and the peptide sheet is easily transferred to a low-noise glass substrate. The thickness of the peptide membrane can be adjusted to approximately 5 nm (or even to 2 nm) by an etching process, and the diameters of the peptide nanopores can be precisely controlled using a focused electron beam with an attuned spot size. The ionic current noise of the peptide nanopore is comparable to those of typical silicon nitride nanopores or multilayer 2D materials. Using this membrane, we successfully observe translocation of 1000 bp double-stranded DNA with a sufficient signal-to-noise ratio of ∼30 and an elongated translocation speed of ∼1 bp µs-1. Our results suggest that the self-assembled peptide film can be used as a sensitive nanopore membrane and employed as a platform for applying biological functionalities to solid-state substrates.

Evaluation of pulmonary toxicity of benzalkonium chloride and triethylene glycol mixtures using in vitro and in vivo systems.

Benzalkonium chloride (BAC) is a widely used disinfectant/preservative, and respiratory exposure to this compound has been reported to be highly toxic. Spray-form household products have been known to contain BAC together with triethylene glycol (TEG) in their solutions. The purpose of this study was to estimate the toxicity of BAC and TEG mixtures to pulmonary organs using in vitro and in vivo experiments. Human alveolar epithelial (A549) cells incubated with BAC (1-10 µg/mL) for 24 hours showed significant cytotoxicity, while TEG (up to 1000 µg/mL) did not affect cell viability. However, TEG in combination with BAC aggravated cell damage and inhibited colony formation as compared to BAC alone. TEG also exacerbated BAC-promoted production of reactive oxygen species (ROS) and reduction of glutathione (GSH) level in A549 cells. However, pretreatment of the cells with N-acetylcysteine (NAC) alleviated the cytotoxicity, indicating oxidative stress could be a mechanism of the toxicity. Quantification of intracellular BAC by LC/MS/MS showed that cellular distribution/absorption of BAC was enhanced in A549 cells when it was exposed together with TEG. Intratracheal instillation of BAC (400 µg/kg) in rats was toxic to the pulmonary tissues while that of TEG (up to 1000 µg/kg) did not show any harmful effect. A combination of nontoxic doses of BAC (200 µg/kg) and TEG (1000 µg/kg) promoted significant lung injury in rats, as shown by increased protein content and lactate dehydrogenase (LDH) activity in bronchoalveolar lavage fluids (BALF). Moreover, BAC/TEG mixture recruited inflammatory cells, polymorphonuclear leukocytes (PMNs), in terminal bronchioles and elevated cytokine levels, tumor necrosis factor α (TNF-α), and interleukin 6 (IL-6) in BALF. These results suggest that TEG can potentiate BAC-induced pulmonary toxicity and inflammation, and thus respiratory exposure to the air mist from spray-form products containing this chemical combination is potentially harmful to humans.

Synthesis of triphenylsulfonium triflate bound copolymer for electron beam lithography.

Photoacid generator (PAG) has been widely used as a key component in photoresist for high-resolution patterning with high sensitivity. A novel acrylic monomer, triphenylsulfonium salt methyl methacrylate (TPSMA), was synthesized and includes triphenylsulfonium triflate as a PAG. The poly(MMA-co-TPSMA) (PMT) as a polymer-bound PAG was synthesized with methyl methacrylate (MMA) and TPSMA for electron beam lithography. Characterization of PMT was carried out by NMR and FTIR. The molecular weight was analyzed by GPC. Thermal properties were studied using TGA and DSC. Thecharacterization results were in good agreement with corresponding chemical compositions and thermal stability. PMT was subsequently employed in electron beam lithography and its lithographic performance was confirmed by FE-SEM. This PMT was accomplished to improve the lithographic performance including sensitivity, line width roughness (LWR) and resolution. We found that PMT was capable of 20 nm negative tone patterns with better sensitivity than hydrogensilsesquioxane (HSQ) which is a conventional negative tone resist.

Evaluation of a Vaccine Candidate Designed for Broad-Spectrum Protection against Type A Foot-and-Mouth Disease in Asia.

Foot-and-mouth disease (FMD) vaccines are currently the most powerful protective and preventive measures used to control FMD. In this study, the chimeric vaccine strain containing antigenic epitopes from the FMD virus serotype A, which belongs to the ASIA topotype, was produced and evaluated. The chimeric vaccine strains contain sea-97/G1 (VP4, VP2, VP3) and A22 Iraq (VP1) or G-VII (VP1) for use in FMD vaccines in Asia. The 50% protective dose was determined in mice. Vaccinated mice were challenged with three different type A viruses (Sea-97/G1, Sea-97/G2, G-VII clade) seven days post-vaccination (dpv), and mice that received the vaccine candidates were protected against the three viruses. The protective capability of one of the vaccine candidates was evaluated in pigs. Vaccinated pigs were challenged with three different type A viruses (Sea-97/G1, Sea-97/G2, G-VII clade) at 28 dpv, and pigs that received the vaccine candidate were protected against the three viruses. The results showed that this vaccine candidate, which was designed to provide protection against FMD in Asia, efficiently protected pigs against virus challenge and thus has potential as a broad-spectrum vaccine for various epidemic FMD viruses.

Evaluation of Vaccine Strains Developed for Efficient, Broad-Range Protection against Foot-and-Mouth Disease Type O.

Foot-and-mouth disease (FMD) type O includes 11 genetic topotypes. The Southeast Asia (SEA), Middle East-South Asia (ME-SA), and Cathay topotypes belong to FMD type O and occur frequently in Asia. Therefore, it is necessary to develop a potent vaccine strain with a broad antigenic coverage in order to provide complete protection against these three topotypes. In this study, an experimental vaccine was produced using chimeric vaccine strains (JC-VP1 or PA2-VP1) that contained VP4, VP2, and VP3 of the ME-SA topotype (O Manisa) and VP1 of the SEA topotype (Mya98 lineage; O/SKR/Jincheon/2014) or ME-SA topotype (PanAsia2 lineage; O/PAK/44). Mice were immunized with the experimental vaccines, and they were fully protected against the three topotypes. The neutralizing antibody titers of PA2-VP1 were significantly higher than those of JC-VP1 in the early vaccination phase in pigs. Here, we confirmed complete protection in pigs vaccinated with JC-VP1 or PA2-VP1, when challenged against the SEA (O/SKR/Jincheon/2014), ME-SA (O/SKR/Boeun/2017) and Cathay (O/Taiwan/97) topotype viruses, with moderately higher protection provided by PA2-VP1 than by JC-VP1.

The C3d-fused foot-and-mouth disease vaccine platform overcomes maternally-derived antibody interference by inducing a potent adaptive immunity.

Vaccination prevents and controls foot-and-mouth disease (FMD). However, the current FMD vaccine remains disadvantageous since it cannot overcome maternally-derived antibody (MDA) interference in weeks-old animals, which suppress active immunity via vaccination. To address this, we developed the immune-enhancing O PA2-C3d and A22-C3d FMD vaccine strains that can stimulate receptors on the surface of B cells by inserting C3d (a B cell epitope) into the VP1 region of O PA2 (FMDV type O) and A22 (FMDV type A). We purified inactivated viral antigens from these vaccine strains and evaluated their immunogenicity and host defense against FMDV infection in mice. We also verified its efficacy in inducing an adaptive immune response and overcome MDA interference in MDA-positive (MDA(+), FMD-seropositive) and -negative (MDA(-), FMD-seronegative) pigs. These results suggest a key strategy for establishing novel FMD vaccine platform to overcome MDA interference and induce a robust adaptive immune response.

Efficient protection against Asia1 type foot-and-mouth disease using a chimeric vaccine strain suitable for East Asia.

The type Asia1 genetic group(G)-V lineage foot-and-mouth disease (FMD) virus was identified in the East-Asian region in 2009. To date, only Shamir has been used as a standard vaccine strain worldwide for type Asia1. To prevent type Asia1 FMD in eastern Asia, two vaccine strains (ASM-R: G-V and ASM-SM: G-V/Shamir fusion) were developed and tested against type Asia1 virus strains. After immunization with the two experimental vaccines, the ASM-SM strain showed a higher level of protection against Shamir virus in mice. Additional immunogenicity tests were carried out in cattle and pigs, revealing sufficient antibody production capable of protecting the animals against the viral challenge. In cattle, the immune response started just 2 weeks after vaccination. Immunogenicity was lower in pigs, but antibody production was greatly increased to a high level after a second vaccination round. In particular, herein, 60 % and 100 % of the vaccinated pigs challenged with the Asia1 Shamir virus were determined to be clinically protected after one and two vaccination rounds with ASM-R, respectively. Pigs vaccinated twice produced sufficient antibody titers with low virus shedding for short time. Moreover, ASM-SM single-vaccinated pigs showed 100 % protection when challenged with the Asia1 Shamir virus. In summary, the vaccine strain ASM-SM designed for the defense of the Asian region efficiently granted protection to pigs against the typical Asia1 virus, Shamir.

The HSP70-fused foot-and-mouth disease epitope elicits cellular and humoral immunity and drives broad-spectrum protective efficacy.

Current foot-and-mouth disease (FMD) vaccines have significant limitations, including side effects due to oil emulsions at the vaccination site, a narrow spectrum of protective efficacy, and incomplete host defenses mediated by humoral immunity alone. To overcome these limitations, new FMD vaccines must ensure improved safety with non-oil-based adjuvants, a broad spectrum of host defenses within/between serotypes, and the simultaneous induction of cellular and humoral immunity. We designed a novel, immune-potent, recombinant protein rpHSP70-AD that induces robust cellular immunity and elicits a broad spectrum of host defenses against FMD virus (FMDV) infections. We demonstrated that an oil emulsion-free vaccine containing rpHSP70-AD mediates early, mid-term, and long-term immunity and drives potent host protection against FMDV type O and A, suggesting its potential as an FMD vaccine adjuvant in mice and pigs. These results suggest a key strategy for establishing next-generation FMD vaccines, including novel adjuvants.

The Potential Adjuvanticity of CAvant®SOE for Foot-and-Mouth Disease Vaccine.

Foot-and-mouth disease (FMD) is a notifiable contagious disease of cloven-hoofed mammals. A high potency vaccine that stimulates the host immune response is the foremost strategy used to prevent disease persistence in endemic regions. FMD vaccines comprise inactivated virus antigens whose immunogenicity is potentiated by immunogenic adjuvants. Oil-based adjuvants have clear advantages over traditional adjuvant vaccines; however, there is potential to develop novel adjuvants to increase the potency of FMD vaccines. Thus, we aimed to evaluate the efficacy of a novel water-in-oil emulsion, called CAvant®SOE, as a novel vaccine adjuvant for use with inactivated FMD vaccines. In this study, we found that inactivated A22 Iraq virus plus CAvant®SOE (iA22 Iraq-CAvant®SOE) induced effective antigen-specific humoral (IgG, IgG1, and IgG2a) and cell-mediated immune responses (IFN-γ and IL-4) in mice. Immunization of pigs with a single dose of iA22 Iraq-CAvant®SOE also elicited effective protection, with no detectable clinical symptoms against challenge with heterologous A/SKR/GP/2018 FMDV. Levels of protection are strongly in line with vaccine-induced neutralizing antibody titers. Collectively, these results indicate that CAvant®SOE-adjuvanted vaccine is a promising candidate for control of FMD in pigs.

Translocation of DNA and protein through a sequentially polymerized polyurea nanopore.

Here, we investigated the translocation of biomolecules, such as DNA and protein, through a sequentially polymerized polyurea nanopore, with a thin (<10 nm) polymer membrane of uniform thickness. The polyurea membrane was synthesized by molecular layer deposition using p-phenylenediisocyanate (PDI) and p-phenylenediamine (PDA) as sequential precursors. The membrane exhibited a hydrophobic surface with a highly negative surface charge density (-51 mC m-2 at pH 8). It was particularly noted that the high surface charge density of the membrane resulted in a highly developed electro-osmotic flow which, in turn, strongly influenced the capture probability of biomolecules, depending on the balance between the electro-osmotic and electrophoretic forces. For instance, the capture frequency of negatively charged DNA was demonstrated to be quite low, since these two forces more or less cancelled each other, whereas that of positively charged MDM2 was much higher, since these two forces were additive. We also identified that the mean translocation time of MDM2 through the polyurea nanopore was 26.1 ± 3.7 µs while that of the SiN nanopore was 14.2 ± 2.0 µs, hence suggesting that the enhanced electrostatic interaction between positively charged MDM2 and the negatively charged pore surface affects the translocation speed.

Synchronized optical and electronic detection of biomolecules using a low noise nanopore platform.

In the past two decades there has been a tremendous amount of research into the use of nanopores as single molecule sensors, which has been inspired by the Coulter counter and molecular transport across biological pores. Recently, the desire to increase structural resolution and analytical throughput has led to the integration of additional detection methods such as fluorescence spectroscopy. For structural information to be probed electronically high bandwidth measurements are crucial due to the high translocation velocity of molecules. The most commonly used solid-state nanopore sensors consist of a silicon nitride membrane and bulk silicon substrate. Unfortunately, the photoinduced noise associated with illumination of these platforms limits their applicability to high-bandwidth, high-laser-power synchronized optical and electronic measurements. Here we present a unique low-noise nanopore platform, composed of a predominately Pyrex substrate and silicon nitride membrane, for synchronized optical and electronic detection of biomolecules. Proof of principle experiments are conducted showing that the Pyrex substrates have substantially lowers ionic current noise arising from both laser illumination and platform capacitance. Furthermore, using confocal microscopy and a partially metallic pore we demonstrate high signal-to-noise synchronized optical and electronic detection of dsDNA.

Investigation of disinfectants for foot-and-mouth disease in the Republic of Korea.

Disinfectants for foot-and-mouth disease were sprayed on livestock barns and roads from early February to May 2011. Although 90% of the disinfectant was concentrated on the roads, 10% was sprayed on cattle sheds and other sites where foot-and-mouth disease occurred. Since the outbreak of foot-and-mouth disease in November 2010, there has been a steady increase in disinfectant use. Consequently, its adverse environmental effects have prompted government officials to take preventive measures. The major chemical components of the disinfectants are citric acid, potassium sulfate base complex, quaternary ammonium compound, malic acid, and glutaraldehyde, ranging in amounts from tons to hundreds of tons. The exact amount of each component of the disinfectants could not be identified because the types of components used in the different commercial formulations overlapped. In this review, we obtained information on disinfectants that are widely used nationwide, including the types of major chemical components and their respective toxicities (both human and ecological).