A Quantitative Approach to Characterize the Surface Modification on Nanoparticles Based on Localized Dielectric Environments.

Nanoparticles (NPs) are utilized for the functionalization of composite materials and nanofluids. Although oxide NPs (e.g., silica (SiO2)) exhibit less dispersibility in organic solvents or polymers due to their hydrophilic surface, the surface modification using silane coupling agents can improve their dispersibility in media with low dielectric constants. Herein, SiO2 NPs were functionalized using octyltriethoxysilane (OTES, C8) and dodecyltriethoxysilane (DTES, C12), wherein the degrees of surface modification of SiO2@C8 and SiO2@C12 were quantitatively evaluated based on the ratio of modifier to surface silanol group (θ) and the volume fraction of organic modifier to total particle volume (ϕR). The variations of surface properties were revealed by analyzing the Hansen solubility parameters (HSP). Particularly, the surface modification using OTES or DTES significantly affected the polarity (δP) of NPs. The local dielectric environments of surface-modified SiO2 NPs were characterized using a solvatochromic dye, Laurdan. By analyzing the peak position of the steady-state emission spectrum of Laurdan in a NP suspension, the apparent dielectric environments surrounding NPs (εapp) were obtained. A good correlation between ϕR and εapp was observed, indicating that ϕR is a reliable quantity for understanding the properties of surface-modified NPs. Furthermore, the generalized polarization (GP) of NPs was investigated. The surface-modified SiO2 NPs with higher ϕR (≥0.15) exhibited GP > 0, suggesting that the modifiers are well-organized on the surface of NPs. The localized dielectric environment surrounding NPs could be predicted by analyzing the volume fraction of nonpolar moieties derived from modifiers. Alternatively, εapp and GP can be utilized for understanding the properties of inorganic-organic hybrid NPs.

Molecular Evolution and Epidemiology of Parechovirus-A3 in Japan, 1997-2019.

Parechovirus-A3 (PeV-A3), first reported in 2004 in Japan, is an emerging pathogen that causes sepsis and meningoencephalitis in neonates and young infants. Although PeV-A3 has been identified worldwide, its epidemiological characteristics differ by region. To investigate the molecular evolution and epidemiology of PeV-A3, we performed genetic analyses of 131 PeV-A3 strains from the years 1997-2019 in Niigata, Japan. During 2016-2019, annual numbers remained steady, in contrast to the PeV-A3 epidemic interval of every 2-3 years that was observed in Japan from 2006. Bayesian evolutionary analysis of the complete viral protein 1 region revealed alternate dominant clusters during years of PeV-A3 epidemics. The branch including the oldest and first isolated PeV-A3 strains in Japan has been disrupted since 2001. The year of PeV-A3 emergence was estimated to be 1991. Continuous surveillance with genetic analyses of different regions will improve understanding of PeV-A3 epidemiology worldwide.

Detection of parechovirus-A in hospitalized children with acute lower respiratory infection in Myanmar, 2017-2018.

Parechovirus-A (PeV-A) causes emerging infection in children, and clinical presentation depends on genotype. The virus has been investigated mainly in developed countries; however, data from developing countries, especially in Asia, are sparse. This study investigated whether PeV-A circulated in children in Myanmar. This retrospective study evaluated PeV-A in nasopharyngeal samples from children aged 1 month to 12 years who were hospitalized with acute lower respiratory infection at Yankin Children Hospital, Yangon, Myanmar, during the period from May 2017 to April 2019. Real-time polymerase chain reaction (PCR) was used to detect PeV-A, and PCR-positive samples were used for genotyping and phylogenetic analysis. In total, 11/570 (1.9%) of samples were positive for PeV-A; 7 were successfully genotyped by sequencing the VP3/VP1 region, as follows: PeV-A1 (n = 4), PeV-A5 (n = 1), PeV-A6 (n = 1), and PeV-A14 (n = 1). Median age was 10.0 months (interquartile range 4.0-12.0 months), and other respiratory viruses were detected in all cases. Phylogenetic analysis showed that all detected PeV-A1 strains were in clade 1 A, which was a minor clade worldwide. Four PeV-A genotypes were detected in Myanmar. The clinical impact of PeV-A in children should be evaluated in future studies.

Autolysin as a fibronectin receptor on the cell surface of Clostridium perfringens.

OBJECTIVE: Clostridium perfringens causes food poisoning and gas gangrene, a serious wound-associated infection. C. perfringens cells adhere to collagen via fibronectin (Fn). We investigated whether the peptidoglycan hydrolase of C. perfringens, i.e., autolysin (Acp), is implicated in Fn binding to C. perfringens cells. METHODS: This study used recombinant Acp fragments, human Fn and knockout mutants (C. perfringens 13 acp::erm and HN13 ΔfbpC ΔfbpD). Ligand blotting, Western blotting analysis, and complementation tests were performed. The Fn-binding activity of each mutant was evaluated by ELISA. RESULTS: From an Fn-binding assay using recombinant Acp fragments, Fn was found to bind to the catalytic domain of Acp. In mutant cells lacking Acp, Fn binding was significantly decreased, but was restored by the complementation of the acp gene. There are three known kinds of Fn-binding proteins in C. perfringens: FbpC, FbpD, and glyceraldehyde-3-phosphate dehydrogenase. We found no difference in Fn-binding activity between the mutant cells lacking both FbpC and FbpD (SAK3 cells) and the wild-type cells, indicating that these Fn-binding proteins are not involved in Fn binding to C. perfringens cells. CONCLUSIONS: We found that the Acp is an Fn-binding protein that acts as an Fn receptor on the surface of C. perfringens cells.

Development of Novel PCR Assays for Improved Detection of Enterovirus D68.

Enterovirus D68 (EV-D68) causes a range of clinical manifestations, including asthma-like illness, severe respiratory disease, and acute flaccid myelitis. EV-D68 has caused worldwide outbreaks since 2014 and is now recognized as a reemerging infection in many countries. EV-D68-specific PCR assays are widely used for the diagnosis of EV-D68 infection; however, assay sensitivity is a concern because of genetic changes in recently circulated EV-D68. To address this, we summarized EV-D68 sequences from previously reported world outbreaks from 2014 through 2020 on GenBank, and found several mutations at the primer and probe binding sites of the existing EV-D68-specific PCR assays. Subsequently, we designed two novel assays corresponding to the recently reported EV-D68 sequences: an EV-D68-specific real-time and seminested PCR. In an analysis of 22 EV-D68 confirmed cases during a recent EV-D68 outbreak in Japan, the new real-time PCR had higher sensitivity than the existing assay (100% versus 45%, P < 0.01) and a lower median CT value (27.8 versus 32.8, P = 0.005). Sensitivity was higher for the new nonnested PCR (91%) than for the existing seminested PCR assay (50%, P < 0.01). The specificity of the new real-time PCR was 100% using samples from non-EV-D68-infected cases (n = 135). In conclusion, our novel assays had higher sensitivity than the existing assay and might lead to more accurate diagnosis of recently circulating EV-D68. To prepare for future EV-D68 outbreaks, EV-D68-specific assays must be continuously monitored and updated.

Multipoint Lock-and-Key Assembly of Particles with Anisotropic Dents toward Modeling Rigid Macromolecules in a Colloidal Scale.

Multipoint lock-and-key particle assembly, consisting of lock particles with multiple anisotropic dents and rod-shaped particles as key particles, is developed for colloidal modeling application. The lock particles were connected with each other at a key particle as their joint in the presence of depletants, forming rigid colloidal molecules imitating rigid polymers (e.g., polymers containing aromatic rings and intramolecular hydrogen bonds). A single-particle level observation was conducted to visualize the colloidal polymerization of the particle assembly. Motion trajectories of the lock particles observed by optical microscopy indicated that the particle diffusivity was dramatically lowered when the lock particle connected with another one, suggesting that particle diffusion was suppressed by particle assembly formation. Because the kinetic and regioselectivity of colloidal polymerization are assumed to be analogous to those at the atomic scale, the proposed lock-and-key assembly can be a promising colloidal model for atomic-scale polymers associated with their micro-Brownian motion.

Temporal coding of insulin action through multiplexing of the AKT pathway.

One of the unique characteristics of cellular signaling pathways is that a common signaling pathway can selectively regulate multiple cellular functions of a hormone; however, this selective downstream control through a common signaling pathway is poorly understood. Here we show that the insulin-dependent AKT pathway uses temporal patterns multiplexing for selective regulation of downstream molecules. Pulse and sustained insulin stimulations were simultaneously encoded into transient and sustained AKT phosphorylation, respectively. The downstream molecules, including ribosomal protein S6 kinase (S6K), glucose-6-phosphatase (G6Pase), and glycogen synthase kinase-3ß (GSK3ß) selectively decoded transient, sustained, and both transient and sustained AKT phosphorylation, respectively. Selective downstream decoding is mediated by the molecules' network structures and kinetics. Our results demonstrate that the AKT pathway can multiplex distinct patterns of blood insulin, such as pulse-like additional and sustained-like basal secretions, and the downstream molecules selectively decode secretion patterns of insulin.

Expression of glyceraldehyde-3-phosphate dehydrogenase on the surface of Clostridium perfringens cells.

During research to identify fibronectin (Fn)-binding proteins (Fbps) on the surface of Clostridium perfringens cells, we identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a candidate Fbp. GAPDH is a glycolytic enzyme found in a wide range of prokaryotes and eukaryotes. The Fn-binding activity of recombinant C. perfringens GAPDH (rGAPDH) was investigated using both ligand blotting analysis and enzyme-linked immunosorbent assay (ELISA). rGAPDH strongly bound plasminogen but not laminin or gelatin. Although GAPDH has no signal sequence, it is expressed on the cell surface of many microorganisms. The presence of GAPDH on the surface of C. perfringens cells was analyzed using ELISA and flow cytometry analyses; purified rGAPDH bound to the surface of C. perfringens cells. As autolysin is reportedly involved in the binding of GAPDH to the cell surface, we evaluated the interaction between rGAPDH and the C. perfringens autolysin Acp by both ELISA and ligand blotting assay. These assays revealed that rGAPDH binds to the catalytic domain of Acp but not the cell wall binding domains. These results suggest that autolysin mediates expression of GAPDH on the surface of C. perfringens cells and indicate a possible moonlighting function for GAPDH in binding both Fn and plasminogen.

Yolk/Shell Colloidal Crystals Incorporating Movable Cores with Their Motion Controlled by an External Electric Field.

Yolk/shell particles composed of a submicrometer-sized movable core and a silica shell are promising building blocks for novel optical colloidal crystals, because the locations of cores in the shell compartment can be reversibly changed by using external stimuli. Two dimensional arrays of yolk/shell particles incorporating movable cores were prepared by a self-assembly method. The movable cores of colloidal crystals in water could be observed with an optical microscope under application of external electric field. The motions of inner silica cores depended on the electric field strength and frequency and were categorized into three cases: (1) Random Brownian motion, (2) anisotropic motion of cores moving in a direction orthogonal to a field, and (3) suppressed motion fixed in the center of shell compartment. Random Brownian motion of cores was scarcely affected by field strength when a high frequency (in the MHz range) electric field was applied. On the other hand, an increase in field strength at low-frequency fields (kHz) transiently changed the core motion from (1) to (2) and a further increase in field strength changed it from (2) to (3). When the silica core was incorporated in a large void a stronger electric field was needed to suppress its motion than when it was in a small void. The high responsivity to electric fields in a low-frequency range indicated the importance of electric double layer (EDL) interaction between core and inner shell in controlling the core location in yolk/shell colloidal crystals. It was also shown that movable titania cores in yolk/shell particles required a low-frequency field with a high strength to change from the random to anisotropic motion. The result suggested that the electrostatic interaction between EDLs of the silica core and the inner silica wall could be stronger than that between EDLs of the titania core and the silica shell.

Role of Maternal Antibodies in Infants with Severe Diseases Related to Human Parechovirus Type 3.

Human parechovirus type 3 (HPeV3) is an emerging pathogen that causes sepsis and meningoencephalitis in young infants. To test the hypothesis that maternal antibodies can protect this population, we measured neutralizing antibody titers (NATs) to HPeV3 and other genotypes (HPeV1 and HPeV6) in 175 cord blood samples in Japan. The seropositivity rate (≥1:32) for HPeV3 was 61%, similar to that for the other genotypes, but decreased significantly as maternal age increased (p<0.001). Furthermore, during the 2014 HPeV3 epidemic, prospective measurement of NATs to HPeV3 in 45 patients with severe diseases caused by HPeV3 infection showed low NATs (≤1:16) at onset and persistently high NATs (≥1:512) until age 6 months. All intravenous immunoglobulin samples tested elicited high NATs to HPeV3. Our findings indicate that maternal antibodies to HPeV3 may help protect young infants from severe diseases related to HPeV3 and that antibody supplementation may benefit these patients.

Polyamine Oxidase5 Regulates Arabidopsis Growth through Thermospermine Oxidase Activity.

The major plant polyamines (PAs) are the tetraamines spermine (Spm) and thermospermine (T-Spm), the triamine spermidine, and the diamine putrescine. PA homeostasis is governed by the balance between biosynthesis and catabolism; the latter is catalyzed by polyamine oxidase (PAO). Arabidopsis (Arabidopsis thaliana) has five PAO genes, AtPAO1 to AtPAO5, and all encoded proteins have been biochemically characterized. All AtPAO enzymes function in the back-conversion of tetraamine to triamine and/or triamine to diamine, albeit with different PA specificities. Here, we demonstrate that AtPAO5 loss-of-function mutants (pao5) contain 2-fold higher T-Spm levels and exhibit delayed transition from vegetative to reproductive growth compared with that of wild-type plants. Although the wild type and pao5 are indistinguishable at the early seedling stage, externally supplied low-dose T-Spm, but not other PAs, inhibits aerial growth of pao5 mutants in a dose-dependent manner. Introduction of wild-type AtPAO5 into pao5 mutants rescues growth and reduces the T-Spm content, demonstrating that AtPAO5 is a T-Spm oxidase. Recombinant AtPAO5 catalyzes the conversion of T-Spm and Spm to triamine spermidine in vitro. AtPAO5 specificity for T-Spm in planta may be explained by coexpression with T-Spm synthase but not with Spm synthase. The pao5 mutant lacking T-Spm oxidation and the acl5 mutant lacking T-Spm synthesis both exhibit growth defects. This study indicates a crucial role for T-Spm in plant growth and development.

Rattle-type colloidal crystals composed of spherical hollow particles containing an anisotropic, movable core.

Controls over the position and orientation of anisotropic particles in their assemblies are intriguing issues for functional colloidal crystals that are switchable with external fields such as electric and magnetic fields. We propose a novel approach for the fabrication of rattle-type colloidal crystals containing an anisotropic, movable core surrounded by a void space that allows rearrangement of the anisotropic core in the assembly. In the fabrication, multilayered core-shell particles composed of a titania core, polystyrene shell, and silica shell were prepared and then heated at 500 °C for 4 h to selectively remove the middle layer of polystyrene. The heating treatment induced deformation of spherical titania cores in the compartment of silica shells, while the void space required for the orientation and relocation of anisotropic core was generated. The rattle particles fabricated were self-assembled by a simple dip-coating to form an arrangement of the spherical yolk/shell particles incorporating an anisotropic core. Brownian motion of the anisotropic cores observed with an optical microscope showed that the assembly of rattle-type particles had the potential to control location and orientation of the anisotropic cores in the shell compartment by application of external fields.

Magnetic field aligned assembly of nonmagnetic composite dumbbells in nanoparticle-based aqueous ferrofluid.

Monodisperse, nonmagnetic, asymmetrical composite dumbbells in a suspension of magnetic nanoparticles (ferrofluid) were aligned by application of an external magnetic field to the ferrofluid. The asymmetrical composite dumbbells were prepared by two-step soap-free emulsion polymerization consisting of the first polymerization to coat spherical silica cores with cross-linked poly(methyl methacrylate) (PMMA) shell and the second polymerization to protrude a polystyrene (PSt) lobe from the core-shell particles. A chain structure of nonmagnetic dumbbells oriented to the applied magnetic field was observed at nanoparticle content of 2.0 vol % and field strengths higher than 1.0 mT. A similar chain structure of the dumbbells was observed under application of alternating electric field at strengths higher than 50 V/mm. Parallel and orthogonally combined applications of the electric and magnetic fields were also conducted to examine independence of the electric and magnetic applications as operational factors in the dumbbell assembling. Dumbbell chains stiffer than those in a single application of external field were formed in the parallel combined application of electric and magnetic fields. The orthogonal combination of the different applied fields could form a magnetically aligned chain structure of the nonmagnetic dumbbells oriented to the electric field. The present work experimentally indicated that the employment of inverse magnetorheological effect for nonmagnetic, anisotropic particles can be a useful method for the simultaneous controls over the orientation and the positon of anisotropic particles in their assembling.

Latent process genes for cell differentiation are common decoders of neurite extension length.

A latent process involving signal transduction and gene expression is needed as a preparation step for cellular function. We previously found that nerve growth factor (NGF)-induced cell differentiation has a latent process, which is dependent on ERK activity and gene expression and required for subsequent neurite extension. A latent process can be considered as a preparation step that decodes extracellular stimulus information into cellular functions; however, molecular mechanisms of this process remain unknown. We identified Metrnl, Dclk1 and Serpinb1a as genes that are induced during the latent process (LP) with distinct temporal expression profiles and are required for subsequent neurite extension in PC12 cells. The LP genes showed distinct dependency on the duration of ERK activity, and they were also induced during the latent process of PACAP- and forskolin-induced cell differentiation. Regardless of neurotrophic factors, expression levels of the LP genes during the latent process (0-12 hours), but not phosphorylation levels of ERK, always correlated with subsequent neurite extension length (12-24 hours). Overexpression of all LP genes together, but not of each gene separately, enhanced NGF-induced neurite extension. The LP gene products showed distinct spatial localization. Thus, the LP genes appear to be the common decoders for neurite extension length regardless of neurotrophic factors, and they might function in distinct temporal and spatial manners during the latent process. Our findings provide molecular insight into the physiological meaning of the latent process as the preparation step for decoding information for future phenotypic change.

MALDI-Q-TOF mass spectrometric determination of gold and platinum in tissues using their diethyldithiocarbamate chelate complexes.

A rapid determination method is presented for gold (Au(3+)) and platinum (Pt(4+)) in tissues using matrix-assisted laser desorption ionization quadrupole time-of-flight mass spectrometry (MALDI-Q-TOF-MS). Au and Pt ions in wet-ashed tissue solution were reacted with diethyldithiocarbamate (DDC), and the resulting chelate complex ions Au(DDC)2 (+) and Pt(DDC)3 (+) were detected by MALDI-Q-TOF-MS using α-cyano-4-hydroxycinnamic acid as a matrix. The limit of detection (LOD) was 0.8 ng/g tissue and the quantification range was 2-400 ng/g for Au, and the LOD was 6 ng/g tissue and the quantification range was 20-4,000 ng/g for Pt. The Pt levels detected by MALDI-Q-TOF-MS in several tissues of a patient overdosed with cisplatin were nearly the same as those detected by flow-injection electrospray ionization mass spectrometry. The LODs of Au and Pt were 0.04 pg per well (sample spot) and 0.3 pg per well, respectively. To our knowledge, this is the first attempt to quantify Au(3+) and Pt(4+) ions in tissues by MALDI-Q-TOF-MS.

Differential Reactivation of Cytomegalovirus and Epstein-Barr Virus in Patients with B Cell Lymphoma.

Although cytomegalovirus (CMV) and Epstein-Barr virus (EBV) are considered latent viruses, their reactivation occurs in immunosuppressed conditions. We previously reported that CMV and EBV are reactivated in patients receiving immunosuppressive therapy and/or chemotherapy. This retrospective, single-center study aimed to determine the frequency of viral reactivation and clinical characteristics of patients with B cell lymphoma (B-ML) receiving chemotherapy. Twenty-four patients (mean age 73 years, range 40-87 years; male-to-female ratio, 15:9) with diffuse large B cell lymphoma (n = 15), follicular lymphoma (n = 8), or mantle cell lymphoma (n = 1) were enrolled. Serum CMV and EBV DNA levels were analyzed using quantitative real-time polymerase chain reaction in patients with B-ML receiving chemotherapy. We determined the cumulative reactivation of each virus and analyzed the relationship between viral reactivation and clinical characteristics. Three patients experienced relapse or refractory (R/R) disease and the others had de novo lymphomas. The frequencies of CMV and EBV reactivations were 54.2% and 37.5%, respectively. CMV reactivation occurred significantly earlier during chemotherapy courses in R/R patients than in de novo patients (p = 0.0038), while EBV reactivation was frequently found before treatment. Baseline serum levels of soluble interleukin-2 receptor were higher (4318.0 vs. 981.1 U/mL, p = 0.010) and hemoglobin levels were lower (11.1 vs. 13.0 g/dL, p = 0.0038) in patients with EBV reactivation than in those without reactivation. These findings were not observed in patients with CMV reactivation. CMV reactivation was associated with iatrogenic immunosuppression, whereas EBV reactivation was related to immunosuppression by lymphoma, indicating that the mechanisms of these viral reactivations differed.

Surface lattice resonance in three-dimensional plasmonic arrays fabricated via self-assembly of silica-coated gold nanoparticles.

HYPOTHESIS: Three-dimensional plasmonic nanoparticle arrays in which the nanoparticles are assembled with a certain distance apart are expected to exhibit unique optical properties attributed to surface lattice resonances because of the interactions between the nanoparticle layers. EXPERIMENTS: Multi-layered gold nanoparticle arrays were created to experimentally prove surface lattice resonances from three-dimensional arrays. Silica-coated gold nanoparticles were employed as building blocks for the array because the distance between the nanoparticles can be tuned by adjusting the thickness of the silica coating. Employing highly monodisperse building blocks enabled to fabricate both single-layered and multi-layered plasmonic arrays via a confined convective assembly method. FINDINGS: Multi-layering of monodisperse building blocks brought about some additional peaks corresponded to Bragg diffraction of gold nanoparticle periodic array and the interactions between layers in a hexagonal close-packed structure of the nanoparticles, respectively. Most importantly, the multi-layered arrays exhibited a distinctive extinction peak at the same wavelength as that observed from the single-layered array, proving the realization of surface lattice resonances from the three-dimensional plasmonic array.

Quantification of olanzapine and its three metabolites by liquid chromatography-tandem mass spectrometry in human body fluids obtained from four deceased, and confirmation of the reduction from olanzapine N-oxide to olanzapine in whole blood in vitro.

PURPOSE: Quantification of olanzapine (OLZ) and its metabolites such as N-desmethylolanzapine (DM-O), 2-hydroxymethylolanzapine (2H-O) and olanzapine N-oxide (NO-O) in five kinds of human body fluids including whole blood by liquid chromatography (LC)-tandem mass spectrometry (MS/MS) has been presented; the quantification methods were carefully devised and validated using the matrix-matched calibration and standard addition methods. METHODS: OLZ and its three metabolites were extracted from 40 µL each of body fluids by two-step liquid-liquid separations. The samples and reagents were pre-cooled in a container filled with ice for the extraction because of the thermal instability of OLZ and its three metabolites especially in whole blood. RESULTS: The limits of quantification (LOQs) of OLZ and 2H-O were 0.05 ng/mL and those of DM-O and NO-O were 0.15 ng/mL in whole blood and urine, respectively. The concentrations of OLZ and its metabolites in heart whole blood, pericardial fluid, stomach contents, bile and urine were determined for two cadavers and those in whole blood and urine for the other two cadavers. The reduction from NO-O to OLZ was observed at 25 â„ƒ in whole blood in vitro. CONCLUSIONS: To our knowledge, this is the first report on the quantification of metabolites of olanzapine in the authentic human body fluids by LC-MS/MS as well as on the confirmation of in vitro reduction from NO-O to OLZ in whole blood that seems to have induced the quick decrease of NO-O.

Myeloid-associated differentiation marker is an essential host factor for human parechovirus PeV-A3 entry.

Human parechovirus (PeV-A) is an RNA virus that belongs to the family Picornaviridae and it is currently classified into 19 genotypes. PeV-As usually cause mild illness in children and adults. Among the genotypes, PeV-A3 can cause severe diseases in neonates and young infants, resulting in neurological sequelae and death. In this study, we identify the human myeloid-associated differentiation marker (MYADM) as an essential host factor for the entry of six PeV-As (PeV-A1 to PeV-A6), including PeV-A3. The infection of six PeV-As (PeV-A1 to PeV-A6) to human cells is abolished by knocking out the expression of MYADM. Hamster BHK-21 cells are resistant to PeV-A infection, but the expression of human MYADM in BHK-21 confers PeV-A infection and viral production. Furthermore, VP0 capsid protein of PeV-A3 interacts with one extracellular domain of human MYADM on the cell membrane of BHK-21. The identification of MYADM as an essential entry factor for PeV-As infection is expected to advance our understanding of the pathogenesis of PeV-As.

Constitutively and highly expressed Oryza sativa polyamine oxidases localize in peroxisomes and catalyze polyamine back conversion.

Polyamine oxidases (PAOs) are FAD-dependent enzymes involved in polyamine (PA) catabolism. Recent studies have revealed that plant PAOs are not only active in the terminal catabolism of PAs as demonstrated for maize apoplastic PAO but also in a polyamine back-conversion pathway as shown for most Arabidopsis PAOs. We have characterized Oryza sativa PAOs at molecular and biochemical levels. The rice genome contains 7 PAO isoforms that are termed OsPAO1 to OsPAO7. Of the seven PAOs, OsPAO3, OsPAO4, and OsPAO5 transcripts were most abundant in 2-week-old seedlings and mature plants, while OsPAO1, OsPAO2, OsPAO6, and OsPAO7 were expressed at very low levels with different tissue specificities. The more abundantly expressed PAOs--OsPAO3, OsPAO4, and OsPAO5--were cloned, and their gene products were produced in Escherichia coli. The enzymatic activities of the purified OsPAO3 to OsPAO5 proteins were examined. OsPAO3 favored spermidine (Spd) as substrate followed by thermospermine (T-Spm) and spermine (Spm) and showed a full PA back-conversion activity. OsPAO4 substrate specificity was similar to that of OsPAO5 preferring Spm and T-Spm but not Spd. Those enzymes also converted Spm and T-Spm to Spd, again indicative of PA back-conversion activities. Lastly, we show that OsPAO3, OsPAO4, and OsPAO5 are localized in peroxisomes. Together, these data revealed that constitutively and highly expressed O. sativa PAOs are localized in peroxisomes and catalyze PA back-conversion processes.