Advanced Analysis to Distinguish between Physical Decrease and Inactivation of Viable Phages in Aerosol by Quantitating Phage-Specific Particles.

　Recent studies have investigated the efficacy of air-cleaning products against pathogens in the air. A standard method to evaluate the reduction in airborne viruses caused by an air cleaner has been established using a safe bacteriophage instead of pathogenic viruses; the reduction in airborne viruses is determined by counting the number of viable airborne phages by culture, after operating the air cleaner. The reduction in the number of viable airborne phages could be because of "physical decrease" or "inactivation". Therefore, to understand the mechanism of reduction correctly, an analysis is required to distinguish between physical decrease and inactivation. The purpose of this study was to design an analysis to distinguish between the physical decrease and inactivation of viable phi-X174 phages in aerosols. We established a suitable polymerase chain reaction (PCR) system by selecting an appropriate primer-probe set for PCR and validating the sensitivity, linearity, and specificity of the primer-probe set to robustly quantify phi-X174-specific airborne particles. Using this quantitative PCR system and culture assay, we performed a behavior analysis of the phage aerosol in a small chamber (1 m3) at different levels of humidity, as humidity is known to affect the number of viable airborne phages. The results revealed that the reduction in the number of viable airborne phages was caused not only by physical decrease but also by inactivation under particular levels of humidity. Our study could provide an advanced analysis to differentiate between the physical decrease and inactivation of viable airborne phages.

A Novel Method of Safely Measuring Influenza Virus Aerosol Using Antigen-Capture Enzyme-Linked Immunosorbent Assay for the Performance Evaluation of Protective Clothing Materials.

Currently, threats caused by pathogens are serious public health problems worldwide. Protective clothing is essential when one is treating infected patients or dealing with unknown pathogens. Therefore, it is necessary to evaluate the performance of protective clothing against pathogens. In Japan, some methods for evaluating the performance of protective clothing have been established in the Japanese Industrial Standards (JIS). However, a test method against virus aerosols has not been established. Because there is a risk of infection from a live virus during the test, it is necessary to devise a safe method for the virus-aerosol-based test. Here, we propose a new method of safely measuring virus aerosols for the performance evaluation of protective clothing materials. To ensure safety, an inactivated virus was used. As a model virus, the influenza virus was selected owing to the proper small diameter of the virus particles. To quantitatively measure the particle-amount of the inactivated influenza virus, we developed an antigen-capture enzyme-linked immunosorbent assay (ELISA) targeting the M1 protein. Furthermore, we evaluated two materials using our method. Significant differences in the protection performance against the virus aerosol were observed between different sample materials, thereby confirming the applicability of our new method for performance evaluation.

Relationship between Virucidal Efficacy and Free Iodine Concentration of Povidone-Iodine in Buffer Solution.

Povidone-iodine solutions prepared to various concentrations (0.01, 0.1, 1 and 10%) with 0.2M phosphate buffer (pH 7.0) (PVP-I PB) were analyzed to determine their free iodine concentrations using membrane permeation cells, and their inactivation effects on three viruses (influenza A virus, poliovirus type 1 and adenovirus type 3) were examined. The free iodine concentrations in the 0.01-10% PVP-I PB were determined to be 1.84, 4.88, 1.58 and 0.17 ppm (approximate values), respectively, with the maximum obtained for the 0.1% solution. The virucidal efficacy of these PVP-I PB against poliovirus type 1 and adenovirus type 3 was found to be generally dependent on free iodine concentration, with the 0.1% solution being the most effective. Influenza A virus was inactivated with an action time of 15 s at all four concentrations examined. The results of this study suggested an association between free iodine concentration and virucidal efficacy for the 0.01-10% PVP-I PB.

Influence of prior pandemic A(H1N1)2009 virus infection on invasion of MDCK cells by community-associated methicillin-resistant Staphylococcus aureus.

Secondary bacterial pneumonia due to community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has become a highly publicized cause of death associated with influenza. In this study, we performed the gentamicin-killing assay using Madin-Darby canine kidney (MDCK) cells and MRSA strains to investigate whether prior infection from pandemic A(H1N1)2009 virus (A[H1N1]pdm09) lead to increased invasion of MDCK cells by MRSA. We found that the invasion rate of two MRSA strains (ATCC BAA-1680 [USA 300] and ATCC BAA-1699 [USA 100]) into intact MDCK cell monolayers was 0.29 ± 0.15% and 0.007 ± 0.002%, respectively (p < 0.01, n ≥ 3). In addition, the relative invasion rate of both ATCC BAA-1680 and ATCC BAA-1699 was significantly increased by prior A(H1N1)pdm09 infection of MDCK monolayers from 1 ± 0.28 to 1.38 ± 0.02 and from 1 ± 0.24 to 1.73 ± 0.29, respectively (p < 0.01). These results indicate that ATCC BAA-1680 displays much stronger invasiveness of MDCK cells than ATCC BAA-1699, although invasion of both strains was increased by prior A(H1N1)pdm09 infection. In conclusion, this study provided the first evidence that prior A(H1N1)pdm09 infection facilitates the invasion of MDCK cells by MRSA, presumably due to cellular injury caused by the virus.

Islet1 selectively promotes peripheral axon outgrowth in Rohon-Beard primary sensory neurons.

We isolated a novel zebrafish mutant, lullaby (llb), and showed that the llb locus encodes the zebrafish orthologue of isl1. Rohon-Beard (RB) primary sensory neurons are multipolar neurons that extend their central axons longitudinally within the spinal cord and also extend their peripheral axons under the skin. In llb embryos, the outgrowth of the peripheral axons of RB neurons was selectively impaired, which correlated with down-regulation of the expression of dihydropyrimidinase-like 3 (dpysl3, also known as collapsin response mediator protein 4, crmp4). Antisense morpholino oligonucleotide (AMO)-mediated knockdown of dpysl3 inhibited the outgrowth of the peripheral axons of RB neurons, and semaphorin 3d (sema3d) AMO enhanced this effect. These data indicate that Dpysl3 is cooperating with Sema3d in the peripheral axon outgrowth, and Isl1 is required for the selective outgrowth of the peripheral axons of RB neurons by maintaining the expression of dpysl3.

Lactoferrin conjugated with 40-kDa branched poly(ethylene glycol) has an improved circulating half-life.

PURPOSE: We developed a lactoferrin conjugate by modifying bovine lactoferrin (bLF) with a 40-kDa branched poly(ethylene glycol) (PEG) molecule (designated 40 k-PEG-bLf), and we evaluated its in vitro activities and pharmacokinetic properties. MATERIALS AND METHODS: We prepared 40k-PEG-bLf by amino conjugation with N-hydroxysuccinimide-activated PEG. This conjugate was purified by cation exchange chromatography and its in vitro biological activities, such as iron binding, anti-inflammatory effects, and resistance to proteolytic enzymes were investigated. In vivo pharmacokinetics analyses, were also performed to examine the rate of clearance from the plasma in rats. RESULTS: The 40k-PEG-bLf conjugate was fully active in iron binding and exhibited 97.1 +/- 5.5% (mean +/- S.E., n = 6) of the original anti-inflammatory activity. The in vitro peptic susceptibility of 40 k-PEG-bLf revealed that the proteolytic half-life increased at least 6-fold that of unmodified LF. This PEGylated conjugate demonstrated a plasma half-life that was 8.7-fold longer than that of the unmodified bLF in rats. CONCLUSIONS: The 40k-PEG-bLf exhibited improved in vitro bioactivity and stability and enhanced pharmacokinetic properties as compared to those of the unmodified bLF and the 20 k-PEG-bLf conjugate, which was recently developed by PEGylation of bLF with a 20-kDa branched PEG [Nojima Y. et al. Bioconjugate Chem. 19:2253-2259 (2008)].

The pH-dependent formation of PEGylated bovine lactoferrin by branched polyethylene glycol (PEG)-N-hydroxysuccinimide (NHS) active esters.

PEGylation is an established method for improving the pharmacokinetic properties and pharmacodynamic effects of therapeutic proteins. Amino conjugation by N-hydroxysuccinimide activated polyethylene glycol (PEG-NHS) represents the most common modification of the target proteins. In this study, we compared the reaction velocities of pH-dependent preparations of bovine lactoferrin modified with branched PEG-NHS conducted at pH 7.4 to pH 9.0. PEGylation reaction rates were dependent on pH value, but not on PEG molecular mass. At pH 7.4, reactions advanced gradually and reached steady state by 2 h, whereas at pH 9.0, reactions advanced very quickly and reached steady state within 10 min. Hydrolysis half-life of PEG-NHS exceeded 120 min at pH 7.4, but was below 9 min at pH 9.0. Thus, the pH value is one of the most important factors to obtain the optimal condition for PEGylation by NHS esters.

Mutation of cGMP phosphodiesterase 6alpha'-subunit gene causes progressive degeneration of cone photoreceptors in zebrafish.

In mammals, the blockade of the phototransduction cascade causes loss of vision and, in some cases, degeneration of photoreceptors. However, the molecular mechanisms that link phototransduction with photoreceptor degeneration remain to be elucidated. Here, we report that a mutation in the gene encoding a central effector of the phototransduction cascade, cGMP phosphodiesterase 6alpha'-subunit (PDE6alpha'), affects not only the vision but also the survival of cone photoreceptors in zebrafish. We isolated a zebrafish mutant, called eclipse (els), which shows no visual behavior such as optokinetic response (OKR). The cloning of the els mutant gene revealed that a missense mutation occurred in the pde6alpha' gene, resulting in a change in a conserved amino acid. The PDE6 expressed in rod photoreceptors is a heterotetramer comprising two closely related similar hydrolytic alpha and beta subunits and two identical inhibitory gamma subunits, while the PDE6 expressed in cone photoreceptors consists of two homodimers of alpha' subunits, each with gamma subunits. The els mutant displays no visual response to bright light, where cones are active, but shows relatively normal OKR to dim light, where only rods function, suggesting that only the cone-specific phototransduction pathway is disrupted in the els mutant. Furthermore, in the els mutant, cones are selectively eliminated but rods are retained at the adult stage, suggesting that cones undergo a progressive degeneration in the els mutant retinas. Taken together, these data suggest that PDE6alpha' activity is important for the survival of cones in zebrafish.

Development of poly(ethylene glycol) conjugated lactoferrin for oral administration.

Lactoferrin (LF) is an iron-binding glycoprotein that possesses multifunctional biological activities. Recent reports from clinical trials suggest that LF is potentially effective as a therapeutic protein against cancer and gangrene. However, pharmaceutical proteins such as LF are unstable in vivo. Therefore, to improve stability, we developed mono-PEGylated bovine LF (20k-PEG-bLf) with branched 20 kDa (2 x 10 kDa) poly(ethylene glycol) (PEG). We examined in vitro activities such as iron binding, IL-6 cell based assay, and resistance to a proteolytic enzyme in artificial gastric fluid. The 20k-PEG-bLf protein was fully active in iron binding and exhibited 69.6 +/- 2.9% (mean +/- S.E., n = 6) of the original anti-inflammatory activity. The proteolytic half-life increased 2-fold over that of unmodified LF. In vivo pharmacokinetic analyses were performed to examine absorption from the intestinal epithelium and serum clearance. Direct administration of 20k-PEG-bLf (30 mg/kg) into rat stomachs demonstrated that the amount of absorption from the intestinal tract increased approximately 10-fold relative to unmodified LF. Intravenous injection of the protein (1 mg/kg) revealed that 20k-PEG-bLf prolongs serum half-life by approximately 5.4-fold, and that the area under the curve (AUC) was increased approximately 9.2-fold compared to that of unmodified LF. PEGylation improved the physical and pharmacokinetic properties of bovine LF. This is the first report on the use of bioconjugation of LF for the development of a promising oral pharmaceutical agent.

Role of Bone morphogenetic protein 4 in zebrafish semicircular canal development.

Bone morphogenetic proteins (BMPs) are known to play roles in inner ear development of higher vertebrates. In zebrafish, there are several reports showing that members of the BMP family are expressed in the otic vesicle. We have isolated a novel zebrafish mutant gallery, which affects the development of the semicircular canal. Gallery merely forms the lateral and the immature anterior protrusion, and does not form posterior and ventral protrusions. We found that the expression of bmp2b and bmp4, both expressed in the normal optic vesicle at the protrusion stage, are extremely upregulated in the otic vesicle of gallery. To elucidate the role of BMPs in the development of the inner ear of zebrafish, we have applied excess BMP to the wild-type otic vesicle. The formation of protrusions was severely affected, and in some cases, they were completely lost in BMP4-treated embryos. Furthermore, the protrusions in gallery treated with Noggin were partially rescued. These data indicate that BMP4 plays an important role in the development of protrusions to form semicircular canals.

Histone deacetylase 1 regulates retinal neurogenesis in zebrafish by suppressing Wnt and Notch signaling pathways.

In the developing vertebrate retina, progenitor cells initially proliferate but begin to produce postmitotic neurons when neuronal differentiation occurs. However, the mechanism that determines whether retinal progenitor cells continue to proliferate or exit from the cell cycle and differentiate is largely unknown. Here, we report that histone deacetylase 1 (Hdac1) is required for the switch from proliferation to differentiation in the zebrafish retina. We isolated a zebrafish mutant, ascending and descending (add), in which retinal cells fail to differentiate into neurons and glial cells but instead continue to proliferate. The cloning of the add gene revealed that it encodes Hdac1. Furthermore, the ratio of the number of differentiating cells to that of proliferating cells increases in proportion to Hdac activity, suggesting that Hdac proteins regulate a crucial step of retinal neurogenesis in zebrafish. Canonical Wnt signaling promotes the proliferation of retinal cells in zebrafish, and Notch signaling inhibits neuronal differentiation through the activation of a neurogenic inhibitor, Hairy/Enhancer-of-split (Hes). We found that both the Wnt and Notch/Hes pathways are activated in the add mutant retina. The cell-cycle progression and the upregulation of Hes expression in the add mutant retina can be inhibited by the blockade of Wnt and Notch signaling, respectively. These data suggest that Hdac1 antagonizes these pathways to promote cell-cycle exit and the subsequent neurogenesis in zebrafish retina. Taken together, these data suggest that Hdac1 functions as a dual switch that suppresses both cell-cycle progression and inhibition of neurogenesis in the zebrafish retina.

Dual roles of zygotic and maternal Scribble1 in neural migration and convergent extension movements in zebrafish embryos.

In the developing vertebrate hindbrain, the characteristic trajectory of the facial (nVII) motor nerve is generated by caudal migration of the nVII motor neurons. The nVII motor neurons originate in rhombomere (r) 4, and migrate caudally into r6 to form the facial motor nucleus. In this study, using a transgenic zebrafish line that expresses green fluorescent protein (GFP) in the cranial motor neurons, we isolated two novel mutants, designated landlocked (llk) and off-road (ord), which both show highly specific defects in the caudal migration of the nVII motor neurons. We show that the landlocked locus contains the gene scribble1 (scrb1), and that its zygotic expression is required for migration of the nVII motor neurons mainly in a non cell-autonomous manner. Taking advantage of the viability of the llk mutant embryos, we found that maternal expression of scrb1 is required for convergent extension (CE) movements during gastrulation. Furthermore, we show a genetic interaction between scrb1 and trilobite(tri)/strabismus(stbm) in CE. The dual roles of the scrb1 gene in both neuronal migration and CE provide a novel insight into the underlying mechanisms of cell movement in vertebrate development.

The zebrafish-secreted matrix protein you/scube2 is implicated in long-range regulation of hedgehog signaling.

The Hedgehog (Hh) signal plays a pivotal role in induction of ventral neuronal and muscle cell types around the midline during vertebrate development [1]. We report that the gene disrupted in zebrafish you mutants, in which Hh signaling is impaired, encodes the secreted matrix protein Scube2. Consistently, epistasis analyses suggested that Scube2 functions upstream of Hh ligands or through a parallel pathway. In addition, overexpression analyses suggested that Scube2 is an essential, but a permissive, mediator of Hh signaling in zebrafish embryos. Surprisingly, the you gene is expressed in the dorsal neural tube, raising the possibility that Scube2 could indirectly act via a long-range regulator of Hh signaling. The dorsal Bmps have a long-range and opposing influence on Hh signaling [2-5]. We show that neural plate patterning is affected in you mutants in a way that is consistent with the aberrant long-range action of a Bmp-dependent signal. We further show that Bmp activity can be attenuated by the coexpression of Scube2. Our data support the idea that Scube2 can modulate the long-range action of Bmp-dependent signaling in the neural tube and somites.

N-cadherin mediates retinal lamination, maintenance of forebrain compartments and patterning of retinal neurites.

The complex, yet highly ordered and predictable, structure of the neural retina is one of the most conserved features of the vertebrate central nervous system. In all vertebrate classes, retinal neurons are organized into laminae with each neuronal class adopting specific morphologies and patterns of connectivity. Using genetic analyses in zebrafish, we demonstrate that N-cadherin (Ncad) has several distinct and crucial functions during the establishment of retinal organization. Although the location of cell division is disorganized in embryos with reduced or no Ncad function, different classes of retinal neurons are generated. However, these neurons fail to organize into correct laminae, most probably owing to compromised adhesion between retinal cells. In addition, amacrine cells exhibit exuberant and misdirected outgrowth of neurites that contributes to severe disorganization of the inner plexiform layer. Retinal ganglion cells also exhibit defects in process outgrowth, with axons exhibiting fasciculation defects and adopting incorrect ipsilateral trajectories. At least some of these defects are likely to be due to a failure to maintain compartment boundaries between eye, optic nerve and brain. Although in vitro studies have implicated Fgf receptors in modulating the axon outgrowth promoting properties of Ncad, most aspects of the Ncad mutant phenotype are not phenocopied by treatments that block Fgf receptor function.

A new assay system for guinea pig interferon biological activity.

We have developed an assay system for guinea pig interferon (IFN) based on reduction of viral cytopathic effect (CPE) in various cell lines. CPE inhibition was detected optimally in the guinea pig fibroblast cell line 104C1 infected with encephalomyocarditis virus (EMCV). The amount of biologically active guinea pig IFN was quantified by estimating viable cell numbers colorimetrically by means of a tetrazolium compound, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-1) and 1-methoxy-5-methylphenazinium methylsulfate (PMS). WST-1 color developed until stopped by the addition of sulfuric acid. This had no effect on the colorimetric assay, and the color was stable for at least 24 h. The acid also inactivated the EMCV and, thus, eliminated the viral hazard. Inhibition of CPE activity was highly correlated with the concentration of culture supernatants from BCG-vaccinated guinea pig splenocytes stimulated in vitro with tuberculin or an immunostimulatory oligoDNA. This assay detected guinea pig IFN and human IFN-alpha, but not IFN-gamma from human, mouse, rat, pig, or dog. This assay system has proved useful for the titration of guinea pig IFN, being easy to perform, free from viral hazard, relatively species specific, highly reproducible, and inexpensive.

Discovery of immunostimulatory CpG-DNA and its application to tuberculosis vaccine development.

DNA containing an unmethylated CpG motif has a potent immunostimulatory effect on the vertebrate immune system. Because such CpG motifs are relatively common in bacterial DNA, but rare in mammalian animal and plant DNA, they may be an evolutionary adaptation augmenting innate immunity, most likely in response to pathogens that replicate within the host cells, such as viruses and intracellular bacteria. Microbial infection induces innate immunity by triggering pattern-recognition systems. The infected cells produce proinflammatory cytokines that directly combat microbial invaders and express costimulating surface molecules, which develop adaptive immunity by inducing distinct T cell differentiation. Bacterial DNA with unmethylated CpG-DNA stimulates vertebrate immature immune cells to induce maturation and to produce TNF-alpha as well as Th1-type cytokines, IL-12 and IFN-gamma. Therefore, CpG-DNA functions as an adjuvant for regulating the initiation of Th1 differentiation. The roles of immunostimulatory CpG motifs in DNA vaccine developments and in therapeutic applications have been discussed.

Isolation and characterization of triacylglycerol-secreting mutant strain from yeast, Saccharomyces cerevisiae.

To establish the molecular bases for development of a microbiological system approaching excretive fermentation of useful lipids, a mutant strain that accumulates lipids in the medium was isolated from the laboratory yeast Saccharomyces cerevisiae. Following the mutagenesis to strain YP1, a long chain fatty acid utilizer with ethylmethane sulfonate, the mutant strain, STG1, was selected from about 80,000 colonies. The analysis of extracellular lipids and the monitoring of leakage of intracellular proteins indicated that strain STG1 secreted lipids containing triacylglycerols into the extracellular space without cell lysis. Genetic studies clarified that this mutation was recessive and was complemented by wild-type genomic DNA fragments. STG1 was considered to be a good tool for elucidation of the molecular mechanism for transmembrane lipid transport.