Inactivation mechanism of SARS-CoV-2 by ozone in aqueous and gas phases.

The comparison of the inactivation rate of SARS-CoV-2 by ozone in water with that in gas, based on data from references and experiments, has indicated the inactivation rate of the former is remarkably higher than that of the latter. To investigate the reason for this difference, we analyzed the reaction rate using a diffusional reaction model, in which ozone is carried by micro spherical viruses to inactivate the target viruses. Using this model, we can evaluate the amount of ozone required to inactivate a virus based on the ct value. We found that inactivation in gas phase requires 1014-1015 ozone molecules per virus virion, while the inactivation in aqueous phase requires 5×1010 to 5×1011 ozone molecules. This implies that the efficiency in gas phase is 200-20,000 times lower than that in aqueous phase. This is not attributed to the lower probability of collision in gas phase than in aqueous phase. Rather, it may be due to the fact that the ozone and radicals generated by ozone react and subsequently dissipate. We proposed the diffusion of ozone into a spherical virus at a steady state and the decomposition reaction model through radicals.

Effect of Theaflavin on Oral Bacteria in Japanese Subjects: A Randomized, Placebo-Controlled, Double-Blind Study.

Black tea is a popular beverage worldwide. Theaflavins (TFs), which are active functional components of black tea, are potentially valuable for preventing and/or treating the progression of periodontal diseases. Our previous pilot study showed that TF intake decreases the number of Porphyromonas gingivalis (P. gingivalis) bacteria in the saliva. In this study, we aimed to determine whether TF intake improves periodontal disease attributed to oral bacteria in a randomized, placebo-controlled, and double-blind study. A total of 56 healthy subjects without periodontal diseases were enrolled and assigned to the placebo and TF groups (n = 28). TF intake for 6 weeks did not significantly alter the clinical evaluation of subjects. There was no significant adverse effect among the subjects. The number of P. gingivalis and Fusobacterium nucleatum (F. nucleatum) bacteria, which was the primary endpoint in this study, was not impacted by TF intake. The change ratio of Prevotella intermedia was significantly decreased by TF intake (P = .043) when compared with the placebo group. Collectively, our findings suggest that TFs have beneficial effects on oral bacteria for the prevention of periodontal disease. The study protocol was registered in the University Hospital Medical Information Network (UMIN000020049).

RXR partial agonist produced by side chain repositioning of alkoxy RXR full agonist retains antitype 2 diabetes activity without the adverse effects.

We previously reported RXR partial agonist CBt-PMN (1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)-1H-benzotriazole-5-carboxylic acid: 5, EC50 = 143 nM, Emax = 75%), which showed a potent glucose-lowering effect without causing serious adverse effects. However, it remains important to elucidate the structural requirements for RXR efficacy and the glucose-lowering effect because RXR-permissive heterodimers such as PPAR/RXR or LXR/RXR are reported to be activated differently depending upon the chemical structure of RXR agonists. In this work, we show that an RXR partial agonist, NEt-4IB (6-[ethyl-(4-isobutoxy-3-isopropylphenyl)amino]pyridine-3-carboxylic acid: 8b, EC50 = 169 nM, Emax = 55%), can be obtained simply by repositioning the side chains (interchanging the isobutoxy and isopropoxy groups) at the hydrophobic moiety of the RXR full agonist NEt-3IB (6-[ethyl-(3-isobutoxy-4-isopropylphenyl)amino]pyridine-3-carboxylic acid: 7b, EC50 = 19 nM). NEt-4IB (8b) showed antitype 2 diabetes activity without the above side effects upon repeated oral administration to mice at 10 mg/kg/day, similarly to 5.

Mechanism of retinoid X receptor partial agonistic action of 1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)-1H-benzotriazole-5-carboxylic acid and structural development to increase potency.

We have reported that retinoid X receptor (RXR) partial agonist 1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)-1H-benzotriazole-5-carboxylic acid (CBt-PMN, 4a) shows a significant antidiabetes effect in the KK-A(y) type 2 diabetes model mice, with reduced side effects compared to RXR full agonists. To elucidate the mechanism of the RXR partial agonist activity of 4a, we synthesized derivatives of 4a, evaluated their RXR agonist activity, and performed structure-activity relationship analysis. Reporter gene assay revealed that though 6b, which possesses an amino group at the 2-position of 5-carboxybenzimidazole, showed RXR full-agonist activity, compounds 6d and 6e, which possess an oxygen atom and a sulfur atom at the corresponding position, respectively, showed weak RXR agonist activity. On the other hand, 6c, which has a trifluoromethyl group at the corresponding position, acts as an RXR partial agonist, having similar Emax (67 ± 2%) and lower EC50 (15 ± 0 nM) compared to those of 4a (Emax = 75 ± 4%, EC50 = 143 ± 2 nM). A fluorescence polarization assay of cofactor recruitment confirmed that fluorescein-labeled D22 coactivator peptide was less efficiently recruited to RXR by 4a and 6c than by LGD1069 (1), a known RXR full agonist. Electrostatic potential field calculations and computational docking studies suggested that full agonists show an electrostatic attraction, which stabilizes the holo structure and favors coactivator recruitment, between the side chain at the benzimidazole 2-position and the α-carbonyl oxygen of asparagine-306 in helix 4 (H4) of the RXR receptor. However, RXR partial agonists 4a and 6c lack this interaction. Like 4a, 6c showed a significant antidiabetes effect in KK-A(y) type 2 diabetes model mice with reduced levels of the side effects associated with RXR full agonists. These findings should aid the design of new RXR partial agonists as antitype 2 diabetes drug candidates.

Fungal mn oxides supporting Mn(II) oxidase activity as effective Mn(II) sequestering materials.

We examined the Mn(II)-oxidizing ability of the biogenic Mn oxide (BMO) formed in cultures ofa Mn(II)-oxidizing fungus, Acremonium strictum strain KR21-2. The newly formed BMO effectively sequestered dissolved Mn(II) mainly by oxidizing Mn(II) to insoluble Mn under air-equilibrated conditions, and this ability lasted for at least 8 days. Deaerating the BMOs, poisoning them with NaN3, or heating them all readily weakened their Mn(II) oxidation ability, indicating the involvement of enzymatic Mn(II) oxidation. There was no Mn(II)-oxidizing ability observed for mycelia cultivated without Mn(II) or for residual mycelia after the BMO phase was dissolved, suggesting the need for the oxide phase. A sodium dodecyl sulphate-polyacrylamide gel electrophoresis assay demonstrated that the oxide phase embeds the Mn(II) oxidase and thereby maintains the enzymatic activity in BMOs. Freezing at -80 degrees C preserved the Mn(II)-oxidizing ability in BMOs for at least 4 weeks, while lyophilization caused a complete loss of this ability. Based on these results, we propose that fungal Mn oxides supporting Mn(II) oxidase activity are an effective Mn(II)-sequestering material capable of oxidizing Mn(II) continuously from solutions containing no additional nutrients to maintain biological activity.

Fluorescent retinoid X receptor ligands for fluorescence polarization assay.

Retinoid X receptor (RXR) agonists are candidate agents for the treatment of metabolic syndrome and type 2 diabetes via activation of peroxisome proliferator-activated receptor (PPAR)/RXR or liver X receptor (LXR)/RXR-heterodimers, which control lipid and glucose metabolism. Reporter gene assays or binding assays with radiolabeled compounds are available for RXR ligand screening, but are unsuitable for high-throughput screening. Therefore, as a first step towards stabilizing a fluorescence polarization (FP) assay system for high-throughput RXR ligand screening, we synthesized fluorescent RXR ligands by modification of the lipophilic domain of RXR ligands with a carbostyril fluorophore, and selected the fluorescent RXR agonist 6-[ethyl(1-isobutyl-2-oxo-4-trifluoromethyl-1,2-dihydroquinolin-7-yl)amino]nicotinic acid 8d for further characterization. Compound 8d showed FP in the presence of RXR and the FP was decreased in the presence of the RXR agonist LGD1069 (2). This compound should be a lead compound for use in high-throughput assay systems for screening RXR ligands.

Proteomic analysis of growth phase-dependent expression of Legionella pneumophila proteins which involves regulation of bacterial virulence traits.

Legionella pneumophila, which is a causative pathogen of Legionnaires' disease, expresses its virulent traits in response to growth conditions. In particular, it is known to become virulent at a post-exponential phase in vitro culture. In this study, we performed a proteomic analysis of differences in expression between the exponential phase and post-exponential phase to identify candidates associated with L. pneumophila virulence using 2-Dimentional Fluorescence Difference Gel Electrophoresis (2D-DIGE) combined with Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry (MALDI-TOF-MS). Of 68 identified proteins that significantly differed in expression between the two growth phases, 64 were up-regulated at a post-exponential phase. The up-regulated proteins included enzymes related to glycolysis, ketone body biogenesis and poly-3-hydroxybutyrate (PHB) biogenesis, suggesting that L. pneumophila may utilize sugars and lipids as energy sources, when amino acids become scarce. Proteins related to motility (flagella components and twitching motility-associated proteins) were also up-regulated, predicting that they enhance infectivity of the bacteria in host cells under certain conditions. Furthermore, 9 up-regulated proteins of unknown function were found. Two of them were identified as novel bacterial factors associated with hemolysis of sheep red blood cells (SRBCs). Another 2 were found to be translocated into macrophages via the Icm/Dot type IV secretion apparatus as effector candidates in a reporter assay with Bordetella pertussis adenylate cyclase. The study will be helpful for virulent analysis of L. pneumophila from the viewpoint of physiological or metabolic modulation dependent on growth phase.

Proteomic identification of serum proteins associated with stress-induced gastric ulcers in fasted rats.

Several physical and psychological stresses frequently become triggers for gastrointestinal disorders such as ulcer. In this study, we tried to identify serum proteins as potential biomarkers for the evaluation of stress-induced gastric ulcer. By proteomic analysis using rats with gastric ulcer induced by water immersion and restraint (WIR) stress as an animal model, we found quantitative changes in several serum proteins, including creatine kinase muscle M chain (CK-M) and apolipoprotein A-IV (ApoA4) in the stressed rats. On western blotting and enzyme-linked immunosorbent assay (ELISA), we confirmed that serum CK-M was remarkably increased by WIR stress. However, ApoA4 appeared to be decreased by fasting, but not WIR stress, which is usually applied prior to WIR stress. The findings suggest that these two serum proteins might be useful as biomarkers, CK-M for stress-induced gastric ulcer and ApoA4 for starvation.

Cancer antineovascular therapy with liposome drug delivery systems targeted to BiP/GRP78.

Angiogenesis is crucial for tumor growth and hematogenous metastasis. Specifically expressed and functional protein molecules in angiogenic endothelial cells, especially on the plasma membrane, may be molecular targets for antiangiogenic drugs and drug delivery systems (DDS) in cancer therapy. To discover such target molecules, we performed subcellular proteome analysis of human umbilical vein endothelial cells (HUVECs) treated with or without vascular endothelial growth factor (VEGF) using 2-dimensional difference in-gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). Among the identified proteins, BiP/GRP78, a molecular chaperone, was highly expressed in the membrane/organelle fraction of HUVECs after VEGF treatment. The involvement of BiP in VEGF-induced angiogenesis was examined by RNA interference. BiP knockdown significantly suppressed VEGF-induced endothelial cell proliferation and VEGF-induced phosphorylation of extracellular-regulated kinase 1/2, phospholipase C-γ, and VEGF receptor-2 in HUVECs. Cell surface biotinylation analysis revealed that the cell surface expression of BiP was elevated in VEGF-activated HUVECs. Aiming to apply BiP to a target molecule in liposomal DDS, we developed liposomes modified with the WIFPWIQL peptide, which has been shown to bind to BiP, and investigated its potential for cancer therapy. The WIFPWIQL-modified liposomes (WIFPWIQL liposomes) were significantly taken up by VEGF-activated HUVECs as compared to peptide-unmodified liposomes. WIFPWIQL liposomes appeared to accumulate in tumor endothelial cells in vivo. WIFPWIQL liposomes containing doxorubicin significantly suppressed tumor growth and prolonged the survival of colon26 NL-17 carcinoma cell-bearing mice. In summary, BiP may regulate VEGF-induced endothelial cell proliferation through VEGFR-2-mediated signaling and be an effective target molecule for cancer antineovascular therapy.

Proteomic analysis of hypoxia-induced tube breakdown of an in vitro capillary model composed of HUVECs: potential role of p38-regulated reduction of HSP27.

We recently reported that hypoxia could induce the breakdown of capillary-like tubes formed by human umbilical vein endothelial cells (HUVECs) and that this breakdown was regulated by p38 and not by a caspase cascade, although the exact molecular mechanisms remain unknown. The aim of this study was to identify proteins that regulated hypoxia-induced tube breakdown through p38-regulated and caspase-independent mechanisms. The involvement of adhesion proteins, integrins, VE-cadherin, PECAM-1, and occludin was first investigated. Although some of these proteins decreased after hypoxia, none of them met the conditions of being quantitatively restored by p38 inhibition but not by caspase inhibition. We then conducted 2-D DIGE coupled with MALDI-TOF/TOF-MS to identify altered protein expression. The differential proteomic analysis of tube-forming HUVECs treated with normoxia or hypoxia and treated with hypoxia in the presence or absence of SB203580, a specific p38 inhibitor, revealed the involvement of heat shock proteins in this tube breakdown. We also confirmed that the amount of HSP27 and HSP70 changed in a p38-regulated and caspase-independent manner during hypoxia. Knocking down HSP27 expression using RNAi further augmented hypoxia-induced tube breakdown. Taken together, it was shown that p38-regulated and caspase-independent reduction of HSP27 plays an important role in hypoxia-induced tube breakdown.

Functional analyses of neutrophil-like differentiated cell lines under a hyperglycemic condition.

Diabetic patients are prone to severe bacterial infections. The functional alterations of neutrophils by hyperglycemia are thought to be partially responsible for such infections. In this study, we investigated the functional changes of neutrophil-like differentiated cell lines (dHL-60, dTHP-1, and dNB-4) by treatment with 5.5 mM, 11 mM, or 35 mM of glucose. In dHL-60 cells, the incubation with high glucose (35 mM) resulted in the enhancement of cell aggregation, the suppression of cellular fragility, the induction of reactive-oxygen species (ROS) production by phorbol myristate acetate (PMA) stimulation, and the impairment of phagocytosis. In dTHP-1 cells, the treatment with higher glucose generated the suppression of cellular fragility and extremely impaired phagocytosis (by 35 mM), and induced ROS production due to PMA stimulation (by 11 mM). Furthermore, the higher glucose exposure to dNB-4 cells enlarged intracellular vacuoles (by 35 mM) and induced ROS production due to PMA stimulation (by 11 mM). Since the ROS generation of those cells was enhanced only after PMA stimulation under the higher glucose conditions, glucose may have a priming effect rather than a triggering effect. These extraordinary sensitivities caused by the higher glucose treatments may reflect the dysfunction or overactivation of neutrophils.

Proteomic characterization of angiogenic endothelial cells stimulated with cancer cell-conditioned medium.

To characterize the protein expression profiles and identify the molecules associated with tumor angiogenesis, the cellular proteins of human umbilical vein endothelial cells (HUVECs) in response to cancer cell-conditioned medium (CM) prepared from HT1080 human fibrosarcoma cells were analyzed using fluorescence-labeled 2D gel-based proteomics. Most differentially expressed proteins in HT1080-CM-stimulated cells were found to be downregulated (88%) rather than upregulated (12%) based on statistical analysis of protein spot signals. Additionally, we examined the effects of vascular endothelial cell growth factor (VEGF), a proangiogenic factor, on cellular protein expression. In contrast, most differentially expressed proteins were found to be upregulated (59%) rather than downregulated (41%) in VEGF-stimulated HUVECs. Comparative analyses of 29 and 35 protein species identified in CM-stimulated and VEGF-stimulated HUVECs, respectively, revealed the remarkable differences between these two stimulations. Only four proteins were differentially expressed by both treatments: annexin A2, enolase 1, and T-plastin (downregulated by CM but upregulated by VEGF), and RAN (downregulated by both CM and VEGF). These findings provide new information regarding the regulation of protein expression associated with tumor angiogenesis.

Molecular identification of Ehrlichia species and 'Candidatus Neoehrlichia mikurensis' from ticks and wild rodents in Shizuoka and Nagano Prefectures, Japan.

A total of 293 ticks and 111 wild rodents that were collected in Shizuoka and Nagano Prefectures, Japan, were examined for infection of Ehrlichia species and 'Candidatus Neoehrlichia mikurensis.' The 16S rDNA or the omp-1 gene of these bacterial DNAs were detected from the spleens of tick-inoculated mice (5 positive/total 29 mice) or from the spleens of wild rodents (25 positive/total 111 rodents) by PCR amplifi-cation. Sequencing of the 16S rDNA revealed Ehrlichia spp. from the 5 tick-inoculated mice and 8 wild rodents, and 'Candidatus N. mikurensis' from 17 wild rodents. The data suggest the presence of additional genetic variants, and potential vectors and/or reservoirs for these bacteria in central Japan.

Anaplasma phagocytophilum-infected ticks, Japan.

We report Anaplasma phagocytophilum infection of Ixodes persulcatus and I. ovatus ticks in Japan. Unique p44/msp2 paralogs (and/or 16S rRNA genes) were detected in tick tissues, salivary glands, and spleens of experimentally infected mice. These findings indicate the public health threat of anaplasmosis in Japan.

Proteome analysis of UV-B-induced anti-apoptotic regulatory factors.

Ultraviolet (UV) irradiation is well known to induce apoptosis, a hallmark event of which is the occurrence of sunburn cells in the epidermis. Keratinocytes in which DNA damaged by UV irradiation is not repaired undergo apoptosis as sunburn cells. However, we have previously reported that low-dose UV-B irradiation (approximately 0.1 J/cm2) suppressed the apoptosis induced by cell detachment and serum depletion. Dysregulation of apoptosis is important in tumor progression and malignancy and in promoting resistance to cancer therapy. To develop a better understanding of the antiapoptotic effect of UV irradiation, and to design the effective induction of apoptosis, we tried the proteome analysis of the molecules regulating apoptosis in low-dose UV-B-irradiated NIH3T3 cells, using two-dimensional difference gel electrophoresis (DIGE). Of a total of 3811 protein spots detected, 42 were found to be different between the cells undergoing apoptosis and cells after the irradiation. Of the spots selected, 25 were identified using MALDI-TOF/TOF-MS, some as structural proteins. Although typical apoptosis-related molecules were not detected, possibly because proteins with low molecular weights were difficult to identify in the gel conditions used in this study, some of the proteins were considered to be involved in apoptosis. The DIGE system used in this experiment has advantages (including a high level of statistical confidence) for discovering new functional proteins related to the regulation of apoptosis.

Characterization of Ehrlichia species from Ixodes ovatus ticks at the foot of Mt. Fuji, Japan.

A total of 390 adult ticks (288 Ixodes ovatus and 102 I. persulcatus ) collected at the foot of Mt. Fuji and two near cities in Shizuoka prefecture, Japan, were examined for Ehrlichia infection by isolation with laboratory mice from whole tick tissues. Ehrlichial DNAs were detected from the spleens of mice inoculated with tissues from I. ovatus, but not I. persulcatus. The prevalence of ehrlichiae in the ticks was estimated to be ca. 3%. The 16S rDNA analysis revealed that the sequences of 8 ehrlichial isolates (termed "Shizuoka" isolates) obtained were identical, and they were very similar, but not identical, to those of two Ehrlichia species strain variants recently isolated in Japan, followed by Ehrlichia chaffeensis in the US. Analysis of parts of the omp-1 multigene family specific for monocytic ehrlichiosis agents showed that the Shizuoka isolates were distinct from other ehrlichial organisms. The Shizuoka isolates caused death in immunocompetent laboratory mice, suggesting that they are highly pathogenic in mice. The data show that the Shizuoka isolates are likely to be a new strain variant of Ehrlichia species in Japan. Further characterization and surveillance will be required in Japan due to the presence of these human ehrlichiosis agent-like organisms.

Influence of TT virus co-infection on IFN-beta therapy in patients with chronic hepatitis C.

Although results of IFN alpha therapy in chronic hepatitis C (C-CH) patients co-infected with TT virus (TTV) have been reported, no results of IFN beta therapy or IFN beta and alpha combination therapy have been reported. In this study, we retrospectively investigated whether co-infection with TTV affects the results of IFN therapy by using stored sera from 60 C-CH patients co-infected with TTV who underwent IFN beta therapy or IFN beta and alpha combination therapy. The stored sera were from 29 complete responders, 10 incomplete responders, and 21 non-responders, and they were used for qualitative and quantitative analysis of HCV RNA, HCV genotype analysis, and qualitative and quantitative analyses of TTV DNA. TTV DNA was detected in 23 (38.3%) of the 60 C-CH sera. The TTV DNA-positive rate was 17.2% among the complete responders to IFN therapy, versus 58.1% in the incomplete responders and non-responders, and the difference was significant (p < 0.01). While the complete response prediction rate based on two factors, HCV RNA level and HCV genotype, was 80.8% (21/26) in the C-CH patients, the prediction rate based on three factors, these two factors plus TTV DNA, was higher, 90.0% (18/20). It was concluded that determination of HCV RNA concentration, HCV genotype, and TTV DNA, before IFN beta therapy or IFN beta and alpha combination therapy is useful for predicting the results of treatment of C-CH patients.

Selection of more appropriate PCR primer pairs for improved efficiency in detecting Norwalk-like virus (NLV) RNA.

A pair of primers, NV35 and NV36, and another pair of primers, NV81 and NV82/SM82, are commonly used for polymerase chain reaction (PCR) detection of Norwalk-like virus (NLV) genome RNA sequences in authorized test laboratories in Japan. However, the efficiency of NLV genome RNA detection with these primer pairs has been less than satisfactory. In the present study, we attempted to establish more appropriately matched primer pairs for improved detection of NLV genome RNA sequences using a combination of primers including NV35, NV36, NV81, NV82/SM82, SR33, and SRs (a mixture of 4 primers SR46, SR48, SR50, and SR52). We also evaluated appropriate primers for improved reverse transcription of NLV genome RNA. Stool samples used for detection of NLV included 18 samples collected from NLV-infected patients who ingested oysters (group 1) and 13 samples collected from those who did not ingest oysters (group 2). Reverse transcription of RNA genome with primer NV35 was less efficient compared with that with primer SR33 or NV81. When PCR products obtained with NV35 and NV36 as a pair of primers were subjected to gel electrophoresis, a strong extra band was detected compared with those obtained with other primer pairs. Since this extra band may represent heterodimeric or homodimeric hybrids, or intramolecular hybrids derived from these primers, this template-independent hybridization could lower the efficiency of primer-dependent polymerase reaction. Of 18 primer pairs, a pair of NV81 and SRs provided the best detection of PCR products following reverse transcription of NLV RNA with SR33 or NV81. The detection rate was 61% for both reverse transcription with SR33 and that with NV81. After reverse transcription using SR33 as a primer, nested PCR using a pair of NV81 and SRs following primary PCR using a pair of NV81 and NV82/SM82 increased the detection rate to 89% in group 1 and 100% in group 2.