Novel Genotype of HA Clade 2.3.4.4b H5N8 Subtype High Pathogenicity Avian Influenza Virus Emerged at a Wintering Site of Migratory Birds in Japan, 2021/22 Winter.

Surveillance of avian influenza virus (AIV) was conducted in the 2021-2022 winter season at a wintering site of migratory Anatidae in Japan. An H5N8 subtype high pathogenicity AIV (HPAIV) with a unique gene constellation and four low pathogenicity AIVs (LPAIVs) were isolated from environmental samples. The genetic origin of the HPAIV (NK1201) was determined with whole-genome sequencing and phylogenetic analyses. Six of NK1201's eight genes were closely related to HA clade 2.3.4.4b H5N8 subtype HPAIVs, belonging to the G2a group, which was responsible for outbreaks in poultry farms in November 2021 in Japan. However, the remaining two genes, PB1 and NP, most closely matched those of the LPAIVs H7N7 and H1N8, which were isolated at the same place in the same 2021-2022 winter. No virus of the NK1201 genotype had been detected prior to the 2021-2022 winter, indicating that it emerged via genetic reassortment among HPAIV and LPAIVs, which were prevalent at the same wintering site. In addition, experimental infection in chickens indicated that NK1201 had slightly different infectivity compared to the reported infectivity of the representative G2a group H5N8 HPAIV, suggesting that the PB1 and NP genes derived from LPAIVs might have affected the pathogenicity of the virus in chickens. Our results directly demonstrate the emergence of a novel genotype of H5N8 HPAIV through gene reassortment at a wintering site. Analyses of AIVs at wintering sites can help to identify the emergence of novel HPAIVs, which pose risks to poultry, livestock, and humans.

Color-tunable bioluminescence imaging portfolio for cell imaging.

The present study describes a color-tunable imaging portfolio together with twelve novel coelenterazine (CTZ) analogues. The three groups of CTZ analogues create diverse hues of bioluminescence (BL) ranging from blue to far red with marine luciferases. We found that the hue completes the whole color palette in the visible region and shows red-shifted BL with a marine luciferase: for example, Renilla luciferase 8 (RLuc8) and Artificial Luciferase 16 (ALuc16) show 187 nm- and 105 nm-redshifted spectra, respectively, by simply replacing the substrate CTZ with 1d. The optical properties of the new CTZ analogues were investigated such as the kinetic parameters, dose dependency, and luciferase specificity. The 2-series CTZ analogues interestingly have specificity to ALucs and are completely dark with RLuc derivatives, and 3d is highly specific to only NanoLuc. We further determined the theoretical background of the red-shifted BL maximum wavelengths (λBL) values according to the extended π conjugation of the CTZ backbone using Density Functional Theory (DFT) calculations. This color-tunable BL imaging system provides a useful multicolor imaging portfolio that efficiently images molecular events in mammalian cells.

Favorable Intercalation of Nitrate Ions with Fluorine-Substituted Layered Double Hydroxides.

Understanding and controlling confined nanospace to accommodate substrates and promote high ion conduction are essential to various fields. Layered double hydroxides (LDHs) have emerged as promising candidates for anion exchangers using the interlayer nanospace in their crystal structures. Miyata reported in 1983 that the affinity of anions for intercalation with most major Mg-Al LDHs increased in the following order: NO3- < Br- < F- < SO42- < HPO32-. Attempts to alter the affinity with different metal cations (M2+ and M3+) have been unsuccessful. Analyses of the crystalline structures of LDHs, positively charged host layers, interlayer anions, and interlayer water molecules indicate that they inevitably interact through hydrogen bonding. In other words, the affinity of LDHs for anions is controlled by tuning the hydrogen bonding. In this study, we prepared fluorine-substituted LDHs (F-LDHs) with different Mg/Al ratios by partially replacing the OH structural groups, which originated from the host layer, with fluorine atoms; the resulting change in affinity was investigated. The distribution coefficient, which is a useful indicator of the affinity of an LDH for a particular anion, was examined. The results showed that only F-LDHs with Mg/Al ratios of 3.5 exhibited high affinity, especially for NO3- ions, and the affinity increased in the following order: HPO42- < SO42- < F- < Br- < NO3-. The separation factors of these specific F-LDHs with respect to both NO3-/F- and NO3-/SO42- were higher than that of LDHs with other compositions by 1 order of magnitude. Raman spectroscopy above 3000 cm-1 revealed that the fluorine substitution of LDHs significantly changed the hydrogen bonding nature in the interlayer space. Highly electronegative fluorine atoms significantly decrease the extent of hydrogen bonding interactions between OH structural groups and both interlayer water molecules and anions, wherein steric effects are induced by the shrunken interlayer space, and van der Waals forces are revealed to be the predominant interaction with anions. Therefore, the highest affinity was observed for NO3- ions in F-LDHs.

Single-cell bioluminescence imaging of deep tissue in freely moving animals.

Bioluminescence is a natural light source based on luciferase catalysis of its substrate luciferin. We performed directed evolution on firefly luciferase using a red-shifted and highly deliverable luciferin analog to establish AkaBLI, an all-engineered bioluminescence in vivo imaging system. AkaBLI produced emissions in vivo that were brighter by a factor of 100 to 1000 than conventional systems, allowing noninvasive visualization of single cells deep inside freely moving animals. Single tumorigenic cells trapped in the mouse lung vasculature could be visualized. In the mouse brain, genetic labeling with neural activity sensors allowed tracking of small clusters of hippocampal neurons activated by novel environments. In a marmoset, we recorded video-rate bioluminescence from neurons in the striatum, a deep brain area, for more than 1 year. AkaBLI is therefore a bioengineered light source to spur unprecedented scientific, medical, and industrial applications.

Visualizing changes in brain-derived neurotrophic factor (BDNF) expression using bioluminescence imaging in living mice.

Brain-derived neurotrophic factor (BDNF) plays a fundamental role in expressing various neural functions including memory consolidation. Alterations of BDNF levels in the brain are associated with neurodegenerative and neuropsychiatric disorders. Therefore, it is important to understand how levels of BDNF are controlled. Recently we generated a novel transgenic mouse strain, termed the Bdnf-Luciferase transgenic (Bdnf-Luc Tg) mouse, to monitor changes in Bdnf expression. In the present study, we detected the bioluminescence signal from living Bdnf-Luc Tg mice after intraperitoneal administration of d-luciferin. Despite high levels of Bdnf expression in the brain, it was difficult to detect a signal from the brain region, probably because of its poorly penetrable (short-wavelength) bioluminescence. However, we could detect the changes in the bioluminescence signal in the brain region using a luciferin analogue generating a near-infrared wavelength of bioluminescence. We also found a strong correlation between increases in body weight and bioluminescence signal in the abdominal region of Tg mice fed a high-fat diet. These results show that changes in Bdnf expression can be visualized using living mice, and that the Tg mouse could be a powerful tool for clarification of the role of Bdnf expression in pathophysiological and physiological conditions.

A luciferin analogue generating near-infrared bioluminescence achieves highly sensitive deep-tissue imaging.

In preclinical cancer research, bioluminescence imaging with firefly luciferase and D-luciferin has become a standard to monitor biological processes both in vitro and in vivo. However, the emission maximum (λmax) of bioluminescence produced by D-luciferin is 562 nm where light is not highly penetrable in biological tissues. This emphasizes a need for developing a red-shifted bioluminescence imaging system to improve detection sensitivity of targets in deep tissue. Here we characterize the bioluminescent properties of the newly synthesized luciferin analogue, AkaLumine-HCl. The bioluminescence produced by AkaLumine-HCl in reactions with native firefly luciferase is in the near-infrared wavelength ranges (λmax=677 nm), and yields significantly increased target-detection sensitivity from deep tissues with maximal signals attained at very low concentrations, as compared with D-luciferin and emerging synthetic luciferin CycLuc1. These characteristics offer a more sensitive and accurate method for non-invasive bioluminescence imaging with native firefly luciferase in various animal models.

Multicolor Bioluminescence Obtained Using Firefly Luciferin.

Firefly bioluminescence is widely used in life science research as a useful analysis tool. For example, the adenosine-5`-triphosphate (ATP)-dependent enzymatic firefly bioluminescence reaction has long been utilized as a microbial monitoring tool. Rapid and sensitive firefly luciferin-luciferase combinations are used not only to measure cell viability but also for reporter-gene assays. Recently, bioluminescence was utilized as a noninvasive, real-time imaging tool for living subjects to monitor cells and biological events. However, the number of commercialized luciferase genes is limited and tissue-permeable near-infrared (NIR) region emitting light is required for in vivo imaging. In this review, recent studies describing synthetic luciferin analogues predicted to have red-shifted bioluminescence are summarized. Luciferase substrates emitting red, green, and blue light that were designed and developed in our laboratory are presented. The longest emission wavelength of the synthesized luciferin analogues was recorded at 675 nm, which is within the NIR region. This compound is now commercially available as "Aka Lumine®".

Lowering of blood pressure improves endothelial dysfunction by increase of nitric oxide production in hypertensive rats.

To investigate the effects of angiotensin converting enzyme inhibitor (ACE-I) and other antihypertensive agents on the nitric oxide (NO) release during hypertension, seven- and fourteen-week-old SHR and deoxycorticosterone acetate (DOCA)-salt rats were treated with hydralazine, manidipine (Ca antagonist) or quinapril (ACE-I) for 3 weeks to lower blood pressure. Systolic blood pressure (SBP) was measured by the tail cuff method once each week. Endothelial cells (ECs) derived from the descending aorta of the treated rats were cultured and NOx levels in culture media were measured with an NO analyzer based on the Griess reaction. In both SHR and DOCA-salt rats, antihypertensive therapy lowered SBP to levels similar to those of control rats. The only exception was quinapril treatment of DOCA-salt rats. Although NOx release by ECs derived from hypertensive rats was improved by antihypertensive therapy, the effect was most pronounced in SHR treated with quinapril. In addition, restoration of NOx release was much more remarkable in younger SHR. NOx release was significantly higher in DOCA-salt rats treated with quinapril than in control rats without reduction of SBP. These results suggest that lowering blood pressure improves release of NO by ECs during hypertension and that the time at which antihypertensive therapy is started is also important to preserve endothelial function. Furthermore, ACE-I is suggested to protect endothelial function by increasing NO production in addition to lowering blood pressure.

Clinical features of hepatocellular carcinoma seronegative for both HBsAg and anti-HCV antibody but positive for anti-HBc antibody in Japan.

OBJECTIVE: We determined the prevalence of patients with hepatocellular carcinoma (HCC) who were positive for only anti-hepatitis B core (anti-HBc) antibody among 284 Japanese patients and compared their clinical features to those who were hepatitis B surface antigen positive [HBsAg(+)]. METHODS: Serum HBsAg and anti-hepatitis C virus (anti-HCV) antibody were examined for all HCC patients. Testing for anti-HBc antibody was performed in the HBsAg(-)/anti-HCV(-) patients. The clinical factors and the survival rate were compared between the HBsAg(+) patients (HCC-B) and those positive for anti-HBc alone (HCC-PB). RESULTS: There were 19 (6.7%) HBsAg(+), 236 (83.1%) anti-HCV(+), seven (2.5%) HBsAg(+)/anti-HCV(+), and 22 (7.7%) HBsAg(-)/anti-HCV(-) among the 284 patients tested. Sixteen (72.7%) of the 22 HBsAg(-)/anti-HCV(-) patients were assigned to the HCC-PB group. The prevalence of positivity for anti-HBc alone among all 284 HCC patients was 5.6%. Significant differences between the HCC-PB and HCC-B groups were that age at diagnosis was higher in the HCC-PB group (72.1 yr) than in the HCC-B group (56.2 yr) (p < 0.001), the serum alpha-fetoprotein concentrations were lower in the HCC-PB group (8.2 ng/ml) than in the HCC-B group (43 ng/ml) (p = 0.0488), and a higher familial history of liver disease was observed in the HCC-B group (p = 0.0373). However, there was no significant difference in the cumulative survival rate. CONCLUSIONS: Positivity for anti-HBc alone is not rare compared to HBsAg(+), and the clinical features of positivity for anti-HBc alone are similar to those of HBsAg(+). Some differences in the clinical features between the two groups may be explained by differences in the time of first exposure to hepatitis B virus. Therefore, the natural course of acute hepatitis B may be reconsidered.