Detection of various DNA and RNA viruses in bats in Yamaguchi Prefecture, Japan.

Bats are important natural hosts of various zoonotic viruses, including Ebola virus, Lyssa virus, and severe acute respiratory syndrome coronavirus (SARS-CoV). Although investigation of bats is valuable for predicting emerging infectious diseases from these animals, few surveys of bat-derived viruses have been conducted in Japan. In the present study, samples were collected from a total of 132 bats of 4 different species from 4 different locations within Yamaguchi Prefecture; these sample were employed for comprehensive detection of bat-derived viruses by polymerase chain reaction (PCR) and reverse transcription (RT)-PCR using primers universal for each of 4 different viral classes. As a result of PCR and RT-PCR, various herpesviruses, astroviruses, coronaviruses, and adenoviruses were identified from a total of 80 bats. The detected herpesviruses belong to the Betaherpesvirinae or Gammaherpesvirinae subfamily, the detected adenoviruses to the genus Mastadenovirus, the detected astroviruses to the genus Mamastrovirus; and the detected coronaviruses belong to the genus Alphacoronavirus. The detected sequences of 12 strains of 4 families showed 100 % amino acid identity with viruses previously detected either in China or South Korea. These findings expand our understanding of viruses carried by bats, and provide insights into the nature of bat-derived viruses in Japan.

Characteristics of pectens in diurnal and nocturnal birds and a new functional proposal relating to non-visual opsins.

The pecten is a fold-structured projection at the ocular fundus in bird eyes, showing morphological diversity between the diurnal and nocturnal species. However, its biological functions remain unclear. This study investigated the morphological and histological characteristics of pectens in wild birds. Additionally, the expression of non-visual opsin genes was studied in chicken pectens. These genes, identified in the chicken retina and brain, perceive light periodicity regardless of visual communication. Similar pleat numbers have been detected among bird taxa; however, pecten size ratios in the ocular fundus showed noticeable differences between diurnal and nocturnal birds. The pectens in nocturnal brown hawk owl show extremely poor vessel distribution and diameters compared with that of diurnal species. RT-PCR analysis confirmed the expression of Opn5L3, Opn4x, Rrh and Rgr genes. In situ hybridization analysis revealed the distribution of Rgr-positive reactions in non-melanotic cells around the pecten vessels. This study suggests a novel hypothesis that pectens develop dominantly in diurnal birds as light acceptors and contribute to continuous visual function or the onset of periodic behaviour.

Appearance of small extracellular vesicles in the mouse pregnant serum and the localization in placentas.

Exosomes or small extracellular vesicles (sEVs) are present in the blood of pregnant mice and considered to be involved in pregnancy physiology. Although sEVs in pregnant periods are proposed to be derived from placentas, sEVs-producing cells are not well known in mouse placentas. We studied the dynamics and localization of sEVs in pregnant serum and placentas, and examined gestational variation of microRNA (miRNA). Serums and placentas were collected from non-pregnant (NP) and pregnant mice throughout the entire gestational day (Gd). EVs were purified from serums and total RNA was isolated from EVs. Nanoparticle-tracking assay (NTA) revealed that the rates of sEVs in EVs are 53% at NP, and increased to 80.1% at Gd 14.5 and 97.5% at Gd 18.5. Western blotting on EVs showed positive reactivity to the tetraspanin markers and clarified that the results using anti-CD63 antibody were most consistent with the sEVs appearance detected by NTA. Serum EVs also showed a positive reaction to the syncytiotrophoblast marker, syncytin-1. Immunohistostaining using anti-CD63 antibody showed positive reactions in mouse placentas at the syncytiotrophoblasts and endothelial cells of the fetal capillaries. Quantitative PCR revealed that significantly higher amounts of miRNAs were included in the sEVs of Gd 18.5. Our results suggested that sEVs are produced in the mouse placenta and transferred to maternal or fetal bloodstreams. sEVs are expected to have a miRNA-mediated physiological effect and become useful biomarkers reflecting the pregnancy status.

Establishment of African pygmy mouse induced pluripotent stem cells using defined doxycycline inducible transcription factors.

Mus minutoides is one of the smallest mammals worldwide; however, the regulatory mechanisms underlying its dwarfism have not been examined. Therefore, we aimed to establish M. minutoides induced pluripotent stem cells (iPSCs) using the PiggyBac transposon system for applications in developmental engineering. The established M. minutoides iPSCs were found to express pluripotency markers and could differentiate into neurons. Based on in vitro differentiation analysis, M. minutoides iPSCs formed embryoid bodies expressing marker genes in all three germ layers. Moreover, according to the in vivo analysis, these cells contributed to the formation of teratoma and development of chimeric mice with Mus musculus. Overall, the M. minutoides iPSCs generated in this study possess properties that are comparable to or closely resemble those of naïve pluripotent stem cells (PSCs). These findings suggest these iPSCs have potential utility in various analytical applications, including methods for blastocyst completion.

Verification of the efficacy of deciduae for determining the genetic type of mouse embryos.

Mouse embryos in the early-implantation stage require manipulation under a microscope. While the extraction of DNA, RNA and proteins from a single sample allows for both determination of genetic type and analysis of gene expression, whole mount analysis is not possible. In this study, we explored the applicability of PCR using extraembryonic tissues, especially the decidual side tissue after isolating the embryos from implantation sites to establish a method for determining the genetic type of embryos. The implantation site was resected at each day from the date of vaginal plug confirmation, separated into embryos and deciduae. Genomic DNA were isolated separately from the embryos and the deciduae. PCR was performed using these genomic DNA, and the band patterns were compared after electrophoresis. As a result, we demonstrated that detecting embryo-derived cells in the decidua allows determination of the sex and presence of transgenes without harming the mouse embryos themselves, from 8.5 days of age. This method enables the determination of the genetic type of mouse embryos without damaging. This technique would expand the adaptations for analysis of mouse implanted embryos.

Specific expression and blood kinetics for relaxin 2, lipocalin 2, and tissue factor pathway inhibitor 2 at the canine placenta and pregnant bloods.

In general, humoral factors released from the placenta influence pregnancy progression, but the involvement of the canine placenta is often unidentified. We investigated specific genes in canine placentas and analyzed the blood dynamics of the translated proteins. Furthermore, RNAs are known to be released from placentas embedding in exosomes, a type of extracellular vesicles. Here, the presence of cell-free RNAs in pregnant serums was also confirmed. RNA specimens were purified from the normal healthy dog placentas and applied to RNA-Seq analysis. Expressions of frequent genes were confirmed by RT-PCR using placentas from other individuals and breeds. Relaxin (RLN) 2, lipocalin (LCN) 2, and tissue factor pathway inhibitor (TFPI) 2 were selected as high-expressed and placenta-specific genes. By western blot, the three factors were clearly detected in the pregnant serums. Quantitative analysis revealed that the amount of RLN2 increased significantly from non-pregnancy to day 41 of pregnancy. Regarding LCN2 and TFPI2, the protein serum levels elevated during pregnancy, but the statistical differences were not detected. Exosomes were found in all pregnant serums; however, the percentage was less than 6% in total extracellular vesicles. The cell-free RNA related to RLN2 was detected, but no elevation was confirmed during pregnancy. We found specific genes in the canine placenta and the transition of their translated protein into the blood. These factors may become useful tools for research on canine pregnancy and monitoring of reproductive management. Exosomes and cell-free RNA could not be found to be valid in canine reproduction.

Degradation of glycosylinositol phosphoceramide during plant tissue homogenization.

A convenient method for the determination of plant sphingolipids (glycosylinositol phosphoceramide, GIPC; glucosylceramide, GluCer; phytoceramide 1-phosphate, PC1P and phytoceramide, PCer) was developed. This method includes the extraction of lipids using 1-butanol, alkali hydrolysis with methylamine and separation by TLC. The amounts of sphingolipids in the sample were determined based on the relative intensities of standard sphingolipids visualized by primulin/UV on TLC. Using this method, we found that almost all GIPCs were degraded in response to tissue homogenization in cruciferous plants (cabbage, broccoli and Arabidopsis thaliana). The decrease in GIPCs was compensated for by increases in PC1P and PCer, indicating that GIPC was degraded by hydrolysis at the D and C positions of GIPC, respectively. In carrot roots and leaves, most of GIPC degradation was compensated for by an increase in PCer. In rice roots, the decrease in GIPCs was not fully explained by the increases in PC1P and PCer, indicating that enzymes other than phospholipase C and D activities operated. As the visualization of lipids on TLC is useful for detecting the appearance or disappearance of lipids, this method will be available for the characterization of metabolism of sphingolipids in plants.

Characteristic amino acid residues in the growth hormone receptor gene on Mus minutoides underlying dwarfism.

The African pygmy mouse ( Mus minutoides ) displays a dwarfism phenotype distinctive from closely related species. This study aimed to investigate the growth hormone receptor (Ghr) gene sequence in M. minutoides . We identified several amino acid variations, including the P469L mutation. Our findings suggest that this mutation affects Ghr protein functionality, decreasing Igf1 expression and contributing to the dwarfism observed in M. minutoides . Further studies utilizing genome editing technology are necessary to elucidate the mechanisms involved in mammalian body size determination.

Expression dynamics of Crry at the implantation sites in normal pregnancy and response against miscarriage induction.

In mammals, immune tolerance against fetal tissue has been established for normal pregnancy progression. It is known that Crry regulates complemental activity to prevent injury of the mouse embryo and extra-embryonic tissue. This study aimed to examine the expression appearance and normal localization sites of Crry in the mouse placenta. Also, the emergency responses of Crry were verified at the time of experimental miscarriage induction. Moreover, we investigated an existing similar protein of Crry in animal placentas other than mice. Crry expression level showed a peak at day 8.5 of pregnancy. Trophoblast giant cells and decidual cells indicated a positive reaction to anti-Crry antibody. After treatments of interferon-γ, Crry expression was increased significantly in the survived implantation sites as compared with the controls. However, there was no significant difference in the miscarriage-initiated sites. It disclosed that Crry distributes from the early to middle periods of the placentas and involves complement regulation at the extraembryonic tissue and decidua basalis. Crry also showed an ability to respond to risk against external initiation for urgent miscarriage. Finally, we found anti-mouse Crry antibody-bound proteins in the placenta of many animals. It suggests a potency of Crry to make an environment of immune tolerance in many types of mammal placentas.

Nonspecific phospholipase C3 of radish has phospholipase D activity towards glycosylinositol phosphoceramide.

Glycosylinositol phosphoceramide (GIPC) is a major sphingolipid in the plasma membranes of plants. Previously, we found an enzyme activity that produces phytoceramide 1-phosphate (PC1P) by hydrolysis of the D position of GIPC in cabbage and named this activity as GIPC-phospholipase D (PLD). Here, we purified GIPC-PLD by sequential chromatography from radish roots. Peptide mass fingerprinting analysis revealed that the potential candidate for GIPC-PLD protein was nonspecific phospholipase C3 (NPC3), which has not been characterized as a PLD. The recombinant NPC3 protein obtained by heterologous expression system in Escherichia coli produced PC1P from GIPC and showed essentially the same enzymatic properties as those we characterized as GIPC-PLD in cabbage, radish and Arabidopsis thaliana. From these results, we conclude that NPC3 is one of the enzymes that degrade GIPC.

Effectiveness of third vaccine dose for coronavirus disease 2019 during the Omicron variant pandemic: a prospective observational study in Japan.

The administration of a third booster dose of messenger ribonucleic acid (mRNA) vaccines against coronavirus disease 2019 (COVID-19) has progressed worldwide. Since January 2022, Japan has faced a nationwide outbreak caused by the Omicron variant, which occurred simultaneously with the progression of mass vaccination with the third booster dose. Therefore, this study evaluated the effectiveness of the third dose of vaccine by reverse transcription-polymerase chain reaction (RT-PCR) test using nasopharyngeal swab samples from adults aged ≥ 18 years tested after having close contact with COVID-19 cases between January and May 2022. Participants who completed only one dose were excluded from the study. Among the 928 enrolled participants, 139 had never been vaccinated, 609 had completed two doses, 180 had completed three doses before the swab test, and the overall RT-PCR test positivity rate in each group was 48.9%, 46.0%, and 32.2%, respectively. The vaccine effectiveness of the third dose to prevent infection after close contact was approximately 40% (95% confidence interval: 20-60%), which was the highest at 10-70 days after receiving the third dose. In conclusion, the effectiveness of the three-dose mRNA COVID-19 vaccine after close contact during the Omicron outbreak is approximately 40%.

COVID-19-Related Symptoms during the SARS-CoV-2 Omicron (B.1.1.529) Variant Surge in Japan.

The exact profiles of the clinical symptoms related to the SARS-CoV-2 Omicron variant (B.1.1.529) remain largely uncertain. Therefore, this study aimed to clarify the clinical manifestations of infection with this variant. We enrolled individuals who were tested by quantitative nasopharyngeal swab reverse transcription-polymerase chain reaction (RT-PCR) test at a large screening center in a city of Japan during the B.1.1.529 Omicron variant wave between January and May 2022, after contact with COVID-19 patients. Swab tests were planned to be performed approximately 4-5 days after contact. The presence of COVID-19-related symptoms was assessed at the swab test site. Among the 2,507 enrolled individuals, 943 (37.6%) were RT-PCR test-positive and 1,564 (62.4%) were test-negative. Among the 943 PCR test-positive participants, the prevalence of the symptoms was as follows: 47.3% with cough, 32.9% with sore throat, 18.4% with fatigability, 12.7% with fever of ≥ 37.5℃, 9.9% with dyspnea, 2.1% with dysosmia, and 1.4% with dysgeusia. The prevalence of cough, sore throat, dyspnea, and fatigability was higher among adults aged ≥ 18 years than among children and adolescents. The prevalence of dysosmia and dysgeusia remarkably decreased during the Omicron wave (1-3%) compared to during the pre-Omicron variant waves (15-25%). In summary, common COVID-19-related symptoms during the Omicron variant wave included cough and sore throat, followed by fatigability, fever, and dyspnea. The prevalence of most of these symptoms was higher in adults than in non-adults. The prevalence of dysosmia and dysgeusia remarkably decreased with the Omicron variant than with pre-Omicron variants.

FKBP52 and FKBP51 differentially regulate the stability of estrogen receptor in breast cancer.

Estrogen receptor α (ERα) is a transcription factor that induces cell proliferation and exhibits increased expression in a large subset of breast cancers. The molecular mechanisms underlying the up-regulation of ERα activity, however, remain poorly understood. We identified FK506-binding protein 52 (FKBP52) as a factor associated with poor prognosis of individuals with ERα-positive breast cancer. We found that FKBP52 interacts with breast cancer susceptibility gene 1 and stabilizes ERα, and is essential for breast cancer cell proliferation. FKBP52 depletion resulted in decreased ERα expression and proliferation in breast cancer cell lines, including MCF7-derived fulvestrant resistance (MFR) cells, suggesting that inhibiting FKBP52 may provide a therapeutic effect for endocrine therapy­resistant breast cancer. In contrast, FKBP51, a closely related molecule to FKBP52, reduced the stability of ERα. Consistent with these findings, FKBP51 was more abundantly expressed in normal tissues than in cancer cells, suggesting that these FKBPs may function in the opposite direction. Collectively, our study shows that FKBP52 and FKBP51 regulate ERα stability in a reciprocal manner and reveals a regulatory mechanism by which the expression of ERα is controlled.

Mouse embryonic stem cells maintain differentiation potency into somatic lineage despite alternation of ploidy.

Vertebrates, including mammals, are considered to have evolved by whole genome duplications. Although some fish have been reported to be polyploids that have undergone additional genome duplication, there have been no reports of polyploid mammals due to abnormal development after implantation. Furthermore, as the number of physiologically existing tetraploid somatic cells is small, details of the functions of these ploidy-altered cells are not fully understood. In this present study, we aimed to clarify the details of the differentiation potency of tetraploids using tetraploid embryonic stem cells. To clarify the differentiation potency, we used mouse tetraploid embryonic stem cells derived from tetraploid embryos. We presented tetraploid embryonic stem cells differentiated into neural and osteocyte lineage in vitro and tetraploid cells that contributed to various tissues of chimeric embryos ubiquitously in vivo. These results revealed that mouse embryonic stem cells maintain differentiation potency after altering the ploidy. Our results provide an important basis for the differentiation dynamics of germ layers in mammalian polyploid embryogenesis.

Effectiveness of mRNA COVID-19 Vaccines in Japan during the Nationwide Pandemic of the Delta Variant.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remained a major global health concern in 2021. To suppress the spread of infection, mass vaccinations have been performed across countries worldwide. In Japan, vaccinations of the first and second doses for most of the nation were performed during the nationwide outbreak of the B.1.617.2 (Delta) variant with the L452R spike protein mutation, and the effectiveness of the vaccinations to suppress the spread of COVID-19 among the people in Japan remains uncertain. In this study, adults aged ≥18 years, who were in contact with patients with COVID-19 and underwent nasopharyngeal swab reverse transcription-polymerase chain reaction (RT-PCR) tests during August and September 2021 at a mass screening test center in Japan, were enrolled. In this period, more than 95% of the COVID-19 infections were reportedly caused by the Delta variant. As a result, a total of 784 adults with recent contact history, including 231 (29.5%) RT-PCR test-positive cases, were enrolled. The test positivity rate was lower in individuals who had been vaccinated twice than in unvaccinated individuals (12.5% vs. 39.0%, p < 0.0001), with the risk ratio of 0.32 (95% confidence interval 0.23-0.46). The vaccine effectiveness was the highest between 7-90 days after the second vaccine dose. In conclusion, two doses of mRNA COVID-19 vaccines effectively suppressed transmission in Japan during the nationwide pandemic of the Delta variant, estimated to have prevented 50-80% of the infection.

Characterization of Ceramides and Glucosylceramides of the Satsuma Mandarin(Citrus unshiu) Fruit.

Ceramide (Cer) and glucosylceramide (GlcCer) were isolated from Satsuma mandarin (Citrus unshiu) fruits and characterized. 2,3-Dihydroxy fatty acids with C20 or longer acyl chains were found in Cer. GlcCers from the flesh of the fruit contained sphingosine (4-trans-sphingenine) as a major component. Notably, the Cer content was 1.5-fold higher than GlcCer content. The ratio of Cer plus GlcCer to the total lipid content in Satsuma mandarin was higher than that in the other citrus fruits analyzed in this study. Collectively, the pomace of the Satsuma mandarin fruit can be a good source of sphingolipids as functional components in foods.

COVID-19 Transmission at Schools in Japan.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a global public health concern in 2021. However, the risk of attending schools during the pandemic remains unevaluated. This study estimated the secondary transmission rate at schools using the results of a real-time reverse transcription-polymerase chain reaction (RT-PCR) screening test performed between July 2020 and April 2021, before starting the nationwide mass vaccination. A total of 1,924 students (20 RT-PCR-positive; 1.0%) from 52 schools or preschools were evaluated, together with 1,379 non-adults (95 RT-PCR-positive; 6.9%) exposed to SARS-CoV-2 in non-school environments. Assuming that the infectious index cases were asymptomatic and the transmission at schools followed a Bernoulli process, we estimated the probability of transmission after each contact at school as approximately 0.005 (0.5% per contact) with the current infection prevention measures at schools in Japan (i.e., hand hygiene, physical distancing, wearing masks, and effective ventilation). Furthermore, assuming that all children are capable of carrying the infection, then contact between an index case and 20-30 students per day at schools would yield the expected value for secondary cases of ≥ 1.0, during the 10 days of the infectious period. In conclusion, with the current infection prevention measures at schools in Japan, secondary transmission at schools would occur in approximately every 200 contacts. When considering this rate, compliance with the current infection prevention measures at schools and early detection and quarantine of the index cases would be effective in preventing the spread of COVID-19 at schools.

The Brachypodium distachyon cold-acclimated plasma membrane proteome is primed for stress resistance.

In order to survive subzero temperatures, some plants undergo cold acclimation (CA) where low, nonfreezing temperatures, and/or shortened day lengths allow cold-hardening and survival during subsequent freeze events. Central to this response is the plasma membrane (PM), where low temperature is perceived and cellular homeostasis must be preserved by maintaining membrane integrity. Here, we present the first PM proteome of cold-acclimated Brachypodium distachyon, a model species for the study of monocot crops. A time-course experiment investigated CA-induced changes in the proteome following two-phase partitioning PM enrichment and label-free quantification by nano-liquid chromatography-mass spectrophotometry. Two days of CA were sufficient for membrane protection as well as an initial increase in sugar levels and coincided with a significant change in the abundance of 154 proteins. Prolonged CA resulted in further increases in soluble sugars and abundance changes in more than 680 proteins, suggesting both a necessary early response to low-temperature treatment, as well as a sustained CA response elicited over several days. A meta-analysis revealed that the identified PM proteins have known roles in low-temperature tolerance, metabolism, transport, and pathogen defense as well as drought, osmotic stress, and salt resistance suggesting crosstalk between stress responses, such that CA may prime plants for other abiotic and biotic stresses. The PM proteins identified here present keys to an understanding of cold tolerance in monocot crops and the hope of addressing economic losses associated with modern climate-mediated increases in frost events.

Discriminatory Value of Self-reported Olfactory Dysfunction in the Prediction of Coronavirus Disease 2019.

Objective Coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), remains the world's largest public health concern in 2021. A history of close contact with infectious patients is a factor that predicts a positive SARS-CoV-2 test result. Meanwhile, the precise predictive value of symptoms suggestive of COVID-19 has not been fully elucidated. This study aimed to clarify the predictive and discriminatory value of each clinical symptom suggestive of COVID-19. Methods This study enrolled participants who were tested for SARS-CoV-2 by reverse transcription polymerase chain reaction using a nasopharyngeal swab between November 2020 and January 2021. All enrolled patients were evaluated for data regarding the presence and closeness of contact with infectious patients and comprehensive clinical features (i.e., fever, cough, dyspnea, fatigue, dysosmia, and dysgeusia). Results Among the 1,744 tested participants, 144 tested positive for SARS-CoV-2. In the test-positive group, self-reported cough, fatigue, dysosmia, and dysgeusia were significant predictors of COVID-19, independent from a history of close contact. In particular, the presence of dysosmia was the strongest predictor of COVID-19 in both univariate and multivariate analyses. Among the 42 patients with self-reported dysosmia, 25 (59.5%) were SARS-CoV-2 test-positive. Self-reported dysosmia was reported by 25 (17.4%) of the 144 patients who tested positive for SARS-CoV-2, and 15 (60.0%) of the 25 COVID-19 patients with dysosmia had accompanying dysgeusia. Conclusion The presence of dysosmia was reported by 10-25% of patients with COVID-19, and is a significant predictor of COVID-19 infection, independent from a history of close contact.

Histological analysis of implanted embryos in large Japanese field mouse (Apodemus speciosus) and estimation of developmental stage.

The large Japanese field mouse (Apodemus speciosus) is a small rodent species endemic to Japan. The genetic characteristics of A. speciosus include different chromosome numbers within the same species. Furthermore, A. speciosus has been used in radiation and genetic research. In the present study, a pregnant A. speciosus was obtained, and histochemical analysis of the implanted embryos was performed and compared with the developmental stages of the mouse (Mus musculus). Although there were some differences, the structures of the implanted embryos, including the primitive streak and placenta of A. speciosus were similar to those of mouse. Our study will be important for the construction of a developmental atlas of A. speciosus.