Gn protein expressed in plants for diagnosis of severe fever with thrombocytopenia syndrome virus.

Severe fever with thrombocytopenia syndrome virus (SFTSV) causes the highly fatal disease in humans. To facilitate diagnosis, the native form of subunit glycoprotein (Gn), a prime target for potential vaccines and therapies, was produced in Nicotiana benthamiana using a Bamboo mosaic virus-based vector system. By fusion with secretory signal tags, SSExt, derived from the extension protein, and the (SP)10 motif, the yield of the recombinant Gn (rGn) was remarkably increased to approximately 7 mg/kg infiltrated leaves. Ultimately, an rGn-based ELISA was successfully established for the detection of SFTSV-specific antibodies in serum samples from naturally infected monkeys. As validated with the reference method, the specificity and sensitivity of rGn-ELISA were 94% and 96%, respectively. In conclusion, utilizing well-suited fusion tags facilitates rGn production and purification in substantial quantities while preserving its antigenic properties. The rGn-ELISA, characterized by its commendable sensitivity and specificity could serve as a viable alternative diagnostic method for assessing SFTSV seroprevalence. KEY POINTS: • SFTSV Gn, fused with secretory signal tags, was expressed by the BaMV-based vector. • The plant fusion tags increased expression levels and eased the purification of rGn. • The rGn-ELISA was established and validated; its specificity and sensitivity > 94%.

Azithromycin, a potent autophagy inhibitor for cancer therapy, perturbs cytoskeletal protein dynamics.

BACKGROUND: Autophagy plays an important role in tumour cell growth and survival and also promotes resistance to chemotherapy. Hence, autophagy has been targeted for cancer therapy. We previously reported that macrolide antibiotics including azithromycin (AZM) inhibit autophagy in various types of cancer cells in vitro. However, the underlying molecular mechanism for autophagy inhibition remains unclear. Here, we aimed to identify the molecular target of AZM for inhibiting autophagy. METHODS: We identified the AZM-binding proteins using AZM-conjugated magnetic nanobeads for high-throughput affinity purification. Autophagy inhibitory mechanism of AZM was analysed by confocal microscopic and transmission electron microscopic observation. The anti-tumour effect with autophagy inhibition by oral AZM administration was assessed in the xenografted mice model. RESULTS: We elucidated that keratin-18 (KRT18) and α/ß-tubulin specifically bind to AZM. Treatment of the cells with AZM disrupts intracellular KRT18 dynamics, and KRT18 knockdown resulted in autophagy inhibition. Additionally, AZM treatment suppresses intracellular lysosomal trafficking along the microtubules for blocking autophagic flux. Oral AZM administration suppressed tumour growth while inhibiting autophagy in tumour tissue. CONCLUSIONS: As drug-repurposing, our results indicate that AZM is a potent autophagy inhibitor for cancer treatment, which acts by directly interacting with cytoskeletal proteins and perturbing their dynamics.

Deletion of gene OV132 attenuates Orf virus more effectively than gene OV112.

Orf virus (ORFV), a Parapoxvirus in Poxviridae, infects sheep and goats resulting in contagious pustular dermatitis. ORFV is regarded as a promising viral vector candidate for vaccine development and oncolytic virotherapy. Owing to their potential clinical application, safety concerns have become increasingly important. Deletion of either the OV132 (encoding vascular endothelial growth factor, VEGF) or OV112 (encoding the chemokine binding protein, CBP) genes reduced ORFV infectivity, which has been independently demonstrated in the NZ2 and NZ7 strains, respectively. This study revealed that the VEGF and CBP gene sequences of the local strain (TW/Hoping) shared a similarity of 47.01% with NZ2 and 90.56% with NZ7. Due to the high sequence divergence of these two immunoregulatory genes among orf viral strains, their contribution to the pathogenicity of Taiwanese ORFV isolates was comparatively characterized. Initially, two ORFV recombinants were generated, in which either the VEGF or CBP gene was deleted and replaced with the reporter gene EGFP. In vitro assays indicated that both the VEGF-deletion mutant ORFV-VEGFΔ-EGFP and the CBP deletion mutant ORFV-CBPΔ-EGFP were attenuated in cells. In particular, ORFV-VEGFΔ-EGFP significantly reduced plaque size and virus yield compared to ORFV-CBPΔ-EGFP and the wild-type control. Similarly, in vivo analysis revealed no virus yield in the goat skin biopsy infected by ORFV-VEGFΔ-EGFP, and significantly reduced the virus yield of ORFV-CBPΔ-EGFP relative to the wild-type control. These results confirmed the loss of virulence of both deletion mutants in the Hoping strain, whereas the VEGF-deletion mutant was more attenuated than the CBP deletion strain in both cell and goat models. KEY POINTS: • VEGF and CBP genes are crucial in ORFV pathogenesis in the TW/Hoping strain • The VEGF-deletion mutant virus was severely attenuated in both cell culture and animal models • Deletion mutant viruses are advantageous vectors for the development of vaccines and therapeutic regimens.

Anisotropic Crystal Growth of Layered Nickel Hydroxide along the Stacking Direction Using Amine Ligands.

Edge surfaces of two-dimensional crystals play crucial roles in their properties, such as intercalation behavior and catalytic activities; however, reports on the preparation of crystals with a high aspect ratio of thickness to lateral size, typically a prism-like crystal morphology composed of stacked layers, are scarce. We report the anisotropic crystal growth of ß-Ni(OH)2 along the stacking direction using bidentate amine ligands, which act as both the base and the reservoir of Ni2+ through the formation of Ni-diamine complexes. Various characterization results of the crystal structure, composition, and crystal orientation indicate the formation of hexagonal prisms of ß-Ni(OH)2 with an unusually high aspect ratio of the thickness to the lateral size higher than 1. A systematic investigation focusing on the molar ratio of amine ligands to Ni2+, the concentration of Ni-diamine complexes, and stability constants of the complexes revealed that anisotropic growth was promoted when the supersaturation was relatively high and was maintained constant for a long time. We clarified the role of amine ligands in controlling supersaturation through the controlled release of metal ions from stable complexes. ß-Co(OH)2 with a hexagonal prism shape was prepared using this protocol. This study provides valuable indications for developing synthetic chemistry for various layered compounds to achieve a controlled aspect ratio.

BRCA1 degradation in response to mitochondrial damage in breast cancer cells.

BRCA1 is a well-studied tumor suppressor involved in the homologous repair of DNA damage, whereas PINK1, a mitochondrial serine/threonine kinase, is known to be involved in mitochondrial quality control. Genetic mutations of PINK1 and Parkin cause autosomal recessive early-onset Parkinson's disease. We found that in breast cancer cells, the mitochondrial targeting reagents, which all induce mitochondrial depolarization along with PINK1 upregulation, induced proteasomal BRCA1 degradation. This BRCA1 degradation was dependent on PINK1, and BRCA1 downregulation upon mitochondrial damage caused DNA double-strand breaks. BRCA1 degradation was mediated through the direct interaction with the E3 ligase Parkin. Strikingly, BRCA1 and PINK1/Parkin expression were inversely correlated in cancerous mammary glands from breast cancer patients. BRCA1 knockdown repressed cancer cell growth, and high BRCA1 expression predicted poor relapse-free survival in breast cancer patients. These observations indicate a novel mechanism by which mitochondrial damage is transmitted to the nucleus, leading to BRCA1 degradation.

LOCAL LEVELS OF RADIATION EXPOSURE DOSES DUE TO RADIOCESIUM FOR RETURNED RESIDENTS IN TOMIOKA TOWN, FUKUSHIMA PREFECTURE.

Tomioka Town is located within a 20-km radius of the Fukushima Daiichi Nuclear Power Station. Radiation dose rates due to radiocesium in residents' living spaces were evaluated from the measurements of ambient dose rates and environmental samples after returning home. The mean ambient dose rates were 0.15-0.18-µSv/h indoors and 0.23-0.26-µSv/h outdoors during 2018 and 2019, and the additional radiation dose rates were calculated to be 1.4 mSv/y in 2018 and 1.1 mSv/y in 2019. Ambient dose equivalent from surface soils within housing sites were estimated to be 0.66 mSv/y in 2018 and 0.54 mSv/y in 2019. Moreover, committed effective doses from local foods were calculated in 19-74 µSv/y for children and 39-100 µSv/y for adults during 2018 and 2019. These findings suggest that current radiation exposure doses have been controlled at the levels close to the public dose limit (1 mSv/y) in residents' living spaces.

Risk perception regarding implementation of iodine thyroid blocking during a nuclear disaster of mothers living near a nuclear power station in Japan.

Pre-emptive evacuation orders following the accident at the Fukushima Daiichi Nuclear Power Station (FDNPS) in March 2011 and subsequent regulatory limits regarding contaminated food, milk, and water minimized the external and internal radiation exposure doses of nearby residents. However, with regard to implementation of iodine thyroid blocking (ITB), residents were confused because no information on the matter was released by the central and/or local governments. Based on lessons learned from the FDNPS accident, many countries have revised their guidelines regarding ITB during nuclear disasters. To adequately revise such guidelines and ensure effective ITB implementation during a nuclear disaster, however, residents' perceptions of ITB must be clarified. In this study, the perception of risks associated with ITB was investigated in mothers residing near the Sendai Nuclear Power Plant (SNPP) in Kagoshima Prefecture, Japan. Of the 520 mothers surveyed, 467 (89.8%) expressed anxiety regarding the administration of potassium iodine (KI) to their children. Logistic regression analysis revealed that the mothers' anxiety regarding the administration of KI to their children was positively correlated with their wish to consult an expert about KI and their hesitation to let their children eat foods produced in Fukushima, and negatively correlated with having confidence about administering KI to their children. Careful communication of potential risks to mothers residing near nuclear power plants is thus critical for implementing effective ITB in children.

Establishment and characterization of transformed goat primary cells by expression of simian virus 40 large T antigen for orf virus propagations.

Due to the limited host range of orf virus (ORFV), primary cells derived from its natural hosts, such as goats and sheep, are recommended for isolation and propagation of wild type ORFV. This situation limits the option for the study of virus-host interaction during ORFV infection since primary cells only support a few numbers of passages. SV40 T antigen is a viral oncoprotein that can abrogate replicative senescence, leading to an extended life span of cells. In this study, the transformation of two goat primary cells, fibroblast (FB) and testis (GT) cells, were achieved by stably expressing SV40 T antigen using the lentiviral technique. The presence of the gene encoding SV40 T antigen was validated by polymerase chain reaction (PCR) and western blot analyses. As evidenced by immunofluorescent microscopy, the two types of cells expressing SV40 T antigen (namely, FBT and GTT) were purified to homogeneity. Moreover, faster growth kinetics and a lower serum dependency were noticed in FBT and GTT, as compared with their counterpart parental cells. FBT and GTT remain permissive and can form plaque of ORFV, despite with different profiles; generally speaking, with SV40 T expression, ORFV forms plaques with smaller size and distinct margin. Most importantly, the prolonged life span of goat FBT and GTT serves as an ideal cell culture resource for ORFV isolation from the field, studies of ORFV pathogenesis and efficient vaccine development.

Evaluation of Environmental Contamination and Estimated Radiation Exposure Dose Rates among Residents Immediately after Returning Home to Tomioka Town, Fukushima Prefecture.

On 1 April 2017, six years have passed since the Fukushima Daiichi Nuclear Power Station (FDNPS) accident, and the Japanese government declared that some residents who lived in Tomioka Town, Fukushima Prefecture could return to their homes. We evaluated environmental contamination and radiation exposure dose rates due to artificial radionuclides in the livelihood zone of residents (living space such as housing sites), including a restricted area located within a 10-km radius from the FDNPS, immediately after residents had returned home in Tomioka town. In areas where the evacuation orders had been lifted, the median air dose rates were 0.20 µSv/h indoors and 0.26 µSv/h outdoors, and the radiation exposure dose rate was 1.6 mSv/y. By contrast, in the "difficult-to-return zone," the median air dose rate was 2.3 µSv/h (20 mSv/y) outdoors. Moreover, the dose-forming artificial radionuclides (radiocesium) in the surface soil were 0.018 µSv/h (0.17 mSv/y) in the evacuation order-lifted areas and 0.73 µSv/h (6.4 mSv/y) in the difficult-to-return zone. These findings indicate that current concentrations of artificial radionuclides in soil samples have been decreasing in the evacuation order-lifted areas of Tomioka town; however, a significant external exposure risk still exists in the difficult-to-return zone. The case of Tomioka town is expected to be the first reconstruction model including the difficult-to-return zone.

Adenosine Deaminase Acting on RNA 1 Associates with Orf Virus OV20.0 and Enhances Viral Replication.

Orf virus (ORFV) infects sheep and goats and is also an important zoonotic pathogen. The viral protein OV20.0 has been shown to suppress innate immunity by targeting the double-stranded RNA (dsRNA)-activated protein kinase (PKR) by multiple mechanisms. These mechanisms include a direct interaction with PKR and binding with two PKR activators, dsRNA and the cellular PKR activator (PACT), which ultimately leads to the inhibition of PKR activation. In the present study, we identified a novel association between OV20.0 and adenosine deaminase acting on RNA 1 (ADAR1). OV20.0 bound directly to the dsRNA binding domains (RBDs) of ADAR1 in the absence of dsRNA. Additionally, OV20.0 preferentially interacted with RBD1 of ADAR1, which was essential for its dsRNA binding ability and for the homodimerization that is critical for intact adenosine-to-inosine (A-to-I)-editing activity. Finally, the association with OV20.0 suppressed the A-to-I-editing ability of ADAR1, while ADAR1 played a proviral role during ORFV infection by inhibiting PKR phosphorylation. These observations revealed a new strategy used by OV20.0 to evade antiviral responses via PKR.IMPORTANCE Viruses evolve specific strategies to counteract host innate immunity. ORFV, an important zoonotic pathogen, encodes OV20.0 to suppress PKR activation via multiple mechanisms, including interactions with PKR and two PKR activators. In this study, we demonstrated that OV20.0 interacts with ADAR1, a cellular enzyme responsible for converting adenosine (A) to inosine (I) in RNA. The RNA binding domains, but not the catalytic domain, of ADAR1 are required for this interaction. The OV20.0-ADAR1 association affects the functions of both proteins; OV20.0 suppressed the A-to-I editing of ADAR1, while ADAR1 elevated OV20.0 expression. The proviral role of ADAR1 is likely due to the inhibition of PKR phosphorylation. As RNA editing by ADAR1 contributes to the stability of the genetic code and the structure of RNA, these observations suggest that in addition to serving as a PKR inhibitor, OV20.0 might modulate ADAR1-dependent gene expression to combat antiviral responses or achieve efficient viral infection.

[The importance of cooperation between nurse and registered dietician toward a QOL improvement of the terminal stage patient--a case of advanced esophageal cancer].

As for the esophageal terminal stage patient with enteral feeding when the patient was hospitalized, an oral nourishment intake support was given with a cooperation of nurse and registered dietician. After that, the patient was transferred to home care and was seceded from PEG. It has been suggested that an early assessment of the problems with the terminal stage patient, ascertainment of home care timing and the team support consisted of multi professionals were all important ingredients.

Progesterone: its occurrence in plants and involvement in plant growth.

Progesterone is a mammalian gonadal hormone. In the current study, we identified and quantified progesterone in a range of higher plants by using GC-MS and examined its effects on the vegetative growth of plants. The growth of Arabidopsis (Arabidopsis thaliana) seedlings was promoted by progesterone at low concentrations but suppressed at higher concentrations under both light and dark growth conditions. The growth of the gibberellin-deficient mutant lh of pea (Pisum sativum) was also promoted by progesterone. An earlier study demonstrated that progesterone binds to MEMBRANE STEROID BINDING PROTEIN 1 (MSBP1) of Arabidopsis. In this work, we cloned the homologous genes of Arabidopsis, MSBP2 and STEROID BINDING PROTEIN (SBP), as well as of rice (Oryza sativa), OsMSBP1, OsMSBP2 and OsSBP and examined their expression in plant tissues. All of these genes, except OsMSBP1, were expressed abundantly in plant tissues. The roles of progesterone in plant growth were also discussed.