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Carotenoids are not naturally accumulated in the endosperms of rice, which is a cereal crop. In our previous study, biofortified rice seeds accumulated two red color carotenoids, ketocarotenoids including astaxanthin and ketoxanthophylls including capsanthin, via four-step pathway engineering. Their biosynthetic pathways require zeaxanthin as a common precursor and then bifurcated. In this study, cross-fertilization between two astaxanthin (BP and sBP) and two capsanthin (C and CB) rice plants were reciprocally performed to elucidate the rice seed system’s metabolic preference. Filial seeds showed different colors in red hues between mutually interbred lines as well as from parents. The strongest driving force for metabolism was caused by the sBP line for astaxanthin regardless of the parent side and proved to be the most efficacious customized synthetic gene for rice crops. It further resulted in the highest astaxanthin levels when paternal sBP mated with maternal CB line, which has the biggest capacity of total carotenoids. Also, reciprocal crosses between CB and BP lines ascertained their metabolically balanced gene efficacy by showing the pathway preference toward either ketoxanthophylls or ketocarotenoids depending on the paternal parental side. Our study suggests that gene efficacy plays the most decisive role in leading the metabolic path and revealed the unexpectedly paternal parent-of-origin effects in the endosperm. Consequently, we simultaneously biofortified rice crops with two functional red carotenoids and finally generated new red-colored rice varieties.

Ketocarotenoid–biofortified crops are required as natural additives with respect to their Rubi-like reddish color and strong antioxidative properties for foods and feeds. They can be an alternative means of microalgal and bacterial systems and do not depend on chemical synthesis, providing safe, sustainable, and processing-free platforms. To reach sufficient commercial demands, different metabolic engineering strategies have been used in diverse crop plants. In this study, we summarized the current achievements for de novo production of ketocarotenoids in major crops and described host crop-specific tactics to optimize the ketocarotenoid biosynthetic pathway, considering the following different levels and types of carotenoid being accumulated as precursors: lutein and zeaxanthin in maize seeds; lutein in rice seeds, canola seeds, soybean seeds, and tobacco flowers; zeaxanthin in potato tubers; β–carotene in carrot roots; and lycopene in tomato fruits. This study suggests prospects for the advanced performance of ketocarotenoid biofortification in crops in two crucial points: host crop cultivar and the elaborately designed genetic strategies. The latter includes an increase in the total carotenoid capacity by co-consideration to supply precursors and stabilizers, the prevention of metabolite loss in competing or catabolic pathways or both pathways, and the sensible choice of genes and genetic elements for optimal expression. Furthermore, progresses in synthetic biology and new biotechnology, such as genome editing, could accelerate the development of high-valued “Rubi Crops,” which satisfy the nutritional and functional needs of humans and animals.

Yes-associated protein (YAP)/WW domain-containing transcription factor (TAZ) is critical for cell proliferation, survival, and self-renewal. It has been shown to play a crucial oncogenic role in many different types of tumors. In this study, we investigated the antitumor effect of the extracts of Perilla frutescens var. acuta (Odash.) Kudo leaves (PLE) on Hippo-YAP/TAZ signaling. PLE induced the phosphorylation of YAP/TAZ, thereby inhibiting their activity. In addition, the treatment suppresses YAP/TAZ transcriptional activity via the dissociation of the YAP/TAZ-TEAD complex. To elucidate the molecular mechanism of PLE in the regulation of YAP activity, we treated WT and cell lines with gene knockout (KO) for Hippo pathway components with PLE. The inhibitory effects of PLE on YAP-TEAD target genes were significantly attenuated in LATS1/2 KO cells. Moreover, we found the antitumor effect of PLE on MDA-MB-231 and BT549, both of which are triple-negative breast cancer (TNBC) cell lines. PLE reduced the viability of TNBC cells in a dose-dependent manner and induced cell apoptosis. Further, PLE inhibited the migration ability in MDA-MB-231 cells. This ability was weakened in YAP and TEAD-activated clones suggesting that the inhibition of migration by PLE is mainly achieved by regulating YAP activity. Taken together, the results of this study indicate that PLE suppressed cell growth and increased the apoptosis of breast cancer (BC) cells via inactivation of YAP activity in a LATS1/2-dependent manner.

We identified clade 2.3.4.4 highly pathogenic avian influenza A(H5N6) viruses from whooper swans (Cygnus cygnus) found dead in Mongolia. The identification of these infections in wild birds in this area is of concern because of the potential for virus dissemination during fall migration.

To increase nutritional values as dietary sources of provitamin A and health-promoting antioxidants in rice grains, carotenoids have been biofortified in a Golden Ricelike variety, the stPAC (stPsy-2A-stCrtI) rice. In both of non-transgenic (NT) and stPAC seeds, total chlorophylls and carotenoids were gradually decreased during seed development while de novo biosynthesized carotenoids being comprised mainly of β-carotene, lutein and zeaxanthin were accumulated from early stage of 10 DAF and peaked at 20 DAF in stPAC seeds. The de novo production of carotenoids coincided with the high levels of transgene expression driven by the rice globulin gene promoter. Interestingly, expression levels of endogenous carotenoid metabolic pathway genes were the highest at 30 DAF in NT seeds whereas they were generally down-regulated in stPAC seeds, suggesting a negative feedback control mechanism of carotenoid metabolism by enhanced carotenoid production. The transgenic protein levels in stPAC seeds were not changed much during seed storage for up to 5 years, while carotenoid contents in the seeds were decreasing after 1 year of storage. The decrease in carotenoid contents was restored when the transgenic plants re-grown, supporting the reliability of transgenic pathways for carotenoid biofortification in rice grains. Thus, our results showed that transgene-driven biofortification of carotenoids was made and maintained over several transgenic generations with a possible negative feed-back control of endogenous carotenoid metabolism during seed development.

Since 2014, H5Nx clade 2.3.4.4 highly pathogenic avian influenza viruses (HPAIV) have caused outbreaks in wild birds and poultry in multiple continents, including Asia, Europe, Africa, and North America. Wild birds were suspected to be the sources of the local and global spreads of HPAIV. This study evaluated the infectivity, pathogenicity, and transmissibility of clade 2.3.4.4 H5N6 HPAIV in mandarin ducks (Aixgalericulata) and domestic pigeons (Columbia livia domestica). None of the birds used in this study, 20 mandarin ducks or 8 pigeons, showed clinical signs or mortality due to H5N6 HPAI infection. Two genotypes of H5N6 HPAIV showed replication and transmission by direct and indirect contact between mandarin ducks. H5N6 HPAIV replicated and transmitted by direct contact between pigeons, although the viral shedding titer and duration were relatively lower and shorter than those in mandarin ducks. Influenza virus antigen was detected in various internal organs of infected mandarin ducks and pigeons, indicating systemic infection. Therefore, our results indicate mandarin ducks and pigeons can be subclinically infected with clade 2.3.4.4 H5N6 HPAIV and transfer the virus to adjacent birds. The role of mandarin ducks and pigeons in the spread and prevalence of clade 2.3.4.4 H5N6 viruses should be carefully monitored.

Foot-and-mouth disease virus (FMDV) 2A constructs have been successfully used for the production of “Golden Rice”, a β-carotene producing rice strain. However, to allay public fears and opposition to plants carrying a mammalian pathogenic viral sequence, 2A-like synthetic sequences from Thosea asigna virus and Infectious myonecrosis virus were used to coordinate the coexpression of carotenoid biosynthetic genes. Here, up to four carotenogenic genes encoding PSY, CRTI, BCH and BKT were concatenated and produced β-carotene, zeaxanthin, and ketocarotenoids (astaxanthin and adonixanthin) in transgenic rice seeds displaying color variation due to the difference in carotenoid content and composition., Highlights • Carotenogenic genes upto four were concatenated via three 2A-like peptides into single cistrons. • Polycistrons stepwisely biosynthesized β-carotene, zeaxanthin and ketocarotenoids in rice. • A Thosea asigna viral 2A peptide is a good alternative to FMDV 2A for plant biotechnology.

We report the first detection of Y280-lineage H9N2 avian influenza viruses in live bird markets in Korea during July 2020. The viruses were isolated from domestic ducks and chickens traded in three markets in two different provinces, indicating dispersal of the newly introduced viruses. Complete genome sequencing and comparative phylogenetic analyses of all eight gene segments of the viruses showed high nucleotide homology to a Y280-lineage H9N2 avian influenza virus isolated in a chicken farm in China, which belongs to one of the most prevalent H9N2 genotypes in China. Increasing human cases of the same genotype H9N2 infection in China and the mammalian specific markers present in the viruses isolated suggest potential implications for public health.

In October 2020, a highly pathogenic avian influenza (HPAI) subtype H5N8 virus was identified from a fecal sample of a wild mandarin duck (Aix galericulata) in South Korea. We sequenced all eight genome segments of the virus, designated as A/Mandarin duck/Korea/K20-551-4/2020(H5N8), and conducted genetic characterization and comparative phylogenetic analysis to track its origin. Genome sequencing and phylogenetic analysis show that the hemagglutinin gene belongs to H5 clade 2.3.4.4 subgroup B. All genes share high levels of nucleotide identity with H5N8 HPAI viruses identified from Europe during early 2020. Enhanced active surveillance in wild and domestic birds is needed to monitor the introduction and spread of HPAI via wild birds and to inform the design of improved prevention and control strategies.

Highly pathogenic avian influenza viruses (HPAIVs) and low pathogenic avian influenza viruses (LPAIVs) represent important threats to the poultry industry and global human health. Due to the high rates of avian influenza virus (AIV) transmission, controlling AIV outbreaks is challenging. HPAIV is known to be transmitted from wild birds to domestic ducks, from which it can be transmitted to layer and broiler chickens. Therefore, surveillance of AIV in domestic ducks and chickens in advance of outbreaks can prevent its spread and enable timely implementation of disease control measures. Certain molecular diagnostic tools can be applied in the field for faster AIV detection. In this study, we evaluated the AIV-detection ability of two insulated isothermal PCR (iiPCR) devices, POCKIT™Micro DUO Nucleic Acid Analyzer (POCKIT DUO) and POCKIT™ Central Nucleic Acid Analyzer (POCKIT Central). We found that the analytical, in vivo and clinical performances of the two POCKIT devices were comparable to those of real-time reverse transcription PCR. Due to their brief protocols and short detection times, POCKIT DUO and POCKIT Central represent promising molecular diagnostic devices for the reliable detection of AIV.

Dynamin-related protein 1 (DRP1) is a mitochondrial membrane GTPase and regulates mitochondrial fission. In this study, we found that the cytokine RANKL increased the expression of DRP1 and its receptor proteins, Fis1, Mid49, and Mid 51, during osteoclast formation in mouse bone marrow-derived macrophages. Inactivation of the kinase GSK3β appeared to induce DRP1 expression. DRP1 knockdown or the DRP1 inhibitor Mdivi1 suppressed osteoclast differentiation via downregulation of c-Fos and NFATc1, the key transcription factor for osteoclast formation. Finally, the DRP1 inhibitor suppressed lipopolysaccharide-induced osteoclast formation in a calvarial model and ovariectomy-induced bone loss in vivo. Taken together, our data demonstrate that DRP1 positively contributes to RANKL-induced osteoclast differentiation by regulating the c-Fos-NFATc1 axis, suggesting the importance of mitochondrial DRP1 in osteoclastogenesis.

Renal fibrosis is one of the characteristic features of chronic kidney disease (CKD). Fibrotic change not only impairs the filtration function of the kidney but is also recognized as a marker of end-stage renal disease (ESRD). The epithelial to mesenchymal transition (EMT) is known to play a role in embryonic development and organ formation, but it is getting much attention for its pathological role in the invasion and metastasis of carcinoma. Recently, it has also been reported that EMT plays a role in the formation of fibrosis during chronic inflammation. EMT contribute to the development of the fibrosis in CKD. Moreover, glomerular podocytes and tubular epithelial cells can also undergo mesenchymal transition in CKD. Hesperetin is a flavonoid present in citrus and is well known for its antioxidant and anti-inflammatory properties. In this study, we investigated the effects of hesperetin on the EMT-elicited podocytes. First, we generated an EMT model by treating transforming growth factor (TGF)-β1, a potent inducer of EMT to the podocytes. TGF-β1 decreased the expression of epithelial markers such as nephrin, zonula occludens-1 (ZO-1), while it increased the mesenchymal markers, including fibronectin (FN), vimentin, and α-smooth muscle actin (α-SMA) in the podocytes. Hesperetin suppressed EMT-like changes elicited by TGF-β1. Interestingly, hesperetin did not interfere with the Smad signaling-the classical TGF-β signaling-pathway, which was confirmed by the experiment with smad 2/3 −/− podocytes. Instead, hesperetin suppressed EMT-like changes by inhibiting the mTOR pathway-one of the alternative TGF-β signaling pathways. In conclusion, hesperetin has a protective effect on the TGF-β1 elicited EMT-like changes of podocytes through regulation of mTOR pathway. It could be a good candidate for the suppression of kidney fibrosis in various CKD.

The wide geographic spread of Eurasian Goose/Guangdong lineage highly pathogenic avian influenza (HPAI) clade 2.3.4.4 viruses by wild birds is of great concern. In December 2014, an H5N8 HPAI clade 2.3.4.4 Group A (2.3.4.4A) virus was introduced to North America. Long-distance migratory wild aquatic birds between East Asia and North America, such as Northern Pintail (Anas acuta), were strongly suspected of being a source of intercontinental transmission. In this study, we evaluated the pathogenicity, infectivity and transmissibility of an H5N8 HPAI clade 2.3.4.4A virus in Northern Pintails and compared the results to that of an H5N1 HPAI clade 2.3.2.1 virus. All of Northern Pintails infected with either H5N1 or H5N8 virus lacked clinical signs and mortality, but the H5N8 clade 2.3.4.4 virus was more efficient at replicating within and transmitting between Northern Pintails than the H5N1 clade 2.3.2.1 virus. The H5N8-infected birds shed high titre of viruses from oropharynx and cloaca, which in the field supported virus transmission and spread. This study highlights the role of wild waterfowl in the intercontinental spread of some HPAI viruses. Migratory aquatic birds should be carefully monitored for the early detection of H5 clade 2.3.4.4 and other HPAI viruses.

An avian influenza A(H6N5) virus with all 8 segments of North American origin was isolated from wild bird feces in South Korea. Phylogenetic analysis suggests that this virus may have been introduced into Asia by wild birds, highlighting the role of wild birds in the dispersal of these viruses.

Although critical in phagocytosis in innate immunity, reactive oxygen species (ROS) collaterally inflict damage to host phagocytes because they indiscriminate targets. Since Nrf2 increases the expression of anti-oxidant enzymes that nullifies ROS, ROS activating Nrf2 is a critical negative regulatory step for countering the deleterious effects of ROS. Here, we postulate whether, along with ROS activating Nrf2, NADPH oxidase components also participate in direct activation of Nrf2, contributing to protection from ROS. Our results show that the p47phox of the NADPH oxidase, but not p65phox or p40phox, physically binds to Nrf2, activating the Nrf2 function. p47phox binding to Nrf2/Keap1 complex suppresses the ubiquitination of Nrf2, while p47phox becomes ubiquitinated by Keap1. p47phox increases the nuclear translocation of Nrf2 and the expression of Nrf2-dependent genes, whereas genetic ablation of p47phox decreases the expression of those genes. In a lipopolysaccharide-induced acute lung inflammation mouse model, selective expression of p47phox in mouse lungs induces the expression of Nrf2-dependent genes and is sufficient to suppress neutrophilic lung inflammation. Therefore, our findings suggest that p47phox is a novel regulator of Nrf2 function.

A β-carotene is the most well-known dietary source as provitamin A carotenoids. Among β-carotene-producing Golden Rice varieties, PAC (Psy:2A:CrtI) rice has been previously developed using a bicistronic recombinant gene that linked the Capsicum Psy and Pantoea CrtI genes by a viral 2A sequence. To enhance β-carotene content by improving this PAC gene, its codon was optimized for rice plants (Oryza sativa L.) by minimizing the codon bias between the transgene donor and the host rice and was then artificially synthesized as stPAC (stPsy:2A:stCrtI) gene. The GC content (58.7 from 50.9%) and codon adaptation index (0.85 from 0.77) of the stPAC gene were increased relative to the original PAC gene with 76% DNA identity. Among 67 T1 seeds of stPAC transformants showing positive correlations between transgene copy numbers (up to three) and carotenoid contents, three stPAC lines with a single intact copy were chosen to minimize unintended insertional effects and compared to the representative line of the PAC transgene with respect to their codon optimization effects. Translation levels were stably increased in all three stPAC lines (3.0-, 2.5-, 2.9-fold). Moreover, a greater intensity of the yellow color of stPAC seeds was correlated with enhanced levels of β-carotene (4-fold, 2.37 μg/g) as well as total carotenoid (2.9-fold, 3.50 μg/g) relative to PAC seeds, suggesting a β-branch preference for the stPAC gene. As a result, the codon optimization of the transgene might be an effective tool in genetic engineering for crop improvement as proven at the enhanced levels of translation and carotenoid production.

A reassortant clade 2.3.4.4 avian influenza A(H5N6) virus was isolated from a fecal sample of a Mandarin duck (Aix galericulata) in South Korea during October 2016. This virus was genetically similar to H5N6 subtype virus isolates from China, Vietnam, Laos, and Hong Kong, including human isolates.

Wild birds play a major role in the evolution, maintenance, and dissemination of highly pathogenic avian influenza viruses (HPAIV). Sub-clinical infection with HPAI in resident wild birds could be a source of dissemination of HPAIV and continuous outbreaks. In this study, the pathogenicity and infectivity of two strains of H5N8 clade 2.3.4.4 virus were evaluated in the Mandarin duck (Aix galericulata) and domestic pigeon (Columba livia domestica). None of the birds experimentally infected with H5N8 viruses showed clinical signs or mortality. The H5N8 viruses efficiently replicated in the virus-inoculated Mandarin ducks and transmitted to co-housed Mandarin ducks. Although relatively high levels of viral shedding were noted in pigeons, viral shedding was not detected in some of the pigeons and the shedding period was relatively short. Furthermore, the infection was not transmitted to co-housed pigeons. Immunohistochemical examination revealed the presence of HPAIV in multiple organs of the infected birds. Histopathological evaluation showed the presence of inflammatory responses primarily in HPAIV-positive organs. Our results indicate that Mandarin ducks and pigeons can be infected with H5N8 HPAIV without exhibiting clinical signs; thus, they may be potential healthy reservoirs of the H5N8 HPAIV.

We isolated new reassortant avian influenza A(H5N6) viruses from feces of wild waterfowl in South Korea during 2017–18. Phylogenetic analysis suggested that reassortment occurred between clade 2.3.4.4b H5N8 and Eurasian low pathogenicity avian influenza viruses circulating in wild birds. Dissemination to South Korea during the 2017 fall migratory season followed.

ER stress is an early event of acute kidney injury and has been linked to accelerate the development of chronic kidney disease. Therefore, the compounds that can mimic ER stress inhibitor may confer regulatory effects on ER stress induced apoptosis. In this study, we investigated the protective effects of flavonoid morin against ER stress induced apoptosis in human renal proximal tubular HK-2 cells. Morin downregulated the expression of GRP78, central regulator of ER stress response, induced by ER stress inducer tunicamycin. Interestingly, morin selectively inhibited the IRE1 pathway among the three major arms of the ER stress responses. The increased expression of XBP1-sp, phosphor-IRE-1α, and phosphor-JNK by TM were markedly suppressed by the pretreatment of morin. Morin also decreased the intracellular ROS production and the apoptosis induced by TM in HK-2 cells. Taken together, our finding show that morin acts as an ER stress inhibitor, and can be a good candidate in various ER-stress associated kidney diseases.

Context:Eclipta prostrata L. (Asteraceae) (EP) has been widely used for the treatment of skin disease in Asian traditional medicine. Objective: This study investigates the potency of EP in promoting hair growth in vivo and in vitro. Materials and methods: C57BL/6N mice were divided into four groups (n = 4) as follows: control (topical treatment of normal saline), topical 3% minoxidil to the dorsal skin of mice for 14 days, and low (1 mg/day) and high (10 mg/day) doses of EP orally administered once a day for 14 days. Dorsal hairs of C57BL/6N mice were depilated to synchronize anagen induction. Hair growth activity was evaluated by gross and microscopic observations. Sections of dorsal skin were stained with haematoxylin and eosin. We also treated the various concentrations of EP (5, 10 and 50 ��g/mL) for 24 h on the human dermal papilla cells (HDPs) and examined the effects of EP on the expression of FGF-7 and mTOR signalling. Results: EP enhanced the induction of anagen in the dorsal skin of mice, characterized by the appearance of inner root sheath along with hair shaft, the emergence of hair shaft through the epidermis. EP increased the expression of FGF-7, while decreased the level of FGF-5 in C57/BL6 mice. EP also increased the expression of FGF-7, activated the mTOR signalling in HDPs. Discussion and conclusions: These results suggest that EP has a potency to enhance the growth of hair follicle, promoting hair growth through regulation of FGF-7 and FGF-5.

The Hippo pathway controls organ size and tissue homeostasis, with deregulation leading to cancer. The core Hippo components in mammals are composed of the upstream serine/threonine kinases Mst1/2, MAPK4Ks and Lats1/2. Inactivation of these upstream kinases leads to dephosphorylation, stabilization, nuclear translocation and thus activation of the major functional transducers of the Hippo pathway, YAP and its paralogue TAZ. YAP/TAZ are transcription co-activators that regulate gene expression primarily through interaction with the TEA domain DNA-binding family of transcription factors (TEAD). The current paradigm for regulation of this pathway centres on phosphorylation-dependent nucleocytoplasmic shuttling of YAP/TAZ through a complex network of upstream components. However, unlike other transcription factors, such as SMAD, NF-κB, NFAT and STAT, the regulation of TEAD nucleocytoplasmic shuttling has been largely overlooked. In the present study, we show that environmental stress promotes TEAD cytoplasmic translocation via p38 MAPK in a Hippo-independent manner. Importantly, stress-induced TEAD inhibition predominates YAP-activating signals and selectively suppresses YAP-driven cancer cell growth. Our data reveal a mechanism governing TEAD nucleocytoplasmic shuttling and show that TEAD localization is a critical determinant of Hippo signalling output.

Ethnopharmacological relevance Mahaenggamseok-tang (MHGST), an herbal formula in traditional Asian medicine, has been used to treat patients with various pulmonary diseases including common cold and influenza. However, the potential therapeutic effect of MHGST on acute lung injury (ALI), a leading cause of death worldwide, and the anti-inflammatory mechanisms of MHGST remained less understood. Materials and methods The methanol extract of MHGST was prepared and fingerprinted by HPLC. For the induction of ALI, C57BL/6 mice (n=5/group) received a single intraperitoneal (i.p.) injection of LPS. Referring to the dose for patients, two different amounts of MHGST were delivered in an aerosol to mouse lungs via trachea 2 h after the i.p. LPS administration. Lung histology, bronchoalveolar lavage fluid, myeloperoxidase (MPO) activity, and the expression of inflammatory and Nrf2-dependent genes were analyzed to determine the effect of MHGST on lung inflammation. For mechanistic studies, western blotting and semi-quantitative RT-PCR were conducted using RAW 264.7 cells. Results When administered 2 h after the onset of ALI, MHGST relieved lung pathology characteristic to ALI, with decreases of neutrophil infiltration and MPO activity. While suppressing the expression of inflammatory genes, MHGST increased the expression of Nrf2-dependent genes in ALI mouse lungs. Concordantly, MHGST activated Nrf2 activity while suppressing NF-κB in RAW 264.7 cells. Conclusion MHGST suppressed neutrophilic lung inflammation, a hallmark of ALI, which was associated with the activation of anti-inflammatory Nrf2 and the suppression of pro-inflammatory NF-κB. Our results suggest that MHGST has a therapeutic potential against ALI.

Highly pathogenic avian influenza (HPAI) viruses cause economic losses in the poultry industry and pose a severe threat to human health. Rapid and accurate diagnostic methods are important because they can help prevent further spread of the virus and reduce the time required for eradication of the virus. We developed a low-density microarray for the rapid detection and identification of avian influenza virus subtypes H5, H7, and H9 and their pathotypes in a previous study. In the present study, we report the development of updated probe sets and evaluation of the diagnostic microarray using H5N8 clade 2.3.4.4 HPAI viruses including clinical samples, without the need for egg propagation. Cy3-labeled DNA targets were obtained by reverse transcription polymerase chain reaction using Cy3-labeled universal primers, and labeled amplicons were hybridized to the microarray. All positive samples from RT-PCR showed H5-specific and highly pathogenic pattern in the microarray, without purification of PCR products. Furthermore, it allowed for specific detection of the subtype and pathotype from low DNA concentration samples that did not allow direct sequence analysis. Therefore, this diagnostic microarray has enormous potential for the rapid subtyping and pathotyping from clinical samples without the need for culture.

Highlights • We used a DIA to assess replicated IBV vaccines during titration in eggs. • We compared the DIA to a clinical sign (dwarfism) determination method. • The DIA was more sensitive and specific than the clinical sign determination method. • DIA is a rapid, sensitive, reliable method for assessing IBV vaccines titration., A sensitive and specific method for measuring the vaccine titer of infectious bronchitis virus (IBV) is important to commercial manufacturers for improving vaccine quality. Typically, IBV is titrated in embryonated chicken eggs, and the infectivity of the virus dilutions is determined by assessing clinical signs in the embryos as evidence of viral propagation. In this study, we used a dot-immunoblotting assay (DIA) to measure the titers of IBV vaccines that originated from different pathogenic strains or attenuation methods in embryonated eggs, and we compared this assay to the currently used method, clinical sign evaluation. To compare the two methods, we used real-time reverse transcription-PCR, which had the lowest limit of detection for propagated IBV. As a clinical sign of infection, dwarfism of the embryo was quantified using the embryo: egg (EE) index. The DIA showed 9.41% higher sensitivity and 15.5% higher specificity than the clinical sign determination method. The DIA was particularly useful for measuring the titer of IBV vaccine that did not cause apparent stunting but propagated in embryonated chicken eggs such as a heat-adapted vaccine strain. The results of this study indicate that the DIA is a rapid, sensitive, reliable method for determining IBV vaccine titer in embryonated eggs at a relatively low cost.

H5Nx clade 2.3.4.4 highly pathogenic avian influenza viruses (HPAIVs) have been disseminated to wide geographic regions since 2014. In 2016, five distinct genotypes (C-1 to C-5) of clade 2.3.4.4c H5N6 HPAIVs were detected in South Korea. In this study, we evaluated the pathogenicity, susceptibility to infection, and transmissibility of the two strains representing the C-1 and C-4 genotypes of the H5N6 viruses, which have different PA and NS gene, in domestic ducks. Although the susceptibility to infection of domestic ducks to the two strains was similar, the C-4 genotype virus induced higher mortality in ducks than C-1 genotype virus. A higher titer of viral shedding were detected in ducks challenged with the C-4 genotype virus compared with the C-1 genotype virus. These results indicated that the reassortment of HPAIVs with prevailing low pathogenic avian influenza viruses could effect on the pathogenicity in ducks.

Carotenoid pigments are valuable components of the human diet. A notable example is β-carotene, or provitamin A, which is converted into the derivatives astaxanthin and capsanthin, via the common intermediate zeaxanthin. To generate rice varieties producing diverse carotenoids beyond β-carotene, we specifically used a Capsicum β-carotene hydroxylase gene, B (Ca B ch) and a codon optimized version of the same gene, stB ( stB ch) to increase zeaxanthin synthesis. We also used a recombinant BAK gene (Ca B ch-2 A -HpB k t), consisting of the CaBch sequence and a Haematococcus β-carotene ketolase gene (HpBkt) linked by a bicistronic 2 A sequence, as well as a codon optimized recombinant stBAK gene ( stB ch-2 A -stB k t) to create astaxanthin synthesis. The four cassettes to seed-specifically express the B, stB, BAK and stBAK genes were individually combined with a PAC gene (Ca P sy-2 A -Pa C rtI) cassette to previously impart β-carotene-enriched trait in rice endosperm. The single T-DNA vectors of B-PAC, stB-PAC, BAK-PAC and stBAK-PAC resulted in the accumulation of zeaxanthin and astaxanthin in the endosperm of the transgenic rice seeds. In addition, an extended version on the carotenoid pathway was introduced into rice to allow the production of capsanthin, by intercrossing a B-PAC rice line with a Ccs rice line, which harbors a Capsicum capsanthin-capsorubin synthase gene. Ultimately, we developed three functional rice varieties: B-PAC (0.8 μg/g zeaxanthin, deep yellow), stBAK-PAC (1.4 μg/g ketocarotenoids, including astaxanthin, pinkish red) and B-PAC x Ccs (0.4 μg/g of ketoxanthophylls, including capsanthin, orange-red) with the similar levels of total carotenoids to PAC rice, suggesting the capacity was dependent on β-carotene levels. Collectively, a combination of genetic engineering and conventional breeding is effective for multi-step metabolic engineering and biochemical pathway extension.

Background: Measuring the size of the anterior cruciate ligament (ACL) tibial footprint on magnetic resonance image (MRI) is common for preoperative planning of ACL reconstruction. However, the accuracy of such measurement has not been well documented. Purpose: To investigate whether the actual size of the ACL tibial footprint could be predicted by its measurement on MRI and to develop equations to improve the accuracy of predicting the actual size based on MRI measurement. Study Design: Cohort study (diagnosis); Level of evidence, 2. Methods: A total of 164 patients with normal visualized ACL in gross evaluation and MRI were included (mean ± SD age, 67.3 ± 8.3 years). Cases with ACL tear, severe mucoid degeneration, osteophyte around the ACL tibial insertion, or intervals >12 months between MRI and actual measurement were excluded. The ACL tibial footprint was carefully dissected and measured during total knee arthroplasty. The length of the ACL tibial footprint on MRI was measured on a sagittal image, while the width was measured on an oblique coronal image. For the ACL tibial footprint, the association between measurement on MRI and actual measurement of length and width was analyzed via univariable and multivariable regression analyses. Reliability of measurements on MRI was also evaluated. Results: The length and width of the ACL tibial footprint as measured on MRI showed strong correlation with the actual length and width (coefficients: ρ = 0.904 and ρ = 0.808, respectively). There were differences between ACL size on MRI and its actual size: length, 12.4 mm (range, 9.7-15.3 mm) vs 13.8 (10.6-17.8) ( P < .001); width, 8.8 mm (range, 7.0-12.1 mm) vs 7.2 (5.8-10.4) ( P < .001). Based on sex, there were also differences between the size per MRI and the actual size ( P < .001 for all): length in men, 12.6 mm (range, 10.9-15.3 mm) vs 14.2 (12.3-17.8); length in women, 12.4 mm (range, 9.7-14.5 mm) vs 13.7 (10.6-15.8); width in men, 9.3 mm (range, 8.0-12.1 mm) vs 7.6 (5.8-10.4); width in women, 8.7 mm (range, 7.0-10.4 mm) vs 7.2 (5.8-9.7). The actual length of the ACL tibial footprint could be predicted by its length on MRI and sex ( R2 = 0.83, P < .001). Similarly, actual width could be predicted by the width on MRI and sex ( R2 = 0.75, P < .001). All intraclass correlation coefficients were >0.8, indicating good reliability. Conclusion: Measurements of the size of the ACL tibial footprint on MRI showed strong correlation with its actual size. Prediction equations showed good concordance correlation coefficients.

In recent years, avian paramyxovirus type 4 (APMV-4) frequently isolated from wild and domestic bird populations particularly waterfowls worldwide. However, molecular characteristics and genetic diversity of APMV-4 are uncertain, owing to the limited availability of sequence information. A total of 11 APMV-4 strains from 9850 fecal, swab, and environmental samples were isolated during the surveillance program in wintering seasons of 2013-2017 in South Korea. We performed genetic characterization and phylogenetic analysis to investigate the genetic diversity and relatedness between isolates from the region. We report high APMV-4 genetic diversity (multiple genotypes and sub-genotypes) among wild bird and poultry populations in Korea and that the potential virus exchange occurs between neighboring countries via wild bird migration. Furthermore, our study results suggest the possibility of transcontinental transmission of APMV-4 between Asia and Europe.

Objectives: Cornu cervi pantotrichum (CCP) has been widely used in Korean and China, as an anti-fatigue, anti-aging, and tonic agent to enhance the functions of the reproductive and the immune systems. Because CCP has various growth factors that play important roles in the development of hair follicles, we examined whether CCP pharmacopuncture solution (CCPPS) was capable of promoting hair growth in an animal model. Methods: One day after hair depilation, CCPPS were topically applied to the dorsal skin of C57BL/6 mice once a day for 15 days. Hair growth activity was evaluated by using macro- and microscopic observations. Dorsal skin tissues were stained with hematoxylin and eosin. Expressions of bromodeoxyuridine (BrdU), proliferating cell nuclear antigen (PCNA), and fibroblast growth factor (FGF)-7 were examined by using immunohistochemical staining. A reverse transcription polymerase chain reaction (RT-PCR) analysis was also conducted to measure the messenger RNA (mRNA) expression of FGF-7. Results: CCPPS induced more active hair growth than normal saline. Histologic analysis showed enlargement of the dermal papilla, elongation of the hair shaft, and expansion of hair thickness in CCPPS treated mice, indicating that CCPPS effectively induced the development of anagen. CCPPS treatment markedly increased the expressions of BrdU and PCNA in the hair follicles of C57BL/6 mice. In addition, CCPPS up regulated the expression of FGF-7, which plays an important role in the development of hair follicles. Conclusion: These results reveal that CCPPS facilitates hair re-growth by proliferation of hair follicular cells and up-regulation of FGF-7 and suggest that CCPPS can potentially be applied as an alternative treatment for patients with alopecia.

Highly pathogenic avian influenza A(H5N8) viruses were isolated from migratory waterfowl in South Korea during fall 2014-winter 2015, a recurrence after initial introduction in winter 2014. These reappeared viruses were phylogenetically distinct from isolates circulating in poultry farms in South Korea.

Avian paramyxoviruses (APMVs) constitute some of the most globally prevalent avian viruses and are frequently isolated from wild migratory bird species. Using 1,907 fresh fecal samples collected during the 2012 avian influenza surveillance program, we identified two serotypes of APMV: APMV-4 ( n=10) and APMV-8 ( n=1). Sequences for these isolates phylogenetically clustered with Asian APMV-4 and APMV-8 recently isolated from wild birds in Korea, Japan, China, and Kazakhstan. Analysis by DNA barcoding indicated that the Mongolian APMV-4 and APMV-8 strains were isolated from Anseriformes species including Mallards ( Anas platyrhynchos) and Whooper Swans ( Cygnus cygnus). The close genetic relatedness to Asian isolates, and to similar host species, suggested that wild bird species in the Anatidae family might play an important role as a natural reservoir in the spread of APMV-4 and APMV-8. However, we did not find conclusive evidence to support this hypothesis owing to the limited number of strains that could be isolated. Enhanced surveillance of poultry and wild bird populations in Asia is therefore crucial for the understanding of global AMPV transmission, ecology, evolution, and epidemiology.

The pathogenicity and transmissibility of a reassortant clade 2.3.4.4 avian influenza A (H5N6) virus were evaluated in ferrets. Virus excretion was detected in the upper respiratory tract, but the ferrets did not show any clinical signs of infection. Transmission did not occur between cohoused or respiratory droplet-contact ferrets.

We report here the first full-genome sequence of an avian paramyxovirus type 4 (APMV-4) strain isolated from a domestic mallard duck at a live bird market in South Korea. Phylogenetic analyses provide genetic information on a new genetic clade, APMV-4, isolated from a domestic duck and evidence of APMV-4 exchange between poultry and wild birds.

Asian-lineage H5 highly pathogenic avian influenza viruses (HPAIV) have caused recurrent outbreaks in poultry and wild birds. In January 2014, H5N8 HPAIV caused outbreaks in South Korea and subsequently spread to East Asia, Europe, and North America. We report the isolation of an H5N8 HPAIV strain from wild birds in Seoul, the most-developed city in South Korea. We analyzed the complete genome sequence of this isolate and estimated its origin using a phylogenetic analysis. The Seoul H5N8 isolate clustered phylogenetically with strains isolated from migratory wild birds but was distinct from Korean poultry isolates. This H5N8 virus was likely introduced into the urbanized city by migratory wild birds. Therefore, wild bird habitats in urbanized areas should be carefully monitored for HPAIV.

Plastoglobules (PGs) are thylakoid membrane microdomains within plastids that are known as specialized locations of carotenogenesis. Three rice phytoene synthase proteins (OsPSYs) involved in carotenoid biosynthesis have been identified. Here, the N-terminal 80-amino-acid portion of OsPSY2 (PTp) was demonstrated to be a chloroplast-targeting peptide by displaying cytosolic localization of OsPSY2(Delta PTp): mCherry in rice protoplast, in contrast to chloroplast localization of OsPSY2: mCherry in a punctate pattern. The peptide sequence of a PTp was predicted to harbor two transmembrane domains eligible for a putative PG-targeting signal. To assess and enhance the PG-targeting ability of PTp, the original PTp DNA sequence (PTp) was modified to a synthetic DNA sequence (stPTp), which had 84.4% similarity to the original sequence. The motivation of this modification was to reduce the GC ratio from 75% to 65% and to disentangle the hairpin loop structures of PTp. These two DNA sequences were fused to the sequence of the synthetic green fluorescent protein (sGFP) and drove GFP expression with different efficiencies. In particular, the RNA and protein levels of stPTp-sGFP were slightly improved to 1.4-fold and 1.3-fold more than those of sGFP, respectively. The green fluorescent signals of their mature proteins were all observed as speckle-like patterns with slightly blurred stromal signals in chloroplasts. These discrete green speckles of PTp-sGFP and stPTp-sGFP corresponded exactly to the red fluorescent signal displayed by OsPSY2: mCherry in both etiolated and greening protoplasts and it is presumed to correspond to distinct PGs. In conclusion, we identified PTp as a transit peptide sequence facilitating preferential translocation of foreign proteins to PGs, and developed an improved PTp sequence, a stPTp, which is expected to be very useful for applications in plant biotechnologies requiring precise micro-compartmental localization in plastids.

In this Letter, the authors neglected to acknowledge funding from the Yonsei University Future-leading Research Initiative of 2017 (2017-22-007) awarded to H.W.P.

International audience; AbstractAkabane virus (AKAV), an arthropod-transmitted bunyavirus, is a major cause of congenital abnormalities and encephalomyelitis in ruminants. In 2010, there was a major outbreak of encephalomyelitis in Korea and fifteen AKAV strains, including AKAV-7, were isolated from cows. To identify the neuropathogenicity of AKAV-7, we performed experimental infection of cows. Six-month-old female Korean Holstein dairy cattle were inoculated with AKAV-7 by various routes, including intracerebral (IC), intrasubarachnoid space (IS), subcutaneous (SC) and intravenous (IV); a separate group was vaccinated before intravenous infection. Five of the six cows in the IC group and two of the six cows in the IS group showed clinical signs such as locomotor ataxia and paralysis of the hind limbs. Three of six cows died after IC infection 9–12 days post infection (dpi). Histopathologic changes such as nonsuppurative encephalomyelitis were confirmed in various parts of the central nervous system in the IC, IS and SC groups. Early onset of neutralizing antibodies in the serum and lower viral mRNA levels in the peripheral blood mononuclear cells (PBMCs) and various tissues in the vaccinated group was noticeable compared to the unvaccinated group (IV group). We suggest that the AKAV vaccine currently used in Korea may be partially effective for protection against AKAV-7 in cows.

Ethnopharmacological relevance Schisandra chinensis ( Turcz. ) Baill. (SC) is a traditional Chinese herbal medicine with diverse pharmacological activities for treatment of various human diseases. Endoplasmic reticulum (ER) stress is associated with the pathogenesis of nonalcoholic fatty liver disease (NAFLD). In this study, we investigated the protective effects of methanol extract of Schisandra chinensis (SC extract) against ER stress-induced NAFLD in vitro and in vivo . Material and methods The protective effects of SC extract were examined in tunicamycin- or palmitate-treated HepG2 cells in vitro , and in tunicamycin-injected mice or high fed diet (HFD) obese mice in vivo . Expression of ER stress markers including glucose regulated protein 78 (GRP78), C/EBP homolog protein (CHOP), and X-box-binding protein-1 (XBP-1), and triglyceride accumulation were measured in HepG2 cells and in the liver of mice. Results SC extract significantly inhibited expression of tunicamycin-induced ER stress markers in tunicamycin-treated HepG2 cells and in the liver of tunicamycin-injected mice, and it also inhibited tunicamycin-induced triglyceride accumulation. Similar observations were made under physiological ER stress conditions such as in palmitate-treated HepG2 cells and in the liver of HFD obese mice. In addition, SC extract repressed the expression of inflammatory genes and lipogenic genes in palmitate-treated HepG2 cells. Schisandrin, an abundant bioactive lignan in SC extract, inhibited the expression of ER stress markers in tunicamycin-or palmitate-treated HepG2 cells, whereas Gomisin J did not affect ER stress markers. Conclusions SC attenuates ER stress and prevents development of NAFLD. SC may be useful as a pharmacological agent for protection against ER stress-induced human diseases.

Background Fatigue is one of the most common presenting symptoms in primary care in Korea. In this study, we aimed to determine the effect of exercise intervention on the severity of fatigue of unknown medical cause during a period of follow-up. Methods We used the data collected from an outpatient fatigue clinic in Seoul National University Bundang Hospital. The study was conducted from March 3, 2010 to May 31, 2014. We measured the body mass index of each patient and evaluated variables including lifestyle factors (smoking, alcohol consumption, and regular exercise), quality of sleep, anxiety, depression, stress severity, and fatigue severity using questionnaires. A total of 152 participants who completed questionnaires to determine changes in fatigue severity and the effect of exercise for each period were evaluated. We used univariate analysis to verify possible factors related to fatigue and then conducted multivariate analysis using these factors and the literature. Results Of 130 patients with the complaint of chronic fatigue for over 6 months, over 90 percent reported moderate or severe fatigue on the Fatigue Severity Scale and Brief Fatigue Inventory questionnaires. The fatigue severity scores decreased and fatigue improved over time. The amount of exercise was increased in the first month, but decreased afterwards. Conclusion There was no significant relationship between changes in the amount of exercise and fatigue severity in each follow-up period. Randomized controlled trials and a cohort study with a more detailed exercise protocol in an outpatient setting are needed in the future.

Asian-lineage H5 highly pathogenic avian influenza (HPAI) viruses have caused continuous outbreaks in poultry and wild birds. Development of rapid and accurate diagnostic methods is needed for preventing further spread of the virus and reducing the time required for eradication of the virus. We developed a low-density microarray for the rapid detection and identification of avian influenza virus subtypes H5, H7, and H9 and their pathotypes in a previous study. In the present study, we evaluated previously developed diagnostic microarray using avian influenza viruses isolated in Mongolia, including H5 HPAI viruses. All H5 HPAI viruses isolated in Mongolia were shown as H5-specific and highly pathogenic pattern in the microarray. H2, H3 and H12 viruses isolated in Mongolia used in this study did not show any H5, H7 and H9 patterns. These results indicated that this diagnostic microarray has enormous potential for the rapid subtyping and pathotyping of influenza viruses, including viruses isolated in Mongolia.

Background: Hominis Placenta (HP) known as a restorative medicine in Traditional Chinese Medicine (TCM), has been widely applied in the clinics of Korea and China as an anti-aging agent to enhance the regeneration of tissue. This study was conducted to investigate whether topical treatment of HP promotes hair regrowth in the animal model. Methods: The dorsal hairs of 8-week-old C57BL/6 mice were depilated to synchronize hair follicles to the anagen phase. HP was applied topically once a day for 15 days. Hair growth was evaluated visually and microscopically. The incorporation of bromodeoxyuridine (BrdU) and expression of proliferating cell nuclear antigen (PCNA), fibroblast growth factor-7 (FGF-7) in dorsal skin tissue was examined by immunohistochemical analysis. Reverse transcription polymerase chain reaction (RT-PCR) was used to measure the mRNA expression of FGF-7. Results: HP exhibited potent hair growth-promoting activity in C57BL/6 mice. Gross examination indicated that HP markedly increased hair regrowth as well as hair density and diameter. Histologic analysis showed that HP treatment enhanced the anagen induction of hair follicles. Immunohistochemical analysis revealed that BrdU incorporation and the expressions of PCNA were increased by treatment of HP. HP treatment significantly increased the expression of FGF-7, which plays pivotal roles to maintain anagen phase both protein and mRNA levels. Conclusions: Taken together, our results indicate that HP has a potent hair growth-promoting activity; therefore, it may be a good candidate for the treatment of alopecia.