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The Manila clam Ruditapes philippinarum on the west coast of Korea harbors several digenetic trematodes. However, most studies in this region have been restricted to a few sampling sites and the current species designation of some trematodes is largely based on morphology, leaving the molecular phylogenetic position among the Digenea unsolved. Thus, we first provide both morphology and molecular phylogeny of some components in the trematodes community in the Manila clam based on a large-scale survey of 26 sites on the west coast, where well-developed tidal flats serve as large commercial clam culture beds. Our study revealed that the trematodes community in the clams consisted of at least 5 species that belong to 3 families (Himasthlidae, Gymnophallidae, Baccigeridae) and 1 superfamily (Monorchioidea). The life mode of the 5 different species included the metacercaria and sporocyst, with one species (Parvatrema duboisi) utilizing the clams as both the first and/or second intermediate host. Trematode infection prevalences were not evenly distributed among the study sites, although the reasons behind this are yet to be determined. Morphological identification was confirmed with the molecular analyses based on ITS and 28S rDNA; phylogenetic analysis also revealed that Cercaria pectinata infecting the clam gonad should be referred to as Bacciger bacciger hereafter. The present preliminary study provides a crucial baseline that could be further developed in a future study on the digenean trematodes community in the Manila clam., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

This study reports the occurrence of Perkinsus marinus associated with wild Pacific oyster (Crassostrea gigas) specimens collected along the west coast of Korea. Confirmation of P. marinus presence was achieved by conventional PCR using World Organization of Animal Health (WOAH)-recommended primers that specifically targeted regions of the rDNA locus (ITS1, 5.8S, and ITS2). Sequencing of 10 samples revealed two distinct sequences differing by a single base pair, indicating potential haplotype variability. One sequence closely resembled the P. marinus strain found in Maryland, USA, whereas the other exhibited divergence, indicative of species diversity in the Korean strain, as was evident from the haplotype network analysis. Further validation involved the Ray's Fluid Thioglycollate Medium (RFTM) assay, which initially yielded inconclusive results, possibly due to low infection intensity. Subsequently, RFTM and 2 M NaOH assays conducted on the isolates in the present study, cultured P. marinus cells in standard DMEM/F12 medium, and a positive P. marinus strain (ATCC 50509), revealed characteristic hypnospores of P. marinus upon Lugol's iodine staining. These comprehensive investigations underscore the conclusive confirmation of P. marinus in Korean waters and mark a significant milestone in our understanding of the distribution and characteristics of this parasite in previously unreported regions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Hypothesis: Ice mitigation has received increasing attention due to the severe safety and economic threats of icing hazards to modern industries. Slippery icephobic surface is a potential ice mitigation approach due to its ultra-low ice adhesion strength, great humidity resistance, and effective delay of ice nucleation. However, this approach currently has limited practical applications because of serious liquid depletion in the icing/de-icing process., Experiments: A new strategy of phase change materials (PCM)-impregnation porous metallic structures (PIPMSs) was proposed to develop phase changeable icephobic surfaces in this study, and aimed to solve the rapid depletion via the phase changeable interfacial interactions., Findings: Evaluation of surface icephobicity and interfacial analysis proved that the phase changeable surfaces (PIPMSs) worked as an effective and durable icephobic platform by significantly delaying ice nucleation, providing long-term humid tolerance, low ice adhesion strength of as-prepared samples (less than 5 kPa), and signally improved maintaining capacity of impregnated PCMs (less than 10 % depletion) after 50 icing/de-icing cycles. To explore the interfacial responses, phase change models consisting of the unfrozen quasi-liquid layer and solid lubricant layer at the ice/PIPMSs interfaces were established, and the involved icephobic mechanisms of PIPMSs were studied based on the analysis of interfacial interactions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Pyrethroids are primarily used for mosquito control in Korea. However, high frequencies of mutations conferring resistance to not only pyrethroids but also to other insecticides have been found in mosquito populations. This study aimed to examine the hypothesis that insecticides used outside of public health may play a role in selection. Briefly, the resistance mutation frequencies to three insecticide groups (pyrethroids, organophosphates, and cyclodienes) were estimated in two representative groups of mosquito species (Anopheles Hyrcanus Group and Culex pipiens complex). The relationship between these frequencies and the land-use status of the collection sites was investigated through multiple regression analysis. In the Anopheles Hyrcanus Group, the frequencies of both ace1 (organophosphate resistance) and rdl (cyclodiene resistance) mutations were positively correlated with 'proximity to golf course', possibly be due to the insecticides used for turf maintenance. They also showed positive correlations with field area and rice paddy area, respectively, suggesting the role of agricultural insecticides in the selection of these resistance traits. For the Cx. pipiens complex, the kdr (pyrethroid resistance), ace1, and rdl mutations were positively correlated with the residential area, field, and rice paddy, respectively. Therefore, pyrethroids used for public health could serve as a direct source of resistance selection pressure against kdr, whereas non-public health insecticides may pose primary selection pressure against the ace1 and rdl traits. The current findings suggest that the insecticides used in agriculture and the golf industry play a significant role in mosquito selection, despite variations in the extent of indirect selection pressure according to the mosquito groups and insecticide classes., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Throughout the recent COVID-19 pandemic, South Korea led national efforts to develop vaccines and therapeutics for SARS-CoV-2. The project proceeded as follows: 1) evaluation system setup (including Animal Biosafety Level 3 (ABSL3) facility alliance, standardized nonclinical evaluation protocol, and laboratory information management system), 2) application (including committee review and selection), and 3) evaluation (including expert judgment and reporting). After receiving 101 applications, the selection committee reviewed pharmacokinetics, toxicity, and efficacy data and selected 32 final candidates. In the nonclinical efficacy test, we used golden Syrian hamsters and human angiotensin-converting enzyme 2 transgenic mice under a cytokeratin 18 promoter to evaluate mortality, clinical signs, body weight, viral titer, neutralizing antibody presence, and histopathology. These data indicated eight new drugs and one repositioned drug having significant efficacy for COVID-19. Three vaccine and four antiviral drugs exerted significant protective activities against SARS-CoV-2 pathogenesis. Additionally, two anti-inflammatory drugs showed therapeutic effects on lung lesions and weight loss through their mechanism of action but did not affect viral replication. Along with systematic verification of COVID-19 animal models through large-scale studies, our findings suggest that ABSL3 multicenter alliance and nonclinical evaluation protocol standardization can promote reliable efficacy testing against COVID-19, thus expediting medical product development., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2023. Published by Elsevier Ltd.)

The observed and predicted changes in climate, as well as the growth in urban population, are creating severe stress on existing water resources in South Korea. By the importation of agricultural products from more water-rich countries through the virtual water concept, a country could save local water resources for other important uses. However, these imports from other countries could lead to certain vulnerabilities in the importing country derived from climate change. Therefore, through the application of the virtual water concept and the climate vulnerability index ((CVI) - measure of a country's vulnerability to indirect climate impacts), this study assessed the implication of virtual water imports and climate change through food trade, on the water, land and food security status of South Korea over the period of 2000-2017. The results showed that significant amounts of national water and land was saved through the importation of major upland crops. Virtual water imports increased significantly over time, rising from 16.2 Bm 3 in 2000 to 16.5, 17.4, and 20.7 Bm 3 in 2005, 2011, and 2017 respectively, with the USA, China, Australia, Brazil and Canada being the major exporters to South Korea. The study also revealed high CVI values for the oils and grains category of imported food, implying the high vulnerability of South Korea to climate change effects resulting from the import of these crops. The quantitative impacts and structural changes in virtual water trade, as well as the link between climate change, food security, international trade, and domestic water consumption could be evaluated for the sustainable management and allocation of resources. This study successfully identified and quantified the status of food trade and its environmental implications in the study area, providing insight into a better allocation of locally available resources., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The members in the family Aplysidae known as sea hares play a crucial role as a grazer in small tidal pools or shallow subtidal hard bottoms. Like other marine gastropods, hemocyte types and functions of sea hares are limitedly known. This study identified and characterized the hemocytes of common Aplysia species Aplysia kurodai, A. juliana, and A. oculifera in rocky tidal pools in Jeju Island off the south coast of Korea using flow cytometry and light microscopy. The flow cytometry identified three different hemocytes in the hemolymph of the three sea hare species: granulocytes, hyalinocytes, and blast-like cells. The granulocytes exhibited pseudopodia on the cell surface and granules in the cytoplasm. Morphology of the hyalinocyte was similar to that of the granulocytes, while they lack cytoplasmic granules. The blast-like cells were small and round, with very thin cytoplasm. The hyalinocytes were the most abundant in the hemolymph, accounting for 89.8-92.6% of the total hemocytes. Flow cytometry indicated that the granulocytes and blast-like cells were less than 5.6% and 5.4% of the total hemocyte populations. Flow cytometry also revealed that the granulocytes and hyalinocytes are engaged in cellular defensive activities such as intra-cellular lysosomal content, phagocytosis, and ROS production. The mean lysosomal contents of the granulocytes (0.4 × 10 5 -0.2 × 10 5 A U.) were 2-3 times higher than that of hyalinocytes (0.2 × 10 5 -0.6 × 10 5 A U.). In addition, the ROS production of the granulocytes (0.98 × 10 6 -1.95 × 10 6 A U.) was about twice higher than that of the hyalinocytes (0.62 × 10 6 -1.14 × 10 6 A U.). Of the three species of sea hares, the granulocytes showed comparatively higher phagocytosis capacity (70.4-92.3%) than that of the hyalinocytes (34.8-46.0%). Flow cytometry and microscopy indicated that the hemocyte types and their functions were identical, regardless of the species., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Spawning in marine bivalves is a great energy-demanding process, and it often results in lethal and sublethal stresses during the post-spawning period, including depressed immune capacity. The blood cockle Tegillarca granosa (Linnaeus, 1758) distributes widely in silty-mud tidal flats on the south coast of Korea, and they spawn in late summer. To understand the impacts of spawning on immune parameters, we analyzed the total hemocyte count (THC), hemocyte mortality, phagocytosis capacity, and reactive oxygen species (ROS) production of T. granosa in pre-, and post-spawning condition using a flow cytometer. Histology indicated that the blood cockles occurring on the south coast of Korea ripe and ready to spawn in July, and they spawned in August and September. The THC in the blood cockle hemolymph declined from pre-spawning (1.2 × 10 8  cell mL -1 ) to post-spawning (0.9 × 10 8  cell mL -1 ), possibly due to the spawning stress and the massive infiltration of hemocytes in the gonad to phagocytose and resorb the residual gametes during the post-spawning period. The hemocyte mortality increased linearly from August (4.1%) to November (9.1%), as the histology revealed that the blood cockle completed spawning, and they resorbed the relict gametes. The granulocyte phagocytosis capacity declined dramatically from July (12.7%) to September (6.0%), when the cockles were engaged in active spawning. The flow cytometry revealed that the production of reactive oxygen species (ROS) from the granulocytes and the erythrocytes type II increased linearly from August (0.8-0.9 × 10 5 A U.) to December (2.1-2.8 × 10 5 A U.), which may cause stresses at a cellular level during this period. As the data indicated, spawning is a stressful activity inducing depressed immunological capacities in the blood cockles., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Inflammation is a form of innate immune response of living organisms to harmful stimuli. In marine bivalves, inflammation is a common defense mechanism. Several studies have investigated the morphological features of inflammation in bivalves, such as hemocyte infiltration. However, the molecular and biochemical responses associated with inflammation in marine bivalves remain unexplored. Here, we investigated changes in nitric oxide (NO) levels, cyclooxygenase 2 (COX-2) activity, and allograft inflammatory factor-1 (AIF-1) gene expression levels in hemolymph samples collected from Manila clam (Ruditapes philippinarum) exposed to pro- and anti-inflammatory substances. These included the pro-inflammatory agent lipopolysaccharide (LPS), and the nonsteroidal anti-inflammatory drugs (NSAIDs) ibuprofen and diclofenac, all widely used in vertebrates. Our study showed that NO levels, COX-2 activity, and AIF-1 expression increased in response to the treatments with LPS and decreased in response to the treatments with NSAIDs in a concentration-dependent manner. These results suggest that the mechanism of inflammatory responses in bivalves is very similar to that of vertebrates, and we propose that inflammatory responses can be quantified using these techniques and used to determine the physiological status of marine bivalves exposed to biotic or abiotic stresses., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Recently, a putative new hyperparasitic haplosporidian in the genus Urosporidium was identified from metacercariae of the trematode Parvatrema duboisi infecting Manila clam Ruditapes philippinarum on the west coast of Korea. In this study, we applied small subunit ribosomal DNA (SSU rDNA) sequences as a marker to substantiate the phylogenetic relationship of the unidentified Urosporidium within the Order Haplosporida. In our phylogenetic analysis, the 1890 bp of SSU rDNA sequences obtained were closely related to a haplosporidian parasite forming a sister clade to Urosporidium group, although the gene sequences were only 89.22-89.70% similar to Urosporidium spp. Such molecular phylogenetic distance within the genus suggested that the unidentified Urosporidium is a new member of the genus. Accordingly, we report the unidentified haplosporidian hyperparasite as Urosporidium tapetis sp. nov., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Pyrethroid (PYR) and organophosphate (OP) insecticides have been extensively used for mosquito control for several decades in South Korea, and has resulted in the rapid development of resistance in the field. In this study, quantitative sequencing (QS) protocols were developed for the frequency prediction of insecticide resistance alleles [e.g., the L1014F/C mutation on the voltage sensitive sodium channel as a PYR resistance allele and the G119S mutation on the acetylcholinesterase 1 as OP resistance alleles] in four regional populations of Anopheles Hyrcanus Group and Culex pipiens complex. Both of the L1014F/C and G119S mutations were observed in all examined regional populations of An. Hyrcanus Group, suggesting a wide distribution of both PYR and OP resistance. In contrast, populations of the Cx. pipiens complex were determined to possess almost no G119S mutation, but relatively higher frequencies of the L1014F mutation, showing differential resistance patterns between different mosquito groups. The mutation frequencies were also monitored throughout a mosquito season (May-October) at one collection site to determine the seasonal changes of resistance mutation frequency in mosquito populations. Dramatic decreases of both L1014F/C and G119S mutation frequencies were observed in the An. Hyrcanus Group toward the fall, with no mutations observed in the early spring, suggesting a connection between the fitness costs of overwintering and insecticide resistance. However, no apparent trends were detectable in the Cx. pipiens complex populations due to low or zero mutation frequencies., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Paramyxean parasites in the genus Marteilia deteriorate digestive tissues of the host organisms, resulting in mortality of oysters, cockles, and mussels. Most reports of infection by Marteilia spp. are from Europe, while a new species of Marteilia was identified recently in Japan. Here, we report a previously unidentified species in the genus Marteilia from digestive diverticula of Manila clam Ruditapes philippinarum from the south coast of Korea. Prevalence of the parasite was low, 0.5-3.3% in the study sites. We characterized this species using light and transmission electron microscopy (TEM), and analyzed the 18S rDNA sequence. Light microscopy revealed the sporulation process from uninucleated stage to spore in the epithelial tissues of the digestive gland. TEM revealed that the parasites produced four secondary cells containing four tri-cellular spores. An electron-dense haplosporosome-like structure and striated inclusions were evident in the spore and the primary cells, respectively, while refringent granules were rarely observed in the secondary cells. Phylogenetic analyses of the 18S rDNA sequence placed this isolate in the genus Marteilia, although it is not identical to other known species in the genus. Based on morphological and molecular characters, we describe this species as Marteilia tapetis sp. nov., the second Marteilia species reported parasitizing Manila clams in Asian waters., (Copyright © 2019. Published by Elsevier Inc.)

Conditions of elevated temperature and CO 2 levels [30 °C and 970 parts-per-million (ppm), respectively] reduced the systemic titers of a potato virus Y (PVY) isolate in Nicotiana benthamiana plants, relative to standard conditions (25 °C, ~405 ppm CO 2 ). Under controlled conditions we studied how these growing environments affected the transmission of infection by aphids. Probabilities of transmission of infection by insects that fed on infected donor plants kept at either standard conditions, or at 30 °C and 970 ppm CO 2 were both determined and found to positively correlate with titers in donor leaves, independently of the ambient conditions in which recipient plantlets would grow. With these data, viral prevalence was simulated under conditions of elevated temperature and CO 2 levels and found that for it to remain comparable to that simulated under standard conditions, insect arrivals to recipient plants in the former scenario would have to increase several-fold in their frequency., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Internal defense of ascidians relies, at least partially, on cells circulating in body fluids and infiltrating in tissues, referred to as hemocytes, although structure and composition of ascidian hemocytes still remain unclear. In the present study, we investigated hemocyte types and their functions of the solitary ascidian Halocynthia roretzi using flow cytometry. Based on morphology, cellular activities and intracellular parameters from the flow cytometry, we identified eight hemocyte types including, three granulocytes (Gr-1, Gr-2, and Gr-3), 4 hyalinocytes (Hy-1, Hy-1', Hy-2, and Hy-3) and lymphocyte-like (Ly-like) cells. The granulocyte Gr-1 accounted for 30% of the total circulating hemocytes and exhibited highest density of lysosomes and oxidative activity. Gr-1 was deeply involved in phagocytosis and degradation of foreign material. Hyalinocytes consist of two main populations, Hy-1 and Hy-2, and each accounted for 30% of the circulating hemocyte. Hy-1 displayed lysosomal content, an inducible oxidative activity, and no proteases, while Hy-2 expressed highest density of intracellular proteases, no lysosomes and a low oxidative activity. It was believed that Hy-2 may represent an important link between cellular and humoral immune reactions. Hy-1 did not show phagocytosis activity. Hy-3 and the Ly-like cells presented a similar profile except for their size and complexity, and Hy-3 may represent an intermediate differentiation/maturation step between Ly-like cells and other hemocyte populations. This first characterization of the hemocyte populations of H. roretzi provides a solid basis to investigate further their respective roles and functions in physiological and pathological contexts., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

In this study, we first report on the occurrence of Urosporidium sp., a haplosporidian hyperparasite infecting the trematode, Parvatrema duboisi, which parasitizes Manila clams, Ruditapes philippinarum on the west and south coasts of Korea. The larval P. duboisi infected by the sporocyst stage of Urosporidium sp. demonstrated numerous small yellowish spores in their tissues. The heavily infected metacercariae exhibited degenerate bodies and the larvae were often motionless. Clams heavily infected by the metacercariae of P. duboisi also displayed abnormal golden spots on the mantle tissue. In histology, different life stages of Urosporidium sp. could be identified, including the uni-nucleate, plasmodial, sporogonic stages and the acid fast mature spores released from the cyst. In scanning electron microscopy (SEM), the mature spore exhibited a semi-circular rim around the apical end and the orifice was covered internally with a flap. Loop-like filaments ornamentation was also identified from Urosporidium sp. in SEM, suggesting that Urosporidium sp. found in this study is a new member in the genus. Prevalence of Urosporidium sp.-infected trematodes in this study ranged from 2.5% to 24.0% in April 2010 and the infection was observed from 8 sampling sites out of the 26 sites surveyed on the west and south coasts., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Reactive oxygen and nitrogen species (ROS and RNS) are naturally produced in all cells and organisms. Modifications of standard conditions alter reactive species generation and may result in oxidative stress. Because of the degradation of marine ecosystems, massive aquaculture productions, global change and pathogenic infections, oxidative stress is highly prevalent in marine bivalve molluscs. Haemocytes of bivalve molluscs produce ROS and RNS as part of their basal metabolism as well as in response to endogenous and exogenous stimuli. However, sources and pathways of reactive species production are currently poorly deciphered in marine bivalves, potentially leading to misinterpretations. Although sources and pathways of ROS and RNS productions are highly conserved between vertebrates and invertebrates, some uncommon pathways seem to only exist in marine bivalves. To understand the biology and pathobiology of ROS and RNS in haemocytes of marine bivalves, it is necessary to characterise their sources and pathways of production. The aims of the present review are to discuss the currently known and unknown intracellular sources of reactive oxygen and nitrogen species in marine bivalve molluscs, in light of terrestrial vertebrates, and to expose principal pitfalls usually encountered., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

In the study, we investigated the effect of dicoumarol, an anti-coagulant agent with the inhibitory activity of NAD(P)H quinone oxidoreductase 1 (NQO1), on TRAIL-induced apoptosis in renal cancer cell. Combined treatment with dicoumarol and TRAIL significantly induced apoptosis in various human renal carcinoma cells including Caki, ACHN, and A498, but not in normal human skin fibroblasts (HSF) and mouse kidney cells (TMCK-1). When we elucidated the relevance of NQO1 in dicoumarol plus TRAIL-mediated apoptosis, both ES936 (a NQO1 inhibitor) and knockdown of NQO1 with siRNA had no effect on TRAIL-mediated apoptosis, suggesting that the stimulating effect of dicoumarol on TRAIL-mediated apoptosis is independent of NQO1 activity. We found that dicoumarol transcriptionally down-regulated Bcl-2 expression via inhibition of NF-κB and CREB activity, whereas it down-regulated Mcl-1 and c-FLIP expression at the post-translational level. Overexpression of Bcl-2, Mcl-1, or c-FLIP overcame the dicoumarol plus TRAIL-induced apoptosis, indicating that down-regualtion of these anti-apoptotic proteins may critically contribute to the sensitizing effect of dicoumarol on TRAIL-mediated apoptosis., (Copyright © 2014 Elsevier Inc. All rights reserved.)

We first characterized the morphology and immune-related activities of hemocytes in the subtropical oysters Saccostrea kegaki, Ostrea circumpicta, and Hyotissa hyotis using light microscopy and flow cytometry. Hemocytes of these three oyster species were classified into three main types: 1) granulocytes containing numerous granules in the cytoplasm, 2) hyalinocytes with no or fewer granules, and 3) blast-like cells characterized by the smallest size and very thin cytoplasm. The percentage of each hemocyte population was similar in all species; hyalinocytes were the most abundant cell in the hemolymph accounting for more than 59%, followed by granulocytes (23-31%) and blast-like cells (3-5%). The size of granulocytes of S. kegaki was smaller (P < 0.05) than those of O. circumpicta and H. hyotis. Light microscopy also allowed the description of vacuolated cells characterized by large vacuoles in the cytoplasm. Flow cytometry analysis confirmed that the granulocytes of the three oyster species were the major hemocytes engaged in cellular defense with the largest lysosome content, and the most active phagocytosis activity and oxidative activity, as was previously reported in several marine bivalves. Phagocytic activity was the lowest in S. kegaki hemocytes, and PMA-stimulated oxidative activity was the lowest in H. hyotis hemocytes. Our results provide the basic information of hemocytes population of three subtropical oysters for further investigations associated with various environmental disease stresses., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The oyster ovarian parasite Marteilioides chungmuensis has been reported from Korea and Japan, damaging the oyster industries. Recently, Marteilioides-like organisms have been identified in other commercially important marine bivalves. In this study, we surveyed Marteilioides infection in the Manila clam Ruditapes philippinarum, Suminoe oyster Crassostrea ariakensis, and Pacific oyster Crassostrea gigas, using histology and Marteilioides-specific small subunit (SSU) rDNA PCR. The SSU rDNA sequence of M. chungmuensis (1716 bp) isolated from C. gigas in Tongyoung bay was 99.9% similar to that of M. chungmuensis reported in Japan. Inclusions of multi-nucleated bodies in the oocytes, typical of Marteilioides infection, were identified for the first time in Suminoe oysters. The SSU rDNA sequence of a Marteilioides-like organism isolated from Suminoe oysters was 99.9% similar to that of M. chungmuensis. Marteilioides sp. was also observed from 7 Manila clams of 1840 individuals examined, and the DNA sequences of which were 98.2% similar to the known sequence of M. chungmuensis. Unlike Marteilioides infection of Pacific oysters, no remarkable pathological symptoms, such as large multiple lumps on the mantle, were observed in infected Suminoe oysters or Manila clams. Distribution of the infected Manila clams, Suminoe oysters and Pacific oysters was limited to small bays on the south coast, suggesting that the southern coast is the enzootic area of Marteilioides infection., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Aging increases the co-incidence of Alzheimer's disease (AD) and type 2 diabetes (T2DM). However, the critical factors that contribute to the age-related increase in AD-T2DM comorbidity have yet to be clarified. In this study, aging effects and their relationship to AD-related pathology and T2DM as well as the underlying mechanisms of this process were investigated using obese rats with chronic T2DM. Tau pathology and its associated signaling pathways in the brain were compared between Otsuka Long-Evans Tokushima Fatty (OLETF) rats and corresponding non-diabetic controls at various ages. Tau phosphorylation at AD-related epitopes, including Thr212, Thr231, Ser262, and Ser396, increased with age in the soluble brain extracts of chronic OLETF rats and were accompanied by synaptic protein loss. There was also a marked age-dependent accumulation of polyubiquitinated substances in diabetic rats. Accordingly, tau proteins were highly polyubiquitinated in aged OLETF rats and a strong degree of co-localization existed between p-tau and ubiquitin in these neurons. In addition, the mRNA and protein levels of p62, a known cargo molecule that transports polyubiquitinated tau to proteasomal and autophagic degradation systems, decreased robustly with age in OLETF rats and there was an inverse correlation between protein levels of p62 and p-tau. The impaired degradation of polyubiquitinated p-tau due to age- and T2DM-dependent decreases in p62 transcription is a primary mechanism underlying increased AD-like pathology in a T2DM rat model as age increases. These results provide novel insight into the mechanisms supporting the age-related increase in AD-T2DM comorbidity., (© 2013.)

TRAIL resistance in many cancer cells is one of the major problems in TRAIL-based cancer therapy. Thus, the agents that can sensitize the tumor cells to TRAIL-mediated apoptosis are strictly needed for the improvement of anti-cancer effect of TRAIL. Acrolein is a byproduct of lipid peroxidation, which has been involved in pulmonary, cardiac and neurodegenerative diseases. We investigated whether acrolein, an α,β-unsaturated aldehyde, can potentiate TRAIL-induced apoptosis in human renal cancer cells. The combined treatment with acrolein and TRAIL significantly induced apoptosis, and stimulated of caspase-3 activity, DNA fragmentation, and cleavage of PARP. We found that acrolein down-regulated the protein level of Bcl-2 and Bcl-2 overexpression inhibited the cell death induced by the combined treatment with acrolein and TRAIL. In addition, acrolein up-regulated C/EBP homologous protein (CHOP) and TRAIL death receptor 5 (DR5) and down-regulation of CHOP or DR5 expression using the respective small interfering RNA significantly attenuated the apoptosis induced by acrolein plus TRAIL. Interestingly, pretreatment with an antioxidant, N-acetylcysteine (NAC), inhibited not only CHOP and DR5 up-regulation but also the cell death induced by acrolein plus TRAIL. Taken together, our results demonstrated that acrolein enhances TRAIL-induced apoptosis in Caki cells through down-regulation of Bcl-2 and ROS dependent up-regulation of DR5., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Molecular and immunological probes were used to identify various life stages of Perkinsus olseni, a protozoan parasite of the Manila clam Ruditapes philippinarum, from a marine environment and decomposing clam tissue. Western blotting revealed that the antigenic determinants of the rabbit anti-P. olseni antibody developed in this study were peptides with molecular masses of 55.9, 24.0, and 19.2kDa. Immunofluorescent assay indicated that the rabbit anti-P. olseni IgG was specific to all life stages, including the prezoosporangium, trophozoite, and zoospore. Perkinsus olseni prezoosporangium-like cells were successfully isolated from marine sediment collected from Hwangdo on the west coast of Korea, where P. olseni-associated clam mortality has recurred for the past decade. Purified cells were positively stained with the rabbit anti-P. olseni antibody in an immunofluorescence assay, confirming for the first time the presence of P. olseni in marine sediment. Actively replicating zoospores inside the prezoosporangia were observed in the decomposing clam tissue collected from Hwangdo. P. olseni was also isolated from the feces and pseudofeces of infected clams and confirmed by PCR. The clams released 1-2 prezoosporangia per day through feces. The data suggested that the fecal discharge and decomposition of the infected clam tissue could be the two major P. olseni transmission routes., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), either alone or in combination with other anticancer drugs, is considered as a new strategy for anticancer therapy. Compound C, a cell-permeable pyrrazolopyrimidine derivative, acts as a potent, selective, reversible ATP-competitive inhibitor of AMP-activated protein kinase (AMPK). In this study, we show that compound C sensitizes Caki human renal cancer cells, but not normal human skin fibroblast cells (HSF) and human mesangial cells, to TRAIL-mediated apoptosis. However, AMPK siRNA failed to affect TRAIL-mediated apoptosis in Caki cells and transduction of dominant negative AMPK rather attenuated TRAIL-induced apoptosis, indicating that the effect of compound C on sensitization of TRAIL-induced apoptosis is independent of AMPK activity. Interestingly, we found that down-regulation of c-FLIP(L) and Mcl-1 contributes to compound C-enhanced TRAIL-induced apoptosis. Reduced expression of c-FLIP(L) and Mcl-1 were caused by the decreased protein stability of c-FLIP(L) and Mcl-1, but not by their transcriptional control, in compound C-treated cells. Pretreatment with N-acetyl-L-cysteine (NAC) significantly inhibited the cell death induced by the combined treatment with compound C and TRAIL as well as recovered the expression levels of c-FLIP(L) and Mcl-1 down-regulated by the combinatory treatment with compound C plus TRAIL, suggesting that compound C-stimulated TRAIL-induced apoptosis appears to be dependent on the generation of reactive oxygen species for down-regulation of c-FLIP(L) and Mcl-1. Taken together, the present study demonstrates that compound C enhances TRAIL-induced apoptosis in human renal cancer cells by ROS-mediated c-FLIP(L) and Mcl-1 down-regulation., (Copyright 2010 Elsevier Inc. All rights reserved.)

The disk abalone Haliotis discus discus and the spiny top shell Turbo cornutus are edible gastropod species of high economic value, mainly in Asia. Mortality outbreaks and variations in worldwide stock abundance have been reported and suggested to be associated, at least in part, with pathogenic infections. Ecology, biology and immunology of both species are currently not well documented. The characterisation of the immune systems of these species is necessary to further assess the responses of H. discus discus and T. cornutus to environmental, chemical and disease stresses. In the present study, we investigated the morphology and immune-related activities of hemocytes in both species using light microscopy and flow cytometry. Two types of hemocytes were identified in the disk abalone hemolymph, blast-like cells and hyalinocytes; whereas four main hemocyte types were distinguished in the spiny top shell, blast-like cells, type I and II hyalinocytes, and granulocytes. Flow cytometric analysis also revealed differences between cell types in immune-related activities. Three subsets of hemocytes, defined by differing lysosomal characteristics, were observed in the hemolymph of the spiny top shell, and only one in the disk abalone. Phagocytic activity was higher in H. discus discus hemocytes than in T. cornutus hemocytes, and the kinetics of PMA-stimulated oxidative activity was different between hemocytes of the disk abalone and the spiny top shell. Finally our results suggest for the first time a predominant mitochondrial origin of oxidative activity in gastropod hemocytes., (Copyright 2009 Elsevier Ltd. All rights reserved.)

The Suminoe oyster Crassostrea ariakensis has been attempted to be introduced in the Chesapeake Bay, USA, as an alternative to the eastern oyster Crassostrea virginica. Commercial production of Suminoe oysters is currently restricted due to the incomplete understanding of their biological, physiological and immunological nature. Accordingly, understanding immune system of C. ariakensis is crucial to prevent disease associated mortality and subsequent management of the Suminoe oyster. We investigated immunological activities and morphology of hemocytes of the Suminoe oyster using flow cytometry and light microscopy. Three types of hemocytes were identified in the hemolymph including hyalinocyte, granulocyte and blast-like cells. Hyalinocytes were the largest cells and the most abundant, while granulocytes were intermediate-size cell containing numerous granules in the cytoplasm. Blast-like cells were the smallest and least numerous. Flow cytometry revealed that the granulocytes are most active in the cell phagocytosis and spontaneous reactive oxygen species (ROS) production. The hyalinocytes also showed a certain level of the phagocytosis and oxidative activity but in a lesser extent than the granulocytes. In contrast, the blast-like cells did not show any phagocytosis or oxidative activity. The flow cytometry used in this study confirmed that as observed from other marine bivalves, the granulocytes are the main hemocytes involved in the cellular defence in the Suminoe oyster.

Objective: We examined the Transtheoretical Model (TTM) of behavioral change based on cervical cancer screening among Korean women. We assessed the stages of Pap smear adoption, positive and negative attitudes toward Pap smear, and contributory factors., Method: This study was based on the 2007 Korean National Cancer Screening Survey, which included a total of 1224 women > or =30 years. Multiple logistic analyses were conducted to determine the factors that influenced the women's stages of adoption., Results: Fifty-seven percent of the women reported on-schedule screening while 43% did not. Those subjects who were 50-64 years old, had higher incomes, married, lived in a rural area, exercised regularly, and had regular checkups were more likely to be in maintenance than in precontemplation. However, the con scores were negatively associated with a transition from precontemplation to maintenance. Further, a high con score was associated with being in relapse risk or relapse rather than in maintenance while the pro scores were not significant across the stages of adoption., Conclusion: Our results confirm the applicability of the TTM to Pap smear screening in an Asian context. Age, income, marital status, residency, regular exercise, health checkups, and con scores were significantly associated with the stage of adoption.

Hepatitis C virus (HCV) RNA replication requires viral nonstructural proteins as well as cellular factors. Recently, a cellular protein, synaptotagmin-binding, cytoplasmic RNA-interacting protein (SYNCRIP), also known as NSAP1, was found to bind HCV RNA and enhance HCV IRES-dependent translation. We investigate whether this protein is also involved in the HCV RNA replication. We found that SYNCRIP was associated with detergent-resistant membrane fractions and colocalized with newly-synthesized HCV RNA. Knock-down of SYNCRIP by siRNA significantly decreased the amount of HCV RNA in the cells containing a subgenomic replicon or a full-length viral RNA. Lastly, an in vitro replication assay after immunodepletion of SYNCRIP showed that SYNCRIP was directly involved in HCV RNA replication. These findings indicate that SYNCRIP has dual functions, participating in both RNA replication and translation in HCV life cycle.

A novel C-type lectin designated Manila clam lectin 3 (MCL3), with a molecular weight of 17.380kDa, was identified among haemocyte expressed sequence tags of Perkinsus olseni-infected Manila clams. MCL3 was expressed in Escherichia coli M15 cells and purified with a Ni-NTA His-binding resin matrix. MCL3 agglutinated rabbit erythrocytes in the presence of Ca(+2). MCL3-induced agglutination was partially inhibited by GalNAc, Man, lactose, and raffinose, whereas the polysaccharides bovine mucin type II and Candida mannan completely inhibited agglutination. MCL3 was expressed in the haemocytes of Manila clams 3 days after infection with P. olseni and 1 day after infection with Vibrio tapetis. Based on real-time PCR analysis, mRNA transcripts of MCL3 were present in the adductor, foot, gill, mantle, palp, and siphon of Perkinsus-infected Manila clams. Furthermore, MCL3 was detected in the foot, gill, mantle, gonad, digestive gland, gonad connective tissue, intestine, and stomach by immunohistochemical localization.

Relationships of 13 species of the genus Glossina (tsetse flies) were inferred from mitochondrial (cytochrome oxidase 1, NADH dehydrogenase 2 and 16S) and nuclear (internal transcribed spacer 1 of rDNA) sequences. The resulting phylogeny confirms the monophyly of the morphologically defined fusca, morsitans and palpalis subgenera. Genetic distances between palpalis and morsitans subspecies suggest that their status needs revision. In particular, cytochrome oxidase 1 sequences showed large geographical differences within G. palpalis palpalis, suggesting the existence of cryptic species within this subspecies. The morphology of palpalis group female genital plates was examined, and individuals were found varying outside the ranges specified by the standard identification keys, making definitive morphological classification impossible. A diagnostic PCR to distinguish G. palpalis palpalis, G. tachinoides and G. palpalis gambiensis based on length differences of internal transcribed spacer 1 sequences is presented.

The pathological condition of the short-neck clam Ruditapes philippinarum was surveyed along the coast of Kumamoto, Japan, in June 2004. DNA sequences of the non-transcribed spacer region and internal transcribed spacer region flanking 5.8S rRNA identified Perkinsus olseni among the clams. Ray's fluid thioglycollate medium assay indicated that 96.7% of the clams surveyed from the Kiguchi River tidal flat (native clams, Stn KR-N) and 96.7% of the clams surveyed from the Midori River tidal flat (Stn MR) were infected with P. olseni with an infection intensity of 464,278 and 199,937 Perkinsus cells/gram tissue wet weight (gWW), respectively. In contrast, 66.7% of the clams imported from China and stored along the Kiguchi River tidal flat (Stn KR-I) and 20.2% of clams from the Arao tidal flat (Stn AT) were infected with P. olseni with an infection intensity of 37,547 and 3382 Perkinsus cells/gWW, respectively. Brown ring disease was detected in the clam population from Stn KR-I at a prevalence of 90.0%. Polymerase chain reaction and the 16S rRNA sequence suggested that the agents of brown ring disease observed at Stn KR-I were Vibrio tapetis-like bacteria. Sporocysts and metacercariae of unidentified trematodes were also observed in the gonads and mantle of the clams from Stn KR-I, Stn MR, and Stn AT, at prevalences of 7.1-42.9%. Metacestodes (larval tapeworms) were found in the foot and digestive gland at a prevalence of 52.5%, 30.0%, and 14.3% in clams from Stns MR, AT, and KR-N, respectively. Histology also showed massive hemocyte infiltration and inflammation among clams heavily infected with P. olseni. Castration of the follicle was typical among clams infected with the trematode. The data indicate that most of the clams along the coast of Kumamoto are infected with various pathogens at various rates of infection, and these pathogens could have negative effects on the clam population in the long term.

This is the first report of the occurrence of Perkinsus olseni in the Venus clam Protothaca jedoensis off the western and southern coasts of South Korea. Histological observations revealed Perkinsus-like organisms in the mantle, gills, digestive tubules, and gonad. Haemocytic infiltration and tissue necrosis were also observed in heavily infected clams. Hypnospore formation of the Perkinsus-like organism was confirmed with Ray's fluid thioglycollate medium assay (RFTM). When incubated in filtered and aerated seawater, the hypnospore gave rise to cell division and subsequently discharged hundreds of motile zoospores. Genus- and species-specific polymerase chain reaction (PCR) assays and the DNA sequences of the internal transcribed spacer region (ITS) of the Perkinsus sp. isolated from the Venus clam were identical to those of P. olseni reported from the Manila clam Venerupis (=Ruditapes)philippinarum. Based on the DNA sequences and microscopic data, the Perkinsus-like pathogen isolated from P. jedoensis was identified as P. olseni, which parasitizes the Manila clam in European and Asian waters and Haliotis rubra (abalone) in Australian waters. The prevalence and infection intensity of a clam population collected from Yosu, Korea, was determined using RFTM and Choi's 2M NaOH digestion technique. The intensities averaged 10,768 and 7438 Perkinsus cells per gram tissue in 2003 and 2004, and the prevalence ranged from 37.0 to 53.9%, respectively.

The characteristics of a lectin from the marine bivalve Ruditapes philippinarum (Manila clam) were investigated in this study. A method was developed for the isolation of the Manila clam lectin (MCL). Affinity chromatography using mucin-Sepharose, ion-exchange chromatography with DEAE-Toyoperl, and gel filtration with Superose 6 were used for MCL isolation. SDS-PAGE showed that the MCL protein had a molecular mass of 138 kDa, and consisted of 74-, 34-, and 30-kDa subunits. The native lectin in solution behaved as a 274-kDa protein in gel filtration chromatography. The lectin activity of MCL was Ca2+ -dependent, and the optimal Ca2+ concentration for MCL activity was 20 mM. MCL activity was stable between pH 6 and pH 9, and was temperature-dependent; incubation of MCL at 90 degrees C led to irreversible denaturation. The activity of MCL was not inhibited by the presence of monosaccharides, such as Man, Fuc, Gal, Glc, GlcNAc, and NeuNAc. In contrast, the lectin activity of MCL was strongly inhibited by the presence of porcine mucins. MCL activity was also inhibited by N-acetyl-d-galactosamine, human embryonic alpha-1-acid glycoprotein, and highly branched mannans from marine halophilic bacteria. It appears that MCLs have unusual carbohydrate specificities for N-acetyl-d-galactosamine, which contains both mucin-type carbohydrate chains and highly branched mannans. Immunofluorescence staining revealed that MCL was bound to the surfaces of purified hypnospores from Perkinsus sp., which is a protozoan parasite of Manila clams.

Polypyrimidine-tract-binding protein (PTB) has been shown to bind specifically to the 5' ends of mouse hepatitis virus (MHV) RNA and its complementary strand. To further characterize the function of PTB in MHV replication, we generated dominant-negative mutant cell lines that express a full-length PTB or a truncated form of PTB, which includes only the N-terminal half of the protein, retaining its protein-dimerization domain. The truncated form of PTB was localized in the cytoplasm, whereas the full-length PTB was present mainly in the nucleus. The truncated form can interact with the full-length PTB in vitro. We observed that both the full-length and the truncated PTB, when overexpressed, functioned in a dominant-negative manner in MHV replication. However, the truncated form exhibited more severe effects on syncytia formation, virus production, and synthesis of viral RNA and viral proteins. To clarify the precise function of PTB in MHV replication, we dissociated the processes of viral transcription from translation by transfecting different types of MHV defective-interfering (DI) RNA that contain various reporter genes into these stable cell lines. Transcription of the DI RNA during MHV infection was greatly inhibited in these cell lines, indicating that PTB modulates MHV transcription. In contrast, translation of the DI RNA was not affected by PTB depletion in in vitro translation in rabbit reticulocyte lysate or by PTB overexpression in in vivo translation experiments in MHV-infected cells. Given that PTB interacts with the viral N protein, which is one of the components of the MHV replication complex, PTB may exert its function on viral replication/transcription by association with viral RNA as well as other viral and cellular factors in the replication complex.

The search for differentially expressed genes in gastric cancer may help define molecular alterations and molecular diagnosis of gastric cancer. Using the differential display PCR technique, we identified 18 genes that are differentially expressed between normal and tumor human gastric tissues. Their expressions were verified with reverse Northern blot analysis and Northern blot analysis. Oxidative phosphorylation-related genes, antizyme inhibitor of ornithine decarboxylase, protein phosphatase-1beta, 35-kDa peroxisomal membrane protein, and cystic fibrosis transmembrane conductance receptor were highly expressed in tumor tissue, whereas pepsinogen A, Na-K ATPase alpha subunit, nerve growth factor receptor, and alpha-tropomyosin were highly expressed in normal tissue. In addition, 3 unknown genes were found to be differentially expressed in paired gastric tissues. These differentially expressed genes may provide significant opportunities for further understanding of gastric carcinogenesis and the molecular diagnosis of gastric cancer., (Copyright 2000 Academic Press.)

Spherical microporous silica powders with a narrow size distribution have been prepared by a precipitation technique involving the hydrolysis reaction of a silicon alkoxide in ethanol. The formation of the important microporosity has been investigated following two templating methods: the co-hydrolysis and condensation of two alkoxides, one of which presents porogen function, and the adsorption of an organic compound (glycerol) as the porogen. In both processes, the organic porogen is removed by a simple calcination. In the first method, the addition of more than 20 mol% of the porogen alkoxide, necessary for generating enough microporosity, disturbs completely the condensation process resulting in microporous, nonuniform silica particles of large size distribution. The best result has been obtained with the glycerol method where submicrometer-sized silica spheres with a very narrow size distribution and about 40 vol% porosity have been synthesized. The presence of glycerol during the synthesis considerably affects the precipitation mechanism, resulting in a larger mean particle size. The use of an aggregative growth model has successfully been employed to explain the effect of the porogen during particle formation. The precipitation mechanism of silica involves the aggregation between particles of similar size until a critical size is reached, resulting in a uniform particle size distribution. In the presence of glycerol, it has been shown that a second aggregative growth between still-nucleating primary particles and large particles occurred with increasing reaction time. This second aggregative growth appears at an intermediate stage of the precipitation process and is due to both the precipitation of smaller primary particles and the destabilization of the colloidal stability of the system. This explains why the final particle size reached in this system is larger compared to silica particles synthesized without glycerol and shows how glycerol is incorporated in the silica particles. The synthesis of silica microporous spheres of narrow size distribution, by varying particle size and porosity, should yield a wide range of aqueous silica slurries for particular chemical mechanical polishing applications. Copyright 2000 Academic Press.

The IE2 gene product of human cytomegalovirus (HCMV) is one of a few viral regulatory proteins expressed immediately upon infection of the host cell. It is a potent transcriptional activator of many viral and cellular promoters. We found that the retinoblastoma susceptibility gene product (Rb) dramatically suppressed this IE2 transactivation of various promoters. However, unlike another tumor suppressor protein, p53, Rb did not have any significant effect on basal levels of transcription, suggesting that Rb specifically interacts with IE2 rather than other cellular factors involved in the general transcription machinery. We found by protein-affinity chromatography that Rb in nuclear extracts or produced by in vitro translation directly bound to IE2. Our results suggest that Rb may regulate the life cycle of HCMV, which is endemic in the human population. Furthermore, these data may provide new insights into the slow rate of HCMV DNA replication in cells and the possible involvement of HCMV in tumorigenesis.