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Purpose: To compare performance of 1-mm deep learning reconstruction (DLR) with 3-mm routine MRI imaging for the delineation of pituitary axis and identification of cavernous sinus invasion for pituitary macroadenoma., Method: This retrospective study included 104 patients (59.4 ± 13.1 years; 46 women) who underwent an MRI protocol including 1-mm deep learning-reconstructed and 3-mm routine images for evaluating pituitary adenoma between August 2019 and October 2020. Five readers (24, 9, 2 years, and <1 year of experience) assessed the delineation of pituitary axis (gland and stalk) and the presence of cavernous sinus invasion for using a pairwise design. The signal-to-noise ratio (SNR) was measured. Diagnostic performance as well as image preference data were analysed and compared according to the readers' experience using the McNemar test., Results: For delineation of normal pituitary axis, all readers preferred thin 1-mm DLR MRI over 3-mm MRI (overall superiority, 55.8 %, P <.001), with this preference being greater in the less experienced readers (92.3 % vs. 55.8 % [expert], P <.001). The readers showed higher diagnostic performance for cavernous sinus invasion on 1-mm (AUC, 0.91 and 0.92) than on 3-mm imaging (AUC, 0.87 and 0.88). The SNR of the 1-mm DLR was 1.21-fold higher than that of the routine 3-mm imaging., Conclusion: Deep learning reconstruction-based 1-mm imaging demonstrates improved image quality and better delineation of microstructure in the sellar fossa and is preferred by both radiologists and non-radiologist physicians, especially in less experienced readers., 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 © 2022 Elsevier B.V. All rights reserved.)

Objectives: To determine whether imaging-based risk stratification enables prognostication in diffuse glioma, NOS (not otherwise specified)., Methods: Data from 220 patients classified as diffuse glioma, NOS, between January 2011 and December 2020 were retrospectively included. Two neuroradiologists analyzed pre-surgical CT and MRI to assign gliomas to the three imaging-based risk types considering well-known imaging phenotypes (e.g., T2/FLAIR mismatch). According to the 2021 World Health Organization classification, the three risk types included (1) low-risk, expecting oligodendroglioma, isocitrate dehydrogenase (IDH)-mutant, and 1p/19q-codeleted; (2) intermediate-risk, expecting astrocytoma, IDH-mutant; and (3) high-risk, expecting glioblastoma, IDH-wildtype. Progression-free survival (PFS) and overall survival (OS) were estimated for each risk type. Time-dependent receiver operating characteristic analysis using 10-fold cross-validation with 100-fold bootstrapping was used to compare the performance of an imaging-based survival model with that of a historical molecular-based survival model published in 2015, created using The Cancer Genome Archive data., Results: Prognostication according to the three imaging-based risk types was achieved for both PFS and OS (log-rank test, p < 0.001). The imaging-based survival model showed high prognostic value, with areas under the curves (AUCs) of 0.772 and 0.650 for 1-year PFS and OS, respectively, similar to the historical molecular-based survival model (AUC = 0.74 for PFS and 0.87 for OS). The imaging-based survival model achieved high long-term performance in both 3-year PFS (AUC = 0.806) and 5-year OS (AUC = 0.812)., Conclusion: Imaging-based risk stratification achieved histomolecular-level prognostication in diffuse glioma, NOS, and could aid in guiding patient referral for insufficient or unsuccessful molecular diagnosis., Key Points: • Three imaging-based risk types enable distinct prognostication in diffuse glioma, NOS (not otherwise specified). • The imaging-based survival model achieved similar prognostic performance as a historical molecular-based survival model. • For long-term prognostication of 3 and 5 years, the imaging-based survival model showed high performance., (© 2022. The Author(s), under exclusive licence to European Society of Radiology.)

Aquaporins (AQPs) are key proteins that regulate fluid homeostasis in cells via modulating osmotic water transport. In the present study, we identified three variants of Aqp1ab transcript (mmAQP1ab x1, mmAQP1ab x2, and mmAQP1ab x3) in mud loaches (Misgurnus mizolepis), and their expression patterns were examined in response to heavy metal and immunostimulant exposure. Mud loach Aqp1ab gene has a somewhat different organizational structure (i.e. five exons interrupted by four introns) compared to most other teleostean Aqp1ab orthologues, which have four exons. The 5'-flanking regulatory region of Aqp gene showed diverse transcription factor binding motifs, particularly those associated with stress/immune responses. Developmental expression patterns indicated that Aqp1ab mRNA was maternally inherited, presumably important for fine-tuning gene expression during embryonic and early larval developments. Expression of mud loach Aqp1ab mRNA was significantly and differentially modulated in several tissues (intestine, kidneys, spleen, and liver) in response to various heavy metal treatments. In addition, Aqp1ab gene expression was highly induced in response to immune challenge (LPS and polyI:C injections). Collectively, our results suggested that AQPs are multifunctional effectors playing diverse roles in cellular pathways relevant to immune and/or stress adaptation responses, in addition to their involvement in osmoregulation., (Copyright © 2022 Elsevier Inc. All rights reserved.)

MicroRNAs (miRNAs) have been implicated in nerve injury and demyelination; however, their functions in peripheral nerves remain unclear. To determine the potential functions of miRNAs, an miRNA array was carried out. Here, miRNA array analysis of neuregulin-treated Schwann cells revealed 18 upregulated (> 2-fold) and 13 downregulated (> 2-fold) miRNAs. After sciatic nerve injury, miR708-5p was highly expressed in neuregulin-treated Schwann cells, whereas it was downregulated during postnatal development. A predicted functional interaction was found between miR708-5p and transcription factor CP2-like protein 1 (TFCP2L1) using a bioinformatics tool. This finding suggested that miR708-5p may regulate TFCP2L1. During sciatic nerve development, TFCP2L1 was upregulated on postnatal days 1 and 4, while it was downregulated after nerve axotomy and crush injury. Notably, TFCP2L1 was upregulated in cAMP-treated Schwann cells. We also found that activity of the myelin protein zero promoter was downregulated in TFCP2L1 siRNA-treated Schwann cells, whereas it was upregulated in TFCP2L1-overexpressing cells. Immunofluorescence analysis showed that TFCP2L1 was localized in Schwann cells. In addition, miR708-5p overexpression promoted migration of Schwann cells, while miR-708-5p inhibitor inhibited migration. miR708-5p inhibitor also blocked the migration of TFCP2L1 siRNA-treated Schwann cells. These findings indicate the functions of miR708-5p in TFCP2L1 regulation in the peripheral nervous system occur via regulation of Schwann cell migration., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Localized adiposity is a serious aesthetic problem and a well-known health risk factor. There is a growing interest in minimally invasive treatment options for excessive fat accumulation, such as pharmacopuncture. LIPOSA is a newly developed pharmacopuncture formula from three natural herbs: The tuber of Pinellia ternata (Thunb.) Breitenb., the whole plant of Taraxacum platycarpum Dahlst. and the root of Astragalus membranaceus Bunge. The present study investigated the effects of pharmacopuncture treatment with LIPOSA on localized adiposity. Male C57BL/6J mice were fed high fat diet for 8 weeks to induce obesity. Then, 100 µl LIPOSA was injected into the left-side inguinal fat pad at various concentrations, including 13.35, 26.7 and 53.4 mg/ml. Normal saline was injected into the right-side inguinal fat pad of each mouse as a control. The treatment was performed three times per week for 2 weeks. The weight and histological changes were analyzed in the inguinal fat pad of the obese mice. The expression levels of adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), autophagy-related gene (ATG)5, ATG7 and LC3-II, as lipophagy-related factors, were evaluated to confirm the lipid-catabolic effects of LIPOSA. LIPOSA pharmacopuncture markedly decreased the weight of the fat tissue and the size of the adipocytes in the inguinal region of the mouse models of obesity in a dose-dependent manner. The expression levels of ATGL, HSL, ATG5, ATG7 and LC3-II were significantly increased by the LIPOSA treatments. In addition, LIPOSA pharmacopuncture was found to decrease the expression levels of ACC, PPAR-γ and PEPCK. The results indicated that subcutaneous injection of LIPOSA can degrade local fat and induce lipophagic and lipase activation effects. In addition, lipid metabolism related to fat accumulation was regulated by the LIPOSA treatment. The present study suggests that LIPOSA pharmacopuncture can be a non-surgical alternative in the treatment of localized adiposity., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Lee et al.)

Even a tiny functioning pituitary adenoma could cause symptoms; hence, accurate diagnosis and treatment are crucial for management. However, it is difficult to diagnose a small pituitary adenoma using conventional MR sequence. Deep learning-based reconstruction (DLR) using magnetic resonance imaging (MRI) enables high-resolution thin-section imaging with noise reduction. In the present single-institution retrospective study of 201 patients, conducted between August 2019 and October 2020, we compared the performance of 1 mm DLR MRI with that of 3 mm routine MRI, using a combined imaging protocol to detect and delineate pituitary adenoma. Four readers assessed the adenomas in a pairwise fashion, and diagnostic performance and image preferences were compared between inexperienced and experienced readers. The signal-to-noise ratio (SNR) was quantitatively assessed. New detection of adenoma, achieved using 1 mm DLR MRI, was not visualised using 3 mm routine MRI (overall: 6.5% [13/201]). There was no significant difference depending on the experience of the readers in new detections. Readers preferred 1 mm DLR MRI over 3 mm routine MRI (overall superiority 56%) to delineate normal pituitary stalk and gland, with inexperienced readers more preferred 1 mm DLR MRI than experienced readers. The SNR of 1 mm DLR MRI was 1.25-fold higher than that of the 3 mm routine MRI. In conclusion, the 1 mm DLR MRI achieved higher sensitivity in the detection of pituitary adenoma and provided better delineation of normal pituitary gland than 3 mm routine MRI., (© 2021. The Author(s).)

Although microRNAs (miRNAs or miRs) have been studied in the peripheral nervous system, their function in Schwann cells remains elusive. In this study, we performed a microRNA array analysis of cyclic adenosine monophosphate (cAMP)-induced differentiated primary Schwann cells. KEGG pathway enrichment analysis of the target genes showed that upregulated miRNAs (mR212-5p, miR335, miR20b-5p, miR146b-3p, and miR363-5p) were related to the calcium signaling pathway, regulation of actin cytoskeleton, retrograde endocannabinoid signaling, and central carbon metabolism in cancer. Several key factors, such as purinergic receptors (P2X), guanine nucleotide-binding protein G(olf) subunit alpha (GNAL), P2RX5, P2RX3, platelet-derived growth factor receptor alpha (PDGFRA), and inositol 1,4,5-trisphosphate receptor type 2 (ITPR2; calcium signaling pathway) are potential targets of miRNAs regulating cAMP. Our analysis revealed that miRNAs were differentially expressed in cAMP-treated Schwann cells; miRNA363-5p was upregulated and directly targeted the P2X purinoceptor 4 (P2RX4)-UTR, reducing the luciferase activity of P2RX4. The expression of miRNA363-5p was inhibited and the expression of P2RX4 was upregulated in sciatic nerve injury. In contrast, miRNA363-5p expression was upregulated and P2RX4 expression was downregulated during postnatal development. Of note, a P2RX4 antagonist counteracted myelin degradation after nerve injury and increased pERK and c-Jun expression. Interestingly, a P2RX4 antagonist increased the levels of miRNA363-5p. This study suggests that a double-negative feedback loop between miRNA363-5p and P2RX4 contributes to the dedifferentiation and migration of Schwann cells after nerve injury.

Objectives: To determine reproducible MRI parameters predictive of molecular subtype and risk stratification in glioma and develop a structured reporting system., Methods: All study patients were initially diagnosed with glioma, 141 from the Cancer Genome Atlas and 131 from our tertiary institution, as training and validation sets, respectively. Images were analyzed by three neuroradiologists with 1-7 years of experience. MRI features including contrast enhancement pattern, necrosis, margin, edema, T2/FLAIR mismatch, internal cyst, and cerebral blood volume higher than normal cortex were reported using a structured reporting system. The pathology was stratified into five risk types: (1) oligodendroglioma, isocitrate dehydrogenase [IDH]-mutant, 1p19q co-deleted; (2) diffuse astrocytoma, IDH-mutant, grade II-III; (3) glioblastoma, IDH-mutant, grade IV; (4) diffuse astrocytoma, IDH-wild, grade II-III; and (5) glioblastoma, IDH-wild, grade IV. Significant predictors were selected using multivariate logistic regression, and diagnostic performance was tested using a validation set., Results: Reproducible imaging parameters exhibiting > 50% agreement across readers included the presence of necrosis, T2/FLAIR mismatch, internal cyst, and predominant contrast enhancement. In the validation set, prediction of risk type 5 exhibited the highest diagnostic performance with AUCs of 0.92 (reader 1) and 0.93 (reader 2) with predominant enhancement, followed by risk type 2 with AUCs of 0.95 and 0.95 with T2/FLAIR mismatch sign and no necrosis, and risk type 1 with AUCs of 0.84 and 0.83 with internal cyst or necrosis. Risk types 3 and 4 were difficult to visually predict., Conclusions: Imaging parameters with high reproducibility enabling prediction of IDH-wild-type glioblastoma, IDH-mutant/1p19q co-deletion oligodendroglioma, and IDH-mutant diffuse astrocytoma were identified., Key Points: • Reproducible MRI parameters for determining molecular subtypes of glioma included the presence of necrosis, T2/FLAIR mismatch, internal cyst, and predominant contrast enhancement. • IDH-wild type glioblastoma, IDH-mutant/1p19q co-deletion oligodendroglioma, and IDH-mutant low-grade astrocytoma were identified using MRI parameters with high inter-reader reproducibility. • Identification of IDH-wild type low-grade glioma and IDH-mutant glioblastoma was difficult by visual analysis., (© 2021. European Society of Radiology.)

Localized fat deposits are associated with health and aesthetic problems that mainly affect a large proportion of individuals. Recently, bioactive constituents of TP have been reported to affect lipid metabolism. In this study, we performed a network pharmacological analysis to assume potential lipolytic effects of TP and investigated the actual lipolytic effects of TP extract injection on local body fat and its underlying mechanism. Using the genes related to active compounds of TP, the network was constructed. Through the Functional Enrichment Analysis, Lipid Metabolism and Fatty Acid Metabolism were expected to be affiliated with the network, which implied possible lipolytic effects of TP. On the comparison between TP network and Obesity-related Gene Sets, about three-fourths of elements were in common with the gene sets, which indicated a high relevance between TP and obesity. Based on the genes in lipolysis-related pathways, Perilipin, CGI-58, ATGL, HSL and MGL were selected to identify the actual lipolytic effects of TP. TP injection reduced the inguinal fat weight. Also, the diameter of the adipocytes was decreased by the TP treatment in HFD-induced obese mice. In addition, TP suppressed lipid accumulation in differentiated 3T3-L1 adipocytes. Moreover, because the expression of Perilipin was increased, CGI-58, ATGL, HSL and MGL were markedly decreased. Furthermore, glycerol release was down-regulated by the TP treatment. TP exerted its lipolytic effects by regulating the lipolysis machinery through stimulation of lipases. Based on the present findings, TP is expected to be a potent component of injection lipolysis for removing localized body fat., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Rare neurovascular diseases (RNVDs) have not been well-recognized in Korea. They involve the central nervous system and greatly affect the patients' lives. However, these diseases are difficult to diagnose and treat due to their rarity and incurability. We established a list of RNVDs by referring to the previous literature and databases worldwide to better understand the diseases and their current management status. We categorized 68 RNVDs based on their pathophysiology and clinical manifestations and estimated the prevalence of each disease in Korea. Recent advances in genetic, molecular, and developmental research have enabled further understanding of these RNVDs. Herein, we review each disease, while considering its classification based on updated pathologic mechanisms, and discuss the management status of RNVD in Korea., Competing Interests: The authors have no potential conflicts of interest to disclose., (Copyright © 2021 The Korean Society of Radiology.)

Background: Pine nut oil (PNO), a standardized and well-defined extract of Pinus koraiensis (Korean pine), has beneficial effects on wound healing, inflammatory diseases, and cancer. However, the explanation for the mechanism by which PNO reduces body fat remains uncertain. We performed a protein-protein interaction network (PPIN) analysis to explore the genes associated with pinolenic acid using the MEDILINE database from PubChem and PubMed. It was concluded through the PPIN analysis that PNO was involved in a neutral lipid biosynthetic process., Purpose: This study evaluated the effects of PNO predicted by the network analysis of fat accumulation in chronic obesity mouse models established by feeding a high fat diet (HFD) to C57BL/6J mice and explored potential mechanisms., Methods: HFD mice were fed only HFD or HFD with PNO at 822 and 1644 mg/kg. After an oral administration of 7 weeks, several body weight and body fat-related parameters were examined, including the following: adipose weight, adipocyte size, serum lipid profiles, adipocyte expression of PPAR-γ, sterol regulatory element binding protein (SREBP)-1c, lipoprotein lipase (LPL) and leptin., Results: We showed that oral administration of PNO to HFD mice reduces body fat weight, fat in tissue, white adipose tissue weight, and adipocyte size. The serum cholesterol was improved in the HFD mice treated with PNO. Additionally, PNO has significantly attenuated the HFD-induced changes in the adipose tissue expression of PPAR-γ, SREBP-1c, LPL, and leptin., Conclusions: The findings from this study based on the PPIN analysis suggest that PNO has potential as drug to reduce body fat through fat regulatory mechanisms by PPAR-γ and SREBP-1c., (Copyright © 2021. Published by Elsevier GmbH.)

With an increasing interest for the use of triploids in abalone aquaculture, it is crucial to understand their physiological responses to environmental stress, particularly such as heat-stress and hypoxia, which are significant factors that cause adverse effects on the efficiency and capacity of farming practice in abalone production. However, nothing is known about gene expression of triploid abalone to modulate physiological responses under different environmental stresses. Transcriptomic response to the acute heat-stress and hypoxia were explored in hepatopancreas of diploid and triploid Pacific abalone (Haliotis discus hannai) juveniles. A total of 316 million clean reads were de novo assembled into 271,039 contigs, of which a transcriptome with 209,974 non-redundant transcripts was produced. Using generalized fold change (GFOLD) algorithm with a cut-off │GFOLD value│ > 4, we identified differentially expressed transcripts (DETs) from diploid and triploid abalone in responses to acute heat-stress and hypoxia treatments, respectively. Comparative analysis of the identified DETs revealed alteration of transcript expression profile, level, and process in triploid abalone compared to their diploid siblings. Thus, our study will provide not only comprehensive insight into understanding of the transcriptional regulation to environmental stresses in triploid abalone but a framework for efficient management of triploid abalone aquaculture., (Copyright © 2020. Published by Elsevier B.V.)

Osteoporosis is a common skeletal disease in post-menopausal women. Palmul -tang, an herbal medicine, has been treated for gynecological disease such as anemia, anorexia, anti-fatigue, unspecified menstruation and female infertility in East Asia. In this study, ameliorative effects of Palmul -tang soft extracts (PMT), a Korean Medicine, on osteoporosis were investigated. Ovariectomized (OVX) osteoporotic ICR mice were intragastrically administrated PMT for 4 weeks. The level of bone mineral density (BMD) was analyzed in bone tissues by dual X-ray absorptiometry. The bone medullary cavity and deposition of collagen were investigated by histological analysis. In addition, the BMP-2 signaling-related molecules, osteoblastic differentiation and formation markers, were determined in femoral tissues. The levels of BMD and bone mineral content were significantly increased in tibia, femurs and LV by treatment of PMT. PMT replenished bone marrow cavity and increased collagen deposition in bone marrow cells of femur. In addition, administration of PMT recovered serum ALP, bALP, osteocalcin and calcium levels in osteoporotic mice. Moreover, PMT treatment up-regulated the expressions of BMP-2, RUNX2 and OSX with its downstream factors, ALP, OPN and BSP-1, in the femoral tissues. Taken together, PMT restored the bone minerals and improvement of bone integrity by bone-forming BMP-2 signaling pathway. These results demonstrate that PMT could be an ameliorative agent for osteoporosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Choi, Kim, Nam, Kim, Cho and Yang.)

Atopic dermatitis (AD) is a chronic cutaneous disorder that is characterized by severe eczematous inflammation, swelling, and lichenification. Activation of T helper (Th)-22 cells by allergens leads to epidermal hyperplasia with hyperkeratosis at the chronic phase of AD. Derma-Hc is composed of five natural herbs with anti-AD effects, such as Astragalus membranaceus BUNGE, Schizonepeta tenuifolia Briq., Cryptotympana pustulata Fabr., Angelica sinensis Diels, Arctium lappa L. In this study, the ameliorative effect of Derma-Hc on cutaneous lichenification in 2,4-dinitrochlorobenzne (DNCB)-induced AD was investigated. The dorsal skin of mice was sensitized with DNCB to induce AD-like skin lesions. The dermatitis score and frequency of scratching were evaluated. Thickness of epidermis and dermis was measured by staining with H&E. In addition, infiltration of the mast cell was observed by staining with toluidine blue. Then, desmosomal cadherin, DSC1 was examined by immunofluorescence. Pathological mechanisms involved in lichenification were analyzed in AD-like skin lesions and TNF-α + IFN-γ-treated with human keratinocytes including keratinocyte differentiation genes and JAK1-STAT3 signaling pathway with IL-22 by RT-PCR and western blotting. Topical treatment of Derma-Hc improved AD-like symptoms such as dryness, edema and lichenefication and decreased the number of scratches. Histopathological analysis demonstrated that Derma-Hc significantly inhibited epidermal hyperplasia, hyperkeratosis, and mast cells infiltration. In addition, the level of DSC1 was highly expressed in the epidermis by Derma-Hc. Moreover, mRNA expression level of FLG, an epidermal differentiation complex gene, was recovered by Derma-Hc treatment. KLK5 and KLK7 were markedly reduced to normalize keratinocyte differentiation in dorsal skin tissues and human keratinocytes. On the other hand, Derma-Hc restored expression level of SPINK5. In addition, Derma-Hc inhibited IL-22 via the blockade of JAK1-STAT3 signal pathway. Taken together, Derma-Hc, a natural herbal formula, regulated keratinocyte differentiation and inhibited epidermal hyperplasia with hyperkeratosis. Therefore, Derma-Hc could be a promising candidate for treating chronic AD through modulating signaling of IL-22-associated skin lichenification.

Three distinct superoxide dismutases (SODs)-copper/zinc-SOD (SOD1), manganese-SOD (SOD2), and extracellular copper/zinc-SOD (SOD3)-were identified from a primitive chondrostean fish, Acipenser baerii , enabling the comparison of their transcriptional regulation patterns during development, prelarval ontogeny, and immune stimulation. Each A. baerii SOD isoform (AbSOD) shared conserved structural features with its vertebrate orthologs; however, phylogenetic analyses hypothesized a different evolutionary history for AbSOD3 relative to AbSOD1 and AbSOD2 in the vertebrate lineage. The AbSOD isoforms showed different tissue distribution patterns; AbSOD1 was predominantly expressed in most tissues. The expression of the AbSOD isoforms showed isoform-dependent dynamic modulation according to embryonic development and prelarval ontogenic behaviors. Prelarval microinjections revealed that lipopolysaccharide only induced AbSOD3 expression, while Aeromonas hydrophila induced the expression of AbSOD2 and AbSOD3. In fingerlings, the transcriptional response of each AbSOD isoform to bacterial infection was highly tissue-specific, and the three isoforms exhibited different response patterns within a given tissue type; AbSOD3 was induced the most sensitively, and its induction was the most pronounced in the kidneys and skin. Collectively, these findings suggest isoform-dependent roles for the multigene SOD family in antioxidant defenses against the oxidative stress associated with development and immune responses in these endangered sturgeon fish.

Maternal genes are important in directing early development and determining egg quality in fish. We here report the de novo transcriptome from four tissue libraries of the cyprinid loach, Misgurnus anguillicaudatus , and for the first time identified maternal gene transcripts in unfertilized eggs and suggest their immune system involvement. Expression profiles and functional enrichment revealed a total 24,116 transcripts were expressed as maternal transcripts in unfertilized eggs, which were involved in a wide range of biological functions and pathways. Comparison expression profiles and analysis of tissue specificity revealed that the large numbers of maternal transcripts were stored in unfertilized eggs near the late phase of ovarian maturation and before ovulation. Functional classification showed a total of 279 maternal immune-related transcripts classified with immune system process GO term and immune system KEGG pathway. qPCR analysis showed that transcript levels of identified maternal immune-related candidate genes were dynamically modulated during development and early ontogeny of M. anguillicaudatus. Taken together, this study could not only provide knowledge on the protective roles of maternal immune-related genes during early life stage of M. anguillicaudatus but could also be a valuable transcriptomic/genomic resource for further analysis of maternally provisioned genes in M. anguillicaudatus and other related teleost fishes.

Exposure to particulate matter (PM) has been known to be one of the risk factors to cause allergic asthma, leading to development of respiratory disease. Banhahubak -tang tablet (BHT), a standardized Korean Medicine, is prescribed for neurasthenia, laryngopharyngitis and asthma. In this study, we investigated therapeutic effects of BHT on airway inflammation in ovalbumin (OVA) and PM smaller than 10 μm (PM 10 )-induced allergic asthma mice. To establish allergic asthma with airway hyper-responsiveness by PM 10 , BALB/c mice were sensitized and challenged with OVA and PM 10 , and orally administered BHT. Histological staining was performed to assess airway remodeling. Serum and bronchoalveolar lavage fluid (BALF) was collected for measuring immunoglobulin levels and counting inflammatory cells, respectively. Expression levels of Janus kinase 1 (JAK1)/signal transducer and activator of transcription 6 (STAT6), pro-inflammatory cytokines and type 2 T-helper (Th2)-related cytokines were analyzed in vivo and in vitro models. Histopathological analysis demonstrated that BHT suppressed inflammatory cell infiltration, mucus hypersecretion and collagen deposition in the airway. BHT administration effectively decreased number of inflammatory cells in BALF. BHT reduced total serum Immunoglobulin E (IgE) and Immunoglobulin G (IgG) levels. In addition, BHT significantly inhibited the phosphorylation of JAK1 and STAT6 expressions. Release of pro-inflammatory cytokines and Th2-related cytokines were down-regulated by BHT. In conclusion, BHT mitigated airway inflammation by down-regulating pro-inflammatory and Th2-related cytokines via JAK1/STAT6 signaling. BHT might be a promising herbal medicine for preventing airway inflammation. Moreover, an intervention study among humans is needed to further evaluate the possible beneficial effects of BHT in allergic asthma.

Testis-specific serine/threonine kinase 1-like (TSSK1-like), which plays important roles in late-phase spermatogenesis and male fertility, was characterized in Pacific abalone Haliotis discus hannai, an important commercial marine gastropod. Further, its expression patterns were assessed in diploid and induced triploid males showing differential degrees of testis maturation. Abalone TSSK1-like shared conserved structural features with mammalian TSSK1s and other potential metazoan orthologs, especially regarding the catalytic STKc domain. Phylogenetically, abalone TSSK1-like displayed a genetic affiliation with its molluscan TSSK1-like orthologs and human TSSK1. Additionally, abalone TSSK1-like gene showed a tetrapartite exon-intron organization, unlike the intronless structure of most amniotic tetrapodian TSSK1s. Molecular phylogenetic analysis in the metazoan lineage suggested a possible revision in the origin of the earliest ancestral TSSK1. Further, abalone TSSK1-like showed testis-predominant expression, which was significantly influenced by both age and seasonal reproductive cycles. Comparative expression analyses between diploid and triploid abalone males suggested that robust TSSK1-like expression occurred primarily at the post-meiotic stage. Additionally, RT-PCR assay indicates that mature abalone sperms retain TSSK1-like transcripts after release. Taken together, this study provides useful insights for further studies to assess male reproduction and sterility and/or partial fertility of induced male triploidy in abalone species., Competing Interests: The authors have declared that no competing interests exist.

In vertebrate reproductive system, estrogen receptor (ER) plays a pivotal role in mediation of estrogenic signaling pathways. In the present study, we report the cDNA cloning, expression analysis, and transcriptional activity of ERβ1 subtype from medaka Oryzias dancena . The deduced O. dancena ERβ1 (odERβ1; 519 amino acids) contained six characteristic A/B to E/F domains with very short activation function 2 region (called AF2). A phylogenetic analysis indicated that odERβ1 was highly conserved among teleost ERβ1 subgroup. A conventional RT-PCR revealed that the odERβ1 transcripts were widely distributed in the multiple tissues, the ovary, brain, gill, intestine, kidney, and muscle. Further, the relatively higher odERβ1 expressions in the ovary and brain were clearly reproduced in RT-qPCR assay. When HA-fused odERβ1 expression vector was transfected into HEK293 cells, an immunoreactivity for odERβ1 was mainly detected in the nucleus part. Finally, an estrogen responsive element driven luciferase reporter assays demonstrated that the transcriptional activity of odERβ1 significantly increased by estradiol-17β (E2) in a dose dependent manner ( p <0.05). However, fold-activation of odERβ1 in the presence of E2 was markedly weak, when it compared with those of O. latipes ERβ1. Taken together, these data suggest that odERβ1 represents a functional variant of teleost ERβ subtype and provides a basic tool allowing future studies examining the function of F domain of ERβ1 subtype and expanding our knowledge of ERβ evolution., (© Copyright 2019 The Korean Society of Developmental Biology.)

Two liver-expressed antimicrobial peptide 2 (LEAP2) isoforms were characterized in a primitive chondrostean sturgeon species, Acipenser baerii (Acipenseriformes). A. baerii LEAP2 isoforms represented essentially common structures shared by their vertebrate orthologs at both genomic (i.e., tripartite organization) and peptide (two conserved disulfide bonds) levels. A. baerii LEAP2 isoforms (designed LEAP2AB and LEAP2C, respectively) phylogenetically occupy the most basal position in the actinopterygian lineage and represent an intermediate character between teleostean and tetrapodian LEAP2s in the sequence alignment. Molecular phylogenetic analysis including LEAP2s from extant primitive fish species indicated that the evolutionary origin of ancestral LEAP2 in vertebrate groups should date back to earlier than the actinopterygian-sarcopterygian split. Gene expression assays under both basal and stimulated conditions suggested that A. baerii LEAP2 isoforms have undergone substantial subfunctionalization in tissue distribution pattern, developmental/ontogenetic expression, and immune responses. LEAP2AB showed a predominant liver expression, while LEAP2C exhibited the highest level of expression in the intestine. LEAP2C was a more dominantly expressed isoform during embryonic development and prelarval ontogeny. The LEAP2AB isoform is more closely associated with innate immune response to microbial invasion, compared with LEAP2C, as evidenced by results from LPS, poly(I:C) and Aeromonas hydrophila challenges. Synthetic mature peptides of LEAP2AB displayed a more potent antimicrobial activity than did LEAP2C. Data from this study could be useful not only to provide deeper insights into the evolutionary mechanism of LEAP2 in the actinopterygian lineage but also to better understand the innate immunity of this commercially important chondrostean species., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Hepcidin, a cysteine-rich antimicrobial peptide (AMP), plays key roles as a regulatory hormone in iron homeostasis, providing a link between iron metabolism and innate immunity. Unlike many other AMPs displaying a high degree of sequence variability among closely related organisms, hepcidin is highly conserved from teleosts to mammals. However, little is known about the early ancestry of hepcidins in the vertebrate lineage. Here, we first report potential a prototype hepcidin from the Siberian sturgeon Acipenser baerii, a primitive chondrostean species. The A. baerii hepcidin (AbHAMP) gene showed a tripartite exon-intron organization, which encoded a precursor protein comprised of three structural signatures containing eight cysteine residues, a common structure in vertebrate hepcidin genes and proteins. mRNA expression by iron-overloading and bacterial infection and antibacterial activity revealed that AbHAMP might play a role in iron metabolism regulator in the liver, and in direct and/or indirect host immune response in the kidney against invading pathogen. Comparison of gene and protein sequences reveled that AbHAMP possesses intermediate characteristics between tetrapodian and teleostean hepcidins (HAMP1s). Phylogenetically, AbHAMP had a closer genetic affiliation to tetrapodian orthologs than to teleostean orthologs, suggesting that the structures of this chondrostean hepcidin may closely reflect the structures of an evolutionarily ancestral form that might have evolved into extant hepcidins in tetrapods and teleosts, respectively. Based on the identification of hepcidin from the chondrostean group, the emergence of the common ancestral hepcidin should be traced back to in early Osteichthyes: no later than sarcopterygian (lobe-finned fishes) - actinopterygian (ray-finned fishes) split., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Early growth response protein 2 (EGR2) is an essential transcription factor for peripheral nerve myelination. Schwann cells (SCs), the peripheral myelin-forming glial cells, express high levels of EGR2 during postnatal myelination. In contrast, SCs exhibit low EGR2 expression during Wallerian degeneration after injury. In this study, we screened 10 potential microRNAs (miRNAs) (20a-5p, 137-5p, 140-5p, 148b-3p, 150-5p, 17-5p, 93-5p, 20b-5p, 106b-5p, and 152-3p) that potentially target EGR2 using miRNA algorithms and identified that miRNAs 106b-5p, 140-5p, 93-5p, and 17-5p target EGR2 in SCs. These miRNAs directly target EGR2 by binding to the 3'-untranslated region to suppress EGR2 mRNA levels. Additionally, the levels of miRNAs 93-5p, 106b-5p, 17-5p, and 140-5p were decreased in the sciatic nerves during postnatal development; however, these miRNAs were increased on day 1 after sciatic nerve injury. Taken together, these findings suggest that the expression of EGR2 during postnatal development and Wallerian degeneration could be regulated by the inverse expression of miRNAs 106b-5p, 140-5p, 93-5p, and 17-5p, which target EGR2.

The optimal transfection conditions for efficient transgene delivery into a specific cell type should be empirically determined, particularly in cases involving unusual cell types. We compared the conditions for effective introduction of transgenes into Siberian sturgeon (Acipenser baerii) cell lines by evaluating the cytotoxicity and transfection efficiency of three commercially available transfection reagents: Lipofectamine 2000, X-tremeGENE HP DNA Transfection Reagent, and GeneJuice Transfection Reagent. Plasmid vectors containing the gene encoding enhanced green fluorescent protein were mixed with each of the transfection reagents using reagent-to-plasmid ratios of 1:1, 2:1, and 4:1. Then, the complexes were used to transfect three Siberian sturgeon cell lines derived from the heart, head kidney, and gonad. Cytotoxicity and transfection efficiency were measured via flow cytometry after propidium iodide staining. No significant cytotoxicity was observed at the optimal treatment conditions in all cases, with the exception of Lipofectamine 2000-treated gonad-derived cells. Although the transfection efficiencies in A. baerii cells were generally low, X-tremeGENE HP DNA Transfection Reagent showed the highest transfection efficiency at ratios of 2:1 or 4:1, depending on the cell type. Hence, X-tremeGENE HP DNA Transfection Reagent can be used to effectively transfer foreign genes into three A. baerii cell lines.

An anesthetic protocol was optimized for microinjection-related handling of Siberian sturgeon (Acipenser baerii; Acipenseriformes) prolarvae, an extant primitive fish species commonly grown in aquaculture. Comparative examinations of three selected anesthetics (clove oil, lidocaine, and MS-222) with a dosage regime of 50, 100, 200, and 400 mg/L indicated that MS-222 was the most efficient agent for Siberian sturgeon prolarvae, as evidenced by the fast induction of anesthesia with quick and uniform recovery. Meanwhile, clove oil should be avoided, due to prolonged recovery times varying widely between individuals. None of the tested anesthetics significantly affected prolarval viability at any of the dosage regimes tested in this study. Based on an analysis of the duration of an unconscious state in air, we recommend a dose of 200 mg/L MS-222 for microinjection. Recovery time after use of this dose was influenced by the prolarval age and the development of gills, in which prolarvae older than 3 days after hatching required longer recovery times than did younger prolarvae. Post-recovery behavioral assessment showed no apparent difference between MS-222-anesthetized and non-anesthetized prolarvae in their swimming behavior and phototactic responses. Applicability of currently developed anesthetic protocol using MS-222 in larval microinjection was demonstrated with the injection of a visible dye to the anesthetized prolarvae, followed by the analysis of post-recovery viability. Taken together, the present anesthetic protocol based on 200 mg/L of MS-222 could provide researchers with practical usefulness with good safety margins for the micromanipulation and other related handlings of Siberian sturgeon prolarvae., Competing Interests: The authors have declared that no competing interests exist.

Development of advanced cell culture methods has gained increasing attention because it allows for efficient genetic engineering and precise regulation of animal reproduction on a cellular basis. Numerous studies have attempted to develop an advanced cell culture method. Previous studies have altered cell culture media and pretreated culture plates with functional molecules. Among them, a mussel-inspired polymer coating has been extensively utilized owing to its wide applicability. For instance, adhesion of human embryonic stem cells and neuronal cells on solid surfaces has been improved. Despite the excellent capability of the mussel-inspired polymer coating, most studies have primarily focused on mammalian cells. However, the efficacy of these coatings on the adhesion of other cell lines is yet unclear. This study aimed to assess the potential of the mussel-inspired polymer coating in the regulation of the adhesion of fish ovarian germline stem cells on solid surfaces. Solid surfaces were coated by polydopamine and poly-L-lysine, and the effect of the coatings on cellular behaviors was investigated., Competing Interests: The authors declare no conflicts of interest.

Background: Schwannoma arising from peripheral nervous sheaths is a benign tumor., Methods: To evaluate cell cytotoxicity, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction and terminal deoxynucleotidyltransferase UTP nick-end labeling (TUNEL) assays were used. A microRNA (miRNA) array was used to identify the miRNAs involved in curcumin-induced apoptosis. To examine miRNA expression, quantitative RT-PCR was used., Results: In this study, curcumin exerted cellular cytotoxicity against RT4 schwannoma cells, with an increase in TUNEL-positive cells. Curcumin also activated the expression of apoptotic proteins, such as polyADP ribose polymerase, caspase-3, and caspase-9. The miRNA array revealed that seven miRNAs (miRNA 350, miRNA 17-2-3p, let 7e-3p, miRNA1224, miRNA 466b-1-3p, miRNA 18a-5p, and miRNA 322-5p) were downregulated following treatment with both 10 and 20 μM curcumin in RT4 cells, while four miRNAs (miRNA122-5p, miRNA 3473, miRNA182, and miRNA344a-3p) were upregulated. Interestingly, transfection with a miRNA 344a-3p mimic downregulated the mRNA expression of Bcl2 and upregulated that of Bax, Curcumin treatment in RT 4 cells also reduced the mRNA expression of Bcl2 and enhanced expression of Bax, Overexpression of miRNA344a-3p mimic combined with curcumin treatment activated the expression of apoptotic proteins, including procaspase-9 and cleaved caspase-3 while inhibition of miRNA 344a-3p using miR344a-3p inhibitor repressed cleaved caspase-3 and -9 in curcumin treated RT-4 cells compared to control., Conclusions: Our findings demonstrate that curcumin induces apoptosis in schwannoma cells via miRNA 344a-3p. Thus, curcumin may serve as a potent therapeutic agent for the treatment of schwannoma.

Background: In Korea, there are two types of medical doctors: one practises conventional medicine (hereafter called a physician), and the other practises traditional medicine (hereafter called a Korean medical doctor). This study aimed to compare the provision of complementary and alternative medicine (CAM) by these providers to CAM use per self-judgement in Korea., Methods: We analysed 1668 Korean people via an internet survey with the Korean adopted version of the I-CAM-Q, namely, the International Questionnaire to measure use of CAM, to understand whether respondents used CAM based either on a prescription or advice from a physician or a Korean medical doctor or on self-judgement., Results: In the previous 12 months, the proportions of respondents who were treated by a physician, who were treated by a Korean medical doctor and who were not treated by anyone were 67.9, 20.7 and 14.2%, respectively. Among the respondents who received CAM based on a prescription or advice from a physician, traditional Korean medicine practices and dietary supplements were commonly used; only a small percentage used other CAM therapies. Respondents who received CAM based on a prescription or advice from a Korean medical doctor showed similar results. Acupuncture and moxibustion, traditional Korean medicines (decoction), or cupping were more commonly used. Korean traditional medicines as over-the-counter (OTC) drugs were more commonly used by respondents who received CAM therapy based on a prescription or advice from a physician than by those who received CAM therapy based on a prescription or advice from a Korean medical doctor. A total of 74% of the responders used any CAM by self-judgement in the previous 12 months., Conclusions: For the use of CAM in Korea, in addition to the Korean traditional medical care provided by Korean medical doctors, general physicians advised people regarding Korean traditional medical care and dietary supplements.

Metaphase spread preparation in adult abalone has not been successful, which has restricted the applications of karyotyping-based technologies. Here, we present a non-lethal method to enable preparation of metaphase spreads from live adult abalone using a tissue culture method. Mantle tissue fragments from live adult abalone were cultured in vitro and the cultured cells were used for metaphase spread preparation. To retrieve a sufficient number of proliferating cells required for metaphase spread preparation, at least 14 days of culture was required, and culturing the marginal zone of mantle was more optimal than culturing other areas. Additionally, it was shown that simple medium consisting of basal medium, fetal bovine serum and antibiotics could stimulate cellular proliferation followed by metaphase spread preparation.

This study was conducted to establish and characterize the clonal-cell lines from Siberian sturgeon Acipenser baerii head-kidney tissues and to evaluate its applicability as a research tool. From the culture of A. baerii head-kidney derived cells, 10 cell lines were established first and then eight clonal-cell lines were derived from clonal growth and colony expansion of two cell lines that showed significant high colony-forming ability. All eight clonal-cell lines were morphologically similar and grew stably under monolayer culture but their growth rates were significantly different. They possessed diploid DNA contents, expressed epithelial cell-related genes and showed strong anchorage dependency to substrates. When a clonal-cell line was transfected separately with three plasmid vectors including fluorescent reporter genes driven by cytomegalovirus, marine medaka Oryzias dancena β-actin or A. baerii β-actin promoter, the cell lines expressed fluorescent signals regardless of promoter types. The cells harbouring foreign genes could be expanded to stable cell lines under drug selection and then they additionally could form the extensively proliferating colonies at low-density culture. Finally, the clonal-cell lines showed the susceptibility to viral haemorrhagic septicaemia virus (VHSV). Collectively, the clonal-cell lines from A. baerii head kidney were established and these cell lines will be able to provide an excellent in vitro system for various biological studies in this fish species., (© 2018 The Fisheries Society of the British Isles.)

BACKGROUND: Miho spine loach (Cobitis choii) is an endangered Korean endemic fish. Whole testis cryopreservation is a good way for species preservation, but needs to the sacrifice of a large number of fish to optimize the freezing condition. Considering this limitation, a surrogate fish species was used for the protocol development., Objective: This study was to establish the effective condition for Miho spine loach whole testis cryopreservation by optimizing the conditions for whole testis cryopreservation in an allied species, mud loach (Misgurnus mizolepis)., Materials and Methods: The condition for whole testis cryopreservation was optimized in mud loach first, and then the optimal condition was applied to Miho spine loach testes., Results: The optimal condition for mud loach testis cryopreservation consists of the freezing medium containing 1.3 M dimethyl sulfoxide, 6% fetal bovine serum and 0.3 M trehalose, -1 C/min cooling rate and 26 degree C thawing temperature, which also permits effective cryopreservation of Miho spine loach testes., Conclusion: An effective cryopreservation condition for whole testis of the endangered Miho spine loach has been established by using mud loach as a surrogate fish.

Ice-binding protein (IBPs) protect cells from cryo-injury during cryopreservation by inhibiting ice recrystallization (IR), which is a main cause of cell death. In the present study, we employed two IBPs, one, designated LeIBP from Arctic yeast, and the other, designated FfIBP from Antarctic sea ice bacterium, in the cryopreservation of three economically valuable marine microalgae, Isochrysis galbana, Pavlova viridis , and Chlamydomonas coccoides . Both of the IBPs showed IR inhibition in f/2 medium containing 10% DMSO, indicating that they retain their function in freezing media. Microalgal cells were frozen in 10% DMSO with or without IBP. Post-thaw viability exhibited that the supplementation of IBPs increased the viability of all cryopreserved cells. LeIBP was effective in P. viridis and C. coccoides, while FfIBP was in I. galbana . The cryopreservative effect was more drastic with P. viridis when 0.05 mg/mL LeIBP was used. These results clearly demonstrate that IBPs could improve the viability of cryopreserved microalgal cells., Competing Interests: The authors declare no conflict of interest.

Optimum concentrations of anesthetic clove oil and anesthetic lidocaine-HCl were determined for a species of adult marine medaka, Oryzias dancena , over a range of salinity conditions, and investigated in a transport simulation experiment by analyzing various water and physiological parameters. Research indicated that the higher the concentration of anesthetic at each salinity, the shorter the anesthesia time at each salinity. At each concentration, fish were anesthetized slower at water salinities over 10 ppt ( P <0.05). Anesthesia time at 10 ppt was faster than any other salinity. In 10 ppt salinity, the dissolved oxygen (DO) concentrations and respiratory frequencies of the clove-oil-administered groups decreased until 48 hours ( P< 0.05), whereas the NH 4 + and CO 2 concentrations increased until 48 hours ( P< 0.05). In same period, the DO, NH 4 + , and CO 2 concentrations and respiratory frequencies all decreased as the clove oil concentration increased ( P< 0.05). The trends in the DO, NH 4 + , and CO 2 concentrations and respiratory frequencies in the lidocaine-HCl-administered groups were similar to those in the clove-oil-administered groups. In conclusion, clove oil and lidocaine-HCl are effective anesthetics, improving the transportation of the marine medaka. The results from this study will contribute to safe laboratory handling of the marine medaka, which are commonly required by many research studies and experiments.

The marine medaka, Oryzias dancena is a suitable sample as a laboratory animal because it has a small size and clearly distinguishes between female and male. Data on the growth and maturity of the diploid and triploid sea cucurbit species suitable for laboratory animals are very useful for studying other species. Triploidy was induced in the marine medaka by cold shock treatment (0°C) of fertilized eggs for 45 min, applied two minutes after fertilization. The diploid and triploid male fish were larger than their female counterparts ( P< 0 . 05), and the concentrations of thyroid stimulating hormone (TSH) and thyroxine (T4) were higher in the induced triploids over 1 year ( P< 0 . 05). In both the diploid and tri-ploid groups the concentrations of TSH and T4 were higher in the male fish than in the females ( P< 0 . 05), while the testo-sterone and estradiol-17ß concentrations in the induced triploids were lower than in the diploids ( P< 0 . 05). The gonadosomatic index (GSI) of the triploid fish was lower than that for the diploids, and the GSI for females in each ploidy group were higher than that for the males. For both groups the GSI was highest at 4 months of age, and decreased thereafter to 12 months. Analysis of the gonads of one-year-old triploid fish suggested that the induction of triploidy probably causes sterility in this species; this effect was more apparent in females than in males.

A novel metallothionein (MT) gene from the Pacific abalone H. discus hannai was characterized and its mRNA expression patterns (tissue distribution, developmental expression and differential expression in responsive to various in vivo stimulatory treatments) were examined. Abalone MT shares conserved structural features with previously known gastropod orthologs at both genomic (i.e., tripartite organization) and amino acid (conserved Cys motifs) levels. The 5'-flanking regulatory region of abalone MT gene displayed various transcription factor binding motifs particularly including ones related with metal regulation and stress/immune responses. Tissue distribution and basal expression patterns of MT mRNAs indicated a potential association between ovarian MT expression and sexual maturation. Developmental expression pattern suggested the maternal contribution of MT mRNAs to embryonic and early larval developments. Abalone MT mRNAs could be significantly induced by various heavy metals in different tissues (gill, hepatopancreas, muscle and hemocyte) in a tissue- and/or metal-dependent fashion. In addition, the abalone MT gene was highly modulated in responsive to other non-metal, stimulatory treatments such as immune challenge (LPS, polyI:C and bacterial injections), hypoxia (decrease from normoxia 8 ppm-2 ppm), thermal elevation (increase from 20 °C to 30 °C), and xenobiotic exposure (250 ppb of 17α-ethynylestradiol and 0.25 ppb of 2,3,7,8-tetrachlorodibenzodioxin) where differential expression patterns were toward either up- or down-regulation depending on types of stimulations and tissues examined. Taken together, our results highlight that MT is a multifunctional effector playing in wide criteria of cellular pathways especially associated with development and stress responses in this abalone species., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

This study was conducted to identify embryonic stem cell (ESC) activities of a long-term cultured embryonic cell line previously derived from blastula-stage Oryzias dancena embryos. Five sub-cell lines were established from the embryonic cell line via clonal expansion of single cells. ESC activities, including clonogenicity, alkaline phosphatase (AP) activity, and differentiation capacity, were examined in the five sub-cell lines. We observed both clonogenicity and AP activity in all five sub-cell lines, but the proportion of cells that exhibited both properties was significantly different among them. Even though we detected different formation rates and sizes of embryoid body (EB) among these cells, all lines were stably able to form EBs and further induction for differentiation showed their capability to differentiate into other cell types in a spontaneous manner. From this study, we determined that the embryonic cell lines examined possessed heterogeneous ESC activities and can be utilized as a marine model system for fish ESC-based research.

This study was conducted to establish the efficient condition for stable derivation of heart-derived cell culture in Siberian sturgeon (Acipenser baerii). Three factors including isolation methods, cell densities in initial seeding, and basal media were evaluated for the derivation of heart-derived cell culture. As the results, enzymatic isolation was more efficient than mechanical isolation in both cell retrieval and further culture. Total 48 trials of culture employing low and middle cell densities of less than 5.5 × 10(4) cells/cm(2) in initial seeding did not induce cell survivals (0%, 0/48), but the trials in high cell density of more than 5.5 × 10(5) cells/cm(2) could induce cell survival and primary cell attachment on the plate (88.9%, 24 in 27 trials). When all initially attached cell populations were continuously cultured in two different media, only five cell populations that were enzymatically isolated and cultured under Leibovitz's L-15 medium could grow up to more than 40th subculture. Each cell population was stably cultured according to its own growth rate and all showed normal diploid DNA contents. Two morphologically different cell types that has an elongated shape or a round shape were identified in culture, which was subsequently identified that two cell types are considered as a fibroblast (an elongated shape) and a vascular endothelial cell (a round shape) on the basis of the results of gene and protein expression analysis. Additionally, the sufficient number of viable cells could be successfully retrieved after freezing and thawing from all five cell populations suggesting the feasibility of long-term cryopreservation of the cells. The data and cells obtained from this study will contribute to development of in vitro model for basic biological studies using sturgeon species.

We have identified six putative aquaporin (AQP) genes from marine medaka Oryzias dancena (named odAQPs 1, 3, 8, 10, 11 and 12). The marine medaka AQP cDNAs encode polypeptides of 259-298 amino acids, respectively. Topology predictions showed six transmembrane domains, five connecting loops, and cytoplasmic N- and C-terminal domains, all of which is conserved among AQP molecules. Although asparagine-proline-alanine (NPA) motifs are highly conserved in most odAQP isoforms, several AQPs revealed variant types of motifs such as asparagine-proline-proline (NPP), asparagine-proline-valine (NPV) or/and asparagine-proline-serine (NPS) motifs. The phylogenic analysis showed that marine medaka AQPs had closet relationship with Japanese ricefish (medaka; Oryzias latipes) counterparts. Reverse transcription (RT)-PCR analyses showed that marine medaka AQP transcripts would be expressed in not only osmoregulatory tissues but also nonosmoregulatory tissues, and also that the expression levels of certain AQP isoforms in nonosmoregulatory tissues were readily comparable or even higher than those in typically known osmoregulatory organs. Although the overall tissue distribution patterns of AQPs were not significantly different between 0- and 30-ppt acclimated fish, the expression levels under different salinities were largely variable among isoforms and tissues. This is the first report to investigate tissue expression profiles of teleostean AQPs 11 and 12 during the long-term acclimation to freshwater and salted water., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The aim of this study was to quantitatively assess the effects of short-term intermittent ethanol intoxication on cerebral metabolite changes among sham controls (CNTL), low-dose ethanol (LDE)-exposed, and high-dose ethanol (HDE)-exposed rats, which were determined with ex vivo high-resolution spectra. Eight-week-old male Wistar rats were divided into three groups. Twenty rats in the LDE (n=10) and the HDE (n=10) groups received ethanol doses of 1.5 and 2.5 g/kg, respectively, through oral gavage every 8h for 4days. At the end of the 4-day intermittent ethanol exposure, one-dimensional ex vivo 500-MHz ¹H nuclear magnetic resonance spectra were acquired from 30 samples of the frontal cortex region (from the three groups). Normalized total N-acetylaspartate (tNAA: NAA+NAAG [N-acetylaspartyl-glutamate]), GABA, and glutathione (GSH) levels were significantly lower in the frontal cortex of the HDE-exposed rats than that of the LDE-exposed rats. Moreover, compared to the CNTL group, the LDE rats exhibited significantly higher normalized GABA levels. The six pairs of normalized metabolite levels were positively (+) or negatively (-) correlated in the rat frontal cortex as follows: tNAA and GABA (+), tNAA and aspartate (Asp) (+), myo-Inositol (mIns) and Asp (-), mIns and alanine (+), mIns and taurine (+), and mIns and tNAA (-). Our results suggested that short-term intermittent ethanol intoxication might result in neuronal degeneration and dysfunction, changes in the rate of GABA synthesis, and oxidative stress in the rat frontal cortex. Our ex vivo(1)H high-resolution magic angle spinning nuclear magnetic resonance spectroscopy results suggested some novel metabolic markers for the dose-dependent influence of short-term intermittent ethanol intoxication in the frontal cortex., (Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.)

Two paralogue genes of warm-temperature-acclimation-associated 65-kDa protein were characterized and their mRNA expression patterns during various experimental stimulations were examined in the rockbream (Oplegnathus fasciatus; Perciformes). Rockbream Wap65 isoforms (rbWap65-1 and rbWap65-2) share basically common structural features with other teleostean orthologues and human hemopexin (HPX) at both amino acid (conserved cysteine and histidine residues) and genomic levels (ten-exon structure), although the rbWap65-2 reveals more homologous characteristics to human HPX than does rbWap65-1 isoform. Southern blot analysis indicates that each rbWap65 isoform exists as a single copy gene in the rockbream genome. Both rbWap65 genes were predicted to possess various transcription factor (TF) binding motifs related with stress and innate immunity in their 5ʹ-upstream regions, in which inflammation-related motifs were more highlighted in the rbWap65-2 than in rbWap65-1. Based on the RT-PCR assay, the liver-predominant expression pattern was more apparent in rbWap65-1 than rbWap65-2 isoform. During thermal elevation, clear upregulation was found only for the rbWap65-1. In contrast, immune stimulations (bacterial challenges, viral infection and iron overload) activated more preferentially the rbWap65-2 isoform in overall, although the inducibility was affected by the kinds of stimulators and tissue types. Taken together, our data suggest that the two paralogue rbWap65 isoforms have experienced subfunctionalization and/or neofunctionalization during their evolutionary history, in which the rbWap65-2 has retained closer, functional orthology to the human HPX while the rbWap65-1 have been diversified to be more related with thermal acclimation physiology., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The purpose of this study was to determine whether metabolic profiling of core needle biopsy (CNB) samples using high-resolution magic angle spinning (HR-MAS) magnetic resonance spectroscopy (MRS) could be used for predicting pathologic response to neoadjuvant chemotherapy (NAC) in patients with locally advanced breast cancer. After institutional review board approval and informed consent were obtained, CNB tissue samples were collected from 37 malignant lesions in 37 patients before NAC treatment. The metabolic profiling of CNB samples were performed by HR-MAS MRS. Metabolic profiles were compared according to pathologic response to NAC using the Mann-Whitney test. Multivariate analysis was performed with orthogonal projections to latent structure-discriminant analysis (OPLS-DA). Various metabolites including choline-containing compounds were identified and quantified by HR-MAS MRS in all 37 breast cancer tissue samples obtained by CNB. In univariate analysis, the metabolite concentrations and metabolic ratios of CNB samples obtained with HR-MAS MRS were not significantly different between different pathologic response groups. However, there was a trend of lower levels of phosphocholine/creatine ratio and choline-containing metabolite concentrations in the pathologic complete response group compared to the non-pathologic complete response group. In multivariate analysis, the OPLS-DA models built with HR-MAS MR metabolic profiles showed visible discrimination between the pathologic response groups. This study showed OPLS-DA multivariate analysis using metabolic profiles of pretreatment CNB samples assessed by HR- MAS MRS may be used to predict pathologic response before NAC, although we did not identify the metabolite showing statistical significance in univariate analysis. Therefore, our preliminary results raise the necessity of further study on HR-MAS MR metabolic profiling of CNB samples for a large number of cancers.

NMR Spectroscopy has been established as a major tool for identification and quantification of metabolites in a living system. Since the metabolomics era began, one-dimensional NMR spectroscopy has been intensively employed due to its simplicity and quickness. However, it has suffered from an inevitable overlap of signals, thus leading to inaccuracy in identification and quantification of metabolites. Two-dimensional (2D) NMR has emerged as a viable alternative because it can offer higher accuracy in a reasonable amount of time. We employed (1) H,(13) C-HSQC to profile metabolites of six different laboratory E. coli strains. We identified 18 metabolites and observed clustering of six strains according to their metabolites. We compared the metabolites among the strains, and found that a) the strains specialized for protein production were segregated; b) XL1-Blue separated itself from others by accumulating amino acids such as alanine, aspartate, glutamate, methionine, proline, and lysine; c) the strains specialized for cloning purpose were spread out from one another; and d) the strains originating from B strain were characterized by succinate accumulation. This work shows that 2D-NMR can be applied to identify a strain from metabolite analysis, offering a possible alternative to genetic analysis to identify E. coli strains., (Copyright © 2013 Verlag Helvetica Chimica Acta AG, Zürich.)

Stable transgenic germlines carrying the red fluorescence protein (RFP) gene (rfp) driven by fast skeletal myosin light chain-2 gene (mlc2f) promoter were established in a truly euryhaline fish species, the marine medaka (Oryzias dancena; Beloniformes). Transgenic lines contained transgene copy numbers varying from a single copy to more than 230 copies per genome. Although the transgenic founders displayed mosaic and/or ectopic expression of the RFP signal, the resultant F1 transgenics and their progeny showed consistently stable transmission of the transgenic locus and uniform RFP signal through several subsequent generations. In adult transgenics, an authentic brilliant red fluorescence was achieved over the skeletal muscles of the transgenic individuals, which might be sufficient for ornamental display. Expression analysis of the transgenic mRNAs indicated that rfp transcripts were predominantly expressed in the skeletal muscles. Different transgenic lines displayed different levels of transgene expression at the mRNA, protein, and phenotypic levels. However, the efficiency of transgene expression was independent of the transgene copy number. The RFP protein levels were consistently stable in the transgenic fish muscles through several generations, up to F5. The results of this study suggest that transgenic marine medaka that acquire strong fluorescent signals in their skeletal muscles can be developed as a promising, novel ornamental fish for display in both freshwater and seawater aquaria.

Transgenic marine medaka (Oryzias dancena) germlines were generated by the microinjection of the red fluorescent protein (RFP) reporter gene (rfp) driven by the endogenous choriogenin H gene (chgH) promoter. The selected transgenic lines contained multiple copies of the transgene (3-42 copies per cell) in their genomes. Although all the founders were mosaic, the transgene was stably transmitted from the F1 generation to all subsequent generations following a Mendelian pattern. Different transgenic lines showed different responsiveness to estradiol-17β (E2) exposure at the mRNA and protein levels, and the expression efficiency was dependent upon the transgene copy number. The induction of RFP was significantly affected by the developmental stage of transgenic larvae: later-stage larvae (older than 7 days post-hatching) showed higher sensitivity to E2 exposure than earlier-stage larvae. The response of transgenic expression to E2 was fairly dependent upon the E2 dose (200-3,200 ng/L) and exposure period (1-7 days), according to both a microscopic examination of RFP intensity and a qRT-PCR assay. The transgenic marine medaka showed similar transgenic responses to E2 under freshwater, brackish, and seawater conditions. In addition to E2, the transgenic RFP signal was also successfully induced during 1-week exposure to various other natural (1 μg/L estrone and 10 μg/L estriol) and synthetic (xeno)estrogens (0.1 μg/L 17α-ethynylestradiol, 1 μg/L diethylstilbestrol, and 10 mg/L bisphenol A). The efficiency of transgene expression varied greatly among the chemicals tested. The results of this study suggest that the chgH-rfp transgenic marine medaka species will be useful in the in vivo detection of waterborne estrogens under a wide range of salinity conditions.

The aim of present study was to quantitatively investigate the neurochemical profile of the frontal cortex region in a rat model of long-term alcohol consumption, by using in vivo proton magnetic resonance spectroscopy ((1)H-MRS) at 4.7 T and ex vivo(1)H high-resolution magic angle spinning (HR-MAS) technique at 11.7 T. Twenty male rats were divided into two groups and fed a liquid diet for 10 weeks. After 10 weeks, in vivo(1)H MRS spectra were acquired from the frontal cortex brain region. After in vivo(1)H MRS experiments, all animals were sacrificed and 20 frontal cortex tissue samples were harvested. All tissue examinations were performed with the 11.7 T HR-MAS spectrometer and high-resolution spectra were acquired. The in vivo and ex vivo spectra were quantified as absolute metabolite concentrations and normalized ratios of total signal-intensity (i.e., metabolitesNorm), respectively. The absolute quantifications of in vivo spectra showed significantly higher glycerophosphocholine plus phosphocholine (GPC+PCh) and lower myo-inositol (mIns) concentrations in ethanol-treated rats compared to controls. The quantifications of ex vivo spectra showed significantly higher PChNorm, ChoNorm and tChoNorm, and lower GPCNorm and mInsNorm ratio levels in ethanol-treated rats compared to controls. Our findings suggest that reduced mIns concentrations caused by the long-term alcohol consumption may lead to hypo-osmolarity syndrome and astrocyte hyponatremia. In addition, increased choline-containing compound concentrations may reflect an increased cell turnover rate of phosphatidylcholine and other phospholipids, indicating an adaptive mechanism. Therefore, these results might be utilized as key markers in chronic alcohol intoxication metabolism., (Copyright © 2013 Elsevier Ltd. All rights reserved.)