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Interferon regulatory factor (IRF) 3 and IRF7 are important innate immune-related genes that induce type I interferon (IFN). In this study, PmIRF3 and PmIRF7 were identified from red sea bream (Pagrus major) infected by red sea bream iridovirus (RSIV), and expression and functional analyses were performed. PmIRF3 and PmIRF7 consisted of 465 and 436 amino acids, respectively, and contained a well-conserved IRF domain, IRF3 domain, and serine-rich domain (SRD). PmIRF3 and PmIRF7 are ubiquitously expressed in the liver and gills in normal fish, and with a high expression being recorded in kidney and spleen during RSIV infection. In P. major fin cells (PMF cells), pAcGFP-PmIRF3 and pAcGFP-PmIRF7 were localized to the cytoplasm, and overexpression of PmIRF3 and PmIRF7 using the pcDNA3.1 vector induced type I IFN. In addition, in the experiment in vitro, PMF cells overexpressed with PmIRF3 or PmIRF7 exhibited antiviral activity against RSIV and significantly reduced viral genome copies and virus titer. Therefore, PmIRF3 and PmIRF7 may have antiviral activity through the induction of type I IFN against RSIV infection of red sea bream.

Lipid is one of the most important nutritional factors affecting growth and survival of larval fish. This study aims to determine optimum level of lipid in formulated microdiets for the 10-day old rockfish (Sebastes schlegeli) larvae. Five granulated microdiets (CL11, CL14, CL17, CL20, and CL23) containing various (11%, 14%, 17%, 20%, and 23%, respectively) levels of lipid were prepared, and their lipid levels were controlled by adding fish oil at the expense of dextrin. The effects of feeding rockfish larvae with these formulated microdiets were compared with two commercial microdiets (Belgium and Japan), and local crumble diet. At the end of the 29-day feeding trial, weight gain (%) and total length (mm) of larval fish fed the CL20 diet were significantly (P 0.05) affected by the experimental diets. Crude lipid content of the whole-body fish was relatively well reflected from lipid levels of the experimental diets. In conclusion, the greatest weight gain and longest total length were obtained in larval fish fed the CL20 diet among the experimentally formulated microdiets. The optimum lipid level in the experimentally formulated microdiet was estimated to be 17.3% for rockfish larvae based on the broken-line analysis.

To delineate a livestock manure-derived groundwater plume (LDGP) in agro-livestock farming areas with the extensive use of chemical fertilizers, multilevel monitoring wells (MLWs) were installed for depth-specific sampling of groundwater, and then hydrochemical, nitrate N-O isotopic and microbiological data of shallow groundwater collected from the MLWs were evaluated with the help of multivariate statistical tools. The LDGP was distinguished from the pervasive agricultural contamination based on δ15Nnitrate (10‰). Fecal coliforms and Escherichia coli were not observed in the LDGP, whereas bovine enterovirus type 2 was detected at a sample collected at a depth of 18 m below ground level (bgl) downgradient manure piles, indicating a potential virus risk. Among hydrochemical parameters, Ca2+, Mg2+, Na+, Cl- and SiO2(aq) were shown to be effective indicators to trace the LDGP. On the other hand, nitrate was not effective to discriminate the LDGP because of denitrification in deep parts (>6 m bgl) within the LDGP, although nitrate contamination was serious at shallow parts (≤6 m bgl; 62.4 to 119.1 mg/L, median 97.9 mg/L). Thus, an integrated hydrochemical index consisting of Ca2+, Mg2+, Na+, Cl- and SiO2(aq) was suggested based on the result of principal component analysis with isometric log-ratio transformed data to delineate the LDGP in shallow unconsolidated aquifers overlying silicate bedrocks, and a threshold of the index was determined to be 1.17. The application of the index to three other agro-livestock farming areas with prevalent agricultural contamination successfully distinguished the groundwater samples within the LDGP (11 of 40 samples) that showed the index values exceeding the threshold and a median δ15Nnitrate of 12.5 ‰. The study results show that the hydrochemical index can be used for the quick evaluation of a LDGP in agro-livestock farming areas given its cost efficiency and easily accessible analysis devices for water chemistry compared to isotopes, although the broad use of the index to determine LDGPs needs to be further verified by application to various geology and land uses. Livestock manures should be carefully managed to protect groundwater resources, including impermeable covers above or beneath the manure piles, given the potential risk of virus at deep parts and nitrate at shallow parts within the LDGP. The methodology to select hydrochemical indicators for distinguishing a LDGP and the combination of the indicators to develop an index suggested in this study will be useful to build new indices adapted to local conditions.

Summary: Despite the recognized importance of antitumor immunity, our understanding of brain tumor immunity is poor. Orthotopic injection models have been widely used for immunological analyses. However, these models have limitations in analysis of antitumor immunity because the approach involves drilling skulls and injecting tumor cells, which can induce adverse effects. We describe a protocol for the induction of spontaneous brain tumor model, isolation of single cells from brain tumor microenvironment, and analysis of the immune responses using scRNA-seq and flow cytometry.For complete details on the use and execution of this protocol, please refer to Park et al. (2021). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Ulcerative colitis (UC) is a severe inflammatory disease that has spread throughout the world. Cirsium japonicum (CJ) and Aralia elata (AE) are natural herbs with potent antioxidative antidiabetics and anti-inflammatory effects. In this investigation, we studied the defensive role of the combination of CJ and AE against LPS-induced inflammation in RAW 264.7 cells, dextran sulfate sodium (DSS)-induced colitis in mice, and acetic acid-induced colitis in dogs. MTT assay was performed to identify the toxic effect of CJ and AE extracts. NO, and MDA level was also measured by NO and MDA assay. To measure the pro-inflammatory protein expression, a western blot was performed. To induce colitis, 3% DSS was used for mice and 6% acetic acid was used for dogs. Histopathology and colonoscopy were executed to detect the effect of extracts. CJ and AE pretreatment reduced the level of NO, MDA, and the expression of pro-inflammatory proteins cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) in RAW 264.7. Compared to the separate doses of CJ and AE, the combined dose of CJ and AE significantly reduced clinical symptoms induced by DSS in mice and acetic acid in dogs including weight loss, bloody stool, shortening of the colon, and the severity of colitis and degree of histological damage in the colon. Therefore, these results indicated that a combined dose of CJ and AE has a protective effect against LPS-induced RAW 264.7 cells, DSS-mediated colonic inflammation in mice, and acetic acid-induced colitis in dogs.

Summary: The homeodomain is found in hundreds of transcription factors that play roles in fate determination via cell-autonomous regulation of gene expression. However, some homeodomain-containing proteins (HPs) are thought to be secreted and penetrate neighboring cells to affect the recipient cell fate. To determine whether this is a general characteristic of HPs, we carried out a large-scale validation for intercellular transfer of HPs. Our screening reveals that intercellular transfer is a general feature of HPs, but it occurs in a cell-context-sensitive manner. We also found the secretion is not solely a function of the homeodomain, but it is supported by external motifs containing hydrophobic residues. Thus, mutations of hydrophobic residues of HPs abrogate secretion and consequently interfere with HP function in recipient cells. Collectively, our study proposes that HP transfer is an intercellular communication method that couples the functions of interacting cells. : Lee et al. evaluate capabilities of homeodomain proteins (HPs) for transfer between cells. They find that intercellular transfer is a general but cell-context-sensitive property of HP. Intercellular HP transfer can be an unconventional way for the cells to communicate with neighboring cells that associate structurally and functionally. Keywords: homeodomain, cell-penetrating protein, protein secretion, intercellular protein transfer

The “ciliopathies” are a newly defined group of disorders characterized by defects in the structure or function of the cellular primary cilium. Patients with these disorders display variably expressive fibrocystic renal disease, retinal blindness, polydactyly, obesity, and brain dysgenesis as well as neurocognitive impairments. Joubert syndrome is a ciliopathy defined by cerebellar vermis hypoplasia, oculomotor apraxia, intermittent hyperventilation, and mental retardation. Recent evidence suggests important roles for the primary cilium in mediating a host of extracellular signaling events such as morphogen, mitogen, homeostatic and polarity signals. Based upon the clinical features of ciliopathies and cilia mediated signaling pathways, the data support a role for the primary cilium in modulating neurogenesis, cell polarity, axonal guidance and possibly adult neuronal function.