Evaluation of MIC distribution and establishment of epidemiological cut-off values for Streptococcus parauberis isolated from aquatic animals.

AIMS: Streptococcus parauberis is responsible for the development of streptococcosis in marine fish. The aim of the current study was to determine the antimicrobial susceptibility of aquatic Strep. parauberis strains, thus establishing laboratory-specific epidemiological cut-off (COWT) values to distinguish wild-type (WT) and nonwild-type (NWT) strains. METHODS AND RESULTS: Using 220 Strep. parauberis isolates obtained from diseased Paralichthys olivaceus, Platichthys stellatus, and Sebastes schlegelii over 6 years from seven different locations in Korea, we established COWT values for eight common antimicrobial agents using the standard broth microdilution method. The COWT values calculated using MIC distribution with the NRI and ECOFFinder methods were the same or within one dilution step for the eight antimicrobials tested. Nine NWT isolates with decreased susceptibility to at least two antimicrobials and one of these isolates exhibited decreased susceptibility to six antimicrobial agents were identified using COWT values based on NRI. CONCLUSIONS: Interpretive criteria for Strep. parauberis have not yet been established, and the findings of this study provide putative COWT values for eight antimicrobial agents frequently used in aquaculture in Korea.

Artificial intelligence-based non-small cell lung cancer transcriptome RNA-sequence analysis technology selection guide.

The incidence and mortality rates of lung cancer are high worldwide, where non-small cell lung cancer (NSCLC) accounts for more than 85% of lung cancer cases. Recent non-small cell lung cancer research has been focused on analyzing patient prognosis after surgery and identifying mechanisms in connection with clinical cohort and ribonucleic acid (RNA) sequencing data, including single-cell ribonucleic acid (scRNA) sequencing data. This paper investigates statistical techniques and artificial intelligence (AI) based non-small cell lung cancer transcriptome data analysis methods divided into target and analysis technology groups. The methodologies of transcriptome data were schematically categorized so researchers can easily match analysis methods according to their goals. The most widely known and frequently utilized transcriptome analysis goal is to find essential biomarkers and classify carcinomas and cluster NSCLC subtypes. Transcriptome analysis methods are divided into three major categories: Statistical analysis, machine learning, and deep learning. Specific models and ensemble techniques typically used in NSCLC analysis are summarized in this paper, with the intent to lay a foundation for advanced research by converging and linking the various analysis methods available.

Red Sea Bream Iridovirus (RSIV) Kinetics in Rock Bream (Oplegnathus fasciatus) at Various Fish-Rearing Seawater Temperatures.

Red sea bream iridoviral disease (RSIVD) causes serious economic losses in the aquaculture industry. In this paper, we evaluated RSIV kinetics in rock bream under various rearing water temperatures and different RSIV inoculation concentrations. High viral copy numbers (approximately 103.7-106.7 RSIV genome copies/L/g) were observed during the period of active fish mortality after RSIV infection at all concentrations in the tanks (25 °C and 20 °C). In the group injected with 104 RSIV genome copies/fish, RSIV was not detected at 21-30 days post-infection (dpi) in the rearing seawater. In rock bream infected at 15 °C and subjected to increasing water temperature (1 °C/d until 25 °C) 3 days later, the virus replication rate and number of viral copies shed into the rearing seawater increased. With the decrease in temperature (1 °C/d) from 25 to 15 °C after the infection, the virus replicated rapidly and was released at high loads on the initial 3-5 dpi, whereas the number of viral copies in the fish and seawater decreased after 14 dpi. These results indicate that the number of viral copies shed into the rearing seawater varies depending on the RSIV infection level in rock bream.

Red sea bream interleukin (IL)-1ß and IL-8 expression, subcellular localization, and antiviral activity against red sea bream iridovirus (RSIV).

Interleukin-1 beta (IL-1ß) is transcribed by monocytes, macrophages, and dendritic cells in response to activation of toll-like receptors (TLRs) by pathogen-associated molecular patterns (PAMPs) or cytokine signalling and causes a rapid inflammatory response to infection. IL-8, also known as chemokine C-X-C motif ligand (CXCL)-8, is regulated by IL-1ß and affects the chemotaxis of macrophages and neutrophils upon pathogen infection. In healthy red sea bream, rsbIL-1ß is most highly distributed in the liver, and rsbIL-8 is most highly distributed in the head kidney. In response to RSIV infection, rsbIL-1ß and rsbIL-8 mRNA are significantly upregulated in the kidney and spleen. This may be because the primary infection targets of RSIV are the kidney and spleen. In the gills, both genes were significantly upregulated at 7 days after RSIV infection and may be accompanied by a cytokine storm. In the liver, both genes were significantly downregulated at most observation points, which may be because the immune cells such as macrophages and dendritic cells expressing rsbIL-1ß or rsbIL-8 migrated to other tissues because the degree of RSIV infection was relatively low. Using a GFP fusion protein, it was confirmed that rsbIL-1ß and rsbIL-8 were localized to the cytoplasm of Pagrus major fin (PMF) cells. RsbIL-1ß overexpression induced the expression of interferon gamma (IFN-γ), myxovirus-resistance protein (Mx) 1, IL-8, IL-10, TNF-α, and MyD88, while rsbIL-8 overexpression induced the expression of IFN-γ, Mx1, rsbIL-1ß and TNF-α. In addition, overexpression of both genes significantly reduced the genome copies of RSIV and significantly reduced the viral titers. Therefore, rsbIL-1ß and rsbIL-8 in red sea bream play an antiviral role against RSIV through their normal signalling.

Molecular characterization and expression analysis of septin gene family and phagocytic function of recombinant septin 2, 3 and 8 of starry flounder (Platichthys stellatus).

Septin is an evolutionarily conserved family of GTP-binding proteins. Septins are known to be involved in a variety of cellular processes, including cell division, chromosome separation, cell polarity, motility, membrane dynamics, exocytosis, apoptosis, phagocytosis, DNA damage responses, and other immune responses. In this study, the sequences of the septin gene family of starry flounder were obtained using NGS sequencing, and the integrity of the sequences was verified through cloning and sequencing. At first, the amino acid sequence was annotated using the cDNA sequence, and then, the gene sequence was verified through multiple sequence alignment and phylogenetic analyses using the related conserved sequences. The septin gene family was classified into three subgroups based on the phylogenetic analysis. High conservation within the domain and homology between the genes reported in different species were confirmed. The expression level of septin gene family mRNA in each tissue of healthy starry flounder was evaluated to confirm the tissue- and gene-specific expression levels. Additionally, as a result of the analysis of mRNA expression after simulated pathogen infection, significant expression changes and characteristics were confirmed upon infection with bacteria (Streptococcus parauberis PH0710) and virus (VHSV). Based on the current results and that of previous studies, to confirm the immunological function, Septin 2, 3, and 8 were produced as recombinant proteins based on the amino acid sequences, and their role in phagocytosis was further investigated. The results of this study indicate that septin gene family plays a complex and crucial role in the host immune response to pathogens of starry flounder.

Dietary Supplementation with Ginger (Zingiber officinale) Residue from Juice Extraction Improves Juvenile Black Rockfish (Sebastes schlegelii) Growth Performance, Antioxidant Enzyme Activity and Resistance to Streptococcus iniae Infection.

Plant-derived feed additives provide cost effective and environmentally friendly alternatives to antibiotics for improving fish performance in aquaculture. An 8-week feeding trial was conducted to evaluate the effects of dietary ginger residue from juice extraction (GRJE) on juvenile black rockfish (Sebastes schlegelii) growth performance, antioxidant enzyme activities, and resistance to Streptococcus iniae infection. Juvenile rockfish (n = 450; initial weight = 2.2 ± 0.01 g) were randomly distributed into 30 L rectangular tanks (30 fish per tank). Five experimental diets with GRJE concentrations of 0% (control), 0.25%, 0.5%, 0.75%, and 1% were prepared in triplicate. Three groups of fish were randomly assigned to each diet and fed to apparent satiation twice daily. After the feeding trial, fish were challenged with S. iniae, and cumulative survival was observed for six days. Growth parameters, feed efficiency, and the protein efficiency ratio showed a quadratic correlation with the GRJE concentration in the fish diet. Proximate composition and plasma chemistry were not significantly affected. Plasma lysozyme, superoxide dismutase, glutathione, and catalase activities linearly increased with increasing GRJE supplementation levels. Moreover, survival in the S. iniae challenge test was significantly higher in fish fed diets supplemented with 0.75-1% GRJE. Our findings demonstrated that 0.75% GRJE dietary supplementation enhanced the growth performance, antioxidant activity, and disease resistance of juvenile black rockfish with no adverse effects.

First report of eosinophil peroxidase in starry flounder (Platichthys stellatus): Gene identification and gene expression profiling.

Eosinophils are granular leukocytes that are evolutionarily preserved in the innate immune system of some invertebrates and vertebrates, and these cells can directly remove invading microorganisms and secrete various cytokines, and are also involved in homeostasis. These eosinophils are made up of specific granular proteins that can be differentiated from other cells, and eosinophil peroxidase (EPX) is a peroxidase released only from eosinophils that plays an important role in maintaining the main function and homeostasis of eosinophils. We obtained the sequence information of EPX for the first time from the starry flounder (Platichthys stellatus), and predicted it by amino acid sequencing to confirm sequence alignment and phylogenetic characteristics with other species. Based on analysis of the expression characteristics of PsEPX mRNA in healthy P. stellatus, it was expressed at the highest level in peripheral blood lymphocytes (PBLs) and was also expressed at a relatively high level in the head kidney and intestine, which are immune-related tissues. After artificial infection with Streptococcus parauberis and viral haemorrhagic septicaemia virus, which are the causes of major pathogenic diseases, the expression level of PsEPX was significantly regulated, which showed specific characteristics of pathogens or tissues. These results suggest that PsEPX is an important component of the immune system of P. stellatus and is considered a basic research case for the study of the immunological function of eosinophils in fish.

Molecular Identification and mRNA Expression Profiles of Galectin-9 Gene in Red Sea Bream (Pagrus major) Infected with Pathogens.

Galectin (Gal) is a member of a family of ß-galactoside-binding lectin. The members of this family play important roles in the recognition of carbohydrate ligands and in various other biological processes. In this study, we identified the gene encoding Gal-9 in Pagrus major (PmGal-9) and analyzed its expression in various tissues after pathogen challenge. Alignment analysis revealed that the two galactose-binding lectin domains of the deduced protein were highly conserved among all the teleosts. Phylogenetic analysis revealed that PmGal-9 is most closely related to the Gal-9 gene of gilthead sea bream. PmGal-9 was ubiquitously expressed in all tissues analyzed but was predominantly expressed in the spleen, head kidney, and intestine. After challenges with major microbial pathogens (Edwardsiella piscicida, Streptococcus iniae, or red sea bream iridovirus) of red sea bream, PmGal-9 mRNA expression was significantly regulated in most immune-related tissues. These results suggested that PmGal-9 not only plays an important role in the immune system of red sea bream but is also a possible inflammatory marker for pathogenic diseases.

Functional characterization and gene expression profile of perforin-2 in starry flounder (Platichthys stellatus).

The membrane attack complex/perforin (MACPF) superfamily consists of multifunctional proteins that form pores on the membrane surface of microorganisms to induce their death and have various immune-related functions. PFN2 is a perforin-like protein with an MACPF domain, and humans with deficient PFN2 levels have increased susceptibility to bacterial infection, which can lead to fatal consequences for some patients. Therefore, in this study, we confirmed the antimicrobial function of PFN2 in starry flounder (Platichthys stellatus). The molecular properties were confirmed based on the verified amino acid sequence of PsPFN2. In addition, the expression characteristics of tissue-specific and pathogen-specific PsPFN2 mRNA were also confirmed. The recombinant protein was produced using Escherichia coli, and the antimicrobial activity was then confirmed. The coding sequence of PFN2 (PsPFN2) in P. stellatus consists of 710 residues. The MACPF domain was conserved throughout evolution, as shown by multiple sequence alignment and phylogenetic analysis. PsPFN2 mRNA is abundantly distributed in immune-related organs such as the spleen and gills of healthy starry flounder, and significant expression changes were confirmed after artificial infection by bacteria or viruses. We cloned the MACPF domain region of PFN2 to produce a recombinant protein (rPFN2) and confirmed its antibacterial effect against a wide range of bacterial species and the parasite (Miamiensis avidus).

The First Detection of Kudoa hexapunctata in Farmed Pacific Bluefin Tuna in South Korea, Thunnus orientalis (Temminck and Schlegel, 1844).

The consumption of fish and shellfish worldwide is steadily increasing, and tuna is a particularly valuable fish species. However, infection caused by Kudoa spp. is causing problems in many fish including the Pacific bluefin tuna (Thunnus orientalis), and there is much controversy about the association of these infections with foodborne disease. In this study, using haematological and histological analyses of the blood and internal organs (liver, spleen, kidney, heart, stomach, intestine, gill, and muscle) of Pacific bluefin tuna cultured in South Korea, infection with Myxosporea was first identified, and molecular biological analysis was conducted. In this study, Kudoa hexapunctata was finally identified. The Pacific bluefin tunas analysed in this study did not show any gross pathology lesions, such as visible cysts and/or myoliquefaction, of infection with this species. The histological analytical results can provide guidelines for the identification of K. hexapunctata. In the case of K. hexapunctata-induced infection, unlike other countries, such as Japan, there have been no reports in South Korea, and this study is the first to detect K. hexapunctata infection in Pacific bluefin tuna cultured in South Korea. The correlation between K. hexapunctata and food poisoning is not yet clear, however, it is thought that continuous observation of its infection is necessary.

The molecular characterization, expression analysis and antimicrobial activity of theromacin from Asian polychaeta (Perinereis linea).

Antimicrobial peptides (AMPs) are molecular factors in innate immunity and are believed to play a key role in invertebrate host defence. We identified theromacin (TM) from an Asian polychaeta, Perinereis linea, using de novo RNA-seq analysis. TM, a typical AMP of invertebrates, is a cysteine-rich AMP with five disulfide bonds consisting of ten cysteine residues. In gene expression analysis, TM genes were constantly upregulated after lipopolysaccharide (LPS) stimulation. In contrast, after peptidoglycan (PGN) stimulation, it was upregulated initially and downregulated after 12 h. We synthesized a peptide based on the macin AMP in the TM amino acid sequence. The synthetic peptide showed antibacterial activity against some Gram-positive and Gram-negative bacteria. Therefore, the AMPs of P. linea might have broad roles in host defence and exhibit different degrees of activity.

Two short antimicrobial peptides derived from prosaposin-like proteins in the starry flounder (Platichthys stellatus).

Prosaposin (PSAP) is a precursor of saposin (SAP), which is present in lysosomal and secreted proteins. PSAP is a member of the SAP-like protein families, which comprise multifunctional proteins. In particular, their antimicrobial activity has been reported. We identified PSAP-like (PsPSAPL) sequences from starry flounder and analysed their expression and antimicrobial activity based on cDNA and amino acid sequences. PsPSAPL showed conservation of three saposin B type domains at high levels, and PsPSAPL mRNA was relatively abundantly distributed in the brain and gills of healthy starry founders. PsPSAPL mRNA showed significant expression changes in response to viral haemorrhagic septicaemia virus and Streptococcus parauberis. Synthetic peptides (PsPSAPL-1 and -2), prepared based on amino acid sequences, were used to confirm as well as analyse the antimicrobial activity against bacteria and parasites. Consequently, PsPSAPL-1 and -2 were found to significantly inhibit the growth of various bacteria and kill the Miamiensis avidus. In addition, bacterial biofilm formation was significantly inhibited. Safety was also confirmed by analysing cell haemolysis. These results indicate the immunological function of PsPSAP and the potential antimicrobial activity of the AMPs PsPSAPL-1 and -2.

Molecular characterization and gene expression data of liver expressed antimicrobial Peptide-2 (LEAP-2) isolated from rock bream (Oplegnathus fasciatus).

Antimicrobial peptides (AMPs) are known to play a role as a first line of defence against microbial invasion. Liver Expressed Antimicrobial Peptides-2 (LEAP-2) is one of the AMPs. LEAP-2 includes four highly conserved cysteine residues and belongs to a cysteine-rich peptides group. We identified and characterized the molecular properties of LEAP-2 in rock bream. The expression levels of rock bream LEAP-2 (RbLEAP-2) in the 12 different tissues of healthy fish and the RbLEAP-2 expression pattern after infections with Edwardsiella piscicida (E. piscicida), Streptococcus iniae (S. iniae) and red seabream iridovirus (RSIV) were examined. This data provide that RbLEAP-2 plays an important role in innate immunity when rock bream is infected with a pathogen.

Efficacy and safety data of ceftiofur antibiotics against Streptococcus parauberis PH0710 infection in starry flounder (Platichthys stellatus).

This article provides efficacy and safety data of ceftiofur antibiotics against streptococcal infection in starry flounder. Ceftiofur, which is a veterinary antibiotics, is effective against fishery bacteria. Ceftiofur can be prescribed and sold by veterinarians. However, it is illegal in South Korea for fishery disease managers to prescribe and sell ceftiofur. Therefore, in order to utilize available antibiotics and prevent illegal use of veterinary antibiotics, it is necessary to perform research to determine the recommended effective dose and administration methods of antibiotics for fisheries. In this article, the appropriate concentration and injection method of antibiotics to treat starry flounder infected with S. parauberis PH0710 were provided. In addition, histopathological examination results were provided to confirm the effect of antibiotics on the host tissue. Accordingly, these data could be used as basic data for the application of ceftiofur antibiotics in disease management for fisheries.

The atypical chemokine receptor 4 in red sea bream (Pagrus major): Molecular characterization and gene expression analysis.

Atypical chemokine receptor 4 (ACKR4) is regulated by cytokines, binds chemokines and regulates the chemokine gradient. We verified the cDNA sequence by confirming ACKR4 from red sea bream (PmACKR4) by next generation sequencing (NGS) and analysed the molecular characteristics and gene expression profile. In the analysis using the predicted amino acid sequence of PmACKR4, a highly conserved G protein-coupled receptor 1 region and two cysteine residues were identified and included in the ACKR4 teleost cluster in the phylogenetic analysis. In healthy red sea bream, PmACKR4 mRNA was expressed at the highest levels in head kidney and was upregulated in all immune -related tissues used in the experiment after challenges with Streptococcus iniae (S. iniae) and red sea bream iridovirus (RSIV). These results suggest that ACKR4 is highly conserved in red sea bream and may play an important role in the immune system as previously reported. It is thought that ACKR4 acts as a regulator of immune -related cells via immune reactions after pathogenic infection.

Data on CXC chemokine ligand 10(CXCL10) expression and activation in red sea bream during bacterial and viral infection.

CXCL10 plays an important role in angiogenesis and inhibits the differentiation of endothelial cells into capillaries. It also plays an important role in the generation and transmission of effector T cell responses and the recruitment of T cells to inflammatory sites. In this article, we constructed cDNAs to identify and analyse the CXCL10 domain, and performed multiple alignments and a phylogenetic analysis to determine homology with other animals. Real-time PCR was performed to confirm construction and expression after bacterial and viral infection.

Functional analysis and gene expression profiling of extracellular cathepsin Z in red sea bream, Pagrus major.

Cathepsin Z (CTSZ) is a lysosomal cysteine protease that is known to be involved in the maintenance of homeostasis and the biological mechanisms of immune cells. In this study, we have confirmed the tissue specific expression of the cathepsin Z (PmCTSZ) gene in Pagrus major, and confirmed its biological function after producing recombinant protein using Escherichia coli (E. coli). Multiple sequence alignment analysis revealed that the active site of the cysteine proteases and three N-glycosylation sites of the deduced protein sequence were highly conserved among all of the organisms. Phylogenetic analysis revealed that PmCTSZ was included in the clusters of CTSZ and the cysteine proteases of other bony fish and is most closely related to Japanese flounder CTSZ. PmCTSZ was distributed in all of the tissues from healthy red sea bream that were used in the experiment and was most abundantly found in the spleen and gill. Analysis of mRNA expression after bacterial (Edwardsiella piscicida: E. piscicida and Streptococcus iniae: S. iniae) or viral (red seabream iridovirus: RSIV) challenge showed significant gene expression regulation in immune-related tissues, but they maintained relatively normal levels of expression. We produced recombinant PmCTSZ (rPmCTSZ) using an E. coli expression system and confirmed the biological function of extracellular rPmCTSZ in vitro. We found that bacterial proliferation was significantly inhibited by rPmCTSZ, and the leukocytes of red sea bream also induced apoptosis and viability reduction.

Characterization of gene expression profiles and functional analysis of peptidoglycan recognition protein 2 from rock bream (Oplegnathus fasciatus).

Peptidoglycan recognition protein 2 (PGRP2) is a Zn2+-dependent peptidase that plays important roles in binding to microbial components of the cell membrane, inducing phagocytosis and antimicrobial activity. Rock bream (Oplegnathus fasciatus) PGRP2 (RbPGRP2) was identified in the intestine by next generation sequencing (NGS) analysis. The open reading frame (ORF) the RbPGRP2 cDNA (470 amino acid residues) contains a peptidoglycan recognition protein domain (residues 300 to 446). Alignment analysis revealed that RbPGRP2 shares 37.6-53.5% overall sequence identity with the PGRP2s of other species. Phylogenetic analysis revealed that RbPGRP2 clustered together with PGRP2s from teleosts. In healthy rock bream, RbPGRP2 was found to be ubiquitously expressed in all of the examined tissues, especially in the liver. RbPGRP2 expression was significantly upregulated in all of the examined tissues of rock bream after infection with Edwardsiella piscicida, Streptococcus iniae and red sea bream iridovirus (RSIV) compared with the control. Purified rRbPGRP2 interactions with bacteria and inhibited the growth of bacteria in the presence of Zn2+. These results indicate that RbPGRP2 plays an important role in the innate immune response against bacterial infection.

Molecular characterization, expression and functional analysis of peptidoglycan recognition protein-SC2 from rock bream, Oplegnathus fasciatus.

Peptidoglycan recognition proteins are members of the family of pattern recognition receptors (PRRs), that play important roles in the recognition of peptidoglycan and various biological processes. In this study, we have characterized peptidoglycan recognition protein-SC2 (PGRP-SC2) in rock bream (Oplegnathus fasciatus) (RbPGRP-SC2) and analysed its expression in various tissues after pathogen challenge. A sequence alignment revealed that the residues essential to zinc binding of the deduced protein were highly conserved among all the organisms. Phylogenetic analysis revealed that RbPGRP-SC2 is most closely related to the large yellow croaker PGRP-SC2. RbPGRP-SC2 was ubiquitously expressed in all tissues analysed, predominantly distributed in muscle and skin. After challenge with microbial pathogens (Edwardsiella piscicida), Streptococcus iniae or red seabream iridovirus [RSIV]), RbPGRP-SC2 was up-regulated in all the tissues examined, especially in liver. We produced recombinant RbPGRP-SC2 (rRbPGRP-SC2) using an Escherichia coli expression system. The rRbPGRP-SC2 had agglutination activity towards both Gram-negative (E. piscicida) and Gram-positive bacteria (S. iniae). In addition, rRbPGRP-SC2 induced leukocyte apoptosis and promoted leukocyte phagocytosis. These results suggest that the RbPGRP-SC2 plays an important role in the immune system and in maintaining cellular homeostasis of rock bream.

The first report of siglec-3/CD33 gene in a teleost (rock bream, Oplegnathus fasciatus): An analysis of its spatial expression during stimulation to red seabream iridovirus (RSIV) and two bacterial pathogens.

Siglec-3/CD33 is a myeloid-specific inhibitory receptor that is expressed on cells of the immune system, where it is believed to play a regulatory role, modulating the inflammatory and immune responses. We characterized CD33 (RbCD33) in rock bream which is a transmembrane protein with two IG-like domains and a cytoplasmic tail. It has a deduced amino acid sequence of 390 residues and has tyrosine-based signaling motifs in the cytoplasmic tail. The RbCD33 mRNA was highly expressed in peripheral blood leukocytes and was also detected in the muscle, spleen, skin, head kidney, gills, trunk kidney, heart, stomach, brain, intestine and liver by quantitative real-time PCR. A temporal variation in expression of RbCD33 was observed in different tissues after stimulating with E. tarda, S. iniae and red seabream iridovirus (RSIV). In the head kidney tissue, E. tarda and S. iniae induced RbCD33, while a down regulation was observed with RSIV. In addition, in spleen tissue, S. iniae caused a very high induction of RbCD33 in comparison with an E. tarda and RSIV challenge. In the liver and gill tissues, all three pathogens induced a high expression of RbCD33. The expression pattern in various tissues and its high induction after pathogen stimulation suggests that RbCD33 plays an important role in initiating the immune response via the inhibition of signal transduction of the myeloid lineage cells.