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Human skin color is largely determined by genetic factors. Recent genome-wide association studies (GWAS) have reported several genetic variants associated with skin color, mostly in European and African populations. In this study, we performed GWAS in 17,019 Korean women to identify genetic variants associated with facial skin color, quantitatively measured as CIELAB color index. We identified variants in three, one, and six genomic loci associated with facial skin color index L*, a*, and b* values, respectively, and replicated the associations (combined analysis P-value

The use of prime editing-a gene-editing technique that induces small genetic changes without the need for donor DNA and without causing double strand breaks-to correct pathogenic mutations and phenotypes needs to be tested in animal models of human genetic diseases. Here we report the use of prime editors 2 and 3, delivered by hydrodynamic injection, in mice with the genetic liver disease hereditary tyrosinemia, and of prime editor 2, delivered by an adeno-associated virus vector, in mice with the genetic eye disease Leber congenital amaurosis. For each pathogenic mutation, we identified an optimal prime-editing guide RNA by using cells transduced with lentiviral libraries of guide-RNA-encoding sequences paired with the corresponding target sequences. The prime editors precisely corrected the disease-causing mutations and led to the amelioration of the disease phenotypes in the mice, without detectable off-target edits. Prime editing should be tested further in more animal models of genetic diseases.

Human milk lipids provide not only energy but also indispensable bioactive components such as essential fatty acids. To establish the recommended daily intake value and guidelines for infant formula, a reference library of fatty acid composition has been generated from 4 Asian countries (South Korea, China, Vietnam, and Pakistan). Regardless of country, palmitic acid (C16:0), linoleic acid (C18:1), and linolenic acid (C18:2) were the 3 most abundant fatty acids in human milk and account for more than 75% of total fatty acids (total FA). However, there were several considerable differences between fatty acids, particularly n-3 and n-6 (omega-3 and omega-6) groups. Chinese mothers' milk had a high concentration of linoleic acid at 24.38 ± 10.02% of total FA, which may be due to maternal diet. Among the 4 countries, Pakistani mothers' milk contained a high amount of saturated fatty acid (56.83 ± 5.96% of total FA), and consequently, polyunsaturated fatty acids, including n-3 and n-6, were significantly lower than in other countries. It is noteworthy that docosahexaenoic acid (DHA) in Pakistani mothers' milk was 44.8 ± 33.3 mg/L, which is only 25 to 30% of the levels in the other 3 countries, suggesting the need for DHA supplementation for infants in Pakistan. Moreover, the ratio of n-6 to n-3 was also remarkably high in Pakistani mothers' milk (15.21 ± 4.96), being 1.4- to 1.7-fold higher than in other countries. The average DHA:ARA ratio in Asian human milk was 1.01 ± 0.79. Korean mothers' milk showed a high DHA:ARA ratio, with a value of 1.30 ± 0.98, but Pakistani mothers' milk had a significantly lower value (0.42 ± 0.12). The fatty acid compositions and anthropometric data of mother (body mass index, age) did not show any correlation. The obtained data might provide information about human milk compositions in the Asian region that could benefit from setting up recommended nutrient intake and infant formula for Asian babies.

Type 2 diabetes mellitus (T2DM) is associated with chronic hyperglycemia and lipid metabolism. A previous genome-wide association study revealed the TOMM40-APOE region as novel locus for T2DM susceptibility. This association study was conducted to determine the genetic effects of APOE single nucleotide polymorphisms (SNPs) on T2DM susceptibility and lipid profiles in a Korean population. A total of 6 tagging SNPs, including rs7412 and rs429358, were selected for e genotype analysis and genotyped in 1436 subjects, consisting of 352 T2DM patients and 1084 unaffected controls. Logistic regression analyses were conducted and there were no significant associations among the APOE 6 tagging SNPs, e genotypes, and haplotypes with T2DM susceptibility. To investigate the association of the APOE tagging SNPs with the lipid profiles, a regression analysis was conducted. As a result, rs7412 was significantly associated with the total cholesterol (TC) and low-density lipoprotein cholesterol (LDL) levels (Pcorr = 2.30 × 10–5 and 3.39 × 10–13, respectively) in the unaffected controls. The e2 allele and e3 allele were significantly associated with the TC (Pcorr = 4.46 × 10–6 and 0.02, respectively) and LDL levels (Pcorr = 3.54 × 10–14 and 0.0006, respectively) in the unaffected controls. Further analysis of only the unaffected controls was conducted. As a result, the APOE alleles e2 and e3 showed a significant association with the TC and LDL levels (P

Variation in skin pigmentation can be affected by both environmental factors and intrinsic factors such as age, gender, and genetic variation. Recent GWASs revealed that genetic variants of genes functionally related to a pigmentation pathway were associated with skin pigmentary traits. However, these GWASs focused on populations with European ancestry, and only a few studies have been performed on Asian populations, limiting our understanding of the genetic basis of skin pigmentary traits in Asians. To evaluate the genetic variants associated with facial pigmented spots, we conducted a GWAS analysis of objectively measured facial pigmented spots in 17,019 Korean women. This large-scale GWAS identified several genomic loci that were significantly associated with facial pigmented spots (five previously reported loci and two previously unreported loci, to our knowledge), which were detected by UV light: BNC2 at 9p22 (rs16935073; P-value = 2.11 × 10−46), PPARGC1B at 5q32 (rs32579; P-value = 9.04 × 10−42), 10q26 (rs11198112; P-value = 9.66 × 10−38), MC1R at 16q24 (rs2228479; P-value = 6.62 × 10−21), lnc01877 at 2q33 (rs12693889; P-value = 1.59 × 10−11), CDKN2B-AS1 at 9p21 (rs643319; P-value = 7.76 × 10−9), and MFSD12 at 19p13 (rs2240751; P-value = 9.70 × 10−9). Further functional characterization of the candidate genes needs to be done to fully evaluate their contribution to facial pigmented spots.

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.

Several Streptococcus pyogenes Cas9 (SpCas9) variants have been developed to improve an enzyme’s specificity or to alter or broaden its protospacer-adjacent motif (PAM) compatibility, but selecting the optimal variant for a given target sequence and application remains difficult. To build computational models to predict the sequence-specific activity of 13 SpCas9 variants, we first assessed their cleavage efficiency at 26,891 target sequences. We found that, of the 256 possible four-nucleotide NNNN sequences, 156 can be used as a PAM by at least one of the SpCas9 variants. For the high-fidelity variants, overall activity could be ranked as SpCas9 ≥ Sniper-Cas9 > eSpCas9(1.1) > SpCas9-HF1 > HypaCas9 ≈ xCas9 >> evoCas9, whereas their overall specificities could be ranked as evoCas9 >> HypaCas9 ≥ SpCas9-HF1 ≈ eSpCas9(1.1) > xCas9 > Sniper-Cas9 > SpCas9. Using these data, we developed 16 deep-learning-based computational models that accurately predict the activity of these variants at any target sequence. Deep-learning models predict the Cas9 variant with optimal activity and specificity for any target sequence.

We isolated and purified an antimicrobial peptide (AMP) from the mantle of the hard-shelled mussel, Mytilus coruscus. The peptide was purified through C18 reversed-phase high-performance liquid chromatography, and displayed antibacterial activity. Total molecular mass of 11,182 Da was determined using matrix-assisted laser desorption ionization time-of-flight mass spectrophotometry. The N-terminal 23-amino acid sequence of its purified peak was obtained through Edman degradation, revealing 82% identity with myticusin-1 of M. coruscus. Complete sequence of the target peptide was determined through cDNA cloning and rapid amplification of cDNA ends. The complete sequence comprised 574 bp with a 387-bp open reading frame (ORF) encoding 24 amino acids of a signal peptide and 104 amino acids of a mature peptide, which was named myticusin-beta. Furthermore, we discovered two novel isoforms of myticusin-beta. We constructed and expressed recombinant myticusin-beta, which displayed antimicrobial activity against gram-positive (Bacillus cereus, Bacillus subtilis, Clostridium perfringens, Staphylococcus aureus, Streptococcus iniae, Streptococcus mutans) and gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Vibrio alginolyticus, Klebsiella pneumoniae). Purified recombinant myticusin-beta also showed anti-parasitic activity at various concentrations. A short AMP analog was designed and synthesized based on the sequence of myticusin-beta, with markedly improved antimicrobial activity. Expression of myticusin-beta was detected in the mantle at the highest level, followed by hemocytes. The results obtained in this work suggest that myticusin-beta is an immune-related AMP of M. coruscus and an effective alternative to antibiotics.

Calpains (CAPNs) belong to the papain superfamily of cysteine proteases, and they are calcium-dependent cytoplasmic cysteine proteases that regulate a variety of physiological processes. We obtained the sequence of CAPN3 from an NGS-based analysis of Pagrus major (PmCAPN3) and confirmed the conserved molecular biological properties in the predicted amino acid sequence. The amino acid sequence and predicted domains of CAPN3 were found to be highly conserved in all of the examined species, and one catalytic domain and four calcium binding sites were identified. In healthy P. major, the PmCAPN3 mRNA was most abundantly expressed in the muscle and skin, and ubiquitously expressed in the other tissues used in the experiment. After artificial infections with fish pathogens, significant changes in its expression levels were found in immune-related tissues, most of showed upregulation. In particular, the highest level of expression was found in the liver, a tissue associated with protease activity. Taken together, these results suggest a physiological activity for PmCAPN3 in P. major and reveal functional possibilities that have not yet been reported in the immune system.

Protopanaxadiol (PPD), an aglycon found in several dammarene-type ginsenosides, has high potency as a pharmaceutical. Nevertheless, application of these ginsenosides has been limited because of the high production cost due to the rare content of PPD in Panax ginseng and a long cultivation time (4–6 years). For the biological mass production of the PPD, de novo biosynthetic pathways for PPD were introduced in Saccharomyces cerevisiae and the metabolic flux toward the target molecule was restructured to avoid competition for carbon sources between native metabolic pathways and de novo biosynthetic pathways producing PPD in S. cerevisiae. Here, we report a CRISPRi (clustered regularly interspaced short palindromic repeats interference)-based customized metabolic flux system which downregulates the lanosterol (a competing metabolite of dammarenediol-II (DD-II)) synthase in S. cerevisiae. With the CRISPRi-mediated suppression of lanosterol synthase and diversion of lanosterol to DD-II and PPD in S. cerevisiae, we increased PPD production 14.4-fold in shake-flask fermentation and 5.7-fold in a long-term batch-fed fermentation.

The diseases caused by Streptococcus sp. are frequently outbreak in olive flounder aquaculture farms in South Korea, and antibiotics are often abused to control the disease. This results in resistant strains that cannot be treated with existing fisheries antibiotics. In order to apply to other antibiotics, research on use, dosage and safety are essential. Tiamulin is an antibiotic that has good activity against mycoplasma and Gram-positive bacteria. It is mainly used in swine, cattle and poultry. Streptococcus parauberis KSP28 infected flounder had the best therapeutic effect at 15 mg/kg when injected intramuscularly, and 15 mg/kg and 30 mg/kg had the same effect when administered orally. However, intramuscular injection showed inflammation and necrosis of the muscles, and glomerulonephritis, renal tubular atrophy, Bowman’s capsule expansion, and reticuloendothelial system activity at the high dose group. Therefore, it is not appropriate to apply tiamulin injection to olive flounder because of the serious side effects. In addition, further studies should identify side effects of oral tiamulin administration.

Butaphosphan {[1-(butylamino)-1-methylethyl]-phosphonic acid (BTP)}-cyanocobalamin [vitamin B12 (C)], a combination drug comprising an organic source of phosphorus and a synthetic form of vitamin B12, is used to attenuate stress responses in livestock. This study was performed to examine the effects of BTP-C in olive flounder Paralichthys olivaceus under normal and stressful conditions. P. olivaceus was intramuscularly injected with BTP-C (50–300 mg BTP-C/kg body weight) under normal conditions and changes in muscle adenosine nucleotides examined. BTP-C increased adenosine 5′-triphosphate (ATP) levels; this effect was more pronounced at 22 °C than at 13 °C. Serum transaminase levels were also elevated at a dose of 300 mg BTP-C/kg, which suggested possible hepatotoxicity of the drug. Effects of BTP-C on stress were examined by injecting it at a rate of 50 mg/kg. BTP-C partially prevented hypothermia- and hypoxia-induced reduction in feeding. Serum immunoglobulin M levels were increased by BTP-C in these stress tests. Although BTP-C did not affect hypothermia-induced cortisol elevation or lysozyme reduction, changes in these parameters due to hypoxia were alleviated by the drug. Stress-attenuating effects of BTP-C were also confirmed in fish farms following injection of an antibiotic. These results indicate that BTP-C may be useful against various forms of stress in fish. Accelerated ATP production following the administration of BTP-C is one possible mechanism resulting in the attenuation of stress effects. The clinically effective dose of BTP-C, 50 mg/kg, did not cause any marked toxicity in P. olivaceus.

Two emerging psychoactive substances, 2-(2,5-dimethoxy-4-methylphenyl)-N-(2-methoxybenzyl)ethanamine (25D-NBOMe) and N-(2-methoxybenzyl)-2,5-dimethoxy-4-chlorophenethylamine (25C-NBOMe), are being abused, leading to fatal and non-fatal intoxications. However, most of their adverse effects have been reported anecdotally. In the present study, cardiotoxicity was evaluated through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, rat electrocardiography (ECG), and human ether-a-go-go-related gene (hERG) assay. Expression levels of p21 (CDC42/RAC)-activated kinase 1 (PAK1), one of known biomarkers for cardiotoxicity, were also analyzed. Both 25D-NBOMe and 25C-NBOMe at 100 μM reduced cell viability in MTT assay. At 2.0 mg/kg and 0.75 mg/kg, they prolonged QT intervals in rat ECG. PAK1 was down-regulated by treatment with these two test compounds. Furthermore, potassium channels were inhibited by 25D-NBOMe treatment in hERG assay. Taken together, these results suggest that both 25D-NBOMe and 25C-NBOMe have potential cardiotoxicity, especially regarding cardiac rhythm. Further studies are needed to confirm the relationship between PAK1 down-regulation and cardiotoxicity.

The objective of this study was to demonstrate the pharmacokinetic–pharmacodynamic profile, bioavailability, and withdrawal time of tylosin tartrate (TT) administered to olive flounder via intramuscular (IM, 10 or 20 mg/kg, n = 240) and intravascular (IV, 10 mg/kg, n = 90) injections. Serum concentrations of tylosin were determined using a validated liquid chromatography-tandem mass spectrometry method. According to the non-compartmental analysis, the bioavailability of TT was 87%. After the IV injection, the terminal half-life, total body clearance, volume of distribution, and mean residence time of TT were 21.07 h, 0.07 L/kg/h, 2.15 L/kg, and 16.39 h, respectively. Rapid absorption (Tmax 0.25 h), prolonged action (terminal half-life, 33.96 and 26.04 h, MRT, 43.66 and 33.09 h), and linear dose–response relationship (AUC0-inf, 123.55 and 246.05 µg/mL*h) were monitored following 10 and 20 mg/kg IM injection. The withdrawal time of TT from muscle (water temperature, 22 °C) was 9.84 days, rounded up to 10 days (220 degree days). Large Cmax/MIC90, AUC0-inf/MIC90, and T &gt, MIC90 values were obtained for Streptococcus isolates and these PK/PD indices satisfied the criteria required for efficacy evaluation. This study lays a foundation for the optimal use of TT and provides valuable information for establishing therapeutic regimens.

American oyster defensin (AOD) was previously purified from acidified gill extract of the American oyster, Crassostrea virginica. AOD is composed of 38 amino acids with three disulfide bonds and exhibits strong antimicrobial activity against Gram-positive bacteria as well as significant activity against Gram-negative bacteria. Here, to develop promising peptides into antibiotic candidates, we designed five arginine-rich analogs (A0, A1, A2, A3, and A4), predicted their loop and extended strand/random structures—including nine amino acids and a disulfide bond derived from the C-terminus of AOD—and described their antimicrobial and cytotoxic effects, as well as their modes of action. In our experimental results, the A3 and A4 analogs exhibited potent antimicrobial activity against all test organisms—including four Gram-positive bacteria, six Gram-negative bacteria, and Candida albicans—without cell toxicity. A sequence of experiments, including a membrane permeabilization assay, DNA binding study, and DNA polymerization inhibition test, indicated that the two analogs (A3 and A4) possibly&nbsp, did not act directly on the bacterial membrane but instead interacted with intracellular components such as DNA or DNA amplification reactions. AOD analogs also showed strong bacterial inhibition activity in the plasma environment. In addition, analog-treated microbial cells clearly exhibited membrane disruption, damage, and leakage of cytoplasmic contents. Collectively, our results suggest that two analogs, A3 and A4, have potent antimicrobial activity via DNA interaction and have the potential for development into novel antimicrobial agents.

Airborne diseases are the leading cause of infectious disease-related deaths in the world. In particular, the influenza virus activates a network of immune cells that leads to clearance or an overzealous response that can be fatal. Tight regulation of the cytokines that enable proper activation and function of immune cells is necessary to clear infections efficiently while minimizing damage to the host. Interleukin-7 (IL-7) is a cytokine known for its importance in T cell development and survival. How IL-7 shapes CD8 T cell responses during an acute viral infection is less understood. We had previously shown that IL-7 signaling deficient mice have reduced accumulation of influenza-specific CD8 T cells following infection. We sought to determine whether IL-7 affects early CD8 T cell expansion in the mediastinal lymph node and effector function in the lungs. Using IL-7Rα signaling deficient mice, we show that IL-7 is required for a normal sized mediastinal lymph node and the early clonal expansion of antigen-specific CD8 T cells therein. Bone marrow chimeric models and adoptive transfer of transgenic TCR CD8 T cells reveal a cell-intrinsic role for IL-7 in the accumulation of NP366–374 and PA224–233-specific CD8 T cells. We also found that IL-7 dictates terminal differentiation, degranulation and cytokine production in PA224–233-specific but not NP366–374-specific CD8 T cells. We further demonstrate that IL-7 is induced in the lung tissue by viral infection and we characterize multiple cellular sources that contribute to IL-7 production. Drugs that manipulate IL-7 signaling are currently under clinical trial for multiple conditions. Our findings on IL-7 and its effects on lower respiratory diseases will be important for expanding the utility of these therapeutics.Author SummaryInterleukin-7 plays an important role in development of immune cells such as lymphocytes. In recent years, its role in the immune system has been expanded beyond the development of immune cells to include revitalizing of lymphocytes during tumor and chronic viral response. We show here that IL-7 is required for accumulation and function of specialized lymphocytes in the lungs during an acute influenza infection.

Crepuscular mosquitoes, which swarm in low light conditions, exhibit a range of adaptations including large aspect-ratio wings, high flapping frequencies and small stroke amplitudes that taken together, facilitate the generation of wing-tones that are well-suited for acoustic communication. In the current study, we employ computational aeroacoustic modeling to conduct a comparative study of wing-tone and flight efficiency in a mosquito (male Culex) and a similar sized flying insect: a fruit fly (Drosophila). Based on this analysis, we show that pound-for-pound, a mosquito generates wing-tones that are a factor of about 3.4 times more intense than a fruit fly, and the mosquito is more efficient by a factor of about 3.7 in converting mechanical power into acoustic power. The wing-tones for the mosquito are also more tilted in the forward direction, a characteristic that would be more conducive for acoustic signaling during a mate chase. The simulation data also shows that the specific power (mechanical power over mean lift) of the mosquito is nearly equal to that of the fruit fly, indicating that the adaptations that facilitate wing-tone based communication in mosquitoes, do not seem to compromise their flight efficiency.

Prime editing can induce any small-sized genetic change without donor DNA or double strand breaks. However, it has not been investigated whether prime editing is possible in postnatal animals. Here we delivered prime editors 2 and 3 into a mouse model of hereditary tyrosinemia, a genetic liver disease, using hydrodynamic injection, which corrected the disease-causing mutation and rescued the phenotype. We also achieved prime editing in the retina and retina pigment epithelium in wild-type mice by delivering prime editor 3 using trans-splicing adeno-associated virus. Deep sequencing showed that unintended edits at or near the target site or off-target effects were not detectable except for low levels (0% to 1.2%) of indels when PE3, but not PE2, was used. Our study suggests that precise, prime editor-mediated genome editing is possible in somatic cells of adult animals.

Although environmental enrichment (EE) is known to reduce oxidative stress in Parkinson&rsquo, s disease (PD), the metabolic alternations for detoxifying endogenous and xenobiotic compounds according to various brain regions are not fully elucidated yet. This study aimed to further understand the role of EE on detoxifying enzymes, especially those participating in phase I of metabolism, by investigating the levels of enzymes in various brain regions such as the olfactory bulb, brain stem, frontal cortex, and striatum. Eight-month-old transgenic PD mice with the overexpression of human A53T &alpha, synuclein and wild-type mice were randomly allocated to either standard cage condition or EE for 2 months. At 10 months of age, the expression of detoxifying enzymes was evaluated and compared with wild-type of the same age raised in standard cages. EE improved neurobehavioral outcomes such as olfactory and motor function in PD mice. EE-treated mice showed that oxidative stress was attenuated in the olfactory bulb, brain stem, and frontal cortex. EE also reduced apoptosis and induced cell proliferation in the subventricular zone of PD mice. The overexpression of detoxifying enzymes was observed in the olfactory bulb and brain stem of PD mice, which was ameliorated by EE. These findings were not apparent in the other experimental regions. These results suggest the stage of PD pathogenesis may differ according to brain region, and that EE has a protective effect on the PD pathogenesis by decreasing oxidative stress.

Background Olive flounder, Paralichthys olivaceus, is an economically important aquaculture species in Korea. Olive flounders have been heavily damaged by streptococcal infections every year and are treated with antibiotics. However, antibiotic abuse is causing the emergence of resistant strains, and to overcome this, research has shown that new antibiotics must be applied. Tylosin is a relatively safe antibiotic and has good activity against Gram-positive bacteria and mycoplasma. We studied the therapeutic effects and side effects of tylosin on Streptococcus parauberis-infected olive flounder. Methods After artificial infection of olive flounder with S. parauberis SPOF18J3, an appropriate dose of tylosin was confirmed by intramuscular injection (I.M.) at 2.5, 5, 10, and 15 mg/kg, and oral administration at 10 and 20 mg/kg. After I.M. and oral administration dosing of tylosin, side effects were confirmed by serological analysis, histopathological analysis, and median lethal dose (LD50) analysis at both an appropriate concentration and a high concentration. Statistical analysis was performed using one-way analysis of variance (ANOVA) and Tukey’s test (p < 0.05). Results The appropriate I.M. and oral administration concentration of tylosin administered to olive flounder infected with S. parauberis SPOF18J3 was found to be 10 mg/kg. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were showed not significantly different between the control group and the experimental groups. The histopathologic results showed mild inflammatory responses in muscle and tubular vacuolization and tubular atrophy appeared, but there were no significant differences between the groups. The LD50 was confirmed to be 461 mg/kg. Conclusion In this study, an effective treatment method was provided by verifying the treatment effects and side effects of tylosin in olive flounder infected with S. parauberis, which can be applied directly to aquaculture sites. In addition, these results may be used as a reference for evaluation required upon request to obtain approval for tylosin antibiotics as fishery antibiotics in Korea. After approval, it is possible that a fishery disease manager will be able to prescribe and sell the antibiotic tylosin.

Mitochondria behave as functional and structural hubs for innate defense against intracellular infection. While the mitochondrial membrane serves as a platform for the assembly of signaling complexes activated by intracellular infection, various danger molecules derived from impaired mitochondria activate innate signaling pathways. Using methionyl-tRNA formyl transferase (MTFMT)-deficient cells, which exhibit impaired mitochondrial activity, we examined the role of mitochondrial integrity in regulating innate defense against infection. Since MTFMT functions at the early steps of mitochondrial translation, its loss was expected to cause defects in mitochondrial activity. Under transient MTFMT gene silencing conditions, we observed shortened mitochondria along with reduced activity. MTFMT-silenced cells were more susceptible to intracellular infection, as examined by infection with RNA viruses and the intracellular bacterium Shigella flexneri. In support of this observation, MTFMT-silenced cells possessed lowered basal NF-κB activity, which remained low after S. flexneri infection. In addition, the mitochondrial accumulation of evolutionarily conserved signaling intermediate in Toll pathway (ECSIT), an adaptor protein for NF-κB activation, was significantly decreased in MTFMT-silenced cells, explaining the reduced NF-κB activity observed in these cells. Since impaired mitochondria likely release mitochondrial molecules, we evaluated the contribution of mitochondrial N-formyl peptides to the regulation of bacterial infection. Transient transfection of mitochondrial-derived N-formyl peptides favored S. flexneri infection, which was accompanied by enhanced bacterial survival, but did not affect host cell viability. However, transient transfection of mitochondrial-derived N-formyl peptides did not affect basal NF-κB activity. Altogether, these data suggest that the integrity of mitochondria is essential to their proper function in protecting against infection, as intact mitochondria not only block the release of danger molecules but also serve as signaling hubs for the downstream NF-κB pathway.

Glycemic carbohydrates, such as starch and sucrose, are fully hydrolyzed into monosaccharides by mucosal α-glucosidases in the small intestine. In this study, we optimized the reaction conditions of mammalian mucosal α-glucosidases to digest various types of glycemic carbohydrates to determine their hydrolytic activity. The results clearly showed that mammalian mucosal α-glucosidases had different hydrolytic properties compared to fungal amyloglucosidase, which is commonly used in vitro assay to determine carbohydrate quality, on various sizes and α-linked carbohydrates. Furthermore, non-gelatinized and gelatinized starches from different botanical sources were differently hydrolyzed to glucose by the prepared solution of mammalian α-glucosidases and porcine pancreatic α-amylase. Thus, it is clearly suggested that the digestion of glycemic carbohydrates by the optimized reaction condition with mammalian mucosal α-glucosidases and pancreatic α-amylase can be applied to determine the carbohydrate quality and digestion properties as it is more reliable to human digestive enzymes compared to fungal or microbial α-glucosidases.

Anti-lipopolysaccharide factors (ALFs) are a representative host defense protein in crustaceans. In this study, we successfully developed two novel antimicrobial peptides (AMPs), named crab-ALF2A and crab-ALF6A, which contain changes to the amino acid sequences of the lipopolysaccharide binding domain and signal peptide, respectively, of the ALF of the swimming crab Portunus trituberculatus. The crab-ALF2A peptide showed potent antimicrobial activity against the Gram-positive bacteria Bacillus cereus, Staphylococcus aureus, and Streptococcus iniae (minimal effective concentration [MEC] 1.51–1.93 μg/mL) and the Gram-negative bacteria Pseudomonas aeruginosa and Escherichia coli (MEC 1.87–1.98 μg/mL), with maximal bactericidal activity at a peptide concentration of 5 μg/mL. The crab-ALF6A peptide also showed potent antimicrobial activity against B. cereus, S. aureus, and S. iniae (MEC 1.49–2.3 μg/mL) and P. aeruginosa and E. coli (MEC 1.72–1.19 μg/mL) at a peptide concentration of 5 μg/mL. Notably, the crab-ALF2A and crab-ALF6A peptides exhibited strong activity against Candida albicans (MECs of 2.11 and 1.95 μg/mL, respectively). These activities were stable following heat treatment. Moreover, the effect of crab-ALF2A and crab-ALF6A peptide treatment on microbe cell morphology was confirmed by scanning electron microscopy. Membrane disruption and damage, and the leakage of cytoplasmic content were clearly observed. A downsizing peptide approach illustrated that the hexapeptide ALF6A8 (RVLLRL) was the shortest peptide showing significant antimicrobial activity. Our approach allows for the generation of novel antimicrobial peptides in a cost effective manner as potential next-generation antibiotics.

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.

In this study, the effect of ammonia derived from different stocking densities on immunological, hematological, and oxidative stress parameters was analyzed in the blood or liver of red seabream. Density- and time-dependent increases in inorganic nitrogen compounds were measured for 20 days by analyzing the three major inorganic nitrogen compounds, total ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen. Three immunity parameters, alternative complement activity, lysozyme activity, and total immunoglobulin content were significantly decreased in the blood at the highest stocking density (10 kg m−3). The concentrations of hemoglobin and white blood cells were significantly decreased at 10 kg m−3, while there was no significant change in red blood cells. The significant increases in cortisol level and the enzymatic activities of alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase at 10 kg m−3 clearly supported inorganic nitrogen compounds-triggered stress. A significant elevation of lipid peroxidation value and depletion of intracellular glutathione were observed at 5 and/or 10 kg m−3 in the liver tissue. The hepatic enzymatic activities of antioxidant defense enzymes, catalase and superoxide dismutase were also significantly increased. When a protein skimmer removes the inorganic nitrogen compounds at the highest density, most parameters showed no significant change. Taken together, these results suggest that accumulated inorganic nitrogen compounds at the highest stocking density inhibit innate immunity and induce oxidative stress in red seabream. This information will be helpful to maintain homeostasis of red seabream by controlling immunity and oxidative status through inorganic nitrogen compounds removal in intensive culture condition.

Epigenetic silencing is considered to be a major mechanism for loss of activity in tumor suppressors. Reversal of epigenetic silencing by using inhibitors of DNA methyltransferase (DNMT) or histone deacetylases (HDACs) such as 5-Aza-CdR and FK228 has shown to enhance cytotoxic activities of several anticancer agents. This study aims to assess the combinatorial effects of gene-silencing reversal agents (5-Aza-CdR and FK228) and oxaliplatin in gastric cancer cells, i.e., Epstein-Barr virus (EBV)-negative SNU-638 and EBV-positive SNU-719 cells. The doublet combinatorial treatment of 5-Aza-CdR and FK228 exhibited synergistic effects in both cell lines, and this was further corroborated by Zta expression induction in SNU-719 cells. Three drug combinations as 5-Aza-CdR/FK228 followed by oxaliplatin, however, resulted in antagonistic effects in both cell lines. Simultaneous treatment with FK228 and oxaliplatin induced synergistic and additive effects in SNU-638 and SNU-719 cells, respectively. Three drug combinations as 5-Aza-CdR prior to FK228/oxaliplatin, however, again resulted in antagonistic effects in both cell lines. This work demonstrated that efficacy of doublet synergistic combination using DNMT or HDACs inhibitors can be compromised by adding the third drug in pre- or post-treatment approach in gastric cancer cells. This implies that the development of clinical trial protocols for triplet combinations using gene-silencing reversal agents should be carefully evaluated in light of their potential antagonistic effects.

Among the various materials found inside microplastic pollution, nanosized microplastics are of particular concern due to difficulties in quantification and detection; moreover, they are predicted to be abundant in aquatic environments with stronger toxicity than microsized microplastics. Here, we demonstrated a stronger accumulation of nanosized microbeads in the marine rotifer Brachionus koreanus compared to microsized ones, which was associated with oxidative stress-induced damages on lipid membranes. In addition, multixenobiotic resistance conferred by P-glycoproteins and multidrug resistance proteins, as a first line of membrane defense, was inhibited by nanoplastic pre-exposure, leading to enhanced toxicity of 2,2',4,4'-tetrabromodiphenyl ether and triclosan in B. koreanus. Our study provides a molecular mechanistic insight into the toxicity of nanosized microplastics toward aquatic invertebrates and further implies the significance of synergetic effects of microplastics with other environmental persistent organic pollutants.

Olive flounder (Paralichthys olivaceus) is one of the most valuable marine aquatic species in South Korea and faces tremendous exposure to the viral hemorrhagic septicemia virus (VHSV). Given the growing importance of flounder, it is therefore essential to understand the host defense of P. olivaceus against VHSV infection, but studies on its immune mechanism are hindered by the lack of genomic resources. In this study, the P. olivaceus was infected with disease-causing VHSV isolates, ADC-VHS2012-11 and ADC-VHS2014-5 which showed moderate virulent (20% mortality) and high virulent (65% mortality), in order to investigate the effect of difference in pathogenicity in head kidney during 1, 3, 7 days of post-infection using Illumina sequencing. After removing low-quality sequences, we obtained 144,933,160 high quality reads from thirty-six libraries which were further assembled into 53,384 unigenes with an average length of 563 bp with a range of 200 to 9605 bp. Transcriptome annotation revealed that 30,475 unigenes with a cut-off e-value of 10−5 were functionally annotated. In total, 10,046 unigenes were clustered into 26 functional categories by searching against the eggNOG database, and 22,233 unigenes to 52 GO terms. In addition, 12,985 unigenes were grouped into 387 KEGG pathways. Among the 13,270 differently expressed genes, 6578 and 6692 were differentially expressed only in moderate and high virulent, respectively. Based on our sequence analysis, many candidate genes with fundamental roles in innate immune system including, pattern recognition receptors (TLRs & RLRs), Mx, complement proteins, lectins, and cytokines (chemokines, IFN, IRF, IL, TRF) were differentially expressed. Furthermore, GO enrichment analysis for these genes revealed gene response to defense response to virus, apoptotic process and transcription factor activity. In summary, this study identifies several putative immune pathways and candidate genes deserving further investigation in the context of novel gene discovery, gene expression and regulation studies and lays the foundation for fish immunology especially in P. olivaceus against VHSV.

Injectable antibiotics have some advantages over oral administration under certain conditions. However, they have not been adopted actively, especially in the aquaculture field. Here, we focused on the effect of intramuscular (i.m.) injection of lincomycin HCl (LH), a lincosamide antibiotic, on a streptococcal infection in olive flounders (Paralichthys olivaceus). In-vitro efficacy tests with 49 Streptococcus parauberis clinical isolates revealed inhibition of 32 isolates (65.3%) by 2 μg/mL LH, as the 50% minimum inhibitory concentration (MIC50). A single LH i.m. injection (10 mg/kg) in-vivo significantly reduced cumulative mortality rates against S. parauberis infection from 100% to 0–20.0% (P MIC50 (time period remained above MIC50) values against S. parauberis. After intravenous injection, the volume of distribution (Vz) was high in olive flounder (2.44 L/kg). LH bioavailability was 88.99% following a single i.m. injection of 10 mg/kg. Analyses of muscle residue depletion demonstrated appropriate withdrawal times of 19.95 days (99% statistical tolerance limit) after a single 10 mg/kg i.m. injection. Overall, our results suggest that a single i.m. injection of LH at 10 mg/kg offers acceptable efficacy with reasonable withdrawal periods for streptococcal infection in olive flounder.

Coinhibitory pathways in the B7-CD28 family provide critical inhibitory signals that regulate immune homeostasis, defense and protect tissue integrity. CD276 (B7-H3) is an important immune checkpoint member of this family, which is induced on antigen-presenting cells (APCs), and plays an important role in the inhibition of T-cell function. We have characterized the CD276 gene of olive flounder, Paralichthys olivaceus. OfCD276 has an ORF of 912 bp that codes for 303 amino acids with a predicted molecular mass of 33 kDa. It is a type I transmembrane protein with a single extracellular V- and C-like Ig domains, a transmembrane region, and a highly diverse cytoplasmic tail. This gene was distinctly expressed in gill, spleen, and skin, and sparsely expressed in other tissues. Pathogen stimulation by VHSV revealed that transcription of OfCD276 was induced on early hours in liver and expressed late in head kidney, spleen, intestine and gill tissues. Flow cytometry analysis of leukocytes revealed the percentage of granulocytes and lymphocytes that expressed OfCD276 molecules on their cell surface was 85.1% and 3.1%, respectively. Our study shows a significant role played by this coinhibitory molecule that participate in the regulation of the cell mediated immune response.

Olive flounder (Paralichthys olivaceus) is one of the most economically important aquaculture fish. However, its production is often affected by various diseases, especially viral hemorrhagic septicemia virus (VHSV) that cause serious economic losses. In this study, we sequenced the whole transcriptome of the P. olivaceus using Illumina RNA-sEq. De novo assembly of control and virus-infected cDNA libraries of head kidney at 13 and 20 °C was accomplished with 2,007,532,438 raw reads, resulting in 244,578 unigenes with an average length of 533 bp and found 65,535 candidate coding unigenes with homology to other species by BLAST analysis. DEG analysis among control and virus-infected head kidney samples of 13 and 20 °C revealed that 1290 up-regulated and 162 down-regulated genes (p ≤ 0.01), linked to metabolism, virulence factors, adhesion and immune-response. We constructed an expressed gene catalog for the P. olivaceus to serve as a resource for marine environmental genomic and immuno-genetic/genomic studies focused on uncovering the molecular mechanisms underlying the responses of P. olivaceus to VHSV under different temperature.

We purified an ∼6.4-kDa antimicrobial peptide from an acidified gill extract of the Pacific oyster, Crassostrea gigas , by cation-exchange and C 18 reversed-phase high performance liquid chromatography (HPLC). The identified peptide was composed of 54 amino acids and had a molecular weight of 6484.6 Da. Comparison of the amino acid sequence and molecular weight with those of other known proteins or peptides revealed that the peptide had high identity with the 60S ribosomal protein L29, and so was named cgRPL29. The full-length cgRPL29 cDNA of the Pacific oyster comprised 325-bp, including a 5'-untranslated region (UTR) of 100-bp, a 3′-UTR of 57-bp, and an open reading frame of 168-bp encoding 55 amino acids, with a Met residue at the N-terminus. The cgRPL29 mRNA tissue distribution suggested that it is constitutively expressed in a non-tissue-specific manner. Secondary structural prediction and homology modeling indicated cgRPL29 have an unordered structure containing two partial α-helical regions. This is to our knowledge the first report of the antimicrobial effect of the 60S ribosomal protein L29 from marine invertebrates.

Triclosan (TCS) is an antimicrobial agent that has been widely dispersed and detected in the marine environment. However, the effects of TCS in marine invertebrates are poorly understood. In this study, the effects of TCS on life cycle history (e.g. mortality and fecundity) along with cellular reactive oxygen species (ROS) levels, GSH content, antioxidant enzymatic activities, and mRNA expression levels of oxidative stress-mediated genes were measured in the copepod Tigriopus japonicus. The no observed effect concentration (NOEC) and median lethal concentration (LC50) of TCS in the adult stage were determined to be 300μg/L and 437.476μg/L, respectively, while in the nauplius stages the corresponding values were 20μg/L, and 51.76μg/L, respectively. Fecundity was significantly reduced (P

In July 2012, philometrid nematodes were discovered in cultured rockfish (Sebastes schlegeli) in Cheonsuman (Bay), the Republic of Korea. The nematodes were detected in the epithelial tissues of the rockfish and were identified as Clavinema mariae based on morphological studies using light and scanning electron microscopy. They revealed the characteristics same as previously identified C. mariae, notably having a long body with narrow posterior half, no caudal projection, a cylindrical-shaped esophagus, a well-developed anterior bulbous part of the esophagus, cephalic papillae, and a dorsal esophageal gland. This is the first confirmation of C. mariae infection in rockfish in Korea.

Background Korean diet has received considerable attention because of the low prevalence of obesity and metabolic disorders in Korea. Although the Korean diet has been shown to have health benefits, these effects have been investigated by analyzing individual nutrients or food components. In this study, we used a dietary pattern approach to investigate the effect of the Korean diet on obesity and glucose homeostasis in mice fed a high-fat diet (HFD). Methods C57BL/6 mice were fed the HFD for 7 weeks to induce obesity and then fed either the HFD or Korean diet for the next 7 weeks. The Korean diet was based on food frequency data obtained from the 4th Korean National Health and Nutrition Examination Survey and adjusted to have the same energy content as the HFD. Serum insulin and leptin were quantified by enzyme-linked immunosorbent assay, and glucose clearance was assessed using the oral glucose tolerance test. Lipogenic gene expression was determined by quantitative reverse transcription-polymerase chain reaction. Results The Korean diet significantly decreased the HFD-induced body weight increase and body fat accumulation and improved serum lipid profiles. Hepatic mRNA levels of lipogenic genes were lower in Korean diet-fed mice, which also showed decreased fasting blood glucose, insulin, and leptin levels, and improved glucose clearance. The Korean diet also ameliorated HFD-induced islet hypertrophy and elevated pancreatic insulin level. Conclusions Korean diet prevented obesity and ameliorated insulin resistance in mice fed a HFD. Therefore, Korean diet may be useful as a therapeutic diet to control metabolic disorders.

The supernatant from rat intestinal acetone powder (RIAP) was used as a source of mammalian glucosidases to determine the digestion properties of glycemic-carbohydrates. We hypothesized that many glucosidases are still anchored to the precipitated-intestinal tissues with available enzymes, and developed a method using the RIAP suspension to optimize the in vitro carbohydrate digestion model. The glucose production from various types of glycemic ingredients by RIAP suspension showed that this carbohydrate-hydrolysis model using the entire spectrum of glucosidases can be applied in an in vitro assay to determine carbohydrate quality from glycemic food products at the mammalian level. This approach better mimics the mammalian situation compared to other assays to determine the glycemic-carbohydrate digestion properties that employ fungal/microbial glucosidases that have different hydrolytic activities compared to mammalian enzymes. The method can also be used to determine the inhibitory effects of α-glucosidase inhibitors to attenuate the post-prandial blood glucose level.