Metabolic Engineering of Escherichia coli for Methyl Parathion Degradation.

Organophosphate compounds are widely used in pesticides to control weeds, crop diseases, and insect pests. Unfortunately, these synthetic compounds are hazardous and toxic to all types of living organisms. In the present work, Escherichia coli was bioengineered to achieve methyl parathion (MP) degradation via the introduction of six synthetic genes, namely, opdS, pnpAS, pnpBS, pnpCS, pnpDS, and pnpES, to obtain a new transformant, BL-MP. MP and its subsequent decomposition intermediates were completely degraded by this transformant to enter the metabolites of multiple anabolic pathways. The MP-degraded strain created in this study may be a promising candidate for the bioremediation of MP and potential toxic intermediates.

Pseudomonas laoshanensis sp. nov., isolated from peanut field soil.

A novel Gram-stain-negative, aerobic strain, designated Y22T, was isolated from peanut field soil in Laoshan Mountain in China. Cells of strain Y22T were rod-shaped and motile by a single flagellum. The strain was found to be oxidase- and catalase-positive. 16S rRNA gene sequence based on phylogenetic analysis indicated that strain Y22T belonged to the genus Pseudomonas, and showed the highest 16S rRNA gene sequence similarity of 99.0% to Pseudomonas pelagia JCM 15562T, followed by Pseudomonas salina JCM 19469T (98.4%), Pseudomonas sabulinigri JCM 14963T (97.9%), Pseudomonas bauzanensis CGMCC 1.9095T (97.6%) and Pseudomonas litoralis KCTC23093T (97.5%). The phylogenetic analysis based on multilocus sequence analyses with concatenated 16S rRNA, gyrB, rpoD and rpoB genes indicated that strain Y22T belonged to Pseudomonas pertucinogena lineage. The average nucleotide identity scores between strain Y22T and closely related species were 74.6-82.8%, and the Genome-to-Genome Distance Calculator scores were 16.4-44.9%. The predominant cellular fatty acids of strain Y22T were C18:1ω7c (29.6%), C17:0 cyclo (17.5%) and summed feature 3 (C16:1ω7c and/or C16:1ω6c) (17.4%). The genomic DNA G+C content was 57.9 mol%. On the basis of phenotypic characteristics, phylogenetic analyses and in silico DNA-DNA relatedness, a novel species, Pseudomonas laoshanensis sp. nov. is proposed. The type strain is Y22T (= JCM 32580T = KCTC 62385T = CGMCC 1.16552T).

Calycosin inhibited autophagy and oxidative stress in chronic kidney disease skeletal muscle atrophy by regulating AMPK/SKP2/CARM1 signalling pathway.

Skeletal muscle atrophy is a common and serious complication of chronic kidney disease (CKD). Oxidative stress and autophagy are the primary molecular mechanisms involved in muscle atrophy. Calycosin, a major component of Radix astragali, exerts anti-inflammatory, anti-oxidative stress and anti-autophagy effects. We investigated the effects and mechanisms of calycosin on skeletal muscle atrophy in vivo and in vitro. 5/6 nephrectomy (5/6 Nx) rats were used as a model of CKD. We evaluated bodyweight and levels of serum creatinine (SCr), blood urea nitrogen (BUN) and serum albumin (Alb). H&E staining, cell apoptosis, oxidative stress biomarkers, autophagosome and LC3A/B levels were performed and evaluated in skeletal muscle of CKD rat. Calycosin treatment improved bodyweight and renal function, alleviated muscle atrophy (decreased the levels of MuRF1 and MAFbx), increased superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activity and reduced malondialdehyde (MDA) levels in skeletal muscle of CKD rats. Importantly, calycosin reduced autophagosome formation, down-regulated the expression of LC3A/B and ATG7 through inhibition of AMPK and FOXO3a, and increased SKP2, which resulted in decreased expression of CARM1, H3R17me2a. Similar results were observed in C2C12 cells treated with TNF-α and calycosin. Our findings showed that calycosin inhibited oxidative stress and autophagy in CKD induced skeletal muscle atrophy and in TNF-α-induced C2C12 myotube atrophy, partially by regulating the AMPK/SKP2/CARM1 signalling pathway.

Pseudomonas phragmitis sp. nov., isolated from petroleum polluted river sediment.

A Gram-stain-negative, rod-shaped bacterium, motile by means of a single polar flagellum, designated S-6-2T, was isolated from petroleum polluted river sediment in Huangdao, Shandong Province, PR China. The 16S rRNA gene sequence analysis revealed that S-6-2T represented a member of the genus Pseudomonas, sharing the highest sequence similarities with Pseudomonas parafulva (97.5 %) and Pseudomonas fulva (97.5 %). Phylogenetic analysis based on 16S rRNA gene, concatenated 16S rRNA, gyrB, rpoB and rpoD genes and genome core-genes indicated that S-6-2T was affiliated with the members of the Pseudomonas pertucinogena group. The average nucleotide identity (ANI) and genome-to-genome distance between the whole genome sequences of S-6-2T and closely related species of the genus Pseudomonas within the P. pertucinogena group were less than 77.94 % and 20.5 %, respectively. Differences in phenotypic characteristics were also found between S-6-2T and the closely related species. The major cellular fatty acids (>10 %) were summed feature 8 (C18 : 1ω7c/ C18  : 1ω6c), C16 : 0, C17 : 0cyclo and C12 : 0. The predominant respiratory quinone was ubiquinone 9. The major polar lipids were diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), one unidentified lipid (L1), two unidentified phospholipids (PL1 and PL2) and an aminophospholipid (APL). The DNA G+C content of the genome of S-6-2T was 60.1 mol%. On the basis of the evidence from the polyphasic taxonomic study, strain S-6-2T can be classified as representative of a novel species of the genus Pseudomonas, for which the name Pseudomonas phragmitis sp. nov. is proposed. The type strain is S-6-2T (=CGMCC 1.15798T=KCTC 52539T).

Metabolic engineering of rice endosperm for betanin biosynthesis.

Betanin has been widely used as an additive for many centuries, and its use has increased because of its market application as an additive, high free radical scavenging activity, and safety, health-promoting properties. The main source of betanin is red beet, but many factors notably affect the yield of betanin from red beets. Betanin is not produced in cereal grains. Thus, developing biofortified crops with betanin is another alternative to health-promoting food additives. Here, rice endosperm was bioengineered for betanin biosynthesis by introducing three synthetic genes (meloS, BvDODA1S, and BvCYP76AD1S). The overexpression of these genes driven by rice endosperm-specific promoter established the betanin biosynthetic pathways in the endosperm, resulting in new types of germplasm - 'Betanin Rice' (BR). The BR grains were enriched with betanin and had relatively high antioxidant activity. Our results proved that betanin can be biosynthesized de novo in rice endosperm by introducing three genes in the committed betanin biosynthetic pathway. The betanin-fortified rice in this study can be used as a functional grain to promote health and as a raw material to process dietary supplements.

Salidroside ameliorates endothelial inflammation and oxidative stress by regulating the AMPK/NF-κB/NLRP3 signaling pathway in AGEs-induced HUVECs.

Endothelial dysfunction plays important roles in vascular dysfunction under diabetic conditions. The generation of advanced glycation end products (AGEs), which can induce inflammation and oxidative stress, is pivotal in endothelial dysfunction. Salidroside, a major active compound in Rhodiola rosea, exerts protective effects against vascular diseases. To study the effects and mechanism of salidroside in diabetes-induced vascular endothelial dysfunction, an in vitro model was established with AGEs-induced human umbilical vein endothelial cells (HUVECs). Then, cell viability, cell apoptosis, pro-inflammatory cytokines and oxidative biomarkers were tested to determine the effects of salidroside at 10, 50 and 100 µM doses on AGEs induced HUVECs. Additionally, RNA-Seq and bioinformatics analyses were used to search for the underlying mechanism of salidroside. The results showed that salidroside promoted cell viability and significantly alleviated cell apoptosis in AGEs-induced HUVECs. Furthermore, salidroside remarkably decreased the levels of the pro-inflammatory cytokines TNF-α, IL-1ß and IL-6 and impeded the expression of VCAM-1 and ICAM-1 induced by AGEs. Additionally, salidroside promoted superoxide dismutase (SOD) activity and increased catalase (CAT) and glutathione peroxidase (GSH-Px) levels while inhibiting the intracellular generation of reactive oxygen species (ROS) and malondialdehyde (MDA) in AGEs-induced HUVECs. Importantly, salidroside alleviated endothelial inflammation and oxidative stress by activating AMPK phosphorylation and inhibiting NF-ĸB p65 and NLRP3 inflammasome activation. Therefore, we used compound C, an accepted AMPK inhibitor, to further demonstrate the mechanism. Interestingly, the phenomenon produced by salidroside was abolished. Our findings suggest that salidroside ameliorates AGEs-induced endothelial inflammation and oxidative stress, partially via the AMPK/NF-κB/NLRP3 signaling pathway.

Pseudomonas qingdaonensis sp. nov., an aflatoxin-degrading bacterium, isolated from peanut rhizospheric soil.

A Gram-stain-negative, aerobic, mobile, and rod-shaped bacterium, designated JJ3T, was isolated from peanut rhizospheric soil in Qingdao, Shandong Province, China, and was characterized using a polyphasic approach. Strain JJ3T grew at 4-40 °C, at pH 5.0-9.0 and 0-4% NaCl. The strain was positive for both catalase and oxidase tests, and was able to degrade aflatoxin B1. According to the 16S rRNA gene sequence comparisons, the strain JJ3T was identified as a member of the genus Pseudomonas and was most closely related to Pseudomonas japonica JCM 21532T and Pseudomonas alkylphenolica JCM 16553T with sequence similarity of 99.0% and 98.9%, respectively. A multilocus sequence analysis (MLSA) of concatenating 16S rRNA, gyrB and rpoD gene sequences showed that strain JJ3T belonged to the Pseudomonas putida subcluster. Genomic comparison of strain JJ3T with its closest phylogenetic type strain using average nucleotide index (ANI) and digital DNA-DNA relatedness revealed 76.7-82.9% and 20.2-37.1%, respectively. All values were distinctly lower than the thresholds established for species differentiation. The predominant cellular fatty acids of strain JJ3T were C17:0 cyclo (24.0%), C16:0 (21.4%), summed features 3 (C16:1ω7c and/or C16:1ω6c) (11.5%) and summed features 8 (C18:1ω7c and/or C18:1ω6c) (10.5%). The major polar lipids of strain JJ3T were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The physiological, biochemical, and genetic characteristics support the assignment of JJ3T to the genus Pseudomonas, but are different to those of phylogenetically neighboring species to represent a novel species. The name Pseudomonas qingdaonensis sp. nov. is proposed, with JJ3T (= JCM 32579T = KCTC 62384T = CGMCC 1.16493T) as the type strain.

Hispaglabridin B, a constituent of liquorice identified by a bioinformatics and machine learning approach, relieves protein-energy wasting by inhibiting forkhead box O1.

BACKGROUND AND PURPOSE: Liquorice is the root of Glycyrrhiza glabra, which is a popular food in Europe and China that has previously shown benefits for skeletal fatigue and nutrient metabolism. However, the mechanism and active ingredients remain largely unclear. The aim of this study was to investigate the active ingredients of liquorice for muscle wasting and elucidate the underlying mechanisms. EXPERIMENTAL APPROACH: RNA-Seq and bioinformatics analysis were applied to predict the main target of liquorice. A machine learning model and a docking tool were used to predict active ingredients. Isotope labelling experiments, immunostaining, Western blots, qRT-PCR, ChIP-PCR and luciferase reporters were utilized to test the pharmacological effects in vitro and in vivo. The reverse effects were verified through recombination-based overexpression. KEY RESULTS: The liposoluble constituents of liquorice improved muscle wasting by inhibiting protein catabolism and fibre atrophy. We further identified FoxO1 as the target of liposoluble constituents of liquorice. In addition, hispaglabridin B (HB) was predicted as an inhibitor of FoxO1. Further studies determined that HB improved muscle wasting by inhibiting catabolism in vivo and in vitro. HB also markedly suppressed the transcriptional activity of FoxO1, with decreased expression of the muscle-specific E3 ubiquitin ligases MuRF1 and Atrogin-1. CONCLUSIONS AND IMPLICATIONS: HB can serve as a novel natural food extract for preventing muscle wasting in chronic kidney disease and possibly other catabolic conditions.

SKA3 promotes cell proliferation and migration in cervical cancer by activating the PI3K/Akt signaling pathway.

BACKGROUND: Cervical cancer (CC) is one of the most common cancers among females worldwide. Spindle and kinetochore-associated complex subunit 3 (SKA3), located on chromosome 13q, was identified as a novel gene involved in promoting malignant transformation in cancers. However, the function and underlying mechanisms of SKA3 in CC remain unknown. Using the Oncomine database, we found that expression of SKA3 mRNA is higher in CC tissues than in normal tissues and is linked with poor prognosis. METHODS: In our study, immunohistochemistry showed increased expression of SKA3 in CC tissues. The effect of SKA3 on cell proliferation and migration was evaluated by CCK8, clone formation, Transwell and wound-healing assays in HeLa and SiHa cells with stable SKA3 overexpression and knockdown. In addition, we established a xenograft tumor model in vivo. RESULTS: SKA3 overexpression promoted cell proliferation and migration and accelerated tumor growth. We further identified that SKA3 is involved in regulating cell cycle progression and the PI3K/Akt signaling pathway via RNA-sequencing (RNA-Seq) and gene set enrichment analyses. Western blotting results revealed that SKA3 overexpression increased levels of p-Akt, cyclin E2, CDK2, cyclin D1, CDK4, E2F1 and p-Rb in HeLa cells. Additionally, the use of an Akt inhibitor (GSK690693) significantly reversed the cell proliferation capacity induced by SKA3 overexpression in HeLa cells. CONCLUSIONS: We suggest that SKA3 overexpression contributes to CC cell growth and migration by promoting cell cycle progression and activating the PI3K-Akt signaling pathway, which may provide potential novel therapeutic targets for CC treatment.

FIBP knockdown attenuates growth and enhances chemotherapy in colorectal cancer via regulating GSK3ß-related pathways.

Colorectal cancer stem cells (CSCs), characterized by self-renewal ability and high expression of proliferative genes, contribute to the chemoresistance of colorectal cancer (CRC). We aimed to identify the molecular mechanisms underlying CRC chemoresistance through comprehensive bioinformatics screenings and experimental confirmation of gene functions. We found that high expression of FGF1 intracellular binding protein (FIBP) was correlated with chemoresistance and poor prognosis in CRC patients. Therefore, the chemoresistant CRC cell line HCT116-CSC with high expression of the stem cell markers CD44 and CD133 was established for further phenotypic tests. FIBP knockdown inhibited proliferation, enhanced chemotherapy effects, and attenuated the stemness markers of CRC cells in vivo and in vitro. Through RNA-seq and gene set enrichment analysis, we identified cyclin D1 as a key downstream target in FIBP-regulated cell cycle progression and proliferation. Moreover, FIBP bound to GSK3ß, inhibited its phosphorylation at Tyr216, and activated ß-catenin/TCF/cyclin D1 signaling in HCT116-CSCs. Additional GSK3ß knockdown reversed the FIBP silencing-induced inhibition of proliferation and decreased stemness marker expression in HCT116-CSCs. Furthermore, DNA methylation profiling suggested that FIBP regulated the stemness of CRC cells via methylation activity that was dependent on GSK3ß but independent of ß-catenin signaling. Our data illuminate the potential of FIBP as a novel therapeutic target for treating chemoresistant CRC through inhibition of GSK3ß-related signaling.

[Screening active ingredients of Shenkangwan that regulate endothelial-mesenchymal transition of endothelial cells in vitro].

OBJECTIVE: To screen the effective components of Shenkangwan that regulate endothelial-mesenchymal transition in endothelial cells for optimizing prescription of Shenkangwan. METHODS: ALK5 was identified as one of the target receptors that regulate endothelial-mesenchymal transition of endothelial cells using molecular docking technique. Nine molecules were screened as the candidate effective components in Shenkangwan, among which calycosin, ononin and stigmasterol were selected for testing. Glomerular epithelial cells were exposed to high glucose and treated with calycosin, ononin, or stigmasterol, and the cellular expressions of α-smooth muscle actin (α-SMA) and vimentin mRNA were detected with real-time fluorescence quantitative PCR. The phosphorylation of SMAD2/3 in the cells was detected using Western blotting. RESULTS: Calycosin, ononin and stigmasterol did not produce significant cytotoxicity in glomerular epithelial cells (P>0.05). The cells exposed to high glucose and calycosin treatment showed significantly decreased mRNA levels of α-SMA and vimentin (P<0.05) and inhibited phosphorylation of SMAD2/3. Ononin and stigmasterol did not produce such effects in the cells. CONCLUSION: In endothelial cells with high glucose-induced injury, calycosin can inhibit the up-regulation of α-SMA and vimentin and inhibit phosphorylation of SMAD2/3 to regulate endothelial-mesenchymal transition and improve diabetic nephropathy.

Dietary fiber intake is associated with chronic kidney disease (CKD) progression and cardiovascular risk, but not protein nutritional status, in adults with CKD.

BACKGROUND AND OBJECTIVES: Evidence suggests that dietary fiber benefits patients with chronic kidney disease (CKD); however, this conclusion requires further validation. In this study, we examined the effects of dietary fiber on kidney function, inflammation, indoxyl sulfate, nutritional status, and cardiovascular risk in patients with advanced CKD. METHODS AND STUDY DESIGN: We performed linear regressions to assess the association between dietary fiber intake and CKD parameters. The aforementioned parameters were compared over an 18-month follow- up period. Kaplan-Meier analysis was used to investigate the association between fiber intake and Cardiac vascular disease (CVD). RESULTS: In total, 157 patients were included in this study. Dietary fiber and inflammatory indices were associated (interleukin [IL]-6: ß=-0.024, p=0.035). The differential estimated glomerular filtration rate (ΔeGFR) as well as levels of C-reactive protein, IL-6, indoxyl sulfate, and serum cholesterol in the higher fiber intake (>=25 g/day) group were lower than those in the lower fiber intake (<25 g/day) group (p<0.05). Differences in IL-6 and indoxyl sulfate levels were more significant in patients in the higher protein intake group (p<0.05). Dietary fiber intake may be a protective factor associated with CVD (hazard ratio=0.537 and 0.305- 0.947). The protein nutritional status was not different between the two groups (p>0.05). CONCLUSIONS: Our results suggest that increasing fiber intake can retard the decrease in the eGFR; can reduce the levels of proinflammatory factors, indoxyl sulfate, and serum cholesterol; and is negatively associated with cardiovascular risk, but does not disrupt the nutritional status of patients with CKD.

Effect of Zinc Supplementation on Maintenance Hemodialysis Patients: A Systematic Review and Meta-Analysis of 15 Randomized Controlled Trials.

We aimed to examine the effects of zinc supplementation on nutritional status, lipid profile, and antioxidant and anti-inflammatory therapies in maintenance hemodialysis (MHD) patients. We performed a systematic review and meta-analysis of randomized, controlled clinical trials of zinc supplementation. Metaregression analyses were utilized to determine the cause of discrepancy. Begg and Egger tests were performed to assess publication bias. Subgroup analysis was utilized to investigate the effects of zinc supplementation in certain conditions. In the crude pooled results, we found that zinc supplementation resulted in higher serum zinc levels (weighted mean difference [WMD] = 28.489; P < 0.001), higher dietary protein intake (WMD = 8.012; P < 0.001), higher superoxide dismutase levels (WMD = 357.568; P = 0.001), and lower levels of C-reactive protein (WMD = -8.618; P = 0.015) and malondialdehyde (WMD = -1.275; P < 0.001). The results showed no differences in lipid profile. In the metaregression analysis, we found that serum zinc levels correlated positively with intervention time (ß = 0.272; P = 0.042) and varied greatly by ethnicity (P = 0.023). Results from Begg and Egger tests showed that there was no significant bias in our meta-analysis (P > 0.1). Results of subgroup analysis supported the above results. Our analysis shows that zinc supplementation may benefit the nutritional status of MHD patients and show a time-effect relationship.

Shewanella inventionis sp. nov., isolated from deep-sea sediment.

A novel Gram-stain-negative, rod-shaped bacterial strain, designated KX27T, was isolated from deep-sea sediment of the Okinawa Trough and characterized on the basis of a polyphasic taxonomic approach. The phenotypic features of strain KX27T were similar to those of members of the genus Shewanella. Strain KX27T exhibited highest 16S rRNA gene sequence similarity (98.37 %) to Shewanellabasaltis JCM 14937T. Levels of gyrB gene sequence similarity between strain KX27T and its closest genetic neighbours were lower than 90 %, the species cut-off value for Shewanella. The DNA-DNA hybridization value between strain KX27T and S. basaltis JCM 14937T (54 %) was lower than the accepted species threshold. The predominant cellular fatty acids of strain KX27T were summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), C17 : 1ω8c, summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c) and iso-C15 : 0. The major polar lipids of strain KX27T were phosphatidylethanolamine and phosphatidylglycerol. On the basis of combined phenotypic, chemotaxonomic and phylogenetic analysis, strain KX27T represents a novel species of the genus Shewanella, for which the name Shewanella inventionis sp. nov. is proposed. The type strain is KX27T (=KCTC 42807T=CGMCC 1.15339T).

Pseudomonas oceani sp. nov., isolated from deep seawater.

In this study, we identified a novel Gram-stain-negative, aerobic, motile, and rod-shaped bacterium, strain KX 20T, isolated from the deep seawater in Okinawa Trough, northwestern Pacific Ocean. Phylogenetic analysis based on 16S rRNA gene sequence showed that strain KX 20T was related to members of the genus Pseudomonas and shares the highest sequence identities with Pseudomonas aestusnigri CECT 8317T (99.4 %) and Pseudomonas pachastrellae JCM 12285T (98.5 %). The 16S rRNA gene sequence identities between strain KX 20T and other members of the genus Pseudomonaswere below 96.6 %. The gyrB and rpoD genes of strain KX 20T shared 82.0 to 89.3 % sequence identity with the gyrB and rpoD genes of the closest phylogenetic neighbours of KX 20T. The predominant cellular fatty acids of strain KX 20T were summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c) (29.2 %), C16 : 0 (24.5 %), summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) (21.5 %) and C12 : 0 (8.2 %). The major polar lipids of strain KX 20T were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and unknown phospholipids. The genomic DNA G+C content of strain KX 20T was 62.9 mol%. On the basis of phylogenetic analysis and phenotypic characteristics, a novel species, Pseudomonas oceani sp. nov. is proposed. The type strain is KX 20T (=CGMCC 1.15195T=DSM 100277T).

Characteristics of the cultivable bacteria from sediments associated with two deep-sea hydrothermal vents in Okinawa Trough.

In this study, different culture-dependent methods were used to examine the cultivable heterotrophic bacteria in the sediments associated with two deep-sea hydrothermal vents (named HV1 and HV2) located at Iheya Ridge and Iheya North in Okinawa Trough. The two vents differed in morphology, with HV1 exhibiting diffuse flows while HV2 being a black smoker with a chimney-like structure. A total of 213 isolates were identified by near full-length 16S rRNA gene sequence analysis. Of these isolates, 128 were from HV1 and 85 were from HV2. The bacterial community structures were, in large parts, similar between HV1 and HV2. Nevertheless, differences between HV1 and HV2 were observed in one phylum, one class, 4 orders, 10 families, and 20 genera. Bioactivity analysis revealed that 25 isolates belonging to 9 different genera exhibited extracellular protease activities, 21 isolates from 11 genera exhibited extracellular lipase activities, and 13 isolates of 8 genera displayed antimicrobial activities. This is the first observation of a large population of bacteria with extracellular bioactivities existing in deep-sea hydrothermal vents. Taken together, the results of this study provide new insights into the characteristics of the cultivable heterotrophic bacteria in deep-sea hydrothermal ecosystems.

A CCL21 chemokine of tongue sole (Cynoglossus semilaevis) promotes host resistance against bacterial infection.

Chemokines are a large family of chemotactic cytokines. Based on the arrangement of the first two cysteine residues, chemokines are divided into four groups, one of which is the CC chemokine group. In this study, we characterized a CC chemokine, CsCCL21, from half-smooth tongue sole (Cynoglossus semilaevis), and analyzed its activity. CsCCL21 contains two conserved N-terminal cysteine residues in a NCCL motif and is phylogenetically related to the CCL19/21/25 subgroup of CC chemokines. CsCCL21 was constitutively expressed in nine tissues and significantly upregulated by bacterial and viral infection. The recombinant CsCCL21 (rCsCCL21) induced migration of peripheral blood leukocytes. When the two conserved cysteine residues in the NCCL motif were mutated, the chemotactic activity of rCsCCL21 was abolished. rCsCCL21 enhanced the resistance of tongue sole against bacterial infection, but the mutant protein with NCCL mutation lacked this antibacterial effect. Taken together, these results suggest that CsCCL21 is a functional CC chemokine with the ability to recruit leukocytes and is involved in antibacterial immunity in a manner that requires the conserved NCCL motif.