IL-2 Signaling Couples the MAPK and mTORC1 Axes to Promote T Cell Proliferation and Differentiation in Teleosts.

IL-2 is a pleiotropic cytokine that is critical for T cell immunity. Although the IL-2-mediated regulation of T cell immunity in mammals is relatively well understood, it remains largely unknown whether and how IL-2 regulates T cell immunity in lower vertebrates. To address this knowledge gap, we investigated the role played by IL-2 in the regulation of T cell response, as well as the associated underlying mechanisms in a teleost fish, large yellow croaker (Larimichthys crocea). We found that large yellow croaker (L. crocea) IL-2 (LcIL-2) significantly promoted T cell proliferation both in vivo and in vitro; significantly induced the differentiation of Th1, Th2, regulatory T, and cytotoxic T cells while inhibiting Th17 differentiation; and participated in the elimination of invading pathogenic bacteria. Mechanistically, the binding of LcIL-2 to its heterotrimer receptor complex (LcIL-15Rα/LcIL-2Rß/Lcγc) triggered the conserved JAK-STAT5 pathway, which in turn regulated the expression of genes involved in T cell expansion, differentiation, and biological function. The MAPK and mammalian target of rapamycin complex 1 (mTORC1) axes, which are involved in TCR-mediated signaling, were also required for LcIL-2-mediated T cell response. Collectively, our results demonstrated that fish IL-2 plays a comprehensive regulatory role in T cell response and highlighted the complex and delicate network regulating T cell-driven immune response. We propose that T cell immunity is regulated by the interplay between TCR signaling and cytokine signaling, and that this basic strategy evolved before the emergence of the tetrapod lineage. Our findings provide valuable insights into the regulatory mechanisms underlying T cell response in teleosts.

Large yellow croaker (Lrimichthys crocea) IL-2 modulates humoral immunity via the conserved JAK-STAT5 signal pathway.

The terminal differentiation of B cells into plasma cells is central to the generation of protective, long-lived humoral immune responses. In mammals, interleukin-2 (IL-2) has been shown to play a role in B cell proliferation and differentiation. However, it remains unclear whether fish IL-2 is involved in B cell proliferation and differentiation. To this end, we investigated the regulatory role of IL-2 in B cell proliferation and differentiation in large yellow croaker (Larimichthys crocea). We found that L. crocea IL-2 (LcIL-2) significantly increased IgM+ B cells proliferation both in vivo and in vitro and facilitated IgM+ B cells differentiation into plasma cells. Furthermore, LcIL-2 increased the production of specific antibodies after immunization with the Vibrio alginolyticus subunit vaccine, recombinant dihydrolipoamide dehydrogenase (rDLD); simultaneous administration of LcIL-2 and rDLD prior to challenge with Vibrio parahaemolyticus or V. alginolyticus significantly increased relative percent survival. Mechanistically, LcIL-2 promoted B cell proliferation and regulated B cell differentiation by triggering the JAK-STAT5 signaling pathway. Collectively, our results demonstrated that LcIL-2 improved B cell proliferation and specific antibody production via the conserved JAK-STAT5 signaling pathway in large yellow croaker, providing valuable insights into the mechanisms underlying the IL-2-mediated regulation of the humoral immune response in fish.

The identification and expression of an interleukin-21 receptor in large yellow croaker (Larimichthys crocea).

BACKGROUND: We identified a homologue of IL-21R (LcIL-21R) in large yellow croaker (Larimichthys crocea, Lc). Our investigation focused on understanding the molecular structural features and immune function of LcIL-21R. METHODS: We cloned the LcIL-21R gene from the genome of Larimichthys crocea by RT‒PCR, and the molecular and structural characteristics of LcIL-21R were analyzed by a series of protein analysis tools. We used real-time PCR to investigate the tissue distribution of LcIL-21R, and LcIL-21R gene expression regulation was also measured in head kidney leukocytes under trivalent bacterial vaccine or poly (I:C) stimulation. RESULTS: The open reading frame (ORF) of the LcIL-21R gene is 1629 bp long and encodes a precursor protein of 542 amino acids (aa), with a 23-aa signal peptide and a 519-aa mature peptide containing four putative N-glycosylation sites. LcIL-21R has two fibronectin type III (FNIII)-like domains (D1 and D2), a transmembrane domain, and a cytoplasmic region. A conserved WSXWS motif was also found in the D2 domain. The predicted structure of the extracellular region of LcIL-21R (LcIL-21R-Ex) is highly similar to that of human IL-21R. LcIL-21R was constitutively expressed in all tissues examined, and LcIL-21R mRNA levels were increased in the head kidney and spleen upon inactivated trivalent bacterial vaccine or poly(I:C) stimulation. CONCLUSIONS: Our results suggest that LcIL-21R shares structural and functional properties with IL-21Rs found in other vertebrates, indicating its potential involvement in the IL-21-mediated immune response to pathogenic infections. These findings contribute to our understanding of the evolutionary conservation of IL-21 signaling and its role in the immune system.

Clinical features and treatment outcomes of infantile appendicitis: a multicentre study.

Appendicitis in infants is a life-threatening condition that is seldom studied. Our purpose was to conduct a comprehensive evaluation of appendicitis in this age group. This was a multicenter retrospective study. Patients aged under one year with surgically confirmed appendicitis during January, 2010 to May, 2022 were identified from four institutional databases. The patients were grouped as neonates or older infants based on their age at the onset of symptoms associated with acute appendicitis. The study encompassed 98 infants, with median age of 66.5 (IQR, 13.8-176.0) days. Neonates were more likely to exhibit abdominal distension (64.9%) and fever (56.8%), while older infants more frequently presented with fever (88.5%) and vomiting (49.2%). Most patients (76.5%) were misdiagnosed during their initial clinical encounter, with a -rate was 3.1% (3 deaths), with neonates exhibiting a rate of 5.4%, and older infants 1.6%. Compared to older infants, neonates showed a higher incidence of appendiceal perforation (OR, 2.9; 95%CI, 1.1-8.1), mechanical ventilation (OR, 9.5; 95%CI, 3.1-29.2), and ICU admission (OR, 16.1; 95%CI, 5.6-45.7). However, there were no significant differences in mortality rates, 30-day readmission rates, and surgical complications between the two groups. CONCLUSION: Although most infants with appendicitis were misdiagnosed during the first clinical encounter, the observed mortality rates were considerably lower than previously reported. While neonates and infants over 28 days displayed differing clinical presentations and disease severity, their outcomes were similar. WHAT IS KNOWN: • Appendicitis in infants is a critical yet underemphasized health concern, often misdiagnosed at initial clinical encounters due to its atypical presentation and non-specific symptoms. • The mortality rates in the neonates with appendicitis was 23% during the past decades. WHAT IS NEW: • The neonates and older infants displayed differing clinical presentations and disease severity. The treatment outcomes were similar. • The mortality rate for infantile appendicitis (3.1%) was significantly lower than historically reported.

Development of monoclonal antibody against IgM of large yellow croaker (Larimichthys crocea) and characterization of IgM+ B cells.

In the present study, a monoclonal antibody (mAb) against large yellow croaker IgM was produced by immunizing mice with purified large yellow croaker serum IgM. Western blotting showed that this mAb could specifically react with the heavy chain of large yellow croaker serum IgM. Indirect immunofluorescence assay (IFA) analysis suggested that the resulting mouse anti-IgM mAb could recognize membrane-bound IgM (mIgM) molecules of large yellow croaker. This mouse anti-IgM mAb also can be used for sorting of large yellow croaker IgM+ B cells through the magnetic-activated cell sorting (MACS) method, which was further confirmed by RT-PCR analysis of specific marker genes for B cells. Flow cytometry analysis showed that the percentages of IgM+ B cells in head kidney, spleen and peripheral blood lymphocytes were 29.00 ±â€¯1.58%, 33.00 ±â€¯1.64%, and 16.50 ±â€¯2.39%, respectively. Additionally, the phagocytosis rates of IgM+ B cells for 0.5 µm beads in head kidney, spleen and peripheral blood were calculated to be 7.56 ±â€¯0.58%, 4.053 ±â€¯0.62% and 23.17 ±â€¯2.26%, respectively, while only 2.36 ±â€¯0.23%, 1.16 ±â€¯0.44% and 6.41 ±â€¯0.45 of IgM+ B cells in these three tissues ingested 1 µm beads. Taken together, our data demonstrated that the mouse anti-IgM mAb produced in this study could be used as a tool to characterize IgM+ B cells and to study functions of IgM in large yellow croaker.

Molecular cloning and bioactivity of an IL-2 homologue in large yellow croaker (Larimichthys crocea).

Interleukin-2 (IL-2), an important immunomodulatory cytokine, plays a crucial role in promoting the proliferation, activation and differentiation of T cells. Here, the cDNA of an IL-2 homologue (LcIL-2) in large yellow croaker (Larimichthys crocea) was cloned by RACE-PCR techniques. The open reading frame (ORF) of LcIL-2 gene is 426 bp long and encoded a precursor protein of 141 amino acids (aa), with a 20-aa signal peptide and a 121-aa mature peptide containing two putative N-glycosylation sites at Asn77 and Asn101. The LcIL-2 is preferentially expressed in lymphocytes-rich tissues, such as spleen and blood, and is increased in head kidney and spleen upon inactivated trivalent bacterial vaccine or poly(I:C) stimulation. LcIL-2 expression could also be detected in primary head kidney leukocytes (PKL), primary head kidney macrophages (PKM) and primary head kidney granulocytes (PKG), with the highest level in PKL. In addition, the expression level of LcIL-2 in PKL was slightly induced by LPS or poly(I:C), while markedly induced by PHA or Con-A. The recombinant LcIL-2 protein produced in Pichia pastoris could increase the expression of genes involved in Th1 (IL-2, IFN-γ and T-bet) and Th2 (IL-4/13A, IL-4/13B and GATA3) development and differentiation, and of the IL-2 downstream transcription factor STAT5B gene, but inhibit the expression of genes related to Th17 (IL-17A/F2 and IL-17A/F3) development and differentiation. Taken together, our results indicated that LcIL-2 possesses similar structural and functional characteristics to other vertebrate IL-2s, and may play a role in T cell development and differentiation.

Risk factors spatial-temporal detection for dengue fever in Guangzhou.

Dengue fever (DF) has been a growing public-health concern in China since its emergence in Guangdong Province in 1978. Of all the regions that have experienced dengue outbreaks in mainland China, the city of Guangzhou is the most affected. This study aims to investigate the potential risk factors for dengue virus (DENV) transmission in Guangzhou, China, from 2006 to 2014. The impact of risk factors on DENV transmission was qualified by the q-values calculated using a novel spatial-temporal method, the GeoDetector model. Both climatic and socioeconomic factors were considered. The impacts on DF incidence of each single factor and the interaction of two factors were analysed. The results show that the number of days with rainfall of the month before last has the highest determinant power, with a q-value of 0.898 (P < 0.01); the q-values of the other factors related to temperature and precipitation were around 0.38-0.50. Integrating a Pearson correlation analysis, nonlinear associations were found between the DF incidence in Guangzhou and the climatic factors considered. The coupled impact of the different variables considered was enhanced compared with their individual effects. In addition, an increased number of tourists in the city were associated with a high incidence of DF. This study demonstrates that the number of rain days in a month has great influence on the DF incidence of the month after next; the temperature and precipitation have nonlinear impacts on the DF incidence in Guangzhou; both the domestic and overseas tourists coming to the city increase the risk of DENV transmission. These findings are useful in the risk assessment of DENV transmission, to predict DF outbreaks and to implement preventive DF reduction strategies.

Genome sequencing of the perciform fish Larimichthys crocea provides insights into molecular and genetic mechanisms of stress adaptation.

The large yellow croaker Larimichthys crocea (L. crocea) is one of the most economically important marine fish in China and East Asian countries. It also exhibits peculiar behavioral and physiological characteristics, especially sensitive to various environmental stresses, such as hypoxia and air exposure. These traits may render L. crocea a good model for investigating the response mechanisms to environmental stress. To understand the molecular and genetic mechanisms underlying the adaptation and response of L. crocea to environmental stress, we sequenced and assembled the genome of L. crocea using a bacterial artificial chromosome and whole-genome shotgun hierarchical strategy. The final genome assembly was 679 Mb, with a contig N50 of 63.11 kb and a scaffold N50 of 1.03 Mb, containing 25,401 protein-coding genes. Gene families underlying adaptive behaviours, such as vision-related crystallins, olfactory receptors, and auditory sense-related genes, were significantly expanded in the genome of L. crocea relative to those of other vertebrates. Transcriptome analyses of the hypoxia-exposed L. crocea brain revealed new aspects of neuro-endocrine-immune/metabolism regulatory networks that may help the fish to avoid cerebral inflammatory injury and maintain energy balance under hypoxia. Proteomics data demonstrate that skin mucus of the air-exposed L. crocea had a complex composition, with an unexpectedly high number of proteins (3,209), suggesting its multiple protective mechanisms involved in antioxidant functions, oxygen transport, immune defence, and osmotic and ionic regulation. Our results reveal the molecular and genetic basis of fish adaptation and response to hypoxia and air exposure. The data generated by this study will provide valuable resources for the genetic improvement of stress resistance and yield potential in L. crocea.

Construction of the High-Density Genetic Linkage Map and Chromosome Map of Large Yellow Croaker (Larimichthys crocea).

High-density genetic maps are essential for genome assembly, comparative genomic analysis and fine mapping of complex traits. In this study, 31,191 single nucleotide polymorphisms (SNPs) evenly distributed across the large yellow croaker (Larimichthys crocea) genome were identified using restriction-site associated DNA sequencing (RAD-seq). Among them, 10,150 high-confidence SNPs were assigned to 24 consensus linkage groups (LGs). The total length of the genetic linkage map was 5451.3 cM with an average distance of 0.54 cM between loci. This represents the densest genetic map currently reported for large yellow croaker. Using 2889 SNPs to target specific scaffolds, we assigned 533 scaffolds, comprising 421.44 Mb (62.04%) of the large yellow croaker assembled sequence, to the 24 linkage groups. The mapped assembly scaffolds in large yellow croaker were used for genome synteny analyses against the stickleback (Gasterosteus aculeatus) and medaka (Oryzias latipes). Greater synteny was observed between large yellow croaker and stickleback. This supports the hypothesis that large yellow croaker is more closely related to stickleback than to medaka. Moreover, 1274 immunity-related genes and 195 hypoxia-related genes were mapped to the 24 chromosomes of large yellow croaker. The integration of the high-resolution genetic map and the assembled sequence provides a valuable resource for fine mapping and positional cloning of quantitative trait loci associated with economically important traits in large yellow croaker.

Guanidine-Based Covalent Organic Frameworks: Cooperation between Cores and Linkers for Chromic Sensing and Efficient CO2 Conversion.

C(sp)-H carboxylation with CO2 is an attractive route of CO2 utilization and is traditionally promoted by transition metal catalysts, and organocatalysis for the conversion remains rarely explored and challenging. In this article, triaminoguanidine-derived covalent organic frameworks (COFs) were used as platforms to develop heterogeneous organocatalysts for the reaction. We demonstrated that the COFs with guanidine cores and pyrazine linkers show high catalytic performance as a result of the cooperation between cores and linkers. The core is vitally important, which is deprotonated to the guanidinato group that binds and activates CO2. The pyrazine linker collaborates with the core to activate the C(sp)-H bond through hydrogen bonding. In addition, the COFs show acid- and base-responsive chromic behaviors thanks to the amphoteric nature of the core and the auxochromic effect of the pyrazine linker. The work opens up new avenues to organocatalysts for C-H carboxylation and chromic materials for sensing and switching applications.

Analysis of the Genetic Model of the Extremely Narrow Channel Shallow Water Delta in DL-A Oilfield, Bohai Bay Basin.

In recent years, the oil and gas reserves discovered in shallow water deltas in China have continued to grow. The research on shallow water delta deposition models and depositional genesis is becoming more and more mature. In this latest discovery, a unique type of extremely narrow channel shallow water delta deposit was found at the top of the V oil group in the lower part of the Minghuazhen Formation during the Neogene period at DL-A Oilfield, located in the Bohai Bay Basin. The width of most single channels in this deposit measures between 100 and 200m, which is relatively rare and differs from existing research. To better understand this unique narrow channel shallow water delta deposit, a range of analysis methods were conducted including trace element analysis, major element analysis, grain size analysis, core observation, casting thin section observation, 3D seismic analysis, and other methods. These analyses were used to determine the sedimentary environment and sedimentary genesis of the deposit in the study area. The results show the following: (1) The top of the V oil group in the lower part of Minghuazhen Formation was deposited with a strong oxidizing environment. In the early stage, the climate was dry and cold, and gradually changed to warm and humid in the late stage. (2) Due to the frequent exposure to the surface, obvious weathered surfaces and sedimentary discontinuities were observed on the cores; the particle size analysis shows that the lamina types developed in the study area are clastic-clay laminae and clay-clastic laminae, which are mostly developed in shallow lakes area. (3) Observations of cores and thin sections also indicated that the hydrodynamic conditions frequently changed in the study area, alternating between strong and weak hydrodynamic conditions in a short period due to the alternating occurrence of flood and dry periods during the rainy season. Weak hydrodynamic conditions and slow water flow result in insufficient undercutting and sidecutting of rivers. The alternating occurrence of flood periods and dry periods has led to the development of crevasse splays and frequent river channel diversions, resulting in the inability of long-term stable development of the river channel. Besides, the change of water level has also led to the rebuilding of the river. Therefore, the multiple effects led to the formation of an extremely narrow channel shallow water delta. The accuracy of the sedimentary model is verified by a comparative study of the Shaliu River and Buha River in the modern Qinghai Lake. The new extremely narrow channels deposition model proposed this time further improves the deposition theory. At the same time, the modern depositional characteristics of the Shaliu River and Buha River also reveal the reservoir deposition between channels that cannot be distinguished by seismic data, providing guidance for the development of oil and gas in the study area.

Low levels of serum vitamin C in children with limb fractures: a case-control study.

Introduction: The role of vitamin C in pediatric fractures has not received much attention, although it is known to be a factor in osteoporotic fractures in the elderly. This case-control study aimed to investigate the changes in serum vitamin C levels among children with limb fractures. Methods: We recruited 325 children with and 316 children without limb fractures hospitalized between January 2021 and December 2021. Following admission, basic demographic data of all participants were collected, and fasting serum vitamin C levels were determined using ultra-high-performance liquid chromatography-tandem mass spectrometry. Results: The mean age of the fracture group was 5.1 years (95% CI, 4.83-5.33). The serum vitamin C levels in the fracture group (4.48 µg/ml) were significantly lower than those in the control group (8.38 µg/ml) (p < 0.0001). Further subgroup analysis of the fracture group revealed that serum vitamin C levels decreased significantly after 4 years of age and there was a significant difference in the duration after injury between <6 and >6 h (p = 0.0224). Spearman's rank correlation coefficient suggested that age and vitamin C levels were negatively correlated in the fracture group. Conclusion: In general, children with limb fractures had lower serum vitamin C levels, especially those aged 4 years and over.

Preterm Birth and Infantile Appendicitis.

OBJECTIVE: To investigate the potential association between preterm birth and infantile appendicitis. METHODS: We conducted a retrospective, multicenter, matched case-control study. This study included consecutive patients <1 year of age with surgery- or autopsy-confirmed appendicitis, admitted between December 2007 and May 2023. For each case, 10 healthy infants were randomly selected and matched by age. Infants were categorized as neonates (0 to 28 days) or older infants (>28 days and <1 year). RESULTS: The study included 106 infants diagnosed with appendicitis (median age 2.4 months) and 1060 age-matched healthy controls. In the univariate analysis, preterm birth was significantly associated with the development of appendicitis within the first year of life (odds ratio [OR], 4.23; 95% confidence interval [CI], 2.67-6.70). Other factors associated with a higher risk of infantile appendicitis included being male (OR, 1.91; 95%CI, 1.25-2.94), weight-for-age z-score (OR, 0.72; 95%CI, 0.64-0.81), and exclusively fed on formula (OR, 2.95; 95%CI, 1.77-4.91). In multivariable analyses, preterm remained significantly associated with appendicitis (adjusted OR, 3.32; 95%CI, 1.76-6.24). Subgroup analysis revealed that a preterm birth history increased the risk of appendicitis in both neonates (adjusted OR, 4.56; 95%CI, 2.14-9.71) and older infants (adjusted OR, 3.63; 95%CI, 1.72-7.65). However, preterm did not significantly influence the incidence of appendiceal perforation. CONCLUSIONS: Preterm infants have an increased risk of appendicitis during the first year of life. A preterm birth history may help improve the timely diagnosis of infantile appendicitis.

Insight into the role of crystallinity in oxide electrolytes enabling high-performance all-solid-state lithium-sulfur batteries.

All-solid-state lithium-sulfur batteries (ASSLSBs) would be a promising candidate for the next-generation batteries due to the utilization of energy-dense electrodes and the non-flammable oxide solid-state electrolytes (SSEs), but still face great challenges such as low ionic conductivity of SSEs, poor interfacial contact and lithium (Li) dendrite propagation. Herein, we regulated the crystallinity degrees of the large-scale-fabricated Li1.5Al0.5Ge1.5(PO4)3 (LAGP) SSEs and explored the critical role of crystallinity optimization in reinforcing the basic properties of LAGP, developing a fundamental explanation for the inherent relation between the crystallinity and the performance of ASSLSBs. Benefiting from the optimized crystallinity (∼99.9 %), the large-scale-fabricated LAGP not only realizes the low surface roughness and high ionic conductivity (2.11 × 10-4 S cm-1) to improve interfacial contact and reduce resistance in ASSLSBs, but also possesses the dense internal structure with low porosity (1.49 %) to physically resist dendritic propagation and penetration. Consequently, the ASSLSB with the optimized LAGP delivers a high reversible capacity of 647.9 mAh/g even after 150 cycles at 0.1 C. This work confirms the significance of crystallinity in understanding the working mechanisms of oxide SSEs and developing future high-performance ASSLSBs.

Advances in functional guest materials for resistive gas sensors.

Resistive gas sensors are considered promising candidates for gas detection, benefiting from their small size, ease of fabrication and operation convenience. The development history, performance index, device type and common host materials (metal oxide semiconductors, conductive polymers, carbon-based materials and transition metal dichalcogenides) of resistive gas sensors are firstly reviewed. This review systematically summarizes the functions, functional mechanisms, features and applications of seven kinds of guest materials (noble metals, metal heteroatoms, metal oxides, metal-organic frameworks, transition metal dichalcogenides, polymers, and multiple guest materials) used for the modification and optimization of the host materials. The introduction of guest materials enables synergistic effects and complementary advantages, introduces catalytic sites, constructs heterojunctions, promotes charge transfer, improves carrier transport, or introduces protective/sieving/enrichment layers, thereby effectively improving the sensitivity, selectivity and stability of the gas sensors. The perspectives and challenges regarding the host-guest hybrid materials-based gas sensors are also discussed.

Physical simulation study on grouting water plugging of flexible isolation layer in coal seam mining.

Deep coal seam mining often leads to water resource loss due to bedrock water entering the workings of the mine and is discharged adjacent to the mining area. Using the geological conditions of the Maiduoshan coal mine, this paper applied a physical simulation experiment. The specified rock above the coal seam was hydraulically fractured in advance to form a postmining grouted fracture network, followed by grouting to construct a flexible isolation layer that blocked the infiltration of groundwater from the aquifer into the water-conducting fracture zone. Stress sensors, flow sensors and strata displacement monitoring technology were deployed inside the experimental material to study the spatial distribution characteristics and evolution law of the water-conducting fracture zone in the overlying rocks. Analysis of the water-conducting fracture zone development law, stress variation, overburden evolution characteristics, fracturing and grouting sequence of the flexible isolation layer and the effect of postmining grouting on the water barrier was conducted. These experiments verified the feasibility of fracture and grouting of the flexible isolation layer. These research results will provide practical guidance for the transition from the current safe and efficient mining methods to safe and green mining methods of deep coal mining in the western mining areas of China.

The genome of a hadal sea cucumber reveals novel adaptive strategies to deep-sea environments.

How organisms cope with coldness and high pressure in the hadal zone remains poorly understood. Here, we sequenced and assembled the genome of hadal sea cucumber Paelopatides sp. Yap with high quality and explored its potential mechanisms for deep-sea adaptation. First, the expansion of ACOX1 for rate-limiting enzyme in the DHA synthesis pathway, increased DHA content in the phospholipid bilayer, and positive selection of EPT1 may maintain cell membrane fluidity. Second, three genes for translation initiation factors and two for ribosomal proteins underwent expansion, and three ribosomal protein genes were positively selected, which may ameliorate the protein synthesis inhibition or ribosome dissociation in the hadal zone. Third, expansion and positive selection of genes associated with stalled replication fork recovery and DNA repair suggest improvements in DNA protection. This is the first genome sequence of a hadal invertebrate. Our results provide insights into the genetic adaptations used by invertebrate in deep oceans.

Identification and expression analysis of IL-2 receptors in large yellow croaker (Larimichthys crocea).

Interleukin-2 (IL-2) signals influence various lymphocyte subsets during differentiation, immune responses and homeostasis. IL-2 acts on different cells by binding to its receptors (IL-2R), which consists of three subunits, IL-2Rα (CD25), IL-2Rß (CD122), and the common gamma chain or γc (CD132). In the present study, three IL-2 receptor subunits, designated as LcCD25-like (LcCD25L), LcIL-2Rß and Lcγc, were characterized in large yellow croaker (Larimichthys crocea). The LcCD25L, like other teleost CD25L or IL-2/IL-15Rα, contains only one sushi domain at N-terminus. The synteny of CD25L from different teleost are conserved. The deduced protein of LcIL-2Rß and Lcγc exhibits a typical class I cytokine receptors architecture, including a cytokine-binding homology domain (CHD) consisting of two fibronectin type-III (FNIII) domains (D1 and D2) and a conserved WSXWS motif in D2 domain. These three IL-2 receptor subunits were constitutively expressed in all tissues and primary immune-related cells examined. The LcCD25L was highly expressed in blood, while LcIL-2Rß and Lcγc were highly expressed in spleen and gill. For immune-related cells, LcCD25L and LcIL-2Rß were highly expressed in PKLs, while the Lcγc exhibited the highest expression in PKMs. These three IL-2 receptor subunits could be dramatically induced by T cell mitogen PHA in PKLs, which mainly composed of T and B lymphocytes. The results presented indicated that large yellow croaker IL-2R might exercise function on lymphocytes, especially on activated T cells.

Large yellow croaker peroxiredoxin IV protect cells against oxidative damage and apoptosis.

Oxidative stress and inflammation lead to cell damage and are implicated in many disease states. High concentrations of hydrogen peroxide (H2O2) may mediate cells apoptosis by increasing intracellular reactive oxygen species (ROS) levels. In this study, we established a LYCK-PrxIV cell line (large yellow croaker head kidney cell line stably expressing peroxiredoxin IV). The level of nitric oxide (NO), superoxide anion and hydrogen peroxide (H2O2) in this LYCK-PrxIV cells were significantly lower than those in control cells of LYCK-pcDNA3.1 (LYCK cell line stably transfected by pcDNA3.1 vector). Additionally, when exposed to H2O2, cell apoptosis was significantly alleviated in LYCK-PrxIV than in control cells. Meanwhile, the ROS level and ATP content were maintained more stable in LYCK-PrxIV than in LYCK-pcDNA3.1. The over-expression of LcPrxIV in LYCK-PrxIV cells induced a declined mRNA expression of LcCXC, LcCC, LcIL-8 and LcTNF-α2, as well as an increase of LcIL-10 mRNA expression, when compared to LYCK-pcDNA3.1. On the other hand, the expression of chemokine LcCXC, LcCC and LcTNF-a2 increased in LYCK-pcDNA3.1 after H2O2 stimulation, while that of LcIL-8 and LcIL-10 decreased. The regualtion of gene expression in LYCK-PrxIV cells was almost the same as that in LYCK-pcDNA3.1, but the change fold was much more moderate. These results suggest that LcPrxIV may be an indispensable ROS scavenger protecting LYCK cells against oxidative damage as well as the subsequent apoptosis and inflammatory response, which provides a clue that LcPrxIV may be an assist in fish immune response.

Functions of TNF-α1 and TNF-α2 in large yellow croaker (Larimichthys crocea) in monocyte/macrophage activation.

Tumor necrosis factor-α (TNF-α) plays crucial roles in cell development, proliferation, apoptosis, inflammation, and immunity. TNF-α genes have been identified in various fish species, however, their biological functions remain to be further clarified. In this study, we identified a novel TNF-α homologue (LcTNF-α2) from large yellow croaker (Larimichthys crocea), which shares a low amino acid sequence identity to the previously reported large yellow croaker TNF-α (LcTNF-α1). The open reading frame of LcTNF-α2 is 714 nucleotides long, encoding a peptide of 237 amino acids (aa). The deduced LcTNF-α2 protein contains a 23-aa transmembrane region, a TACE restriction site at residues T71/L72, a TNF family signature (I108- F135), and two conserved cysteine residues (C39 and C179), as found in other known TNF-α sequences. Both LcTNF-α1 and LcTNF-α2 genes were constitutively expressed in all examined tissues and significantly up-regulated in the spleen and head kidney by Vibrio alginolyticus. Their transcripts were also detected in primary head kidney monocytes/macrophages (MO/Mϕs), lymphocytes (PKLs), granulocytes (PKGs), and large yellow croaker head kidney (LYCK) cell line and significantly increased in these cell types by inactivated Vibrio alginolyticus. Recombinant LcTNF-α1 and LcTNF-α2 proteins (rLcTNF-α1 and rLcTNF-α2) produced in Pichia pastoris not only significantly increased the production of reactive oxygen species (ROS), but also promoted the expression of proinflammatory cytokines (IL-1ß, IL-6,IL-8, and TNF-α1) in MO/Mϕs from large yellow croaker. Even more, after stimulation with rLcTNF-α1 and rLcTNF-α2, the production of nitrogen oxide (NO) and the expression of inducible NO synthase (iNOS) gene were significantly up-regulated. However, only rLcTNF-α1 remarkedly enhanced the phagocytosis of MO/Mϕs and increased the expression of TNF-α2 in MO/Mϕs. These results therefore indicated that LcTNF-α1 and LcTNF-α2 both play roles in promoting activation of head kidney MO/Mϕs.