Promiscuous G-protein activation by the calcium-sensing receptor.

The human calcium-sensing receptor (CaSR) detects fluctuations in the extracellular Ca2+ concentration and maintains Ca2+ homeostasis1,2. It also mediates diverse cellular processes not associated with Ca2+ balance3-5. The functional pleiotropy of CaSR arises in part from its ability to signal through several G-protein subtypes6. We determined structures of CaSR in complex with G proteins from three different subfamilies: Gq, Gi and Gs. We found that the homodimeric CaSR of each complex couples to a single G protein through a common mode. This involves the C-terminal helix of each Gα subunit binding to a shallow pocket that is formed in one CaSR subunit by all three intracellular loops (ICL1-ICL3), an extended transmembrane helix 3 and an ordered C-terminal region. G-protein binding expands the transmembrane dimer interface, which is further stabilized by phospholipid. The restraint imposed by the receptor dimer, in combination with ICL2, enables G-protein activation by facilitating conformational transition of Gα. We identified a single Gα residue that determines Gq and Gs versus Gi selectivity. The length and flexibility of ICL2 allows CaSR to bind all three Gα subtypes, thereby conferring capacity for promiscuous G-protein coupling.

The extracellular vesicle proteomes of Sorghum bicolor and Arabidopsis thaliana are partially conserved.

Plant extracellular vesicles (EVs) are membrane-bound organelles involved mainly in intercellular communications and defense responses against pathogens. Recent studies have demonstrated the presence of proteins, nucleic acids including small RNAs, and lipids along with other metabolites in plant EVs. Here, we describe the isolation and characterization of EVs from sorghum (Sorghum bicolor). Nanoparticle tracking analysis, dynamic light scattering, and cryo-electron tomography showed the presence of a heterogeneous population of EVs isolated from the apoplastic wash of sorghum leaves. Cryo-electron microscopy revealed that EVs had a median size of 110 nm and distinct populations of vesicles with single or multiple lipid bilayers and low or high amounts of contents. The heterogeneity was further supported by data showing that only a subset of EVs that were stained with a membrane dye, Potomac Gold, were also stained with the membrane-permeant esterase-dependent dye, calcein acetoxymethyl ester. Proteomic analysis identified 437 proteins that were enriched in multiple EV isolations, with the majority of these also found in the EV proteome of Arabidopsis (Arabidopsis thaliana). These data suggest a partial conservation of EV contents and function between the monocot, sorghum, and a distantly related eudicot, Arabidopsis.

Bmp4 in Zebrafish Enhances Antiviral Innate Immunity through p38 MAPK (Mitogen-Activated Protein Kinases) Pathway.

Bone morphogenetic proteins (BMPs) are a group of structurally and functionally related signaling molecules that comprise a subfamily, belonging to the TGF-ß superfamily. Most BMPs play roles in the regulation of embryonic development, stem cell differentiation, tumor growth and some cardiovascular and cerebrovascular diseases. Although evidence is emerging for the antiviral immunity of a few BMPs, more BMPs are needed to determine whether this function is universal. Here, we identified the zebrafish bmp4 ortholog, whose expression is up-regulated through challenge with grass carp reovirus (GCRV) or its mimic poly(I:C). The overexpression of bmp4 in epithelioma papulosum cyprini (EPC) cells significantly decreased the viral titer of GCRV-infected cells. Moreover, compared to wild-type zebrafish, viral load and mortality were significantly increased in both larvae and adults of bmp4-/- mutant zebrafish infected with GCRV virus. We further demonstrated that Bmp4 promotes the phosphorylation of Tbk1 and Irf3 through the p38 MAPK pathway, thereby inducing the production of type I IFNs in response to virus infection. These data suggest that Bmp4 plays an important role in the host defense against virus infection. Our study expands the understanding of BMP protein functions and opens up new targets for the control of viral infection.

Establishment and identification of a skin cell line from Chinese tongue sole (Cynoglossus semilaevis) and analysis of the changes in its transcriptome upon LPS stimulation.

The Chinese tongue sole (Cynoglossus semilaevis) holds significant economic importance within the fishing industry along the eastern coasts of China. In recent years, the frequent outbreaks of bacterial diseases have become a common concern as the aquaculture scale expands. The majority of the diseased fish exhibit symptoms such as skin congestion, damage and skin ulceration. As the skin serves as the first line of defense against bacterial infections, establishing a skin cell line for immunological research on Chinese tongue sole's response to bacterial infection is of utmost importance. In this study, a cell line named CSS (derived from the skin of the Chinese tongue sole) was successfully established. The cells have demonstrated stability during passages and exhibit a multipolar fibroblast-like morphology. They were cultured in L-15 medium with 20% serum and have been successfully passed through 60 passages over a period of 20 months. The identification of the mitochondrial CO1 gene confirmed that the cell originated from Chinese tongue sole. The karyotype detection revealed that the cell had a chromosome number of 2n = 42. After being stored in liquid nitrogen for 15 months, the cells can maintain more than 75% viability upon recovery. After transfecting with cy3-labeled scramble siRNA and pEGFP-N3 plasmid, clear fluorescence was observed in the transfected cells. We observed that lipopolysaccharide (LPS) from Escherichia coli significantly upregulate the gene expression of various immune-related pathways at 2 h in the CSS cell line. Additionally, the differentially expressed genes showed a higher enrichment in immune-related pathways at 2 and 6 h after stimulation compared to the 24 h point. Moreover, we identified 347 genes that exhibited a gradual increase in expression during the 0-24 h stimulation period. These genes were primarily enriched in pathways related to Autophagy, GABAergic synapse, Apelin signaling and Ferroptosis. In general, the CSS cell line established in this study exhibits stable growth and can serve as a valuable tool for in vitro studies of immunology and other basic biologies of Chinese tongue sole.

Expression analysis of TRAF2­ and NCK­interacting protein kinase (TNIK) and phosphorylated TNIK in papillary thyroid carcinoma.

The aim of the present study was to evaluate the expression of TRAF2- and NCK-interacting kinase (TNIK) and the levels of the active form of TNIK, phosphorylated (p)-TNIK, in papillary thyroid carcinoma (PTC), and to identify and compare the levels of TNIK and p-TNIK among PTC, benign thyroid tumors and normal tissues. The levels of TNIK and p-TNIK were examined by reverse transcription-quantitative (RT-q)PCR and immunohistochemical analysis (IHC) in PTC, benign thyroid tumors and normal tissues, and their association with clinicopathological features was evaluated. First, analysis of the Gene Expression Profiling Interactive Analysis and The Cancer Genome Atlas datasets suggested that the mRNA expression of TNIK was markedly increased in PTC tissues compared with that in normal tissues. RT-qPCR analyses then indicated that the relative mRNA expression of TNIK in PTC tissues was 4.47±6.16, which was significantly higher than that in adjacent tissues 2.57±5.83. The IHC results suggested that the levels of TNIK and p-TNIK in PTC tissues were markedly elevated compared with those in benign thyroid tumors and normal tissues. The levels of p-TNIK in patients with PTC were significantly associated with extrathyroidal extension (χ2=4.199, P=0.040). Positive staining for TNIK was observed in 187 out of 202 (92.6%) cases in the cytoplasm, nucleus or cytomembrane of PTC cells. Among the 187 positive cases, cytoplasm expression was identified in 162 cases (86.6%), nuclear expression in 17 cases (9.1%) and cytomembrane expression in 8 cases (4.3%). Positive staining for p-TNIK was observed in 179 out of 202 (88.6%) cases in the nuclei, cytoplasm or cytomembrane of PTC cells. In the 179 p-TNIK-positive cases, localization in the nuclei plus cytoplasm was identified in 142 cases (79.3%), nuclear localization in 9 cases (5.0%), presence in the cytoplasm in 21 cases (11.7%) and cytomembrane localization in 7 cases (3.9%). Both TNIK and p-TNIK were upregulated in PTC tissues and p-TNIK was significantly associated with extrathyroidal extension. It may act as a crucial oncogene to participate in PTC carcinogenesis and progression.

RESPIRATORY VARIABILITY OF VALVULAR PEAK SYSTOLIC VELOCITY AS A NEW INDICATOR OF FLUID RESPONSIVENESS IN PATIENTS WITH SEPTIC SHOCK.

ABSTRACT: Objective: The aim of this study was to evaluate the reliability and feasibility of pulse Doppler measurements of peak velocity respiratory variability of mitral and tricuspid valve rings during systole as new dynamic indicators of fluid responsiveness in patients with septic shock. Methods: Transthoracic echocardiography (TTE) was performed to measure the respiratory variability of aortic velocity-time integral (∆VTI), respiratory variability of tricuspid annulus systolic peak velocity (∆RVS), respiratory variability of mitral annulus systolic peak velocity (∆LVS), and other related indicators. Fluid responsiveness was defined as a 10% increase in cardiac output after fluid expansion, assessed by TTE. Results: A total of 33 patients with septic shock were enrolled in this study. First, there was no significant difference in the population characteristics between the fluid responsiveness positive group (n = 17) and the fluid responsiveness negative group (n = 16) ( P > 0.05). Second, Pearson correlation test showed that ∆RVS, ∆LVS, and TAPSE with the relative increase in cardiac output after fluid expansion ( R = 0.55, P = 0.001; R = 0.40, P = 0.02; R = 0.36, P = 0.041). Third, multiple logistic regression analysis demonstrated that ∆RVS, ∆LVS, and TAPSE were significantly correlated with fluid responsiveness in patients with septic shock. Fourth, receiver operating characteristic (ROC) curve analysis revealed that ∆VTI, ∆LVS, ∆RVS, and TAPSE had good predictive ability for fluid responsiveness in patients with septic shock. The area under the curve (AUC) of ∆VTI, ∆LVS, ∆RVS, and TAPSE for predicting fluid responsiveness was 0.952, 0.802, 0.822, and 0.713, respectively. The sensitivity (Se) values were 1.00, 0.73, 0.81, and 0.83, whereas the specificity (Sp) values were 0.84, 0.91, 0.76, and 0.67, respectively. The optimal thresholds were 0.128, 0.129, 0.130, and 13.9 mm, respectively. Conclusion: Tissue Doppler ultrasound evaluation of respiratory variability of mitral and tricuspid annular peak systolic velocity could be a feasible and reliable method for the simple assessment of fluid responsiveness in patients with septic shock.

Identification and functional analysis of histone 1.2-like in red sea bream (Pagrus major).

Histone H1 acts as an essential chromatin component and participates in the formation of higher chromatin structures together with core histones. In addition, H1 also has important functions in physiological processes such as gene expression regulation, DNA repair, and the immune response. In this study, the histone homologous protein Pm-H1.2-like was identified from the transcriptome database of Pagrus major we studied previously. Conservatism of evolution was investigated by sequence alignment and phylogenetic analysis. Transcripts of Pm-H1.2-like were detected in P. major tissues. The highest expression level was found in gill and skin tissues. Consistent with the data from the transcriptome database, we observed that the expression of Pm-H1.2-like was rapidly induced in nonspecific cytotoxic cells (NCCs) infected with inactivated Vibrio anguillarum. Gene silencing of Pm-H1.2-like by RNAi significantly suppressed the expression of NK-lysin and GZMB in NCCs at 12 h after pathogen stimulation, but had no significant effect on IFN-γ expression. Next, we obtained the fusion proteins rPm-H1.2-like and rPm-H1.2-like (36-80) through prokaryotic expression. ELISA showed that rPm-H1.2-like bound to oligonucleotide (ODN) in a concentration-dependent manner, while no binding activity of rPm-H1.2-like (36-80) with ODN was observed. This study confirmed that Pm-H1.2-like actively participates in the immune response of NCCs to bacterial infection, deepening the understanding of the immune features of histone H1 in fish.

Transcriptome analysis of turbot (Scophthalmus maximus) head kidney and liver reveals immune mechanism in response to Vibrio anguillarum infection.

The diseases triggered by Vibrio anguillarum infection have created huge economic losses to the turbot (Scophthalmus maximus) farming industry. However, the immune mechanism of turbot to V. anguillarum infection has not been deeply investigated. To better understand the immune response of turbot to V. anguillarum infection, transcriptome analysis of the head kidney and liver of turbot was performed. A total of 15,948 and 11,494 differentially expressed genes (DEGs) were obtained from the turbot head kidney and liver, respectively. Transcriptome analysis revealed that the head kidney and liver of turbot have some differences in the gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis of the DEGs for the different functions of these two organs. Although there are many uncertain factors in this immune process, such as the occurrence of alternative splicing (AS) events and the differences in the protein structure of the DEGs, the NFκB signalling pathway, MKK-dependent AP-1 activation, JAK-STAT signalling pathway, the signal transmission of MHC Ⅰ and a series of DEGs including HSP90 driving NLRP3 to produce inflammatory factors (IL-1ß, IL-8, TNFα, etc.) were possible important immune response pathways for turbot to V. anguillarum infection. Overall, our research has conducted a preliminary exploration of the immune mechanism of turbot in response to V. anguillarum infection.

Improvement of Lithium Storage Performance of Silica Anode by Using Ketjen Black as Functional Conductive Agent.

In this paper, SiO2 aerogels were prepared by a sol-gel method. Using Ketjen Black (KB), Super P (SP) and Acetylene Black (AB) as a conductive agent, respectively, the effects of the structure and morphology of the three conductive agents on the electrochemical performance of SiO2 gel anode were systematically investigated and compared. The results show that KB provides far better cycling and rate performance than SP and AB for SiO2 anode electrodes, with a reversible specific capacity of 351.4 mA h g-1 at 0.2 A g-1 after 200 cycles and a stable 311.7 mA h g-1 at 1.0 A g-1 after 500 cycles. The enhanced mechanism of the lithium storage performance of SiO2-KB anode was also proposed.

Hybrid Structures of Sisal Fiber Derived Interconnected Carbon Nanosheets/MoS2/Polyaniline as Advanced Electrode Materials in Lithium-Ion Batteries.

In this work, we designed and successfully synthesized an interconnected carbon nanosheet/MoS2/polyaniline hybrid (ICN/MoS2/PANI) by combining the hydrothermal method and in situ chemical oxidative polymerization. The as-synthesized ICNs/MoS2/PANI hybrid showed a "caramel treat-like" architecture in which the sisal fiber derived ICNs were used as hosts to grow "follower-like" MoS2 nanostructures, and the PANI film was controllably grown on the surface of ICNs and MoS2. As a LIBs anode material, the ICN/MoS2/PANI electrode possesses excellent cycling performance, superior rate capability, and high reversible capacity. The reversible capacity retains 583 mA h/g after 400 cycles at a high current density of 2 A/g. The standout electrochemical performance of the ICN/MoS2/PANI electrode can be attributed to the synergistic effects of ICNs, MoS2 nanostructures, and PANI. The ICN framework can buffer the volume change of MoS2, facilitate electron transfer, and supply more lithium inset sites. The MoS2 nanostructures provide superior rate capability and reversible capacity, and the PANI coating can further buffer the volume change and facilitate electron transfer.

Spatial variations in trophic-functional patterns of periphytic ciliates and indications to water quality in coastal waters of the Yellow Sea.

To evaluate the water quality status using ecological features of the periphytic ciliate communities, a 1-year (Jan. to Dec., 2016) investigation was conducted in coastal waters of the Yellow Sea, northern China. Four trophic-functional groups (TFgrs) were recorded from a total of 141 species-abundance dataset: algivores (A); bacterivores (B); non-selectives (N); and predators (R), comprising of 65, 34, 26, and 16 species, respectively. In terms of species number, TFgr A was predominant in clean areas while TFgrs B and N were dominant in heavy polluted areas and TFgr R was dominant in slightly polluted area. The trophic-functional patterns of the periphytic ciliate communities showed a clear spatial variation within the pollution gradient. Trophic-functional trait diversity measures represented a clear increasing trend from polluted stations to the clean area regarding the pollution gradients. Multivariate correlation and best matching analysis revealed that the spatial pattern of the trophic-functional groupings were significantly shaped by environmental variable nutrients and chemical oxygen demand, alone or in combination with pH, dissolved oxygen, salinity, and transparency. Thus, we suggest that the ecological features based on the trophic-functional patterns of periphytic ciliate communities might be used for bioassessment of water quality in marine ecosystems.

Molecular cloning and expression study on Toll-like receptor 5M in turbot, Scophthalmus maximus.

Toll-like receptor 5 (TLR5) is responsible for the recognition of bacterial flagellin in mammals and play an important role in innate immunity. In the present study, a TLR5M gene was cloned from turbot, Scophthalmus maximus, and its immune responsive expression was subsequently studied in vivo. The Scophthalmus maximus (Sm)TLR5M gene is 4268 bp in length, consists of four exons and three introns and encodes a peptide of 892 amino acids (aa). The deduced protein possesses a signal peptide sequence, a leucine-rich repeat (LRR) domain composed of 23 LRR motifs, a transmembrane (TM) domain and a Toll/interleukin-1 receptor (TIR) domain. Phylogenetic analysis grouped SmTLR5M with other teleost TLR5Ms. A number of binding sites for transcription factors involved in immune response regulation were predicted in the 5'-flanking region of SmTLR5M. Quantitative real-time PCR (qPCR) analysis demonstrated that SmTLR5M mRNA was expressed ubiquitously with higher levels in head kidney and spleen. Its expression following stimulation with flagellin and lipopolysaccharide (LPS) was further tested in gills, spleen, head kidney and muscle. The maximum increases of SmTLR5M transcript levels ranged from 1.3 to 6.8-fold and appeared at 3 h to 5 day post-injection depending on different organs and stimuli. These findings suggest that SmTLR5M may play an important role in immune responses to infections with bacterial pathogens.

Cloning and expression study of an IRF4a gene and its two transcript variants in turbot, Scophthalmus maximus.

Interferon regulatory factor 4 (IRF4) is known to be involved in antiviral response as well as regulation of functional and developmental processes in lymphomyeloid cell lineages in mammals. In this study, the gene of IRF4a and its two transcript variants (named IRF4a1 and -2) were cloned from turbot, Scophthalmus maximus, the tissue distributions and in vivo immune responsive expression patterns of the two transcripts were subsequently examined. The Scophthalmus maximus (Sm)IRF4a gene is 8367 nucleotide (nt) in length, consisting of eight exons and seven introns. The SmIRF4a1 transcript is 3185 nt long, containing an open reading frame (ORF) of 1401 nt that encodes a polypeptide of 466 amino acids (aa). The SmIRF4a2 transcript is 2265 nt long and identical with the SmIRF4a1 from position 1 to 1171, containing an ORF of 1164 nt that encodes a truncated protein of 387 aa as a result of a frame shift in exon 6 which introduces a premature stop codon. The deduced aa sequence of SmIRF4a1 posses a DNA-binding domain (DBD), a nuclear localization signal (NLS), a serine-rich domain (SRD) and an IRF association domain (IAD), while SmIRF4a2 lacks the C-terminal 52 residues of the IAD and the downstream C-terminal extension, instead, they are replaced by a 8-aa segment although the three upstream domains are intact. Quantitative real-time PCR analysis revealed a broad tissue expression for both SmIRF4a1 and -2 with the former showing a significantly higher expression in all examined tissues except skin. Expressions of two transcript variants after stimulation with polyinosinic:polycytidylic acid [poly(I:C)] and turbot reddish body iridovirus (TRBIV) were tested in gills, spleen, head kidney and muscle. A two-wave of induced expression pattern was observed for both transcripts with either stimulus treatment during a 7-day time course. SmIRF4a2 responded more promptly to the stimuli and showed a higher level of inducibility in the early phase while SmIRF4a1 was strongly detected in the later phase. These data suggest an important role of SmIRF4a2 in the fast immune response under a background of SmIRF4a1-dominant antiviral response in the IRF4a system of turbot.

Cloning and expression analysis of a Toll-like receptor 21 (TLR21) gene from turbot, Scophthalmus maximus.

Toll-like receptor 21 (TLR21) is a non-mammalian TLR recognizing unmethylated CpG DNA and considered as a functional homolog of mammalian TLR9. In the present study, a TLR21 gene was cloned from turbot, Scophthalmus maximus, its immune responsive expression was subsequently studied in vivo. The turbot (Sm)TLR21 gene is an intronless gene with a length of 3527 bp and encodes a peptide of 984 amino acids. The deduced protein possesses a signal peptide sequence, a leucine-rich repeat (LRR) domain composed of 16 LRR motifs, a transmembrane (TM) region and a Toll/interleukin-1 receptor (TIR) domain. Phylogenetic analysis grouped it with other teleost TLR21s. Quantitative real-time PCR (qPCR) analysis demonstrated the constitutive expression of SmTLR21 mRNA in all twelve examined tissues with higher levels in the lymphomyeloid-rich tissues like spleen and head kidney. Further, upon stimulation with polyinosinic: polycytidylic acid [poly(I:C)], turbot reddish body iridovirus (TRBIV) and CpG oligodeoxynucleotides (CpG-ODN) 2395, the SmTLR21 mRNA expression was up-regulated in the gills, head kidney, spleen and muscle. The maximum increases of SmTLR21 transcript levels ranged from 1.3 to 8.1-fold and appeared at 3 h to 5 day post-injection depending on different organs and stimuli. These findings suggest that SmTLR21 may play an important role in the immune responses to the infections of a broad range of pathogens that include RNA and DNA viruses and bacteria.

Cloning and expression study of a Toll-like receptor 2 (tlr2) gene from turbot, Scophthalmus maximus.

Toll-like receptor 2 (TLR2) in mammals is a member of the ancient Toll-like family of receptors that predominantly recognizes conserved components of Gram-positive bacteria. In the present study, a tlr2 gene and its 5'-flanking sequence were cloned from turbot, Scophthalmus maximus, its responsive expressions to various immunostimulants were subsequently studied in vivo. The turbot (sm)tlr2 gene spans over 9.0 kb with a structure of 12 exon-11 intron and encodes 816 amino acids. The deduced protein shows the highest sequence identity (76.1%) to Japanese flounder Tlr2 and possesses a signal peptide sequence, a leucine-rich repeat (LRR) domain composed of 19 LRR motifs, a transmembrane region and a Toll/interleukin-1 receptor (TIR) domain. Phylogenetic analysis grouped it with other neoteleostei Tlr2as. A number of transcription factor binding sites known to be important for the basal transcriptional activity of TLR3 and response of TLR2 to lipopolysaccharide (LPS) signalling in mammals were predicted in the 5'-flanking sequence of smtlr2. Quantitative real-time PCR (qPCR) analysis demonstrated the constitutive expression of smtlr2 mRNA in all twelve examined tissues with higher levels in the lymphomyeloid-rich tissues and liver. Further, smtlr2 expression was up-regulated following stimulation with LPS, peptidoglycan (PGN) or polyinosinic: polycytidylic acid [poly(I:C)] in the gills, head kidney, spleen and muscle. Finally, for all three immunostimulants, a two-wave induced smtlr2 expression was observed in the head kidney and spleen in a 7-day time course and the strongest inducibility in the head kidney. These findings suggest a possible role of Smtlr2 in the immune responses to the infections of a broad range of pathogens that include Gram-positive and Gram-negative bacteria and RNA virus.

Interferon regulatory factor 4b (IRF4b) in Japanese flounder, Paralichthys olivaceus: Sequencing, ubiquitous tissue distribution and inducible expression by poly(I:C) and DNA virus.

Interferon regulatory factor 4 (IRF4) in mammals is known to be critical in regulation of development and functions of lymphomyeloid cell lineages. Recent studies have demonstrated its involvement in immune responses to bacterial and viral challenges in teleosts. In this study, an IRF4 gene was cloned from Japanese flounder (Paralichthys olivaceus) and its expression in response to polyinosinic:polycytidylic acid [poly(I:C)] and lymphocystis disease virus (LCDV) stimulations was studied in vivo. The cloned gene spans over 5.9 kb, comprises eight exons and seven introns and encodes a putative protein of 456 amino acids. The deduced amino acid sequence possesses a conserved DNA-binding domain (DBD), an IRF-association domain (IAD) and a nuclear localization signal (NLS). Phylogenetic analysis clustered it into the teleost IRF4b clade and, thus, it was named Paralichthys olivaceus (Po)IRF4b. The constitutive expression of PoIRF4b transcripts was detectable in all examined organs, with highest levels found in lymphomyeloid-rich tissues. They were induced by both poly(I:C) and LCDV with a similar inducibility in immune or non-immune organs. Two waves of induced expression of PoIRF4b were observed with the two stimuli during a 7-day time course in the immune organs, with the early-phase induction being stronger. The maximum increases of PoIRF4b transcript levels ranged from 1.3 to 4.0-fold and appeared at day 1-5 post-injection depending on different organs and stimuli. In both stimulation cases, the strongest induction was detected in spleen and the weakest in muscle. These results indicate that PoIRF4b may participate in regulation of immune responses of flounders to both RNA and DNA virus infections.

Molecular cloning and expression analysis of interferon stimulated gene 15 (ISG15) in turbot, Scophthalmus maximus.

The interferon stimulated gene 15 (ISG15) is strongly induced in many cell types by double-stranded RNA (polyinosinic: polycytidylic acid, poly I:C) and viral infection. In this study, we described the nucleotide, mRNA tissue distribution and regulation of an ISG15 gene from turbot, Scophthalmus maximus (SmISG15). SmISG15 gene is 862 bp in length, composed of two exons and one intron, and encodes 158 amino acids. The deduced protein exhibits the highest homology (44.7-71.2% identity) with ISG15s from other fishes and possesses two conserved tandem ubiquitin-like (UBL) domains and a C-terminal RLRGG conjugating motif known to be important for the functions of ISG15s in vertebrates. Phylogenetic analysis grouped SmISG15 into fish ISG15. SmISG15 mRNA was constitutively expressed in all tissues examined, with higher levels observed in immune organs. Gene expression analysis was performed for SmISG15 in the spleen, head kidney, gills and muscle of turbots challenged with poly I:C or turbot reddish body iridovirus (TRBIV) over a 7-day time course. The result showed that SmISG15 was upregulated by both stimuli in all four tissues, with induction by poly I:C apparently stronger and initiated more quickly. A two-wave induced expression of SmISG15 was seen in the spleen, head kidney and gills, suggesting an induction of SmISG15 either by IFN-dependent or -independent pathway. These results provide insights into the roles of fish ISG15 in antiviral immunity.

Molecular cloning and expression studies of the adapter molecule myeloid differentiation factor 88 (MyD88) in turbot (Scophthalmus maximus).

Myeloid differentiation factor 88 (MyD88) is an adapter protein involved in the interleukin-1 receptor (IL-1R) and Toll-like receptor (TLR)-mediated activation of nuclear factor-kappaB (NF-κB). In this study, a full length cDNA of MyD88 was cloned from turbot, Scophthalmus maximus. It is 1619 bp in length and contains an 858-bp open reading frame that encodes a peptide of 285 amino acid residues. The putative turbot (Sm)MyD88 protein possesses a N-terminal death domain and a C-terminal Toll/IL-1 receptor (TIR) domain known to be important for the functions of MyD88 in mammals. Phylogenetic analysis grouped SmMyD88 with other fish MyD88s. SmMyD88 mRNA was ubiquitously expressed in all examined tissues of healthy turbots, with higher levels observed in immune-relevant organs. To explore the role of SmMyD88, its gene expression profile in response to stimulation of lipopolysaccharide (LPS), CpG oligodeoxynucleotide (CpG-ODN) or turbot reddish body iridovirus (TRBIV) was studied in the head kidney, spleen, gills and muscle over a 7-day time course. The results showed an up-regulation of SmMyD88 transcript levels by the three immunostimulants in all four examined tissues, with the induction by CpG-ODN strongest and initiated earliest and inducibility in the muscle very weak. Additionally, TRBIV challenge resulted in a quite high level of SmMyD88 expression in the spleen, whereas the two synthetic immunostimulants induced the higher levels in the head kidney. These data provide insights into the roles of SmMyD88 in the TLR/IL-1R signaling pathway of the innate immune system in turbot.

Cloning and expression analysis of a Toll-like receptor 22 (tlr22) gene from turbot, Scophthalmus maximus.

Toll-like receptor 22 (TLR22) exists exclusively in aquatic animals and recognizes double stranded RNA (dsRNA). In the present study, a tlr22 gene and its 5'-flanking sequence were cloned from turbot, Scophthalmus maximus, its immune responsive expression was subsequently studied in vivo. The turbot (sm)tlr22 gene spans over 5.6 kb with a structure of 4 exon-3 intron and encodes 962 amino acids. The deduced protein shows the highest sequence identity (76.7%) to Japanese flounder Tlr22 and possesses a signal peptide sequence, a leucine-rich repeat (LRR) domain composed of 27 LRR motifs, a transmembrane region and a Toll/interleukin-1 receptor (TIR) domain. Phylogenetic analysis grouped it with other teleost Tlr22s. The interferon-stimulated response element (ISRE) and signal transducer and activator of transcription (STAT) binding site important for the basal transcriptional activity of TLR3 were predicted in the 5'-flanking sequence of smtlr22 gene. Quantitative real-time PCR (qPCR) analysis demonstrated the constitutive expression of smtlr22 mRNA in all examined tissues with higher levels in the head kidney, kidney and spleen. Further, smtlr22 expression was significantly up-regulated following challenge with polyinosinic: polycytidylic acid (poly I:C), lipopolysaccharide (LPS) or turbot reddish body iridovirus (TRBIV) in the gills, head kidney, spleen and muscle, with maximum increases ranging from 2.56 to 6.24 fold upon different immunostimulants and organs. These findings suggest a possible role of Smtlr22 in the immune responses to the infections of a broad range of pathogens that include DNA and RNA viruses and Gram-negative bacteria.

Whole cell cryo-electron tomography suggests mitochondria divide by budding.

Eukaryotes rely on mitochondrial division to guarantee that each new generation of cells acquires an adequate number of mitochondria. Mitochondrial division has long been thought to occur by binary fission and, more recently, evidence has supported the idea that binary fission is mediated by dynamin-related protein (Drp1) and the endoplasmic reticulum. However, studies to date have depended on fluorescence microscopy and conventional electron microscopy. Here, we utilize whole cell cryo-electron tomography to visualize mitochondrial division in frozen hydrated intact HeLa cells. We observe a large number of relatively small mitochondria protruding from and connected to large mitochondria or mitochondrial networks. Therefore, this study provides evidence that mitochondria divide by budding.