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Antarctic organisms are consistently suffering from multiple environmental pressures, especially the strong UV radiation caused by the loss of the ozone layer. The mosses and lichens dominate the vegetation of the Antarctic continent, which grow and propagate in these harsh environments. However, the molecular mechanisms and related regulatory networks of these Antarctic plants against UV-B radiation are largely unknown. Here, we used an integrated multi-omics approach to study the regulatory mechanism of long non-coding RNAs (lncRNAs) of an Antarctic moss (Pohlia nutans) in response to UV-B radiation. We identified a total of 5729 lncRNA sequences by transcriptome sequencing, including 1459 differentially expressed lncRNAs (DELs). Through functional annotation, we found that the target genes of DELs were significantly enriched in plant-pathogen interaction and the flavonoid synthesis pathway. In addition, a total of 451 metabolites were detected by metabonomic analysis, and 97 differentially change metabolites (DCMs) were found. Flavonoids account for 20% of the total significantly up-regulated metabolites. In addition, the comprehensive transcriptome and metabolome analyses revealed the co-expression pattern of DELs and DCMs of flavonoids. Our results provide insights into the regulatory network of lncRNA under UV-B radiation and the adaptation of Antarctic moss to the polar environments.

Persistent organic pollutants (POPs) have received significant and ongoing attention. To establish favorable regulatory policies, it is vital to investigate the occurrence, source, and budgets of POPs worldwide. POPs including phthalic acid esters (PAEs), organophosphate esters (OPEs), brominated flame retardants (BFRs), and highly chlorinated flame retardants (HFRs) have not yet been examined in the Eastern Indian Ocean (EIO). In this study, the distribution of POPs has been investigated from surface sediments with the depth of 4369-5742 m in the Central Indian Ocean Basin (CIOB) and Wharton Basin (WB) of EIO. The average (±SD) concentrations of ∑

12-oxo-phytodienoic acid (OPDA) is a biosynthetic precursor of jasmonic acid and triggers multiple biological processes from plant development to stress responses. However, the OPDA signaling and relevant regulatory networks were largely unknown in basal land plants. Using an integrated multi-omics technique, we investigated the global features in metabolites and transcriptional profiles of an Antarctic moss (

Gadus macrocephalus (Pacific cod) is an economically important species on the northern coast of the Pacific. Although numerous studies on G. macrocephalus exist, there are few reports on its genomic data. Here, we used whole-genome sequencing data to elucidate the genomic characteristics and phylogenetic relationship of G. macrocephalus. From the 19-mer frequency distribution, the genome size was estimated to be 658.22 Mb. The heterozygosity, repetitive sequence content and GC content were approximately 0.62%, 27.50% and 44.73%, respectively. The draft genome sequences were initially assembled, yielding a total of 500,760 scaffolds (N50 = 3565 bp). A total of 789,860 microsatellite motifs were identified from the genomic data, and dinucleotide repeat was the most dominant simple sequence repeat motif. As a byproduct of whole-genome sequencing, the mitochondrial genome was assembled to investigate the evolutionary relationships between G. macrocephalus and its relatives. On the basis of 13 protein-coding gene sequences of the mitochondrial genome of Gadidae species, the maximum likelihood phylogenetic tree showed that complicated relationships and divergence times among Gadidae species. Demographic history analysis revealed changes in the G. macrocephalus population during the Pleistocene by using the pairwise sequentially Markovian coalescent model. These findings supplement the genomic data of G. macrocephalus, and make a valuable contribution to the whole-genome studies on G. macrocephalus.

The Antarctic continent has extreme natural environment and fragile ecosystem. Mosses are one of the dominant floras in the Antarctic continent. However, their genomic features and adaptation processes to extreme environments remain poorly understood. Here, we assembled the high-quality genome sequence of the Antarctic moss (

An additive- and pollution-free method for the preparation of biogenic silver and silver chloride nanoparticles (Ag@AgCl NPs) was developed from the bacteria Shewanella sp. Arc9-LZ, which was isolated from the deep sea of the Arctic Ocean. The optimal synthesizing conditions were explored, including light, pH, Ag+ concentration and time. The nanoparticles were studied by means of ultraviolet-visible (UV-Vis) spectrophotometry, energy dispersive spectrometry (EDS), X-ray diffraction (XRD) and inductively coupled plasma optical emission spectrometers (ICP-OES). The transmission electron microscope (TEM) showed that the nanoparticles were spherical and well dispersed, with particle sizes less than 20.00 nm. With Ag@AgCl nanoparticles, the kinetic rate constants for congo red (CR) and rhodamine B (RhB) dye degradation were 2.74 × 10−1 min−1 and 7.78 × 10−1 min−1, respectively. The maximum decolourization efficiencies of CR and RhB were 93.36% and 99.52%, respectively. Ag@AgCl nanoparticles also showed high antibacterial activities against the Gram-positive and Gram-negative bacteria. The Fourier transform infrared spectroscopy (FTIR) spectrum indicated that the O-H, N-H and -COO- groups in the supernatant of Arc9-LZ might participate in the reduction, stabilization and capping of nanoparticles. We mapped the schematic diagram on possible mechanisms for synthesizing Ag@AgCl NPs.

The δ- and λ-variants of COVID-19 are blowing to globle economy, human life and health. The variants put the world in a harsher state. Lockdown is powerful in stopping the spread and infection. We evaluate the lockdown impact on NO2, SO2, O3, PM2.5 and PM10 in Wuhan, which reported the first COVID-19 case. Data before, during and after blokade in year 2020 were all analyzed. Lockdown significantly decreased four of the five pollutants. The decreasing reasons are discussed in social policies, people's living habits and Chinese characters. Wuhan is a transportation hub in central China. Viruses spread through it to all over the country. Although COVID-19 mainly bring bad social effects, people get chance to rethink the value of life: We should sustainablely develop and pursuing spiritual instead of excessive material enjoyment. These ultimately built more stable societies and benefit mankind itself.

The δ -variant of COVID-19 is blowing to globle economy, hummen’s life and health. The infection rates of δ -variant is twice of the original variants. It is putting the world in a harsher state. Lockdown policy is still powerful in stopping spread of virus. This paper aims to evaluate the lockdown impact on NO 2 , SO 2 , O 3 , PM 2.5 and PM 10 in Wuhan, which reported the first OVID-19 infection case. We analyzed data of the whole periods including before, during and after blokade. Generally, lockdown has significant decreasing effects on all the 5 pollutants except for surface ozone. The reasons for the pollutants’ changes are discussed from the aspects including social policy, people's living habits and Chinese character. To assess the extensive impact of lockdown policy on urban environment, we also studied the same pollutants in Shanghai, which is one of the biggest and central city in China and almost locates at the same latitude with Wuhan. Better results were obtained compared with those of Wuhan with all the pollutants decreasing their concentration and a better air quality. This effects even last after the blockade. This is mainly because Shanghainese have formed more prudent living habits and attach more importance to environmental protection during the lockdown period. Althgough COVID-19 brings bad effect on globle socio-economy and humen’s health, people get chance to rethink the value of life: We should sustainablely develop and pursuing spiritual instead of excessive material enjoyment. It can sustain a healthy living enviroment.

Three new cyclic lipopeptides (compounds 1–3) were isolated from Bacillus sp. SY27F, which collected from the deep-sea hydrothermal vent in the Indian Ocean. The planar structures of compounds 1–3 were characterized by LC–MS, GC–MS and NMR, whereas their absolute configurations were determined by modified Marfey’s analysis and optical rotation contrast. They shared the same heptapeptide chain (N-L-Gln1-D-Leu2-L-Leu3-L-Val4-L-Asp5-D-Leu6-L-Ile7-C), differing only by the length of the fatty acid chain. Compounds 1–3 displayed significant cytotoxic activity against DU145, MCF-7 and HepG2 cancer cell lines with IC50 values of 96.8–190.7 nM and moderate to weak cytotoxicity against A549, HeLa and HT1080, with IC50 values of 9.3–43.2 μM, respectively. However, none of the three compounds showed any antibacterial activity against Escherichia coli, Staphylococcus aureus and Monilia albicans.

Leukocyte cell-derived chemotaxin 2 (LECT2) is a multi-functional protein that is mainly synthesized by the liver. However, its role in roughskin scalping is less known. Here, we cloned a leukocyte cell-derived chemotaxin 2 (TfLECT2) genes in the liver of roughskin scalping, Trachidermus fasciatus, and studied its possible role involved in the immune response against Vibrio anguillarum (V. anguillarum) of peritoneal lecukocytes under in vivo conditions. The cDNA sequence of TfLECT2 is 566 bp in size. Its deduced amino acid (aa) sequence comprises 151 residues, of which the first 16 residues form a putative signal peptide and 101 residues compose a typical peptidase M23 domain in the C-terminal region. The domain structure is conserved in all LECT2 proteins, which suggests a close phylogenetic relationship between TfLECT2 and LECT2 in other fish species. Real-time quantitative PCR analysis revealed that TfLECT2 gene expression was dramatically increased in liver after V. anguillarum stimulation. Subsequently, TfLECT2 was prokaryotic expressed and purified to prepare anti-TfLECT2 antibody. After V. anguillarum challenge, leukocytes recruitment and LECT2 levels in peritoneal exudates were increased, and positively correlated with each other. Moreover, recombinant TfLECT2 administration significantly improved immune responses after infection, principally in stimulating the recruitment, phagocytosis and respiratory burst of leukocytes at the site of infection; however, anti-TfLECT2 treatment neutralized these abilities. Therefore, TfLECT2 may trigger the early immune events of peritoneal leukocytes and it will be useful to induce innate immune response of fish.

COVID-19 and its variants have been changing the world. The spread of variants brings severe effects to the global economy and to human’s lives and health, as well as to society. Lockdown is proven to be effective in stopping the spread. It also provides a chance to study natural environmental changes with humanity’s limited interference. This paper aims to evaluate the impact of lockdown on five major airborne pollutants, i.e., NO2, SO2, O3, PM2.5 and PM10, in the three different functional regions of Chongming, Xuhui and Jinshan of Shanghai. Changes in the same pollutants from the three regions over the same/different periods were all studied and compared. Overall, the COVID-19 lockdown has changed pollutant concentrations in the long and short terms. Concentrations of four pollutants decreased, except for that of earth surface O3, which increased. SO2 had significant correlations with all other pollutants. PM2.5 and PM10 are more externally input than locally produced. NO2, SO2 and PM levels sharply reduced in Jinshan and Xuhui due to the limited usage of fossil fuel. Lockdown improved the air quality. People now have a chance to rethink the value of life and the harmony between economic progress and environmental protection. This is helpful to establish sustainable societies.

Background With the growing demand for fossil fuels and the severe energy crisis, lignocellulose is widely regarded as a promising cost-effective renewable resource for ethanol production, and the use of lignocellulose residues as raw material is remarkable. Polar organisms have important value in scientific research and development for their novelty, uniqueness and diversity. Results In this study, a fungus Aspergillus sydowii MS-19, with the potential for lignocellulose degradation was screened out and isolated from an Antarctic region. The growth profile of Aspergillus sydowii MS-19 was measured, revealing that Aspergillus sydowii MS-19 could utilize lignin as a sole carbon source. Its ability to synthesize low-temperature lignin peroxidase (Lip) and manganese peroxidase (Mnp) enzymes was verified, and the properties of these enzymes were also investigated. High-throughput sequencing was employed to identify and characterize the transcriptome of Aspergillus sydowii MS-19. Carbohydrate-Active Enzymes (CAZyme)-annotated genes in Aspergillus sydowii MS-19 were compared with those in the brown-rot fungus representative species, Postia placenta and Penicillium decumbens. There were 701CAZymes annotated in Aspergillus sydowii MS-19, including 17 cellulases and 19 feruloyl esterases related to lignocellulose-degradation. Remarkably, one sequence annotated as laccase was obtained, which can degrade lignin. Three peroxidase sequences sharing a similar structure with typical lignin peroxidase and manganese peroxidase were also found and annotated as haem-binding peroxidase, glutathione peroxidase and catalase-peroxidase. Conclusions In this study, the fungus Aspergillus sydowii MS-19 was isolated and shown to synthesize low-temperature lignin-degrading enzymes: lignin peroxidase (Lip) and manganese peroxidase (Mnp). These findings provide useful information to improve our understanding of low-temperature lignocellulosic enzyme production by polar microorganisms and to facilitate research and applications of the novel Antarctic Aspergillus sydowii strain MS-19 as a potential lignocellulosic enzyme source. Electronic supplementary material The online version of this article (doi:10.1186/s12866-017-1028-0) contains supplementary material, which is available to authorized users.

Potassium ion channels are one of the most diversely and widely distributed channels, which are involved in all kinds of physiological functions in both excitable and non-excitable cells. The expression of voltage-gated potassium ion (Kv) channels is highly variable according to the state of macrophages activation. Macrophages have an important function in innate immunity against intruding pathogens. They produce a variety of inflammatory and immunoactive molecules that modulate imflammatory responses. Here we show that blockade of K+ channels by non-selective Kv channel inhibitor tetraethylammonium chloride (TEA), and 4-aminopyridine (4-AP) inhibited proinflammatory cytokines expression, cell proliferation, and reactive oxygen species (ROS) production in LPS-stimulated macrophages of Sea perch (Lateolabrax japonicas). Then we isolated four Kv channels genes (spKv1.1, spKv1.2, spKv1.5 and spKv3.1) in LPS-activated fish macrophages. These channels genes were up-regulated after LPS stimulation except spKv3.1, which remained unchanged during the test. The results of this study indicate that Kv channels could be required for modulating the immune function of fish macrophages.

Plant leucine-rich repeats receptor-like kinases (LRR-RLKs) play key roles in plant growth, development, and responses to environmental stresses. However, the functions of LRR-RLKs in bryophytes are still not well documented. Here, a putative LRR-RLK gene, PnLRR-RLK, was cloned and characterized from the Antarctic moss Pohlia nutans. Phylogenetic analysis revealed that PnLRR-RLK protein was clustered with the Arabidopsis thaliana LRR XI family proteins. Subcellular localization analysis of PnLRR-RLK revealed that it was mainly localized on plasma membrane. The expression of PnLRR-RLK was induced by mock high salinity, cold, drought, and exogenously supplied abscisic acid (ABA) and methyl jasmonate (MeJA). Meanwhile, the overexpression of PnLRR-RLK showed an increased tolerance of transgenic Arabidopsis to salt and ABA stresses than that of the wild type (WT) plants. Furthermore, the expression levels of several salt tolerance genes (AtHKT1, AtSOS3, AtP5CS1, and AtADH1) and an ABA negatively regulating gene AtABI1 were significantly increased in transgenic plants. Meanwhile, the expression levels of ABA biosynthesis genes (AtNCED3, AtABA1, and AtAAO3) and ABA early response genes (AtMYB2, AtRD22, AtRD29A, and AtDREB2A) were decreased in transgenic Arabidopsis after salt stress treatment. Therefore, these results suggested that PnLRR-RLK might involve in regulating salt stress-related and ABA-dependent signaling pathway, thereby contribute to the salinity tolerance of the Antarctic moss P. nutans.

Dimethyl phthalate (DMP) is a widespread environmental contaminant that poses potential toxicity risks for animals and humans. However, the toxicological effects of DMP on fish have not been adequately examined. In this study, the acute toxicity, oxidative damage, antioxidant enzyme activities, and relative gene expression patterns were investigated in the liver of adult zebrafish (Danio rerio) exposed to DMP. We found that the lethal concentration (LC50) of DMP for zebrafish after 96 h of exposure was 45.8 mg/L. The zebrafish that were exposed to low, medium and high concentrations of DMP (0.5, 4.6, and 22.9 mg/L, respectively) for 96 h had an increased malondialdehyde (MDA) content and a lower antioxidant capacity compared with the control solvent group. The total superoxide dismutase (SOD) activity was significantly higher than 0 h after initial exposure for 24 h at low concentrations, and then decreased at high concentrations after exposure for 96 h. The catalase (CAT) and glutathione S-transferase (GST) activities were significantly reduced after 96 h of exposure to high concentrations of DMP, with the up- or down-regulation of the related transcriptional expression. These findings indicated that DMP could cause physiological effects in zebrafish by disturbing the expression levels of antioxidant enzymes. These results might contribute to the identification of biomarkers to monitor phthalate pollution.

Annotated unigenes associated with carbohydrate metabolism and xenobiotic biodegradation. Annotated unigenes, pathways and corresponding members number associated with carbohydrate metabolism and xenobiotic biodegradation after the CAZyme annotation of A. sydowii MS-19 transcriptome. (DOCX 18Â kb)

Phylogeny of Antarctic fungal isolates. Neighbour-joining tree showing the relationship between the ITS sequences from 15 Antarctic native isolates and their closest relatives as well as common fungi in Antarctic. The bootstrap values of the neighbor-joining analysis with 1000 replications are shown on the branches. The scale bar represents 0.05 substitutions per amino acid site. The isolated strains can be classified into 4 classes: Eurotiomycetes, Leotiomycetes, Sordariomycetes and Dothideomycetes. All the 18S/ITS sequences of isolated fungus have been submitted to GenBank and the GenBank accession No. can be available in the brackets. (PPTX 76Â kb)

Plant receptor-like kinases (RLKs) are a group of conserved signaling components that regulate developmental programs and responses to biotic and abiotic stresses. However, only limited information is available on the functions of RLKs in bryophytes. Here, we described the isolation and functional characterization of a RLK gene (PnRLK-1) from the Antarctic moss Pohlia nutans. Sequence alignment and phylogenetic analysis showed that PnRLK-1 was homologous to the cytoplasmic-type RLKs. In addition, subcellular localization analysis in which the transient expression of PnRLK-1-green fluorescent protein was observed in Arabidopsis thaliana mesophyll protoplasts revealed that PnRLK-1 was localized mainly in cytoplasm and a little on plasma membrane. Real-time PCR analysis showed that PnRLK-1 transcriptions were upregulated by mock cold, drought, high salinity, and exogenously supplied abscisic acid (ABA) or methyl jasmonate (MeJA). Overexpression of PnRLK-1 in transgenic Arabidopsis resulted in enhanced plant tolerance to high salinity and H2O2 stresses, and increased plant sensitivity to ABA. Furthermore, the expression levels of stress-responsive genes in transgenic Arabidopsis were also upregulated, including the genes of ABA-dependent signaling pathway (AtABI4, AtABI5, and AtMYB2) and reactive oxygen species (ROS)-scavenging enzymes (AtAPX1, AtZAT10, and AtCAT1). Therefore, we proposed that PnRLK-1 was a novel RLK involving in the adaptation of the Antarctic moss to the polar environment, increasing the salt and oxidative tolerance as well as the ABA sensitivity.

Under stress conditions, the mitochondrial respiratory chain generates reactive oxygen species that cause oxidative damage to lipids, proteins and nucleic acids. Mitochondrial chaperones are essential for repairing denatured proteins in plants confronted with diverse stresses. However, their functions have not been well characterized in lower plants such as bryophytes. Here, we cloned a heat-shock 70 protein gene (designated PnHSP70) from the Antarctic moss Pohlia nutans and investigated its transcription levels under stress conditions. The PnHSP70 cDNA encoded a polypeptide of 678 amino acids with an ATPase domain, a substrate peptide-binding domain and a C-terminal domain. Sequence alignment and phylogenetic analysis showed that PnHSP70 was homologous to other known mitochondrial HSP70 proteins. In addition, subcellular localization analysis in which the transient expression of PnHSP70-green fluorescent protein was observed in Arabidopsis thaliana mesophyll protoplasts revealed that PnHSP70 was targeted to mitochondria. Real-time PCR analysis showed that low or high temperature, salinity, drought or PEG treatments as well as UV-B radiation could induce up-regulation of PnHSP70 expression levels. The plant hormones abscisic acid and jasmonic acid also up-regulated the expression levels of PnHSP70. Together, our results revealed that PnHSP70, as a mitochondrial chaperone, might play an important role in the adaptation of the Antarctic moss P. nutans to the polar environment.

Bacilohydrin A, a new cyclic lipopeptide composed of seven mixed D/L-amino acids (L-Ile1—L-Leu2—D-Asp3—L-Val4—L-Leu5—L-Leu6—L-Gln7) and a ( R)-3-hydroxy-12-methyltetradecanoic acid (HMT) residue, was isolated from the crude culture extract of Bacillus sp. SY27F, which was obtained from the Indian Ocean hydrothermal vent. Its planar structure was elucidated by spectroscopic analyses, including 1D and 2D NMR spectroscopy, mass spectrometry (MS), and secondary ion mass spectrometry (MS/MS), whereas its absolute configuration was determined by modified Marfey's analysis. Bacilohydrin A displayed significant cytotoxicities against DU-145, MCF-7 and HepG2 cancer cell lines with IC50 values of 50.3-175.1 nM. Additionally, this is the first time to determine the absolute configuration of a D-aspartic acid residue in surfactin class.

Genome-wide characterization of the Pohlia nutans transcriptome is essential for clarifying the role of stress-relevant genes in Antarctic moss adapting to the extreme polar environment. High-throughput Illumina sequencing was used to analyze the gene expression profile of P. nutans after cold treatment. A total of 93,488 unigenes, with an average length of 405 bp, were obtained. Gene annotation showed that 16,781 unigenes had significant similarity to known functional protein-coding genes, most of which were annotated using the GO, KOG and KEGG pathway databases. Global profiling of the differentially expressed genes revealed that 3,796 unigenes were significantly upregulated after cold treatment, while 1,405 unigenes were significantly downregulated. In addition, 816 receptor-like kinases and 1,309 transcription factors were identified from P. nutans. This overall survey of transcripts and stress-relevant genes can contribute to understanding the stress-resistance mechanism of Antarctic moss and will accelerate the practical exploitation of the genetic resources for this organism.

Potassium ion channels are one of the most diversely and widely distributed channels, which are involved in all kinds of physiological functions in both excitable and non-excitable cells. The expression of voltage-gated potassium ion (Kv) channels is highly variable according to the state of macrophages activation. Macrophages have an important function in innate immunity against intruding pathogens. They produce a variety of inflammatory and immunoactive molecules that modulate imflammatory responses. Here we show that blockade of K

Voltage-gated potassium (Kv) channels on cell plasma membrane play an important role in both excitable cells and non-excitable cells and Kv1 subfamily is most extensively studied channel in mammalian cells. Recently, this potassium channel was reported to control processes inside mammalian T lymphocytes such as cell proliferation and volume regulation. Little is known about Kv1 channels in fish. We have postulated the presence of such a channel in lymphocytes and speculated its potential role in immunoregulation in fish. Employing specific primers and RNA template, we cloned a segment of a novel gene from sea perch blood sample and subsequently obtained a full cDNA sequence using RACE approach. Bioinformatic analysis revealed structural and phylogenetic characteristics of a novel Kv channel gene, designated as spKv1.3, which exhibits homologous domains to the members of Kv1.3 family, but it differs notably from some other members of that family at the carboxyl terminus. Full-length of spKv1.3 cDNA is 2152 bp with a 1440 bp open reading frame encoding a protein of 480 amino acids. SpKv1.3 gene is expressed in all of the tested organs and tissues of sea perch. To assess the postulated immune function of spKv1.3, we stimulated lymphocytes with LPS and/or channel blocker 4-AP. Expression levels of messenger RNA (mRNA) of spKv1.3 under stimulation conditions were measured by quantitative RT-PCR. The results showed that LPS can motivate the up-regulation of spKv1.3 expression significantly. Interestingly, we found for the first time that 4-AP with LPS can also increase the spKv1.3 mRNA expression levels in time course. Although 4-AP could block potassium channels physically, we speculated that its effect on blockage of potassium channel may start up an alternative mechanism which feed back and evoke the spKv1.3 mRNA expression.

To study the molecular mechanism of the Antarctic sea–ice alga in adaptation to polar sea–ice environments, the RNA was prepared for cDNA library construction of Chlamydomonas sp. ICE-L. Three different methods were tested to prepare total RNA from this psychrophilic, unicellular green alga rich in protein and polysaccharide. Lauryl sodium sulfate- based method allowed a most effective extraction of high-quality total RNA compared to the other methods. Total RNA extracted with this protocol was used for cDNA library construction. The recombination rate of constructed cDNA library was 98.60%, the primary titer was 7.15 × 106 pfu, and an average sequence length was 1.2 kb. These results show that with a high-quality RNA preparation, a cDNA library can be constructed successfully for Chlamydomonas sp. ICE-L.

In order to provide pertinent evidence of ion channel with immune response in the fish, whole cell patch-clamp technique was employed for potassium ion channel study in turbot (Scophthalmus maximus). Lymphocytes were isolated by Percoll density gradient centrifugation from peripheral blood samples, and electrophysiological characters of the channel were analyzed. In the recorded cells, activated voltage of the channels was −42.5±3.7 mV and the average peak current was 313.12±28.2 pA. The channel was identified as voltage dependent, the current was outward and it could be inhibited by 10 mmol/dm3 TEA or 5 mmol/dm3 4-AP, a specific potassium channel inhibitor, identifying the existence of potassium channel in peripheral lymphocytes of the turbot.

Whole-cell patch-clamp techniques were used to study ion channels of a marine alga. High quality protoplasts suitable for electrophysiological studies were isolated from the green marine alga, Ulva pertusa, using enzyme mixtures consisting of cellulase and abalone power and identified by calcofluor fluorescence. The vitality of protoplasts varied depending on the alga growth stage, and those isolated from younger tissue in March maintained a high vitality with high sealing success rate compared with protoplasts isolated from mature or non-growing plants in August or November. In the whole-cell configuration, large inward currents were elicited by negative voltage pulses. The voltage-dependent component was predominantly carried by Cl−, as confirmed by the use of the Cl− channel inhibitor DIDS and reversal potential of current-voltage plots. This evidence suggests that hyperpolarization-activated Cl− permeable channels are responsible for the influx of Cl− into U. pertusa cells. Voltage-dependent outward currents were also recorded in several protoplasts, and their properties need further investigation.

The present study was initiated to provide a systematic protocol for producing protoplasts from the Antarctic sea ice alga Chlamydomonas sp. ICE-L suitable for physiological studies. The results showed that the mixtures of 3.0% Cellulase R-10 and 2.0% Macerozyme R-10 were most effective for isolating protoplasts from this alga. Optimum pH and temperature for hydrolytic enzyme reaction were pH 6.0 and 15∘C, respectively. Mannitol and sorbitol were found to be the excellent osmotic stabilizers. Growth conditions of the algae prior to enzyme treatment also influenced the yield of protoplasts greatly. At the optimized condition, protoplast production was 47.8%, and the viability of isolated protoplasts was more than 97.6% as confirmed by Evan's blue staining.

Vibrio anguillarum is a major pathogen threatening the fish aquaculture in China. Infection of cultivated turbot (Scophthalmus maximus) with V. anguillarum induced rapid synthesis and secretion of IL-1beta, which initiates the innate immune response. SNARE proteins are known to regulate vesicular trafficking and fusion in all eukaryotes. Here, we determined whether SNARE proteins, specifically vesicle-associated membrane protein-2 (VAMP-2), are involved in regulated exocytosis of IL-1beta of leukocytes in marine fish. We show that VAMP-2 is present in turbot blood leukocytes, with nucleotide sequence identity of 88.2% and 93.0% to those of zebra fish and sea bass, respectively. After Vibrio infection, turbot leukocyte VAMP-2 was increased at the levels of transcription and translation in a temporal pattern coinciding with leukocyte IL-1beta secretion. Confocal microscopy localized VAMP-2 to vesicle structures in leukocytes. Taken together, our results suggest that VAMP-2 is involved in regulated exocytosis of cytokines in immunocytes in fish.

Antarctic ice alga Chlamydomonas sp. ICE-L can endure extreme low temperature and high salinity stress under freezing conditions. To elucidate the molecular acclimation mechanisms using gene expression analysis, the expression stabilities of ten housekeeping genes of Chlamydomonas sp. ICE-L during freezing stress were analyzed. Some discrepancies were detected in the ranking of the candidate reference genes between geNorm and NormFinder programs, but there was substantial agreement between the groups of genes with the most and the least stable expression. RPL19 was ranked as the best candidate reference genes. Pairwise variation (V) analysis indicated the combination of two reference genes was sufficient for qRT-PCR data normalization under the experimental conditions. Considering the co-regulation between RPL19 and RPL32 (the most stable gene pairs given by geNorm program), we propose that the mean data rendered by RPL19 and GAPDH (the most stable gene pairs given by NormFinder program) be used to normalize gene expression values in Chlamydomonas sp. ICE-L more accurately. The example of FAD3 gene expression calculation demonstrated the importance of selecting an appropriate category and number of reference genes to achieve an accurate and reliable normalization of gene expression during freeze acclimation in Chlamydomonas sp. ICE-L.

The ability of Antarctic ice algae, Chlamydomonas sp. ICE-L, to survive and proliferate at low temperature and high salinity implies that they have overcome key barriers inherent in Antarctic environments. A full-length complementary DNA (cDNA) sequence of omega-3 fatty acid desaturase, designated CiFAD3, was isolated via reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends methods. The full-length of CiFAD3 cDNA contained an open reading frame of 1,302 bp with 5′-terminal untranslated region (UTR) of 36 bp and 3′-terminal UTR of 507 bp encoding a fatty acid desaturase protein of 434 amino acids. Sequence alignment and phylogenetic analysis showed that the gene was homologous to known chloroplastic omega-3 fatty acid desaturase. Meanwhile, CiFAD3 sequence showed typical features of membrane-bound desaturase such as three conserved histidine boxes along with four membrane spanning regions that were universally present among plant desaturases. Under different stress conditions, messenger RNA (mRNA) expression levels of CiFAD3 were measured by quantitative RT-PCR. The results showed that both temperature and salinity could motivate the upregulation of CiFAD3 expression. The mRNA accumulation of CiFAD3 increased 2.6-fold at 0°C and 1.8-fold at 12°C compared to the algae at 6°C. Similarly, mRNA expression levels of CiFAD3 increased 3.8-fold after 62‰ NaCl treatment for 2 h. However, CiFAD3 mRNA expression levels were partially decreased after UV radiation. These data suggest that CiFAD3 is the enzyme responsible for the omega-3 fatty acid desaturation involved in ice algae Chlamydomonas sp. ICE-L acclimatizing to cold temperature and high salinity in Antarctic environment.

A cDNA encoding heat shock protein 70 of Antarctic ice algae Chlamydomonas sp. ICE-L (designated as CiHsp70) was identified by RT-PCR and rapid amplification of cDNA ends approaches. The full-length cDNA of CiHsp70 was 2,232 bp, consisting of a 5'-terminal untranslated region (UTR) of 76 bp, a 3'-terminal UTR of 203 bp with a poly (A) tail, and an open reading frame of 1,953 bp. The CiHsp70 cDNA encoded a polypeptide of 651 amino acids with an ATPase domain of 388 amino acids, the substrate peptide binding domain of 246 amino acids and a C-terminus domain of 17 amino acids. The inducible CiHsp70 cDNA was highly homologous to other plant cytosolic Hsp70 genes and clustered together with green algae and higher plant rather than brown algae, diatom and Cryptophyta. Antarctic ice algae were treated with different stress conditions and messenger RNA (mRNA) expression levels of CiHsp70 were quantified by quantitative RT-PCR. The results showed that both cold and heat shock treatments could stimulate CiHsp70 mRNA expression. Meanwhile, CiHsp70 mRNA expression level increased 2.9-fold in response to UV-B radiation for 6 h, while the expression levels of CiHsp70 were remarkably increased after removing the UV-B radiation and immediately providing additional 6 h visible light. Furthermore, treating with 62 or 93 per thousand NaCl for 2 h, CiHsp70 mRNA expression level increased 3.0- and 2.1-fold, respectively. Together, our observations revealed that CiHsp70 as a molecular chaperone might play an important role in Antarctic ice algae Chlamydomonas sp. ICE-L acclimatizing to polar environment.

Calcium-activated potassium channels on plasma membrane enable potassium influx into the cell with ensuing changes in plasma membrane potential and consequent effects on cellular metabolic functions. Recently, this potassium channel was reported to regulate the cellular responses of mammalian immune cells. We have postulated the presence of such a channel in fish immune cells and its potential role in immunoregulation in fish. Employing specific primers and RNA template, we cloned a segment of a novel gene from turbot blood sample and subsequently obtained a full cDNA sequence using RACE approaches. Bioinformatic analysis revealed structural and phylogenetic characteristics of a novel small conductance calcium-activated potassium channel gene, we called TSKCa, which exhibits homologous domains to other species particularly in the transmembrane regions. Full-length TSKCa cDNA is 1698 bp with a 1632 bp open reading frame encoding a protein of 544 amino acids. TSKCa gene is expressed in majority of the tested organs and tissues of turbot. To assess the postulated immune function of TSKCa, we infected turbot with the pathogen Vibrio anguillarum. Here, semi-quantitative RT-PCR analysis demonstrated increased mRNA expression of TSKCa in head kidney, spleen and blood, indicating an important role of TSKCa in these organ tissues that mediate the immune defense response of turbot. In contrast, there was less change in expression in the turbot intestines and liver which were less implicated in the immune response in present study.