Lamprey VDAC2: Suppressing hydrogen peroxide-induced 293T cell apoptosis by downregulating BAK expression.

The voltage-dependent anion channel 2 (VDAC2) is the abundant protein in the outer mitochondrial membrane. Opening VDAC2 pores leads to the induction of mitochondrial energy and material transport, facilitating interaction with various mitochondrial proteins implicated in essential processes such as cell apoptosis and proliferation. To investigate the VDAC2 in lower vertebrates, we identified Lr-VDAC2, a homologue of VDAC2 found in lamprey (Lethenteron reissneri), sharing a sequence identity of greater than 50 % with its counterparts. Phylogenetic analysis revealed that the position of Lr-VDAC2 aligns with the lamprey phylogeny, indicating its evolutionary relationship within the species. The Lr-VDAC2 protein was primarily located in the mitochondria of lamprey cells. The expression of the Lr-VDAC2 protein was elevated in high energy-demanding tissues, such as the gills, muscles, and myocardial tissue in normal lampreys. Lr-VDAC2 suppressed H2O2 (hydrogen peroxide)-induced 293 T cell apoptosis by reducing the expression levels of Caspase 3, Caspase 9, and Cyt C (cytochrome c). Further research into the mechanism indicated that the Lr-VDAC2 protein inhibited the pro-apoptotic activity of BAK (Bcl-2 antagonist/killer) protein by downregulating its expression at the protein translational level, thus exerting an anti-apoptotic function similar to the role of VDAC2 in humans.

Evolutionary analysis of SLC10 family members and insights into function and expression regulation of lamprey NTCP.

The Na ( +)-taurocholate cotransporting polypeptide (NTCP) is a member of the solute carrier family 10 (SLC10), which consists of 7 members (SLC10a1-SLC10a7). NTCP is a transporter localized to the basolateral membrane of hepatocytes and is primarily responsible for the absorption of bile acids. Although mammalian NTCP has been extensively studied, little is known about the lamprey NTCP (L-NTCP). Here we show that L-NTCP follows the biological evolutionary history of vertebrates, with conserved domain, motif, and similar tertiary structure to higher vertebrates. L-NTCP is localized to the cell surface of lamprey primary hepatocytes by immunofluorescence analysis. HepG2 cells overexpressing L-NTCP also showed the distribution of L-NTCP on the cell surface. The expression profile of L-NTCP showed that the expression of NTCP is highest in lamprey liver tissue. L-NTCP also has the ability to transport bile acids, consistent with its higher vertebrate orthologs. Finally, using a farnesoid X receptor (FXR) antagonist, RT-qPCR and flow cytometry results showed that L-NTCP is negatively regulated by the nuclear receptor FXR. This study is important for understanding the adaptive mechanisms of bile acid metabolism after lamprey biliary atresia based on understanding the origin, evolution, expression profile, biological function, and expression regulation of L-NTCP.

Lamprey prohibitin 2 inhibits non-small cell lung carcinoma cell proliferation by down-regulating the expression and phosphorylation levels of cell cycle-associated proteins.

Prohibitin 2 (PHB2) is an evolutionarily conserved and functionally diverse protein that plays an important role in multiple cellular functions, including cell proliferation, cell migration, and apoptosis, and is also known to participate in the process of tumorigenesis and development. In this study, the lamprey PHB2 (Lm-PHB2) gene was over-expressed in KRAS (kirsten rat sarcoma viral oncogene homolog)-mutated non-small cell lung carcinoma (NSCLC) cells to investigate its effect on cell proliferation. The effects of Lm-PHB2 protein on the proliferation of NSCLC cells were determined by treating cells with the purified recombinant Lm-PHB2 protein (rLm-PHB2) followed by cell counting kit (CCK) assays and flow cytometry. Analysis showed that rLm-PHB2 blocked cells in the G2 phase and inhibited the cell proliferation of A549, Calu-1, and NCI-H226 to various degrees. The effect on Calu-1 cells was the most obvious and was concentration- and time-dependent. Similarly, cells transfected with the pEGFP-N1-Lm-PHB2 plasmid also resulted in the suppression of proliferation in A549 cells and Calu-1 cells. Quantitative real-time polymerase chain reaction (qRT-PCR) showed that Lm-PHB2 inhibited cell proliferation by repressing the transcription of PLK1 (polo-like kinase 1), Wee1 (wee1 kinase), CCNB1 (cyclin B1), and CDC25C (cell division control protein 25C). According to western blot analysis, Lm-PHB2 not only down-regulated the expression of PLK1, Wee1, CCNB1, and CDC25C but also reduced the phosphorylation levels of CCNB1 and CDC25C, thus blocking Calu-1 cells in G2/M phase. Our findings demonstrate a function of lamprey PHB2 that may inhibit the proliferation of some NSCLC cells by down-regulating the expression and phosphorylation of cell cycle-associated proteins.

Loss of insulin signaling may contribute to atrial fibrillation and atrial electrical remodeling in type 1 diabetes.

Atrial fibrillation (AF) is prevalent in diabetes mellitus (DM); however, the basis for this is unknown. This study investigated AF susceptibility and atrial electrophysiology in type 1 diabetic Akita mice using in vivo intracardiac electrophysiology, high-resolution optical mapping in atrial preparations, and patch clamping in isolated atrial myocytes. qPCR and western blotting were used to assess ion channel expression. Akita mice were highly susceptible to AF in association with increased P-wave duration and slowed atrial conduction velocity. In a second model of type 1 DM, mice treated with streptozotocin (STZ) showed a similar increase in susceptibility to AF. Chronic insulin treatment reduced susceptibility and duration of AF and shortened P-wave duration in Akita mice. Atrial action potential (AP) morphology was altered in Akita mice due to a reduction in upstroke velocity and increases in AP duration. In Akita mice, atrial Na+ current (INa) and repolarizing K+ current (IK) carried by voltage gated K+ (Kv1.5) channels were reduced. The reduction in INa occurred in association with reduced expression of SCN5a and voltage gated Na+ (NaV1.5) channels as well as a shift in INa activation kinetics. Insulin potently and selectively increased INa in Akita mice without affecting IK Chronic insulin treatment increased INa in association with increased expression of NaV1.5. Acute insulin also increased INa, although to a smaller extent, due to enhanced insulin signaling via phosphatidylinositol 3,4,5-triphosphate (PIP3). Our study reveals a critical, selective role for insulin in regulating atrial INa, which impacts susceptibility to AF in type 1 DM.

Prediction of ATPase cation transporting 13A2 molecule in Petromyzon marinus and pan-cancer analysis into human tumors from an evolutionary perspective.

The ATPase cation transporting 13A2 protein (ATP13A2), which maintains the homeostasis of mitochondria and lysosomes, plays a significant role in human neurodegenerative diseases and cancer. Through constructing a lamprey proteome database, employing multiple sequence alignment and phylogenetic analysis, 5 ATP13A2 proteins from Petromyzon marinus (Pm-ATP13A2) were identified based on the evolutionary perspective. The motif and domain analysis showed that the ATP13A2 protein was conserved. The multiple phosphorylation sites and transmembrane structures highlighted the characteristics of ATP13A2 as the P-ATPase-V cation transporting protein. Based on the information provided by the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases, this study was conducted as a preliminary investigation of the carcinogenic effects of the ATP13A2 gene in a variety of tumors. The ATP13A2 was strongly expressed in most tumors, except in two types of nervous system tumors glioblastoma multiforme (GBM) and brain lower grade glioma (LGG). Moreover, the expression of ATP13A2 was strongly correlated with the prognosis of tumor patients. The high expression of ATP13A2 was obviously related to the poor prognosis of LGG. The poor prognosis of LGG patients may affect the ATP13A2 expression through the immune cells and radiotherapy. Also, cancer-related fibroblast infiltration was observed. All in all, this work offers more insights into the molecular evolution of the ATP13A2 protein and facilitates the understanding of the carcinogenic effects of the ATP13A2 in different tumors. Our discussion also promotes the study into the successful evolution of the vertebrate brain and the mechanism of clinical brain-related diseases.

A novel protein upstream stimulatory factor 2 identified in lamprey, Lethenteron reissneri.

Upstream stimulatory factors are kinds of multi-functional transcription factors, which are expressed in eukaryotes widely, including Upstream stimulatory factor 1 (USFl) and upstream stimulatory factor 2 (USF2). USF protein has a typical basic helix-loop-helix leucine zipper (b-HLH-LZ) structure, which is involved in cell cycle, cell proliferations, glucose and lipid metabolism, and other biochemical processes. Although the USF family is an important regulator of cellular processes, little is known about the USF genes of lampreys, especially their evolutionary relationships, expression profiles, and biological functions. Here, an upstream stimulatory factor 2 (USF2) homolog from lamprey (Lethenteron reissneri) was identified and characterized (designated as L-USF2) because it is closer to USF2 subfamily than to USF1 subfamily. The cDNA fragment of L-USF2 has an open reading frame (ORF) of 765-bp length, encodes 254 amino acids, and contains an HLH domain at the c-terminal of amino acids. Meanwhile, motifs and genetic structure analysis reveal that USF2 gene exons are conserved. Moreover, the 3D structure analysis indicates that L-USF2 adopts the general USF2 folding and has a high structural similarity with H-USF2. The synteny results showed that the L-USF2 adjacent gene changed greatly compared with the jaw vertebrates. By real-time quantitative experiment and Western blot analysis, we found that L-USF2 gene played a significant role in the immune responses. This study not only provides us with a further understanding of the evolution and function of the USF gene family but also provides a basis for exploring its immune responses and immune defenses in lampreys.

Lamprey PHB2 maintains mitochondrial stability by tanslocation to the mitochondria under oxidative stress.

Before we have reported lamprey PHB2 could enhance the cellular oxidative-stressed tolerance, here the aim was to explore its mechanisms. We used flow cytometry analysis to identify a Lampetra morii homologue of PHB2 (Lm-PHB2) that could significantly decrease the levels of ROS generation in HEK293T cells. According to confocal microscopy observations, Lm-PHB2 contributed to maintain the mitochondrial morphology of HEK293T cells, and then both cellular nuclear location and translocation from the nucleus to mitochondria of Lm-PHB2 were also examined in HEK293T cells under oxidative stress. We also examined the expressions and locations of various Lm-PHB2 deletion mutants and the amino acid mutant by confocal microscopy and the results showed that the translocation of Lm-PHB2 into mitochondria was dependent on the Lm-PHB21-50aa region and the 17th, 48th and 57th three arginines (R) of N-terminal were very critical. In addition, the analyses of QRT-PCR and Western blot demonstrated that Lm-PHB2 increased the expression levels of OPA1 and HAX1 in HEK293T cells treated with H2O2. The analyses of immunofluorescence and immunoprecipitation showed that Lm-PHB2 could interact with OPA1 and HAX1, respectively. The above mentioned results indicate that Lm-PHB2 could assist OPA1 and HAX1 to maintain mitochondrial morphology and decrease ROS levels by the translocation from the nucleus to mitochondria under oxidative stress.

Proteomic analysis of buccal gland secretion from fasting and feeding lampreys (Lampetra morii).

BACKGROUND: Previous studies have shown that lamprey buccal glands contain some regulators related to anticoagulation, nociception, and immune responses due to the blood sucking habit. Regrettably, the protein expression profile in the buccal glands of feeding lampreys has never been reported yet. The present study was performed in order to further identify more proteins which are closely associated with lamprey feeding process. METHODS: 2D-PAGE, NanoLC-MS/MS with higher resolution, Ensembl lamprey and NCBI protein databases, as well as western blot was used to compare the proteomics of buccal gland secretion from China northeast lampreys (Lampetra morii) which had been fed for 0, 10, and 60 min, respectively. RESULTS: In the present study, the number of identified protein species in the buccal glands of feeding groups (60 min) was increased significantly, nearly ten times of that in the fasting group. During the feeding stage, novel proteins emerged in the buccal gland secretion of lampreys. According to gene ontology (GO) analysis and function predictions, these proteins were summarized and discussed based on their potential roles during feeding process. Furthermore, some of the identified proteins were confirmed to express during the feeding time of lampreys. CONCLUSION: When lampreys attack host fishes to suck blood and flesh, their buccal glands could secrete enough proteins to suppress blood coagulation, nociception, oxidative stress, immune response, as well as other adverse effects encountered during their parasitic lives. The present study would provide clues to clarify the feeding mechanism of the bloodsucking lampreys.

Label-Free Quantitative Proteomic Reveals Differentially Expressed Proteins in Aeromonas-Immunostimulated Leukocytes of Lampetra japonica.

Lamprey was considered to be one of the most basal jawless vertebrate representatives for studying vertebrate evolution, embryo development, and the origin of adaptive immunity. Here we investigated the effect of the gut-derived Aeromonas on the lamprey leukocytes proteome using the label-free liquid chromatography-tandem mass spectrometry for quantitative proteomics analysis. Significant difference was observed in the regulation of 34 out of 755 proteins in Aeromonas-immunized lamprey. 31 proteins were only identified in saline solution-immunized lamprey and 47 proteins were only identified in Aeromonas-immunized lamprey. Quantitative real-time polymerase chain reaction was used to validate the results of the proteomic analysis. The differentially expressed proteins were found to be associated with several different biological processes. The identification of leukocytes proteins essential for lamprey adaptive immune response induced by gut-derived Aeromonas strain could supply important information on lamprey-Aeromonas interactions and VLR-based adaptive immune signal pathways.

Identification and characterisation of the anti-oxidative stress properties of the lamprey prohibitin 2 gene.

The highly conserved protein prohibitin 2 (PHB2) has been implicated as a cell-surface receptor in the regulation of proliferation, apoptosis, transcription, and mitochondrial protein folding. In the present study, we identified a Lampetra morii homologue of PHB2, Lm-PHB2, showing greater than 61.8% sequence identity with its homologues. Phylogenetic analysis indicated that the position of Lm-PHB2 is consistent with lamprey phylogeny. Expression of the Lm-PHB2 protein was nearly equivalent in the heart, liver, kidneys, intestines, and muscles of normal lampreys. However, the Lm-PHB2 protein was down-regulated in the myocardia of lampreys challenged for 5 days with adriamycin (Adr), followed by a significant up-regulation 10 days after treatment. In vitro, recombinant Lm-PHB2 (rLm-PHB2) protein could significantly enhance the H2O2-induced oxidative stress tolerance in Chang liver (CHL) cells. Further mechanism studies indicated that the nucleus-to-mitochondria translocation of Lm-PHB2 was closely involved in the oxidative stress protection. Our results suggests that the strategies to modulate Lm-PHB2 levels may constitute a novel therapeutic approach for myocardial injury and liver inflammatory diseases, conditions in which oxidative stress plays a critical role in tissue injury and inflammation.

[The identification and verification of species-specific microRNAs and their precursors in Lampetra japonica].

MicroRNAs (miRNAs) negatively regulate genes which are involved in various biological processes of metabolism at both transcriptional and post-transcriptional levels. In recent years, the existence and function of miRNAs have been extensively studied in plants and animals with the application of deep sequencing and microarray technology. In this study, small RNAs from leucocytes of Lampetra japonica (L. japonica) were sequenced using the second generation high-throughput sequencing technology. A total of 5 207 787 small RNA sequences were identified, and 4 739 346 of them assembled into 10 989 variants. Based on sequence similarity analysis, the sequences of these variants matched known miRNAs of 306 conserved families, among which 6 conserved miRNA family members expressed at an extremely high level which reflected the conservatism of miRNAs among species. In addition, 70 unannotated sequences were predicted to be new miRNAs, and 34 of them were further verified expressing in antigen-treated L. japonica leucocytes by miRNA microarray assay. Moreover, the minimal folding free energy indexes for 16 of the 34 miRNA precursors exceed 0.85, indicating the existence of species-specific miRNAs in L. japonica which may play important roles in regulating, growth, development and disease response of L. japonica leukocytes.

The prostaglandin E2 type 4 receptor participates in the response to acute oxidant stress in airway epithelial cells.

Oxidative stress is implicated in the pathogenesis of many inflammatory pulmonary diseases, including cystic fibrosis (CF). Delineating how oxidative stress stimulates CF transmembrane conductance regulator (CFTR) in airway epithelial cells is useful, both to increase the understanding of airways host defense and suggest therapeutic approaches to reduce the oxidant stress burden in the CF lung. Using the airway epithelial cell line Calu-3, we investigated the hypothesis that hydrogen peroxide (H2O2), which stimulates anion efflux through CFTR, does so via the production of prostaglandin E2 (PGE2). Using iodide efflux as a biochemical marker of CFTR activity and short circuit current (I(sc)) recordings, we found that the H2O2-stimulated efflux was abolished by cyclooxygenase-1 inhibition and potentially also involves microsomal prostaglandin E synthase-1 activity, implicating a role for PGE2 production. Furthermore, H2O2 application resulted in a rapid release of PGE2 from Calu-3 cells. We additionally hypothesized that the PGE2 subtype 4 (EP(4)) receptor was involved in mediating this response. In the presence of (4Z)-7-[(rel-1S,2S,5R)-5-((1,1'-biphenyl-4-yl)methoxy)-2-(4-morpholinyl)-3-oxocyclopentyl]-4-heptenoic acid (AH23848) (which blocks the EP4 receptor), the H2O2-stimulated response was abolished. To investigate this finding in a polarized system, we measured the increase in I(sc) induced by H2O2 addition in the presence and absence of AH23848. H2O2 induced a robust increase in I(sc), which was significantly attenuated in the presence of AH23848, suggesting some role for the EP4 receptor. In conclusion, with H2O2 as a model oxidant stress, stimulation of CFTR seems to involve PGE2 production and likely EP4 receptor activation in Calu-3 airway epithelial cells. This mechanism would be compromised in the CF airways.

Experiment and Simulation of Non-Reactive Solute Transport in Porous Media.

To more accurately predict the migration behavior of pollutants in porous media, we conduct laboratory scale experiments and model simulation. Aniline (AN) is used in one-dimensional soil column experiments designed under various media and hydrodynamic conditions. The advection-dispersion equation (ADE) and the continuous-time random walk (CTRW) were used to simulate the breakthrough curves (BTCs) of the solute transport. The results show that the media and hydrodynamic conditions are two important factors affecting solute transport and are related to the degree of non-Fickian transport. The simulation results show that CTRW can more effectively describe the non-Fickian phenomenon in the solute transport process than ADE. The sensitive parameter in the CTRW simulation process is ß , which can reflect the degree of non-Fickian diffusion in the solute transport. Understanding the relationship of ß with velocity and media particle size is conducive to improving the reactive solute transport model. The results of this study provide a theoretical basis for better prediction of pollutant transport in groundwater.

Activation of the EP4 prostanoid receptor induces prostaglandin E2 and pro-inflammatory cytokine production in human airway epithelial cells.

Prostaglandin (PG)E2 mediates its effects via activation of four distinct PGE2 receptors, termed EP1₋4, all of which are present on the model human airway epithelial cell line, Calu-3. We previously reported that acute activation of the EP4 subtype of the PGE2 receptor is associated with increased anion efflux from these cells, via the CFTR chloride channel. In the present study we examine the effects of longer term activation of the EP4 receptor in Calu-3 cells in an attempt to determine whether this would prove beneficial or detrimental to the airway epithelial cell environment. Using PGE1-OH, an EP4 receptor selective agonist, we determined that EP4 receptor activation was associated with increased phosphorylation of extracellular signal-related kinases (ERKs) and induction of the transcription factor early growth response factor-1 (Egr-1). Additionally, using specific enzyme-linked immunosorbent assays and quantitative PCR, we detected increased production of PGE2, IL-6, IL-8 and the chemokine monocyte chemotactic protein-1 (MCP-1) at both the protein and gene level in response to EP4 receptor activation. Intriguingly, the enhanced production of PGE2 in response to EP4 receptor activation raises the possibility of a positive feedback situation. Generally, within the airways, PGE2 is considered to have pro-inflammatory effects, whilst the enhanced production of IL-6, IL-8 and MCP-1 would be associated with the recruitment and activation of inflammatory cells to the airways. Thus, we conclude that chronic activation of the EP4 receptor is associated with increased production of mediators likely to increase the pro-inflammatory milieu of airway epithelial cells.

Genetic and Functional Characterization of Novel Brown-Like Adipocytes Around the Lamprey Brain.

During the process of vertebrate evolution, many thermogenic organs and mechanisms have appeared. Mammalian brown adipose tissue (BAT) generates heat through the uncoupling oxidative phosphorylation of mitochondria, acts as a natural defense against hypothermia and inhibits the development of obesity. Although the existence, cellular origin and molecular identity of BAT in humans have been well studied, the genetic and functional characteristics of BAT from lampreys remain unknown. Here, we identified and characterized a novel, naturally existing brown-like adipocytes at the lamprey brain periphery. Similar to human BAT, the lamprey brain periphery contains brown-like adipocytes that maintain the same morphology as human brown adipocytes, containing multilocular lipid droplets and high mitochondrion numbers. Furthermore, we found that brown-like adipocytes in the periphery of lamprey brains responded to thermogenic reagent treatment and cold exposure and that lamprey UCP2 promoted precursor adipocyte differentiation. Molecular mapping by RNA-sequencing showed that inflammation in brown-like adipocytes treated with LPS and 25HC was enhanced compared to controls. The results of this study provide new evidence for human BAT research and demonstrate the multilocular adipose cell functions of lampreys, including: (1) providing material energy and protecting structure, (2) generating additional heat and contributing to adaptation to low-temperature environments, and (3) resisting external pathogens.

Lamprey Prohibitin2 Arrest G2/M Phase Transition of HeLa Cells through Down-regulating Expression and Phosphorylation Level of Cell Cycle Proteins.

Prohibitin 2(PHB2) is a member of the SFPH trans-membrane family proteins. It is a highly conserved and functionally diverse protein that plays an important role in preserving the structure and function of the mitochondria. In this study, the lamprey PHB2 gene was expressed in HeLa cells to investigate its effect on cell proliferation. The effect of Lm-PHB2 on the proliferation of HeLa cells was determined by treating the cells with pure Lm-PHB2 protein followed by MTT assay. Using the synchronization method with APC-BrdU and PI double staining revealed rLm-PHB2 treatment induced the decrease of both S phase and G0/G1 phase and then increase of G2/M phase. Similarly, cells transfected with pEGFP-N1-Lm-PHB2 also exhibited remarkable reduction in proliferation. Western blot and quantitative real-time PCR(qRT-PCR) assays suggested that Lm-PHB2 caused cell cycle arrest in HeLa cells through inhibition of CDC25C and CCNB1 expression. According to our western blot analysis, Lm-PHB2 was also found to reduce the expression level of Wee1 and PLK1 and the phosphorylation level of CCNB1, CDC25C and CDK1 in HeLa cells. Lamprey prohibitin 2 could arrest G2/M phase transition of HeLa cells through down-regulating expression and phosphorylation level of cell cycle proteins.

Development of anti-PRL (prolactin) autoantibodies by homologous PRL in rats: a model for macroprolactinemia.

Macroprolactinemia is hyperprolactinemia in humans mainly due to anti-PRL (prolactin) autoantibodies and is a pitfall for the differential diagnosis of hyperprolactinemia. Despite its high prevalence, the pathogenesis remains unclear. In this study, we examined whether anti-PRL autoantibodies develop via immunization with homologous rat pituitary PRL in rats to elucidate what mechanisms are involved and whether they cause hyperprolactinemia with low PRL bioactivity, as seen in human macroprolactinemia. Anti-PRL antibodies were developed in 19 of 20 rats immunized with homologous rat pituitary PRL and 29 of 30 rats with heterogeneous bovine or porcine pituitary PRL but did not develop in 25 control rats. In rats with anti-PRL antibodies, the basal serum PRL levels were elevated, and a provocative test for PRL secretion using dopamine D2 receptor antagonist (metoclopramide) showed a normal rising response with a slower clearance of PRL because of the accumulation of macroprolactin in blood. Antibodies developed by porcine or rat pituitary PRL reduced the bioactivity of rat serum PRL, and gonadal functions in these rats were normal despite hyperprolactinemia. Anti-PRL antibodies were stable and persisted for at least 5 wk after the final injection of PRL. These findings suggest that pituitary PRL, even if homologous, has antigenicity, leading to the development of anti-PRL autoantibodies. We successfully produced an animal model of human macroprolactinemia, with which we can explain the mechanisms of its clinical characteristics, i.e. asymptomatic hyperprolactinemia.

Down-regulation of Cl- pump ClP55 subunit induced enhancement of glutamate neurotoxicity in cultured rat hippocampal neurons.

To test whether the increased intracellular Cl- concentration ([Cl-]i) is responsible for the enhanced glutamate toxicity, antisense oligonucleotide of ClP55, a Cl- -ATPase/pump associated protein, was transfected in cultured rat hippocampal neurons. Neuronal [Cl-]i in the antisense oligonucleotide-transfected culture increased to a level 3- to 4-fold higher than that in control. Glutamate exposure (10 microM, 10 min) increased neuronal apoptosis and decreased Akt-pS473 level in the antisense oligonucleotide-transfected neurons, but not in control or sense oligonucleotide-transfected ones, suggesting the responsibility of elevated [Cl-]i in the enhancement of glutamate neurotoxicity.

Bacterial community composition in the gut content of Lampetra japonica revealed by 16S rRNA gene pyrosequencing.

The composition of the bacterial communities in the hindgut contents of Lampetrs japonica was surveyed by Illumina MiSeq of the 16S rRNA gene. An average of 32385 optimized reads was obtained from three samples. The rarefaction curve based on the operational taxonomic units tended to approach the asymptote. The rank abundance curve representing the species richness and evenness was calculated. The composition of microbe in six classification levels was also analyzed. Top 20 members in genera level were displayed as the classification tree. The abundance of microorganisms in different individuals was displayed as the pie charts at the branch nodes in the classification tree. The differences of top 50 genera in abundance between individuals of lamprey are displayed as a heatmap. The pairwise comparison of bacterial taxa abundance revealed that there are no significant differences of gut microbiota between three individuals of lamprey at a given rarefied depth. Also, the gut microbiota derived from L. japonica displays little similarity with other aquatic organism of Vertebrata after UPGMA analysis. The metabolic function of the bacterial communities was predicted through KEGG analysis. This study represents the first analysis of the bacterial community composition in the gut content of L. japonica. The investigation of the gut microbiota associated with L. japonica will broaden our understanding of this unique organism.