Population genomics of an icefish reveals mechanisms of glacier-driven adaptive radiation in Antarctic notothenioids.

BACKGROUND: Antarctica harbors the bulk of the species diversity of the dominant teleost fish suborder-Notothenioidei. However, the forces that shape their evolution are still under debate. RESULTS: We sequenced the genome of an icefish, Chionodraco hamatus, and used population genomics and demographic modelling of sequenced genomes of 52 C. hamatus individuals collected mainly from two East Antarctic regions to investigate the factors driving speciation. Results revealed four icefish populations with clear reproduction separation were established 15 to 50 kya (kilo years ago) during the last glacial maxima (LGM). Selection sweeps in genes involving immune responses, cardiovascular development, and photoperception occurred differentially among the populations and were correlated with population-specific microbial communities and acquisition of distinct morphological features in the icefish taxa. Population and species-specific antifreeze glycoprotein gene expansion and glacial cycle-paced duplication/degeneration of the zona pellucida protein gene families indicated fluctuating thermal environments and periodic influence of glacial cycles on notothenioid divergence. CONCLUSIONS: We revealed a series of genomic evidence indicating differential adaptation of C. hamatus populations and notothenioid species divergence in the extreme and unique marine environment. We conclude that geographic separation and adaptation to heterogeneous pathogen, oxygen, and light conditions of local habitats, periodically shaped by the glacial cycles, were the key drivers propelling species diversity in Antarctica.

Gut Microbial Divergence Between Three Hadal Amphipod Species from the Isolated Hadal Trenches.

Amphipods are the dominant scavenging metazoan species in the hadal trenches at water depths below 6,000 m. The gut microbiota have been considered to be contribution to the adaptation of deep-sea organisms; however, few comparative analyses of animal gut microbiota between different isolated hadal environments have been done so far. Here, we employed high-throughput 16S rRNA sequencing to compare the gut microbial taxonomic composition and functional potential diversity of three hadal amphipod species, Hirondellea gigas, Bathycallisoma schellenbergi, and Alicella gigantea, collected from the Mariana Trench, Marceau Trench, and New Britain Trench in the Pacific Ocean, respectively. Results showed that different community compositions were detected across all the amphipod specimens based on the analyses of alpha-diversity, hierarchical cluster tree, and PCoA (principal coordinate analysis). Moreover, almost no correlation was observed between genera overrepresented in different amphipods by microbe-microbe correlations analysis, which suggested that the colonization of symbionts were host-specific. At genus level, Psychromonas was dominant in H. gigas, and Candidatus Hepatoplasma was overall dominant in A. gigantea and B. schellenbergi. Comparison of the functional potential showed that, though three hadal amphipod species shared the same predominant functional pathways, the abundances of those most shared pathways showed distinct differences across all the specimens. These findings pointed to the enrichment of particular functional pathways in the gut microbiota of the different isolated trench amphipods. Moreover, in terms of species relative abundance, alpha-diversity and beta-diversity, there was high similarity of gut microbiota between the two A. gigantea populations, which dwelled in two different localities of the same hadal trench. Altogether, this study provides an initial investigation into the gut-microbial interactions and evolution at the hadal depths within amphipod. Each of these three amphipod species would be a model taxa for future studies investigating the influence habitat difference and geography on gut-microbial communities.

Characterization of EPO H131S as a key mutation site in the hypoxia-adaptive evolution of Gymnocypris dobula.

Erythropoietin (EPO) is a glycoprotein hormone involved in proerythropoiesis, antioxidation, and antiapoptosis. It also contributes to cellular immune function in high-altitude species, such as the schizothoracine fish Gymnocypris dobula. Six mutation sites previously identified in EPO from G. dobula (GD-EPO) were injected into zebrafish embryos, and their effects were compared with EPO from the low-altitude schizothoracine Schizothorax prenanti. The key mutation site in GD-EPO was identified as H131S. Under hypoxic conditions, the levels of superoxide dismutase and malondialdehyde were decreased, whereas that of nitric oxide was increased in zebrafish injected with GD-EPO compared with those injected with S. prenanti-EPO (SP-EPO). The results suggest that EPO in high-altitude schizothoracine species is both antioxidative and antiapoptotic, driven by the H131S mutation site. Thus, this enhanced the ability of this species to adapt to the high-altitude hypoxic environment. These results provide a basis for investigating further the hypoxia adaptation mechanisms of teleosts.

Research progress of microRNAs involved in hematopoiesis.

Hematopoiesis plays an important role(s) in maintenance and physiology of life. Hematopoiesis in vertebrates mainly includes self-renewal of the hematopoietic stem cells, proliferation and differentiation of the hematopoietic progenitor cells, and maturation of the blood cells. The regulation of hematogenesis involves a variety of transcription factors, membrane receptors, hematopoietic growth factors, and microRNAs, which interact with each other and form a variety of signaling pathways and signal networks. MicroRNAs are a class of non-coding RNAs widely distributed in eukaryotic cells and play important roles in the hematopoietic process. The expression of microRNAs is regulated by transcription factors involved in hematopoietic signaling pathways. In turn, their expression can inhibit or down-regulate those of transcription factors involved in hematopoietic related signaling pathways and other related regulatory factors, thereby affecting the signaling pathways related to hematopoiesis and ultimately the hematopoietic process. In this review, we introduce the hematopoiesis processes and related signal pathways in vertebrates, focusing on the relationships between microRNAs and hematopoietic transcription factors and signal pathways, and summarizing the recent research progress of microRNAs in hematopoiesis.

Specific immunity proteomic profile of the skin mucus of Antarctic fish Chionodraco hamatus and Notothenia coriiceps.

The white-blooded Antarctic icefish is the only known vertebrate lacking oxygen-transporting haemoglobins. Fish skin mucus, as the first line of defence against pathogens, can reflect fish welfare. In this study, we analysed the skin mucus proteome profiles of the two Antarctic fish species, the white-blooded Antarctic icefish, Chionodraco hamatus, and the red-blooded Antarctic fish, Notothenia coriiceps, unfolding the different proteins by liquid chromatography coupled with tandem mass spectrometry isobaric tags for relative and absolute quantitation (iTRAQ) technology. Of the 4444 totally identified proteins, 227 differentially expressed proteins (DEPs) were found in the comparison between C. hamatus and N. coriiceps, of which 121 were upregulated and 106 were downregulated in the icefish. In the Kyoto Encyclopedia of Genes and Genomes pathway annotation, we found two pathways "Legionellosis" and "Complement and coagulation cascades" were significantly enriched, among of which innate immune candidate proteins such as C3, CASP1, ASC, F3 and C9 were significantly upregulated, suggesting their important roles in C. hamatus immune system. Additionally, the DEP protein-protein interaction network analysis and "Response to stress" GO category provided candidate biomarkers for deep understanding of the distinct immune response of the two Antarctic fish underlying the cold adaptation.

Genetic Diversity and Population Structure Analysis of Three Deep-Sea Amphipod Species from Geographically Isolated Hadal Trenches in the Pacific Ocean.

Amphipods of the superfamily Lysianassoidea that inhabit the hadal zone ( > 6000 m) have large bathymetric ranges and play a key role in deep ocean ecosystems. The endemism of these amphipod species makes them a good model for investigating potent natural selection and restricted dispersal in deep ocean trenches. Here, we describe genetic diversity and intraspecific population differentiation among three amphipod species from four Pacific trenches based on a mtDNA concatenated dataset (CO Ι and 16S rRNA genes) from 150 amphipod individuals. All amphipod populations had low genetic diversity, as indicated by haplotype and nucleotide diversity values. Population geographic relationship analysis of two Alicella gigantea populations revealed no genetic differentiation between these two localities (pairwise genetic differentiation coefficient = 0.00032, gene flow = 784.58), and the major variation (99.97%) was derived from variation within the populations. Historical demographic events were investigated using Tajima's D and Fu's F neutrality tests and analysis of mismatch distribution. Consistent results provided strong evidence to support the premise that demographic expansion occurred only for the Mariana population of Hirondellea gigas, possibly within the last 2.1-3.4 million years. These findings suggest that the formation of amphipod population structure might be the result of multiple factors including high hydrostatic pressure, food distribution, trench topographic forcing and potential ecological interactions.

Hypoxia-inducible factor 1α from a high-altitude fish enhances cytoprotection and elevates nitric oxide production in hypoxic environment.

Hypoxia-inducible factors (HIFs) are master transcription factor regulating hypoxic responses in vertebrates. Species of Schizothoracine, a sub-family of cyprinidae, are highly endemic to the hypoxic Qinghai-Tibetan Plateau (QTP). What roles the HIFs play in hypoxic adaptation in the Schizothoracine fish is little known. In this study, the HIF-1α/B gene from Gymnocypris dobula (Gd) was characterized. The predicted protein for Gd-HIF-1α/B contains the main domains (bHLH, PAS, PAC, ODD, N-TAD, and C-TAD). Moreover, a specific mutation that the proline hydroxylation motif (LXXLAP) mutated into PxxLAP was observed in Gd-HIF-1α/B CODD domain, which may lead to changes in the function. To clarify whether HIF-1α/B of G. dobula possesses hypoxic adaptive features, Gd-HIF1α/B and Schizothorax prenanti-HIF1α/B (Sp-HIF1α/B) were cloned into an expression vector and transfected into 293T cells. Cell viability was found to be significantly higher in cells transfected with Gd-HIF-1α/B than those transfected with Sp-HIF-1α/B under hypoxic conditions. In addition, G. dobula HIF-1α/B showed stronger activity in transactivating the expression of nitric oxide (NO)-synthesizing enzyme, NOS2B under hypoxia stresses than the orthologous gene from S. prenanti, which were accompanied with upregulated expressions of NOS2B in heart of G. dobula, which may attribute to elevated NO levels detected in G. dobula than the lower land species. These results indicated that the HIF-1α plays an important role in mediating the iNOS signaling system in the process of evolutionary adaptation of the Schizothoracine to the highland environment.

GC bias lead to increased small amino acids and random coils of proteins in cold-water fishes.

BACKGROUND: Temperature adaptation of biological molecules is fundamental in evolutionary studies but remains unsolved. Fishes living in cold water are adapted to low temperatures through adaptive modification of their biological molecules, which enables their functioning in extreme cold. To study nucleotide and amino acid preference in cold-water fishes, we investigated the substitution asymmetry of codons and amino acids in protein-coding DNA sequences between cold-water fishes and tropical fishes., The former includes two Antarctic fishes, Dissostichus mawsoni (Antarctic toothfish), Gymnodraco acuticeps (Antarctic dragonfish), and two temperate fishes, Gadus morhua (Atlantic cod) and Gasterosteus aculeatus (stickleback), and the latter includes three tropical fishes, including Danio rerio (zebrafish), Oreochromis niloticus (Nile tilapia) and Xiphophorus maculatus (Platyfish). RESULTS: Cold-water fishes showed preference for Guanines and cytosines (GCs) in both synonymous and nonsynonymous codon substitution when compared with tropical fishes. Amino acids coded by GC-rich codons are favored in the temperate fishes, while those coded by AT-rich codons are disfavored. Similar trends were discovered in Antarctic fishes but were statistically weaker. The preference of GC rich codons in nonsynonymous substitution tends to increase ratio of small amino acid in proteins, which was demonstrated by biased small amino acid substitutions in the cold-water species when compared with the tropical species, especially in the temperate species. Prediction and comparison of secondary structure of the proteomes showed that frequency of random coils are significantly larger in the cold-water fish proteomes than those of the tropical fishes. CONCLUSIONS: Our results suggested that natural selection in cold temperature might favor biased GC content in the coding DNA sequences, which lead to increased frequency of small amino acids and consequently increased random coils in the proteomes of cold-water fishes.

miRNA-152 targets GATA1 to regulate erythropoiesis in Chionodraco hamatus.

Chionodraco hamatus is a teleost within the suborder Notothenioidei, the members of which are known to lack functional erythrocytes with modified hematopoiesis. Hematopoiesis is an essential process during the development of animals, where it is tightly regulated by many different transcription factors, signaling proteins, chromatin modifications, and microRNAs (miRNAs). The miRNAs are known to regulate the expression of their target genes at the post-transcriptional level. However, little is known about the miRNA-mediated regulation of hematopoiesis. In this study, we confirmed that miR-152 plays a crucial role in hematopoiesis during the development of C. hamatus. The overexpression of miR-152 reduced hematopoiesis according to the decreased expression of GATA1 and reduced o-dianisidine staining of hemoglobin. Mechanistically, reduced hematopoiesis was regulated by the miR-152-mediated down-regulated expression of GATA1. Bioinformatics analysis was used to predict the target gene of miR-152. Western blotting as well as dual luciferase and EGFP reporter assays were employed to investigate the expression of GATA1 mediated by miR-152. Finally, verification experiments in the zebrafish autologous model strongly supported the effect of miR-152 on hematopoiesis. In conclusion, we suggest that miR-152 is a novel molecular factor that regulates hematopoiesis during the development of C. hamatus by down-regulating the expression of GATA1.

[Research progress on microRNAs involved in vascular development].

The vertebrate vascular system development is a very important and complicated process. MicroRNAs regulate gene expression at transcriptional and post-transcriptional levels and play important roles in many physiological and pathological processes. MicroRNAs mainly participate in the regulation of vascular smooth muscle cell and vascular endothelial cell development. In this paper, we summarize the recent progress regarding the microRNAs involved in the vascular development. In particular, we focus on the microRNAs including miR-126, miR-17/92 family in endothelial cell's regulation, and miR-143/145 family, miR-21 in vascular smooth muscle cell's regulation. The future research on the role of microRNAs in vascular development is also prospected.

Analysis of the erythropoietin of a Tibetan Plateau schizothoracine fish (Gymnocypris dobula) reveals enhanced cytoprotection function in hypoxic environments.

BACKGROUND: Erythropoietin (EPO) is a glycoprotein hormone that plays a principal regulatory role in erythropoiesis and initiates cell homeostatic responses to environmental challenges. The Qinghai-Tibet Plateau is a natural laboratory for hypoxia adaptation. Gymnocypris dobula is a highly specialized plateau schizothoracine fish that is restricted to > 4500 m high-altitude freshwater rivers and ponds in the Qinghai-Tibet Plateau. The role of EPO in the adaptation of schizothoracine fish to hypoxia is unknown. RESULTS: The EPO and EPO receptor genes from G. dobula and four other schizothoracine fish from various altitudinal habitats were characterized. Schizothoracine EPOs are predicted to possess 2-3 N-glycosylation (NGS) sites, 4-5 casein kinase II phosphorylation (CK2) sites, 1-2 protein kinase C (PKC) phosphorylation sites, and four conserved cysteine residues within four helical domains, with variations in the numbers of NGS and CK2 sites in G. dobula. PAML analysis indicated a d N/d S value (ω) = 1.112 in the G. dobula lineage, and a few amino acids potentially under lineage-specific positive selection were detected within the G. dobula EPO. Similarly, EPO receptors of the two high-altitude schizothoracines (G. dobula and Ptychobarbus kaznakovi), were found to be statistically on the border of positive selection using the branch-site model (P-value = 0.096), and some amino acids located in the ligand-binding domain and the fibronectin type III domain were identified as potentially positive selection sites. Tissue EPO expression profiling based on transcriptome sequencing of three schizothoracines (G. dobula, Schizothorax nukiangensis Tsao, and Schizothorax prenanti) showed significant upregulation of EPO expression in the brain and less significantly in the gill of G. dobula. The elevated expression together with the rapid evolution of the EPO gene in G. dobula suggested a possible role for EPO in adaptation to hypoxia. To test this hypothesis, Gd-EPO and Sp-EPO were cloned into an expression vector and transfected into the cultured cell line 293 T. Significantly higher cell viability was observed in cells transfected with Gd-EPO than cells harboring Sp-EPO when challenged by hypoxia. CONCLUSION: The deduced EPO proteins of the schizothoracine fish contain characteristic structures and important domains similar to EPOs from other taxa. The presence of potentially positive selection sites in both EPO and EPOR in G. dobula suggest possible adaptive evolution in the ligand-receptor binding activity of the EPO signaling cascade in G. dobula. Functional study indicated that the EPO from high-altitude schizothoracine species demonstrated features of hypoxic adaptation by reducing toxic effects or improving cell survival when expressed in cultured cells, providing evidence of molecular adaptation to hypoxic conditions in the Qinghai-Tibet Plateau.

Evolutionary suppression of erythropoiesis via the modulation of TGF-ß signalling in an Antarctic icefish.

The Antarctic icefish, a family (Channichthyidae) of teleosts within the perciform suborder Notothenioidei, are the only known vertebrates without oxygen-transporting haemoglobins and that are largely devoid of circulating erythrocytes. To elucidate the evo-devo mechanisms underpinning the suppressed erythropoiesis in the icefish, we conducted comparative studies on the transcriptomes and microRNAomes of the primary haematopoietic tissues between an icefish (Chionodraco hamatus) and two red-blooded notothenioids (Trematomus bernacchii and Gymnodraco acuticeps). We identified substantial remodelling of the haematopoietic programs in the icefish through which erythropoiesis is selectively suppressed. Experimental verification showed that erythropoietic suppression in the icefish may be attributable to the upregulation of TGF-ß signalling, which coincides with reductions in multiple transcription factors essential for erythropoiesis and the upregulation of hundreds of microRNAs, the majority (> 80%) of which potentially target erythropoiesis regulating factors. Of the six microRNAs selected for verification, three miRNAs (miR-152, miR-1388 and miR-16b) demonstrated suppressive functions on GATA1 and ALAS2, which are two factors important for erythroid differentiation, resulting in reduced numbers of erythroids in microinjected zebra fish embryos. Codon substitution analyses of the genes of the TGF-ß superfamily revealed signs of positive selection in TGF-ß1 and endoglin in the lineages leading to Antarctic notothenioids. Both genes are previously known to function in erythropoietic suppression. These findings implied a general trend of erythropoietic suppression in the cold-adapted notothenioid lineages through evolutionary modulation of the multi-functional TGF-ß signalling pathway. This trend is more pronounced in the haemoglobin-less icefish, which may pre-emptively hinder the otherwise defective erythroids from production.

Genetic Population Structure of Thunnus albacares in the Central Pacific Ocean Based on mtDNA COI Gene Sequences.

Thunnus albacares is an important fishery species throughout the world. Polymorphisms of sequence variations in mtDNA COI genes were assessed to explore the genetic differentiations among 11 populations of T. albacares sampled from the central Pacific Ocean. Sixty-one mtDNA haplotypes and 38 variable sites were detected. Analysis of mtDNA COI sequences revealed that tuna from the 11 localities were characterized by moderately high haplotype diversity (h = 0.650 ± 0.040), while sequence divergence between haplotypes was relatively low (π = 0.00364 ± 0.00044). Analyses of molecular variance and FST analysis supported that significant genetic differentiations existed between some of the sampled populations. Tests of neutral evolution and mismatch distribution analysis suggested that T. albacares might have experienced a population expansion, which possibly occurred within the last 0.82 million years. Our study unraveled the genetic structure of the extant population of T. albacares and addressed the related fishery management issues including fishery stock identification and management.

Genome sequencing of Coryphaenoides yaquinae reveals convergent and lineage-specific molecular evolution in deep-sea adaptation.

Abyssal (3501-6500 m) and hadal (>6500 m) fauna evolve under harsh abiotic stresses, characterized by high hydrostatic pressure, darkness and food shortage, providing unique opportunities to investigate mechanisms underlying environmental adaptation. Genomes of several hadal species have recently been reported. However, the genetic adaptation of deep sea species across a broad spectrum of ocean depths has yet to be thoroughly investigated, due to the challenges imposed by collecting the deep sea species. To elucidate the correlation between genetic innovation and vertical distribution, we generated a chromosome-level genome assembly of the macrourids Coryphaenoides yaquinae, which is widely distributed in the abyssal/hadal zone ranging from 3655 to 7259 m in depth. Genomic comparisons among shallow, abyssal and hadal-living species identified idiosyncratic and convergent genetic alterations underlying the extraordinary adaptations of deep-sea species including light perception, circadian regulation, hydrostatic pressure and hunger tolerance. The deep-sea fishes (Coryphaenoides Sp. and Pseudoliparis swirei) venturing into various ocean depths independently have undergone convergent amino acid substitutions in multiple proteins such as rhodopsin 1, pancreatic and duodenal homeobox 1 and melanocortin 4 receptor which are known or verified in zebrafish to be related with vision adaptation and energy expenditure. Convergent evolution events were also identified in heat shock protein 90 beta family member 1 and valosin-containing protein genes known to be related to hydrostatic pressure adaptation specifically in fishes found around the hadal range. The uncovering of the molecular convergence among the deep-sea species shed new light on the common genetic innovations required for deep-sea adaptation by the fishes.

Identification of two miRNAs regulating cardiomyocyte proliferation in an Antarctic icefish.

The hemoglobinless Antarctic icefish develop large hearts to compensate for reduced oxygen-carrying capacity, which serves as a naturally occurred model to explore the factors regulating cardiogenesis. Through miRNAome and microRNAome comparisons between an icefish (Chionodraco hamatus) and two red-blooded notothenioids, we discovered significant upregulation of factors in the BMP signaling pathways and altered expression of many miRNAs, including downregulation of 14 miRNAs in the icefish heart. Through knocking down of these miRNAs, we identified two of them, miR-458-3p and miR-144-5p, involved in enlarged heart development. The two miRNAs were found to regulate cardiomyocyte proliferation by targeting bone morphogenetic protein-2 (bmp2). We further validated that activation of the miRNA-bmp2 signaling in the fish heart could be triggered by hypoxic exposure. Our study suggested that a few miRNAs play important roles in the hypoxia-induced cardiac remodeling of the icefish which shed new light on the mechanisms regulating cardiomyocyte proliferation in heart.

Proteomic characterization and evolutionary analyses of zona pellucida domain-containing proteins in the egg coat of the cephalochordate, Branchiostoma belcheri.

BACKGROUND: Zona pellucida domain-containing proteins (ZP proteins) have been identified as the principle constituents of the egg coat (EC) of diverse metazoan taxa, including jawed vertebrates, urochordates and molluscs that span hundreds of millions of years of evolutionary divergence. Although ZP proteins generally contain the zona pellucida (ZP) structural modules to fulfill sperm recognition and EC polymerization functions during fertilization, the primary sequences of the ZP proteins from the above-mentioned animal classes are drastically different, which makes it difficult to assess the evolutionary relationships of ZP proteins. To understand the origin of vertebrate ZP proteins, we characterized the egg coat components of Branchiostoma belcheri, an invertebrate species that belongs to the chordate subphylum Cephalochordata. RESULTS: Five ZP proteins (BbZP1-5) were identified by mass spectrometry analyses using the egg coat extracts from both unfertilized and fertilized eggs. In addition to the C-terminal ZP module in each of the BbZPs, the majority contain a low-density lipoprotein receptor domain and a von Willebrand factor type A (vWFA) domain, but none possess an EGF-like domain that is frequently observed in the ZP proteins of urochordates. Fluorescence in situ hybridization and immuno-histochemical analyses of B. belcheri ovaries showed that the five BbZPs are synthesized predominantly in developing eggs and deposited around the extracellular space of the egg, which indicates that they are bona fide egg coat ZP proteins. BbZP1, BbZP3 and BbZP4 are significantly more abundant than BbZP2 and BbZP5 in terms of gene expression levels and the amount of mature proteins present on the egg coats. The major ZP proteins showed high polymorphism because multiple variants are present with different molecular weights. Sequence comparison and phylogenetic analysis between the ZP proteins from cephalochordates, urochordates and vertebrates showed that BbZP1-5 form a monophyletic group and share no significant sequence similarities with the ZP proteins of urochordates and the ZP3 subtype of jawed vertebrates. By contrast, small regions of homology were identifiable between the BbZP and ZP proteins of the non-jawed vertebrate, the sea lamprey Petromyzon marinus. The lamprey ZP proteins were highly similar to the ZP1 and ZP2 subtypes of the jawed vertebrates, which suggests that the ZP proteins of basal chordates most likely shared a recent common ancestor with vertebrate ZP1/2 subtypes and lamprey ZP proteins. CONCLUSIONS: The results document the spectra of zona pellucida domain-containing proteins of the egg coat of basal chordates. Particularly, the study provides solid evidence for an invertebrate origin of vertebrate ZP proteins and indicates that there are diverse domain architectures in ZP proteins of various metazoan groups.

MicroRNA-3148 inhibits glioma by decreasing DCUN1D1 and inhibiting the NF-kB pathway.

Glioma, which originates in the brain, is the most aggressive tumor of the central nervous system. It has been shown that microRNA (miRNA) controls the proliferation, migration and apoptosis of glioma cells. The objective of the present study was to measure microRNA-3148 (miR-3148) expression and investigate its impact on the pathogenetic mechanism of glioma. In the present study, reverse transcription-quantitative real-time PCR was employed to detect miR-3148 expression levels in glioma tissues and cell lines. Cell Counting Kit-8 assay, 5-ethynyl-2'-deoxyuridine assay, and Transwell migration assay were performed to assess the influence of miR-3148 on the malignant biological behavior of glioma cells. The biological functions of miR-3148 in glioma were examined via a xenograft tumor growth assay. Furthermore, the association between miR-3148 and DCUN1D1 was investigated via immunohistochemistry, dual-luciferase reporter assay and western blotting. It was observed that miR-3148 was expressed at low levels in glioma cells, and this represented a poor survival rate. In addition, an increased level of miR-3148 in cells and animal models inhibited glioma cell migration and proliferation. Moreover, miR-3148 decreased DCUN1D1 and curbed the nuclear factor κ enhancer binding protein (NF-κB) signaling pathway, thus decreasing the growth of glioma. Thus, miR-3148 is expressed within glioma tissues at low levels where it suppresses glioma by curbing the NF-κB pathway and lowering DCUN1D1.

Iron supplementation inhibits hypoxia-induced mitochondrial damage and protects zebrafish liver cells from death.

Acute hypoxia in water has always been a thorny problem in aquaculture. Oxygen and iron play important roles and are interdependent in fish. Iron is essential for oxygen transport and its concentration tightly controlled to maintain the cellular redox homeostasis. However, it is still unclear the role and mechanism of iron in hypoxic stress of fish. In this study, we investigated the role of iron in hypoxic responses of two zebrafish-derived cell lines. We found hypoxia exposed zebrafish liver cells (ZFL) demonstrated reduced expression of Ferritin and the gene fth31 for mitochondrial iron storage, corresponding to reduction of both intracellular and mitochondrial free iron and significant decrease of ROS levels in multiple cellular components, including mitochondrial ROS and lipid peroxidation level. In parallel, the mitochondrial integrity was severely damaged. Addition of exogenous iron restored the iron and ROS levels in cellular and mitochondria, reduced mitochondrial damage through enhancing mitophagy leading to higher cell viability, while treated the cells with iron chelator (DFO) or ferroptosis inhibitor (Fer-1) showed no improvements of the cellular conditions. In contrast, in hypoxia insensitive zebrafish embryonic fibroblasts cells (ZF4), the expression of genes related to iron metabolism showed opposite trends of change and higher mitochondrial ROS level compared with the ZFL cells. These results suggest that iron homeostasis is important for zebrafish cells to maintain mitochondrial integrity in hypoxic stress, which is cell type dependent. Our study enriched the hypoxia regulation mechanism of fish, which helped to reduce the hypoxia loss in fish farming.

Identification of Antibacterial Activity of Hepcidin From Antarctic Notothenioid Fish.

Hepcidin is a small peptide composed of signal peptide, propeptide, and the bioactive mature peptide from N terminal to C terminal. Mature hepcidin is an antibacterial peptide and iron regulator with eight highly conserved cysteines forming four intramolecular disulfide bonds, giving it a ß sheet hairpin-like structure. Hepcidin homologs are found in a variety of vertebrates, especially fish, and their diversity may be associated with different habitats and different levels of pathogens. Dissostichus mawsoni, an Antarctic notothenioid fish that lives in the coldest water unlike most places of the world, with at least two hepcidin variants with eight cysteines. We confirmed the formation process of activated mature hepcidins from D. mawsoni in Chinese hamster ovary (CHO) cell line, obtained recombinant hepcidin protein from prokaryotes, and characterized its binding ability and antibacterial activity against varying bacteria. The expression of hepcidin in CHO cell line showed that the prepropeptide of Dmhep_8cysV1 and Dmhep_8cysV2 cleavage into smaller mature peptide. The antibacterial assay and flow cytometry showed that Dmhep_8cysV1, Dmhep_8cysV2, and Drhep bound to different bacteria and killed them with different minimum inhibitory concentration. These data suggest that hepcidin plays an important role in the innate immunity of D. mawsoni and is of great value in improving resistance to pathogens.

Trace Elemental Analysis of the Exoskeleton, Leg Muscle, and Gut of Three Hadal Amphipods.

Hadal trenches are the deepest areas worldwide. Amphipods are considered a key factor in hadal ecosystems because of their important impacts on the hadal environment. Amphipods have benthic habits, and therefore, serve as good metal biomonitors. However, little is known about the hadal amphipod metal accumulations. In the present study, Alicella gigantea, Hirondellea gigas, and Scopelocheirus schellenbergi were sampled from the New Britain Trench (8824m, 7.02S 149.16E), Mariana Trench (10,839m, 11.38N 142.42E), and Marceau Trench (6690m, 1.42N 148.74E) in the West Pacific Ocean, respectively. The elemental concentrations of the three hadal amphipods were subsequently investigated. Nine trace elements (V, Cr, Mn, Co, Ni, Se, Mo, Ag, and Cd) of three tissues (exoskeleton, leg muscle, and gut) of the hadal amphipods were detected by using inductively coupled plasma mass spectrometry (ICP-MS) method. The concentrations of Cr, Cd, and Mn were comparably higher among those nine examined elements. The greatest accumulations of the elements Cr, Ag, and V in the exoskeleton and leg muscle were observed in H. gigas, and elements Mn, Co, and Se showed the highest accumulations in the gut in H. gigas among the three hadal amphipods. In addition, comparisons of the leg muscle trace element accumulation between the hadal amphipods and non-abyssal and shallow water decapoda and amphipoda species showed that the hadal amphipods possessed comparably higher concentrations of the trace elements Cd, Co, Mo, Ag, and V. This finding suggested a bottom-up effect of food availability and indicated the effects of human activities within the hadal environments. This study reveals the trace element bio-accumulation of three hadal amphipods, and suggests that deep-sea amphipods are potential indicator species for trace element bioavailability in the deep-sea environment.