Comparative Transcriptome Analysis of the Skin and the Peritoneal Wall Layer of Triplophysa stenura Distributed in High Elevations.

A total of 81,868 All-Unigenes were sequenced and assembled by the transcriptome in the dorsal skin, the lateral skin, and the peritoneal wall layer of Triplophysa stenura with a total assembly length of 123,827,585 bp, and 68,750 unigenes were annotated to seven functional databases. A total of 588 DEGs were screened between the dorsal and lateral skin, 17,097 DEGs were screened between the dorsal skin and the peritoneal wall layer, and 16,598 DEGs were screened between the lateral skin and the peritoneal wall layer. Most of DEGs in three tissues were annotated to GO terms related to cellular structures, binding, cellular processes, and catalytic activity. They were also annotated to KEGG pathways such as the MAPK signaling pathway, PI3K-Akt signaling pathway, Wnt signaling pathway, melanogenesis, tyrosine metabolism, and cell cycle. A total of twenty-three DEGs were found to be enriched in the melanin synthesis pathway by a local Blast comparison, of which nine DEGs were significantly upregulated in the peritoneal wall layer and six DEGs were significantly upregulated in the dorsal and lateral skin. The results suggest that these genes may be associated with the molecular mechanism of melanin synthesis in T. stenura, and the differential regulation of genes may be related to the differences in UVR intensity and tissue sites of melanin synthesis. Further investigation is needed on how these genes specifically regulate melanin synthesis.

Sexual dimorphism in Odontobutis sinensis brain-pituitary-gonad axis and liver highlighted by histological and transcriptomic approach.

In this study, sexual dimorphism in Chinese dark sleeper (Odontobutis sinensis) brain-pituitary-gonad axis and liver was highlighted by histological and transcriptomic approach. The results showed that there were two significant differences between males and females. Firstly, males grew larger and faster than females. Transcriptomic analysis and qPCR validation indicated that two key growth genes, insulin-like growth factor (igf) and 25-hydroxyvitamin D-1 alpha hydroxylase (cyp27b), were more highly detected in male liver than that in female liver. Secondly, histological analysis displayed that the liver in males showed an obvious ivory fatty phenomenon with more fat vacuoles and lipid droplet aggregation compared to that in females. Transcriptomic analysis indicated that the transcript level of vitellogenin (vtg) in male liver were significantly lower than that in female liver. After 17ß-estradiol (E2) treatment of primary cultured hepatocytes, the vtg mRNA expression was induced significantly, while dihydrotestosterone (DHT) treatment had little effect on it. Generally, this study will provide some ideas for further exploring the mechanism of sexual dimorphism in Odontobutis sinensis.

[Distribution and comparison of melanin in different tissues and organs of Triplophysa stenura and T. orientalis]. / ç»†å°¾é«˜åŽŸé³ å’Œä¸œæ–¹é«˜åŽŸé³ ä¸åŒç»„ç»‡å™¨å®˜é»‘è‰²ç´ çš„åˆ†å¸ƒå’Œæ¯”è¾ƒ.

We examined the distribution characteristics of melanin in different tissues and organs of Triplophysa stenura and T. orientalis by using histological method. The results showed that melanin was distributed in the head skin, dorsal skin, lateral skin, peritoneal kidney layer, spinal cord cavity wall, peritoneal wall layer, pericardial cavity wall, neurocranial cavity wall and eyes of both Triplophysa species. Melanin was not found in the abdomen skin, liver chorion, gonad capsule and spleen capsule. Melanin was distributed in the dermis layer and subcutaneous layer in the skin, and in the endothelial layer or wall layer in other tissues and organs. Melanin was mainly distributed on the back, with sparse and symmetrical distribution on both sides of the body. Melanin was more abundant and densely distributed on the dorsal and lateral skin with stripes than areas without stripes. Melanin in the no stripes skin was only partially aggregated or formed intermittently distributed melanin patches. The distribution of melanin in different tissues and organs of the same Triplophysa species was different, and the percentage of distribution area and thickness of melanin layer were significantly different. However, the distribution characteristics of melanin in the same tissues and organs were similar in two Triplophysa species. The distribution of melanin in the tissues and organs of both species were related to the intensity of ultraviolet radiation they received, which was an adaptation to the intense ultraviolet radiation environment of the plateau.

[Distribution of melanin in the larvae of Schizothorax o'connori]. / å¼‚é½¿è£‚è ¹é±¼ä»”é±¼çš„é»‘è‰²ç´ åˆ†å¸ƒ.

We examined the distribution of melanin during the development of the larvae of Schizothorax o'connori except the eyes with histological method. The results showed that after hatching, the appearance sequence of melanin in different organs were following an order of the outer membrane of neurocranium, the pericardial cavity and the dorsal skin, and the peritoneum and the spinal cord. Specifically, melanin appeared in the outer membrane of neurocranium around 5 DAH (days after hatching), in the pericardial cavity and the back skin at 7 DAH, and in the peritoneum and the spinal cord at 10 DAH. Melanin was found in the skin and internal organs (the outer membrane of neurocranium, the pericardial cavity, the peritoneum, the spinal cord) of S. o'connori at 10 DAH, which was mainly distributed on the back. The appearance and distribution of melanin in the postembryonic development of S. o'connori might be related to the high ultraviolet radiation. Our results could provide reference for further research on the UV protection mechanism of melanin for fish and provide theoretical support for the optimization of rearing conditions for larvae in the plateau.

Genome and population evolution and environmental adaptation of Glyptosternon maculatum on the Qinghai-Tibet Plateau.

Persistent uplift means the Qinghai-Tibet Plateau (QTP) is an ideal natural laboratory to investigate genome evolution and adaptation within highland environments. However, how paleogeographic and paleoclimatic events influence the genome and population of endemic fish species remains unclear. Glyptosternon maculatum is an ancient endemic fish found on the QTP and the only critically endangered species in the Sisoridae family. Here, we found that major transposons in the G. maculatum genome showed episodic bursts, consistent with contemporaneous geological and climatic events during the QTP formation. Notably, histone genes showed significant expansion in the G. maculatum genome, which may be mediated by long interspersed nuclear elements (LINE) repetitive element duplications. Population analysis showed that ancestral G. maculatum populations experienced two significant depressions 2.6 million years ago (Mya) and 10 000 years ago, exhibiting excellent synchronization with Quaternary glaciation and the Younger Dryas, respectively. Thus, we propose that paleogeography and paleoclimate were dominating driving forces for population dynamics in endemic fish on the QTP. Tectonic movements and temperature fluctuation likely destroyed the habitat and disrupted the drainage connectivity among populations. These factors may have caused severe bottlenecks and limited migration among ancestral G. maculatum populations, resulting in the low genetic diversity and endangered status of the species today.

The complete mitochondrial genomes of two Chinese endemic cave fishes, Sinocyclocheilus longibarbarus and Sinocyclocheilus punctatus (Cypriniformes: Cyprinidae).

Sinocyclocheilus longibarbarus and Sinocyclocheilus punctatus were collected from a karst cave Libo County, southwest of China. The two Sinocyclocheilus species can be distinguished obviously by external morphological characteristics. In this study, the complete mitochondrial genome sequences of two species were assembled, and both sequences reflected gene organization typical for mitochondrial DNA of the genus Sinocyclocheilus, comprising of 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs), and a large non-coding control region. Phylogenetic analysis showed that S. punctatus was first clustered together with S. mutipunctatus, and S. longibarbarus was closely related to S. yishanensis. The complete mitogenome of two species may provide useful information for the further taxonomic and phylogenetic studies.

Effect of pre-strain on microstructure and micro-yield properties of Al-Cu-Li alloy.

In this study, Al-Cu-Li alloys were pre-strained to various plastic strains before ageing. The T1 (Al2CuLi) precipitates and dislocation density in various pre-strained Al-Cu-Li alloys were analyzed with transmission electron microscopy (TEM) and X-ray diffraction (XRD) technology respectively. The micro-yield strength (Micro-YS) of tested alloys was measured and the strengthening mechanism was discussed. It is found that the pre-strain increases the dislocation density, promotes the precipitation and inhibits the growth of T1 precipitates. The precipitation strengthening of T1 precipitates is higher than strain strghening of dislocations and it first increase then decrease whearas the strain strenthening continuously increase with pre-strain. The improvement in strength of Al-Cu-Li alloy caused by pre-straining is mainly due to strain strengthening rather than precipitation strengthening. Therefore, although pre-strain improves the macro-yield strength (Macro-YS) as well as the Micro-YS, excessive pre-strains are not conducive to the improvement of the Micro-YS. The pre-strain of 2% is a promising method to simultaneously improve the Micro-YS and Macro-YS of Al-Cu-Li alloys due to its synergistic improvement effect on precipitation strengthening and strain strengthening. Macro-YS and Micro-YS of pre-strained Al-Cu-Li alloys can be estimated with a simple strength mode. However, the larger error between the estimated and measured Micro-YS suggests that the accurate estimation of Micro-YS requires further to consider the adverse effect of mobile dislocations caused by pre-strain on the Micro-YS.

Effect of external stress on the microstructure and mechanical properties of creep-aged Al-Cu-Li-Ag alloy.

The effects of external stress on the precipitation of T1 precipitates and mechanical properties of creep-aged Al-Cu-Li-Ag alloys are investigated. Promotion mechanisms of external stress to the precipitation of T1 precipitates are discussed. It is found that external stress significantly promotes the precipitation and improves the distribution of the T1 precipitates in the creep-aged alloys. There is a threshold stress, close to the yield stress, that has only a limited promotion effect on the precipitation of T1 precipitates. The external stress below and above the threshold stress promotes the precipitation of T1 precipitates by two different mechanisms. One is the promotion mechanism of lattice distortion produced by the elastic stress. Another is the promotion mechanism of multiplication of dislocations produced by the plastic stress. Both elastic and plastic external stress can synergistically improve the strength and ductility. Especially, the plastic external stress resulted in the best improvement to ductility of creep-aged alloys. Hence, the creep ageing with plastic external stress is an alternative method to synergistically improve the strength and ductility of Al-Cu-Li-Ag alloys. However, it is necessary to avoid using excessive plastic stress for the creep ageing because it may cause creep damage and degrade its mechanical properties.

Genome of Tetraploid Fish Schizothorax o'connori Provides Insights into Early Re-diploidization and High-Altitude Adaptation.

Whole-genome duplications (WGDs) of Schizothoracinae are believed to have played a significant role in speciation and environmental adaptation on the Qinghai-Tibet Plateau (QTP). Here, we present a genome for Schizothorax o'connori, a QTP endemic fish and showed the species as a young tetraploid with a recent WGD later than ∼1.23 mya. We exhibited that massive insertions between duplicated genomes caused by transposon bursts could induce mutagenesis in adjacent sequences and alter the expression of neighboring genes, representing an early re-diploidization process in a polyploid genome after WGD. Meanwhile, we found that many genes involved in DNA repair and folate transport/metabolism experienced natural selection and might contribute to the environmental adaptation of this species. Therefore, the S. o'connori genome could serve as a young tetraploid model for investigations of early re-diploidization in polyploid genomes and offers an invaluable genetic resource for environmental adaptation studies of the endemic fish of the QTP.

Atractylodes macrocephala polysaccharides regulate the innate immunity of colorectal cancer cells by modulating the TLR4 signaling pathway.

BACKGROUND: It has been well-recognized that the polysaccharides from Atractylodes macrocephala (PAM) are immune system enhancers, which can facilitate the proliferation of lymphocytes and stimulate immune cells. Nevertheless, the antitumor effects of PAM and their molecular mechanisms remain unclear. AIM: Our research aimed to evaluate the anti-cancer effects of PAM on colorectal cancer (CRC). METHODS: We tested the effects of PAM on the growth and proliferation of CRC cells and macrophages by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The pro-inflammatory cytokines expression and secretion was analyzed by real-time RT-PCR and ELISA assay. We also used MC38 cells xenograft model to test the anti-cancer effects of PAM in vivo. RESULTS: We found that although PAM treatment did not significantly affect the growth of CRC cells or enhance the proliferation of bone marrow-derived macrophages (BMDMs), it could enhance the phagocytosis of BMDMs by CRC cells. Biochemical tests and immunoblotting assays revealed that exposing BMDMs to PAM promoted the production of interleukin-6 (IL-6), interferon λ (IFN λ), tumor necrosis factor α (TNF-α), and nitric oxide (NO) through the MyD88/TLR4-dependent signaling pathway. One noteworthy observation is that PAM treatment could significantly prevent tumorigenesis of MC38 cells in C57BL/6J mice and increase the survival duration of mice with tumors, without influence on the weight of those mice. However, the anti-cancer effects of PAM were compromised in TLR4 KO mice, further suggesting that TLR4 signaling plays a vital role in the anti-cancer effects of PAM. CONCLUSION: Therefore, PAM may prove to be a potential candidate in cancer immunotherapy.

Morphological Characteristics and Comparative Transcriptome Analysis of Three Different Phenotypes of Pristella maxillaris.

Pristella maxillaris is known as the X-ray fish based on its translucent body. However, the morphological characteristics and the molecular regulatory mechanisms of these translucent bodies are still unknown. In this study, the following three phenotypes, a black-and-gray body color or wild-type (WT), a silvery-white body color defined as mutant I (MU1), and a fully transparent body with a visible visceral mass named as mutant II (MU2), were investigated to analyze their chromatophores and molecular mechanisms. The variety and distribution of pigment cells in the three phenotypes of P. maxillaris significantly differed by histological assessment. Three types of chromatophores (melanophores, iridophores, and xanthophores) were observed in the WT, whereas MU1 fish were deficient in melanophores, and MU2 fish lacked melanophores and iridophores. Transcriptome sequencing of the skin and peritoneal tissues of P. maxillaris identified a total of 166,089 unigenes. After comparing intergroup gene expression levels, more than 3,000 unigenes with significantly differential expression levels were identified among three strains. Functional annotation and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of the differentially expressed genes (DEGs) identified a number of candidates melanophores and iridophores genes that influence body color. Some DEGs that were identified using transcriptome analysis were confirmed by quantitative real-time PCR. This study serves as a global survey of the morphological characteristics and molecular mechanism of different body colors observed in P. maxillaris and thus provides a valuable theoretical foundation for the molecular regulation of the transparent phenotype.

Chromosome-level genome assembly of Triplophysa tibetana, a fish adapted to the harsh high-altitude environment of the Tibetan Plateau.

Triplophysa is an endemic fish genus of the Tibetan Plateau in China. Triplophysa tibetana, which lives at a recorded altitude of ~4,000 m and plays an important role in the highland aquatic ecosystem, serves as an excellent model for investigating high-altitude environmental adaptation. However, evolutionary and conservation studies of T. tibetana have been limited by scarce genomic resources for the genus Triplophysa. In the present study, we applied PacBio sequencing and the Hi-C technique to assemble the T. tibetana genome. A 652-Mb genome with 1,325 contigs with an N50 length of 3.1 Mb was obtained. The 1,137 contigs were further assembled into 25 chromosomes, representing 98.7% and 80.47% of all contigs at the base and sequence number level, respectively. Approximately 260 Mb of sequence, accounting for ~39.8% of the genome, was identified as repetitive elements. DNA transposons (16.3%), long interspersed nuclear elements (12.4%) and long terminal repeats (11.0%) were the most repetitive types. In total, 24,372 protein-coding genes were predicted in the genome, and ~95% of the genes were functionally annotated via a search in public databases. Using whole genome sequence information, we found that T. tibetana diverged from its common ancestor with Danio rerio ~121.4 million years ago. The high-quality genome assembled in this work not only provides a valuable genomic resource for future population and conservation studies of T. tibetana, but it also lays a solid foundation for further investigation into the mechanisms of environmental adaptation of endemic fishes in the Tibetan Plateau.

The sequence and de novo assembly of Oxygymnocypris stewartii genome.

Animal genomes in the Qinghai-Tibetan Plateau provide valuable resources for scientists to understand the molecular mechanism of environmental adaptation. Tibetan fish species play essential roles in the local ecology; however, the genomic information for native fishes was still insufficient. Oxygymnocypris stewartii, belonging to Oxygymnocypris genus, Schizothoracinae subfamily, is a native fish in the Tibetan plateau living within the elevation from roughly 3,000 m to 4,200 m. In this report, PacBio and Illumina sequencing platform were used to generate ~385.3 Gb genomic sequencing data. A genome of about 1,849.2 Mb was obtained with a contig N50 length of 257.1 kb. More than 44.5% of the genome were identified as repetitive elements, and 46,400 protein-coding genes were annotated in the genome. The assembled genome can be used as a reference for future population genetic studies of O. stewartii and will improve our understanding of high altitude adaptation of fishes in the Qinghai-Tibetan Plateau.

Polysaccharide of atractylodes macrocephala inhibits the growth of mice in-situ colon cancer HT-29 cell xenograft via activating immune cells / 中国肿瘤生物治疗杂志

@# Objective: To investigate the effect and mechanism of polysaccharide of atractylodes macrocephala (PAM) on the growth of colon cancer cells in mice bearing in-situ colon cancer transplantation tumor. Methods: 1×107 colon cancer HT-29 cells labeled with luciferase were injected into colon serosa of the mice to establish the in-situ colon cancer transplantation tumor model. When the tumor volume reached 230 mm3, the mice were given 30 mg/kg PAM (PAM group) or equal volume of normal saline (Control group) by gavage for 10 consecutive days. The effect of PAM on the growth of colon cancer cells in mice was tested by in vivo tumor imaging technology. The expressions of MHCII and IL-12 in granulocytes, dendritic cells and macrophages, the activation of lymphocytes, and IFN-γ expression in CD4+ and CD8+ cells of tumor tissues were detected by Flow cytometry. Results: PAM significantly inhibited the growth of colon cancer cells in mice bearing in-situ colon cancer transplantation tumor (P<0.01). PAM activated immune cells though increasing the expression levels of MHCII and IL-12 in dendritic cells and macrophages (both P<0.01). PAM significantly increased the frequency of CD8+ cells, NK cells, CD44+/NK cells and CD44+/CD4+ cells in tumor tissues and the number of CD8+ cells and NK cells per unit volume (all P<0.01). PAM significantly increased the IFN-γ secretion of CD4+ and CD8+ cells (both P<0.01), too. Conclusion: PAM inhibits the growth of colon cancer by activating immune cells in tumor tissues of mice bearing in-situ colon cancer transplantation tumor.

The draft genome of blunt snout bream (Megalobrama amblycephala) reveals the development of intermuscular bone and adaptation to herbivorous diet.

The blunt snout bream Megalobrama amblycephala is the economically most important cyprinid fish species. As an herbivore, it can be grown by eco-friendly and resource-conserving aquaculture. However, the large number of intermuscular bones in the trunk musculature is adverse to fish meat processing and consumption. As a first towards optimizing this aquatic livestock, we present a 1.116-Gb draft genome of M. amblycephala, with 779.54 Mb anchored on 24 linkage groups. Integrating spatiotemporal transcriptome analyses, we show that intermuscular bone is formed in the more basal teleosts by intramembranous ossification and may be involved in muscle contractibility and coordinating cellular events. Comparative analysis revealed that olfactory receptor genes, especially of the beta type, underwent an extensive expansion in herbivorous cyprinids, whereas the gene for the umami receptor T1R1 was specifically lost in M. amblycephala. The composition of gut microflora, which contributes to the herbivorous adaptation of M. amblycephala, was found to be similar to that of other herbivores. As a valuable resource for the improvement of M. amblycephala livestock, the draft genome sequence offers new insights into the development of intermuscular bone and herbivorous adaptation.

Phylogeography and population genetics of Schizothorax o'connori: strong subdivision in the Yarlung Tsangpo River inferred from mtDNA and microsatellite markers.

The Qinghai-Tibet Plateau (QTP) is a biodiversity hotspot, resulting from its geological history, contemporary environment and isolation. Uplift of the QTP and Quaternary climatic oscillations are hypothesised to have influenced the genetic diversity, population structure and dynamics of all QTP endemic species. In this study, we tested this hypothesis by assaying variation at two mitochondrial DNA regions (cytochrome b and control region) and at 12 microsatellite loci of seven populations of the endemic fish, Schizothorax o'connori from the Yarlung Tsangpo River (YLTR) on the QTP. Analyses revealed one group of six populations to the west, above the Yarlung Tsangpo Grand Canyon (YTGC), and a second group to the east below the YTGC. Estimates of the timing of this east-west split indicate that these groups represent evolutionarily significant units that have evolved separately and rapidly in the middle Pleistocene, at the time of the Kunlun-Huanghe Movement A Phase and the Naynayxungla glaciation. Population dynamic analyses indicate that S. o'connori experienced a pronounced late Pleistocene expansion during the last interglacial period. The results of this study support the hypotheses that the QTP uplift and Quaternary climatic oscillations have played important roles in shaping the population genetics and dynamics of this endemic fish.

Molecular Characterization and Expression Analyses of the Complement Component C8α, C8ß and C9 Genes in Yellow Catfish (Pelteobagrus fulvidraco) after the Aeromonas hydrophila Challenge.

The complement components C8α, C8ß and C9 have important roles in the innate immune system against invading microorganisms. Partial cDNA sequences of the Pf_C8α, Pf_C8ß and Pf_C9 genes (Pf: abbreviation of Pelteobagrus fulvidraco) were cloned from yellow catfish. The Pf_C8α, Pf_C8ß and Pf_C9 genes showed the greatest amino acid similarity to C8α (54%) and C8ß (62%) of zebrafish and to C9 (52%) of grass carp, respectively. Ontogenetic expression analyses using real-time quantitative PCR suggested that the three genes may play crucial roles during embryonic and early larval development. The mRNA expressions of the three genes were all at the highest levels in liver tissue, and at lower or much lower levels in 16 other tissues, demonstrating that the liver is the primary site for the protein synthesis of Pf_C8α, Pf_C8ß and Pf_C9. Injection of Aeromonas hydrophila led to up-regulation of the three genes in the spleen, head kidney, kidney, liver and blood tissues, indicating that the three genes may contribute to the host's defense against invading pathogenic microbes. An increased understanding of the functions of the Pf_C8α, Pf_C8ß and Pf_C9 genes in the innate immunity of yellow catfish will help enhance production of this valuable freshwater species.

The complete mitochondrial genome of perch Perca fluviatilis (Perciformes: Percidae).

In this study, we cloned and sequenced the complete mitochondrial genome of Perca fluviatilis. It was a circular double-stranded DNA molecule of 16,537 base pairs (bp) in length and consists of 13 protein-coding genes, 22 tRNA genes, two ribosomal RNA genes and two main non-coding regions (the control region and the origin of the light strand replication). The mitogenome of shared common features with those of other toleosts in terms of gene order and base composition. All protein-coding genes were initiated with ATG except for COX 1, which began with GTG instead. However, the termination codons of 13 protein-coding genes are varied with TAG, TAA or T. This mitogenome sequence data would contribute to better understanding phylogenetic relationships and population genetics of the family Percidae.

Complete mitochondrial genome of the Chinese bitterling Acheilognathus macropterus (Cypriniformes: cyprinidae).

Acheilognathus macropterus belongs to the family Acheilognathinae. In the present study, we obtain the complete mitochondrial genome of the Chinese bitterling A. macropterus by PCR amplification and DNA sequencing. It is a circular double-stranded DNA molecule of 16,773 bp in length, containing 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and two main non-coding regions (the control region and the origin of the light strand replication). The gene composition and order of which are similar to most other vertebrates. Compared with Acanthorhodeus macropterus, they share 98.90% nucleotide sequences similarity, and the biggest nucleotide sequence discrepancy between homologous genes are observed in ND5 gene. The molecular data we presented here could provide useful information for the studies on species identification, evolutionary relationships and population genetics of the Acheilognathinae.

Complete mitochondrial genome of Macropodus ocellatus (Perciformes: Anabantidae: Macropodusinae).

In this study, we cloned and sequenced the complete mitochondrial genome of Macropodus ocellatus. The genome is a circular molecule of 16,501 bp in length and consists of 13 protein-coding genes, 22 tRNA genes, 2 ribosomal RNA genes and 2 main non-coding regions (the control region and the origin of the light strand replication). The gene composition and order of which were similar to most other vertebrates. All protein-coding genes are initiated with ATG except for COX 1, which begin with GTG instead. However, the termination codons of 13 protein-coding genes are varied with TAA, TA, T or AGG. This mitogenome sequenc data would contribute to better understanding species identification, evolutionary relationships and population genetics of the Macropodus.