Integrated transcriptome and miRNA sequencing analyses reveal that hypoxia stress induces immune and metabolic disorders in gill of genetically improved farmed tilapia (GIFT, Oreochromis niloticus).

The survival and growth of fish are significantly impacted by a hypoxic environment (low dissolved oxygen). In this study, we compared tissue structure, physiological changes, and mRNA/miRNA transcriptome, in gills of genetically improved farmed tilapia (GIFT, Oreochromis niloticus) between the hypoxic group (DO: 0.55 mg/L, HG) and the control group (DO: 5 mg/L, CG). The results showed that the gill filaments in the hypoxic group showed curling, engorgement, and apoptotic cells increased, and that exposure for 96 h resulted in a reduction in the antioxidant capacity. We constructed and sequenced miRNA and mRNA libraries from gill tissues of GIFT at 96 h of hypoxia stress. Between the HG and CG, a total of 14 differentially expressed (DE) miRNAs and 1557 DE genes were obtained. GO and KEGG enrichment showed that DE genes were mainly enriched in immune and metabolic pathways such as natural killer cell mediated cytotoxicity, steroid biosynthesis, primary immunodeficiency, and synthesis and degradation of ketone bodies. Based on the results of mRNA sequencing and screening for miRNA-mRNA pairs, we selected and verified six DE miRNAs and their probable target genes. The sequencing results were consistent with the qRT-PCR validation results. The result showed that under hypoxia stress, the innate immune response was up-regulated, and the adaptive immune response was down-regulated in the gill of GIFT. The synthesis of cholesterol in gill cells is reduced, which is conducive to the absorption of solvent oxygen. These findings offer fresh information about the processes of fish adaptation to hypoxic stress.

Transcriptome changes of Takifugu obscurus liver after acute exposure to phenanthrene.

Phenanthrene (Phe) is a model compound in polycyclic aromatic hydrocarbon (PAH) research. Reportedly, Phe treatment induced oxidative stress and histological disorders to Takifugu obscurus liver. In this study, to further explore the molecular responses of T. obscurus liver to Phe exposure, transcriptome sequencing was applied to compare mRNA transcription profiles between Phe treatment and the control. Compared with the control, 1,581 and 1,428 genes were significantly upregulated and downregulated in Phe treatment, respectively. Further analysis revealed that Phe treatment mainly upregulated genes in Ras-MAPK and PI3K-akt signaling pathways, which represented insulin resistance and further activated the FOXO signaling pathway. The triacylglycerol biosynthesis was promoted but the gluconeogenesis process was inhibited in response to Phe treatment, demonstrating that Phe exposure disturbed the sugar and lipid metabolism. Moreover, Phe treatment upregulated the Apelin-APJ and ErbB signaling pathways, promoting angiogenesis in T. obscurus liver. Insulin resistance, promoted triacylglycerol biosynthesis, and angiogenesis might explain the molecular mechanisms underlying carcinogenic toxicity of Phe. Overall, this study provides new insights to understand the environmental risk of Phe to fishes.

Reappraise role of No. 10 lymphadenectomy for proximal gastric cancer in the era of minimal invasive surgery during total gastrectomy: a pooled analysis of 4 prospective trial.

BACKGROUND: For patients with locally advanced proximal gastric cancer (LAPGC), the individualized selection of patients with highly suspected splenic hilar (No. 10) lymph node (LN) metastasis to undergo splenic hilar lymphadenectomy, is a clinical dilemma. This study aimed to re-evaluate the feasibility and safety of laparoscopic spleen-preserving splenic hilar lymphadenectomy (LSPSHL) and to identify the population who would benefit from it. METHODS: A total of 1068 patients (D2 group = 409; D2 + No. 10 group = 659) who underwent laparoscopic total gastrectomy from four prospective trials between January 2015 and July 2019 were analyzed. RESULTS: No significant difference in the incidence (16.9% vs. 16.4%; P = 0.837) of postoperative complications were found between the two groups. The metastasis rate of No. 10 LN among patients in the D2 + No. 10 group was 10.3% (68/659). Based on the decision tree, patients with LAPGC with tumor invading the greater curvature (Gre), patients with non-Gre-invading LAPGC with a tumor size > 5 cm and clinical positive locoregional LNs were defined as the high-priority No. 10 dissection group. The metastasis rate of No. 10 LNs in the high-priority group was 19.4% (41/211). In high-priority group, the 3-year overall survival of the D2 + No. 10 group was better than that of the D2 group (74.4% vs. 42.1%; P = 0.005), and the therapeutic index of No. 10 was higher than the indices of most suprapancreatic stations. CONCLUSIONS: LSPSHL for LAPGC is safe and feasible when performed by experienced surgeons. LSPSHL could be recommended for the high-priority group patients even without invasion of the Gre.

Toll-like receptors (TLRs) respond to tributyltin chloride (TBT-Cl) exposure in the river pufferfish (Takifugu obscurus): Evidences for its toxic injury function.

Tributyltin chloride (TBT-Cl) residual in water body had become a noticeable ecological problem for aquatic ecosystems. Toll-like receptors (TLRs) are an ancient family of pattern recognition receptors that play key roles in detecting nonself antigens and immune system activation. In this study, we explored the effect of TBT-Cl exposure on four TLRs expression in river pufferfish, Takifugu obscurus. The four T. obscurus Toll-like receptors (To-TLRs) contained different types of domains such as leucine-rich repeats (LRRs), leucine-rich repeats, typical subfamily (LRR_TYP) and other special domains. The To-TLRs mRNA transcripts expressed in all tissues, also To-TLR2 was investigated with higher level in kidney, as well as To-TLR3 in kidney, while To-TLR18 in liver and To-TLR22 in intestine. After the acute and chronic exposure of TBT-Cl, To-TLR2 and To-TLR3 mRNA transcripts were significantly down-regulated in gill. However, To-TLR18 and To-TLR22 were significantly up-regulated in gill and liver. Moreover, the histology and immunohistochemistry (IHC) results showed the different injury degrees of TBT-Cl in liver and gill and implied the cytoplasm reorganization after TBT-Cl stress and the function of immunoregulation for To-TLRs to TBT-Cl exposure. All the results indicated that To-TLRs might involve in sensing and mediating innate immune responses caused by TBT-Cl for keeping detoxification homeostasis.

Molecular cloning and characteristics of DnaJa1and DnaJb1 in Coilia nasus: possible function involved in oogenesis during spawning migration.

BACKGROUND: Coilia nasus oogenesis/spawning migration is a well-defined synchronous arrangement process. DnaJs are indispensable molecular chaperones for oogenesis process. However, how DnaJs involved the anadromous spawning migration mechanism is outstanding and plausible. RESULTS: In this regard, two DnaJs (Cn-DnaJa1 and Cn-DnaJb1) are cloned from the Coilia nasus's ovary. Their structure both contains J domain, G/F domain and ZF domain. Their mRNA transcripts were found extensively expressed in all the sampled tissues and significantly highly in gonads, which probably mean that DnaJs involved in C. nasus's gonad development basal metabolic processes. In the process of spawning migration, Cn-DnaJa1 and Cn-DnaJb1 mRNA transcripts were also expressed with significant differences during oogenesis with highest levels in the development phase, and maintaining high levels during the multiplication, mature and spawning phase. Further study showed that the DnaJa1and DnaJb1protein have high distribution in the onset phase and mainly distributed in the oocyte cytoplasm especially during the migration development phase's. CONCLUSIONS: This experiment study demonstrated that DnaJs participate in reproductive regulation during the spawning migration process in C. nasus and possibly play a vital role in the ovary development process. These findings also provided a base knowledge for further molecular mechanism study of spawning migration.

Comparative transcriptomics analysis of the river pufferfish (Takifugu obscurus) by tributyltin exposure: Clues for revealing its toxic injury mechanism.

TBT residual in water had become a noticeable ecological problem for aquatic ecosystems. The river pufferfish (Takifugu obscurus) is a kind of an anadromous fish species and widely distributed in the East China Sea and the Yellow Sea. Because of the water contamination, the pufferfish wild resource had a sudden decline in recent years. Therefore, the study on the response of pufferfish to the TBT exposure may contribute to reveal toxic injury mechanism of T. obscurus under TBT exposure. In this study, the transcriptional library of T. obscurus liver and gill was constructed and sequenced by an improved Illumina HiseqX10 high-throughput sequencing platform under different concentrations of TBT acute stress. The blood cell numbers distinctly decreased after TBT exposure, showing the adverse effects of TBT invasion and self-adjusting ability of the pufferfish. The production of reactive oxygen species increased, demonstrating the oxidation resistance of T. obscurus when exposed to TBT. The obtained data were compared with the genome data of Takifugu rubripes and transcriptional resource database. On this basis, gene function annotation, analysis and classification were carried out by bioinformatics method, and differential genes related to toxic injury function were screened out. Meanwhile, new toxic related genes and related signal pathways were sought to provide new theoretical guidance for the pathogenesis of T. obscurus exposed to TBT. This study not only enriched the transcriptome data of T. obscurus under environmental stress, but also provided a new research method for the response mechanism of T. obscurus under the stimulation of environmental factors.

Changes of gonadotropin-releasing hormone receptor 2 during the anadromous spawning migration in Coilia nasus.

BACKGROUND: An increase in the activity of the pituitary-gonad axis (PG-axis) and gonad development are essential for the onset of spawning migration in teleosts. In the fish Coilia nasus, gonad development and spawning migration up the Yangtze River occurs by the end of each summer. We hypothesized that gonadotropin releasing hormones receptor 2 (GnRH-R2), which together produce a signal that interacts with the PG-axis, may help to regulate spawning migration processes. RESULTS: In this regard, we (1) characterized the gonadosomatic index (GSI) in the anadromous fish C. nasus; (2) analyzed the GnRH-R2 mRNA expression levels in ovary and brain, and concentrations in the serum; and (3) identified the GnRH-R2 protein distribution in the brain and ovaries. We found strong relationships between all of these indices. CONCLUSIONS: The results indicate that GnRH-R2 could act together to promote spawning during the anadromous migration. There is some evidence that the GnRH-R2 gene expression levels and protein distributions change in association with the migratory behavior.

Molecular cloning and expression analysis of chymotrypsin-like serine protease from the redclaw crayfish (Cherax quadricarinatus): a possible role in the junior and adult innate immune systems.

A novel chymotrypsin-like serine protease (CLSP) was isolated from the hepatopancreas of the redclaw crayfish Cherax quadricarinatus (Cq-chy). The full-length cDNA of Cq-chy contains 951 nucleotides encodes a peptide of 270 amino acids. The mature peptide comprising 223 amino acids contains the conserved catalytic triad (H, D, and S). Similarity analysis showed that Cq-chy shares high identity with chymotrypsins from the fiddler crab; Uca pugilator. Cq-chy mRNA expression in C. quadricarinatus was shown to be: (a) tissue-related with the highest expression in the hepatotpancreas and widely distributed, (b) highly responsive in the hepatopancreas to White Spot Syndrome Virus (WSSV) challenge, and (c) differently regulated in immature and adult crayfish. In this study we successfully isolated Cq-chy. Our observations indicate that Cq-chy is differently involved in the immature and adult innate immune reactions, thus suggesting a role for CLSPs in the invertebrate innate immune system.

Discovery of the indicator role of period 2 in yellow catfish (Pelteobagrus fulvidraco) food intake during early life development stages.

The early development stages of fish are a highly ordered and tightly regulated, involving many circadian rhythm-related gene and protein processes. Nonetheless, there are few reports on the effects of circadian clock genes on the early development stages of fish. We studied Pelteobagrus fulvidraco Period 2 (Pf-Per 2) gene structures and expression patterns during the early life stages of development, including the fertilized embryo, yolk absorption, preliminary food, rotifer breeding, and mixed food stages. cDNA of Pf-Per 2 is 4593 bp in length, with 357 bp 5'-untranslated region (5'UTR), 216 bp 3'UTR. The 4020 bp open reading frame consists of 1339 encoded amino acids. By multiple sequence alignment and phylogenetic analysis, the sequence was found to demonstrate high similarity to humans, rodents, microorganisms, and other fish species. Expression patterns of mRNA transcripts showed existence of rhythmic oscillations in the yellow catfish during the early development phase. The higher expression level of Per 2 is obviously present in the early embryonic development stage; the continuous downward trend of Per 2 was observed in the embryonic development and yolk nutrition absorption stages; additionally, the expression of Per 2 mRNA was significantly increased during individual development, rotifer breeding, and mixed food stages. Moreover, immunohistochemistry studies revealed strongest immune-labeled positive signals of Per 2 proteins mainly located in the cytoplasm of the olfactory bulb cell. Our findings reveal Pf-Per 2 serves important functions and may be useful as an indicator of P. fulvidraco early life development and initial food intake process stages.

FoxL2 combined with Cyp19a1a regulate the spawning upstream migration in Coilia nasus.

FoxL2 is a member of the forkhead/HNF-3-related family of transcription factors which provides tissue specific gene regulation. It is known to regulate ovarian aromatase, which plays a crucial role in ovarian development and mature. To understand the role of FoxL2/ovarian aromatase encoded gene Cyp19a1a during ovarian development and recrudescence, we identified cDNA characteristics of FoxL2 and Cyp19a1a, analyzed its temporal expression both at transcript and protein levels in the anadromous fish, Coilia nasus. Tissue distribution pattern revealed that FoxL2 mRNA expression level was highest in ovary, while Cyp19a1a mRNA was highest in brain. During the upstream migration cycle, in ovary, the FoxL2 mRNA temporal expression peaked at the multiplication stage (stage III in May), the Cyp19a1a mRNA expression peaked at the onset stage (stage I in March). It was found that their mRNA transcripts were maintained at high level during the migration stage (from stage I in March to stage VI in July). Additionally, the strongest immunolabeling positive signals of Cyp19a1a and FoxL2 proteins were mainly found in the cytoplasm of olfactory bulb cell, stratum granulare and neurogliocyte cells and development stage oocytes. Data indicated that FoxL2 and Cyp19a1a were inducible and functional in the C. nasus ovary development and migration process. Therefore, the present results can be regarded as evidence for indispensable roles of FoxL2 and Cyp19a1a in the ovary development and migratory behavior at gene expression patterns and encoded protein distribution level.

Toxic function of CD28 involving in the TLR/MyD88 signal pathway in the river pufferfish (Takifugu obscurus) after exposed to tributyltin chloride (TBT-Cl).

Cluster of differentiation 28 (CD28) is a co-stimulatory receptor found on the surface of T cells. Takifugu obscurus is a kind of anadromous fish species. In this study, the full-length sequence of To-CD28 was obtained, including a 672-bp open reading frame that encodes a peptide chain of 223 amino acids. The phylogenetic analysis showed that To-CD28 is similar to the CD28 protein in Takifugu rubripes. The total hematocyte count distinctly decreased after TBT-Cl exposure, showing the adverse effects of TBT-Cl invasion and self-adjusting ability upon To-CD28 accumulation. The production of reactive oxygen species increased, demonstrating the oxidation resistance of T. obscurus when exposed to TBT-Cl. The tissue expression patterns indicated To-CD28 is a widely distributed receptor in T. obscurus. Its high expression in the liver and gill suggests that To-CD28 could be potentially functioned in TBT-Cl toxic process. The mRNA levels of To-CD28 and relative genes in the TLR-MyD88 signal pathway were significantly up-regulated under TBT-Cl exposure. The immunohistochemistry also showed that the To-CD28 protein signal was enhanced under TBT-Cl exposure, which proved that the positive protection of To-CD28 for maintaining homeostasis. Our study indicated that To-CD28 might participate in the toxicity mechanism upon TBT-Cl exposure and regulate homeostasis stability of T. obscurus.

Developmental Expression of HSP60 and HSP10 in the Coilia nasus Testis during Upstream Spawning Migration.

Heat shock protein 60 (HSP60) and heat shock protein 10 (HSP10) are important chaperones, which have been proven to have essential roles in mediating the correct folding of nuclear encoded proteins imported to mitochondria. Mitochondria are known as the power house of the cell, with which it produces energy and respires aerobically. In this regard, the obtained HSP60 and HSP10 have typical characteristics of the HSP60/10 family signature. Their mRNA transcripts detected were highest during the developmental phase (in April), while the lowest levels were found in the resting phase (after spawning in late July). Additionally, the strongest immunolabeling positive signals were found in the primary spermatocyte, with lower positive staining in secondary sperm cells, and a weak or absent level in the mature sperm. At the electron microscopic level, immunogold particles were localized in the mitochondrial matrix. Data indicated that HSP10 and HSP60 were inducible and functional in the Coilia nasus testis development and migration process, suggesting their essential roles in this process. The results also indicated that HSP60 may be one indicator of properly working mitochondrial import and refolding in the fish testis. This study also provides an expanded perspective on the role of heat shock protein families in spawning migration biology.

Molecular Characteristic, Protein Distribution and Potential Regulation of HSP90AA1 in the Anadromous Fish Coilia nasus.

Heat shock proteins play essential roles in basic cellular events. Spawning migration is a complex process, with significant structural and biochemical changes taking place in the adult gonad. To date, the molecular mechanisms underlying migration reproductive biology remain undetermined. In this regard, a full length HSP90AA1 comprising 2608 nucleotides from the anadromous fish Coilia nasus was characterized, encoding 742 amino acid (aa) residues with potential phosphorylation sites. HSP90AA1 mRNA transcripts were detected in all organs, especially in the gonad. Furthermore, the greatest transcript levels were found during the developmental phase, while the lowest levels were found during the resting phase. In addition, the strongest immunolabeling positive signal was found in the primary spermatocyte and oocyte, with lower positive staining in secondary germ cells, and a weak or absent level in the mature sperm and oocyte. Interestingly, HSP90AA1 was mainly located in the cytoplasm of germ cells. These results are important for understanding the molecular mechanism of anadromous migration reproductive biology. In combination with data from other fish species, the result of this present study may facilitate further investigations on the spawning migration mechanism.

Testes transcriptome profiles of the anadromous fish Coilia nasus during the onset of spermatogenesis.

RNA-Seq technology has been widely applied to transcriptomics, genomics and functional gene study. Here, we performed de novo transcriptome sequencing to produce 23,842,172 clean reads representing a total of 4,815,798,404 (4.8 Gb) nucleotides from comprehensive transcript dataset for testis of Coilia nasus. Over 20 million Illumina reads were assembled into 194,636 unigenes, and 42,642 annotated genes were predicted by Blastx and ESTScan, respectively. Applying Blast analysis and functional annotation (e.g., GO, COG, SwissProt and KEGG) using the assembled gene models from catalogs of other species, we have sampled an extensive and diverse expressed gene catalog for C. nasus representing a large proportion of the genes involved in the onset of spermatogenesis. The results will provide a general clue to the potential spermatogenesis molecular mechanisms for this species.

Ovary transcriptome profiling of Coilia nasus during spawning migration stages by Illumina sequencing.

Coilia nasus is an anadromous kind of small to moderate size fish species, and limited transcriptomics research has been performed. In the present study, we performed Illumina sequencing to produce a 22,996,612 clean reads representing with a total of 4,599,079,601 (4.5Gb) nucleotides comprehensive transcript dataset for ovary of C. nasus. Over 20 million total reads were assembled into 63,141 unigenes, and 27,027 annotated genes were predicted by Blastx and ESTScan, respectively. Applying Blast analysis and functional annotation (e.g., GO, COG, Swissprot and KEGG), we have sampled an extensive and diverse expressed gene catalog for C. nasus representing a large proportion of the genes expressed in the ovary development process. This approach will assist in the discovery and annotation of novel genes that play key roles in anadromous fish spawning migration process.

[Expression of PTEN protein and its correlation with p27kip1 and cyclin D1 expression in primary breast cancer].

OBJECTIVE: To study the expression of phosphatase and tensin homology deleted on chromosometen ten (PTEN) protein, a tumor suppressor gene in breast cancer and its correlation with p27(kip1) and cyclin D1 expression. METHODS: PTEN protein expression, p27(kip1) and cyclin D1 protein expression were detected by immunohistochemical method in paraffin sections from 61 women with primary breast cancer. PTEN protein expression was compared with clinico-pathologic parameters as related to p27(kip1) and cyclin D1. RESULTS: PTEN, being shown in the cytoplasm, was negative in 6.6% (4/61), reduced in 41.0% (25/61) and positive in 52.5% (32/61) samples. PTEN expression level was correlated with axillary lymph node status, loss of estrogen receptor stain, recurrence and metastasis. On univariate analysis, the disease-free survival rate of patients with higher PTEN expression (> 50% cells stained) was better than those with lower expression (P = 0.0101). However, there was no correlation between p27(kip1), cyclin D1 expression or PTEN expression. CONCLUSION: PTEN, its lower expression being correlated with poor outcome of breast cancer patients, plays a prominent role in breast cancer. p27(kip1) or cyclin D1 may not be the primary downstream genes of PTEN in breast cancer.