Identification and Differential Expression of MicroRNAs during Metamorphosis of the Japanese Flounder (Paralichthys olivaceus)

BACKGROUND: MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs of 20-25 nucleotides that play a key role in diverse biological processes. Japanese flounder undergo dramatic metamorphosis in their early development. The metamorphosis is characterized by morphological transformation from a bilaterally symmetrical to an asymmetrical body shape concomitant with extensive morphological and physiological remodeling of organs. So far, only a few miRNAs have been identified in fish and there are very few reports about the Japanese flounder miRNA. METHODOLOGY/PRINCIPAL FINDINGS: Solexa sequencing technology was used to perform high throughput sequencing of the small RNA library from the metamorphic period of Japanese flounder. Subsequently, aligning these sequencing data with metazoan known miRNAs, we characterized 140 conserved miRNAs and 57 miRNA: miRNA* pairs from the small RNA library. Among these 57 miRNA: miRNA* pairs, twenty flounder miRNA precursors were amplified from genomic DNA. We also demonstrated evolutionary conservation of Japanese flounder miRNAs and miRNA* in the animal evolution process. Using miRNA microarrays, we identified 66 differentially expressed miRNAs at two metamorphic stages (17 and 29 days post hatching) of Japanese flounder. The results show that miRNAs might play a key role in regulating gene expression during Japanese flounder metamorphosis. CONCLUSIONS/SIGNIFICANCE: We identified a large number of miRNAs during flounder metamorphosis, some of which are differentially expressed at two different metamorphic stages. The study provides an opportunity for further understanding of miRNA function in the regulation of flounder metamorphosis and gives us clues for further studies of the mechanisms of metamorphosis in Japanese flounder

In Situ Frost-Heaving Characteristics of Shallow Layer of Soil Slopes in Intermittently Frozen Region Based on PFC3D

The in situ frost-heaving (FH) process and characteristics of the shallow layer of a residual soil slope in the intermittently frozen zone were simulated by a modified three-dimensional particle flow code (PFC3D) model, of which the mesoscopic parameters of soil and ice particles were calibrated through the indoor experiments. In this model, the in situ FH process was gradually achieved by expanding the volume of ice particles (divided into 24 times of expansion), and the process was terminated when the monitored porosity was stable. These countermeasures avoided the stress accumulation and effectively realized the simulation of the in situ FH process. The results found that the displacement occurred firstly and got the largest final value at the surface angle (SA) under the in situ FH effect, followed by that at the foot, and it gradually extended to the interior based on these two regions. The vertical tension was present at the SA, and the major force type in the lateral interlayer was pressure. In addition, the FH effect seemed to be strongly related to the frozen depth, and a sliding surface was found in a steeper slope. Finally, the smaller stone appeared to be favorable to the slope stability, but it was reduced by the larger stone to some extent

Identification and characterization of TOR in Macrobrachium rosenbergii and its role in muscle protein and lipid production

Abstract The recent scarcity of fishmeal and other resources means that studies on the intrinsic mechanisms of nutrients in the growth and development of aquatic animals at the molecular level have received widespread attention. The target of rapamycin (TOR) pathway has been reported to receive signals from nutrients and environmental stresses, and regulates cellular anabolism and catabolism to achieve precise regulation of cell growth and physiological activities. In this study, we cloned and characterized the full-length cDNA sequence of the TOR gene of Macrobrachium rosenbergii (MrTOR). MrTOR was expressed in all tissues, with higher expression in heart and muscle tissues. In situ hybridization also indicated that MrTOR was expressed in muscle, mainly around the nucleus. RNA interference decreased the expression levels of MrTOR and downstream protein synthesis-related genes (S6K, eIF4E, and eIF4B) (P < 0.05) and the expression and enzyme activity of the lipid synthesis-related enzyme, fatty acid synthase (FAS), and increased enzyme activity of the lipolysis-related enzyme, lipase (LPS). In addition, amino acid injection significantly increased the transcript levels of MrTOR and downstream related genes (S6K, eIF4E, eIF4B, and FAS), as well as triglyceride and total cholesterol tissue levels and FAS activity. Starvation significantly increased transcript levels and enzyme activities of adenylate-activated protein kinase and LPS and decreased transcript levels and enzyme activities of FAS, as well as transcript levels of MrTOR and its downstream genes (P < 0.05), whereas amino acid injection alleviated the starvation-induced decreases in transcript levels of these genes. These results suggested that arginine and leucine activated the TOR signaling pathway, promoted protein and lipid syntheses, and alleviated the pathway changes induced by starvation

Identification and Functional Analysis of the Cell Proliferation Regulator, Insulin-like Growth Factor 1 (IGF1) in Freshwater Pearl Mussel (<i>Hyriopsis cumingii</i>)

Insulin-like growth factor 1 (IGF1) plays an important regulatory role in the regulation of growth, differentiation, and anabolism in a variety of cells. In this study, the full-length cDNA of the IGF1 gene was cloned from Hyriopsis cumingii, named HcIGF1. The expression level of HcIGF1 in six tissues (adductor muscle, foot, hepatopancreas, gill, mantle, and gonad) was determined. In addition, the localization of HcIGF1 in the mantle was analyzed by in situ hybridization, and finally the function of HcIGF1 was explored by RNA interference and prokaryotic expression. The results showed that the amino acid sequence contained a typical IIGF structural domain. The phylogenetic tree showed that HcIGF1 clustered with other marine bivalve sequences. Quantitative real-time PCR and in situ hybridization analysis showed that HcIGF1 was expressed in all tissues. The highest expression was in the foot and the lowest was in the mantle. In the mantle tissue, the hybridization signal was mainly concentrated in the outer mantle. After RNA interference, the expression of IGF1 was found to be significantly decreased (p IGF1R, AKT1, and cyclin D2 were downregulated, while MAPK1 were upregulated. The recombinant HcIGF1 protein was purified and its growth-promoting effect was investigated. The results showed that the recombinant HcIGF1 protein could significantly promote the proliferative activity of the mantle cells of mussels, with the best proliferative effect at 12.5 μg/mL. The results of this study provide a new method to solve the problem of weak proliferation of shellfish cells in vitro and lay the foundation for further understanding of the growth regulation mechanism of H. cumingii, as well as a better understanding of the physiological function of IGF1 in mollusks

A New Cell Line Derived from the Spleen of the Japanese Flounder (Paralichthys olivaceus) and Its Application in Viral Study

A new cell line Japanese flounder spleen (JFSP) derived from the spleen of Japanese flounder (Paralichthys olivaceus) was established and characterized in this study. The JFSP cells grew rapidly at 29 &deg;C, and the optimum fetal bovine serum concentration in the L-15 medium was 15%. Cells were subcultured for more than 80 passages. The JFSP cells have a diploid chromosome number of 2n = 68, which differs from the chromosome number of normal diploid Japanese flounder. The established cells were susceptible to Bohle virus (BIV), Viral hemorrhagic septicemia virus (VHSV), Hirame rhabdovirus (HIRRV), Infectious hematopoietic necrosis virus (IHNV), and Lymphocystis disease virus (LCDV), as evidenced by varying degrees of cytopathic effects (CPE). Replication of the virus in JFSP cells was confirmed by qRT-PCR and transmission electron microscopy. In addition, the expression of four immune-related genes, TRAF3, IL-1&beta;, TNF-&alpha;, and TLR2, was differentially altered following viral infection. The results indicated that the cells underwent an antiviral immune response. JFSP cell line is an ideal tool in vitro for virology. The use of fish cell lines to study the immune genes and immune mechanism of fish and to clarify the immune mechanism of fish has important theoretical significance and practical application value for the fundamental prevention and treatment of fish diseases

Silicalite‑1 Layer Secures the Bifunctional Nature of a CO₂ Hydrogenation Catalyst

Close proximity usually shortens the travel distance of reaction intermediates, thus able to promote the catalytic performance of CO2 hydrogenation by a bifunctional catalyst, such as the widely reported In2O3/H-ZSM-5. However, nanoscale proximity (e.g., powder mixing, PM) more likely causes the fast deactivation of the catalyst, probably due to the migration of metals (e.g., In) that not only neutralizes the acid sites of zeolites but also leads to the reconstruction of the In2O3 surface, thus resulting in catalyst deactivation. Additionally, zeolite coking is another potential deactivation factor when dealing with this methanol-mediated CO2 hydrogenation process. Herein, we reported a facile approach to overcome these three challenges by coating a layer of silicalite-1 (S-1) shell outside a zeolite H-ZSM-5 crystal for the In2O3/H-ZSM-5-catalyzed CO2 hydrogenation. More specifically, the S-1 layer (1) restrains the migration of indium that preserved the acidity of H-ZSM-5 and at the same time (2) prevents the over-reduction of the In2O3 phase and (3) improves the catalyst lifetime by suppressing the aromatic cycle in a methanol-to-hydrocarbon conversion step. As such, the activity for the synthesis of C2+ hydrocarbons under nanoscale proximity (PM) was successfully obtained. Moreover, an enhanced performance was observed for the S-1-coated catalyst under microscale proximity (e.g., granule mixing, GM) in comparison to the S-1-coating-free counterpart. This work highlights an effective shielding strategy to secure the bifunctional nature of a CO2 hydrogenation catalyst

Tandem catalysis with double-shelled hollow spheres

Metal-zeolite composites with metal (oxide) and acid sites are promising catalysts for integrating multiple reactions in tandem to produce a wide variety of wanted products without separating or purifying the intermediates. However, the conventional design of such materials often leads to uncontrolled and non-ideal spatial distributions of the metal inside/on the zeolites, limiting their catalytic performance. Here we demonstrate a simple strategy for synthesizing double-shelled, contiguous metal oxide@zeolite hollow spheres (denoted as MO@ZEO DSHSs) with controllable structural parameters and chemical compositions. This involves the self-assembly of zeolite nanocrystals onto the surface of metal ion-containing carbon spheres followed by calcination and zeolite growth steps. The step-by-step formation mechanism of the material is revealed using mainly in situ Raman spectroscopy and X-ray diffraction and ex situ electron microscopy. We demonstrate that it is due to this structure that an Fe2O3@H-ZSM-5 DSHSs-showcase catalyst exhibits superior performance compared with various conventionally structured Fe2O3-H-ZSM-5 catalysts in gasoline production by the Fischer-Tropsch synthesis. This work is expected to advance the rational synthesis and research of hierarchically hollow, core-shell, multifunctional catalyst materials

Tandem catalysis with double-shelled hollow spheres

Metal-zeolite composites with metal (oxide) and acid sites are promising catalysts for integrating multiple reactions in tandem to produce a wide variety of wanted products without separating or purifying the intermediates. However, the conventional design of such materials often leads to uncontrolled and non-ideal spatial distributions of the metal inside/on the zeolites, limiting their catalytic performance. Here we demonstrate a simple strategy for synthesizing double-shelled, contiguous metal oxide@zeolite hollow spheres (denoted as MO@ZEO DSHSs) with controllable structural parameters and chemical compositions. This involves the self-assembly of zeolite nanocrystals onto the surface of metal ion-containing carbon spheres followed by calcination and zeolite growth steps. The step-by-step formation mechanism of the material is revealed using mainly in situ Raman spectroscopy and X-ray diffraction and ex situ electron microscopy. We demonstrate that it is due to this structure that an Fe2O3@H-ZSM-5 DSHSs-showcase catalyst exhibits superior performance compared with various conventionally structured Fe2O3-H-ZSM-5 catalysts in gasoline production by the Fischer-Tropsch synthesis. This work is expected to advance the rational synthesis and research of hierarchically hollow, core-shell, multifunctional catalyst materials