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Nonalcoholic fatty liver disease (NAFLD) is a global health concern with no approved drugs. High-protein dietary intervention is currently the most effective treatment. However, its underlying mechanism is unknown. Here, using Drosophila oenocytes, the specialized hepatocyte-like cells, we find that dietary essential amino acids ameliorate hepatic steatosis by inducing polyubiquitination of Plin2, a lipid droplet-stabilizing protein. Leucine and isoleucine, two branched-chain essential amino acids, strongly bind to and activate the E3 ubiquitin ligase Ubr1, targeting Plin2 for degradation. We further show that the amino acid-induced Ubr1 activity is necessary to prevent steatosis in mouse livers and cultured human hepatocytes, providing molecular insight into the anti-NAFLD effects of dietary protein/amino acids. Importantly, split-intein-mediated trans-splicing expression of constitutively active UBR2, an Ubr1 family member, significantly ameliorates obesity-induced and high fat diet-induced hepatic steatosis in mice. Together, our results highlight activation of Ubr1 family proteins as a promising strategy in NAFLD treatment.

In recent years, the availability of reverse genetics systems for porcine reproductive and respiratory syndrome virus (PRRSV) has created new perspectives for the use of recombinant viruses as expression vectors. Most of these recombinant PRRSV vectors express foreign genes through either an independent transcription unit inserted in ORF1b and ORF2, or in ORF7 and the 3' UTR. The aim of this study was to find an alternative site for foreign gene insertion into the PRRSV genome. Here, we constructed an infectious cDNA clone for a cell-adapted PRRSV strain, GXNN1396-P96. This cDNA-clone-derived recombinant virus (rGXAM) was comparable in its growth kinetics in MARC-145 cells to the parental virus, GX1396-P96. Using the infectious cDNA-clone, we inserted an independent transcription unit in ORF4 and ORF5a to generate a novel PRRSV-based recombinant virus expressing the green fluorescent protein (GFP) gene. Biological characterization of the recombinant virus, rGX45BSTRS-GFP, showed that it maintained similar growth characteristics but produced fewer infectious virions than the parental PRRSV. These data demonstrate that the ORF4 and ORF5a site is able to tolerate the insertion of foreign genes.

Nickel (Ni)-contamination impairs soil ecosystem, threatening human health. A laboratory simulation of Ni-polluted farmland soil study, in the presence or absence of earthworm, was carried out to investigate the toxic responses of soil microorganisms, including microbial biomass C (MBC), soil basal respiration (SBR), metabolic quotient (qCO2), urease (UA) and dehydrogenase activities (DHA). Additionally, the variations of Ni bioavailability were also explored. Results manifested that MBC and SBR were stimulated at 50 and 100 mg·kg−1 of Ni but inhibited by further increasing Ni level, showing a Hormesis effect. Earthworm input delayed the occurrence of a maximum SBR inhibition rate under the combined double-factors of time and dose. No specific effect of Ni concentration on the qCO2 was observed. UA was significantly suppressed at 800 mg·kg−1 Ni (P

Purpose The purpose of this study was to systematically characterize and correlate the transcriptome and DNA methylome signatures of mouse Muller cells that may underlie the development, physiological functions, and regeneration capacity of these cells. Methods Mouse Muller cells under normal, injury, and aging conditions were sorted from Muller cell-specific green fluorescent protein (GFP)-expressing mice. RNA sequencing was used to sequence transcriptomes, and reduced representation bisulfite sequencing was used to sequence DNA methylomes. Various bioinformatics tools were used to compare and correlate the transcriptomes and DNA methylomes. Results Muller cells express a distinct transcriptome that is in line with their retinal supporting roles and dormant retinogenic status. Injury changes the Muller cell transcriptome dramatically but fails to stimulate the cell cycle machinery and retinogenic factors to the states observed in early retinal progenitor cells (RPCs). Muller cells exhibit a less methylated genome than that of early RPCs, but most regulatory elements for Muller cell- and RPC-specific genes are similarly hypomethylated in both Muller cells and RPCs, except for a subset of Muller cell-specific functional genes. Aging only subtly affects the transcriptome and DNA methylome of Muller cells. Conclusions Failure to reactivate the cell cycle machinery and retinogenic factors to necessary levels might be key barriers blocking Muller cells from entering an RPC-like regeneration state. DNA methylation might regulate the expression of a subset of Muller cell-specific functional genes during development but is likely not involved in restricting the regeneration activity of Muller cells.

Background Porcine respiratory and reproductive syndrome virus (PRRSV) is one of the most economically significant pathogens in the Chinese swine industry. ORF5 and nsp2 are highly variable regions of the PRRSV genome. Therefore, nsp2 and GP5 are often selected for investigation of variations and phylogenetic analyses for their genetic diversities. Knowledge of the molecular evolution of PRRSV field strains may contribute to the control of PRRS in China. Results The results of multiple sequence alignments of GP5 showed that there is 84.5–100% aa identity among the 56 strains in this study. These strains shared 84.5–99.0% aa identity with the prototypical type 2 PRRSV VR-2332 and 56.6–59.2% with strain LV, prototypical type 1 PRRSV. Phylogenetic analysis showed there is considerable diversity among PRRSV ORF5 and the existence of two lineages (5 and 8). Most of the strains were classified into lineage 8 with multiple sub-lineages (3, 4 and 6). Moreover, PRRSV strains with 5 novel patterns of deletions or insertions in the nsp2 region were found. Conclusions Phylogenetic analysis based on ORF5 sequences indicated the diversity of PRRSV in southern parts of China and the strains with 30 aa deletion in nsp2 are dominant in the porcine population. Also, new PRRSV strains with different patterns of deletions or insertions in nsp2 are emerging. The data presented here constitute a useful basis for further epidemiological studies regarding the heterogeneity of PRRSV strains in China and provide a basis for the prevention of PRRS in southern parts of China. Electronic supplementary material The online version of this article (10.1186/s12917-019-1906-9) contains supplementary material, which is available to authorized users.

Previously, a spontaneous 88-amino-acid (aa) deletion in nsp2 was associated with cell-adaptation of porcine reproductive and respiratory syndrome virus (PRRSV) strain JXM100, which arose during passaging of the highly pathogenic PRRSV (HP-PRRSV) strain JX143 in MARC-145 cells. Here, to elucidate the biological role of this deletion, we specifically deleted the region of a cDNA clone of HP-PRRSV strain JX143 (pJX143) corresponding to these 88 amino acids. The effect of the deletion on virus replication in cultured cells and transcriptional activation of inflammatory cytokines and chemokines in pulmonary alveolar macrophages (PAMs) was examined. Mutant virus with the 88-aa deletion in nsp2 (rJX143-D88) had faster growth kinetics and produced larger plaques in MARC-145 cells than the parental virus (rJX143), suggesting that the deletion enhanced virus replication in MARC-145 cells. In contrast, the overall yield of rJX143 was almost 1 log higher than that of rJX143-D88, suggesting that the 88-aa deletion in nsp2 decreased the production of infectious viruses in PAMs. Infection with the mutant virus with the 88-aa deletion resulted in increased mRNA expression of type I interferon (IFN-α and IFN-β) and chemokines genes. In addition, the mRNA expression of antiviral genes (ISG15, ISG54 and PKR) regulated by the IFN response was upregulated in PAMs infected with the mutant virus rJX143-D88. Our results demonstrate that virus-specific host immunity can be enhanced by modifying certain nsp2 epitope regions. These findings provide important insights for understanding virus pathogenesis and development of future vaccines.

Purpose The optic fissure (OF) is a transient opening in the ventral optic cup (OC) that acts as a passage for blood vessels and retinal ganglion cell axons during early eye development. Failure to close the OF is the developmental basis for uveal coloboma, a congenital blinding eye disease that significantly contributes to childhood blindness. Genes specifically expressed in the OF region may play important roles in OF development and function. The aim of this study was to characterize the transcriptome of OC cells in the OF region and investigate the function of OF-specific genes during OF closure. Methods Laser-assisted microdissection was used to collect different regions of OC tissues. Microarray analysis was used to obtain and compare gene expression profiles of different OC regions. RNA in situ hybridization (ISH) was used to further characterize OF-specific gene expression patterns. Morpholino knockdown in zebrafish was used to study the function of a newly discovered OF-specific gene during OF closure. Results Microarray comparison revealed that the OC at the OF region exhibited a unique gene expression profile. OC expression patterns of a number of newly discovered OF-specific genes were confirmed by ISH. Morpholino knockdown and downstream target expression and function analysis demonstrated that afap1l2, a newly discovered OF-specific gene, controls OF closure by regulating pax2a expression. Conclusions Our study characterized the unique transcriptome of the OF region of the OC and demonstrated the essential role of a newly discovered OF-specific gene in OF closure. This study provides a valuable foundation for future mechanism dissection in OF development and physiology, and for human coloboma etiology exploration.

Both the spotted scat Scatophagus argus and rabbitfish Siganus canaliculatus belong to the few cultured herbivorous marine teleost, however, their fatty acyl desaturase (Fad) system involved in long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis is different. The S. argus has a △6 Fad, while the rabbitfish has △4 and △6/△5 Fads, which were the first report in vertebrate and marine teleost, respectively. In order to compare the characteristics of elongases of very long-chain fatty acids (Elovl) between them, two Elovl cDNAs were cloned from S. argus in the present study. One has 885bp of open read fragment (ORF) encoding a protein with 294 amino acid (aa) showing Elovl5 activity functionally characterized by heterologous expression in yeast, which was primarily active for the elongation of C18 and C20 PUFAs. The other has 915bp of ORF coding for a 305 aa protein showing Elovl4 activity, which was more efficient in the elongation of C20 and C22 PUFAs. Tissue distribution analyses by RT-PCR showed that elovl5 was highly expressed in the liver compared to other tissues determined, whereas elovl4 transcripts were only detected in the eye. The expression of elovl5 and elovl4 were significantly affected by dietary fatty acid composition, with highest expression of mRNA in the liver and eye of fish fed a diet with an 18:3n−3/18:2n−6 ratio of 1.7:1. These results indicated that the S. argus has a similar Elovl system in the LC-PUFA biosynthetic pathway to that of rabbitfish although their Fad system was different, suggesting that the diversification of fish LC-PUFA biosynthesis specificities is more associated with its Fad system. These new insights expand our knowledge and understanding of the molecular basis and regulation of LC-PUFA biosynthesis in fish.

Purpose Coloboma is a sight-threatening congenital eye disease caused by a failure in optic fissure (OF) closure. The aim of this study was to investigate the role of Adamts16, a metalloproteinase, in OF closure. Methods RNA in situ hybridization was used to examine the expression of Adamts16 in developing mouse and zebrafish eyes. Morpholino knockdowns were performed to study adamts16 function during zebrafish eye development. Additionally, immunofluorescent staining, RNA in situ hybridization, bromodeoxyuridine (BrdU) labeling, TUNEL assays, and high-throughput sequencing were used to examine altered cellular and molecular events in adamts16-morphant optic cups (OCs). Results Adamts16 is expressed at the edges of the closing OF in both mice and zebrafish eyes. Zebrafish adamts16 knockdown resulted in coloboma formation. In adamts16-morphant eyes, the basement membrane failed to disassemble at the closing OF edges, OC cells exhibited decreased proliferation and increased apoptosis, and fibroblast growth factor 8 (fgf8) was ectopically upregulated in the OC. Conclusions adamts16 is required for proper OF closure in zebrafish eyes. adamts16 controls OF closure possibly through the combined functions of degrading the basement membrane at the closing OF edges, promoting cell proliferation and survival, and restricting fgf8 expression. Our study linked a metalloproteinase to OF closure, which may facilitate future etiologic studies on human coloboma cases.

Using next-generation sequencing technology alone, we have successfully generated and assembled a draft sequence of the giant panda genome. The assembled contigs (2.25 gigabases (Gb)) cover approximately 94% of the whole genome, and the remaining gaps (0.05 Gb) seem to contain carnivore-specific repeats and tandem repeats. Comparisons with the dog and human showed that the panda genome has a lower divergence rate. The assessment of panda genes potentially underlying some of its unique traits indicated that its bamboo diet might be more dependent on its gut microbiome than its own genetic composition. We also identified more than 2.7 million heterozygous single nucleotide polymorphisms in the diploid genome. Our data and analyses provide a foundation for promoting mammalian genetic research, and demonstrate the feasibility for using next-generation sequencing technologies for accurate, cost-effective and rapid de novo assembly of large eukaryotic genomes. © 2010 Macmillan Publishers Limited. All rights reserved., link_to_OA_fulltext

Marine fish are generally unable or have low ability for the biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFA) from C18 PUFA precursors, with some notable exceptions including the herbivorous marine teleost Siganus canaliculatus in which such a capability was recently demonstrated. To determine whether this is a unique feature of S. canaliculatus or whether it is common to the herbivorous marine teleosts, LC-PUFA biosynthetic pathways were investigated in the herbivorous euryhaline Scatophagus argus. A putative desaturase gene was cloned and functionally characterized, and tissue expression and nutritional regulation were investigated. The full-length cDNA was 1972 bp, containing a 1338 bp open-reading frame encoding a polypeptide of 445 amino acids, which possessed all the characteristic features of fatty acyl desaturase (Fad). Functional characterization by heterologous expression in yeast showed the protein product of the cDNA efficiently converted 18:3n-3 and 18:2n-6 to 18:4n-3 and 18:3n-6, respectively, indicating Δ6 desaturation activity. Quantitative real-time PCR showed that highest Δ6 fad mRNA expression was detected in liver followed by brain, with lower expression in other tissues including intestine, eye, muscle, adipose, heart kidney and gill, and lowest expression in stomach and spleen. The expression of Δ6 fad was significantly affected by dietary lipid and, especially, fatty acid composition, with highest expression of mRNA in liver of fish fed a diet with a ratio of 18:3n-3/18:2n-6 of 1.72:1. The results indicated that S. argus may have a different LC-PUFA biosynthetic system from S. canaliculatus despite possessing similar habitats and feeding habits suggesting that LC-PUFA biosynthesis may not be common to all marine herbivorous teleosts.