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To explore genetic diversity and estimate the genetic differences among populations of Lutraria maxima in the coastal waters off south to southeast China, the morphology of the species of five different geographical populations (Beihai, Weizhou Island, Zhanjiang, Xiamen, and Fuzhou) in Guangxi, Guangdong, and Fujian provinces was studied statistically in combination with the microsatellite markers. As revealed by morphological principal component analysis (PCA), the cumulative contribution rate of the first three principal components was 72.596%. The discrimination accuracy ranged from 47.5% to 80.0%, and the scatter plots of principal component and discriminant analysis were consistent in overall, showing that the Xiamen and Fuzhou populations were overlapped obviously. For microsatellite markers, 10 pairs of polymorphic primers were obtained by high-throughput transcriptome sequencing, and used for genetic diversity analysis. It was showed that the average number of alleles and effective alleles observed in each population ranged from 8.100 to 10.900, and from 3.497 to 4.228, respectively. The average observed heterozygosity (Ho) and expected heterozygosity (He) in the five populations ranged from 0.541 to 0.615, and from 0.642 to 0.733, respectively. The genetic distance (DA) ranged from 0.078 to 0.523, and the population genetic differentiation index (FST) ranged from 0.027 to 0.139. The unweighted pair-population method with arithmetic means (UPGMA) and structure analysis showed that the five populations could be divided into two main clusters, the Beibu Gulf group (Beihai and Weizhou Island) and the Southeast China Sea group (Zhanjiang, Xiamen, and Fuzhou), suggesting that L. maxima has been separated geographically by the barrier of the Leizhou Peninsula into two groups in evolution, which provided us with a scientific clue to better protect the bioresource and establish an appropriate fishery management stocks for L. maxima populations in south China.

Porcine reproductive and respiratory syndrome (PRRS) is the most economically important infectious disease of pigs worldwide. Our previous study revealed that Tongcheng (TC) pigs display higher resistance to PRRS than Largewhite (LW) pigs, but the genetic mechanism remains unknown. Here, we first confirmed that CXCL14 was downregulated in lungs and porcine alveolar macrophages (PAMs) responding to PRRS virus (PRRSV) infection, but the decline in LW pigs was more obvious than that in TC pigs. Then, we found that the overexpression of CXCL14 activated type-I interferon (IFN-I) signaling by upregulating interferon beta (IFNB), which plays a major role in the antiviral effect. To further decipher the mechanism underlying its differential expression, we characterized the core promoter of CXCL14 as being located from -145 to 276 bp of the transcription start site (TSS) and identified two main haplotypes that displayed significant differential transcriptional activities. We further identified two coupled point mutations that altered the binding status of CEBPB and were responsible for the differential expression in TC and LW pigs. The regulatory effect of CEBPB on CXCL14 was further confirmed by RNA interference (RNAi) and chromatin immunoprecipitation (ChIP), providing crucial clues for deciphering the mechanism of CXCL14 downregulation in unusual conditions. The present study revealed the potential antiviral effect of CXCL14, occurring via activation of interferon signaling, and suggested that CXCL14 contributes to the PRRS resistance of TC pigs.

Complement factor D (Df) is a serine protease, which can activate the alternative pathway by cleaving complement factor B, and involves in the innate defense against pathogens infection in teleost. In this study, we cloned, characterized the Df gene from blunt snout bream (Megalobrama amblycephala) (Mamdf), and examined its expression pattern and antimicrobial activity. The open reading frame (ORF) of Mamdf was 753 bp, encoding 250 amino acids with a molecular mass of 27.2 kDa. Mamdf consisted of a single serine protease trypsin superfamily domain, 3 substrate binding sites and 3 active sites, but no potential N-glycosylation site. Pairwise alignment showed that Mamdf shared the highest identity (94%) with grass carp (Ctenopharyngodon idellus). Phylogenetic analysis indicated that Mamdf and other vertebrate Df had a common ancestral origin. Mamdf structured with 4 introns and 5 exons. The Mamdf mRNA expressed relatively high at the intestine appearance stage during early development and constitutively expressed in various tissues with the highest expression in the kidney in healthy adults. After challenged with Aeromonas hydrophila, significant changes of Mamdf at both mRNA and protein levels in the kidney, spleen, liver and head-kidney were observed. The recombinant Mamdf protein showed antimicrobial activity against both gram-positive bacteria and gram-negative bacteria. The above results suggested the immune function of Mamdf, and would benefit further detailed Df function research in the immune process in teleost.

Nitrite stress is a major environmental factor that limits aquatic animal growth, reproduction and survival. Even so, some shrimp can withstand somewhat high concentrations of nitrite environment as our previous studies indicated. Therefore, we hypothesized that these shrimps might have specific adaptive strategies to adapt to high concentrations of nitrite environment. To identify the genes and pathways involved in the regulation of nitrite tolerance, we performed comparative transcriptomic analysis in the Litopenaeus vannamei nitrite-tolerant (NT) and nitrite-sensitive (NS) families, and untreated shrimps were used as the control group. After 24 h of nitrite exposure (NaNO2, 112.5 mg/L), a total of 1521 and 868 differentially expressed genes (DEGs) were obtained from NT compared with NS and control group, respectively. Functional enrichment analysis revealed that most of these DEGs were involved in immune defense, energy metabolism processes and endoplasmic reticulum (ER) stress. Nitrite stress significantly enhanced energy metabolism in NT by activating the expression of genes encoding key enzymes in oxidative phosphorylation (OXPHOS) pathway and tricarboxylic acid (TCA) cycle. Furthermore, some DEGs involved in innate immunity- related genes and pathways, and ER stress responses were highly expressed in NT. Therefore, we speculate that accelerated energy metabolism, higher expression of immunity and ER related genes might be the important adaptive strategies for NT in relative to NS under nitrite stress. These results will provide new sights on the potential tolerant molecular mechanisms and the breeding of new varieties of nitrite tolerant L. vannamei, with possible implications for other aquatic animals.

Background As non-coding RNA molecules of more than 200 bp in length, long non-coding RNAs (lncRNAs) play a variety of roles in biological processes, including regulating the immune responses to bacterial infections. In recent years, there have been many in-depth studies on mammalian lncRNAs, but the relevant studies in fish are very limited. Meanwhile, since lncRNAs are not conserved among species, it is difficult to apply the existing results directly to unstudied species. Results To obtain the information of lncRNAs in Megalobrama amblycephala, one of the most economically important freshwater fish in China, also to better understand the biological significance of lncRNAs in the immunity system, the fish liver at 0, 4, 12, 24, and 72 h post Aeromonas hydrophila infection (hpi) were obtained for lncRNA-sequencing (lncRNA-seq). A total of 14,849 lncRNAs were identified, and 2196 lncRNAs showed significant differences at different time points post A. hydrophila infection. Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that the target genes of the differentially expressed lncRNAs were enriched in several pathways related to immune such as apoptosis, inflammation, and immune response. Time-specific modules were then identified, using weighted correlation network analysis (WGCNA), and 28 modules significantly correlated with different time point after infection were found. Furthermore, four immune-related genes and six lncRNAs in the time-specific modules were subsequently verified by RT-qPCR. Conclusions The above findings reveal the discovery of widespread differentially expressed lncRNAs in the M. amblycephala liver post A. hydrophila infection, suggesting that lncRNAs might participate in the regulation of host response to bacterial infection, enriching the information of lncRNAs in teleost and providing a resources basis for further studies on the immune function of lncRNAs.

Circular RNAs (circRNAs), a class of non-coding RNAs, play an important role in regulating various biological processes. In the present study, circRNAs from the Megalobrama amblycephala liver were identified at five different time points post Aeromonas hydrophila using RNA-seq technology. A total of 250 circRNAs were identified, of which 106 were differentially expressed (DE) in ten pairwise comparisons. GO and KEGG analyses showed that the parental genes of DE circRNAs were enriched in phagocytosis, complement and coagulation cascades, and Fc gamma R-mediated phagocytosis pathways. According to ceRNA hypothesis, the interaction network of circRNAs, miRNAs and mRNAs was constructed. Moreover, WGCNA was conducted, and five specific modules significantly related to bacterial infection were identified. All the above results reveal the important role of circRNAs in immune response, which enriches the information of circRNAs in teleost, and helps to understand the immune response mechanism of M. amblycephala to A. hydrophila.

BackgroundNitrite is a major environmental toxin in aquaculture systems disrupting multiple physiological functions in aquatic animals. Although nitrite tolerance in shrimp is closely related to successful industrial production, few genetic studies of this trait are available. ResultsIn this study, we constructed a high-density genetic map of L. vannamei with 17,242 single nucleotide polymorphism markers spanning 6,828.06 centimorgans (cM) and an average distance of 0.4 cM between adjacent markers on 44 linkage groups (LGs). Using this genetic map, we identified 2 quantitative trait loci (QTLs) involved in nitrite tolerance on two LGs explaining 8.42–10.31% of the phenotypic variance. We then sequenced the transcriptomes of the most nitrite-tolerant and the most nitrite-sensitive individuals from each of four genetically distinct L. vannamei families and found 2,002, 1,983, 1,954, and 1,867 differentially expressed genes in the four families, respectively. By integrating QTL and transcriptomics analyses, we identified a candidate gene associated with nitrite tolerance. This gene was annotated as solute carrier family 26 member 6 (SLC26A6). RNA interference (RNAi) analysis demonstrated that SLC26A6 was critical for nitrite tolerance in L. vannamei. ConclusionsThe present study increases our understanding of the molecular mechanisms underlying nitrite tolerance in shrimp, and provides a basis for molecular-marker-assisted shrimp breeding.

BackgroundAmmonia is one of the most common toxicological environment factors affecting shrimp health. Although ammonia tolerance in shrimp is closely related to successful industrial production, few genetic studies of this trait are available.ResultsIn this study, we constructed a high-density genetic map of the Pacific white shrimp (Litopenaeus vannamei) using specific length amplified fragment sequencing (SLAF-seq). The constructed genetic map contained 17,338 polymorphic markers spanning 44 linkage groups, with a total distance of 6360.12 centimorgans (cM) and an average distance of 0.37 cM. Using this genetic map, we identified a quantitative trait locus (QTL) that explained 7.41–8.46% of the phenotypic variance inL. vannameisurvival time under acute ammonia stress. We then sequenced the transcriptomes of the most ammonia-tolerant and the most ammonia-sensitive individuals from each of four genetically distinctL. vannameifamilies. We found that 7546 genes were differentially expressed between the ammonia-tolerant and ammonia-sensitive individuals. Using QTL analysis and the transcriptomes, we identified one candidate gene (annotated as an ATP synthase g subunit) associated with ammonia tolerance.ConclusionsIn this study, we constructed a high-density genetic map ofL. vannameiand identified a QTL for ammonia tolerance. By combining QTL and transcriptome analyses, we identified a candidate gene associated with ammonia tolerance. Our work provides the basis for future genetic studies focused on molecular marker-assisted selective breeding.

Chinese horseshoe crab, Tachypleus tridentatus, is an ancient marine arthropod with a long evolutionary history. As a kind of living fossil species, the pathogen defenses of horseshoe crabs entirely depend on the innate immune system. Although, there are abundant immune molecules found in the horseshoe crab hemolymph, the biological mechanisms underlying their abilities of distinguishing and defending against invading microbes are still unclear. In this study, we used high-throughput sequencing at mRNA and protein levels and bioinformatics analysis methods to systematically analyze the innate immune response to Gram-negative bacteria in hemolymph of Chinese horseshoe crab. These results showed that many genes in the complement and coagulation cascades, Toll, NF-κB, C-type lectin receptor, JAK-STAT, and MAPK signaling pathways, and antimicrobial substances were activated at 12 and 24 h post-infection, suggesting that Gram-negative bacteria could activate the hemolymph coagulation cascade and antibacterial substances release via the above pathways. In addition, we conjectured that Toll and NF-κB signaling pathway were most likely to participate in the immune response to Gram-negative bacteria in hemolymph of horseshoe crab through an integral signal cascade. These findings will provide a useful reference for exploring the ancient original innate immune mechanism.

HIF prolyl hydroxylase 1 (PHD1) functions in prolyl hydroxylation on mammal hypoxia-inducible factors (HIF), important transcription factors involved in hypoxia, however the roles of Phd1 in fish remain unclear. In this study, the full-length cDNA and promoter sequences of blunt snout bream ( Megalobrama amblycephala ) phd1 gene were isolated by a modified RACE strategy. The phd1 cDNA was 2672 bp for encoding 481 amino acid residues. In silico assays indicated that phd1 had 5 exons, and a 348 bp CpG island in the exon1, and several transcription factor binding sites (CAAT box, HRE, ARNT, FOX, etc) were also found on the promoter. The quantitative real-time PCR results suggested that phd1 mRNA was constitutively expressed in all detected tissues, and higher in the blood, brain and heart in normoxia, but significantly decreased after hypoxia in all detected tissues except for gill. Western blot assays indicated that two Phd1 isoforms were generated by alternative translation initiation. Moreover, these two isoforms were both localized in the nucleus, therein only the senior isoform promoted cell proliferation. Taken together, the present study firstly describes the functions of M. amblycephala two Phd1 isoforms in hypoxia and cell proliferation.

Sex reversal in insects, amphibians, reptiles, and fishes is a complicated and interesting biological phenomenon. Sex reversal changes the sex ratio of populations and may complicate breeding schemes. In the Chinese tongue sole (Cynoglossus semilaevis), genetic females may change into pseudomales, thereby increasing aquaculture costs because of the lower growth rate of the males than that of the females. Here we identify a new locus associated with sex reversal; this single nucleotide polymorphism (SNP) is located in the third intron of the doublesex and mab-3 related transcription factor 1 (Dmrt1) gene on the Z chromosome (named Cyn_Z_8564889) and has two alleles, A and G. Cyn_Z_8564889 regulates sex reversal interactively with our previously detected SNP (Cyn_Z_6676874), with the genetic females simultaneously carrying the T allele of Cyn_Z_6676874 and the A allele of Cyn_Z_8564889 changing into pseudomales. Other Dmrt1 polymorphisms were detected, which formed two haplotypes. Two SNPs in the second exon of Dmrt1 result in amino acid changes, suggesting that Dmrt1 is essential in sex reversal. We also verified that pseudomales produce no or little W sperm. The interaction and linkage between Cyn_Z_6676874 and Cyn_Z_8564889 and the absence of W sperm from pseudomales unravel the genetic architecture of sex reversal in C. semilaevis.

Background: Ammonia is one of the most common toxicological environment factors affecting shrimp health. Although ammonia tolerance in shrimp is closely related to successful industrial production, few genetic studies of this trait are available. Results: In this study, we constructed a high-density genetic map of the Pacific white shrimp (Litopenaeus vannamei) using specific length amplified fragment sequencing (SLAF-seq). The constructed genetic map contained 807,505 polymorphic markers spanning 44 linkage groups, with a total distance of 6,360.12 centimorgans (cM) and an average distance of 0.37 cM. Using this genetic map, we identified a quantitative trait locus (QTL) that explained 7.41–8.46% of the phenotypic variance in L. vannamei survival time under acute ammonia stress. We then sequenced the transcriptomes of the most ammonia-tolerant and the most ammonia-sensitive individuals from each of four genetically distinct L. vannamei families. We found that 7546 genes were differentially expressed between the ammonia-tolerant and ammonia-sensitive individuals. Using QTL analysis and the transcriptomes, we identified one candidate gene (annotated as an ATP synthase g subunit) associated with ammonia tolerance. Conclusions: In this study, we constructed a high-density genetic map of L. vannamei and identified a QTL for ammonia tolerance. By combining QTL and transcriptome analyses, we identified a candidate gene associated with ammonia tolerance. Our work provides the basis for future genetic studies focused on molecular marker-assisted selective breeding.

MicroRNAs (miRNAs), as important regulators of post-transcriptional gene expression, play important roles in the occurrence and function of organs. In this study, morpholino (MO) knockdown of miR-462/miR-731 was used to investigate the potential mechanisms of the miR-462-731 cluster during zebrafish liver development. The results showed significant reduction of digestive organs, especially liver and exocrine pancreas after the miR-462/miR-731 knockdown, and those phenotypes could be partially rescued by corresponding miRNA duplex. Acinar cells of the exocrine pancreas were also severely affected with pancreatic insufficiency. In particular, knockdown of miR-462 caused pancreas morphogenesis abnormity with specific bilateral exocrine pancreas. Additionally, it was found that miR-731 played a role in liver and exocrine pancreas development by directly targeting dkk3b, instead of the down-regulation of Wnt/β-catenin signaling. These findings contribute significantly to our understanding of molecular mechanisms of miR-462-731 cluster in development of digestive organs.

Common carp (Cyprinus carpio) is one of the major aquaculture species in the world and accounts for approximately 8% of the total aquaculture production. The presence of intermuscular bones (IMBs) dramatically affects the economic value of this fish in the market. To understand the genetic variation in IMBs in common carp, in this study, we estimated the heritability and carried out quantitative trait locus (QTL) mapping of IMBs in mirror carp. We used 30 full-sib families including 871 individuals to calculate the heritability and genetic correlation and found that the heritability of the total IMB count, Y-shaped (YS) IMB count and I-shaped (IS) IMB count were moderate and significant (P

Outbreaks of spring viraemia of carp virus (SVCV) in several carp species and other cultivated fish can cause significant mortality and jeopardize the billion-dollar worldwide fish industry. Spring viraemia of carp virus, also known as Rhabdovirus carpio, is a bullet-shaped RNA virus that enters and amplifies in gill epithelium and later spreads to internal organs. Young fish under stressed conditions (spring cold water, etc.) are more vulnerable to SVCV-induced lethality because of their lack of a mature immune system. Currently, the host response of SVCV remains largely unknown. Here, we observed that autophagy is activated in SVCV-infected epithelioma papulosum cyprini (EPC) cells. We demonstrated that the SVCV glycoprotein, rather than viral replication, activates the autophagy pathway. In addition, SVCV utilized the autophagy pathway to facilitate its own genomic RNA replication and to enhance its titres in the supernatants. Autophagy promoted the survival of SVCV-infected cells by eliminating damaged mitochondrial DNA generated during viral infection. We further showed that SVCV induces autophagy in EPC cells through the ERK/mTOR signalling pathway. Our results reveal a connection between autophagy and SVCV replication and propose autophagy suppression as a novel means to restrict SVCV viral replication.

Hyperplasia and hypertrophy are two distinct processes of skeletal muscle growth regulated by four myogenic regulatory factors (MRFs, contains MyoD, Myf5, Mrf4 and myogenin (MyoG)) and myostatin (MSTN). In this study, characterization of muscle morphology and satellite cells in juvenile (1-year-old) and adult (2-year-old) Megalobrama amblycephala was described. Compared with 1-year-old M. amblycephala, the diameter scope of epaxial, horizontal septum and hypaxial muscle fibers including red and white muscle in 2-year-old fish exhibited broader with dramatic reduction in frequency distribution of20 μm diameter, nevertheless observable increase in frequency distribution of50 μm diameter. Intermyofibrillar (IM) nuclei were also found except numerous subsarcolemmal (SS) nuclei in 2-year-old fish, whereas only SS nuclei were observed in 1-year-old fish. Immunofluorescence results showed that more satellite cells existed in red muscle than white muscle in 1-year-old fish, rather than 2-year-old fish. Moreover, we observed predominant increase in the mRNA levels of MyoD, Myf5, Mrf4, and MSTN during muscle development of fish in 2-year-old fish except MyoG.

Objectives Nevirapine (NVP), a still widely used nonnucleoside reverse transcriptase inhibitor, can cause severe hepatotoxicity. Previous studies suggest that CD4 cell counts more than 250 cells per microliter in women and more than 400 cells per microliter in men are risk factors for NVP-related hepatotoxicity. These studies have informed Chinese national treatment guidelines. We evaluate whether current Chinese guidelines for NVP use are appropriate. Methods Longitudinal data were pooled from 2 clinical trials between 2005 and 2009 across mainland China. Five hundred sixty-six antiretroviral therapy-naive Chinese patients were given NVP-containing antiretroviral therapy for 24 weeks. Hepatotoxicity was defined as alanine aminotransferase, aspartate transaminase, or total bilirubin level greater than 1.25 times the upper limit of normal range. Severe hepatotoxicity was defined as greater than 5 times the upper limit of normal range. Results One hundred ninety-seven (36.1%) patients developed hepatotoxicity during treatment, including 42 (7.7%) patients with severe hepatotoxicity. CD4 cell count more than 250 cells per microliter was an independent predictor for hepatotoxicity both in men [relative risk = 1.22 (95% confidence interval: 1.04 to 1.44)] and in women [relative risk = 1.72 (95% confidence interval: 1.20 to 2.46)]. Severe hepatotoxicity was also more common among all persons with CD4 >250 cells per microliter. Conclusions Hepatotoxicity was a common adverse effect of NVP among men and women with CD4 >250 cells per microliter. Chinese treatment guidelines should be considered to reflect this risk.

The economic and biological significance of blunt snout bream (Megalobrama amblycephala) makes this species important to explore the underlying molecular mechanism of hypoxia response. In the present study, we compared the transcriptional responses to serious hypoxia in skeletal muscle among hypoxia tolerant (MT), sensitive (MS) and control (without hypoxia treatment, MC) M. amblycephala obtained according to the time difference of losing balance after hypoxia treatment. A total of 88,200,889 clean reads were generated and assembled into 44,493 unigenes. Transcriptomic comparison revealed 463 genes differentially expressed among different groups. A similar hypoxia-induced transcription patterns suggested a common hypoxia response involved in cell cycle, p53 signaling pathway, apoptosis, heart contraction and blood circulation. Interesting, four genes, heat shock protein beta-8 (hspb8), cysteine/serine-rich nuclear protein 1 (csrnp1), salt-inducible kinase 1 (sik1), and visinin-like 1a (vsnl1a) were up-regulated in MT Vs MC but down-regulated in MS Vs MC. Additionally, FoxO signaling pathway was significantly enriched only in MT Vs MC. These results not only provided the first insights into the mechanism that muscle tissue coped with the hypoxia stress in cyprinid species, but offered a theory base for breeding of M. amblycephala with hypoxia-resistant traits.

A total of 14 novel dinucleotide microsatellite loci were developed using genomic DNA enrichment protocol and characterized for one Glyptosternoid catfish, Glyptosternum maculatum. Polymorphism was explored using 36 individuals from one natural population. The observed number of alleles ranged from 2 to 9. The ranges of observed and expected heterozygosity were 0.0278 to 1 and 0.4319 to 0.8498, respectively and the average of PIC was 0.5754. In addition, successful cross-species amplification of these set of microsatellite markers in the related taxa, Pseudechenneis sulcatus (McClelland) suggested that they would provide a useful tool for the genetic and conservation studies of Sisoridae species.

Hsp70 (70-kDa heat shock protein) plays a key regulatory role in muscle development. In order to evaluate whether recombinant Hsp70 protein (rMaHsp70) of M. amblycephala could affect muscle growth by inducing the expression of Myogenin (MyoG), Myogenic Differentiation Antigen (MyoD), Myogenic factor 5 (Myf5), myogenic regulatory factor 4 (MRF4) and myostatin (MSTN) in muscle, Hsp70 was injected intraperitoneally (IP) at two concentrations: 1.0 mg/mL and 3.4 mg/mL. The mRNA and protein levels of the transcription factor Pax-7 and the protein Hsp70 initially increased, then as concentration levels of injected recombinant M. amblycephala Hsp70 protein rMaHsp70 further increased, the protein Hsp70 decreased. In order to investigate the relationship between muscle hyperplasia, hypertrophy and satellite cell growth following rMaHsp70 administration, the number of satellite cells were analyzed and revealed that the numbers of satellite cells in low concentration group (1 mg/mL) was predominantly higher than in the other concentration groups in both red and white muscle. These results suggest that low concentrations of rMaHsp70 could decrease muscle-related gene expression and increase the number of satellite cells. Our research will be helpful in adapting Hsp70 as a feed additive to enhance M. amblycephala growth.

Epithelial protein lost in neoplasm (EPLIN) is a cytoskeleton-associated protein that is down-regulated in transformed cells. Two EPLIN isoforms (alpha and beta) are generated by alternative promoter usage from a single gene. In pigs EPLIN was preferentially expressed in the muscle of splay-legged piglets, but not in the healthy ones, suggesting that it plays an important role in muscle development. A precise mRNA expression analysis through muscle development could shed some light on the EPLIN function associated with splayed legs. This article describes the isolation of the two alternative splice variants of EPLIN mRNA in pigs. The chromosome assignment and several polymorphism sites were also identified to lay a foundation for potential breeding applications. Gene expression analysis by real-time polymerase chain reaction (PCR) showed that both of transcripts were expressed in almost all tissues examined but in variable amounts in adult pigs. The temporal expression analysis indicated that they are not coexpressional through muscle development: EPLIN-alpha was detected in developing skeletal muscle, but EPLIN-beta was not.

PHD3 is a hydroxylase that hydroxylates prolyl residues on hypoxia-inducible factors (HIFs) in mammals. In this study, the full-length cDNA and promoter sequences of Megalobrama amblycephala PHD3 gene were isolated by a modified RACE method. PHD3 cDNA was 1622 bp in length, including an ORF of 717 bp encoding 238 amino acid residues. The semi-quantitative PCR results suggested that PHD3 was highly expressed in liver in the normal condition, while after hypoxia treatment this gene was significantly increased in all analyzed tissues. PHD3 was detected only in the initial stages of M. amblycephala embryo development. In addition, the presence of another alternatively processed PHD3 transcript, designated PHD3Δ1 was observed in the process of analyzing the expression of PHD3. Both PHD3 and PHD3Δ1 were up-regulated under hypoxia, and had five the hypoxia response elements (HREs) by in silico scanning on the promoter. Further luciferase assay indicated that all HREs significantly responded to hypoxia. Taken together, these results suggest that PHD3 plays important roles in hypoxia response and early embryo development of M. amblycephala.

Skeletal muscle growth is regulated by both positive and negative factors, such as myogenic regulatory factors (MRFs) and myostatin (MSTN), and involves both hyperplasia and hypertrophy. In the present study, morphological changes during muscle development in Megalobrama amblycephala were characterized and gene expression levels were measured by quantitative real-time polymerase chain reaction (qRT-PCR) analysis in juvenile [60, 90, 120, and 180 days post-hatching (dph)] and adult fish. Our results show that during muscle development, the frequency of muscle fibers with a diameter20 μm dramatically decreased in both red and white muscles, with a concomitant increase in the frequency of30 μm fibers in red muscle and50 μm fibers in white muscle. At 90-120 dph, the ratio of hyperplastic to hypertrophic areas in red and white muscles increased, but later decreased at 120-180 dph. The effect of hypertrophy was significantly larger than hyperplasia during these phases. qRT-PCR indicated MRF and MSTN (MSTNa and MSTNb) genes had similar expression patterns that peaked at 120 dph, with the exception of MSTNa. This new information on the molecular regulation of muscle growth and rapid growth phases will be of value to the cultivation of M. amblycephala.

Eleven microsatellite markers were isolated and characterized from enriched genomic libraries of Channa argus. The loci were scored in 40 specimens from one natural population. We explored 6–17 alleles per locus with observed and expected heterozygosities ranging from 0.8000 to 1.0000 and 0.7269 to 0.9551, respectively, containing 0.830 average polymorphic information content. Significant deviations from Hardy–Weinberg equilibrium were detected only at one loci and linkage disequilibrium between three loci was found to be significant after applying Bonferroni correction (P < 0.005). These newly developed markers should be significant in population genetic assessment studies or construction of genetic linkage map for this important snakehead fish species in future.

BACKGROUND The molecular epidemiology of the HIV-1 CRF01_AE subtype as a risk factor for fast HIV-1 progression remains poorly understood. METHODS We analyzed HIV-1 tropism by utilizing samples from 201 treatment-naive patients in our multicenter cohort (12 research centers in different provinces of China). Tropism was determined by V3 loop sequencing. Data from 235 treatment-naive patients infected sexually (including aforementioned 201 patients) in this cohort with date of estimated seroconversion (EDS) were retrospectively evaluated. Median time from EDS to AIDS was analyzed by Kaplan-Meier curves. Hazard ratios were determined by Cox proportional model. RESULTS CRF01_AE subtype was predominant (46.0%), especially in the MSM group. Further analysis revealed that the proportion of X4 tropism was higher in the CRF01_AE subtype (45.5%) than in others (C/CRF07_BC/CRF08_BC, 4.3%; B, 6.1%; P

The ubiquitously expressed hypoxia-inducible factor-1α (HIF-1α) acts as a key transcription factor in regulating metabolism, development, cellular survival, proliferation and pathology under hypoxia condition. Compared to mammals, fish are more vulnerable to hypoxia stress and contamination; however, the regulation of HIF-1α in fish remains obscure. In this study, zebrafish HIF-1α promoter was cloned and found to possess a CpG island located at -97 to +403, but the canonical TATA-box was absent. Aligning 240-bp HIF-1α proximal promoter region of zebrafish with other vertebrates showed more than 82 % identity with cyprinid fishes. Further luciferase analysis suggested that the minimal core promoter might locate at -134 to +97, and several putative transcription factor binding sites were found in this region by bioinformatic analysis. Moreover, it was shown that the zebrafish HIF-1α mRNA was significantly activated by 10 μg/mL lipopolysaccharide (LPS) under hypoxia condition and peaked at 8 h after treatment, suggesting LPS- and hypoxia-regulated zebrafish HIF-1α transcriptional activity in a synergistic pattern. This synergistic effect was closely related to the living environment of fish, indicating that this mechanism would be more conducive to fish survival.

The 2009 flu pandemic involved the emergence of a new strain of a swine-origin H1N1 influenza virus (S-OIV H1N1) that infected almost every country in the world. Most infections resulted in respiratory illness and some severe cases resulted in acute lung injury. In this report, we are the first to describe a mouse model of S-OIV virus infection with acute lung injury and immune responses that reflect human clinical disease. The clinical efficacy of the antiviral oseltamivir (Tamiflu) administered in the early stages of S-OIV H1N1 infection was confirmed in the mouse model. Moreover, elevated levels of IL-17, Th-17 mediators and IL-17-responsive cytokines were found in serum samples of S-OIV-infected patients in Beijing. IL-17 deficiency or treatment with monoclonal antibodies against IL-17-ameliorated acute lung injury induced by the S-OIV H1N1 virus in mice. These results suggest that IL-17 plays an important role in S-OIV-induced acute lung injury and that monoclonal antibodies against IL-17 could be useful as a potential therapeutic remedy for future S-OIV H1N1 pandemics.

Histone deacetylase 4 (HDAC4) is an important modifier enzyme for chromatin remodeling and plays an essential role in regulating gene expression. Spatio-temporal expression spectrum revealed that zebrafish hdac4 mRNA, ubiquitously distributed in various tissues, were significantly higher at 36 hpf (hours post-fertilization) and 6 dpf (days post-fertilization) than other periods. Trichostatin A (TSA) inhibited the development of zebrafish embryos and transcription of hdac4 and mef2a (myocyte enhancer factor-2A). Moreover, five vectors containing different promoter regions of hdac4 were constructed in order to analyze promoter activity. The vector containing the region from -125 to +160 exhibited maximum luciferase activity that was approximately 30.3-fold and 58.9-fold higher than the control in two kinds of cells, respectively. By comparing the luciferase activities between the region from -302 to +30 and -698 to +30, it was suggested that the region between -698 and -302 might contain mild negative regulatory elements.

CapZ is a widely distributed and highly conserved actin-binding protein that caps the barbed end of actin filaments and nucleates actin polymerization in a Ca(2+)-independent manner. In myofibrils, it is localized in the Z-lines. In this study, we cloned and characterized Capz subunit genes from the pig muscle. The nucleotide sequences and the predicted protein sequences share high sequence identity with other mammalian orthologs. The reverse transcriptase polymerase chain reaction (RT-PCR) revealed that porcine Capzbeta, Capzalpha1, and Capzalpha2 genes are expressed in all 11 tissues studied (liver, spleen, small intestine, large intestine, lymph node, kidney, heart, skeletal muscle, brain, fat, and lung) but in variable amounts. Radiation hybrid mapping data indicated that Capzbeta, Capzalpha1, and Capzalpha2 map to q2.1-2.6 of pig chromosome 6 (SSC6), q1.6-q2.2 of pig chromosome 4 (SSC4), and q1.3-2.3 of pig chromosome 18 (SSC18), respectively. An A/C single nucleotide polymorphism in Capzbeta intron 4 was identified with a HhaI PCR restriction fragment length polymorphism, which showed great allele frequency differences between Guizhou Xiang, Guangxi Bama, Wuzhishan, Tongcheng, Landrace, and Yorkshire pigs. The association analysis suggested that the Capzbeta genotype was associated with leaf fat (P0.05) in our experimental population.

Calsarcins comprise a novel family of muscle-specific calcineurin-interacting proteins and play an important role in modulating both the function and substrate specificity of calcineurin in muscle cells. In this study, we cloned and characterized calsarcins from pig muscle. The deduced amino acid sequences of porcine calsarcin-1 (CS-1), calsarcin-2 (CS-2), and calsarcin-3 (CS-3) share the same putative calcineurin and alpha-actinin binding regions. Radiation hybrid mapping data indicate that CS-1 and CS-2 map to q2.1-2.5 of pig chromosome 8 (SSC8) and q2.4 of pig chromosome 14 (SSC14), respectively. The mRNA expressions of both CS-1 and CS-2 are regulated in skeletal muscle similarly during postnatal development but not during prenatal development, indicating differences in function, additionally demonstrated by minute differences in cellular localization within Pig Kidney Epithelial cells (PK15). We provide the first evidence that CS-1 is abundantly expressed in porcine heart and has an expression pattern similar to the human gene. This result suggests that the pig may be a suitable animal model to study the function of calsarcins in human heart disease.

Background In a previous screen to identify differentially expressed genes associated with embryonic development, the porcine PNAS-4 gene had been found. Considering differentially expressed genes in early stages of muscle development are potential candidate genes to improve meat quality and production efficiency, we determined how porcine PNAS-4 gene regulates meat production. Therefore, this gene has been sequenced, expression analyzed and associated with meat production traits. Results We cloned the full-length cDNA of porcine PNAS-4 gene encoding a protein of 194 amino acids which was expressed in the Golgi complex. This gene was mapped to chromosome 10, q11–16, in a region of conserved synteny with human chromosome 1 where the human homologous gene was localized. Real-time PCR revealed that PNAS-4 mRNA was widely expressed with highest expression levels in skeletal muscle followed by lymph, liver and other tissues, and showed a down-regulated expression pattern during prenatal development while a up-regulated expression pattern after weaning. Association analysis revealed that allele C of SNP A1813C was prevalent in Chinese indigenous breeds whereas A was dominant allele in Landrace and Large White, and the pigs with homozygous CC had a higher fat content than those of the pigs with other genotypes (P < 0.05). Conclusion Porcine PNAS-4 protein tagged with green fluorescent protein accumulated in the Golgi complex, and its mRNA showed a widespread expression across many tissues and organs in pigs. It may be an important factor affecting the meat production efficiency, because its down-regulated expression pattern during early embryogenesis suggests involvement in increase of muscle fiber number. In addition, the SNP A1813C associated with fat traits might be a genetic marker for molecular-assisted selection in animal breeding.