Molecular cloning and expression pattern of oriental river prawn (Macrobrachium nipponense) nitric oxide synthase.

Nitric oxide synthase (NOS) produces nitric oxide (NO) by catalyzing the conversion of l-arginine to l-citrulline, with the concomitant oxidation of nicotinamide adenine dinucleotide phosphate. Recently, various studies have verified the importance of NOS invertebrates and invertebrates. However, the NOS gene family in the oriental river prawn Macrobrachium nipponense is poorly understood. In this study, we cloned the full-length NOS complementary DNA from M. nipponense (MnNOS) and characterized its expression pattern in different tissues and at different developmental stages. Real-time quantitative polymerase chain reaction (RT-qPCR) showed the MnNOS gene to be expressed in all investigated tissues, with the highest levels observed in the androgenic gland (P < 0.05). Our results revealed that the MnNOS gene may play a key role in M. nipponense male sexual differentiation. Moreover, RT-qPCR revealed that MnNOS mRNA expression was significantly increased in post-larvae 10 days after metamorphosis (P < 0.05). The expression of this gene in various tissues indicates that it may perform versatile biological functions in M. nipponense.

Molecular cloning, characterization, and expression analysis of a Broad-Complex homolog during development in the oriental river prawn Macrobrachium nipponense.

Broad-Complex (BR-C) is an early ecdysone-responsive gene encoding a family of zinc-finger transcription factors. In this study, we isolated the full-length cDNA of a BR-C homolog from the testes of the oriental river prawn (Macrobrachium nipponense), according to established expressed sequence tag information, using the rapid amplification of cDNA ends technique. The homolog was designated as MnBR-C. The full-length cDNA of MnBR-C contained a 1095-bp open reading frame encoding a precursor protein of 365 amino acid residues. Comparative and bioinformatic analyses revealed that MnBR-C exhibited a high degree of homology with BR-C proteins, and contained the BTB and Zf-H2C2-2 domains. Real-time quantitative polymerase chain reaction (qPCR) analysis revealed that the MnBR-C expression level varied significantly in the developing embryo, postembryonic larva, and adult tissue. Real-time qPCR showed that the MnBR-C gene was expressed in all of the tissues investigated, with the highest level of expression in the brain. In addition, MnBR-C was more abundantly expressed in the testes than in the ovaries.

Genomic cloning, expression, and single nucleotide polymorphism association analysis of the insulin-like androgenic gland hormone gene in the oriental river prawn (Macrobrachium nipponense).

Increasing evidence suggests that the insulin-like androgenic gland hormone (IAG) gene plays an important role in male sexual differentiation, metabolism, and growth in crustaceans. In the present study, we isolated the full-length genome sequence of IAG by genome walking based on the cDNA sequence in Macrobrachium nipponense. Four novel single nucleotide polymorphisms (SNPs) were studied, including 509G>T, 529G>T, 590A>T in intron 1, and 2226A>G in intron 2. The association of genetic variation with growth traits [body length (BL) and body weight (BW)] was analyzed. Individuals with GG geno- type at locus 2226A>G maintained higher mean BL (P < 0.01) and BW (P < 0.05) than AA and GA individuals. These results suggest that IAG SNPs may be useful molecular markers for selecting growth traits in M. nipponense.

Characterization of the male-specific lethal 3 gene in the oriental river prawn, Macrobrachium nipponense.

In this study, male-specific lethal 3 homolog (Mnmsl3) was cloned and characterized from the freshwater prawn Macrobrachium nipponense (Crustacea: Decapoda: Palaemonidae) by rapid amplification of cDNA ends. The deduced amino acid sequences of Mnmsl3 showed high-sequence homology to the insect Msl3 and contained a conserved chromatin organization modifier domain and an MORF4-related gene domain. Real-time quantitative reverse transcription-polymerase chain reaction showed that the Mnmsl3 gene was expressed in all the investigated tissues, with the highest level of expression in the testis. The expression level of Mnmsl3 between males and females was different in the gonad (testis or ovary), abdominal ganglion, and heart. The results revealed that the Mnmsl3 gene might play roles in regulating chromatin and in dosage compensation of M. nipponense. Real-time quantitative reverse transcription-polymerase chain reaction also revealed that Mnmsl3 mRNA expression was significantly increased in both 5 and 20 days post-larvae after metamorphosis, suggesting that Mnmsl3 plays complex and important roles in the early embryonic development and sex differentiation of M. nipponense.

Genetic analysis of QTL for eye cross and eye diameter in common carp (Cyprinus carpio L.) using microsatellites and SNPs.

A group of 107 F1 hybrid common carp was used to construct a linkage map using JoinMap 4.0. A total of 4877 microsatellite and single nucleotide polymorphism (SNP) markers isolated from a genomic library (978 microsatellite and 3899 SNP markers) were assigned to construct the genetic map, which comprised 50 linkage groups. The total length of the linkage map for the common carp was 4775.90 cM with an average distance between markers of 0.98 cM. Ten quantitative trait loci (QTL) were associated with eye diameter, corresponding to 10.5-57.2% of the total phenotypic variation. Twenty QTL were related to eye cross, contributing to 10.8-36.9% of the total phenotypic variation. Two QTL for eye diameter and four QTL for eye cross each accounted for more than 20% of the total phenotypic variation and were considered to be major QTL. One growth factor related to eye diameter was observed on LG10 of the common carp genome, and three growth factors related to eye cross were observed on LG10, LG35, and LG44 of the common carp genome. The significant positive relationship of eye cross and eye diameter with other commercial traits suggests that eye diameter and eye cross can be used to assist in indirect selection for many commercial traits, particularly body weight. Thus, the growth factor for eye cross may also contribute to the growth of body weight, implying that aggregate breeding could have multiple effects. These findings provide information for future genetic studies and breeding of common carp.

Molecular cloning and expression analysis of female sterile homeotic gene (fsh) in the oriental river prawn Macrobrachium nipponense.

The gene female sterile homeotic (fsh) plays crucial roles in molecular function, including protein kinase activity and DNA binding, which are involved in biological processes such as terminal region determination and negative regulation of DNA-dependent transcription. Although fsh has been found in Drosophila melanogaster, little is known regarding its expression in crustaceans. In this study, a fsh gene homologue, designated as Mnfsh, was cloned and characterized from the testis of the oriental river prawn, Macrobrachium nipponense, by using EST analysis and the RACE approach for the first time. The full-length cDNA of Mnfsh was 2029 bp, consisting of a 5' UTR of 361 bp, a 3' UTR of 216 bp, and an ORF of 1452 bp encoding 484 amino acids. qRT-PCR analysis showed that the Mnfsh gene was expressed in the testis, ovary, muscle, heart, eyestalk, and abdominal ganglion, with the highest level of expression in the ovary and the lowest in the heart. qRT-PCR analyses showed that the expression levels of Mnfsh mRNA both significantly increased in the zoea stage, the VII larvae, and 1st day post-larvae after metamorphosis. In conclusion, the results of the present study indicate that Mnfsh is an arthropod fsh homologue and probably also plays important roles in embryogenesis, organogenesis, and morphological differentiation of M. nipponense.

Gene expression profile analysis of testis and ovary of oriental river prawn, Macrobrachium nipponense, reveals candidate reproduction-related genes.

This study utilized high-throughput RNA sequencing technology to identify reproduction- and development-related genes of Macrobrachium nipponense by analyzing gene expression profiles of testis and ovary. More than 20 million 1 x 51-bp reads were obtained by Illumina sequencing, generating more than 7.7 and 11.7 million clean reads in the testis and ovary library, respectively. As a result, 10,018 unitags were supposed to be differentially expressed genes (DEGs) between ovary and testis. Compared to the ovary library, 4563 (45.5%) of these DEGs exhibited at least 6-fold upregulated expression, while 5455 (54.5%) DEGs exhibited at least 2-fold downregulated expression in the testis. The Gene Ontology (GO) enrichment analysis showed that 113 GO terms had potential molecular functions in reproduction. The Kyoto Encyclopedia of Genes and Genomes results revealed that the most important pathways may be relevant to reproduction and included 7 pathways. Forty-two genes were identified as reproduction-, development-, and sex-related genes based on GO classification and sequence comparison with other publications, including male reproductive-related LIM protein, spermatogenesis-associated protein, gametocyte-specific factor 1, VASA-like protein, vitellogenin, sex-determining protein fem-1, and other potential candidates. These results will advance research in the field of molecular genetics in M. nipponense and offer a valuable resource for further research related to reproduction in crustaceans.

Identification of androgenic gland microRNA and their target genes to discover sex-related microRNA in the oriental river prawn, Macrobrachium nipponense.

The oriental river prawn, Macrobrachium nipponense, is an important aquaculture species in China. The androgenic gland produces hormones that play crucial roles in the differentiation of crustaceans to the male sex. MicroRNA (miRNA) post-transcriptionally regulates many protein-coding genes, influencing important biological and metabolic processes. However, currently, there is no published data identifying miRNA in M. nipponense. In this study, we identified novel miRNA in the androgenic gland of M. nipponense. Using the high-throughput Illumina Solexa system, 1077 miRNA were identified from small RNA libraries by aligning with the de novo androgenic gland transcriptome of M. nipponense (obtained from RNA-Seq) and the sequences in the miRBase21 database. A total of 8,248, 76,011, and 78,307 target genes were predicted in the EST and SRA sequences provided in the NCBI database, and the androgenic gland transcriptome of M. nipponense, respectively. Some potential sex-related miRNA were identified based on the function of the predicted target genes. The results of our study provide new information regarding the miRNA expression in M. nipponense, which could be the basis for further genetic studies on decapod crustaceans.

Comparative transcriptome analysis of testes and ovaries for the discovery of novel genes from Amur sturgeon (Acipenser schrenckii).

Sturgeons (Acipenser schrenckii) are of high evolutionary, economic, and conservation value, and caviar isone of the most valuable animal food products in the world. The Illumina HiSeq2000 sequencing platform was used to construct testicular and ovarian transcriptomes to identify genes involved in reproduction and sex determination in A. schrenckii. A total of 122,381 and 114,527 unigenes were obtained in the testicular and ovarian transcriptomes, respectively, with average lengths of 748 and 697 bp. A total of 46,179 genes were matched to the non-redundant nr database. GO (31,266), KEGG (39,712), and COG analyses (20,126) were performed to identify potential genes and their functions. Twenty-six gene families involved in reproduction and sex determination were identified from the A. schrenckii testicular and ovarian transcriptomes based on functional annotation of non-redundant transcripts and comparisons with the published literature. Furthermore, 1309 unigenes showed significant differences between the testes and ovaries, including 782 genes that were up-regulated in the testes and 527 that were up-regulated in the ovaries. Eleven genes were involved in reproduction and sex determination mechanisms. Furthermore, 19,065 simple sequence repeats (SSRs) were identified in the expressed sequence tagged dataset, and 190,863 and 193,258 single nucleotide polymorphisms (SNPs) were obtained from the testicular and ovarian transcriptomic databases, respectively. This study provides new sequence information about A. schrenckii, which will provide a basis for the further study of reproduction and sex determination mechanisms in Acipenser species. The potential SSR and SNP markers isolated from the transcriptome may shed light on the evolution and molecular ecology of Acipenser species.

Genetic diversity analysis of oriental river prawn, Macrobrachium nipponense, in Yellow River using microsatellite marker.

To assess the genetic status of this species, the genetic diversity of wild Macrobrachium nipponense from seven geographic locations in the Yellow River basin were investigated using 20 polymorphic microsatellite DNA loci. The genetic diversity between populations was indicated by the mean number of alleles per locus and mean observed heterozygosity (H) and the expected H, which was arranged from 2 to 10, from 0.4705 to 0.5731, and from 0.5174 to 0.6146, respectively. Hardy-Weinberg equilibrium analysis indicated that a deficiency of heterozygotes existed in all seven populations. Both the F(ST) and AMOVA analyses showed that there is significant difference on population differentiation among populations. The UPGMA clustering tree demonstrated that their close relationship is consistent with their geographic proximity. The data suggest that this Yellow River population has a wide genetic base that is suitable for breeding.

Molecular cloning and expression analysis of two sex-lethal homolog genes during development in the oriental river prawn, Macrobrachium nipponense.

In this study, two Sxl gene homologs, designated as Mnsxl1 and Mnsxl2, were cloned and characterized from the freshwater prawn Macrobrachium nipponense by rapid amplification of cDNA ends. The deduced amino acid sequences of Mnsxl1 and Mnsxl2 showed high sequence homology to the insect Sxl and contained conserved domains in two RNA-binding motifs. Real-time quantitative reverse transcription-polymerase chain reaction (RT-QPCR) showed that the Mnsxl1 and Mnsxl2 genes were expressed in all investigated tissues, with the highest level of expression in the intestine and liver. RT-QPCR also revealed that Mnsxl1 and Mnsxl2 mRNAs expressions were both significantly increased at 5 and 20 days post-larvae after metamorphosis. Thus, the results of the present study imply that Mnsxl1 and Mnsxl2 play complex and important roles in the sex differentiation of M. nipponense.

Hybrid Ag@TiO2 core-shell nanostructures with highly enhanced photocatalytic performance.

A new synthetic approach has been developed to prepare silver@titanium dioxide (Ag@TiO2) core-shell nanostructures with controllable size, shape, crystal phase and function at ambient conditions (e.g. in water, ≤100 ° C). This approach shows a few unique features, including short reaction time (a few minutes) for forming core-shell nanostructures, no requirement of high temperature calcinations for generating TiO2 (e.g. at ~100 ° C in our case), tunable TiO2 shell thickness, high yield and good reproducibility. The experimental results show that the Ag@TiO2 core-shell nanostructures exhibit excellent photocatalytic activity compared to the commercial TiO2 (P25) and Ag-doped TiO2 nanocomposite in the degradation of organic dye molecules (e.g. methyl orange) with ultraviolet (UV) irradiation. This could be attributed to the large surface area of TiO2 nanoparticles for maximum harvesting of UV light, mixed anatase and rutile crystalline phases in the TiO2 shell and the effective charge separation between Ag and TiO2 that can reduce the possible recombination of electron-hole (e(-)-h(+)) pairs within TiO2 generated under UV radiation. To further understand the charge separation situation within Ag-TiO2 composites, theoretical simulation (e.g. density functional theory, DFT) was employed in this study. The DFT simulation results indicate that for the Ag@TiO2 core-shell nanostructures, photo-generated electrons transfer readily from the external TiO2 layer to the internal Ag layer with heavy accumulation compared to those doping Ag on TiO2 surfaces, which may reduce the recombination of e(-)-h(+) pairs and thus enhance the photocatalytic efficiency. The findings may open a new strategy to synthesize TiO2-based photocatalysts with highly enhanced efficiency for environmental remediation applications.

[Study on the rule of retention value in weak acid anion chromatography].

In chromatography, if analytes are strong acid anions or strong alkali cations, the dependence of a logarithm of the adjusted retention value on the logarithm of the concentration of the eluent is given by log tR' = const - (j/i).log C, where j is the charge of the analyte ion, i is the charge of the eluent ion, const is a constant that depends on the column, eluent and analyte ion. But in weak acid anion chromatography, existing forms of the sample anion change with the change of the concentration of the eluent. So the study on the relationship between retention value of weak acid anion and concentration of the eluent is more difficult. Relationship between adjusted retention value of some weak acid anion and concentration of the eluent was studied with sodium hydroxide as eluent. The nonlinear fitting function has been set up. We transform it into multiple linear fitting to give the answer. The correlation coefficients for phosphate defined by the function were over 0.99. The relative errors with the use of function were below 5%.

Presence of serum anti-p53 antibodies is associated with pleural effusion and poor prognosis in lung cancer patients.

This study was designed prospectively to evaluate the development of anti-p53 antibodies (Abs) in lung cancer patients in relation to their clinical outcome. Sera, derived from 125 lung cancer patients, consisting of 14 small cell lung cancers (SCLC) and 111 non-SCLCs (NSCLC), were surveyed. The p53-null human NSCLC cell line, NCI-H1299, transfected with a human mutant p53 gene was prepared as the source of p53 antigen for immunoblotting analyses to detect the presence of serum anti-p53 Abs. The control group included sera from 10 healthy adults and 14 patients with benign pulmonary diseases. Clinical data including staging and survival were recorded for statistical analyses. The anti-p53 Abs were found in 8% (10 of 125) of the lung cancer patients studied (8.1% of NSCLC versus 7.1% of SCLC patients), whereas none of the control sera had detectable anti-p53 Abs. The presence of anti-p53 Abs was closely associated with malignant pleural effusions (P = 0.001). The p53 Ab-positive patients had a worse prognosis than the p53 Ab-negative patients (P < 0.02; median survival, 20 versus 41 weeks). In both univariate and multivariate analyses, the tumor extension and probably the presence of anti-p53 Abs were significant predictors for cancer death. The development of anti-p53 Abs (n = 9) was also a predictor for poor survival in patients with malignant effusions (n = 51). In conclusion, the presence of serum anti-p53 Abs is closely associated with malignant pleural effusions in lung cancer patients. It may serve as a negative prognostic factor for survival independent of malignant pleural effusions and tumor staging.