Transcriptome and metabolome analyses reveal gender-specific expression genes in sea cucumber (Holothuria leucospilota).

The sea cucumber Holothuria leucospilota, a nutritive and commercial marine species, has a high protein and low lipid content. To date, the mechanisms underlying gender determination and differentiation in sea cucumbers remain unclear. Identifying gender-specific molecular markers is an effective method of revealing the genetic basis of gender determination and differentiation. The inability to distinguish between male and female individuals causes reproductive efficiency to decline in aquaculture. In this study, we used the gonads of the sea cucumber H. leucospilota as samples to conduct the experiment. The differentially abundant metabolites (DAMs) detected by liquid chromatography-mass spectrometry were enriched in pathways associated with prolactin metabolism, insulin metabolism, hypoxia-inducible factor-1 signaling, and calcium signaling. At the transcriptome level, Illumina sequencing was performed on H. leucospilota, demonstrating that gender-specific expression genes were enriched in the retinoic acid-inducible gene I-like receptor signaling pathway, C-type lectin receptor signaling pathway, alpha-linolenic acid metabolism, and ether lipid metabolism by the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. By analyzing the common pathways between DAMs and differentially expressed genes, we found that gender-related genes of H. leucospilota were mostly enriched in the necroptosis pathway and the cysteine and methionine metabolism pathways. According to the common pathways, uch-sc1 and uch-sc2 are male-specific expression genes, and uch-sc3 and bhmt are female-specific expression genes at the mRNA level. These results provide information on gender differences in H. leucospilota.

Aesculin offers increased resistance against oxidative stress and protective effects against Aß-induced neurotoxicity in Caenorhabditis elegans.

Aesculin, a coumarin compound, is one of the major active ingredients of traditional Chinese herbal medicine Qinpi (Cortex Fraxini), which has been reported to exhibit antioxidative, anti-inflammatory and neuroprotective properties against oxidative stress and cellular apoptosis. However, the regulatory mechanisms remain poorly characterized in vivo. This research was performed to explore the underlying molecular mechanisms behind aesculin response conferring oxidative stress resistance, and the protective effects on amyloid-ß (Aß)-mediated neurotoxicity in Caenorhabditis elegans. Study indicated that aesculin plays the protective roles for C. elegans against oxidative stress and Aß-mediated neurotoxicity and reduces the elevated ROS and MDA contents through enhancement of antioxidant defenses. The KEGG pathway analysis suggested that the differentially expressed genes are mainly involved in longevity regulating pathway, and the nuclear translocation of DAF-16 and the RNAi of daf-16 and hsf-1 indicated that DAF-16 and HSF-1 play critical roles in integrating upstream signals and inducing the expressions of stress resistance-related genes. Furthermore, the up-regulated expressions of their target genes such as sod-3 and hsp-16.2 were confirmed in transgenic GFP reporter strains CF1553 and CL2070, respectively. These results indicated that the regulators DAF-16 and HSF-1 elevate the stress resistance of C. elegans by modulating stress-responsive genes. Further experiments revealed that aesculin is capable of suppressing Aß-induced oxidative stress and apoptosis and improves chemosensory behavior dysfunction in Aß-transgenic nematodes. In summary, this study suggested that aesculin offers increased resistance against oxidative stress and protective effects against Aß-induced neurotoxicity through activation of stress regulators DAF-16 and HSF-1 in nematodes.

Neuroprotective Effects of Palmatine via the Enhancement of Antioxidant Defense and Small Heat Shock Protein Expression in Aß-Transgenic Caenorhabditis elegans.

Palmatine is a naturally occurring isoquinoline alkaloid that has been reported to display neuroprotective effects against amyloid-ß- (Aß-) induced neurotoxicity. However, the mechanisms underlying the neuroprotective activities of palmatine remain poorly characterized in vivo. We employed transgenic Caenorhabditis elegans models containing human Aß 1-42 to investigate the effects and possible mechanisms of palmatine-mediated neuroprotection. Treatment with palmatine significantly delayed the paralytic process and reduced the elevated reactive oxygen species levels in Aß-transgenic C. elegans. In addition, it increased oxidative stress resistance without affecting the lifespan of wild-type C. elegans. Pathway analysis suggested that the differentially expressed genes were related mainly to aging, detoxification, and lipid metabolism. Real-time PCR indicated that resistance-related genes such as sod-3 and shsp were significantly upregulated, while the lipid metabolism-related gene fat-5 was downregulated. Further studies demonstrated that the inhibitory effects of palmatine on Aß toxicity were attributable to the free radical-scavenging capacity and that the upregulated expression of resistance-related genes, especially shsp, whose expression was regulated by HSF-1, played crucial roles in protecting cells from Aß-induced toxicity. The research showed that there were significantly fewer Aß deposits in transgenic CL2006 nematodes treated with palmatine than in control nematodes. In addition, our study found that Aß-induced toxicity was accompanied by dysregulation of lipid metabolism, leading to excessive fat accumulation in Aß-transgenic CL4176 nematodes. The alleviation of lipid disorder by palmatine should be attributed not only to the reduction in fat synthesis but also to the inhibition of Aß aggregation and toxicity, which jointly maintained metabolic homeostasis. This study provides new insights into the in vivo neuroprotective effects of palmatine against Aß aggregation and toxicity and provides valuable targets for the prevention and treatment of AD.

Antioxidant Peptides from Sepia esculenta Hydrolyzate Attenuate Oxidative Stress and Fat Accumulation in Caenorhabditis elegans.

The hydrolysate of golden cuttlefish (Sepia esculenta) was prepared by using papain, and then, it was further separated by ultrafiltration, gel filtration chromatography, and reverse-phase high-performance liquid chromatography (RP-HPLC). The peptide components of the active fraction were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and then two novel peptides, SeP2 (DVEDLEAGLAK, 1159.27 Da) and SeP5 (EITSLAPSTM, 1049.22 Da), were obtained and displayed significant alleviation effects on oxidative stress in Caenorhabditis elegans. Studies indicated that S. esculenta antioxidant peptides (SePs) increase superoxide dismutase (SOD) activity but reduce reactive oxygen species (ROS) and malondialdehyde (MDA) levelsin oxidation-damaged nematodes. Using transgenic CF1553 nematodes, the sod-3p::GFP expression in the worms treated with SePs was significantly higher than that of the control nematodes. Real-time PCR also demonstrated that the expression of stress-related genes such as sod-3 is up-regulated by SePs. Furthermore, studies showed that SePs could obviously decrease fat accumulation as well as reduce the elevated ROS and MDA levels in high-fat nematodes. Taken together, these results indicated that SePs are capable of the activation of antioxidant defense and the inhibition of free radicals and lipid peroxidation, play important roles in attenuating oxidative stress and fat accumulation in C. elegans, and might have the potential to be used in nutraceutical and functional foods.

Novel Bioactive Peptides from Meretrix meretrix Protect Caenorhabditis elegans against Free Radical-Induced Oxidative Stress through the Stress Response Factor DAF-16/FOXO.

The hard clam Meretrix meretrix, which has been traditionally used as medicine and seafood, was used in this study to isolate antioxidant peptides. First, a peptide-rich extract was tested for its protective effect against paraquat-induced oxidative stress using the nematode model Caenorhabditis elegans. Then, three novel antioxidant peptides; MmP4 (LSDRLEETGGASS), MmP11 (KEGCREPETEKGHR) and MmP19 (IVTNWDDMEK), were identified and were found to increase the resistance of nematodes against paraquat. Circular dichroism spectroscopy revealed that MmP4 was predominantly in beta-sheet conformation, while MmP11 and MmP19 were primarily in random coil conformation. Using transgenic nematode models, the peptides were shown to promote nuclear translocation of the DAF-16/FOXO transcription factor, a pivotal regulator of stress response and lifespan, and induce the expression of superoxide dismutase 3 (SOD-3), an antioxidant enzyme. Analysis of DAF-16 target genes by real-time PCR reveals that sod-3 was up-regulated by MmP4, MmP11 and MmP19 while ctl-1 and ctl-2 were also up-regulated by MmP4. Further examination of daf-16 using RNA interference suggests that the peptide-increased resistance of C. elegans to oxidative stress was DAF-16 dependent. Taken together, these data demonstrate the antioxidant activity of M. meretrix peptides, which are associated with activation of the stress response factor DAF-16 and regulation of the antioxidant enzyme genes.

Bioactive Peptides from Angelica sinensis Protein Hydrolyzate Delay Senescence in Caenorhabditis elegans through Antioxidant Activities.

Since excessive reactive oxygen species (ROS) is known to be associated with aging and age-related diseases, strategies modulating ROS level and antioxidant defense systems may contribute to the delay of senescence. Here we show that the protein hydrolyzate from Angelica sinensis was capable of increasing oxidative survival of the model animal Caenorhabditis elegans intoxicated by paraquat. The hydrolyzate was then fractionated by ultrafiltration, and the antioxidant fraction (<3 kDa) was purified by gel filtration to obtain the antioxidant A. sinensis peptides (AsiPeps), which were mostly composed of peptides with <20 amino acid residues. Further studies demonstrate that AsiPeps were able to reduce the endogenous ROS level, increase the activities of the antioxidant enzymes superoxide dismutase and catalase, and decrease the content of the lipid peroxidation product malondialdehyde in nematodes treated with paraquat or undergoing senescence. AsiPeps were also shown to reduce age pigments accumulation and extend lifespan but did not affect the food-intake behavior of the nematodes. Taken together, our results demonstrate that A. sinensis peptides (AsiPeps) are able to delay aging process in C. elegans through antioxidant activities independent of dietary restriction.

Caenorhabditis elegans in Chinese medicinal studies: making the case for aging and neurodegeneration.

Aging is a progressive process with degenerative changes of various tissues and organs. As a classic model organism in genetics and neurobiology, Caenorhabditis elegans is also a powerful system in aging and behavioral studies and can be used at both the molecular and organismal levels to evaluate potential therapeutics for age-related neurodegeneration, owing to its short life span, relative simplicity, and high degree of experimental tractability as well as significant conservation of disease genes and signaling pathways with humans. We attempt here to summarize the use of C. elegans models in exploring traditional Chinese medicine for potential remedies against aging and neurodegeneration.

[Analysis of SSR information in EST resource of sweet wormwood (Artemisia annua) and development of EST-SSR marker].

OBJECTIVE: To study the distribution frequency and characteristics of nucleotide repeat of 94 923 ESTs for developing microsatellite primers, providing a theoretical basis and technical support for appropriate conservation and application of sweet wormwood (Artemisia annua). METHOD: EST-SSR detection was performed using Perl program MISA. Gene Ontology (GO) annotations were formatted for input into the GOSlim program and the output was parsed to count the occurrence of each GO category. Primer 3 software was used to design 18 pairs primers, amplified products were separated on a 6% denaturing polyacrylamide gel using silver staining. RESULT: By searching with Active Perl, totally 2 110 SSRs were detected, accounting for 8.6%. The frequency of occurrence of dinucleotide and trinucleotide was 28% and 50.4%, respectively. The most common repeat motifs of trinucleotide were ACC/GGT, accounting for 9.8%. Three hundred and twelve SSR-ESTs were annotated using GO terms. The suitable PCR system of 15 pairs primers was established, and revealed microsatellite polymorphism in 36 individuals. CONCLUSION: There are a variety of motifs at EST-SSR locus in sweet wormwood, and more effective amplification and polymorphism in 18 pairs detected primers. Therefore, EST resource is an effective and feasible approach to develop SSR markers, and EST-SSRs will a powerful tool for studies of sweet wormwood genetic resources.

Molecular structural and functional characterization of STAT1 gene regulatory region in teleost Channa argus.

The transcription factor STAT1 is involved in signal transduction of type I and II interferons (IFNs). However, the molecular characteristics of the STAT1 regulatory region still remain to be elucidated in teleosts. In the present study, the complete cDNA and the regulatory region of the STAT1 gene were isolated from snakehead (Channa argus). More than 2.4kb 5'-flanking region of STAT1 shares the regulatory elements of IFN-stimulated response element (ISRE) and IFN-gamma activation site (GAS). Consensus ISRE and GAS were located from -373 to -361 and -716 to -724 in the promoter region, respectively. Moreover, it is noticeable that the crucial elements of ISRE (+698 to +710) and GAS (+294 and +301) are present in the first intron of snakehead STAT1. Comparisons of six vertebrate STAT1 5'-flanking regions all present the common sequence characteristics of IFN-induced gene promoter, which include ISRE, GAS and Sp1 sites. In order to further characterize the snakehead STAT1 regulatory region, six reporter constructs of snakehead STAT1 promoter and first intron were generated to examine the specificity to human interferon-gamma (hIFN-gamma). Only those constructs containing the ISRE element showed notable reporter activity after stimulation of Hela cells with hIFN-gamma. However, sequential deletions of putative transcription factor binding sites indicated that GAS elements have little effect on the promoter and intronic activity in response to hIFN-gamma. Taken together, these results suggest that the regulatory mechanisms of IFN-signalling appear to be mediated in a similar manner in fish and mammals.

Gene structures and promoter characteristics of interferon regulatory factor 1 (IRF-1), IRF-2 and IRF-7 from snakehead Channa argus.

Three interferon regulatory factor (IRF) genes, CaIRF-1, CaIRF-2 and CaIRF-7, and their promoters of snakehead (Channa argus) were cloned and characterized. The CaIRF-1 gene consists of ten exons, spans 4.3 kb and encodes a putative peptide of 299 aa. The CaIRF-2 gene consists of nine exons, spans 8 kb and encodes a putative peptide of 328 aa. The gene organizations of CaIRF-1 and CaIRF-2 are very similar to that of human IRF-1 and IRF-2 except more compact. Comparison of exon-intron organization of the two genes indicated a common evolutionary structure, notably within the exons encoding the DNA binding domain (DBD) of the two factors. The CaIRF-7 gene spans 4.1 kb and encodes a putative peptide of 437 aa. However, the gene organization of CaIRF-7 consisting of ten exons is different to human IRF-7a gene which has an intron in 5' UTR. Three CaIRFs share homology in N-terminal encompassing the DBD that contains a characteristic repeat of tryptophan residues. The promoters of CaIRF-1 and CaIRF-2 genes contain the conserved sites for NF-kappaB and Sp1. The gamma-IFN activation sites (GAS) were found in the promoters of CaIRF-1 and CaIRF-7. The promoter of CaIRF-7 contains conserved interferon stimulating response element (ISRE) which is characteristic of IFN-induced gene promoter, and suggests that there also exist intracellular amplifier circuit in fish IFN signal pathway. Moreover, the element GAAANN oriented in both directions is repeated in CaIRF promoter regions, which confers to further inducibility by IFN. The constitutive expression of CaIRF genes were found to increase obviously in response to induction by the known IFN-inducer poly I:C.