Inhibitor AN3661 reveals biological functions of Arabidopsis CLEAVAGE and POLYADENYLATION SPECIFICITY FACTOR 73.

Cleavage and polyadenylation specificity factor (CPSF) is a protein complex that plays an essential biochemical role in mRNA 3'-end formation, including poly(A) signal recognition and cleavage at the poly(A) site. However, its biological functions at the organismal level are mostly unknown in multicellular eukaryotes. The study of plant CPSF73 has been hampered by the lethality of Arabidopsis (Arabidopsis thaliana) homozygous mutants of AtCPSF73-I and AtCPSF73-II. Here, we used poly(A) tag sequencing to investigate the roles of AtCPSF73-I and AtCPSF73-II in Arabidopsis treated with AN3661, an antimalarial drug with specificity for parasite CPSF73 that is homologous to plant CPSF73. Direct seed germination on an AN3661-containing medium was lethal; however, 7-d-old seedlings treated with AN3661 survived. AN3661 targeted AtCPSF73-I and AtCPSF73-II, inhibiting growth through coordinating gene expression and poly(A) site choice. Functional enrichment analysis revealed that the accumulation of ethylene and auxin jointly inhibited primary root growth. AN3661 affected poly(A) signal recognition, resulted in lower U-rich signal usage, caused transcriptional readthrough, and increased the distal poly(A) site usage. Many microRNA targets were found in the 3' untranslated region lengthened transcripts; these miRNAs may indirectly regulate the expression of these targets. Overall, this work demonstrates that AtCPSF73 plays important part in co-transcriptional regulation, affecting growth, and development in Arabidopsis.

Effects of Collagen Hydrolysate From Large Hybrid Sturgeon on Mitigating Ultraviolet B-Induced Photodamage.

Ultraviolet B (UVB) radiation leads to the excessive accumulation of reactive oxygen species (ROS), which subsequently promote inflammation, degradation of the extracellular matrix, and photoaging in skin. Thus antioxidant activity is particularly important when screening for active substances to prevent or repair photodamage. Marine fish-derived bioactive peptides have become a trend in cosmetics and functional food industries owing to their potential dermatological benefits. In this study, 1-diphenyl- 2-pycryl-hydrazyl (DPPH) scavenging activity was selected to optimize the hydrolysis conditions of sturgeon skin collagen peptides with antioxidant activity. The optimal hydrolysis conditions for sturgeon skin collagen hydrolysate (SSCH) were determined by response surface methodology, which comprised an enzyme dosage of flavorzyme at 6,068.4 U/g, temperature of 35.5°C, pH of 7, and hydrolysis time of 6 h. SSCH showed good radical-scavenging capacities with a DPPH scavenging efficiency of 95%. Then, the effect of low-molecular-weight SSCH fraction (SSCH-L) on UVB irradiation-induced photodamage was evaluated in mouse fibroblast L929 cells and zebrafish. SSCH-L reduced intracellular ROS levels and the malondialdehyde content, thereby alleviating the oxidative damage caused by UVB radiation. Moreover SSCH-L inhibited the mRNA expression of genes encoding the pro-inflammatory cytokines IL-1ß, IL-6, TNF-α, and Cox-2. SSCH-L treatment further increased the collagen Ⅰα1 content and had a significant inhibitory effect on matrix metalloproteinase expression. The phosphorylation level of JNK and the expression of c-Jun protein were significantly reduced by SSCH-L. Additionally, SSCH-L increased the tail fin area at 0.125 and 0.25 mg/ml in a zebrafish UVB radiation model, which highlighted the potential of SSCH-L to repair UVB-irradiated zebrafish skin damage. Peptide sequences of SSCH-L were identified by liquid chromatography-tandem mass spectrometry. Based on the 3D-QSAR modeling prediction, six total peptides were selected to test the UVB-protective activity. Among these peptides, DPFRHY showed good UVB-repair activity, ROS-scavenging activity, DNA damage-protective activity and apoptosis inhibition activity. These results suggested that DPFRHY has potential applications as a natural anti-photodamage material in cosmetic and functional food industries.

Photoprotective effects of Sargassum thunbergii on ultraviolet B-induced mouse L929 fibroblasts and zebrafish.

BACKGROUND: Chronic exposure to ultraviolet B (UVB) causes a series of adverse skin reactions, such as erythema, sunburn, photoaging, and cancer, by altering signaling pathways related to inflammation, oxidative stress, and DNA damage. Marine algae have abundant amounts and varieties of bioactive compounds that possess antioxidant and anti-inflammatory properties. Thus, the objective of this study was to investigate the photoprotective effects of an ethanol extract of Sargassum thunbergii. METHODS: Sargassum thunbergii phenolic-rich extract (STPE) was prepared, and its activity against UVB damage was evaluated using L929 fibroblast cells and zebrafish. STPE was extracted and purified by 40% ethanol and macroporous resin XDA-7. Reactive oxygen species (ROS) and antioxidant markers, such as superoxide dismutase (SOD), catalase (CAT) activities, and malondialdehyde (MDA) content were analyzed. The effect of STPE on UVB-induced inflammation was determined by inflammatory cytokine gene and protein expression. The expression of signaling molecules in the Nuclear Factor KappaB (NF-κB) pathway was determined by western blotting. DNA condensation was analyzed and visualized by Hoechst 33342 staining. In vivo evaluation was performed by tail fin area and ROS measurement using the zebrafish model. RESULTS: The total polyphenol content of STPE was 72%. STPE reduced ROS content in L929 cells, improved SOD and CAT activities, and significantly reduced MDA content, thereby effectively alleviating UVB radiation-induced oxidative damage. STPE inhibited the mRNA and protein expression of TNF-α, IL-6, and IL-1α. STPE reversed DNA condensation at concentrations of 20 and 40 µg/mL compared with the UVB control. Moreover, STPE inhibited NF-κB signaling pathway activation and alleviated DNA agglutination in L929 cells after UVB irradiation. Additionally, 1.67 µg/mL STPE significantly increased the tail fin area in zebrafish, and 0.8-1.6 µg/mL STPE effectively eliminated excessive ROS after UVB radiation. CONCLUSIONS: STPE inhibited UVB-induced oxidative stress, inflammatory cytokine expression, and DNA condensation via the downregulation of the NF-κB signaling pathway, suggesting that it prevents UVB-induced photodamage, and has potential for clinical development for skin disease treatment.

Uninterrupted embryonic growth leading to viviparous propagule formation in woody mangrove.

Vivipary is a rare sexual reproduction phenomenon where embryos germinate directly on the maternal plants. However, it is a common genetic event of woody mangroves in the Rhizophoraceae family. The ecological benefits of vivipary in mangroves include the nurturing of seedlings in harsh coastal and saline environments, but the genetic and molecular mechanisms of vivipary remain unclear. Here we investigate the viviparous embryo development and germination processes in mangrove Kandelia obovata by a transcriptomic approach. Many key biological pathways and functional genes were enriched in different tissues and stages, contributing to vivipary. Reduced production of abscisic acid set a non-dormant condition for the embryo to germinate directly. Genes involved in the metabolism of and response to other phytohormones (gibberellic acid, brassinosteroids, cytokinin, and auxin) are expressed precociously in the axis of non-vivipary stages, thus promoting the embryo to grow through the seed coat. Network analysis of these genes identified the central regulatory roles of LEC1 and FUS3, which maintain embryo identity in Arabidopsis. Moreover, photosynthesis related pathways were significantly up-regulated in viviparous embryos, and substance transporter genes were highly expressed in the seed coat, suggesting a partial self-provision and maternal nursing. We conclude that the viviparous phenomenon is a combinatorial result of precocious loss of dormancy and enhanced germination potential during viviparous seed development. These results shed light on the relationship between seed development and germination, where the continual growth of the embryo replaces a biphasic phenomenon until a mature propagule is established.

Global transcriptomic analysis of zebrafish in response to embryonic exposure to three antidepressants, amitriptyline, fluoxetine and mianserin.

Antidepressants are among the most commonly detected pharmaceuticals in aqueous systems, and, as emerging organic pollutants, may exert negative effects on non-target aquatic organisms. Previously, it has been revealed that antidepressant exposure significantly inhibits the growth and development of fish during their early developmental stages. Thus, in the present study, we aimed to identify and compare the underlying mechanisms of action of different antidepressants at the transcriptional level using zebrafish (Danio rerio) embryos. Through high-throughput RNA sequencing (RNA-Seq) data analysis, 32, 34, and 130 differentially expressed genes (DEGs) were obtained from zebrafish larvae after 120h of embryonic exposure to sublethal concentrations of amitriptyline, fluoxetine, and mianserin, respectively. The expression profiles of the identified DEGs showed similar trends in response to the three antidepressant treatments, suggesting consistent toxic effects of low concentrations of these three drugs on the regulation of gene expression in fish. Several metabolic and signaling pathways, including glycolysis/gluconeogenesis and the insulin pathway, were affected in the exposed fish larvae. The expression profiles of selected DEGs were then verified by the qRT-PCR method, which indicated significant positive correlations with the RNA-Seq results. Next, we determined the concentration-dependent expression patterns of 6 selected DEGs in fish larvae exposed to three antidepressants at a series of environmentally relevant concentrations. The results revealed a significant concentration-dependent reduction in the levels of dual-specificity phosphatase 5 (dusp5) mRNA, as well as a non-concentration-dependent gene expression inhibition of prostaglandin D2 synthase b (ptgdsb); the circadian rhythm-related genes, i.e. those encoding nuclear receptor subfamily 1, group D, member 1 (nr1d1) and period 2 (per2); and genes encoding early growth response factors (egr1 and egr4), in the antidepressant-treated fish larvae. In summary, to our knowledge, our findings demonstrate, for the first time, that the three different categories of antidepressants have common effects on the gene expression involved in multiple biological processes and signaling pathways during the early development of fish and thus provide information for characterizing the adverse outcome pathways and on the ecological risk assessment of these pharmaceutical pollutants in the aquatic environment.

Benzo[a]pyrene modulation of acute immunologic responses in Red Sea bream pretreated with lipopolysaccharide.

The effects of polycyclic aromatic hydrocarbons (PAHs) have been reported to modulate the immune response in aquatic animals, but the collected information of their effects on fish immunity is so far ambiguous. This study demonstrated that Benzo[a]pyrene (BaP) exposure altered the expression pattern of an antimicrobial peptide hepcidin (PM-hepc) gene and the activities of some immune-associated parameters in the lipopolysaccharide (LPS)-challenged red sea bream (Pagrus major). It was observed that LPS could increase respiratory burst, lysozyme and antibacterial activity in P. major. However when the P. major was exposed to different concentrations of BaP (1, 4, or 8 µg L(-1) ) for 14 days and then challenged with LPS there was no significant change in the lysozyme and antibacterial activity. It was further observed that LPS could induce the PM-hepc mRNA expression at 3, 6, and 12-h post-LPS challenge. However, when P. major was exposed first to BaP for 14 days and then challenged with LPS, the expression of PM-hepc mRNA was delayed in the liver until 24 h and not significantly induced until 48 and 96 h. The mRNA expression pattern was completely different from that only with LPS challenge, showing that BaP exposure changed the PM-hepc mRNA expression pattern of fish with LPS challenge. This study demonstrated that BaP exposure can weaken or inhibit the induction of lysozyme and antibacterial activity in the LPS-challenged P. major; conversely BaP exposure could enhance the mRNA expression of PM-hepc gene, indicating that the effect of BaP has different modulatory mechanism on hepcidin genes and immune-associated parameters.

Molecular cloning, recombinant expression, and antimicrobial activity of EC-hepcidin3, a new four-cysteine hepcidin isoform from Epinephelus coioides.

Hepcidin, a cysteine-rich antimicrobial peptide, is widespread in fish and shows multiple activities, including antimicrobial, antivirus, and antitumor. Here, a new four-cysteine hepcidin isoform gene, EC-hepcidin3, was cloned from the marine-cultured orange-spotted grouper (Epinephelus coioides). The complete cDNA sequence consisted of 603 bases with an open reading frame (ORF) of 270 bases. The genomic DNA sequence was composed of two introns and three exons, and its 312-bp upstream region had multiple putative transcription factor binding sites. Soluble recombinant protein EC-proHep3 containing a His-tag at the C-terminus was obtained from expression plasmid pET-28a/EC-proHep3 in Escherichia coli Rosetta. It was purified by immobilized metal affinity chromatography (IMAC), and it showed antibacterial activity in vitro. Kinetic studies indicated that recombinant EC-proHep3 has strong, rapid activity against Staphylococcus aureus and Pseudomonas stutzeri. The results indicate that EC-hepcidin3 might be an effective component in the innate immune system of groupers.

17ß estradiol induced ROS generation, DNA damage and enzymatic responses in the hepatic tissue of Japanese sea bass.

The importance of endocrine disrupting chemicals and their effects on fish has been documented in recent years. However, little is known about whether the estrogenic compound 17ß estradiol (E2) causes oxidative stress in the hepatic tissue of fish. Therefore, this work tested the hypothesis that E2 might cause oxidative stress in the Japanese sea bass Lateolabrax japonicus liver. To test this hypothesis, its effects on reactive oxygen species (ROS) production, DNA damage, antioxidants and biotransformation enzyme were investigated in two different size groups (fingerling and juvenile groups) following 30 days exposure. Results showed that there was a good relationship between the E2 exposure concentration, plasma E2 level and ROS generation. In addition ROS production correlated negatively with 7-ethoxyresorufin-O-deethylase activity and positively with DNA damage and lipid peroxidation (LPO). Antioxidant enzymes such as superoxide dismutase and catalase did not show any significant relation with ROS, LPO and DNA damage. In contrast, glutathione mediated enzymes showed a good relationship with the above parameters suggesting that the glutathione system in fish might be responsible for protection against the impact of E2 and also indicating a possible adaptive response during exposure periods. In addition, it was observed that fingerling was more susceptible to E2 exposure than juvenile fish. The present study provided strong evidence that the ROS level increased significantly in the liver of E2 exposed fish, and that ROS might serve as a biomarker to indicate estrogen contamination.

Soluble expression and purification of a crab antimicrobial peptide scygonadin in different expression plasmids and analysis of its antimicrobial activity.

Scygonadin is an anionic antimicrobial peptide recently identified from the seminal plasma of Scylla serrata. To gain more detailed information on its antimicrobial activity, scygonadin mature peptide was expressed in Escherichia coli in order to obtain a large quantity of biologically active product. An approximately 43 kDa fusion protein CKS-scygonadin was obtained in a highly stable and soluble form. The soluble component of the fusion CKS-scygonadin was purified by immobilized metal affinity chromatography (IMAC). A single 11 kDa recombinant scygonadin was cleaved from CKS-scygonadin and purified from the cleavage mixture using an affinity chromatography column with a yield of 10.6 mg/L. Alternatively, a recombinant scygonadin was purified from pET28-scygonadin by one-step Ni(2+) affinity chromatography and 65.9 mg/L pure recombinant scygonadin was obtained which was higher than that purified from pTrc-CKS/scygonadin in bacteria culture. The recombinant scygonadin was confirmed using SDS-PAGE analysis and MS-fingerprinting. Both recombinant products of scygonadin from different expressed plasmids showed the activity against both Gram-positive and Gram-negative bacteria, but no activity against yeast and fungi tested. The kinetic studies showed that the recombinant scygonadin was strong active against Staphylococcus aureus and the killing of S. aureus appeared time and dose dependent. Considering the quantity of recombinant product and the applicability of purification, the pET28-scygonadin expression system is a better choice to produce large quantities of recombinant scygonadin for commercial use in future. This is the first report on the heterologous expression of antimicrobial peptide scygonadin in E. coli.

Cloning and expression of a hepcidin gene from a marine fish (Pseudosciaena crocea) and the antimicrobial activity of its synthetic peptide.

Hepcidin gene is widely expressed in various fish, suggesting that this antimicrobial peptide is a very important component in the innate immune system. Large yellow croaker (Pseudosciaena crocea) is one of the important economic species of marine-cultured fish but knowledge of its innate immune mechanism is lacking. In this study, we characterize a P. crocea hepcidin gene named as PC-hepc. It consists of an open reading frame of 258 bases encoding 85 amino acids and has a conserved sequence in common with other known hepcidins. The genomic DNA of PC-hepc contains three exons and two introns, the same organization as other reported hepcidins, indicating that PC-hepc is one member of the hepcidin family in fish. The tissue-specific expression of PC-hepc gene in normal fish and the expression pattern in LPS-challenged fish at the time course of stimulation were investigated. The expression of PC-hepc mRNA was significantly increased in the spleen, heart and stomach but not significantly induced in the liver after LPS challenge. An interesting finding is the demonstration of high amounts of PC-hepc transcripts in the kidney in normal fish and their maintenance through 48h exposure to LPS challenge. The synthetic PC-hepc demonstrated a rather wide spectrum of antimicrobial activity in vitro against bacteria and fungi tested, and particularly showed strong activity against the principal fish pathogens, Aeromonas hydrophila, Vibrio parahaemloyticus, Vibrio alginolyticus and Vibrio harvryi. The study indicates that PC-hepc may play a role with a tissue-specific mode in the innate immunity of P. crocea.

Genomic organization and tissue-specific expression analysis of hepcidin-like genes from black porgy (Acanthopagrus schlegelii B).

Hepcidin is an antimicrobial peptide and putative iron regulatory hormone previously described in mice and humans. Dozens of fish hepcidins have been isolated and characterized so far. Here we present seven hepcidin-like cDNA sequences named AS-hepc1-7, amplified from the normal commercially cultured fish (black porgy) by RACE-PCR. Sequence analysis reveals that these seven potential hepcidin peptides have highly conserved sequences with other known hepcidins, but they are different from each other in constitution and characteristics of predicted mature amino acids. Based on the study, it is deduced that AS-hepc1-7 represent different variants of a family of hepcidin genes in black porgy. To understand the organization of these hepcidin-like genes, we sequenced AS-hepc2 DNA, AS-hepc3 DNA, AS-hepc4 DNA, AS-hepc7 DNA and AS-hepc2 upstream region; and all of the four genomic DNAs consisted of two introns and three exons, the same organization as other reported hepcidins. The tissue-specific gene expression of hepcidins in normal black porgy was evaluated using RT-PCR and dot blot approaches. RT-PCR showed that transcripts of hepcidin-like mRNAs were present in each tested tissue of normal juvenile black porgy, including liver, spleen, kidney, heart, brain, stomach, intestine, gill, skin and blood, but abundant hepcidin-like mRNA transcripts were only detected in the liver, kidney, spleen, intestine and stomach by dot blot assay. In addition, using dot blot and Northern blot approach, a significant increase of hepcidin mRNA transcription was observed in the liver within 48 h after immersion in a suspension of live bacteria, which suggested that the expression pattern of hepcidin-like genes in black porgy might be different in the liver from the other tissues as previously reported in several hepcidin studies.