Transcriptome Sequencing and Comparative Analysis of Saccharina japonica (Laminariales, Phaeophyceae) under Blue Light Induction

BACKGROUND: Light has significant effect on the growth and development of Saccharina japonica, but there are limited reports on blue light mediated physiological responses and molecular mechanism. In this study, high-throughput paired-end RNA-sequencing (RNA-Seq) technology was applied to transcriptomes of S. japonica exposed to blue light and darkness, respectively. Comparative analysis of gene expression was designed to correlate the effect of blue light and physiological mechanisms on the molecular level. PRINCIPAL FINDINGS: RNA-seq analysis yielded 70,497 non-redundant unigenes with an average length of 538 bp. 28,358 (40.2%) functional transcripts encoding regions were identified. Annotation through Swissprot, Nr, GO, KEGG, and COG databases showed 25,924 unigenes compared well (E-value <10(-5)) with known gene sequences, and 43 unigenes were putative BL photoreceptor. 10,440 unigenes were classified into Gene Ontology, and 8,476 unigenes were involved in 114 known pathways. Based on RPKM values, 11,660 (16.5%) differentially expressed unigenes were detected between blue light and dark exposed treatments, including 7,808 upregulated and 3,852 downregulated unigenes, suggesting S. japonica had undergone extensive transcriptome re-orchestration during BL exposure. The BL-specific responsive genes were indentified to function in processes of circadian rhythm, flavonoid biosynthesis, photoreactivation and photomorphogenesis. SIGNIFICANCE: Transcriptome profiling of S. japonica provides clues to potential genes identification and future functional genomics study. The global survey of expression changes under blue light will enhance our understanding of molecular mechanisms underlying blue light induced responses in lower plants as well as facilitate future blue light photoreceptor identification and specific responsive pathways analysis

Effect of Diet Supplemented With Rapeseed Meal or Hydrolysable Tannins on the Growth, Nutrition, and Intestinal Microbiota in Grass Carp (Ctenopharyngodon idellus)

Grass carp (Ctenopharyngodon idellus; n = 320) were received four different diets for 56 days. The experimental diets were: fishmeal (FM) containing 10% fishmeal (without rapeseed meal), and rapeseed meal (RM) containing 50% rapeseed meal (without fishmeal), and two semi-purified diets either without (T0) or with 1.25% (T1) supplemental hydrolysable tannin. The approximate content of tannin in the RM diet was 1.31%, which was close to that of T1, while the tannin content of FM was close to that of T0. The weight gain rate of grass carp of the RM group was significantly lower than that of the FM group, while the feeding conversion ratio and the feeding rate were significantly higher in the T1 group than in T0. The muscle lipid content was significantly lower in RM than in FM, while T1 was lower than T0. Intestinal activities of trypsin and α-amylase were significantly higher in T1 and RM groups compared with the other treatments. The hepatic activities of aspartate aminotransferase and alanine aminotransferase were lower in T1 and RM groups compared with the other treatments, while hepatic glycogen, and malonaldehyde were significantly higher in T1 and RM groups. In serum, the total protein and globulin contents were significantly higher in T1 and RM groups, while albumin was significantly lower in the RM group compared to the FM group. High-throughput sequencing showed that Proteobacteria, Firmicutes, and Actinobacteria were the dominant bacterial phyla among groups. The intestinal microbial diversity was higher in T1 and RM. Redundancy analysis showed that tannin, rapeseed meal, and intestinal trypsin activity were positively or negatively correlated with the relative abundance of several different intestinal microbiota at phylum and/or genus levels. The results indicated that 1.25% tannin could not be the main reason for the poor growth of grass carp of the RM group; however, the protein metabolism was disturbed, the absorption of carbohydrate was improved, and the accumulation of lipid had decreased. Furthermore, tannin and rapeseed meal supplementations modulated the intestinal microbiota, and may sequentially regulate the intestinal function by fermenting dietary nutrition, producing digestive enzymes, and modulating probiotics

Effect of triptolide on proliferation and apoptosis of angiotensin II-induced cardiac fibroblasts in vitro: a preliminary study

Background: The effect of triptolide (TPL) on cardiac fibroblasts (CFbs) and cardiac fibrosis remain unknown till now. This study was conducted to explore the effects of TPL on proliferation and apoptosis of angiotensin II (Ang II)-induced CFbs.Materials and Methods: Ang II was used to promote proliferation of CFbs. Two dosages of TPL (10ng/ml and 100ng/ml) were chosen. MTT assay was used to detect cell survival rate in vitro. Flow cytometer was performed to analyze apoptosis of CFbs. Hydroxyproline concentration was detected with hydroxyproline assay kit. Quantitative real-time PCR was used to detect the expression of TGF-β1 and Smad3 mRNA.Results: Ang II promoted CFbs proliferation significantly. Compared to Ang II group, TPL markedly reduced the viability of CFbs and its Hydroxyproline concentration (P&lt;0.05). Besides, TPL can significantly promote apoptosis of CFbs (P&lt;0.05). Furthermore, TPL reduced the expressions of TGF-β1 and Smad3 mRNA in Ang II-induced CFbs (P&lt;0.05).Conclusion: TPL can inhibit the proliferation of CFbs in rats by down-regulating TGF-β1/Smad3 signaling pathway. TPL might be a promising therapeutic drug for myocardial fibrosis.Keywords: Cardiac fibroblast; triptolide; proliferation; apoptosis; angiotensi

Cloning and Comparative Studies of Seaweed Trehalose-6-Phosphate Synthase Genes

The full-length cDNA sequence (3219 base pairs) of the trehalose-6-phosphate synthase gene of Porphyra yezoensis (PyTPS) was isolated by RACE-PCR and deposited in GenBank (NCBI) with the accession number AY729671. PyTPS encodes a protein of 908 amino acids before a stop codon, and has a calculated molecular mass of 101,591 Daltons. The PyTPS protein consists of a TPS domain in the N-terminus and a putative TPP domain at the C-terminus. Homology alignment for PyTPS and the TPS proteins from bacteria, yeast and higher plants indicated that the most closely related sequences to PyTPS were those from higher plants (OsTPS and AtTPS5), whereas the most distant sequence to PyTPS was from bacteria (EcOtsAB). Based on the identified sequence of the PyTPS gene, PCR primers were designed and used to amplify the TPS genes from nine other seaweed species. Sequences of the nine obtained TPS genes were deposited in GenBank (NCBI). All 10 TPS genes encoded peptides of 908 amino acids and the sequences were highly conserved both in nucleotide composition (>94%) and in amino acid composition (>96%). Unlike the TPS genes from some other plants, there was no intron in any of the 10 isolated seaweed TPS genes

Facile One-Pot Synthesis of Self-Assembled Folate-Biotin-Pullulan Nanoparticles for Targeted Intracellular Anticancer Drug Delivery

The self-assembled folate-biotin-pullulan (FBP) nanoparticles (NPs) were prepared by facile one-pot synthesis and their physicochemical properties were characterized. The self-assembled FBP NPs were used as an anticancer drug nanocarrier entrapping doxorubicin (DOX) for targeting folate-receptors-overexpressing cancer cells. The identification of prepared NPs to folate-receptor-expressing cancer cells (KB cells) was affirmed by cell viability measurement, folate competition test, and flow cytometric analysis. Compared with the naked DOX and DOX/BP NPs, the DOX/FBP NPs had lower IC50 value compared to KB cells as a result of the folate-receptor-mediated endocytosis process. The cytotoxicity of DOX/FBP NPs to KB cells could be inhibited competitively by free folate. The cellular intake pattern of naked DOX and drug-loaded NPs was identified by confocal laser scanning microscopy (CLSM) observation and the higher cellular uptake of drug for DOX/FBP NPs over naked DOX was observed. The prepared FBP NPs had the potential to be used as a powerful carrier to target anticancer drugs to folate-receptor-expressing tumor cells and reduce cytotoxicity to normal tissues

Genome-Wide Mapping of Cytosine Methylation Revealed Dynamic DNA Methylation Patterns Associated with Sporophyte Development of Saccharina japonica

Cytosine methylation plays vital roles in regulating gene expression and plant development. However, the function of DNA methylation in the development of macroalgae remains unclear. Through the genome-wide bisulfite sequencing of cytosine methylation in holdfast, stipe and blade, we obtained the complete 5-mC methylation landscape of Saccharina japonica sporophyte. Our results revealed that the total DNA methylation level of sporophyte was less than 0.9%, and the content of CHH contexts was dominant. Moreover, the distribution of CHH methylation within the genes exhibited exon-enriched characteristics. Profiling of DNA methylation in three parts revealed the diverse methylation pattern of sporophyte development. These pivotal DMRs were involved in cell motility, cell cycle and cell wall/membrane biogenesis. In comparison with stipe and blade, hypermethylation of mannuronate C5-epimerase in holdfast decreased the transcript abundance, which affected the synthesis of alginate, the key component of cell walls. Additionally, 5-mC modification participated in the regulation of blade and holdfast development by the glutamate content respectively via glutamine synthetase and amidophosphoribosyl transferase, which may act as the epigenetic regulation signal. Overall, our study revealed the global methylation characteristics of the well-defined holdfast, stipe and blade, and provided evidence for epigenetic regulation of sporophyte development in brown macroalgae

The 40S Ribosomal Protein S6 Response to Blue Light by Interaction with SjAUREO in <i>Saccharina japonica</i>

Blue light (BL) plays an important role in regulation of the growth and development of aquatic plants and land plants. Aureochrome (AUREO), the recent BL photoreceptor identified in photosynthetic stramenopile algae, is involved in the photomorphogenesis and early development of Saccharina japonica porophytes (kelp). However the factors that interact with the SjAUREO under BL conditions specifically are not clear. Here in our study, three high quality cDNA libraries with CFU over 5 &#215; 106 and a recombination rate of 100% were constructed respectively through white light (WL), BL and darkness (DK) treatments to the juvenile sporophytes. Based on the constructed cDNA libraries, the interactors of SjAUREO were screened and analyzed. There are eighty-four genes encoding the sixteen predicted proteins from the BL cDNA library, sixty-eight genes encoding eighteen predicted proteins from the DK cDNA library, and seventy-four genes encoding nineteen proteins from the WL cDNA library. All the predicted proteins are presumed to interact with SjAUREO when co-expressed with SjAUREO seperately. The 40S ribosomal protein S6 (RPS6), which only exists in the BL treated cDNA library except for two other libraries, and which is essential for cell proliferation and is involved in cell cycle progression, was selected for detailed analysis. We showed that its transcription was up-regulated by BL, and was highly transcribed in the basal blade (meristem region) of juvenile sporophytes but less in the distal part. Taken together, our results indicated that RPS6 was highly involved in BL-mediated kelp cellular division and photomorphogenesis by interacting with SjAUREO

Metro and elderly health in Hong Kong: protocol for a natural experiment study in a high-density city

Introduction Public transport accessible to older people may offer a transformative solution to achieving healthy ageing. However, the evidence to support such transport infrastructure modifications is unclear. Previous studies on public transport use and elderly health were mostly observational studies using cross-sectional data. Few studies have examined the before-and-after effects of a new metro, for example, to see if it leads to improved elderly health.Methods and analysis We use a new metro line in Hong Kong as a natural experiment to examine the impact of the metro-led public transport intervention on elderly health. In Hong Kong, more than 90% of daily travels are made by public transport. The public transport modifications consist of the new metro line with eight stations and changes in the walking environment and bus services around the stations. We will look at the before-and-after differences in public transport use and health outcomes between elderly participants living in treatment neighbourhoods (400 m walking buffered areas of the new metro stations) and in control groups (living in comparable areas but unaffected by the new metro). Questionnaire-based baseline data were collected in 2019 before the COVID-19 pandemic, while some qualitative interviews are ongoing. Amid the pandemic, we conducted a quick telephone-based survey of COVID-19’s potential impact on public transport use behaviours of our elderly cohort in September 2020. Note there is no lockdown in Hong Kong until the writing of the paper (January 2021). After the new metro opens, we will conduct a follow-up survey, tentatively in late 2022. We aim to investigate if the new metro and the associated changes in the built environment have any effects on public transport use behaviours, physical activity and wider health outcomes among the elderly (eg, social inclusion, quality of life, subjective well-being).Ethics and dissemination The Human Research Ethics Committee of the University of Hong Kong reviewed and approved the study procedures and materials (reference number: EA1710040). Results will be communicated through scientific papers and research reports

Non-Coding RNAs Participate in the Regulation of CRY-DASH in the Growth and Early Development of Saccharina japonica (Laminariales, Phaeophyceae)

CRY-DASH, a new cryptochrome blue light receptor, can repair damaged DNA and regulate secondary metabolism and development of fungus. However, its role in regulation during the growth of Saccharina japonica is still unclear. After cloning the full-length of CRY-DASH from S. japonica (sjCRY-DASH), we deduced that its open reading frame was 1779 bp long and encoded 592 amino acids. sjCRY-DASH transcription was rapidly upregulated within 30 min in response to blue light and exhibited 24 h periodicity with different photoperiods. Moreover, sjCRY-DASH maintained the same periodicity in suitable growth temperature, suggesting a close relationship between this periodicity and circadian rhythm regulation. Novel-m3234-5p, which was targeted to sjCRY-DASH, decreased with increasing sjCRY-DASH transcription, acting as a negative modulator of sjCRY-DASH. Six long non-coding RNAs classified as long intergenic non-coding RNAs (lincRNAs) exhibited co-expression with sjCRY-DASH. A miRNA sjCRY DASH lincRNA network was consequently identified. By predicting the endogenous competing mRNAs of novel-m3234-5p, we found that sjCRY-DASH indirectly participated in the regulation of DNA damage repair, protein synthesis and processing, and actin transport. In conclusion, our results revealed that non-coding RNAs participate in the regulation of sjCRY-DASH, which played vital roles in the growth and early development of S. japonica