Reconstruction of Long-Chain Polyunsaturated Acid Synthesis Pathways in Marine Red Microalga Porphyridium cruentum Using Lipidomics and Transcriptomics.

The marine red microalga Porphyridium can simultaneously synthesize long-chain polyunsaturated fatty acids, including eicosapentaenoic acid (C20:5, EPA) and arachidonic acid (C20:4, ARA). However, the distribution and synthesis pathways of EPA and ARA in Porphyridium are not clearly understood. In this study, Porphyridium cruentum CCALA 415 was cultured in nitrogen-replete and nitrogen-limited conditions. Fatty acid content determination, transcriptomic, and lipidomic analyses were used to investigate the synthesis of ARA and EPA. The results show that membrane lipids were the main components of lipids, while storage lipids were present in a small proportion in CCALA 415. Nitrogen limitation enhanced the synthesis of storage lipids and ω6 fatty acids while inhibiting the synthesis of membrane lipids and ω3 fatty acids. A total of 217 glycerolipid molecular species were identified, and the most abundant species included monogalactosyldiglyceride (C16:0/C20:5) (MGDG) and phosphatidylcholine (C16:0/C20:4) (PC). ARA was mainly distributed in PC, and EPA was mainly distributed in MGDG. Among all the fatty acid desaturases (FADs), the expressions of Δ5FAD, Δ6FAD, Δ9FAD, and Δ12FAD were up-regulated, whereas those of Δ15FAD and Δ17FAD were down-regulated. Based on these results, only a small proportion of EPA was synthesized through the ω3 pathway, while the majority of EPA was synthesized through the ω6 pathway. ARA synthesized in the ER was likely shuttled into the chloroplast by DAG and was converted into EPA by Δ17FAD.

The inhibition of enterovirus 71 induced apoptosis by Durvillaea antarctica through P53 and STAT1 signaling pathway.

The infection of enterovirus 71 (EV71) resulted in hand, foot, and mouth disease and may lead to severe nervous system damage and even fatalities. There are no effective drugs to treat the EV71 virus and it is crucial to find novel drugs against it. Polysaccharide isolated from Durvillaea antarctica green algae has an antiviral effect. In this study, D. antarctica polysaccharide (DAPP) inhibited the infection of EV71 was demonstrated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), reverse transcription polymerase chain reaction, flow cytometry, and western blot. MTT assay showed that DAPP had no toxicity on Vero cells at the concentration 250 µg/ml. Furthermore, DAPP significantly reduced the RNA level of EV71 in a dose-dependent manner. Moreover, DAPP inhibited the Vero cells apoptosis induced by EV71 via the P53 signaling pathway. Meanwhile, the expression of signal transducer and activator of transcription 1 and mammalian target of rapamycin were increased and the proinflammatory cytokines were significantly inhibited by DAPP. Taken together, these results suggested that DAPP could be a potential pharmaceutical against the infection of EV71 virus.

Inhibition of enterovirus 71 infection by polysaccharides extracted from Picochlorum sp. 122 via the AKT and ATM/ATR signaling pathways.

Enterovirus 71 (EV71) poses a major threat to public health globally due to severe and even fatal hand, foot, and mouth disease (HFMD). However, no effective antiviral agents are available to treat HFMD caused by this virus. Polysaccharides have been shown to exhibit antiviral activity, and polysaccharides extracted from Picochlorum sp. 122 (PPE) could potentially be used to treat HFMD, but reports on their antiviral activity are limited. In this study, the antiviral activity of PPE against EV71 was verified in Vero cells. PPE was shown to limit EV71 infection, as demonstrated using an MTT assay and by observing the cellular cytopathic effect. In addition, a decrease in VP1 RNA and protein levels indicated that PPE effectively inhibits proliferation of EV71 in Vero cells. An annexin V affinity assay also indicated that PPE protects host cells from apoptosis through the AKT and ATM/ATR signalling pathways. These results demonstrate that PPE has potential as an antiviral drug to treat HFMD caused by EV71.

Growth and Biochemical Composition of Porphyridium purpureum SCS-02 under Different Nitrogen Concentrations.

Microalgae of the genus Porphyridium show great potential for large-scale commercial cultivation, as they accumulate large quantities of B-phycoerythrin (B-PE), long chain polyunsaturated fatty acids (LC-PUFAs) and exopolysaccharide (EPS). The present study aimed to adjust culture nitrogen concentrations to produce Porphyridium biomass rich in B-PE, LC-PUFAs and EPS. Porphyridium purpureum SCS-02 was cultured in ASW culture medium with low nitrogen supply (LN, 3.5 mM), medium nitrogen supply (MN, 5.9 mM) or high nitrogen supply (HN, 17.6 mM). HN significantly enhanced the accumulation of biomass, intracellular protein, B-PE and eicosapentaenoic acid. LN increased the intracellular carbohydrate and arachidonic acid content, and promoted the secretion of EPS. The total lipids content was almost unaffected by nitrogen concentration. Based on these results, a semi-continuous two-step process was proposed, which included the production of biomass rich in B-PE and LC-PUFAs with sufficient nitrogen, and induced EPS excretion with limited nitrogen and strong light.

Maribacter flavus sp. nov., isolated from a cyanobacterial culture pond.

A Gram-stain-negative, yellow, non-spore-forming, strictly aerobic bacterium, designated C3T, was isolated from a cyanobacterial culture pond. Cells were halophilic, rod-shaped and able to move by gliding. Growth of strain C3T was observed at 15-30 °C (optimum 25 °C), pH 6.0-9.0 (optimum pH 7.5), and in the presence of 1-7 % (w/v) NaCl (optimum 2-3 %). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain C3T formed a distinct lineage within the family Flavobacteriaceae and exhibited the highest similarity (95.21 %) to the type strains of Maribacter dokdonensis, Maribacter arcticus, Maribacter orientalis and Maribacter stanieri, and 'Maribacter caenipelagi' HD-44. The only isoprenoid quinone present within strain C3T was menaquinone 6 (MK-6). The G+C content of genomic DNA was 41.5 mol%. The major polar lipids were phosphatidylethanolamine and three unidentified lipids. The predominant cellular fatty acids (>5 % of the total fatty acids) were iso-C15 : 1 G, iso-C15 : 0, iso-C15 : 0 3-OH, iso-C17 : 0 3-OH, and summed feature 3 (comprising C16 : 1ω7c and/or C16 : 1ω6c). On the basis of the phenotypic, chemotaxonomic and phylogenetic characteristics, strain C3T represents a novel species of the genus Maribacter, for which the name Maribacter flavus sp. nov. is proposed. The type strain is C3T ( = KCTC 42508T = CGMCC 1.15112T).

[Prokaryote diversity in the surface sediment of northern South China Sea].

OBJECTIVE: In order to investigate the composition and diversity of prokaryotes in marine surface sediment from site XSCS13 at northern South China Sea. METHOD: We extracted environment total DNA directly from the sediment and amplified 16S rRNA genes from the total DNA, thereby constructing 16S rRNA clone libraries of both archaea and bacteria. Then we selected positive clones randomly from the library and identified them by the method of restriction fragment length polymorphism (RFLP). After that, the unique RFLP pattern corresponded sequences were sequenced, BLAST and then constructed into a phylogenetic tree. RESULTS: Most of the clones were sequences from uncultured microbes. For the Archaea part: the community was mainly comprised of 3 phyla: Crenarchaeota, Thaumarchaeota and Euryarchaeota, among which Crenarchaeota was the dominant class with the percentage of 71%, while Euryarchaeota took the least with only 3 clones. The main group of Crenarchaeota was Marine Group I, taking 61% of all. For the bacteria part, all together 9 phyla were included: 32.6% of Proteobacteria, 3% of Verrucomicrobia, 5.2% of Bacteroidete, 4.44% of Acidobacteria, 6% of Chloroflexi, 3.7% of Firmicute, 5.2% of Planctomycete, 11.1% of Gemmatimonadete and 4.44% of Actinobacteria. Unlike archaea, there's no overwhelming preponderant phylum in bacterium, each phylum was relatively distributed in balanced proportions. Proteobacteria had 3 classes involved: alpha-Proteobacteri, gamma-Proteobacteria and delta-Proteobacteria, and gamma-Proteobacteria stood out with 54.5% of proportion. In addition, over half of all the clones were related to reduction reaction of sulfate and generation of methane. CONCLUSION: The research indicated that prokaryote diversity in marine surface sediment from site XSCS13 at northern South China Sea was quite plentiful, and a great mass of abundant microbial resources still remained unknown. Furthermore, the community structure of archaea and bacteria showed that the sampling site may be in a cold spring area with abundant methane.

Rapid Screening of Microalgae as Potential Sources of Natural Antioxidants.

In order to rapidly screen microalgae species as feedstocks for antioxidants, extracts were obtained from 16 microalgae strains (under 11 genera, 7 classes) using two methods: a one-step extraction with ethanol/water and a three-step fractionating procedure using hexane, ethylacetate, and water successively. Measuring the total phenol content (TPC), total carotenoid content (TCC), and antioxidant activity of the extracts, indicating TPC and TCC, played an important role in determining the antioxidant activity of the microalgae. A weighted scoring system was used to evaluate the antioxidant activity, and the scores of microalgal samples from two extraction methods were calculated using the same system. Among the investigated microalgae, Euglena gracilis SCSIO-46781 had the highest antioxidant score, contributing to high TPC and TCC, followed by Arthrospira platensis SCSIO-44012, Nannochloropsis sp. SCSIO-45224, Phaeodactylum tricornutum SCSIO-45120, and Nannochloropsis sp. SCSIO-45006, respectively. Additionally, the above-mentioned five strains are currently being applied in commercial production, indicating this system could be effective not only for screening microalgal antioxidants, but also for screening microalgal species/strains with strong adaptation to environmental stress, which is a critical trait for their commercial cultivation.

Comparison of Production and Fluorescence Characteristics of Phycoerythrin from Three Strains of Porphyridium.

Phycoerythrin, a special photosynthetic pigment, is widely used as fluorescent dye and has lots of underlying beneficial effects on health. A marine red microalga Porphyridium is considered as the potential feedstock for phycoerythrin production. However, the phycoerythrin-related properties of Porphyridium have not been systematically evaluated, especially between the species of P. cruentum and P. purpureum. The present study aimed to evaluate the production and fluorescence characteristics of phycoerythrin of three strains of Porphyridium. The results showed that P. purpureum SCS-02 presented the highest biomass, phycoerythrin content and yield were 6.43 g L-1, 9.18% DW and 0.288 g L-1, respectively. There was no significant difference between P. purpureum and P. cruentum in α and ß subunits amino acid sequences of phycoerythrin and in fluorescence characteristics. The high gene expression level of the key enzymes in phycoerythrobilin synthesis (porphobilinogen synthase and oxygen-dependent coproporphyrinogen-III oxidase) could be related to the high phycoerythrin content of Porphyridium. Based on systematic evaluation, P. purpureum SCS-02 was selected due to its high biomass and phycoerythrin yield. P. purpureum and P. cruentum were highly similar in the phylogenetic tree, as well as in fluorescence characteristics; therefore, it was speculated that they might be the same Porphyridium species.

Extraction optimization, physicochemical characterization, and antioxidant activity of polysaccharides from Rhodosorus sp. SCSIO-45730.

Microalgal polysaccharides have been reported in many studies due to their uniqueness, biocompatibility, and high value, and Rhodosorus sp. SCSIO-45730 was an excellent source of polysaccharides and ß-glucans. However, the polysaccharides from the red unicellular alga Rhodosorus sp. SCSIO-45730 have barely been studied. In this work, hot water extraction of Rhodosorus sp. SCSIO-45730 polysaccharides (RSP) was optimized using response surface methodology (RSM) based on Box-Behnken design (BBD). The maximum RSP yield (9.29%) was achieved under the optimum extraction conditions: liquid-solid ratio of 50.00 mL g-1; extraction temperature of 84 °C; extraction time of 2 h; and extraction times of 5 times. The results of physicochemical characterization showed that RSP had high sulfate and uronic acid with content of 19.58% and 11.57%, respectively, rough layered structure, and mainly contained glucose, galactose, xylose, and galacturonic acid with mass percentages of 34.08%, 28.70%, 12.46%, and 12.10%. Furthermore, four kinds of antioxidant assays were carried out, and the results indicated that RSP had strong scavenging activities on ABTS and hydroxyl radical and moderate scavenging activities on DPPH and ferrous chelating ability. These results indicated that RSP showed potential as a promising source of antioxidants applied in food, pharmaceutical, and cosmetics industry. Supplementary Information: The online version contains supplementary material available at 10.1007/s10811-021-02646-2.

Complete genome analysis of a novel E3-partial-deleted human adenovirus type 7 strain isolated in Southern China.

Human adenovirus (HAdV) is a causative agent of acute respiratory disease, which is prevalent throughout the world. Recently there are some reports which found that the HAdV-3 and HAdV-5 genomes were very stable across 50 years of time and space. But more and more recombinant genomes have been identified in emergent HAdV pathogens and it is a pathway for the molecular evolution of types. In our paper, we found a HAdV-7 GZ07 strain isolated from a child with acute respiratory disease, whose genome was E3-partial deleted. The whole genome was 32442 bp with 2864 bp deleted in E3 region and was annotated in detail (GenBank: HQ659699). The growth character was the same as that of another HAdV-7 wild strain which had no gene deletion. By comparison with E3 regions of the other HAdV-B, we found that only left-end two proteins were remained: 12.1 kDa glycoprotein and 16.1 kDa protein. E3 MHC class I antigen-binding glycoprotein, hypothetical 20.6 kDa protein, 20.6 kDa protein, 7.7 kDa protein., 10.3 kDa protein, 14.9 kDa protein and E3 14.7 kDa protein were all missing. It is the first report about E3 deletion in human adenovirus, which suggests that E3 region is also a possible recombination region in adenovirus molecular evolution.

A method to type the potential angucycline producers in actinomycetes isolated from marine sponges.

Angucyclines are aromatic polyketides with antimicrobial, antitumor, antiviral and enzyme inhibition activities. In this study, a new pair of degenerate primers targeting the cyclase genes that are involved in the aromatization of the first and/or second ring of angucycline, were designed and evaluated in a PCR protocol targeting the jadomycin cyclase gene of Streptomyces venezuelae ISP5230. The identity of the target amplicon was confirmed by sequencing. After validation, the primers were used to screen 49 actinomycete isolates from three different marine sponges to identify putative angucycline producers. Seven isolates were positively identified using this method. Sequence analysis of the positive amplicons confirmed their identity as putative angucycline cyclases with sequence highly similar to known angucycline cyclases. Phylogenetic analysis clustered these positives into the angucycline group of cyclases. Furthermore, amplifications of the seven isolates using ketosynthase-specific primers were positive, backing the results using the cyclase primers. Together these results provided strong support for the presence of angucycline biosynthetic genes in these isolates. The specific primer set targeting the cyclase can be used to identify putative angucycline producers among marine actinobacteria, and aid in the discovery of novel angucyclines.

[A series of excellent strategy to analyze environmental sample archaeal diversity].

OBJECTIVE: Finding a series of archaeal 16S rRNA gene universal primer applied strategies to detect complex microbial diversity in environmental samples, especially with rapidly development of next generation sequencing technology challenge. METHODS: We used Oligocheck soft to simulate two pairs of archaeal 16S rRNA gene universal primers with RDP (Ribosomal database project) database 16S rRNA gene sequences matching percentage. In succession, the sediment sample was constructed for two clone libraries by using two pairs of archaeal 16S rRNA gene universal primers. RESULTS: The soft simulation matched percentage result suggests that primer f109/r958 is better than primer f21/r958. This result is consistent with RFLP and diversity index analyses by two clone libraries. CONCLUSION: Multi-primers and properly primer are used to recovery environmental microbiology diversity, which will be advanced in environmental microbial resolution.

[Comparison of bacterial diversity of polluted and unpolluted sediment by brominated flame retardant].

OBJECTIVE: By using brominated flame retardant we compared the bacterial diversity of highly polluted river sediment with that of nearby unpolluted lake. METHOD: Total DNA was extracted from unpolluted and highly polluted sediment sample by brominated flame retardant in Guiyu of China. The 16S rRNA gene was amplified by PCR using bacterial primer 27F and 1500R. The plasmid libraries of the amplicons were constructed. The positive clones with insert were screened on plates with IPTG/X-gal/Ap. Amplified ribosmal DNA restriction analysis (ARDRA) was carried out with restriction enzymes Hha I and Hinf I. Representative clones of each operational taxonomic unit based on the ARDRA patterns were selected to be sequenced. After proof reading and careful comparison to remove the chimeric sequences, the partial sequence of 16S rRNA gene were used for construction of the phylogenetic tree. RESULT: The result of blast searching showed that clones from unpolluted sediment sample belonged to alpha-Proteobacteria, beta-Proteobacteria, gamma-Proteobacteria, delta-Proteobacteria, Planctomycetes, Acidobacteria, Actinobacteria, Chloroflexi, Verrucomicrobia, Firmicutes, the predominant bacteria (30.2% of total clones) is Acidobacteria; most clones from polluted sediment belonged to alpha-Proteobacteria, beta-Proteobacteria, epsilon-Proteobacteria, delta-Proteobacteria, Planctomycetes, Acidobacteria, Actinobacteria, Chloroflexi, Bacteroidetes, Firmicutes, candidate division 0P01, candidate division OP08, the predominant bacteria (44.9% of total clones) are epsilon-Proteobacteria and Chloroflexi. CONCLUSION: Bacterial community structure of polluted sediment has distinguished feature and obviously different from the unpolluted sediment sample, which is mainly reflected in the dominant position of epsilon-Proteobacteria and Chloroflexi in the bacterial flora.

A Novel Three-Step Extraction Strategy for High-Value Products from Red Algae Porphyridium purpureum.

The microalga Porphyridium accumulates high-value compounds such as phycoerythrin, polyunsaturated fatty acids, and polysaccharides, and thus, the extraction of these compounds could significantly expand the value of Porphyridium biomass. In the present study, a novel fractional extraction strategy based on the characteristics of these compounds was established using cold water, 95% ethanol, and hot water. The yield of phycoerythrin, lipids, and polysaccharides was 63.3, 74.3, and 75.2%, respectively. The phycoerythrin exhibited excellent fluorescence characteristics but had low purity. The crude lipid was dark with poor fluidity. Digalactosyldiacylglycerol and sulphoquinovosyldiacylglycerol containing C20:5 and C20:4 were the most abundant glycerolipids, while glucose, xylose, and galactose constituted the intracellular polysaccharides that had covalently bound to proteins (8.01%), uronic acid (4.13%), and sulfate (8.31%). Compared with polysaccharides and crude lipids, crude phycoerythrin showed the best antioxidant activity. Overall, the three-step fractional extraction process was feasible for Porphyridium; however, further purification is necessary for downstream applications.

Growth and Biochemical Composition Characteristics of Arthrospira platensis Induced by Simultaneous Nitrogen Deficiency and Seawater-Supplemented Medium in an Outdoor Raceway Pond in Winter.

Arthrospira platensis, a well-known cyanobacterium, is widely applied not only in human and animal nutrition but also in cosmetics for its high amounts of active products. The biochemical composition plays a key role in the application performance of the Arthrospira biomass. The present study aimed to evaluate the growth and biochemical composition characteristics of A. platensis, cultured with a nitrogen-free and seawater-supplemented medium in an outdoor raceway pond in winter. The results showed that the biomass yield could achieve 222.42 g m-2, and the carbohydrate content increased by 247% at the end of the culture period (26 d), compared with that of the starter culture. The daily and annual areal productivities were 3.96 g m-2 d-1 and 14.44 ton ha-1 yr-1 for biomass and 2.88 g m-2 d-1 and 10.53 ton ha-1 yr-1 for carbohydrates, respectively. On the contrary, a profound reduction was observed in protein, lipid, and pigment contents. Glucose, the main monosaccharide in the A. platensis biomass, increased from 77.81% to 93.75% of total monosaccharides. Based on these results, large-scale production of carbohydrate-rich A. platensis biomass was achieved via a low-cost culture, involving simultaneous nitrogen deficiency and supplementary seawater in winter.

Culture-dependent and culture-independent diversity of Actinobacteria associated with the marine sponge Hymeniacidon perleve from the South China Sea.

In this report, the diversity of Actinobacteria associated with the marine sponge Hymeniacidon perleve collected from a remote island of the South China Sea was investigated employing classical cultivation and characterization, 16S rDNA library construction, 16S rDNA-restriction fragment length polymorphism (rDNA-RFLP) and phylogenetic analysis. A total of 184 strains were isolated using seven different media and 24 isolates were selected according to their morphological characteristics for phylogenetic analysis on the basis of their 16S rRNA gene sequences. Results showed that the 24 isolates were assigned to six genera including Salinispora, Gordonia, Mycobacterium, Nocardia, Rhodococcus and Streptomyces. This is the first report that Salinispora is present in a marine sponge from the South China Sea. Subsequently, 26 rDNA clones were selected from 191 clones in an Actinobacteria-specific 16S rDNA library of the H. perleve sample, using the RFLP technique for sequencing and phylogenetic analysis. In total, 26 phylotypes were clustered in eight known genera of Actinobacteria including Mycobacterium, Amycolatopsis, Arthrobacter, Brevibacterium, Microlunatus, Nocardioides, Pseudonocardia and Streptomyces. This study contributes to our understanding of actinobacterial diversity in the marine sponge H. perleve from the South China Sea.

Differential mantle transcriptomics and characterization of growth-related genes in the diploid and triploid pearl oyster Pinctada fucata.

To explore the molecular mechanism of triploidy effect in the pearl oyster Pinctada fucata, two RNA-seq libraries were constructed from the mantle tissue of diploids and triploids by Roche-454 massive parallel pyrosequencing. The identification of differential expressed genes (DEGs) between diploid and triploid may reveal the molecular mechanism of triploidy effect. In this study, 230 down-regulated and 259 up-regulated DEGs were obtained by comparison between diploid and triploid libraries. The gene ontology and KEGG pathway analysis revealed more functional activation in triploids and it may due to the duplicated gene expression in transcriptional level during whole genome duplication (WGD). To confirm the sequencing data, a set of 11 up-regulated genes related to growth and development control and regulation were analyzed by RT-qPCR in independent experiment. According to the validation and annotation of these genes, it is hypothesized that the set of up-regulated expressed genes had the correlated expression pattern involved in shell building or other interactive probable functions during triploidization. The up- regulation of growth-related genes may support the classic hypotheses of 'energy redistribution' from early research. The results provide valuable resources to understand the molecular mechanism of triploidy effect in both shell building and producing high-quality seawater pearls.

Exploring and exploiting microbial diversity through metagenomics for natural product drug discovery.

Microorganisms of millions species exist in every corner of the Earth, and form a dynamic genetic reservoir that are not clearly revealed and categorized due to barrier in current cultivation technology. Their applications in biomedical and environmental aspects are more than satisfactory. However, the situation has drastically changed during the turn of the century because of the rapid development of phylogenetic studies based on rRNA sequencing independent of standard laboratory cultivation. More recently, high throughput sequencing technology which enables direct sequencing of community DNA for metagenomic analyses are making a direct impact on our understanding of microbial diversity, ecology, and secondary metabolism. In this review, we highlight some recent progress and innovation on metagenomic research with an emphasis on natural product drug discovery. The rapid path of accumulating decoded metagenomics would be an efficient guide on direct access to the genomes of numerous non-culturable microorganisms for their genomic diversity and associated chemical prosperity for potential medicinal applications.

[Epidemiological study of human type 5 adenovirus in Guangzhou using chemiluminescence for neutralizing antibody assay].

OBJECTIVE: To establish a high-throughput chemiluminescence assay of serotype 5 specific neutralizing antibody and understand the epidemiology of this antibody in the healthy adults and children in Guangzhou. METHODS: Using rAd5 carrying the reporter gene of secreted alkaline phosphatases (SEAP), serum samples from 116 healthy adults and 94 healthy children were examined with chemiluminescence assay to detect the presence of Ad5 neutralizing antibody. The reliability of this assay was tested against conventional cytopathic effect observation. RESULTS: The chemiluminescence assay using secreted alkaline phosphatases (SEAP) as the reporter allowed rapid, sensitive, specific and reproducible detection of serotype 5 specific neutralizing antibody for epidemiological study of Ad5, which was positive in 26.72% of the adults and 17.02% of the children in this study. CONCLUSION: Ad5 neutralizing antibody is prevalent in the population of Guangzhou, suggesting the necessity of developing other serotype adenovectors for better vaccination and therapeutic effects.