A key gene, violaxanthin de-epoxidase-like 1, enhances fucoxanthin accumulation in Phaeodactylum tricornutum.

BACKGROUND: Fucoxanthin has been widely investigated owing to its beneficial biological properties, and the model diatom Phaeodactylum tricornutum, possessing fucoxanthin (Fux) chlorophyll proteins as light-harvesting systems, is considered to have the potential to become a commercial cell factory for the pigment production. RESULTS: Here, we compared the pigment contents in 10 different P. tricornutum strains from the globe, and found that strain CCMP631 (Pt6) exhibited the highest Fux content but with a low biomass. Comparison of mRNA levels revealed that higher Fux content in Pt6 was related with the higher expression of gene violaxanthin de-epoxidase-like (VDL) protein 1 (VDL1), which encodes the enzyme catalyzing the tautomerization of violaxanthin to neoxanthin in Fux biosynthesis pathway. Single nucleotide variants of VDL1 gene and allele-specific expression in strains Pt1 (the whole genome sequenced strain CCMP632) and Pt6 were analyzed, and overexpressing of each of the 4 VDL1 alleles, two from Pt1 and two from Pt6, in strain Pt1 leads to an increase in downstream product diadinoxanthin and channels the pigments towards Fux biosynthesis. All the 8 VDL1 overexpression (OE) lines showed significant increases by 8.2 to 41.7% in Fux content without compromising growth, and VDL1 Allele 2 OE lines even exhibited the higher cell density on day 8, with an increase by 24.2-28.7% in two Pt1VDL1-allele 2 OE lines and 7.1-11.1% in two Pt6VDL1-allele 2 OE lines, respectively. CONCLUSIONS: The results reveal VDL1, localized in the plastid stroma, plays a key role in Fux over-accumulation in P. tricornutum. Overexpressing VDL1, especially allele 2, improved both the Fux content and growth rate, which provides a new strategy for the manipulation of Fux production in the future.

Two plastidial lysophosphatidic acid acyltransferases differentially mediate the biosynthesis of membrane lipids and triacylglycerols in Phaeodactylum tricornutum.

Lysophosphatidic acid acyltransferases (LPAATs) catalyze the formation of phosphatidic acid (PA), a central metabolite in both prokaryotic and eukaryotic organisms for glycerolipid biosynthesis. Phaeodactylum tricornutum contains at least two plastid-localized LPAATs (ptATS2a and ptATS2b), but their roles in lipid synthesis remain unknown. Both ptATS2a and ptATS2b could complement the high temperature sensitivity of the bacterial plsC mutant deficient in LPAAT. In vitro enzyme assays showed that they prefer lysophosphatidic acid over other lysophospholipids. ptATS2a is localized in the plastid inner envelope membrane and CRISPR/Cas9-generated ptATS2a mutants showed compromised cell growth, significantly changed plastid and extra-plastidial membrane lipids at nitrogen-replete condition and reduced triacylglycerols (TAGs) under nitrogen-depleted condition. ptATS2b is localized in thylakoid membranes and its knockout led to reduced growth rate and TAG content but slightly altered molecular composition of membrane lipids. The changes in glycerolipid profiles are consistent with the role of both LPAATs in the sn-2 acylation of sn-1-acyl-glycerol-3-phosphate substrates harboring 20:5 at the sn-1 position. Our findings suggest that both LPAATs are important for membrane lipids and TAG biosynthesis in P. tricornutum and further highlight that 20:5-Lyso-PA is likely involved in the massive import of 20:5 back to the plastid to feed plastid glycerolipid syntheses.

Proteomes reveal the lipid metabolic network in the complex plastid of Phaeodactylum tricornutum.

Phaeodactylum tricornutum plastid is surrounded by four membranes, and its protein composition and function remain mysterious. In this study, the P. tricornutum plastid-enriched fraction was obtained and 2850 proteins were identified, including 92 plastid-encoded proteins, through label-free quantitative proteomic technology. Among them, 839 nuclear-encoded proteins were further determined to be plastidial proteins based on the BLAST alignments within Plant Proteome DataBase and subcellular localization prediction, in spite of the strong contamination by mitochondria-encoded proteins and putative plasma membrane proteins. According to our proteomic data, we reconstructed the metabolic pathways and highlighted the hybrid nature of this diatom plastid. Triacylglycerol (TAG) hydrolysis and glycolysis, as well as photosynthesis, glycan metabolism, and tocopherol and triterpene biosynthesis, occur in the plastid. In addition, the synthesis of long-chain acyl-CoAs, elongation, and desaturation of fatty acids (FAs), and synthesis of lipids including TAG are confined in the four-layered-membrane plastid based on the proteomic and GFP-fusion localization data. The whole process of generation of docosahexaenoic acid (22:6) from palmitic acid (16:0), via elongation and desaturation of FAs, occurs in the chloroplast endoplasmic reticulum membrane, the outermost membrane of the plastid. Desaturation that generates 16:4 from 16:0 occurs in the plastid stroma and outer envelope membrane. Quantitative analysis of glycerolipids between whole cells and isolated plastids shows similar composition, and the FA profile of TAG was not different. This study shows that the diatom plastid combines functions usually separated in photosynthetic eukaryotes, and differs from green alga and plant chloroplasts by undertaking the whole process of lipid biosynthesis.

Allele-dependent expression and functionality of lipid enzyme phospholipid:diacylglycerol acyltransferase affect diatom carbon storage and growth.

In the acyl-CoA-independent pathway of triacylglycerol (TAG) synthesis unique to plants, fungi, and algae, TAG formation is catalyzed by the enzyme phospholipid:diacylglycerol acyltransferase (PDAT). The unique PDAT gene of the model diatom Phaeodactylum tricornutum strain CCMP2561 boasts 47 single nucleotide variants within protein coding regions of the alleles. To deepen our understanding of TAG synthesis, we observed the allele-specific expression of PDAT by the analysis of 87 published RNA-sequencing (RNA-seq) data and experimental validation. The transcription of one of the two PDAT alleles, Allele 2, could be specifically induced by decreasing nitrogen concentrations. Overexpression of Allele 2 in P. tricornutum substantially enhanced the accumulation of TAG by 44% to 74% under nutrient stress; however, overexpression of Allele 1 resulted in little increase of TAG accumulation. Interestingly, a more serious growth inhibition was observed in the PDAT Allele 1 overexpression strains compared with Allele 2 counterparts. Heterologous expression in yeast (Saccharomyces cerevisiae) showed that enzymes encoded by PDAT Allele 2 but not Allele 1 had TAG biosynthetic activity, and 7 N-terminal and 3 C-terminal amino acid variants between the 2 allele-encoded proteins substantially affected enzymatic activity. P. tricornutum PDAT, localized in the innermost chloroplast membrane, used monogalactosyldiacylglycerol and phosphatidylcholine as acyl donors as demonstrated by the increase of the 2 lipids in PDAT knockout lines, which indicated a common origin in evolution with green algal PDATs. Our study reveals unequal roles among allele-encoded PDATs in mediating carbon storage and growth in response to nitrogen stress and suggests an unsuspected strategy toward lipid and biomass improvement for biotechnological purposes.

Transcriptome analysis reveals immune regulation in the spleen of koi carp (Cyprinus carpio Koi) during Aeromonas hydrophila infection.

A. hydrophila (Aeromonas hydrophila) is one of the most hazardous pathogenic microorganisms threatening the aquaculture industry and exhibits zoonotic-like characteristics. This study was designed to investigate the differential gene expression and pathway enrichment in the spleen of koi carp (Cyprinus carpio koi) upon A. hydrophila infection. The Illumina NovaSeq 6000 sequencing platform was used to identify 252 DEGs (differentially expressed genes), including 112 upregulated genes and 140 downregulated genes, in the spleens of koi carp challenged with A. hydrophila compared to those in the spleens of koi carp treated with PBS (phosphate-buffered saline). DEGs were shown to be involved in 133 pathways by KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis. Numerous immunological disease-related pathways, such as the immune defense network for IgA production, Staphylococcus aureus infection, and antigen processing and presentation, were enriched in the DEGs. In addition, the expression levels of 10 randomly screened DEGs, including the inflammatory factor nlrp3 (NOD-like receptor family pyrin domain containing 3), cytokine il-8 (interleukin-8), c2 (complement c2), c3 (complement c3), and the lipid mediator cox1 (cyclooxygenase-1), were compared by qPCR. The results showed that six genes, including il-8, cox1, and nlrp3, were upregulated according to both RNA-seq and qPCR validation, while four, including c2 and c3, showed downregulated expression. This result verified a strong correlation between the RNA-seq and qPCR datasets at the expression level. Moreover, this study provided splenic transcriptome data for koi carp during A. hydrophila infection and provided theoretical support for future drug development.

Zeaxanthin epoxidase is involved in the carotenoid biosynthesis and light-dependent growth of the marine alga Nannochloropsis oceanica.

BACKGROUND: The marine alga Nannochloropsis oceanica, an emerging model belonging to Heterokont, is considered as a promising light-driven eukaryotic chassis for transforming carbon dioxide to various compounds including carotenoids. Nevertheless, the carotenogenic genes and their roles in the alga remain less understood and to be further explored. RESULTS: Here, two phylogenetically distant zeaxanthin epoxidase (ZEP) genes from N. oceanica (NoZEP1 and NoZEP2) were functionally characterized. Subcellular localization experiment demonstrated that both NoZEP1 and NoZEP2 reside in the chloroplast yet with differential distribution patterns. Overexpression of NoZEP1 or NoZEP2 led to increases of violaxanthin and its downstream carotenoids at the expense of zeaxanthin in N. oceanica, with the extent of changes mediated by NoZEP1 overexpression being greater as compared to NoZEP2 overexpression. Suppression of NoZEP1 or NoZEP2, on the other hand, caused decreases of violaxanthin and its downstream carotenoids as well as increases of zeaxanthin; similarly, the extent of changes mediated by NoZEP1 suppression was larger than that by NoZEP2 suppression. Interestingly, chlorophyll a dropped following violaxanthin decrease in a well-correlated manner in response to NoZEP suppression. The thylakoid membrane lipids including monogalactosyldiacylglycerol also correlated with the violaxanthin decreases. Accordingly, NoZEP1 suppression resulted in more attenuated algal growth than NoZEP2 suppression did under either normal light or high light stage. CONCLUSIONS: The results together support that both NoZEP1 and NoZEP2, localized in the chloroplast, have overlapping roles in epoxidating zeaxanthin to violaxanthin for the light-dependent growth, yet with NoZEP1 being more functional than NoZEP2 in N. oceanica. Our study provides implications into the understanding of carotenoid biosynthesis and future manipulation of N. oceanica for carotenoid production.

Acanthopanax trifoliatus (L.) Merr polysaccharides ameliorates hyperglycemia by regulating hepatic glycogen metabolism in type 2 diabetic mice.

Introduction: Drug monotherapy was inadequate in controlling blood glucose levels and other comorbidities. An agent that selectively tunes multiple targets was regarded as a new therapeutic strategy for type 2 diabetes. Acanthopanax trifoliatus (L.) Merr polysaccharide (ATMP) is a bio-macromolecule isolated from Acanthopanax trifoliatus (L.) Merr and has therapeutic potential for diabetes management due to its anti-hyperglycemia activity. Methods: Type 2 diabetes mellitus was induced in mice using streptozotocin, and 40 and 80 mg/kg ATMP was administered daily via the intragastric route for 8 weeks. Food intake, water intake, and body weight were recorded. The fasting blood glucose (FBG), fasting insulin (FINS) and an oral glucose tolerance test (OGTT) were performed. Histological changes in the liver and pancreas were analyzed by H&E staining. The mRNA and the protein levels of key factors involved in glycogen synthesis, glycogenolysis, and gluconeogenesis were measured by quantitative real time PCR and Western blotting. Results: In this study, we found that ATMP could effectively improve glucose tolerance and alleviate insulin resistance by promoting insulin secretion and inhibiting glucagon secretion. In addition, ATMP decreases glycogen synthesis by inhibiting PI3K/Akt/GSK3ß signaling, reduces glycogenolysis via suppressing cAMP/PKA signaling, and suppresses liver gluconeogenesis by activating AMPK signaling. Conclusion: Together, ATMP has the potential to be developed as a new multitargets therapeutics for type 2 diabetes.

CGI-58 Protein Acts as a Positive Regulator of Triacylglycerol Accumulation in Phaeodactylum tricornutum.

Comparative gene identification-58 (CGI-58) is an activating protein of triacylglycerol (TAG) lipase. It has a variety of catalytic activities whereby it may play different roles in diverse organisms. In this study, a homolog of CGI-58 in Phaeodactylum tricornutum (PtCGI-58) was identified. PtCGI-58 was localized in mitochondria by GFP fusion protein analysis, which is different from the reported subcellular localization of CGI-58 in animals and plants. Respectively, PtCGI-58 overexpression resulted in increased neutral lipid content and TAG accumulation by 42-46% and 21-32%. Likewise, it also increased the relative content of eicosapentaenoic acid (EPA), and in particular, the EPA content in TAGs almost doubled. Transcript levels of genes involved in de novo fatty acid synthesis and mitochondrial ß-oxidation were significantly upregulated in PtCGI-58 overexpression strains compared with wild-type cells. Our findings suggest that PtCGI-58 may mediate the breakdown of lipids in mitochondria and the recycling of acyl chains derived from mitochondrial ß-oxidation into TAG biosynthesis. Moreover, this study potentially illuminates new functions for CGI-58 in lipid homeostasis and provides a strategy to enrich EPA in algal TAGs.

Chemical profile and antioxidant activity of bidirectional metabolites from Tremella fuciformis and Acanthopanax trifoliatus as assessed using response surface methodology.

This study aimed to establish a bidirectional fermentation system using Tremella fuciformis and Acanthopanax trifoliatus to promote the transformation and utilization of the synthesized antioxidant metabolites from fermentation supernatant. The effect of fermentation conditions on the total phenolic content was investigated using response surface methodology in terms of three factors, including temperature (22-28°C), pH (6-8), and inoculum size (2-8%, v/v). The optimized fermentation parameters were: 28°C, pH 8, and an inoculum size of 2%, which led to a maximum total phenolic content of 314.79 ± 6.89 µg/mL in the fermentation supernatant after 24 h culture. The content of total flavonoids and polysaccharides reached 78.65 ± 0.82 µg/mL and 9358.08 ± 122.96 µg/mL, respectively. In addition, ABTS+, DPPH⋅, and ⋅OH clearance rates reached 95.09, 88.85, and 85.36% at 24 h under optimized conditions, respectively. The content of total phenolics, flavonoids and polysaccharides in the optimized fermentation supernatant of T. fuciformis-Acanthopanax trifoliatus increased by 0.88 ± 0.04, 0.09 ± 0.02, and 33.84 ± 1.85 times that of aqueous extracts from A. trifoliatus, respectively. Simultaneously, 0.30 ± 0.00, 0.26 ± 0.01, and 1.19 ± 0.12 times increase of antioxidant activity against ABTS+, DPPH⋅, and ⋅OH clearance rates were observed, respectively. Additionally, the metabolite composition changes caused by fermentation were analyzed using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) based on untargeted metabolomics and the phytochemical profile of fermentation supernatant differentiated significantly based on unsupervised principal component analysis (PCA) during fermentation from 24 to 96 h. Furthermore, a significant increase in antioxidant phenolic and flavonoid compounds, such as ellagic acid, vanillin, luteolin, kaempferol, myricetin, isorhamnetin, and (+)-gallocatechin, was observed after fermentation. Thus, these results indicated that the fermentation broth of T. fuciformis and A. trifoliatus had significant antioxidant activity, and may have potential application for health products such as functional beverages, cosmetics, and pharmaceutical raw materials.

PDAT regulates PE as transient carbon sink alternative to triacylglycerol in Nannochloropsis.

Triacylglycerols (TAGs) are the main storage lipids in photosynthetic organisms under stress. In the oleaginous alga Nannochloropsis oceanica, while multiple acyl CoA:diacylglycerol (DAG) acyltransferases (NoDGATs) are involved in TAG production, the role of the unique phospholipid:DAG acyltransferase (NoPDAT) remains unknown. Here, we performed a functional complementation assay in TAG-deficient yeast (Saccharomyces cerevisiae) and an in vitro assay to probe the acyltransferase activity of NoPDAT. Subcellular localization, overexpression, and knockdown (KD) experiments were also conducted to elucidate the role of NoPDAT in N. oceanica. NoPDAT, residing at the outermost plastid membrane, does not phylogenetically fall into the clades of algae or plants and uses phosphatidylethanolamine (PE) and phosphatidylglycerol with 16:0, 16:1, and 18:1 at position sn-2 as acyl-donors in vivo. NoPDAT KD, not triggering any compensatory mechanism via DGATs, led to an ∼30% decrease of TAG content, accompanied by a vast accumulation of PEs rich in 16:0, 16:1, and 18:1 fatty acids (referred to as "LU-PE") that was positively associated with CO2 availability. We conclude that the NoPDAT pathway is parallel to and independent of the NoDGAT pathway for oil production. LU-PE can serve as an alternative carbon sink for photosynthetically assimilated carbon in N. oceanica when PDAT-mediated TAG biosynthesis is compromised or under stress in the presence of high CO2 levels.

Impaired photoprotection in Phaeodactylum tricornutum KEA3 mutants reveals the proton regulatory circuit of diatoms light acclimation.

Diatoms are successful phytoplankton clades able to acclimate to changing environmental conditions, including e.g. variable light intensity. Diatoms are outstanding at dissipating light energy exceeding the maximum photosynthetic electron transfer (PET) capacity via the nonphotochemical quenching (NPQ) process. While the molecular effectors of NPQ as well as the involvement of the proton motive force (PMF) in its regulation are known, the regulators of the PET/PMF relationship remain unidentified in diatoms. We generated mutants of the H+ /K+ antiporter KEA3 in the model diatom Phaeodactylum tricornutum. Loss of KEA3 activity affects the PET/PMF coupling and NPQ responses at the onset of illumination, during transients and in steady-state conditions. Thus, this antiporter is a main regulator of the PET/PMF coupling. Consistent with this conclusion, a parsimonious model including only two free components, KEA3 and the diadinoxanthin de-epoxidase, describes most of the feedback loops between PET and NPQ. This simple regulatory system allows for efficient responses to fast (minutes) or slow (e.g. diel) changes in light environment, thanks to the presence of a regulatory calcium ion (Ca2+ )-binding domain in KEA3 modulating its activity. This circuit is likely tuned by the NPQ-effector proteins, LHCXs, providing diatoms with the required flexibility to thrive in different ocean provinces.

Metabolic engineering of the oleaginous alga Nannochloropsis for enriching eicosapentaenoic acid in triacylglycerol by combined pulling and pushing strategies.

The marine alga Nannochloropsis oceanica has been considered as a promising photosynthetic cell factory for synthesizing eicosapentaenoic acid (EPA), yet the accumulation of EPA in triacylglycerol (TAG) is restricted to an extreme low level. Poor channeling of EPA to TAG was observed in N. oceanica under TAG induction conditions, likely due to the weak activity of endogenous diacylglycerol acyltransferases (DGATs) on EPA-CoA. Screening over thirty algal DGATs revealed potent enzymes acting on EPA-CoA. Whilst overexpressing endogenous DGATs had no or slight effect on EPA abundance in TAG, introducing selected DGATs with strong activity on EPA-CoA, particularly the Chlamydomonas-derived CrDGTT1, which resided at the outermost membrane of the chloroplast and provided a strong pulling power to divert EPA to TAG for storage and protection, led to drastic increases in EPA abundance in TAG and TAG-derived EPA level in N. oceanica. They were further promoted by additional overexpression of an elongase gene involved in EPA biosynthesis, reaching 5.9- and 12.3-fold greater than the control strain, respectively. Our results together demonstrate the concept of applying combined pulling and pushing strategies to enrich EPA in algal TAG and provide clues for the enrichment of other desired fatty acids in TAG as well.

Δ6 Fatty Acid Elongase is Involved in Eicosapentaenoic Acid Biosynthesis Via the ω6 Pathway in the Marine Alga Nannochloropsis oceanica.

Nannochloropsis oceanica represents a promising sunlight-driven alga for producing eicosapentaenoic acid (EPA, 20:5Δ5,8,11,14,17), a value-added very long-chain polyunsaturated fatty acid (VLC-PUFA). Here, we unraveled the function and roles of a Δ6 fatty acid elongase (NoΔ6-FAE) in N. oceanica. Heterologous expression of NoΔ6-FAE in yeast confirmed its function in elongating C18 Δ6-PUFAs rather than others. Subcellular localization experiments suggested that NoΔ6-FAE resides in the chloroplast endoplasmic reticulum. NoΔ6-FAE knockdown attenuated C20:3Δ8,11,14, C20:4Δ5,8,11,14, and EPA yet enhanced C18:3Δ6,9,12, leading to overall decreases in total fatty acids, triacylglycerol, diacylglycerol, free fatty acids, and polar membrane lipids. In contrast, NoΔ6-FAE overexpression in N. oceanica caused nearly opposite phenotypes. Moreover, N. oceanica lacked detectable C18:3Δ9,12,15, C18:4Δ6,9,12,15, and C20:4Δ8,11,14,17 even under NoΔ6-FAE knockdown or overexpression. Our results reveal the involvement of NoΔ6-FAE in EPA biosynthesis via the ω6 pathway in N. oceanica and highlight the potential of manipulating NoΔ6-FAE for improved lipid production.

Immunoregulatory Effect of Acanthopanax trifoliatus (L.) Merr. Polysaccharide on T1DM Mice.

BACKGROUND: Acanthopanax trifoliatus (L.) Merr. is a medicinal plant found in Southeast Asia, and its young leaves and shoots are consumed as a vegetable. The main bioactive components of this herb are polysaccharides that have significant anti-diabetic effects. The aim of this study was to evaluate the immunoregulatory effect of A. trifoliatus (L.) Merr. polysaccharide (ATMP) on a mouse model of type 1 diabetes mellitus (T1DM). METHODS: The monosaccharide composition and mean molecular mass of ATMP were determined by HPLC and HPGPC. T1DM was induced in mice using STZ, and 35, 70 and 140mg/kg ATMP was administered daily via the intragastric route for six weeks. Untreated and metformin-treated positive control groups were also included. The body weight of the mice, food and water intake and fasting glucose levels were monitored throughout the 6-week regimen. Histological changes in the pancreas and spleen were analyzed by H&E staining. Oral glucose tolerance was evaluated with the appropriate test. Peroxisome proliferator-activated receptor γ (PPARγ) mRNA and protein levels in the spleen were measured by quantitative real time PCR and Western blotting. IL-10, IFN-γ and insulin levels in the sera were determined by ELISA. The CD4+ and CD8+T cells in spleen tissues were detected by immunohistochemistry (IHC). RESULTS: ATMP and metformin significantly decreased fasting blood glucose, and the food and water intake after 6 weeks of treatment. In contrast, serum insulin levels, glucose tolerance and body weight improved considerably in the high and medium-dose ATMP and metformin groups. T1DM was associated with pancreatic and splenic tissue damage. The high dose (140mg/kg) of ATMP reduced infiltration of inflammatory cells into the pancreas and restored the structure of islet ß-cells in the diabetic mice. Consistent with this, 35, 70 and 140mg/kg ATMP increased IL-10 levels and decreased that of IFN-γ, thereby restoring the CD4+/CD8+ and Th1/Th2 cytokine ratio. At the molecular level, high-dose ATMP up-regulated PPARγ in the splenic cells. CONCLUSION: ATMP exerts a hypoglycemic effect in diabetic mice by restoring the immune balance in the spleen.

Correction: FengLiao affects gut microbiota and the expression levels of Na+/H+ exchangers, aquaporins and acute phase proteins in mice with castor oil-induced diarrhea.

[This corrects the article DOI: 10.1371/journal.pone.0236511.].

FengLiao affects gut microbiota and the expression levels of Na+/H+ exchangers, aquaporins and acute phase proteins in mice with castor oil-induced diarrhea.

The severe side effects of chemosynthetic anti-diarrhea drugs have created an interest in low-toxic alternative plant-derived compounds. FengLiao consists of Polygonum hydropiper Linn. and Daphniphyllum calycinum Bench., and is widely used in China to treat diarrhea due to low levels of toxicity. In this study, the effects of FengLiao were analyzed in a castor oil-induced diarrhea model, using the anti-diarrhea drug, loperamide, as the positive control. The effects were evaluated using stool characteristics and the expression levels of various diarrhea-related factors in the jejunum and liver, as well as changes in the microbiota of the jejunum. The symptoms of diarrhea and stool consistency were improved through FengLiao and loperamide treatment. Furthermore, FengLiao down-regulated alpha 1-acid glycoprotein (AGP) and C-reactive protein (CRP) levels, and up-regulated transferrin (TRF) mRNA levels in the liver, and down-regulated Aquaporin 3 (AQP3) and Na+/H+ exchanger isoform 8 (NHE8) expression in the epithelial cells of the jejunum. It also increased the relative abundance of Bifidobacterium, Aerococcus, Corynebacterium_1 and Pseudomonas, and lowered the Firmicutes/Bacteroidetes (F/B) ratio, which maintained the balance between immunity and intestinal health. Taken together, FengLiao alleviated castor oil-induced diarrhea by altering gut microbiota, and levels of jejunum epithelial transport proteins and acute phase proteins.

Incorporation of ion exchange functionalized-montmorillonite into solid lipid nanoparticles with low irritation enhances drug bioavailability for glaucoma treatment.

Montmorillonite-loaded solid lipid nanoparticles with good biocompatibility, using Betaxolol hydrochloride as model drug, were prepared by the melt-emulsion sonication and low temperature-solidification methods and drug bioavailability was significantly improved in this paper for the first time to application to the eye. The appropriate physical characteristics were showed, such as the mean particle size, Zeta potential, osmotic pressure, pH values, entrapping efficiency (EE%) and drug content (DC%), all showed well suited for possible ocular application. In vitro release experiment indicated that this novel system could continuously release 57.83% drugs within 12 h owing to the dual drug controlled-release effect that was achieved by ion-exchange feature of montmorillonite and structure of solid lipid nanoparticles. Low irritability and good compatibility of nanoparticles were proved by both CAM-TBS test and cytotoxicity experiment. We first discovered from the results of Rose Bengal experiment that the hydrophilicity of the drug-loaded nanoparticles surface was increased during the loading and releasing of the hydrophilic drug, which could contribute to prolong the ocular surface retention time of drug in the biological interface membrane of tear-film/cornea. The results of in vivo pharmacokinetic and pharmacodynamics studies further confirmed that increased hydrophilicity of nanoparticles surface help to improve the bioavailability of the drug and reduce intraocular pressure during administration. The results suggested this novel drug delivery system could be potentially used as an in situ drug controlled-release system for ophthalmic delivery to enhance the bioavailability and efficacy.

Screening of High Temperature-Tolerant Oleaginous Diatoms.

Screening suitable strains with high temperature adaptability is of great importance for reducing the cost of temperature control in microalgae cultivation, especially in summer. To obtain high temperature-tolerant diatoms, water samples were collected in summer from 7 different regions of China across the Northeast, North and East. A total of 731 water samples was collected and from them 131 diatom strains were isolated and identified based on the 18S rRNA sequences. Forty-nine strains out of the 131 diatoms could survive at 30°C, and 6 strains with relatively high biomass and lipid content at high temperature were selected and were found to be able to grow at 35°C. Cyclotella sp. HB162 had the highest dry biomass of 0.46 g/l and relatively high triacylglycerol (TAG) content of 237.4 mg/g dry biomass. The highest TAG content of 246.4 mg/g dry biomass was obtained in Fistulifera sp. HB236, while Nitzschia palea HB170 had high dry biomass (0.33 g/l) but relatively low TAG content (105.9 mg/g dry biomass). N. palea HB170 and Fistulifera sp. HB236 presented relatively stable growth rates and lipid yields under fluctuating temperatures ranging from 28 to 35°C, while Cyclotella HB162 maintained high lipid yield at temperatures below 25°C. The percentage of saturated fatty acids and monounsaturated fatty acids in all the 6 strains was 84-91% in total lipids and 90-94% in TAGs, which makes them the ideal feedstock for biodiesel.

Amino Acid Catabolism During Nitrogen Limitation in Phaeodactylum tricornutum.

Diatoms can accumulate high levels of triacylglycerols (TAGs) under nitrogen depletion and have attracted increasing attention as a potential system for biofuel production. In Phaeodactylum tricornutum, a model diatom, about 40% of lipid is synthesized from the breakdown of cellular components under nitrogen starvation. Our previous studies indicated that carbon skeletons from enhanced branched-chain amino acid (BCAA) degradation under nitrogen deficiency contribute to TAG biosynthesis in P. tricornutum. In this review, we outlined the catabolic pathways of all 20 amino acids based on the genome, transcriptome, proteome, and metabolome data. The contribution of these amino acid catabolic pathways to TAG accumulation was also analyzed.

Electroporation Transformation Protocol for Phaeodactylum tricornutum.

Genetic transformation system is very important for both basic biological research and commercial exploitation of diatoms. Here we describe a high-efficiency nuclear transformation method for the model diatom Phaeodactylum tricornutum using an electroporation system, and the maximum transformation frequency obtained is about 3 × 10-5 cells. The described protocol also provides some clue for developing electroporation transformation system in other eukaryotic microalgae.