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1.
Mar Pollut Bull ; 161(Pt B): 111806, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33126142

RESUMO

The brown tide formed by a microscopic alga called Aureococcus anophagefferens has a devastating effect on filter-feeding bivalves, however, the related toxic principle remains an open question. In this study, we found that A. anophagefferens cells could motivate detoxification associated genes including CYP450, GST, P-gp and MVP, and induce SOD activity in the mussel Perna viridis. D1-like and D2-like receptors were expressed at high level in the gills of P. viridis, however, D2-like receptor transcript was too low to detect in digestive gland. The exposure of A. anophagefferens did not lead to any significant alterations in the expression of D1-like and D2-like receptors in both gills and digestive gland. These findings suggested that A. anophagefferens exhibited cytotoxicity toward bivalves, but did not obviously disrupt the dopamine system at transcriptional level in the acute exposure. Further studies are warranted to explore the nature of toxic compounds in A. anophagefferens affected bivalves.


Assuntos
Perna (Organismo) , Estramenópilas , Animais , Sistema Enzimático do Citocromo P-450/genética , Brânquias , Alimentos Marinhos
2.
Skeletal Radiol ; 2020 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-33112978

RESUMO

OBJECTIVE: To identify a diagnostic indicator of lumbar spondylolysis visible in plain X-ray films. METHODS: One hundred and seventy-two patients with low back pain who received X-ray and computerized tomography (CT) examinations were identified and studied. They were divided into three groups: the spondylosis without spondylolisthesis (SWS) group, comprising 67 patients with bilateral pars interarticularis defects at L5 and without spondylolisthesis, the isthmic spondylolisthesis (IS) group, comprising 74 patients with L5/S1 spondylolisthesis and bilateral L5 pars interarticularis defects, and the control group, comprising 31 patients with low back pain but without spondylolysis. The sagittal diameters of the vertebral arch (SDVAs) of L4 and L5 were measured in lateral X-ray image, and the differences in SDVA between L4 and L5 (DSL4-5) in each case were calculated and analyzed. RESULTS: There were no significant differences in demographic characteristics among the three groups. In the SWS and IS groups, the SDVA of L5 was significantly longer than the SDVA of L4 (p < 0.001), whereas no significant difference found in the control group (p > 0.05). DSL4-5, in which the SDVA of L4 was subtracted from the SDVA of L5, significantly differed among the three groups (p < 0.001), and the normal threshold was provisionally determined to be 1.55 mm. CONCLUSIONS: In bilateral L5 spondylolysis, the SDVA of L5 is wider than the SDVA of L4, and this difference is greater in isthmic spondylolisthesis. This sign in lateral X-rays may provide a simple and convenient aid for the diagnosis of spondylolysis.

3.
Biotechnol Biofuels ; 13: 160, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32944076

RESUMO

Background: Despite the great potential of marine diatoms in biofuel sector, commercially viable biofuel production from native diatom strain is impractical. Targeted engineering of TAG pathway represents a promising approach; however, recruitment of potential candidate has been regarded as critical. Here, we identified a glycerol-3-phosphate acyltransferase 2 (GPAT2) isoform and overexpressed in Phaeodactylum tricornutum. Results: GPAT2 overexpression did not impair growth and photosynthesis. GPAT2 overexpression reduced carbohydrates and protein content, however, lipid content were significantly increased. Specifically, TAG content was notably increased by 2.9-fold than phospho- and glyco-lipids. GPAT2 overexpression elicited the push-and-pull strategy by increasing the abundance of substrates for the subsequent metabolic enzymes, thereby increased the expression of LPAAT and DGAT. Besides, GPAT2-mediated lipid overproduction coordinated the expression of NADPH biosynthetic genes. GPAT2 altered the fatty acid profile in TAGs with C16:0 as the predominant fatty acid moieties. We further investigated the impact of GPAT2 on conferring abiotic stress, which exhibited enhanced tolerance to hyposaline (70%) and chilling (10 ºC) conditions via altered fatty acid saturation level. Conclusions: Collectively, our results exemplified the critical role of GPAT2 in hyperaccumulating TAGs with altered fatty acid profile, which in turn uphold resistance to abiotic stress conditions.

4.
Spinal Cord ; 58(12): 1310-1316, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32561846

RESUMO

STUDY DESIGN: Retrospective analysis. SETTING: China Rehabilitation Research Center, Beijing, China. OBJECTIVE: To explore possible mechanisms underlying spinal cord injury (SCI) in children caused by hyperextension of the spine while dancing. METHODS: The clinical records of 88 children with SCI (mean age, 5.97 years; age range, 4-10 years) admitted to our hospital from January 1989 to October 2019 were retrospectively reviewed. Computed tomography and magnetic resonance imaging were performed on the day of injury. The time from injury to development of paralysis, as well as post-injury activities were surveyed, while abnormal patterns on images, the range of the involved vertebrae, and the extents of edema and atrophy were assessed. RESULTS: Among the 88 patients, 6 (6.8%) were unable to move immediately after SCI, while paralysis occurred in 42, 23, and 17 patients at <30, 30-60, and >60 min after SCI, respectively. The neurological level of injury of 84 patients was between T4 and T12. On sagittal T2-weighted images (T2WIs), the longitudinal range of spinal cord edema was more than one vertebral body in 65 patients, while spinal cord atrophy below T8 was found in 40 patients. On axial T2WIs, although three patients had none, long T2 signals were found in the central gray matter of seven patients. Meanwhile, necrosis of the central area combined with the peripheral white matter was observed in 57 patients, while three patients had total involvement on a cross section. CONCLUSION: Ischemia-related damage, rather than direct trauma to the spinal cord, may play an important role in SCI due to spinal hyperextension during dancing.

5.
Chemosphere ; 254: 126874, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32361543

RESUMO

Okadaic acid (OA), one of the most important phycotoxins, is widely distributed around the world, concerning diarrheic shellfish poisoning (DSP), and even colorectal cancer. Here, we found that long-term exposure of OA at a low dose (80 µg kg-1 body weight) had certain effects on colonic microbiotas and tract in rat. In the OA-exposed rat, colonic epithelium layer was damaged, and relative abundance of some microbiotas were significantly changed, especially genera in Clostridiales. However, no intestinal inflammation or significant disease was observed. Combined with the increase in relative abundance of some genera in Clostridiales induced by OA in the fermentation experiment, we proposed that OA could cause damage to the intestinal epithelium and increase the relative abundance of pathogenic bacteria, thereby increasing the probability of contact between intestinal epithelium and pathogenic bacteria and leading to an easier pathogenicity.


Assuntos
Carcinógenos/toxicidade , Microbioma Gastrointestinal/efeitos dos fármacos , Ácido Okadáico/toxicidade , Animais , Colo , Inflamação , Mucosa Intestinal , Intestinos , Microbiota , Ratos , Intoxicação por Frutos do Mar , Testes de Toxicidade Crônica
6.
Ecotoxicol Environ Saf ; 192: 110265, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32045784

RESUMO

Diarrheic shellfish poisoning (DSP) toxins are produced by harmful microalgae and accumulate in bivalve mollusks, causing various toxicity. These toxic effects appear to abate with increasing DSP concentration and longer exposure time, however, the underlying mechanisms remain unclear. To explore the underlying molecular mechanisms, de novo transcriptome analysis of the digestive gland of Perna viridis was performed after Prorocentrum lima exposure. RNA-seq analysis showed that 1886 and 237 genes were up- and down-regulated, respectively after 6 h exposure to P. lima, while 265 genes were up-regulated and 217 genes were down-regulated after 96 h compared to the control. These differentially expressed genes mainly involved in Nrf2 signing pathways, immune stress, apoptosis and cytoskeleton, etc. Combined with qPCR results, we speculated that the mussel P. viridis might mainly rely on glutathione S-transferase (GST) and ABC transporters to counteract DSP toxins during short-term exposure. However, longer exposure of P. lima could activate the Nrf2 signaling pathway and inhibitors of apoptosis protein (IAP), which in turn reduced the damage of DSP toxins to the mussel. DSP toxins could induce cytoskeleton destabilization and had some negative impact on the immune system of bivalves. Collectively, our findings uncovered the crucial molecular mechanisms and the regulatory metabolic nodes that underpin the defense mechanism of bivalves against DSP toxins and also advanced our current understanding of bivalve defense mechanisms.


Assuntos
Dinoflagelados/metabolismo , Expressão Gênica/efeitos dos fármacos , Toxinas Marinhas/toxicidade , Perna (Organismo)/efeitos dos fármacos , Animais , Regulação para Baixo , Perfilação da Expressão Gênica , Toxinas Marinhas/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Perna (Organismo)/genética , Perna (Organismo)/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Alimentos Marinhos , Intoxicação por Frutos do Mar , Regulação para Cima
7.
Biotechnol J ; 15(2): e1900135, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31464064

RESUMO

Microalgae have long been considered as potential biological feedstock for the production of wide array of bioproducts, such as biofuel feedstock because of their lipid accumulating capability. However, lipid productivity of microalgae is still far below commercial viability. Here, a glucose-6-phosphate dehydrogenase from the oleaginous microalga Nannochloropsis oceanica is identified and heterologously expressed in the green microalga Chlorella pyrenoidosa to characterize its function in the pentose phosphate pathway. It is found that the G6PD enzyme activity toward NADPH production is increased by 2.19-fold in engineered microalgal strains. Lipidomic analysis reveals up to 3.09-fold increase of neutral lipid content in the engineered strains, and lipid yield is gradually increased throughout the cultivation phase and saturated at the stationary phase. Moreover, cellular physiological characteristics including photosynthesis and growth rate are not impaired. Collectively, these results reveal the pivotal role of glucose-6-phosphate dehydrogenase from N. oceanica in NADPH supply, demonstrating that provision of reducing power is crucial for microalgal lipogenesis and can be a potential target for metabolic engineering.


Assuntos
Chlorella/enzimologia , Glucosefosfato Desidrogenase/metabolismo , Metabolismo dos Lipídeos , Lipídeos/análise , Lipogênese/genética , Microalgas/enzimologia , Biocombustíveis , Chlorella/genética , Chlorella/crescimento & desenvolvimento , Glucosefosfato Desidrogenase/genética , Engenharia Metabólica , Microalgas/genética , Microalgas/crescimento & desenvolvimento , NADP/metabolismo , Via de Pentose Fosfato , Fotossíntese
8.
Bioresour Technol ; 296: 122351, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31708386

RESUMO

This study investigates the prospective of utilizing kitchen wastewater and food wastes, biofuels industry byproducts as alternative water and carbon sources. Kitchen wastewater did not impede cellular growth rate of the evolved Phaeodactylum strain E70, which indicates its potential as an alternative to freshwater resources. Among the organic wastes assessed, food waste hydrolysate significantly increased cell growth. Supplement of crude glycerol in cultivation medium enhances the total fatty acid content. Mixed food waste hydrolysate and crude glycerol remarkably increased both the cell density and total fatty acid content. Also, the supplement of butylated hydroxytoluene alleviated the oxidative stress induced by impurities in organic wastes and concomitantly increased microalgal total fatty acids and polyunsaturated fatty acids content. The experimental results reported in this study show that a waste-based biorefinery could lead to utilization of organic waste resources for the efficient production of value-added products.


Assuntos
Eliminação de Resíduos , Entorses e Distensões , Biocombustíveis , Ácidos Graxos Insaturados , Alimentos , Humanos , Estudos Prospectivos
9.
Methods Mol Biol ; 2050: 175-179, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31468492

RESUMO

Electroporation refers to the application of high strength electric pulse to create transient pores in the membrane, thereby enabling the passage of hydrophilic molecules into the cells. Based on the properties of cell and cell wall, the electroporation parameters vary among the algal species. Here, we demonstrated the optimized protocol for successful introduction of recombinant DNA (~5000 bp) into Nannochloropsis oceanica. The linearized recombinant plasmid that harbors eGFP and Bh-sle as the reporter and marker gene, respectively, was electroporated into the electrocompetent N. oceanica cells at voltage of 2200 V, 50 µF, resistance at 600 Ω using electroporator, and the transformed cells were then screened by molecular analysis. The report exemplifies a straightforward and reliable electroporation strategy for generating transgenic N. oceanica cells.


Assuntos
DNA/genética , Eletroporação/métodos , Estramenópilas/genética , Genes Reporter , Proteínas de Fluorescência Verde/genética , Organismos Geneticamente Modificados/crescimento & desenvolvimento , Estramenópilas/crescimento & desenvolvimento , Transformação Genética
10.
Bioresour Technol ; 289: 121720, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31271916

RESUMO

Haematococcus pluvialis is a main biological resource for the antioxidant astaxanthin production, however, potential modulators and molecular mechanisms underpinning astaxanthin accumulation remain largely obscured. We discovered that provision of ethanol (0.4%) significantly triggered the cellular astaxanthin content up to 3.85% on the 4th day of treatment. Amongst, 95% of the accumulated astaxanthin was esterified, particularly enriched with monoesters. Ultrastructural analysis revealed that ethanol altered cell wall structure and physiological properties. Antioxidant analyses revealed that astaxanthin accumulation offset the ethanol induced oxidative stress. Ethanol treatment reduced carbohydrates while increased lipids and jasmonic acid production. Transcriptomic analysis uncovered that ethanol orchestrated the expression of crucial genes involved in carotenogenesis, e.g. PSY, BKT and CRTR-b were significantly upregulated. Moreover, methyl jasmonic acid synthesis was induced and played a major role in regulating the carotenogenic genes. The findings uncovered the novel viewpoint in the intricate transcriptional regulatory mechanisms of astaxanthin biosynthesis.


Assuntos
Clorofíceas/metabolismo , Ciclopentanos/metabolismo , Etanol/farmacologia , Oxilipinas/metabolismo , Clorofíceas/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Xantofilas/biossíntese
11.
Biotechnol Bioeng ; 116(11): 3006-3015, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31282986

RESUMO

There has been growing interest in using microalgae as production hosts for a wide range of value-added compounds. However, microalgal genetic improvement is impeded by lack of genetic tools to concurrently control multiple genes. Here, we identified two novel strong promoters, designated Pt202 and Pt667, and delineated their potential role on simultaneously driving the expression of key lipogenic genes in Phaeodactylum tricornutum. In silico analyses of the identified promoter sequences predicted the presence of essential core cis elements such as TATA and CAAT boxes. Regulatory role of the promoters was preliminarily assessed by using GUS reporter which demonstrated strong GUS expression. Thereafter, two key lipogenic genes including malic enzyme (PtME) and 5-desaturase (PtD5b), were overexpressed by the two promoters Pt202 and Pt667, respectively, in P. tricornutum. Combinatorial gene overexpression did not impair general physiological performance, meanwhile neutral lipid content was remarkably increased by 2.4-fold. GC-MS analysis of fatty acid methyl esters revealed that eicosapentaenoic acid (EPA; C20:5) was increased significantly. The findings augment a crucial kit to microalgal genetic tools that could facilitate the multiple-gene expression driven by various promoters, and promote microalgae for industrial bioproduction.


Assuntos
Diatomáceas , Regulação da Expressão Gênica/fisiologia , Lipogênese/fisiologia , Microalgas , Regiões Promotoras Genéticas , Diatomáceas/genética , Diatomáceas/metabolismo , Microalgas/genética , Microalgas/metabolismo
12.
Spinal Cord ; 57(11): 972-978, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31239531

RESUMO

STUDY DESIGN: Retrospective analysis. SETTING: China Rehabilitation Research Center, Beijing, China. OBJECTIVE: A retrospective study that documents the modalities and clarifies the heterogeneity among spinal cord injuries (SCIs) caused by trauma to the thoracolumbar vertebral junction. METHODS: X-ray and MRI imaging, neurological records, and the urodynamics results of 190 patients were reviewed and used to categorize different SCI modalities. First, injuries were divided into complete and incomplete injuries using the International Standard for Neurological Classification of Spinal Cord Injury. Next, the complete injuries were further grouped using the neurological level of injury and Long T2 signal from mid-sagittal MRI images, whereas the bulboconvernosus reflexes were also used as a reference to detect injury to the sacral cord. RESULTS: The SCI modalities were classified into five categories: pure complete epiconus lesion with caudal cord intact (G1), complete epiconus injury with conus medullaris (CM) totally involved in the lesion (G2), CM syndrome, cauda equine syndrome without sacral sparing (G3 and G4), and incomplete injury (G5). CONCLUSIONS: The heterogeneity of SCIs at the thoracolumbar junction was documented, a criterion we propose to be of great significance when selecting patients for clinical trials. In particular, the G2 group, which comprises nearly one third of the patients with epiconus lesions, is sometimes mistaken as G1, an observation that has thus far received insufficient attention.


Assuntos
Ensaios Clínicos como Assunto/métodos , Vértebras Lombares/diagnóstico por imagem , Seleção de Pacientes , Traumatismos da Medula Espinal/diagnóstico por imagem , Traumatismos da Medula Espinal/reabilitação , Vértebras Torácicas/diagnóstico por imagem , Adolescente , Adulto , Idoso , Feminino , Humanos , Vértebras Lombares/lesões , Imagem por Ressonância Magnética/métodos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Traumatismos da Medula Espinal/epidemiologia , Vértebras Torácicas/lesões , Adulto Jovem
13.
Mar Drugs ; 17(5)2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-31052268

RESUMO

Prorocentrum lima is a typical benthic toxic dinoflagellate, which can produce phycotoxins such as okadaic acid (OA). In this study, we identified three ABC transporter genes (ABCB1, ABCC1 and ABCG2) and characterized their expression patterns, as well as OA production under different environmental conditions in P. lima. We found that the three ABC transporters all showed high identity with related ABC proteins from other species, and contained classical features of ABC transport proteins. Among them, ABCG2 was a half size transporter. The three ABC transporter genes displayed various expression profiles under different conditions. The high concentration of Cu2+ could up-regulate ABCB1, ABCC1 and ABCG2 transcripts in P. lima, suggesting the potential defensive role of ABC transporters against metal ions in surrounding waters. Cu2+, in some concentration, could induce OA production; meanwhile, tributyltin inhibited OA accumulation. The grazer Artemia salina could induce OA production, and P. lima displayed some toxicity to the grazer, indicating the possibility of OA as an anti-grazing chemical. Collectively, our results revealed intriguing data about OA production and the expression patterns of three ABC transporter genes. However, we could not find any significant correlation between OA production and expression pattern of the three ABC transporters in P. lima. Our results might provide new molecular insights on the defensive responses of P. lima to the surrounding environment.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Dinoflagelados/metabolismo , Dinoflagelados/crescimento & desenvolvimento , Meio Ambiente , Toxinas Marinhas/química , Ácido Okadáico/metabolismo , RNA Mensageiro
14.
Ecotoxicol Environ Saf ; 180: 192-201, 2019 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-31085430

RESUMO

As a main marine phycotoxin, okadaic acid (OA) is mainly responsible for diarrheic shellfish poisoning (DSP), through specifically inhibiting phosphatase (PP1 and PP2A). It has been shown that isotope labelled-OA could cross the placental barrier in mice. However, it remains obscure how OA exposure could affect the formation of neural crest cells (NCCs), especially cranial NCCs in early embryo development. Here, we explored the effects of OA exposure on the generation of neural crest cells during embryonic development using the classic chick embryo model. We found that OA exposure at 100 nM (80.5 µg/L) could cause craniofacial bone defects in the developing chick embryo and delay the development of early chick embryos. Immunofluorescent staining of HNK-1, Pax7, and Ap-2α demonstrated that cranial NCC generation was inhibited by OA exposure. Double immunofluorescent staining with Ap-2α/PHIS3 or Pax7/c-Caspase3 manifested that both NCC proliferation and apoptosis were restrained by OA exposure. Furthermore, the expression of Msx1 and BMP4 were down-regulated in the developing chick embryonic neural tubes, which could contribute the inhibitive production of NCCs. We also discovered that expression of EMT-related adhesion molecules, such as Cadherin 6B (Cad6B) and E-cadherin, was altered following OA exposure. In sum, OA exposure negatively affected the development of embryonic neural crest cells, which in turn might result in cranial bone malformation.


Assuntos
Inibidores Enzimáticos/toxicidade , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Crista Neural/efeitos dos fármacos , Ácido Okadáico/toxicidade , Animais , Apoptose/efeitos dos fármacos , Caderinas/metabolismo , Embrião de Galinha , Regulação para Baixo , Desenvolvimento Embrionário/efeitos dos fármacos , Crista Neural/citologia , Crista Neural/embriologia , Tubo Neural/efeitos dos fármacos , Tubo Neural/metabolismo , Crânio/anormalidades
15.
Fish Shellfish Immunol ; 90: 173-179, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31059815

RESUMO

It is well documented that diarrhetic shellfish poisoning (DSP) toxins have strong genetic toxicity, cytotoxicity and oxidative damage to bivalve species. However, these toxic effects seem to decrease with the extension of exposure time and the increment of the toxin concentration, the mechanism involved remained unclear, though. In this paper, we found that expression of the genes related to cytoskeleton and Nrf2 signaling pathway displayed different changes over time in the gill of Perna viridis after exposure to DSP toxins-producing microalga Prorocentrum lima. During the short-term exposure (3 h and 6 h), KEAP1 gene expression was significantly up-regulated, coupled with up-regulation of MRP, ABCB1 and CAT transcriptions and down-regulation of GPx1 and NQO1 mRNA. After longer exposure to high density of P. lima, Nrf2 was significantly up-regulated, accompanied with up-regulation of Nrf2 pathway related genes such as NQO1, SOD, GST-ω and ABCB1, whereas KEAP1 was down-regulated. TUBA1C and TUBB1 transcripts were significantly down-regulated after short-term exposure of P. lima, but both of them were up-regulated at 96 h after exposure to high density of P. lima. Paraffin section demonstrated that P. lima had a strong damage on the gill of mussels during the short-term exposure. However, the negative effect to the gill decreased, and the gill restored after longer exposure (96 h). Taking together, we proposed that P. lima had a negative impact on cytoskeleton of mussel gill tissue, could cause oxidative damage to the gills. However, longer exposure of P. lima in high density could activate Nrf2 signaling pathway, thereby reducing the influence of toxin on mussel. Our study might provide a novel clue for the resistance mechanism of shellfish to DSP toxins.


Assuntos
Antioxidantes/metabolismo , Dinoflagelados/fisiologia , Toxinas Marinhas/efeitos adversos , Fator 2 Relacionado a NF-E2/genética , Perna (Organismo)/genética , Animais , Elementos de Resposta Antioxidante/imunologia , Fator 2 Relacionado a NF-E2/metabolismo , Perna (Organismo)/efeitos dos fármacos , Perna (Organismo)/enzimologia , Perna (Organismo)/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/imunologia , Regulação para Cima
16.
Ecotoxicol Environ Saf ; 176: 178-185, 2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-30927639

RESUMO

Diarrhetic shellfish poisoning (DSP) toxins are key shellfish toxins that cause diarrhea, vomiting and even tumor. Interestingly, bivalves such as Perna viridis have been reported to exhibit some resistances to alleviate toxic effects of DSP toxins in a species-specific manner. Nevertheless, the molecular mechanisms underlying the resistance phenomenon to DSP toxins, particularly the mechanistic role of CYP450 is scant despite its crucial role in detoxification. Here, we exposed P. viridis to Prorocentrum lima and examined the expression pattern of the CYP450 and our comprehensive analyses revealed that P. lima exposure resulted in unique expression pattern of key CYP450 genes in bivalves. Exposure to P. lima (2 × 105 cells/L) dramatically orchestrated the relative expression of CYP450 genes. CYP2D14-like mRNA was significantly down-regulated at 6 h in gill, but up-regulated at 2 h in digestive gland compared with control counterparts (p < 0.05), while CYP3A4 mRNA was increased at 12 h in gill. After exposure to P. lima at 2 × 106 cells/L, the expression of CYP3A4 mRNA was significantly increased in digestive gland at 2 h and 12 h, while CYP2D14-like was up-regulated at 6 h. Besides, CYP3L3 and CYP2C8 also exhibited differential expression. These data suggested that CYP3A4, CYP2D14-like, and even CYP3L3 and CYP2C8 might be involved in DSP toxins metabolism. Besides, provision of ketoconazole resulted in significant decrement of CYP3A4 in digestive gland at 2 h and 12 h, while the OA content significantly decreased at 2 h and 6 h compared to control group without ketoconazole. These findings indicated that ketoconazole could depress CYP3A4 activity in bivalves thereby altering the metabolic activities of DSP toxins in bivalves, and also provided novel insights into the mechanistic role of CYP3A4 on DSP toxins metabolism in bivalves.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Dinoflagelados/metabolismo , Toxinas Marinhas/toxicidade , Perna (Organismo)/enzimologia , Intoxicação por Frutos do Mar , Poluentes da Água/toxicidade , Animais , Sistema Enzimático do Citocromo P-450/genética , Brânquias/efeitos dos fármacos , Brânquias/enzimologia , Perna (Organismo)/efeitos dos fármacos , Alimentos Marinhos/análise
17.
Sci Adv ; 5(1): eaau3795, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30729156

RESUMO

Commercialization of algal lipids and biofuels is still impractical owing to the unavailability of lipogenic strains and lack of economically viable oil extraction strategies. Because lipogenesis is governed by multiple factors, success in generating industrial-suitable algal strains using conventional strategies has been limited. We report the discovery of a novel bZIP1 transcription factor, NobZIP1, whose overexpression results in a remarkable elevation of lipid accumulation and lipid secretion in a model microalga Nannochloropsis oceanica, without impairing other physiological properties. Chromatin immunoprecipitation-quantitative PCR analysis revealed that the key genes up- and down-regulated by NobZIP1 are involved in lipogenesis and cell wall polymer synthesis, respectively, which, in turn, induce lipid overproduction and secretion. Among these regulated genes, UDP-glucose dehydrogenase was shown to alter cell wall composition, thus also boosting lipid secretion. In summary, these results offer a comprehensive strategy for concurrent lipid overproduction and secretion, strongly increasing the commercial potential of microalgae.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/genética , Ácidos Graxos/biossíntese , Microalgas/genética , Microalgas/metabolismo , Estramenópilas/genética , Estramenópilas/metabolismo , Transcrição Genética , Biocombustíveis , Metabolismo dos Carboidratos/genética , Parede Celular/genética , Imunoprecipitação da Cromatina/métodos , Inativação Gênica , Metabolismo dos Lipídeos/genética , Lipogênese/genética , Desidrogenase do Álcool de Açúcar/genética
18.
Bioresour Technol ; 277: 204-210, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30630660

RESUMO

Microalgae have emerged as the potential source for value-added products such as polyunsaturated fatty acids (PUFAs). Metabolic engineering of multiple metabolic pathways has promoted eicosapentaenoic acid (EPA) production in microalgae, however, further improvement is warranted owing to the burgeoning demand. Here we improved the microalgal strains by adaptive evolution under hyposalinity treatment, which showed that 70% salinity potentiated the algae to enhance PUFAs. To exploit the maximal PUFA production potential of evolved strains, we subjected evolved algae to light, temperature and fulvic acid treatment. Amongst, fulvic acid (15 mg/L) enhanced growth and achieved the highest EPA content (13.9%) in the evolved diatom. Fulvic acid enhanced antioxidant potential and unprecedently governed the expression of PUFA and lipid biosynthetic genes. Collectively, this investigation demonstrates the efficacy of adaptive evolution empowered by fulvic acid and exemplifies a feasible strain improving strategy to harness the biotechnological potential of microalgae.


Assuntos
Benzopiranos/metabolismo , Ácido Eicosapentaenoico/análogos & derivados , Microalgas/metabolismo , Diatomáceas/metabolismo , Ácido Eicosapentaenoico/biossíntese , Temperatura
19.
Biotechnol J ; 14(3): e1800220, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30076758

RESUMO

Chrysolaminarin, the primary polysaccharide reservoir in some marine algae, has attracted much attention due to its broad health properties. However, its biosynthetic pathway and regulation mechanisms have rarely been reported which hinders the improvement of production efficiency. Therefore, this study aims to identify key metabolic nodes in the chrysolaminarin biosynthetic pathway. A phosphoglucomutase (PGM) in the model microalga Phaeodactylum tricornutum, revealing its critical role in chrysolaminarin biosynthesis is identified. PGM overexpression significantly elevates chrysolaminarin content by 2.54-fold and reaches 25.6% of cell dry weight; while algal growth and photosynthesis are not impaired. Besides, PGM overexpression up- and down-regulates the expression of chrysolaminarin and lipid biosynthetic genes, respectively. Microscopic analysis of aniline blue stained cells reveals that overproduced chrysolaminarin localized predominantly in vacuoles. Lipidomic analyses reveal that PGM overexpression significantly reduces the lipid content. The findings reveal the critical role of PGM in regulating the carbon flux between carbohydrate and lipid biosynthesis in microalgae, and provide a promising candidate for high efficiency production of chrysolaminarin.


Assuntos
Ciclo do Carbono/fisiologia , Carbono/metabolismo , Microalgas/metabolismo , Fosfoglucomutase/metabolismo , Polímeros/metabolismo , Vias Biossintéticas/fisiologia , Regulação para Baixo/fisiologia , Metabolismo dos Lipídeos/fisiologia , Lipídeos/química , Fotossíntese/fisiologia , Polissacarídeos/metabolismo , Regulação para Cima/fisiologia
20.
Biotechnol Biofuels ; 11: 318, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30479663

RESUMO

Background: Metabolic engineering has emerged as a potential strategy for improving microalgal lipid content through targeted changes to lipid metabolic networks. However, the intricate nature of lipogenesis has impeded metabolic engineering. Therefore, it is very important to identify the crucial metabolic nodes and develop strategies to exploit multiple genes for transgenesis. In an attempt to unravel the microalgal triacylglycerol (TAG) pathway, we overexpressed two key lipogenic genes, glycerol-3-phosphate acyltransferase (GPAT1) and lysophosphatidic acid acyltransferase (LPAT1), in oleaginous Phaeodactylum tricornutum and determined their roles in microalgal lipogenesis. Results: Engineered P. tricornutum strains showed enhanced growth and photosynthetic efficiency compared with that of the wild-type during the growth phase of the cultivation period. However, both the cell types reached stationary phase on day 7. Overexpression of GPAT1 and LPAT1 increased the TAG content by 2.3-fold under nitrogen-replete conditions without compromising cell growth, and they also orchestrated the expression of other key genes involved in TAG synthesis. The transgenic expression of GPAT1 and LPAT1 influenced the expression of malic enzyme and glucose 6-phosphate dehydrogenase, which enhanced the levels of lipogenic NADPH in the transgenic lines. In addition, GPAT1 and LPAT1 preferred C16 over C18 at the sn-2 position of the glycerol backbone. Conclusion: Overexpression of GPAT1 together with LPAT1 significantly enhanced lipid content without affecting growth and photosynthetic efficiency, and they orchestrated the expression of other key photosynthetic and lipogenic genes. The lipid profile for elevated fatty acid content (C16-CoA) demonstrated the involvement of the prokaryotic TAG pathway in marine diatoms. The results suggested that engineering dual metabolic nodes should be possible in microalgal lipid metabolism. This study also provides the first demonstration of the role of the prokaryotic TAG biosynthetic pathway in lipid overproduction and indicates that the fatty acid profile can be tailored to improve lipid production.

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