RESUMEN
Plant cystatins are involved in the regulation of protein turnover and play important roles in defense mechanisms. We cloned the ApCystatin gene from Agapanthus praecox ssp. orientalis, a famous ornamental and medical plant. The complete cDNA sequence of ApCystatin is comprised of 1439 nucleotides with a 423 bp ORF encoding 140 amino acids. The mRNA level of ApCystatin was significantly up-regulated under various abiotic stress, such as salt, osmosis, oxidative and cold stresses, which suggested that ApCystatin participated in the plant's resistance to stress. The recombinant ApCystatin fusion protein expressed in E. coli transetta (DE3) cells was approximate 18â¯kDa. 25⯵g of ApCystatin inhibited more than 95% activity of papain, suggesting ApCystatin as a papain-like protease inhibitor. As an exogenous substance, 1.60⯵g/mL ApCystatin protein improved the regrowth percentage of Arabidopsis 60-h seedlings after cryopreservation from 30% to 47%. In addition, the relative survival rate of A. praecox embryogenic callus after cryopreservation also increased for 30% with addition of 1.20⯵g/mL ApCystatin protein. This indicated that ApCystatin performed protective property against cryoinjury to Arabidopsis 60-h seedlings and A. praecox embryogenic callus during cryopreservation. Under various abiotic stress conditions, the recombinant ApCystatin protein showed significant advantage in growth rates at NaCl, mannitol, PEG6000, cold, acidic and alkaline conditions, compared to control. In conclusion, ApCystatin as a new member of plant cystatins exhibited protective property against cryoinjury in plant cryopreservation and abiotic stress in E. coli.
Asunto(s)
Amaryllidaceae/genética , Crioprotectores/farmacología , Cistatinas/genética , Estrés Fisiológico/efectos de los fármacos , Secuencia de Aminoácidos , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Secuencia de Bases , Clonación Molecular , Criopreservación , Cistatinas/farmacología , Inhibidores de Cisteína Proteinasa/farmacología , ADN Complementario/genética , Escherichia coli , Regulación de la Expresión Génica de las Plantas , Vectores Genéticos , Proteínas de Plantas/genética , Proteínas de Plantas/farmacología , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologíaRESUMEN
Aims: To investigate the white matter (WM) integrity and hippocampal functional connectivity (FC) in type 2 diabetes mellitus (T2DM) patients without mild cognitive impairment (MCI) by using diffusion tensor imaging (DTI) and resting-state functional magnetic resonance imaging (rs-fMRI), respectively. Methods: Twelve T2DM patients without MCI and 24 age, sex and education matched healthy controls (HC) were recruited. DTI and rs-fMRI data were subsequently acquired on a 3.0T MR scanner. Tract-based spatial statistics (TBSS) combining region of interests (ROIs) analysis was used to investigate the alterations of DTI metrics (fractional anisotropy (FA), mean diffusivity (MD), λ1 and λ23) and FC measurement was performed to calculate hippocampal FC with other brain regions. Cognitive function was evaluated by using Mini-Mental State Examination (MMSE) and Montreal Cognitive Assessment (MoCA). Brain volumes were also evaluated among these participants. Results: There were no difference of MMSE and MoCA scores between two groups. Neither whole brain nor regional brain volume decrease was revealed in T2DM patients without MCI. DTI analysis revealed extensive WM disruptions, especially in the body of corpus callosum (CC). Significant decreases of hippocampal FC with certain brain structures were revealed, especially with the bilateral frontal cortex. Furthermore, the decreased FA in left posterior thalamic radiation (PTR) and increased MD in the splenium of CC were closely related with the decreased hippocampal FC to caudate nucleus and frontal cortex. Conclusions: T2DM patients without MCI showed extensive WM disruptions and abnormal hippocampal FC. Moreover, the WM disruptions and abnormal hippocampal FC were closely associated. Highlights -T2DM patients without MCI demonstrated no obvious brain volume decrease.-Extensive white matter disruptions, especially within the body of corpus callosum, were revealed with DTI analysis among the T2DM patients.-Despite no MCI in T2DM patients, decreased functional connectivity between hippocampal region and some critical brain regions were detected.-The alterations in hippocampal functional connectivity were closely associated with those of the white matter structures in T2DM patients. This trial was registered to ClinicalTrials.gov (NCT02420470, https://www.clinicaltrials.gov/).
RESUMEN
We analyzed topology of brain functional networks in type 2 diabetes mellitus (T2DM) patients without mild cognitive impairment. We recruited T2DM patients without mild cognitive impairment (4 males and 8 females) and healthy control subjects (8 males and 16 females) to undergo cognitive testing and resting-state functional magnetic resonance imaging. Graph theoretical analysis of functional brain networks revealed abnormal small-world architecture in T2DM patients as compared to control subjects. The functional brain networks of T2DM patients showed increased path length, decreased global efficiency and disrupted long-distance connections. Moreover, reduced nodal characteristics were distributed in the frontal, parietal and temporal lobes, while increased nodal characteristics were distributed in the frontal, occipital lobes, and basal ganglia in the T2DM patients. The disrupted topological properties correlated with cognitive performance of T2DM patients. These findings demonstrate altered topological organization of functional brain networks in T2DM patients without mild cognitive impairment.
RESUMEN
Network analysis has been widely used in studying Alzheimer's disease (AD). However, how the white matter network changes in cognitive impaired patients with subjective cognitive decline (SCD) (a symptom emerging during early stage of AD) and amnestic mild cognitive impairment (aMCI) (a pre-dementia stage of AD) is still unclear. Here, structural networks were constructed respectively based on FA and FN for 36 normal controls, 21 SCD patients, and 33 aMCI patients by diffusion tensor imaging and graph theory. Significantly lower efficiency was found in aMCI patients than normal controls (NC). Though not significant, the values in those with SCD were intermediate between aMCI and NC. In addition, our results showed significantly altered betweenness centrality located in right precuneus, calcarine, putamen, and left anterior cingulate in aMCI patients. Furthermore, association was found between network metrics and cognitive impairment. Our study suggests that the structural network properties might be preserved in SCD stage and disrupted in aMCI stage, which may provide novel insights into pathological mechanisms of AD.
Asunto(s)
Trastornos del Conocimiento/patología , Disfunción Cognitiva/patología , Sustancia Blanca/patología , Anciano , Mapeo Encefálico , Estudios de Casos y Controles , Imagen de Difusión Tensora , Femenino , Humanos , Procesamiento de Imagen Asistido por Computador , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Modelos BiológicosRESUMEN
BACKGROUND: The ε4 allele of the Apolipoprotein E gene (APOE-ε4) is a potent genetic risk factor for sporadic Alzheimer's disease (AD). Amnestic mild cognitive impairment (aMCI) is an intermediate state between normal cognitive aging and dementia, which is easy to convert to AD dementia. It is an urgent problem in the field of cognitive neuroscience to reveal the conversion of aMCI-ε4 to AD. Based on our preliminary work, we will study the neuroimaging features in the special group of aMCI-ε4 with multi-modality magnetic resonance imaging (structural MRI, resting state-fMRI and diffusion tensor imaging) longitudinally. METHODS/DESIGN: In this study, 200 right-handed subjects who are diagnosed as aMCI with APOE-ε4 will be recruited at the memory clinic of the Neurology Department, XuanWu Hospital, Capital Medical University, Beijing, China. All subjects will undergo the neuroimaging and neuropsychological evaluation at a 1 year-interval for 3 years. The primary outcome measures are 1) Microstructural alterations revealed with multimodal MRI scans including structure MRI (sMRI), resting state functional MRI (rs-fMRI), diffusion tensor imaging (DTI); 2) neuropsychological evaluation, including the World Health Organization-University of California-LosAngeles Auditory Verbal Learning Test (WHO-UCLA AVLT), Addenbrook's cognitive examination-revised (ACE-R), mini-mental state examination (MMSE), Montreal Cognitive Assessment (MoCA), Clinical Dementia Rating scale (CDR). DISCUSSION: This study is to find out the neuroimaging biomarker and the changing laws of the marker during the progress of aMCI-ε4 to AD, and the final purpose is to provide scientific evidence for new prevention, diagnosis and treatment of AD. TRIAL REGISTRATION: This study has been registered to ClinicalTrials.gov (NCT02225964, https://www.clinicaltrials.gov/ ) in August 24, 2014.
Asunto(s)
Enfermedad de Alzheimer/diagnóstico por imagen , Disfunción Cognitiva/diagnóstico por imagen , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/psicología , Apolipoproteína E4/genética , Biomarcadores , China , Disfunción Cognitiva/genética , Disfunción Cognitiva/psicología , Imagen de Difusión Tensora/métodos , Progresión de la Enfermedad , Estudios de Seguimiento , Neuroimagen Funcional , Humanos , Procesamiento de Imagen Asistido por Computador , Imagen por Resonancia Magnética , Pruebas Neuropsicológicas , Estudios ProspectivosRESUMEN
KEY MESSAGE: Elevated antioxidant status and positive abiotic stress response in dehydration enhance cell resistance to cryoinjury, and controlling oxidative damage via reactive oxygen species homeostasis maintenance leads to high survival. Cryoprotectants are important for cell survival in cryopreservation, but high concentrations can also cause oxidative stress. Adding vitamin C to the cryoprotectant doubled the survival ratio in Arabidopsis thaliana 60-h seedlings (seedlings after 60-h germination) cryopreservation. In this study, the metabolites and transcriptional profiling of 60-h seedlings were analyzed in both the control cryopreservation procedure (CCP) and an improved cryopreservation procedure (ICP) to reveal the mechanism of plant cell response to oxidative stress from cryopreservation. Reactive oxygen species (ROS) and peroxidation levels reached a peak after rapid cooling-warming in CCP, which were higher than that in ICP. In addition, gene regulation was significantly increased in CCP and decreased in ICP during rapid cooling-warming. Before cryogenic treatment, the number of specifically regulated genes was nearly 10 times higher in ICP dehydration than CCP dehydration. Among these genes, DREBs/CBFs were beneficial to cope with cryoinjury, and calcium-binding protein, OXI1, WRKY and MYB family members as key factors in ROS signal transduction activated the ROS-producing and ROS-scavenging networks including AsA-GSH and GPX cycles involved in scavenging H2O2. Finally, elevated antioxidant status and oxidative stress response in the improved dehydration enhanced seedling resistance to cryogenic treatment, maintained ROS homeostasis and improved cell recovery after cryopreservation.
Asunto(s)
Arabidopsis/genética , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Plantones/genética , Transcriptoma , Antioxidantes/metabolismo , Arabidopsis/fisiología , Ácido Ascórbico/metabolismo , Criopreservación , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Peróxido de Hidrógeno/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Plantones/fisiología , Estrés FisiológicoRESUMEN
KEY MESSAGE: Oxidative stress and apoptosis-like programmed cell death, induced in part by H 2 O 2 , are two key factors that damage cells during plant cryopreservation. Their inhibition can improve cell viability. We hypothesized that oxidative stress and apoptosis-like event induced by ROS seriously impact plant cell viability during cryopreservation. This study documented changes in cell morphology and ultrastructure, and detected dynamic changes in ROS components (O 2 (·-) , H2O2 and OH·), antioxidant systems, and programmed cell death (PCD) events during embryonic callus cryopreservation of Agapanthus praecox. Plasmolysis, organelle ultrastructure changes, and increases in malondialdehyde (a membrane lipid peroxidation product) suggested that oxidative damage and PCD events occurred at several early cryopreservation steps. PCD events including autophagy, apoptosis-like, and necrosis also occurred at later stages of cryopreservation, and most were apoptosis. H2O2 is the most important ROS molecule mediating oxidative damage and affecting cell viability, and catalase and AsA-GSH cycle are involved in scavenging the intracellular H2O2 and protecting the cells against stress damage in the whole process. Gene expression studies verified changes of antioxidant system and PCD-related genes at the main steps of the cryopreservation process that correlated with improved cell viability. Reducing oxidative stress or inhibition of apoptosis-like event by deactivating proteases improved cryopreserved cell viability from 49.14 to 86.85 % and 89.91 %, respectively. These results verify our model of ROS-induced oxidative stress and apoptosis-like event in plant cryopreservation. This study provided a novel insight into cell stress response mechanisms in cryopreservation.
Asunto(s)
Apoptosis/efectos de los fármacos , Criopreservación , Liliaceae/citología , Liliaceae/embriología , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/toxicidad , Semillas/citología , Antioxidantes/metabolismo , Autofagia/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Etiquetado Corte-Fin in Situ , Liliaceae/genética , Liliaceae/ultraestructura , Peroxidación de Lípido/efectos de los fármacos , Malondialdehído/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Semillas/efectos de los fármacos , Semillas/ultraestructuraRESUMEN
Plant recovery status after cryopreservation by vitrification had a negative relationship to the oxidative stress induced by reactive oxygen species (ROS). Arabidopsis thaliana seedlings germinated for 48 h or 72 h with different survival tolerances were examined at five steps of cryopreservation, to determine the role of ROS (O2(-), H2O2 and OH) and antioxidant systems (SOD, POD, CAT, AsA and GSH) in cryo-injury. In addition, the effects of the steps on membrane lipid peroxidation were studied using malondialdehyde (MDA) as an indicator. The results indicated that H2O2-induced oxidative stress at the steps of dehydration and rapid warming was the main cause of cryo-injury of 48-h seedlings (high survival rate) and 72-h seedlings (no survival). The H2O2 was mainly generated in cotyledons, shoot tips and roots of seedlings as indicated by Amplex Red staining. Low survival of 72-h seedlings was associated with severe membrane lipid peroxidation, which was caused by increased OH generation activity and decreased SOD activity. The antioxidant-related gene expression by qRT-PCR and physiological assays suggested that the antioxidant system of 48-h seedlings were activated by ROS, and they mounted a defense against oxidative stress. A high level of ROS led to the weakening of the antioxidant system of 72-h seedlings. Correlation analysis indicated that enhanced antioxidant enzymes activities contributed to the high survival rate of 48-h seedlings, which could reflect by cryopreservation of antioxidant mutant seedlings. This model system indicated that elevated CAT activity and AsA content were determinants of cryogenic stress tolerance, whose manipulation could improve the recovery of seedlings after cryopreservation.
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Arabidopsis/fisiología , Criopreservación , Estrés Oxidativo/fisiología , Especies Reactivas de Oxígeno/metabolismo , Catalasa/metabolismo , Membrana Celular/patología , Cotiledón/metabolismo , Calor , Peróxido de Hidrógeno/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Malondialdehído/metabolismo , Raíces de Plantas/metabolismo , Brotes de la Planta/metabolismo , Plantones/fisiología , Superóxido Dismutasa/metabolismo , VitrificaciónRESUMEN
Eighteen strains of thraustochytrids were newly isolated from Hong Kong mangroves, and their fatty acid and squalene contents were analyzed. All strains could grow well heterotrophically with glucose as the sole carbon source. All of them had the typical fatty acid profile of thraustchytrids and could produce a large amount of docosahexaenoic acid. The cell dry weight ranged from 5.49 to 15.62 g/L and squalene content from 0.02 to 0.18 mg/g at 72 h. The highest squalene-producing strain, BR-MP4-A1 was identified as a new strain of Aurantiochytrium species through sequence comparison of the 18S rRNA gene. The highest biomass of Aurantiochytrium sp. BR-MP4-A1 was achieved at 72 h, whereas its squalene content reached the maximum of 0.567 mg/g at 36 h but decreased rapidly thereafter. The production of squalene by thraustochytrids might be highly influenced by culture conditions.
Asunto(s)
Escualeno/análisis , Estramenopilos/química , ADN/análisis , Ácidos Docosahexaenoicos/análisis , Ácidos Grasos/análisis , Hong Kong , Filogenia , ARN Ribosómico 18S/genética , Escualeno/metabolismo , Estramenopilos/genética , Estramenopilos/crecimiento & desarrolloRESUMEN
Although phosphatidylserine synthase (PSS) from Escherichia coli is an ideal enzyme for phospholipid production, its application in the food industry has been limited because of the low PSS yield. In this study, the pss gene was cloned from E. coli K(12) and expressed in Bacillus subtilis DB104, and the recombinant PSS was characterized subsequently. PSS was purified to 39.59-fold, and the highest activity was detected as 13.62 U/mg. The enzyme was found to be stable in a pH range of 6.5-9.5, with optimal pH values of 8.0 for hydrolysis and 7.0 for transphosphatidylation, respectively. The optimal temperature for PSS activity was 35 degrees C. The enzyme activity could be detected after 1 h of heating at 65 degrees C. Among the detected detergents and metal ions, Triton X-100, Ca(2+), Mn(2+), and Co(2+) could improve PSS activity. The transformation of phosphatidylcholine to phosphatidylserine under PSS catalyzation was carried out in a biphasic system, which confirmed the actual catalyzing ability of the recombinant protein.
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Bacillus subtilis/genética , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/genética , Escherichia coli K12/enzimología , Expresión Génica , Secuencia de Aminoácidos , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/aislamiento & purificación , Clonación Molecular , Estabilidad de Enzimas , Calor , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Transformación BacterianaRESUMEN
Natamycin has been widely used as a natural preservative to prevent mold contamination in food. In this study, statistically based experimental designs were employed for the optimization of medium components for natamycin production by Streptomyces gilvosporeus. After glucose, yeast extract, and soy peptone were screened as suitable carbon and nitrogen sources, a full factorial design was used to evaluate the effects of various factors on natamycin production. Glucose and pH were identified as having significant effects (with confidence level >90%). Glucose concentration and initial pH were subsequently optimized by use of a central composite design. The result indicated that glucose and pH had a significant interactive effect on natamycin production. The optimal glucose concentration and initial pH value were 38.2 g/L and 7.8, respectively. This optimization strategy led to a natamycin yield of 2.45 g/L, which was nearly 90% higher than that in the original medium.
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Microbiología Industrial , Modelos Estadísticos , Natamicina/metabolismo , Streptomyces/metabolismo , Medios de Cultivo/química , Glucosa/metabolismo , Concentración de Iones de Hidrógeno , Streptomyces/crecimiento & desarrolloRESUMEN
The lipid composition and the distribution of fatty acids in the lipid pool were determined in eicosapentaenoic acid (EPA)-producing microalga (Nitzschia laevis) grown under different temperatures. Both the relative amounts of lipid classes and the degree of fatty acid unsaturation in various lipid species were not greatly changed under tested growth conditions. Higher temperature up to 23°C benefited the growth of N. laevis but only had a slight influence on EPA and lipid contents. Further increasing the culture temperature caused a serious inhibition of both the cell growth and fatty acid biosynthesis. Under all temperatures tested, triacylglycerol (TAG) was the predominant lipid constituent (64.5-69.1% of total lipid) and was highly saturated. Lower temperature favored the formation of polar lipids. The highest content of phosphatidylcholine (PC), the major phospholipids component, was reached at 15°C (10.9% of total lipid). In sharp contrast to TAG, PC was highly unsaturated and contained a higher amount of EPA under lower temperature. The highest EPA content in polar lipid was achieved at 19°C. The results from this investigation suggested that the low temperature could improve the distribution of polyunsaturated fatty acids in phospholipids, though it could not significantly influence their amount, especially in PC.
RESUMEN
The diatom Nitzschia laevis Hust. is a potential producer of eicosapentaenoic acid (EPA). To elucidate its cellular response to salt stress, the effects of salinity on EPA production, lipid composition, and fatty acid distribution in the lipid pool were investigated. The highest contents of total fatty acids, EPA, and polar lipids were all obtained at NaCl of 20 g · L(-1) , under which 71.3% of total EPA existed in polar lipid fractions. In N. laevis, high salt concentration might induce the decrease in neutral lipids (NLs), whereas the production of polar lipids, including phospholipids (PLs) and glycolipids (GLs), was enhanced. The degree of fatty acid unsaturation of both neutral and polar lipid fractions increased sharply when NaCl concentration increased from 10 to 20 g · L(-1) but decreased at NaCl concentration of 30 g · L(-1) . The amount of total free sterols was increased with the increase in salt concentration. All these changes in lipid and fatty acids suggested a decrease in membrane permeability and fluidity under high salt concentration, which could help the alga acclimate to the salinity stress.
RESUMEN
Many phototrophic microorganisms contain large quantities of high-value products such as n-3 polyunsaturated fatty acids and carotenoids but phototrophic growth is often slow due to light limitation. Some phototrophic microorganisms can also grow on cheap organic substrate heterotrophically. Heterotrophic cultivation can be well controlled and provides the possibility to achieve fast growth and high yield of valuable products on a large scale. Several strategies have been investigated for cultivation of phototrophic microorganisms without light. These include trophic conversion of obligate photoautotrophic microorganisms by genetic engineering, development of efficient cultivation systems and optimization of culture conditions. This paper reviews recent advances in heterotrophic cultivation of phototrophic cells with an emphasis on microalgae.