RESUMEN
We investigated the influence of 40 days of drought on growth, storage processes and primary photosynthetic processes in 3-month-old Scots pine and Norway spruce seedlings growing in perlite culture. Water stress significantly affected seedling water status, whereas absolute dry biomass growth was not substantially influenced. Water stress induced an increase in non-structural carbohydrate content (sugars, sugar alcohols, starch) in the aboveground part of pine seedlings in contrast to spruce seedlings. Due to the relatively low content of sugars and sugar alcohols in seedling organs, their expected contribution to osmotic potential changes was quite low. In contrast to biomass accumulation and storage, photosynthetic primary processes were substantially influenced by water shortage. In spruce seedlings, PSII was more sensitive to water stress than PSI. In particular, electron transport in PSI was stable under water stress despite the substantial decrease of electron transport in PSII. The increase in thermal energy dissipation due to enhancement of non-photochemical quenching (NPQ) was evident in both species under water stress. Simultaneously, the yields of non-regulated energy dissipation in PSII were decreased in pine seedlings under drought. A relationship between growth, photosynthetic activities and storage processes is analysed under weak water deficit.
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Fotosíntesis/fisiología , Picea/crecimiento & desarrollo , Picea/fisiología , Plantones/crecimiento & desarrollo , Plantones/fisiología , Fotoquímica , Complejo de Proteína del Fotosistema I/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , AguaRESUMEN
The inability to inspect metabolic activities within distinct subcellular compartments has been a major barrier to our understanding of eukaryotic cell metabolism. Previous work addressed this challenge by analyzing metabolism in isolated organelles, which grossly bias metabolic activity. Here, we describe a method for inferring physiological metabolic fluxes and metabolite concentrations in mitochondria and cytosol based on isotope tracing experiments performed with intact cells. This is made possible by computational deconvolution of metabolite isotopic labeling patterns and concentrations into cytosolic and mitochondrial counterparts, coupled with metabolic and thermodynamic modelling. Our approach lowers the uncertainty regarding compartmentalized fluxes and concentrations by one and three orders of magnitude compared to existing modelling approaches, respectively. We derive a quantitative view of mitochondrial and cytosolic metabolic activities in central carbon metabolism across cultured cell lines without performing cell fractionation, finding major variability in compartmentalized malate-aspartate shuttle fluxes. We expect our approach for inferring metabolism at a subcellular resolution to be instrumental for a variety of studies of metabolic dysfunction in human disease and for bioengineering.
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Respiración de la Célula , Mitocondrias , Humanos , Citosol/metabolismo , Mitocondrias/metabolismo , Línea Celular , Isótopos/metabolismo , Marcaje IsotópicoRESUMEN
Metastases are often the direct cause of death from pancreatic ductal adenocarcinoma (PDAC). The role of genomic alterations (GA) in mediating tropism and metastasis formation by PDAC cells is currently unknown. We aimed to identify GAs predisposing colonization of PDAC cells to the liver and decipher mechanisms enabling this process. In order to reveal specific genes, we studied the frequency of GA in 8,880 local and 7,983 metastatic PDAC samples. We observed differential pattern of GA in the local tumor and specific metastatic sites, with liver metastases characterized by deletion of CDKN2A/B (encoding p16/p15, respectively). The role of CDKN2A/B in promoting liver metastasis was evidenced by enhanced tumorigenic phenotype of p15/p16-deleted PDAC cells when exposed to hepatocytes conditioned media. The liver is characterized by high-ammonia low-glutamine environment and transcriptomic assays indicated unique adaptation of PDAC cells to these conditions, including regulation of genes leading to reduced glutaminolysis, like overexpression of GLUL and reduction in GLS2. Furthermore, metabolic assays indicated an increase in glutamate derived from [U-13C]-glucose in p15/p16-deleted cells. Importantly, these cells thrived under high ammonia condition. These data suggest a unique role for genomic alterations in mediating tropism of PDAC. Among these alterations, p15/16 deletion was identified as a promoter of liver metastases. Further studies indicated a unique role for p15/16 in regulating glutaminolysis. These findings reveal vulnerabilities in PDAC cells, which may pave the way for the development of novel therapeutic strategies aiming at the prevention of liver metastases formation.
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Adenocarcinoma , Carcinoma Ductal Pancreático , Neoplasias Hepáticas , Neoplasias Pancreáticas , Amoníaco/uso terapéutico , Carcinoma Ductal Pancreático/patología , Inhibidor p15 de las Quinasas Dependientes de la Ciclina , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Pancreáticas/metabolismo , Tropismo , Neoplasias PancreáticasRESUMEN
The folic acid cycle mediates the transfer of one-carbon (1C) units to support nucleotide biosynthesis. While the importance of serine as a mitochondrial and cytosolic donor of folate-mediated 1C units in cancer cells has been thoroughly investigated, a potential role of glycine oxidation remains unclear. We developed an approach for quantifying mitochondrial glycine cleavage system (GCS) flux by combining stable and radioactive isotope tracing with computational flux decomposition. We find high GCS flux in hepatocellular carcinoma (HCC), supporting nucleotide biosynthesis. Surprisingly, other than supplying 1C units, we found that GCS is important for maintaining protein lipoylation and mitochondrial activity. Genetic silencing of glycine decarboxylase inhibits the lipoylation and activity of pyruvate dehydrogenase and impairs tumor growth, suggesting a novel drug target for HCC. Considering the physiological role of liver glycine cleavage, our results support the notion that tissue of origin plays an important role in tumor-specific metabolic rewiring.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Ácido Fólico/metabolismo , Glicina/metabolismo , Glicina-Deshidrogenasa (Descarboxilante)/metabolismo , Humanos , Lipoilación/genética , Proteínas Mitocondriales/metabolismo , Nucleótidos/metabolismoRESUMEN
Folate metabolism supplies one-carbon (1C) units for biosynthesis and methylation and has long been a target for cancer chemotherapy. Mitochondrial serine catabolism is considered the sole contributor of folate-mediated 1C units in proliferating cancer cells. Here, we show that under physiological folate levels in the cell environment, cytosolic serine-hydroxymethyltransferase (SHMT1) is the predominant source of 1C units in a variety of cancers, while mitochondrial 1C flux is overly repressed. Tumor-specific reliance on cytosolic 1C flux is associated with poor capacity to retain intracellular folates, which is determined by the expression of SLC19A1, which encodes the reduced folate carrier (RFC). We show that silencing SHMT1 in cells with low RFC expression impairs pyrimidine biosynthesis and tumor growth in vivo. Overall, our findings reveal major diversity in cancer cell utilization of the cytosolic versus mitochondrial folate cycle across tumors and SLC19A1 expression as a marker for increased reliance on SHMT1.
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Citosol/metabolismo , Ácido Fólico/metabolismo , Glicina Hidroximetiltransferasa/genética , Mitocondrias/metabolismo , Neoplasias/metabolismo , Proteína Portadora de Folato Reducido/genética , Animales , Sistemas CRISPR-Cas/genética , Ciclo del Carbono/genética , Línea Celular , Ácido Fólico/genética , Glicina Hidroximetiltransferasa/deficiencia , Glicina Hidroximetiltransferasa/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Neoplasias/patología , Proteína Portadora de Folato Reducido/metabolismoRESUMEN
Mass spectrometry based metabolomics is a widely used approach in biomedical research. However, current methods coupling mass spectrometry with chromatography are time-consuming and not suitable for high-throughput analysis of thousands of samples. An alternative approach is flow-injection mass spectrometry (FI-MS) in which samples are directly injected to the ionization source. Here, we show that the sensitivity of Orbitrap FI-MS metabolomics methods is limited by ion competition effect. We describe an approach for overcoming this effect by analyzing the distribution of ion m/z values and computationally determining a series of optimal scan ranges. This enables reproducible detection of ~9,000 and ~10,000 m/z features in metabolomics and lipidomics analysis of serum samples, respectively, with a sample scan time of ~15 s and duty time of ~30 s; a ~50% increase versus current spectral-stitching FI-MS. This approach facilitates high-throughput metabolomics for a variety of applications, including biomarker discovery and functional genomics screens.
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Análisis de Inyección de Flujo/métodos , Espectrometría de Masas/métodos , Metabolómica/métodos , Línea Celular Tumoral , Análisis de Inyección de Flujo/normas , Ensayos Analíticos de Alto Rendimiento , Humanos , Iones/química , Lipidómica/métodos , Espectrometría de Masas/normas , Metabolómica/normas , Suero/química , Suero/metabolismoRESUMEN
In this research an accumulation of nerve agent markers in garden cress (Lepidium sativum) as a model plant object was studied using LC-QTOF hybrid system. For the determination of methylphosphonic acid and alkyl methylphosphonates, which are specific markers of sarin, soman, VR and VX, simple and sensitive approach was developed. Direct analysis of aqueous extracts on the reversed phase column with polar endcapping allowed to achieve satisfactory retention factor for methylphosphonic acid, which has high polarity and is usually very weakly retained on the ordinary reversed phase columns. Application of the QTOF mass spectrometer with high mass resolution led to the increase in the accuracy of the conducted measurements. The HPLC-HRMS technique developed exclusively for this study has been validated for linearity, limit of detection, limit of quantification, precision, accuracy and matrix effect prior to the analysis of plant extract samples. Hydroponic growth model was employed to examine accumulation of nerve agent markers in garden cress. It was found that after elimination of nerve agent markers from the plant growth medium, garden cress was able to store these substances for at least 5 weeks providing high retrospectivity of the analysis. Moreover, during the cress growth, no metabolization of alkyl methylphosphonates was observed. This allows not only to reveal the fact of nerve agents release into environment, but also to define its type after a long period of time.
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Biomarcadores Ambientales , Monitoreo del Ambiente/métodos , Lepidium sativum/química , Agentes Nerviosos/análisis , Cromatografía Líquida de Alta Presión , Lepidium sativum/metabolismo , Agentes Nerviosos/metabolismo , Compuestos Organofosforados/análisis , Extractos Vegetales/químicaRESUMEN
In this paper, the ultrasound assisted extraction method for isolation of steroidal glycosides from D. deltoidea plant cell suspension culture with a subsequent HPLC-MS determination was developed. After the organic solvent was selected via a two-factor experiment the optimization via Latin Square 4â¯×â¯4 experimental design was carried out for the following parameters: extraction time, organic solvent concentration in extraction solution and the ratio of solvent to sample. It was also shown that the ultrasound assisted extraction method is not suitable for isolation of steroidal glycosides from the D. deltoidea plant material. The results were double-checked using the multiple successive extraction method and refluxing extraction. Optimal conditions for the extraction of steroidal glycosides by the ultrasound assisted extraction method were: extraction time, 60â¯min; acetonitrile (water) concentration in extraction solution, 50%; the ratio of solvent to sample, 400â¯mL/g. Also, the developed method was tested on D. deltoidea cell suspension cultures of different terms and conditions of cultivation. The completeness of the extraction was confirmed using the multiple successive extraction method.