ABSTRACT
Viral hemorrhagic septicemia virus (VHSV) is one of the most serious viral pathogen that infects farmed fish. In this study, we measured the replication of VHSV increased steadily at 9, 24, 72, and 120 h after infection and progression of necrosis was observed at 72 hpi. We performed transcriptomic and metabolomics profiling of kidney tissues collected at each infection time using Illumina HiSeq2000 and ultra-performance liquid chromatography/quadrupole time-of-flight mass spectroscopy to investigate the mechanisms of VHSV infection in the kidneys of olive flounder. A total of 13,862 mRNA molecules and 72 metabolites were selected to identify the mechanisms of infection and they were integrated using KEGG pathway database. Six KEGG metabolic pathways, including carbohydrate metabolism, amino acid metabolism, lipid metabolism, transport and catabolism, metabolism of cofactors and vitamins, and energy metabolism, were significantly suppressed, whereas the immune system was activated by VHSV infection. A decrease in levels of amino acids such as valine, leucine, and isoleucine, as well as in their derivative carnitines, was observed after VHSV infection. In addition, an increase in arachidonic acid level was noted. Integrated analysis of transcriptome and metabolome using KEGG pathway database revealed four types of responses in the kidneys of olive flounder to VHSV infection. Among these, the mechanisms related to the immune system and protein synthesis were activated, whereas ATP synthesis and the antioxidant system activity were suppressed. This is the first study describing the mechanisms of metabolic responses to VHSV infection in olive flounder. The results suggest that the suppression of ATP synthesis and antioxidant systems, such as glutathione and peroxisome signaling, could be the cause of necrosis, whereas the activation of the immune system could result in the inflammation of kidney tissue in VHSV-infected olive flounder.
Subject(s)
Fish Diseases/immunology , Flatfishes/genetics , Flatfishes/immunology , Hemorrhagic Septicemia, Viral/immunology , Metabolome/immunology , Transcriptome/immunology , Animals , Flatfishes/metabolism , Flounder/immunology , Kidney/immunology , Novirhabdovirus/physiology , Time FactorsABSTRACT
BACKGROUND & AIMS: Genomic instability promotes colon carcinogenesis by inducing genetic mutations, but not all genes affected by this process have been identified. We investigated whether genomic instability in human colorectal cancer (CRC) cells produces mutations in the hepatocyte growth factor (HGF) gene. METHODS: We genotyped human colon tumor tissues and adjacent nontumor tissues collected from 78 patients University of Pittsburgh Health Sciences and Veterans Hospital, along with 40 human CRC and adjacent nontumor tissues in a commercial microarray. We used cellular, biochemical, and molecular biological techniques to investigate the factors that alter HGF signaling in colon cancer cells and its effects on cell proliferation and survival. RESULTS: All tested human CRC tissues and cell lines that had microsatellite instability contained truncations in the regulatory deoxyadenosine tract element (DATE) of the HGF gene promoter. The DATE was unstable in 14% (11 of 78) of CRC samples; DATE truncation was also polymorphic and detected in 18% (13 of 78) of CRC tissues without microsatellite instability. In CRC cell lines, truncation of DATE activated expression of HGF, resulting in its autocrine signaling via MET. This promoted cell proliferation and resistance to necroptosis. HGF signaling via MET reduced levels of the receptor-interacting serine-threonine kinase 1, a mediator of necroptosis, in CRC cells. High levels of HGF protein in tumor tissues correlated with lower levels of receptor-interacting serine-threonine kinase 1 and shorter survival times of patients. CONCLUSIONS: Thirty-one percent of CRC samples contain alterations in the DATE of the HGF promoter. Disruption of the DATE increased HGF signaling via MET and reduced levels of receptor-interacting serine-threonine kinase 1 and CRC cell necroptosis. DATE alteration might be used as a prognostic factor or to select patients for therapies that target HGF-MET signaling.
Subject(s)
Adenocarcinoma/genetics , Apoptosis , Colonic Neoplasms/genetics , Genomic Instability , Hepatocyte Growth Factor/genetics , Transcriptional Activation , Adenocarcinoma/metabolism , Adenocarcinoma/mortality , Adenocarcinoma/pathology , Aged , Aged, 80 and over , Autocrine Communication , Cell Proliferation , Cell Survival , Colonic Neoplasms/metabolism , Colonic Neoplasms/mortality , Colonic Neoplasms/pathology , DNA Mismatch Repair , Female , Gene Expression Profiling/methods , Gene Expression Regulation, Neoplastic , Genetic Predisposition to Disease , HCT116 Cells , HT29 Cells , Hepatocyte Growth Factor/metabolism , Humans , Male , Microsatellite Instability , Middle Aged , Necrosis , Oligonucleotide Array Sequence Analysis , Phenotype , Prognosis , Promoter Regions, Genetic , Proto-Oncogene Proteins c-met/metabolism , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Signal Transduction , Time Factors , TransfectionABSTRACT
BACKGROUND: Rhizomes of Polygonatum species are commonly used as herbal supplements in Asia. They have different medicinal effects by species but have been misused and mixed owing to their similar taste and smell. Therefore accurate and reliable analytical methods to discriminate between Polygonatum species are required. RESULTS: In this study, global and targeted metabolite profiling using (1) H nuclear magnetic resonance ((1) H NMR) spectroscopy and ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS) was applied to discriminate between different Polygonatum species. Partial least squares discriminant analysis (PLS-DA) models were used to classify and predict species of Polygonatum. Cross-validation derived from PLS-DA revealed good predictive accuracy. Polygonatum species were classified into unique patterns based on K-means clustering analysis. 4-Hydrobenzoic acid and trigonelline were identified as novel marker compounds and quantified accurately. CONCLUSION: The results demonstrate that metabolite profiling approaches coupled with chemometric analysis can be used to classify and discriminate between different species of various herbal medicines. © 2015 Society of Chemical Industry.
Subject(s)
Dietary Supplements/analysis , Food Inspection/methods , Polygonatum/chemistry , Rhizome/chemistry , Alkaloids/analysis , Alkaloids/metabolism , Benzoates/analysis , Benzoates/metabolism , Biomarkers/analysis , Chromatography, High Pressure Liquid , Cluster Analysis , Dietary Supplements/standards , Discriminant Analysis , Ethnopharmacology , Least-Squares Analysis , Medicine, Korean Traditional , Nuclear Magnetic Resonance, Biomolecular , Pattern Recognition, Automated , Polygonatum/metabolism , Principal Component Analysis , Republic of Korea , Rhizome/metabolism , Species Specificity , Spectrometry, Mass, Electrospray Ionization , Tandem Mass SpectrometryABSTRACT
UNLABELLED: Met, the transmembrane tyrosine kinase receptor for hepatocyte growth factor (HGF), is known to function as a potent antiapoptotic mediator in normal and neoplastic cells. Herein we report that the intracellular cytoplasmic tail of Met has evolved to harbor a tandem pair of caspase-3 cleavage sites, which bait, trap, and disable the active site of caspase-3, thereby blocking the execution of apoptosis. We call this caspase-3 cleavage motif the Death Defying Domain (DDD). This site consists of the following sequence: DNAD-DEVD-T (where the hyphens denote caspase cleavage sites). Through functional and mechanistic studies, we show that upon DDD cleavage by caspase-3 the resulting DEVD-T peptide acts as a competitive inhibitor and entraps the active site of caspase-3 akin to DEVD-CHO, which is a potent, synthetic inhibitor of caspase-3 activity. By gain- and loss-of-function studies using restoration of DDD expression in DDD-deficient hepatocytic cells, we found that both caspase-3 sites in DDD are necessary for inhibition of caspase-3 and promotion of cell survival. Employing mutagenesis studies, we show that DDD could operate independently of Met's enzymatic activity as determined by using kinase-dead human Met mutant constructs. Studies of both human liver cancer tissues and cell lines uncovered that DDD cleavage and entrapment of caspase-3 by DDD occur in vivo, further proving that this site has physiological and pathophysiological relevance. CONCLUSION: Met can directly inhibit caspase-3 by way of a novel mechanism and promote hepatocyte survival. The results presented here will further our understanding of the mechanisms that control not only normal tissue homeostasis but also abnormal tissue growth such as cancer and degenerative diseases in which apoptotic caspases are at play.
Subject(s)
Apoptosis , Caspase 3/chemistry , Hepatocytes/physiology , Proto-Oncogene Proteins c-met/physiology , Amino Acid Sequence , Animals , Binding Sites , Caspase 3/physiology , Caspase Inhibitors/pharmacology , Cytoprotection , Humans , Mice , Molecular Sequence Data , Oligopeptides/pharmacology , Protein Structure, Tertiary , Proto-Oncogene Proteins c-met/chemistryABSTRACT
This study was designed to classify and identify closely related thistle species in the genus Cirsium, as well as Carduus and Cephalonoplos species, which are also thistles. The comprehensive and untargeted metabolite profiles of nine Korean thistles were determined using ultra high performance liquid chromatography combined with hybrid quadrupole time-of-flight mass spectrometry. The difference in metabolite profiles among species was explored using principal component analysis and hierarchical clustering analysis. The significantly different metabolites (Bonferroni-corrected P-value < 0.001) were used to construct a partial least squares discriminant analysis model to predict the species of thistle. Nine species were successfully classified using a partial least squares discriminant analysis model and confirmed using a cross-validation method. Species with similar features were grouped based on unique patterns in variable clusters. The present study suggests that liquid chromatography with quadrupole time-of-flight mass spectrometry untargeted metabolomic profiling with chemometric analysis is an efficient and powerful tool for discriminating between different species of medicinal herbs.
Subject(s)
Cirsium/chemistry , Chromatography, High Pressure Liquid , Cirsium/metabolism , Cluster Analysis , Discriminant Analysis , Mass Spectrometry , Principal Component Analysis , Republic of Korea , Time FactorsABSTRACT
Pb toxicity is linked to cardiovascular and nephrotoxicity issues. Exposure to this heavy metal can occur through food and drinking water. Therefore, this study aimed to evaluate Pb exposure and assess health risks in Korean adults using a physiologically based toxicokinetic (PBTK) model. Human blood Pb concentrations were monitored using the Korean National Environmental Health Survey (KoNEHS) Cycle 4. The average Pb exposure in Korean adults was 0.520 µg/kg bw/day. The PBTK results were compared with scenario-based results from the 2021 risk assessment report of five heavy metals, including Pb, conducted by the MFDS. Exposure determined through reverse dosimetry was approximately two times higher than scenario-based exposure (0.264 µg/kg bw/day). The higher exposure levels obtained during PBTK analysis may be attributed to sustained exposure within historically more contaminated living environments and the long half-life of Pb. These findings suggest that the PBTK-based method can quantify aggregated exposure levels in the body over time, potentially serving as a complementary tool to address the constraints of scenario-based assessment methods for integrated risk assessment. Moreover, this model is convenient and cost-effective compared with scenario-based exposure estimation. These findings can facilitate the application of model for tracking continuous national changes in hazardous substance levels.
Subject(s)
Biological Monitoring , Lead , Humans , Lead/blood , Lead/toxicity , Lead/pharmacokinetics , Republic of Korea , Adult , Male , Biological Monitoring/methods , Risk Assessment/methods , Toxicokinetics , Female , Middle Aged , Models, Biological , Environmental Exposure , Young Adult , Environmental Pollutants/toxicity , Environmental Pollutants/blood , Environmental Pollutants/pharmacokineticsABSTRACT
To investigate the effects of changes in extracellular osmolality on the function of kidney collecting duct cells, particularly on water and sodium reabsorption in the conditions of diuresis and antidiuresis, we generated transcriptome and metabolome profiles of primary cultured inner medullary collecting duct (IMCD) cells. They were grown in hyperosmolar culture medium (640 mOsm) for 4 days and then exposed to either reduced (300 mOsm) or same osmolality for 1 or 2 days more. Integrated analysis of the transcriptome and metabolome revealed that decreased extracellular osmolality was associated with decreased levels of organic osmolytes, glucose, intermediates of citric acid cycle, and branched-chain amino acids (BCAA) in IMCD cells, along with significantly decreased gene expression and protein abundance of P-type transporters (ATP1B1), ABC transporters (ABCC5 and ABCG1), and insulin signaling pathways (IRS2). Quantitative real-time RT-PCR and semiquantitative immunoblotting confirmed the changes of transcript levels of differentially expressed genes and protein levels. Taken together, integrated analysis of omics data demonstrated that water and sodium reabsorption could be reduced by decreased extracellular osmolality per se, through decreased levels of ABC transporters and IRS2, which play a potential role in the transport of organic osmolytes, BCAA, glucose, and trafficking of epithelial sodium channel.
Subject(s)
Kidney Tubules, Collecting/metabolism , Metabolome , Transcriptome , Animals , Cells, Cultured , Cluster Analysis , Culture Media , Gene Expression Profiling , Gene Expression Regulation , Kidney Tubules, Collecting/cytology , Kidney Tubules, Collecting/physiology , Magnetic Resonance Spectroscopy , Male , Metabolic Networks and Pathways , Multivariate Analysis , Osmolar Concentration , Pattern Recognition, Automated , Principal Component Analysis , Proteome/genetics , Proteome/metabolism , Rats , Rats, Sprague-Dawley , Real-Time Polymerase Chain Reaction , Salt Tolerance , Statistics, NonparametricABSTRACT
Many aspects of plant metabolism that are involved in plant growth and development are influenced by light-regulated diurnal rhythms as well as endogenous clock-regulated circadian rhythms. To identify the rhythmic proteins in rice, periodically grown (12h light/12h dark cycle) seedlings were harvested for three days at six-hour intervals. Continuous dark-adapted plants were also harvested for two days. Among approximately 3000 reproducible protein spots on each gel, proteomic analysis ascertained 354 spots (~12%) as light-regulated rhythmic proteins, in which 53 spots showed prolonged rhythm under continuous dark conditions. Of these 354 ascertained rhythmic protein spots, 74 diurnal spots and 10 prolonged rhythmic spots under continuous dark were identified by MALDI-TOF MS analysis. The rhythmic proteins were functionally classified into photosynthesis, central metabolism, protein synthesis, nitrogen metabolism, stress resistance, signal transduction and unknown. Comparative analysis of our proteomic data with the public microarray database (the Plant DIURNAL Project) and RT-PCR analysis of rhythmic proteins showed differences in rhythmic expression phases between mRNA and protein, suggesting that the clock-regulated proteins in rice are modulated by not only transcriptional but also post-transcriptional, translational, and/or post-translational processes.
Subject(s)
Circadian Rhythm , Oryza/metabolism , Plant Proteins/metabolism , Proteomics/methods , Seedlings/metabolism , Circadian Rhythm/genetics , Darkness , Electrophoresis, Gel, Two-Dimensional , Gene Expression Profiling , Gene Expression Regulation, Plant , Oligonucleotide Array Sequence Analysis , Oryza/genetics , Plant Proteins/genetics , Proteome/genetics , Proteome/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Seedlings/geneticsABSTRACT
A thiol-reactive membrane-associated protein (TRAP) binds covalently to the cytoplasmic domain of the human insulin receptor (IR) beta-subunit when cells are treated with the homobifunctional cross-linker reagent 1,6-bismaleimidohexane. Here, TRAP was found to be phospholipase C gamma1 (PLCgamma1) by mass spectrometry analysis. PLCgamma1 associated with the IR both in cultured cell lines and in a primary culture of rat hepatocytes. Insulin increased PLCgamma1 tyrosine phosphorylation at Tyr-783 and its colocalization with the IR in punctated structures enriched in cortical actin at the dorsal plasma membrane. This association was found to be independent of PLCgamma1 Src homology 2 domains, and instead required the pleckstrin homology (PH)-EF-hand domain. Expression of the PH-EF construct blocked endogenous PLCgamma1 binding to the IR and inhibited insulin-dependent phosphorylation of mitogen-activated protein kinase (MAPK), but not AKT. Silencing PLCgamma1 expression using small interfering RNA markedly reduced insulin-dependent MAPK regulation in HepG2 cells. Conversely, reconstitution of PLCgamma1 in PLCgamma1-/- fibroblasts improved MAPK activation by insulin. Our results show that PLCgamma1 is a thiol-reactive protein whose association with the IR could contribute to the activation of MAPK signaling by insulin.
Subject(s)
Blood Proteins/metabolism , Phosphoproteins/metabolism , Receptor, Insulin/metabolism , Type C Phospholipases/chemistry , Type C Phospholipases/metabolism , Animals , Blood Platelets/metabolism , CHO Cells , Cell Membrane/metabolism , Cells, Cultured , Cricetinae , Cross-Linking Reagents/pharmacology , Enzyme Activation , Fibroblasts/enzymology , Fibroblasts/metabolism , Hepatocytes/metabolism , Humans , Insulin/pharmacology , Male , Maleimides/pharmacology , Mice , Mice, Knockout , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinases/drug effects , Mitogen-Activated Protein Kinases/metabolism , Molecular Weight , Phospholipase C gamma , Phosphorylation , RNA, Small Interfering/pharmacology , Rats , Rats, Inbred F344 , Receptor, Insulin/antagonists & inhibitors , Type C Phospholipases/drug effects , Type C Phospholipases/isolation & purificationABSTRACT
Light signal transduction in plants involves an intricate series of pathways which is finely regulated by interactions between specific signalling proteins, as well as by protein modifications such as phosphorylation and ubiquitination. The identification of novel phytochrome-interacting proteins and the precise signalling mechanisms that they mediate is still ongoing. In our present study, we show that the newly identified putative phytochrome-associated protein, PAPP2C (phytochrome-associated protein phosphatase type 2C), interacts in the nucleus with phyA (phytochrome A) and phyB, both in vitro and in vivo. Moreover, the phosphatase activity of PAPP2C and its association with phytochromes were found to be enhanced by red light, indicating that it plays a role in mediating phytochrome signalling. In particular, PAPP2C specifically binds to the N-terminal PHY domain of the phytochromes. We thus speculate that this interaction reflects a unique regulatory function of this phosphatase toward established phytochrome-associated proteins. We also show that PAPP2C effectively dephosphorylates phytochromes in vitro. Interestingly, PAPP2C indirectly mediates the dephosphorylation of PIF3 (phytochrome-interacting factor 3) in vitro. Taken together, we suggest that PAPP2C functions as a regulator of PIF3 by dephosphorylating phytochromes in the nucleus.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Phytochrome/metabolism , Plants, Genetically Modified/metabolism , Arabidopsis/genetics , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/genetics , Cell Nucleus/metabolism , Gene Expression Regulation, Plant , Phenotype , Phosphorylation , Phytochrome A/metabolism , Phytochrome B/metabolism , Plants, Genetically Modified/genetics , Protein Binding , Protein Structure, Tertiary , Signal Transduction/genetics , Signal Transduction/physiologyABSTRACT
Bacteriophytochrome from Deinococcus radiodurans (DrBphP) is a plant phytochrome homolog. To investigate the interaction of chromophore and protein structure, we purified recombinant DrBphP and performed biochemical analyses. Differences of apo- and holo-protein in electrophoretic properties in native gels and their susceptibility to trypsin indicate changes in both the conformation and surface topography of this protein as a result of chromophore assembly. Furthermore, proteolysis to Pr and Pfr conformers displayed distinctive cleavage patterns with a noticeable Pr-specific tryptic fragment. Of interest, a prolonged tryptic digestion showed a more severe impact upon the Pfr form. Most importantly, when we assessed the extent of dark reversion to evaluate the role of the cleaved part, a rapidly accelerated reversion was observed upon cleavage at residues 329-505 corresponding to the PHY domain. Our data thus show that the PHY domain is necessary for the Pfr stabilization and spectral integrity of DrBphP.
Subject(s)
Bacterial Proteins/chemistry , Deinococcus/metabolism , Phytochrome/chemistry , Amino Acid Sequence , Protein Folding , Protein Structure, Tertiary , Spectrum Analysis , Trypsin/chemistryABSTRACT
BACKGROUND: The renal dysfunction of chronic kidney disease (CKD) alters serum metabolite levels, but it is not clear how diabetes mellitus (DM) affects the metabolic changes in CKD. METHODS: Serum metabolites from pre-dialysis CKD patients (n=291) with or without DM and from healthy controls (n=56) was measured using nuclear magnetic resonance. RESULTS: Initial principal components analysis and partial least squares-discriminant analysis score plots segregated the CKD patients according to CKD stage and separated DM from non-DM patients. In the CKD patients, associations were seen with clinical characteristics, hyperglycemia, altered amino acid metabolism, accumulated uremic toxins, and dyslipidemia. Of interest, diabetes more strongly affected the metabolic signature during early stage CKD. Furthermore, serum metabolite profiles were successfully applied to the PLS regression model to predict the estimated glomerular filtration rate. The R(2) values from the PLS models for CKD patients with DM were higher than those for CKD without DM. CONCLUSIONS: Metabolomics is useful clinically for providing a metabolic signature that is associated with the CKD phenotype and diabetes more seriously affects patients with early stage CKD compared to those with advanced CKD.
Subject(s)
Diabetes Mellitus/blood , Diabetes Mellitus/metabolism , Renal Insufficiency, Chronic/blood , Renal Insufficiency, Chronic/metabolism , Female , Humans , Magnetic Resonance Spectroscopy , Male , Middle AgedABSTRACT
To determine whether or not FT-IR spectroscopy could be used for taxonomic and metabolic discrimination of African yam lines, tuber samples from African and Asian yam species were subjected to FT-IR. Most remarkable spectral differences between African and Asian yams were found in the 1750-1700 cm(-1) region, polysaccharide (1200-900 cm(-1)) and protein/amide I and II (1700-1500 cm(-1)) regions of FT-IR spectra. A hierarchical dendrogram based on partial least square-discriminant analysis (PLS-DA) of FT-IR data from 7 African yam species show phylogenetic relationship. In addition, the content of dioscin, a steroidal saponin found in yam tuber, was predicted using a PLS regression model with regression coefficient R(2)=0.7208 indicated that prediction model had average accuracy. Thus, considering these results we suggest that FT-IR combined with multivariate analysis could be applied as a novel tool for metabolic evaluation and high-throughput screening of African yam lines with higher content of dioscin.
Subject(s)
Dioscorea/chemistry , Diosgenin/analogs & derivatives , Multivariate Analysis , Spectroscopy, Fourier Transform Infrared/methods , Diosgenin/chemistry , Least-Squares AnalysisABSTRACT
ICP-MS and (1)H NMR are commonly used to determine the geographical origin of food and crops. In this study, data from multielemental analysis performed by ICP-AES/ICP-MS and metabolomic data obtained from (1)H NMR were integrated to improve the reliability of determining the geographical origin of medicinal herbs. Astragalus membranaceus and Paeonia albiflora with different origins in Korea and China were analysed by (1)H NMR and ICP-AES/ICP-MS, and an integrated multivariate analysis was performed to characterise the differences between their origins. Four classification methods were applied: linear discriminant analysis (LDA), k-nearest neighbour classification (KNN), support vector machines (SVM), and partial least squares-discriminant analysis (PLS-DA). Results were compared using leave-one-out cross-validation and external validation. The integration of multielemental and metabolomic data was more suitable for determining geographical origin than the use of each individual data set alone. The integration of the two analytical techniques allowed diverse environmental factors such as climate and geology, to be considered. Our study suggests that an appropriate integration of different types of analytical data is useful for determining the geographical origin of food and crops with a high degree of reliability.
Subject(s)
Magnetic Resonance Imaging/methods , Magnetic Resonance Spectroscopy/methods , Plants, Medicinal/chemistry , Crops, Agricultural , GeographyABSTRACT
To determine whether Fourier transform (FT)-IR spectral analysis combined with multivariate analysis of whole-cell extracts from ginseng leaves can be applied as a high-throughput discrimination system of cultivation ages and cultivars, a total of total 480 leaf samples belonging to 12 categories corresponding to four different cultivars (Yunpung, Kumpung, Chunpung, and an open-pollinated variety) and three different cultivation ages (1 yr, 2 yr, and 3 yr) were subjected to FT-IR. The spectral data were analyzed by principal component analysis and partial least squares-discriminant analysis. A dendrogram based on hierarchical clustering analysis of the FT-IR spectral data on ginseng leaves showed that leaf samples were initially segregated into three groups in a cultivation age-dependent manner. Then, within the same cultivation age group, leaf samples were clustered into four subgroups in a cultivar-dependent manner. The overall prediction accuracy for discrimination of cultivars and cultivation ages was 94.8% in a cross-validation test. These results clearly show that the FT-IR spectra combined with multivariate analysis from ginseng leaves can be applied as an alternative tool for discriminating of ginseng cultivars and cultivation ages. Therefore, we suggest that this result could be used as a rapid and reliable F1 hybrid seed-screening tool for accelerating the conventional breeding of ginseng.
ABSTRACT
Leachate, generated by the decomposition of animal carcasses, presents many environmental, sanitary, and food safety hazards. However, research on the characteristics of leachate is lacking. In this study, we performed biochemical profiling of leachate from two animal species (pig and cattle) in two soil types (sandy loam and sandy soil) using (1)H-NMR-based profiling, followed by multivariate data analysis. The leachate was collected from a well-controlled artificial burial site over a 31-week period. Principal components analysis (PCA) of the NMR data showed similar patterns between species and soil types. Organic components, including organic acids and phenols, predominated, and their levels increased with time. The methylamine level in leachate from pig carcasses 18 weeks following burial was significantly higher than that from cattle carcasses; leachate from cattle carcasses in sandy soil 1 week after burial contained unique components (specifically ethanol, formate, alanine, N-methylation, and taurine), in contrast with those from sandy loam soil. This study suggests that a NMR-based profiling approach is useful to characterize the organic components in leachate from animal carcasses over time.
Subject(s)
Death , Soil/chemistry , Water Pollutants, Chemical/analysis , Animals , Cattle , Methylamines/analysis , Multivariate Analysis , Phenols/analysis , Principal Component Analysis , Proton Magnetic Resonance Spectroscopy , Species Specificity , SwineABSTRACT
Chronic kidney disease (CKD) is characterized by the gradual loss of the kidney function to excrete wastes and fluids from the blood. (1)H NMR-based metabolomics was exploited to investigate the altered metabolic pattern in rats with CKD induced by surgical reduction of the renal mass (i.e., 5/6 nephrectomy (5/6 Nx)), particularly for identifying specific metabolic biomarkers associated with early of CKD. Plasma metabolite profiling was performed in CKD rats (at 4- or 8-weeks after 5/6 Nx) compared to sham-operated rats. Principle components analysis (PCA), partial least squares-discriminant analysis (PLS-DA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) score plots showed a significant separation between the groups. The resulting metabolic profiles demonstrated significantly increased plasma levels of organic anions, including citrate, ß-hydroxybutyrate, lactate, acetate, acetoacetate, and formate in CKD. Moreover, levels of alanine, glutamine, and glutamate were significantly higher. These changes were likely to be associated with complicated metabolic acidosis in CKD for counteracting systemic metabolic acidosis or increased protein catabolism from muscle. In contrast, levels of VLDL/LDL (CH2)n and N-acetylglycoproteins were decreased. Taken together, the observed changes of plasma metabolite profiles in CKD rats provide insights into the disturbed metabolism in early phase of CKD, in particular for the altered metabolism of acid-base and/or amino acids.
Subject(s)
Magnetic Resonance Spectroscopy/methods , Renal Insufficiency, Chronic/blood , Renal Insufficiency, Chronic/metabolism , Animals , Kidney/metabolism , Male , Metabolome/physiology , Rats , Rats, Sprague-Dawley , Transcriptome/geneticsABSTRACT
Radiation exposure is a threat to public health because it causes many diseases, such as cancers and birth defects, due to genetic modification of cells. Compared with the past, a greater number of people are more frequently exposed to higher levels of radioactivity today, not least due to the increased use of diagnostic and therapeutic radiation-emitting devices. In this study, ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS)-based metabolic profiling was used to investigate radiation- induced metabolic changes in human fibroblasts. After exposure to 1 and 5 Gy of γ-radiation, the irradiated fibroblasts were harvested at 24, 48, and 72 h and subjected to global metabolite profiling analysis. Mass spectral peaks of cell extracts were analyzed by pattern recognition using principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA). The results showed that the cells irradiated with 1 Gy returned to control levels at 72 h post radiation, whereas cells irradiated with 5 Gy were quite unlike the controls; therefore, cells irradiated with 1 Gy had recovered, whereas those irradiated with 5 Gy had not. Lipid and amino acid levels increased after the higher-level radiation, indicating degradation of membranes and proteins. These results suggest that MS-based metabolite profiling of γ-radiation-exposed human cells provides insight into the global metabolic alterations in these cells.
Subject(s)
Fibroblasts/radiation effects , Gamma Rays , Metabolome/radiation effects , Metabolomics , Cell Proliferation/radiation effects , Cell Survival/radiation effects , Cells, Cultured , Chromatography, High Pressure Liquid , Dermis/cytology , Discriminant Analysis , Dose-Response Relationship, Radiation , Fibroblasts/metabolism , Humans , Least-Squares Analysis , Mass Spectrometry/methods , Multivariate Analysis , Principal Component Analysis , Time FactorsABSTRACT
Marine mussels (Mytilus) are widely used as bioindicators to measure pollution in marine environments. In this study, (1)H NMR spectroscopy and multivariate statistical analyses were used to differentiate mussel groups from a heavy metal-polluted area (Onsan Bay) and a clean area (Dokdo area). Principal component analysis and orthogonal projection to latent structure-discriminant analysis revealed significant separation between extracts of mussels from Onsan Bay and from the Dokdo area. Organic osmolytes (betaine and taurine) and free amino acids (alanine, arginine, glutamine, phenylalanine, and threonine) were more highly accumulated in Onsan Bay mussels compared with Dokdo mussels. These results demonstrate that NMR-based metabolomics can be used as an efficient method for characterizing heavy metal contamination derived from polluted area compared to clean area and to identify metabolites related to environments that are contaminated with heavy metals.
Subject(s)
Environmental Monitoring , Metabolome/physiology , Metals, Heavy/toxicity , Mytilus/drug effects , Water Pollutants, Chemical/toxicity , Animals , Magnetic Resonance Spectroscopy , Metabolomics , Metals, Heavy/analysis , Metals, Heavy/metabolism , Multivariate Analysis , Mytilus/metabolism , Principal Component Analysis , Water Pollutants, Chemical/analysis , Water Pollutants, Chemical/metabolismABSTRACT
Zebrafish (Danio rerio) is becoming a popular developmental biology model to study diseases and for drug discovery. In this study, we performed proton nuclear magnetic resonance spectroscopy ((1)H-NMR)- and gas chromatography-mass spectrometry (GC/MS)-based metabolic profiling of an alcoholic fatty liver using a zebrafish disease model. We examined metabolic differences between the control and alcoholic fatty liver groups in zebrafish to determine how metabolism in an alcoholic fatty liver is regulated. Multivariate statistical analysis showed a significant difference between the control and alcoholic fatty liver groups. The alcoholic fatty liver group showed increased excretion of isoleucine, acetate, succinate, choline, creatine, acetoacetate, 3-hydroxybutyrate (3HB), ethyl glucuronide (EtG), lactate/pyruvate ratio, fatty acids, and cholesterol, and decreased excretion of citrate, aspartate, tyrosine, glycine, glucose, alanine, betaine, and maltose. Metabolites identified in the fatty liver groups were associated with long-term alcohol consumption, which causes both oxidation-reduction (redox) changes and oxidative stress. This study suggests that global metabolite profiling in a zebrafish model can provide insights into the metabolic changes in an alcoholic fatty liver.