ABSTRACT
OBJECTIVES: We aimed to assess the relationship of left atrial appendage (LAA) fibrosis with atrial fibrillation (AF) and postoperative events in patients receiving coronary artery bypass graft surgery (CABG). BACKGROUND: Increased atrial fibrosis has been associated with AF and worse outcome following catheter ablation. Only limited data exists focusing on the impact of LAA fibrosis on AF after CABG. METHODS: LAA tissue from 164 CABG-patients was stained with Masson-Goldner trichrome. The histological landscape was scanned and segmented into superpixels for software analysis (QuPath). A classification algorithm was extensively trained to detect fibrotic superpixels for quantification. In 43 propensity score matched pairs with AF or sinus rhythm (SR), LAA fibrosis was compared. Moreover, subgroups of mitral valve regurgitation (MR) were analyzed as follows: SR, SR + MR, AF and AF + MR. The predictive value of LAA fibrosis postoperative stroke, postoperative AF and mortality was assessed. RESULTS: Fibrotic remodeling (%) showed no significant difference for the total cohort between the SR and AF group (SR: 30.8 ± 11.4% and AF: 33.8 ± 16.0%, respectively, p = .32). However, significant fibrotic remodeling was observed for SR and AF subgroups (SR: 27.2 ± 12.2% vs. AF: 35.3 ± 13.7%; respectively, p = .049) and between SR and SR + MR subgroups (SR: 27.2 ± 12.2% vs. SR + MR: 34.9 ± 9.1%, respectively, p = .027). LAA fibrosis was not significantly associated with postoperative stroke, postoperative AF or overall mortality (all p > .05). CONCLUSION: LAA fibrosis may contribute to an individual arrhythmia substrate for AF in patients with AF but also in those with SR and coincidence of MR. LAA fibrosis was not found to be predictive for clinical events in patients after CABG.
Subject(s)
Atrial Appendage , Atrial Fibrillation , Mitral Valve Insufficiency , Stroke , Atrial Appendage/diagnostic imaging , Atrial Fibrillation/complications , Atrial Fibrillation/etiology , Coronary Artery Bypass/adverse effects , Fibrosis , Humans , Mitral Valve Insufficiency/diagnosis , Mitral Valve Insufficiency/etiology , Mitral Valve Insufficiency/surgery , Stroke/etiologyABSTRACT
PURPOSE: Most cancer-related deaths worldwide are associated with lung cancer. Subtyping of non-small cell lung cancer (NSCLC) into adenocarcinoma (AC) and squamous cell carcinoma (SqCC) is of importance, as therapy regimes differ. However, conventional staining and immunohistochemistry have their limitations. Therefore, a spatial metabolomics approach was aimed to detect differences between subtypes and to discriminate tumor and stroma regions in tissues. METHODS: Fresh-frozen NSCLC tissues (n = 35) were analyzed by matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) of small molecules (< m/z 1000). Measured samples were subsequently stained and histopathologically examined. A differentiation of subtypes and a discrimination of tumor and stroma regions was performed by receiver operating characteristic analysis and machine learning algorithms. RESULTS: Histology-guided spatial metabolomics revealed differences between AC and SqCC and between NSCLC tumor and tumor microenvironment. A diagnostic ability of 0.95 was achieved for the discrimination of AC and SqCC. Metabolomic contrast to the tumor microenvironment was revealed with an area under the curve of 0.96 due to differences in phospholipid profile. Furthermore, the detection of NSCLC with rarely arising mutations of the isocitrate dehydrogenase (IDH) gene was demonstrated through 45 times enhanced oncometabolite levels. CONCLUSION: MALDI-MSI of small molecules can contribute to NSCLC subtyping. Measurements can be performed intraoperatively on a single tissue section to support currently available approaches. Moreover, the technique can be beneficial in screening of IDH-mutants for the characterization of these seldom cases promoting the development of treatment strategies.
Subject(s)
Carcinoma, Non-Small-Cell Lung/classification , Lung Neoplasms/classification , Metabolomics/methods , Aged , Aged, 80 and over , Biomarkers, Tumor/metabolism , Carcinoma, Non-Small-Cell Lung/diagnosis , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Cohort Studies , Cytological Techniques/methods , Female , Germany , Humans , Immunohistochemistry/methods , Isocitrate Dehydrogenase/genetics , Isocitrate Dehydrogenase/metabolism , Lung Neoplasms/diagnosis , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Machine Learning , Male , Middle Aged , Mutation , Neoplasm Staging , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
BACKGROUND: Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) seem to be highly transmissible, often infect otherwise healthy humans and frequently occur in hospital outbreaks. METHODS: Refugees, living in accommodations in Germany were screened for nasal carriage of S. aureus. The isolates were investigated regarding resistance and virulence, phenotypically and by whole genome data analysis. RESULTS: 5.6% (9/161) of the refugees are carriers of S. aureus. 2.5% (4/161) are MRSA carriers. Among the refugees, spa-types t021, t084, t304, t991 and t4983 were detected, as well as the new spa-types t18794 and t18795. t304 and t991 are assumed to be local spa-types from the middle east. The isolates are less resistant and marginal biofilm formers. Each isolate has a remarkable set of virulence genes, although genes, encoding for proteins strongly associated with invasive S. aureus infections, like Panton-Valentine leucocidin, were not detected. CONCLUSION: The detection of strains from the middle east, supports the assumption that strains co-travel with the refugees and persist despite a transition of the host's living conditions. Whole genome data analysis does not permit to finally evaluate a germ's virulence. Nevertheless, an impression of the virulence potential of the strains, regarding skills in colonization, resistance, immune evasion, and host cell damaging can be pictured.
Subject(s)
Methicillin-Resistant Staphylococcus aureus , Refugees , Staphylococcal Infections , Anti-Bacterial Agents , Humans , Methicillin , Methicillin-Resistant Staphylococcus aureus/genetics , Microbial Sensitivity Tests , Staphylococcal Infections/epidemiology , Staphylococcus aureus/genetics , Virulence/genetics , Virulence Factors/geneticsABSTRACT
The combined occurrence of salt stress and hypoxia leads to increased growth reduction and severe toxic effects compared to salt stress alone. In the present work, we analyzed the metabolic response of sugar beet (Beta vulgaris L.) to salt stress combined with hypoxia in roots as well as in young and mature leaves. B. vulgaris plants were grown in a hydroponic culture under low and high salt concentrations combined with normoxic and hypoxic conditions. A non-targeted metabolic approach was used to identify the biochemical pathways underlying the metabolic and physiological adaptation mechanisms. Young and mature leaves showed a similar metabolic response to salt stress alone and combined stresses, accumulating sugar compounds. Osmoprotectants such as proline and pinitol were accumulated under combined stress. Roots exposed to hypoxic conditions showed increased TCA (tricarboxylic acid cycle) intermediates levels such as succinate, fumarate and malate. During hypoxia, the concentration of free amino acids as well as intermediates of the GABA (gamma-aminobutyric acid) shunt increased in roots as well as in leaves. The combination of salt stress and hypoxia results in a severe stress response in roots and leaves. A partial flux of the TCA cycle linked with the GABA shunt might be activated during hypoxia to regain reduction equivalents.
Subject(s)
Beta vulgaris , Hypoxia , Plant Roots/physiology , Salinity , Stress, Physiological , Beta vulgaris/metabolism , Gene Expression Regulation, Plant , Plant Leaves/metabolism , Sodium Chloride/pharmacology , Sugars , gamma-Aminobutyric AcidABSTRACT
Rice is known to contain limiting amino acids. Synthesis of GABA in plants is an adaptive response by initiating glutamic acid. A higher rate of GABA production was observed in samples enriched with glutamic acid and vacuum impregnation (VI) with longer germination time. Heat map profiles classified GABA and essential amino acids into 1) small increments consisting of Arg, His and Met, 2) moderate increments consisting of GABA, Trp, Lys, Phe and Thr, and 3) large increments consisting of Ile, Leu and Val. In Jasmine rice, highest essential amino acids were found in samples soaked with water, enriched with glutamic acid, and germinated for 72-96 h. Highest GABA (44.8 mg/100 g) was noticed after VI for 20-40 min and germinated for 72-96 h. In Riceberry, highest GABA (74.2 mg/100 g) and essential amino acids were associated with samples treated with VI for 20-40 min and germinated for 96 h.
Subject(s)
Oryza , Amino Acids, Essential , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Vacuum , gamma-Aminobutyric AcidABSTRACT
Urachal adenocarcinomas (UrC) are rare but aggressive. Despite being of profound therapeutic relevance, UrC cannot be differentiated by histomorphology alone from other adenocarcinomas of differential diagnostic importance. As no reliable tissue-based diagnostic biomarkers are available, we aimed to detect such by integrating mass-spectrometry imaging-based metabolomics and digital pathology, thus allowing for a multimodal approach on the basis of spatial information. To achieve this, a cohort of UrC (n = 19) and colorectal adenocarcinomas (CRC, n = 27) as the differential diagnosis of highest therapeutic relevance was created, tissue micro-arrays (TMAs) were constructed, and pathological data was recorded. Hematoxylin and eosin (H&E) stained tissue sections were scanned and annotated, enabling an automized discrimination of tumor and non-tumor areas after training of an adequate algorithm. Spectral information within tumor regions, obtained via matrix-assisted laser desorption/ionization (MALDI)-Orbitrap-mass spectrometry imaging (MSI), were subsequently extracted in an automated workflow. On this basis, metabolic differences between UrC and CRC were revealed using machine learning algorithms. As a result, the study demonstrated the feasibility of MALDI-MSI for the evaluation of FFPE tissue in UrC and CRC with the potential to combine spatial metabolomics data with annotated histopathological data from digitalized H&E slides. The detected Area under the curve (AUC) of 0.94 in general and 0.77 for the analyte taurine alone (diagnostic accuracy for taurine: 74%) makes the technology a promising tool in this differential diagnostic dilemma situation. Although the data has to be considered as a proof-of-concept study, it presents a new adoption of this technology that has not been used in this scenario in which reliable diagnostic biomarkers (such as immunohistochemical markers) are currently not available.
Subject(s)
Metabolomics/methods , Molecular Imaging/methods , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Urinary Bladder Neoplasms , Aged , Biomarkers, Tumor/analysis , Biomarkers, Tumor/metabolism , Colorectal Neoplasms/diagnostic imaging , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Female , Humans , Male , Metabolome/physiology , Middle Aged , Multivariate Analysis , Urinary Bladder Neoplasms/diagnostic imaging , Urinary Bladder Neoplasms/metabolism , Urinary Bladder Neoplasms/pathologyABSTRACT
The nitrogen-fixing α-proteobacterium Sinorhizobium meliloti genome codifies at least 50 response regulator (RR) proteins mediating different and, in many cases, unknown processes. RR-mutant library screening allowed us to identify genes potentially implicated in survival to acid conditions. actJ mutation resulted in a strain with reduced growth rate under mildly acidic conditions as well as a lower capacity to tolerate a sudden shift to lethal acidic conditions compared with the parental strain. Mutation of the downstream gene actK, which encodes for a histidine kinase, showed a similar phenotype in acidic environments suggesting a functional two-component system. Interestingly, even though nodulation kinetics, quantity, and macroscopic morphology of Medicago sativa nodules were not affected in actJ and actK mutants, ActK was required to express the wild-type nitrogen fixation phenotype and ActJK was necessary for full bacteroid development and nodule occupancy. The actJK regulatory system presented here provides insights into an evolutionary process in rhizobium adaptation to acidic environments and suggests that actJK-controlled functions are crucial for optimal symbiosis development.
Subject(s)
Sinorhizobium meliloti , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Medicago sativa/metabolism , Nitrogen Fixation , Sinorhizobium meliloti/genetics , Sinorhizobium meliloti/metabolism , Symbiosis/geneticsABSTRACT
Mass Spectrometry Imaging (MSI) is an established and still evolving technique for the spatial analysis of molecular co-location in biological samples. Nowadays, MSI is expanding into new domains such as clinical pathology. In order to increase the value of MSI data, software for visual analysis is required that is intuitive and technique independent. Here, we present QUIMBI (QUIck exploration tool for Multivariate BioImages) a new tool for the visual analysis of MSI data. QUIMBI is an interactive visual exploration tool that provides the user with a convenient and straightforward visual exploration of morphological and spectral features of MSI data. To improve the overall quality of MSI data by reducing non-tissue specific signals and to ensure optimal compatibility with QUIMBI, the tool is combined with the new pre-processing tool ProViM (Processing for Visualization and multivariate analysis of MSI Data), presented in this work. The features of the proposed visual analysis approach for MSI data analysis are demonstrated with two use cases. The results show that the use of ProViM and QUIMBI not only provides a new fast and intuitive visual analysis, but also allows the detection of new co-location patterns in MSI data that are difficult to find with other methods.
Subject(s)
Diagnostic Imaging/methods , Image Processing, Computer-Assisted/methods , Mass Spectrometry/methods , Animals , Humans , Kidney/anatomy & histology , Male , Mice , Pseudoxanthoma Elasticum/pathology , Skin/pathology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Vibrissae/anatomy & histologyABSTRACT
Nitrogen-fixing rhizobia and legumes have developed complex mutualistic mechanism that allows to convert atmospheric nitrogen into ammonia. Signalling by mitogen-activated protein kinases (MAPKs) seems to be involved in this symbiotic interaction. Previously, we reported that stress-induced MAPK (SIMK) shows predominantly nuclear localization in alfalfa root epidermal cells. Nevertheless, SIMK is activated and relocalized to the tips of growing root hairs during their development. SIMK kinase (SIMKK) is a well-known upstream activator of SIMK. Here, we characterized production parameters of transgenic alfalfa plants with genetically manipulated SIMK after infection with Sinorhizobium meliloti. SIMKK RNAi lines, causing strong downregulation of both SIMKK and SIMK, showed reduced root hair growth and lower capacity to form infection threads and nodules. In contrast, constitutive overexpression of GFP-tagged SIMK promoted root hair growth as well as infection thread and nodule clustering. Moreover, SIMKK and SIMK downregulation led to decrease, while overexpression of GFP-tagged SIMK led to increase of biomass in above-ground part of plants. These data suggest that genetic manipulations causing downregulation or overexpression of SIMK affect root hair, nodule and shoot formation patterns in alfalfa, and point to the new biotechnological potential of this MAPK.
Subject(s)
Medicago sativa , Plant Proteins , Biomass , Cluster Analysis , Medicago sativa/genetics , Mitogen-Activated Protein Kinase Kinases , Plant Proteins/genetics , Symbiosis/geneticsABSTRACT
BACKGROUND: Plant growth-promoting rhizobacteria (PGPR) are known to improve plant growth and are used as biofertilizers, thanks to their numerous benefits to agriculture such as phosphorus solubilization and phytohormone production. In this paper, four rhizospheric bacteria (Phyllobacterium sp., Bacillus sp., Agrobacterium sp., and Rhizobium sp.) isolated from surface-sterilized root nodules of Acacia cyanophylla were tested for their ability to solubilize inorganic phosphate and to produce indole-3-acetic acid (IAA) under laboratory conditions. Then, the best IAA producer (Rhizobium sp.) was selected to test optimized conditions for IAA production. Finally, the effect of the four strains on plant growth for A. cyanophylla was evaluated in vivo. RESULTS: The results showed that the totality of the tested isolates had solubilized inorganic phosphate (P) in both NBRIP (National Botanical Research Institute Phosphate) and PVK (Pikovskaya) media. Bacillus sp. was a high P-solubilizer and showed maximum solubilization in PVK (519 µg ml-1) and NBRIP (782 µg ml-1). The optimization of maximum phosphate solubilization was done using different sources of carbon (1%) and nitrogen (0.1%). Glucose and ammonium sulfate were selected to be the best carbon and nitrogen source for phosphate solubilization by all tested strains, except for Phyllobacterium sp., which recorded the highest phosphate solubilization with ammonium nitrate. The IAA production by the tested strains indicated that Rhizobium sp. produced the highest amount of IAA (90.21 µg ml-1) in culture media supplemented with L-tryptophan. The best production was observed with L-Trp concentration of 0.2% (116.42 µg ml-1) and at an initial pH of 9 (116.07 µg ml-1). The effect of NaCl on IAA production was tested at concentrations of 0 to 5% and the maximum production of 89.43 µg ml-1 was found at 2% NaCl. The extraction of crude IAA from this strain was done and purity was confirmed with Thin Layer Chromatography (TLC) analysis. A specific spot from the extracted IAA production was found to correspond with a standard spot of IAA with the same Rf value. Finally, the tested PGPR demonstrated growth stimulatory effects on Acacia cyanophylla seedlings in vivo, with a great increase of shoots' and roots' dry weights, and shoot length compared to control. The rhizobacterial isolates were identified by 16S rDNA sequence analysis as Agrobacterium sp. NA11001, Phyllobacterium sp. C65, Bacillus sp. CS14, and Rhizobium sp. V3E1. CONCLUSION: This study highlights the importance of the use of phosphate solubilizing and IAA producer microorganisms as biofertilizers to increase crop yields. The studied strains showed a significant phosphate solubilization potential and IAA production. The use of selected strains as inoculants would be interesting, in particular with a view of promoting sustainable agriculture. However, further studies to verify the efficacy of the best isolates in situ is certainly required.
ABSTRACT
Cisplatin is a widely used chemotherapeutic agent. However, it is causing nephrotoxic side effects including a reduced glomerular filtration rate and acute kidney injury. Although kidneys can recover to an extent from the treatment, long-term damage is possible. While a lot of research is focusing on short-term effects, little is known about adverse metabolic effects in the process of recovery. In this study, male Han Wistar rats were dosed with a single intraperitoneal injection of 3 mg/kg cisplatin. Urine and kidney samples were harvested 3, 8 and 26 days after administration. Tubular injury was demonstrated through urinary biomarkers. Complementing this, mass spectrometry imaging gives insight on molecular alterations on a spatial level, thus making it well suited to analyze short- and long-term disturbances. Various metabolic pathways seem to be affected, as changes in a wide range of metabolites were observed between treated and control animals. Besides previously reported early changes in kidney metabolism, unprecedented long-term effects were detected including deviation in nucleotides, antioxidants, and phospholipids.
Subject(s)
Antineoplastic Agents/toxicity , Antioxidants/metabolism , Cisplatin/toxicity , Energy Metabolism/drug effects , Kidney/drug effects , Animals , Biomarkers/metabolism , Injections, Intraperitoneal , Kidney/metabolism , Kidney/pathology , Kidney Tubules/drug effects , Kidney Tubules/metabolism , Kidney Tubules/pathology , Male , Rats, Wistar , Time FactorsABSTRACT
Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor. High infiltration rates and poor therapy responses make it the deadliest glioma. The tumor metabolism is known to differ from normal one and is influenced through various factors which can lead to longer survival. Metabolites are small molecules (< 1500 Da) that display the metabolic pathways in the tissue. To determine the metabolic alterations between tumor and peritumoral tissue in human GBMs, mass spectrometry imaging (MSI) was performed on thin sections from 25 resected tumors. In addition, the GBMs were compared with six gliomas harboring a mutation in the isocitrate dehydrogenase (IDH1) gene (IDH1). With this technique, a manifold of analytes can be easily visualized on a single tissue section. Metabolites were annotated based on their accurate mass using high resolution MSI. Differences in their mean intensities in the tumor and peritumoral areas were statistically evaluated and abundances were visualized on the tissue. Enhanced levels of the antioxidants ascorbic acid, taurine, and glutathione in tumor areas suggest protective effects on the tumor. Increased levels of purine and pyrimidine metabolism compounds in GBM areas indicate the high energy demand. In accordance with these results, enhanced abundances of lactate and glutamine were detected. Moreover, decreased abundance of N-acetylaspartate, a marker for neuronal health, was measured in tumor areas. Obtained metabolic information could potentially support and personalize therapeutic approaches, hence emphasizing the suitability of MSI for GBM research.
Subject(s)
Brain Neoplasms/metabolism , Glioblastoma/metabolism , Brain Neoplasms/genetics , Brain Neoplasms/pathology , Glioblastoma/genetics , Glioblastoma/pathology , Humans , Isocitrate Dehydrogenase/genetics , Mutation , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
As a consequence of the detoxification process, drugs and drug related metabolites can accumulate in the liver, resulting in drug induced liver injury (DILI), which is the major cause for dose limitation. Amitriptyline, a commonly used tricyclic anti-depressant, is known to cause DILI. The mechanism of Amitriptyline induced liver injury is not yet completely understood. However, as it undergoes extensive hepatic metabolism, unraveling the molecular changes in the liver upon Amitriptyline treatment can help understand Amitriptyline's mode of toxicity. In this study, Amitriptyline treated male rat liver tissue was analyzed using Matrix Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging (MALDI-MSI) to investigate the spatial abundances of Amitriptyline, lipids, and bile acids. The metabolism of Amitriptyline in liver tissue was successfully demonstrated, as the spatial distribution of Amitriptyline and its metabolites localize throughout treatment group liver samples. Several lipids appear upregulated, from which nine were identified as distinct phosphatidylcholine (PC) species. The detected bile acids were found to be lower in Amitriptyline treatment group. The combined results from histological findings, Oil Red O staining, and lipid zonation by MSI revealed lipid upregulation in the periportal area indicating drug induced macrovesicular steatosis (DIS).
Subject(s)
Amitriptyline/toxicity , Antidepressive Agents, Tricyclic/toxicity , Bile Acids and Salts/metabolism , Chemical and Drug Induced Liver Injury/metabolism , Lipid Metabolism/drug effects , Animals , Chemical and Drug Induced Liver Injury/pathology , Liver/chemistry , Liver/metabolism , Liver/pathology , Male , Mass Spectrometry , Organ Size/drug effects , Phosphatidylcholines/metabolism , Rats , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Up-Regulation/drug effectsABSTRACT
Automated matrix deposition for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is crucial for producing reproducible analyte ion signals. Here we report an innovative method employing an automated immersion apparatus, which enables a robust matrix deposition within 5 minutes and with scalable throughput by using MAPS matrix and non-polar solvents. MSI results received from mouse heart and rat brain tissues were qualitatively similar to those from nozzle sprayed samples with respect to peak number and quality of the ion images. Overall, the immersion-method enables a fast and careful matrix deposition and has the future potential for implementation in clinical tissue diagnostics.
Subject(s)
Molecular Imaging/methods , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Animals , Brain Chemistry , Histological Techniques , Maleic Anhydrides/chemistry , Mice , Myocardium/chemistry , Rats , Reproducibility of Results , RotationABSTRACT
Alkaloids are common secondary metabolites found also in plants of the large family of Solanaceae, and they contribute frequently to the economic importance of these plants as food, spices, drugs, or pharmaceuticals. Matrix-assisted laser desorption/ionization mass spectrometry imaging was applied to visualize the spatial localization of the main steroidal alkaloids in diverse plant tissues of Lycopersicon esculentum, Solanum nigrum, and Solanum dulcamara. Among others, the basic aglycons, tomatidenol, tomatidine, solasodine, and soladulcine, along with their corresponding glycoalkaloids, were identified with distinct distributions within plant tissue structures and plant parts and with respect to the degree of ripeness. The alkaloids are identified by their mass and fragmentation pattern. Multivariate unsupervised principal compound analysis and the k-means clustering analysis were calculated on the basis of all peaks, automatically picked from all selected regions, with total ion count normalization resulting in characterization of the tissues and organs with respect to their chemical similarity. It can therefore be concluded that the tissue-specific localizations of alkaloids in nightshades depend upon the ripeness status and the developmental stage of the plants.
Subject(s)
Alkaloids/chemistry , Solanum lycopersicum/metabolism , Solanum nigrum/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Alkaloids/metabolism , Solanum lycopersicum/chemistry , Metabolomics/methods , Solanum nigrum/chemistry , Tissue DistributionABSTRACT
Consumers are constantly exposed to chemical mixtures such as multiple residues of different pesticides via the diet. This raises questions concerning potential combination effects, especially because these substances are tested for regulatory purposes on an individual basis. With approximately 500 active substances approved as pesticides, there are too many possible combinations to be tested in standard animal experiments generally requested for regulatory purposes. Therefore, the development of in vitro tools and alternative testing strategies for the assessment of mixture effects is extremely important. As a first step in the development of such in vitro tools, we used (tri)azoles as model substances in a set of different cell lines derived from the primary target organ of these substances, the liver (human: HepaRG, rat: H4IIE). Concentrations were reconciled with measured tissue concentrations obtained from in vivo experiments to ensure comparable effect levels. The effects of the substances were subsequently analyzed by transcriptomics and metabolomics techniques and compared to data from corresponding in vivo studies. The results show that similar toxicity pathways are affected by substances and combinations, thus indicating a similar mode of action and additive effects. Two biomarkers obtained by the approach, CAR and Cyp1A1, were used for mixture toxicity modeling and confirmed the concentration-additive effects, thus supporting the selected testing strategy and raising hope for the development of in vitro methods suitable to detect combination effects and prioritize mixtures of concern for further testing.
Subject(s)
Gene Expression Profiling/methods , Liver/drug effects , Metabolomics/methods , Pesticides/toxicity , Toxicity Tests/methods , Triazoles/toxicity , Animals , Cell Line , Hep G2 Cells , Humans , Rats , Risk Assessment , Species SpecificityABSTRACT
BACKGROUND: The spatial distribution and colocalization of functionally related metabolites is analysed in order to investigate the spatial (and functional) aspects of molecular networks. We propose to consider community detection for the analysis of m/z-images to group molecules with correlative spatial distribution into communities so they hint at functional networks or pathway activity. To detect communities, we investigate a spectral approach by optimizing the modularity measure. We present an analysis pipeline and an online interactive visualization tool to facilitate explorative analysis of the results. The approach is illustrated with synthetical benchmark data and two real world data sets (barley seed and glioblastoma section). RESULTS: For the barley sample data set, our approach is able to reproduce the findings of a previous work that identified groups of molecules with distributions that correlate with anatomical structures of the barley seed. The analysis of glioblastoma section data revealed that some molecular compositions are locally focused, indicating the existence of a meaningful separation in at least two areas. This result is in line with the prior histological knowledge. In addition to confirming prior findings, the resulting graph structures revealed new subcommunities of m/z-images (i.e. metabolites) with more detailed distribution patterns. Another result of our work is the development of an interactive webtool called GRINE (Analysis of GRaph mapped Image Data NEtworks). CONCLUSIONS: The proposed method was successfully applied to identify molecular communities of laterally co-localized molecules. For both application examples, the detected communities showed inherent substructures that could easily be investigated with the proposed visualization tool. This shows the potential of this approach as a complementary addition to pixel clustering methods.
Subject(s)
Data Visualization , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Brain Neoplasms/pathology , Cluster Analysis , Glioblastoma/pathology , Hordeum , Humans , Principal Component Analysis , Seeds/anatomy & histology , Seeds/chemistryABSTRACT
The leafy green vegetable Lactuca sativa, L. is susceptible to the soil-born fungus Rhizoctonia solani AG1-IB. In a previous study, we reported on the transcriptional response of R. solani AG1-IB (isolate 7/3/14) during the interspecies interaction with L. sativa cv. Tizian by means of RNA sequencing. Here we present the L. sativa transcriptome and metabolome from the same experimental approach. Three distinct interaction zones were sampled and compared to a blank (non-inoculated) sample: symptomless zone 1, zone 2 showing light brown discoloration, and a dark brown zone 3 characterized by necrotic lesions. Throughout the interaction, we observed a massive reprogramming of the L. sativa transcriptome, with 9231 unique genes matching the threshold criteria for differential expression. The lettuce transcriptome of the light brown zone 2 presents the most dissimilar profile compared to the uninoculated zone 4, marking the main stage of interaction. Transcripts putatively encoding several essential proteins that are involved in maintaining jasmonic acid and auxin homeostasis were found to be negatively regulated. These and other indicator transcripts mark a potentially inadequate defence response, leading to a compatible interaction. KEGG pathway mapping and GC-MS metabolome data revealed large changes in amino acid, lignin and hemicellulose related pathways and related metabolites.
Subject(s)
Lactuca/genetics , Rhizoctonia/physiology , Transcriptome , Calcium Signaling/genetics , Lactuca/metabolism , Lactuca/microbiology , Metabolome , Plant Diseases/genetics , Plant Diseases/microbiology , Plant Leaves/genetics , Plant Leaves/metabolism , Plant Leaves/microbiology , Plant Proteins/genetics , Plant Proteins/metabolism , Principal Component Analysis , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and MALDI MS imaging are ubiquitous analytical methods in medical, pharmaceutical, biological, and environmental research. Currently, there is a strong interest in the investigation of low molecular weight compounds (LMWCs), especially to trace and understand metabolic pathways, requiring the development of new matrix systems that have favorable optical properties and a high ionization efficiency and that are MALDI silent in the LMWC area. In this paper, five conjugated polymers, poly{[ N, N'-bis(2-octyldodecyl)-naphtalene-1,4,5,8-bis(dicarboximide)-2,6-diyl]- alt-5,5'(2,2'-bithiophene)} (PNDI(T2)), poly(3-dodecylthiophene-2,5-diyl) (P3DDT), poly{[2,3-bis(3-octyloxyphenyl)quinoxaline-5,8-diyl]- alt-(thiophene-2,5-diyl)} (PTQ1), poly{[ N, N'-bis(2-octyldodecyl)-isoindigo-5,5'-diyl] -alt-5,5'(2,2'-bithiophene)} (PII(T2)), and poly(9,9-di- n-octylfluorenyl-2,7-diyl) (P9OFl) are investigated as matrices. The polymers have a strong optical absorption, are solution processable, and can be coated into thin films, allowing a vast reduction in the amount of matrix used. All investigated polymers function as matrices in both positive and negative mode MALDI, classifying them as rare dual-mode matrices, and show a very good analyte ionization ability in both modes. PNDI(T2), P3DDT, PTQ1, and PII(T2) are MALDI silent in the full measurement range (> m/ z = 150k), except at high laser intensities. In MALDI MS experiments of single analytes and a complex biological sample, the performance of the polymers was found to be as good as two commonly used matrices (2,5-DHB for positive and 9AA for negative mode measurements). The detection limit of two standard analytes was determined as being below 164 pmol for reserpine and below 245 pmol for cholic acid. Additionally P3DDT was used successfully in first MALDI MS imaging experiments allowing the visualization of the tissue morphology of rat brain sections.