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In JoVE (1)
Other Publications (15)
- International Journal of Molecular Medicine
- Anticancer Research
- Cancer Epidemiology, Biomarkers & Prevention : a Publication of the American Association for Cancer Research, Cosponsored by the American Society of Preventive Oncology
- Molecular Medicine Reports
- NMR in Biomedicine
- Journal of Agricultural and Food Chemistry
- Magnetic Resonance in Medicine : Official Journal of the Society of Magnetic Resonance in Medicine / Society of Magnetic Resonance in Medicine
- Journal of Proteome Research
- Oncology Reports
- International Journal of Molecular Medicine
- International Journal of Oncology
- International Journal of Molecular Medicine
- Journal of Magnetic Resonance Imaging : JMRI
- Lipids
- Journal of Agricultural and Food Chemistry
Articles by Valeria Righi in JoVE
JoVE Neuroscience
Magnetic Resonance Spectroscopy of live Drosophila melanogaster using Magic Angle Spinning
1NMR Surgical Laboratory, Department of Surgery, Massachusetts General Hospital, Harvard Medical School, 2Shriners Burn Institute, 3Department of Radiology, Athinoula A. Martinos Center of Biomedical Imaging, Harvard Medical School, 4Molecular Surgery Laboratory, Department of Surgery, Massachusetts General Hospital, Harvard Medical School
This technique enables the use of high-resolution magic angle spinning proton MR spectroscopy (HRMAS 1H-MRS) for molecular characterization of live Drosophila melanogaster with a conventional 14.1 tesla spectrometer equipped with an HRMAS probe.
Other articles by Valeria Righi on PubMed
Ex Vivo HR-MAS MRS of Human Meningiomas: a Comparison with in Vivo 1H MR Spectra
We report on the magnetic resonance spectroscopy (MRS) characterisation of different human meningiomas. Three histological subtypes of meningiomas (meningothelial, fibrous and oncocytic) were analysed both through in vivo and ex vivo MRS experiments. The ex vivo high-resolution magic angle spinning (HR-MAS) investigations, permitting an accurate description of the metabolic profile, are very helpful for the assignment of the resonances in vivo of human meningiomas and for the validation of the quantification procedure of in vivo MR spectra. By using one- and two-dimensional experiments, we were able to identify several metabolites in different histological subtypes of meningiomas. Our spectroscopic data confirmed the presence of the typical metabolites of these benign neoplasms and, at the same time, that meningomas with different morphological characteristics have different metabolic profiles, particularly regarding macromolecules and lipids. The ex vivo spectra allowed a better understanding and interpretation of the in vivo MR spectra, showing that the HR-MAS MRS technique could be a complementary method to strongly support the in vivo MR spectroscopy and increase its clinical potentiality.
Ex Vivo HR-MAS Magnetic Resonance Spectroscopy of Normal and Malignant Human Renal Tissues
The aim of the present study was to examine the metabolic profile of normal and tumoral renal tissues by ex vivo high resolution magic angle spinning magnetic resonance spectroscopy (HR-MAS MRS).
Biochemical Alterations from Normal Mucosa to Gastric Cancer by Ex Vivo Magnetic Resonance Spectroscopy
The metabolic profile and morphologic aspects of normal and pathologic human gastric mucosa were studied. The aim of the present research was the application of ex vivo high-resolution magic angle spinning magnetic resonance spectroscopy (HR-MAS MRS) to the human gastric tissue to get information on the molecular steps involved in gastric carcinogenesis and the identification of biochemical markers useful for the development of in vivo MRS methodologies to diagnose gastric pathologies in clinical situations.
Burn Trauma in Skeletal Muscle Results in Oxidative Stress As Assessed by in Vivo Electron Paramagnetic Resonance
Using a mouse model, we tested the hypotheses that severe burn trauma causes metabolic disturbances in skeletal muscle, and that these can be measured and repeatedly followed by in vivo electron paramagnetic resonance (EPR). We used a 1.2-GHz (L-band) EPR spectrometer to measure partial pressure of oxygen (pO(2)) levels, redox status and oxidative stress following a non-lethal burn trauma model to the left hind limbs of mice. Results obtained in the burned mouse gastrocnemius muscle indicated a significant decrease in tissue pO(2) immediately (P=0.032) and at 6 h post burn (P=0.004), compared to the gastrocnemius of the unburned hind limb. The redox status of the skeletal muscle also peaked at 6 h post burn (P=0.027) in burned mice. In addition, there was an increase in the EPR signal of the nitroxide produced by oxidation of the hydroxylamine (CP-H) probe at 12 h post burn injury, indicating a burn-induced increase in mitochondrial reactive oxygen species (ROS). The nitroxide signal continued to increase between 12 and 24 h, suggesting a further increase in ROS generation post burn. These results confirm genomic results, which indicate a downregulation of antioxidant genes and therefore strongly suggest the dysfunction of the mitochondrial oxidative system. We believe that the direct measurement of tissue parameters such as pO(2), redox and ROS by EPR may be used to complement measurements by nuclear magnetic resonance (NMR) in order to assess tissue damage and the therapeutic effectiveness of antioxidant agents in severe burn trauma.
1H HR-MAS and Genomic Analysis of Human Tumor Biopsies Discriminate Between High and Low Grade Astrocytomas
We investigate the profile of choline metabolites and the expression of the genes of the Kennedy pathway in biopsies of human gliomas (n = 23) using (1)H High Resolution Magic Angle Spinning (HR-MAS, 11.7 Tesla, 277 K, 4000 Hz) and individual genetic assays. (1)H HR-MAS spectra allowed the resolution and relative quantification by the LCModel of the resonances from choline (Cho), phosphocholine (PC) and glycerophosphorylcholine (GPC), the three main components of the combined tCho peak observed in gliomas by in vivo (1)H NMR spectroscopy. All glioma biopsies depicted a prominent tCho peak. However, the relative contributions of Cho, PC, and GPC to tCho were different for low and high grade gliomas. Whereas GPC is the main component in low grade gliomas, the high grade gliomas show a dominant contribution of PC. This circumstance allowed the discrimination of high and low grade gliomas by (1)H HR-MAS, a result that could not be obtained using the tCho/Cr ratio commonly used by in vivo (1)H NMR spectroscopy. The expression of the genes involved in choline metabolism has been investigated in the same biopsies. High grade gliomas depict an upregulation of the beta gene of choline kinase and phospholipase C, as well as a downregulation of the cytidyltransferase B gene, the balance of these being consistent with the accumulation of PC. In the low grade gliomas, phospholipase A(1) and lysophospholipase are upregulated and phospholipase D is downregulated, supporting the accumulation of GPC. The present findings offer a promising procedure that will potentially help to accurately grade glioma tumors using (1)H HR-MAS, providing in addition the genetic background for the alterations of choline metabolism observed in high and low grade gliomas.
Structural Characteristics of 'Hayward' Kiwifruits from Elephantiasis-affected Plants Studied by DRIFT, FT-Raman, NMR, and SEM Techniques
This is the first study on structural and ultrastructural changes taking place in Actinidia deliciosa kiwifruits affected by "elephantiasis syndrome", by means of DRIFT, FT-Raman, NMR, and SEM techniques. The fruits arising from elephantiasis-affected plants assume a round and smaller shape, limiting their marketing. Despite etiological studies on this disease, so far no information is available on the structural and ultrastructural characteristics of the fruits. The SEM and spectroscopic data showed significant modifications regarding the polysaccharide fraction in kiwifruits from diseased plants. The pectins seem to be the polysaccharide fraction more involved in the structural variations of the fruits. These structural and ultrastructural variations are related to the elephantiasis syndrome, and they could be adopted as markers for early diagnosis of the disease.
Cerebral Activation by Fasting Induces Lactate Accumulation in the Hypothalamus
Carbon-13 ((13)C) high-resolution magic angle spinning (HR-MAS) spectroscopy was used to investigate the neuroglial coupling mechanisms underlying appetite regulation in the brain of C57BL/6J mice metabolizing [1-(13)C]glucose. Control fed or overnight fasted mice received [1-(13)C]glucose (20 micromol/g intraperitoneally [i.p.]), 15 min prior to brain fixation by focused microwaves. The hypothalamic region was dissected from the rest of the brain and (13)C HR-MAS spectra were obtained from both biopsies. Fasting resulted in a significant increase in hypothalamic [3-(13)C]lactate and [2-(13)C]gamma-aminobutyric acid (GABA) relative to the remaining brain. Administration of the orexigenic peptide ghrelin (0.3 nmol/g i.p.) did not increase hypothalamic [3-(13)C]lactate or [2-(13)C]GABA, suggesting that ghrelin signaling is not sufficient to elicit all the metabolic consequences of hypothalamic activation by fasting. Our results indicate that the hypothalamic regulation of appetite involves, in addition to the well-known neuropeptide signaling, increased neuroglial lactate shuttling and augmented GABA concentrations.
Discrimination of Healthy and Neoplastic Human Colon Tissues by Ex Vivo HR-MAS NMR Spectroscopy and Chemometric Analyses
The metabolic profile of human healthy and neoplastic colorectal tissues was obtained using ex vivo High-Resolution Magic Angle Spinning (HR-MAS) NMR spectroscopy. Principal Components Analysis (PCA) and Partial Least Squares Discriminant Analysis (PLS-DA) were applied to NMR data in order to highlight the biochemical differences between healthy and neoplastic colorectal tissues. The synergic combination of ex vivo HR-MAS NMR spectroscopy with Multivariate Data Analysis enables discrimination between healthy and tumoral colorectal tissues and identification of the increase of taurine, acetate, lactate, and lipids, and the decrease of polyols and sugars as tumoral characteristics. Moreover, it was found that macroscopically/histologically normal colorectal tissues, collected at least 15 cm from the adenocarcinoma, are characterized by a metabolic pattern quite similar to that typical of tumoral lesions. It was shown that ex vivo HR-MAS NMR spectroscopy, performed on intact specimens, may be of great potentiality in the clinical evaluation of human neoplastic colorectal tissues and that the biochemical data represent the molecular basis for an accurate and noninvasive clinical applications of in vivo NMR spectroscopy.
Identification of Mobile Lipids in Human Cancer Tissues by Ex Vivo Diffusion Edited HR-MAS MRS
Magnetic Resonance Spectroscopy visible mobile lipids are considered important markers in the diagnosis of human cancer and are thought to be closely involved in various aspects of tumour transformation, such as cell proliferation, necrosis, apoptosis, hypoxia and drug resistance. A method allowing the straightforward identification of the lipid classes contributing to the mobile lipids in human malignant tissues is highly advisable. Ex vivo High Resolution Magic Angle Spinning Magnetic Resonance Spectroscopy was done directly on human cerebral, renal and colorectal malignant tissue specimens. A diffusion edited sequence, based on stimulated echo and bipolar gradient pulses, was used to characterize molecules with low diffusion rates, arising from mobile lipid components. Cholesterol, triglycerides and phosphatidylcholine are simultaneously detected and all contribute to the mobile lipid resonances present in malignant glioma and clear cell renal carcinoma tissue specimens spectra. On the contrary, papillary cell renal carcinoma spectrum is predominated by phosphatidylcholine resonances and that of colorectal adenocarcinoma is characterized by signals arising from triglycerides. Ex vivo diffusion edited High Resolution Magic Angle Spinning Magnetic Resonance Spectroscopy, done on intact tissue, is a powerful analytical tool to obtain a simple and immediate identification of mobile lipid components. This can offer a significant contribution to better understanding their involvement in cancer tissues. Furthermore, ex vivo high resolution spectroscopic measurements allow to improve the interpretation of in vivo Magnetic Resonance spectra, increasing its clinical potentiality.
Molecular Characterization and Quantification Using State of the Art Solid-state Adiabatic TOBSY NMR in Burn Trauma
We describe a novel solid-state nuclear magnetic resonance (NMR) method that maximizes the advantages of high-resolution magic-angle-spinning (HRMAS), relative conventional liquid-state NMR approaches, when applied to intact biopsies of skeletal muscle specimens collected from burn trauma patients. This novel method, termed optimized adiabatic TOtal through Bond correlation SpectroscopY (TOBSY) solid-state NMR pulse sequence for two-dimensional (2D) 1H-1H homonuclear scalar-coupling longitudinal isotropic mixing, was demonstrated to provide a 40-60% improvement in signal-to-noise ratio (SNR) relative to its liquid-state analogue TOCSY (TOtal Correlation SpectroscopY). Using 1- and 2-dimensional HRMAS NMR experiments, we identified several metabolites in burned tissues. Quantification of metabolites in burned tissues showed increased levels of lipid compounds, intracellular metabolites (e.g., taurine and phosphocreatine) and substantially decreased water-soluble metabolites (e.g., glutathione, carnosine, glucose, glutamine/glutamate and alanine). These findings demonstrate that HRMAS NMR Spectroscopy using TOBSY is a feasible technique that reveals new insights into the pathophysiology of burn trauma. Moreover, this method has applications that facilitate the development of novel therapeutic strategies.
High-resolution Magic Angle Spinning Magnetic Resonance Spectroscopy Detects Glycine As a Biomarker in Brain Tumors
The non-essential amino acid neurotransmitter glycine (Gly) may serve as a biomarker for brain tumors. Using 36 biopsies from patients with brain tumors [12 glioblastoma multiforme (GBM); 10 low-grade (LG), including 7 schwannoma and 3 pylocytic astrocytoma; 7 meningioma (MN); 7 brain metastases (MT), including 3 adenocarcinoma and 4 breast cancer] and 9 control biopsies from patients undergoing surgery for epilepsy, we tested the hypothesis that the presence of glycine may distinguish among these brain tumor types. Using high-resolution magic angle spinning (HRMAS) 1H magnetic resonance spectroscopy (MRS), we determined a theoretically optimum echo time (TE) of 50 ms for distinguishing Gly signals from overlapping myo-inositol (Myo) signals and tested our methodology in phantom and biopsy specimens. Quantitative analysis revealed higher levels of Gly in tumor biopsies (all combined) relative to controls; Gly levels were significantly elevated in LG, MT and GBM biopsies (P
In Vivo High-resolution Magic Angle Spinning Magnetic Resonance Spectroscopy of Drosophila Melanogaster at 14.1 T Shows Trauma in Aging and in Innate Immune-deficiency is Linked to Reduced Insulin Signaling
In vivo magnetic resonance spectroscopy (MRS), a non-destructive biochemical tool for investigating live organisms, has yet to be used in the fruit fly Drosophila melanogaster, a useful model organism for investigating genetics and physiology. We developed and implemented a high-resolution magic-angle-spinning (HRMAS) MRS method to investigate live Drosophila at 14.1 T. We demonstrated, for the first time, the feasibility of using HRMAS MRS for molecular characterization of Drosophila with a conventional MR spectrometer equipped with an HRMAS probe. We showed that the metabolic HRMAS MRS profiles of injured, aged wild-type (wt) flies and of immune deficient (imd) flies were more similar to chico flies mutated at the chico gene in the insulin signaling pathway, which is analogous to insulin receptor substrate1-4 (IRS1-4) in mammals and less to those of adipokinetic hormone receptor (akhr) mutant flies, which have an obese phenotype. We thus provide evidence for the hypothesis that trauma in aging and in innate immune-deficiency is linked to insulin signaling. This link may explain the mitochondrial dysfunction that accompanies insulin resistance and muscle wasting that occurs in trauma, aging and immune system deficiencies, leading to higher susceptibility to infection. Our approach advances the development of novel in vivo non-destructive research approaches in Drosophila, suggests biomarkers for investigation of biomedical paradigms, and thus may contribute to novel therapeutic development.
Combined Off-resonance Imaging and T2 Relaxation in the Rotating Frame for Positive Contrast MR Imaging of Infection in a Murine Burn Model
To develop novel magnetic resonance (MR) imaging methods to monitor accumulation of macrophages in inflammation and infection. Positive-contrast MR imaging provides an alternative to negative-contrast MRI, exploiting the chemical shift induced by ultra-small superparamagnetic iron-oxide (USPIO) nanoparticles to nearby water molecules. We introduce a novel combination of off-resonance (ORI) positive-contrast MRI and T(2ρ) relaxation in the rotating frame (ORI-T(2ρ)) for positive-contrast MR imaging of USPIO.
EPA or DHA Supplementation Increases Triacylglycerol, but Not Phospholipid, Levels in Isolated Rat Cardiomyocytes
It is well recognized that a high dietary intake of long-chain polyunsaturated fatty acids (LC-PUFA) has profound benefits on health and prevention of chronic diseases. In particular, in recent years there has been a dramatic surge of interest in the health effects of n-3 LC-PUFA derived from fish, eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids. Notwithstanding, the metabolic fate and the effects of these fatty acids once inside the cell has seldom been comprehensively investigated. Using cultured neonatal rat cardiomyocytes as model system we have investigated for the first time, by means of high-resolution magic-angle spinning nuclear magnetic resonance (HR-MAS NMR) spectroscopy in combination with gas chromatography (GC), the modification occurring in the cell lipid environment after EPA and DHA supplementation. The most important difference between control and n-3 LC-PUFA-supplemented cardiomyocytes highlighted by HR-MAS NMR spectroscopy is the increase of signals from mobile lipids, identified as triacylglycerols (TAG). The observed increase of mobile TAG is a metabolic response to n-3 LC-PUFA supplementation, which leads to an increased lipid storage. The sequestration of mobile lipids in lipid bodies provides a deposit of stored energy that can be accessed in a regulated fashion according to metabolic need. Interestingly, while n-3 LC-PUFA supplementation to neonatal rat cardiomyocytes causes a huge variation in the cell lipid environment, it does not induce detectable modifications in water-soluble metabolites, suggesting negligible interference with normal metabolic processes.
Effect of Commercial Lignosulfonate-humate on Zea Mays L. Metabolism
Lignosulfonate-humate a and lignosulfonate-humate b, derived by an industrial process from lignin, were studied chemically and biologically, and their effects on maize metabolism compared with the responses induced by humic substances obtained from leonardite. Lignosulfonate-humate a and lignosulfonate-humate b elicited hormonelike activity and leonardite displayed giberellin properties. To improve our understanding of their biological action, lignosulfonate-humate a, lignosulfonate-humate b and leonardite were supplied to maize plants and their effect was studied on growth, nitrogen metabolism and photosynthesis. All products increased root and leaf growth. Glutamine-synthetase, glutamate-synthase enzyme activities and protein content were all increased. The treatments also increased chlorophyll content, glucose, fructose and rubisco enzyme activity, suggesting a positive role of lignosulfonate-humate a, lignosulfonate-humate b and leonardite in the photosynthetic process. In addition, an increase in phenol content was observed. In light of these results, being environmentally friendly products, lignosulfonate-humate a and lignosulfonate-humate b could be used to increase crop yield.
