A crucial and distinctive feature of tuberculosis infection is that Mycobacterium tuberculosis (Mtb) resides in granulomatous lesion at various stages of disease development and necrosis, an aspect that is little understood. We used a novel approach, applying high resolution magic angle spinning nuclear magnetic resonance spectroscopy (HRMAS NMR) directly to infected tissues, allowing us to study the development of tuberculosis granulomas in guinea pigs in an untargeted manner. Significant up-regulation of lactate, alanine, acetate, glutamate, oxidized and the reduced form of glutathione, aspartate, creatine, phosphocholine, glycerophosphocholine, betaine, trimethylamine N-oxide, myo-inositol, scyllo-inositol, and dihydroxyacetone was clearly visualized and was identified as the infection progressed. Concomitantly, phosphatidylcholine was down-regulated. Principal component analysis of NMR data revealed clear group separation between infected and uninfected tissues. These metabolites are suggestive of utilization of alternate energy sources by the infiltrating cells that generate much of the metabolites in the increasingly necrotic and hypoxic developing granuloma through the glycolytic, pentose phosphate, and tricarboxylic acid pathways. The most relevant changes seen are, surprisingly, very similar to metabolic changes seen in cancer during tumor development.
High-resolution magic-angle spinning (HR-MAS) proton NMR spectroscopy is used to explore the metabolic signatures of head and neck squamous cell carcinoma (HNSCC) which included matched normal adjacent tissue (NAT) and tumor originating from tongue, lip, larynx and oral cavity, and associated lymph-node metastatic (LN-Met) tissues. A total of 43 tissues (18 NAT, 18 Tumor and 7 LN-Met) from twenty-two HNSCC patients were analyzed. Principal Component Analysis of NMR data showed a clear classification between NAT and tumor tissues, however, LN-Met tissues were classified among tumor. A partial least squares discriminant analysis model generated from NMR metabolic profiles was used to differentiate normal from tumor samples (Q2 > 0.80, Receiver Operator Characteristic area under the curve > 0. 86, using 7-fold cross validation). HNSCC and LN-Met tissues showed elevated levels of lactate, amino acids including leucine, isoleucine, valine, alanine, glutamine, glutamate, aspartate, glycine, phenylalanine and tyrosine, choline containing compounds, creatine, taurine, glutathione and decreased levels of triglycerides. These elevated metabolites were associated with highly active glycolysis, increased amino acids influx (anaplerosis) into the TCA cycle, altered energy metabolism, membrane choline phospholipid metabolism, and oxidative and osmotic defense mechanisms. Moreover, decreased levels of triglycerides may indicate lipolysis followed by β-oxidation of fatty acids that may exist to deliver bioenergy for rapid tumor cell proliferation and growth.
Phosphatidyl-myo-inositol mannosides (PIMs) are key glycolipids of the mycobacterial cell envelope. They are considered not only essential structural components of the cell but also important molecules implicated in host-pathogen interactions. Although their chemical structures are well established, knowledge of the enzymes and sequential events leading to their biosynthesis is still incomplete. Here we show for the first time that although both mannosyltransferases PimA and PimB (MSMEG_4253) recognize phosphatidyl-myo-inositol (PI) as a lipid acceptor, PimA specifically catalyzes the transfer of a Manp residue to the 2-position of the myo-inositol ring of PI, whereas PimB exclusively transfers to the 6-position. Moreover, whereas PimB can catalyze the transfer of a Manp residue onto the PI-monomannoside (PIM 1 ) product of PimA, PimA is unable in vitro to transfer Manp onto the PIM 1 product of PimB. Further assays using membranes from Mycobacterium smegmatis and purified PimA and PimB indicated that the acylation of the Manp residue transferred by PimA preferentially occurs after the second Manp residue has been added by PimB. Importantly, genetic evidence is provided that pimB is an essential gene of M. smegmatis. Altogether, our results support a model wherein Ac 1 PIM 2 , a major form of PIMs produced by mycobacteria, arises from the consecutive action of PimA, followed by PimB, and finally the acyltransferase MSMEG_2934. The essentiality of these three enzymes emphasizes the interest of novel anti-tuberculosis drugs targeting the initial steps of PIM biosynthesis. PIMs3 are unique mannolipids found in abundant quantities in the inner and outer membranes of the cell envelope of Mycobacterium spp. and a few other actinomycetes. 4 They are based on a phosphatidyl-myo-inositol (PI) lipid anchor carrying one to six Manp residues and up to four acyl chains (for review see Refs. 1, 2). Based on a conserved mannosyl-PI anchor, they are also thought to be the precursors of the two major mycobacterial lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM) (1, 2). PIMs, LM, and LAM are considered not only essential structural components of the mycobacterial cell envelope (3-6), but also important molecules implicated in host-pathogen interactions in the course of tuberculosis and leprosy (1).Although the chemical structure of PIMs is now well established, knowledge of the enzymes and sequential events leading to their biosynthesis is still fragmentary. According to the currently accepted model, the biosynthetic pathway is initiated by the transfer of two Manp residues and a fatty acyl chain to PI in the cytoplasmic leaflet of the plasma membrane. Based on genetic and biochemical evidence, Korduláková et al. (5) identified PimA (MSMEG_2935 in Mycobacterium smegmatis mc 2 155) as the enzyme that catalyzes the first mannosylation step of the pathway transferring a Manp residue most likely to the 2-position of the myo-inositol (myo-Ins) ring of PI. In contrast, the identity of PimBЈ, the enzyme responsible for the tran...
Effective diagnosis and surveillance of Bladder Cancer (BCa) is currently challenged by detection methods that are of poor sensitivity, particularly for low-grade tumors, resulting in unnecessary invasive procedures and economic burden. We performed HR-MAS NMR-based global metabolomic profiling and applied unsupervised principal component analysis (PCA) and hierarchical clustering performed on NMR dataset of bladder derived tissues and identified metabolic signatures that differentiate BCa from benign disease. A partial least-square discriminant analysis (PLS-DA) model (leave-one-out cross-validation) was used as diagnostic model to distinguish benign and BCa tissues. Receiver operating characteristic curve generated either from PC1 loadings of PCA or from predicted Y-values resulted in an area under curve of 0.97. Relative quantification of more than fifteen tissue metabolites derived from HR-MAS NMR showed significant differences (P < 0.001) between benign and BCa samples. Noticeably, striking metabolic signatures were observed even for early stage BCa tissues (Ta-T1) demonstrating the sensitivity in detecting BCa. With the goal of cross-validating metabolic signatures derived from HR-MAS NMR, we utilized the same tissue samples to analyze eight metabolites through gas chromatography-mass spectrometry (GC-MS)-targeted analysis, which undoubtedly complements HR-MAS NMR derived metabolomic information. Cross-validation through GC-MS clearly demonstrates the utility of straightforward, non-destructive and rapid HR-MAS NMR technique for clinical diagnosis of BCa with even greater sensitivity. In addition to its utility as a diagnostic tool, these studies will lead to a better understanding of aberrant metabolic pathways in cancer as well as the design and implementation of personalized cancer therapy through metabolic modulation.
A better understanding of molecular pathways involved in malignant transformation of head and neck squamous cell carcinoma (HNSCC) is essential for the development of novel and efficient anti-cancer drugs. To delineate the global metabolism of HNSCC, we report 1H NMR-based metabolic profiling of HNSCC cells from five different patients that were derived from various sites of the upper aerodigestive tract, including the floor of mouth, tongue and larynx. Primary cultures of normal human oral keratinocytes (NHOK) from three different donors were used for comparison. 1H NMR spectra of polar and non-polar extracts of cells were used to identify more than thirty-five metabolites. Principal component analysis performed on the NMR data revealed a clear classification of NHOK and HNSCC cells. HNSCC cells exhibited significantly altered levels of various metabolites that clearly revealed dysregulation in multiple metabolic events, including Warburg effect, oxidative phosphorylation, energy metabolism, TCA cycle anaplerotic flux, glutaminolysis, hexosamine pathway, osmo-regulatory and anti-oxidant mechanism. In addition, significant alterations in the ratios of phosphatidylcholine/lysophosphatidylcholine and phosphocholine/glycerophosphocholine, and elevated arachidonic acid observed in HNSCC cells reveal an altered membrane choline phospholipid metabolism (MCPM). Furthermore, significantly increased activity of phospholipase A2 (PLA2), particularly cytosolic PLA2 (cPLA2) observed in all the HNSCC cells confirm an altered MCPM. In summary, the metabolomic findings presented here can be useful to further elucidate the biological aspects that lead to HNSCC, and also provide a rational basis for monitoring molecular mechanisms in response to chemotherapy. Moreover, cPLA2 may serve as a potential therapeutic target for anti-cancer therapy of HNSCC.
In the long term, diabetes profoundly affects multiple organs, such as the kidney, heart, brain, liver, and eyes. The gradual loss of function in these vital organs contributes to mortality. Nonetheless, the effects of diabetes on the lung tissue are not well understood. Clinical and experimental data from our studies revealed that diabetes induces inflammatory and fibrotic changes in the lung. These changes were mediated by TGF-β-activated epithelial-to-mesenchymal transition (EMT) signaling pathways. Our studies also found that glucose restriction promoted mesenchymal-to-epithelial transition (MET) and substantially reversed inflammatory and fibrotic changes, suggesting that diabetes-induced EMT was mediated in part by the effects of hyperglycemia. Additionally, the persistent exposure of diabetic cells to high glucose concentrations (25 mM) promoted the upregulation of caveolin-1, N-cadherin, SIRT3, SIRT7 and lactate levels, suggesting that long-term diabetes may promote cell proliferation. Taken together, our results demonstrate for the first time that diabetes induces fibrotic changes in the lung via TGF-β1-activated EMT pathways and that elevated SMAD7 partially protects the lung during the initial stages of diabetes. These findings have implications for the management of patients with diabetes.
The unconjugated bile acids cholic acid, deoxycholic acid, and chenodeoxycholic acid; their glycine and taurine conjugates glycocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, and taurochenodeoxycholic acid; and a taurine conjugated ursodeoxycholic acid, tauroursodeoxycholic acid, were characterized through 1H and 13C NMR in aqueous media under the physiological pH region (7.4 +/- 0.1). Assignments of 1H and 13C signals of all the bile acids were made using a combination of several one- and two-dimensional, homonuclear (1H-1H) and heteronuclear (1H-13C) correlations as well as spectral editing NMR methods. Stereochemical assignment of the five-membered ring of the bile acids is reported here for the first time. The complete characterization of various bile acids in aqueous media presented here may have implications in the study of the pathophysiology of biliary diseases through human biliary fluids using NMR spectroscopy.
With the understanding that the laboratory propagated strain of Mycobacterium tuberculosis H37Rv is of modest virulence and is drug susceptible, in the present study, we performed a nuclear magnetic resonance-based metabolomic analysis of lung tissues and serum obtained from guinea pigs infected by low dose aerosol exposure to clinical isolates of Mycobacterium tuberculosis. High Resolution Magic Angle Spinning NMR coupled with multivariate statistical analysis of 159 lung tissues obtained from multiple locations of age-matched naïve and 30 and 60 days of infected guinea pig lungs revealed a wide dispersal of metabolic patterns, but within these, distinct clusters of signatures could be seen that differentiated between naive control and infected animals. Several metabolites were identified that changed in concert with the progression of each infection. Major metabolites that could be interpreted as indicating host glutaminolysis were consistent with activated host immune cells encountering increasingly hypoxic conditions in the necrotic lung lesions. Moreover, glutathione levels were constantly elevated, probably in response to oxygen radical production in these lesions. Additional distinct signatures were also seen in infected serum, with altered levels of several metabolites. Multivariate statistical analysis clearly differentiated the infected from the uninfected sera; in addition, Receiver Operator Characteristic curve generated with principal component 1 scores showed an area under the curve of 0.908. These data raise optimism that discrete metabolomic signatures can be defined that can predict the progression of the tuberculosis disease process, and form the basis of an innovative and rapid diagnostic process.
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