Glioblastomas and brain metastases are highly proliferative brain tumors with short survival times. Previously, using 13C-NMR analysis of brain tumors resected from patients during infusion of 13C-glucose, we demonstrated that there is robust oxidation of glucose in the citric acid cycle, yet glucose contributes less than 50% of the carbons to the acetyl-CoA pool. Here we show that primary and metastatic mouse orthotopic brain tumors have the capacity to oxidize [1,2-13C]acetate and can do so simultaneously with [1,6-13C]glucose oxidation. The tumors do not oxidize [U-13C]glutamine. In vivo oxidation of [1,2-13C]acetate was validated in brain tumor patients and was correlated with expression of acetyl-CoA synthetase enzyme 2, ACSS2. Together the data demonstrate a strikingly common metabolic phenotype in diverse brain tumors that includes the ability to oxidize acetate in the citric acid cycle. This adaptation may be important for meeting the high biosynthetic and bioenergetic demands of malignant growth.

Clear cell renal cell carcinoma (ccRCC) is the most common form of human kidney cancer. Histological and molecular analyses suggest that ccRCCs have significantly altered metabolism. Recent human studies of lung cancer and intracranial malignancies demonstrated an unexpected preservation of carbohydrate oxidation in the tricarboxylic acid (TCA) cycle. To test the capacity of ccRCC to oxidize substrates in the TCA cycle, we infused C-labeled fuels in ccRCC patients and compared labeling patterns in tumors and adjacent kidney. After infusion with [U-C]glucose, ccRCCs displayed enhanced glycolytic intermediate labeling, suppressed pyruvate dehydrogenase flow, and reduced TCA cycle labeling, consistent with the Warburg effect. Comparing C labeling among ccRCC, brain, and lung tumors revealed striking differences. Primary ccRCC tumors demonstrated the highest enrichment in glycolytic intermediates and lowest enrichment in TCA cycle intermediates. Among human tumors analyzed by intraoperativeC infusions, ccRCC is the first to demonstrate a convincing shift toward glycolytic metabolism.

The contributions of backbone NH group dynamics to the conformational heat capacity of the B1 domain of Streptococcal protein G have been estimated from the temperature dependence of 15 N NMR-derived order parameters. Longitudinal~R 1 ! and transverse~R 2 ! relaxation rates, transverse cross-relaxation rates~h xy !, and steady state $ 1 H%-15 N nuclear Overhauser effects were measured at temperatures of 0, 10, 20, 30, 40, and 50 8C for 89-100% of the backbone secondary amide nitrogen nuclei in the B1 domain. The ratio R 2 0h xy was used to identify nuclei for which conformational exchange makes a significant contribution to R 2 . Relaxation data were fit to the extended model-free dynamics formalism, incorporating an axially symmetric molecular rotational diffusion tensor. The temperature dependence of the order parameter~S 2 ! was used to calculate the contribution of each NH group to conformational heat capacity~C p ! and a characteristic temperature~T *!, representing the density of conformational energy states accessible to each NH group. The heat capacities of the secondary structure regions of the B1 domain are significantly higher than those of comparable regions of other proteins, whereas the heat capacities of less structured regions are similar to those in other proteins. The higher local heat capacities are estimated to contribute up to ;0.8 kJ0mol K to the total heat capacity of the B1 domain, without which the denaturation temperature would be ;9 8C lower~78 8C rather than 87 8C!. Thus, variation of backbone conformational heat capacity of native proteins may be a novel mechanism that contributes to high temperature stabilization of proteins.Keywords: B1 domain; entropy; heat capacity; NMR relaxation; order parameter; protein dynamics; protein stability Most globular proteins are marginally stable because the factors that favor formation of the native state, primarily desolvation of hydrophobic groups and formation of intramolecular hydrogen bonds and salt bridges, are almost equally balanced against those that favor denaturation, primarily the higher conformational entropy of the unfolded protein chain relative to that of the native state~Creigh-ton, 1993; Fersht, 1999!. At high temperatures, the balance between these factors is altered such that many proteins exhibit reversible thermal denaturation with a characteristic midpoint, the melting temperature~T m !. The free energy of unfolding of a proteiñ DG NϪU ! depends upon the changes in enthalpy, entropy, and heat capacity that occur upon unfolding and the temperature~T ! according to the equation~Creighton, 1993; Fersht, 1999!:in which DH 0 and DS 0 are the enthalpy and entropy changes, respectively, at a reference temperature T 0 and DC p,NϪU is the change in heat capacity at constant pressure, which is assumed to be invariant with temperature. Equation 1 indicates that native proteins may be stabilized by an increase in DH 0 or by a decrease in either DS 0 or DC p,NϪU . Thus, one possible strategy for a protein to achieve high thermal ...

2-hydroxyglutarate (2HG) is produced in gliomas with mutations of isocitrate dehydrogenases (IDH) 1 and 2. The 1H resonances of the J-coupled spins of 2HG are extensively overlapped with signals from other metabolites. Here we report a comparative study at 3T of the utility of the PRESS (point-resolved spectroscopy) sequence with a standard short TE (35 ms) and a long TE (97 ms) which had been theoretically designed for detecting the 2HG 2.25 ppm resonance. The performance of the methods is evaluated using data from phantoms, 7 healthy volunteers, and 22 subjects with IDH-mutated gliomas. The results indicate that TE = 97 ms provides higher detectability of 2HG than TE = 35 ms, and that this improved capability is gained when data are analyzed with basis spectra that include the effects of the volume localizing radio-frequency and gradient pulses.

A major rate-limiting step in developing more effective immunotherapies for GBM is our inadequate understanding of the cellular complexity and the molecular heterogeneity of immune infiltrates in gliomas. Here, we report an integrated analysis of 201,986 human glioma, immune, and other stromal cells at the single cell level. In doing so, we discover extensive spatial and molecular heterogeneity in immune infiltrates. We identify molecular signatures for nine distinct myeloid cell subtypes, of which five are independent prognostic indicators of glioma patient survival. Furthermore, we identify S100A4 as a regulator of immune suppressive T and myeloid cells in GBM and demonstrate that deleting S100a4 in non-cancer cells is sufficient to reprogram the immune landscape and significantly improve survival. This study provides insights into spatial, molecular, and functional heterogeneity of glioma and glioma-associated immune cells and demonstrates the utility of this dataset for discovering therapeutic targets for this poorly immunogenic cancer.

The synchronization (correlation) of conformational fluctuations in folded proteins may influence the rates of enzyme catalysis and ligand binding as well as the stabilities of native proteins and their complexes. However, experimental detection of correlated motions remains difficult. Herein, we present an analysis of the covariation of NMR-derived backbone dynamical parameters among a family of ten mutants of a small protein. Both the spatial restriction and the time scales of backbone motions exhibit a higher degree of covariation than would be expected if the internal motions of each group were independent, providing experimental support for correlated dynamics. Application of this approach to other proteins may reveal dynamical correlations that influence catalysis, ligand-binding and/or protein stability.

Glucose is the ultimate substrate for most brain activities that use carbon, including synthesis of the neurotransmitters glutamate and γ-aminobutyric acid via mitochondrial tricarboxylic acid (TCA) cycle. Brain metabolism and neuronal excitability are thus interdependent. However, the principles that govern their relationship are not always intuitive because heritable defects of brain glucose metabolism are associated with the paradoxical coexistence, in the same individual, of episodic neuronal hyperexcitation (seizures) with reduced basal cerebral electrical activity. One such prototypic disorder is pyruvate dehydrogenase (PDH) deficiency (PDHD). PDH is central to metabolism because it steers most of the glucose-derived flux into the TCA cycle. To better understand the pathophysiology of PDHD, we generated mice with brain-specific reduced PDH activity that paralleled salient human disease features, including cerebral hypotrophy, decreased amplitude electroencephalogram (EEG), and epilepsy. The mice exhibited reductions in cerebral TCA cycle flux, glutamate content, spontaneous, and electrically evoked in vivo cortical field potentials and gamma EEG oscillation amplitude. Episodic decreases in gamma oscillations preceded most epileptiform discharges, facilitating their prediction. Fast-spiking neuron excitability was decreased in brain slices, contributing to in vivo action potential burst prolongation after whisker pad stimulation. These features were partially reversed after systemic administration of acetate, which augmented cerebral TCA cycle flux, glutamate-dependent synaptic transmission, inhibition and gamma oscillations, and reduced epileptiform discharge duration. Thus, our results suggest that dysfunctional excitability in PDHD is consequent to reduced oxidative flux, which leads to decreased neuronal activation and impaired inhibition, and can be mitigated by an alternative metabolic substrate.

Chemical shift anisotropy (CSA) tensors are essential in the structural and dynamic studies of proteins using NMR spectroscopy. Results from relaxation studies in biomolecular solution and solid-state NMR experiments on aligned samples are routinely interpreted using well-characterized CSA tensors determined from model compounds. Since CSA tensors, particularly the 15N CSA, highly depend on a number of parameters including secondary structure, electrostatic interaction and the amino acid sequence, there is a need for accurately determined CSA tensors from proteins. In this study we report the backbone amide-15N CSA tensors for a 16.7-kDa membrane-bound and paramagnetic-heme containing protein, rabbit cytochrome b5 (cytb5), determined using the 15N CSA/15N-1H dipolar transverse cross-correlation rates. The mean values of 15N CSA determined for residues in helical, sheet and turn regions are −187.9, −166.0, and −161.1 ppm, respectively, with an overall average value of −171.7 ppm. While the average CSA value determined from this study is in good agreement with previous solution NMR experiments on small globular proteins, the CSA value determined for residues in helical conformation is slightly larger which may be attributed to the paramagnetic effect from Fe(III) of the heme unit in cytb5. However, like in previous solution NMR studies, the CSA values reported in this study are larger than the values measured from solid-state NMR experiments. We believe that the CSA parameters reported in this study will be useful in determining the structure, dynamics and orientation of proteins, including membrane proteins, using NMR spectroscopy.