Single-cell analysis of human glioma and immune cells identifies S100A4 as an immunotherapy target.

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

Proton Magnetic Resonance Spectroscopy Characterization of Rathke’s Cleft Cysts (RCCs): Relevance to the Differential Diagnosis of Pituitary Adenomas and RCCs

Background: Rathke’s Cleft Cysts (RCCs) are rare epithelial cysts arising from remnants of the Rathke pouch in the pituitary gland. A subset of these lesions enlarge and produce a mass effect with consequent hypopituitarism, and may result in visual loss. Moreover, some RCCs with a high intra-cystic protein content may mimic cystic pituitary adenoma, which makes their differential diagnosis ambiguous. Currently, medical professionals have no definitive way to distinguish RCCs from pituitary adenomas. Therefore, preoperative confirmation of RCCs would be of help to medical professionals for the management and proper surgical decision making. The goal of this study is to identify molecular markers in RCCs. Methods: We characterized aqueous and chloroform extracts of surgically resected RCCs and pituitary adenomas using ex vivo 1H NMR spectroscopy. Results: All RCCs exclusively showed the presence of mucopolysaccharides which are glycosaminoglycans (GAGs) made up of disaccharides of aminosugars and uronic sugars. Conclusion: GAGs can be used as metabolite marker for the detection of RCCs and this knowledge will lay the groundwork for the development of a non-invasive, in vivo magnetic resonance spectroscopy methodology for the differential diagnosis of RCCs and pituitary adenomas using clinical MRI scanners

Effects of hydrogen bonding on amide-proton chemical shift anisotropy in a proline-containing model peptide

Longitudinal relaxation due to cross-correlation between dipolar ((HN-1H alpha)-H-1) and amide-proton chemical shift anisotropy (H-1(N) CSA) has been measured in a model tripeptide Piv-(L)Pro-(L)Pro-(L)Phe-OMe. The peptide bond across diproline segment is known to undergo cis/trans isomerization and only in the cis form does the lone Phe amide-proton become involved in intramolecular hydrogen bonding. The strength of the cross correlated relaxation interference is found to be significantly different between cis and trans forms, and this difference is shown as an influence of intramolecular hydrogen bonding on the amide-proton CSA. (C) 2015 Elsevier B.V. All rights reserved

Determination of ¹⁵N Chemical Shift Anisotropy from a Membrane-Bound Protein by NMR Spectroscopy

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 <i>b</i>₅ (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

Evaluation of surface properties of low density polyethylene (LDPE) films tailored by atmospheric pressure non-thermal plasma (APNTP) assisted co-polymerization and immobilization of chitosan for improvement of antifouling properties

This work describes the development of antifouling functional coatings on the surface of low density polyethylene (LDPE) films by means of atmospheric pressure non-thermal plasma (APNTP) assisted copolymerization using a mixture of acrylic acid and poly (ethylene glycol). The aim of the study was to investigate the antifouling properties of the plasma copolymerized LDPE films and the same was carried out as a function of deposition time with fixed applied potential of 14 kV. In a second stage, the plasma copolymerized LDPE films were functionalized with chitosan (CHT) to further enhance its antifouling properties. The surface hydrophilicity, structural, topographical and chemistry of the plasma copolymerized LDPE films were examined by contact angle (CA), X-ray diffraction (XRD), atomic force microscopy (AFM), Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). Coating stability was also studied in detail over a storage time of 15 days by storing in water and air. The antifouling properties of the plasma copolymerized LDPE films were examined via protein adsorption and platelet adhesion studies. CA study showed significant changes in surface wettability after the coating process. XPS and FTIR analysis proved the presence of a dense multifunctional coating and an efficient immobilization of CHT. Substantial amendments in surface topography were observed, positively enhancing the overall surface hydrophilicity. Finally, in-vitro analysis showed excellent antifouling behavior of the surface modified LDPE films

Acetate Is a Bioenergetic Substrate for Human Glioblastoma and Brain Metastases

SummaryGlioblastomas 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 while simultaneously oxidizing [1,6-13C]glucose. 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

Novel application of complementary imaging techniques to examine in vivo glucose metabolism in the kidney

The metabolic status of the kidney is a determinant of injury susceptibility and a measure of progression for many disease processes; however, noninvasive modalities to assess kidney metabolism are lacking. In this study, we employed positron emission tomography (PET) and intravital multiphoton microscopy (MPM) to assess cortical and proximal tubule glucose tracer uptake, respectively, following experimental perturbations of kidney metabolism. Applying dynamic image acquisition PET with 2-(18)fluoro-2-deoxyglucose ((18)F-FDG) and tracer kinetic modeling, we found that an intracellular compartment in the cortex of the kidney could be distinguished from the blood and urine compartments in animals. Given emerging literature that the tumor suppressor protein p53 is an important regulator of cellular metabolism, we demonstrated that PET imaging was able to discern a threefold increase in cortical (18)F-FDG uptake following the pharmacological inhibition of p53 in animals. Intravital MPM with the fluorescent glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG) provided increased resolution and corroborated these findings at the level of the proximal tubule. Extending our observation of p53 inhibition on proximal tubule glucose tracer uptake, we demonstrated by intravital MPM that pharmacological inhibition of p53 diminishes mitochondrial potential difference. We provide additional evidence that inhibition of p53 alters key metabolic enzymes regulating glycolysis and increases intermediates of glycolysis. In summary, we provide evidence that PET is a valuable tool for examining kidney metabolism in preclinical and clinical studies, intravital MPM is a powerful adjunct to PET in preclinical studies of metabolism, and p53 inhibition alters basal kidney metabolism