Sex differences in brain tumor glutamine metabolism reveal sex-specific vulnerabilities to treatment.

BACKGROUND: Brain cancer incidence and mortality rates are greater in males. Understanding the molecular mechanisms that underlie those sex differences could improve treatment strategies. Although sex differences in normal metabolism are well described, it is currently unknown whether they persist in cancerous tissue. METHODS: Using positron emission tomography (PET) imaging and mass spectrometry, we assessed sex differences in glioma metabolism in samples from affected individuals. We assessed the role of glutamine metabolism in male and female murine transformed astrocytes using isotope labeling, metabolic rescue experiments, and pharmacological and genetic perturbations to modulate pathway activity. FINDINGS: We found that male glioblastoma surgical specimens are enriched for amino acid metabolites, including glutamine. Fluoroglutamine PET imaging analyses showed that gliomas in affected male individuals exhibit significantly higher glutamine uptake. These sex differences were well modeled in murine transformed astrocytes, in which male cells imported and metabolized more glutamine and were more sensitive to glutaminase 1 (GLS1) inhibition. The sensitivity to GLS1 inhibition in males was driven by their dependence on glutamine-derived glutamate for α-ketoglutarate synthesis and tricarboxylic acid (TCA) cycle replenishment. Females were resistant to GLS1 inhibition through greater pyruvate carboxylase (PC)-mediated TCA cycle replenishment, and knockdown of PC sensitized females to GLS1 inhibition. CONCLUSION: Our results show that clinically important sex differences exist in targetable elements of metabolism. Recognition of sex-biased metabolism may improve treatments through further laboratory and clinical research. FUNDING: This work was supported by NIH grants, Joshua's Great Things, the Siteman Investment Program, and the Barnard Research Fund.

Opioid-Prescribing Practices Between Total Knee and Hip Arthroplasty in an Outpatient Versus Inpatient Setting.

ABSTRACT: Despite trends showing increases in the utilization of outpatient (OP) ambulatory surgery centers (ASCs) and decreases in the utilization of inpatient (IP) facilities for total knee arthroplasty (TKA) and total hip arthroplasty (THA), little is known about opioid prescribing for these procedures between each setting. This study evaluated differences in opioid prescribing and consumption between OP ASC and IP settings for elective TKA and THA surgeries over a 1-year period. Data collection also included pain and satisfaction of pain control postsurgery. In an OP ASC, analysis revealed a significant decrease in pills prescribed (p < .001, p < .001) and consumed (p < .001, p < .001) for TKA and THA, respectively. There was a significant decrease in the morphine equivalence units prescribed (p < .001, p < .001) and consumed (p < .001, p < .001) for TKA and THA, respectively. For TKA, pain was significantly lower (p = .018) and satisfaction of pain control was significantly higher (p = .007). For THA, pain (p = .374) and satisfaction of pain control (p = .173) were similar between the settings. Benefits of performing these surgeries in an OP ASC setting are patients having similar or lower levels of pain and having similar or higher satisfaction of pain control. Patients are also prescribed and consume less opioids. This has important implications for healthcare systems.

Implementation of formal prescription guidelines reduces the economic cost of unused opioids after orthopedic surgery.

BACKGROUND: Prior literature has reported on the concerning emergence of opioid overprescribing, yet there remains a lack of knowledge in understanding the cost of waste of this over-prescription and underconsumption of opioids. As such, further investigating the cost of waste of opioids following orthopedic surgery is of interest to patients, providers, and payors. In one of the largest private orthopedic practices in the United States, opioid prescribing and consumption patterns were tracked prior to, and after the implementation of, formal prescription guidelines. OBJECTIVES: To (1) establish the cost of waste of unused opioids before the implementation of formal prescription guidelines and (2) examine how the cost of unused opioids may be reduced after implementation of formal internal prescription guidelines. METHODS: Two separate phases (Phase I and Phase II) were implemented at different time intervals throughout a two-year period. Implementation of prescription guidelines occurred between Phases I and II, and data from Phase I (pre-implementation) was compared to that from Phase II (postimplementation). Data collection included type, dosage, quantity of opioids prescribed and consumed after elective outpatient procedures in ambulatory surgery centers, in addition to patient interviews/surveys within two weeks after surgery to measure consumption. From these data, the cost of waste was calculated by taking the total cost of prescribed opioids (sum of each prescription × Average Wholesale Price (AWP) minus 60%) per 1,000 patients, and subtracting the total cost of consumed opioids per 1,000 patients, calculated in a similar manner. Further analysis was performed to describe differences in the cost of waste of individual opioids between each of the phases. RESULTS: In Phase I, prior to implementation of formal internal prescription guidelines, there was a sizable cost of waste of unused opioids (per 1,000 patients, AWP minus 60%) of $11,299.51. The cost of waste in Phase II, after implementation of formal internal prescription guidelines, was $6,117.12, which was a significant decrease of 45.9% ($5,182.39) from Phase I (P < 0.001). Furthermore, both the average number of morphine equivalent units prescribed and consumed per patient decreased from Phase I to Phase II (294.6 vs 187.8, P < 0.001; and 144.9 vs 96.0, P < 0.001, respectively). Finally, in describing individual medications, there was a significant decrease in cost of waste (per 1,000 patients, AWP minus 60%) between Phases I and II for- Hydrocodone with APAP 5/525 mg (P< 0.001), Oxycodone CR 10 mg (P< 0.001), Morphine CR 15 mg (P=0.001), and Tramadol 50 mg (P = 0.014). CONCLUSIONS: The results of this study suggest that there is a significant cost of waste associated with differences in prescribed versus consumed opioids following elective orthopedic surgery. This cost of waste was significantly reduced following the introduction and implementation of formal prescription guidelines. DISCLOSURES: This study was funded internally by Revo Health and Twin Cities Orthopedics. Giveans reports consulting fees from Medtrak, Inc., and Superior Medical Experts. The other authors have nothing to disclose.

Left ventricle assist device pulsatility index at the time of implantation is associated with follow-up pulmonary hemodynamics.

HeartMate II left ventricular assist device controllers provide data including pulsatility index, reflecting the relationship between pump function and hemodynamics. We propose that a higher pulsatility index at hospital discharge following implant may be associated with less vascular congestion and improved clinical outcomes. A retrospective analysis of 40 patients (age 59.2 ± 10.3 years) supported with the HeartMate II devices was conducted. Data revealed moderate Pearson correlations between pulsatility index at discharge and right atrial pressure, pulmonary artery systolic pressure, pulmonary artery diastolic pressure, mean pulmonary arterial pressure, and pulmonary capillary wedge pressure, respectively, post-surgery (median of 377 days), demonstrating a stronger relationship when analyzed for the EPC controller (n = 28) only (r = -.57, p < .01; r = -.38, p < .05; r = -.59, p < .01; r = -.47, p = .01 and r = -.53, p < .01, respectively). The pulsatility index derived from the EPC controller was associated with the significant risk of re-hospitalization within 1 and 2 years after the implantation of left ventricular assist device; hazard ratio = 0.557 with 95% confidence interval (0.315-0.983), p = .04 and hazard ratio = .579 (0.341-0.984), p = .04. A higher pulsatility index at discharge was associated with greater volume unloading, lower pulmonary pressures, and lower risk of all-cause re-hospitalizations within 1 and 2 years post-surgery. As such, pump-derived data may provide additional value in predicting left ventricular assist device hemodynamics.

REDOR NMR Reveals Multiple Conformers for a Protein Kinase C Ligand in a Membrane Environment.

Bryostatin 1 (henceforth bryostatin) is in clinical trials for the treatment of Alzheimer's disease and for HIV/AIDS eradication. It is also a preclinical lead for cancer immunotherapy and other therapeutic indications. Yet nothing is known about the conformation of bryostatin bound to its protein kinase C (PKC) target in a membrane microenvironment. As a result, efforts to design more efficacious, better tolerated, or more synthetically accessible ligands have been limited to structures that do not include PKC or membrane effects known to influence PKC-ligand binding. This problem extends more generally to many membrane-associated proteins in the human proteome. Here, we use rotational-echo double-resonance (REDOR) solid-state NMR to determine the conformations of PKC modulators bound to the PKCδ-C1b domain in the presence of phospholipid vesicles. The conformationally limited PKC modulator phorbol diacetate (PDAc) is used as an initial test substrate. While unanticipated partitioning of PDAc between an immobilized protein-bound state and a mobile state in the phospholipid assembly was observed, a single conformation in the bound state was identified. In striking contrast, a bryostatin analogue (bryolog) was found to exist exclusively in a protein-bound state, but adopts a distribution of conformations as defined by three independent distance measurements. The detection of multiple PKCδ-C1b-bound bryolog conformers in a functionally relevant phospholipid complex reveals the inherent dynamic nature of cellular systems that is not captured with single-conformation static structures. These results indicate that binding, selectivity, and function of PKC modulators, as well as the design of new modulators, are best addressed using a dynamic multistate model, an analysis potentially applicable to other membrane-associated proteins.

Mechanistic Basis for ATP-Dependent Inhibition of Glutamine Synthetase by Tabtoxinine-ß-lactam.

Tabtoxinine-ß-lactam (TßL), also known as wildfire toxin, is a time- and ATP-dependent inhibitor of glutamine synthetase produced by plant pathogenic strains of Pseudomonas syringae. Here we demonstrate that recombinant glutamine synthetase from Escherichia coli phosphorylates the C3-hydroxyl group of the TßL 3-(S)-hydroxy-ß-lactam (3-HßL) warhead. Phosphorylation of TßL generates a stable, noncovalent enzyme-ADP-inhibitor complex that resembles the glutamine synthetase tetrahedral transition state. The TßL ß-lactam ring remains intact during enzyme inhibition, making TßL mechanistically distinct from traditional ß-lactam antibiotics such as penicillin. Our findings could enable the design of new 3-HßL transition state inhibitors targeting enzymes in the ATP-dependent carboxylate-amine ligase superfamily with broad therapeutic potential in many disease areas.

Lactate metabolism is associated with mammalian mitochondria.

It is well established that lactate secreted by fermenting cells can be oxidized or used as a gluconeogenic substrate by other cells and tissues. It is generally assumed, however, that within the fermenting cell itself, lactate is produced to replenish NAD+ and then is secreted. Here we explore the possibility that cytosolic lactate is metabolized by the mitochondria of fermenting mammalian cells. We found that fermenting HeLa and H460 cells utilize exogenous lactate carbon to synthesize a large percentage of their lipids. Using high-resolution mass spectrometry, we found that both 13C and 2-2H labels from enriched lactate enter the mitochondria. The lactate dehydrogenase (LDH) inhibitor oxamate decreased respiration of isolated mitochondria incubated in lactate, but not of isolated mitochondria incubated in pyruvate. Additionally, transmission electron microscopy (TEM) showed that LDHB localizes to the mitochondria. Taken together, our results demonstrate a link between lactate metabolism and the mitochondria of fermenting mammalian cells.

Exogenous Fatty Acids Are the Preferred Source of Membrane Lipids in Proliferating Fibroblasts.

Cellular proliferation requires the formation of new membranes. It is often assumed that the lipids needed for these membranes are synthesized mostly de novo. Here, we show that proliferating fibroblasts prefer to take up palmitate from the extracellular environment over synthesizing it de novo. Relative to quiescent fibroblasts, proliferating fibroblasts increase their uptake of palmitate, decrease fatty acid degradation, and instead direct more palmitate to membrane lipids. When exogenous palmitate is provided in the culture media at physiological concentrations, de novo synthesis accounts for only a minor fraction of intracellular palmitate in proliferating fibroblasts as well as proliferating HeLa and H460 cells. Blocking fatty acid uptake decreased the proliferation rate of fibroblasts, HeLa, and H460 cells, while supplementing media with exogenous palmitate resulted in decreased glucose uptake and rendered cells less sensitive to glycolytic inhibition. Our results suggest that cells scavenging exogenous lipids may be less susceptible to drugs targeting glycolysis and de novo lipid synthesis.

Fluctuating asymmetry in Menidia beryllina before and after the 2010 Deepwater Horizon oil spill.

Assessing the impacts of the Deepwater Horizon oil spill with a dependable baseline comparison can provide reliable insight into environmental stressors on organisms that were potentially affected by the spill. Fluctuating asymmetry (small, non-random deviations from perfect bilateral symmetry) is an informative metric sensitive to contaminants that can be used to assess environmental stress levels. For this study, the well-studied and common Gulf of Mexico estuarine fish, Menidia beryllina, was used with pre and post-oil spill collections. Comparisons of fluctuating asymmetry in three traits (eye diameter, pectoral fin length, and pelvic fin length) were made pre and post-oil spill across two sites (Old Fort Bayou and the Pascagoula River), as well as between years of collection (2011, 2012)--one and two years, respectfully, after the spill in 2010. We hypothesized that fluctuating asymmetry would be higher in post-Deepwater Horizon samples, and that this will be replicated in both study areas along the Mississippi Gulf coast. We also predicted that fluctuating asymmetry would decrease through time after the oil spill as the oil decomposed and/or was removed. Analyses performed on 1135 fish (220 pre and 915 post Deepwater Horizon) showed significantly higher post spill fluctuating asymmetry in the eye but no difference for the pectoral or pelvic fins. There was also higher fluctuating asymmetry in one of the two sites both pre and post-spill, indicating observed asymmetry may be the product of multiple stressors. Fluctuating asymmetry decreased in 2012 compared to 2011. Fluctuating asymmetry is a sensitive measure of sub lethal stress, and the observed variability in this study (pre vs. post-spill or between sites) could be due to a combination of oil, dispersants, or other unknown stressors.

Differential incorporation of glucose into biomass during Warburg metabolism.

It is well established that most cancer cells take up an increased amount of glucose relative to that taken up by normal differentiated cells. The majority of this glucose carbon is secreted from the cell as lactate. The fate of the remaining glucose carbon, however, has not been well-characterized. Here we apply a novel combination of metabolomic technologies to track uniformly labeled glucose in HeLa cancer cells. We provide a list of specific intracellular metabolites that become enriched after being labeled for 48 h and quantitate the fraction of consumed glucose that ends up in proteins, peptides, sugars/glycerol, and lipids.

Characterization of structural variations in the peptidoglycan of vancomycin-susceptible Enterococcus faecium: understanding glycopeptide-antibiotic binding sites using mass spectrometry.

Enterococcus faecium, an opportunistic pathogen that causes a significant number of hospital-acquired infections each year, presents a serious clinical challenge because an increasing number of infections are resistant to the so-called antibiotic of last resort, vancomycin. Vancomycin and other new glycopeptide derivatives target the bacterial cell wall, thereby perturbing its biosynthesis. To help determine the modes of action of glycopeptide antibiotics, we have developed a bottom-up mass spectrometry approach complemented by solid-state nuclear magnetic resonance (NMR) to elucidate important structural characteristics of vancomycin-susceptible E. faecium peptidoglycan. Using accurate-mass measurements and integrating ion-current chromatographic peaks of digested peptidoglycan, we identified individual muropeptide species and approximated the relative amount of each. Even though the organism investigated is susceptible to vancomycin, only 3% of the digested peptidoglycan has the well-known D-Ala-D-Ala vancomycin-binding site. The data are consistent with a previously proposed template model of cell-wall biosynthesis where D-Ala-D-Ala stems that are not cross-linked are cleaved in mature peptidoglycan. Additionally, our mass-spectrometry approach allowed differentiation and quantification of muropeptide species seen as unresolved chromatographic peaks. Our method provides an estimate of the extent of muropeptides containing O-acetylation, amidation, hydroxylation, and the number of species forming cyclic imides. The varieties of muropeptides on which the modifications are detected suggest that significant processing occurs in mature peptidoglycan where several enzymes are active in editing cell-wall structure.

Rotational-echo double-resonance NMR distance measurements for the tubulin-bound Paclitaxel conformation.

The important anticancer drug Taxol (paclitaxel, PTX) owes its unique activity to its ability to bind to tubulin in a stoichiometric ratio and promote its assembly into microtubules. The conformation of the microtubule-bound drug has been the focus of numerous research efforts, since the inability of polymerized tubulin to form crystals precludes structure proof by X-ray crystallography. Likewise, although the alpha,beta-tubulin dimer structure has been solved by electron crystallography, the 3.7 A resolution is too low to permit direct determination of either ligand conformation or binding pose. In this article, we present experimental results from 2H{19F} REDOR NMR that provide direct confirmation that paclitaxel adopts a T-shaped conformation when it is bound to tubulin.

Peptide antibiotics in action: investigation of polypeptide chains in insoluble environments by rotational-echo double resonance.

Rotational-echo double resonance (REDOR) is a solid-state NMR technique that has the capability of providing intra- and intermolecular distance and orientational restraints in non-crystallizable, poorly soluble heterogeneous molecular systems such as cell membranes and cell walls. In this review, we will present two applications of REDOR: the investigation of a magainin-related antimicrobial peptide in lipid bilayers and the study of a vancomycin-like glycopeptide in the cell walls of Staphylococcus aureus.

Secondary structure and lipid contact of a peptide antibiotic in phospholipid bilayers by REDOR.

The chemical shifts of specific (13)C and (15)N labels distributed throughout KIAGKIA-KIAGKIA-KIAGKIA (K3), an amphiphilic 21-residue antimicrobial peptide, prove that the peptide is in an all alpha-helical conformation in the bilayers of multilamellar vesicles (MLVs) containing dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylglycerol (1:1). Rotational-echo double-resonance (REDOR) (13)C[(31)P] and (15)N[(31)P] experiments on the same labeled MLVs show that on partitioning into the bilayer, the peptide chains remain in contact with lipid headgroups. The amphipathic lysine side chains of K3 in particular appear to play a key role in the electrostatic interactions with the acidic lipid headgroups. In addition to the extensive peptide-headgroup contact, (13)C[(19)F] REDOR experiments on MLVs containing specifically (19)F-labeled lipid tails suggest that a portion of the peptide is surrounded by a large number of lipid acyl chains. Complementary (31)P[(19)F] REDOR experiments on these MLVs show an enhanced headgroup-lipid tail contact resulting from the presence of K3. Despite these distortions, static (31)P NMR lineshapes indicate that the lamellar structure of the membrane is preserved.

Structure of (KIAGKIA)3 aggregates in phospholipid bilayers by solid-state NMR.

The interchain (13)C-(19)F dipolar coupling measured in a rotational-echo double-resonance (REDOR) experiment performed on mixtures of differently labeled KIAGKIA-KIAGKIA-KIAGKIA (K3) peptides (one specifically (13)C labeled, and the other specifically (19)F labeled) in multilamellar vesicles of dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylglycerol (1:1) shows that K3 forms close-packed clusters, primarily dimers, in bilayers at a lipid/peptide molar ratio (L/P) of 20. Dipolar coupling to additional peptides is weaker than that within the dimers, consistent with aggregates of monomers and dimers. Analysis of the sideband dephasing rates indicates a preferred orientation between the peptide chains of the dimers. The combination of the distance and orientation information from REDOR is consistent with a parallel (N-N) dimer structure in which two K3 helices intersect at a cross-angle of approximately 20 degrees. Static (19)F NMR experiments performed on K3 in oriented lipid bilayers show that between L/P = 200 and L/P = 20, K3 chains change their absolute orientation with respect to the membrane normal. This result suggests that the K3 dimers detected by REDOR at L/P = 20 are not on the surface of the bilayer but are in a membrane pore.