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The global HIV/AIDS epidemic still currently affects approximately 38 million individuals globally. The protease enzyme of the human immunodeficiency virus is a major drug target in antiviral therapy, however, under the influence of reverse transcriptase and in the context of drug pressure, the rapid PR mutation rate contributes significantly to clinical failure. The set of cooperative non-active site mutations, I13V/I62V/V77I, have been associated with reduced inhibitor susceptibility and are the focus of the current study. When compared to the wild-type protease the mutant protease exhibited decreased binding affinities towards ATV and DRV by 64- and 12-fold, respectively, and decreased the overall favourable Gibbs free energy for ATV, DRV, RTV and SQV. Moreover, these mutations decreased the thermal stability of the protease when in complex with ATV and DRV by approximately 6.4 and 4.2 °C, respectively. The crystal structure of the mutant protease revealed that the location of these mutations and their effect on the hydrophobic sliding mechanism may be crucial in their role in resistance.

Summary statistics for kpl, the apparent rate of conversion for HP Pyruvate to lactate, under various measurement conditions and parameter constraints due to differing prior information.

Hyperpolarized [1-13C]-pyruvate has shown tremendous promise as an agent for imaging tumor metabolism with unprecedented sensitivity and specificity. Imaging hyperpolarized substrates by magnetic resonance is unlike traditional MRI because signals are highly transient and their spatial distribution varies continuously over their observable lifetime. Therefore, new imaging approaches are needed to ensure optimal measurement under these circumstances. Constrained reconstruction algorithms can integrate prior information, including biophysical models of the substrate/target interaction, to reduce the amount of data that is required for image analysis and reconstruction. In this study, we show that metabolic MRI with hyperpolarized pyruvate is biased by tumor perfusion and present a new pharmacokinetic model for hyperpolarized substrates that accounts for these effects. The suitability of this model is confirmed by statistical comparison with alternates using data from 55 dynamic spectroscopic measurements in normal animals and murine models of anaplastic thyroid cancer, glioblastoma, and triple-negative breast cancer. The kinetic model was then integrated into a constrained reconstruction algorithm and feasibility was tested using significantly undersampled imaging data from tumor-bearing animals. Compared with naïve image reconstruction, this approach requires far fewer signal-depleting excitations and focuses analysis and reconstruction on new information that is uniquely available from hyperpolarized pyruvate and its metabolites, thus improving the reproducibility and accuracy of metabolic imaging measurements. Cancer Res; 75(22); 4708–17. ©2015 AACR.

Animation of the dynamic conversion of hyperpolarized pyruvate into lactate in a murine model of anaplastic thyroid cancer. Heat maps for (left) HP pyruvate and (right) HP lactate overlay T2-weighted MRI. This animation can be estimated with arbitrary temporal resolution following model-based reconstruction from highly undersampled data.

Magnetic resonance imaging is a powerful clinical imaging technique that allows for noninvasive tomographic visualization of anatomic structures with high spatial resolution and soft tissue contrast. However, its application in molecular imaging of cancer has been limited by the lack of sensitivity and detection accuracy in depicting the biochemical expression of these diseases. Here, we combine an ultrasensitive design of superparamagnetic polymeric micelles (SPPM) and an off-resonance saturation (ORS) method to enhance the imaging efficacy of tumor biomarkers in vivo. SPPM nanoparticles encoded with cyclic(RGDfK) were able to target the αvβ3-expressing microvasculature in A549 non–small cell lung tumor xenografts in mice. ORS greatly improved tumor detection accuracy over the conventional T2*-weighted method by its ability to turn “ON” the contrast of SPPM. This combination of ORS imaging with a tumor vasculature–targeted, ultrasensitive SPPM design offers new opportunities in molecular imaging of cancer. [Cancer Res 2009;69(4):1651–8]

Cellular redox is intricately linked to energy production and normal cell function. Although the redox states of mitochondria and cytosol are connected by shuttle mechanisms, the redox state of mitochondria may differ from redox in the cytosol in response to stress. However, detecting these differences in functioning tissues is difficult. Here, we employed 13C magnetic resonance spectroscopy (MRS) and co-polarized [1-13C]pyruvate and [1,3-13C2]acetoacetate ([1,3-13C2]AcAc) to monitor production of hyperpolarized (HP) lactate and β-hydroxybutyrate as indicators of cytosolic and mitochondrial redox, respectively. Isolated rat hearts were examined under normoxic conditions, during low-flow ischemia, and after pretreatment with either aminooxyacetate (AOA) or rotenone. All interventions were associated with an increase in [Pi]/[ATP] measured by 31P NMR. In well-oxygenated untreated hearts, rapid conversion of HP [1-13C]pyruvate to [1-13C]lactate and [1,3-13C2]AcAc to [1,3-13C2]β-hydroxybutyrate ([1,3-13C2]β-HB) was readily detected. A significant increase in HP [1,3-13C2]β-HB but not [1-13C]lactate was observed in rotenone-treated and ischemic hearts, consistent with an increase in mitochondrial NADH but not cytosolic NADH. AOA treatments did not alter the productions of HP [1-13C]lactate or [1,3-13C2]β-HB. This study demonstrates that biomarkers of mitochondrial and cytosolic redox may be detected simultaneously in functioning tissues using co-polarized [1-13C]pyruvate and [1,3-13C2]AcAc and 13C MRS and that changes in mitochondrial redox may precede changes in cytosolic redox.

The role of Zn

PURPOSE This study is to investigate time-resolved 13 C MR spectroscopy (MRS) as an alternative to imaging for assessing pyruvate metabolism using hyperpolarized (HP) [1-13 C]pyruvate in the human brain. METHODS Time-resolved 13 C spectra were acquired from four axial brain slices of healthy human participants (n = 4) after a bolus injection of HP [1-13 C]pyruvate. 13 C MRS with low flip-angle excitations and a multichannel 13 C/1 H dual-frequency radiofrequency (RF) coil were exploited for reliable and unperturbed assessment of HP pyruvate metabolism. Slice-wise areas under the curve (AUCs) of 13 C-metabolites were measured and kinetic analysis was performed to estimate the production rates of lactate and HCO3- . Linear regression analysis between brain volumes and HP signals was performed. Region-focused pyruvate metabolism was estimated using coil-wise 13 C reconstruction. Reproducibility of HP pyruvate exams was presented by performing two consecutive injections with a 45-minutes interval. RESULTS [1-13 C]Lactate relative to the total 13 C signal (tC) was 0.21-0.24 in all slices. [13 C] HCO3- /tC was 0.065-0.091. Apparent conversion rate constants from pyruvate to lactate and HCO3- were calculated as 0.014-0.018 s-1 and 0.0043-0.0056 s-1 , respectively. Pyruvate/tC and lactate/tC were in moderate linear relationships with fractional gray matter volume within each slice. White matter presented poor linear regression fit with HP signals, and moderate correlations of the fractional cerebrospinal fluid volume with pyruvate/tC and lactate/tC were measured. Measured HP signals were comparable between two consecutive exams with HP [1-13 C]pyruvate. CONCLUSIONS Dynamic MRS in combination with multichannel RF coils is an affordable and reliable alternative to imaging methods in investigating cerebral metabolism using HP [1-13 C]pyruvate.

PURPOSE This study aimed to investigate the role of regional f0 inhomogeneity in spiral hyperpolarized 13 C image quality and to develop measures to alleviate these effects. METHODS Field map correction of hyperpolarized 13 C cardiac imaging using spiral readouts was evaluated in healthy subjects. Spiral readouts with differing duration (26 and 45 ms) but similar resolution were compared with respect to off-resonance performance and image quality. An f0 map-based image correction based on the multifrequency interpolation (MFI) method was implemented and compared to correction using a global frequency shift alone. Estimation of an unknown frequency shift was performed by maximizing a sharpness objective based on the Sobel variance. The apparent full width half at maximum (FWHM) of the myocardial wall on [13 C]bicarbonate was used to estimate blur. RESULTS Mean myocardial wall FWHM measurements were unchanged with the short readout pre-correction (14.1 ± 2.9 mm) and post-MFI correction (14.1 ± 3.4 mm), but significantly decreased in the long waveform (20.6 ± 6.6 mm uncorrected, 17.7 ± 7.0 corrected, P = .007). Bicarbonate signal-to-noise ratio (SNR) of the images acquired with the long waveform were increased by 1.4 ± 0.3 compared to those acquired with the short waveform (predicted 1.32). Improvement of image quality was observed for all metabolites with f0 correction. CONCLUSIONS f0 -map correction reduced blur and recovered signal from dropouts, particularly along the posterior myocardial wall. The low image SNR of [13 C]bicarbonate can be compensated with longer duration readouts but at the expense of increased f0 artifacts, which can be partially corrected for with the proposed methods.

Dynamic nuclear polarization (DNP) coupled with 15N magnetic resonance imaging (MRI) provides an opportunity to image quantitative levels of biologically important metal ions such as Zn2+, Mg2+ or Ca2+ using appropriately designed 15N enriched probes. For example, a Zn-specific probe could prove particularly valuable for imaging the tissue distribution of freely available Zn2+ ions, an important known metal ion biomarker in the pancreas, in prostate cancer, and in several neurodegenerative diseases. In the present study, we prepare the cell-permeable, 15N-enriched, d6-deuterated version of the well-known Zn2+ chelator, tris(2-pyridylmethyl)amine (TPA) and demonstrate that the polarized ligand had favorable T1 and linewidth characteristics for 15N MRI. Examples of how polarized TPA can be used to quantify freely available Zn2+ in homogenized human prostate tissue and intact cells are presented.

Extracellular pH is an important biomarker in many diseases yet a routine method for imaging tissue pH has not been established in the clinic. Paramagnetic chemical exchange saturation transfer (paraCEST) agents are well-suited for imaging tissue pH because the basis of CEST, chemical exchange, is inherently sensitive to pH. Several previous pH-sensitive paraCEST agents were based upon with an exchanging Ln 3+ bound water molecule as the CEST antenna but this design often added additional line-broadening of the bulk water signal due to T 2 exchange. We report here a pH-sensitive paraCEST agent that lacks an inner-sphere water molecule but contains one Ln-bound -OH group for CEST activation. The Yb 3+ - complex, Yb( 1 ), displayed a single, highly-shifted CEST peak originating from the exchangeable Yb-OH proton, the frequency of which changed over the biologically relevant pH range. CEST images of phantoms ranging in pH from 6-8 demonstrate the potential of this agent for imaging pH. Initial rodent imaging studies showed that Gd( 1 ) remains in the vascular system much longer than anticipated but is cleared slowing via renal filtration.

Purpose To develop a simplified method for quantitative measurement of NAD+ /NADH (nicotinamide adenine dinucleotides) levels in human brain by 31 P MRS without interference from the α-ATP signal and with inclusion of multiple UDP-sugar components. Methods Simple pulse-acquire 31 P MR spectra were collected at 7T with and without a frequency-selective inversion pulse to remove the dominant α-ATP signal from the underlying NAD(H) signal. Careful inspection of the 31 P signal at -9.8 ppm previously assigned to UDP-glucose revealed multiple UDP-sugar components that must also be considered when deconvoluting the NAD(H) signal to quantify NAD+ and NADH. Finally, the overlapping NAD(H) and UDP(G) resonances were deconvoluted into individual components using Voigt lineshape analysis and UDP(G) modeling. Results The inversion-based spectral editing method enabled clean separation of the NAD(H) signal from the otherwise dominant α-ATP signal. In addition, the upfield signal near -9.8 ppm appears more "quartet-like" than a simple doublet consistent with contributions from other nucleotide sugars such as UDP-galactose, UDP-N-acetyl-galactosamine, and UDP-N-acetyl-glucosamine in addition to UDP-glucose. Deconvolution of the combined NAD(H) and UDP(G) signals showed that the measured NAD+ /NAD ratio was heavily influenced by UDP(G) modeling (7.5 ± 1.8 when the UDP(G) signal was fitted as multiple doublets versus 5.3 ± 0.6 when a simplified pseudo doublet model was used). In a test/re-test experiments separated by 2 weeks, consistent NAD+ /NADH ratios were measured in the brain of seven human subjects. Conclusions The NAD+ /NADH ratio in human brain can be measured using 31 P MR spectra simplified by spectral editing and with inclusion of multiple UDP-sugar components in the data analysis.

The neonatal mammalian heart is capable of regeneration for a brief window of time after birth. However, this regenerative capacity is lost within the first week of life, which coincides with a postnatal shift from anaerobic glycolysis to mitochondrial oxidative phosphorylation, particularly towards fatty-acid utilization. Despite the energy advantage of fatty-acid beta-oxidation, cardiac mitochondria produce elevated rates of reactive oxygen species when utilizing fatty acids, which is thought to play a role in cardiomyocyte cell-cycle arrest through induction of DNA damage and activation of DNA-damage response (DDR) pathway. Here we show that inhibiting fatty-acid utilization promotes cardiomyocyte proliferation in the postnatatal heart. First, neonatal mice fed fatty-acid deficient milk showed prolongation of the postnatal cardiomyocyte proliferative window, however cell cycle arrest eventually ensued. Next, we generated a tamoxifen-inducible cardiomyocyte-specific, pyruvate dehydrogenase kinase 4 (PDK4) knockout mouse model to selectively enhance oxidation of glycolytically derived pyruvate in cardiomyocytes. Conditional PDK4 deletion resulted in an increase in pyruvate dehydrogenase activity and consequently an increase in glucose relative to fatty-acid oxidation. Loss of PDK4 also resulted in decreased cardiomyocyte size, decreased DNA damage and expression of DDR markers and an increase in cardiomyocyte proliferation. Following myocardial infarction, inducible deletion of PDK4 improved left ventricular function and decreased remodelling. Collectively, inhibition of fatty-acid utilization in cardiomyocytes promotes proliferation, and may be a viable target for cardiac regenerative therapies.

Amide proton transfer-weighted (APTw) MR imaging shows promise as a biomarker of brain tumor status. Currently used APTw MRI pulse sequences and protocols vary substantially among different institutes, and there are no agreed-on standards in the imaging community. Therefore, the results acquired from different research centers are difficult to compare, which hampers uniform clinical application and interpretation. This paper reviews current clinical APTw imaging approaches and provides a rationale for optimized APTw brain tumor imaging at 3 T, including specific recommendations for pulse sequences, acquisition protocols, and data processing methods. We expect that these consensus recommendations will become the first broadly accepted guidelines for APTw imaging of brain tumors on 3 T MRI systems from different vendors. This will allow more medical centers to use the same or comparable APTw MRI techniques for the detection, characterization, and monitoring of brain tumors, enabling multi-center trials in larger patient cohorts and, ultimately, routine clinical use.

The concept of using paramagnetic metal ion complexes as chemical exchange saturation transfer agents (paraCEST) for molecular imaging of various biological processes first appeared in the literature about 20 years ago. The first paraCEST agent was based on a highly shifted, inner-sphere, slowly exchanging water molecule that could be activated at a frequency far away from bulk water, a substantial advantage for selective activation of the agent alone. Many other paraCEST agent designs followed that were based on activation of exchanging -NH or -OH proton on the chelate itself. Both types of paraCEST designs are attractive for molecular imaging because the rates of water molecule or ligand proton exchange can be designed to be sensitive to a biological or physiological property such as pH, enzyme activity, or redox. Hence, the intensity or frequency of the resulting CEST signal provides a direct readout of that property. Many molecular designs have appeared in the literature over the past 20 years, mostly reported as proof-of-concept designs but, unfortunately, only a few reports have explored the limitations of paraCEST agents for imaging a biological process in vivo. As a community, we now know that the sensitivity of paraCEST agents is lower than one might anticipate based upon simple chemical exchange principles and, in general, it appears the sensitivity of paraCEST agents is even lower in vivo than in vitro. In this short review, we address some of the factors that contribute to the limited sensitivity of paraCEST agents in vivo, offer some thoughts on approaches that could lead to dramatically improved paraCEST sensitivity, and challenge the scientific community to perform more in vivo experiments designed to test these ideas.

Cellular redox is intricately linked to energy production and normal cell function. Although the redox state of mitochondria and cytosol are connected by shuttle mechanisms, the redox state of mitochondria may differ from redox in the cytosol in response to stress. However, detecting these differences in functioning tissue is difficult. Here, we employed (13)C magnetic resonance spectroscopy (MRS) and co-polarized [1-(13)C]pyruvate and [1,3-(13)C(2)]acetoacetate ([1,3-(13)C(2)]AcAc) to monitor production of hyperpolarized (HP) lactate and β-hydroxybutyrate as indicators of cytosolic and mitochondrial redox, respectively. Isolated rat hearts were examined under normoxic conditions, during low-flow ischemia, and after pretreatment with either aminooxyacetate (AOA) or rotenone. All interventions were associated with an increase in [Pi]/[ATP] measured by (31)P NMR. In well-oxygenated untreated hearts, rapid conversion of HP [1-(13)C]pyruvate to [1-(13)C]lactate and [1,3-(13)C(2)]AcAc to [1,3-(13)C(2)]β-hydroxybutyrate ([1,3-(13)C(2)]β-HB) was readily detected. A significant increase in HP [1,3-(13)C(2)]β-HB but not [1-(13)C]lactate was observed in rotenone-treated and ischemic hearts, consistent with an increase in mitochondrial NADH but not cytosolic NADH. AOA treatments did not alter the productions of HP [1-(13)C]lactate or [1,3-(13)C(2)]β-HB. This study demonstrates that biomarkers of mitochondrial and cytosolic redox may be detected simultaneously in functioning tissue using co-polarized [1-(13)C]pyruvate and [1,3-(13)C(2)]AcAc and (13)C MRS, and that changes in mitochondrial redox may precede changes in cytosolic redox.

An imaging method for detecting β-cell function in real-time in the rodent pancreas could provide new insights into the biological mechanisms involving loss of β-cell function during development of type 2 diabetes and for testing of new drugs designed to modulate insulin secretion. In this study, we used a zinc-responsive MRI contrast agent and an optimized 2D MRI method to show that glucose stimulated insulin and zinc secretion can be detected as functionally active “hot spots” in the tail of the rat pancreas. A comparison of functional images with histological markers show that insulin and zinc secretion does not occur uniformly among all pancreatic islets but rather that some islets respond rapidly to an increase in glucose while others remain silent. Zinc and insulin secretion was shown to be altered in streptozotocin and exenatide treated rats thereby verifying that this simple MRI technique is responsive to changes in β-cell function.

Background Excessive lactate production, a hallmark of cancer, is largely formed by the reduction of pyruvate via lactate dehydrogenase (LDH) to l-lactate. Although d-lactate can also be produced from glucose via the methylglyoxal pathway in small amounts, less is known about the amount of d-lactate produced in cancer cells. Since the stereoisomers of lactate cannot be distinguished by conventional 1H NMR spectroscopy, a chiral NMR shift reagent was used to fully resolve the 1H NMR resonances of d- and l-lactate. Methods The production of l-lactate from glucose and d-lactate from methylglyoxal was first demonstrated in freshly isolated red blood cells using the chiral NMR shift reagent, YbDO3A-trisamide. Then, two different cell lines with high GLO1 expression (H1648 and H 1395) were selected from a panel of over 80 well-characterized human NSCLC cell lines, grown to confluence in standard tissue culture media, washed with phosphate-buffered saline, and exposed to glucose in a buffer for 4 h. After 4 h, a small volume of extracellular fluid was collected and mixed with YbDO3A-trisamide for analysis by 1H NMR spectroscopy. Results A suspension of freshly isolated red blood cells exposed to 5mM glucose produced l-lactate as expected but very little d-lactate. To evaluate the utility of the chiral NMR shift reagent, methylglyoxal was then added to red cells along with glucose to stimulate the production of d-lactate via the glyoxalate pathway. In this case, both d-lactate and l-lactate were produced and their NMR chemical shifts assigned. NSCLC cell lines with different expression levels of GLO1 produced both l- and d-lactate after incubation with glucose and glutamine alone. A GLO1-deleted parental cell line (3553T3) showed no production of d-lactate from glucose while re-expression of GLO1 resulted in higher production of d-lactate. Conclusions The shift-reagent-aided NMR technique demonstrates that d-lactate is produced from glucose in NSCLC cells via the methylglyoxal pathway. The biological role of d-lactate is uncertain but a convenient method for monitoring d-lactate production could provide new insights into the biological roles of d- versus l-lactate in cancer metabolism.

PURPOSE: This study is to investigate time-resolved (13)C MR spectroscopy as an alternative to imaging for assessing pyruvate metabolism using hyperpolarized [1-(13)C]pyruvate in the human brain. METHODS: Time-resolved (13)C spectra were acquired from four axial brain slices of healthy human participants (n=4) after a bolus injection of hyperpolarized [1-(13)C]pyruvate. (13)C MR spectroscopy with low flip-angle excitations and a multichannel (13)C/(1)H dual-frequency RF coil were exploited for reliable and unperturbed assessment of hyperpolarized pyruvate metabolism. Slice-wise area under the curves (AUCs) of (13)C-metabolites were measured and kinetic analysis was performed to estimate the production rates of lactate and HCO(3)(–). Linear regression analysis between brain volumes and hyperpolarized signals was performed. Region-focused pyruvate metabolism was estimated using coil-wise (13)C reconstruction. Reproducibility of hyperpolarized pyruvate exams was presented by performing two consecutive injections with a 45-min interval. RESULTS: [1-(13)C]Lactate relative to the total (13)C signal (tC) was 0.21–0.24 in all slices. [(13)C]HCO(3)(–)/tC was 0.065–0.091. Apparent conversion rate constants from pyruvate to lactate and HCO(3)(–) were calculated as 0.014–0.018s(−1) and 0.0043–0.0056s(−1), respectively. Pyruvate/tC and lactate/tC were in moderate linear relationships with fractional grey matter volume within each slice. White matter presented poor linear regression fit with hyperpolarized signals, and moderate correlations of the fractional cerebrospinal fluid volume with pyruvate/tC and lactate/tC were measured. Measured hyperpolarized signals were comparable between two consecutive exams with hyperpolarized [1-(13)C]pyruvate. CONCLUSIONS: Dynamic MRS in combination with multichannel RF coils is an affordable and reliable alternative to imaging methods in investigating cerebral metabolism using hyperpolarized [1-(13)C]pyruvate.

The TCA cycle is a central metabolic pathway for energy production and biosynthesis. A major control point of metabolic flux through the cycle is the decarboxylation of 2-ketoglutarate by the TCA cycle enzyme 2-ketoglutarate dehydrogenase (2-KGDH). In this project, we developed (13)C labeled 2-ketoglutarate derivatives to monitor 2-KGDH activity in vivo. (13)C NMR analysis of liver extracts revealed that uniformly (13)C labeled 2-ketogutarate, in its cell permeable ester form, was rapidly taken up and hydrolyzed in liver and underwent extensive metabolism to produce labeled glutamate, succinate, lactate and other metabolites. Diethyl [1,2-(13)C(2)]-2-ketoglutarate was successfully polarized by dynamic nuclear polarization and within seconds after injection into rats, the probe produced hyperpolarized [(13)C]bicarbonate in the liver reflecting flux through the TCA cycle. These experiments demonstrate that this tracer offers the possibility of directly monitoring flux through 2-KGDH in vivo.

HIV protease plays a critical role in the life cycle of the virus through the generation of mature and infectious virions. Detailed knowledge of the structure of the enzyme and its substrate has led to the development of protease inhibitors. However, the development of resistance to all currently available protease inhibitors has contributed greatly to the decreased success of antiretroviral therapy. When therapy failure occurs, multiple mutations are found within the protease sequence starting with primary mutations, which directly impact inhibitor binding, which can also negatively impact viral fitness and replicative capacity by decreasing the binding affinity of the natural substrates to the protease. As such, secondary mutations which are located outside of the active site region accumulate to compensate for the recurrently deleterious effects of primary mutations. However, the resistance mechanism of these secondary mutations is not well understood, but what is known is that these secondary mutations contribute to resistance in one of two ways, either through increasing the energetic penalty associated with bringing the protease into the closed conformation, or, through decreasing the stability of the protein/drug complex in a manner that increases the dissociation rate of the drug, leading to diminished inhibition. As a result, the elasticity of the enzyme-substrate complex has been implicated in the successful recognition and catalysis of the substrates which may be inferred to suggest that the elasticity of the enzyme/drug complex plays a role in resistance. A realistic representation of the dynamic nature of the protease may provide a more powerful tool in structure-based drug design algorithms.

Prostatic zinc content is a known biomarker for discriminating normal healthy tissue from benign prostatic hyperplasia (BPH) and prostate cancer (PCa). Given that zinc content is not readily measured without a tissue biopsy, we have been exploring noninvasive imaging methods to detect these diagnostic differences using a zinc-responsive MRI contrast agent. During imaging studies in mice, we observed that a bolus of glucose stimulates secretion of zinc from the prostate of fasted mice. This discovery allowed the use of a Gd-based zinc sensor to detect differential zinc secretion in regions of healthy versus malignant prostate tissue in a transgenic adenocarcinoma mouse model of PCa. Here, we used a zinc-responsive MRI agent to detect zinc release across the prostate during development of malignancy and confirm the loss of total tissue zinc by synchrotron radiation X-ray fluorescence (μSR-XRF). Quantitative μSR-XRF results show that the lateral lobe of the mouse prostate uniquely accumulates high concentrations of zinc, 1.06 ± 0.08 mM, and that the known loss of zinc content in the prostate is only observed in the lateral lobe during development of PCa. Additionally, we confirm that lesions identified by a loss of zinc secretion indeed represent malignant neoplasia and that the relative zinc concentration in the lesion is reduced to 0.370 ± 0.001 mM. The μSR-XRF data also provided insights into the mechanism of zinc secretion by showing that glucose promotes movement of zinc pools (∼1 mM) from the glandular lumen of the lateral lobe of the mouse prostate into the stromal/smooth muscle surrounding the glands. Co-localization of zinc and gadolinium in the stromal/smooth muscle areas as detected by μSR-XRF confirm that glucose initiates secretion of zinc from intracellular compartments into the extracellular spaces of the gland where it binds to the Gd-based agent and albumin promoting MR image enhancement.

PURPOSE For efficient and integrative analysis of de novo adenosine triphosphate (ATP) synthesis, creatine-kinase-mediated ATP synthesis, T1 relaxation time, and ATP molecular motion dynamics in human skeletal muscle at rest. METHODS Four inversion-transfer modules differing in center inversion frequency were combined to generate amplified magnetization transfer (MT) effects in targeted MT pathways, including Pi ↔ γ-ATP, PCr ↔ γ-ATP, and 31 Pγ(α)ATP ↔ 31 PβATP . MT effects from both forward and reverse exchange kinetic pathways were acquired to reduce potential bias and confounding factors in integrated data analysis. RESULTS Kinetic data collected using 4 wideband inversion modules (8 minutes each) yielded the forward exchange rate constants, kPCr→γATP = 0.31 ± 0.05 s-1 and kPi→γATP = 0.064 ± 0.012 s-1 , and the reverse exchange rate constants, kγATP→Pi = 0.034 ± 0.006 s-1 and kγATP→PCr = 1.37 ± 0.22 s-1 , respectively. The cross-relaxation rate constant, σγ(α) ↔ βATP was -0.20 ± 0.03 s-1 , corresponding to ATP rotational correlation time τc of 0.8 ± 0.1 × 10-7 seconds. The intrinsic T1 relaxation times were Pi (9.2 ± 1.4 seconds), PCr (6.2 ± 0.4 seconds), γ-ATP (1.8 ± 0.1 seconds), α-ATP (1.4 ± 0.1 seconds), and β-ATP (1.1 ± 0.1 seconds). Muscle ATP T1 values were found to be significantly longer than those previously measured in the brain using a similar method. CONCLUSION A combination of multiple inversion transfer modules provides a comprehensive and integrated analysis of ATP metabolism and molecular motion dynamics. This relatively fast technique could be potentially useful for studying metabolic disorders in skeletal muscle.

Guanosine diphosphate mannose (GDP-Man) is the donor substrate required for mannosylation in the synthesis of glycoproteins, glycolipids and the newly discovered glycoRNA. Normal GDP-Man biosynthesis plays a crucial role in support of a variety of cellular functions, including cell recognition, cell communication and immune responses against viruses. Here, we report the detection of GDP-Man in human brain for the first time, using 31 P MRS at 7 T. The presence of GDP-Man is evidenced by the detection of a weak 31 P doublet at -10.7 ppm that can be assigned to the phosphomannosyl group (Pβ) of the GDP-Man molecule. This weak but well-resolved signal lies 0.9 ppm upfield of UDP(G) Pβ-multiplet from a mixture of UDP-Glc, UDP-Gal, UDP-GlcNAc and UDP-GalNAc. In reference to ATP (2.8 mM), the concentration of GDP-Man in human brain was estimated to be 0.02 ± 0.01 mM, about 15-fold lower than the total concentration of UDP(G) (0.30 ± 0.04, N = 17) and consistent with previous reports of UDP-Man in cells and brain tissue extracts measured by high-performance liquid chromatography. The reproducibility of the measured GDP-Man between test and 2-week retest was 21% ± 15% compared with 5% ± 4% for UDP(G) (N = 7). The measured concentrations of GDP-Man and UDP(G) are linearly correlated ([UDP(G)] = 4.3 [GDP-Man] + 0.02, with R = 0.66 and p = 0.0043), likely reflecting the effect of shared sugar precursors, which may vary among individuals in response to variation in nutritional intake and consumption. Given that GDP-Man has another set of doublet (Pα) at -8.3 ppm that overlaps with NAD(H) and UDP(G)-Pα signals, the amount of GDP-Man could potentially interfere with the deconvolution of these mixed signals in composition analysis. Importantly, this new finding may be useful in advancing our understanding of glycosylation and its role in the development of cancer, as well as infectious and neurodegenerative diseases.

Nucleotide sugars are required for the synthesis of glycoproteins and glycolipids, which play crucial roles in many cellular functions such as cell communication and immune responses. Uridine diphosphate-glucose (UDP-Glc) was previously believed to be the only nucleotide sugar detectable in brain by 31 P-MRS. Using spectra of high SNR and high resolution acquired at 7 T, we showed that multiple nucleotide sugars are coexistent in brain and can be measured simultaneously. In addition to UDP-Glc, these also include UDP-galactose (UDP-Gal), -N-acetyl-glucosamine (UDP-GlcNAc) and -N-acetyl-galactosamine (UDP-GalNAc), collectively denoted as UDP(G). Coexistence of these UDP(G) species is evident from a quartet-like multiplet at -9.8 ppm (M-9.8 ), which is a common feature seen across a wide age range (24-64 years). Lineshape fitting of M-9.8 allows an evaluation of all four UDP(G) components, which further aids in analysis of a mixed signal at -8.2 ppm (M-8.2 ) for deconvolution of NAD+ and NADH. For a group of seven young healthy volunteers, the concentrations of UDP(G) species were 0.04 ± 0.01 mM for UDP-Gal, 0.07 ± 0.03 mM for UDP-Glc, 0.06 ± 0.02 mM for UDP-GalNAc and 0.08 ± 0.03 mM for UDP-GlcNA, in reference to ATP (2.8 mM). The combined concentration of all UDP(G) species (average 0.26 ± 0.06 mM) was similar to the pooled concentration of NAD+ and NADH (average 0.27 ± 0.06 mM, with a NAD+ /NADH ratio of 6.7 ± 2.1), but slightly lower than previously found in an older cohort (0.31 mM). The in vivo NMR analysis of UDP-sugar composition is consistent with those from tissue extracts by other modalities in the literature. Given that glycosylation is dependent on the availability of nucleotide sugars, assaying multiple nucleotide sugars may provide valuable insights into potential aberrant glycosylation, which has been implicated in certain diseases such as cancer and Alzheimer's disease.

Carbon isotope tracers have been used to determine relative rates of tricarboxylic acid cycle (TCA) cycle pathways since the 1950s. Steady-state experimental data are typically fit to a single mathematical model of metabolism to determine metabolic fluxes. Whether the chosen model is appropriate for the biological system has generally not been evaluated systematically. An overly-simple model omits known pathways while an overly-complex model may produce incorrect results due to overfitting.The objectives were to develop and study a method that systematically evaluates multiple TCA cycle mathematical models as part of the fitting process.The problem of choosing overly-simple or overly-complex models was approached by developing software that automatically explores all possible combinations of flux through pyruvate dehydrogenase, pyruvate kinase, pyruvate carboxylase and anaplerosis at propionyl-CoA carboxylase, and equivalent pathways, all relative to TCA cycle flux. Typical TCA cycle metabolic tracer experiments that useWhen fitting alternative models of the TCA cycle metabolism, the SSRE may identify more than one model that fits the data well. Among those models, the AIC provides guidance as to which is the simplest of the candidate models is sufficient to describe the observed data. However under some conditions, AIC used alone inappropriately discriminates against necessary metabolic complexity.In combination, the SSRE and AIC help the investigator identify the model that best describes the metabolism of a biological system.

OBJECTIVES: In the United States, prostate cancer (PCa) is the most common cancer in men. Multi-parametric magnetic resonance imaging (MRI) is increasingly being relied upon for the diagnosis and characterization of PCa, but differentiating malignancy from benign prostatic hyperplasia (BPH) in the transition zone using MRI can be challenging. The characteristically high levels of zinc in human prostate tissue and a close relationship between malignant proliferation and zinc homeostatic dysregulation create opportunities to visualize PCa with novel contrast media. In mouse models, glucose-stimulated zinc secretion (GSZS) can be preferentially observed in healthy prostate tissue compared with malignant tissue; in vivo, these differences can be captured with MRI by using Gdl1, a gadolinium-based zinc-responsive contrast agent. In this study, we examined whether this technology can be applied in a large animal model by imaging older dogs with clinically diagnosed BPH. MATERIALS AND METHODS: Four intact male dogs 6 years or older with enlarged prostates were imaged (T1-weighted turbo spin-echo, TE/TR, 12/400 milliseconds and T2-weighted, TE/TR, 112/5000 milliseconds) using a 3 T scanner before and at multiple time points after intravenous injection of 0.05 mmol/kg GdL1 plus either (a) 2 mL/kg of 50% dextrose in 1 session or (b) 2 mL/kg normal saline in another session. The two sessions were one week apart, and their order was randomly determined for each dog. During postprocessing, regions of interest were generated in prostate tissue and in paraspinal muscles to evaluate the contrast-to-noise ratio (CNR). The ratio of CNR at any postinjection time point compared with baseline CNR was defined as r-CNR. After the second imaging session, the dogs were euthanized, and their prostates were harvested for histopathological examination. Baseline and postintervention plasma and urine samples were analyzed for total zinc by inductively coupled plasma mass spectrometry. RESULTS: The mean ± SD r-CNR values at 13 minutes postinjection in the dextrose versus saline imaging sessions were 134% ± 10% and 127% ± 7%, respectively (P < 0.01). The histopathologic evaluation of prostate tissues confirmed BPH in all dogs. Interestingly, prostatic intraepithelial neoplasia was detected in 1 animal, and a suspicious mass was found in the same region on T2-weighted scans. The r-CNR of the mass was calculated as 113% ± 4% and 111% ± 6% in the dextrose and saline groups, respectively, with no significant differences between the 2 interventions (P = 0.54), whereas there was a statistically significant difference between the r-CNR of the whole prostate in the dextrose (130% ±11%) and saline (125% ± 9%) interventions (P = 0.03). Inductively coupled plasma mass spectrometry analyses showed a significantly higher urinary zinc in the dextrose versus saline groups, but no differences were found in plasma zinc levels. CONCLUSIONS: T1-weighted MRI of the enlarged canine prostate showed higher r-CNR after injection of GdL1 plus dextrose compared with GdL1 plus saline, consistent with GSZS from BPH tissues. One small region of neoplastic tissue was identified in a single dog on the basis of less GSZS from that region by MRI. These findings suggest a new method for the detection of PCa by MRI that could facilitate the differentiation of BPH from PCa in the transition zone.

INTRODUCTION: As the most common type of kidney cancer, renal cell carcinoma (RCC) emerges in the form of highly vascularized tumors. Our team has recently demonstrated the therapeutic effects of two novel heme-targeting agents (heme-sequestering peptide 2 (HSP2) and cyclopamine tartrate (CycT)) which effectively suppress angiogenic activities and normalize tumor vasculatures in non-small cell lung cancer (NSCLC) cells and mouse tumor models [1,2]. We have now applied non-invasive multimodal imaging to evaluate these heme related treatments of kidney cancer. METHODS: Magnetic Resonance Imaging (MRI) and MultiSpectral Optoacoustic Tomography (MSOT) are ideally suited for investigations of the kidney and RCC given their extensive vasculature. MRI was performed at 9.4-T using high-resolution axial T2-weighted multi-slice images encompassing the entire kidney tumor were obtained with a fat suppressed fast spin-echo sequence in about 8 minutes. The MSOT system illuminates the mouse from all sides, providing effective excitation (700, 780, 800, 820, 850 nm) even at depth, revealing rudimentary anatomy, as well as functional oxy- and deoxyhemoglobin. We used MSOT and MRI modalities to determine the degree to which CycT modified hypoxia in orthotopic PDX renal XP 334 tumor xenografts in mice. RESULTS: Both MSOT and MRI showed anatomical differences between treated and control tumors with treated tumors being significantly smaller (p < 0.002). Dynamic hemoglobin changes to oxygen-breathing challenge through MSOT as a response to control and CycT group in kidney tumors. CONCLUSION: MR and MSOT imaging allowed us to monitor and evaluate the effects of the novel heme targeting drugs CycT, which significantly suppressed orthotopic kidney PDX 334 tumor growth. MSOT demonstrates dynamic hemoglobin changes to oxygen-breathing challenge in response to CycT and untreated control groups in kidney tumors. Combining MR imaging with other non-invasive imaging modalities provides a clear guide on monitoring and targeting treatments for tumors in mice. REFERENCES: [1] S. Sohoni, et al., Elevated Heme Synthesis and Uptake Underpin Intensified Oxidative Metabolism and Tumorigenic Functions in Non-Small Cell Lung Cancer Cells, Cancer Res, 79 (2019) 2511-2525. [2] P. Ghosh, et al., Oxygen-Enhanced Optoacoustic Tomography Reveals the Effectiveness of Targeting Heme and Oxidative Phosphorylation at Normalizing Tumor Vascular Oxygenation, Cancer Research, 80 (2020) 3542. Acknowledgments: CPRIT RP200021 & RP160617 and Infrastructure of Kidney SPORE Brugarolas for PDX tissues well as small animal imaging resource of P30 CA142543 and Shared Instrumentation grants S10 OD018094-01A1, 1S10 RR024757 and S10 RR031859 Citation Format: Li Liu, Hashini Wanniarachchi, Tianyuan Wang, Xiaojing Wang, Dean Sherry, Ralph P. Mason, Li Zhang. Evaluation of therapeutic strategy for the treatment of kidney cancer using multimodal imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6288.

A Mn(II)-based zinc-sensitive MRI contrast agent, MnPyC3A–BPEN, was prepared, characterized, and applied in imaging experiments to detect glucose-stimulated zinc secretion (GSZS) from the mouse pancreas and prostate in vivo. Thermodynamic and kinetic stability tests showed that MnPy-C3A–BPEN has superior kinetic inertness compared to GdDTPA, is less susceptible to transmetalation in the presence of excess Zn(2+) ions, and less susceptible to transchelation by albumin. In comparison with other gadolinium-based zinc sensors bearing a single zinc binding moiety, MnPyC3A–BPEN appears to be a reliable alternative for imaging β-cell function in the pancreas and glucose-stimulated zinc secretion from the prostate.

Much of our knowledge of the biochemical pathways we refer to as intermediary metabolism was derived from studies of isolated enzymes, organelles, and cells using radioactive tracers. With advancements in NMR technology and the commercial availability of 13C-enriched compounds as biochemical tracers, we are gaining new insights into the complexities of metabolism not only at the cellular level but now in human subjects. After the demonstration that dynamic nuclear polarization methods can be used to create highly polarized 13C samples ready for injection into animals or human subjects, one can begin to obtain enough kinetic details to measure flux not only through extended pathways but also through individual enzyme-catalyzed steps along a metabolic pathway. In this chapter, we illustrate several examples of using hyperpolarized 13C tracers to measure flux through competing pathways in isolated perfused rodent hearts and livers and in living animals. Even though multiple oxidizable substrates (glucose, fats, and ketones) are always available to tissues in vivo, presentation of a highly polarized substrate such as pyruvate to tissues while imaging downstream metabolic products derived from pyruvate can provide novel insights into metabolic regulation of competing pathways under variable nutritional states. This powerful feature of hyperpolarized 13C metabolic imaging offers considerable potential as a rapid, noninvasive tool for monitoring changes in metabolic flux as a tissue responds to dietary alterations or drugs. This new imaging method will add unique diagnostic information to the study of human diseases in the future.

HIV protease plays a critical role in the life cycle of the virus through the generation of mature and infectious virions. Detailed knowledge of the structure of the enzyme and its substrate has led to the development of protease inhibitors. However, the development of resistance to all currently available protease inhibitors has contributed greatly to the decreased success of antiretroviral therapy. When therapy failure occurs, multiple mutations are found within the protease sequence starting with primary mutations, which directly impact inhibitor binding, which can also negatively impact viral fitness and replicative capacity by decreasing the binding affinity of the natural substrates to the protease. As such, secondary mutations which are located outside of the active site region accumulate to compensate for the recurrently deleterious effects of primary mutations. However, the resistance mechanism of these secondary mutations is not well understood, but what is known is that these secondary mutations contribute to resistance in one of two ways, either through increasing the energetic penalty associated with bringing the protease into the closed conformation, or, through decreasing the stability of the protein/drug complex in a manner that increases the dissociation rate of the drug, leading to diminished inhibition. As a result, the elasticity of the enzyme-substrate complex has been implicated in the successful recognition and catalysis of the substrates which may be inferred to suggest that the elasticity of the enzyme/drug complex plays a role in resistance. A realistic representation of the dynamic nature of the protease may provide a more powerful tool in structure-based drug design algorithms.

Introduction and Hypothesis: Conventional metabolic imaging methods such as PET, dobutamine stress echo or late gadolinium enhancement do not directly detect the functional state of the critical organelle in oxidative metabolism, the mitochondrion. Hyperpolarized (HP) 13 C-metabolic imaging has been used to assess mitochondrial function in preclinical models and is an exciting new imaging method for evaluating the ischemic myocardium noninvasively. This study aims to evaluate myocardial response to dobutamine stress in ischemic rat hearts using HP-[1- 13 C]pyruvate. Methods: Metabolism of HP[1- 13 C]pyruvate was investigated in male SD rats in four groups: 1. normal myocardium (NM), 2. ischemic myocardium (IM), 3. NM+dobutamine, 4. IM+dobutamine. The myocardial ischemic model was developed by partial occlusion of the coronary artery as described in earlier studies by others. For dobutamine stress HP-MRS, intra-peritoneal injection of 1.5 mg/kg of dobutamine was administered before positioning the animal in the MRI scanner. 13 C data were acquired 7 days after the surgery in a 4.7 T MRI scanner using a Varian 60 mm dual-tuned 1 H/ 13 C linear volume coil. Anatomical 1 H images were acquired for slice planning and positioning. 13 C intensity maps were generated and displayed as overlays on grayscale 1 H images. In separate experiments, in vivo HP- 13 C-MRS data acquisition was initiated immediately after the pyruvate injection. Results: The HP signals from [1- 13 C]lactate, [1- 13 C]alanine, and 13 C-bicarbonate were detected in real-time 13 C spectra from hearts following injection of HP[1- 13 C]pyruvate. In hearts with partial coronary ligation, the MRS data were acquired from a mixture of ischemic and nonischemic myocardium. A 27% decrease in bicarbonate production was observed in hearts with ischemic myocardium compared to control hearts. After stimulation of NM hearts with dobutamine, less bicarbonate was produced from HP-pyruvate likely due to an increased contribution of other endogenous substrates to acetyl-CoA. Production of bicarbonate was unchanged in IM hearts following adrenergic stimulation. Conclusions: Our results demonstrated that decreased oxidative metabolism of pyruvate as a result of myocardial ischemia and dobutamine stress.