Technologist-Based Implementation of Total Metabolic Tumor Volume into Clinical Practice.

Metabolic tumor volume (MTV) is defined as the total metabolically active tumor volume seen on 18F-FDG PET/CT examinations. Calculating MTV is often time-consuming, requiring a high degree of manual input. In this study, the MTV calculations of a board-certified nuclear radiologist were compared with those of 2 nuclear medicine technologists. As part of the technologists' educational program, after their classroom time they were trained by the radiologist for 30 min. The technologists calculated MTV within 7.5% of the radiologist's calculations in a set of patients who had diffuse large B-cell lymphoma and were undergoing initial staging 18F-FDG PET/CT. These findings suggest that nuclear medicine technologists may help accelerate implementation of MTV into clinical practice with favorable accuracy, possibly as an initial step followed by validation by the interpreting physician. The aim of this study was to explore whether efficiency is improved by integrating nuclear medicine technologists into a semiautomated workflow to calculate total MTV.

Tailoring Trehalose for Biomedical and Biotechnological Applications.

Trehalose is a non-reducing sugar whose ability to stabilize biomolecules has brought about its widespread use in biological preservation applications. Trehalose is also an essential metabolite in a number of pathogens, most significantly the global pathogen Mycobacterium tuberculosis, though it is absent in humans and other mammals. Recently, there has been a surge of interest in modifying the structure of trehalose to generate analogues that have applications in biomedical research and biotechnology. Non-degradable trehalose analogues could have a number of advantages as bioprotectants and food additives. Trehalose-based imaging probes and inhibitors are already useful as research tools and may have future value in the diagnosis and treatment of tuberculosis, among other uses. Underlying the advancements made in these areas are novel synthetic methods that facilitate access to and evaluation of trehalose analogues. In this review, we focus on both aspects of the development of this class of molecules. First, we consider the chemical and chemoenzymatic methods that have been used to prepare trehalose analogues and discuss their prospects for synthesis on commercially relevant scales. Second, we describe ongoing efforts to develop and deploy detectable trehalose analogues, trehalose-based inhibitors, and non-digestible trehalose analogues. The current and potential future uses of these compounds are discussed, with an emphasis on their roles in understanding and combatting mycobacterial infection.

Rapid One-step Enzymatic Synthesis and All-aqueous Purification of Trehalose Analogues.

Chemically modified versions of trehalose, or trehalose analogues, have applications in biology, biotechnology, and pharmaceutical science, among other fields. For instance, trehalose analogues bearing detectable tags have been used to detect Mycobacterium tuberculosis and may have applications as tuberculosis diagnostic imaging agents. Hydrolytically stable versions of trehalose are also being pursued due to their potential for use as non-caloric sweeteners and bioprotective agents. Despite the appeal of this class of compounds for various applications, their potential remains unfulfilled due to the lack of a robust route for their production. Here, we report a detailed protocol for the rapid and efficient one-step biocatalytic synthesis of trehalose analogues that bypasses the problems associated with chemical synthesis. By utilizing the thermostable trehalose synthase (TreT) enzyme from Thermoproteus tenax, trehalose analogues can be generated in a single step from glucose analogues and uridine diphosphate glucose in high yield (up to quantitative conversion) in 15-60 min. A simple and rapid non-chromatographic purification protocol, which consists of spin dialysis and ion exchange, can deliver many trehalose analogues of known concentration in aqueous solution in as little as 45 min. In cases where unreacted glucose analogue still remains, chromatographic purification of the trehalose analogue product can be performed. Overall, this method provides a "green" biocatalytic platform for the expedited synthesis and purification of trehalose analogues that is efficient and accessible to non-chemists. To exemplify the applicability of this method, we describe a protocol for the synthesis, all-aqueous purification, and administration of a trehalose-based click chemistry probe to mycobacteria, all of which took less than 1 hour and enabled fluorescence detection of mycobacteria. In the future, we envision that, among other applications, this protocol may be applied to the rapid synthesis of trehalose-based probes for tuberculosis diagnostics. For instance, short-lived radionuclide-modified trehalose analogues (e.g., 18F-modified trehalose) could be used for advanced clinical imaging modalities such as positron emission tomography-computed tomography (PET-CT).

Deoxyfluoro-d-trehalose (FDTre) analogues as potential PET probes for imaging mycobacterial infection.

Mycobacterium tuberculosis, the etiological agent of human tuberculosis, requires the non-mammalian disaccharide trehalose for growth and virulence. Recently, detectable trehalose analogues have gained attention as probes for studying trehalose metabolism and as potential diagnostic imaging agents for mycobacterial infections. Of particular interest are deoxy-[(18)F]fluoro-d-trehalose ((18)F-FDTre) analogues, which have been suggested as possible positron emission tomography (PET) probes for in vivo imaging of M. tuberculosis infection. Here, we report progress toward this objective, including the synthesis and conformational analysis of four non-radioactive deoxy-[(19)F]fluoro-d-trehalose ((19)F-FDTre) analogues, as well as evaluation of their uptake by M. smegmatis. The rapid synthesis and purification of several (19)F-FDTre analogues was accomplished in high yield using a one-step chemoenzymatic method. Conformational analysis of the (19)F-FDTre analogues using NMR and molecular modeling methods showed that fluorine substitution had a negligible effect on the conformation of the native disaccharide, suggesting that fluorinated analogues may be successfully recognized and processed by trehalose metabolic machinery in mycobacteria. To test this hypothesis and to evaluate a possible route for delivery of FDTre probes specifically to mycobacteria, we showed that (19)F-FDTre analogues are actively imported into M. smegmatis via the trehalose-specific transporter SugABC-LpqY. Finally, to demonstrate the applicability of these results to the efficient preparation and use of short-lived (18)F-FDTre PET radiotracers, we carried out (19)F-FDTre synthesis, purification, and administration to M. smegmatis in 1 hour.

Genome-wide profiling of DNA methylation and gene expression in Crassostrea gigas male gametes.

DNA methylation patterns and functions are variable across invertebrate taxa. In order to provide a better understanding of DNA methylation in the Pacific oyster (Crassostrea gigas), we characterized the genome-wide DNA methylation profile in male gamete cells using whole-genome bisulfite sequencing. RNA-Seq analysis was performed to examine the relationship between DNA methylation and transcript expression. Methylation status of over 7.6 million CpG dinucleotides was described with a majority of methylated regions occurring among intragenic regions. Overall, 15% of the CpG dinucleotides were determined to be methylated and the mitochondrial genome lacked DNA methylation. Integrative analysis of DNA methylation and RNA-Seq data revealed a positive association between methylation status, both in gene bodies and putative promoter regions, and expression. This study provides a comprehensive characterization of the distribution of DNA methylation in the oyster male gamete tissue and suggests that DNA methylation is involved in gene regulatory activity.

Molecular characterization of Pseudo-nitzschia community structure and species ecology in a hydrographically complex estuarine system (Puget Sound, Washington, USA).

Species within the toxic marine diatom genus Pseudo-nitzschia coexist in coastal and estuarine waters globally and are difficult to distinguish by microscopy. Here, we describe a sensitive, high throughput PCR-based Automated Ribosomal Intergenic Spacer Analysis (ARISA) approach to determine the relative abundance of Pseudo-nitzschia species within natural communities over space and time. The method was quantitatively validated using simplified mixtures of DNA or ITS1 standards from isolates of P. pungens, P. multiseries, and P. delicatissima. Relative abundance calculations based on ARISA profiles from these mixtures reflected input ratios, with minor deviations resulting from intraspecific variability. ARISA was used to identify and quantify at least eight species within Puget Sound and the eastern Strait of Juan de Fuca, Washington, USA: P. americana, P. australis/P. seriata, P. cuspidata, P. delicatissima, P. fraudulenta, P. fryxelliana, P. multiseries, and P. pungens; genotypes corresponding to P. pungens var. pungens and P. pungens var. cingulata were identified by environmental sequencing. The different species were significantly correlated with physical (temperature, salinity), biological (chlorophyll a fluorescence, oxygen), and/or chemical (ammonium, nutrient ratios) factors. The ability to determine shifts in the relative abundance of Pseudo-nitzschia species over spatial and temporal scales relevant to dispersion and selection facilitates dissection of the varied mechanisms driving vertical and horizontal species distribution patterns in hydrographically complex systems.

Gamma knife radiosurgery of tentorial meningiomas.

OBJECTIVE: Tentorial meningiomas are complex lesions that may not always be completely resected without significant morbidity or mortality. In this study, we evaluate the outcomes of tentorial meningiomas treated with Gamma Knife radiosurgery (GKRS). METHODS: We performed a retrospective review of a prospectively compiled database evaluating the outcomes of 35 patients with tentorial meningiomas treated at the University of Virginia from 1990 to 2006. There were 29 females and 6 males with a median age of 60 years (range 21-82). Twenty were treated with primary radiosurgery, and 15 patients were treated with adjuvant radiosurgery after surgical resection. Patients were assessed clinically and radiologically at routine intervals following GKRS. Kaplan-Meier analysis was used to assess tumor progression. RESULTS: The mean follow-up was 5 years (range 2-16 years). The mean pre-radiosurgery tumor volume was 5.1 cc (range 0.7-27.3cc). At last follow-up, 31 patients (89%) displayed either no growth or a decrease in tumor volume. Four (11%) patients displayed an increase in volume. Kaplan Meier analysis demonstrated radiographic progression free survival at 3, 5, and 10 years to be 96%, 91%, and 73% respectively. At the last clinical follow-up, 33 patients (94%) demonstrated no change or improvement in their neurological condition and 2 patients clinically declined (6%). CONCLUSION: GKRS offers an acceptable rate of tumor control for tentorial meningiomas, and accomplishes this with a low incidence of new or worsening neurological deficits.

Radiation dose and incidence of new metastasis in the anterior temporal lobe structures of radiosurgically treated patients.

OBJECT: Gamma Knife surgery (GKS) is frequently used to treat patients with metastasis to the brain. Radiosurgery seeks to limit radiation to the brain tissue surrounding the metastatic deposits. In patients with such lesions, a low radiation dose to the eloquent brain may help to prevent adverse effects. In this study the authors aimed to quantify the radiosurgical dose delivered to the anterior temporal structures in cases of metastatic brain lesions. They also evaluated the incidence and timing of new metastases in the anterior temporal lobes (ATLs) in patient cohorts that underwent GKS with or without whole-brain radiation therapy (WBRT). METHODS: The authors retrospectively analyzed 100 patients with metastatic brain lesions treated with GKS at the University of Virginia Health System. The anterior 5 cm of the temporal lobes and the hippocampi within the ATLs were contoured on the Gamma Knife planning station. Using the dose-volume histogram function in GammaPlan, treatment parameters for the metastases as well as radiation doses to the contoured ATLs and hippocampi were measured. Patients had clinical and MR imaging follow-ups at 3-month intervals. The ATLs and hippocampal regions were evaluated for the formation of new metastases on follow-up imaging. RESULTS: The demographic data--age, sex, Karnofsky Performance Scale score, number of temporal metastases at the time of GKS, total volume of metastatic tumors per patient, and number of intracranial metastatic deposits--were similar in the 2 cohorts. In patients without an ATL metastasis at the time of GKS, the mean maximum, 50% volume, and integral doses of radiation to the anterior temporal structures were very low: 2.6 Gy, 0.6 Gy, and 36.3 mJ in the GKS cohort and 2.1 Gy, 0.6 Gy, and 40.9 mJ in the GKS+WBRT cohort, respectively. Among the ATLs that had not shown a brain metastasis at the time of GKS, 8 of 92 temporal lobes in the GKS cohort and 10 of 89 in the GKS+WBRT cohort demonstrated a new anterior temporal lesion on follow-up MR imaging. CONCLUSIONS: Gamma Knife surgery delivered a low dose of background radiation to the ATLs and hippocampi. The incidence of a new ATL metastasis in the GKS cohort was not higher than in the GKS+WBRT cohort. Gamma Knife surgery in the management of brain metastases limits the delivery of radiation to eloquent brain tissue without evidence of an appreciable propensity to develop new metastatic disease in the ATLs or hippocampi. This therapeutic approach may help to avoid unintended neurological dysfunction due to nonspecific delivery of radiation to eloquent brain tissues.

Radiosurgery for intracranial hemangiopericytomas: outcomes after initial and repeat Gamma Knife surgery.

OBJECT: Intracranial hemangiopericytoma is a rare CNS tumor that exhibits a high incidence of local recurrence and distant metastasis. The purpose of this study was to evaluate the role of Gamma Knife surgery (GKS) in the management of intracranial hemangiopericytomas. METHODS: In a review of the University of Virginia radiosurgery database between 1989 and 2008, the authors found recurrent or residual hemangiopericytomas after resection in 21 patients in whom radiosurgery was performed to treat 28 discrete tumors. The median age of this population was 47 years (range 31-61 years) at the time of the initial GKS. Prior treatments included embolization (6), transcranial resection (39), transsphenoidal resection (2), and fractionated radiotherapy (8). The mean prescription and maximum radiosurgical doses to the tumors were 17.0 and 40.3 Gy, respectively. Repeat radiosurgery was used to treat 13 tumors. The median follow-up period was 68 months (range 2-138 months). RESULTS: At last follow-up, local tumor control was demonstrated in 47.6% of the patients (10 of 21 patients) with hemangiopericytomas. Of the 28 tumors treated, 8 decreased in size on follow-up imaging (28.6%), 5 remained unchanged (17.9%), and 15 ultimately progressed (53.6%). The progression-free survival rates were 90, 60.3, and 28.7% at 1, 3, and 5 years after initial GKS. The progression-free survival rate improved to 95, 71.5, and 71.5% at 1, 3, and 5 years after multiple GKS treatments. The 5-year survival rate after radiosurgery was 81%. Prior fractionated irradiation or radiosurgical prescription dose did not correlate with tumor control. In 4 (19%) of 21 patients extracranial metastases developed. CONCLUSIONS: Radiosurgery is a reasonable treatment option for recurrent hemangiopericytomas. Long-term close clinical and imaging follow-up is necessary because of the high probability of local recurrence and distant metastases. Repeat radiosurgery may be used to treat new or recurrent hemangiopericytomas over a long follow-up course.

Trans-sodium crocetinate enhancing survival and glioma response on magnetic resonance imaging to radiation and temozolomide.

OBJECT: Glioblastoma (GB) tumors typically exhibit regions of hypoxia. Hypoxic areas within the tumor can make tumor cells less sensitive to chemotherapy and radiation therapy. Trans-sodium crocetinate (TSC) has been shown to transiently increase oxygen to hypoxic brain tumors. The authors examined whether this improvement in intratumor oxygenation translates to a therapeutic advantage when delivering standard adjuvant treatment to GBs. METHODS: The authors used C6 glioma cells to create a hypoxic GB model. The C6 glioma cells were stereotactically injected into the rat brain to create a tumor. Fifteen days later, MR imaging was used to confirm the presence of a glioma. The animals were randomly assigned to 1 of 3 groups: 1) temozolomide alone (350 mg/m(2)/day for 5 days); 2) temozolomide and radiation therapy (8 Gy); or 3) TSC (100 microg/kg for 5 days), temozolomide, and radiation therapy. Animals were followed through survival studies, and tumor response was assessed on serial MR images obtained at 15-day intervals during a 2-month period. RESULTS: Mean survival (+/- SEM) of the temozolomide-alone and the temozolomide/radiotherapy groups was 23.2 +/- 0.9 and 29.4 +/- 4.4 days, respectively. Mean survival in the TSC/temozolomide/radiotherapy group was 39.8 +/- 6 days, a statistically significant improvement compared with either of the other groups (p < 0.05). Although tumor size was statistically equivalent in all groups at the time of treatment initiation, the addition of TSC to temozolomide and radiotherapy resulted in a statistically significant reduction in the MR imaging-documented mean tumor size at 30 days after tumor implantation. The mean tumor size in the TSC/temozolomide/radiotherapy group was 18.9 +/- 6.6 mm(2) compared with 42.1 +/- 2.7 mm(2) in the temozolomide-alone group (p = 0.047) and 35.8 +/- 5.1 mm(2) in the temozolomide/radiation group (p = 0.004). CONCLUSIONS: In a hypoxic GB model, TSC improves the radiological and clinical effectiveness of temozolomide and radiation therapy. Further investigation of this oxygen diffusion enhancer as a radiosensitizer for hypoxic brain tumors seems warranted.

Effect of trans sodium crocetinate on brain tumor oxygenation. Laboratory investigation.

OBJECT: Glioblastoma multiforme tumors typically exhibit regions of hypoxia. Hypoxic regions within the tumor make cells less sensitive to radiosurgery and radiation therapy. Trans sodium crocetinate (TSC) has been shown to be a radiosensitizer. The goal of this research was to elucidate the underlying mechanism of TSC's radiosensitizing effect. METHODS: A rat C6 glioma model was used. The C6 glioma cells were stereotactically injected into the rat brain to create a tumor. Two weeks later, MR imaging was used to confirm the presence of a glioma. Following demonstration on MR imaging of a brain tumor, animals were randomized into 1 of 2 groups: 1) TSC alone (100 microg/kg), or 2) saline control. Licox probes were inserted into the brain tumor and contralateral cerebral hemisphere. Tissue oxygenation measurements were recorded before and after intravenous infusion of either TSC or saline. RESULTS: Not surprisingly, tissue oxygenation measurements revealed that the brain tumor was hypoxic relative to the contralateral cerebral hemisphere brain tissue. Two to 8 minutes after TSC was infused, tissue oxygenation measurements in the brain tumor increased above baseline by as much as 60%. After this temporary elevation following TSC infusion, tumor oxygenation measurements returned to baseline. No significant elevations in tissue oxygenation were seen on the contralateral side. Similarly, the saline vehicle was not observed to increase tissue oxygenation in either the brain tumor or the contralateral brain tissue. CONCLUSIONS: Administration of TSC transiently improves tissue oxygenation in hypoxic gliomas. Such an effect is one potential mechanism for the radiosensitization previously observed after addition of TSC.

Implications of early versus late bilateral pulmonary infiltrates in patients with aneurysmal subarachnoid hemorrhage.

INTRODUCTION: Bilateral pulmonary infiltrates occur frequently following aneurysmal subarachnoid hemorrhage (SAH), and may be associated with worse outcomes. The etiology, natural history, and prognosis of infiltrates occurring soon after SAH may differ from the characteristics of infiltrates developing at a later time. METHODS: We performed a retrospective cohort study involving 245 consecutive patients with a ruptured cerebral aneurysm to assess the association between "early" (< or = 72 h) or "late" (>72 h) bilateral pulmonary infiltrates and subsequent death or neurologic impairment. We used logistic regression models to adjust for baseline differences in age, level of consciousness, amount of blood on computed tomography, and the presence or absence of clinical vasospasm. RESULTS: Sixty-seven patients (27%) developed bilateral pulmonary infiltrates. Of these, 36 (54%) had early infiltrates, 24 (36%) had late infiltrates, and 7 (10%) had both. Twenty-eight patients (11% of entire cohort) met criteria for acute respiratory distress syndrome (ARDS). Patients with early infiltrates were more likely to have presented with stupor or coma than patients who developed infiltrates later (64% vs. 29%, P < 0.01). In multivariable analysis, late pulmonary infiltrates were strongly predictive of poor outcome (OR 5.0, 95% CI 1.9-13.6, P < 0.01), while early infiltrates were not (OR 1.2, 95% CI 0.5-3.0, P = 0.66). CONCLUSIONS: Bilateral pulmonary infiltrates after SAH most often occur within three days of aneurysm rupture. However, only infiltrates occurring beyond this time are independently associated with poor outcome. Increased emphasis on the prevention of late pulmonary complications has the potential to improve outcomes in SAH.

Applications of radiotherapy and radiosurgery in the management of pediatric Cushing's disease: a review of the literature and our experience.

Surgical extirpation of pituitary adenomas is considered the mainstay of therapy in pediatric patients with Cushing's disease. However, a small subset of patients will require adjuvant therapy either due to tumor invasiveness, or disease recurrence. Conventional radiation therapy (or radiotherapy) delivers ionizing radiation to control hormonally active cells in fractionated doses (spread out over time) in order to give normal cells time to recover, while radiosurgery involves focusing a high dose of radiation structures in a single treatment session to the adenoma while generally sparing the normal gland and surrounding of any substantial amount of radiation. This paper reviews the effectiveness of radiation in the treatment of pediatric Cushing's disease.