9.4 T MR microscopy of the substantia nigra with pathological validation in controls and disease.

BACKGROUND: The anatomy of the substantia nigra on conventional MRI is controversial. Even using histological techniques it is difficult to delineate with certainty from surrounding structures. We sought to define the anatomy of the SN using high field spin-echo MRI of pathological material in which we could study the anatomy in detail to corroborate our MRI findings in controls and Parkinson's disease and progressive supranuclear palsy. METHODS: 23 brains were selected from the Queen Square Brain Bank (10 controls, 8 progressive supranuclear palsy, 5 Parkinson's disease) and imaged using high field 9.4 Tesla spin-echo MRI. Subsequently brains were cut and stained with Luxol fast blue, Perls stain, and immunohistochemistry for substance P and calbindin. Once the anatomy was defined on histology the dimensions and volume of the substantia nigra were determined on high field magnetic resonance images. RESULTS: The anterior border of the substantia nigra was defined by the crus cerebri. In the medial half it was less distinct due to the deposition of iron and the interdigitation of white matter and the substantia nigra. The posterior border was flanked by white matter bridging the red nucleus and substantia nigra and seen as hypointense on spin-echo magnetic resonance images. Within the substantia nigra high signal structures corresponded to confirmed nigrosomes. These were still evident in Parkinson's disease but not in progressive supranuclear palsy. The volume and dimensions of the substantia nigra were similar in Parkinson's disease and controls, but reduced in progressive supranuclear palsy. CONCLUSIONS: We present a histologically validated anatomical description of the substantia nigra on high field spin-echo high resolution magnetic resonance images and were able to delineate all five nigrosomes. In accordance with the pathological literature we did not observe changes in the nigrosome structure as manifest by volume or signal characteristics within the substantia nigra in Parkinson's disease whereas in progressive supranuclear palsy there was microarchitectural destruction.

Reprogramming of hepatic fat accumulation and 'browning' of adipose tissue by the short-chain fatty acid acetate.

BACKGROUND/OBJECTIVES: Short-chain fatty acids, produced by microbiome fermentation of carbohydrates, have been linked to a reduction in appetite, body weight and adiposity. However, determining the contribution of central and peripheral mechanisms to these effects has not been possible. SUBJECTS/METHODS: C57BL/6 mice fed with either normal or high-fat diet were treated with nanoparticle-delivered acetate, and the effects on metabolism were investigated. RESULTS: In the liver, acetate decreased lipid accumulation and improved hepatic function, as well as increasing mitochondrial efficiency. In white adipose tissue, it inhibited lipolysis and induced 'browning', increasing thermogenic capacity that led to a reduction in body adiposity. CONCLUSIONS: This study provides novel insights into the peripheral mechanism of action of acetate, independent of central action, including 'browning' and enhancement of hepatic mitochondrial function.

Acquired resistance to EGFR tyrosine kinase inhibitors alters the metabolism of human head and neck squamous carcinoma cells and xenograft tumours.

BACKGROUND: Acquired resistance to molecularly targeted therapeutics is a key challenge in personalised cancer medicine, highlighting the need for identifying the underlying mechanisms and early biomarkers of relapse, in order to guide subsequent patient management. METHODS: Here we use human head and neck squamous cell carcinoma (HNSCC) models and nuclear magnetic resonance (NMR) spectroscopy to assess the metabolic changes that follow acquired resistance to EGFR tyrosine kinase inhibitors (TKIs), and which could serve as potential metabolic biomarkers of drug resistance. RESULTS: Comparison of NMR metabolite profiles obtained from control (CAL(S)) and EGFR TKI-resistant (CAL(R)) cells grown as 2D monolayers, 3D spheroids or xenograft tumours in athymic mice revealed a number of differences between the sensitive and drug-resistant models. In particular, we observed elevated levels of glycerophosphocholine (GPC) in CAL(R) relative to CAL(S) monolayers, spheroids and tumours, independent of the growth rate or environment. In addition, there was an increase in alanine, aspartate and creatine+phosphocreatine in resistant spheroids and xenografts, and increased levels of lactate, branched-chain amino acids and a fall in phosphoethanolamine only in xenografts. The xenograft lactate build-up was associated with an increased expression of the glucose transporter GLUT-1, whereas the rise in GPC was attributed to inhibition of GPC phosphodiesterase. Reduced glycerophosphocholine (GPC) and phosphocholine were observed in a second HNSCC model probably indicative of a different drug resistance mechanism. CONCLUSIONS: Our studies reveal metabolic signatures associated not only with acquired EGFR TKI resistance but also growth pattern, microenvironment and contributing mechanisms in HNSCC models. These findings warrant further investigation as metabolic biomarkers of disease relapse in the clinic.

Dichloroacetate induces autophagy in colorectal cancer cells and tumours.

BACKGROUND: Dichloroacetate (DCA) has been found to have antitumour properties. METHODS: We investigated the cellular and metabolic responses to DCA treatment and recovery in human colorectal (HT29, HCT116 WT and HCT116 Bax-ko), prostate carcinoma cells (PC3) and HT29 xenografts by flow cytometry, western blotting, electron microscopy, (1)H and hyperpolarised (13)C-magnetic resonance spectroscopy. RESULTS: Increased expression of the autophagy markers LC3B II was observed following DCA treatment both in vitro and in vivo. We observed increased production of reactive oxygen species (ROS) and mTOR inhibition (decreased pS6 ribosomal protein and p4E-BP1 expression) as well as increased expression of MCT1 following DCA treatment. Steady-state lactate excretion and the apparent hyperpolarised [1-(13)C] pyruvate-to-lactate exchange rate (k(PL)) were decreased in DCA-treated cells, along with increased NAD(+)/NADH ratios and NAD(+). Steady-state lactate excretion and k(PL) returned to, or exceeded, control levels in cells recovered from DCA treatment, accompanied by increased NAD(+) and NADH. Reduced k(PL) with DCA treatment was found in HT29 tumour xenografts in vivo. CONCLUSIONS: DCA induces autophagy in cancer cells accompanied by ROS production and mTOR inhibition, reduced lactate excretion, reduced k(PL) and increased NAD(+)/NADH ratio. The observed cellular and metabolic changes recover on cessation of treatment.

High resolution MR anatomy of the subthalamic nucleus: imaging at 9.4 T with histological validation.

Using conventional MRI the subthalamic nucleus (STN) is not clearly defined. Our objective was to define the anatomy of the STN using 9.4 T MRI of post mortem tissue with histological validation. Spin-echo (SE) and 3D gradient-echo (GE) images were obtained at 9.4 T in 8 post mortem tissue blocks and compared directly with corresponding histological slides prepared with Luxol Fast Blue/Cresyl Violet (LFB/CV) in 4 cases and Perl stain in 3. The variability of the STN anatomy was studied using internal reference points. The anatomy of the STN and surrounding structures was demonstrated in all three anatomical planes using 9.4 T MR images in concordance with LFB/CV stained histological sections. Signal hypointensity was seen in 6/8 cases in the anterior and medial STN that corresponded with regions of more intense Perl staining. There was significant variability in the volume, shape and location of the borders of the STN. Using 9.4 T MRI, the internal signal characteristics and borders of the STN are clearly defined and significant anatomical variability is apparent. Direct visualisation of the STN is possible using high field MRI and this is particularly relevant, given its anatomical variability, for planning deep brain stimulation.

Quantification of the interceptor action of caffeine on the in vitro biological effect of the anti-tumour agent topotecan.

Using published in vitro data on the dependence of the percentage of apoptosis induced by the anti-cancer drug topotecan in a leukaemia cell line on the concentration of added caffeine, and a general model of competitive binding in a system containing two aromatic drugs and DNA, it has been shown to be possible to quantify the relative change in the biological effect just using a set of component concentrations and equilibrium constants of the complexation of the drugs. It is also proposed that a general model of competitive binding and parameterization of that model may potentially be applied to any system of DNA-targeting aromatic drugs under in vitro conditions. The main reasons underpinning the proposal are the general feature of the complexation of aromatic drugs with DNA and their interaction in physiological media via hetero-association.

Structural basis for the binding affinity of a homologous series of synthetic phenoxazone drugs with DNA: NMR and molecular mechanics analysis.

The molecular basis of the marked structure-activity relationship for a homologous series of DNA-binding phenoxazone drugs (ActII-ActIV) has been investigated by NMR spectroscopy and molecular mechanics. The spatial structures of the complexes between the drugs and a model deoxytetranucleotide, 5'-d(TpGpCpA), have been determined by molecular mechanics methods using homonuclear (1)H-(1)H 2D-NOESY and heteronuclear (1)H-(31)P (HMBC) NMR spectroscopic data. Observed intermolecular NOE contacts and equilibrium binding studies confirm that the binding affinity of the synthetic phenoxazone derivatives with d(TGCA) decreases with an increase in the number of CH(2) groups in the dimethylaminoalkyl side chains, i.e., ActII > ActIII > ActIV, in agreement with the observed biological activity of these compounds. Molecular mechanics calculations of the spatial structures of the intercalated complexes of ActII-ActIV with d(TGCA) indicate that the different binding constants of the phenoxazone derivatives with the DNA oligomer are due to the different degrees of intercalation of the chromophore and the different steric arrangements of aminoalkyl side chains in the minor groove of the tetramer duplex; this results in different distances between the negatively-charged phosphates of the DNA duplex and the terminal positively-charged N(CH(3))(2) groups of the side chains.

A comparison of bile composition from heart-beating and non-heart-beating rabbit organ donors during normothermic extracorporeal liver perfusion: experimental evaluation using proton magnetic resonance spectroscopy.

BACKGROUND: Organs retrieved from marginal and non-heart-beating donors (NHBDs) have sustained variable degrees of preretrieval damage that result in an increased incidence of complications. Normothermic extracorporeal liver perfusion (NELP) provides an opportunity to evaluate and resuscitate such organs. The aim of this study was to identify markers of ischemic injury in bile during perfusion of livers from heart-beating donors (HBDs) and NHBDs. METHODS: Livers were retrieved from New Zealand white rabbits. The HBD group (n=4) had no in situ warm ischemia before retrieval and the NHBD group (n=4), 45 minutes of in situ warm ischemia before liver retrieval. After 40 minutes of postretrieval cold ischemia, all livers were dual vessel reperfused, normothermically with oxygenated buffer solution supplemented with rabbit red blood cells, for 6 hours. Bile was collected and examined with 1HMRS. RESULTS: Perfusion bile from HBD group showed an increased concentration of bile acids, lactate, glucose, and phosphatidylcholine, but a decreased concentration of acetate as compared to retrieval bile. This trend was further enhanced in NHBD group. The mean +/- SD (in micromol/L) were bile acids (10.48 +/- 2.8 vs 26.05 +/- 12.1 vs 44.5 +/- 44.5), lactate (10.66 +/- 4.5 vs 14.66 +/- 5.2 vs 13.22 +/- 1.8), glucose (5.37 +/- 2 vs 21.2 +/- 5.0 vs 29.09 +/- 15.3), phosphatidylcholine (0.21 +/- 0.02 vs 5.57 +/- 1.7 vs 6.42 +/- 0.3), and acetate (1.8 +/- 0.5 vs 0.39 +/- 0.1 vs 0.38 +/- 0.09) for retrieval bile, HBD perfusion bile, and NHBD perfusion bile, respectively. One animal from each group did not produce any bile during perfusion. CONCLUSIONS: 1HMRS of biliary constituents revealed differences with the type of ischemia. These indices may be potential markers of the extent of warm ischemic injury and the functional activity of an extracorporeally perfused liver.

High resolution MRI of the brain at 4.7 Tesla using fast spin echo imaging.

Over recent years, high field MR scanners (3 T and above) have become increasingly widespread due to potential advantages such as higher signal-to-noise ratio. However, few examples of high resolution images covering the whole brain in reasonable acquisition times have been published to date and none have used fast spin echo (FSE), a sequence commonly employed for the acquisition of T(2) weighted images at 1.5 T. This is mostly due to the increased technical challenges associated with uniform signal generation and the increasingly restrictive constraints of current safety guidelines at high field. We investigated 10 volunteers using an FSE sequence optimized to the 4.7 T environment. This sequence allows the acquisition of 17- and 34-slice data sets with an in-plane resolution of approximately 500 microm x 500 microm and a slice thickness of 2 mm, in 5 min 40 s and 11 min 20 s, respectively. The images appear T(2) weighted, although the contrast is due to the combined effects of chosen echo time, magnetization transfer, direct radio frequency saturation and diffusion as well as the T(1) and T(2) relaxation times of the tissue. The result is an excellent detailed visualization of anatomical structures, demonstrating the great potential of 4.7 T MRI for clinical applications. This paper shows that, with careful optimization of sequence parameters, FSE imaging can be used at high field to generate images with high spatial resolution and uniform contrast across the whole brain within the prescribed power deposition limits.

Disseminated melioidosis in two cats.

Melioidosis was diagnosed in two cats at necropsy. The first cat presented with jaundice and anaemia but died of overwhelming sepsis soon after admission, despite blood transfusion and other supportive measures. The second cat died several days after developing neurological signs; an infected digital wound may have been the primary focus of infection in this patient. The cats had presumably acquired the infection in Malaysia and northern Australia, respectively, and in both cases disease may have represented reactivation of a latent infection brought on by the stress of relocation. The epidemiology, clinical presentation, diagnosis and treatment of melioidosis are discussed from a feline perspective.

Proton nuclear magnetic resonance analysis of hepatic bile from donors and recipients in human liver transplantation.

The current shortage of donor organs in liver transplantation has led experienced transplant centers to use more "marginal" grafts. The development of a reliable technique of bile collection gives access to hepatic bile from donors and recipients for bile analysis to characterize the grafts. Proton nuclear magnetic resonance analysis has been applied to the study of bile for more than 30 years, showing encouraging results. This is the first study where proton nuclear magnetic resonance analysis has been applied to hepatic bile from selected liver grafts to evaluate its potential role in graft assessment. Hepatic bile was collected from eight liver donors (four with normal and four with steatotic grafts) during organ retrieval and four transplant recipients (two with good early graft function and two with primary dysfunction) immediately after graft reperfusion. A Varian Unity+ NMR spectrometer, operating at 11.7 Tesla (500 MHz for 1H), was used to obtain the proton nuclear magnetic resonance spectra. The results showed that the hepatic bile from steatotic grafts collected before transplantation had more intense phosphatidylcholine head group resonance than bile from normal grafts. It also showed slower clearance of University of Wisconsin solution in grafts with subsequent primary graft dysfunction, suggesting a slower recovery of bile secretion. These preliminary findings suggest that proton nuclear magnetic resonance analysis might help to differentiate the characteristics of bile acids and biliary lipids from normal and steatotic grafts. The monitoring of the resonance signal of University of Wisconsin solution washout, bile acid, and biliary lipid secretion may help to predict the development of primary graft dysfunction and avoid the need for retransplantation.

Emerging homogeneous DNA-based technologies in the clinical laboratory.

BACKGROUND: Advances in molecular diagnostic technologies have enabled genetic testing in single closed-tube reactions. The purpose of this review is to highlight some of the platforms and technologies currently available for the homogeneous detection of targets and the application of the technologies in the clinical setting. Validation issues surrounding the technologies, which may need to be addressed before they can become widely accepted, will also be discussed. APPROACH: This review discusses the principles of several of the major technologies available for performing homogeneous genetic analyses. Publications arising from the application of the technologies in a wide range of clinical areas are used to highlight and compare the potential advantages and shortcomings of the various technologies. CONTENT: This review is descriptive and focuses on three areas: the technologies available for performing homogeneous analysis, the clinical applications where the technologies are being used, and validation issues surrounding the acceptance of the technologies in the general clinical setting. SUMMARY: This review intends to give the reader a greater understanding of the various technologies available for performing homogeneous genetic testing in the clinical laboratory. Through insight into the principles and performance characteristics underlying these technologies, the end user can evaluate their value and limitations in the clinical diagnostic setting.

Interlaboratory study on thermal cycler performance in controlled PCR and random amplified polymorphic DNA analyses.

BACKGROUND: Intercomparisons of PCR-based data between laboratories require an assurance of assay reproducibility. We performed an interlaboratory study to investigate the contribution made by a variety of thermal cyclers to PCR performance as measured by interblock reproducibility and intrablock repeatability. METHODS: Two standardized assays designed to minimize the introduction of non-thermal-cycler-dependent variations were evaluated by 18 laboratories in the United Kingdom, using 33 thermal cyclers of various makes and models. We used a single-product (590 bp) PCR, established in our laboratory as a robust and specific reaction. The second reaction, a multiproduct random amplified polymorphic DNA (RAPD) PCR, was known to be more susceptible to small changes in block temperature and was therefore considered a way of assessing block uniformity with respect to temperature. Assay repeatability data were analyzed with respect to temperature calibration status, the type of temperature control mechanism, thermal cycler age, and the presence of oil overlay or heated lid systems. RESULTS: All (100%) of the laboratories produced the correct target for the single-product PCR assay, although substantial variation in yield in replicate reactions was observed in 9.4% of these. The RAPD reaction generated results that varied extensively both within the same block and between different thermal cyclers. For eight replicates of a positive sample, 88% intrablock repeatability was demonstrated in calibrated thermal cyclers, which decreased to 63% in noncalibrated instruments. CONCLUSIONS: Irrespective of the make and model of thermal cycler, temperature-calibrated instruments consistently generated more repeatable RAPD data than noncalibrated instruments. Guidelines are offered on optimizing and monitoring thermal cycler performance.

Reduction of alcohol drinking and upregulation of opioid receptors by oral naltrexone in AA rats.

Rats of the high-drinking AA line were given 1 mg/kg naltrexone (NTX) or vehicle orally with a stress-free procedure just before 1 h of access to 10% ethanol daily for 8 days and again, 8 h later on the first 7 days. Forebrain homogenate binding studies using 0.03-6.00 nM [3H] naloxone were conducted from 1 to 4 days following treatment. NTX significantly suppressed alcohol intake, with the effect becoming progressively greater over days and continuing during the post-treatment period. Saturation binding studies in brain homogenate revealed that NTX had increased the B(max) for opioid receptors by 93%, 74%, 49%, and 28%, respectively, from post-treatment days 1 to 4 without altering K(d). B(max) was negatively correlated (r=-0.510, p=0.008) with alcohol intake during the preceding hour, but in control rats, it was positively correlated with changes in alcohol intake over time (r=+0.790, p=0.020). These results are consistent with the hypothesis that opioid receptors mediate reinforcement from alcohol and that NTX reduces subsequent alcohol drinking by extinction. Opioid receptor upregulation can develop simultaneously with suppression of drinking and may partially counteract the clinical benefits from NTX in the treatment of alcoholism.

A new system for the metabolic investigation of the isolated, perfused rat heart.

NMR spectroscopy is an invaluable technique in metabolic investigations of isolated, perfused hearts. Most studies employ global perfusion methods together with an NMR coil that surrounds the heart and thus detects signals from its entirety. The present report describes the construction and testing of a novel, two surface-coil probe, in combination with a dual-perfused heart preparation, that enables spectra to be collected independently from the two coronary beds of the rat heart.(31)P NMR spectra of perfused rat hearts in which the septum and right ventricle have been made ischaemic, while the free left ventricular wall is fully perfused, demonstrate the powerful potential of this new system.

Altered brain metabolism in the C57BL/Wld mouse strain detected by magnetic resonance spectroscopy: association with delayed Wallerian degeneration?

In the C57BL/Wld(s) (Wld) mouse strain, both PNS and CNS axonal disintegration during Wallerian degeneration is dramatically slowed, with isolated axons being able to conduct compound action potentials (CAPs) for several weeks post-transection. The ability to conduct a CAP signifies the presence of an intact plasma membrane, normal ion gradients, and functioning ion channels. In neurons, ion homeostasis is primarily regulated by the Na(+)-K(+)-ATPase, which utilizes approximately 50% of neuronal energy output. To investigate the possibility that the Wld mutation prolongs axonal degeneration by conferring a more favorable energetic status to neurons or alters metabolism, we used 31P and 1H magnetic resonance spectroscopy (MRS) to compare the cerebral and muscle energy metabolism, membrane phospholipid contents, and water-soluble metabolites of Wld and wild-type (C57BL/6J [6J], and BALB/c) mouse strains. We first demonstrate that, with advancing age, transected Wld CNS nerves degenerate faster, paralleling previous findings in the PNS. We found significantly decreased phosphocreatine and phosphomonoester concentrations in the brains of Wld mice at 1- and 2-months of age compared to both 6J and BALB/c mice, but we failed to find differences in the adenylate (ATP, ADP, or AMP) or phospholipid concentrations. In another excitable tissue, skeletal muscle, no differences in energy-containing metabolites were detected. High resolution 1H MRS indicated that at 1 month of age, Wld brains have cytosolic levels of glutamate and phosphocholine that are significantly decreased, relative to total N-acetyl aspartate content. Our results demonstrate that delayed Wallerian degeneration in the C57BL/Wld mouse strain is associated with altered cerebral metabolism, although these changes may be secondary to the mutation.

Giant toads Bufo marinus in Australia and Venezuela have antibodies against 'ranaviruses'.

A serological survey was conducted for antibodies against 'ranaviruses' in the giant toad Bufo marinus in Australia and Venezuela. Sera containing antibodies against 'ranaviruses' were found in both countries. In Australia positive antibodies were identified in populations throughout most of the known range of B. marinus. Results were confirmed by immunofluorescence and immunoelectron microscopy where a characteristic staining pattern of 'ranaviruses' in infected cells was observed. Whilst a 'ranavirus(es)' has been isolated from populations of B. marinus in Venezuela, no virus has been isolated from Australian B. marinus populations. The significance of 'ranavirus' sero-positive B. marinus in Australia is discussed.

Localised sequence regions possessing high melting temperatures prevent the amplification of a DNA mimic in competitive PCR.

The polymerase chain reaction is an immensely powerful technique for identification and detection purposes. Increasingly, competitive PCR is being used as the basis for quantification. However, sequence length, melting temperature and primary sequence have all been shown to influence the efficiency of amplification in PCR systems and may therefore compromise the required equivalent co-amplification of target and mimic in competitive PCR. The work discussed here not only illustrates the need to balance length and melting temperature when designing a competitive PCR assay, but also emphasises the importance of careful examination of sequences for GC-rich domains and other sequences giving rise to stable secondary structures which could reduce the efficiency of amplification by serving as pause or termination sites. We present data confirming that under particular circumstances such localised sequence, high melting temperature regions can act as permanent termination sites, and offer an explanation for the severity of this effect which results in prevention of amplification of a DNA mimic in competitive PCR. It is also demonstrated that when Taq DNA polymerase is used in the presence of betaine or a proof reading enzyme, the effect may be reduced or eliminated.

Chytridiomycosis causes amphibian mortality associated with population declines in the rain forests of Australia and Central America.

Epidermal changes caused by a chytridiomycete fungus (Chytridiomycota; Chytridiales) were found in sick and dead adult anurans collected from montane rain forests in Queensland (Australia) and Panama during mass mortality events associated with significant population declines. We also have found this new disease associated with morbidity and mortality in wild and captive anurans from additional locations in Australia and Central America. This is the first report of parasitism of a vertebrate by a member of the phylum Chytridiomycota. Experimental data support the conclusion that cutaneous chytridiomycosis is a fatal disease of anurans, and we hypothesize that it is the proximate cause of these recent amphibian declines.

PET and NMR dual acquisition (PANDA): applications to isolated, perfused rat hearts.

Positron emission tomography and nuclear magnetic resonance spectroscopy are non-invasive techniques that allow serial metabolic measurements to be obtained in a single subject. Significant advantages could be obtained if both types of scans could be acquired with a single machine. A small-scale PET scanner, designed to operate in a high magnetic field, was therefore constructed and inserted into the top half of a 7.3 cm bore, 9.4 T NMR magnet and its performance characterized. The magnetic field did not significantly affect either the sensitivity (approximately 3 kcps/MBq) or the spatial resolution (2.0 mm full width at half maximum, measured using a 0.25 mm diameter line source) of the scanner. However, the presence of the PET scanner resulted in a small decrease in field homogeneity. The first, simultaneous 31P NMR spectra (200, 80 degrees pulses collected at 6 s intervals) and PET images (transverse, mid-ventricular slices at the level of the mitral value) from isolated, perfused rat hearts were acquired using a specially designed NMR probe inserted into the bottom half of the magnet. The PET images were of excellent quality, enabling the left ventricular wall and interventricular septum to be clearly seen. In conclusion, we have demonstrated the simultaneous acquisition of PET and NMR data from perfused rat hearts; we believe that the combination of these two powerful techniques has tremendous potential in both the laboratory and the clinic.