Neurochemical properties measured by 1H magnetic resonance spectroscopy may predict cognitive behaviour therapy outcome in paediatric OCD: a pilot study.

To identify neurochemical factors measured pre-treatment that may predict cognitive behavioural treatment (CBT) outcome, aiming at understanding possible causes of poor CBT response. 1H magnetic resonance spectroscopy was used before treatment with CBT in treatment naïve 11-18 year-old patients with moderate-severe OCD. Diagnoses and assessment of OCD severity were based on semi-structured interviews. Linear mixed effects models were used to analyse the association between metabolite level and treatment outcome. Worse CBT outcome was associated with higher concentration of glutamine and glutamate combined (Glx) in middle cingulate cortex (MCC) (F = + 3.35, p = 0.004) and of N-acetylaspartate and N-acetylaspartylglutamate combined (tNAA) (F = + 2.59, p = 0.019). Also, we noted a tendency towards higher thalamic Glx concentration (F = + 1.91, p = 0.077) to be associated with worse CBT outcome. In general, the findings of the current pilot study are compatible with the hypothesis of an overweight of excitatory to inhibitory factors in brain circuits driving goal-directed behaviours (GDB). Higher MCC Glx and tNAA may be involved in the selection of GDB. A more detailed understanding of how these brain areas function in health and illness is needed.

Clinical validation of a novel automated cell-free DNA screening assay for trisomies 21, 13, and 18 in maternal plasma.

OBJECTIVE: To evaluate clinical performance of a new automated cell-free (cf)DNA assay in maternal plasma screening for trisomies 21, 18, and 13, and to determine fetal sex. METHOD: Maternal plasma samples from 1200 singleton pregnancies were analyzed with a new non-sequencing cfDNA method, which is based on imaging and counting specific chromosome targets. Reference outcomes were determined by either cytogenetic testing, of amniotic fluid or chorionic villi, or clinical examination of neonates. RESULTS: The samples examined included 158 fetal aneuploidies. Sensitivity was 100% (112/112) for trisomy 21, 89% (32/36) for trisomy 18, and 100% (10/10) for trisomy 13. The respective specificities were 100%, 99.5%, and 99.9%. There were five first pass failures (0.4%), all in unaffected pregnancies. Sex classification was performed on 979 of the samples and 99.6% (975/979) provided a concordant result. CONCLUSION: The new automated cfDNA assay has high sensitivity and specificity for trisomies 21, 18, and 13 and accurate classification of fetal sex, while maintaining a low failure rate. The study demonstrated that cfDNA testing can be simplified and automated to reduce cost and thereby enabling wider population-based screening.

1H magnetic resonance spectroscopy evidence for occipital involvement in treatment-naive paediatric obsessive-compulsive disorder.

OBJECTIVE: Obsessive-compulsive disorder (OCD) is a chronic psychiatric disorder leading to considerable distress and disability. Therapies are effective in a majority of paediatric patients, however, many only get partial response. It is therefore important to study the underlying pathophysiology of the disorder. METHODS: 1H magnetic resonance spectroscopy (MRS) was used to study the concentration of brain metabolites in four different locations (cingulate gyrus and sulcus, occipital cortex, thalamus and right caudate nucleus). Treatment-naive children and adolescents with OCD (13 subjects) were compared with a group of healthy age- and gender-matched subjects (11 subjects). Multivariate analyses were performed on the concentration values. RESULTS: No separation between controls and patients was found. However, a correlation between metabolite concentrations and symptom severity as measured with the Children's Yale-Brown Obsessive-Compulsive Scale (CY-BOCS) was found. Strongest was the correlation with the CY-BOCS obsession subscore and aspartate and choline in the caudate nucleus (positively correlated with obsessions), lipids at 2 and 0.9 ppm in thalamus, and occipital glutamate+glutamine, N-acetylaspartate and myo-inosytol (negatively correlated with obsessions). CONCLUSIONS: The observed correlations between 1H MRS and CY-BOCS in treatment-naive patients further supports an occipital involvement in OCD. The results are consistent with our previous study on adult OCD patients. The 1H MRS data were not supportive of a separation between the patient and control groups.

Laboratory-Evolved Enzymes Provide Snapshots of the Development of Enantioconvergence in Enzyme-Catalyzed Epoxide Hydrolysis.

Engineered enzyme variants of potato epoxide hydrolase (StEH1) display varying degrees of enrichment of (2R)-3-phenylpropane-1,2-diol from racemic benzyloxirane. Curiously, the observed increase in the enantiomeric excess of the (R)-diol is not only a consequence of changes in enantioselectivity for the preferred epoxide enantiomer, but also to changes in the regioselectivity of the epoxide ring opening of (S)-benzyloxirane. In order to probe the structural origin of these differences in substrate selectivity and catalytic regiopreference, we solved the crystal structures for the evolved StEH1 variants. We used these structures as a starting point for molecular docking studies of the epoxide enantiomers into the respective active sites. Interestingly, despite the simplicity of our docking analysis, the apparent preferred binding modes appear to rationalize the experimentally determined regioselectivities. The analysis also identifies an active site residue (F33) as a potentially important interaction partner, a role that could explain the high conservation of this residue during evolution. Overall, our experimental, structural, and computational studies provide snapshots into the evolution of enantioconvergence in StEH1-catalyzed epoxide hydrolysis.

Furaldehyde substrate specificity and kinetics of Saccharomyces cerevisiae alcohol dehydrogenase 1 variants.

BACKGROUND: A previously discovered mutant of Saccharomyces cerevisiae alcohol dehydrogenase 1 (Adh1p) was shown to enable a unique NADH-dependent reduction of 5-hydroxymethylfurfural (HMF), a well-known inhibitor of yeast fermentation. In the present study, site-directed mutagenesis of both native and mutated ADH1 genes was performed in order to identify the key amino acids involved in this substrate shift, resulting in Adh1p-variants with different substrate specificities. RESULTS: In vitro activities of the Adh1p-variants using two furaldehydes, HMF and furfural, revealed that HMF reduction ability could be acquired after a single amino acid substitution (Y295C). The highest activity, however, was reached with the double mutation S110P Y295C. Kinetic characterization with both aldehydes and the in vivo primary substrate acetaldehyde also enabled to correlate the alterations in substrate affinity with the different amino acid substitutions. CONCLUSIONS: We demonstrated the key role of Y295C mutation in HMF reduction by Adh1p. We generated and kinetically characterized a group of protein variants using two furaldehyde compounds of industrial relevance. Also, we showed that there is a threshold after which higher in vitro HMF reduction activities do not correlate any more with faster in vivo rates of HMF conversion, indicating other cell limitations in the conversion of HMF.

Obtaining optical purity for product diols in enzyme-catalyzed epoxide hydrolysis: contributions from changes in both enantio- and regioselectivity.

Enzyme variants of the plant epoxide hydrolase StEH1 displaying improved stereoselectivities in the catalyzed hydrolysis of (2,3-epoxypropyl)benzene were generated by directed evolution. The evolution was driven by iterative saturation mutagenesis in combination with enzyme activity screenings where product chirality was the decisive selection criterion. Analysis of the underlying causes of the increased diol product ratios revealed two major contributing factors: increased enantioselectivity for the corresponding epoxide enantiomer(s) and, in some cases, a concomitant change in regioselectivity in the catalyzed epoxide ring-opening half-reaction. Thus, variant enzymes that catalyzed the hydrolysis of racemic (2,3-epoxypropyl)benzene into the R-diol product in an enantioconvergent manner were isolated.

Degraded water suppression in small volume ¹H MRS due to localised shimming.

OBJECT: High quality, artifact free, ¹H MRS require a homogeneous magnetic field over the volume of interest (VOI) and an effective global water suppression (WS). However, the shim coils act globally and, hence, affect the resonance frequency over the entire object, making the frequency selective WS spatially selective. Unsuppressed water signal may cause spurious echo artifacts. AIM: Study and visualise the effect localised shimming has on WS. MATERIALS AND METHODS: A water suppression imaging (WSI) scan for visualising regions unaffected by the WS was designed and applied in vivo for two different VOI localisations, at two field strengths. Two shim optimisation methods, and first and second order shims were compared. In addition, shim settings for six VOI localisations were retrospectively investigated. RESULTS: The WSI-scan effectively visualised the spatial extent of the WS. The coverage decreased when the shim was optimised on a smaller VOI. Second order shims accentuated the problem, resulting, on average, in a WS coverage of only 35% of the head volume. CONCLUSION: Localised shimming can result in large regions of unsuppressed water, which can lead to spurious echo artifacts in the spectrum. To help overcome these problems globally optimised shims can be used during WS.

Adipose tissue morphology, imaging and metabolomics predicting cardiometabolic risk and family history of type 2 diabetes in non-obese men.

We evaluated the importance of body composition, amount of subcutaneous and visceral fat, liver and heart ectopic fat, adipose tissue distribution and cell size as predictors of cardio-metabolic risk in 53 non-obese male individuals. Known family history of type 2 diabetes was identified in 25 individuals. The participants also underwent extensive phenotyping together with measuring different biomarkers and non-targeted serum metabolomics. We used ensemble learning and other machine learning approaches to identify predictors with considerable relative importance and their intricate interactions. Visceral fat and age were strong individual predictors of ectopic fat accumulation in liver and heart along with markers of lipid oxidation and reduced glucose tolerance. Subcutaneous adipose cell size was the strongest individual predictor of whole-body insulin sensitivity and also a marker of visceral and ectopic fat accumulation. The metabolite 3-MOB along with related branched-chain amino acids demonstrated strong predictability for family history of type 2 diabetes.

Epoxide hydrolysis as a model system for understanding flux through a branched reaction scheme.

The epoxide hydrolase StEH1 catalyzes the hydrolysis of trans-methylstyrene oxide to 1-phenyl-propane-1,2-diol. The (S,S)-epoxide is exclusively transformed into the (1R,2S)-diol, while hydrolysis of the (R,R)-epoxide results in a mixture of product enantiomers. In order to understand the differences in the stereoconfigurations of the products, the reactions were studied kinetically during both the pre-steady-state and steady-state phases. A number of closely related StEH1 variants were analyzed in parallel, and the results were rationalized by structure-activity analysis using the available crystal structures of all tested enzyme variants. Finally, empirical valence-bond simulations were performed in order to provide additional insight into the observed kinetic behaviour and ratios of the diol product enantiomers. These combined data allow us to present a model for the flux through the catalyzed reactions. With the (R,R)-epoxide, ring opening may occur at either C atom and with similar energy barriers for hydrolysis, resulting in a mixture of diol enantiomer products. However, with the (S,S)-epoxide, although either epoxide C atom may react to form the covalent enzyme intermediate, only the pro-(R,S) alkylenzyme is amenable to subsequent hydrolysis. Previously contradictory observations from kinetics experiments as well as product ratios can therefore now be explained for this biocatalytically relevant enzyme.

Imaging single DNA molecules for high precision NIPT.

Cell-free DNA analysis is becoming adopted for first line aneuploidy screening, however for most healthcare programs, cost and workflow complexity is limiting adoption of the test. We report a novel cost effective method, the Vanadis NIPT assay, designed for high precision digitally-enabled measurement of chromosomal aneuploidies in maternal plasma. Reducing NIPT assay complexity is achieved by using novel molecular probe technology that specifically label target chromosomes combined with a new readout format using a nanofilter to enrich single molecules for imaging and counting without DNA amplification, microarrays or sequencing. The primary objective of this study was to assess the Vanadis NIPT assay with respect to analytical precision and clinical feasibility. Analysis of reference DNA samples indicate that samples which are challenging to analyze with low fetal-fraction can be readily detected with a limit of detection determined at <2% fetal-fraction. In total of 286 clinical samples were analysed and 30 out of 30 pregnancies affected by trisomy 21 were classified correctly. This method has the potential to make cost effective NIPT more widely available with more women benefiting from superior detection and false positive rates.

fMRI after Phalloplasty with Nerve Anastomosis in a Trans-Man Patient.

We report on a case of a trans-man patient, who underwent penile reconstruction with the use of a radial forearm flap, urethroplasty, vaginectomy and scrotoplasty, insertion of testicle implants, and penile erection implants, similar to previously described methods. One of the requirements for an ideal phalloplasty is the preservation of erogenous sensitivity, which is often demanded by the patients for fulfilling their sexual well-being. For the first time known to us, we use a functional magnetic resonance imaging following radial forearm flap phalloplasty with nerve anastomosis to assess the cortical activation after clitoral stimulation. The patient was poked with a plastic pen on the neophallus and the groin. Regular block design with T1 and BOLD-T2* images were used. The results contradict the classic Penfield and Rasmussen homunculus, that is, the activations in the primary somatosensory cortex (S1) were bilateral with a left-sided dominance in the lateral parts of the medial postcentral gyrus (same region as the groin), and no activations were observed in the mesial parts of the postcentral gyrus. We also reported bilateral activations with a left-sided dominance in the secondary somatosensory cortex (S2) and near Broca's area at the sylvian fissure just posterior to ramus ascendens. Our findings are similar to previous studies reporting on imaging related to genital sensitivity.

The influence of cardiac triggering time and an optimization strategy for improved cardiac MR spectroscopy.

PURPOSE: To study how cardiac motion affects the spectral quality in cardiac MR spectroscopy and to establish an optimization strategy for the cardiac triggering time for improved quality and success rate of cardiac MRS. METHOD: Water spectra were acquired while the cardiac triggering time was varied over the cardiac cycle, and five different spectral quality parameters were studied (frequency, phase, linewidth, amplitude and noise). Furthermore, three different optimization strategies for the cardiac triggering time were tested, and finally, a comparison was made between water suppressed lipid spectra acquired in systole and diastole. RESULTS: The cardiac triggering time had a high impact on the spectral quality, especially on the mean signal amplitude and the standard deviation of the signal amplitude, phase and linewidth. Generally, the highest spectral quality was observed for spectra acquired in mid to end systole, at approximately 23% of the cardiac cycle. The exact optimal triggering time differed between subjects and needed to be individually optimized. To optimize the triggering time with our proposed MRS-method gave in average 13% higher signal than when the triggering time was determined through imaging. Lipid spectra acquired in systole demonstrated higher quality with improved SNR compared with acquisitions made in diastole. CONCLUSION: This study shows that the spectral quality in cardiac MRS is strongly dependent on the cardiac triggering time, and that the spectral quality as well as the repeatability between acquisitions is greatly improved when the cardiac triggering time is individually optimized in mid to end systole using MRS.

Accurate and sensitive measurements of magnetic susceptibility using echo planar imaging.

Susceptibility differences are common causes for artifacts in magnetic resonance (MR); therefore, it is important to choose phantom materials in a way that these artifacts are kept at a minimum. In this study, a previously proposed MR imaging (MRI) method [Beuf O, Briguet A, Lissac M, Davis R. Magnetic resonance imaging for the determination of magnetic susceptibility of materials. J Magn Reson 1996; Series B(112):111-118] was improved to facilitate sensitive in-house measurements of different phantom materials so that such artifacts can more easily be minimized. Using standard MRI protocols and distilled water as reference, we measured magnetic volume susceptibility differences with a clinical MR system. Two imaging techniques, echo planar imaging (EPI) and spin echo, were compared using liquid samples whose susceptibilities were verified by MR spectroscopy. The EPI sequence has a very narrow bandwidth in the phase-encoding direction, which gives an increased sensitivity to magnetic field inhomogeneities. All MRI measurements were evaluated in two ways: (1) manual image analysis and (2) model fitting. The narrow bandwidth of the EPI made it possible to detect very small susceptibility differences (equivalent susceptibility difference, Deltachi(e)> or =0.02 ppm), and even plastics could be measured. Model fitting yielded high accuracy and high sensitivity and was less sensitive to other image artifacts as compared with manual image analysis.

Expanding the Catalytic Triad in Epoxide Hydrolases and Related Enzymes.

Potato epoxide hydrolase 1 exhibits rich enantio- and regioselectivity in the hydrolysis of a broad range of substrates. The enzyme can be engineered to increase the yield of optically pure products as a result of changes in both enantio- and regioselectivity. It is thus highly attractive in biocatalysis, particularly for the generation of enantiopure fine chemicals and pharmaceuticals. The present work aims to establish the principles underlying the activity and selectivity of the enzyme through a combined computational, structural, and kinetic study using the substrate trans-stilbene oxide as a model system. Extensive empirical valence bond simulations have been performed on the wild-type enzyme together with several experimentally characterized mutants. We are able to computationally reproduce the differences between the activities of different stereoisomers of the substrate and the effects of mutations of several active-site residues. In addition, our results indicate the involvement of a previously neglected residue, H104, which is electrostatically linked to the general base H300. We find that this residue, which is highly conserved in epoxide hydrolases and related hydrolytic enzymes, needs to be in its protonated form in order to provide charge balance in an otherwise negatively charged active site. Our data show that unless the active-site charge balance is correctly treated in simulations, it is not possible to generate a physically meaningful model for the enzyme that can accurately reproduce activity and selectivity trends. We also expand our understanding of other catalytic residues, demonstrating in particular the role of a noncanonical residue, E35, as a "backup base" in the absence of H300. Our results provide a detailed view of the main factors driving catalysis and regioselectivity in this enzyme and identify targets for subsequent enzyme design efforts.

Cost of care for patients treated with lipid-lowering drugs.

OBJECTIVE: To investigate the relationship between attainment of treatment goals with lipid-lowering therapy and healthcare costs. PARTICIPANTS: 9789 patients who received treatment with a lipid-lowering agent at any time between 1 January 1993 and 14 April 2003. DESIGN AND METHODS: A cohort study using linkage of patient medical records from 29 Swedish primary care centres and the Swedish national inpatient register. The primary outcomes of interest were the total costs of medical care and costs of cardiovascular-related inpatient care during the year before treatment initiation and during years 1, 2 and 3 of treatment. The cost data were analysed with a two-part random-effects regression model. RESULTS: Of the 9789 patients identified in the database for the study, 6316 had at least one cholesterol measurement during the year after the index prescription and were included in the analysis. 37% of the patients attained the goal of low-density lipoprotein cholesterol < 3.0 mmol/L and total cholesterol < 5.0 mmol/L. Patients who attained treatment goal had 44% higher pre-treatment costs of care. During the first year of treatment, patients who attained treatment goal had 28% higher costs of care. After the first year, costs for goal-attaining patients were 17% higher. However, the cost of cardiovascular-related inpatient care in patients attaining cholesterol treatment goal was twice as high as in patients not achieving goal before treatment start and 40% lower 2-3 years after treatment start. CONCLUSION: Patients reaching target cholesterol levels showed a trend of cost reductions over time, whereas no such trend could be found for patients not reaching goal levels. Reductions in costs were substantial for cardiovascular-related inpatient care for patients attaining cholesterol goals compared with patients not attaining cholesterol goals.