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Human milk oligosaccharides are of high interest as active ingredients in infant formulas and dietary food supplements. Full characterization of members of this compound class is challenging due to the intrinsic complexity of byproducts during synthesis by fermentation. Moreover, when method validation is targeted for a regulated environment, a robust chromatographic separation of the highly polar oligosaccharides needs to be addressed, including isomers and compounds relevant for potential product adulteration. We present a combined approach of validated chromatography and NMR spectroscopy, which allows for full mass balancing of industrially produced 2'-fucosyl-d-lactose. A combination of NMR spectroscopy, mass spectrometry, and action IR spectroscopy tackles structural elucidation of monoacetylated species as a new class of byproducts.

Importance: β-Adrenergic stimulation elicits heart palpitations and dyspnea, key features of acute anxiety and sympathetic arousal, yet no neuroimaging studies have examined how the pharmacologic modulation of interoceptive signals is associated with fear-related neurocircuitry in individuals with generalized anxiety disorder (GAD)., Objective: To examine the neural circuitry underlying autonomic arousal induced via isoproterenol, a rapidly acting, peripheral β-adrenergic agonist akin to adrenaline., Design, Setting, and Participants: This crossover randomized clinical trial of 58 women with artifact-free data was conducted from January 1, 2017, to November 31, 2019, at the Laureate Institute for Brain Research in Tulsa, Oklahoma., Exposures: Functional magnetic resonance imaging was used to assess neural responses during randomized intravenous bolus infusions of isoproterenol (0.5 and 2.0 μg) and saline, each administered twice in a double-blind fashion., Main Outcomes and Measures: Blood oxygen level-dependent responses across the whole brain during isoproterenol administration in patients with GAD vs healthy comparators. Cardiac and respiratory responses, as well as interoceptive awareness and anxiety, were also measured during the infusion protocol., Results: Of the 58 female study participants, 29 had GAD (mean [SD] age, 26.9 [6.8] years) and 29 were matched healthy comparators (mean [SD] age, 24.4 [5.0] years). During the 0.5-μg dose of isoproterenol, the GAD group exhibited higher heart rate responses (b = 5.34; 95% CI, 2.06-8.61; P = .002), higher intensity ratings of cardiorespiratory sensations (b = 8.38; 95% CI, 2.05-14.71; P = .01), higher levels of self-reported anxiety (b = 1.04; 95% CI, 0.33-1.76; P = .005), and significant hypoactivation in the ventromedial prefrontal cortex (vmPFC) that was evident throughout peak response (Cohen d = 1.55; P < .001) and early recovery (Cohen d = 1.52; P < .001) periods. Correlational analysis of physiological and subjective indexes and percentage of signal change extracted during the 0.5-μg dose revealed that vmPFC hypoactivation was inversely correlated with heart rate (r56 = -0.51, adjusted P = .001) and retrospective intensity of both heartbeat (r56 = -0.50, adjusted P = .002) and breathing (r56 = -0.44, adjusted P = .01) sensations. Ventromedial prefrontal cortex hypoactivation correlated inversely with continuous dial ratings at a trend level (r56 = -0.38, adjusted P = .051), whereas anxiety (r56 = -0.28, adjusted P = .27) and chronotropic dose 25 (r56 = -0.14, adjusted P = .72) showed no such association., Conclusions and Relevance: In this crossover randomized clinical trial, women with GAD exhibited autonomic hypersensitivity during low levels of adrenergic stimulation characterized by elevated heart rate, heightened interoceptive awareness, increased anxiety, and a blunted neural response localized to the vmPFC. These findings support the notion that autonomic hyperarousal may be associated with regulatory dysfunctions in the vmPFC, which could serve as a treatment target to help patients with GAD more appropriately appraise and regulate signals of sympathetic arousal., Trial Registration: ClinicalTrials.gov Identifier: NCT02615119.

Blunted anterior insula activation during interoceptive perturbations has been associated with stimulant (cocaine and amphetamine) use disorder (SUD) and is related to risk for and prognosis of SUD. However, little is known whether these interoceptive alterations extend to opioid use disorder (OUD). This exploratory study used the same experimental probe during functional magnetic resonance imaging (fMRI) to test the hypothesis that SUD and OUD exhibit interoceptive discrepancies characterized by subjective ratings and activation within the insula. Recently, abstinent individuals diagnosed with current SUD (n = 40) or current OUD (n = 20) were compared with healthy individuals (CTL; n = 30) on brain and self-report responses during an interoceptive attention task known to elicit insula activation. Participants selectively attended to interoceptive (heartbeat and stomach) and exteroceptive signals during blood-oxygen-level-dependent fMRI recording. Groups and conditions were compared on (a) activation within probabilistic cytoarchitectonic segmentations of the insula and (b) self-reported stimulus intensity. First, SUD showed amplified ratings of heart-related sensations but attenuation of dorsal dysgranular insula activity relative to CTL. Amplified ratings were linked to drug use recency, while attenuation was normalized with greater past-year stimulant use. Second, SUD and OUD showed attenuation of dorsal dysgranular insula activity during attention to stomach sensations relative to CTL. Taken together, these results are consistent with altered neural processing of interoceptive signals in drug addiction, particularly as a function of SUD. Future studies will need to determine whether interoceptive metrics help to explain substance use disorder pathophysiology and are useful for predicting outcomes., (© 2019 Society for the Study of Addiction.)

Several studies have recently suggested that an abnormal processing of respiratory interoceptive and nociceptive (painful) stimuli may contribute to eating disorder (ED) pathophysiology. Mood and anxiety disorders (MA) are also characterized by abnormal respiratory symptoms, and show substantial comorbidity with ED. However, no studies have examined both respiratory and pain processing simultaneously within ED and MA. The present study systematically evaluated responses to perturbations of respiratory and nociceptive signals across the levels of physiology, behavior, and symptom report in a transdiagnostic ED sample (n = 51) that was individually matched to MA individuals (n = 51) and healthy comparisons (HC; n = 51). Participants underwent an inspiratory breath-holding challenge as a probe of respiratory interoception and a cold pressor challenge as a probe of pain processing. We expected both clinical groups to report greater stress and fear in response to respiratory and nociceptive perturbation than HCs, in the absence of differential physiological and behavioral responses. During breath-holding, both the ED and MA groups reported significantly more stress, feelings of suffocation, and suffocation fear than HC, with the ED group reporting the most severe symptoms. Moreover, anxiety sensitivity was related to suffocation fear only in the ED group. The heightened affective responses in the current study occurred in the absence of group differences in behavioral (breath hold duration, cold pressor duration) and physiological (end-tidal carbon dioxide, end-tidal oxygen, heart rate, skin conductance) responses. Against our expectations, there were no group differences in the response to cold pain stimulation. A matched-subgroup analysis focusing on individuals with anorexia nervosa (n = 30) produced similar results. These findings underscore the presence of abnormal respiratory interoception in MA and suggest that hyperreactivity to respiratory signals may be a potentially overlooked clinical feature of ED., Competing Interests: The authors have declared that no competing interests exist.

Objective: The aim of the study is to develop a workflow to establish geometrical quality criteria for 3D printed anatomical models as a guidance for selecting the most suitable 3D printing technologies available in a clinical environment., Methods: We defined the 3D geometry of a 25-year-old male patient's L4 vertebra and the geometry was then printed using two technologies, which differ in printing resolution and affordability: Fused Deposition Modelling (FDM) and Digital Light Processing (DLP). In order to measure geometrical accuracy, the 3D scans of two physical models were compared to the virtual input model. To compare surface qualities of these printing technologies we determined surface roughness for two regions of interest. Finally, we present our experience in the clinical application of a physical model in a congenital deformity case., Results: The analysis of the distribution of the modified Hausdorff distance values along the vertebral surface meshes (99% of values <1 mm) of the 3D printed models provides evidence for high printing accuracy in both printing techniques. Our results demonstrate that the surface qualities, measured by roughness are adequate (~99% of values <0.1 mm) for both physical models. Finally, we implemented the FDM physical model for surgical planning., Conclusion: We present a workflow capable of determining the quality of 3D printed models and the application of a high quality and affordable 3D printed spine physical model in the pre operative planning. As a result of the visual guidance provided by the physical model, we were able to define the optimal trajectory of the screw insertion during surgery., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Distorted perception of one's body and appearance, in general, is a core feature of several psychiatric disorders including anorexia nervosa and body dysmorphic disorder and is operative to varying degrees in nonclinical populations. Yet, body image perception is challenging to assess, given its subjective nature and variety of manifestations. The currently available methods have several limitations including restricted ability to assess perceptions of specific body areas. To address these limitations, we created Somatomap, a mobile tool that enables individuals to visually represent their perception of body-part sizes and shapes as well as areas of body concerns and record the emotional valence of concerns., Objective: This study aimed to develop and pilot test the feasibility of a novel mobile tool for assessing 2D and 3D body image perception., Methods: We developed a mobile 2D tool consisting of a manikin figure on which participants outline areas of body concern and indicate the nature, intensity, and emotional valence of the concern. We also developed a mobile 3D tool consisting of an avatar on which participants select individual body parts and use sliders to manipulate their size and shape. The tool was pilot tested on 103 women: 65 professional fashion models, a group disproportionately exposed to their own visual appearance, and 38 nonmodels from the general population. Acceptability was assessed via a usability rating scale. To identify areas of body concern in 2D, topographical body maps were created by combining assessments across individuals. Statistical body maps of group differences in body concern were subsequently calculated using the formula for proportional z-score. To identify areas of body concern in 3D, participants' subjective estimates from the 3D avatar were compared to corresponding measurements of their actual body parts. Discrepancy scores were calculated based on the difference between the perceived and actual body parts and evaluated using multivariate analysis of covariance., Results: Statistical body maps revealed different areas of body concern between models (more frequently about thighs and buttocks) and nonmodels (more frequently about abdomen/waist). Models were more accurate at estimating their overall body size, whereas nonmodels tended to underestimate the size of individual body parts, showing greater discrepancy scores for bust, biceps, waist, hips, and calves but not shoulders and thighs. Models and nonmodels reported high ease-of-use scores (8.4/10 and 8.5/10, respectively), and the resulting 3D avatar closely resembled their actual body (72.7% and 75.2%, respectively)., Conclusions: These pilot results suggest that Somatomap is feasible to use and offers new opportunities for assessment of body image perception in mobile settings. Although further testing is needed to determine the applicability of this approach to other populations, Somatomap provides unique insight into how humans perceive and represent the visual characteristics of their body., (©Christina Ralph-Nearman, Armen C Arevian, Maria Puhl, Rajay Kumar, Diane Villaroman, Nanthia Suthana, Jamie D Feusner, Sahib S Khalsa. Originally published in JMIR Mental Health (http://mental.jmir.org), 29.10.2019.)

Rationale: Adolescents are often described as "lacking brakes" resulting in an increase in several behaviors associated with risk for addiction. Prefrontal cortex dopamine and cortico-limbic interaction play an important role in addiction, and we have previously shown that the dopamine D1 receptor is elevated on prelimbic prefrontal output neurons in adolescent rats. We hypothesized that a constellation of risk-related behaviors is mediated by prefrontal output neuron expression of D1., Objectives: We aimed to determine the role of the dopamine D1 receptor in behavioral and neural correlates of risk for addiction that are often observed in adolescents. Therefore, high-risk behaviors as well as subcortical D2 receptor expression were investigated in adult animals with experimentally elevated D1 on prefrontal glutamatergic neurons., Methods: A lentiviral vector that selectively expressed the D1 receptor within glutamate neurons was injected in the prelimbic prefrontal cortex of adult male rats. Place conditioning to cocaine, alcohol, and nicotine, as well as delay discounting, novelty preferences, anxiety, cocaine self-administration, and sucrose preferences were assessed., Results: Virally mediated D1 over-expression in adults leads to stronger drug-cue associations and greater consumption of sweet solutions, elevates bias towards immediate satisfaction rather than delaying gratification, decreases anxiety, and causes rats to work harder for and take more cocaine. Furthermore, elevated cortical D1 reduces D2 receptors in the accumbens (a putative risk marker)., Conclusions: Together, these data suggest a common mechanism for increased motivational drive to seek and consume substances with hedonic value, consistent with adolescent addictive processes.

Background: The spontaneous osteoporotic fracture of the sacrum, known as a sacral insufficiency fracture (SIF) was first described as an unrecognized syndrome of the elderly by Laurie, in 1982. Numerous case histories and a few series of cases have been discussed in medical journals; however, none have been reported in Hungary., Goal: To delineate the leading diagnostic steps in the recognition of SIF and review the therapeutic guidelines. CASE HISTORIES, METHODS: Between January 2009 and the first six months of 2010 11 cases of SIF were diagnosed at the National Center for Spinal Disorders. We examined the clinical aspects of the illness, the radiological modalities, the fracture markings, the pace of recovery and duration., Results: The 11 patients were found to have various SIF predestining etiological factors and the following classic fractures--H-type, unilateral, horizontal, unilateral-horizontal and vertical as well as a bilateral pattern. In cases often not showing obvious clinical symptoms and in cases resulting in conventional radiological examinations of low sensitivity and specificity, we used mapping techniques in setting up the exact diagnosis., Conclusion: If we consider SIF from patient history and known risk factors, diagnostic procedure (primer original) may be shortened and a number of unnecessary tests (biopsy) may be avoided.

Berberine bridge enzyme (BBE) is a member of the recently discovered family of bicovalently flavinylated proteins. In this group of enzymes, the FAD cofactor is linked via its 8alpha-methyl group and the C-6 atom to conserved histidine and cysteine residues, His-104 and Cys-166 for BBE, respectively. 6-S-Cysteinylation has recently been shown to have a significant influence on the redox potential of the flavin cofactor; however, 8alpha-histidylation evaded a closer characterization due to extremely low expression levels upon substitution. Co-overexpression of protein disulfide isomerase improved expression levels and allowed isolation and purification of the H104A protein variant. To gain more insight into the functional role of the unusual dual mode of cofactor attachment, we solved the x-ray crystal structures of two mutant proteins, H104A and C166A BBE, each lacking one of the covalent linkages. Information from a structure of wild type enzyme in complex with the product of the catalyzed reaction is combined with the kinetic and structural characterization of the protein variants to demonstrate the importance of the bicovalent linkage for substrate binding and efficient oxidation. In addition, the redox potential of the flavin cofactor is enhanced additively by the dual mode of cofactor attachment. The reduced level of expression for the H104A mutant protein and the difficulty of isolating even small amounts of the protein variant with both linkages removed (H104A-C166A) also points toward a possible role of covalent flavinylation during protein folding.

Tobacco smoking is a major public health issue and a better understanding of tobacco addiction represents an important challenge. Many factors are involved in tobacco addiction, including genetic factors. Taking them into account in smoking cessation programs would allow to better adapt these programs to individual characteristics and improve their rate of success. Given enzymatic induction by tobacco smoke, smoking cessation can nevertheless have important consequences on the metabolism of some drugs, that have to be taken into consideration. Here we present different clinical and genetic aspects of smoking and of smoking cessation. A dose adjustment of drugs influenced by tobacco smoke is proposed when quitting smoking.

Berberine bridge enzyme catalyzes the stereospecific oxidation and carbon-carbon bond formation of (S)-reticuline to (S)-scoulerine. In addition to this type of reactivity the enzyme can further oxidize (S)-scoulerine to the deeply red protoberberine alkaloid dehydroscoulerine albeit with a much lower rate of conversion. In the course of the four electron oxidation, no dihydroprotoberberine species intermediate was detectable suggesting that the second oxidation step leading to aromatization proceeds at a much faster rate. Performing the reaction in the presence of oxygen and under anoxic conditions did not affect the kinetics of the overall reaction suggesting no strict requirement for oxygen in the oxidation of the unstable dihydroprotoberberine intermediate. In addition to the kinetic characterization of this reaction we also present a structure of the enzyme in complex with the fully oxidized product. Combined with information available for the binding modes of (S)-reticuline and (S)-scoulerine a possible mechanism for the additional oxidation is presented. This is compared to previous reports of enzymes ((S)-tetrahydroprotoberberine oxidase and canadine oxidase) showing a similar type of reactivity in different plant species.

Berberine bridge enzyme catalyzes the conversion of (S)-reticuline to (S)-scoulerine by formation of a carbon-carbon bond between the N-methyl group and the phenolic ring. We elucidated the structure of berberine bridge enzyme from Eschscholzia californica and determined the kinetic rates for three active site protein variants. Here we propose a catalytic mechanism combining base-catalyzed proton abstraction with concerted carbon-carbon coupling accompanied by hydride transfer from the N-methyl group to the N5 atom of the FAD cofactor.

The objective of this study was to evaluate cross-sectional relationships among symptoms of psychological stress, sleep, and physiological arousal during non-rapid eye movement (NREM) sleep in a sample of 30 patients with chronic, primary insomnia (mean age, 30.2 years, 60% female). Study measures included indexes of subjective stress, visually scored sleep, and physiological arousal during NREM sleep: quantitative electroencephalogram (QEEG) and quantitative electrocardiogram (QEKG) measures. Psychological stress was more strongly related to indexes of physiological arousal during NREM sleep than to visually scored measures of sleep. Higher levels of perceived stress were associated with decreased EEG delta power (rho = -0.50, p < .01) and increased EEG beta power (rho = 0.38, p < .05). Increased frequency of stress-related avoidance behaviors was associated with decreased EKG high-frequency power (rho = -0.46, p < .05). Although QEEG measures were significantly correlated with sleep maintenance (QEEG delta power rho = 0.45, p < .01; QEEG beta power rho = -0.54, p < .01) and time spent in delta sleep (QEEG delta power rho = 0.65, p < .001; QEEG beta power rho = -0.65, p < .001), QEKG measures were unrelated to visually scored measures of sleep. Perceived stress and stress-related avoidance behaviors were associated with multiple indexes of physiological arousal during NREM sleep in patients with chronic, primary insomnia.

Major liver resection cannot be performed when the remaining liver mass is too small. Preoperative embolization of the portal vein (PVE) helps to increase the volume of the non-tumorous liver segments, and patients' liver function will remain stable postoperatively. CT, MRI, CTAP examinations help to decide about surgery. Volume measurements are performed based on data of CT scans. PVE is indicated, when the remaining, non-tumorous liver volume is too small. The procedure starts with percutaneous portal vein catheterization, then selected portal vein branches are embolized with a mixture of contrast material, antibiotics and Gelfoam particles. The patients stay in the hospital 3-5 days after the procedure. Control CT-volumetry is done after 3-6 weeks. From November 2001 to April 2002 3 patients were selected to have this procedure. PVE of the right portal branches were performed successfully in two cases. Control CT-volumetry showed significant increase of the volume of the left liver lobe. One patient underwent successful right hemihepatectomy. On the third patient we could not catheterize the portal vein. PVE is a relatively safe and tolerable procedure. The increased remaining liver volume helps in performing major liver resection.