Enzymatic one-step synthesis of natural 2-pyrones and new-to-nature derivatives from coenzyme A esters.

The 2-pyrone moiety is present in a wide range of structurally diverse natural products with various biological activities. The plant biosynthetic routes towards these compounds mainly depend on the activity of either type III polyketide synthase-like 2-pyrone synthases or hydroxylating 2-oxoglutarate dependent dioxygenases. In the present study, the substrate specificity of these enzymes is investigated by a systematic screening using both natural and artificial substrates with the aims of efficiently forming (new) products and understanding the underlying catalytic mechanisms. In this framework, we focused on the in vitro functional characterization of a 2-pyrone synthase Gh2PS2 from Gerbera x hybrida and two dioxygenases AtF6'H1 and AtF6'H2 from Arabidopsis thaliana using a set of twenty aromatic and aliphatic CoA esters as substrates. UHPLC-ESI-HRMSn based analyses of reaction intermediates and products revealed a broad substrate specificity of the enzymes, enabling the facile "green" synthesis of this important class of natural products and derivatives in a one-step/one-pot reaction in aqueous environment without the need for halogenated or metal reagents and protective groups. Using protein modeling and substrate docking we identified amino acid residues that seem to be important for the observed product scope.

Fourteen years of follow-up after first arthroscopic Bankart repair in athletes: functional outcomes and magnetic resonance imaging findings.

BACKGROUND: The arthroscopic Bankart procedure is the most performed surgery for shoulder stabilization. Short-term to midterm results are well studied; however, long-term results over 10 years are rare. PURPOSE: This study evaluates the long-term results and magnetic resonance imaging (MRI) findings in athletes at a mean follow-up of 14 years after an arthroscopic Bankart stabilization as well as risk factors for osteoarthritis. METHODS: A total of 63 athletes had an arthroscopic Bankart repair between 2001 and 2008, of whom 46 patients (73.0%) participated in the final follow-up. The Constant, Rowe, and Western Ontario Shoulder Instability Index (WOSI) score and the rate of return to sports were evaluated. Glenohumeral osteoarthritis was assessed using the Samilson-Prieto classification. Known risk factors for osteoarthritis were analyzed. MRI findings (bone marrow edema, cysts, and joint effusion) were analyzed. RESULTS: The average follow-up was 14 years. Assessment was performed on 46 athletes with an average age of 21.6 at the time of surgery. The overall redislocation rate was 21.7%. The Constant score was 96.7, the Rowe score was 83.4, and the Western Ontario Shoulder Instability Index score was 90.7 out of 100. A total of 84.8% of the athletes returned to their initial sports level. Glenohumeral osteoarthritis occurred in 28.1%. Known risk factors for osteoarthritis were confirmed. Further MRI findings were rare. CONCLUSIONS: Arthroscopic Bankart repair in athletes shows good long-term clinical results. However, this is only in patients without osteoarthritis, which was rare, but was confirmed as a risk factor. We assume that resorption of anchors differs in patients. If it does have an impact on developing arthrosis, this should be confirmed in further studies.

Acute Post-Concussive Assessments of Brain Tissue Magnetism Using Magnetic Resonance Imaging.

Recent studies have demonstrated the promising capabilities of magnetic resonance imaging (MRI)-based quantitative susceptibility maps (QSM) in producing biomarkers of brain injury. The present study aims to further explore acute QSM changes in athletes after sports concussion and investigate prognostication capabilities of QSM-derived imaging metrics. The QSM were derived from neurological MRI data acquired on a cohort (n = 78) of concussed male American football athletes within 48 h of injury. The MRI-derived QSM values in subcortical gray and white matter compartments after concussion showed differences relative to a matched uninjured control group (white matter: z = 3.04, p = 0.002, subcortical gray matter: z = -2.07, p = 0.04). Subcortical gray matter QSM MRI measurements also correlated strongly with duration of symptoms (ρ = -0.46, p = 0.002) within a subcohort of subjects who had symptom durations for at least one week (n = 39). The acute QSM MRI metrics showed promising prognostication capabilities, with subcortical gray matter compartment QSM values yielding a mean classification area under the curve performance of 0.78 when predicting symptoms of more than two weeks in duration. The results of the study reproduce previous acute post-concussion group QSM findings and provide promising initial prognostication capabilities of acute QSM measurements in a post-concussion setting.

Optogenetic augmentation of the hypercholinergic endophenotype in DYT1 knock-in mice induced erratic hyperactive movements but not dystonia.

BACKGROUND: The most prevalent inherited form of generalized dystonia is caused by a mutation in torsinA (DYT1, ∆GAG) with incomplete penetrance. Rodent models with mutated torsinA do not develop dystonic symptoms, but previous ex vivo studies indicated abnormal excitation of cholinergic interneurons (ChI) and increased striatal acetylcholine. METHODS: We used in vivo optogenetics to exacerbate this endophenotype in order to determine its capacity to trigger dystonic symptoms in freely behaving mice. Tor1a+/Δgag DYT1 mice and wildtype littermates expressing channelrhodopsin2 under the Chat promotor were implanted bilaterally with optical LED cannulae and stimulated with blue light pulses of varied durations. FINDINGS: Six months old DYT1 KI mice but not wildtype controls responded with hyperactivity to blue light specifically at 25 ms pulse duration, 10 Hz frequency. Neuronal activity (c-Fos) in cholinergic interneurons was increased immediately after light stimulation and persisted only in DYT1 KI over 15 min. Substance P was increased specifically in striosome compartments in naïve DYT1 KI mice compared to wildtype. Under optogenetic stimulation substance P increased in wildtype to match levels in Dyt1 KI, and acetylcholinesterase was elevated in the striatum of stimulated DYT1 KI. No signs of dystonic movements were observed under stimulation of up to one hour in both genotypes and age groups, and the sensorimotor deficit previously observed in 6 months old DYT1 KI mice persisted under stimulation. INTERPRETATION: Overall this supports an endophenotype of dysregulated cholinergic activity in DYT1 dystonia, but depolarizing cholinergic interneurons was not sufficient to induce overt dystonia in DYT1 KI mice.

Uranium(VI) Complexes with a Calix[4]arene-Based 8-Hydroxyquinoline Ligand: Thermodynamic and Structural Characterization Based on Calorimetry, Spectroscopy, and Liquid-Liquid Extraction.

The environmental aspects of ore processing and waste treatment call for an optimization of applied technologies. There, understanding of the structure and complexation mechanism on a molecular scale is indispensable. Here, the complexation of UVI with a calix[4]arene-based 8-hydroxyquinoline ligand was investigated by applying a wide range of complementary methods. In solution, the formation of two complex species was proven with stability constants of log ß1:1=5.94±0.02 and log ß2:1=6.33±0.01, respectively. The formation of the 1:1 complex was found to be enthalpy driven [ΔH1:1=(-71.5±10.0) kJ mol-1; TΔS1:1=(-37.57±10.0) kJ mol-1], whereas the second complexation step was found to be endothermic and entropy driven [ΔH2:1=(32.8±4.0) kJ mol-1; TΔS2:1=(68.97±4.0) kJ mol-1]. Moreover, the molecular structure of [UO2(H6L)(NO3)](NO3) (1) was determined by single-crystal X-ray diffraction. Concluding, radiotoxic UVI was separated from a EuIII-containing solution by the calix[4]arene-based ligand in solvent extractions.

Polyethylenimine Nanoparticle-Mediated siRNA Delivery to Reduce α-Synuclein Expression in a Model of Parkinson's Disease.

RNA interference (RNAi)-based strategies that mediate the specific knockdown of target genes by administration of small interfering RNAs (siRNAs) could be applied for treatment of presently incurable neurodegenerative diseases such as Parkinson's disease. However, inefficient delivery of siRNA into neurons hampers in vivo application of RNAi. We have previously established the 4-12 kDa branched polyethylenimine (PEI) F25-LMW with superior transfection efficacy for delivery of siRNA in vivo. Here, we present that siRNA complexed with this PEI extensively distributes across the CNS down to the lumbar spinal cord after a single intracerebroventricular infusion. siRNA against α-synuclein (SNCA), a pre-synaptic protein that aggregates in Parkinson's disease, was complexed with PEI F25-LMW and injected into the lateral ventricle of mice overexpressing human wild-type SNCA (Thy1-aSyn mice). Five days after the single injection of 0.75 µg PEI/siRNA, SNCA mRNA expression in the striatum was reduced by 65%, accompanied by reduction of SNCA protein by ∼50%. Mice did not show signs of toxicity or adverse effects. Moreover, ependymocytes and brain parenchyma were completely preserved and free of immune cell invasion, astrogliosis, or microglial activation. Our results support the efficacy and safety of PEI nanoparticle-mediated delivery of siRNA to the brain for therapeutic intervention.

Cognitive Performance Under Electroconvulsive Therapy (ECT) in ECT-Naive Treatment-Resistant Patients With Major Depressive Disorder.

OBJECTIVES: Although electroconvulsive therapy (ECT) is considered a safe and highly effective treatment option for major depressive disorder, there are still some reservations with regard to possible adverse cognitive adverse effects. This is the case despite a large body of evidence showing that these deficits are transient and that there even seems to be a long-term improvement of cognitive functioning level. However, most data concerning cognitive adverse effects stem from studies using mixed samples of treatment-resistant and non-treatment-resistant as well as ECT-naive and non-ECT-naive subjects. Furthermore, neurocognitive measures might partly be sensitive to practice effects and improvements in depressive symptom level. METHODS: We examined neurocognitive performance in a sample of 20 treatment-resistant and ECT-naive subjects using repeatable neurocognitive tests, whereas changes in depressive symptom level were controlled. Cognitive functioning level was assessed before (baseline), 1 week, and 6 months (follow-up 1 and 2) after (12 to) 15 sessions of unilateral ECT treatment. RESULTS: No adverse cognitive effects were observed in any of the cognitive domains examined. Instead, a significant improvement in verbal working memory performance was found from baseline to follow-up 2. When changes in depressive symptom levels were controlled statistically, this improvement was no longer seen. CONCLUSIONS: Although findings that ECT does not lead to longer lasting cognitive deficits caused by ECT were confirmed, our study adds evidence that previous results of a beneficial effect of ECT on cognition might be questioned.

Sensorimotor tests unmask a phenotype in the DYT1 knock-in mouse model of dystonia.

Hereditary generalized dystonia is often caused by a GAG deletion in TOR1A (DYT1) that encodes for the protein torsinA. Although mutation carriers show alterations in neuronal connectivity and sensorimotor deficits, only 30% develop dystonia. Uncovering the factors triggering the dystonic symptoms and underlying pathophysiology would greatly benefit the development of more effective therapies. In DYT1 knock-in (KI) mice, the expression of torsinA mutant alters the connectivity of neurons and the function of striatal cholinergic interneurons. We aimed to determine if heterozygous DYT1 KI mice exhibit deficits in behavioural tests that explore the connectivity of the sensory and motor system. DYT1 KI mice were tested in cognitive tests and challenging motor paradigms, followed by the adhesive removal test and the adaptive rotating beam test which both require sensorimotor integration. DYT1 KI mice did not exhibit cognitive deficits and were able to perform similarly to wild type mice even in challenging motor tests with relatively stable sensory input. Conversely, DYT1 KI mice spent more time on sensing and removing an adhesive sticker from the back of the nose; they exhibited difficulty to traverse rotating rods, especially if the surface was smooth and the diameter small. Our observations further support a role of sensorimotor integration in manifestation of this movement disorder. Future studies in DYT1 KI mice will explore the involved neurocircuitry and underlying molecular mechanisms.

Altered postnatal maturation of striatal GABAergic interneurons in a phenotypic animal model of dystonia.

GABAergic disinhibition has been suggested to play a critical role in the pathophysiology of several basal ganglia disorders, including dystonia, a common movement disorder. Previous studies have shown a deficit of striatal GABAergic interneurons (IN) in the dtsz mutant hamster, one of the few phenotypic animal models of dystonia. However, mechanisms underlying this deficit are largely unknown. In the present study, we investigated the migration and maturation of striatal IN during postnatal development (18days of age) and at age of highest severity of dystonia (33days of age) in this hamster model. In line with previous findings, the density of GAD67-positive IN and the level of parvalbumin mRNA, a marker for fast spiking GABAergic IN, were lower in the dtsz mutant than in control hamsters. However, an unaltered density of Nkx2.1 labeled cells and Nkx2.1 mRNA level suggested that the migration of GABAergic IN into the striatum was not retarded. Therefore, different factors that indicate maturation of GABAergic IN were determined. While mRNA of the KCC2 cation/chloride transporters and the cytosolic carboanhydrase VII, used as markers for the so called GABA switch, as well as BDNF were unaltered, we found a reduced number of IN expressing the alpha1 subunit of the GABAA-receptor (37.5%) in dtsz hamsters at an age of 33days, but not after spontaneous remission of dystonia at an age of 90days. Since IN shift expression from alpha2 to alpha1 subunits during postnatal maturation, this result together with a decreased parvalbumin mRNA expression suggest a delayed maturation of striatal GABAergic IN in this animal model, which might underlie abnormal neuronal activity and striatal plasticity.

The α2ß3γ2 GABAA receptor preferring agonist NS11394 aggravates dystonia in the phenotypic dtsz model.

Dystonia is a movement disorder, characterized by involuntary muscle contractions resulting in abnormal movements and/or postures. Antidystonic effects of benzodiazepines in patients with different types of dystonia could be replicated in the dtsz mutant hamster, a phenotypic model of paroxysmal dystonia. Compounds with preferred binding at specific subunits of the gamma aminobutyric acid type A (GABAA) receptor may provide a more beneficial spectrum of effects in comparison with benzodiazepines. We therefore examined the effects of the α1ß3γ2 GABAA receptor preferring compound zolpidem (2.0-10.0mg/kg i.p.) and of the α2ß3γ2 GABAA receptor preferring compound NS11394 (3.0-30mg/kg i.p.) on the severity of dystonia in the dtsz mutant in comparison with the benzodiazepine clonazepam (0.5-1.0mg/kg i.p.). As expected, clonazepam exerted pronounced antidystonic effects. While zolpidem showed moderate beneficial effects, NS11394 significantly increased the severity of dystonia. The present results indicate for the first time that positive GABAA receptor modulators show contrary effects on dystonia dependent on their preference for alpha-subunits. The potential link between alterations in GABAA receptor subunits and GABAergic disinhibition in dystonia deserves further attention in research on the pathophysiology and therapeutic targets.

Speciation Studies of Metals in Trace Concentrations: The Mononuclear Uranyl(VI) Hydroxo Complexes.

A direct luminescence spectroscopic experimental setup for the determination of complex stability constants of mononuclear uranyl(VI) hydrolysis species is presented. The occurrence of polynuclear species is prevented by using a low uranyl(VI) concentration of 10­8 M (2.4 ppb). Time-resolved laser-induced fluorescence spectra were recorded in the pH range from 3 to 10.5. Deconvolution with parallel factor analysis (PARAFAC) resulted in three hydrolysis complexes. A tentative assignment was based on thermodynamic calculations: UO22+, UO2(OH)+, UO2(OH)2, UO2(OH)3­. An implementation of a Newton­Raphson algorithm into PARAFAC allowed a direct extraction of complex stability constants during deconvolution yielding log(ß1M,1°C)1:1 = −4.6, log(ß1M,1°C)1:2 = −12.2, log(ß1M,1°C)1:3 = −22.3. Extrapolation to standard conditions gave log(ß0)1:1 = −3.9, log(ß0)1:2 = −10.9, and log(ß0)1:3 = −20.7. Luminescence characteristics (band position, lifetime) of the individual mononuclear hydroxo species were derived to serve as a reference data set for further investigations. A correlation of luminescence spectroscopic features with Raman frequencies was demonstrated for the mononuclear uranyl(VI) hydroxo complexes for the first time. Thereby a signal-to-structure correlation was achieved and the complex assignment validated.

The novel adaptive rotating beam test unmasks sensorimotor impairments in a transgenic mouse model of Parkinson's disease.

Development of disease modifying therapeutics for Parkinson's disease (PD), the second most common neurodegenerative disorder, relies on availability of animal models which recapitulate the disease hallmarks. Only few transgenic mouse models, which mimic overexpression of alpha-synuclein, show dopamine loss, behavioral impairments and protein aggregation. Mice overexpressing human wildtype alpha-synuclein under the Thy-1 promotor (Thy1-aSyn) replicate these features. However, female mice do not exhibit a phenotype. This was attributed to a potentially lower transgene expression located on the X chromosome. Here we support that female mice overexpress human wildtype alpha-synuclein only about 1.5 fold in the substantia nigra, compared to about 3 fold in male mice. Since female Thy1-aSyn mice were shown previously to exhibit differences in corticostriatal communication and synaptic plasticity similar to their male counterparts we hypothesized that female mice use compensatory mechanisms and strategies to not show overt motor deficits despite an underlying endophenotype. In order to unmask these deficits we translated recent findings in PD patients that sensory abnormalities can enhance motor dysfunction into a novel behavioral test, the adaptive rotating beam test. We found that under changing sensory input female Thy1-aSyn mice showed an overt phenotype. Our data supports that the integration of sensorimotor information is likely a major contributor to symptoms of movement disorders and that even low levels of overexpression of human wildtype alpha-synuclein has the potential to disrupt processing of these information. The here described adaptive rotating beam test represents a sensitive behavioral test to detect moderate sensorimotor alterations in mouse models.

Characterization of identity, metabolism and androgenic activity of 17-hydroxyandrosta-3,5-diene by GC-MS and a yeast transactivation system.

Anabolic-androgenic steroids are frequently misused compounds in sports, and they belong to the controlled substances according to the requirements of the World Anti-Doping Agency. The classical techniques of steroid detection are mass spectrometry coupled to gas or liquid chromatography. Biological methods that base on the ability of substances to bind the steroid receptor are not applied in routine doping control procedures so far, but they appear to be useful for characterization of steroid androgenic potential. In this study we used the yeast androgen receptor reporter system (YAS), which in the past has already successfully been applied to both various androgenic substances and also urine samples. Giving attention to the androgenic potential of steroidal dietary supplements, we exemplified the analysis using both mass spectrometry techniques and the YAS-based assay on the product "Syntrax Tetrabol" which was a confiscated dietary supplement and marketed as a steroid precursor. Identification, structure and the kinetic behavior of its excreted metabolites were analyzed by NMR, GC-MS and LC-MS/MS. The androgenic potential of the parent compound as well as its metabolites in urine was evaluated with the help of the YAS. The application of urine samples with a previous deconjugation and the inclusion of urine density values were carried out and led to increased responses on the YAS. Further, the possibility of a complementary application of structure-based instrumental analysis and biological detection of androgenicity with the help of the YAS seems to be desirable and is discussed.

How are they really doing? Observation of inclusionary classroom participation for children with mild-to-moderate deafness.

Direct observation was utilized to study how 5 children with mild-to-moderate deafness participated within inclusive classroom settings. Responses to practice and prompt opportunities, levels of prompting required to follow classroom directions, and engagement were analyzed across students with mild-to-moderate deafness and were compared to students with normal hearing. Similar responses to practice and prompt opportunities were observed across students, and engagement data indicated that 4 children with mild-to-moderate deafness had similar rates to their peers. However, children with mild-to-moderate deafness required higher levels of prompting and were less accurate at following classwide verbal prompts. Agreement data on variables ranged between 83% and 99%, with the exception of 2 prompting levels. Social validity judgments indicated that the information was useful and important. Potential uses for data include consultation with teachers regarding interventions to increase student engagement and research regarding inclusionary practices.

Rapid access to N-substituted diketopiperazines by one-pot Ugi-4CR/deprotection+activation/cyclization (UDAC).

The most efficient diversity generating approaches to heterocycles are combinations of a multicomponent (MCR) with a cyclization reaction, for example, by Ugi-deprotection-cylization (UDC) protocols. If the desired post-Ugi reaction requires more than one deprotection, for example of two initially protected Ugi-reactive groups, or if it requires additional activation, for example, by an Ugi-activation-cyclization (UAC), either the isolation of intermediates or a sequential process or both become necessary. A recently introduced convertible isonitrile reagent allows a mild and chemoselective in situ post-Ugi activation of the isonitrile-born carboxylate with simultaneous deprotection of the nucleophilic amine, that is, liberation and activation of two Ugi-reactive groups, if desired also under subsequent lactam formation. This is exemplified by the synthesis of peptide-peptoid diketopiperazines.