Optimisation of imidazole compounds as selective TAAR1 agonists: Discovery of RO5073012

A series of imidazole compounds has been identified which affords potent and selective partial and full agonists of the TAAR1 receptor. Starting from 2-benzyl-imidazoline screening hits, a series of structurally related 2-benzyl- and 4-benzyl-imidazoles was investigated first, but it proved highly challenging to obtain compounds having sufficient selectivity against the adrenergic alpha 2 receptor. This issue could be successfully addressed by modification of the linker region and SAR exploration led to the discovery of highly selective isopropyl-substituted 4-aminomethyl-imidazole compounds. The work culminated in the identification of the selective TAAR1 partial agonist RO5073012 (4-chlorophenyl)-(1H-imidazol-4-ylmethyl)-isopropyl-amine, 24), which has a good pharmacokinetic profile after oral administration in rodents. RO5073012 has been found to be active in a behavioural rat model which is considered indicative for schizophrenia.     

Label-free LC-MSMS analysis of vitreous from autoimmune uveitis reveals a significant decrease in secreted Wnt signalling inhibitors DKK3 and SFRP2

Equine recurrent uveitis is a severe and frequent blinding disease in horses which presents with auto-reactive invading T-cells, resulting in the destruction of the inner eye. Infiltration of inflammatory cells into the retina and vitreous is driven by currently unknown guidance cues, however surgical removal of the vitreous (vitrectomy) has proven therapeutically successful. Therefore, proteomic analyses of vitrectomy samples are likely to result in detection of proteins contributing to disease pathogenesis. Vitreous from healthy and ERU diseased horses were directly compared by quantitative mass spectrometry based on label-free quantification of peak intensities across samples. We found a significant upregulation of complement and coagulation cascades and downregulation of negative paracrine regulators of canonical Wnt signalling including the Wnt signalling inhibitors DKK3 and SFRP2. Based on immunohistochemistry, both proteins are expressed in equine retina and suggest localisation to retinal Müller glial cells (RMG), which may be the source cells for these proteins. Furthermore, retinal expression levels and patterns of DKK3 change in response to ERU. Since many other regulated proteins identified here are associated with RMG cells, these cells qualify as the prime responders to autoimmune triggers.     

Relation of left ventricular mass to QTc in normotensive severely obese patients

Prolongation of the corrected QT interval (QTc) has been described in obese subjects. This study assesses the relation of left ventricular (LV) mass to QTc in normotensive severely obese subjects. Fifty normotensive patients whose BMI was ≥40 kg/m(2) (mean age: 38 ± 7 years) were studied. QTc was derived using Bazett's formula. LV mass was calculated using the formula of Devereux et al. and was indexed to height(2.7). Mean QTc was 428.8 ± 19.0 ms and was significantly longer in those with than in those without LV hypertrophy (P < 0.01) QTc correlated positively and significantly with BMI (r = 0.392, P < 0.025), LV mass/height(2.7) (r = 0.793, P < 0.0005), systolic blood pressure (r = 0.742, P < 0.001), LV end - systolic wall stress (r = 0.746, P < 0.001) and LV internal dimension in diastole (r = 0.788, P < 0.0005). Among five variables tested, LV mass/height(2.7) was identified as the sole predictor of QTc by multivariate analysis. In conclusion, LV mass and loading conditions that may affect LV mass are important determinants of QTc in normotensive severely obese subjects.     

Anti-type II collagen antibodies are associated with early radiographic destruction in rheumatoid arthritis

Introduction

We have previously reported that high levels of antibodies specific for native human type II collagen (anti-CII) at the time of RA diagnosis were associated with concurrent but not later signs of inflammation. This was associated with CII/anti-CII immune complex (IC)-induced production of pro-inflammatory cytokines in vitro. In contrast, anti-cyclic citrullinated peptide antibodies (anti-CCP) were associated both with late inflammation and late radiological destruction in the same RA cohort. We therefore hypothesized that anti-CII are also associated with early erosions.

Methods

Two-hundred-and-fifty-six patients from an early RA cohort were included. Baseline levels of anti-CII, anti-CCP and anti-mutated citrullinated vimentin were analyzed with ELISA, and rheumatoid factor levels were determined by nephelometry. Radiographs of hands and feet at baseline, after one and after two years were quantified using the 32-joints Larsen erosion score.

Results

Levels of anti-CII were bimodally distributed in the RA cohort, with a small (3.1%, 8/256) group of very high outliers with a median level 87 times higher than the median for the healthy control group. Using a cut-off discriminating the outlier group that was associated with anti-CII IC-induced production of proinflammatory cytokines in vitro, baseline anti-CII antibodies were significantly (p = 0.0486) associated with increased radiographic damage at the time of diagnosis. Anti-CII-positive patient had also significantly increased HAQ score (p = 0.0303), CRP (p = 0.0026) and ESR (p = 0.0396) at the time of diagnosis but not during follow-up. The median age among anti-CII-positive subjects was 12 years higher than among the anti-CII-negative patients.

Conclusion

In contrary to anti-CCP, anti-CII-positive patients with RA have increased joint destruction and HAQ score at baseline. Anti-CII thus characterizes an early inflammatory/destructive phenotype, in contrast to the late appearance of an inflammatory/destructive phenotype in anti-CCP positive RA patients. The anti-CII phenotype might account for part of the elderly acute onset RA phenotype with rather good prognosis.     

Improbable but true: the invasive inbreeding ambrosia beetle Xylosandrus morigerus has generalist genotypes

The wide distribution and dominance of invasive inbreeding species in many forest ecosystems seems paradoxical in face of their limited genetic variation. Successful establishment of invasive species in new areas is nevertheless facilitated by clonal reproduction: parthenogenesis, regular self-fertilization, and regular inbreeding. The success of clonal lineages in variable environments has been explained by two models, the frozen niche variation (FNV) model and the general-purpose genotype (GPG) model. We tested these models on a widely distributed forest pest that has been recently established in Costa Rica-the sibling-mating ambrosia beetle Xylosandrus morigerus. Two deeply diverged mitochondrial haplotypes coexist at multiple sites in Costa Rica. We find that these two haplotypes do not differ in their associations with ecological factors. Overall the two haplotypes showed complete overlap in their resource utilization; both genotypes have broad niches, supporting the GPG model. Thus, probable or not, our findings suggest that X. morigerus is a true ecological generalist. Clonal aspects of reproduction coupled with broad niches are doubtless important factors in the successful colonization of new habitats in distant regions.     

Succinic acid fermentation in a stationary-basket bioreactor with a packed bed of immobilized Actinobacillus succinogenes: 1. Influence of internal diffusion on substrate mass transfer and consumption rate

This paper is dedicated to the study on external and internal mass transfers of glucose for succinic fermentation under substrate and product inhibitions using a bioreactor with a stationary basket bed of immobilized Actinobacillus succinogenes cells. By means of the substrate mass balance for a single particle of biocatalysts, considering the Jerusalimsky kinetic model including both inhibitory effects, specific mathematical expressions have been developed for describing the profiles of the substrate concentrations and mass flows in the outer and inner regions of biocatalyst particles, as well as for estimating the influence of internal diffusion on glucose consumption rate. The results indicated that very low values of internal mass flow could be reached in the particles center. The corresponding region was considered biologically inactive, with its extent varying from 0.24% to 44% from the overall volume of each biocatalyst. By immobilization of bacterial cells and use of a basket bed, the rate of glucose consumption is reduced up to 200 times compared with the succinic fermentation system containing free cells.     

Retinal Neurodegeneration in Wilson’s Disease Revealed by Spectral Domain Optical Coherence Tomography

Background/Objective

In addition to cirrhosis of the liver, Wilson’s disease leads to copper accumulation and widespread degeneration of the nervous system. Delayed visual evoked potentials (VEPs) suggest changes to the visual system and potential structural changes of the retina.

Methods

We used the latest generation of spectral domain optical coherence tomography to assess the retinal morphology of 42 patients with Wilson’s disease and 76 age- and sex-matched controls. We measured peripapillary retinal nerve fiber layer (RNFL) thickness and total macular thickness and manually segmented all retinal layers in foveal scans of 42 patients with Wilson’s disease and 76 age- and sex-matched controls. The results were compared with VEPs and clinical parameters.

Results

The mean thickness of the RNFL, paramacular region, retinal ganglion cell/inner plexiform layer and inner nuclear layer was reduced in Wilson’s disease. VEPs were altered with delayed N75 and P100 latencies, but the N140 latency and amplitude was unchanged. An analysis of the laboratory parameters indicated that the serum concentrations of copper and caeruloplasmin positively correlated with the thickness of the outer plexiform layer and with N75 and P100 VEP latencies.

Conclusion

Neuronal degeneration in Wilson’s disease involves the retina and changes can be quantified by optical coherence tomography. While the VEPs and the thickness of the outer plexiform layer appear to reflect the current copper metabolism, the thicknesses of the RNFL, ganglion cell/inner plexiform layer, inner nuclear layer and the total paramacular thickness may be the best indicators of chronic neuronal degeneration.     

An Experimental Test of the Accumulated Copying Error Model of Cultural Mutation for Acheulean Handaxe Size

Archaeologists interested in explaining changes in artifact morphology over long time periods have found it useful to create models in which the only source of change is random and unintentional copying error, or ‘cultural mutation’. These models can be used as null hypotheses against which to detect non-random processes such as cultural selection or biased transmission. One proposed cultural mutation model is the accumulated copying error model, where individuals attempt to copy the size of another individual''s artifact exactly but make small random errors due to physiological limits on the accuracy of their perception. Here, we first derive the model within an explicit mathematical framework, generating the predictions that multiple independently-evolving artifact chains should diverge over time such that their between-chain variance increases while the mean artifact size remains constant. We then present the first experimental test of this model in which 200 participants, split into 20 transmission chains, were asked to faithfully copy the size of the previous participant''s handaxe image on an iPad. The experimental findings supported the model''s prediction that between-chain variance should increase over time and did so in a manner quantitatively in line with the model. However, when the initial size of the image that the participants resized was larger than the size of the image they were copying, subjects tended to increase the size of the image, resulting in the mean size increasing rather than staying constant. This suggests that items of material culture formed by reductive vs. additive processes may mutate differently when individuals attempt to replicate faithfully the size of previously-produced artifacts. Finally, we show that a dataset of 2601 Acheulean handaxes shows less variation than predicted given our empirically measured copying error variance, suggesting that other processes counteracted the variation in handaxe size generated by perceptual cultural mutation.     

Mass spectrometric identification of novel posttranslational modification sites in Huntingtin

Huntington's disease (HD) is caused by a CAG triplet repeat expansion in exon 1 of the Huntingtin (Htt) gene, encoding an abnormal expanded polyglutamine (polyQ) tract that confers toxicity to the mutant Htt (mHtt) protein. Recent data suggest that posttranslational modifications of mHtt modulate its cytotoxicity. To further understand the cytotoxic mechanisms of mHtt, we have generated HEK293 cell models stably expressing Strep- and FLAG-tagged Htt containing either 19Q (wild-type Htt), 55Q (mHtt), or 94Q (mHtt) repeats. Following tandem affinity purification, the tagged Htt and associated proteins were subjected to tandem mass spectrometry or 2D nano-LC tandem mass spectrometry and several novel modification sites of mHtt containing 55Q or 94Q were identified. These were phosphorylation sites located at Ser431 and Ser432, and ubiquitination site located at Lys444. The two phosphorylation sites were confirmed by Western blot analysis using phosphorylation site-specific antibodies. In addition, prevention of phosphorylation at the two serine sites altered mHtt toxicity and accumulation. These modifications of mHtt may provide novel therapeutic targets for effective treatment of the disorder.     

Probing exchange kinetics and atomic resolution dynamics in high-molecular-weight complexes using dark-state exchange saturation transfer NMR spectroscopy

We present the protocol for the measurement and analysis of dark-state exchange saturation transfer (DEST), a novel solution NMR method for characterizing, at atomic resolution, the interaction between an NMR-'visible' free species and an NMR-'invisible' species transiently bound to a very high-molecular-weight (>1 MDa) macromolecular entity. The reduced rate of reorientational motion in the bound state that precludes characterization by traditional NMR methods permits the observation of DEST. (15)N-DEST profiles are measured on a sample comprising the dark state in exchange with an NMR-visible species; in addition, the difference (ΔR(2)) in (15)N transverse relaxation rates between this sample and a control sample comprising only the NMR-visible species is also obtained. The (15)N-DEST and ΔR(2) data for all residues are then fitted simultaneously to the McConnell equations for various exchange models describing the residue-specific dynamics in the bound state(s) and the interconversion rate constants. Although the length of the experiments depends strongly on sample conditions, approximately 1 week of NMR spectrometer time was sufficient for full characterization of samples of amyloid-β (Aβ) at concentrations of ~100 μM.