Relative accuracy of spatial predictive models for lynx Lynx canadensis derived using logistic regression-AIC,multiple criteria evaluation and Bayesian approaches

We compared probability surfaces derived using one set of environmental variables in three Geographic Information Systems (GIS) -based approaches: logistic regression and Akaike's Information Criterion (AIC),Multiple Criteria Evaluation (MCE),and Bayesian Analysis (specifically Dempster-Shafer theory). We used lynx Lynx canadensis as our focal species,and developed our environment relationship model using track data collected in Banff National Park,Alberta,Canada,during winters from 1997 to 2000. The accuracy of the three spatial models were compared using a contingency table method. We determined the percentage of cases in which both presence and absence points were correctly classified (overall accuracy),the failure to predict a species where it occurred (omission error) and the prediction of presence where there was absence (commission error). Our overall accuracy showed the logistic regression approach was the most accurate (74.51% ). The multiple criteria evaluation was intermediate (39.22%),while the Dempster-Shafer (D-S) theory model was the poorest (29.90%). However,omission and commission error tell us a different story: logistic regression had the lowest commission error,while D-S theory produced the lowest omission error. Our results provide evidence that habitat modellers should evaluate all three error measures when ascribing confidence in their model. We suggest that for our study area at least,the logistic regression model is optimal. However,where sample size is small or the species is very rare,it may also be useful to explore and/or use a more ecologically cautious modelling approach (e.g. Dempster-Shafer) that would over-predict,protect more sites,and thereby minimize the risk of missing critical habitat in conservation plans.     

Identification of a small molecule yeast TORC1 inhibitor with a multiplex screen based on flow cytometry

TOR (target of rapamycin) is a serine/threonine kinase, evolutionarily conserved from yeast to human, which functions as a fundamental controller of cell growth. The moderate clinical benefit of rapamycin in mTOR-based therapy of many cancers favors the development of new TOR inhibitors. Here we report a high-throughput flow cytometry multiplexed screen using five GFP-tagged yeast clones that represent the readouts of four branches of the TORC1 signaling pathway in budding yeast. Each GFP-tagged clone was differentially color-coded, and the GFP signal of each clone was measured simultaneously by flow cytometry, which allows rapid prioritization of compounds that likely act through direct modulation of TORC1 or proximal signaling components. A total of 255 compounds were confirmed in dose-response analysis to alter GFP expression in one or more clones. To validate the concept of the high-throughput screen, we have characterized CID 3528206, a small molecule most likely to act on TORC1 as it alters GFP expression in all five GFP clones in a manner analogous to that of rapamycin. We have shown that CID 3528206 inhibited yeast cell growth and that CID 3528206 inhibited TORC1 activity both in vitro and in vivo with EC(50)'s of 150 nM and 3.9 μM, respectively. The results of microarray analysis and yeast GFP collection screen further support the notion that CID 3528206 and rapamycin modulate similar cellular pathways. Together, these results indicate that the HTS has identified a potentially useful small molecule for further development of TOR inhibitors.     

High-Speed Cell Sorting of Infectious Trophic and Cystic Forms of Pneumocystis carinii

ABSTRACT. The separation of Pneumocystis carinii life-cycle stages while preserving infectivity is a hitherto unresolved challenge. We describe an original, reproducible, and efficient method for separating trophic from cystic forms of P. carinii using a high-speed cell sorter. The large amounts of highly purified (99.6±0.3%) infectious trophic and cystic forms can now be used to elucidate the poorly understood P. carinii life cycle.     

Voltage-Activated Calcium Signals in Myotubes Loaded with High Concentrations of EGTA

In the present study we describe the analysis of optically recorded whole cell Ca²⁺ transients elicited by depolarization in cultured skeletal myotubes. Myotubes were obtained from the mouse muscle-derived cell line C2C12 and from mouse satellite cells. The cells were voltage-clamped and perfused with an artificial intracellular solution containing 15 mM EGTA to ensure that the bulk of the Ca²⁺ mobilized by depolarization is bound to this extrinsic buffer. The apparent on- and off-rate constants of EGTA and the dissociation rate constant of fura-2 in the cell were estimated by investigating the Ca²⁺-dependence of kinetic components of the fluorescence decay after repolarization. These parameters were used to calculate the time course of the total voltage-controlled flux of Ca²⁺ to the myoplasmic space (Ca²⁺ input flux). The validity of the procedure was confirmed by model simulations using artificial Ca²⁺ input fluxes. Both C2C12 and primary-cultured myotubes showed a very similar phasic-tonic time course of the Ca²⁺ input flux. In most measurements, the input flux was considerably larger and showed a different time course than the estimated Ca²⁺ flux carried by the L-type Ca²⁺ channels, indicating that it consists mainly of voltage-controlled Ca²⁺ release from the sarcoplasmic reticulum. In cells with extremely small fluorescence transients, the calculated input fluxes matched the kinetic characteristics of the Ca²⁺ inward current, indicating that Ca²⁺ release was absent. These measurements served as a control for the fidelity of the fluorimetric flux analysis. The procedures promise a deeper insight into alterations of Ca²⁺ release gating in studies employing myotube expression systems for mutant or chimeric protein components of excitation-contraction coupling.     

α7 and β2 Nicotinic Acetylcholine Receptor Subunits Form Heteromeric Receptor Complexes that Are Expressed in the Human Cortex and Display Distinct Pharmacological Properties

The existence of α7β2 nicotinic acetylcholine receptors (nAChRs) has recently been demonstrated in both the rodent and human brain. Since α7-containing nAChRs are promising drug targets for schizophrenia and Alzheimer’s disease, it is critical to determine whether α7β2 nAChRs are present in the human brain, in which brain areas, and whether they differ functionally from α7 nAChR homomers. We used α-bungarotoxin to affinity purify α7-containing nAChRs from surgically excised human temporal cortex, and found that α7 subunits co-purify with β2 subunits, indicating the presence of α7β2 nAChRs in the human brain. We validated these results by demonstrating co-purification of β2 from wild-type, but not α7 or β2 knock-out mice. The pharmacology and kinetics of human α7β2 nAChRs differed significantly from that of α7 homomers in response to nAChR agonists when expressed in Xenopus oocytes and HEK293 cells. Notably, α7β2 heteromers expressed in HEK293 cells display markedly slower rise and decay phases. These results demonstrate that α7 subunits in the human brain form heteromeric complexes with β2 subunits, and that human α7β2 nAChR heteromers respond to nAChR agonists with a unique pharmacology and kinetic profile. α7β2 nAChRs thus represent an alternative mechanism for the reported clinical efficacy of α7 nAChR ligands.     

A Pan-GTPase Inhibitor as a Molecular Probe

Overactive GTPases have often been linked to human diseases. The available inhibitors are limited and have not progressed far in clinical trials. We report here a first-in-class small molecule pan-GTPase inhibitor discovered from a high throughput screening campaign. The compound CID1067700 inhibits multiple GTPases in biochemical, cellular protein and protein interaction, as well as cellular functional assays. In the biochemical and protein interaction assays, representative GTPases from Rho, Ras, and Rab, the three most generic subfamilies of the GTPases, were probed, while in the functional assays, physiological processes regulated by each of the three subfamilies of the GTPases were examined. The chemical functionalities essential for the activity of the compound were identified through structural derivatization. The compound is validated as a useful molecular probe upon which GTPase-targeting inhibitors with drug potentials might be developed.     

Association of the IL-10 Gene Family Locus on Chromosome 1 with Juvenile Idiopathic Arthritis (JIA)

Background

The cytokine IL-10 and its family members have been implicated in autoimmune diseases and we have previously reported that genetic variants in IL-10 were associated with a rare group of diseases called juvenile idiopathic arthritis (JIA). The aim of this study was to fine map genetic variants within the IL-10 cytokine family cluster on chromosome 1 using linkage disequilibrium (LD)-tagging single nucleotide polymorphisms (tSNPs) approach with imputation and conditional analysis to test for disease associations.

Methodology/Principal Findings

Fifty-three tSNPs were tested for association between Caucasian paediatric cohorts [219 systemic JIA (sJIA), 187 persistent oligoarticular JIA (pOJIA), and 139 extended OJIA (eOJIA) patients], and controls (Wellcome Trust control cohort, WTCCC2). Significant association with sJIA was detected at rs1400986 in the promoter of IL-20 (odds ratio 1.53; 95% CI 1.21–1.93; p = 0.0004), but in no other subtypes. Imputation analysis identified additional associated SNPs for pOJIA at IL-20 and IL-24, including a rare, functional, missense variant at IL-24 with a p = 0.0002. Penalised logistic regression analysis with HyperLasso and conditional analysis identified several further associations with JIA subtypes. In particular, haplotype analysis refined the sJIA association, with a joint effect at rs1400986 and rs4129024 in intron 1 of MAPKAPK2 (p = 3.2E−5). For pOJIA, a 3-SNP haplotype including rs1878672 in intron 3 of IL-10 showed evidence for association (p = 0.0018). In eOJIA, rs10863962 (3′UTR of FCAMR) and rs12409577 (intron of IL-19) haplotype showed some evidence of association (p = 0.0003).

Conclusions

This study supports previous association of IL-20 with sJIA. Haplotype analyses provided stronger association signals than single point analyses, while a penalised logistic regression approach also suggested multiple independent association signals. Replication studies are required to confirm or refute these findings. The results indicate that combined effects with unknown/rare variants remain to be characterised in JIA, and represent a possible example of synthetic association in this region.     

Synthesis and optimization of arylsulfonylpiperazines as a novel class of inhibitors of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1)

The synthesis and SAR of a series of arylsulfonylpiperazine inhibitors of 11β-HSD1 are described. Optimization rapidly led to potent, selective, and orally bioavailable inhibitors demonstrating efficacy in a cynomolgus monkey ex vivo enzyme inhibition model.     

International guide to prescribing psychotropic medication for the management of problem behaviours in adults with intellectual disabilities

Psychotropic medications are used regularly to manage problem behaviours among people with intellectual disabilities. This causes concern because often these medications are used out of their licensed indications in this context. The WPA Section on Psychiatry of Intellectual Disability has recently developed an evidence and consensus-based international guide for practitioners for the use of psychotropic medications for problem behaviours among adults with intellectual disabilities. This guide advises on assessment of behaviours, producing a formulation, initiation of treatment, assessment of out-come and adverse effects, follow-up arrangements, and possibility of discontinuation of treatment.     

Dissociation between hippocampal neuronal loss, astroglial and microglial reactivity after pharmacologically induced reverse glutamate transport

The inflammatory central nervous system response that involves activated microglia and reactive astrocytes may both heal and harm neurons, as inflammatory mediators lead to neuroprotection or excitation at one dose but to injury at a different concentration. To investigate these complex cellular interactions, L-trans-pyrrolidine-2,4-dicarboxylate (PDC), a selective substrate inhibitor of glutamate transport, was infused during 14 days in the rat hippocampus at three different rates: 5, 10 and 25 nmol/h. A microglial reaction appeared at the 5 nmol/h PDC rate in absence of astroglial reaction and neuronal loss. Microgliosis and neuronal death were observed at PDC 10 nmol/h in absence of astrogliosis and calcium precipitation, whereas all four aspects were present at the highest rate. This dissociation between neuronal loss and astroglial reactivity took place in presence of a permanent microglial reaction. These data suggest a specific response of microglia to PDC whose neuronal effects may differ with the infused dose.