Binding of Kif23-iso1/CHO1 to 14-3-3 Is Regulated by Sequential Phosphorylations at Two LATS Kinase Consensus Sites

Kif23 kinesin is an essential actor of cytokinesis in animals. It exists as two major isoforms, known as MKLP1 and CHO1, the longest of which, CHO1, contains two HXRXXS/T NDR/LATS kinase consensus sites. We demonstrate that these two sites are readily phosphorylated by NDR and LATS kinases in vitro, and this requires the presence of an upstream -5 histidine residue. We further show that these sites are phosphorylated in vivo and provide evidence revealing that LATS1,2 participate in the phosphorylation of the most C-terminal S814 site, present on both isoforms. This S814 phosphosite was previously reported to constitute a 14-3-3 binding site, which plays a role in Kif23 clustering during cytokinesis. Surprisingly, we found that phosphorylation of the upstream S716 NDR/LATS consensus site, present only in the longest Kif23 isoform, is required for efficient phosphorylation at S814, thus revealing sequential phosphorylation at these two sites, and differential regulation of Kif23-14-3-3 interaction for the two Kif23 isoforms. Finally, we provide evidence that Kif23 is largely unphosphorylated on S814 in post-abscission midbodies, making this Kif23 post-translational modification a potential marker to probe these structures.     

Dietary Supplement Enriched in Antioxidants and Omega-3 Protects from Progressive Light-Induced Retinal Degeneration

In the present study, we have evaluated one of the dietary supplements enriched with antioxidants and fish oil used in clinical care for patient with age-related macular degeneration. Rats were orally fed by a gastric canula daily with 0.2 ml of water or dietary supplement until they were sacrificed. After one week of treatment, animals were either sacrificed for lipid analysis in plasma and retina, or used for evaluation of rod-response recovery by electroretinography (ERG) followed by their sacrifice to measure rhodopsin content, or used for progressive light-induced retinal degeneration (PLIRD). For PLIRD, animals were transferred to bright cyclic light for one week. Retinal damage was quantified by ERG, histology and detection of apoptotic nuclei. Animals kept in dim-cyclic-light were processed in parallel. PLIRD induced a thinning of the outer nuclear layer and a reduction of the b-wave amplitude of the ERG in the water group. Retinal structure and function were preserved in supplemented animals. Supplement induced a significant increase in omega-3 fatty acids in plasma by 168% for eicosapentaenoic acid (EPA), 142% for docosapentaenoic acid (DPA) and 19% for docosahexaenoic acid (DHA) and a decrease in the omega-6 fatty acids, DPA by 28%. In the retina, supplement induced significant reduction of linolenic acid by 67% and an increase in EPA and DPA by 80% and 72%, respectively, associated with significant decrease in omega-6 DPA by 42%. Supplement did not affect rhodopsin content or rod-response recovery. The present data indicate that supplement rapidly modified the fatty acid content and induced an accumulation of EPA in the retina without affecting rhodopsin content or recovery. In addition, it protected the retina from oxidative stress induced by light. Therefore, this supplement might be beneficial to slow down progression of certain retinal degeneration.     

Shifts of Gamma Phase across Primary Visual Cortical Sites Reflect Dynamic Stimulus-Modulated Information Transfer

Distributed neural processing likely entails the capability of networks to reconfigure dynamically the directionality and strength of their functional connections. Yet, the neural mechanisms that may allow such dynamic routing of the information flow are not yet fully understood. We investigated the role of gamma band (50–80 Hz) oscillations in transient modulations of communication among neural populations by using measures of direction-specific causal information transfer. We found that the local phase of gamma-band rhythmic activity exerted a stimulus-modulated and spatially-asymmetric directed effect on the firing rate of spatially separated populations within the primary visual cortex. The relationships between gamma phases at different sites (phase shifts) could be described as a stimulus-modulated gamma-band wave propagating along the spatial directions with the largest information transfer. We observed transient stimulus-related changes in the spatial configuration of phases (compatible with changes in direction of gamma wave propagation) accompanied by a relative increase of the amount of information flowing along the instantaneous direction of the gamma wave. These effects were specific to the gamma-band and suggest that the time-varying relationships between gamma phases at different locations mark, and possibly causally mediate, the dynamic reconfiguration of functional connections.     

Risks and Benefits of Nalmefene in the Treatment of Adult Alcohol Dependence: A Systematic Literature Review and Meta-Analysis of Published and Unpublished Double-Blind Randomized Controlled Trials

Background

Nalmefene is a recent option in alcohol dependence treatment. Its approval was controversial. We conducted a systematic review and meta-analysis of the aggregated data (registered as PROSPERO 2014:CRD42014014853) to compare the harm/benefit of nalmefene versus placebo or active comparator in this indication.

Methods and Findings

Three reviewers searched for published and unpublished studies in Medline, the Cochrane Library, Embase, ClinicalTrials.gov, Current Controlled Trials, and bibliographies and by mailing pharmaceutical companies, the European Medicines Agency (EMA), and the US Food and Drug Administration. Double-blind randomized clinical trials evaluating nalmefene to treat adult alcohol dependence, irrespective of the comparator, were included if they reported (1) health outcomes (mortality, accidents/injuries, quality of life, somatic complications), (2) alcohol consumption outcomes, (3) biological outcomes, or (4) treatment safety outcomes, at 6 mo and/or 1 y. Three authors independently screened the titles and abstracts of the trials identified. Relevant trials were evaluated in full text. The reviewers independently assessed the included trials for methodological quality using the Cochrane Collaboration tool for assessing risk of bias. On the basis of the I2 index or the Cochrane’s Q test, fixed or random effect models were used to estimate risk ratios (RRs), mean differences (MDs), or standardized mean differences (SMDs) with 95% CIs. In sensitivity analyses, outcomes for participants who were lost to follow-up were included using baseline observation carried forward (BOCF); for binary measures, patients lost to follow-up were considered equal to failures (i.e., non-assessed patients were recorded as not having responded in both groups). Five randomized controlled trials (RCTs) versus placebo, with a total of 2,567 randomized participants, were included in the main analysis. None of these studies was performed in the specific population defined by the EMA approval of nalmefene, i.e., adults with alcohol dependence who consume more than 60 g of alcohol per day (for men) or more than 40 g per day (for women). No RCT compared nalmefene with another medication. Mortality at 6 mo (RR = 0.39, 95% CI [0.08; 2.01]) and 1 y (RR = 0.98, 95% CI [0.04; 23.95]) and quality of life at 6 mo (SF-36 physical component summary score: MD = 0.85, 95% CI [−0.32; 2.01]; SF-36 mental component summary score: MD = 1.01, 95% CI [−1.33; 3.34]) were not different across groups. Other health outcomes were not reported. Differences were encountered for alcohol consumption outcomes such as monthly number of heavy drinking days at 6 mo (MD = −1.65, 95% CI [−2.41; −0.89]) and at 1 y (MD = −1.60, 95% CI [−2.85; −0.35]) and total alcohol consumption at 6 mo (SMD = −0.20, 95% CI [−0.30; −0.10]). An attrition bias could not be excluded, with more withdrawals for nalmefene than for placebo, including more withdrawals for safety reasons at both 6 mo (RR = 3.65, 95% CI [2.02; 6.63]) and 1 y (RR = 7.01, 95% CI [1.72; 28.63]). Sensitivity analyses showed no differences for alcohol consumption outcomes between nalmefene and placebo, but the weight of these results should not be overestimated, as the BOCF approach to managing withdrawals was used.

Conclusions

The value of nalmefene for treatment of alcohol addiction is not established. At best, nalmefene has limited efficacy in reducing alcohol consumption.     

AutoDockFR: Advances in Protein-Ligand Docking with Explicitly Specified Binding Site Flexibility

Automated docking of drug-like molecules into receptors is an essential tool in structure-based drug design. While modeling receptor flexibility is important for correctly predicting ligand binding, it still remains challenging. This work focuses on an approach in which receptor flexibility is modeled by explicitly specifying a set of receptor side-chains a-priori. The challenges of this approach include the: 1) exponential growth of the search space, demanding more efficient search methods; and 2) increased number of false positives, calling for scoring functions tailored for flexible receptor docking. We present AutoDockFR–AutoDock for Flexible Receptors (ADFR), a new docking engine based on the AutoDock4 scoring function, which addresses the aforementioned challenges with a new Genetic Algorithm (GA) and customized scoring function. We validate ADFR using the Astex Diverse Set, demonstrating an increase in efficiency and reliability of its GA over the one implemented in AutoDock4. We demonstrate greatly increased success rates when cross-docking ligands into apo receptors that require side-chain conformational changes for ligand binding. These cross-docking experiments are based on two datasets: 1) SEQ17 –a receptor diversity set containing 17 pairs of apo-holo structures; and 2) CDK2 –a ligand diversity set composed of one CDK2 apo structure and 52 known bound inhibitors. We show that, when cross-docking ligands into the apo conformation of the receptors with up to 14 flexible side-chains, ADFR reports more correctly cross-docked ligands than AutoDock Vina on both datasets with solutions found for 70.6% vs. 35.3% systems on SEQ17, and 76.9% vs. 61.5% on CDK2. ADFR also outperforms AutoDock Vina in number of top ranking solutions on both datasets. Furthermore, we show that correctly docked CDK2 complexes re-create on average 79.8% of all pairwise atomic interactions between the ligand and moving receptor atoms in the holo complexes. Finally, we show that down-weighting the receptor internal energy improves the ranking of correctly docked poses and that runtime for AutoDockFR scales linearly when side-chain flexibility is added.     

Individualizing Pharmacotherapy in Patients with Renal Impairment: The Validity of the Modification of Diet in Renal Disease Formula in Specific Patient Populations with a Glomerular Filtration Rate below 60 Ml/Min. A Systematic Review

Background

The Modification of Diet in Renal Disease (MDRD) formula is widely used in clinical practice to assess the correct drug dose. This formula is based on serum creatinine levels which might be influenced by chronic diseases itself or the effects of the chronic diseases. We conducted a systematic review to determine the validity of the MDRD formula in specific patient populations with renal impairment: elderly, hospitalized and obese patients, patients with cardiovascular disease, cancer, chronic respiratory diseases, diabetes mellitus, liver cirrhosis and human immunodeficiency virus.

Methods and Findings

We searched for articles in Pubmed published from January 1999 through January 2014. Selection criteria were (1) patients with a glomerular filtration rate (GFR) < 60 ml/min (/1.73m²), (2) MDRD formula compared with a gold standard and (3) statistical analysis focused on bias, precision and/or accuracy. Data extraction was done by the first author and checked by a second author. A bias of 20% or less, a precision of 30% or less and an accuracy expressed as P₃₀% of 80% or higher were indicators of the validity of the MDRD formula. In total we included 27 studies. The number of patients included ranged from 8 to 1831. The gold standard and measurement method used varied across the studies. For none of the specific patient populations the studies provided sufficient evidence of validity of the MDRD formula regarding the three parameters. For patients with diabetes mellitus and liver cirrhosis, hospitalized patients and elderly with moderate to severe renal impairment we concluded that the MDRD formula is not valid. Limitations of the review are the lack of considering the method of measuring serum creatinine levels and the type of gold standard used.

Conclusion

In several specific patient populations with renal impairment the use of the MDRD formula is not valid or has uncertain validity.     

Establishment and Characterization of Rat Portal Myofibroblast Cell Lines

The major sources of scar-forming myofibroblasts during liver fibrosis are activated hepatic stellate cells (HSC) and portal fibroblasts (PF). In contrast to well-characterized HSC, PF remain understudied and poorly defined. This is largely due to the facts that isolation of rodent PF for functional studies is technically challenging and that PF cell lines had not been established. To address this, we have generated two polyclonal portal myofibroblast cell lines, RGF and RGF-N2. RGF and RGF-N2 were established from primary PF isolated from adult rat livers that underwent culture activation and subsequent SV40-mediated immortalization. Specifically, Ntpdase2/Cd39l1-sorted primary PF were used to generate the RGF-N2 cell line. Both cell lines were functionally characterized by RT-PCR, immunofluorescence, immunoblot and bromodeoxyuridine-based proliferation assay. First, immortalized RGF and RGF-N2 cells are positive for phenotypic myofibroblast markers alpha smooth muscle actin, type I collagen alpha-1, tissue inhibitor of metalloproteinases-1, PF-specific markers elastin, type XV collagen alpha-1 and Ntpdase2/Cd39l1, and mesenchymal cell marker ecto-5’-nucleotidase/Cd73, while negative for HSC-specific markers desmin and lecithin retinol acyltransferase. Second, both RGF and RGF-N2 cell lines are readily transfectable using standard methods. Finally, RGF and RGF-N2 cells attenuate the growth of Mz-ChA-1 cholangiocarcinoma cells in co-culture, as previously demonstrated for primary PF. Immortalized rat portal myofibroblast RGF and RGF-N2 cell lines express typical markers of activated PF-derived myofibroblasts, are suitable for DNA transfection, and can effectively inhibit cholangiocyte proliferation. Both RGF and RGF-N2 cell lines represent novel in vitro cellular models for the functional studies of portal (myo)fibroblasts and their contribution to the progression of liver fibrosis.     

R2OBBIE-3D,a Fast Robotic High-Resolution System for Quantitative Phenotyping of Surface Geometry and Colour-Texture

While recent imaging techniques provide insights into biological processes from the molecular to the cellular scale, phenotypes at larger scales remain poorly amenable to quantitative analyses. For example, investigations of the biophysical mechanisms generating skin morphological complexity and diversity would greatly benefit from 3D geometry and colour-texture reconstructions. Here, we report on R²OBBIE-3D, an integrated system that combines a robotic arm, a high-resolution digital colour camera, an illumination basket of high-intensity light-emitting diodes and state-of-the-art 3D-reconstruction approaches. We demonstrate that R²OBBIE generates accurate 3D models of biological objects between 1 and 100 cm, makes multiview photometric stereo scanning possible in practical processing times, and enables the capture of colour-texture and geometric resolutions better than 15 μm without the use of magnifying lenses. R²OBBIE has the potential to greatly improve quantitative analyses of phenotypes in addition to providing multiple new applications in, e.g., biomedical science.