A high-density screen for linkage in multiple sclerosis

To provide a definitive linkage map for multiple sclerosis, we have genotyped the Illumina BeadArray linkage mapping panel (version 4) in a data set of 730 multiplex families of Northern European descent. After the application of stringent quality thresholds, data from 4,506 markers in 2,692 individuals were included in the analysis. Multipoint nonparametric linkage analysis revealed highly significant linkage in the major histocompatibility complex (MHC) on chromosome 6p21 (maximum LOD score [MLS] 11.66) and suggestive linkage on chromosomes 17q23 (MLS 2.45) and 5q33 (MLS 2.18). This set of markers achieved a mean information extraction of 79.3% across the genome, with a Mendelian inconsistency rate of only 0.002%. Stratification based on carriage of the multiple sclerosis–associated DRB1*1501 allele failed to identify any other region of linkage with genomewide significance. However, ordered-subset analysis suggested that there may be an additional locus on chromosome 19p13 that acts independent of the main MHC locus. These data illustrate the substantial increase in power that can be achieved with use of the latest tools emerging from the Human Genome Project and indicate that future attempts to systematically identify susceptibility genes for multiple sclerosis will have to involve large sample sizes and an association-based methodology.     

Comprehensive thermodynamic analysis of 3' double-nucleotide overhangs neighboring Watson-Crick terminal base pairs

Thermodynamic parameters are reported for duplex formation of 48 self-complementary RNA duplexes containing Watson–Crick terminal base pairs (GC, AU and UA) with all 16 possible 3′ double-nucleotide overhangs; mimicking the structures of short interfering RNAs (siRNA) and microRNAs (miRNA). Based on nearest-neighbor analysis, the addition of a second dangling nucleotide to a single 3′ dangling nucleotide increases stability of duplex formation up to 0.8 kcal/mol in a sequence dependent manner. Results from this study in conjunction with data from a previous study [A. S. O'Toole, S. Miller and M. J. Serra (2005) RNA, 11, 512.] allows for the development of a refined nearest-neighbor model to predict the influence of 3′ double-nucleotide overhangs on the stability of duplex formation. The model improves the prediction of free energy and melting temperature when tested against five oligomers with various core duplex sequences. Phylogenetic analysis of naturally occurring miRNAs was performed to support our results. Selection of the effector miR strand of the mature miRNA duplex appears to be dependent upon the identity of the 3′ double-nucleotide overhang. Thermodynamic parameters for 3′ single terminal overhangs adjacent to a UA pair are also presented.     

Comparative protein interactomics of neuroglobin and myoglobin

Neuroglobin is a hypoxia-inducible O(2) -binding protein with neuroprotective effects in cell and animal models of stroke and Alzheimer's disease. The mechanism underlying neuroglobin's cytoprotective action is unknown, although several possibilities have been proposed, including anti-oxidative and anti-apoptotic effects. We used affinity purification-mass spectrometry methods to identify neuroglobin-interacting proteins in normoxic and hypoxic murine neuronal (HN33) cell lysates, and to compare these interactions with those of a structurally and functionally related protein, myoglobin. We report that the protein interactomes of neuroglobin and myoglobin overlap substantially and are modified by hypoxia. In addition, neuroglobin-interacting proteins include partners consistent with both anti-oxidative and anti-apoptotic functions, as well as with a relationship to several neurodegenerative diseases.     

Systematic review of effective strategies for reducing screen time among young children

Screen-media use among young children is highly prevalent, disproportionately high among children from lower-income families and racial/ethnic minorities, and may have adverse effects on obesity risk. Few systematic reviews have examined early intervention strategies to limit TV or total screen time; none have examined strategies to discourage parents from putting TVs in their children's bedrooms or remove TVs if they are already there. In order to identify strategies to reduce TV viewing or total screen time among children <12 years of age, we conducted a systematic review of seven electronic databases to June 2011, using the terms "intervention" and "television," "media," or "screen time." Peer-reviewed intervention studies that reported frequencies of TV viewing or screen-media use in children under age 12 were eligible for inclusion. We identified 144 studies; 47 met our inclusion criteria. Twenty-nine achieved significant reductions in TV viewing or screen-media use. Studies utilizing electronic TV monitoring devices, contingent feedback systems, and clinic-based counseling were most effective. While studies have reduced screen-media use in children, there are several research gaps, including a relative paucity of studies targeting young children (n = 13) or minorities (n = 14), limited long-term (>6 month) follow-up data (n = 5), and few (n = 4) targeting removing TVs from children's bedrooms. Attention to these issues may help increase the effectiveness of existing strategies for screen time reduction and extend them to different populations.     

Protein interaction profiling of the p97 adaptor UBXD1 points to a role for the complex in modulating ERGIC-53 trafficking

UBXD1 is a member of the poorly understood subfamily of p97 adaptors that do not harbor a ubiquitin association domain or bind ubiquitin-modified proteins. Of clinical importance, p97 mutants found in familial neurodegenerative conditions Inclusion Body Myopathy Paget's disease of the bone and/or Frontotemporal Dementia and Amyotrophic Lateral Sclerosis are defective at interacting with UBXD1, indicating that functions regulated by a p97-UBXD1 complex are altered in these diseases. We have performed liquid chromatography-mass spectrometric analysis of UBXD1-interacting proteins to identify pathways in which UBXD1 functions. UBXD1 displays prominent association with ERGIC-53, a hexameric type I integral membrane protein that functions in protein trafficking. The UBXD1-ERGIC-53 interaction requires the N-terminal 10 residues of UBXD1 and the C-terminal cytoplasmic 12 amino acid tail of ERGIC-53. Use of p97 and E1 enzyme inhibitors indicate that complex formation between UBXD1 and ERGIC-53 requires the ATPase activity of p97, but not ubiquitin modification. We also performed SILAC-based quantitative proteomic profiling to identify ERGIC-53 interacting proteins. This analysis identified known (e.g. COPI subunits) and novel (Rab3GAP1/2 complex involved in the fusion of vesicles at the cell membrane) interactions that are also mediated through the C terminus of the protein. Immunoprecipitation and Western blotting analysis confirmed the proteomic interaction data and it also revealed that an UBXD1-Rab3GAP association requires the ERGIC-53 binding domain of UBXD1. Localization studies indicate that UBXD1 modules the sub-cellular trafficking of ERGIC-53, including promoting movement to the cell membrane. We propose that p97-UBXD1 modulates the trafficking of ERGIC-53-containing vesicles by controlling the interaction of transport factors with the cytoplasmic tail of ERGIC-53.     

An anillin-Ect2 complex stabilizes central spindle microtubules at the cortex during cytokinesis

Cytokinesis occurs due to the RhoA-dependent ingression of an actomyosin ring. During anaphase, the Rho GEF (guanine nucleotide exchange factor) Ect2 is recruited to the central spindle via its interaction with MgcRacGAP/Cyk-4, and activates RhoA in the central plane of the cell. Ect2 also localizes to the cortex, where it has access to RhoA. The N-terminus of Ect2 binds to Cyk-4, and the C-terminus contains conserved DH (Dbl homologous) and PH (Pleckstrin Homology) domains with GEF activity. The PH domain is required for Ect2's cortical localization, but its molecular function is not known. In cultured human cells, we found that the PH domain interacts with anillin, a contractile ring protein that scaffolds actin and myosin and interacts with RhoA. The anillin-Ect2 interaction may require Ect2's association with lipids, since a novel mutation in the PH domain, which disrupts phospholipid association, weakens their interaction. An anillin-RacGAP50C (homologue of Cyk-4) complex was previously described in Drosophila, which may crosslink the central spindle to the cortex to stabilize the position of the contractile ring. Our data supports an analogous function for the anillin-Ect2 complex in human cells and one hypothesis is that this complex has functionally replaced the Drosophila anillin-RacGAP50C complex. Complexes between central spindle proteins and cortical proteins could regulate the position of the contractile ring by stabilizing microtubule-cortical interactions at the division plane to ensure the generation of active RhoA in a discrete zone.