A Relational Event Approach to Modeling Behavioral Dynamics

This chapter provides an introduction to the analysis of relational event data (i.e., actions, interactions, or other events involving multiple actors that occur over time) within the R/statnet platform. We begin by reviewing the basics of relational event modeling, with an emphasis on models with piecewise constant hazards. We then discuss estimation for dyadic and more general relational event models using the relevent package, with an emphasis on hands-on applications of the methods and interpretation of results. Statnet is a collection of packages for the R statistical computing system that supports the representation, manipulation, visualization, modeling, simulation, and analysis of relational data. Statnet packages are contributed by a team of volunteer developers, and are made freely available under the GNU Public License. These packages are written for the R statistical computing environment, and can be used with any computing platform that supports R (including Windows, Linux, and Mac).

Improving a Natural CaMKII Inhibitor by Random and Rational Design

CaM-KIIN has evolved to inhibit stimulated and autonomous activity of the Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) efficiently, selectively, and potently (IC50 ∼100 nM). The CN class of peptides, derived from the inhibitory region of CaM-KIIN, provides powerful new tools to study CaMKII functions. The goal of this study was to identify the residues required for CaMKII inhibition, and to assess if artificial mutations could further improve the potency achieved during evolution.First, the minimal region with full inhibitory potency was identified (CN19) by determining the effect of truncated peptides on CaMKII activity in biochemical assays. Then, individual residues of CN19 were mutated. Most individual Ala substitutions decreased potency of CaMKII inhibition, however, P3A, K13A, and R14A increased potency. Importantly, this initial Ala scan suggested a specific interaction of the region around R11 with the CaMKII substrate binding site, which was exploited for further rational mutagenesis to generate an optimized pseudo-substrate sequence. Indeed, the potency of the optimized peptide CN19o was >250fold improved (IC50 <0.4 nM), and CN19o has characteristics of a tight-binding inhibitor. The selectivity for CaMKII versus CaMKI was similarly improved (to almost 100,000fold for CN19o). A phospho-mimetic S12D mutation decreased potency, indicating potential for regulation by cellular signaling. Consistent with importance of this residue in inhibition, most other S12 mutations also significantly decreased potency, however, mutation to V or Q did not.These results provide improved research tools for studying CaMKII function, and indicate that evolution fine-tuned CaM-KIIN not for maximal potency of CaMKII inhibition, but for lower potency that may be optimal for dynamic regulation of signal transduction

Stroke genetics: prospects for personalized medicine.

Epidemiologic evidence supports a genetic predisposition to stroke. Recent advances, primarily using the genome-wide association study approach, are transforming what we know about the genetics of multifactorial stroke, and are identifying novel stroke genes. The current findings are consistent with different stroke subtypes having different genetic architecture. These discoveries may identify novel pathways involved in stroke pathogenesis, and suggest new treatment approaches. However, the already identified genetic variants explain only a small proportion of overall stroke risk, and therefore are not currently useful in predicting risk for the individual patient. Such risk prediction may become a reality as identification of a greater number of stroke risk variants that explain the majority of genetic risk proceeds, and perhaps when information on rare variants, identified by whole-genome sequencing, is also incorporated into risk algorithms. Pharmacogenomics may offer the potential for earlier implementation of 'personalized genetic' medicine. Genetic variants affecting clopidogrel and warfarin metabolism may identify non-responders and reduce side-effects, but these approaches have not yet been widely adopted in clinical practice

Expanding the clinical phenotype in patients with disease causing variants associated with atypical Usher syndrome

Atypical Usher syndrome (USH) is poorly defined with a broad clinical spectrum. Here, we characterize the clinical phenotype of disease caused by variants in CEP78, CEP250, ARSG, and ABHD12. Chart review evaluating demographic, clinical, imaging, and genetic findings of 19 patients from 18 families with a clinical diagnosis of retinal disease and confirmed disease-causing variants in CEP78, CEP250, ARSG, or ABHD12. CEP78-related disease included sensorineural hearing loss (SNHL) in 6/7 patients and demonstrated a broad phenotypic spectrum including: vascular attenuation, pallor of the optic disc, intraretinal pigment, retinal pigment epithelium mottling, areas of mid-peripheral hypo-autofluorescence, outer retinal atrophy, mild pigmentary changes in the macula, foveal hypo-autofluorescence, and granularity of the ellipsoid zone. Nonsense and frameshift variants in CEP250 showed mild retinal disease with progressive, non-congenital SNHL. ARSG variants resulted in a characteristic pericentral pattern of hypo-autofluorescence with one patient reporting non-congenital SNHL. ABHD12-related disease showed rod-cone dystrophy with macular involvement, early and severe decreased best corrected visual acuity, and non-congenital SNHL ranging from unreported to severe. This study serves to expand the clinical phenotypes of atypical USH. Given the variable findings, atypical USH should be considered in patients with peripheral and macular retinal disease even without the typical RP phenotype especially when SNHL is noted. Additionally, genetic screening may be useful in patients who have clinical symptoms and retinal findings even in the absence of known SNHL given the variability of atypical USH

A Significant but Rather Mild Contribution of T286 Autophosphorylation to Ca2+/CaM-Stimulated CaMKII Activity

Autophosphorylation of the Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) at T286 generates partially Ca(2+)/CaM-independent "autonomous" activity, which is thought to be required for long-term potentiation (LTP), a form of synaptic plasticity thought to underlie learning and memory. A requirement for T286 autophosphorylation also for efficient Ca(2+)/CaM-stimulated CaMKII activity has been described, but remains controversial.In order to determine the contribution of T286 autophosphorylation to Ca(2+)/CaM-stimulated CaMKII activity, the activity of CaMKII wild type and its phosphorylation-incompetent T286A mutant was compared. As the absolute activity can vary between individual kinase preparations, the activity was measured in six different extracts for each kinase (expressed in HEK-293 cells). Consistent with measurements on purified kinase (from a baculovirus/Sf9 cell expression system), CaMKII T286A showed a mildly but significantly reduced rate of Ca(2+)/CaM-stimulated phosphorylation for two different peptide substrates (to ~75-84% of wild type). Additional slower CaMKII autophosphorylation at T305/306 inhibits stimulation by Ca(2+)/CaM, but occurs only minimally for CaMKII wild type during CaM-stimulated activity assays. Thus, we tested if the T286A mutant may show more extensive inhibitory autophosphorylation, which could explain its reduced stimulated activity. By contrast, inhibitory autophosphorylation was instead found to be even further reduced for the T286A mutant under our assay conditions. On a side note, the phospho-T305 antibody showed some basal background immuno-reactivity also with non-phosphorylated CaMKII, as indicated by T305/306A mutants.These results indicate that Ca(2+)/CaM-stimulated CaMKII activity is mildly (~1.2-1.3fold) further increased by additional T286 autophosphorylation, but that this autophosphorylation is not required for the major part of the stimulated activity. This indicates that the phenotype of CaMKII T286A mutant mice is indeed due to the lack of autonomous activity, as the T286A mutant showed no dramatic reduction in stimulated activity

Localisation of RNAs into the germ plasm of vitellogenic xenopus oocytes

We have studied the localisation of mRNAs in full-grown Xenopus laevis oocytes by injecting fluorescent RNAs, followed by confocal microscopy of the oocyte cortex. Concentrating on RNA encoding the Xenopus Nanos homologue, nanos1 (formerly Xcat2), we find that it consistently localised into aggregated germ plasm ribonucleoprotein (RNP) particles, independently of cytoskeletal integrity. This implies that a diffusion/entrapment-mediated mechanism is active, as previously reported for previtellogenic oocytes. Sometimes this was accompanied by localisation into scattered particles of the “late”, Vg1/VegT pathway; occasionally only late pathway localisation was seen. The Xpat RNA behaved in an identical fashion and for neither RNA was the localisation changed by any culture conditions tested. The identity of the labelled RNP aggregates as definitive germ plasm was confirmed by their inclusion of abundant mitochondria and co-localisation with the germ plasm protein Hermes. Further, the nanos1/Hermes RNP particles are interspersed with those containing the germ plasm protein Xpat. These aggregates may be followed into the germ plasm of unfertilized eggs, but with a notable reduction in its quantity, both in terms of injected molecules and endogenous structures. Our results conflict with previous reports that there is no RNA localisation in large oocytes, and that during mid-oogenesis even germ plasm RNAs localise exclusively by the late pathway. We find that in mid oogenesis nanos1 RNA also localises to germ plasm but also by the late pathway. Late pathway RNAs, Vg1 and VegT, also may localise into germ plasm. Our results support the view that mechanistically the two modes of localisation are extremely similar, and that in an injection experiment RNAs might utilise either pathway, the distinction in fates being very subtle and subject to variation. We discuss these results in relation to their biological significance and the results of others

The epidemiology and factors associated with nocturnal enuresis among boarding and daytime school children in southeast of Turkey: a cross sectional study

<p>Abstract</p> <p>Background</p> <p>Nocturnal enuresis is an important problem among young children living in Turkey. The purpose of this study was to determine the possible differences in the prevalence of enuresis between children in boarding school and daytime school and the association of enuresis with sociodemographic factors.</p> <p>Methods</p> <p>This was a cross-sectional survey. A total of 562 self-administered questionnaires were distrubuted to parents from two different types of schools. One of them was a day-time school and the other was a boarding school. To describe enuresis the ICD-10 definition of at least one wet night per month for three consecutive months was used. Chi-square test and a logistic regression model was used to identify significant predictive factors for enuresis.</p> <p>Results</p> <p>The overall prevalence of nocturnal enuresis was 14.9%. The prevalence of nocturnal enuresis declined with age. Of the 6 year old children 33.3% still wetted their beds, while the ratio was 2.6% for 15 years-olds. There was no significant difference in prevalence of nocturnal enuresis between boys and girls (14.3% versus 16. 8%). Enuresis was reported as 18.5% among children attending day time school and among those 11.5% attending boarding school (p < 0.05). Prevalence of enuresis was increased in children living in villages, with low income and with positive family history (p < 0.05). After multivariate analysis, history of urinary tract infection (OR = 2.02), age (OR = 1.28), low monthly income (OR = 2.86) and family history of enuresis (OR = 3.64) were factors associated with enuresis. 46.4% of parents and 57.1% of enuretic children were significantly concerned about the impact of enuresis.</p> <p>Conclusion</p> <p>Enuresis was more frequent among children attending daytime school when compared to boarding school. Our findings suggest that nocturnal enuresis is a common problem among school children, especially with low income, smaller age, family history of enuresis and history of urinary tract infection. Enuresis is a pediatric public health problem and efforts at all levels should be made such as preventive, etiological and curative.</p

A Non-Death Role of the Yeast Metacaspase: Yca1p Alters Cell Cycle Dynamics

Caspase proteases are a conserved protein family predominantly known for engaging and executing apoptotic cell death. Nevertheless, in higher eukaryotes, caspases also influence a variety of cell behaviors including differentiation, proliferation and growth control. S. cerevisiae expresses a primordial caspase, yca1, and exhibits apoptosis-like death under certain stresses; however, the benefit of a dedicated death program to single cell organisms is controversial. In the absence of a clear rationale to justify the evolutionary retention of a death only pathway, we hypothesize that yca1 also influences non-apoptotic events. We report that genetic ablation and/or catalytic inactivation of Yca1p leads to a longer G1/S transition accompanied by slower growth in fermentation conditions. Downregulation of Yca1p proteolytic activity also results in failure to arrest during nocodazole treatment, indicating that Yca1p participates in the G2/M mitotic checkpoint. 20s proteasome activity and ROS staining of the Δyca1 strain is indistinguishable from its isogenic control suggesting that putative regulation of the oxidative stress response by Yca1p does not instigate the cell cycle phenotype. Our results demonstrate multiple non-death roles for yca1 in the cell cycle

Developmental Robustness by Obligate Interaction of Class B Floral Homeotic Genes and Proteins

DEF-like and GLO-like class B floral homeotic genes encode closely related MADS-domain transcription factors that act as developmental switches involved in specifying the identity of petals and stamens during flower development. Class B gene function requires transcriptional upregulation by an autoregulatory loop that depends on obligate heterodimerization of DEF-like and GLO-like proteins. Because switch-like behavior of gene expression can be displayed by single genes already, the functional relevance of this complex circuitry has remained enigmatic. On the basis of a stochastic in silico model of class B gene and protein interactions, we suggest that obligate heterodimerization of class B floral homeotic proteins is not simply the result of neutral drift but enhanced the robustness of cell-fate organ identity decisions in the presence of stochastic noise. This finding strongly corroborates the view that the appearance of this regulatory mechanism during angiosperm phylogeny led to a canalization of flower development and evolution