Role of Inn1 and its interactions with Hof1 and Cyk3 in promoting cleavage furrow and septum formation in S. cerevisiae

Cytokinesis requires coordination of actomyosin ring (AMR) contraction with rearrangements of the plasma membrane and extracellular matrix. In Saccharomyces cerevisiae, new membrane, the chitin synthase Chs2 (which forms the primary septum [PS]), and the protein Inn1 are all delivered to the division site upon mitotic exit even when the AMR is absent. Inn1 is essential for PS formation but not for Chs2 localization. The Inn1 C-terminal region is necessary for localization, and distinct PXXP motifs in this region mediate functionally important interactions with SH3 domains in the cytokinesis proteins Hof1 (an F-BAR protein) and Cyk3 (whose overexpression can restore PS formation in inn1Δ cells). The Inn1 N terminus resembles C2 domains but does not appear to bind phospholipids; nonetheless, when overexpressed or fused to Hof1, it can provide Inn1 function even in the absence of the AMR. Thus, Inn1 and Cyk3 appear to cooperate in activating Chs2 for PS formation, which allows coordination of AMR contraction with ingression of the cleavage furrow

Isolation and characterization of cytokinesis mutants in Saccharomyces cerevisiae

Cytokinesis in Saccharomyces cerevisiae can occur via the contraction of an actomyosin ring or through septum formation. Although the actomyosin contractile ring is not essential, S. cerevisiae strains deleted for hofl, a gene important for septum formation, are dependent on the actomyosin contractile ring for cytokinesis and viability (Vallen et al. 2000). A screen for mutants synthetically lethal with hofl was utilized to identify genes required for the function of the actomyosin pathway. Forty-one HOFl - dependent mutants were identified by screening 33,000 mutagenized colonies using a sectoring assay and other genetic tests.\ud Genetic analysis suggests that the 34 recessive mutants fall into 18 complementation groups, 11 of which are single alleles. Seven mutants have dominant mutations or exhibit unlinked non-complementation; it is not yet known how many loci this represents. Complementation testing and plasmid-linked suppression indicates that three of the complementation groups are MYOl, BNIl, and CYK3. Interestingly, some of the myol and cyk3 mutants appear to exhibit unlinked non-complementation. Phenotypic analysis including Hoechst staining of DNA, rhodamine-phalliodin staining of the actin cytoskeleton, DIC microscopic analysis, and zymolyase treatment was used to characterize mutants. Results indicate that four mutants that may affect the actin-myosin pathway and cytokinesis are worthy of further analysis

Isolation and characterization of cytokinesis mutants in Saccharomyces cerevisiae

Cytokinesis in Saccharomyces cerevisiae can occur via the contraction of an actomyosin ring or through septum formation. Although the actomyosin contractile ring is not essential, S. cerevisiae strains deleted for hof1, a gene important for septum formation, are dependent on the actomyosin contractile ring for cytokinesis and viability (Vallen et al. 2000). A screen for mutants synthetically lethal with hof1 was utilized to identify genes required for the function of the actomyosin pathway. Forty-one HOF1-dependent mutants were identified by screening 33,000 mutagenized colonies using a sectoring assay and other genetic tests. Genetic analysis suggests that the 34 recessive mutants fall into 18 complementation groups, 11 of which are single alleles. Seven mutants have dominant mutations or exhibit unlinked non-complementation; it is not yet known how many loci this represents. Complementation testing and plasmid-linked suppression indicates that three of the complementation groups are MYO1, BNI1, and CYK3. Interestingly, some of the myo1 and cyk3 mutants appear to exhibit unlinked non-complementation. Phenotypic analysis including Hoechst staining of DNA, rhodamine-phalliodin staining of the actin cytoskeleton, DIC microscopic analysis, and zymolyase treatment was used to characterize mutants. Results indicate that four mutants that may affect the actin-myosin pathway and cytokinesis are worthy of further analysis

Primary care physician time utilization before and after implementation of an electronic health record: A time-motion study

AbstractDespite benefits associated with the use of electronic health records (EHRs), one major barrier to adoption is the concern that EHRs may take longer for physicians to use than paper-based systems. To address this issue, we performed a time-motion study in five primary care clinics. Twenty physicians were observed and specific activities were timed during a clinic session before and after EHR implementation. Surveys evaluated physicians’ perceptions regarding the EHR. Post-implementation, the adjusted mean overall time spent per patient during clinic sessions decreased by 0.5min (p=0.86; 95% confidence interval [−5.05, 6.04]) from a pre-intervention adjusted average of 27.55min (SE=2.1) to a post-intervention adjusted average of 27.05min (SE=1.6). A majority of survey respondents believed EHR use results in quality improvement, yet only 29% reported that EHR documentation takes the same amount of time or less compared to the paper-based system. While the EHR did not require more time for physicians during a clinic session, further studies should assess the EHR’s potential impact on non-clinic time

The genetic architecture of sporadic and multiple consecutive miscarriage

Miscarriage is a common, complex trait affecting similar to 15% of clinically confirmed pregnancies. Here we present the results of large-scale genetic association analyses with 69,054 cases from five different ancestries for sporadic miscarriage, 750 cases of European ancestry for multiple (>= 3) consecutive miscarriage, and up to 359,469 female controls. We identify one genome-wide significant association (rs146350366, minor allele frequency (MAF) 1.2%, P=3.2 x 10(-8), odds ratio (OR)=1.4) for sporadic miscarriage in our European ancestry meta-analysis and three genome-wide significant associations for multiple consecutive miscarriage (rs7859844, MAF=6.4%, P=1.3 x 10(-8), OR=1.7; rs143445068, MAF=0.8%, P=5.2 x 10(-9), OR=3.4; rs183453668, MAF=0.5%, P=2.8 x 10(-8), OR=3.8). We further investigate the genetic architecture of miscarriage with biobank-scale Mendelian randomization, heritability, and genetic correlation analyses. Our results show that miscarriage etiopathogenesis is partly driven by genetic variation potentially related to placental biology, and illustrate the utility of large-scale biobank data for understanding this pregnancy complication.Peer reviewe

Determinants of Power in Gene-Based Burden Testing for Monogenic Disorders

Whole-exome sequencing has enabled new approaches for discovering genes associated with monogenic disorders. One such approach is gene-based burden testing, in which the aggregate frequency of “qualifying variants” is compared between case and control subjects for each gene. Despite substantial successes of this approach, the genetic causes for many monogenic disorders remain unknown or only partially known. It is possible that particular genetic architectures lower rates of discovery, but the influence of these factors on power has not been rigorously evaluated. Here, we leverage large-scale exome-sequencing data to create an empirically based simulation framework to evaluate the impact of key parameters (background variation rates, locus heterogeneity, mode of inheritance, penetrance) on power in gene-based burden tests in the context of monogenic disorders. Our results demonstrate that across genes, there is a wide range in sample sizes needed to achieve power due to differences in the background rate of rare variants in each gene. Increasing locus heterogeneity results in rapid increases in sample sizes needed to achieve adequate power, particularly when individual genes contribute to less than 5% of cases under a dominant model. Interestingly, incomplete penetrance as low as 10% had little effect on power due to the low prevalence of monogenic disorders. Our results suggest that moderate incomplete penetrance is not an obstacle in this gene-based burden testing approach but that dominant disorders with high locus heterogeneity will require large sample sizes. Our simulations also provide guidance on sample size needs and inform study design under various genetic architectures