Distinct self-interaction domains promote Multi Sex Combs accumulation in and formation of the Drosophila histone locus body

Nuclear bodies (NBs) are structures that concentrate proteins, RNAs, and ribonucleoproteins that perform functions essential to gene expression. How NBs assemble is not well understood. We studied the Drosophila histone locus body (HLB), a NB that concentrates factors required for histone mRNA biosynthesis at the replication-dependent histone gene locus. We coupled biochemical analysis with confocal imaging of both fixed and live tissues to demonstrate that the Drosophila Multi Sex Combs (Mxc) protein contains multiple domains necessary for HLB assembly. An important feature of this assembly process is the self-interaction of Mxc via two conserved N-terminal domains: a LisH domain and a novel self-interaction facilitator (SIF) domain immediately downstream of the LisH domain. Molecular modeling suggests that the LisH and SIF domains directly interact, and mutation of either the LisH or the SIF domain severely impairs Mxc function in vivo, resulting in reduced histone mRNA accumulation. A region of Mxc between amino acids 721 and 1481 is also necessary for HLB assembly independent of the LisH and SIF domains. Finally, the C-terminal 195 amino acids of Mxc are required for recruiting FLASH, an essential histone mRNA-processing factor, to the HLB. We conclude that multiple domains of the Mxc protein promote HLB assembly in order to concentrate factors required for histone mRNA biosynthesis.     

Connective tissue growth factor is required for normal follicle development and ovulation

Connective tissue growth factor (CTGF) is a cysteine-rich protein the synthesis and secretion of which are hypothesized to be selectively regulated by activins and other members of the TGF-β superfamily. To investigate the in vivo roles of CTGF in female reproduction, we generated Ctgf ovarian and uterine conditional knockout (cKO) mice. Ctgf cKO mice exhibit severe subfertility and multiple reproductive defects including disrupted follicle development, decreased ovulation rates, increased numbers of corpus luteum, and smaller but functionally normal uterine horns. Steroidogenesis is disrupted in the Ctgf cKO mice, leading to increased levels of serum progesterone. We show that disrupted follicle development is accompanied by a significant increase in granulosa cell apoptosis. Moreover, despite normal cumulus expansion, Ctgf cKO mice exhibit a significant decrease in oocytes ovulated, likely due to impaired ovulatory process. During analyses of mRNA expression, we discovered that Ctgf cKO granulosa cells show gene expression changes similar to our previously reported granulosa cell-specific knockouts of activin and Smad4, the common TGF-β family intracellular signaling protein. We also discovered a significant down-regulation of Adamts1, a progesterone-regulated gene that is critical for the remodeling of extracellular matrix surrounding granulosa cells of preovulatory follicles. These findings demonstrate that CTGF is a downstream mediator in TGF-β and progesterone signaling cascades and is necessary for normal follicle development and ovulation.     

Structure based evolution of a novel series of positive modulators of the AMPA receptor

Starting from compound 1, we utilized biostructural data to successfully evolve an existing series into a new chemotype with a promising overall profile, exemplified by 19.     

Inhibition of the cellular function of perforin by 1-amino-2,4-dicyanopyrido[1,2-a]benzimidazoles

A high throughput screen showed the ability of a 1-amino-2,4-dicyanopyrido[1,2-a]benzimidazole analogue to directly inhibit the lytic activity of the pore-forming protein perforin. A series of analogues were prepared to study structure-activity relationships (SAR) for the this activity, either directly added to cells or released in situ by KHYG-1 NK cells, at non-toxic concentrations. These studies showed that the pyridobenzimidazole moiety was required for effective activity, with strongly basic centres disfavoured. This class of compounds was relatively unaffected by the addition of serum, which was not the case for a previous class of direct inhibitors.     

Transmission characteristics of the 2009 H1N1 influenza pandemic: comparison of 8 Southern hemisphere countries

While in Northern hemisphere countries, the pandemic H1N1 virus (H1N1pdm) was introduced outside of the typical influenza season, Southern hemisphere countries experienced a single wave of transmission during their 2009 winter season. This provides a unique opportunity to compare the spread of a single virus in different countries and study the factors influencing its transmission. Here, we estimate and compare transmission characteristics of H1N1pdm for eight Southern hemisphere countries/states: Argentina, Australia, Bolivia, Brazil, Chile, New Zealand, South Africa and Victoria (Australia). Weekly incidence of cases and age-distribution of cumulative cases were extracted from public reports of countries'' surveillance systems. Estimates of the reproduction numbers, R ₀, empirically derived from the country-epidemics'' early exponential phase, were positively associated with the proportion of children in the populations (p = 0.004). To explore the role of demography in explaining differences in transmission intensity, we then fitted a dynamic age-structured model of influenza transmission to available incidence data for each country independently, and for all the countries simultaneously. Posterior median estimates of R ₀ ranged 1.2–1.8 for the country-specific fits, and 1.29–1.47 for the global fits. Corresponding estimates for overall attack-rate were in the range 20–50%. All model fits indicated a significant decrease in susceptibility to infection with age. These results confirm the transmissibility of the 2009 H1N1 pandemic virus was relatively low compared with past pandemics. The pattern of age-dependent susceptibility found confirms that older populations had substantial – though partial - pre-existing immunity, presumably due to exposure to heterologous influenza strains. Our analysis indicates that between-country-differences in transmission were at least partly due to differences in population demography.     

Cdk1-dependent destruction of Eco1 prevents cohesion establishment after S phase

Accurate genome segregation depends on cohesion mechanisms that link duplicated sister chromatids, thereby allowing their tension-dependent biorientation in metaphase. In Saccharomyces cerevisiae, cohesion is established during DNA replication when Eco1 acetylates the cohesin subunit Smc3. Cohesion establishment is restricted to S phase of the cell cycle, but the molecular basis of this regulation is unknown. Here, we show that Eco1 is negatively regulated by the protein kinase Cdk1. Phosphorylation of Eco1 after S phase targets it to SCF(Cdc4) for ubiquitination and subsequent degradation. A nonphosphorylatable mutant of Eco1 establishes cohesion after DNA replication, suggesting that Cdk1-dependent phosphorylation of Eco1 is a key factor limiting establishment to S phase. We also show that deregulation of Eco1 results in chromosome separation defects in anaphase. We conclude that this regulatory mechanism helps optimize the level of sister chromatid cohesion, ensuring a robust and efficient anaphase.     

Development and initial characterization of a HAPPY panel for mapping the X. tropicalis genome

HAPPY mapping was designed to pursue the analysis of approximately random HAPloid DNA breakage samples using the PolYmerase chain reaction for mapping genomes. In the present study, we improved the method and integrated two other molecular techniques into the process: whole genome amplification and the Sequenom SNP (single nucleotide polymorphism) genotyping assay in order to facilitate whole genome mapping of X. tropicalis. The former technique amplified enough DNA materials to genotype a large number of markers, while the latter allowed for relatively high throughput marker genotyping with multiplex assays on the HAPPY lines. A total of 58 X. tropicalis genes were genotyped on an initial panel of 383 HAPPY lines, which contributed to formation of a working panel of 146 lines. Further genotyping of 29 markers on the working panel led to construction of a HAPPY map for the X. tropicalis genome. We believe that our improved HAPPY method described in the present study has paved the way for the community to map different genomes with a simple, but powerful approach.     

A novel form of oxytocin in New World monkeys

Oxytocin is widely believed to be present and structurally identical in all placental mammals. Here, we report that multiple species of New World monkeys possess a novel form of oxytocin, [P8] oxytocin. This mutation arises from a substitution of a leucine to a proline in amino acid position 8. Further analysis of this mutation in Saimiri sciureus (squirrel monkey) indicates that [P8] oxytocin is transcribed and translated properly. This mutation is specific to oxytocin, as the peptide sequence for arginine vasopressin, a structurally related nonapeptide, is unaltered. These findings dispel the notion that all placental mammals possess a 'universal' oxytocin sequence, and highlight the need for research on the functional significance of this novel nonapeptide in New World monkeys.