Computational models for neurogenic gene expression in the Drosophila embryo

The early Drosophila embryo is emerging as a premiere model system for the computational analysis of gene regulation in development because most of the genes, and many of the associated regulatory DNAs, that control segmentation and gastrulation are known. The comprehensive elucidation of Drosophila gene networks provides an unprecedented opportunity to apply quantitative models to metazoan enhancers that govern complex patterns of gene expression during development. Models based on the fractional occupancy of defined DNA binding sites have been used to describe the regulation of the lac operon in E. coli and the lysis/lysogeny switch of phage lambda. Here, we apply similar models to enhancers regulated by the Dorsal gradient in the ventral neurogenic ectoderm (vNE) of the early Drosophila embryo. Quantitative models based on the fractional occupancy of Dorsal, Twist, and Snail binding sites raise the possibility that cooperative interactions among these regulatory proteins mediate subtle differences in the vNE expression patterns. Variations in cooperativity may be attributed to differences in the detailed linkage of Dorsal, Twist, and Snail binding sites in vNE enhancers. We propose that binding site occupancy is the key rate-limiting step for establishing localized patterns of gene expression in the early Drosophila embryo.     

Population structure of North Atlantic and North Pacific sei whales (<Emphasis Type="Italic">Balaenoptera borealis</Emphasis>) inferred from mitochondrial control region DNA sequences and microsatellite genotypes

Currently, three stocks of sei whales (Balaenoptera borealis) are defined in the North Atlantic; the Nova Scotian, Iceland-Denmark Strait and Eastern North Atlantic stocks, which are mainly based upon historical catch and sighting data. We analyzed mitochondrial control region DNA (mtDNA) sequences and genotypes from 7 to 11 microsatellite loci in 87 samples from three sites in the North Atlantic; Iceland, the Gulf of Maine and the Azores, and compared against the North Pacific using 489 previously published samples. No statistically significant deviations from homogeneity were detected among the North Atlantic samples at mtDNA or microsatellite loci. The genealogy estimated from the mtDNA sequences revealed a clear division of the haplotypes into a North Atlantic and a North Pacific clade, with the exception of one haplotype detected in a single sample from the Azores, which was included in the North Pacific clade. Significant genetic divergence between the North Atlantic and North Pacific Oceans was detected (mtDNA Φ_ST?=?0.72, microsatellite Weir and Cockerham’s ? = 0.20; p?<?0.001). The coalescent-based estimate of the population divergence time between the North Atlantic and North Pacific populations from the sequence variation among the mtDNA sequences was at 163,000 years ago. However, the inference was limited by an absence of samples from the Southern Hemisphere and uncertainty regarding mutation rates and generation times. The estimates of inter-oceanic migration rates were low (Nm at 0.007 into the North Pacific and at 0.248 in the opposite direction). Although estimates of genetic divergence among the current North Atlantic stocks were low and consistent with the extensive range of movement observed in satellite tagged sei whales, the high uncertainty of the genetic divergence estimates precludes rejection of multiple stocks in the North Atlantic.     

Incidence and Predictors of End Stage Renal Disease among Low-Income Blacks and Whites

We evaluated whether black race is associated with higher incidence of End Stage Renal Disease (ESRD) among a cohort of blacks and whites of similar, generally low socioeconomic status, and whether risk factor patterns differ among blacks and whites and explain the poorly understood racial disparity in ESRD. Incident diagnoses of ESRD among 79,943 black and white participants in the Southern Community Cohort Study (SCCS) were ascertained by linkage with the United States Renal Data System (USRDS) from 2002 through 2009. Person-years of follow up were calculated from date of entry into the SCCS until date of ESRD diagnosis, date of death, or September 1, 2009, whichever occurred first. Cox proportional hazards models were used to estimate hazard ratios (HRs) and 95% confidence intervals (CI) for incident ESRD among black and white participants in relation to baseline characteristics. After 329,003 person-years of follow-up, 687 incident cases of ESRD were identified in the cohort. The age-adjusted ESRD incidence rate was 273 (per 100,000) among blacks, 3.5-fold higher than the rate of 78 among whites. Risk factors for ESRD included male sex (HR = 1.6; 95% CI 1.4–1.9), low income (HR = 1.5; 95% CI 1.2–1.8 for income below vs. above $15,000), smoking (HR = 1.2; 95% CI 1.02–1.4) and histories of diabetes (HRs increasing to 9.4 (95% CI 7.4–11.9) among those with ≥20 years diabetes duration) and hypertension (HR = 2.9; 95% CI 2.3–3.7). Patterns and magnitudes of association were virtually identical among blacks and whites. After adjustment for these risk factors, blacks continued to have a higher risk for ESRD (HR = 2.4; 95% CI = 1.9–3.0) relative to whites. The black-white disparity in risk of ESRD was attenuated but not eliminated after control for known risk factors in a closely socioeconomically matched cohort. Further research characterizing biomedical factors, including CKD progression, in ESRD occurrence in these two racial groups is needed.     

Isosteric ramatroban analogs: selective and potent CRTH-2 antagonists

The chemoattractant receptor-homologous molecule expressed on T(H)2 cells (CRTH-2), also found on eosinophils and basophils, is a prostaglandin D2 receptor involved in the recruitment of these cell types during an inflammatory response. In this report, we describe the synthesis and optimization of a ramatroban isostere that is a selective and potent antagonist of CRTH-2 which may be useful in the treatment of certain diseases.     

Launch of a new report: “Road testing organizational life cycle assessment around the world: applications,experiences and lessons learned”

Purpose

Organizational life cycle assessment (O-LCA) is still a rather young proposal, but moving towards becoming more broadly accepted as a scientifically mature and practical method. The UNEP/SETAC flagship project “LCA of organizations” concluded its “road-testing” phase and is glad to announce the publication of the final report “Road testing organizational life cycle assessment around the world: applications, experiences and lessons learned.” The full report can be accessed at http://www.lifecycleinitiative.org/download/6060. This article shortly summarizes the flagship project phases and main outcomes, particularly the report recently launched, and pinpoints future actions.

Methods

In 2015, the “Guidance on Organizational Life Cycle Assessment” was published. During the following 2 years, the flagship project accompanied 12 organizations in the road testing of that O-LCA Guidance. They represent four world regions, different sectors and sizes. The road testers’ case studies and their feedback are the basis of the Road-testing Report.

Results and discussion

The Road-testing Report aims to complement the O-LCA Guidance through the road testers’ experience, thus delivering advice for future practitioners and inspiration to method developers. It includes executive summaries of the O-LCA road testers’ case studies and the main results of a comprehensive survey through which the road testers share their experience, feedback, and lessons learned. The road testing confirmed the application potential of the O-LCA method and the positive outcomes of the road testing have shown that no immediate updates to the O-LCA Guidance are needed, but some priority actions were identified in order to further ease the application of O-LCA.

Conclusions

Three main tasks for the coming years are identified by the authors: firstly, the challenges highlighted during the road testing should be addressed in the future by the LCA community; specific methodological difficulties of certain kinds of organizations, like the service sector, should be targeted; and finally, the potential revealed by the organizational perspective can be deployed in adjacent LCA fields. The flagship project team hopes that this second publication, together with the great acceptance of the O-LCA Guidance and the contribution of third parties, will pave the way to make O-LCA a mainstream tool.

     

Cardiac Myosin Is a Substrate for Zipper-interacting Protein Kinase (ZIPK)

Zipper-interacting protein kinase (ZIPK) is a member of the death-associated protein kinase family associated with apoptosis in nonmuscle cells where it phosphorylates myosin regulatory light chain (RLC) to promote membrane blebbing. ZIPK mRNA and protein are abundant in heart tissue and isolated ventricular neonatal rat cardiac myocytes. An unbiased substrate search performed with purified ZIPK on heart homogenates led to the discovery of a prominent 20-kDa protein substrate identified as RLC of ventricular myosin. Biochemical analyses showed ZIPK phosphorylated cardiac RLC at Ser-15 with a V_max value 2-fold greater than the value for smooth/nonmuscle RLC; cardiac RLC is a favorable biochemical substrate. Knockdown of ZIPK in cardiac myocytes by small interfering RNA significantly decreased the extent of RLC Ser-15 phosphorylation. Thus, ZIPK may act as a cardiac RLC kinase and thereby affect contractility.     

Azadirachtin disrupts formation of organised microtubule arrays during microgametogenesis of Plasmodium berghei

Transmission of malaria parasites from vertebrate blood to the mosquito vector depends critically on the differentiation of the gametocytes into gametes. This occurs in response to environmental stimuli encountered by the parasite in the mosquito bloodmeal. Male gametogenesis involves three rounds of DNA replication and endomitosis, and the assembly de novo of 8 motile axonemes. Azadirachtin, a plant limnoid and insecticide with an unkown mode of action, specifically inhibits the release of motile gametes from activated microgametocytes but does not inhibit growth and replication of a sexual blood stages. We have combined confocal laser scanning microscopy and transmission electron microscopy to examine the effect of azadirachtin on the complex reorganisation of the microtubule cytoskeleton during gametogenesis in Plasmodium berghei. Neither the replication of the genome nor the ability of tubulin monomers to assemble into microtubules upon gametocyte activation were prevented by azadirachtin. However, the drug interfered with the formation of mitotic spindles and with the assembly of microtubules into typical axonemes. Our observations suggest that azadarachtin specifically disrupts the patterning of microtubules into more complex structures, such as mitotic spindles and axonemes.     

A genetic map of tetraploid <Emphasis Type="Italic">Paspalum notatum</Emphasis> Flügge (bahiagrass) based on single-dose molecular markers

Paspalum notatum Flügge is a warm-season forage grass with mainly diploid (2n = 20) and autotetraploid (2n = 40) representatives. Diploid races reproduce sexually and require crosspollination due to a self-incompatible mating system, while autotetraploids reproduce by aposporous apomixis. The objectives of this work were to develop a genetic linkage map of Paspalum notatum Flügge at the tetraploid level, identify the linkage/s group/s associated with apomixis and carry out a general characterization of its mode of inheritance. A pseudo test-cross F₁ family of 113 individuals segregating for the mode of reproduction was obtained by crossing a synthetic completely sexual tetraploid plant (Q4188) as female parent with a natural aposporous individual (Q4117) as pollen donor. Map construction was based on single-dose markers (SDAFs) segregating from both parents. Two linkage maps (female and male) were constructed. Within each map, homologous groups were assembled by detecting repulsion-phase linked SDAFs. Putative Q4188 and Q4117 homolog groups were identified by mapping shared single dose markers (BSDF). The Q4188 map consisted of 263 markers distributed on 26 co-segregation groups over a total genetic distance of 1.590.6 cM, while the Q4117 map contained 216 loci dispersed on 39 co-segregation groups along 2.265.7 cM, giving an estimated genome coverage of 88% and 83%, respectively. Seven and 12 putative homologous chromosomes were detected within Q4188 and Q4117 maps, respectively. Afterward, ten female and male homologous chromosomes were identified by mapping BSDFs. In the Q4117 map, a single linkage group was associated with apospory. It was characterized by restriction in recombination and preferential chromosome pairing. A BPSD marker mapping within this group allowed the detection of the female homolog and the putative four male groups of the set carrying apospory.     

Optogenetic manipulation of cyclic guanosine monophosphate to probe phosphodiesterase activities in megakaryocytes

Cyclic guanosine monophosphate (cGMP) signalling plays a fundamental role in many cell types, including platelets. cGMP has been implicated in platelet formation, but mechanistic detail about its spatio-temporal regulation in megakaryocytes (MKs) is lacking. Optogenetics is a technique which allows spatio-temporal manipulation of molecular events in living cells or organisms. We took advantage of this method and expressed a photo-activated guanylyl cyclase, Blastocladiella emersonii Cyclase opsin (BeCyclop), after viral-mediated gene transfer in bone marrow (BM)-derived MKs to precisely light-modulate cGMP levels. BeCyclop-MKs showed a significantly increased cGMP concentration after illumination, which was strongly dependent on phosphodiesterase (PDE) 5 activity. This finding was corroborated by real-time imaging of cGMP signals which revealed that pharmacological PDE5 inhibition also potentiated nitric oxide-triggered cGMP generation in BM MKs. In summary, we established for the first-time optogenetics in primary MKs and show that PDE5 is the predominant PDE regulating cGMP levels in MKs. These findings also demonstrate that optogenetics allows for the precise manipulation of MK biology.