Screening drug effects in patient‐derived cancer cells links organoid responses to genome alterations

Cancer drug screening in patient‐derived cells holds great promise for personalized oncology and drug discovery but lacks standardization. Whether cells are cultured as conventional monolayer or advanced, matrix‐dependent organoid cultures influences drug effects and thereby drug selection and clinical success. To precisely compare drug profiles in differently cultured primary cells, we developed DeathPro, an automated microscopy‐based assay to resolve drug‐induced cell death and proliferation inhibition. Using DeathPro, we screened cells from ovarian cancer patients in monolayer or organoid culture with clinically relevant drugs. Drug‐induced growth arrest and efficacy of cytostatic drugs differed between the two culture systems. Interestingly, drug effects in organoids were more diverse and had lower therapeutic potential. Genomic analysis revealed novel links between drug sensitivity and DNA repair deficiency in organoids that were undetectable in monolayers. Thus, our results highlight the dependency of cytostatic drugs and pharmacogenomic associations on culture systems, and guide culture selection for drug tests.     

Quantifying effects of soil heterogeneity on groundwater pollution at four sites in USA

Four sites located in the north-eastern region of the United States of America have been chosen to investigate the impacts of soil heterogeneity in the transport of solutes (bromide and chloride) through the vadose zone (the zone in the soil that lies below the root zone and above the permanent saturated groundwater). A recently proposed mathematical model based on the cumulative beta distribution has been deployed to compare and contrast the regions' heterogeneity from multiple sample percolation experiments. Significant differences in patterns of solute leaching were observed even over a small spatial scale, indicating that traditional sampling methods for solute transport, for example the gravity pan or suction lysimeters, or more recent inventions such as the multiple sample percolation systems may not be effective in estimating solute fluxes in soils when a significant degree of soil heterogeneity is present. Consequently, ignoring soil heterogeneity in solute transport studies will likely result in     

A genome-wide RNAi screen to dissect centriole duplication and centrosome maturation in Drosophila

Centrosomes comprise a pair of centrioles surrounded by an amorphous pericentriolar material (PCM). Here, we have performed a microscopy-based genome-wide RNA interference (RNAi) screen in Drosophila cells to identify proteins required for centriole duplication and mitotic PCM recruitment. We analysed 92% of the Drosophila genome (13,059 genes) and identified 32 genes involved in centrosome function. An extensive series of secondary screens classified these genes into four categories: (1) nine are required for centriole duplication, (2) 11 are required for centrosome maturation, (3) nine are required for both functions, and (4) three genes regulate centrosome separation. These 32 hits include several new centrosomal components, some of which have human homologs. In addition, we find that the individual depletion of only two proteins, Polo and Centrosomin (Cnn) can completely block centrosome maturation. Cnn is phosphorylated during mitosis in a Polo-dependent manner, suggesting that the Polo-dependent phosphorylation of Cnn initiates centrosome maturation in flies.     

From drought sensing to developmental control: evolution of cyclic AMP signaling in social amoebas

Amoebas and other protists commonly encyst when faced with environmental stress. Although little is known of the signaling pathways that mediate encystation, the analogous process of spore formation in dictyostelid social amoebas is better understood. In Dictyostelium discoideum, secreted cyclic AMP (cAMP) mediates the aggregation of starving amoebas and induces the differentiation of prespore cells. Intracellular cAMP acting on cAMP-dependent protein kinase (PKA) triggers the maturation of spores and prevents their germination under the prevalent conditions of high osmolality in the spore head. The osmolyte-activated adenylate cyclase, ACG, produces cAMP for prespore differentiation and inhibition of spore germination. To retrace the origin of ACG function, we investigated ACG gene conservation and function in species that span the dictyostelid phylogeny. ACG genes, osmolyte-activated ACG activity, and osmoregulation of spore germination were detected in species that represent the 4 major groups of Dictyostelia. Unlike the derived species D. discoideum, many basal Dictyostelia have retained the ancestral mechanism of encystation from solitary amoebas. In these species and in solitary amoebas, encystation is independently triggered by starvation or by high osmolality. Osmolyte-induced encystation was accompanied by an increase in cAMP and prevented by inhibition of PKA, indicating that ACG and PKA activation mediate this response. We propose that high osmolality signals drought in soil amoebas and that developmental cAMP signaling in the Dictyostelia has evolved from this stress response.     

Birth Weight,Adult Body Composition,and Subcutaneous Fat Distribution

Objectives: To investigate if birth weight is related to both body mass index (BMI) and distribution of subcutaneous fat at adult age. Research Methods and Procedures: A 9‐year longitudinal study was performed in 229 subjects (192 women) with ages ranging from 27 to 36 years. Birth weight was retrieved by a questionnaire, and adult weight, height, skinfold thicknesses, and waist‐to‐hip ratio (WHR) were repeatedly measured at mean ages 27, 29, 31, and 36 years. BMI, sum of four skinfolds (S4S), the ratio between two truncal skinfolds and S4S (SS/S4S), and the ratio between WHR and the cross‐sectional area of the left thigh were calculated with the available data. Results: The adjusted model showed that in women, birth weight was significantly negatively related to adult S4S [β = ?5.211; (?9.768 to ?0.654)], waist circumference [β = ?1.449; (?2.829 to ?0.069)], and SS/S4S ratio [β = ?3.579; (?5.296 to ?1.862)]. In men, a significant negative association was observed between birth weight and adult WHR [β = ?1.096; (?2.092 to ?0.100)] only. Other relationships showed, although not significantly, the same negative trend, namely that lower birth weight is related to higher adult body fat mass (S4S) and a more truncal subcutaneous fat distribution (SS/S4S). No associations were found between birth weight and either adult BMI or the cross‐sectional area of the thigh. Discussion: Lower birth weight is, in both adult men and women, related to a higher adult subcutaneous fat mass and a more truncal distribution of subcutaneous fat, indicating a higher risk for obesity.     

POLLINATOR-MEDIATED SELECTION ON FLORAL DISPLAY AND FLOWERING TIME IN THE PERENNIAL HERB ARABIDOPSIS LYRATA

The evolution of floral display and flowering time in animal-pollinated plants is commonly attributed to pollinator-mediated selection. Yet, the causes of selection on flowering phenology and traits contributing to floral display have rarely been tested experimentally in natural populations. We quantified phenotypic selection on morphological and phenological characters in the perennial, outcrossing herb Arabidopsis lyrata in two years using female reproductive success as a proxy of fitness. To determine whether selection on floral display and flowering phenology can be attributed to interactions with pollinators, selection was quantified both for open-pollinated controls and for plants receiving supplemental hand-pollination. We documented directional selection for many flowers, large petals, late start of flowering, and early end of flowering. Seed output was pollen-limited in both years and supplemental hand-pollination reduced the magnitude of selection on number of flowers, and reversed the direction of selection on end of flowering. The results demonstrate that interactions with pollinators may affect the strength of selection on floral display and the direction of selection on phenology of flowering in natural plant populations. They thus support the contention that pollinators can drive the evolution of both floral display and flowering time.     

Developmental changes in the effects of prostaglandin E<Subscript>2</Subscript> in the chicken ductus arteriosus

Prostaglandin E₂ (PGE₂) is the major vasodilator prostanoid of the mammalian ductus arteriosus (DA). In the present study we analyzed the response of isolated DA rings from 15-, 19- and 21-day-old chicken embryos to PGE₂ and other vascular smooth muscle relaxing agents acting through the cyclic AMP signaling pathway. PGE₂ exhibited a relaxant response in the 15-day DA, but not in the 19- and 21-day DA. Moreover, high concentrations of PGE₂ (≥3 μM in 15-day and ≥1 μM in 19-day and 21-day DA) induced contraction of the chicken DA. The presence of the TP receptor antagonist SQ29,548, unmasked a relaxant effect of PGE₂ in the 19- and 21-day DA and increased the relaxation induced by PGE₂ in the 15-day DA. The presence of the EP receptor antagonist AH6809 abolished PGE₂-mediated relaxation. The relaxant responses induced by PGE₂ and the β-adrenoceptor agonist isoproterenol, but not those elicited by the adenylate cyclase activator forskolin or the phosphodiesterase 3 inhibitor milrinone, decreased with maturation. High oxygen concentrations (95%) decreased the relaxation to PGE₂. The relaxing potency and efficacy of isoproterenol and milrinone were higher in the pulmonary than in the aortic side of the DA, whereas no regional differences were found in the response to PGE₂. We conclude that, in contrast to the mammalian situation, PGE₂ is a weak relaxant agent of the chicken DA and, with advancing incubation, it even stimulates TP vasoconstrictive receptors.     

Lipase Maturation Factor LMF1, Membrane Topology and Interaction with Lipase Proteins in the Endoplasmic Reticulum

Lipase maturation factor 1 (LMF1) is predicted to be a polytopic protein localized to the endoplasmic reticulum (ER) membrane. It functions in the post-translational attainment of enzyme activity for both lipoprotein lipase and hepatic lipase. By using transmembrane prediction methods in mouse and human orthologs, models of LMF1 topology were constructed and tested experimentally. Employing a tagging strategy that used insertion of ectopic glycan attachment sites and terminal fusions of green fluorescent protein, we established a five-transmembrane model, thus dividing LMF1 into six domains. Three domains were found to face the cytoplasm (the amino-terminal domain and loops B and D), and the other half was oriented to the ER lumen (loops A and C and the carboxyl-terminal domain). This representative model shows the arrangement of an evolutionarily conserved domain within LMF1 (DUF1222) that is essential to lipase maturation. DUF1222 comprises four of the six domains, with the two largest ones facing the ER lumen. We showed for the first time, using several naturally occurring variants featuring DUF1222 truncations, that Lmf1 interacts physically with lipoprotein lipase and hepatic lipase and localizes the lipase interaction site to loop C within DUF1222. We discuss the implication of our results with regard to lipase maturation and DUF1222 domain structure.