Structural optimization and in vitro profiling of N-phenylbenzamide-based farnesoid X receptor antagonists

Activation of the nuclear farnesoid X receptor (FXR) which acts as cellular bile acid sensor has been validated as therapeutic strategy to counter liver disorders such as non-alcoholic steatohepatitis by the clinical efficacy of obeticholic acid. FXR antagonism, in contrast, is less well studied and potent small molecule FXR antagonists are rare. Here we report the systematic optimization of a novel class of FXR antagonists towards low nanomolar potency. The most optimized compound antagonizes baseline and agonist induced FXR activity in a full length FXR reporter gene assay and represses intrinsic expression of FXR regulated genes in hepatoma cells. With this activity and a favorable toxicity-, stability- and selectivity-profile it appears suitable to further study FXR antagonism in vitro and in vivo.     

Ecological and geographic modes of species divergence in wild tomatoes

Understanding the role of geography and ecology in species divergence is central to the study of evolutionary diversification. We used climatic, geographic, and biological data from nine wild Andean tomato species to describe each species' ecological niche and to evaluate the likely ecological and geographical modes of speciation in this clade. Using data from >1000 wild accessions and publicly available data derived from geographic information systems for various environmental variables, we found most species pairs were significantly differentiated for one or more environmental variables. By comparing species' predicted niches generated by species distribution modeling (SDM), we found significant niche differentiation among three of four sister-species pairs, suggesting ecological divergence is consistently associated with recent divergence. In comparison, based on age-range correlation (ARC) analysis, there was no evidence for a predominant geographical (allopatric vs. sympatric) context for speciation in this group. Overall, our results suggest an important role for environmentally mediated differentiation, rather than simply geographical isolation, in species divergence.     

Mutations in the N‐terminal kinase‐like domain of the repressor of photomorphogenesis SPA1 severely impair SPA1 function but not light responsiveness in Arabidopsis

The COP1/SPA complex is an E3 ubiquitin ligase that acts as a key repressor of photomorphogenesis in dark‐grown plants. While both COP1 and the four SPA proteins contain coiled‐coil and WD‐repeat domains, SPA proteins differ from COP1 in carrying an N‐terminal kinase‐like domain that is not present in COP1. Here, we have analyzed the effects of deletions and missense mutations in the N‐terminus of SPA1 when expressed in a spa quadruple mutant background devoid of any other SPA proteins. Deletion of the large N‐terminus of SPA1 severely impaired SPA1 activity in transgenic plants with respect to seedling etiolation, leaf expansion and flowering time. This ΔN SPA1 protein showed a strongly reduced affinity for COP1 in vitro and in vivo, indicating that the N‐terminus contributes to COP1/SPA complex formation. Deletion of only the highly conserved 95 amino acids of the kinase‐like domain did not severely affect SPA1 function nor interactions with COP1 or cryptochromes. In contrast, missense mutations in this part of the kinase‐like domain severely abrogated SPA1 function, suggesting an overriding negative effect of these mutations on SPA1 activity. We therefore hypothesize that the sequence of the kinase‐like domain has been conserved during evolution because it carries structural information important for the activity of SPA1 in darkness. The N‐terminus of SPA1 was not essential for light responsiveness of seedlings, suggesting that photoreceptors can inhibit the COP1/SPA complex in the absence of the SPA1 N‐terminal domain. Together, these results uncover an important, but complex role of the SPA1 N‐terminus in the suppression of photomorphogenesis.     

Construction and Rescue of a Molecular Clone of Deformed Wing Virus (DWV)

European honey bees are highly important in crop pollination, increasing the value of global agricultural production by billions of dollars. Current knowledge about virulence and pathogenicity of Deformed wing virus (DWV), a major factor in honey bee colony mortality, is limited. With this study, we close the gap between field research and laboratory investigations by establishing a complete in vitro model for DWV pathogenesis. Infectious DWV was rescued from a molecular clone of a DWV-A genome that induces DWV symptoms such as crippled wings and discoloration. The expression of DWV proteins, production of infectious virus progeny, and DWV host cell tropism could be confirmed using newly generated anti-DWV monoclonal antibodies. The recombinant RNA fulfills Koch’s postulates circumventing the need of virus isolation and propagation of pure virus cultures. In conclusion, we describe the development and application of a reverse genetics system for the study of DWV pathogenesis.     

Nonsense-mediated mRNA decay in Drosophila: at the intersection of the yeast and mammalian pathways

The nonsense-mediated mRNA decay (NMD) pathway promotes the rapid degradation of mRNAs containing premature stop codons (PTCs). In Caenorhabditis elegans, seven genes (smg1-7) playing an essential role in NMD have been identified. Only SMG2-4 (known as UPF1-3) have orthologs in Saccharomyces cerevisiae. Here we show that the Drosophila orthologs of UPF1-3, SMG1, SMG5 and SMG6 are required for the degradation of PTC-containing mRNAs, but that there is no SMG7 ortholog in this organism. In contrast, orthologs of SMG5-7 are encoded by the human genome and all three are required for NMD. In human cells, exon boundaries have been shown to play a critical role in defining PTCs. This role is mediated by components of the exon junction complex (EJC). Contrary to expectation, however, we show that the components of the EJC are dispensable for NMD in Drosophila cells. Consistently, PTC definition occurs independently of exon boundaries in DROSOPHILA: Our findings reveal that despite conservation of the NMD machinery, different mechanisms have evolved to discriminate premature from natural stop codons in metazoa.     

Estrogen Receptor and HER2 Status on Disseminated Tumor Cells and Primary Tumor in Patients with Early Breast Cancer

BACKGROUND: We evaluated both estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2) status on disseminated tumor cells (DTCs) in the bone marrow of 54 patients with early breast cancer and compared these with the corresponding primary tumor (PT). MATERIALS AND METHODS: Bone marrow aspirates were obtained at the time of first surgery, and ER and HER2 status on DTCs was assessed simultaneously by immunocytochemistry using a triple fluorescence staining method. RESULTS: The median number of DTCs was 13 (range 1-95). The concordance rate between ER status on DTC and PT was 74%. Patients with an ER-positive PT were significantly more likely to have at least one ER-positive DTC (34 out of 42) than patients with an ER-negative PT (6 out of 12; P = .031). Thirty-nine (93%) of the 42 patients with ER-positive PT had at least one ER-negative DTC. The concordance rate between HER2 status on DTC and PT was 52%. The probability of having at least one HER2-positive DTC was not related to the HER2 status of the PT (P = 0.56). Twenty-two (46%) of the 48 patients with a HER2-negative PT had at least one HER2-positive DTC. All the six patients with a HER2-positive PT had at least one HER2-negative DTC. CONCLUSION: Taken together, our study confirms that ER and/or HER2 status may differ between DTC and PT. This discordance could be important for patients lacking ER or HER2 expression on the PT but showing ER-positive or HER2-positive DTC because they might benefit from an endocrine and/or HER2-targeted therapy.     

Truncation Error Estimates in Process Life Cycle Assessment Using Input‐Output Analysis

Process life cycle assessment (PLCA) is widely used to quantify environmental flows associated with the manufacturing of products and other processes. As PLCA always depends on defining a system boundary, its application involves truncation errors. Different methods of estimating truncation errors are proposed in the literature; most of these are based on artificially constructed system complete counterfactuals. In this article, we review the literature on truncation errors and their estimates and systematically explore factors that influence truncation error estimates. We classify estimation approaches, together with underlying factors influencing estimation results according to where in the estimation procedure they occur. By contrasting different PLCA truncation/error modeling frameworks using the same underlying input‐output (I‐O) data set and varying cut‐off criteria, we show that modeling choices can significantly influence estimates for PLCA truncation errors. In addition, we find that differences in I‐O and process inventory databases, such as missing service sector activities, can significantly affect estimates of PLCA truncation errors. Our results expose the challenges related to explicit statements on the magnitude of PLCA truncation errors. They also indicate that increasing the strictness of cut‐off criteria in PLCA has only limited influence on the resulting truncation errors. We conclude that applying an additional I‐O life cycle assessment or a path exchange hybrid life cycle assessment to identify where significant contributions are located in upstream layers could significantly reduce PLCA truncation errors.     

Genetics of hybrid incompatibility between Lycopersicon esculentum and L. hirsutum

We examined the genetics of hybrid incompatibility between two closely related diploid hermaphroditic plant species. Using a set of near-isogenic lines (NILs) representing 85% of the genome of the wild species Lycopersicon hirsutum (Solanum habrochaites) in the genetic background of the cultivated tomato L. esculentum (S. lycopersicum), we found that hybrid pollen and seed infertility are each based on 5-11 QTL that individually reduce hybrid fitness by 36-90%. Seed infertility QTL act additively or recessively, consistent with findings in other systems where incompatibility loci have largely been recessive. Genetic lengths of introgressed chromosomal segments explain little of the variation for hybrid incompatibility among NILs, arguing against an infinitesimal model of hybrid incompatibility and reinforcing our inference of a limited number of discrete incompatibility factors between these species. In addition, male (pollen) and other (seed) incompatibility factors are roughly comparable in number. The latter two findings contrast strongly with data from Drosophila where hybrid incompatibility can be highly polygenic and complex, and male sterility evolves substantially faster than female sterility or hybrid inviability. The observed differences between Lycopersicon and Drosophila might be due to differences in sex determination system, reproductive and mating biology, and/or the prevalence of sexual interactions such as sexual selection.     

Reproductive biology of the andromonoecious Cucumis melo subsp. agrestis (Cucurbitaceae)

Background and Aims

Cucumis melo subsp. agrestis (Cucurbitaceae) is cultivated in many African regions for its edible kernels used as a soup thickener. The plant, an annual, andromonoecious, trailing-vine species, is of high social, cultural and economic value for local communities. In order to improve the yield of this crop, the first step and our aim were to elucidate its breeding system.

Methods

Eight experimental pollination treatments were performed during three growing seasons to assess spontaneous selfing, self-compatibility and effects of pollen source (hermaphroditic vs. male flowers). Pollination success was determined by pollen tube growth and reproductive success was assessed by fruit, seed and seedling numbers and characteristics. The pollinator guild was surveyed and the pollination distance determined both by direct observations and by indirect fluorescent dye dispersal.

Key Results

The species is probably pollinated by several Hymenoptera, principally by Hypotrigona para. Pollinator flight distances varied from 25 to 69 cm. No evidence for apomixis or spontaneous self-pollination in the absence of insect visitors was found. The self-fertility index (SFI = 0) indicated a total dependence on pollinators for reproductive success. The effects of hand pollination on fruit set, seed number and seedling fitness differed among years. Pollen tube growth and reproductive success did not differ between self- and cross-pollinations. Accordingly, a high self-compatibility index for the fruit set (SCI = 1·00) and the seed number (SCI = 0·98) and a low inbreeding depression at all developmental stages (cumulative δ = 0·126) suggest a high selfing ability. Finally, pollen origin had no effect on fruit and seed sets.

Conclusions

This andromonoecious species has the potential for a mixed mating system with high dependence on insect-mediated pollination. The selfing rate through geitonogamy should be important.