ExonSurfer: a web-tool to design primers at exon-exon junctions.

BACKGROUND: Reverse transcription quantitative PCR (RT-qPCR) with intercalating dyes is one of the main techniques to assess gene expression levels used in basic and applied research as well as in diagnostics. However, primer design for RT-qPCR can be complex due to the high demands on primer quality. Primers are best placed on exon junctions, should avoid polymorphic regions, be specific to the target transcripts and also prevent genomic amplification accurately, among others. Current software tools manage to meet all the necessary criteria only insufficiently. Here, we present ExonSurfer, a novel, user-friendly web-tool for qPCR primer design. RESULTS: ExonSurfer combines the different steps of the primer design process, encompassing target selection, specificity and self-complementarity assessment, and the avoidance of issues arising from polymorphisms. Amplification of potentially contaminating genomic DNA is avoided by designing primers on exon-exon junctions, moreover, a genomic alignment is performed to filter the primers accordingly and inform the user of any predicted interaction. In order to test the whole performance of the application, we designed primer pairs for 26 targets and checked both primer efficiency, amplicon melting temperature and length and confirmed the targeted amplicon by Sanger sequencing. Most of the tested primers accurately and selectively amplified the corresponding targets. CONCLUSION: ExonSurfer offers a comprehensive end-to-end primer design, guaranteeing transcript-specific amplification. The user interface is intuitive, providing essential specificity and amplicon details. The tool can also be used by command line and the source code is available. Overall, we expect ExonSurfer to facilitate RT-qPCR set-up for researchers in many fields.

Phyllobilins - Bioactive Natural Products Derived from Chlorophyll - Plant Origins, Structures, Absorption Spectra, and Biomedical Properties.

Phyllobilins are open-chain products of the biological degradation of chlorophyll a in higher plants. Recent studies reveal that phyllobilins exert anti-oxidative and anti-inflammatory properties, as well as activities against cancer cells, that contribute to the human health benefits of numerous plants. In general, phyllobilins have been overlooked in phytochemical analyses, and - more importantly - in the analyses of medicinal plant extracts. Nevertheless, over the past three decades, > 70 phyllobilins have been identified upon examination of more than 30 plant species. Eight distinct chromophoric classes of phyllobilins are known: phyllolumibilins (PluBs), phylloleucobilins (PleBs), phylloxanthobilins (PxBs), and phylloroseobilins (PrBs)-each in type-I or type-II groups. Here, we present a database of absorption and fluorescence spectra that has been compiled of 73 phyllobilins to facilitate identification in phytochemical analyses. The spectra are provided in digital form and can be viewed and downloaded at www.photochemcad.com. The present review describes the plant origin, molecular structure, and absorption and fluorescence features of the 73 phyllobilins, along with an overview of key medicinal properties. The review should provide an enabling tool for the community for the straightforward identification of phyllobilins in plant extracts, and the foundation for deeper understanding of these ubiquitous but underexamined plant-derived micronutrients for human health.

A Chlorophyll-Derived Phylloxanthobilin Is a Potent Antioxidant That Modulates Immunometabolism in Human PBMC.

Phyllobilins are natural products derived from the degradation of chlorophyll, which proceeds via a common and strictly controlled pathway in higher plants. The resulting tetrapyrrolic catabolites-the phyllobilins-are ubiquitous in nature; despite their high abundance, there is still a lack of knowledge about their physiological properties. Phyllobilins are part of human nutrition and were shown to be potent antioxidants accounting with interesting physiological properties. Three different naturally occurring types of phyllobilins-a phylloleucobilin, a dioxobilin-type phylloleucobilin and a phylloxanthobilin (PxB)-were compared regarding potential antioxidative properties in a cell-free and in a cell-based antioxidant activity test system, demonstrating the strongest effect for the PxB. Moreover, the PxB was investigated for its capacity to interfere with immunoregulatory metabolic pathways of tryptophan breakdown in human blood peripheral mononuclear cells. A dose-dependent inhibition of tryptophan catabolism to kynurenine was observed, suggesting a suppressive effect on pathways of cellular immune activation. Although the exact mechanisms of immunomodulatory effects are yet unknown, these prominent bioactivities point towards health-relevant effects, which warrant further mechanistic investigations and the assessment of the in vivo extrapolatability of results. Thus, phyllobilins are a still surprisingly unexplored family of natural products that merit further investigation.

Using the yeast three-hybrid system for the identification of small molecule-protein interactions with the example of ethinylestradiol.

Since the first report on a yeast three-hybrid system, several approaches have successfully utilized different setups for discovering targets of small molecule drugs. Compared to broadly applied MS based target identification approaches, the yeast three-hybrid system represents a complementary method that allows for the straightforward identification of direct protein binders of selected small molecules. One major drawback of this system, however, is that the drug has to be taken up by the yeast cells in sufficient concentrations. Here, we report the establishment of a yeast three-hybrid screen in the deletion strain ABC9Δ, which is characterized by being highly permeable to small molecules. We used this system to screen for protein binding partners of ethinylestradiol, a widely used drug mainly for contraception and hormone replacement therapy. We identified procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 (PLOD2 or lysyl hydroxylase, LH2) as a novel direct target and were able to confirm the interaction identified with the yeast three-hybrid system by a complementary method, affinity chromatography, to prove the validity of the hit. Furthermore, we provide evidence for an interaction between the drug and PLOD2 in vitro and in cellulo.

Rising levels of antioxidative phyllobilins in stored agricultural produce and their impact on consumer acceptance.

Consumers often throw away faded greens, because taste and appearance are less appealing compared to fresh ones. We report here a family of antioxidants, the phyllobilins, which increase during storage in iceberg lettuce and cucumber. We show that informing consumers about rising levels of phyllobilins leads to a longer willingness to consume faded lettuce and to an improved health and safety perception.

Tetrapyrrolic Pigments from Heme- and Chlorophyll Breakdown are Actin-Targeting Compounds.

Chlorophyll and heme are among the "pigments of life", tetrapyrrolic structures, without which life on Earth would not be possible. Their catabolites, the phyllobilins and the bilins, respectively, share not only structural features, but also a similar story: Long considered waste products of detoxification processes, important bioactivities for both classes have now been demonstrated. For phyllobilins, however, research on physiological roles is sparse. Here, we introduce actin, the major component of the cytoskeleton, as the first discovered target of phyllobilins and as a novel target of bilins. We demonstrate the inhibition of actin dynamics in vitro and effects on actin and related processes in cancer cells. A direct interaction with G-actin is shown by in silico studies and confirmed by affinity chromatography. Our findings open a new chapter in bioactivities of tetrapyrroles-especially phyllobilins-for which they form the basis for broad implications in plant science, ecology, and physiology.

Phyllobilins as a challenging diverse natural product class: Exploration of pharmacological activities.

Phyllobilins are a group of chlorophyll-derived bilin-type linear tetrapyrroles, generated in the process of chlorophyll breakdown. Since the first phyllobilin was isolated and characterized in 1991, more and more structures of these chlorophyll catabolites were identified alongside the biochemical players involved in chlorophyll breakdown. In the meantime, phyllobilins are known to occur in a large natural structural variety, and new modifications are still being discovered. Phyllobilins have been regarded as products of chlorophyll detoxification for a very long time, hence they have been completely overlooked as a natural product class in terms of their biological role or pharmacological activity. A change of this paradigm, however, is long overdue. Here, we review the current knowledge of the pharmacological activities of phyllobilins and give an overview of the diverse structural modifications, laying the groundwork for analyzing their role(s) as active components in medicinal plants.

A yellow chlorophyll catabolite in leaves of Urtica dioica L.: An overlooked phytochemical that contributes to health benefits of stinging nettle.

Stinging nettle is appreciated for its antioxidant and anti-inflammatory properties, which renders the plant a popular ingredient in a healthy diet in form of salads or smoothies. The most common use, presumably, is of dried leaves as ingredient in tea mixtures. The plant's health benefits are attributed primarily to phenolic phytochemicals. Here we describe the characterization and quantification of a phylloxanthobilin (PxB), a yellow chlorophyll catabolite, in nettle tea. Despite their abundance in the plant kingdom, chlorophyll catabolites have been overlooked as phytochemicals and as part of human nutrition. Our investigations of tea reveal that one cup of nettle tea contains about 50 µg of PxB with large variations depending on the supplier. When investigating the bioactivities of PxB, our observations show that PxB has antioxidative and anti-inflammatory activities comparable to known bioactive small molecules found in nettle, indicating the phylloxanthobilin to be an overlooked ingredient of nettle tea.

Re-opening the stage for Echinacea research - Characterization of phylloxanthobilins as a novel anti-oxidative compound class in Echinacea purpurea.

BACKGROUND: Phylloxanthobilins are tetrapyrrolic natural products that arise from the degradation of chlorophyll. Phylloxanthobilins have been discovered roughly 10 years ago in the leaves of deciduous trees, and are now considered a compound class with high and still unexplored potential of bioactivities. To date, however, there are no reports on the occurrence of phylloxanthobilins in parts of a medicinal plant used for pharmaceutical preparations. PURPOSE: The relevance of Echinacea purpurea as medicinal plant is undoubtedly high, and a large variety of pharmaceutical preparations is available on the market, mostly for the treatment of the common cold. Nevertheless, its phytochemical profiling has been limited to analysis for previously characterized substances, and this has not explained all its pharmacological efficacies. We therefore set out to investigate the occurrence of phylloxanthobilins in Echinacea purpurea. METHODS: Phylloxanthobilins in leaf extracts of Echinacea purpurea were detected using analytical HPLC. Identified phyllobilins were purified from plant material and characterized by UV/Vis, mass spectrometry, MS/MS, and confirmed by co-injections with previously published phyllobilins from different sources. The anti-oxidant activity of selected isolated phylloxanthobilins was assessed by an in vitro ferric reducing antioxidant power (FRAP) assay; in addition, the ability to scavenge ROS in cells caused by hydrogen peroxide stimulation was determined by measuring H2DCF-DA fluorescence and by assessing cellular GSH levels. RESULTS: In extracts of Echinacea purpurea leaves, an unprecedented diversity of phylloxanthobilins was detected; surprisingly, not only in senescent yellow leaves, but also in green leaves with no visible chlorophyll degradation. Six phylloxanthobilins were identified and structurally characterized. The uptake of phylloxanthobilins by human endothelial kidney cells was demonstrated. When investigating the anti-oxidative activity of these natural products, a potent in vitro activity was demonstrated; in addition, phylloxanthobilins possess intracellular ROS scavenging ability and can prevent oxidative stress as assessed by total cellular GSH levels. CONCLUSION: Phylloxanthobilins are important constituents of Echinacea purpurea extracts, and our first exploratory studies hint towards promising bioactivities of these natural products, which may be relevant for understanding Echinacea efficacies.