Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Network Oncolgy Specialist Advisory Service - A Survey of the Psychosocial Situation of Long Term Cancer Survivors During Childhood or Adolescents

Zusammenfassun

Diagnosis of toxoplasmosis using a synthetic glycosylphosphatidylinositol glycan

Around 2 billion people worldwide are infected with the apicomplexan parasite Toxoplasma gondii which induces a variety of medical conditions. For example, primary infection during pregnancy can result in fetal death or mental retardation of the child. Diagnosis of acute infections in pregnant women is challenging but crucially important as the drugs used to treat T. gondii infections are potentially harmful to the unborn child. Better, faster, more reliable, and cheaper means of diagnosis by using defined antigens for accurate serological tests are highly desirable. Synthetic pathogen-specific glycosylphosphatidylinositol (GPI) glycan antigens are diagnostic markers and have been used to distinguish between toxoplasmosis disease states using human sera

Investigation of the protective properties of glycosylphosphatidylinositol-based vaccine candidates in a Toxoplasma gondii

Vaccination against the ubiquitous parasite Toxoplasma gondii would provide the most efficient prevention against toxoplasmosis-related congenital, brain, and eye diseases in humans. We investigated the immune response elicited by pathogen-specific glycosylphosphatidylinositol (GPI) glycoconjugates using carbohydrate microarrays in a BALB/c mouse model. We further examined the protective properties of the glycoconjugates in a lethal challenge model using the virulent T. gondii RH strain. Upon immunization, mice raised antibodies that bind to the respective GPIs on carbohydrate microarrays, but were mainly directed against an unspecific GPI epitope including the linker. The observed immune response, though robust, was unable to provide protection in mice when challenged with a lethal dose of viable tachyzoites. We demonstrate that anti-GPI antibodies raised against the here described semi-synthetic glycoconjugates do not confer protective immunity against T. gondii in BALB/c mice

Combining Activity Profiling with Advanced Annotation to Accelerate the Discovery of Natural Products Targeting Oncogenic Signaling in Melanoma.

The discovery of bioactive natural products remains a time-consuming and challenging task. The ability to link high-confidence metabolite annotations in crude extracts with activity would be highly beneficial to the drug discovery process. To address this challenge, HPLC-based activity profiling and advanced UHPLC-HRMS/MS metabolite profiling for annotation were combined to leverage the information obtained from both approaches on a crude extract scaled down to the submilligram level. This strategy was applied to a subset of an extract library screening aiming to identify natural products inhibiting oncogenic signaling in melanoma. Advanced annotation and data organization enabled the identification of compounds that were likely responsible for the activity in the extracts. These compounds belonged to two different natural product scaffolds, namely, brevipolides from a Hyptis brevipes extract and methoxylated flavonoids identified in three different extracts of Hyptis and Artemisia spp. Targeted isolation of these prioritized compounds led to five brevipolides and seven methoxylated flavonoids. Brevipolide A (1) and 6-methoxytricin (9) were the most potent compounds from each chemical class and displayed AKT activity inhibition with an IC50 of 17.6 ± 1.6 and 4.9 ± 0.2 μM, respectively

High-Content Screening Pipeline for Natural Products Targeting Oncogenic Signaling in Melanoma.

The incidence of melanoma, the most fatal dermatological cancer, has dramatically increased over the last few decades. Modern targeted therapy with kinase inhibitors induces potent clinical responses, but drug resistance quickly develops. Combination therapy improves treatment outcomes. Therefore, novel inhibitors targeting aberrant proliferative signaling in melanoma via the MAPK/ERK and PI3K/AKT pathways are urgently needed. Biosensors were combined that report on ERK/AKT activity with image-based high-content screening and HPLC-based activity profiling. An in-house library of 2576 plant extracts was screened on two melanoma cell lines with different oncogenic mutations leading to pathological ERK/AKT activity. Out of 140 plant extract hits, 44 were selected for HPLC activity profiling. Active thymol derivatives and piperamides from Arnica montana and Piper nigrum were identified that inhibited pathological ERK and/or AKT activity. The pipeline used enabled an efficient identification of natural products targeting oncogenic signaling in melanoma

Heparan sulfate proteoglycans present PCSK9 to the LDL receptor

Coronary artery disease is the main cause of death worldwide and accelerated by increased plasma levels of cholesterol-rich low-density lipoprotein particles (LDL). Circulating PCSK9 contributes to coronary artery disease by inducing lysosomal degradation of the LDL receptor (LDLR) in the liver and thereby reducing LDL clearance. Here, we show that liver heparan sulfate proteoglycans are PCSK9 receptors and essential for PCSK9-induced LDLR degradation. The heparan sulfate-binding site is located in the PCSK9 prodomain and formed by surface-exposed basic residues interacting with trisulfated heparan sulfate disaccharide repeats. Accordingly, heparan sulfate mimetics and monoclonal antibodies directed against the heparan sulfate-binding site are potent PCSK9 inhibitors. We propose that heparan sulfate proteoglycans lining the hepatocyte surface capture PCSK9 and facilitates subsequent PCSK9: LDLR complex formation. Our findings provide new insights into LDL biology and show that targeting PCSK9 using heparan sulfate mimetics is a potential therapeutic strategy in coronary artery disease

Comparative characterization of two galectins excreted-secreted from intestine-dwelling parasitic versus free-living females of the soil-transmitted nematode Strongyloides

Helminths are complex pathogens that ensure their long-term survival by influencing the immune responses of their host. Excretory/secretory products (ESP) can exert immunoregulatory effects which foster parasite survival. Galectins represent a widespread group of β-galactoside-binding proteins which are involved in a multitude of biological processes operative in parasite-host interaction. We had earlier identified seven galectins in Strongyloides ratti, four of them detected in the ESP of distinct developmental stages of the parasite. In the present report, we focused on the characterization of two of them, Sr-galectin-1 (Sr-Gal-1) and Sr-galectin-3 (Sr-Gal-3). While Sr-Gal-3 expression was strongest in parasitic females, Sr-Gal-1 was predominantly expressed in free-living females. Both proteins were cloned and recombinantly expressed in an E. coli expression system. Their glycan-binding activity was verified by haemagglutination and glycan array analysis. Furthermore, primary immunological activities of the Sr-galectins were initially investigated by the application of an in vitro mucosal 3D-culture model, comprising of mucosa-associated epithelial and dendritic cells. The Sr-galectins stimulated preferentially the release of the type 2 cytokines thymic stromal lymphopoietin and IL-22, a first indication for immunoregulatory activity. In addition, the Sr-galectins dose-dependently fostered cell migration. Our results confirm the importance of these carbohydrate-binding proteins in host-parasite-interaction by indicating possible interaction with the host mucosa-associated cells

Epitope Recognition of Antibodies against a <i>Yersinia pestis</i> Lipopolysaccharide Trisaccharide Component

Today, the process of selecting carbohydrate antigens as a basis for active vaccination and the generation of antibodies for therapeutic and diagnostic purposes is based on intuition combined with trial and error experiments. In efforts to establish a rational process for glycan epitope selection, we employed glycan array screening, surface plasmon resonance, and saturation transfer difference (STD)-NMR to elucidate the interactions between antibodies and glycans representing the <i>Yersinia pestis</i> lipopolysaccharide (LPS). A trisaccharide epitope of the LPS inner core glycan and different LPS-derived oligosaccharides from various Gram-negative bacteria were analyzed using this combination of techniques. The antibody-glycan interaction with a heptose substructure was determined at atomic-level detail. Antibodies specifically recognize the <i>Y. pestis</i> trisaccharide and some substructures with high affinity and specificity. No significant binding to LPS glycans from other bacteria was observed, which suggests that the epitopes for just one particular bacterial species can be identified. On the basis of these results we are beginning to understand the rules for structure-based design and selection of carbohydrate antigens

Microbe-focused glycan array screening platform

International audienceInteractions between glycans and glycan binding proteins are essential for numerous processes in all kingdoms of life. Glycan microarrays are an excellent tool to examine protein-glycan interactions. Here, we present a microbe-focused glycan microarray platform based on oligosaccharides obtained by chemical synthesis. Glycans were generated by combining different carbohydrate synthesis approaches including automated glycan assembly, solution-phase synthesis, and chemoenzymatic methods. The current library of more than 300 glycans is as diverse as the mammalian glycan array from the Consortium for Functional Glycomics and, due to its microbial focus, highly complementary. This glycan platform is essential for the characterization of various classes of glycan binding proteins. Applications of this glycan array platform are highlighted by the characterization of innate immune receptors and bacterial virulence factors as well as the analysis of human humoral immunity to pathogenic glycans