Dopaminergic Progenitors Derived From Epiblast Stem Cells Function Similarly to Primary VM-Derived Progenitors When Transplanted Into a Parkinson's Disease Model.

Neural transplantation in neurodegenerative diseases such as Parkinson's disease (PD) offers to replace cells lost during the progression of the disease process. Primary fetal ventral mesencephalon (VM), the origin of bona fide midbrain dopaminergic (DAergic) precursors, is currently the gold standard source of cells for transplantation in PD. However, the use of tissue from this source raises ethical and logistical constraints necessitating the need for alternative supplies of donor cells. The requirement of any alternative donor cell source is to have the capability to generate authentic mature DAergic neurons, which could be utilized in cell-replacement strategies. Mouse pluripotent stem cells can efficiently generate electrochemically mature midbrain DAergic precursors in vitro using a stepwise control of FGF signaling. Here, we have compared DAergic transplants derived from two progenitor cell sources in an allograft system: mouse epiblast stem cells (EpiSC) and primary fetal mouse VM tissue. Cells were transplanted into the striatum of 6-OHDA lesioned mice pre-treated with L-DOPA. Drug-induced rotations, a number of motor tests and drug-induced abnormal involuntary movements (AIMs) were assessed. Functional improvements were demonstrated post-transplantation in some behavioral tests, with no difference in graft volume or the number of TH immuno-positive cells in the grafts of the two transplant groups. L-DOPA-induced AIMs and amphetamine-induced AIMs were observed in both transplant groups, with no differences in rate or severity between the two groups. Collectively, in this mouse-to-mouse allograft system, we report no significant differences in the functional ability between the gold standard primary VM derived and pluripotent stem cell-derived DAergic transplants.

Hierarchical folding and reorganization of chromosomes are linked to transcriptional changes in cellular differentiation.

Mammalian chromosomes fold into arrays of megabase-sized topologically associating domains (TADs), which are arranged into compartments spanning multiple megabases of genomic DNA. TADs have internal substructures that are often cell type specific, but their higher-order organization remains elusive. Here, we investigate TAD higher-order interactions with Hi-C through neuronal differentiation and show that they form a hierarchy of domains-within-domains (metaTADs) extending across genomic scales up to the range of entire chromosomes. We find that TAD interactions are well captured by tree-like, hierarchical structures irrespective of cell type. metaTAD tree structures correlate with genetic, epigenomic and expression features, and structural tree rearrangements during differentiation are linked to transcriptional state changes. Using polymer modelling, we demonstrate that hierarchical folding promotes efficient chromatin packaging without the loss of contact specificity, highlighting a role far beyond the simple need for packing efficiency.

Mapping discontinuous protein-binding sites via structure-based peptide libraries: combining in silico and in vitro approaches.

To perform their various functions, protein surfaces often have to interact with each other in a specific way. Usually, only parts of a protein are accessible and can act as binding sites. Because proteins consist of polypeptide chains that fold into complex three-dimensional shapes, binding sites can be divided into two different types: linear sites that follow the primary amino acid sequence and discontinuous binding sites, which are made up of short peptide fragments that are adjacent in spatial proximity. Such discontinuous binding sites dominate protein-protein interactions, but are difficult to identify. To meet this challenge, we combined a computational, structure-based approach and an experimental, high-throughput method. SUPERFICIAL is a program that uses protein structures as input and generates peptide libraries to represent the protein's surface. A large number of the predicted peptides can be simultaneously synthesised applying the SPOT technology. The results of a binding assay subsequently help to elucidate protein-protein interactions; the approach is applicable to any kind of protein. The crystal structure of the complex of hen egg lysozyme with the well-characterised murine IgG1 antibody HyHEL-5 is available, and the complex is known to have a discontinuous binding site. Using SUPERFICIAL, the entire surface of lysozyme was translated into a peptide library that was synthesised on a cellulose membrane using the SPOT technology and tested against the HyHEL-5 antibody. In this way, it was possible to identify two peptides (longest common sequence and peptide 19) that represented the discontinuous epitope of lysozyme.

Temporally controlled modulation of FGF/ERK signaling directs midbrain dopaminergic neural progenitor fate in mouse and human pluripotent stem cells.

Effective induction of midbrain-specific dopamine (mDA) neurons from stem cells is fundamental for realizing their potential in biomedical applications relevant to Parkinson's disease. During early development, the Otx2-positive neural tissues are patterned anterior-posteriorly to form the forebrain and midbrain under the influence of extracellular signaling such as FGF and Wnt. In the mesencephalon, sonic hedgehog (Shh) specifies a ventral progenitor fate in the floor plate region that later gives rise to mDA neurons. In this study, we systematically investigated the temporal actions of FGF signaling in mDA neuron fate specification of mouse and human pluripotent stem cells and mouse induced pluripotent stem cells. We show that a brief blockade of FGF signaling on exit of the lineage-primed epiblast pluripotent state initiates an early induction of Lmx1a and Foxa2 in nascent neural progenitors. In addition to inducing ventral midbrain characteristics, the FGF signaling blockade during neural induction also directs a midbrain fate in the anterior-posterior axis by suppressing caudalization as well as forebrain induction, leading to the maintenance of midbrain Otx2. Following a period of endogenous FGF signaling, subsequent enhancement of FGF signaling by Fgf8, in combination with Shh, promotes mDA neurogenesis and restricts alternative fates. Thus, a stepwise control of FGF signaling during distinct stages of stem cell neural fate conversion is crucial for reliable and highly efficient production of functional, authentic midbrain-specific dopaminergic neurons. Importantly, we provide evidence that this novel, small-molecule-based strategy applies to both mouse and human pluripotent stem cells.

Rmst is a novel marker for the mouse ventral mesencephalic floor plate and the anterior dorsal midline cells.

The availability of specific markers expressed in different regions of the developing nervous system provides a useful tool to illuminate their development, regulation and function. We have identified by expression profiling a putative non-coding RNA, Rmst, that exhibits prominent expression in the midbrain floor plate region, the isthmus and the roof plate of the anterior neural tube. At the developmental stage when the ventral dopaminergic neuron territory is being established, Rmst expression appears to be restricted to the presumptive dopaminergic neurons of the ventral tegmental area that lies close to the ventral midline. Thus this study presents Rmst as a novel marker for the developing dopaminergic neurons in the mesencephalic floor plate as well as a marker for the dorsal midline cells of the anterior neural tube and the isthmic organizer.

Survivin minigene DNA vaccination is effective against neuroblastoma.

The inhibitor of apoptosis protein survivin is highly expressed in neuroblastoma (NB) and survivin-specific T cells were identified in Stage 4 patients. Therefore, we generated a novel survivin minigene DNA vaccine (pUS-high) encoding exclusively for survivin-derived peptides with superior MHC class I (H2-K(k)) binding affinities and tested its efficacy to suppress tumor growth and metastases in a syngeneic NB mouse model. Vaccination was performed by oral gavage of attenuated Salmonella typhimurium SL7207 carrying pUS-high. Mice receiving the pUS-high in the prophylactic setting presented a 48-52% reduction in s.c. tumor volume, weight and liver metastasis level in contrast to empty vector controls. This response was as effective as a survivin full-length vaccine and was associated with an increased target cell lysis, increased presence of CD8(+) T-cells at the primary tumor site and enhanced production of proinflammatory cytokines by systemic CD8(+) T cells. Furthermore, depletion of CD8(+) but not CD4(+) T-cells completely abrogated the pUS-high mediated primary tumor growth suppression, demonstrating a CD8(+) T-cell mediated effect. Therapeutic vaccination with pUS-high led to complete NB eradication in over 50% of immunized mice and surviving mice showed an over 80% reduction in primary tumor growth upon rechallenge in contrast to controls. In summary, survivin-based DNA vaccination is effective against NB and the rational minigene design provides a promising approach to circumvent potentially hazardous effects of using full length antiapoptotic genes as DNA vaccines.

SuperToxic: a comprehensive database of toxic compounds.

Within our everyday life, we are confronted with a variety of toxic substances of natural or artificial origin. Toxins are already used, e.g. in medicine, but there is still an increasing number of toxic compounds, representing a tremendous potential to extract new substances. Since predictive toxicology gains in importance, the careful and extensive investigation of known toxins is the basis to assess the properties of unknown substances. In order to achieve this aim, we have collected toxic compounds from literature and web sources in the database SuperToxic. The current version of this database compiles about 60,000 compounds and their structures. These molecules are classified according to their toxicity, based on more than 2 million measurements. The SuperToxic database provides a variety of search options like name, CASRN, molecular weight and measured values of toxicity. With the aid of implemented similarity searches, information about possible biological interactions can be gained. Furthermore, connections to the Protein Data Bank, UniProt and the KEGG database are available, to allow the identification of targets and those pathways, the searched compounds are involved in. This database is available online at: http://bioinformatics.charite.de/supertoxic.

SuperScent--a database of flavors and scents.

Volatiles are efficient mediators of chemical communication acting universally as attractant, repellent or warning signal in all kingdoms of life. Beside this broad impact volatiles have in nature, scents are also widely used in pharmaceutical, food and cosmetic industries, so the identification of new scents is of great industrial interest. Despite this importance as well as the vast number and diversity of volatile compounds, there is currently no comprehensive public database providing information on structure and chemical classification of volatiles. Therefore, the database SuperScent was established to supply users with detailed information on the variety of odor components. The version of the database presented here comprises the 2D/3D structures of approximately 2100 volatiles and around 9200 synonyms as well as physicochemical properties, commercial availability and references. The volatiles are classified according to their origin, functionality and odorant groups. The information was extracted from the literature and web resources. SuperScent offers several search options, e.g. name, Pubchem ID number, species, functional groups, or molecular weight. SuperScent is available online at: http://bioinformatics.charite.de/superscent.

Toxicity versus potency: elucidation of toxicity properties discriminating between toxins, drugs, and natural compounds.

Within our everyday life we are confronted with a variety of toxic substances. A number of these compounds are already used as lead structures for the development of new drugs, but the amount of toxic substances is still a rich resource of new bioactive compounds. During the identification and development of new potential drugs, risk estimation of health hazards is an essential and topical subject in pharmaceutical industry. To face this challenge, an extensive investigation of known toxic compounds is going to be helpful to estimate the toxicity of potential drugs. "Toxicity properties" found during those investigations will also function as a guideline for the toxicological classification of other unknown substances. We have compiled a dataset of approximately 50,000 toxic compounds from literature and web sources. All compounds were classified according to their toxicity. During this study the collection of toxic compounds was investigated extensively regarding their chemical, functional, and structural properties and compared with a dataset of drugs and natural compounds. We were able to identify differences in properties within the toxic compounds as well as in comparison to drugs and natural compounds. These properties include molecular weight, hydrogen bond donors and acceptors, and functional groups which can be regarded as "toxicity properties", i.e. attributes defining toxicity.

Characterization of GD2 peptide mimotope DNA vaccines effective against spontaneous neuroblastoma metastases.

Disialoganglioside GD2 is an established target for immunotherapy in neuroblastoma. We tested the hypothesis that active immunization against the glycolipid GD2 using DNA vaccines encoding for cyclic GD2-mimicking decapeptides (i.e., GD2 mimotopes) is effective against neuroblastoma. For this purpose, two GD2 peptide mimotopes (MA and MD) were selected based on docking experiments to anti-GD2 antibody ch14.18 (binding free energy: -41.23 kJ/mol for MA and -48.06 kJ/mol for MD) and Biacore analysis (K(d) = 12.3 x 10(-5) mol/L for MA and 5.3 x 10(-5) mol/L for MD), showing a higher affinity of MD over MA. These sequences were selected for DNA vaccine design based on pSecTag2-A (pSA) also including a T-cell helper epitope. GD2 mimicry was shown following transfection of CHO-1 cells with pSA-MA and pSA-MD DNA vaccines, with twice-higher signal intensity for cells expressing MD over MA. Finally, these DNA vaccines were tested for induction of tumor protective immunity in a syngeneic neuroblastoma model following oral DNA vaccine delivery with attenuated Salmonella typhimurium (SL 7207). Only mice receiving the DNA vaccines revealed a reduction of spontaneous liver metastases. The highest anti-GD2 humoral immune response and natural killer cell activation was observed in mice immunized with the pSA-MD, a finding consistent with superior calculated binding free energy, dissociation constant, and GD2 mimicry potential for GD2 mimotope MD over MA. In summary, we show that DNA immunization with pSA-MD may provide a useful strategy for active immunization against neuroblastoma.

Design and biological evaluation of photo-switchable inhibitors.

Photo-switchable compounds are becoming increasingly popular for a series of biological applications based on the reversible photo-control of structure and function of biomolecules. Three applications for the usage of BODTCM and hemithioindigo as photo-reactive compounds are described here. The structure of the villin headpiece was modified by replacing a part of the backbone with hemithioindigo, aiming at induction of the folding process by irradiation with a defined wavelength. The E-isomer of BODTCM was applied as potential inhibitor of the 12/15-lipoxygenase (12/15-LOX), which is implicated in the pathogenesis of inflammatory diseases. A required death domain for the binding of proapoptotic proteins (e.g. Bak) to the hydrophobic groove of antiapoptotic proteins is the BH3 helix. Inserting hemithioindigo into this short peptide, stabilization towards proteolytic degradation is achieved. Such photo-reactive compounds might be developed as potential drugs for a great variety of diseases.

SUPERFICIAL--surface mapping of proteins via structure-based peptide library design.

BACKGROUND: The determination of protein surfaces and the detection of binding sites are essential to our understanding of protein-protein interactions. Such binding sites can be characterised as linear and non-linear, the non-linear sites being prevailant. Conventional mapping techniques with arrays of synthetic peptides have limitations with regard to the location of discontinuous or non-linear binding sites of proteins. RESULTS: We present a structure-based approach to the design of peptide libraries that mimic the whole surface or a particular region of a protein. Neighbouring sequence segments are linked by short spacers to conserve local conformation. To this end, we have developed SUPERFICIAL, a program that uses protein structures as input and generates library proposals consisting of linear and non-linear peptides. This process can be influenced by a graphical user interface at different stages, from the surface computation up to the definition of spatial regions. CONCLUSION: Based on 3D structures, SUPERFICIAL may help to negotiate some of the existing limitations, since binding sites consisting of several linear pieces can now be detected.