Peptide-based probes for cancer imaging

Receptors for regulatory peptides are overexpressed in a variety of human cancers. They represent the molecular basis for in vivo imaging with radiolabeled peptide probes. Somatostatin-derived tracers, designed to image the sst2-overexpressing neuroendocrine tumors, have enjoyed almost 2 decades of successful development and extensive clinical applications. More recent developments include second- and third-generation somatostatin analogs, with a broader receptor subtype profile or with antagonistic properties. Emerging tracers for other peptide receptors, including cholecystokinin/gastrin and GLP-1 analogs for neuroendocrine tumors, bombesin and neuropeptide-Y analogs for prostate or breast cancers, or Arg-Gly-Asp peptides for neoangiogenesis labeling, are also in current development. Application fields include both SPECT/CT and PET/CT.

Ligation dependent allele specific quantification (LASQ) of CFTR cDNA on the LightCycler using MLPA hybridization probes

BACKGROUND: As for Cystic Fibrosis (CF) and many other hereditary diseases there is still a lack in understanding the relationship between genetic (e.g. allelic) and phenotypic diversity. Therefore methods which allow fine quantification of allelic proportions of mRNA transcripts are of high importance. METHODS: We used either genomic DNA (gDNA) or total RNA extracted from nasal cells as starting nucleic acid template for our assay. The subjects included in this study were 9 CF patients compound heterozygous for the F508del mutation and each one F508del homozygous and one wild type homozygous respectively. We established a novel ligation based quantification method which allows fine quantification of the allelic proportions of ss and ds CFTR cDNA. To verify reliability and accuracy of this novel assay we compared it with semiquantitative fluorescent PCR (SQF-PCR). RESULTS: We established a novel assay for allele specific quantification of gene expression which combines the benefits of the specificity of the ligation reaction and the accuracy of quantitative real-time PCR. The comparison with SQF-PCR clearly demonstrates that LASQ allows fine quantification of allelic proportions. CONCLUSION: This assay represents an alternative to other fine quantitative methods such as ARMS PCR and Pyrosequencing.

DESIGN AND DEVELOPMENT OF BODIPY-BASED FLUORESCENT PROBES FOR SENSING AND IMAGING OF CYANIDE, Zn (II) IONS, LYSOSOMAL pH AND CANCER CELLS

BODIPY (4,4-Difluoro-3a,4a-diaza-s-indacene) dyes have gained lots of attention in application of fluorescence sensing and imaging in recent years because they possess many distinctive and desirable properties such as high extinction coefficient, narrow absorption and emission bands, high quantum yield and low photobleaching effect. However, most of BODIPY-based fluorescent probes have very poor solubilities in aqueous solution, emit less than 650 nm fluorescence that can cause cell and tissue photodamages compared with bio-desirable near infrared (650-900 nm) light. These undesirable properties extremely limit the applications of BODIPY-based fluorescent probes in sensing and imaging applications. In order to overcome these drawbacks, we have developed a very effective strategy to prepare a series of neutral highly water- soluble BODIPY dyes by enhancing the water solubilities of BODIPY dyes via incorporation of tri(ethylene glycol)methyl ether (TEG) and branched oligo(ethylene glycol)methyl ether (BEG) residues onto BODIPY dyes at 1,7-, 2,6-, 3,5-, 4- and meso- positions. We also have effectively tuned absorptions and emissions of BOIDPY dyes to red, deep red and near infrared regions via significant extension of π-conjugation of BODIPY dyes by condensation reactions of aromatic aldehydes with 2,6-diformyl BODIPY dyes at 1,3,5,7-positions. Based on the foundation that we built for enhancing water solubility and tuning wavelength, we have designed and developed a series of water-soluble, BODIPY-based fluorescent probes for sensitive and selective sensing and imaging of cyanide, Zn (II) ions, lysosomal pH and cancer cells. We have developed three BODIPY-based fluorescent probes for sensing of cyanide ions by incorporating indolium moieties onto the 6-position of TEG- or BEG-modified BOIDPY dyes. Two of them are highly water-soluble. These fluorescent probes showed selective and fast ratiometric fluorescent responses to cyanide ions with a dramatic fluorescence color change from red to green accompanying a significant increase in fluorescent intensity. The detection limit was measured as 0.5 mM of cyanide ions. We also have prepared three highly water-soluble fluorescent probes for sensing of Zn (II) ions by introducing dipicoylamine (DPA, Zn ion chelator) onto 2- and/or 6-positions of BEG-modified BODIPY dyes. These probes showed selective and sensitive responses to Zn (II) ion in the range from 0.5 mM to 24 mM in aqueous solution at pH 7.0. Particularly, one of the probes displayed ratiometric responses to Zn (II) ions with fluorescence quenching at 661 nm and fluorescence enhancement at 521 nm. This probe has been successfully applied to the detection of intracellular Zn (II) ions inside the living cells. Then, we have further developed three acidotropic, near infrared emissive BODIPY- based fluorescent probes for detection of lysosomal pH by incorporating piperazine moiety at 3,5-positions of TEG- or BEG-modified BODIPY dyes as parts of conjugation. The probes have low auto-fluorescence at physiological neutral condition while their fluorescence intensities will significant increase at 715 nm when pH shift to acidic condition. These three probes have been successfully applied to the in vitro imaging of lysosomes inside two types of living cells. At the end, we have synthesized one water- soluble, near infrared emissive cancer cell targetable BODIPY-based fluorescent polymer bearing cancer homing peptide (cRGD) residues for cancer cell imaging applications. This polymer exhibited excellent water-solubility, near infrared emission (712 nm), good biocompatibility. It also showed low nonspecific interactions to normal endothelial cells and can effectively detect breast tumor cells.

Null-wave giant gravitons from thermal spinning brane probes

We construct and analyze thermal spinning giant gravitons in type II/M-theory based on spherically wrapped black branes, using the method of thermal probe branes originating from the blackfold approach. These solutions generalize in different directions recent work in which the case of thermal (non-spinning) D3-brane giant gravitons was considered, and reveal a rich phase structure with various new properties. First of all, we extend the construction to M-theory, by constructing thermal giant graviton solutions using spherically wrapped M2- and M5-branes. More importantly, we switch on new quantum numbers, namely internal spins on the sphere, which are not present in the usual extremal limit for which the brane world volume stress tensor is Lorentz invariant. We examine the effect of this new type of excitation and in particular analyze the physical quantities in various regimes, including that of small temperatures as well as low/high spin. As a byproduct we find new stationary dipole-charged black hole solutions in AdS m × S n backgrounds of type II/M-theory. We finally show, via a double scaling extremal limit, that our spinning thermal giant graviton solutions lead to a novel null-wave zero-temperature giant graviton solution with a BPS spectrum, which does not have an analogue in terms of the conventional weakly coupled world volume theory.

Measurements of a water potential and water content in unsaturated crystalline rock

A water desaturation zone develops around a tunnel in water-saturated rock when the evaporative water loss at the rock surface is larger than the water flow from the surrounding saturated region of restricted permeability. We describe the methods with which such water desaturation processes in rock materials can be quantified. The water retention characteristic theta(psi) of crystalline rock samples was determined with a pressure membrane apparatus. The negative water potential, identical to the capillary pressure, psi, below the tensiometric range (psi < -0.1 MPa) can be measured with thermocouple psychrometers (TP), and the volumetric water contents, theta, by means of time domain reflectometry (TDR). These standard methods were adapted for measuring the water status in a macroscopically unfissured granodiorite with a total porosity of approximately 0.01. The measured water retention curve of granodiorite samples from the Grimsel test site (central Switzerland) exhibits a shape which is typical for bimodal pore size distributions. The measured bimodality is probably an artifact of a large surface ratio of solid/voids. The thermocouples were installed without a metallic screen using the cavity drilled into the granodiorite as a measuring chamber. The water potentials observed in a cylindrical granodiorite monolith ranged between -0.1 and -3.0 MPa; those near the wall in a ventilated tunnel between -0.1 and -2.2 MPa. Two types of three-rod TDR Probes were used, one as a depth probe inserted into the rock, the other as a surface probe using three copper stripes attached to the surface for detecting water content changes in the rock-to-air boundary. The TDR signal was smoothed with a low-pass filter, and the signal length determined based on the first derivative of the trace. Despite the low porosity of crystalline rock these standard methods are applicable to describe the unsaturated zone in solid rock and may also be used in other consolidated materials such as concrete.

Synthesis and Characterization of Photoaffinity Probes that Target the 5-HT3 Receptor

Abstract: The 5-HT3 receptor is one of several ion channels responsible for the transmission of nerve impulses in the peripheral and central nervous systems. Until now, it has been difficult to characterize transmembrane receptors with classical structural biology approaches like X-ray crystallography. The use of photoaffinity probes is an alternative approach to identify regions in the protein where small molecules bind. To this end, we present two photoaffinity probes based on granisetron, a well known antagonist of the 5-HT3 receptor. These new probes show nanomolar binding affinity for the orthosteric binding site. In addition, we investigated their reactivity using irradiation experiments.

Synthesis and testing of photoaffinity probes for the site-selectivechemical modifications of the 5-HT3 receptor

The 5-HT3 receptor (5-HT3R) is an important ion channel responsible for the transmission of nerve impulses in the central nervous system.1 It is difficult to characterize transmembrane dynamic receptors with classical structural biology approaches like crystallization and x-ray. The use of photoaffinity probes is an alternative approach to identify regions in the protein that are important for the binding of small molecules. Therefore we synthesized a small library of photoaffinity probes by conjugating photophores via various linkers to granisetron which is a known antagonist of the 5-HT3R. We were able to obtain several compounds with diverse linker lengths and different photolabile moieties that show nanomolar binding affinities for the orthosteric binding site. Furthermore we established a stable h5-HT3R expressing cell line and a purification protocol to yield the receptor in a high purity. Currently we are investigating the photo crosslinking of these ligands with the 5-HT3R.

Synthesis and testing of photoaffinity probes for the site-selective chemical modifications of the 5-HT3 receptor

The 5-HT3 receptor (5-HT3R) is an important ion channel responsible for the transmission of nerve impulses in the central nervous system.[1] It is difficult to characterize transmembrane dynamic receptors with classical structural biology approaches like crystallization and x-ray. The use of photoaffinity probes is an alternative approach to identify regions in the protein that are important for the binding of small molecules. Therefore we synthesized a small library of photoaffinity probes by conjugating photolabile building blocks via various linkers to granisetron which is a known antagonist of the 5-HT3R. We were able to obtain several compounds with diverse linker lengths and different photo-labile moieties that show nanomolar binding affinities for the orthosteric binding site. Further on we developed a stable 5-HT3R overexpressing cell line and a purification method to yield the receptor in a high purity. Currently we are investigating crosslinking experiments and subsequent MS – analysis.

Synthesis and testing of photoaffinity probes for the site-selectivechemical modifications of the 5-HT3 receptor

The 5-HT3 receptor (5-HT3R) is an important ion channel responsible for the transmission of nerve impulses in the central nervous system.1 It is difficult to characterize transmembrane dynamic receptors with classical structural biology approaches like crystallization and x-ray. The use of photoaffinity probes is an alternative approach to identify regions in the protein that are important for the binding of small molecules. Therefore we synthesized a small library of photoaffinity probes by conjugating photophores via various linkers to granisetron which is a known antagonist of the 5-HT3R. We were able to obtain several compounds with diverse linker lengths and different photolabile moieties that show nanomolar binding affinities for the orthosteric binding site. Furthermore we established a stable h5-HT3R expressing cell line and a purification protocol to yield the receptor in a high purity. Currently we are investigating the photo crosslinking of these ligands with the 5-HT3R.

Synthesis and testing of photoaffinity probes for the site-selective chemical modifications of the 5-HT3 receptor

The 5-HT3 receptor (5-HT3R) is an important ion channel responsible for the transmission of nerve impulses in the central nervous system.1 It is difficult to characterize transmembrane dynamic receptors with classical structural biology approaches like crystallization and x-ray. The use of photoaffinity probes is an alternative approach to identify regions in the protein that are important for the binding of small molecules. Therefore we synthesized a small library of photoaffinity probes by conjugating photophores via various linkers to granisetron which is a known antagonist of the 5-HT3R. We were able to obtain several compounds with diverse linker lengths and different photolabile moieties that show nanomolar binding affinities for the orthosteric binding site. Furthermore we established a stable h5-HT3R expressing cell line and a purification protocol to yield the receptor in a high purity. Currently we are investigating the photo crosslinking of these ligands with the 5-HT3R.

Black probes of Schrödinger spacetimes

We consider black probes of Anti-de Sitter and Schrödinger spacetimes embedded in string theory and M-theory and construct perturbatively new black hole geometries. We begin by reviewing black string configurations in Anti-de Sitter dual to finite temperature Wilson loops in the deconfined phase of the gauge theory and generalise the construction to the confined phase. We then consider black strings in thermal Schrödinger, obtained via a null Melvin twist of the extremal D3-brane, and construct three distinct types of black string configurations with spacelike as well as lightlike separated boundary endpoints. One of these configurations interpolates between the Wilson loop operators, with bulk duals defined in Anti-de Sitter and another class of Wilson loop operators, with bulk duals defined in Schrödinger. The case of black membranes with boundary endpoints on the M5-brane dual to Wilson surfaces in the gauge theory is analysed in detail. Four types of black membranes, ending on the null Melvin twist of the extremal M5-brane exhibiting the Schrödinger symmetry group, are then constructed. We highlight the differences between Anti-de Sitter and Schrödinger backgrounds and make some comments on the properties of the corresponding dual gauge theories.