Strategies for signal amplification in nucleic acid detection

Many aspects of molecular genetics necessitate the detection of nucleic acid sequences. Current approaches involving target amplification (in situ PCR, Primed in situ Labeling, Self-Sustained Sequence Replication, Strand Displacement Amplification), probe amplification (Ligase Chain Reaction, Padlock Probes, Rolling Circle Amplification) and signal amplification (Tyramide Signal Amplification, Branched DNA Amplification) are summarized in the present review, together with their advantages and limitations.     

Methods to identify protein targets of metal-based drugs

Metal-based anticancer agents occupy a distinct chemical space due to their particular coordination geometry and reactivity. Despite the initial DNA-targeting paradigm for this class of compounds, it is now clear that they can also be tuned to target proteins in cells, depending on the metal and ligand scaffold. Since metallodrug discovery is dominated by phenotypic screenings, tailored proteomics strategies were crucial to identify and validate protein targets of several investigative and clinically advanced metal-based drugs. Here, such experimental approaches are discussed, which showed that metallodrugs based on ruthenium, gold, rhenium and even platinum, can selectively and specifically target proteins with clear-cut down-stream effects. Target identification strategies are expected to support significantly the mechanism-driven clinical translation of metal-based drugs.     

Molecular probes for the human adenosine receptors

Adenosine receptors, G protein–coupled receptors (GPCRs) that are activated by the endogenous ligand adenosine, have been considered potential therapeutic targets in several disorders. To date however, only very few adenosine receptor modulators have made it to the market. Increased understanding of these receptors is required to improve the success rate of adenosine receptor drug discovery. To improve our understanding of receptor structure and function, over the past decades, a diverse array of molecular probes has been developed and applied. These probes, including radioactive or fluorescent moieties, have proven invaluable in GPCR research in general. Specifically for adenosine receptors, the development and application of covalent or reversible probes, whether radiolabeled or fluorescent, have been instrumental in the discovery of new chemical entities, the characterization and interrogation of adenosine receptor subtypes, and the study of adenosine receptor behavior in physiological and pathophysiological conditions. This review summarizes these applications, and also serves as an invitation to walk another mile to further improve probe characteristics and develop additional tags that allow the investigation of adenosine receptors and other GPCRs in even finer detail.

     

Determination of Trace Element Stability in Sediments Using Redox Gel Probes: Probe Construction and Theoretical Performance

A simple and inexpensive technique is described that can be used to assess the stability of redox-sensitive compounds in the sediments of wetlands and other shallow water environments. In this method, solid redox-sensitive compounds, such as manganese dioxide (MnO 2 ), are incorporated into agar gels held in rigid plastic holders. One surface of the gel remains exposed along the length of the resulting probe. The probes are pushed vertically into sediments and are left in situ for a period of time (days to weeks), after which they are visually inspected and chemically analyzed. The diffusion of nonreactive solutes (e.g., sulfate) in 2% (wt/vol) agar was unaffected by the presence of immobilized MnO 2 particles. The rate of dissolution of particulate MnO 2 in agar gels in the presence of an external diffusing reductant (L-ascorbic acid) could be quantified by digital analysis of pixel density on gel images. Redox gel probes incubated in the sediment of a wetland built to remove manganese from circumneutral pH coal mine drainage demonstrated different patterns of depth-dependent MnO 2 stability along a 15-m transect. MnO 2 gel probe results were consistent with data obtained using sediment cores and porewater diffusion samplers.     

TaqMan-MGB 探针检测GSTP1外显子5SNP可行性研究

目的：评估TaqMan-MGB探针基因分型方法检测已知SNP的可行性,并与传统的PCR-RFLP方法比较。方法：高通量的TaqMan-MGB探针基因分型方法已被用来检测单核苷酸多态性（SNP）。在321倒样本中,同时用TaqMan-MGB探针基因分型方法和PCR—RFLP方法检测GSTP1外显子5SNP。结果：2种方法所得结果完全一致。野生型（AA）226例（70．4％）,杂合子（AG）92例（28．7％）,纯合突变型3例（O．9％）。结论：TaqMan-MGB探针基因分型方法是一种能快速、高度特异性、高度自动化检测SNP的方法。可用于大规模的基因分型。     

Familial Alzheimer disease presenilin-1 mutations alter the active site conformation of γ-secretase

Presenilin-1 (PS1) is the catalytic subunit of γ-secretase, and mutations in this protein cause familial Alzheimer Disease (FAD). However, little is known about how these mutations affect the active site of γ-secretase. Here, we show that PS1 mutations alter the S2 subsite within the active site of γ-secretase using a multiple photoaffinity probe approach called "photophore walking." Moreover, we developed a unique in vitro assay with a biotinylated recombinant Notch1 substrate and demonstrated that PS1 FAD mutations directly and significantly reduced γ-secretase activity for Notch1 cleavage. Substitution of the Notch Cys-1752 residue, which interacts with the S2 subsite, with Val, Met, or Ile has little effect on wild-type PS1 but leads to more efficient substrates for mutant PS1s. This study indicates that alteration of this S2 subsite plays an important role in determining the activity and specificity of γ-secretase for APP and Notch1 processing, which provides structural basis and insights on how certain PS1 FAD mutations lead to AD pathogenesis.     

Distribution of repetitive DNA sequences in chromosomes of five opisthorchid species (Trematoda, Opisthorchiidae)

Genomes of opisthorchid species are characterized by small size, suggesting a reduced amount of repetitive DNA in their genomes. Distribution of repetitive DNA sequences in the chromosomes of five species of the family Opisthorchiidae (Opisthorchis felineus 2n = 14 (Rivolta, 1884), Opisthorchis viverrini 2n = 12 (Poirier, 1886), Metorchis xanthosomus 2n = 14 (Creplin, 1846), Metorchis bilis 2n = 14 (Braun, 1890), Clonorchis sinensis 2n = 14 (Cobbold, 1875)) was studied with C- and AgNOR-banding, generation of microdissected DNA probes from individual chromosomes and fluorescent in situ hybridization on mitotic and meiotic chromosomes. Small-sized C-bands were discovered in pericentric regions of chromosomes. Ag-NOR staining of opisthorchid chromosomes and FISH with ribosomal DNA probe showed that karyotypes of all studied species were characterized by the only nucleolus organizer region in one of small chromosomes. The generation of DNA probes from chromosomes 1 and 2 of O. felineus and M. xanthosomus was performed with chromosome microdissection followed by DOP-PCR. FISH of obtained microdissected DNA probes on chromosomes of these species revealed chromosome specific DNA repeats in pericentric C-bands. It was also shown that microdissected DNA probes generated from chromosomes could be used as the Whole Chromosome Painting Probes without suppression of repetitive DNA hybridization. Chromosome painting using microdissected chromosome specific DNA probes showed the overall repeat distribution in opisthorchid chromosomes.     

Biological staining: mechanisms and theory

New staining techniques continue to be introduced, and older ones continue to be used and improved. Several factors control specificity, selectivity and visibility of the end product in any procedure using dyes, fluorochromes, inorganic reagents or histochemical reactions applied to sections or similar preparations. Local concentration of the tissue target often determines the intensity of the observed color, as does the fine structure within the object being stained, which may facilitate or impede diffusion of dyes and other reagents. Several contributions to affinity control the specificity of staining. These include electrical forces, which result in accumulation of dye ions in regions of oppositely charged tissue polyions. Weaker short-range attractions (hydrogen bonding, van der Waals forces or hydrophobic bonding, depending on the solvent) hold dyes ions and histochemical end products in contact with their macromolecular substrates. Nonionic forces can also increase visibility of stained sites by causing aggregation of dye molecules. Covalent bonds between dye and tissue result in the strongest binding, such as in methods using Schiff's reagent and possibly also some mordant dyes. The rate at which a reagent gains access to or is removed from targets in a section or other specimen affect what is stained, especially when more then one dye is used, together or sequentially. Rate-controlled staining is greatly influenced by the presence and type of embedding medium, such as a resin, that infiltrates the tissue. The rates of chemical reactions are major determinants of outcome in many histochemical techniques. Selective staining of different organelles within living cells is accomplished mainly with fluorochromes and is controlled by mechanisms different from those that apply to fixed tissues. Quantitative structure-activity relations (QSAR) of such reagents can be derived from such molecular properties as hydrophilic-hydrophobic balance, extent of conjugated bond systems, acid-base properties and ionic charge. The QSAR correlates with staining of endoplasmic reticulum, lysosomes, mitochondria, DNA, or the plasma membranes of living cells.     

Specificity and kinetics of alpha-synuclein binding to model membranes determined with fluorescent excited state intramolecular proton transfer (ESIPT) probe

Parkinson disease is characterized cytopathologically by the deposition in the midbrain of aggregates composed primarily of the presynaptic neuronal protein α-synuclein (AS). Neurotoxicity is currently attributed to oligomeric microaggregates subjected to oxidative modification and promoting mitochondrial and proteasomal dysfunction. Unphysiological binding to membranes of these and other organelles is presumably involved. In this study, we performed a systematic determination of the influence of charge, phase, curvature, defects, and lipid unsaturation on AS binding to model membranes using a new sensitive solvatochromic fluorescent probe. The interaction of AS with vesicular membranes is fast and reversible. The protein dissociates from neutral membranes upon thermal transition to the liquid disordered phase and transfers to vesicles with higher affinity. The binding of AS to neutral and negatively charged membranes occurs by apparently different mechanisms. Interaction with neutral bilayers requires the presence of membrane defects; binding increases with membrane curvature and rigidity and decreases in the presence of cholesterol. The association with negatively charged membranes is much stronger and much less sensitive to membrane curvature, phase, and cholesterol content. The presence of unsaturated lipids increases binding in all cases. These findings provide insight into the relation between membrane physical properties and AS binding affinity and dynamics that presumably define protein localization in vivo and, thereby, the role of AS in the physiopathology of Parkinson disease.     

Chemical probes for tagging mycobacterial lipids

Mycobacteria, which cause tuberculosis and related diseases, possess a diverse set of complex envelope lipids that provide remarkable tolerance to antibiotics and are major virulence factors that drive pathogenesis. Recently, metabolic labeling and bio-orthogonal chemistry have been harnessed to develop chemical probes for tagging specific lipids in live mycobacteria, enabling a range of new basic and translational research avenues. A toolbox of probes has been developed for labeling mycolic acids and their derivatives, including trehalose-, arabinogalactan-, and protein-linked mycolates, as well as newer probes for labeling phthiocerol dimycocerosates (PDIMs) and potentially other envelope lipids. These lipid-centric tools have yielded fresh insights into mycobacterial growth and host interactions, provided new avenues for drug target discovery and characterization, and inspired innovative diagnostic and therapeutic strategies.