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Transforming growth factor-[beta] (TGF-[beta]) elicits its signals through two transmembrane serine/threonine kinase receptors, type II (T[beta]RII) and type I receptors. It is generally believed that the initial receptor dimerization is an essential event for receptor activation. However, previous studies suggested that TGF-[beta] signals by binding to the preexisting T[beta]RII homodimer. Here, using single molecule microscopy to image green fluorescent protein (GFP)-labeled T[beta]RII on the living cell surface, we demonstrated that the receptor could exist as monomers at the low expression level in resting cells and dimerize upon TGF-[beta] stimulation. This work reveals a model in which the activation of serine-threonine kinase receptors is also accomplished via dimerization of monomers, suggesting that receptor dimerization is a general mechanism for ligand-induced receptor activation. serine/threonine kinase receptor | subunit stoichiometry

Liver cancer is the third most deadly cancers in the world. Unfortunately, there is no effective treatment. One of the major problems is that most cancers are diagnosed in the later stage, when surgical resection is not feasible. Thus, accurate early diagnosis would significantly improve the clinical outcome of liver cancer. Currently, there are no effective molecular probes to recognize biomarkers that are specific for liver cancer. The objective of our current study is to identify liver cancer cell-specific molecular probes that could be used for liver cancer recognition and diagnosis. We applied a newly developed cell-SELEX (Systematic Evolution of Ligands by EXponential enrichment) method for the generation of molecular probes for specific recognition of liver cancer cells. The cell-SELEX uses whole live cells as targets to select aptamers (designed DNA/RNA) for cell recognition. In generating aptamers for liver cancer recognition, two liver cell lines were used: a liver cancer cell line BNL 1ME A.7R.1 (MEAR) and a noncancer cell line, BNL CL.2 (BNL). Both cell lines were originally derived from Balb/cJ mice. Through multiple rounds of selection using BNL as a control, we have identified a panel of aptamers that specifically recognize the cancer cell line MEAR with [K.sub.d] in the nanomolar range. We have also demonstrated that some of the selective aptamers could specifically bind liver cancer cells in a mouse model. There are two major new results (compared with our reported cell-SELEX methodology) in addition to the generation of aptamers specifically for liver cancer. The first one is that our current study demonstrates that cell-based aptamer selection can select specific aptamers for multiple cell lines, even for two cell lines with minor differences (MEAR cell is derived from BNL by chemical inducement); and the second result is that cell-SELEX can be used for adhesive cells and thus open the door for solid tumor selection and investigation. The newly generated cancer-specific aptamers hold great promise as molecular probes for cancer early diagnosis and basic mechanism studies.

To popularize the efficient application of the passenger flow simulation technology in urban rail transit transfer stations, and make the simulation evaluation systematic, comprehensive and reasonable, a passenger flow simulation evaluation index system of transfer stations was constructed, based on the characteristics of transfer stations. Then the calculation method, evaluation scope and evaluation standard of each evaluation index were proposed. Finally, through Anhuaqiao station of Beijing urban rail transit, the evaluation index system and evaluation standards were verified feasible, comprehensive and effective.

With the increasing applications of nucleic acid aptamers as a new class of molecular recognition probes in bioanalysis and biosensor development, the development of general and simple signaling strategies to transduce aptamer-target binding events to detectable signals is demanding. We have developed a new signaling method based on aptamers and a DNA molecular light switching complex, [[[Ru(phen).sub.2](dppz)].sup.2+], for sensitive protein detection. In this work, we have demonstrated the applicability of this signaling mechanism to small-molecule detection using ATP as a model target. Our results have shown that upon ATP binding to the folded aptamer where [[[Ru(phen).sub.2](dppz)].sup.2+] intercalated, the conformational change or distortion of the aptamer is large enough to cause a significant luminescence change of [[[Ru(phen).sub.2](dppz)].sup.2+]. By monitoring the ATP-dependent luminescence intensity change, we have achieved ATP detection with high selectivity and high sensitivity down to 1 nM in homogeneous solution. The method is very simple without the needs for covalently labeling aptamers or using costly enzymes and multistep analysis as other reported fluorescence/luminescence assays for ATP. The successful detection of ATP indicates that using the signaling aptamers with [[[Ru(phen).sub.2](dppz)].sup.2+] is expected to be a general method for aptamer-based target detection.

A novel method of signaling aptamer/protein binding for aptamer-based protein detection has been developed using a molecular light switch complex, [[Ru(phen).sub.2-]-[(dppz)].sub.2+]. The method takes advantage of the sensitive luminescence signal change of [[Ru(phen).sub.2][(dppz)].sup.2+] intercalating to the aptamer upon protein/aptamer binding. A 37-nt DNA aptamer against immunoglobulin E (IgE) was first tested as a model system. The luminescence of the [[Ru(phen).sub.2][(dppz)].sup.2+]/IgE aptamer decreased with the increase of IgE. By monitoring the luminescence change, we were able to detect the binding events between the aptamer and IgE for IgE quantitation in homogeneous solutions as well as in serum. The assay was highly selective and sensitive with a detection limit of 100 pM for IgE. This new method is very simple and without the need for the covalent coupling of fluorophores to aptamers. The generalizability of the method was demonstrated by the direct detection of two other proteins, oncoprotein platelet derived growth factor-BB (PDGF-BB) using its DNA aptamer and [alpha]-thrombin using its RNA aptamer. This new approach is expected to promote the exploitation of aptamer-based biosensors for protein assays in biochemical and biomedical studies.

Aptamers are a new class of synthetic DNA/RNA oligonucleotides generated from in vitro selection to selectively bind with various molecules. Due to their molecular recognition capability for proteins, aptamers are becoming promising reagents in protein detection and new drug development. In this study, the specific interaction between the protein immunoglobulin E (IgE) and its 37-nt aptamer has been measured directly by atomic force microscopy. The single-molecule unbinding force between IgE and the aptamer is determined using the Poisson statistical method. The individual unbinding force between IgE and its monoclonal antibody has also been obtained and compared to that between IgE and the aptamer. The results reveal the high affinity of the aptamer to protein, which could match or even surpass that of the antibody to its antigen.

Monitoring proteins in real time and in homogeneous solution has always been a difficult task. We have applied a fluorophore-labeled molecular probe based on a high-affinity platelet-derived growth factor (PDGF) aptamer for the ultrasensitive detection of PDGF in homogeneous solutions. The aptamer is labeled with fluorescein to specifically bind with the PDGF protein. Fluorescence anisotropy is used for the real-time monitoring of the binding between the aptamer and the protein. When the labeled aptamer is bound with its target protein, the rotational motion of the fluorophore attached to the complex becomes much slower because of an increased molecular weight after binding, resulting in a significant fluorescence anisotropy change. Using the anisotropy change, we are able to detect the binding events between the aptamer and the protein in real time and in homogeneous solutions (detection without separation). This assay is highly selective and ultrasensitive. It can detect PDGF in the subnanomolar range. The new method for protein detection is simple and inherits all of the advantages of molecular aptamers. Efficient oncoprotein detection using aptamer-based fluorescence anisotropy measurement will find wide applications in protein monitoring, in cancer diagnosis as well as other studies in which protein analysis is important.

The interactions between two key macromolecular species, nucleic acids and proteins, control many important biological processes. There have been limited effective methodologies to study these interactions in real time. In this work, we have applied a newly developed molecular beacon (MB) DNA probe for the analysis of an enzyme, lactate dehydrogenase (LDH), and for the investigation of its properties of binding with single-stranded DNA. Moleoalar beacons are single-stranded oligonucleotide probes designed to report the presence of specific complementary nucleic acids by fluorescence detection. The interaction between LDH and MB has resulted in a significant fluorescence signal enhancement, which is used for the elucidation of MB/LDH binding properties. The processes of binding between MB and different isoenzymes of LDH have been studied. The results show that the stoichiometry of LDH-5/MB binding is 1:1, and the binding constant is 1.9 x 10(super -7) M(super -1). We have also studied salt effects, binding sites, temperature effects, pH effects, and the binding specificities for different isoenzymes. Our results demonstrate that MB can be effectively used for sensitive protein quantitation and for efficient protein - DNA interaction studies. MB has a signal transduction mechanism built within the molecule and can thus be used for the development of rapid protein assays and for real-time measurements.

We have developed a new fluorescent method for single-molecule detection (SMD) and imaging using an optical fiber probe. The fluorophores were excited by the evanescent wave field produced on the core surface of the optical fiber. This was achieved by exposing a section of the core of the optical fiber probe to the fluorophore solution. Both cylindrical and square optical fiber probes were used for SMD. The fluorescent signals were detected by an intensified charge-coupled device. Single rhodamine 6G molecules have been detected. The number of rhodamine 6G molecules imaged by the optical fiber probe showed an excellent linear relationship with the concentrations of the fluorophores. The SMD scheme was also applied to the imaging of biomolecules, such as molecular beacon DNA molecules, labeled with tetramethylrhodamine. Our results have shown that using an optical fiber is an easy yet effective approach to SMD. It represents a simpler fluorescent method for the detection of single-molecules in solution and at an interface.