Trapping single ions inside single ion channels.

Single Ca++-activated K+ channels from rat muscle plasma membranes are inhibited by Ba++. A single Ba++ entering the channel's conduction pore induces a long-lived blocked state. This study employs Ba++ as a probe of the channel's conduction pathway to show that the channel can be forced to close with a single Ba++ ion inside the pore. A Ba++ ion inside the closed channel is trapped and cannot escape until the channel opens. The results demonstrate that in the channel's closed state, the cytoplasmic side of the conduction pore is obstructed to the passage of ions.     

Quantitative single cell and single molecule proteomics for clinical studies

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A single phantom,a single statistical method for low-contrast detectability assessment

PurposeThe assessment of low-contrast-details is a part of the quality control (QC) program in digital radiology. It generally consists of evaluating the threshold contrast (Cth) detectability details for different-sized inserts, appropriately located in dedicated QC test tools. This work aims to propose a simplified method, based on a statistical model approach for threshold contrast estimation, suitable for different modalities in digital radiology.MethodsA home-made low-contrast phantom, made of a central aluminium insert with a step-wedge, was assembled and tested. The reliability and robustness of the method were investigated for Mammography, Digital Radiography, Fluoroscopy and Angiography. Images were analysed using our dedicated software developed on Matlab®. The Cth is expressed in the same unit (mmAl) for all studied modalities.ResultsThis method allows the collection of Cth information from different modalities and equipment by different vendors, and it could be used to define typical values. Results are summarized in detail. For 0.5 diameter detail, Cth results are in the range of: 0.018–0.023 mmAl for 2D mammography and 0.26–0.34 mmAl DR images. For angiographic images, for 2.5 mm diameter detail, the Cth s median values are 0.55, 0.4, 0.06, 0.12 mmAl for low dose fluoroscopy, coronary fluorography, cerebral and abdominal DSA, respectively.ConclusionsThe statistical method proposed in this study gives a simple approach for Low-Contrast-Details assessment, and the typical values proposed can be implemented in a QA program for digital radiology modalities.     

miRNA相关单核苷酸多态性

miRNA相关单核苷酸多态性(miRNA-related single nucleotide polymorphisms或mirSNP)是可以导致miRNA基因调控功能缺失或紊乱的一类功能型SNP的总称。不论是miRNA靶基因结合位点,还是miRNA基因或miRNA加工基因上的mirSNP,都有可能影响miRNA对靶基因的调控。miRNA基因及miRNA加工基因上的mirSNP主要通过阻碍miRNA的生物合成而发挥功能,而靶基因结合位点上的mirSNP主要通过导致自由能的改变或功能构象的消失,影响miRNA与靶序列结合而丧失其原有的调控功能。mirSNP大多位于人类基因组基因间区和内含子区,与包括肿瘤在内的众多复杂性疾病密切关联。mirSNP不论对于复杂性疾病发病机制研究还是诊疗预后分子标志的确定都具有极其重要的研究价值。     

Biophysics of single molecules

The modern methods of research on biological molecules which have led to the development of a new field of science, biophysics of single molecules, are reviewed. The measurement of the characteristics of single molecules enables one to reveal their individual features, and it is just for this reason that much more information can be obtained from one molecule than from the entire ensemble of molecules. The high sensitivity of the methods considered in detail makes it possible to come close to solving the basic problem of practical importance, namely, the determination of the nucleotide sequence of a single DNA molecule.     

Force unfolding single RNAs

We develop a continue time Monte Carlo algorithm to simulate single RNAs unfolded by a time-dependent external force on the secondary structure level. Two recent unfolding RNA experiments carried out by Bustamante group are mainly investigated. We find that, in contrast to popular two-state assumption about the RNAs free energy landscape along the molecular extension, the molecules used in the experiments do not present apparent energy barriers. The strong cooperative folding and unfolding transitions of the RNAs observed in the experiments and in our simulations arise from the interaction of the molecules and the light trap. In addition, we also investigate the properties of Jarzynski's remarkable equality, whose experimental test has received considerable attention.     

Chromatin under mechanical stress: from single 30 nm fibers to single nucleosomes

About a decade ago, the elastic properties of a single chromatin fiber and, subsequently, those of a single nucleosome started to be explored using optical and magnetic tweezers. These techniques have allowed direct measurements of several essential physical parameters of individual nucleosomes and nucleosomal arrays, including the forces responsible for the maintenance of the structure of both the chromatin fiber and the individual nucleosomes, as well as the mechanism of their unwinding under mechanical stress. Experiments on the assembly of individual chromatin fibers have illustrated the complexity of the process and the key role of certain specific components. Nevertheless a substantial disparity exists in the data reported from various experiments. Chromatin, unlike naked DNA, is a system which is extremely sensitive to environmental conditions, and studies carried out under even slightly different conditions are difficult to compare directly. In this review we summarize the available data and their impact on our knowledge of both nucleosomal structure and the dynamics of nucleosome and chromatin fiber assembly and organization.     

Detection of single nucleotide mutations in wheat using single strand conformation polymorphism gels

We show that single strand conformation polymorphism (SSCP) analysis, using the mutation detection enhancement (MDE^TM) matrix, is efficient at detecting sequence polymorphisms in PCR amplicons. Four independent wheat genomic fragments were amplified from two contrasting templates and sequenced. The allelic fragments were differentiated at 1–6 single nucleotide positions, but MDE-SSCP was able to unequivocally distinguish each allelic pair. The approach is therefore considered a powerful way of identifying single nucleotide polymorphisms (SNPs) without extensive amplicon sequencing.     

Multiplex single nucleotide polymorphisms genotyping using solid-phase single base extension on magnetic nanoparticles

To fulfill the increasing need for large-scale genetic research, we have developed a new solid-phase single base extension (SBE) protocol on magnetic nanoparticles (MNPs) for multiplex SNP detection using adapter polymerase chain reaction (PCR) products as templates. Extension primers were covalently immobilized on the MNPs, and allele-specific extension took place along the stretch of target DNA for one-color ddNTP incorporation. The MNPs with fluorophores were spotted on a glass slide to fabricate a “bead array” to discriminate their genotypes. Eight SNP loci of three DNA samples were interrogated, and the experiment demonstrated that it is an efficient method for large-scale SNP genotyping.     

Detection of a single base substitution in a single cell using the LightCycler

For known mutations, real time polymerase chain reaction followed by melting curve analysis, using hybridization probes, is highly sensitive, rapid and an efficient approach to mutation detection. We have used this approach on the LightCycler for the detection of single base mutations in a single cell, without nested PCR. Hybridization probes were designed for two sequences in the BRCA1 gene containing a single base substitution and deletion, respectively. Polymerase chain reactions of small fragments (100-200 bp) containing the probe sequences were optimized using SYBR Green1, before using hybridization probes. The 5'-probes were 3'-labeled with FITC, whereas the 3'-probes, covering the mutation, were 5'-labeled with LC-Red640 (wild type probes) or LC-Red705 (mutant probes). Dual color detection of wild type and mutant sequences in a single tube was tested on single cells. The reaction mix was prepared in reaction capillaries and a single cell, picked by micromanipulation, was added to this mix. The DNA from the cell is released during the 5-min preheating step of the PCR, using the FastStart hybridization kit (Roche). Reproducible results were obtained, without the need of nested PCR. The technique is useful for microdissected tumors and, with other genes, has great potential for pre-implantation diagnosis in IVF and analysis of residual disease in cancer.     

Characterization of genomic single nucleotide polymorphism via colorimetric detection using a single gold nanoprobe

Identification of specific nucleic acid sequences mediated by gold nanoparticles derivatized thiol-modified oligonucleotides (Au–nanoprobes) has been proven to be a useful tool in molecular diagnostics. Here, we demonstrate that, on optimization, detection may be simplified via the use of a single Au–nanoprobe to detect a single nucleotide polymorphism (SNP) in homo- or heterozygote condition. We validated this non-cross-linking approach through the analysis of 20 clinical samples using a single specific Au–nanoprobe for an SNP in the FTO (fat mass and obesity-associated) gene against direct DNA sequencing. Sensitivity, specificity, and limit of detection (LOD) were determined, and statistical differences were calculated by one-way analysis of variance (ANOVA) and a post hoc Tukey’s test to ascertain whether there were any differences between Au–nanoprobe genotyped groups. For the first time, we show that the use of a single Au–nanoprobe can detect SNP for each genetic status (wild type, heterozygous, or mutant) with high degrees of sensitivity (87.50%) and specificity (91.67%).