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.     

Growth of a single cell

A theory of growth of a cell which takes up nutrients by diffusion or active transport is discussed. The main conclusion is that the volume should grow at least as fast as the third power of the time. Existing experimental evidence is not a conclusive test of the theory, and further experiments to test it are proposed.     

Efficient solid-phase synthesis of a large peptide by a single coupling protocol with a single HPLC purification step

Summary Peptide chain assembly is now routinely performed by the use of automated synthesizers, although purification and characterization of large peptides still requires knowledge and experience. Structural biology has recently become closely involved in molecular recognition studies that often require the analysis of relatively large peptides using high-resolution NMR spectroscopy, for which synthesis of high-quality peptides in 5–10 mg amounts is of prime importance. The present study describes a solid-phase synthesis of a 7 kDa peptide related to the recently characterized ethylene-responsive element binding protein of tobacco, which is the conserved sequence among these proteins. The rapid and efficient preparation was carried out through a single coupling in combination with a single HPLC separation step. Assembly was performed in 63 h. Different coupling chemistries were employed and compared, involving benzotriazol-1-yloxy-tris(pyrrolidino)phosphonium hexafluorophosphate, 1-hydroxy-7-azabenzotriazole and/or the recently introduced reagent,N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphateN-oxide. After each synthesis, purified material was characterized by mass spectrometry, sequencing and enzymatic mapping and shown to contain a high proportion of the desired peptide.     

Efficient solid-phase synthesis of a large peptide by a single coupling protocol with a single HPLC purification step

Peptide chain assembly is now routinely performed by the use of automated synthesizers, although purification and characterization of large peptides still requires knowledge and experience. Structural biology has recently become closely involved in molecular recognition studies that often require the analysis of relatively large peptides using high-resolution NMR spectroscopy, for which synthesis of high-quality peptides in 5–10 mg amounts is of prime importance. The present study describes a solid-phase synthesis of a 7 kDa peptide related to the recently characterized ethylene-responsive element binding protein of tobacco, which is the conserved sequence among these proteins. The rapid and efficient preparation was carried out through a single coupling in combination with a single HPLC separation step. Assembly was performed in 63 h. Different coupling chemistries were employed and compared, involving benzotriazol-1-yloxy-tris(pyrrolidino)phosphonium hexafluorophosphate, 1-hydroxy-7-azabenzotriazole and/or the recently introduced reagent, N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide. After each synthesis, purified material was characterized by mass spectrometry, sequencing and enzymatic mapping and shown to contain a high proportion of the desired peptide.     

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.     

Voltage clamping with a single microelectrode

A technique is described which allows neurons to be voltage clamped with a single microelectrode, and the advantages of this circuit with respect to conventional bridge techniques are discussed. In this circuit, the single micro electrode is rapidly switched from a current passing to a recording mode. The circuitry consists of: (1) an electronic switch; (2) a high impedance, ultralow input capacity amplifier; (3) a sample-and-hold module; (4) conventional voltage clamping circuitry. The closed electronic switch allows current to flow through the electrode. The switch then opens, and the electrode is in a recording mode. The low input capacity of the preamplifier allows the artifact from the current pulse to rapidly abate, after which time the circuit samples the membrane potential. This cycle is repeated at rates up to 10 kHz. The voltage clamping amplifier senses the output of the sample-and-hold module and adjusts the current pulse amplitude to maintain the desired membrane potential. The system was evaluated in Aplysia neurons by inserting two microelectrodes into a cell. One electrode was used to clamp the cell and the other to independently monitor membrane potential at a remote location in the soma.     

Can a single injury cause cancer

In the absence of positive evidence that a single injury can cause cancer, the tendency is growing to award compensation to persons (particularly employees) with cancers alleged to have originated in trauma, even cancers which are generally conceded to be congenital in origin.Experimental attempts to induce cancer through trauma have been unsuccessful or doubtful in result. War-wounded persons, boxers and railroad accident victims have no higher incidence of cancer than other groups.Physicians and others in a position to educate the public should strive to dispel the error that cancer following injury is demonstrably or even probably the cause.     

Modeling electroporation in a single cell

Electroporation uses electric pulses to promote delivery of DNA and drugs into cells. This study presents a model of electroporation in a spherical cell exposed to an electric field. The model determines transmembrane potential, number of pores, and distribution of pore radii as functions of time and position on the cell surface. For a 1-ms, 40 kV/m pulse, electroporation consists of three stages: charging of the cell membrane (0-0.51 micros), creation of pores (0.51-1.43 micros), and evolution of pore radii (1.43 micros to 1 ms). This pulse creates approximately 341,000 pores, of which 97.8% are small ( approximately 1 nm radius) and 2.2% are large. The average radius of large pores is 22.8 +/- 18.7 nm, although some pores grow to 419 nm. The highest pore density occurs on the depolarized and hyperpolarized poles but the largest pores are on the border of the electroporated regions of the cell. Despite their much smaller number, large pores comprise 95.3% of the total pore area and contribute 66% to the increased cell conductance. For stronger pulses, pore area and cell conductance increase, but these increases are due to the creation of small pores; the number and size of large pores do not increase.     

Engineering a novel beta-lactamase by a single point mutation

beta-Lactamases are widespread and efficient bacterial enzymes which play a major role in bacterial resistance to penicillins and cephalosporins. In order to elucidate the role of the residues lying in a conserved loop of the enzymatic cavity of the active-site serine Streptomyces albus G beta-lactamase, modified proteins were produced by oligo-directed mutagenesis. Mutation of Asn116, which lies on one side of the active site cavity pointing to the substrate-binding site, into a serine residue resulted in spectacular modifications of the specificity profile of the enzyme. That replacement yielded an enzyme with a nearly unchanged activity towards good penicillin substrates. In sharp contrast its efficiency in hydrolysing cephalosporins was drastically reduced, the best substrates suffering the largest decrease in the second-order rate constant for serine acylation. In fact that single mutation generated a truly new enzyme behaving exclusively as a penicillinase, a situation which is never encountered to the same degree in any of the numerous naturally occurring variants of class A beta-lactamases.     

Tracking a single pigeon using a shadowing filter algorithm

Miniature GPS devices now allow for measurement of the movement of animals in real time and provide high‐ quality and high‐resolution data. While these new data sets are a great improvement, one still encounters some measurement errors as well as device failures. Moreover, these devices only measure position and require further reconstruction techniques to extract the full dynamical state space with the velocity and acceleration. Direct differentiation of position is generally not adequate. We report on the successful implementation of a shadowing filter algorithm that (1) minimizes measurement errors and (2) reconstructs at the same time the full phase‐space from a position recording of a flying pigeon. This filter is based on a very simple assumption that the pigeon's dynamics are Newtonian. We explore not only how to choose the filter's parameters but also demonstrate its improvements over other techniques and give minimum data requirements. In contrast to competing filters, the shadowing filter's approach has not been widely implemented for practical problems. This article addresses these practicalities and provides a prototype for such application.     

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%).     

Whole genome sequencing of a single Bos taurus animal for single nucleotide polymorphism discovery

Background  

The majority of the 2 million bovine single nucleotide polymorphisms (SNPs) currently available in dbSNP have been identified in a single breed, Hereford cattle, during the bovine genome project. In an attempt to evaluate the variance of a second breed, we have produced a whole genome sequence at low coverage of a single Fleckvieh bull.     

Some strategies for harvesting a single species

Then-stage harvesting strategy of Elizarov and Svirezhev is examined. As a result, some important new features appear. A discussion is presented on whether or not one should harvest a species at one time stage or wait until a later time. The paper is concerned with contributions which are primarily mathematical formulations and results for continuous, as well as discrete time, logistic growth of a single species being harvested. Age class structure is ignored.     

Impotence induced by a single gene mutation

Inbred male mice homozygous for the stubby gene mutation were reported to be sterile nineteen years ago. However, the basis of the sterility has not been identified since testicular steroidogenesis and spermatogenesis are normal. In the present studies, the sexual behavior of stubby mice and their coisogenic, normal siblings was compared quantitatively. The experimental observations provided unequivocal evidence that the phenotypic basis of the sterility of stubby mice is impotency. Stubby mice represent the first animal model for the study of impotence and provide the first evidence of an autosomal gene mutation that has a primary effect on male sexual behavior.