Real-Time Duplex Applications of Loop-Mediated AMPlification (LAMP) by Assimilating Probes

Isothermal nucleic-acid amplification methods such as Loop-Mediated isothermal AMPlification (LAMP) are increasingly appealing alternatives to PCR for use in portable diagnostic system due to the low cost, weight, and power requirements of the instrumentation. As such, interest in developing new probes and other functionality based on the LAMP reaction has been intense. Here, we report on the development of duplexed LAMP assays for pathogen detection using spectrally unique Assimilating Probes. As proof of principle, we used a reaction for Salmonella enterica as a model coupled with a reaction for λ-phage DNA as an internal control, as well as a duplexed assay to sub-type specific quarantine strains of the bacterial wilt pathogen Ralstonia solanacearum. Detection limits for bacterial DNA analyzed in individual reactions was less than 100 genomic equivalents in all cases, and increased by one to two orders of magnitude when reactions were coupled in duplexed formats. Even so, due to the more robust activity of newly available strand-displacing polymerases, the duplexed assays reported here were more powerful than analogous individual reactions reported only a few years ago, and represent a significant advance for incorporation of internal controls to validate assay results in the field.     

Estimating signal strengths in the design of an indoor wireless network

One issue in the design and implementation of a wireless local area network is the selection of access point (AP) locations. Proper AP placement is necessary to provide adequate signal coverage and also to minimize cochannel coverage overlap. The impact of incorrect placement of APs is significant. Placing APs too far apart can lead to gaps in coverage. On the other hand, placing the units too close together leads to excessive cochannel coverage overlap, degrading system performance. Currently, AP placement involves a "trial and error" technique. When a technician tests the effect of moving an AP from one location to another, it is necessary to spend considerable time manually measuring signal strengths in order to determine how this move affects the AP's coverage area. In this letter, we describe a procedure for estimating the coverage areas of relocated APs. The procedure can be used as part of a manual design process or as part of an automated design tool.     

The murine immune response to the male-specific antigen mouse testicular cytochrome c

Male and female A/J mice were examined for their ability to elicit T lymphocyte and antibody (Ab) responses to the male-specific Ag, mouse testicular cytochrome c (Mt cyt). T lymphocytes from both male and female mice primed in vivo responded to the Ag in in vitro proliferation assays, and the dose-response curves were statistically indistinguishable. In addition, similar levels of Ab to Mt cyt were observed in immunized male and female mice. The B cells producing the Ab had switched isotypes to IgG1 and IgG2a, indicating that the self-reactive T helper (Th) cells in male mice were functional. Thus, male mice do not appear to be immunologically tolerant to Mt cyt, at least at the Th and B lymphocyte levels. No evidence for disease was found in male mice primed with Mt cyt. Major histocompatibility complex (MHC) class II-positive antigen-presenting cells are present in the testes and these were shown in vitro to process and present Mt cyt to a T cell hybridoma specific for the synthetic peptide Mt cyt 93-104. However, the hybridoma was not activated in the absence of exogenous Mt cyt 93-104 or Mt cyt, indicating that endogenous Mt cyt is not normally processed in sufficient quantity to effectively load MHC class II molecules with this particular Mt cyt-derived peptide. Notwithstanding any immunologic privilege of the testes, the lack of tolerance to Mt cyt and its failure to elicit an autoimmune disease could extend from the low levels of processed Mt cyt Ag available for T cell recognition. The T cell response elicited by Mt cyt contrasts the lack of response to mouse somatic cytochrome c which differs from Mt cyt at 13 amino acid residues and is expressed in most tissues and at higher levels.     

Patterning of the chick forebrain anlage by the prechordal plate

We analysed the role of the prechordal plate in forebrain development of chick embryos in vivo. After transplantation to uncommitted ectoderm a prechordal plate induces an ectopic, dorsoventrally patterned, forebrain-like vesicle. Grafting laterally under the anterior neural plate causes ventralization of the lateral side of the forebrain, as indicated by a second expression domain of the homeobox gene NKX2.1. Such a lateral ventralization cannot be induced by the secreted factor Sonic Hedgehog alone, as this is only able to distort the ventral forebrain medially. Removal of the prechordal plate does not reduce the rostrocaudal extent of the anterior neural tube, but leads to significant narrowing and cyclopia. Excision of the head process results in the caudal expansion of the NKX2.1 expression in the ventral part of the anterior neural tube, while PAX6 expression in the dorsal part remains unchanged. We suggest that there are three essential steps in early forebrain patterning, which culminate in the ventralization of the forebrain. First, anterior neuralization occurs at the primitive streak stage, when BMP-4-antagonizing factors emanate from the node and spread in a planar fashion to induce anterior neural ectoderm. Second, the anterior translocation of organizer-derived cells shifts the source of neuralizing factors anteriorly, where the relative concentration of BMP-4-antagonists is thus elevated, and the medial part of the prospective forebrain becomes competent to respond to ventralizing factors. Third, the forebrain anlage is ventralized by signals including Sonic Hedgehog, thereby creating a new identity, the prospective hypothalamus, which splits the eye anlage into two lateral domains.     

Kinetic analysis of the coding properties of O6-methylguanine in DNA: the crucial role of the conformation of the phosphodiester bond

Production by N-nitroso compounds of O6-alkylguanine (O6-alkylG) in DNA directs the misincorporation of thymine during DNA replication, leading to G:C to A:T transition mutations, despite the fact that DNA containing O6-alkylG:T base pairs is less stable than that containing O6-alkylG:C pairs. We have examined the kinetics of incorporation by Klenow fragment (KF) of Escherichia coli DNA polymerase I of thymine (T) and of cytosine (C) opposite O6-MeG in the template DNA strand. Both T and C were incorporated opposite O6-MeG much slower than nucleotides forming regular A:T or G:C base pairs. Using various concentrations of dTTP, dCTP, or their phosphorothioate (Sp)-dNTP alpha S analogues, or a mixture of dTTP and dCTP, the progress of incorporation of a single nucleotide in a single catalytic cycle of a preformed KF-DNA complex was measured (pre-steady-state kinetics). The results were consistent with the kinetic scheme (Kuchta, R. D., Benkovic, P., & Benkovic, S. J. (1988) Biochemistry 27, 6716-6725): (1) binding of dNTP to polymerase-DNA; (2) conformational change in polymerase; (3) formation of phosphodiester between the dNTP and the 3'-OH of the primer; (4) conformational change of polymerase; (5) release of pyrophosphate. The results were analyzed mathematically to identify the steps at which the rate constants differ significantly between the incorporation of T and C. The only significant difference was the 5-fold difference in the rates of formation of the phosphodiester bond (for dTTP, kforward = 3.9 s-1 and kback = 1.9 s-1; for dCTP, kforward = 0.7 s-1 and kback = 0.9 s-1). These pre-steady-state progress curves were biphasic with a rapid initial burst followed by an apparently steady-state rise. Deconvolution of these curves gave direct evidence for the importance of the conformational change after polymerization by showing that the curves represented the sum of the rapid accumulation of the product of step 3 followed by the slow conversion of that to the product of step 5 (because of the rapidity of the release of pyrophosphate there was no significant accumulation of the product of step 4). The equilibrium constants for each step suggest that the greatest change in the Gibbs free energy occurs at the conformational change after polymerization and that while the formation of the phosphodiester bond to T is slightly exothermic, that to C is slightly endothermic.(ABSTRACT TRUNCATED AT 400 WORDS)     

Implicit theories and semantic similarities: Reply to Marsh and Groves (1987).

The role of implicit theories in behavioral judgments continues to be a source of debate. In a study exploring the factorial validity of student ratings of instruction, Cadwell and Jenkins (1985) suggested that students' implicit theories of instructor behavior may account for the robust factor structure underlying student ratings. They argued that students rely on the semantic similarity of the rating items, inflating the observed relations among the items. Marsh and Groves cite methodological and conceptual difficulties with the Cadwell and Jenkins study, but most of these difficulties may arise from Marsh and Groves's misconceptions about the role of implicit theories and semantic similarities and, more generally, the cognitive processes underlying judgment tasks. I review these issues and address various points of Marsh and Groves's critique. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Contamination of facial memory through exposure to misleading composite pictures.

In Exp I, 46 male and 51 female undergraduates witnessed a filmed incident and later described the target. Results show that Ss who observed a composite containing misleading information (either incorrect hair or an added moustache) were significantly more likely to misreport hairstyle and the presence of a moustache than those who did not. In Exp II, 210 17–65 yr old Ss observed a filmed incident and were tested for their recall and recognition of the target either immediately or after delays of 2 or 7 days. Significant bias, consistent with the content of the misleading composite, was present for both cued recall and choice of mug shot in a photo spread. The effect did not increase over delay but was greater when the composite was seen just prior to recall than immediately after the incident. Implications concerning interference with the memory of the witness who produced the composite and for other witnesses exposed to the misleading composite are discussed. (33 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

The role of benchmarking for yardstick competition

With the increasing interest in yardstick regulation, there is a need to understand the most appropriate method for realigning tariffs at the outset. Benchmarking is the tool used for such realignment and is therefore a necessary first-step in the implementation of yardstick competition. A number of concerns have been raised about the application of benchmarking, making some practitioners reluctant to move towards yardstick based regimes. We assess five of the key concerns often discussed and find that, in general, these are not as great as perceived. The assessment is based on economic principles and experiences with applying benchmarking to regulated sectors, e.g. in the electricity and water industries in the UK, The Netherlands, Austria and Germany in recent years. The aim is to demonstrate that clarity on the role of benchmarking reduces the concern about its application in different regulatory regimes. We find that benchmarking can be used in regulatory settlements, although the range of possible benchmarking approaches that are appropriate will be small for any individual regulatory question. Benchmarking is feasible as total cost measures and environmental factors are better defined in practice than is commonly appreciated and collusion is unlikely to occur in environments with more than 2 or 3 firms (where shareholders have a role in monitoring and rewarding performance). Furthermore, any concern about companies under-recovering costs is a matter to be determined through the regulatory settlement and does not affect the case for using benchmarking as part of that settlement.     

Metastatic parathyroid carcinoma in the MEN2A syndrome

We report a patient with a metastatic parathyroid carcinoma and medullary carcinoma of the thyroid. This patient represents a variation of the multiple endocrine neoplasia syndrome (MEN) type 2A. There was no evidence of a phaeochromocytoma. The case illustrates the difficulties that may be encountered in localising the source of PTH secretion; the patient underwent four unsuccessful exploratory operations of the neck and mediastinum before further investigations revealed a single metastatic deposit of parathyroid carcinoma involving the first thoracic vertebra. PCR amplification and sequencing of the RET oncogene from the metastatic parathyroid carcinoma and genomic DNA revealed a heterozygous mutation (Cys634Tyr) in exon 11, as has previously been described to occur in MEN 2A. In addition, loss of tumour heterozygosity was demonstrated at loci from chromosomes 1, 2, 3p, 13q and 16p. This represents the first report of a parathyroid carcinoma in a MEN2A patient, in which the multiple allelic deletions are consistent with the generalised losses observed in aggressive tumours.     

Measurement of PLL phase error caused by power supply noise

The measurement of the accumulated phase error of phase-locked loops (PLLs) in microprocessor systems is discussed. A system which creates controlled power supply noise and measures the PLL response is described. Examples of the use of this technique are shown for a PLL used in a 400 MHz microprocessor