DNA nanoswitch as a biosensor

We present a new type of DNA switch, based on the Holliday junction, that uses a combination of binding and conformational switching to enable specific label-free detection of DNA and RNA. We show that a single RNA oligonucleotide species can be detected in a complex mixture of extracted cellular RNA and demonstrate that by exploiting different aspects of the switch characteristics we can achieve 30-fold discrimination between single-nucleotide mismatches in a DNA oligonucleotide.     

Fluorescence absorbance inner-filter decomposition: the role of emission shape on estimates of free Ca(2+) using Rhod-2

A method for decomposing complex emission spectra by correcting for known inner-filter effects is described. This approach builds on previous work using a linear combination of model emission spectra and combines the known absorption characteristics of the system to fit the composite emission spectrum. Rhod-2, which has a small Stokes shift and significant self-absorption, was used as the model system. By adding the absorption characteristics of Rhod-2 to the model, the degree of fit was significantly improved, thus minimizing residuals, and accurately predicted the spectral shape changes with increasing concentration, [Rhod-2]. More complex studies were conducted with Rhod-2 in isolated cardiac mitochondria with multiple emission and absorption elements. By including known absorbances to the spectral decomposition, the overall precision increased almost four fold. Moreover, this approach eliminated the significant [Rhod-2] dependence on the apparent K(50) and therefore improved the accuracy of free [Ca(2+)] calculations. These data demonstrate that secondary inner-filter correction can significantly improve spectral decomposition of complex emission spectra, which are used in a variety of biological applications.     

Temperature and energy performance of refrigerated retail display and commercial catering cabinets under test conditions

An analysis of the performance of well freezers, chest freezers, frozen and chilled door cabinets (solid or glass door) and open fronted chilled cabinets under EN441 test conditions demonstrated that maximum temperatures in cabinets were generally in the most exposed (to ambient) areas and that minimum temperatures were located in the least exposed areas. Detailed positions of maximum and minimum temperature varied between cabinet types. In chest freezers 95% of the maximum temperature positions were located in the top layer and 95% of the minimum temperature positions were located in the middle layer of the cabinets. In full door frozen cabinets the maximum temperature position was in the majority of cases on the top shelf (64%) with most maximum packs being at the front of the top shelf (53%). In the chilled full door cabinets 94% of the maximum temperature packs were situated at the front of the cabinet. In open fronted cabinets the majority of maximum temperature packs (97%) were located at the front of the cabinet, the largest number (60%) being at the front of the base of the cabinet. In well cabinets the majority of maximum temperature packs (81%) were located in the top layer of the cabinet and the majority (91%) of minimum temperature packs were located in the bottom of the cabinet.Large differences in energy consumed by cabinets of similar size and temperature performance were found indicating that large reductions in energy and CO₂ emissions could be achieved by selection of the most efficient cabinets.     

Performance of the radphi detector and trigger in a high rate tagged photon beam

We describe the design and operation of a detector system for measuring all-photon decays of mesons photoproduced in a tagged photon beam with energies between 4.3 and 5.4 GeV and a flux of 5×10⁷ tagged photons per second. Photons from meson decays were detected with a lead-glass calorimeter with an energy resolution of 11% at 1 GeV. Various veto and trigger components were also present. Final states with as many as six photons were successfully detected and reconstructed.     

Chemical introduction of reactive thiols into a viral nanoscaffold: a method that avoids virus aggregation

The use of viral nanoparticles (VNPs) as building blocks for material fabrication has received particular attention in recent years. In earlier studies we showed the applicability of native gel electrophoresis in an agarose matrix as a useful method for the characterization of chemically modified VNPs. Here, we extend these studies and analyze the observed band pattern of intact Cowpea mosaic virus (CPMV) VNPs in agarose gels and show the applicability of native agarose gels for monitoring interparticle linkage of thiol-containing CPMV mutant particles. In addition, we report a protocol that allows the introduction of acetate-protected thiols to CPMV by means of a chemical reaction (rather than genetic modification). The advantage of this approach is that, by incorporating protected thiol groups, the formation of disulfide bonds leading to interparticle linkage is prevented. The resulting thiol-modified CPMV-SH(n) particles are stable, and following deprotection, the introduced thiols are reactive and can be labeled with thiol-selective reagents. They therefore provide a useful additional building block in the CPMV toolbox.     

Molecular plasmonics with tunable exciton-plasmon coupling strength in J-aggregate hybridized Au nanorod assemblies

Controlling coherent electromagnetic interactions in molecular systems is a problem of both fundamental interest and important applicative potential in the development of photonic and opto-electronic devices. The strength of these interactions determines both the absorption and emission properties of molecules coupled to nanostructures, effectively governing the optical properties of such a composite metamaterial. Here we report on the observation of strong coupling between a plasmon supported by an assembly of oriented gold nanorods (ANR) and a molecular exciton. We show that the coupling is easily engineered and is deterministic as both spatial and spectral overlap between the plasmonic structure and molecular aggregates are controlled. We think that these results in conjunction with the flexible geometry of the ANR are of potential significance to the development of plasmonic molecular devices.     

Progesterone can block transmission of the estradiol-induced signal for luteinizing hormone surge generation during a specific period of time immediately after activation of the gonadotropin-releasing hormone surge-generating system

The preovulatory GnRH/LH surge in the ewe is stimulated by a rise in the circulating estradiol concentration that occurs in conjunction with preovulatory ovarian follicle development. In the presence of high levels of progesterone, such as during the luteal phase of the estrous/menstrual cycle, the stimulatory effects of elevated estradiol on GnRH/LH secretion are blocked. Recent work in the ewe has shown that a relatively short period of estradiol exposure can stimulate a GnRH/LH surge that begins after estrogenic support has been removed. This result suggests that surge generation is characterized by an estradiol-dependent period (during which the signal is read) and an estradiol-independent period (during which a cascade of neuronal events transmits the stimulatory signal to the GnRH neurosecretory system, which releases a surge of GnRH). In this series of studies, we addressed the hypothesis that progesterone can block transmission of the stimulatory estradiol signal after it has been read. Nine ovariectomized ewes were run through repeated artificial estrous cycles by sequential addition and removal of exogenous steroids. In study one, ewes received three treatments in a randomized cross-over design. Exposure to a follicular phase estradiol concentration for 10 h (positive control treatment) stimulated an LH surge in all ewes, as determined in hourly jugular blood samples. Maintenance of luteal phase progesterone concentrations throughout the artificial follicular phase (2 x CIDR-G devices, negative control) blocked the stimulatory effects of a 10-h estradiol signal, and no ewes that received this treatment expressed an LH surge. In the experimental group, exposure to luteal phase levels of progesterone, during the period after the surge generating system had been activated by estradiol, blocked the LH surge in six of nine ewes. This result demonstrates that progesterone can block the surge, even when applied after the surge-generating system has been activated and, therefore, that it inhibits either the transmission of the estradiol signal and/or the release of the GnRH/LH surge. In study 2, we assessed whether sensitivity to the inhibitory effects of progesterone was confined to a specific stage of the transmission of the estradiol signal. Eight ewes were exposed to four treatments, over successive artificial estrous cycles. Positive and negative controls were similar to those described in Study 1, except the duration of the stimulatory estradiol signal was reduced to 8 h. The two experimental groups consisted of an EARLY P (progesterone) treatment, in which progesterone was given from hours 8-13 after estradiol insertion (immediately after estradiol removal), and a LATE P treatment, in which progesterone was given from hours 13-18 (immediately before LH surge secretion). As expected, LH surges were stimulated and blocked, in response to the positive and negative controls, respectively. Whereas the EARLY P treatment blocked the LH surge in seven of eight ewes, the LATE P treatment was only successful in inhibiting a surge in one of eight animals. This result demonstrates that progesterone can block the estradiol-induced surge-generating signal soon after the onset of signal transmission (immediately after estradiol removal) but not during the later stages of signal transmission (at the time of GnRH/LH surge onset).     

Analysis of amino and carboxy terminal GLUT-4 targeting motifs in 3T3-L1 adipocytes using an endosomal ablation technique

The targeting of the insulin-responsive glucose transporter, GLUT-4, to an intracellular compartment in adipocytes and muscle is one of the key features responsible for the unique insulin sensitivity of this transporter. Through expression of epitope-tagged GLUT-4 mutants in 3T3-L1 adipocytes, two motifs have been identified as playing a central role in GLUT-4 targeting: FQQI in the amino terminus and a di-leucine motif in the carboxy terminus. The goal of this study was to explore the role of these targeting motifs in the intracellular sorting of GLUT-4 using the Tf-HRP ablation technique. This technique provides a quantitative assessment of the amount of GLUT-4 located in recycling endosomes. In basal adipocytes, we find that approximately 40% of GLUT-4 is ablated following Tf-HRP loading. In contrast, here we demonstrate that the intracellular pool of a mutant in which F5 was mutated to A5 is localized to the recycling endosomal pathway, suggesting that the amino terminal FQQI motif functions in trafficking GLUT-4 from early endosomes. In contrast, GLUT-4 in which L489L490 was mutated to A489A490 was localized predominantly to a nonablated compartment. These data imply a role for the di-leucine motif in sorting from a separate intracellular compartment, such as the TGN. Our findings are discussed within the context of a revised multicompartment model for GLUT-4 trafficking in adipocytes, in which mutations in either the FQQI or LL motifs result in the altered subcellular trafficking of GLUT-4 between multiple intracellular compartments.