Rapid molecular detection of salmonella from produce using real-time PCR

Recent outbreaks of Salmonella linked to fresh produce emphasize the need for rapid and sensitive assays to help control outbreaks. Reverse-transcriptase PCR (RT-PCR) detects the presence of mRNA (shorter half-life than DNA), with greater potential of detecting viable cells. Rapid real-time methods using fluorescent dyes and probes simultaneously detect and confirm the presence of target nucleic acid, eliminating the need for gel electrophoresis. The objective of this research was to rapidly detect Salmonella Typhimurium from spiked lettuce, tomatoes, and peppers using real-time RT-PCR. Washed and ultraviolet light treated lettuce (~25gram), tomato (~100g), and peppers (~130g) samples were inoculated with high (10⁸ to 10⁶ CFU) and low (10³ to 10¹CFU) Salmonella Typhimurium overnight cultures. Samples were then rinsed or hand massaged with 225 ml 0.05 M glycine-saline buffer containing 0.05% Tween and 3% beef extract.Un-inoculated washed produce and sterile buffer were used as negative controls; with S. Typhimurium as a positive control. RNA was extracted from each sample using the Qiagen RNeasy Mini Kit. RT-PCR was carried out using a SYBR Green I RT-PCR kit with previously described Salmonella specific invA gene primers and an internal amplification control (IAC) to eliminate false negatives. Reaction conditions were RT at 50 degree Celsius/40min; PCR at 95 degree Celsius /45s, 58 degree Celsius /45s, 72 degree Celsius /45s for 40 cycles followed by melt temperature(Tm) analysis in a BioRad iCycler to determine specific invA product (~Tm=87.5 degree Celsius) and IAC (Tm=82 degree Celsius). To improve detection sensitivity of low inocula, spiked lettuce, tomatoes, and peppers were pre-enriched in buffered peptone water for 6 hours at 37 degree Celsius, followed by RNA extraction and RT-PCR detection. Each experiment was repeated twice.Real-time RT-PCR after 6-h pre-enrichment, Qiagen RNA extraction and the SYBR Green I kit gave Salmonella detection up to 10³ CFU/25g from lettuce, 10⁴ CFU/~130g from tomatoes, and 10⁴ CFU/25g from peppers. Without enrichment, detection limits were 10⁶ CFU/25g for lettuce, 10⁷ CFU/25g for tomatoes, and 10⁷ CFU/25g for peppers. Sensitive and rapid detection of Salmonella from spiked lettuce, tomatoes, and peppers could still be obtained within one day (~2 working shifts)

Distinguishing Attacks on Stream Ciphers Based on Arrays of Pseudo-random Words

In numerous modern stream ciphers, the internal state consists of a large array of pseudo-random words, and the output key-stream is a relatively simple function of the state. In [Paul-Preneel], it was heuristically shown that in various cases this structure may lead to distinguishing attacks on the cipher. In this paper we further investigate this structural attack. We present a rigorous proof of the main probabilistic claim used in the attack in the basic cases, and demonstrate by examining a concrete example (the cipher SN3) that the heuristic assumptions of the attack are remarkably precise in more complicated cases. Furthermore, we use the general technique to devise a distinguishing attack on the stream cipher MV3 requiring $2^{82}$ words of key-stream. Unlike the attacks in [Paul-Preneel], our attack does not concentrate on the least significant bits of the words, thus allowing to handle the combination of more operations (XORs, modular additions and multiplications, and rotations by a fixed number of bits) in the update and output rules of the cipher

On the Progenitor System of the Type Iax Supernova 2014dt in M61

We present pre-explosion and post-explosion Hubble Space Telescope images of the Type Iax supernova (SN Iax) 2014dt in M61. After astrometrically aligning these images, we do not detect any stellar sources at the position of the SN in the pre-explosion images to relatively deep limits (3 sigma limits of M_F438W > -5.0 mag and M_F814W > -5.9 mag). These limits are similar to the luminosity of SN 2012Z's progenitor system (M_F435W = -5.43 +/- 0.15 and M_F814W = -5.24 +/- 0.16 mag), the only probable detected progenitor system in pre-explosion images of a SN Iax, and indeed, of any white dwarf supernova. SN 2014dt is consistent with having a C/O white-dwarf primary/helium-star companion progenitor system, as was suggested for SN 2012Z, although perhaps with a slightly smaller or hotter donor. The data are also consistent with SN 2014dt having a low-mass red giant or main-sequence star companion. The data rule out main-sequence stars with M_init > 16 M_sun and most evolved stars with M_init > 8 M_sun as being the progenitor of SN 2014dt. Hot Wolf-Rayet stars are also allowed, but the lack of nearby bright sources makes this scenario unlikely. Because of its proximity (D = 12 Mpc), SN 2014dt is ideal for long-term monitoring, where images in ~2 years may detect the companion star or the luminous bound remnant of the progenitor white dwarf.Comment: 5 pages, 3 figures, submitted to ApJ

Constraints in Quantum Geometrodynamics

We compare different treatments of the constraints in canonical quantum gravity. The standard approach on the superspace of 3--geometries treats the constraints as the sole carriers of the dynamic content of the theory, thus rendering the traditional dynamical equations obsolete. Quantization of the constraints in both the Dirac and ADM square root Hamiltonian approaches leads to the well known problems of time evolution. These problems of time are of both an interpretational and technical nature. In contrast, the geometrodynamic quantization procedure on the superspace of the true dynamical variables separates the issues of quantization from the enforcement of the constraints. The resulting theory takes into account states that are off-shell with respect to the constraints, and thus avoids the problems of time. We develop, for the first time, the geometrodynamic quantization formalism in a general setting and show that it retains all essential features previously illustrated in the context of homogeneous cosmologies.Comment: 36 pages, no figures, submitted to IJMPA, Rewording, Fixed Typo

Hybrid metal oxide cycle water splitting

Hybrid thermochemical water splitting cycles are provided in which thermally reduced metal oxides particles are used to displace some but not all of the electrical requirements in a water splitting electrolytic cell. In these hybrid cycles, the thermal reduction temperature is significantly reduced compared to two-step metal-oxide thermochemical cycles in which only thermal energy is required to produce hydrogen from water. Also, unlike the conventional higher temperature cycles where the reduction step must be carried out under reduced oxygen pressure, the reduction step in the proposed hybrid cycles can be carried out in air, allowing for thermal input by a solar power tower with a windowless, cavity receiver

Ternatin and improved synthetic variants kill cancer cells by targeting the elongation factor-1A ternary complex.

Cyclic peptide natural products have evolved to exploit diverse protein targets, many of which control essential cellular processes. Inspired by a series of cyclic peptides with partially elucidated structures, we designed synthetic variants of ternatin, a cytotoxic and anti-adipogenic natural product whose molecular mode of action was unknown. The new ternatin variants are cytotoxic toward cancer cells, with up to 500-fold greater potency than ternatin itself. Using a ternatin photo-affinity probe, we identify the translation elongation factor-1A ternary complex (eEF1A·GTP·aminoacyl-tRNA) as a specific target and demonstrate competitive binding by the unrelated natural products, didemnin and cytotrienin. Mutations in domain III of eEF1A prevent ternatin binding and confer resistance to its cytotoxic effects, implicating the adjacent hydrophobic surface as a functional hot spot for eEF1A modulation. We conclude that the eukaryotic elongation factor-1A and its ternary complex with GTP and aminoacyl-tRNA are common targets for the evolution of cytotoxic natural products

Hybrid metal oxide cycle water splitting

Hybrid thermochemical water splitting systems are disclosed that thermally reduces metal oxides particles to displace some but not all of the electrical requirements in a water splitting electrolytic cell. In these hybrid systems, the thermal reduction temperature is significantly reduced compared to two-step metal-oxide thermochemical cycles in which only thermal energy is required to produce hydrogen from water. Also, unlike conventional higher temperature systems where the reduction step must be carried out under reduced oxygen pressure, the reduction step in the proposed hybrid systems can be carried out in air, allowing for thermal input by a solar power tower with a windowless, cavity receiver

Intrahistiocytic Storage of Clofazimine Crystals in a Cat

A 13-year-old castrated male Maine coon cat with a 5-year history of atypical mycobacteriosis was euthanized and submitted for necropsy. The cat had been kept in clinical remission since diagnosis using a combination of the antimycobacterial drug clofazimine and additional multimodal antimicrobial therapy. Grossly, tissues were diffusely discolored red-brown to yellow. Histologically, the myocardial interstitum was expanded by numerous, often multinucleated cells, which were distended by uniformly shaped acicular cytoplasmic spaces. These cells were immunopositive for CD18 and immunonegative for desmin, suggesting a histiocytic rather than muscular origin. Macrophages in other tissues contained similar acicular spaces. Ultrastructurally, the spaces were surrounded by 2 lipid membranes, resembling an autophagosome. Based on the clinical history and histologic, immunohistochemical, and ultrastructural data, we diagnosed clofazimine crystal storage. To our knowledge, this is the first report of clofazimine storage in a cat or within myocardial interstitial macrophages