Pan-serotypic detection of foot-and-mouth disease virus using a minor groove binder probe reverse transcription polymerase chain reaction assay

A novel assay for the pan-serotypic detection of foot-and-mouth disease virus (FMDV) was designed using a 5' conjugated minor groove binder (MGB) probe real-time RT-PCR system. This assay targets the 3D region of the FMDV genome and is capable of detecting 20 copies of a transcribed RNA standard. The linear range of the test was eight logs from 2 x 10(1) to 2 x 10(8) copies and amplification time was approximately 2 h. Using a panel of 83 RNA samples from representative FMDV isolates, the diagnostic sensitivity of this test was shown to be equivalent to a TaqMan real-time RT-PCR that targets the 5' untranslated region of FMDV. Furthermore, the assay does not detect viruses causing similar clinical diseases in pigs such as swine vesicular disease virus and vesicular stomatitis virus, nor does it detect marine caliciviruses causing vesicular exanthema. The development of this assay provides a useful tool for the differential diagnosis of FMD, potentially for use in statutory or emergency testing programmes, or for detection of FMDV RNA in research applications. (C) 2011 Elsevier B.V. All rights reserved.

Measurement of absolute charge-exchange cross sections for He2+ collisions with He and H-2

Reported are total, absolute charge-exchange cross sections for collisions of 3He(2+) ions with He and H-2. Measurements are reported at fixed energies between 0.33 and 4.67 keV/amu. Both the present results and earlier results of others are analyzed in terms of available experimental small-angle differential cross sections as a function of collision energy, and hence the geometry of the exit aperture of the gas-collision cells used by the various experimental groups. In addition, the effective length of gas-collision cells is studied using fluid dynamic and molecular flow simulations to address the density patterns near the cell entrance and exit apertures. When small acceptance-angle corrections were applied, the results of present and previous measurements for the single electron capture in these systems were brought into good accord in the relevant energy ranges. Taken in their entirety, the present data for 3He(2+) with He and H-2 lend themselves to new theoretical calculations of the multichannel charge-exchange cross sections.

Collisions Strengths and Effective Collision Strengths for transitions within the ground state configuration of S III

We have carried out a 29-state R-matrix calculation in order to calculate collision strengths and effective collision strengths for the electron impact excitation of S III. The recently developed parallel RMATRX II suite of codes have been used, which perform the calculation in intermediate coupling. Collision strengths have been generated over an electron energy range of 0-12 Ryd, and effective collision strength data have been calculated from these at electron temperatures in the range 1000-100,000 K. Results are here presented for the fine-structure transitions between the ground-state configurations of 3s(2)3p(2) P-3(0,1,2), D-1(2), and S-1(0), and the values given resolve a discrepancy between two previous R-matrix calculations.

Photon collisions with atoms and ions within an intermediate-energy R-matrix framework

Recent experimental advances in light technology necessitate the availability of sophisticated theoretical models which can incorporate an accurate treatment of double-electron continua. We describe here a new intermediate-energy R-matrix approach to photoionisation and photo-double-ionisation and illustrate its feasibilty by application to photoionisation and photo-double-ionisation of He, and photodetachment and photo-double-detachment of H^-. Results are shown to be in excellent agreement with previous theoretical and experimental studies. This work is a key step in the development of a multipurpose R-matrix code for multiple-electron ejection. © 2012 American Physical Society

RpoS and RpoN are involved in the growth-dependent regulation of rfaH transcription and O antigen expression in Salmonella enterica serovar Typhi

We reported earlier that the production of O antigen lipopolysaccharide (LPS) by Salmonella enterica serovar Typhi (Salmonella typhi) increases at the onset of stationary phase and correlates with a growth-regulated expression of the rfaH gene under the control of the alternative sigma factor RpoN (Microbiology 148 (2002) 3789). In this study, we demonstrate that RpoS also modulates rfaH promoter activity as revealed by the absence of growth-dependent regulation of an rfaH-lacZ transcriptional fusion and O antigen production in a S. typhi rpoS mutant. Introduction of a constitutively expressed rpoN gene into the rpoS mutant restored increased production of O antigen during stationary phase, suggesting that constitutive production of RpoN could overcome the RpoS defect. Similar results were observed when an rpoS rpoN double mutant was transformed with the intact rpoN gene. Thus, we conclude that both RpoS and RpoN control the rfaH promoter activity and concomitantly, the production of O-specific LPS in S. typhi.

The Escherichia coli transcriptional regulator MarA directly represses transcription of purA and hdeA

The Escherichia coli MarA protein mediates a response to multiple environmental stresses through the activation or repression in vivo of a large number of chromosomal genes. Transcriptional activation for a number of these genes has been shown to occur via direct interaction of MarA with a 20-bp degenerate asymmetric "marbox" sequence. It was not known whether repression by MarA was also direct. We found that purified MarA was sufficient in vitro to repress transcription of both purA and hdeA. Transcription and electrophoretic mobility shift experiments in vitro using mutant promoters suggested that the marbox involved in the repression overlapped the -35 promoter motif and was in the "backward" orientation. This organization contrasts with that of the class II promoters activated by MarA, in which the marbox also overlaps the -35 motif but is in the "forward" orientation. We conclude that MarA, a member of the AraC/XylS family, can act directly as a repressor or an activator, depending on the position and orientation of the marbox within a promoter.

Old drug, New target:Ellipticines selectively inhibit RNA Polymerase I transcription

Transcription byRNApolymerase I (Pol-I) is the main driving force behind ribosome biogenesis, a fundamental cellular process that requires the coordinated transcription of all three nuclear polymerases. Increased Pol-I transcription and the concurrent increase in ribosome biogenesis has been linked to the high rates of proliferation in cancers. The ellipticine family contains a number of potent anticancer therapeutic agents, some having progressed to stage I and II clinical trials; however, the mechanism by which many of the compounds work remains unclear. It has long been thought that inhibition of Top2 is the main reason behind the drugs antiproliferative effects. Here we report that a number of the ellipticines, including 9-hydroxyellipticine, are potent and specific inhibitors of Pol-I transcription, with IC50 in vitro and in cells in the nanomolar range. Essentially, the drugs did not affect Pol-II and Pol-III transcription, demonstrating a high selectivity.Wehave shown that Pol-I inhibition occurs by a p53-, ATM/ATR-, and Top2-independent mechanism. We discovered that the drug influences the assembly and stability of preinitiation complexes by targeting the interaction between promoter recognition factor SL1 and the rRNA promoter. Our findings will have an impact on the design and development of novel therapeutic agents specifically targeting ribosome biogenesis.

Time-dependent density functional theory study of charge transfer in collisions

We study the charge transfer between colliding ions, atoms, or molecules, within time-dependent density functional theory. Two particular cases are presented, the collision between a proton and a Helium atom, and between a gold atom and a butane molecule. In the first case, proton kinetic energies between 16 keV and 1.2 MeV are considered, with impact parameters between 0.31 and 1.9 angstrom. The partial transfer of charge is monitored with time. The total cross-section is obtained as a function of the proton kinetic energy. In the second case, we analyze one trajectory and discuss spin-dependent charge transfer between the different fragments.

MTERF1 Binds mtDNA to Prevent Transcriptional Interference at the Light-Strand Promoter but Is Dispensable for rRNA Gene Transcription Regulation

Mitochondrial transcription termination factor 1, MTERF1, has been reported to couple rRNA gene transcription initiation with termination and is therefore thought to be a key regulator of mammalian mitochondrial ribosome biogenesis. The prevailing model is based on a series of observations published over the last two decades, but no in vivo evidence exists to show that MTERF1 regulates transcription of the heavy-strand region of mtDNA containing the rRNA genes. Here, we demonstrate that knockout of Mterf1 in mice has no effect on mitochondrial rRNA levels or mitochondrial translation. Instead, loss of Mterf1 influences transcription initiation at the light-strand promoter, resulting in a decrease of de novo transcription manifested as reduced 7S RNA levels. Based on these observations, we suggest that MTERF1 does not regulate heavy-strand transcription, but rather acts to block transcription on the opposite strand of mtDNA to prevent transcription interference at the light-strand promoter.

Topoisomerase IIα promotes activation of RNA polymerase I transcription by facilitating pre-initiation complex formation

Type II DNA topoisomerases catalyse DNA double-strand cleavage, passage and re-ligation to effect topological changes. There is considerable interest in elucidating topoisomerase II roles, particularly as these proteins are targets for anti-cancer drugs. Here we uncover a role for topoisomerase IIa in RNA polymerase I-directed ribosomal RNA gene transcription, which drives cell growth and proliferation and is upregulated in cancer cells. Our data suggest that topoisomerase IIa is a component of the initiation-competent RNA polymerase Iß complex and interacts directly with RNA polymerase I-associated transcription factor RRN3, which targets the polymerase to promoter-bound SL1 in pre-initiation complex formation. In cells, activation of rDNA transcription is reduced by inhibition or depletion of topoisomerase II, and this is accompanied by reduced transient double-strand DNA cleavage in the rDNA-promoter region and reduced pre-initiation complex formation. We propose that topoisomerase IIa functions in RNA polymerase I transcription to produce topological changes at the rDNA promoter that facilitate efficient de novo pre-initiation complex formation.