Cryptic AUG is important for 48S ribosomal assembly during internal initiation of translation of coxsackievirus B3 RNA

We have investigated the possible role of a conserved cis-acting element, the cryptic AUG, present in the 5' UTR of coxsackievirus B3 (CVB3) RNA. CVB3 5' UTR contains multiple AUG codons upstream of the initiator AUG, which are not used for the initiation of translation. The 48S ribosomal assembly takes place upstream of the cryptic AUG. We show here that mutation in the cryptic AUG results in reduced efficiency of translation mediated by the CVB3 IRES; mutation also reduces the interaction of mutant IRES with a well characterized IRES trans-acting factor, the human La protein. Furthermore, partial silencing of the La gene showed a decrease in IRES activity in the case of both the wild-type and mutant. We have demonstrated here that the interaction of the 48S ribosomal complex with mutant RNA was weaker compared with wild-type RNA by ribosome assembly analysis. We have also investigated by chemical and enzymic modifications the possible alteration in secondary structure in the mutant RNA. Results suggest that the secondary structure of mutant RNA was only marginally altered. Additionally, we have demonstrated by generating compensatory and non-specific mutations the specific function of the cryptic AUG in internal initiation. Results suggest that the effect of the cryptic AUG is specific and translation could not be rescued. However, a possibility of tertiary interaction of the cryptic AUG with other cis-acting elements cannot be ruled out. Taken together, it appears that the integrity of the cryptic AUG is important for efficient translation initiation by the CVB3 IRES RNA.

Investigations on the RNA binding and phosphorylation of groundnut bud necrosis virus nucleocapsid protein

Groundnut bud necrosis virus belongs to the genus Tospovirus, infects a wide range of crop plants and causes severe losses. To understand the role of the nucleocapsid protein in the viral life cycle, the protein was overexpressed in E. coli and purified by Ni-NTA chromatography. The purified N protein was well folded and was predominantly alpha-helical. Deletion analysis revealed that the C-terminal unfolded region of the N protein was involved in RNA binding. Furthermore, the N protein could be phosphorylated in vitro by Nicotiana benthamiana plant sap and by purified recombinant kinases such as protein kinase CK2 and calcium-dependent protein kinase. This is the first report of phoshphorylation of a nucleocapsid protein in the family Bunyaviridae. The possible implications of the present findings for the viral life cycle are discussed.

Inhibition of Mycobacterium tuberculosis RNA Polymerase by Binding of a Gre Factor Homo log to the Secondary Channel

Because of its essential nature, each step of transcription, viz., initiation, elongation, and termination, is subjected to elaborate regulation. A number of transcription factors modulate the rates of transcription at these different steps, and several inhibitors shut down the process. Many modulators, including small molecules and proteinaceous inhibitors, bind the RNA polymerase (RNAP) secondary channel to control transcription. We describe here the first small protein inhibitor of transcription in Mycobacterium tuberculosis. Rv3788 is a homolog of the Gre factors that binds near the secondary channel of RNAP to inhibit transcription. The factor also affected the action of guanosine pentaphosphate (pppGpp) on transcription and abrogated Gre action, indicating its function in the modulation of the catalytic center of RNAP. Although it has a Gre factor-like domain organization with the conserved acidic residues in the N terminus and retains interaction with RNAP, the factor did not show any transcript cleavage stimulatory activity. Unlike Rv3788, another Gre homolog from Mycobacterium smegmatis, MSMEG_6292 did not exhibit transcription-inhibitory activities, hinting at the importance of the former in influencing the lifestyle of M. tuberculosis.

Unzipping and binding of small interfering RNA with single walled carbon nanotube: A platform for small interfering RNA delivery

In an effort to design efficient platform for siRNA delivery, we combine all atom classical and quantum simulations to study the binding of small interfering RNA (siRNA) by pristine single wall carbon nanotube (SWCNT). Our results show that siRNA strongly binds to SWCNT surface via unzipping its base-pairs and the propensity of unzipping increases with the increase in the diameter of the SWCNTs. The unzipping and subsequent wrapping events are initiated and driven by van der Waals interactions between the aromatic rings of siRNA nucleobases and the SWCNT surface. However, molecular dynamics (MD) simulations of double strand DNA (dsDNA) of the same sequence show that the dsDNA undergoes much less unzipping and wrapping on the SWCNT in the simulation time scale of 70 ns. This interesting difference is due to smaller interaction energy of thymidine of dsDNA with the SWCNT compared to that of uridine of siRNA, as calculated by dispersion corrected density functional theory (DFT) methods. After the optimal binding of siRNA to SWCNT, the complex is very stable which serves as one of the major mechanisms of siRNA delivery for biomedical applications. Since siRNA has to undergo unwinding process with the effect of RNA-induced silencing complex, our proposed delivery mechanism by SWCNT possesses potential advantages in achieving RNA interference. (C) 2012 American Institute of Physics. [http://dx.doi.org/10.1063/1.3682780]

Ribosome-RNA interaction: a potential target for developing antiviral against hepatitis C virus

Hepatitis C virus (HCV), a member of Flaviviridae, encoding a positive-sense single-stranded RNA translates by cap-independent mechanism using the internal ribosome entry site (IRES) present in the 5' UTR of the virus. The IRES has complex stem loop structures and is capable of recruiting the 40S ribosomal subunit in a factor-independent fashion. As the IRES sequence is highly conserved throughout the HCV genotypes and the translation is the first obligatory step of the HCV life cycle, the IRE'S-mediated translation, or more specifically, the ribosome HCV RNA interaction is an attractive target to design effective antivirals. This article will focus on the mechanism of the HCV IRES translation and the various ways in which the interaction of ribosome and IRES has been targeted.

Targeting ribosome assembly on the HCV RNA using a small RNA molecule

Translation initiation of hepatitis C virus (HCV) RNA is the initial obligatory step of the viral life cycle, mediated through the internal ribosome entry site (IRES) present in the 5'-untranslated region (UTR). Initiation on the HCV IRES is mediated by multiple structure-specific interactions between IRES RNA and host 40S ribosomal subunit. In the present study we demonstrate that the SLIIIef domain, in isolation from other structural elements of HCV IRES, retain the ability to interact with 40S ribosome subunit. A small RNA SLRef, mimicking the SLIIIef domain was found to interact specifically with human La protein and the ribosomal protein S5 and selectively inhibit HCV RNA translation. More importantly, SLRef RNA showed significant suppression of replication in HCV monocistronic replicon and decrease of negative strand synthesis in HCV cell culture system. Finally, using Sendai virus based virosome, the targeted delivery of SLRef RNA into mice liver succeeded in selectively inhibiting HCV IRES mediated translation in vivo.

Theory and Algorithms for Hop-Count-Based Localization with Random Geometric Graph Models of Dense Sensor Networks

Wireless sensor networks can often be viewed in terms of a uniform deployment of a large number of nodes in a region of Euclidean space. Following deployment, the nodes self-organize into a mesh topology with a key aspect being self-localization. Having obtained a mesh topology in a dense, homogeneous deployment, a frequently used approximation is to take the hop distance between nodes to be proportional to the Euclidean distance between them. In this work, we analyze this approximation through two complementary analyses. We assume that the mesh topology is a random geometric graph on the nodes; and that some nodes are designated as anchors with known locations. First, we obtain high probability bounds on the Euclidean distances of all nodes that are h hops away from a fixed anchor node. In the second analysis, we provide a heuristic argument that leads to a direct approximation for the density function of the Euclidean distance between two nodes that are separated by a hop distance h. This approximation is shown, through simulation, to very closely match the true density function. Localization algorithms that draw upon the preceding analyses are then proposed and shown to perform better than some of the well-known algorithms present in the literature. Belief-propagation-based message-passing is then used to further enhance the performance of the proposed localization algorithms. To our knowledge, this is the first usage of message-passing for hop-count-based self-localization.

Plasmodium falciparum Prp16 homologue and its role in splicing

Large numbers of Plasmodium genes have been predicted to have introns. However, little information exists on the splicing mechanisms in this organism. Here, we describe the DExD/DExH-box containing Pre-mRNA processing proteins (Prps), PfPrp2p, PfPrp5p, PfPrp16p, PfPrp22p, PfPrp28p, PfPrp43p and PfBrr2p, present in the Plasmodium falciparum genome and characterized the role of one of these factors, PfPrp16p. It is a member of DEAH-box protein family with nine collinear sequence motifs, a characteristic of helicase proteins. Experiments with the recombinantly expressed and purified PfPrp16 helicase domain revealed binding to RNA, hydrolysis of ATP as well as catalytic helicase activities. Expression of helicase domain with the C-terminal helicase-associated domain (HA2) reduced these activities considerably, indicating that the helicase-associated domain may regulate the PfPrp16 function. Localization studies with the PfPrp16 GFP transgenic lines suggested a role of its N-terminal domain (1-80 amino acids) in nuclear targeting. Immunodepletion of PfPrp16p, from nuclear extracts of parasite cultures, blocked the second catalytic step of an in vitro constituted splicing reaction suggesting a role for PfPrp16p in splicing catalysis. Further we show by complementation assay in yeast that a chimeric yeast-Plasmodium Prp16 protein, not the full length PfPrp16, can rescue the yeast prp16 temperature-sensitive mutant. These results suggest that although the role of Prp16p in catalytic step II is highly conserved among Plasmodium, human and yeast, subtle differences exist with regards to its associated factors or its assembly with spliceosomes. (C) 2012 Elsevier B.V. All rights reserved.

Multiple transmitter localization and communication footprint identification using sparse reconstruction techniques

This paper considers the problem of identifying the footprints of communication of multiple transmitters in a given geographical area. To do this, a number of sensors are deployed at arbitrary but known locations in the area, and their individual decisions regarding the presence or absence of the transmitters' signal are combined at a fusion center to reconstruct the spatial spectral usage map. One straightforward scheme to construct this map is to query each of the sensors and cluster the sensors that detect the primary's signal. However, using the fact that a typical transmitter footprint map is a sparse image, two novel compressive sensing based schemes are proposed, which require significantly fewer number of transmissions compared to the querying scheme. A key feature of the proposed schemes is that the measurement matrix is constructed from a pseudo-random binary phase shift applied to the decision of each sensor prior to transmission. The measurement matrix is thus a binary ensemble which satisfies the restricted isometry property. The number of measurements needed for accurate footprint reconstruction is determined using compressive sampling theory. The three schemes are compared through simulations in terms of a performance measure that quantifies the accuracy of the reconstructed spatial spectral usage map. It is found that the proposed sparse reconstruction technique-based schemes significantly outperform the round-robin scheme.

Multi-script and multi-oriented text localization from scene images

This paper describes a new method of color text localization from generic scene images containing text of different scripts and with arbitrary orientations. A representative set of colors is first identified using the edge information to initiate an unsupervised clustering algorithm. Text components are identified from each color layer using a combination of a support vector machine and a neural network classifier trained on a set of low-level features derived from the geometric, boundary, stroke and gradient information. Experiments on camera-captured images that contain variable fonts, size, color, irregular layout, non-uniform illumination and multiple scripts illustrate the robustness of the method. The proposed method yields precision and recall of 0.8 and 0.86 respectively on a database of 100 images. The method is also compared with others in the literature using the ICDAR 2003 robust reading competition dataset.

Strain localization effect on system reliability based design of bridge abutments under earthquake loading

The component and system reliability based design of bridge abutments under earthquake loading is presented in the paper. Planar failure surface has been used in conjunction with pseudo-dynamic approach to compute seismic active earth pressures on an abutment. The pseudo-dynamic method, considers the effect of phase difference in shear waves, soil amplification along with the horizontal seismic accelerations, strain localization in backfill soil and associated post-peak reduction in the shear resistance from peak to residual values along a previously formed failure plane. Four modes of stability viz. sliding, overturning, eccentricity and bearing capacity of the foundation soil are considered in the analysis. The series system reliability is computed with an assumption of independent failure modes. The lower and upper bounds of system reliability are also computed by taking into account the correlations between four failure modes, which is evaluated using the direction cosines of the tangent planes at the most probable points of failure.

Influence of strain localization on reliability based design of bridge abutments using pseudo-dynamic method

The paper focuses on reliability based design of bridge abutments when subjected to earthquake loading. Planar failure surface has been used in conjunction with pseudo-dynamic approach to compute the seismic active earth pressures on the bridge abutment. The proposed pseudo dynamic method, considers the effects of strain localization in the backfill soil and associated post-peak reduction in the shear resistance from peak to residual values along a previously formed failure plane, phase difference in shear waves and soil amplification along with the horizontal seismic accelerations. Four modes of stability viz. sliding, overturning, eccentricity and bearing capacity of the foundation soil are considered for the reliability analysis. The influence of various design parameters on the seismic reliability indices against four modes of failure is presented, following the suggestions of Japan Road Association, Caltrans Bridge Design Specifications and U.S Department of the Army.