Investigation of a unique short open reading frame within the 3' untranslated region of the canine distemper virus matrix messenger RNA

Increasing evidence suggest that the long "untranslated" region (UTR) between the matrix (M) and the fusion (F) proteins of morbilliviruses has a functional role. In canine distemper virus (CDV), the F 5' UTR was recently shown to code for a long F signal peptide (Fsp). Subsequently, it was reported that the M/F UTRs combined with the long Fsp were synergistically regulating the F mRNA and protein expression, thereby modulating virulence. Unique to CDV, a short putative open reading frame (ORF) has been identified within the wild-type CDV-M 3' UTR (termed M2). Here, we investigated whether M2 was expressed from the genome of the virulent and demyelinating A75/17-CDV strain. An expression plasmid encoding the M2 ORF tagged both at its N-terminal (HA) and C-terminal domains (RFP), was first constructed. Then, a recombinant virus with its putative M2 ORF replaced by HA-M2-RFP was successfully recovered from cDNA (termed recA75/17(green)-HA-M2-RFP). M2 expression in cells transfected or infected with these mutants was studied by immunoprecipitation, immunofluorescence, immunoblot and flow cytometry analyses. Although fluorescence was readily detected in HA-M2-RFP-transfected cells, absence of red fluorescence emission in several recA75/17(green)-HA-M2-RFP-infected cell types suggested lack of M2 biosynthesis, which was confirmed by the other techniques. Consistent with these data, no functional role of the short polypeptide was revealed by infecting various cell types with HA-M2-RFP over-expressing or M2-knockout recombinant viruses. Thus, in sharp contrast to the CDV-F 5' UTR reported to translate a long Fsp, our data provided evidence that the CDV-M 3' UTR does not express any polypeptides.

The RYR2-encoded ryanodine receptor/calcium release channel in patients diagnosed previously with either catecholaminergic polymorphic ventricular tachycardia or genotype negative, exercise-induced long QT syndrome: a comprehensive open reading frame mutational analysis.

OBJECTIVES This study was undertaken to determine the spectrum and prevalence of mutations in the RYR2-encoded cardiac ryanodine receptor in cases with exertional syncope and normal corrected QT interval (QTc). BACKGROUND Mutations in RYR2 cause type 1 catecholaminergic polymorphic ventricular tachycardia (CPVT1), a cardiac channelopathy with increased propensity for lethal ventricular dysrhythmias. Most RYR2 mutational analyses target 3 canonical domains encoded by <40% of the translated exons. The extent of CPVT1-associated mutations localizing outside of these domains remains unknown as RYR2 has not been examined comprehensively in most patient cohorts. METHODS Mutational analysis of all RYR2 exons was performed using polymerase chain reaction, high-performance liquid chromatography, and deoxyribonucleic acid sequencing on 155 unrelated patients (49% females, 96% Caucasian, age at diagnosis 20 +/- 15 years, mean QTc 428 +/- 29 ms), with either clinical diagnosis of CPVT (n = 110) or an initial diagnosis of exercise-induced long QT syndrome but with QTc <480 ms and a subsequent negative long QT syndrome genetic test (n = 45). RESULTS Sixty-three (34 novel) possible CPVT1-associated mutations, absent in 400 reference alleles, were detected in 73 unrelated patients (47%). Thirteen new mutation-containing exons were identified. Two-thirds of the CPVT1-positive patients had mutations that localized to 1 of 16 exons. CONCLUSIONS Possible CPVT1 mutations in RYR2 were identified in nearly one-half of this cohort; 45 of the 105 translated exons are now known to host possible mutations. Considering that approximately 65% of CPVT1-positive cases would be discovered by selective analysis of 16 exons, a tiered targeting strategy for CPVT genetic testing should be considered.

Open reading frame P--a herpes simplex virus gene repressed during productive infection encodes a protein that binds a splicing factor and reduces synthesis of viral proteins made from spliced mRNA.

The open reading frame P (ORF P) is located in the domain and on the DNA strand of the herpes simplex virus 1 transcribed during latent infection. ORF P is not expressed in productively infected cells as a consequence of repression by the binding of the major viral regulatory protein to its high-affinity binding site. In cells infected with a mutant virus carrying a derepressed gene, ORF P protein is extensively posttranslationally processed. We report that ORF P interacts with a component of the splicing factor SF2/ASF, pulls down a component of the SM antigens, and colocalizes with splicing factors in nuclei of infected cells. The hypothesis that ORF P protein may act to regulate viral gene expression, particularly in situations such as latently infected sensory neurons in which the major regulatory protein is not expressed, is supported by the evidence that in cells infected with a mutant in which the ORF P gene was derepressed, the products of the regulatory genes alpha 0 and alpha 22 are reduced in amounts early in infection but recover late in infection. The proteins encoded by these genes are made from spliced mRNAs, and the extent of recovery of these proteins late in infection correlates with the extent of accumulation of post-translationally processed forms of ORF P protein.

The open reading frame of bamboo mosaic potexvirus satellite RNA is not essential for its replication and can be replaced with a bacterial gene.

A satellite RNA of 836 nt depends on the bamboo mosaic potexvirus (BaMV) for its replication and encapsulation. The BaMV satellite RNA (satBaMV) contains a single open reading frame encoding a 20-kDa nonstructural protein. A full-length infectious cDNA clone has been generated downstream of the T7 RNA polymerase promoter. To investigate the role of the 20-kDa protein encoded by satBaMV, satBaMV transcripts containing mutations in the open reading frame were tested for their ability to replicate in barley protoplasts and in Chenopodium quinoa using BaMV RNA as a helper genome. Unlike other large satellite RNAs, mutants in the open reading frame did not block their replication, suggesting that the 20-kDa protein is not essential for satBaMV replication. Precise replacement of the open reading frame with sequences encoding chloramphenicol acetyltransferase resulted in high level expression of chloramphenicol acetyltransferase in infected C. quinoa, indicating that satBaMV is potentially useful as a satellite-based expression vector.

Expression of protein encoded by varicella-zoster virus open reading frame 63 in latently infected human ganglionic neurons.

The ganglionic cell type in which varicella-zoster virus (VZV) is latent in humans was analyzed by using antibodies raised against in vitro-expressed VZV open reading frame 63 protein. VZV open reading frame 63 protein was detected exclusively in the cytoplasm of neurons of latently infected human trigeminal and thoracic ganglia. This is, to our knowledge, the first identification of a herpesvirus protein expressed during latency in the human nervous system.

Production of infectious human respiratory syncytial virus from cloned cDNA confirms an essential role for the transcription elongation factor from the 5' proximal open reading frame of the M2 mRNA in gene expression and provides a capability for vaccine development.

Infectious human respiratory syncytial virus (RSV) was produced by the intracellular coexpression of five plasmid-borne cDNAs. One cDNA encoded a complete positive-sense version of the RSV genome (corresponding to the replicative intermediate RNA or antigenome), and each of the other four encoded a separate RSV protein, namely, the major nucleocapsid N protein, the nucleocapsid P phosphoprotein, the major polymerase L protein, or the protein from the 5' proximal open reading frame of the M2 mRNA [M2(ORF1)]. RSV was not produced if any of the five plasmids was omitted. The requirement for the M2(ORF1) protein is consistent with its recent identification as a transcription elongation factor and confirms its importance for RSV gene expression. It should thus be possible to introduce defined changes into infectious RSV. This should be useful for basic studies of RSV molecular biology and pathogenesis; in addition, there are immediate applications to the development of live attenuated vaccine strains bearing predetermined defined attenuating mutations.

Hydrophobic cluster analysis predicts an amino-terminal domain of varicella-zoster virus open reading frame 10 required for transcriptional activation.

Varicella-zoster virus open reading frame 10 (ORF10) protein, the homolog of the herpes simplex virus protein VP16, can transactivate immediate-early promoters from both viruses. A protein sequence comparison procedure termed hydrophobic cluster analysis was used to identify a motif centered at Phe-28, near the amino terminus of ORF10, that strongly resembles the sequence of the activating domain surrounding Phe-442 of VP16. With a series of GAL4-ORF10 fusion proteins, we mapped the ORF10 transcriptional-activation domain to the amino-terminal region (aa 5-79). Extensive mutagenesis of Phe-28 in GAL4-ORF10 fusion proteins demonstrated the importance of an aromatic or bulky hydrophobic amino acid at this position, as shown previously for Phe-442 of VP16. Transactivation by the native ORF10 protein was abolished when Phe-28 was replaced by Ala. Similar amino-terminal domains were identified in the VP16 homologs of other alphaherpesviruses. Hydrophobic cluster analysis correctly predicted activation domains of ORF10 and VP16 that share critical characteristics of a distinctive subclass of acidic activation domains.

Autumn Training School. Development and Promotion of Open Access to Scientific Information and Research (September 19–20, 2014, Veliko Tarnovo, Bulgaria), organized in the frame of DiPP2014

The autumn training school Development and Promotion of Open Access to Scientific Information and Research is organized in the frame of the Fourth International Conference on Digital Presentation and Preservation of Cultural and Scientific Heritage—DiPP2014 (September 18–21, 2014, Veliko Tarnovo, Bulgaria, http://dipp2014.math.bas.bg/), organized under the UNESCO patronage. The main organiser is the Institute of Mathematics and Informatics, Bulgarian Academy of Sciences with the support of EU project FOSTER (http://www.fosteropenscience.eu/) and the P. R. Slaveykov Regional Public Library in Veliko Tarnovo, Bulgaria.

Visual motion estimation for an autonomous underwater reef monitoring robot

Performing reliable localisation and navigation within highly unstructured underwater coral reef environments is a difficult task at the best of times. Typical research and commercial underwater vehicles use expensive acoustic positioning and sonar systems which require significant external infrastructure to operate effectively. This paper is focused on the development of a robust vision-based motion estimation technique using low-cost sensors for performing real-time autonomous and untethered environmental monitoring tasks in the Great Barrier Reef without the use of acoustic positioning. The technique is experimentally shown to provide accurate odometry and terrain profile information suitable for input into the vehicle controller to perform a range of environmental monitoring tasks.

Visual motion estimation for an autonomous underwater reef monitoring robot

Performing reliable localisation and navigation within highly unstructured underwater coral reef environments is a difficult task at the best of times. Typical research and commercial underwater vehicles use expensive acoustic positioning and sonar systems which require significant external infrastructure to operate effectively. This paper is focused on the development of a robust vision-based motion estimation technique using low-cost sensors for performing real-time autonomous and untethered environmental monitoring tasks in the Great Barrier Reef without the use of acoustic positioning. The technique is experimentally shown to provide accurate odometry and terrain profile information suitable for input into the vehicle controller to perform a range of environmental monitoring tasks.

Use of ITS to improve level crossings safety : open issues

Level crossing crashes have been shown to result in enormous human and financial cost to society. According to the Australian Transport Safety Bureau (ATSB) [5] a total of 632 Railway Level crossing (RLX) collisions, between trains and road vehicles, occurred in Australia between 2001 and June 2009. The cost of RLX collisions runs into the tens of millions of dollars each year in Australia [6]. In addition, loss of life and injury are commonplace in instances where collisions occur. Based on estimates that 40% of rail related fatalities occur at level crossings [12], it is estimated that 142 deaths between 2001 and June 2009 occurred at RLX. The aim of this paper is to (i) summarise crash patterns in Australia, (ii) review existing international ITS interventions to improve level crossing and (iii) highlights open human factors research related issues. Human factors (e.g., driver error, lapses or violations) have been evidenced as a significant contributing factor in RLX collisions, with drivers of road vehicles particularly responsible for many collisions. Unintentional errors have been found to contribute to 46% of RLX collisions [6] and appear to be far more commonplace than deliberate violations. Humans have been found to be inherently inadequate at using the sensory information available to them to facilitate safe decision-making at RLX and tend to underestimate the speed of approaching large objects due to the non-linear increases in perceived size [6]. Collisions resulting from misjudgements of train approach speed and distance are common [20]. Thus, a fundamental goal for improved RLX safety is the provision of sufficient contextual information to road vehicle drivers to facilitate safe decision-making regarding crossing behaviours.

Estimation and control for an open-source quadcopter

This paper describes modelling, estimation and control of the horizontal translational motion of an open-source and cost effective quadcopter — the MikroKopter. We determine the dynamics of its roll and pitch attitude controller, system latencies, and the units associated with the values exchanged with the vehicle over its serial port. Using this we create a horizontal-plane velocity estimator that uses data from the built-in inertial sensors and an onboard laser scanner, and implement translational control using a nested control loop architecture. We present experimental results for the model and estimator, as well as closed-loop positioning.

A simulation study of unified GPS and roadside ranging technologies for vehicle positioning

The future vehicle navigation for safety applications requires seamless positioning at the accuracy of sub-meter or better. However, standalone Global Positioning System (GPS) or Differential GPS (DGPS) suffer from solution outages while being used in restricted areas such as high-rise urban areas and tunnels due to the blockages of satellite signals. Smoothed DGPS can provide sub-meter positioning accuracy, but not the seamless requirement. A disadvantage of the traditional navigation aids such as Dead Reckoning and Inertial Measurement Unit onboard vehicles are either not accurate enough due to error accumulation or too expensive to be acceptable by the mass market vehicle users. One of the alternative technologies is to use the wireless infrastructure installed in roadside to locate vehicles in regions where the Global Navigation Satellite Systems (GNSS) signals are not available (for example: inside tunnels, urban canyons and large indoor car parks). The examples of roadside infrastructure which can be potentially used for positioning purposes could include Wireless Local Area Network (WLAN)/Wireless Personal Area Network (WPAN) based positioning systems, Ultra-wide band (UWB) based positioning systems, Dedicated Short Range Communication (DSRC) devices, Locata’s positioning technology, and accurate road surface height information over selected road segments such as tunnels. This research reviews and compares the possible wireless technologies that could possibly be installed along roadside for positioning purposes. Models and algorithms of integrating different positioning technologies are also presented. Various simulation schemes are designed to examine the performance benefits of united GNSS and roadside infrastructure for vehicle positioning. The results from these experimental studies have shown a number of useful findings. It is clear that in the open road environment where sufficient satellite signals can be obtained, the roadside wireless measurements contribute very little to the improvement of positioning accuracy at the sub-meter level, especially in the dual constellation cases. In the restricted outdoor environments where only a few GPS satellites, such as those with 45 elevations, can be received, the roadside distance measurements can help improve both positioning accuracy and availability to the sub-meter level. When the vehicle is travelling in tunnels with known heights of tunnel surfaces and roadside distance measurements, the sub-meter horizontal positioning accuracy is also achievable. Overall, simulation results have demonstrated that roadside infrastructure indeed has the potential to provide sub-meter vehicle position solutions for certain road safety applications if the properly deployed roadside measurements are obtainable.

System identification, estimation and control for a cost effective open-source quadcopter

This paper describes system identification, estimation and control of translational motion and heading angle for a cost effective open-source quadcopter — the MikroKopter. The dynamics of its built-in sensors, roll and pitch attitude controller, and system latencies are determined and used to design a computationally inexpensive multi-rate velocity estimator that fuses data from the built-in inertial sensors and a low-rate onboard laser range finder. Control is performed using a nested loop structure that is also computationally inexpensive and incorporates different sensors. Experimental results for the estimator and closed-loop positioning are presented and compared with ground truth from a motion capture system.

Control design for positioning of underwater vehicles with control allocation

In this paper, we address the control design problem of positioning of over-actuated underwater vehicles. The proposed design is based on a control architecture with combined position and velocity loops and a control tuning method based on the decoupled models. We derive analytical tuning rules based on requirements of closed-loop stability, positioning performance, and the vehicle velocity dynamic characteristics. The vehicle modelling is considered from force to motion with appropriate simplifications related to low-speed manoeuvring hydrodynamics and vehicle symmetry. The control design is considered together with a control allocation mapping. This approach makes the control tuning independent of the characteristics of the force actuators and provides the basis for control reconfiguration in the presence of actuator failure. We propose an anti-wind-up implementation of the controller, which ensures that the constraints related to actuation capacity are not violated. This approach simplifies the control allocation problem since the actuator constraints are mapped into generalised force constraints.