Framework Programmable Platform for the advanced software development workstation: Framework processor design document

The design of the Framework Processor (FP) component of the Framework Programmable Software Development Platform (FFP) is described. The FFP is a project aimed at combining effective tool and data integration mechanisms with a model of the software development process in an intelligent integrated software development environment. Guided by the model, this Framework Processor will take advantage of an integrated operating environment to provide automated support for the management and control of the software development process so that costly mistakes during the development phase can be eliminated

Mutations in the E2 glycoprotein and the 3\u27 untranslated region enhance chikungunya virus virulence in mice

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes debilitating musculoskeletal pain and inflammation and can persist for months to years after acute infection. Although studies of humans and experimentally infected animals suggest that CHIKV infection persists in musculoskeletal tissues, the mechanisms for this remain poorly understood. To evaluate this further, we isolated CHIKV from the serum of persistently infected Rag1 -/- mice at day 28. When inoculated into naive wild-type (WT) mice, this persistently circulating CHIKV strain displayed a capacity for earlier dissemination and greater pathogenicity than the parental virus. Sequence analysis revealed a nonsynonymous mutation in the E2 glycoprotein (E2 K200R) and a deletion within the 3' untranslated region (3'-UTR). The introduction of these changes into the parental virus conferred enhanced virulence in mice, although primary tropism for musculoskeletal tissues was maintained. The E2 K200R mutation was largely responsible for enhanced viral dissemination and pathogenicity, although these effects were augmented by the 3'- UTR deletion. Finally, studies with Irf3/Irf7 -/- and Ifnar1 -/- mice suggest that the E2 K200R mutation enhances viral dissemination from the site of inoculation independently of interferon regulatory factor 3 (IRF3)-, IRF7-, and IFNAR1-mediated responses. As our findings reveal viral determinants of CHIKV dissemination and pathogenicity, their further study should help to elucidate host-virus interactions that determine acute and chronic CHIKV infection

Complete genome sequences of 15 chikungunya virus isolates from Puerto Rico

Here, we report the complete genome sequences of 15 chikungunya virus strains isolated from human plasma from infected patients in Puerto Rico. The results show that currently circulating chikungunya strains in Puerto Rico are closely related

Primary and Secondary Sequence Structure Requirements for Recognition and Discrimination of Target RNAs by Pseudomonas aeruginosa RsmA and RsmF

ABSTRACT CsrA family RNA-binding proteins are widely distributed in bacteria and regulate gene expression at the posttranscriptional level. Pseudomonas aeruginosa has a canonical member of the CsrA family (RsmA) and a novel, structurally distinct variant (RsmF). To better understand RsmF binding properties, we performed parallel systematic evolution of ligands by exponential enrichment (SELEX) experiments for RsmA and RsmF. The initial target library consisted of 62-nucleotide (nt) RNA transcripts with central cores randomized at 15 sequential positions. Most targets selected by RsmA and RsmF were the expected size and shared a common consensus sequence (CANGGAYG) that was positioned in a hexaloop region of the stem-loop structure. RsmA and RsmF also selected for longer targets (≥96 nt) that were likely generated by rare PCR errors. Most of the long targets contained two consensus-binding sites. Representative short (single consensus site) and long (two consensus sites) targets were tested for RsmA and RsmF binding. Whereas RsmA bound the short targets with high affinity, RsmF was unable to bind the same targets. RsmA and RsmF both bound the long targets. Mutation of either consensus GGA site in the long targets reduced or eliminated RsmF binding, suggesting a requirement for two tandem binding sites. Conversely, RsmA bound long targets containing only a single GGA site with unaltered affinity. The RsmF requirement for two binding sites was confirmed with tssA1 , an in vivo regulatory target of RsmA and RsmF. Our findings suggest that RsmF binding requires two GGA-containing sites, while RsmA binding requirements are less stringent. IMPORTANCE The CsrA family of RNA-binding proteins is widely conserved in bacteria and plays important roles in the posttranscriptional regulation of protein synthesis. P. aeruginosa has two CsrA proteins, RsmA and RsmF. Although RsmA and RsmF share a few RNA targets, RsmF is unable to bind to other targets recognized by RsmA. The goal of the present study was to better understand the basis for differential binding by RsmF. Our data indicate that RsmF binding requires target RNAs with two consensus-binding sites, while RsmA recognizes targets with just a single binding site. This information should prove useful to future efforts to define the RsmF regulon and its contribution to P. aeruginosa physiology and virulence

Structural divergence creates new functional features in alphavirus genomes

Alphaviruses are mosquito-borne pathogens that cause human diseases ranging from debilitating arthritis to lethal encephalitis. Studies with Sindbis virus (SINV), which causes fever, rash, and arthralgia in humans, and Venezuelan equine encephalitis virus (VEEV), which causes encephalitis, have identified RNA structural elements that play key roles in replication and pathogenesis. However, a complete genomic structural profile has not been established for these viruses. We used the structural probing technique SHAPE-MaP to identify structured elements within the SINV and VEEV genomes. Our SHAPE-directed structural models recapitulate known RNA structures, while also identifying novel structural elements, including a new functional element in the nsP1 region of SINV whose disruption causes a defect in infectivity. Although RNA structural elements are important for multiple aspects of alphavirus biology, we found the majority of RNA structures were not conserved between SINV and VEEV. Our data suggest that alphavirus RNA genomes are highly divergent structurally despite similar genomic architecture and sequence conservation; still, RNA structural elements are critical to the viral life cycle. These findings reframe traditional assumptions about RNA structure and evolution: rather than structures being conserved, alphaviruses frequently evolve new structures that may shape interactions with host immune systems or co-evolve with viral proteins

Disruption of the Opal Stop Codon Attenuates Chikungunya Virus-Induced Arthritis and Pathology

ABSTRACT Chikungunya virus (CHIKV) is a mosquito-borne alphavirus responsible for several significant outbreaks of debilitating acute and chronic arthritis and arthralgia over the past decade. These include a recent outbreak in the Caribbean islands and the Americas that caused more than 1 million cases of viral arthralgia. Despite the major impact of CHIKV on global health, viral determinants that promote CHIKV-induced disease are incompletely understood. Most CHIKV strains contain a conserved opal stop codon at the end of the viral nsP3 gene. However, CHIKV strains that encode an arginine codon in place of the opal stop codon have been described, and deep-sequencing analysis of a CHIKV isolate from the Caribbean identified both arginine and opal variants within this strain. Therefore, we hypothesized that the introduction of the arginine mutation in place of the opal termination codon may influence CHIKV virulence. We tested this by introducing the arginine mutation into a well-characterized infectious clone of a CHIKV strain from Sri Lanka and designated this virus Opal524R. This mutation did not impair viral replication kinetics in vitro or in vivo . Despite this, the Opal524R virus induced significantly less swelling, inflammation, and damage within the feet and ankles of infected mice. Further, we observed delayed induction of proinflammatory cytokines and chemokines, as well as reduced CD4 + T cell and NK cell recruitment compared to those in the parental strain. Therefore, the opal termination codon plays an important role in CHIKV pathogenesis, independently of effects on viral replication. IMPORTANCE Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes significant outbreaks of viral arthralgia. Studies with CHIKV and other alphaviruses demonstrated that the opal termination codon within nsP3 is highly conserved. However, some strains of CHIKV and other alphaviruses contain mutations in the opal termination codon. These mutations alter the virulence of related alphaviruses in mammalian and mosquito hosts. Here, we report that a clinical isolate of a CHIKV strain from the recent outbreak in the Caribbean islands contains a mixture of viruses encoding either the opal termination codon or an arginine mutation. Mutating the opal stop codon to an arginine residue attenuates CHIKV-induced disease in a mouse model. Compared to infection with the opal-containing parental virus, infection with the arginine mutant causes limited swelling and inflammation, as well as dampened recruitment of immune mediators of pathology, including CD4 + T cells and NK cells. We propose that the opal termination codon plays an essential role in the induction of severe CHIKV disease

Multiple conformations are a conserved and regulatory feature of the RB1 5' UTR

Folding to a well-defined conformation is essential for the function of structured ribonucleic acids (RNAs) like the ribosome and tRNA. Structured elements in the untranslated regions (UTRs) of specific messenger RNAs (mRNAs) are known to control expression. The importance of unstructured regions adopting multiple conformations, however, is still poorly understood. High-resolution SHAPE-directed Boltzmann suboptimal sampling of the Homo sapiens Retinoblastoma 1 (RB1) 5' UTR yields three distinct conformations compatible with the experimental data. Private single nucleotide variants (SNVs) identified in two patients with retinoblastoma each collapse the structural ensemble to a single but distinct well-defined conformation. The RB1 5' UTRs from Bos taurus (cow) and Trichechus manatus latirostris (manatee) are divergent in sequence from H. sapiens (human) yet maintain structural compatibility with high-probability base pairs. SHAPE chemical probing of the cow and manatee RB1 5' UTRs reveals that they also adopt multiple conformations. Luciferase reporter assays reveal that 5' UTR mutations alter RB1 expression. In a traditional model of disease, causative SNVs disrupt a key structural element in the RNA. For the subset of patients with heritable retinoblastoma-associated SNVs in the RB1 5' UTR, the absence of multiple structures is likely causative of the cancer. Our data therefore suggest that selective pressure will favor multiple conformations in eukaryotic UTRs to regulate expression

U-CAN-seq: A Universal Competition Assay by Nanopore Sequencing

RNA viruses quickly evolve subtle genotypic changes that can have major impacts on viral fitness and host range, with potential consequences for human health. It is therefore important to understand the evolutionary fitness of novel viral variants relative to well-studied genotypes of epidemic viruses. Competition assays are an effective and rigorous system with which to assess the relative fitness of viral genotypes. However, it is challenging to quickly and cheaply distinguish and quantify fitness differences between very similar viral genotypes. Here, we describe a protocol for using reverse transcription PCR in combination with commercial nanopore sequencing services to perform competition assays on untagged RNA viruses. Our assay, called the Universal Competition Assay by Nanopore Sequencing (U-CAN-seq), is relatively cheap and highly sensitive. We used a well-studied N24A mutation in the chikungunya virus (CHIKV) nsp3 gene to confirm that we could detect a competitive disadvantage using U-CAN-seq. We also used this approach to show that mutations to the CHIKV 5′ conserved sequence element that disrupt sequence but not structure did not affect the fitness of CHIKV. However, similar mutations to an adjacent CHIKV stem loop (SL3) did cause a fitness disadvantage compared to wild-type CHIKV, suggesting that structure-independent, primary sequence determinants in this loop play an important role in CHIKV biology. Our novel findings illustrate the utility of the U-CAN-seq competition assay

Aptamer-based multiplexed proteomic technology for biomarker discovery

Interrogation of the human proteome in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology. We present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 [mu]L of serum or plasma). Our current assay allows us to measure ~800 proteins with very low limits of detection (1 pM average), 7 logs of overall dynamic range, and 5% average coefficient of variation. This technology is enabled by a new generation of aptamers that contain chemically modified nucleotides, which greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the aptamers are selected. Proteins in complex matrices such as plasma are measured with a process that transforms a signature of protein concentrations into a corresponding DNA aptamer concentration signature, which is then quantified with a DNA microarray. In essence, our assay takes advantage of the dual nature of aptamers as both folded binding entities with defined shapes and unique sequences recognizable by specific hybridization probes. To demonstrate the utility of our proteomics biomarker discovery technology, we applied it to a clinical study of chronic kidney disease (CKD). We identified two well known CKD biomarkers as well as an additional 58 potential CKD biomarkers. These results demonstrate the potential utility of our technology to discover unique protein signatures characteristic of various disease states. More generally, we describe a versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations. This unbiased and highly multiplexed search engine will enable the discovery of novel biomarkers in a manner that is unencumbered by our incomplete knowledge of biology, thereby helping to advance the next generation of evidence-based medicine

Compendium of Single Event Effects Results for Candidate Spacecraft Electronics for NASA

Susceptibility of a variety of candidate spacecraft electronics to proton and heavy ion induced single event effects is studied. Devices tested include digital, linear bipolar, and hybrid devices