Measuring Confidence of Assurance Cases in Safety-Critical Domains

Evaluation of assurance cases typically requires certifiers’ domain knowledge and experience, and, as such, most software certification has been conducted manually. Given the advancement in uncertainty theories and software traceability, we envision that these technologies can synergistically be combined and leveraged to offer some degree of automation to improve the certifiers’ capability to perform software certification. To this end, we present DS4AC, a novel confidence calculation framework that 1) applies the Dempster-Shafer theory to calculate the confidence between a parent claim and its children claims; and 2) uses the vector space model to evaluate the confidence for the evidence items using traceability information. We illustrate our approach on two different applications, where safety is the key property of interest for both systems. In both cases, we use the Goal Structuring Notation to represent the respective assurance cases and provide proof of concept results that demonstrate the DS4AC framework can automate portions of the evaluation of assurance cases, thereby reducing the burden of manual certification process

SARS-CoV Regulates Immune Function-Related Gene Expressions in Human Monocytic Cells

Background: Severe Acute Respiratory Syndrome (SARS) is characterized by acute respiratory distress (ARDS) and pulmonary fibrosis, and the monocyte/macrophage is the key player in the pathogenesis of SARS.
 
Methods: In this study, we compared the transcriptional profiles of SARS coronavirus (SARS-CoV) infected monocytic cells against that infected by coronavirus 229E (CoV-229E). Total RNA was extracted from infected DC-SIGN transfected monocytes (THP-1-DC-SIGN) at 6 and 24 h after infection and the gene expression was profiled by oligonucleotide-based microarray. 

Results: Analysis of immune-related gene expression profiles showed that 24 h after SARS-CoV infection, (i) IFN-alpha/beta-inducible and cathepsin/proteosome genes were down-regulated; (ii) the hypoxia/hyperoxia-related genes were up-regulated; and (iii) the TLR/TLR-signaling, cytokine/cytokine receptor-related, chemokine/chemokine receptor-related, the lysosome-related, MHC/chaperon-related, and fibrosis-related genes were differentially regulated. 

Conclusion: These results elucidate that monocyte/macrophage dysfunction and dysregulation of fibrosis-related genes are two important pathogenic events of SARS. 
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Discovery of frameshifting in Alphavirus 6K resolves a 20-year enigma.

BACKGROUND: The genus Alphavirus includes several potentially lethal human viruses. Additionally, species such as Sindbis virus and Semliki Forest virus are important vectors for gene therapy, vaccination and cancer research, and important models for virion assembly and structural analyses. The genome encodes nine known proteins, including the small '6K' protein. 6K appears to be involved in envelope protein processing, membrane permeabilization, virion assembly and virus budding. In protein gels, 6K migrates as a doublet--a result that, to date, has been attributed to differing degrees of acylation. Nonetheless, despite many years of research, its role is still relatively poorly understood. RESULTS: We report that ribosomal -1 frameshifting, with an estimated efficiency of approximately 10-18%, occurs at a conserved UUUUUUA motif within the sequence encoding 6K, resulting in the synthesis of an additional protein, termed TF (TransFrame protein; approximately 8 kDa), in which the C-terminal amino acids are encoded by the -1 frame. The presence of TF in the Semliki Forest virion was confirmed by mass spectrometry. The expression patterns of TF and 6K were studied by pulse-chase labelling, immunoprecipitation and immunofluorescence, using both wild-type virus and a TF knockout mutant. We show that it is predominantly TF that is incorporated into the virion, not 6K as previously believed. Investigation of the 3' stimulatory signals responsible for efficient frameshifting at the UUUUUUA motif revealed a remarkable diversity of signals between different alphavirus species. CONCLUSION: Our results provide a surprising new explanation for the 6K doublet, demand a fundamental reinterpretation of existing data on the alphavirus 6K protein, and open the way for future progress in the further characterization of the 6K and TF proteins. The results have implications for alphavirus biology, virion structure, viroporins, ribosomal frameshifting, and bioinformatic identification of novel frameshift-expressed genes, both in viruses and in cellular organisms

Powerful sequence similarity search methods and in-depth manual analyses can identify remote homologs in many apparently "orphan" viral proteins

The genome sequences of new viruses often contain many "orphan" or "taxon-specific" proteins apparently lacking homologs. However, because viral proteins evolve very fast, commonly used sequence similarity detection methods such as BLAST may overlook homologs. We analyzed a data set of proteins from RNA viruses characterized as "genus specific" by BLAST. More powerful methods developed recently, such as HHblits or HHpred (available through web-based, user-friendly interfaces), could detect distant homologs of a quarter of these proteins, suggesting that these methods should be used to annotate viral genomes. In-depth manual analyses of a subset of the remaining sequences, guided by contextual information such as taxonomy, gene order, or domain cooccurrence, identified distant homologs of another third. Thus, a combination of powerful automated methods and manual analyses can uncover distant homologs of many proteins thought to be orphans. We expect these methodological results to be also applicable to cellular organisms, since they generally evolve much more slowly than RNA viruses. As an application, we reanalyzed the genome of a bee pathogen, Chronic bee paralysis virus (CBPV). We could identify homologs of most of its proteins thought to be orphans; in each case, identifying homologs provided functional clues. We discovered that CBPV encodes a domain homologous to the Alphavirus methyltransferase-guanylyltransferase; a putative membrane protein, SP24, with homologs in unrelated insect viruses and insect-transmitted plant viruses having different morphologies (cileviruses, higreviruses, blunerviruses, negeviruses); and a putative virion glycoprotein, ORF2, also found in negeviruses. SP24 and ORF2 are probably major structural components of the virionsd

Effect of 5'UTR introns on gene expression in Arabidopsis thaliana.

BACKGROUND: The majority of introns in gene transcripts are found within the coding sequences (CDSs). A small but significant fraction of introns are also found to reside within the untranslated regions (5'UTRs and 3'UTRs) of expressed sequences. Alignment of the whole genome and expressed sequence tags (ESTs) of the model plant Arabidopsis thaliana has identified introns residing in both coding and non-coding regions of the genome. RESULTS: A bioinformatic analysis revealed some interesting observations: (1) the density of introns in 5'UTRs is similar to that in CDSs but much higher than that in 3'UTRs; (2) the 5'UTR introns are preferentially located close to the initiating ATG codon; (3) introns in the 5'UTRs are, on average, longer than introns in the CDSs and 3'UTRs; and (4) 5'UTR introns have a different nucleotide composition to that of CDS and 3'UTR introns. Furthermore, we show that the 5'UTR intron of the A. thaliana EF1alpha-A3 gene affects the gene expression and the size of the 5'UTR intron influences the level of gene expression. CONCLUSION: Introns within the 5'UTR show specific features that distinguish them from introns that reside within the coding sequence and the 3'UTR. In the EF1alpha-A3 gene, the presence of a long intron in the 5'UTR is sufficient to enhance gene expression in plants in a size dependent manner

Transcriptional slippage in the positive-sense RNA virus family Potyviridae.

The family Potyviridae encompasses ~30% of plant viruses and is responsible for significant economic losses worldwide. Recently, a small overlapping coding sequence, termed pipo, was found to be conserved in the genomes of all potyvirids. PIPO is expressed as part of a frameshift protein, P3N-PIPO, which is essential for virus cell-to-cell movement. However, the frameshift expression mechanism has hitherto remained unknown. Here, we demonstrate that transcriptional slippage, specific to the viral RNA polymerase, results in a population of transcripts with an additional "A" inserted within a highly conserved GAAAAAA sequence, thus enabling expression of P3N-PIPO. The slippage efficiency is ~2% in Turnip mosaic virus and slippage is inhibited by mutations in the GAAAAAA sequence. While utilization of transcriptional slippage is well known in negative-sense RNA viruses such as Ebola, mumps and measles, to our knowledge this is the first report of its widespread utilization for gene expression in positive-sense RNA viruses.Work in the AEF laboratory was funded by grants from the WellcomeTrust [088789], [106207] and Biotechnology and Biological ResearchCouncil (BBSRC) [BB/J007072/1], [BB/J015652/1]. Work in the JPC laboratorywas funded by BBSRC grants [BB/J015652/1], [BB/J011762/1]. BYWC wassupported by a Sir Henry Wellcome Postdoctoral Fellowship [096082]and an EMBL long-term postdoctoral fellowship

Students of Color and COVID-19: Experiences, Coping Strategies, and Supports

The coronavirus disease of 2019, known as the COVID-19 pandemic, is a disaster event that posed significant physical, social, financial, and mental health risks to college students. Disproportionate experiences of stressors position students of color as a population particularly vulnerable to the negative impacts of COVID-19, thus, the current study assessed the impact of COVID-19 on undergraduate students of color in the United States. Students participated in semi-structured in-depth interviews about their experiences with stressors during the pandemic. Data were analyzed using conventional content analysis and revealed themes including (a) the pandemic’s impact on students; (b) basic needs as college students; (c) strategies used to cope with stressors; and (d) supports desired from institutions and faculty. Findings inform colleges and healthcare providers of the most salient concerns for students of color and the sources found helpful. As such, these findings may guide effective prevention and intervention strategies to minimize the effects of future disasters

High-Resolution Analysis of Coronavirus Gene Expression by RNA Sequencing and Ribosome Profiling.

Members of the family Coronaviridae have the largest genomes of all RNA viruses, typically in the region of 30 kilobases. Several coronaviruses, such as Severe acute respiratory syndrome-related coronavirus (SARS-CoV) and Middle East respiratory syndrome-related coronavirus (MERS-CoV), are of medical importance, with high mortality rates and, in the case of SARS-CoV, significant pandemic potential. Other coronaviruses, such as Porcine epidemic diarrhea virus and Avian coronavirus, are important livestock pathogens. Ribosome profiling is a technique which exploits the capacity of the translating ribosome to protect around 30 nucleotides of mRNA from ribonuclease digestion. Ribosome-protected mRNA fragments are purified, subjected to deep sequencing and mapped back to the transcriptome to give a global "snap-shot" of translation. Parallel RNA sequencing allows normalization by transcript abundance. Here we apply ribosome profiling to cells infected with Murine coronavirus, mouse hepatitis virus, strain A59 (MHV-A59), a model coronavirus in the same genus as SARS-CoV and MERS-CoV. The data obtained allowed us to study the kinetics of virus transcription and translation with exquisite precision. We studied the timecourse of positive and negative-sense genomic and subgenomic viral RNA production and the relative translation efficiencies of the different virus ORFs. Virus mRNAs were not found to be translated more efficiently than host mRNAs; rather, virus translation dominates host translation at later time points due to high levels of virus transcripts. Triplet phasing of the profiling data allowed precise determination of translated reading frames and revealed several translated short open reading frames upstream of, or embedded within, known virus protein-coding regions. Ribosome pause sites were identified in the virus replicase polyprotein pp1a ORF and investigated experimentally. Contrary to expectations, ribosomes were not found to pause at the ribosomal frameshift site. To our knowledge this is the first application of ribosome profiling to an RNA virus.NI was supported by a Sir Henry Wellcome Postdoctoral Fellowship (Wellcome Trust, 092334/Z/10/Z). Work in the AEF lab was funded by grants from the Wellcome Trust (088789 and 106207), the U.K. Biotechnology and Biological Research Council (BBSRC) (BB/J007072/1 and BB/J015652/1), and the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No [646891]). Work in the IB laboratory was supported by the Medical Research Council (MRC) (MR/M011747/1) and the Biotechnology and Biological Sciences Research Council (BBSRC) (BB/L000334/1).This is the final version of the article. It first appeared from PLOS via http://dx.doi.org/10.1371/journal.ppat.100547

Powerful sequence similarity search methods and in-depth manual analyses can identify remote homologs in many apparently "orphan" viral proteins.

The genome sequences of new viruses often contain many "orphan" or "taxon-specific" proteins apparently lacking homologs. However, because viral proteins evolve very fast, commonly used sequence similarity detection methods such as BLAST may overlook homologs. We analyzed a data set of proteins from RNA viruses characterized as "genus specific" by BLAST. More powerful methods developed recently, such as HHblits or HHpred (available through web-based, user-friendly interfaces), could detect distant homologs of a quarter of these proteins, suggesting that these methods should be used to annotate viral genomes. In-depth manual analyses of a subset of the remaining sequences, guided by contextual information such as taxonomy, gene order, or domain cooccurrence, identified distant homologs of another third. Thus, a combination of powerful automated methods and manual analyses can uncover distant homologs of many proteins thought to be orphans. We expect these methodological results to be also applicable to cellular organisms, since they generally evolve much more slowly than RNA viruses. As an application, we reanalyzed the genome of a bee pathogen, Chronic bee paralysis virus (CBPV). We could identify homologs of most of its proteins thought to be orphans; in each case, identifying homologs provided functional clues. We discovered that CBPV encodes a domain homologous to the Alphavirus methyltransferase-guanylyltransferase; a putative membrane protein, SP24, with homologs in unrelated insect viruses and insect-transmitted plant viruses having different morphologies (cileviruses, higreviruses, blunerviruses, negeviruses); and a putative virion glycoprotein, ORF2, also found in negeviruses. SP24 and ORF2 are probably major structural components of the virions