Six RNA Viruses and Forty-One Hosts: Viral Small RNAs and Modulation of Small RNA Repertoires in Vertebrate and Invertebrate Systems

We have used multiplexed high-throughput sequencing to characterize changes in small RNA populations that occur during viral infection in animal cells. Small RNA-based mechanisms such as RNA interference (RNAi) have been shown in plant and invertebrate systems to play a key role in host responses to viral infection. Although homologs of the key RNAi effector pathways are present in mammalian cells, and can launch an RNAi-mediated degradation of experimentally targeted mRNAs, any role for such responses in mammalian host-virus interactions remains to be characterized. Six different viruses were examined in 41 experimentally susceptible and resistant host systems. We identified virus-derived small RNAs (vsRNAs) from all six viruses, with total abundance varying from “vanishingly rare” (less than 0.1% of cellular small RNA) to highly abundant (comparable to abundant micro-RNAs “miRNAs”). In addition to the appearance of vsRNAs during infection, we saw a number of specific changes in host miRNA profiles. For several infection models investigated in more detail, the RNAi and Interferon pathways modulated the abundance of vsRNAs. We also found evidence for populations of vsRNAs that exist as duplexed siRNAs with zero to three nucleotide 3′ overhangs. Using populations of cells carrying a Hepatitis C replicon, we observed strand-selective loading of siRNAs onto Argonaute complexes. These experiments define vsRNAs as one possible component of the interplay between animal viruses and their hosts

Bypass Suppression of Small-Plaque Phenotypes by a Mutation in Poliovirus 2A That Enhances Apoptosis ▿

The rate of protein secretion in host cells is inhibited during infection with several different picornaviruses, with consequences likely to have significant effects on viral growth, spread, and pathogenesis. This Sin+ (secretion inhibition) phenotype has been documented for poliovirus, foot-and-mouth disease virus, and coxsackievirus B3 and can lead to reduced cell surface expression of major histocompatibility complex class I and tumor necrosis factor receptor as well as reduced extracellular secretion of induced cytokines such as interleukin-6 (IL-6), IL-8, and beta interferon. The inhibition of protein secretion is global, affecting the movement of all tested cargo proteins through the cellular secretion apparatus. To test the physiological significance of the Sin− and Sin+ phenotypes in animal models, Sin− mutant viruses are needed that fail to inhibit host protein secretion and also exhibit robust growth properties. To identify such Sin− mutant polioviruses, we devised a fluorescence-activated cell sorter-based screen to select virus-infected cells that nevertheless expressed newly synthesized surface proteins. After multiple rounds of selection, candidate Sin− mutant viruses were screened for genetic stability, increased secretion of cargo molecules and wild-type translation and growth properties. A newly identified Sin− mutant poliovirus that contained coding changes in nonstructural proteins 2A (N32D) and 2C (E253G) was characterized. In this virus, the 2C mutation is responsible for the Sin− phenotype and the 2A mutation suppresses a resulting growth defect by increasing the rate of cell death and therefore the rate of viral spread. The 2A-N32D suppressor mutation was not allele specific and, by increasing the rate of cellular apoptosis, affected a completely different pathway than the 2C-E253G Sin− mutation. Therefore, the 2A mutation suppresses the 2C-E253G mutant phenotype by a bypass suppression mechanism

Improving Clinical Practice Using a Novel Engagement Approach: Measurement, Benchmarking and Feedback, A Longitudinal Study.

BACKGROUND: Poor clinical outcomes are caused by multiple factors such as disease progression, patient behavior, and structural elements of care. One other important factor that affects outcome is the quality of care delivered by a provider at the bedside. Guidelines and pathways have been developed with the promise of advancing evidence-based practice. Yet, these alone have shown mixed results or fallen short in increasing adherence to quality of care. Thus, effective, novel tools are required for sustainable practice change and raising the quality of care. METHODS: The study focused on benchmarking and measuring variation and improving care quality for common types of breast cancer at four sites across the United States, using a set of 12 Clinical Performance and Value(®) (CPV(®)) vignettes per site. The vignettes simulated online cases that replicate a typical visit by a patient as the tool to engage breast cancer providers and to identify and assess variation in adherence to evidence-based practice guidelines and pathways. RESULTS: Following multiple rounds of CPV measurement, benchmarking and feedback, we found that scores had increased significantly between the baseline round and the final round (P < 0.001) overall and for all domains. By round 4 of the study, the overall score increased by 14% (P < 0.001), and the diagnosis with treatment plan domain had an increase of 12% (P < 0.001) versus baseline. CONCLUSION: We found that serially engaging breast cancer providers with a validated clinical practice engagement and measurement tool, the CPVs, markedly increased quality scores and adherence to clinical guidelines in the simulated patients. CPVs were able to measure differences in clinical skill improvement and detect how fast improvements were made

Low Rates of Genetic Testing in Children With Developmental Delays, Intellectual Disability, and Autism Spectrum Disorders

To explore the routine and effective use of genetic testing for patients with intellectual disability and developmental delay (ID/DD), we conducted a prospective, randomized observational study of 231 general pediatricians (40%) and specialists (60%), using simulated patients with 9 rare pediatric genetic illnesses. Participants cared for 3 randomly assigned simulated patients, and care responses were scored against explicit evidence-based criteria. Scores were calculated as a percentage of criteria completed. Care varied widely, with a median overall score of 44.7% and interquartile range of 36.6% to 53.7%. Diagnostic accuracy was low: 27.4% of physicians identified the correct primary diagnosis. Physicians ordered chromosomal microarray analysis in 55.7% of cases. Specific gene sequence testing was used in 1.4% to 30.3% of cases. This study demonstrates that genetic testing is underutilized, even for widely available tests. Further efforts to educate physicians on the clinical utility of genetic testing may improve diagnosis and care in these patients