Absolute quantitation of individual SARS-CoV-2 RNA molecules provides a new paradigm for infection dynamics and variant differences

Despite an unprecedented global research effort on SARS-CoV-2, early replication events remain poorly understood. Given the clinical importance of emergent viral variants with increased transmission, there is an urgent need to understand the early stages of viral replication and transcription. We used single-molecule fluorescence in situ hybridisation (smFISH) to quantify positive sense RNA genomes with 95% detection efficiency, while simultaneously visualising negative sense genomes, subgenomic RNAs, and viral proteins. Our absolute quantification of viral RNAs and replication factories revealed that SARS-CoV-2 genomic RNA is long-lived after entry, suggesting that it avoids degradation by cellular nucleases. Moreover, we observed that SARS-CoV-2 replication is highly variable between cells, with only a small cell population displaying high burden of viral RNA. Unexpectedly, the B.1.1.7 variant, first identified in the UK, exhibits significantly slower replication kinetics than the Victoria strain, suggesting a novel mechanism contributing to its higher transmissibility with important clinical implications

Effects of feeding field peas in combination with distillers grains plus solubles in finishing and growing diets on cattle performance and carcass characteristics

Two studies were conducted to evaluate field peas and wet or dry corn distillers grains with solubles (WDGS and DDGS, respectively) in finishing and growing diets. In Exp. 1, British crossbred steers (n = 352, initial BW 356 ± 27 kg) were used in a randomized block design with factors being 0 or 20% field peas and 0 or 30% WDGS in dry-rolled corn (DRC) based finishing diets (DM basis). There was an interaction (P \u3c 0.01) for DMI and G:F. Feeding WDGS increased ADG (P \u3c 0.01), whereas peas had no effect on ADG (P = 0.33). Including WDGS increased G:F in diets without peas (P \u3c 0.01), but had no impact (P = 0.12) in diets containing peas. Peas increased G:F (P = 0.04) in diets without WDGS, but decreased G:F (P = 0.03) with WDGS. Feeding WDGS increased HCW (P \u3c 0.01). In Exp. 2, Continental crossbred heifers (yr. 1; n = 108, initial BW 338 ± 14 kg) and British crossbred steers (yr. 2; n = 90, initial BW 321 ± 10 kg) were assigned randomly to 1 of 9 pastures. Treatments were supplementation with loose DDGS meal on the ground (GROUND), in a bunk (BUNK) or a 25% field peas, 75% DDGS cube on the ground (CUBE) at equal CP. Final BW and ADG were less (P \u3c 0.01) for GROUND than for CUBE and BUNK, which were similar. These data indicate up to 50% DRC could be replaced by peas and WDGS, and peas are an acceptable binder for DDGS range cubes

Single molecule fluorescence in situ hybridisation for quantitating post-transcriptional regulation in Drosophila brains.

RNA in situ hybridization is a powerful method to investigate post-transcriptional regulation, but analysis of intracellular mRNA distributions in thick, complex tissues like the brain poses significant challenges. Here, we describe the application of single-molecule fluorescent in situ hybridization (smFISH) to quantitate primary nascent transcription and post-transcriptional regulation in whole-mount Drosophila larval and adult brains. Combining immunofluorescence and smFISH probes for different regions of a single gene, i.e., exons, 3'UTR, and introns, we show examples of a gene that is regulated post-transcriptionally and one that is regulated at the level of transcription. Our simple and rapid protocol can be used to co-visualise a variety of different transcripts and proteins in neuronal stem cells as well as deep brain structures such as mushroom body neuropils, using conventional confocal microscopy. Finally, we introduce the use of smFISH as a sensitive alternative to immunofluorescence for labelling specific neural stem cell populations in the brain

Systematic analysis of YFP traps reveals common mRNA/protein discordance in neural tissues

While post-transcriptional control is thought to be required at the periphery of neurons and glia, its extent is unclear. Here, we investigate systematically the spatial distribution and expression of mRNA at single molecule sensitivity and their corresponding proteins of 200 YFP trap lines across the intact Drosophila nervous system. 97.5% of the genes studied showed discordance between the distribution of mRNA and the proteins they encode in at least one region of the nervous system. These data suggest that post-transcriptional regulation is very common, helping to explain the complexity of the nervous system. We also discovered that 68.5% of these genes have transcripts present at the periphery of neurons, with 9.5% at the glial periphery. Peripheral transcripts include many potential new regulators of neurons, glia, and their interactions. Our approach is applicable to most genes and tissues and includes powerful novel data annotation and visualization tools for post transcriptional regulation.Acknowledgments. We are very grateful to the Bloomington, Vienna, and Kyoto Drosophila Stock Centres (fly stocks), Flybase and Flymine (Lyne et al., 2007) for their reagents and open data, which were invaluable to this work. We are grateful to David Ish-Horowicz, Alfredo Castello, and members of the Davis laboratory for critical reading of the manuscript and feedback on the project. We thank Zegami Ltd. for their help, advice, and hosting the collection. This work was generously supported by a Wellcome Senior Research Fellowship (096144) and Wellcome Investigator Award (209412) to I. Davis, which funded A.I. Jarvelin, R.M. Parton, J.S. Titlow, and M.K. Thompson. Advanced microscopy facilities and technical advice as well as support to D.M. Susano Pinto were provided by Micron Oxford (https://micronoxford.com), supported by Wellcome Strategic Awards (091911 and 107457) and a Medical Research Council/Engineering and Physical Sciences Research Council/Biotechnology and Biological Sciences Research Council next-generation imaging award to I. Davis as the principal investigator. J.S. Titlolw and M.K. Thompson were supported by a Leverhulme Trust grant to I. Davis. Department of Biochemistry DPhil studentships supported J.Y. Lee and D.S. Gala. M. Kiourlappou was supported by the Biotechnology and Biosciences Research Council, grant numbers: BB/M011224/1 and BB/S507623/1, by A.G. Leventis Foundation, and by Zegami Ltd

An Empty Toolbox? Changes in Health Plans’ Approaches for Managing Costs and Care

OBJECTIVE: To examine how health plans have changed their approaches for managing costs and utilization in the wake of the recent backlash against managed care. DATA SOURCES/STUDY SETTING: Semistructured interviews with health plan executives, employers, providers, and other health care decision makers in 12 metropolitan areas that were randomly selected to be nationally representative of communities with more than 200,000 residents. Longitudinal data were collected as part of the Community Tracking Study during three rounds of site visits in 1996–1997, 1998–1999, and 2000–2001. STUDY DESIGN: Interviews probed about changes in the design and operation of health insurance products—including provider contracting and network development, benefit packages, and utilization management processes—and about the rationale and perceived impact of these changes. DATA COLLECTION/EXTRACTION METHODS: Data from more than 850 interviews were coded, extracted, and analyzed using computerized text analysis software. PRINCIPAL FINDINGS: Health plans have begun to scale back or abandon their use of selected managed care tools in most communities, with selective contracting and risk contracting practices fading most rapidly and completely. In turn, plans increasingly have sought cost savings by shifting costs to consumers. Some plans have begun to experiment with new provider networks, payment systems, and referral practices designed to lower costs and improve service delivery. CONCLUSIONS: These changes promise to lighten administrative and financial burdens for physicians and hospitals, but they also threaten to increase consumers’ financial burdens