Efficient transcription through an intron requires the binding of an Sm-type U1 snRNP with intact stem loop II to the splice donor

The mechanism behind the positive action of introns upon transcription and the biological significance of this positive feedback remains unclear. Functional ablation of splice sites within an HIV-derived env cDNA significantly reduced transcription that was rescued by a U1 snRNA modified to bind to the mutated splice donor (SD). Using this model we further characterized both the U1 and pre-mRNA structural requirements for transcriptional enhancement. U1 snRNA rescued as a mature Sm-type snRNP with an intact stem loop II. Position and sequence context for U1-binding is crucial because a promoter proximal intron placed upstream of the mutated SD failed to rescue transcription. Furthermore, U1-rescue was independent of promoter and exon sequence and is partially replaced by the transcription elongation activator Tat, pointing to an intron-localized block in transcriptional elongation. Thus, transcriptional coupling of U1 snRNA binding to the SD may licence the polymerase for transcription through the intron

Cas13b-dependent and Cas13b-independent RNA knockdown of viral sequences in mosquito cells following guide RNA expression

Aedes aegypti and Aedes albopictus mosquitoes are vectors of the RNA viruses chikungunya (CHIKV) and dengue that currently have no specific therapeutic treatments. The development of new methods to generate virus-refractory mosquitoes would be beneficial. Cas13b is an enzyme that uses RNA guides to target and cleave RNA molecules and has been reported to suppress RNA viruses in mammalian and plant cells. We investigated the potential use of the Prevotella sp. P5-125 Cas13b system to provide viral refractoriness in mosquito cells, using a virus-derived reporter and a CHIKV split replication system. Cas13b in combination with suitable guide RNAs could induce strong suppression of virus-derived reporter RNAs in insect cells. Surprisingly, the RNA guides alone (without Cas13b) also gave substantial suppression. Our study provides support for the potential use of Cas13b in mosquitoes, but also caution in interpreting CRISPR/Cas data as we show that guide RNAs can have Cas-independent effects.</p

Cas13b-dependent and Cas13b-independent RNA knockdown of viral sequences in mosquito cells following guide RNA expression

Aedes aegypti and Aedes albopictus mosquitoes are vectors of the RNA viruses chikungunya (CHIKV) and dengue that currently have no specific therapeutic treatments. The development of new methods to generate virus-refractory mosquitoes would be beneficial. Cas13b is an enzyme that uses RNA guides to target and cleave RNA molecules and has been reported to suppress RNA viruses in mammalian and plant cells. We investigated the potential use of the Prevotella sp. P5-125 Cas13b system to provide viral refractoriness in mosquito cells, using a virus-derived reporter and a CHIKV split replication system. Cas13b in combination with suitable guide RNAs could induce strong suppression of virus-derived reporter RNAs in insect cells. Surprisingly, the RNA guides alone (without Cas13b) also gave substantial suppression. Our study provides support for the potential use of Cas13b in mosquitoes, but also caution in interpreting CRISPR/Cas data as we show that guide RNAs can have Cas-independent effects

Understanding misimplementation in U.S. state health departments: An agent-based model

INTRODUCTION: The research goal of this study is to explore why misimplementation occurs in public health agencies and how it can be reduced. Misimplementation is ending effective activities prematurely or continuing ineffective ones, which contributes to wasted resources and suboptimal health outcomes. METHODS: The study team created an agent-based model that represents how information flow, filtered through organizational structure, capacity, culture, and leadership priorities, shapes continuation decisions. This agent-based model used survey data and interviews with state health department personnel across the U.S. between 2014 and 2020; model design and analyses were conducted with substantial input from stakeholders between 2019 and 2021. The model was used experimentally to identify potential approaches for reducing misimplementation. RESULTS: Simulations showed that increasing either organizational evidence-based decision-making capacity or information sharing could reduce misimplementation. Shifting leadership priorities to emphasize effectiveness resulted in the largest reduction, whereas organizational restructuring did not reduce misimplementation. CONCLUSIONS: The model identifies for the first time a specific set of factors and dynamic pathways most likely driving misimplementation and suggests a number of actionable strategies for reducing it. Priorities for training the public health workforce include evidence-based decision making and effective communication. Organizations will also benefit from an intentional shift in leadership decision-making processes. On the basis of this initial, successful application of agent-based model to misimplementation, this work provides a framework for further analyses

Automated Assessment of T2-Weighted MRI to Differentiate Malignant and Benign Primary Solid Liver Lesions in Noncirrhotic Livers Using Radiomics

Rationale and Objectives: Distinguishing malignant from benign liver lesions based on magnetic resonance imaging (MRI) is an important but often challenging task, especially in noncirrhotic livers. We developed and externally validated a radiomics model to quantitatively assess T2-weighted MRI to distinguish the most common malignant and benign primary solid liver lesions in noncirrhotic livers. Materials and Methods: Data sets were retrospectively collected from three tertiary referral centers (A, B, and C) between 2002 and 2018. Patients with malignant (hepatocellular carcinoma and intrahepatic cholangiocarcinoma) and benign (hepatocellular adenoma and focal nodular hyperplasia) lesions were included. A radiomics model based on T2-weighted MRI was developed in data set A using a combination of machine learning approaches. The model was internally evaluated on data set A through cross-validation, externally validated on data sets B and C, and compared to visual scoring of two experienced abdominal radiologists on data set C. Results: The overall data set included 486 patients (A: 187, B: 98, and C: 201). The radiomics model had a mean area under the curve (AUC) of 0.78 upon internal validation on data set A and a similar AUC in external validation (B: 0.74 and C: 0.76). In data set C, the two radiologists showed moderate agreement (Cohen's κ: 0.61) and achieved AUCs of 0.86 and 0.82. Conclusion: Our T2-weighted MRI radiomics model shows potential for distinguishing malignant from benign primary solid liver lesions. External validation indicated that the model is generalizable despite substantial MRI acquisition protocol differences. Pending further optimization and generalization, this model may aid radiologists in improving the diagnostic workup of patients with liver lesions.</p

Gill Damage to Atlantic Salmon (Salmo salar) Caused by the Common Jellyfish (Aurelia aurita) under Experimental Challenge

Peer-reviewed. Copyright © 2011 E.J. Baxter et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Background: Over recent decades jellyfish have caused fish kill events and recurrent gill problems in marine-farmed salmonids. Common jellyfish (Aurelia spp.) are among the most cosmopolitan jellyfish species in the oceans, with populations increasing in many coastal areas. The negative interaction between jellyfish and fish in aquaculture remains a poorly studied area of science. Thus, a recent fish mortality event in Ireland, involving Aurelia aurita, spurred an investigation into the effects of this jellyfish on marine-farmed salmon. Methodology/Principal Findings: To address the in vivo impact of the common jellyfish (A. aurita) on salmonids, we exposed Atlantic salmon (Salmo salar) smolts to macerated A. aurita for 10 hrs under experimental challenge. Gill tissues of control and experimental treatment groups were scored with a system that rated the damage between 0 and 21 using a range of primary and secondary parameters. Our results revealed that A. aurita rapidly and extensively damaged the gills of S. salar, with the pathogenesis of the disorder progressing even after the jellyfish were removed. After only 2 hrs of exposure, significant multi-focal damage to gill tissues was apparent. The nature and extent of the damage increased up to 48 hrs from the start of the challenge. Although the gills remained extensively damaged at 3 wks from the start of the challenge trial, shortening of the gill lamellae and organisation of the cells indicated an attempt to repair the damage suffered. Conclusions: Our findings clearly demonstrate that A. aurita can cause severe gill problems in marine-farmed fish. With aquaculture predicted to expand worldwide and evidence suggesting that jellyfish populations are increasing in some areas, this threat to aquaculture is of rising concern as significant losses due to jellyfish could be expected to increase in the future

Comprehensive Rare Variant Analysis via Whole-Genome Sequencing to Determine the Molecular Pathology of Inherited Retinal Disease

Inherited retinal disease is a common cause of visual impairment and represents a highly heterogeneous group of conditions. Here, we present findings from a cohort of 722 individuals with inherited retinal disease, who have had whole-genome sequencing (n = 605), whole-exome sequencing (n = 72), or both (n = 45) performed, as part of the NIHR-BioResource Rare Diseases research study. We identified pathogenic variants (single-nucleotide variants, indels, or structural variants) for 404/722 (56%) individuals. Whole-genome sequencing gives unprecedented power to detect three categories of pathogenic variants in particular: structural variants, variants in GC-rich regions, which have significantly improved coverage compared to whole-exome sequencing, and variants in non-coding regulatory regions. In addition to previously reported pathogenic regulatory variants, we have identified a previously unreported pathogenic intronic variant in $\textit{CHM}$ in two males with choroideremia. We have also identified 19 genes not previously known to be associated with inherited retinal disease, which harbor biallelic predicted protein-truncating variants in unsolved cases. Whole-genome sequencing is an increasingly important comprehensive method with which to investigate the genetic causes of inherited retinal disease.This work was supported by The National Institute for Health Research England (NIHR) for the NIHR BioResource – Rare Diseases project (grant number RG65966). The Moorfields Eye Hospital cohort of patients and clinical and imaging data were ascertained and collected with the support of grants from the National Institute for Health Research Biomedical Research Centre at Moorfields Eye Hospital, National Health Service Foundation Trust, and UCL Institute of Ophthalmology, Moorfields Eye Hospital Special Trustees, Moorfields Eye Charity, the Foundation Fighting Blindness (USA), and Retinitis Pigmentosa Fighting Blindness. M.M. is a recipient of an FFB Career Development Award. E.M. is supported by UCLH/UCL NIHR Biomedical Research Centre. F.L.R. and D.G. are supported by Cambridge NIHR Biomedical Research Centre

Understanding conflict among experts working on controversial species: A case study on the Australian dingo

Expert elicitation can be valuable for informing decision-makers on conservation and wildlife management issues. To date, studies eliciting expert opinions have primarily focused on identifying and building consensus on key issues. Nonetheless, there are drawbacks of a strict focus on consensus, and it is important to understand and emphasize dissent, too. This study adopts a dissensus-based Delphi to understand conflict among dingo experts. Twenty-eight experts participated in three rounds of investigation. We highlight disagreement on most of the issues explored. In particular, we find that disagreement is underpinned by what we call “conflict over values” and “conflict over evidence.” We also note the broader role played by distrust in influencing such conflicts. Understanding and recognizing the different elements shaping disagreement is critical for informing and improving decision-making and can also enable critique of dominant paradigms in current practices. We encourage greater reflexivity and open deliberation on these aspects and hope our study will inform similar investigations in other contexts

A point-of-care lateral flow assay for neutralising antibodies against SARS-CoV-2

Background: As vaccines against SARS-CoV-2 are now being rolled out, a better understanding of immunity to the virus, whether from infection, or passive or active immunisation, and the durability of this protection is required. This will benefit from the ability to measure antibody-based protection to SARS-CoV-2, ideally with rapid turnaround and without the need for laboratory-based testing. Methods: We have developed a lateral flow POC test that can measure levels of RBD-ACE2 neutralising antibody (NAb) from whole blood, with a result that can be determined by eye or quantitatively on a small instrument. We compared our lateral flow test with the gold-standard microneutralisation assay, using samples from convalescent and vaccinated donors, as well as immunised macaques. Findings: We show a high correlation between our lateral flow test with conventional neutralisation and that this test is applicable with animal samples. We also show that this assay is readily adaptable to test for protection to newly emerging SARS-CoV-2 variants, including the beta variant which revealed a marked reduction in NAb activity. Lastly, using a cohort of vaccinated humans, we demonstrate that our whole-blood test correlates closely with microneutralisation assay data (specificity 100% and sensitivity 96% at a microneutralisation cutoff of 1:40) and that fingerprick whole blood samples are sufficient for this test. Interpretation: Taken together, the COVID-19 NAb-testTM device described here provides a rapid readout of NAb based protection to SARS-CoV-2 at the point of care

Sequence Imputation of HPV16 Genomes for Genetic Association Studies

Human Papillomavirus type 16 (HPV16) causes over half of all cervical cancer and some HPV16 variants are more oncogenic than others. The genetic basis for the extraordinary oncogenic properties of HPV16 compared to other HPVs is unknown. In addition, we neither know which nucleotides vary across and within HPV types and lineages, nor which of the single nucleotide polymorphisms (SNPs) determine oncogenicity.A reference set of 62 HPV16 complete genome sequences was established and used to examine patterns of evolutionary relatedness amongst variants using a pairwise identity heatmap and HPV16 phylogeny. A BLAST-based algorithm was developed to impute complete genome data from partial sequence information using the reference database. To interrogate the oncogenic risk of determined and imputed HPV16 SNPs, odds-ratios for each SNP were calculated in a case-control viral genome-wide association study (VWAS) using biopsy confirmed high-grade cervix neoplasia and self-limited HPV16 infections from Guanacaste, Costa Rica.HPV16 variants display evolutionarily stable lineages that contain conserved diagnostic SNPs. The imputation algorithm indicated that an average of 97.5±1.03% of SNPs could be accurately imputed. The VWAS revealed specific HPV16 viral SNPs associated with variant lineages and elevated odds ratios; however, individual causal SNPs could not be distinguished with certainty due to the nature of HPV evolution.Conserved and lineage-specific SNPs can be imputed with a high degree of accuracy from limited viral polymorphic data due to the lack of recombination and the stochastic mechanism of variation accumulation in the HPV genome. However, to determine the role of novel variants or non-lineage-specific SNPs by VWAS will require direct sequence analysis. The investigation of patterns of genetic variation and the identification of diagnostic SNPs for lineages of HPV16 variants provides a valuable resource for future studies of HPV16 pathogenicity