Double-stranded RNA elements associated with the MVX disease of Agaricus bisporus

Double-stranded RNA (dsRNA) has been isolated from Agaricus bisporus fruit bodies exhibiting a wide range of disease symptoms. The symptoms which occurred singularly or in combination included; bare cropping areas on commercial beds (primordia disruption), crop delay, premature veil opening, off- or brown-coloured mushrooms, sporophore malformations and loss of crop yield. All symptoms were associated with loss of yield and/or product quality. Collectively, these symptoms are described as mushroom virus X (MVX) disease. The dsRNA titre was much lower than that previously encountered with the La France viral disease of mushrooms and a modified cellulose CF11 protocol was used for their detection. A broad survey of cultivated mushrooms from the British industry identified dsRNA elements ranging between 640 bp and 20.2 kbp; the majority have not previously been described in A. bisporus. 26 dsRNA elements were identified with a maximum of 17, apparently non-encapsidated dsRNA elements, in any one sample. Three dsRNAs (16.2, 9.4 and 2.4 kbp) were routinely found in mushrooms asymptomatic for MVX. Previously, La France disease was effectively contained and controlled by minimising the on-farm production and spread of basidiospores. Our on-farm observations suggest that MVX could be spread by infected spores and/or mycelial fragments

Oligonucleotide sequences forming short self-complimentary hairpins can expedite the down-regulation of Coprinopsis cinerea genes

Gene silencing in fungi is often induced by dsRNA hairpin forming constructs the preparation of which can require multiple cloning steps. To simplify gene silencing in the filamentous fungi we have evaluated a high throughput cloning method for target sequences using the homobasidiomycete Coprinopsis cinerea, the GFP reporter and a commercially available vector system. The pSUPER RNAi System™, which was developed for mammalian experiments, exploits the human H1 Polymerase III (Pol III) RNA gene promoter and expedites cloning/expression of specific user-defined oligonucleotide sequences to form short self-complimentary hairpins. Transformation of C. cinerea with pSUPER constructs harboring specific oligonucleotides (19 nt stem length) enabled recovery of transformants with reduced transcripts of the GFP transgene, that were less fluorescent in protein assays and microscopic phenotypes. This technological advance should expedite functional genomic studies in C. cinerea and has wider potential for utility in other homobasidiomycete and filamentous fungi

A flexible method for optimising sharing of healthcare resources and demand in the context of the COVID-19 pandemic.

As the number of cases of COVID-19 continues to grow, local health services are at risk of being overwhelmed with patients requiring intensive care. We develop and implement an algorithm to provide optimal re-routing strategies to either transfer patients requiring Intensive Care Units (ICU) or ventilators, constrained by feasibility of transfer. We validate our approach with realistic data from the United Kingdom and Spain. In the UK, we consider the National Health Service at the level of trusts and define a 4-regular geometric graph which indicates the four nearest neighbours of any given trust. In Spain we coarse-grain the healthcare system at the level of autonomous communities, and extract similar contact networks. Through random search optimisation we identify the best load sharing strategy, where the cost function to minimise is based on the total number of ICU units above capacity. Our framework is general and flexible allowing for additional criteria, alternative cost functions, and can be extended to other resources beyond ICU units or ventilators. Assuming a uniform ICU demand, we show that it is possible to enable access to ICU for up to 1000 additional cases in the UK in a single step of the algorithm. Under a more realistic and heterogeneous demand, our method is able to balance about 600 beds per step in the Spanish system only using local sharing, and over 1300 using countrywide sharing, potentially saving a large percentage of these lives that would otherwise not have access to ICU

Influence of salinity on SAV distribution in a series of intermittently connected coastal lakes

Intermittently closed and open lakes and lagoons (ICOLLs) are coastal lakes that intermittently exchange water with the sea and experience saline intrusions. Understanding effects of seawater exchange on local biota is important to preserve ecosystem functioning and ecological integrity. Coastal dune lakes of northwest Florida are an understudied group of ICOLLs in close geographic proximity and with entrance regimes operating along a frequency continuum. We exploited this natural continuum and corresponding water chemistry gradient to determine effects of water chemistry on resident submersed aquatic vegetation (SAV) distributions in these ecosystems. SAV distribution decreased with increases in salinity, but was unaffected by variation in nitrogen, phosphorous, and turbidity. Salinity perturbations corresponding with water exchange with the Gulf of Mexico were associated with reductions in SAV in coastal dune lakes. Potential impacts associated with changes in global climate may increase the frequency of seawater exchange across all coastal dune lakes and potentially reduce the distribution of oligohaline macrophytes among these ecosystems

Artificial intelligence, bias and clinical safety

This is the final version. Available on open access from BMJ Publishing group via the DOI in this recordEngineering and Physical Sciences Research Council (EPSRC

Meta-analysis of the severe acute respiratory syndrome coronavirus 2 serial intervals and the impact of parameter uncertainty on the coronavirus disease 2019 reproduction number

This is the final version. Available on open access from SAGE Publications via the DOI in this recordThe serial interval of an infectious disease, commonly interpreted as the time between the onset of symptoms in sequentially infected individuals within a chain of transmission, is a key epidemiological quantity involved in estimating the reproduction number. The serial interval is closely related to other key quantities, including the incubation period, the generation interval (the time between sequential infections), and time delays between infection and the observations associated with monitoring an outbreak such as confirmed cases, hospital admissions, and deaths. Estimates of these quantities are often based on small data sets from early contact tracing and are subject to considerable uncertainty, which is especially true for early coronavirus disease 2019 data. In this paper, we estimate these key quantities in the context of coronavirus disease 2019 for the UK, including a meta-analysis of early estimates of the serial interval. We estimate distributions for the serial interval with a mean of 5.9 (95% CI 5.2; 6.7) and SD 4.1 (95% CI 3.8; 4.7) days (empirical distribution), the generation interval with a mean of 4.9 (95% CI 4.2; 5.5) and SD 2.0 (95% CI 0.5; 3.2) days (fitted gamma distribution), and the incubation period with a mean 5.2 (95% CI 4.9; 5.5) and SD 5.5 (95% CI 5.1; 5.9) days (fitted log-normal distribution). We quantify the impact of the uncertainty surrounding the serial interval, generation interval, incubation period, and time delays, on the subsequent estimation of the reproduction number, when pragmatic and more formal approaches are taken. These estimates place empirical bounds on the estimates of most relevant model parameters and are expected to contribute to modeling coronavirus disease 2019 transmission.Engineering and Physical Sciences Research Council (EPSRC)NHS EnglandAlan Turing InstituteMedical Research Council (MRC)National Institute for Health Research (NIHR

Divergent effects of DNMT3A and TET2 mutations on hematopoietic progenitor cell fitness

The DNA methylation regulators DNMT3A and TET2 are recurrently mutated in hematological disorders. Despite possessing antagonistic biochemical activities, loss-of-function murine models show overlapping phenotypes in terms of increased hematopoietic stem cell (HSC) fitness. Here, we directly compared the effects of these mutations on hematopoietic progenitor function and disease initiation. In contrast to Dnmt3a-null HSCs, which possess limitless self-renewal in vivo, Tet2-null HSCs unexpectedly exhaust at the same rate as control HSCs in serial transplantation assays despite an initial increase in self-renewal. Moreover, loss of Tet2 more acutely sensitizes hematopoietic cells to the addition of a common co-operating mutation (Flt

Reprogrammable CRISPR/Cas9-based system for inducing site-specific DNA methylation

Advances in sequencing technology allow researchers to map genome-wide changes in DNA methylation in development and disease. However, there is a lack of experimental tools to site-specifically manipulate DNA methylation to discern the functional consequences. We developed a CRISPR/Cas9 DNA methyltransferase 3A (DNMT3A) fusion to induce DNA methylation at specific loci in the genome. We induced DNA methylation at up to 50% of alleles for targeted CpG dinucleotides. DNA methylation levels peaked within 50 bp of the short guide RNA (sgRNA) binding site and between pairs of sgRNAs. We used our approach to target methylation across the entire CpG island at the CDKN2A promoter, three CpG dinucleotides at the ARF promoter, and the CpG island within the Cdkn1a promoter to decrease expression of the target gene. These tools permit mechanistic studies of DNA methylation and its role in guiding molecular processes that determine cellular fate