Preliminary optimisation of a simplified sample preparation method to permit direct detection of SARS-CoV-2 within saliva samples using reverse-transcription loop-mediated isothermal amplification (RT-LAMP).

We describe the optimisation of a simplified sample preparation method which permits rapid and direct detection of SARS-CoV-2 RNA within saliva, using reverse-transcription loop-mediated isothermal amplification (RT-LAMP). Treatment of saliva samples prior to RT-LAMP by dilution 1:1 in Mucolyse™, followed by dilution in 10 % (w/v) Chelex© 100 Resin and a 98 °C heat step for 2 min enabled detection of SARS-CoV-2 RNA in positive saliva samples. Using RT-LAMP, SARS-CoV-2 RNA was detected in as little as 05:43 min, with no amplification detected in 3097 real-time reverse transcription PCR (rRT-PCR) negative saliva samples from staff tested within a service evaluation study, or for other respiratory pathogens tested (n = 22). Saliva samples can be collected non-invasively, without the need for skilled staff and can be obtained from both healthcare and home settings. Critically, this approach overcomes the requirement for, and validation of, different swabs and the global bottleneck in obtaining access to extraction robots and reagents to enable molecular testing by rRT-PCR. Such testing opens the possibility of public health approaches for effective intervention during the COVID-19 pandemic through regular SARS-CoV-2 testing at a population scale, combined with isolation and contact tracing.

A highly effective reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid detection of SARS-CoV-2 infection.

The COVID-19 pandemic has illustrated the importance of simple, rapid and accurate diagnostic testing. This study describes the validation of a new rapid SARS-CoV-2 RT-LAMP assay for use on extracted RNA or directly from swab offering an alternative diagnostic pathway that does not rely on traditional reagents that are often in short supply during a pandemic. Analytical specificity (ASp) of this new RT-LAMP assay was 100% and analytical sensitivity (ASe) was between 1 × 101 and 1 × 102 copies per reaction when using a synthetic DNA target. The overall diagnostic sensitivity (DSe) and specificity (DSp) of RNA RT-LAMP was 97% and 99% respectively, relative to the standard of care rRT-PCR. When a CT cut-off of 33 was employed, above which increasingly evidence suggests there is a low risk of patients shedding infectious virus, the diagnostic sensitivity was 100%. The DSe and DSp of Direct RT-LAMP (that does not require RNA extraction) was 67% and 97%, respectively. When setting CT cut-offs of ≤33 and ≤25, the DSe increased to 75% and 100%, respectively, time from swab-to-result, CT < 25, was < 15 min. We propose that RNA RT-LAMP could replace rRT-PCR where there is a need for increased sample throughput and Direct RT-LAMP as a near-patient screening tool to rapidly identify highly contagious individuals within emergency departments and care homes during times of increased disease prevalence ensuring negative results still get laboratory confirmation.

The development of two field-ready reverse transcription loop-mediated isothermal amplification assays for the rapid detection of Seneca Valley virus 1.

Seneca Valley virus 1 (SVV-1) has been associated with vesicular disease in swine, with clinical signs indistinguishable from those of other notifiable vesicular diseases such as foot-and-mouth disease. Rapid and accurate detection of SVV-1 is central to confirm the disease causing agent, and to initiate the implementation of control processes. The development of rapid, cost-effective diagnostic assays that can be used at the point of sample collection has been identified as a gap in preparedness for the control of SVV-1. This study describes the development and bench validation of two reverse transcription loop-mediated amplification (RT-LAMP) assays targeting the 5'-untranslated region (5'-UTR) and the VP3-1 region for the detection of SVV-1 that may be performed at the point of sample collection. Both assays were able to demonstrate amplification of all neat samples diluted 1/100 in negative pig epithelium tissue suspension within 8 min, when RNA was extracted prior to the RT-LAMP assay, and no amplification was observed for the other viruses tested. Simple sample preparation methods using lyophilized reagents were investigated, to negate the requirement for RNA extraction. Only a small delay in the time to amplification was observed for these lyophilized reagents, with a time from sample receipt to amplification achieved within 12 min. Although diagnostic validation is recommended, these RT-LAMP assays are highly sensitive and specific, with the potential to be a useful tool in the rapid diagnosis of SVV-1 in the field.

SUMO Suppresses the Activity of the Jasmonic Acid Receptor CORONATINE INSENSITIVE1.

Plants respond rapidly to sudden environmental cues, often responding prior to changes in the hormone levels that coordinate these responses. How this is achieved is not fully understood. The integrative role of the phytohormone jasmonic acid (JA) relies upon the plant's ability to control the levels of JASMONATE ZIM (JAZ) domain-containing repressor proteins. Here, we demonstrate that regardless of intrinsic JA levels, Small Ubiquitin-like Modifier (SUMO)-conjugated JAZ proteins inhibit the JA receptor CORONATINE INSENSITIVE1 (COI1) from mediating non-SUMOylated JAZ degradation. The SUMO-deconjugating proteases OVERLY TOLERANT TO SALT1 (OTS1) and OTS2 regulate JAZ protein SUMOylation and stability. The ots1 ots2 double mutants accumulate SUMOylated and non-SUMOylated JAZ repressor proteins but show no change in endogenous JA levels compared with wild-type plants. SUMO1-conjugated JAZ proteins bind to COI1 independently of the JA mimic coronatine. SUMO inhibits JAZ binding to COI1. We identify the SUMO interacting motif in COI1 and demonstrate that this is vital to SUMO-dependent inhibition of COI1. Necrotroph infection of Arabidopsis thaliana promotes SUMO protease degradation, and this increases JAZ SUMOylation and abundance, which in turn inhibits JA signaling. This study reveals a mechanism for rapidly regulating JA responses, allowing plants to adapt to environmental changes.

Psychedelics and cognitive liberty: Reimagining drug policy through the prism of human rights.

This paper reimagines drug policy--specifically psychedelic drug policy--through the prism of human rights. Challenges to the incumbent prohibitionist paradigm that have been brought from this perspective to date--namely by calling for exemptions from criminalisation on therapeutic or religious grounds--are considered, before the assertion is made that there is a need to go beyond such reified constructs, calling for an end to psychedelic drug prohibitions on the basis of the more fundamental right to cognitive liberty. This central concept is explicated, asserted as being a crucial component of freedom of thought, as enshrined within Article 9 of the European Convention on Human Rights (ECHR). It is argued that the right to cognitive liberty is routinely breached by the existence of the system of drug prohibition in the United Kingdom (UK), as encoded within the Misuse of Drugs Act 1971 (MDA). On this basis, it is proposed that Article 9 could be wielded to challenge the prohibitive system in the courts. This legal argument is supported by a parallel and entwined argument grounded in the political philosophy of classical liberalism: namely, that the state should only deploy the criminal law where an individual's actions demonstrably run a high risk of causing harm to others. Beyond the courts, it is recommended that this liberal, rights-based approach also inform psychedelic drug policy activism, moving past the current predominant focus on harm reduction, towards a prioritization of benefit maximization. How this might translate in to a different regulatory model for psychedelic drugs, a third way, distinct from the traditional criminal and medical systems of control, is tentatively considered. However, given the dominant political climate in the UK--with its move away from rights and towards a more authoritarian drug policy--the possibility that it is only through underground movements that cognitive liberty will be assured in the foreseeable future is contemplated.

Ubiquitin chain topology in plant cell signaling: a new facet to an evergreen story.

Ubiquitin is a peptide modifier able to form polymers of varying length and linkage as part of a powerful signaling system. Perhaps the best-known aspect of this protein's function is as the driver of targeted protein degradation through the Ubiquitin Proteasome System (UPS). Through the formation of lysine 48-linked polyubiquitin chains, it is able to direct the degradation of tagged proteins by the 26S proteasome, indirectly controlling many processes within the cell. However, recent research has indicated that ubiquitin performs a multitude of other roles within the cell beyond protein degradation. It is able to form 6 other "atypical" linkages though lysine residues at positions 6, 11, 27, 29, 33, and 63. These atypical chains perform a range of diverse functions, including the regulation of iron uptake in response to perceived deficiency, repair of double stranded breaks in the DNA, and regulation of the auxin response through the non-proteasomal degradation of auxin efflux carrier protein PIN1. This review explores the role ubiquitin chain topology plays in plant cellular function. We aim to highlight the importance of these varying functions and the future challenges to be encountered within this field.

Identifying attributes required by Foundation Year 1 doctors in multidisciplinary teams: a tool for performance evaluation.

BACKGROUND: Effective working in multidisciplinary teams (MDTs) is promoted as essential in ensuring good healthcare outcomes, suggesting that an understanding exists of the relationship between outcomes and the attributes needed by individuals to function effectively in the MDT. While the characteristics of effective teamwork have been described, the attributes needed by individual MDT members have not been investigated. To address this, the study focuses on newly qualified Foundation Year 1 (FY1) doctors, creating and testing a tool to evaluate their performance in the MDT. METHODS: Repertory grid technique was used to elicit attributes needed by FY1 doctors to function effectively in the MDT. Study participants (all experienced MDT members) used these to evaluate MDT working by FY1 doctor colleagues. Data on 57 FY1 doctors were collected from 95 MDT members working in five hospitals. Participants also ranked the attributes in terms of importance for effective team functioning and rated an 'Ideal' FY1 doctor. RESULTS: The repertory grid permitted differentiation between groups of FY1 doctors' MDT performance. FY1 doctors who undertook interprofessional training were rated no differently than UK-trained graduates without such training. UK-trained graduates were rated significantly higher on all attributes than non-UK-trained graduates. Overall, FY1 doctors were rated lower than the 'Ideal'. Factor analysis and rankings suggested tensions between clinical attributes needed for good team functioning and more 'social' attributes. CONCLUSIONS: This study demonstrates the potential of repertory grid methodology in eliciting attributes that are important for effective teamworking, and using these to evaluate MDT working by FY1 doctors.

Drugs, the Internet and change.

This article investigates the symbiotic relationship between drugs and the Internet, focusing (though not exclusively) on psychedelics. Programming on psychedelics in Silicon Valley from the 1960s to date is detailed, as are the twinned conceptualizations of drugs as a technology and technology as a drug. The correlation between drugs, the Internet, and consumerism is explored: the Internet is a medium through which "white," "grey" and "black" drug markets flourish. Thus, this article details the burgeoning online trades in pharmaceuticals, recreational, and "life-style" drugs that turn the Internet into a veritable candy store. Drug forums transmogrify into street corners, threatening the continued existence of the current system of global prohibition. However, it is arguably the use of the Web as an information source that may offer the greatest challenge to the incumbent paradigm, with experiential discourses offering alternatives to the hegemonic narrative, as the relationships between drugs, those who sell drugs and drug takers are reconfigured online.

Missing celiac disease in family medicine: the importance of hypothesis generation.

PURPOSE: Delays in diagnosing celiac disease average 13 years. We aimed to identify reasons for misdiagnosis in family medicine. BACKGROUND: During a larger study on diagnosis, a scenario describing a 30-year-old female with 3-month abdominal pain, diarrhea, and microcytic anemia consistent with celiac disease was presented on a computer to 84 family physicians. Their information gathering and diagnoses were recorded. Fifty physicians misdiagnosed, and 38 of these took part in "stimulated recall'': they were asked to recall their hypotheses and inferences step by step, aided by a record of their information gathering. They were unaware of the misdiagnosis. ANALYSES: Transcripts were analyzed to identify whether celiac disease was mentioned and how information was interpreted. Two raters independently assessed information interpretation against the published evidence (kappa = 0.85). RESULTS: Physicians did not change their diagnoses during stimulated recall. Only 10 physicians mentioned celiac disease as a hypothesis (26%). "Diarrhea'' and "pain relief by defecation,'' consistent with both celiac disease and irritable bowel syndrome (IBS), were only linked to IBS. "Absence of weight loss'' led to rejecting celiac disease, although weight loss is characteristic of advanced disease. A complete blood count was requested as a routine test and not specifically for celiac disease. Thus, the unexpected result of "microcytic anemia,'' inconsistent with IBS, did not trigger the correct diagnosis. CONCLUSIONS: Most physicians never considered celiac disease. Information inconsistent with the favorite IBS diagnosis was overlooked. Reviewing the case did not prompt physicians to consider celiac disease, re-evaluate the evidence, or rethink the IBS diagnosis.

Public knowledge and attitudes towards Human Papilloma Virus (HPV) vaccination.

BACKGROUND: Human Papilloma Virus (HPV) vaccine has undergone successful trials and has recently been approved for use for the primary prevention of cervical cancer. The aim of this study was to determine knowledge and attitudes towards HPV vaccination. METHODS: Semi-structured interview and questionnaire delivered in a street survey. Standardised HPV-related statements used to measure HPV knowledge and attitudes to vaccination. The setting was three different areas of Birmingham, to target a mix of social class and ethnicity. The sample population was composed of 16-54 year olds. RESULTS: A total of 420 participants were recruited. Poor knowledge of HPV and its links with cervical cancer were observed. 81% had a knowledge score of zero. Knowledge about HPV was associated with different ethnic group and socio-economic group. The majority (88%) of participants were in favour of vaccination, with 83.6% indicating that they would allow a child under their care to be vaccinated. CONCLUSION: Initial responses to the proposed HPV vaccination within the UK public are favourable. However, knowledge levels are poor and media and health professional promotion are required to raise awareness.

On the threshold: how relevant should quantity be in determining intent to supply?

BACKGROUND: In 2005, the English Government announced plans to introduce thresholds into drugs legislation with a view to simplifying proof of intent to supply. METHODS: During the consultation process, the proposal was vigorously attacked as unjust and impractical. Drawing upon European and international experience, this paper critiques the government's decision to shelve this proposal. RESULTS\ CONCLUSIONS: While the use of thresholds to create a presumption that an individual has intent to supply is problematic, thresholds could usefully have been adopted to facilitate a policy of diversion for those who fell below them.