Systematic mutagenesis of the mouse prion protein to identify critical regions for the efficient propagation of prions

The aim of this study was to systematically investigate the contributions of various amino acids within the prion protein, on prion propagation. To test this in a cellular system, we used a sub-cloned population of N2a cells (PK1) that are highly susceptible to RML mouse prions. A library of stable PK1 cells was generated, which expressed the full length mouse prion protein (moPrP) bearing either point, double, or triple alanine replacements. The effects these changes in the prion protein sequence had on the ability of PK1 cells to propagate RML was tested using a previously established cell based assay. We found that: (i) in the unstructured region of the protein, alanine replacements in CC2 region 90-111 of the prion protein severely diminish, but do not abrogate the ability of cells to propagate prions whilst substitutions K23A.K24A.R25A and Q41A exerted a moderate inhibitory effect on propagation; (ii) alanine replacements in CC2 displayed a dominant negative effect by imposing their propagation inhibition phenotype in the presence of the wild-type protein; (iii) the diminished propagation abilities of cells expressing CC2 alanine mutants were a result of these cells being less susceptible to infection than their wild-type counterparts (iv) all alanine replacements tested in the structured region of the protein appeared to hamper prion propagation, regardless of their positioning within this globular domain. Taken together, these results suggest that integrity of the structured region is vital for successful prion propagation, and that although the flexible region of the prion protein alone (residues 23-111), does not exclusively confer infectivity and/or propagative capacity, charge interactions in this region govern the efficacy with which propagation ensues

Impact of the coronavirus pandemic (COVID-19) on the professional practice and personal well-being of community pharmacy teams in the UK

Objectives: Community pharmacy teams (CPTs) were at the frontline of dealing with patients throughout the COVID-19 pandemic. This study aimed to explore the impact on professional practice and personal well-being of CPTs, in the UK. // Methods: A 25-item survey was designed including a range of open and closed questions. The survey was piloted before being published online via SurveyMonkey and distributed using social media platforms. A combination of opportunity and snowball sampling was employed to recruit participants who worked in community pharmacy (CP) during the pandemic. // Results: In total 758 participants (75% completion rate) including pharmacists, owners, managers, technicians, dispensers, healthcare assistants and pre-registration pharmacists took part. Increased workloads and working hours coupled with staff shortages compromised professional practice (n = 257, 35%). Some of the key challenges of working in CP during the pandemic included: a fear of contracting and passing the virus to others (n = 578, 78%), patients stockpiling medicines (n = 530, 71%) and doctors’ surgeries being closed (n = 517, 70%) The impact on emotional well-being (n = 433, 76%) included stress, anxiety, depression and loneliness; physically (n = 322, 56%) it affected sleep, pain and weight. The effects of the pandemic left 45% (n = 258/569) of participants reconsidering their future in CP as they felt demotivated, unsupported and undervalued. // Conclusion: Despite the enhanced pressures and lack of initial recognition CPTs played a vital role in caring for the population during the pandemic. Resources to better support pharmacy teams in the future not only rely on more funding for better provisions but also investing in CPTs’ well-being

Covid-19 and herbal practice: A United Kingdom practitioner survey

Objectives: To identify the effect of the Covid-19 pandemic on UK herbal medicine practice and how herbal medicine practitioners are supporting people with Covid-19. / Design: Mixed-methods e-survey. / Methods: The survey link was distributed through professional associations and social media. Quantitative data were descriptively summarised and qualitative data were analysed using content analysis. / Results: Results from 59 responses indicated a profound effect of the pandemic on herbal medicine practice, with a move to remote working and a reduction in client numbers. Practitioners reported prescribing a wide range of medicinal plants, chiefly Glycyrrhiza glabra L. and Echinacea spp. alongside providing information and advice. Few reported inter-professional collaboration. / Conclusions: Herbal practitioners need to build on current collaborations, research and experience to develop consistent approaches to support people with mild-moderate Covid-19 symptoms. More systematic exploration of herbal medicine practice during and as a consequence of the pandemic is needed

The Cultural and Commercial Value of Tulsi (Ocimum tenuiflorum L.): Multidisciplinary Approaches Focusing on Species Authentication

Tulsi (Holy basil, Ocimum tenuiflorum L., Lamiaceae), native to Asia, has become globalised as the cultural, cosmetic, and medicinal uses of the herb have been popularised. DNA barcoding, a molecular technique used to identify species based on short regions of DNA, can discriminate between different species and identify contaminants and adulterants. This study aimed to explore the values associated with Tulsi in the United Kingdom (UK) and authenticate samples using DNA barcoding. A mixed methods approach was used, incorporating social research (i.e., structured interviews) and DNA barcoding of Ocimum samples using the ITS and trnH-psbA barcode regions. Interviews revealed the cultural significance of Tulsi: including origins, knowledge exchange, religious connotations, and medicinal uses. With migration, sharing of plants and seeds has been seen as Tulsi plants are widely grown in South Asian (SA) households across the UK. Vouchered Ocimum specimens (n = 33) were obtained to create reference DNA barcodes which were not available in databases. A potential species substitution of O. gratissimum instead of O. tenuiflorum amongst SA participants was uncovered. Commercial samples (n = 47) were difficult to authenticate, potentially due to DNA degradation during manufacturing processes. This study highlights the cultural significance of Tulsi, despite a potential species substitution, the plant holds a prestigious place amongst SA families in the UK. DNA barcoding was a reliable way to authenticate Ocimum species

COVID-19 and herbal practice: A United Kingdom practitioner survey

Objectives: To identify the effect of the COVID-19 pandemic on UK herbal medicine practice and how herbal medicine practitioners are supporting people with COVID-19. Design: Mixed-methods e-survey. Methods: The survey link was distributed through professional associations and social media. Quantitative data were descriptively summarised and qualitative data were analysed using content analysis. Results: Results from 59 responses indicated a profound effect of the pandemic on herbal medicine practice, with a move to remote working and a reduction in client numbers. Practitioners reported prescribing a wide range of medicinal plants, chiefly Glycyrrhiza glabra L. and Echinacea spp. alongside providing information and advice. Few reported inter-professional collaboration. Conclusions: Herbal practitioners need to build on current collaborations, research and experience to develop consistent approaches to support people with mild-moderate COVID-19 symptoms. More systematic exploration of herbal medicine practice during and as a consequence of the pandemic is needed. What is already known about the topic: • The COVID-19 pandemic has had a large impact on all types of healthcare • The impact on herbal medicine practice is unclear What this paper adds: • The COVID-19 pandemic has substantially affected UK herbal medicine practice • A wide range of medicinal plants are currently used by herbal practitioners to support people with COVID-19 • Herbal practitioners need to develop consistent holistic approaches to support people with mild-moderate symptoms of COVID-1

Prognostic model to predict postoperative acute kidney injury in patients undergoing major gastrointestinal surgery based on a national prospective observational cohort study.

Background: Acute illness, existing co-morbidities and surgical stress response can all contribute to postoperative acute kidney injury (AKI) in patients undergoing major gastrointestinal surgery. The aim of this study was prospectively to develop a pragmatic prognostic model to stratify patients according to risk of developing AKI after major gastrointestinal surgery. Methods: This prospective multicentre cohort study included consecutive adults undergoing elective or emergency gastrointestinal resection, liver resection or stoma reversal in 2-week blocks over a continuous 3-month period. The primary outcome was the rate of AKI within 7 days of surgery. Bootstrap stability was used to select clinically plausible risk factors into the model. Internal model validation was carried out by bootstrap validation. Results: A total of 4544 patients were included across 173 centres in the UK and Ireland. The overall rate of AKI was 14·2 per cent (646 of 4544) and the 30-day mortality rate was 1·8 per cent (84 of 4544). Stage 1 AKI was significantly associated with 30-day mortality (unadjusted odds ratio 7·61, 95 per cent c.i. 4·49 to 12·90; P < 0·001), with increasing odds of death with each AKI stage. Six variables were selected for inclusion in the prognostic model: age, sex, ASA grade, preoperative estimated glomerular filtration rate, planned open surgery and preoperative use of either an angiotensin-converting enzyme inhibitor or an angiotensin receptor blocker. Internal validation demonstrated good model discrimination (c-statistic 0·65). Discussion: Following major gastrointestinal surgery, AKI occurred in one in seven patients. This preoperative prognostic model identified patients at high risk of postoperative AKI. Validation in an independent data set is required to ensure generalizability

Purine-mediated signalling triggers eye development

A conserved network of eye field transcription factors (EFTFs) underlies the development of the eye in vertebrates and invertebrates(1). To direct eye development, Pax6, a key gene in this network(2,3), interacts with genes encoding other EFTFs such as Rx1 and Six3 (refs 4-6). However, the mechanisms that control expression of the EFTFs remain unclear(7). Here we show that purine-mediated signalling triggers both EFTF expression and eye development in Xenopus laevis. Overexpression of ectonucleoside triphosphate diphosphohydrolase 2 (E-NTPDase2)(8), an ectoenzyme that converts ATP to ADP(9), caused ectopic eye-like structures, with occasional complete duplication of the eye, and increased expression of Pax6, Rx1 and Six3. In contrast, down-regulation of endogenous E-NTPDase2 decreased Rx1 and Pax6 expression. E-NTPDase2 therefore acts upstream of these EFTFs. To test whether ADP (the product of E-NTPDase2) might act to trigger eye development through P2Y1 receptors, selective in Xenopus for ADP(10,11), we simultaneously knocked down expression of the genes encoding E-NTPDase2 and the P2Y1 receptor. This could prevent the expression of Rx1 and Pax6 and eye formation completely. We next measured ATP release(12-14) in the presumptive eye field, demonstrating a transient release of ATP at a time that could plausibly trigger (once converted to ADP) expression of the EFTFs. This surprising role for transient purine-mediated signalling in eye development may be widely conserved, because alterations to the locus of E-NTPDase2 on human chromosome 9 cause severe head and eye defects, including microphthalmia(15-18). Our results suggest a new mechanism for the initiation of eye development