Middle Jurassic fossils document an early stage in salamander evolution.

Salamanders are an important group of living amphibians and model organisms for understanding locomotion, development, regeneration, feeding, and toxicity in tetrapods. However, their origin and early radiation remain poorly understood, with early fossil stem-salamanders so far represented by larval or incompletely known taxa. This poor record also limits understanding of the origin of Lissamphibia (i.e., frogs, salamanders, and caecilians). We report fossils from the Middle Jurassic of Scotland representing almost the entire skeleton of the enigmatic stem-salamander Marmorerpeton. We use computed tomography to visualize high-resolution three-dimensional anatomy, describing morphologies that were poorly characterized in early salamanders, including the braincase, scapulocoracoid, and lower jaw. We use these data in the context of a phylogenetic analysis intended to resolve the relationships of early and stem-salamanders, including representation of important outgroups alongside data from high-resolution imaging of extant species. Marmorerpeton is united with Karaurus, Kokartus, and others from the Middle Jurassic-Lower Cretaceous of Asia, providing evidence for an early radiation of robustly built neotenous stem-salamanders. These taxa display morphological specializations similar to the extant cryptobranchid "giant" salamanders. Our analysis also demonstrates stem-group affinities for a larger sample of Jurassic species than previously recognized, highlighting an unappreciated diversity of stem-salamanders and cautioning against the use of single species (e.g., Karaurus) as exemplars for stem-salamander anatomy. These phylogenetic findings, combined with knowledge of the near-complete skeletal anatomy of Mamorerpeton, advance our understanding of evolutionary changes on the salamander stem-lineage and provide important data on early salamanders and the origins of Batrachia and Lissamphibia.

Feasibility and acceptability of a community pharmacy referral service for suspected lung cancer symptoms.

BACKGROUND: Lung cancer survival rates in the UK are among the lowest in Europe, principally due to late-stage diagnosis. Alternative routes to earlier diagnosis of lung cancer are needed in socioeconomically deprived communities that are disproportionately affected by poor lung cancer outcomes. We assessed the feasibility and acceptability of a community-based pharmacy referral service to encourage earlier symptomatic referral for chest X-rays. METHODS: Seventeen community pharmacies located in a deprived area of Wales participated between March 2019 and March 2020. Stakeholder interviews were conducted with four patients, seven pharmacy professionals and one general practitioner. Four focus groups were conducted, including one with healthcare professionals (n=6) and three with members of the public who were current and former smokers (n=13). Quantitative data regarding patient characteristics and clinical outcomes were collected from hospital records and patient referral questionnaires completed by pharmacists and analysed using descriptive statistics. Qualitative data sets were analysed thematically and triangulated. RESULTS: Twelve patients used the pharmacy referral service, all of whom were male. Average length of the pharmacy consultation was 13 min, with a mean 3 days to accessing chest X-rays in secondary care. Patients experienced a mean 46-day wait for results, with no lung cancer detected. Participants found the service to be acceptable and considered the pharmacy element to be broadly feasible. Perceived barriers included low awareness of the service and concerns about the role and capacity of pharmacists to deliver the service. Facilitators included perceived approachability and accessibility of pharmacists. A well-publicised, multifaceted awareness campaign was recommended. CONCLUSIONS: A community pharmacy referral service for lung symptoms was considered an acceptable alternative pathway to symptomatic diagnosis of lung cancer in deprived communities. Wider implementation of the service would require workforce capacity and training to be addressed to ensure optimum utilisation and promotion of the service.

Evaluation of Galleria mellonella larvae for measuring the efficacy and pharmacokinetics of antibiotic therapies against Pseudomonas aeruginosa infection.

The aim of this study was to determine whether Galleria mellonella larvae can be used (i) as an in vivo infection model for Pseudomonas aeruginosa and (ii) for evaluating the pharmacokinetics and efficacy of antipseudomonal antibiotics. Two strains of P. aeruginosa were employed, NCTC 10662 (antibiotic-susceptible) and NCTC 13437 (multidrug-resistant). Larvae were infected with increasing doses of either P. aeruginosa strain to investigate the effect of inoculum size on survival. Subsequently, infected larvae were treated with a range of antibiotics to examine whether these agents were effective against P. aeruginosa infection in vivo and whether the efficacy of these drugs matched the known susceptibilities of each bacterial strain. Larval burden of P. aeruginosa was also determined after infection and treatment with cefotaxime. Pharmacokinetic properties of the antibiotics tested were measured using a well diffusion assay to determine the concentration of antibiotics in larval haemolymph over time. Galleria mellonella larvae were sensitive to P. aeruginosa infection, and increasing inoculum doses of live cells resulted in greater larval mortality. Heat-killed bacteria had no detrimental effect on survival. Antibiotic efficacy against P. aeruginosa-infected G. mellonella correlated with the measured in vitro sensitivities of the two strains tested. The therapeutic benefit arising from administration of cefotaxime correlated with a reduced burden of bacteria present in the haemolymph. There was a clear correlation between measured antibiotic pharmacokinetics and the therapeutic effect. This study strongly supports future application of the G. mellonella infection model to initial studies of novel antipseudomonal treatments.

Acceptability and practicality of a Spanish translation of paediatric Gait Arms Legs and Spine (pGALS) in Peruvian children.

BACKGROUND: The paediatric Gait, Arms, Legs and Spine (pGALS) musculoskeletal examination tool is validated for use in school-aged English Speaking children and shown to be practical and effective in acute paediatric practice in the UK and Malawi. Our aim was to assess the acceptability and practicality of a Spanish translation of pGALS in an acute paediatric setting in Peru. FINDINGS: Fifty-three school-aged children presenting to Hospital Regional de Loreto, Peru were recruited to undergo a pGALS examination using a Spanish translation of the instructions. The pGALS examination was completed in 92.5% (49/53), with the time taken (median 4.42 minutes) being acceptable to most parents (98.1%, 52/53). Most children (88.7%, 47/53), found the pGALS examination caused 'little' or 'no additional discomfort'. Using pGALS, significant findings were observed in 18/53 (34%) children; these related to fractures (4/18), hypermobility (4/18), infectious causes (5/18) and soft tissue trauma (5/18). CONCLUSION: Using this Spanish translation, pGALS assessment was practical, acceptable and effective in detecting musculoskeletal changes in many children.

Malignancy without immortality? Cellular immortalization as a possible late event in melanoma progression.

Cell senescence is a permanent growth arrest following extended proliferation. Cultured cancer cells including metastatic melanoma cells often appear immortal (proliferate indefinitely), while uncultured benign nevi (moles) show senescence markers. Here, with new explantation methods, we investigated which classes of primary pigmented lesions are typically immortal. Nevi yielded a few proliferating cells, consistent with most nevus cells being senescent. No nevus culture (0/28) appeared immortal. Some thin and thick melanoma cultures proved immortal under these conditions, but surprisingly few (4/37). All arrested cultures displayed three senescence markers in some cells: ß-galactosidase, nuclear p16, and heterochromatic foci/aggregates. However, melanoma cultures also showed features of telomeric crisis (arrest because of ultrashort telomeres). Moreover, crisis markers including anaphase bridges were frequent in uncultured vertical growth-phase (VGP) melanomas. Conversely, all immortal melanoma cultures expressed telomerase reverse transcriptase and telomerase, showing aneuploidy. The findings suggest that primary melanomas are typically precrisis, with immortalization/telomere maintenance as a late event.

p16/cyclin-dependent kinase inhibitor 2A deficiency in human melanocyte senescence, apoptosis, and immortalization: possible implications for melanoma progression.

BACKGROUND: The melanoma susceptibility locus cyclin-dependent kinase inhibitor 2A encodes two unrelated cell growth inhibitors, p16 and alternative reading frame (ARF). In fibroblasts, both proteins are implicated in cellular senescence, a key barrier to tumor development. The p16 coding sequence is more often mutated in melanoma families than is the ARF sequence. To investigate the role of p16 in melanocytes, we assessed aspects of growth, apoptosis, and immortalization in melanocytes cultured from two melanoma patients, both of whom had two inactive p16 alleles but functional ARF. METHODS: Growth and senescence were evaluated by cumulative population-doubling curves, and apoptosis by terminal deoxytransferase labeling. Expression of p53 and p21, which are associated with fibroblast senescence, was assessed by immunoblotting. Amphotropic retroviruses were used to transfer exogenous gene sequences into the melanocytes. RESULTS: Both melanocyte cultures showed high rates of apoptosis, which were reduced when the cells were grown in the presence of keratinocyte feeder cells or human stem cell factor plus endothelin 1. With these growth factors, both cultures proliferated for 45-55 net population doublings, markedly longer than the maximum of 10 net population doublings of normal adult human melanocytes in similar media, indicating impaired senescence. One of the cultures developed chromosomal aberrations, with numerous dicentric chromosomes at senescence, consistent with telomere dysfunction. p53 and p21 levels were not elevated in senescent normal melanocytes but were elevated in senescent p16-deficient melanocytes. Interference with p53 function by transfer of human papillomavirus 16-E6 further extended the lifespan of p16-deficient melanocytes. Human telomerase reverse transcriptase was sufficient to immortalize both these cell strains but not normal melanocytes. CONCLUSION: Normal senescence in human melanocytes requires p16 activity. p53 contributes to a delayed form of senescence that requires telomere shortening, in p16-deficient melanocytes. These findings provide some basis for the role of p16 in melanoma susceptibility.