Lifetime imaging microscopy with sub-micron resolution provides essential understanding of living systems by allowing both the visualisation of their structure, and the sensing of bio-relevant analytes in vivo using external probes. Chemistry is pivotal for the development of the next generation of bio-tools, where contrast, sensitivity, and molecular specificity facilitate observation of processes fundamental to life. A fundamental limitation at present is the nanosecond lifetime of conventional fluorescent probes which typically confines the sensitivity to sub-nanosecond changes, whilst nanosecond background autofluorescence compromises the contrast. High-resolution visualization with complete background rejection and simultaneous mapping of bio-relevant analytes including oxygenwith sensitivity orders of magnitude higher than that currently attainablecan be achieved using time-resolved emission imaging microscopy (TREM) in conjunction with probes with microsecond (or longer) lifetimes. Yet the microsecond timescale has so far been incompatible with available multiphoton excitation/detection technologies. Here we realize for the first time microsecondimaging with multiphoton excitation whilst maintaining the essential sub-micron spatial resolution. The new method is background-free and expands available imaging and sensing timescales 1000-fold. Exploiting the first engineered water-soluble member of a family of remarkably emissive platinum-based, microsecond-lived probes amongst others, we demonstrate (i) the first instance of background-free multiphoton-excited microsecond depth imaging of live cells and histological tissues, (ii) over an order-of-magnitude variation in the probe lifetime in vivo in response to the local microenvironment. The concept of two-photon TREM can be seen as "FLIM + PLIM" as it can be used on any timescale, from ultrafast fluorescence of organic molecules to slower emission of transition metal complexes or lanthanides/actinides, and combinations thereof. It brings together transition metal complexes as versatile emissive probes with the new multiphoton-excitation/ microsecond-detection approach to create a transformative framework for multiphoton imaging and sensing across biological, medicinal and material sciences.
Background & AimsPancreatic ductal adenocarcinoma (PDAC) is often lethal because it is highly invasive and metastasizes rapidly. The actin-bundling protein fascin has been identified as a biomarker of invasive and advanced PDAC and regulates cell migration and invasion in vitro. We investigated fascin expression and its role in PDAC progression in mice.MethodsWe used KRasG12D p53R172H Pdx1-Cre (KPC) mice to investigate the effects of fascin deficiency on development of pancreatic intraepithelial neoplasia (PanIn), PDAC, and metastasis. We measured levels of fascin in PDAC cell lines and 122 human resected PDAC samples, along with normal ductal and acinar tissues; we associated levels with patient outcomes.ResultsPancreatic ducts and acini from control mice and early-stage PanINs from KPC mice were negative for fascin, but approximately 6% of PanIN3 and 100% of PDAC expressed fascin. Fascin-deficient KRasG12D p53R172H Pdx1-Cre mice had longer survival times, delayed onset of PDAC, and a lower PDAC tumor burdens than KPC mice; loss of fascin did not affect invasion of PDAC into bowel or peritoneum in mice. Levels of slug and fascin correlated in PDAC cells; slug was found to regulate transcription of Fascin along with the epithelial−mesenchymal transition. In PDAC cell lines and cells from mice, fascin concentrated in filopodia and was required for their assembly and turnover. Fascin promoted intercalation of filopodia into mesothelial cell layers and cell invasion. Nearly all human PDAC samples expressed fascin, and higher fascin histoscores correlated with poor outcomes, vascular invasion, and time to recurrence.ConclusionsThe actin-bundling protein fascin is regulated by slug and involved in late-stage PanIN and PDAC formation in mice. Fascin appears to promote formation of filopodia and invasive activities of PDAC cells. Its levels in human PDAC correlate with outcomes and time to recurrence, indicating it might be a marker or therapeutic target for pancreatic cancer.
BackgroundHealth care professionals with positive attitudes towards the functional abilities of patients with low back pain are more likely to encourage activity and avoidance of rest as per recommended guidelines. This study investigated whether medical student training fosters positive attitudes towards patients with back pain and their ability to function.MethodsFirst (n = 202) and final (n = 146) year medical students at the University of Glasgow completed the Health Care Professionals' Pain and Impairment Relationship Scale (HC-PAIRS) questionnaire. This measures attitudes of clinicians towards the functional ability of patients with back pain. A group of first (n = 62) and final year (n = 61) business students acted as non-health care controls. Attitudes were compared using two-way ANOVA with year of study and discipline of degree as independent variables.ResultsBoth year of study [F(1,465) = 39.5, p < 0.01] and discipline of degree [F(1,465) = 43.6, p < 0.01] had significant effects on total HC-PAIRS scores and there was a significant interaction effect [F(1,465) = 9.5, p < 0.01]. Medical students commenced their course with more positive attitudes than non-health care students (65.7 vs. 69.2 respectively; p < 0.01) - lower scores translating into more positive attitudes. In their final year, the difference between the two student groups had widened (56.4 vs. 65.3; p < 0.01).ConclusionsUndergraduate medical training promotes positive attitudes towards the functional ability of patients with back pain, suggesting that students may be more likely to develop an evidence-based approach to this patient group after qualification. Some adjustments to training may be warranted to encourage a more positive shift in attitudes.
The formation of epithelial tissues allows organisms to specialise and form tissues with diverse functions and compartmentalised environments. The tight controls on cell growth and migration required to maintain epithelia can present problems such as the development and spread of cancer when normal pathways are disrupted. By attaining a deeper understanding of how cell migration is suppressed to maintain the epithelial organisation and how it is reactivated when epithelial tissues become mesenchymal, new insights into both cancer and development can be gained. Here we discuss recent developments in our understanding of epithelial and mesenchymal regulation of the actin cytoskeleton in normal and cancerous tissue, with a focus on the pancreas and intestinal tract.
The evolution of cellular pathology as a specialty has always been driven by technological developments and the clinical relevance of incorporating novel investigations into diagnostic practice. In recent years, the molecular characterisation of cancer has become of crucial relevance in patient treatment both for predictive testing and subclassification of certain tumours. Much of this has become possible due to the availability of next-generation sequencing technologies and the whole-genome sequencing of tumours is now being rolled out into clinical practice in England via the 100 000 Genome Project. The effective integration of cellular pathology reporting and genomic characterisation is crucial to ensure the morphological and genomic data are interpreted in the relevant context, though despite this, in many UK centres molecular testing is entirely detached from cellular pathology departments. The CM-Path initiative recognises there is a genomics knowledge and skills gap within cellular pathology that needs to be bridged through an upskilling of the current workforce and a redesign of pathology training. Bridging this gap will allow the development of an integrated ‘morphomolecular pathology’ specialty, which can maintain the relevance of cellular pathology at the centre of cancer patient management and allow the pathology community to continue to be a major influence in cancer discovery as well as playing a driving role in the delivery of precision medicine approaches. Here, several alternative models of pathology training, designed to address this challenge, are presented and appraised.
N‐WASP (WASL) is a widely expressed cytoskeletal signalling and scaffold protein also implicated in regulation of Wnt signalling and homeostatic maintenance of skin epithelial architecture. N‐WASP mediates invasion of cancer cells in vitro and its depletion reduces invasion and metastatic dissemination of breast cancer. Given this role in cancer invasion and universal expression in the gastrointestinal tract, we explored a role for N‐WASP in the initiation and progression of colorectal cancer. While deletion of N‐wasp is not detectably harmful in the murine intestinal tract, numbers of Paneth cells increased, indicating potential changes in the stem cell niche, and migration up the crypt–villus axis was enhanced. Loss of N‐wasp promoted adenoma formation in an adenomatous polyposis coli (Apc) deletion model of intestinal tumourigenesis. Thus, we establish a tumour suppressive role of N‐WASP in early intestinal carcinogenesis despite its later pro‐invasive role in other cancers. Our study highlights that while the actin cytoskeletal machinery promotes invasion of cancer cells, it also maintains normal epithelial tissue function and thus may have tumour suppressive roles in pre‐neoplastic tissues. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
Background: Healthcare professionals' (HCPs') attitudes towards pain influence their pain management. Attitudes about pain should be aligned with the evidence-base at the pre-registration stage of an HCP's career but pain education at undergraduate level is often lacking, and negative attitudes can pervade HCP practise. Previous studies investigating change in pain attitudes in undergraduate HCPs are crosssectional in nature and frequently report minimal change in pain attitudes.Objectives: To investigate medical students' attitudes and beliefs towards people with chronic pain over the course of their Scottish undergraduate programme. Design: Five year observational study.Setting: A Scottish university medical school.Participants: Medical students were recruited in first year and followed up to their final year (year one n=205/244, year two n=190/245, year three n=132/279, year four n=110/262, year four n=159/260) for five years.Outcome Measure: The Health Care Providers' Pain and Impairment Relationship Scale (HC-PAIRS with scores ranging from 15-105) was completed annually.Results: A two-way ANOVA found that attitudes and beliefs improved significantly (p<0.01) from first to final year (10.0±10.0). Medics showed a gradual reduction in scores (signifying improved attitudes) annually.Conclusions: This is the first known published study to chart changes in the same cohort of medical students' attitudes and beliefs towards people with chronic pain over time. Changes in attitudes improved steadily over the five year medical degree course. Future work should explore which aspects of degree courses, if any, impact upon attitudes and beliefs towards people with chronic pain so that courses can be enhanced accordingly. Contribution of the Paper:• This is the first known published observational study of pain attitudes in the same group of medical students from first to final year of undergraduate training.• It confirms the findings from previous cross-sectional literature that there is an improvement in attitudes amongst medical students during the course of their usual undergraduate training.• The change in attitudes whilst of a meaningful magnitude, is relatively modest and there is clear potential for improvement.
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