Corrected Super-Resolution Microscopy Enables Nanoscale Imaging of Autofluorescent Lung Macrophages.

Observing the cell surface and underlying cytoskeleton at nanoscale resolution using super-resolution microscopy has enabled many insights into cell signaling and function. However, the nanoscale dynamics of tissue-specific immune cells have been relatively little studied. Tissue macrophages, for example, are highly autofluorescent, severely limiting the utility of light microscopy. Here, we report a correction technique to remove autofluorescent noise from stochastic optical reconstruction microscopy (STORM) data sets. Simulations and analysis of experimental data identified a moving median filter as an accurate and robust correction technique, which is widely applicable across challenging biological samples. Here, we used this method to visualize lung macrophages activated through Fc receptors by antibody-coated glass slides. Accurate, nanoscale quantification of macrophage morphology revealed that activation induced the formation of cellular protrusions tipped with MHC class I protein. These data are consistent with a role for lung macrophage protrusions in antigen presentation. Moreover, the tetraspanin protein CD81, known to mark extracellular vesicles, appeared in ring-shaped structures (mean diameter 93 ± 50 nm) at the surface of activated lung macrophages. Thus, a moving median filter correction technique allowed us to quantitatively analyze extracellular secretions and membrane structure in tissue-derived immune cells.

Tracking the Evolution of Non-Small-Cell Lung Cancer.

BACKGROUND: Among patients with non-small-cell lung cancer (NSCLC), data on intratumor heterogeneity and cancer genome evolution have been limited to small retrospective cohorts. We wanted to prospectively investigate intratumor heterogeneity in relation to clinical outcome and to determine the clonal nature of driver events and evolutionary processes in early-stage NSCLC. METHODS: In this prospective cohort study, we performed multiregion whole-exome sequencing on 100 early-stage NSCLC tumors that had been resected before systemic therapy. We sequenced and analyzed 327 tumor regions to define evolutionary histories, obtain a census of clonal and subclonal events, and assess the relationship between intratumor heterogeneity and recurrence-free survival. RESULTS: We observed widespread intratumor heterogeneity for both somatic copy-number alterations and mutations. Driver mutations in EGFR, MET, BRAF, and TP53 were almost always clonal. However, heterogeneous driver alterations that occurred later in evolution were found in more than 75% of the tumors and were common in PIK3CA and NF1 and in genes that are involved in chromatin modification and DNA damage response and repair. Genome doubling and ongoing dynamic chromosomal instability were associated with intratumor heterogeneity and resulted in parallel evolution of driver somatic copy-number alterations, including amplifications in CDK4, FOXA1, and BCL11A. Elevated copy-number heterogeneity was associated with an increased risk of recurrence or death (hazard ratio, 4.9; P=4.4×10-4), which remained significant in multivariate analysis. CONCLUSIONS: Intratumor heterogeneity mediated through chromosome instability was associated with an increased risk of recurrence or death, a finding that supports the potential value of chromosome instability as a prognostic predictor. (Funded by Cancer Research UK and others; TRACERx ClinicalTrials.gov number, NCT01888601 .).

Programmed death-ligand 1 expression influenced by tissue sample size. Scoring based on tissue microarrays' and cross-validation with resections, in patients with, stage I-III, non-small cell lung carcinoma of the European Thoracic Oncology Platform Lungscape cohort.

PD-L1, as assessed by immunohistochemistry, is a predictive biomarker for immuno-oncology treatment in lung cancer. Different scoring methods have been used to assess its status, resulting in a wide range of positivity rates. We use the European Thoracic Oncology Platform Lungscape non-small cell lung carcinoma cohort to explore this issue. PD-L1 expression was assessed via immunohistochemistry on tissue microarrays (up to four cores per case), using the DAKO 28-8 immunohistochemistry assay, following a two-round external quality assessment procedure. All samples were analyzed under the same protocol. Cross-validation of scoring between tissue microarray and whole sections was performed in 10% randomly selected samples. Cutoff points considered: ≥1, 50 (primarily), and 25%. At the two external quality assessment rounds, tissue microarray scoring agreement rates between pathologists were: 73% and 81%. There were 2008 cases with valid immunohistochemistry tissue microarray results (50% all cores evaluable). Concordant cases at 1, 25, and 50% were: 85, 91, and 93%. Tissue microarray core results were identical for 70% of cases. Sensitivity of the tissue microarray method for 1, 25, and 50% was: 80, 78, and 79% (specificity: 90, 95, 98%). Complete agreement between tissue microarrays and whole sections was achieved for 60% of the cases. Highest sensitivity rates for 1% and 50% cutoffs were detected for higher number of cores. Underestimation of PD-L1 expression on small samples is more common than overestimation. We demonstrated that classification of PD-L1 on small biopsy samples does not represent the overall expression of PD-L1 in all non-small cell cancer carcinoma cases, although the majority of cases are 'correctly' classified. In future studies, sampling more and larger biopsies, recording the biopsy size and tumor load may permit further refinement, increasing predictive accuracy.

The pathology of small airways disease in COPD: historical aspects and future directions.

Small airways disease (SAD) is a cardinal feature of chronic obstructive pulmonary disease (COPD) first recognized in the nineteenth century. The diverse histopathological features associated with SAD underpin the heterogeneous nature of COPD. Our understanding of the key molecular mechanisms which drive the pathological changes are not complete. In this article we will provide a historical overview of key histopathological studies which have helped shape our understanding of SAD and discuss the hallmark features of airway remodelling, mucous plugging and inflammation. We focus on the relationship between SAD and emphysema, SAD in the early stages of COPD, and the mechanisms which cause SAD progression, including bacterial colonization and exacerbations. We discuss the need to specifically target SAD to attenuate the progression of COPD.

Cilia have high cAMP levels that are inhibited by Sonic Hedgehog-regulated calcium dynamics.

Protein kinase A (PKA) phosphorylates Gli proteins, acting as a negative regulator of the Hedgehog pathway. PKA was recently detected within the cilium, and PKA activity specifically in cilia regulates Gli processing. Using a cilia-targeted genetically encoded sensor, we found significant basal PKA activity. Using another targeted sensor, we measured basal ciliary cAMP that is fivefold higher than whole-cell cAMP. The elevated basal ciliary cAMP level is a result of adenylyl cyclase 5 and 6 activity that depends on ciliary phosphatidylinositol (3,4,5)-trisphosphate (PIP3), not stimulatory G protein (Gαs), signaling. Sonic Hedgehog (SHH) reduces ciliary cAMP levels, inhibits ciliary PKA activity, and increases Gli1. Remarkably, SHH regulation of ciliary cAMP and downstream signals is not dependent on inhibitory G protein (Gαi/o) signaling but rather Ca2+ entry through a Gd3+-sensitive channel. Therefore, PIP3 sustains high basal cAMP that maintains PKA activity in cilia and Gli repression. SHH activates Gli by inhibiting cAMP through a G protein-independent mechanism that requires extracellular Ca2+ entry.

Can community health workers increase modern contraceptive use among young married women? A cross-sectional study in rural Niger.

BACKGROUND: The Republic of Niger has the highest rate of early marriage and adolescent fertility in the world. Recent global health initiatives, such as Family Planning 2020, have reinvigorated investments in family planning in low- and middle-income countries (LMICs). As part of this initiative, Niger has implemented ambitious plans to increase contraceptive prevalence through policies designed to increase coverage and access to family planning services. One strategy involves the deployment of volunteer community health workers (relais communautaires) in rural settings to improve access to family planning services, especially among adolescents and youth. The objective of this article is to determine if visits by relais are associated with increased use of modern contraception among young married women in rural Niger. METHODS: Cross-sectional data from a household survey were collected from young married women between the ages of 13 and 19 in three rural districts in the region of Dosso, Niger from May to August 2016. Multivariate logistic regression was conducted to assess the odds of married female youth reporting current use of modern contraceptive methods based on being visited by a relais in the past three months. RESULTS: A total of 956 young married women were included in the final analysis. Among study participants, 9.3% reported a relais visit to discuss health issues in the past three months and 11.4% reported currently using a modern method of contraception. Controlling for socio-demographic variables, the odds of current use of modern contraceptive methods were higher among young married women who were visited by a relais in the last three months compared to those not visited by a relais during this period (AOR = 1.94[95% CI 1.07-3.51]). In this study setting, relais were less likely to visit nulliparous women and women that worked in the past 12 months. CONCLUSION: Young married women visited by relais were more likely to use modern contraceptive methods compared to those not visited by a relais. These results are consistent with similar family planning studies from sub-Saharan Africa and suggest that relais in Niger may be able to provide access to essential family planning services in rural and hard-to-reach areas. Additional efforts to understand the contraceptive barriers faced by nulliparous women and working women should be a key research priority in Niger. TRIAL REGISTRATION: Clinical trial registration number 2016-1430 ; registered on October 7, 2016 (retrospectively registered).

Development and evaluation of a primary care interprofessional education intervention to support people with dementia.

International policy documents emphasize the need to develop interprofessional education (IPE) to support collaborative dementia care in the community. The aim of this short report is to describe the development and pilot evaluation of an interprofessional dementia education workshop for primary care healthcare professionals. A three-hour workshop was iteratively developed through consultation with an expert reference group and people with dementia. The workshop was piloted with three separate primary care teams. A total of fifty-four primary care based healthcare staff who represented fourteen different health care roles in primary care participated in the pilot evaluation. The pilot workshops were evaluated using a mixed method approach which included post-workshop questionnaires for participants (n= 54)and a post-workshop focus group (n=8) with the program design team and workshop facilitators. The results of the pilot phase indicated that the workshop was useful and feasible. The workshop improved participants' self-reported knowledge, understanding and confidence to support people with dementia and their families.Areas for improvement were identified and will be used to inform improvements to the workshop content and delivery in advance of a national roll-out. Future evaluations of the implementation of this interprofessional educational workshop will focus on its impact on healthcare professional behavior and outcomes for the person with dementia and their families.

Ratiometric biosensors based on dimerization-dependent fluorescent protein exchange.

We have developed a versatile new class of genetically encoded fluorescent biosensor based on reversible exchange of the heterodimeric partners of green and red dimerization-dependent fluorescent proteins. We demonstrate the use of this strategy to construct both intermolecular and intramolecular ratiometric biosensors for qualitative imaging of caspase activity, Ca(2+) concentration dynamics and other second-messenger signaling activities.

Dielectric characterisation of human adrenal glands and adrenal tumours for the development of microwave ablation technologies for hypertension treatment.

Adrenal gland-induced hypertension results from underlying adrenal gland disorders including Conn's syndrome, Cushing's syndrome, and Pheochromocytoma. These adrenal disorders are a risk for cardiovascular and renal morbidity and mortality. Clinically, treatment for adrenal gland-induced hypertension involves a pharmaceutical or surgical approach. The former presents very significant side effects whereas the latter can be ineffective in cases where the adrenal disorder reoccurs in the remaining contralateral adrenal gland. Due to the limitations of existing treatment methods, minimally invasive treatment options like microwave ablation (MWA) have received significant attention for treating adrenal gland disorders. A precise comprehension of the dielectric properties of human adrenal glands will help to tailor energy delivery for MWA therapy, thus offering the potential to optimise treatments and minimise damage to surrounding tissues. This study reports the ex vivo dielectric properties of human adrenal glands, including the cortex, medulla, capsule, and tumours, based on the data obtained from four patients (diagnosed with Conn's syndrome, Cushing's syndrome, and Pheochromocytoma) who underwent unilateral adrenalectomy at the University Hospital Galway, Ireland. An open-ended coaxial probe measurement technique was used to measure the dielectric properties for a frequency range of 0.5-8.5 GHz. The dielectric properties were fitted using a two-pole Debye model, and a weighted least squares method was employed to optimise the model parameters. Moreover, the dielectric properties of adrenal tissues and tumours were compared across frequencies commonly used in MWA, including 915 MHz, 2.45 GHz, and 5.8 GHz. The study found that the dielectric properties of adrenal tumours were influenced by the presence of lipid-rich adenomas, and the dielectric properties of Cushing's syndrome tumour were lowest in comparison to the tumours in patients diagnosed with Conn's syndrome and Pheochromocytoma. Furthermore, a notable difference was observed in the dielectric properties of the medulla and cortex among patients diagnosed with Conn's syndrome, Cushing's syndrome, and Pheochromocytoma. These findings have significant implications for the diagnosis and treatment of adrenal tumours, including the optimisation of MWA therapy for precise ablation of adrenal masses.

Histology-validated electromagnetic characterization of ex-vivo ovine lung tissue for microwave-based medical applications.

Microwave thermal ablation is an established therapeutic technique for treating malignant tissue in various organs. Its success greatly depends on the knowledge of dielectric properties of the targeted tissue and on how they change during the treatment. Innovation in lung navigation has recently increased the clinical interest in the transbronchial microwave ablation treatment of lung cancer. However, lung tissue is not largely characterized, thus its dielectric properties investigation prior and post ablation is key. In this work, dielectric properties of ex-vivo ovine lung parenchyma untreated and ablated at 2.45 GHz were recorded in the 0.5-8 GHz frequency range. The measured dielectric properties were fitted to 2-pole Cole-Cole relaxation model and the obtained model parameters were compared. Based on observed changes in the model parameters, the physical changes of the tissue post-ablation were discussed and validated through histology analysis. Additionally, to investigate the link of achieved results with the rate of heating, another two sets of samples, originating from both ovine and porcine tissues, were heated with a microwave oven for different times and at different powers. Dielectric properties were measured in the same frequency range. It was found that lung tissue experiences a different behavior according to heating rates: its dielectric properties increase post-ablation while a decrease is found for low rates of heating. It is hypothesized, and validated by histology, that during ablation, although the tissue is losing water, the air cavities deform, lowering air content and increasing the resulting tissue properties.

Successful Treatment of Refractory Cutaneous Protothecosis With MAT2203, an Oral Lipid Nanocrystal Formulation of Amphotericin B.

Prototheca wickerhamii is a rare cause of cutaneous and systemic infection that requires long treatment courses with potentially toxic medications. We describe a patient with cutaneous protothecosis refractory to triazole monotherapy who experienced clinical and radiographic improvement with the novel oral lipid nanocrystal formulation of amphotericin B without experiencing toxicity.

Unique correlation between GTF2I mutation and spindle cell morphology in thymomas (type A and AB thymomas).

AIM: Recent study has revealed frequent GTF2I mutation in thymomas, with the frequency being highest in types A and AB, followed by B1, B2, B3 and thymic carcinoma. This has led to the conclusion that GTF2I mutation correlates with more indolent histology subtype and better prognosis. In our study, the GTF2I mutation was tested in thymic epithelial tumours to investigate the relation between the mutation status and histology subtype. METHODS: The GTF2I mutation was tested in 111 thymic epithelial tumours by Sanger sequencing. Correlations between GTF2I mutation status and clinicopathological parameters were evaluated. RESULTS: There were 16 cases of type A, including atypical type, 37 type AB, 13 B1, 23 B2, 9 B3, 6 micronodular type, 2 metaplastic type and 5 thymic carcinomas. GTF2I mutation was seen in 78.6% of type A and 83.9% of type AB, while it was not expressed in type B, metaplastic type or thymic carcinoma (p<0.001). 75% of micronodular type also showed the mutation. Both thymoma histotype and stage were significantly associated with GTF2I mutation by univariate analysis. The presence of GTF2I mutation showed a trend towards a favourable prognosis, but this is likely due to their strong association with more indolent histologic subtypes (types A and AB). CONCLUSIONS: GTF2I mutation appears unique in type A and AB thymomas, including those with atypical features and micronodular type, all of which share spindle cell morphology, indicating they represent a group biologically distinct from type B thymomas.

A pleural based mass in a post-partum woman.

Can you diagnose this case of a 27-year-old female who presented 1-week post-partum with an incidental finding of intrathoracic masses and probable hilar lymphadenopathy? https://bit.ly/3S3ejVK.

Histology-Validated Dielectric Characterisation of Lung Carcinoma Tissue for Microwave Thermal Ablation Applications.

Microwave thermal ablation is a promising emerging treatment for early-stage lung cancer. Applicator design optimisation and treatment planning rely on accurate knowledge of dielectric tissue properties. Limited dielectric data are available in the literature for human lung tissue and pulmonary tumours. In this work, neoplastic and non-neoplastic lung dielectric properties are characterised and correlated with gross and histological morphology. Fifty-six surgical specimens were obtained from twelve patients undergoing lung resection for lung cancer in University Hospital of Galway, Ireland. Dielectric spectroscopy in the microwave frequency range (500 MHz-8.5 GHz) was performed on the ex vivo lung specimens with the open-ended coaxial probe technique (in the Department of Pathology). Dielectric data were analysed and correlated with the tissue histology. The dielectric properties of twelve lung tumours (67% non-small cell carcinoma (NSCC)) and uninvolved lung parenchyma were obtained. The values obtained from the neoplastic lung specimens (relative permittivity: 52.0 ± 5.4, effective conductivity: 1.9 ± 0.2 S/m, at 2.45 GHz) were on average twice the value of the non-neoplastic lung specimens (relative permittivity: 28.3 ± 6.7, effective conductivity: 1.0 ± 0.3 S/m, at 2.45 GHz). Dense fibrosis was comparable with tumour tissue (relative permittivity 49.3 ± 4.6, effective conductivity: 1.8 ± 0.1 S/m, at 2.45 GHz).

Extrapulmonary small cell carcinoma: a clinicopathological study with identification of potential diagnostic mimics.

AIMS: To evaluate the clinicopathological features of small cell carcinoma arising outside the lung. METHODS AND RESULTS: Thirty-seven cases with a pathology diagnosis of extrapulmonary small cell carcinoma (EPSCC) were selected. The clinical notes were reviewed and tumour blocks were selected for a fresh haematoxylin and eosin (H&E) section and immunohistochemical stains. The most common tumour locations were cervix and bladder. Twenty-five cases (68%) were finally diagnosed as EPSCC, nine of which were found with coexisting non small cell carcinoma. Two cases (5%) were diagnosed as large cell neuroendocrine carcinoma (LCNEC) of the cervix. The remainder was classified as 10 poorly differentiated carcinomas (PDCs) (27%). Positive staining for thyroid transcription factor 1 (TTF-1) was noted in nine cases of EPSCC and in none of the cases of PDC (P = 0.034). Synaptophysin immunoreactivity was found in 20 cases of EPSCC and two cases of PDC with neuroendocrine differentiation (P = 0.002), as well as two cases of LCNEC. 34ßE12 was positive in eight cases of SCC and two cases of PDC. CONCLUSIONS: Based on this series, EPSCC may be overdiagnosed. Immunohistochemistry for TTF-1, used in combination with synaptophysin, may help to discriminate EPSCC from PDC.

Giant Nonfunctioning Parathyroid Cyst: A Case Report and Review of the Literature.

Parathyroid cysts are a rare clinical entity that may arise in the neck or mediastinum. They are more common in women and generally present in the fourth and fifth decades of life. Diagnosis of parathyroid cysts is challenging, and despite thorough radiological and cytological investigation, they are often mistaken for thyroid pathology. Definitive diagnosis is often only confirmed following complete surgical resection and histopathological analysis. We present the case of a woman who was referred to our outpatient clinic with a left-sided neck mass and associated compressive symptoms. Initial examination and investigation appeared consistent with a large thyroid nodule. Following surgical resection, the lesion was found to be a parathyroid cyst. Subsequently, we review the available literature on parathyroid cysts with particular emphasis on the diagnostic challenge they pose to clinicians.

My Life, My Story: Integrating a Life Story Narrative Component Into Medical Student Curricula.

INTRODUCTION: Medical students experience burnout, depersonalization, and decreases in empathy throughout medical training. My Life, My Story (MLMS) is a narrative medicine project that aims to combat these adverse outcomes by teaching students to interview patients about their life story, with the goal of improving patient-centered care competencies, such as empathy. METHODS: The MLMS project was started in the Veterans Affairs (VA) system and has since spread to dozens of VA sites. We adapted and integrated this project into the Warren Alpert Medical School of Brown University curriculum. As part of the required curriculum, first- and third-year medical students participated in a life story interview with a community-based volunteer or a patient in the inpatient hospital setting, transcribed the story, and reviewed the written story with the patient. We assessed student perceptions of the project, changes in empathy, and changes in burnout symptoms. RESULTS: A total of 240 students participated in this project. Students spent an average of 70.7 minutes interviewing patients. A majority of the students believed MLMS was a good use of time (77%), fostered connection with patients (79%), and was effective in recognizing patients' thoughts and feelings (69%). DISCUSSION: To our knowledge, this is one of the first life story interview interventions to be implemented into a required medical school curriculum and outside the VA setting. MLMS may assist students in improving clinical empathy skills and create a structure for medical trainees to better understand their patients.

Heterogeneity in extracellular vesicle secretion by single human macrophages revealed by super-resolution microscopy.

The diverse origins, nanometre-scale and invasive isolation procedures associated with extracellular vesicles (EVs) mean they are usually studied in bulk and disconnected from their parental cell. Here, we used super-resolution microscopy to directly compare EVs secreted by individual human monocyte-derived macrophages (MDMs). MDMs were differentiated to be M0-, M1- or M2-like, with all three secreting EVs at similar densities following activation. However, M0-like cells secreted larger EVs than M1- and M2-like macrophages. Proteomic analysis revealed variations in the contents of differently sized EVs as well as between EVs secreted by different MDM phenotypes. Super resolution microscopy of single-cell secretions identified that the class II MHC protein, HLA-DR, was expressed on ∼40% of EVs secreted from M1-like MDMs, which was double the frequency observed for M0-like and M2-like EVs. Strikingly, human macrophages, isolated from the resected lungs of cancer patients, secreted EVs that expressed HLA-DR at double the frequency and with greater intensity than M1-like EVs. Quantitative analysis of single-cell EV profiles from all four macrophage phenotypes revealed distinct secretion types, five of which were consistent across multiple sample cohorts. A sub-population of M1-like MDMs secreted EVs similar to lung macrophages, suggesting an expansion or recruitment of cells with a specific EV secretion profile within the lungs of cancer patients. Thus, quantitative analysis of EV heterogeneity can be used for single cell profiling and to reveal novel macrophage biology.

Quantifying the Kinetics of Signaling and Arrestin Recruitment by Nervous System G-Protein Coupled Receptors.

Neurons integrate inputs over different time and space scales. Fast excitatory synapses at boutons (ms and µm), and slow modulation over entire dendritic arbors (seconds and mm) are all ultimately combined to produce behavior. Understanding the timing of signaling events mediated by G-protein-coupled receptors is necessary to elucidate the mechanism of action of therapeutics targeting the nervous system. Measuring signaling kinetics in live cells has been transformed by the adoption of fluorescent biosensors and dyes that convert biological signals into optical signals that are conveniently recorded by microscopic imaging or by fluorescence plate readers. Quantifying the timing of signaling has now become routine with the application of equations in familiar curve fitting software to estimate the rates of signaling from the waveform. Here we describe examples of the application of these methods, including (1) Kinetic analysis of opioid signaling dynamics and partial agonism measured using cAMP and arrestin biosensors; (2) Quantifying the signaling activity of illicit synthetic cannabinoid receptor agonists measured using a fluorescent membrane potential dye; (3) Demonstration of multiplicity of arrestin functions from analysis of biosensor waveforms and quantification of the rates of these processes. These examples show how temporal analysis provides additional dimensions to enhance the understanding of GPCR signaling and therapeutic mechanisms in the nervous system.