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.

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.

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.

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.

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.

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.

Germline FFPE inherited cancer panel testing in deceased family members: implications for clinical management of unaffected relatives.

Where previously, germline genetic testing in deceased affected relatives was not possible due to the absence of lymphocytic DNA, the North-West-Genomic-Laboratory Hub (NWGLH) has developed and validated next-generation sequencing based gene panels utilising formalin-fixed-paraffin-embedded (FFPE) tissue DNA from deceased individuals. This technology has been utilised in the clinical setting for the management of unaffected relatives seen in the Clinical Genetics Service (CGS). Here we assess the clinical impact. At the time of data collection, the NWGLH had analysed 180 FFPE tissue samples from deceased affected individuals: 134 from breast and/or ovarian cancer cases for germline variants in the BRCA1/BRCA2 genes and 46 from colorectal, gastric, ovarian and endometrial cancer cases for germline variants in a panel of 13 genes implicated in inherited colorectal cancer and gastric cancer conditions. Successful analysis was achieved in 140/180 cases (78%). In total, 29 germline pathogenic/likely pathogenic variants were identified in autosomal dominant cancer predisposition genes where the gene was pertinent to the cancer family history (including BRCA1/BRCA2, the mismatch-repair genes and APC). Of the 180 cases, the impact of the result on clinical management of unaffected relatives was known in 143 cases. Of these, the results in 54 cases (38%) directly impacted the clinical management of relatives seen by the CGS. This included changes to risk assessments, screening recommendations and the availability of predictive genetic testing to unaffected relatives. Our data demonstrate how FFPE testing in deceased relatives is an accurate and informative tool in the clinical management of patients referred to the CGS.

Prognostic Impact of KRAS G12C Mutation in Patients With NSCLC: Results From the European Thoracic Oncology Platform Lungscape Project.

INTRODUCTION: KRAS mutations, the most frequent gain-of-function alterations in NSCLC, are currently emerging as potential predictive therapeutic targets. The role of KRAS-G12C (Kr_G12C) is of special interest after the recent discovery and preclinical analyses of two different Kr_G12C covalent inhibitors (AMG-510, MRTX849). METHODS: KRAS mutations were evaluated in formalin-fixed, paraffin-embedded tissue sections by a microfluidic-based multiplex polymerase chain reaction platform as a component of the previously published European Thoracic Oncology Platform Lungscape 003 Multiplex Mutation study, of clinically annotated, resected, stage I to III NSCLC. In this study, -Kr_G12C mutation prevalence and its association with clinicopathologic characteristics, molecular profiles, and postoperative patient outcome (overall survival, relapse-free survival, time-to-relapse) were explored. RESULTS: KRAS gene was tested in 2055 Lungscape cases (adenocarcinomas: 1014 [49%]) with I or II or III stage respective distribution of 53% or 24% or 22% and median follow-up of 57 months. KRAS mutation prevalence in the adenocarcinoma cohort was 38.0% (95% confidence interval (CI): 35.0% to 41.0%), with Kr_G12C mutation representing 17.0% (95% CI: 14.7% to 19.4%). In the "histologic-subtype" cohort, Kr_G12C prevalence was 10.5% (95% CI: 9.2% to 11.9%). When adjusting for clinicopathologic characteristics, a significant negative prognostic effect of Kr_G12C presence versus other KRAS mutations or nonexistence of KRAS mutation was identified in the adenocarcinoma cohort alone and in the "histologic-subtype" cohort. For overall survival in adenocarcinomas, hazard ratio (HR)G12C versus other KRAS is equal to 1.39 (95% CI: 1.03 to 1.89, p = 0.031) and HRG12C versus no KRAS is equal to 1.32 (95% CI: 1.03 to 1.69, p = 0.028) (both also significant in the "histologic-subtype" cohort). For time-to-relapse, HRG12C versus other KRAS is equal to 1.41 (95% CI: 1.03 to 1.92, p = 0.030). In addition, among all patients, for relapse-free survival, HRG12C versus no KRAS is equal to 1.27 (95% CI: 1.04 to 1.54, p = 0.017). CONCLUSIONS: In this large, clinically annotated stage I to III NSCLC cohort, the specific Kr_G12C mutation is significantly associated with poorer prognosis (adjusting for clinicopathologic characteristics) among adenocarcinomas and in unselected NSCLCs.

Nature of the energy landscape for gated electron transfer in a dynamic redox protein.

Conformational control limits most electron transfer (ET) reactions in biology, but we lack general insight into the extent of conformational space explored, and specifically the properties of the associated energy landscape. Here we unite electron-electron double resonance (ELDOR) studies of the diradical (disemiquinoid) form of human cytochrome P450 reductase (CPR), a nicotinamide adenine phosphate dinucleotide (NADPH)-linked diflavin oxidoreductase required for P450 enzyme reduction, with functional studies of internal ET to gain new insight into the extent and properties of the energy landscape for conformationally controlled ET. We have identified multiple conformations of disemiquinoid CPR, which point to a rugged energy landscape for conformational sampling consistent with functional analysis of ET using high-pressure stopped-flow, solvent, and temperature perturbation studies. Crystal structures of CPR have identified discrete "closed" and "open" states, but we emphasize the importance of a continuum of conformational states across the energy landscape. Within the landscape more closed states that favor internal ET are formed by nucleotide binding. Open states that enable P450 enzymes to gain access to electrons located in the FMN-domain are favored in the absence of bound coenzyme. The extent and nature of energy landscapes are therefore accessible through the integration of ELDOR spectroscopy with functional studies. We suggest this is a general approach that can be used to gain new insight into energy landscapes for biological ET mediated by conformational sampling mechanisms.

A kinetic method for measuring agonist efficacy and ligand bias using high resolution biosensors and a kinetic data analysis framework.

The kinetics/dynamics of signaling are of increasing value for G-protein-coupled receptor therapeutic development, including spatiotemporal signaling and the kinetic context of biased agonism. Effective application of signaling kinetics to developing new therapeutics requires reliable kinetic assays and an analysis framework to extract kinetic pharmacological parameters. Here we describe a platform for measuring arrestin recruitment kinetics to GPCRs using a high quantum yield, genetically encoded fluorescent biosensor, and a data analysis framework to quantify the recruitment kinetics. The sensor enabled high temporal resolution measurement of arrestin recruitment to the angiotensin AT1 and vasopressin V2 receptors. The analysis quantified the initial rate of arrestin recruitment (kτ), a biologically-meaningful kinetic drug efficacy parameter, by fitting time course data using routine curve-fitting methods. Biased agonism was assessed by comparing kτ values for arrestin recruitment with those for Gq signaling via the AT1 receptor. The kτ ratio values were in good agreement with bias estimates from existing methods. This platform potentially improves and simplifies assessment of biased agonism because the same assay modality is used to compare pathways (potentially in the same cells), the analysis method is parsimonious and intuitive, and kinetic context is factored into the bias measurement.

Analyzing kinetic signaling data for G-protein-coupled receptors.

In classical pharmacology, bioassay data are fit to general equations (e.g. the dose response equation) to determine empirical drug parameters (e.g. EC50 and Emax), which are then used to calculate chemical parameters such as affinity and efficacy. Here we used a similar approach for kinetic, time course signaling data, to allow empirical and chemical definition of signaling by G-protein-coupled receptors in kinetic terms. Experimental data are analyzed using general time course equations (model-free approach) and mechanistic model equations (mechanistic approach) in the commonly-used curve-fitting program, GraphPad Prism. A literature survey indicated signaling time course data usually conform to one of four curve shapes: the straight line, association exponential curve, rise-and-fall to zero curve, and rise-and-fall to steady-state curve. In the model-free approach, the initial rate of signaling is quantified and this is done by curve-fitting to the whole time course, avoiding the need to select the linear part of the curve. It is shown that the four shapes are consistent with a mechanistic model of signaling, based on enzyme kinetics, with the shape defined by the regulation of signaling mechanisms (e.g. receptor desensitization, signal degradation). Signaling efficacy is the initial rate of signaling by agonist-occupied receptor (kτ), simply the rate of signal generation before it becomes affected by regulation mechanisms, measurable using the model-free analysis. Regulation of signaling parameters such as the receptor desensitization rate constant can be estimated if the mechanism is known. This study extends the empirical and mechanistic approach used in classical pharmacology to kinetic signaling data, facilitating optimization of new therapeutics in kinetic terms.

Internal electron transfer in multi-site redox enzymes is accessed by laser excitation of thiouredopyrene-3,6,8-trisulfonate (TUPS).

We demonstrate that thiouredopyrene-3,6,8-trisulfonate (TUPS), a photoactivatable reagent, can rapidly inject electrons into complex redox enzymes, enabling studies of the kinetics of internal electron that are not accessible using conventional rapid mixing, stopped-flow methods.