Lung extracellular matrix modulates KRT5+ basal cell activity in pulmonary fibrosis.

Aberrant expansion of KRT5+ basal cells in the distal lung accompanies progressive alveolar epithelial cell loss and tissue remodelling during fibrogenesis in idiopathic pulmonary fibrosis (IPF). The mechanisms determining activity of KRT5+ cells in IPF have not been delineated. Here, we reveal a potential mechanism by which KRT5+ cells migrate within the fibrotic lung, navigating regional differences in collagen topography. In vitro, KRT5+ cell migratory characteristics and expression of remodelling genes are modulated by extracellular matrix (ECM) composition and organisation. Mass spectrometry- based proteomics revealed compositional differences in ECM components secreted by primary human lung fibroblasts (HLF) from IPF patients compared to controls. Over-expression of ECM glycoprotein, Secreted Protein Acidic and Cysteine Rich (SPARC) in the IPF HLF matrix restricts KRT5+ cell migration in vitro. Together, our findings demonstrate how changes to the ECM in IPF directly influence KRT5+ cell behaviour and function contributing to remodelling events in the fibrotic niche.

Graphene Sensor Arrays for Rapid and Accurate Detection of Pancreatic Cancer Exosomes in Patients' Blood Plasma Samples.

Biosensors based on graphene field effect transistors (GFETs) have the potential to enable the development of point-of-care diagnostic tools for early stage disease detection. However, issues with reproducibility and manufacturing yields of graphene sensors, but also with Debye screening and unwanted detection of nonspecific species, have prevented the wider clinical use of graphene technology. Here, we demonstrate that our wafer-scalable GFETs array platform enables meaningful clinical results. As a case study of high clinical relevance, we demonstrate an accurate and robust portable GFET array biosensor platform for the detection of pancreatic ductal adenocarcinoma (PDAC) in patients' plasma through specific exosomes (GPC-1 expression) within 45 min. In order to facilitate reproducible detection in blood plasma, we optimized the analytical performance of GFET biosensors via the application of an internal control channel and the development of an optimized test protocol. Based on samples from 18 PDAC patients and 8 healthy controls, the GFET biosensor arrays could accurately discriminate between the two groups while being able to detect early cancer stages including stages 1 and 2. Furthermore, we confirmed the higher expression of GPC-1 and found that the concentration in PDAC plasma was on average more than 1 order of magnitude higher than in healthy samples. We found that these characteristics of GPC-1 cancerous exosomes are responsible for an increase in the number of target exosomes on the surface of graphene, leading to an improved signal response of the GFET biosensors. This GFET biosensor platform holds great promise for the development of an accurate tool for the rapid diagnosis of pancreatic cancer.

Chloroplasts alter their morphology and accumulate at the pathogen interface during infection by Phytophthora infestans.

Upon immune activation, chloroplasts switch off photosynthesis, produce antimicrobial compounds and associate with the nucleus through tubular extensions called stromules. Although it is well established that chloroplasts alter their position in response to light, little is known about the dynamics of chloroplast movement in response to pathogen attack. Here, we report that during infection with the Irish potato famine pathogen Phytophthora infestans, chloroplasts accumulate at the pathogen interface, associating with the specialized membrane that engulfs the pathogen haustorium. The chemical inhibition of actin polymerization reduces the accumulation of chloroplasts at pathogen haustoria, suggesting that this process is partially dependent on the actin cytoskeleton. However, chloroplast accumulation at haustoria does not necessarily rely on movement of the nucleus to this interface and is not affected by light conditions. Stromules are typically induced during infection, embracing haustoria and facilitating chloroplast interactions, to form dynamic organelle clusters. We found that infection-triggered stromule formation relies on BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1)-mediated surface immune signaling, whereas chloroplast repositioning towards haustoria does not. Consistent with the defense-related induction of stromules, effector-mediated suppression of BAK1-mediated immune signaling reduced stromule formation during infection. On the other hand, immune recognition of the same effector stimulated stromules, presumably via a different pathway. These findings implicate chloroplasts in a polarized response upon pathogen attack and point to more complex functions of these organelles in plant-pathogen interactions.

The acid injury and repair (AIR) model: A novel ex-vivo tool to understand lung repair.

Research into mechanisms underlying lung injury and subsequent repair responses is currently of paramount importance. There is a paucity of models that bridge the gap between in vitro and in vivo research. Such intermediate models are critical for researchers to decipher the mechanisms that drive repair and to test potential new treatments for lung repair and regeneration. Here we report the establishment of a new tool, the Acid Injury and Repair (AIR) model, that will facilitate studies of lung tissue repair. In this model, injury is applied to a restricted area of a precision-cut lung slice using hydrochloric acid, a clinically relevant driver. The surrounding area remains uninjured, thus mimicking the heterogeneous pattern of injury frequently observed in lung diseases. We show that in response to injury, the percentage of progenitor cells (pro surfactant protein C, proSP-C and TM4SF1 positive) significantly increases in the injured region. Whereas in the uninjured area, the percentage of proSP-C/TM4SF1 cells remains unchanged but proliferating cells (Ki67 positive) increase. These effects are modified in the presence of inhibitors of proliferation (Cytochalasin D) and Wnt secretion (C59) demonstrating that the AIR model is an important new tool for research into lung disease pathogenesis and potential regenerative medicine strategies.

Effect of the CXCR4 antagonist plerixafor on endogenous neutrophil dynamics in the bone marrow, lung and spleen.

Treatment with the CXCR4 antagonist, plerixafor (AMD3100), has been proposed for clinical use in patients with WHIM (warts, hypogammaglobulinemia, infections, and myelokathexis) syndrome and in pulmonary fibrosis. However, there is controversy with respect to the impact of plerixafor on neutrophil dynamics in the lung, which may affect its safety profile. In this study, we investigated the kinetics of endogenous neutrophils by direct imaging, using confocal intravital microscopy in mouse bone marrow, spleen, and lungs. Neutrophils are observed increasing their velocity and exiting the bone marrow following plerixafor administration, with a concomitant increase in neutrophil numbers in the blood and spleen, while the marginated pool of neutrophils in the lung microvasculature remained unchanged in terms of numbers and cell velocity. Use of autologous radiolabeled neutrophils and SPECT/CT imaging in healthy volunteers showed that plerixafor did not affect GM-CSF-primed neutrophil entrapment or release in the lungs. Taken together, these data suggest that plerixafor causes neutrophil mobilization from the bone marrow but does not impact on lung marginated neutrophil dynamics and thus is unlikely to compromise respiratory host defense both in humans and mice.

Live imaging of alveologenesis in precision-cut lung slices reveals dynamic epithelial cell behaviour.

Damage to alveoli, the gas-exchanging region of the lungs, is a component of many chronic and acute lung diseases. In addition, insufficient generation of alveoli results in bronchopulmonary dysplasia, a disease of prematurity. Therefore visualising the process of alveolar development (alveologenesis) is critical for our understanding of lung homeostasis and for the development of treatments to repair and regenerate lung tissue. Here we show live alveologenesis, using long-term, time-lapse imaging of precision-cut lung slices. We reveal that during this process, epithelial cells are highly mobile and we identify specific cell behaviours that contribute to alveologenesis: cell clustering, hollowing and cell extension. Using the cytoskeleton inhibitors blebbistatin and cytochalasin D, we show that cell migration is a key driver of alveologenesis. This study reveals important novel information about lung biology and provides a new system in which to manipulate alveologenesis genetically and pharmacologically.

Time-lapse Imaging of Alveologenesis in Mouse Precision-cut Lung Slices.

Alveoli are the gas-exchange units of lung. The process of alveolar development, alveologenesis, is regulated by a complex network of signaling pathways that act on various cell types including alveolar type I and II epithelial cells, fibroblasts and the vascular endothelium. Dysregulated alveologenesis results in bronchopulmonary dysplasia in neonates and in adults, disrupted alveolar regeneration is associated with chronic lung diseases including COPD and pulmonary fibrosis. Therefore, visualizing alveologenesis is critical to understand lung homeostasis and for the development of effective therapies for incurable lung diseases. We have developed a technique to visualize alveologenesis in real-time using a combination of widefield microscopy and image deconvolution of precision-cut lung slices. Here, we describe this live imaging technique in step-by-step detail. This time-lapse imaging technique can be used to capture the dynamics of individual cells within tissue slices over a long time period (up to 16 h), with minimal loss of fluorescence or cell toxicity.

Protein stability versus function: effects of destabilizing missense mutations on BRCA1 DNA repair activity.

Mutations in breast cancer susceptibility gene BRCA1 (breast cancer early-onset 1) are associated with increased risk of developing breast and ovarian cancers. BRCA1 is a large protein of 1863 residues with two small structured domains at its termini: a RING domain at the N-terminus and a BRCT (BRCA1 C-terminus domain) repeat domain at the C-terminus. Previously, we quantified the effects of missense mutations on the thermodynamic stability of the BRCT domains, and we showed that many are so destabilizing that the folded functional state is drastically depopulated at physiological temperature. In the present study, we ask whether and how reduced thermodynamic stability of the isolated BRCT mutants translates into loss of function of the full-length protein in the cell. We assessed the effects of missense mutants on different stages of BRCA1-mediated DNA repair by homologous recombination using chicken lymphoblastoid DT40 cells as a model system. We found that all of the mutations, regardless of how profound their destabilizing effects, retained some DNA repair activity and thereby partially rescued the chicken BRCA1 knockout. By contrast, the mutation R1699L, which disrupts the binding of phosphorylated proteins (but which is not destabilizing), was completely inactive. It is likely that both protein context (location of the BRCT domains at the C-terminus of the large BRCA1 protein) and cellular environment (binding partners, molecular chaperones) buffer these destabilizing effects such that at least some mutant protein is able to adopt the folded functional state.