Small Airway Reduction and Fibrosis Is an Early Pathologic Feature of Idiopathic Pulmonary Fibrosis.

Rationale: To improve disease outcomes in idiopathic pulmonary fibrosis (IPF), it is essential to understand its early pathophysiology so that it can be targeted therapeutically. Objectives: Perform three-dimensional assessment of the IPF lung microstructure using stereology and multiresolution computed tomography (CT) imaging. Methods: Explanted lungs from patients with IPF (n = 8) and donor control subjects (n = 8) were inflated with air and frozen. CT scans were used to assess large airways. Unbiased, systematic uniform random samples (n = 8/lung) were scanned with microCT for stereological assessment of small airways (count number, and measure airway wall and lumen area) and parenchymal fibrosis (volume fraction of tissue, alveolar surface area, and septal wall thickness). Measurements and Main Results: The total number of airways on clinical CT was greater in IPF lungs than control lungs (P < 0.01), owing to an increase in the wall (P < 0.05) and lumen area (P < 0.05) resulting in more visible airways with a lumen larger than 2 mm. In IPF tissue samples without microscopic fibrosis, assessed by the volume fraction of tissue using microCT, there was a reduction in the number of the terminal (P < 0.01) and transitional (P < 0.001) bronchioles, and an increase in terminal bronchiole wall area (P < 0.001) compared with control lungs. In IPF tissue samples with microscopic parenchymal fibrosis, terminal bronchioles had increased airway wall thickness (P < 0.05) and dilated airway lumens (P < 0.001) leading to honeycomb cyst formations. Conclusions: This study has important implications for the current thinking on how the lung tissue is remodeled in IPF and highlights small airways as a potential target to modify IPF outcomes.

Proteomic Characterization of Damaged Single Hairs Recovered after an Explosion for Protein-Based Human Identification.

Evidence recovery is challenging where an explosion has occurred. Though hair evidence may be sufficiently robust to be recovered at the site, forensic analysis underutilizes the matrix by relying on morphological analysis. Where DNA is compromised, particularly in hair, protein-based human identification presents a promising alternative. Detection of amino acid polymorphisms in hair proteins as genetically variant peptides (GVPs) permits the inference of individualizing single nucleotide polymorphisms for identification. However, an explosive blast may damage hair proteins and compromise GVP identification. This work assesses effects of an explosive blast on the hair proteome and GVP identification, investigates microscopy as a predictor of proteome profiling success in recovered hairs to improve analysis throughput, and quantifies discriminative power in damaged hairs. The proteomics dataset has been deposited into the ProteomeXchange Consortium (PXD017427). With the exception of degradation in keratins K75, K80, K40, and keratin-associated protein KAP10-11 as markers of hair cuticular damage, corroborated by scanning electron microscopic analysis, minimal hair proteome degradation following explosion allowed successful proteome profiling of single hairs regardless of morphological damage. Finally, GVP identification remained independent of explosion conditions, permitting similar discriminative power between exploded and undamaged hairs. These findings lend greater confidence to GVP analysis in one-inch hairs for forensic identification and provide information about hair protein localization.

Automated analysis of scanning electron microscopic images for assessment of hair surface damage.

Mechanical damage of hair can serve as an indicator of health status and its assessment relies on the measurement of morphological features via microscopic analysis, yet few studies have categorized the extent of damage sustained, and instead have depended on qualitative profiling based on the presence or absence of specific features. We describe the development and application of a novel quantitative measure for scoring hair surface damage in scanning electron microscopic (SEM) images without predefined features, and automation of image analysis for characterization of morphological hair damage after exposure to an explosive blast. Application of an automated normalization procedure for SEM images revealed features indicative of contact with materials in an explosive device and characteristic of heat damage, though many were similar to features from physical and chemical weathering. Assessment of hair damage with tailing factor, a measure of asymmetry in pixel brightness histograms and proxy for surface roughness, yielded 81% classification accuracy to an existing damage classification system, indicating good agreement between the two metrics. Further ability of the tailing factor to score features of hair damage reflecting explosion conditions demonstrates the broad applicability of the metric to assess damage to hairs containing a diverse set of morphological features.

Transcriptional regulatory model of fibrosis progression in the human lung.

To develop a systems biology model of fibrosis progression within the human lung we performed RNA sequencing and microRNA analysis on 95 samples obtained from 10 idiopathic pulmonary fibrosis (IPF) and 6 control lungs. Extent of fibrosis in each sample was assessed by microCT-measured alveolar surface density (ASD) and confirmed by histology. Regulatory gene expression networks were identified using linear mixed-effect models and dynamic regulatory events miner (DREM). Differential gene expression analysis identified a core set of genes increased or decreased before fibrosis was histologically evident that continued to change with advanced fibrosis. DREM generated a systems biology model (www.sb.cs.cmu.edu/IPFReg) that identified progressively divergent gene expression tracks with microRNAs and transcription factors that specifically regulate mild or advanced fibrosis. We confirmed model predictions by demonstrating that expression of POU2AF1, previously unassociated with lung fibrosis but proposed by the model as regulator, is increased in B lymphocytes in IPF lungs and that POU2AF1-knockout mice were protected from bleomycin-induced lung fibrosis. Our results reveal distinct regulation of gene expression changes in IPF tissue that remained structurally normal compared with moderate or advanced fibrosis and suggest distinct regulatory mechanisms for each stage.

Hair Proteome Variation at Different Body Locations on Genetically Variant Peptide Detection for Protein-Based Human Identification.

Human hair contains minimal intact nuclear DNA for human identification in forensic and archaeological applications. In contrast, proteins offer a pathway to exploit hair evidence for human identification owing to their persistence, abundance, and derivation from DNA. Individualizing single nucleotide polymorphisms (SNPs) are often conserved as single amino acid polymorphisms in genetically variant peptides (GVPs). Detection of GVP markers in the hair proteome via high-resolution tandem mass spectrometry permits inference of SNPs with known statistical probabilities. To adopt this approach for forensic investigations, hair proteomic variation and its effects on GVP identification must first be characterized. This research aimed to assess variation in single-inch head, arm, and pubic hair, and discover body location-invariant GVP markers to distinguish individuals. Comparison of protein profiles revealed greater body location-specific variation in keratin-associated proteins and intracellular proteins, allowing body location differentiation. However, robust GVP markers derive primarily from keratins that do not exhibit body location-specific differential expression, supporting GVP identification independence from hair proteomic variation at the various body locations. Further, pairwise comparisons of GVP profiles with 8 SNPs demonstrated greatest interindividual variation and high intraindividual consistency, enabling similar differentiative potential of individuals using single hairs irrespective of body location origin.

Defective Fibrillar Collagen Organization by Fibroblasts Contributes to Airway Remodeling in Asthma.

Rationale: Histologic stains have been used as the gold standard to visualize extracellular matrix (ECM) changes associated with airway remodeling in asthma, yet they provide no information on the biochemical and structural characteristics of the ECM, which are vital to understanding alterations in tissue function.Objectives: To demonstrate the use of nonlinear optical microscopy (NLOM) and texture analysis algorithms to image fibrillar collagen (second harmonic generation) and elastin (two-photon excited autofluorescence), to obtain biochemical and structural information on the remodeled ECM environment in asthma.Methods: Nontransplantable donor lungs from donors with asthma (n = 13) and control (n = 12) donors were used for the assessment of airway collagen and elastin fibers by NLOM, and extraction of lung fibroblasts for in vitro experiments.Measurements and Main Results: Fibrillar collagen is not only increased but also highly disorganized and fragmented within large and small asthmatic airways compared with control subjects, using NLOM imaging. Furthermore, such structural alterations are present in pediatric and adult donors with asthma, irrespective of fatal disease. In vitro studies demonstrated that asthmatic airway fibroblasts are deficient in their packaging of fibrillar collagen-I and express less decorin, important for collagen fibril packaging. Packaging of collagen fibrils was found to be more disorganized in asthmatic airways compared with control subjects, using transmission electron microscopy.Conclusions: NLOM imaging enabled the structural assessment of the ECM, and the data suggest that airway remodeling in asthma involves the progressive accumulation of disorganized fibrillar collagen by airway fibroblasts. This study highlights the future potential clinical application of NLOM to assess airway remodeling in vivo.

Development of a Protein-based Human Identification Capability from a Single Hair.

Shed human hair (lacking root nuclear DNA) frequently contributes important information to forensic investigations involving human identification. Detection of genetic variation observed in amino acid sequences of hair proteins provides a new suite of identity markers that augment microscopic hair analysis and mitochondrial DNA sequencing. In this study, a new method that completely dissolves single hairs using a combination of heat, ultrasonication, and surfactants was developed. Dissolved proteins were digested and genetically variant peptide (GVP) profiles were obtained for single hairs (25 mm) via high-resolution nanoflow liquid chromatography-based mass spectrometry and a novel exome-driven bioinformatic approach. Overall, 6519 unique peptides were identified and a total of 57 GVPs were confirmed. Random match probabilities ranged between 2.6 × 10-2 and 6.0 × 10-9 . The new bioinformatic strategy and ability to analyze GVPs in forensically relevant samples sizes demonstrate applicability of this approach to distinguish individuals in forensic contexts.

In vivo characterization of doxycycline-mediated protection of aortic function and structure in a mouse model of Marfan syndrome-associated aortic aneurysm.

Aortic aneurysm is the most life-threatening complication in Marfan syndrome (MFS) patients. Doxycycline, a nonselective matrix metalloproteinases inhibitor, was reported to improve the contractile function and elastic fiber structure and organization in a Marfan mouse aorta using ex vivo small chamber myography. In this study, we assessed the hypothesis that a long-term treatment with doxycycline would reduce aortic root growth, improve aortic wall elasticity as measured by pulse wave velocity, and improve the ultrastructure of elastic fiber in the mouse model of MFS. In our study, longitudinal measurements of aortic root diameters using high-resolution ultrasound imaging display significantly decreased aortic root diameters and lower pulse wave velocity in doxycycline-treated Marfan mice starting at 6 months as compared to their non-treated MFS counterparts. In addition, at the ultrastructural level, our data show that long-term doxycycline treatment corrects the irregularities of elastic fibers within the aortic wall of Marfan mice to the levels similar to those observed in control subjects. Our findings underscore the key role of matrix metalloproteinases during the progression of aortic aneurysm, and provide new insights into the potential therapeutic value of doxycycline in blocking MFS-associated aortic aneurysm.

Loss of Vascular CD34 Results in Increased Sensitivity to Lung Injury.

Survival during lung injury requires a coordinated program of damage limitation and rapid repair. CD34 is a cell surface sialomucin expressed by epithelial, vascular, and stromal cells that promotes cell adhesion, coordinates inflammatory cell recruitment, and drives angiogenesis. To test whether CD34 also orchestrates pulmonary damage and repair, we induced acute lung injury in wild-type (WT) and Cd34-/- mice by bleomycin administration. We found that Cd34-/- mice displayed severe weight loss and early mortality compared with WT controls. Despite equivalent early airway inflammation to WT mice, CD34-deficient animals developed interstitial edema and endothelial delamination, suggesting impaired endothelial function. Chimeric Cd34-/- mice reconstituted with WT hematopoietic cells exhibited early mortality compared with WT mice reconstituted with Cd34-/- cells, supporting an endothelial defect. CD34-deficient mice were also more sensitive to lung damage caused by influenza infection, showing greater weight loss and more extensive pulmonary remodeling. Together, our data suggest that CD34 plays an essential role in maintaining vascular integrity in the lung in response to chemical- and infection-induced tissue damage.

A direct comparison of active and passive amplification electrodes in the same amplifier system.

BACKGROUND: Active amplification electrodes are becoming more popular for ERP data collection, as they amplify the EEG at the scalp and thereby potentially decrease the influence of ambient electrical noise. However, the performance of active electrodes has not been directly compared with that of passive electrodes in the context of collecting ERPs from a cognitive task. Here, the performance of active and passive amplification electrodes in the same digitizing amplifier system was examined. METHOD: In Experiment 1, interelectrode impedance in an electrically quiet setting was manipulated to determine whether, in such recording conditions, active electrodes can outperform passive ones. In Experiment 2, the performance of active electrodes at the limits of natural skin impedance was explored, as was the relationship between active amplification circuitry and voltage stability in averaged EOG. RESULTS: Results reveal a complex pattern of interrelations between electrode type, impedance, and voltage stability, indicating that which type of electrode is "best" depends non-trivially on the circumstances in which data are being collected. COMPARISON WITH EXISTING METHODS: Traditional, passive electrodes acquired the cleanest data observed in any of the acquisition conditions at very low impedance, but not at any impedance >2 kΩ. CONCLUSION: Active electrodes perform better than passive ones at all impedances other than very low ones; however, this is qualified by the additional finding that during fast voltage fluctuations, such as those most desirable in most ERP studies, active electrodes are less able to accurately follow the EEG than passive ones.