Biology subjects

Herrera, J. A.

Publications and source records attributed to Herrera, J. A..

6 recordsLinked to original sources

bioRxiv2025

Spatial transcriptomic profiling reveals distinct signatures in acute versus chronic wounds through hypergraph modelling and transcriptomic entropy analysis.

IntroductionThe financial burden of wounds to healthcare systems continues to increase, with little progression on advanced treatment options. Single cell RNA sequencing studies have predominantly focused on phenotypically different cell types and are generally descriptive in their analyses. Coordination within the transcriptome can be modelled using hypergraphs, which quantify higher order interactions (coordination between two or more genes) in transcriptomic data. Hypergraph modelling and analysis of transcriptomic entropy of wound samples will further elucidate differences between healing and non-healing wounds. MethodsSpatial transcriptomic analysis (Visium Spatial Gene Expression) was performed on three acute wounds, three chronic wounds and two healthy, unwounded skin samples. Standard transcriptomic analyses were performed in Python using the packages Scanpy and Squidpy. Hypergraph modelling and transcriptomic entropy analyses were performed in R. ResultsAcross the samples, twenty-nine Leiden clusters were defined, with keratinocytes, fibroblasts and adipocytes subclusters present. Fifteen marker genes for acute and chronic wounds were identified, with pathway analysis identifying immune responses and cellular homeostasis present in acute wounds, whilst in chronic wounds, pathways included immune responses and extracellular matrix structure. Spatial transcriptomics allowed for a descriptive analysis of the Leiden clusters within the samples, with each tissue sample being divided up into upper, middle and lower layer for this purpose. Hypergraph analyses of the whole transcriptome from the pathology groups and the independent transcriptomic Leiden clustering, revealed differences in underlying transcriptomic coordination. The top 1000 highly connected genes from each pathology group were analysed by Over Representation Analysis, with integrated stress response signalling, maintenance of cell number and myeloid and mononuclear cell differentiation pathways present in acute samples (all FDR <0.05) compared to regulation of vasculature development, mononuclear cell differentiation and epithelial cell proliferation (all FDR <0.05) in the chronic samples. Analysis of transcriptomic entropy identified further differences between acute and chronic wounds, with decreasing entropy from control to acute to chronic samples (all p-adjusted <0.001). When comparing Leiden clusters that were present in at least two sample groups, there was a statistically significant difference in entropy, although Leiden clusters varied in high or low entropy between acute and chronic wound samples (all p< 2.2 x 10-16). By mapping high and low entropy values to spatial images of each sample, lower transcriptomic entropy within the wound bed was identified compared to the rest of the tissue sample. ConclusionsSpatial transcriptomic analysis allowed for distinct spatial patterning to be observed which delineated acute and chronic wounds. Analysis of higher order interactions within the wound sample transcriptome identified key pathways that were not identified using traditional analyses. Transcriptomic entropy analysis further highlighted the differences between the wound samples. This work has implications for the future development of biomarkers of chronic wounds.

physiology↗

bioRxiv2023

Modelling collagen fibril self-assembly from extracellular medium in embryonic tendon

Collagen is a key structural component of multicellular organisms and is arranged in a highly organised manner. In structural tissues such as tendons, collagen forms bundles of parallel fibres between cells, which appear within a 24 hour window between E13.5 and E14.5 during mouse embryonic development. Current models assume that the organised structure of collagen requires direct cellular control, whereby cells actively lay down collagen fibrils from cell surfaces. However, such models appear incompatible with the time- and length-scales of fibril formation. We propose a phase-transition model to account for the rapid development of ordered fibrils in embryonic tendon, reducing reliance on active cellular processes. We develop phase-field crystal simulations of collagen fibrillogenesis in domains derived from electron micrographs of inter-cellular spaces in embryonic tendon and compare results qualitatively and quantitatively to observed patterns of fibril formation. To test the prediction of this phase-transition model that free protomeric collagen should exist in the intercellular spaces prior to the formation of observable fibrils, we use laser-capture microdissection, coupled with mass spectrometry, which demonstrates steadily increasing free collagen in intercellular spaces up to E13.5, followed by a rapid reduction of free collagen that coincides with the appearance of less soluble collagen fibrils. The model and measurements together provide evidence for extracellular self-assembly of collagen fibrils in embryonic mouse tendon, supporting an additional mechanism for rapid collagen fibril formation during embryonic development.

biophysics↗

bioRxiv2022

The UIP honeycomb airway cells are the site of mucin biogenesis with deranged cilia

Honeycombing (HC) is a histological pattern consistent with Usual Interstitial Pneumonia (UIP). HC refers to cystic airways (HC airways) located at sites of dense fibrosis with marked mucus accumulation. Utilizing laser capture microdissection coupled mass spectrometry (LCM-MS), we interrogated the fibrotic HC airway cells and fibrotic uninvolved airway cells (distant from sites of UIP and morphologically intact) in 10 UIP specimens; 6 non-fibrotic airway cell specimens served as controls. Furthermore, we performed LCM-MS on the mucus plugs found in 6 UIP and 6 mucinous adenocarcinoma (MA) specimens. The mass spectrometry data were subject to both qualitative and quantitative analysis and validated by immunohistochemistry. Surprisingly, fibrotic uninvolved airway cells share a similar protein profile to HC airway cells, showing deregulation of SLITs and ROBO pathway as the strongest category. We find that BPIFB1 is the most significantly increased secretome-associated protein in UIP, whereas MUC5AC is the most significantly increased in MA. We conclude that spatial proteomics demonstrates that the fibrotic uninvolved airway cells are abnormal. In addition, fibrotic HC airway cells are enriched in mucin biogenesis proteins with a marked derangement in proteins essential for ciliogenesis. This unbiased spatial proteomic approach will generate novel and testable hypotheses to decipher fibrosis progression.

pathology↗

bioRxiv2021

The IPF fibroblastic focus is an active collagen biosynthesis factory embedded in a distinct extracellular matrix

BackgroundThe Fibroblastic Focus (FF) is the signature lesion of Idiopathic Pulmonary Fibrosis (IPF) where myofibroblasts accumulate and extracellular matrix (ECM) is produced. However, the molecular composition and function of the FF and surrounding tissue remain undefined. MethodsUtilizing laser capture microdissection coupled mass spectrometry (LCM-MS), we interrogated the FF, adjacent mature scar, and adjacent alveoli in 6 IPF specimens plus 6 non-fibrotic alveolar specimens as controls. The data were subject to qualitative and quantitative analysis, and validation by immunohistochemistry. ResultsWe found that the protein signature of IPF alveoli is defined by immune deregulation as the strongest category. The IPF mature scar was classified as end-stage fibrosis whereas the FF contained an overabundance of a distinctive ECM compared to non-fibrotic control. ConclusionSpatial proteomics demonstrated distinct protein compositions in the histologically defined regions of IPF tissue. These data revealed that the FF is the main site of collagen biosynthesis and that the alveoli adjacent to the FF are abnormal. This new and essential information will inform future mechanistic studies on mechanisms of IPF progression.

pathology↗

bioRxiv2020

IL-13 deficiency exacerbates lung damage and impairs epithelial-derived type 2 molecules during nematode infection

IL-13 plays a key role during protective type 2 immune responses at mucosal sites, such as during infection with nematodes. However, dysregulation of IL-13 can also contribute to the pathogenesis of atopic and fibrotic diseases such as allergic asthma. Matrix remodelling is an important component of repair processes in the lung but also a hallmark of chronic conditions involving fibrosis. Hence, understanding the role of IL-13 in tissue remodelling has important clinical implications. Since IL-13 shares receptors and signalling pathways with IL-4, disentangling the relative contributions of these type 2 cytokines has been challenging. Additionally, little is known about the singular role of IL-13 following acute tissue injury. In this study, we used Nippostrongylus brasiliensis infection as a model of acute lung tissue damage comparing responses between WT and IL-13-deficient mice, in which IL-4 signalling is intact. Importantly, we found that IL-13 played a critical role in limiting tissue injury and haemorrhaging in the lung following infection. Through proteomic and transcriptomic profiling, we identified IL-13-dependent changes in matrix and associated regulators. We further showed that IL-13 is required for the induction of epithelial-derived type 2 effector molecules such as RELM- and surfactant protein D. Pathway analyses predicted that IL-13 was heavily involved in the induction of cellular stress responses and regulation of lung epithelial cell differentiation by suppression of Foxa2 pathways. Thus, we propose that IL-13 has tissue-protective functions during lung injury and regulates epithelial cell responses during type 2 immunity in this acute setting.

immunology↗

bioRxiv2019

Laser capture microdissection coupled mass spectrometry (LCM-MS) for spatially resolved analysis of formalin-fixed and stained human lung tissues

Haematoxylin and eosin (H&E) - which respectively stain nuclei blue and other cellular and stromal material pink - are routinely used for clinical diagnosis based on the identification of morphological features. A richer characterization can be achieved by laser capture microdissection coupled to mass spectrometry (LCM-MS), giving an unbiased assay of the proteins that make up the tissue. However, the process of fixing, and H&E staining of tissues is poorly compatible with standard sample preparation methods for mass spectrometry, resulting in low protein yield. Here we describe a microproteomics technique optimized to analyze H&E-stained, formalin-fixed paraffin-embedded (FFPE) tissues. We advance our methodology by combining 3 techniques shown to individually enhance protein yields (heat extraction, physical disruption, and in column digestion) into one optimized pipeline for the analysis of H&E stained FFPE tissues. Micro-dissected morphologically normal human lung alveoli (0.082 mm3) and human lung blood vessels (0.094 mm3) from FFPE fixed section from Idiopathic Pulmonary Fibrosis (IPF) specimens were then subject to comparative proteomics using this methodology. This approach yielded 1252 differentially expressed proteins including 137 extracellular matrix (ECM) proteins. In addition, we offer proof of principal that MS can identify distinct, characteristic proteomic compositions of anatomical features within complex tissues.

biochemistry↗