IntroductionIdiopathic pulmonary fibrosis (IPF) is a fatal disease of lung parenchymal scarring triggered by alveolar epithelial cell dysfunction. Inherited forms of pulmonary fibrosis, including ...those caused by mutant forms of surfactant protein C (SFTPC), offer an opportunity to study early pathogenic events which remain poorly understood.ObjectivesWe wished to interrogate specific factors involved in SFTPC trafficking to understand the pathological mistrafficking phenotype caused by the commonest pathogenic mutant, SFTPC-I73T, which aberrantly localises to the plasma membrane.MethodsCRISPR-Cas9 forward genetic screens were employed in an immortalised cell model system to interrogate SFTPC processing and trafficking and a FACS-based readout used to identify altered SFTPC localisation. Alveolar organoids derived from human embryonic lung tissue were transduced with an inducible CRISPRi system for the validation of screen hits.ResultsWe identified several novel targets including candidate proteases and ubiquitin ligases in addition to validating core complexes suspected to be involved in SFTPC trafficking. Of particular note is the E3 ligase ITCH which, when depleted, altered SFTPC localisation in a manner that phenocopies the pathogenic I73T mutant. Further validation is underway using genetic manipulation of a novel human embryonic alveolar organoid system.Abstract S108 Figure 1ITCH depletion results in redistribution of SFTPC to the plasma membrane. (A) Subcellular localisation of GFP-SFTPC WT and I73T expressed in HeLa cells. (B) GFP-SFTPC localises at the plasma membrane in ITCH knockout HeLa cellsConclusionsA cell line model of SFTPC trafficking has allowed us to identify important factors in SFTPC trafficking using forward genetic screens which we can test in a physiological system to understand SFTPC handling in health and disease.
Introduction and ObjectivesInfluenza A virus (IAV) infections are a significant cause of mortality worldwide. The concept that stromal cells are permanently altered by insults is termed trained ...immunity. Whether these cells contribute to protection or pathology is unclear. We hypothesise that trained stromal cells may participate in protective immune responses by rapidly reactivating local memory T cells.MethodsC57BL/6 mice were infected intranasally with IAV (WSN, 150PFU) for 30 days and subsequently re-challenged with IAV (X31, 200PFU) for either 2 or 5 days. Mice were sacrificed at day 0, 2, 5, 30, 32, 35 post infection. To detect infected cells, ex vivo, IAV-Nucleoprotein (NP) expression was measured using flow cytometry and the location of NP+ stromal cells was determined using immunofluorescence. Further phenotyping of infection experienced stromal cell subsets was conducted using RNA-scope, immunohistochemistry, and qPCR. To assess the consequences of T cell depletion on NP expression by lung stromal cells following IAV re-challenge, anti-CD4/CD8 blockade was performed during the memory phase of infection.ResultsFollowing influenza virus infection, NP+ epithelial cells were detected at primary (day 2, 5) and to a lesser extent at recall timepoints (day 32, 35). Importantly, NP+ epithelial cells expressed more MHCII compared to NP-negative cells, suggesting enhanced capability to communicate with T cells via enhanced antigen processing/presentation. Using RNAscope, SpiB, a transcription factor that regulates genes involved in antigen processing/presentation, was detected in lung epithelial cells of infected mice. SpiB+ cells were in close proximity to immune cells that form dense clusters containing a mixture of T/B cells and myeloid populations. Interestingly, these microenvironmental changes were dependent on viral replication. Increased frequencies of Ki67+ lung fibroblasts coincided with significant increases in interferon-responsive fibroblasts at early timepoints post infection, compared to naive controls. These two fibroblasts populations did not express NP. Following IAV re-challenge both fibroblast subsets were reduced compared to primary timepoints, suggesting high numbers of infected cells may be required to promote their expansion post infection.ConclusionsInfection experienced stromal cell subsets may promote immune protection upon re-infection, through antigen presentation to T cells and/or alteration of the local lung microenvironment.Please refer to page A211 for declarations of interest related to this abstract.
In the era of competing for core competence, the increasing inputs of knowledge factors have brought the issue of “efficiency-enhancing and quality-improving” into focus; and concerns about the ...“quality” perspective of knowledge require mining information related to complex knowledge hidden in the economic system. In order to quantify knowledge complexity at both the national (or regional) and technological levels, this article combines the Fitness and Complexity algorithm with matrix-estimation exercises based on the framework of the bipartite network. On the basis of these measurements, this article analyzes and discusses the economic implications and evolutionary features while considering the “expiration” of patents; additionally, community detection is conducted to discuss the evolution of the “location” of complex knowledge. The results show that knowledge complexity depends on the structural similarity and specialization of patents; furthermore, the timeliness of patents may affect knowledge complexity conspicuously; moreover, the significance of the “location” of complex knowledge in the past has been downplayed over the past few decades.
•Adopt the bipartite network, giving more attention to the “heterogeneity” and the “interconnectedness” of knowledge.•Combine the matrix-estimation exercise with the Fitness and Complexity algorithm to solve the coupled equations.•Investigate how the “expiration” of patents affects the evaluation of complex knowledge.•Conduct community detection for analyzing the location with its evolution of complex knowledge.
I will try to explain in the context of the Matrix and philosophy what the truth is and whether there is the free will or not. Everyone wants to know the truth, but no one apparently explains what it ...is. The matrix is the desert of the truth, and human mind remains therein thirsty. Can we know that we are alive or dead, that is to say, what is the difference between reality and imagination? Never can this problem only be solved by our minds. So long as we try to arrive at necessity, we remain in possibility.
IntroductionSOX9 (SRY-box transcription factor 9) controls extracellular matrix (ECM) deposition in chondrogenesis, and epithelial differentiation in alveologenesis. We hypothesised that SOX9 drives ...lung fibrosis progression by regulates ECM and alveolar epithelial injury and can provide downstream biomarkers for idiopathic pulmonary fibrosis (IPF).MethodsWe used a Wild type (WT) time-course bleomycin model to study spatio-temporal expression of SOX9, where lungs were harvested at regular intervals (1–28 days) after injury. For functional studies we induced lung fibrosis using bleomycin in inducible global Sox9 Knockout (Sox9KO, Sox9fl/fl;ROSACreER+/−) and Control (Sox9fl/fl;ROSACreER-/−) mice. Fibrosis was assessed at 14 days, using immunohistochemistry (IHC), qPCR, Western blot and hydroxyproline assays (HPA). Bronchoalveolar lavage (BAL) was analysed by flow cytometry and proteomics. Healthy and IPF human tissue were analysed by IHC. Serum from healthy controls and IPF patients was analysed by luminex assay.ResultsUsing WT bleomycin time-course model we observed, using IHC, that SOX9 is upregulated in stroma during the fibroproliferative and fibrotic period (days 7–28). SOX9 co-localised to SFTPC and recently discovered KRT17 expressing pro-fibrotic epithelium. Sox9KO reduced fibrosis severity (HPA and COL1 expression) and reduced KRT17 epithelial cell expression (IHC). Sox9 SiRNA treatment in vitro reduced MRC-5 fibroblast SOX9, α-SMA and COL1 expression. Western blot showed in vitro SOX9 expression was upregulated in TGF-β1 and PDGF-DD treated MRC-5 fibroblasts. In human IPF lung, SOX9 co-localised to SFTPC and KRT17 as well as α-SMA, PDGFR-β and COL1. Overall findings are consistent with SOX9 regulating a fibrotic phenotype in both fibrotic epithelium and fibroblasts.K-means cluster analysis of mouse BAL proteomics identified known promising (SPP1, IGFBP-2) and novel (BM1, biomarker 1) fibrosis biomarkers. In human IPF serum (n=12) BM1, OPN and IGFBP-2 were upregulated (p<0.05) compared to controls (n=8). BM1 was significantly (p<0.05) lower in patients with stable rather than progressive IPF, indicated by lung function and radiology.ConclusionSOX9 co-localises with, and regulates in vivo, critical components of the fibrotic niche: 1) myofibroblasts and ECM secretion; 2) recently discovered KRT17 epithelial cells that have a fibrotic gene signature. Identification of downstream known, and novel, promising predictive biomarkers highlight the translational importance of SOX9.Please refer to page A211 for declarations of interest related to this abstract.
BackgroundIdiopathic pulmonary fibrosis (IPF) is a progressive disease where the lungs become progressively scarred, reducing lung capacity and impairing gas transfer. Genome-wide association studies ...(GWAS) have identified a number of genetic loci associated with risk of IPF.AimTo identify genetic loci associated with declining lung capacity or declining gas transfer.MethodsWe performed a GWAS of longitudinal measures of forced vital capacity (FVC, a measure of lung capacity) and diffusing capacity for lung of carbon monoxide (DLco, a measure of gas transfer) using a linear mixed effects model. This was performed in individuals diagnosed with IPF across three studies and identified variants for further follow-up in an additional independent study. Variants were defined as significantly associated if they had p<5×10−8 in a meta-analysis of all four studies, had consistent direction of effects across and were nominally significant (p<0.05) in each study.Results1,048 individuals with measures of longitudinal FVC and 729 individuals with longitudinal measures of DLco passed quality control. In total, 4,560 measures of FVC and 2,795 measures of DLco and over 7 million genetic variants were included in the analysis. One variant located in an antisense RNA gene for Protein Kinase N2 (PKN2) showed a genome-wide significant association with FVC decline (−140 ml/year per risk allele, 95% CI −180, −100, p=9.14×10−12) with consistent effects across all four studies.ConclusionThese results identify a possible druggable target involved in promoting IPF disease progression.Please refer to page A211 for declarations of interest related to this abstract.
Introduction and ObjectivesPulmonary fibroblasts respond to environmental signals triggered by injury or infections, shaping subsequent responses in the lung. Fibroblasts may contribute to enhanced ...immune protection, or chronic pathogenic inflammation and fibrosis. We hypothesise that molecules upregulated by lung fibroblasts early following influenza A virus (IAV) infection and bleomycin-induced injury persist, in order to generate/maintain immune memory, via altered stromal-immune cell communication.MethodsTo address this, we performed RNA-seq on FACS sorted lung fibroblasts from naïve animals and at early (day 10) and late time points (day 40) following intranasal IAV infection. Transcriptional changes were compared with the bleomycin model of early lung injury (publicly available RNA-seq). The functional profile and location of injury altered lung stromal and immune cells was determined using flow cytometry and immunohistochemistry.ResultsAnalysis of differentially expressed genes demonstrated an enrichment in cell cycle and extracellular matrix genes at day 10 post IAV infection (FDR < 0.05), consistent with fibroblast activation profiles in the bleomycin-injured lung. Three distinct lung fibroblast populations were identified using flow cytometry: damage-responsive (DRF), interferon-responsive (IRF), and antigen-presenting fibroblasts (APF). DRF were significantly elevated in both models at day 10 post injury, while IRF were only detectable in the IAV lung. Interestingly, APF were reduced in the bleomycin lung compared to naïve controls. Furthermore, immunohistochemistry demonstrated that expression of the immunomodulatory molecule, podoplanin, was found in close proximity to immune cell infiltrates in the lung in both models.ConclusionsThese data have important implications for understanding the altered communications between immune and stromal cells during and following subsequent lung infections and injury/fibrotic responses.
IntroductionLung repair requires controlled transforming growth factor-β (TGFβ) signalling and extracellular matrix (ECM) production. We previously demonstrated that constitutive and ...adulthood-induced mesenchymal Gαq/11 knockout alters lung ECM properties, including lung elastin and TGFβ2 deposition, causing abnormal lung development and emphysema, respectively.1 However, the role of the Gαq/11-deficient fibroblast-generated ECM in these phenotypes is unclear.AimUnderstand how wild-type and Gαq/11 -/- fibroblast-deposited ECM influence lung repair.MethodsWild-type (WT) and Gnaq-/-;Gna11-/- (Gαq/11 -/-) murine embryonic fibroblasts (MEFs) were cultured on ECM generated by WT or Gαq/11 -/- MEFs. Wound healing and TGFβ signalling were assessed using scratch wound and conditioned media-stimulated transformed mink lung cell (TMLC) assays.WT and Gαq/11-knockout MEFs were stimulated with 2 ng/ml TGFβ2, and elastin, TGFβ2, and platelet-derived growth factor (PDGF) signalling component expression assessed.ResultsWT MEFs exhibited lower TGFβ signalling and wound healing when cultured on Gαq/11 -/- ECM than on WT ECM (0.53 relative TMLC luciferase activity (RLA); 31.7% vs 46.4% 8 hour healing). Conversely, Gαq/11 -/- MEFs activated more TGFβ on WT ECM than on Gαq/11 -/- ECM (1.8 RLA). When cultured on ECM alone, TMLC TGFβ signalling was reduced on Gαq/11 -/- ECM (0.44 RLA), suggesting that Gαq/11 -/- fibroblast-deposited ECM itself alters repair.Gαq/11 -/- MEFs had lower ECM component (Col3a1, Col1a1, Eln) and growth factor (Pdgfa, Pdgfb, Pdgfd) mRNA expression than WT MEFs. Gαq/11 -/- MEFs stimulated with TGFβ2 had a time-dependent increase in Eln and Pdgfa mRNA expression, (15.8- and 3.6-fold increases, respectively), and elastin protein approached near WT levels by 48 hours. Wound healing was slower in Gαq/11 -/- than WT MEFs (33.2% vs 58.1% 8 hour healing), and was enhanced by exogenous TGFβ2 (to 38.6% and 73.3% healing, respectively). However, TGFβ2 did not restore Gαq/11 -/- MEF healing to WT levels.ConclusionFibroblast Gαq/11 regulates ECM production, and ECM generated by Gαq/11 -/- cells is less supportive of repair than WT ECM. Reduced TGFβ2 content of Gαq/11 -/- ECM may further alter ECM generation, but restoration of TGFβ2 signalling does not fully re-establish repair. A greater understanding of these mechanisms may identify methods of manipulating lung repair to therapeutic potential.Please refer to page A211 for declarations of interest related to this abstract.ReferenceGoodwin AT et al. BioRxiv doi: https://doi.org/10.1101/2020.09.06.284778).
In scientific and engineering fields, the solutions to many problems can be transformed into finding the solutions to Sylvester equation, for which various computational methods (e.g., recurrent ...neural network, RNN) have been presented and investigated. RNN models are frequently used to solve computational problems due to the prevalent exploitation of the gradient-based RNN. However, the overlong convergent time and the too large residual error restrict the widespread applications of the RNN model in solving time-varying problems. Further, a special type of RNN named zeroing neural network (ZNN) is able to solve the time-varying Sylvester equation, which breaks the limitations mentioned above, but fails to handle complex time-varying problems owing to the sharp increment of the calculated amount in matrix inversion involved. To remedy the limitation, a modified gradient-based RNN (MGRNN) model is proposed to generate more accurate computational solutions with less convergent time and adaptive coefficients for solving the time-varying Sylvester equation, which replaces the matrix inversion problem with the matrix transposition problem. Besides, theoretical analyses and mathematical verifications are presented to validate the efficiency and superiority of the proposed MGRNN model compared with the traditional gradient-based recurrent neural network (GRNN) and ZNN models. Furthermore, simulation experiments are conducted to substantiate the properties of the newly proposed MGRNN model for solving the time-varying Sylvester equation.
Objective. To improve the results of treatment in patients, suffering insufficiency of sutures of intestinal anastomoses, using analysis of rate in the genes polymorphous variants of the matrix ...metalloproteinase Type 2 (C-1306 →T) and the tissue inhibitor of the matrix metalloproteinase Type 2 (G303 →A), as well as the result of elaboration of genetic diagnosis and prognostication of such complication.
Materials and methods. In 32 patients, suffering insufficiency of intestinal sutures, which were treated in Shalimov National Institute of Surgery and Transplantology during 2016 - 2021 yrs, there were conducted the laboratory, genetic, immunohistochemical and statistical investigations.
Results. Genetic and statistical analysis for the genes polymorphism of the matrix metalloproteinase Type 2 (C-1306 →T) and the tissue inhibitor of the matrix metalloproteinase Type 2 (G303 →A) have permitted to determine the genotypes variants, associated with risk for the sutures insufficiency occurrence in the hollow gut organs anastomoses. Basing on the data obtained, the prognostication method was elaborated for the sutures insufficiency occurrence in intestinal anastomoses. Such complications are occurred in 1,36 times more frequently in carriers of homozygous СС genotype in gene of the matrix metalloproteinase Type 2 and in two times lesser (5.9%) in carriers the minor homozygotes ТТ, than in the control - 10% (p>0.05). Among the patients with the sutures insufficiency of intestinal anastomoses a statistically significant in 1,6 times more frequent rate of carriers of the homozygous GG gene variant the tissue inhibitor of the matrix metalloproteinase Type 2 was revealed. Carriers of the minor homozygotes АА among the patients with the sutures insufficiency in intestinal anastomoses were absent, while the same genotype was revealed in the control with the 10% (p<0.05) rate. With objective to study the occurrence risk for the sutures insufficiency in intestinal anastomoses in presence of association in the studied genotypes we have analyzed several clinic-laboratory indices. There was revealed the pathogenetic significance of alleles of the genes polymorphic variants of the matrix metalloproteinase Type 2 and the tissue inhibitor of the matrix metalloproteinase Type 2, which were accompanied by hypoproteinemia, high indices of biochemical markers of collagen biodegradation and lowered expression of monoclonal antibodies for α-гладкоmuscular actin and collagen IV, and, finally. have evolved as the risk factors for development of the sutures insufficiency in intestinal anastomoses.
Conclusion. The method proposed consists of genetic investigation of the genes polymorphism of the matrix metalloproteinase Type 2 (C-1306 →T) and of the tissue inhibitor of the matrix metalloproteinase Type 2 (G303 →A). It permits to prognosticate the probability of the sutures insufficiency development in intestinal anastomoses, and, basing on this, to improve the choice of the patients’ treatment tactic.
