Genomic Analysis of Lymphoma Risk in Bullmastiff Dogs.

Lymphoma is the most common haematological malignancy affecting dogs and has a high incidence in the Bullmastiff breed. The aim of this study was to identify risk loci predisposing this breed to the disease. The average age of lymphoma diagnosis in 55 cases was less than 6 years, similar to the median age of 64 cases from our clinical and pathology databases. When fine-scale population structure was explored using NETVIEW, cases were distributed throughout an extended pedigree. When genotyped cases (n = 49) and dogs from the control group (n = 281) were compared in a genome-wide association analysis of lymphoma risk, the most prominent associated regions were detected on CFA13 and CFA33. The top SNPs in a 5.4 Mb region on CFA13 were significant at a chromosome-wide level, and the region was fine-mapped to ~1.2 Mb (CFA13: 25.2-26.4 Mb; CanFam3.1) with four potential functional candidates, including the MYC proto-oncogene bHLH transcription factor (MYC) and a region syntenic with the human and mouse lncRNA Pvt1 oncogene (PVT1). A 380 Kb associated region at CFA33: 7.7-8.1 Mb contained the coding sequence for SUMO specific peptidase7 (SENP7) and NFK inhibitor zeta (NFKBIZ) genes. These genes have annotations related to cancer, amongst others, and both have functional links to MYC regulation. Genomic signatures identified in lymphoma cases suggest that increased risk contributed by the regions identified by GWAS may complement a complex predisposing genetic background.

Identification of Genomic Signatures in Bullmastiff Dogs Using Composite Selection Signals Analysis of 23 Purebred Clades.

Dog breeds represent canine sub-populations with distinctive phenotypic features and limited genetic diversity. We have established a resource to study breed-specific genetic diversity. Utilising genetic resources within our laboratory biobank, public domain genotype data and the phylogenetic framework of 23 breed clades, the primary objective for this study was to identify genomic regions that differentiate the Bullmastiff breed. Through application of a composite index analysis (CSS), genomic signatures were identified in Bullmastiffs when compared to the formative breeds, Mastiffs and Bulldogs, and to 22 other breed groups. Significant regions were identified on 15 chromosomes, with the most differentiated regions found on CFA1, CFA9, and CFA18. These regions may reflect genetic drift following establishment of the breed or the effects of selective breeding during development of the modern Bullmastiff. This was supported by analysis of genes from the identified genomic regions, including 458 genes from the multi-clade analysis, which revealed enriched pathways that may be related to characteristic traits and distinct morphology of the breed. The study demonstrates the utility of the CSS method in breed-specific genome analysis and advances our understanding of genetic diversity in Bullmastiff dogs.

Alchemical Metadynamics: Adding Alchemical Variables to Metadynamics to Enhance Sampling in Free Energy Calculations.

Performing alchemical transformations, in which one molecular system is nonphysically changed to another system, is a popular approach adopted in performing free energy calculations associated with various biophysical processes, such as protein-ligand binding or the transfer of a molecule between environments. While the sampling of alchemical intermediate states in either parallel (e.g., Hamiltonian replica exchange) or serial manner (e.g., expanded ensemble) can bridge the high-probability regions in the configurational space between two end states of interest, alchemical methods can fail in scenarios where the most important slow degrees of freedom in the configurational space are, in large part, orthogonal to the alchemical variable, or if the system gets trapped in a deep basin extending in both the configurational and alchemical space. To alleviate these issues, we propose to use alchemical variables as an additional dimension in metadynamics, making it possible to both sample collective variables and to enhance sampling in free energy calculations simultaneously. In this study, we validate our implementation of "alchemical metadynamics" in PLUMED with test systems and alchemical processes with varying complexities and dimensionalities of collective variable space, including the interconversion between the torsional metastable states of a toy system and the methylation of a nucleoside both in the isolated form and in a duplex. We show that multidimensional alchemical metadynamics can address the challenges mentioned above and further accelerate sampling by introducing configurational collective variables. The method can trivially be combined with other metadynamics-based algorithms implemented in PLUMED. The necessary PLUMED code changes have already been released for general use in PLUMED 2.8.

Analysis of dog breed diversity using a composite selection index.

During breed development, domestic dogs have undergone genetic bottlenecks and sustained selective pressures, as a result distinctive genomic diversity occurs to varying degrees within and between breed groups. This diversity can be identified using standard methods or combinations of these methods. This study explored the application of a combined selection index, composite selection signals (CSS), derived from multiple methods to an existing genotype dataset from three breed groups developed in distinct regions of Asia: Qinghai-Tibet plateau dogs (adapted to living at altitude), Xi dogs (with superior running ability) and Mountain hounds (used for hunting ability). The CSS analysis confirmed top ranked genomic regions on CFA10 and CFA21 in Qinghai-Tibet plateau dogs, CFA1 in Xi dogs and CFA5 in Mountain hounds. CSS analysis identified additional significant genomic regions in each group, defined by a total of 1,397, 1,475 and 1,675 significant SNPs in the Qinghai-Tibetan Plateau dogs, Xi dogs and Mountain hounds, respectively. Chitinase 3 Like 1 (CHI3L1) and Leucine Rich Repeat Containing G Protein-Coupled Receptor 6 (LGR6) genes were located in the top ranked region on CFA7 (0.02-1 Mb) in the Qinghai-Tibetan Plateau dogs. Both genes have been associated with hypoxia responses or altitude adaptation in humans. For the Xi dogs, the top ranked region on CFA25 contained the Transient Receptor Potential Cation Channel Subfamily C Member 4 (TRPC4) gene. This calcium channel is important for optimal muscle performance during exercise. The outstanding signals in the Mountain dogs were on CFA5 with 213 significant SNPs that spanned genes involved in cardiac development, sight and generation of biochemical energy. These findings support the use of the combined index approach for identifying novel regions of genome diversity in dogs. As with other methods, the results do not prove causal links between these regions and phenotypes, but they may assist in focusing future studies that seek to identify functional pathways that contribute to breed diversity.

physical_validation: A Python package to assess the physical validity of molecular simulation results.

Molecular simulations such as molecular dynamics (MD) and Monte Carlo (MC) simulations are powerful tools allowing the prediction of experimental observables in the study of systems such as proteins, membranes, and polymeric materials. The quality of predictions based on molecular simulations depend on the validity of the underlying physical assumptions. physical_validation allows users of molecular simulation programs to perform simple yet powerful tests of physical validity on their systems and setups. It can also be used by molecular simulation package developers to run representative test systems during development, increasing code correctness. The theoretical foundation of the physical validation tests were established by Merz & Shirts (2018), in which the physical_validation package was first mentioned.

Quality of Life Among Geriatric Community Members Following the Kaohsiung Gas Explosion: A 5-Year Cross-Sectional Study.

OBJECTIVE: The aim was to conduct a follow-up study of the quality of life (QoL) and to measure lifestyle factors among the aging survivors of a petrochemical gas explosion in Taiwan 5 years after the event. METHODS: A community-based survey with cross-sectional assessments was conducted with residents of a community who experienced a petrochemical gas explosion 5 years after the disaster. Short Form 12v2 (SF-12v2) was used to survey a representative sample of participants. RESULTS: The risk factors for different QoL subscales were stressors and chronic physical illness, and the protective factors were higher income and better diet and exercise habits. The elderly participants had better diet and exercise habits than the younger participants in this survey. Aging had a negative impact on the physical QoL subscales [physical functioning (PF), bodily pain (BP), general health (GH), Physical Component Summary (PCS)] but a positive impact on psychological QoL subscales [vitality (VT), social functioning (SF), emotional problems (RE), mental health (MH), Mental Health Component Summary (MCS)]. CONCLUSIONS: While participants' psychiatric status had improved after 5 years, their QoL continued to be affected, especially in those with stress and physical illness. The elderly groups maintained a relatively acceptable QoL in terms of psychological aspects. Postdisaster treatment and follow-up should be addressed to a greater degree, especially in victims with mental illness or chronic illness and those with fewer socioeconomic resources.

Identifying signatures of proteolytic stability and monomeric propensity in O-glycosylated insulin using molecular simulation.

Insulin has been commonly adopted as a peptide drug to treat diabetes as it facilitates the uptake of glucose from the blood. The development of oral insulin remains elusive over decades owing to its susceptibility to the enzymes in the gastrointestinal tract and poor permeability through the intestinal epithelium upon dimerization. Recent experimental studies have revealed that certain O-linked glycosylation patterns could enhance insulin's proteolytic stability and reduce its dimerization propensity, but understanding such phenomena at the molecular level is still difficult. To address this challenge, we proposed and tested several structural determinants that could potentially influence insulin's proteolytic stability and dimerization propensity. We used these metrics to assess the properties of interest from [Formula: see text] aggregate molecular dynamics of each of 12 targeted insulin glyco-variants from multiple wild-type crystal structures. We found that glycan-involved hydrogen bonds and glycan-dimer occlusion were useful metrics predicting the proteolytic stability and dimerization propensity of insulin, respectively, as was in part the solvent-accessible surface area of proteolytic sites. However, other plausible metrics were not generally predictive. This work helps better explain how O-linked glycosylation influences the proteolytic stability and monomeric propensity of insulin, illuminating a path towards rational molecular design of insulin glycoforms.

A Community Survey of Quality of Life and Psychiatric Disorders among Residents Following the Kaohsiung Gas Explosion: a 5-Year Cross-Sectional Follow-Up Study.

To investigate factors associated with quality of life (QoL) and disaster-related psychiatric disorders, including posttraumatic stress disorder (PTSD) and major depressive episode (MDE), in the survivors of a gas explosion in Taiwan 5 years after the event. A community-based cross-sectional study of residents from an area that experienced a gas explosion was conducted 5 years after the event. The Short Form 12v2 (SF-12v2) was used to screen 2511 participants. The Disaster-Related Psychological Screening Test (DRPST) was used to assess probable MDE and PTSD. A total of 2511 participants, including 604 males and 1907 females, completed the QoL survey. The average age was 56.02 ± 16.78 years, and most participants were in the ≧65 age group (39.7%). The males had better QoL in the physical dimensions. Lifestyle was significantly positively associated with QoL. A total of 894 participants completed the DRPST, which showed some individuals with probable MDE (n = 93, 10.4%), probable PTSD (n = 22, 2.5%), or probable MDE and PTSD (n = 49, 5.5%); most people had no MDE or PTSD (n = 730, 81.7%). Those in the probable PTSD or MDE groups were significantly more likely to be female or to be experiencing stressors (p < 0.001). The participants continued to be affected by the disaster based on their QoL, even 5 years later. Females had a higher risk of probable psychiatric disorders and poorer QoL in the physical dimensions. Long-term follow-up, interventions and investigations after a disaster are needed.

Evaluation of genetic diversity and management of disease in Border Collie dogs.

Maintaining genetic diversity in dog breeds is an important consideration for the management of inherited diseases. We evaluated genetic diversity in Border Collies using molecular and genealogical methods, and examined changes to genetic diversity when carriers for Trapped Neutrophil Syndrome (TNS) and Neuronal Ceroid Lipofuscinosis (NCL) are removed from the genotyped population. Genotype data for 255 Border Collies and a pedigree database of 83,996 Border Collies were used for analysis. Molecular estimates revealed a mean multi-locus heterozygosity (MLH) of 0.311 (SD 0.027), 20.79% of the genome consisted of runs of homozygosity (ROH ) > 1 Mb, effective population size (Ne) was 84.7, and mean inbreeding (F) was 0.052 (SD 0.083). For 227 genotyped Border Collies that had available pedigree information (GenoPed), molecular and pedigree estimates of diversity were compared. A reference population (dogs born between 2005 and 2015, inclusive; N = 13,523; RefPop) and their ancestors (N = 12,478) were used to evaluate the diversity of the population that are contributing to the current generation. The reference population had a Ne of 123.5, a mean F of 0.095 (SD 0.082), 2276 founders (f), 205.5 effective founders (fe), 28 effective ancestors (fa) and 10.65 (SD 2.82) founder genomes (Ng). Removing TNS and NCL carriers from the genotyped population had a small impact on diversity measures (ROH > 1 Mb, MLH, heterozygosity), however, there was a loss of > 10% minor allele frequency for 89 SNPs around the TNS mutation (maximum loss of 12.7%), and a loss of > 5% for 5 SNPs around the NCL mutation (maximum 5.18%). A common ancestor was identified for 38 TNS-affected dogs and 64 TNS carriers, and a different common ancestor was identified for 33 NCL-affected dogs and 28 carriers, with some overlap of prominent individuals between both pedigrees. Overall, Border Collies have a high level of genetic diversity compared to other breeds.

Bilateral pedal edema associated with lurasidone: a case report.

Lurasidone, an atypical antipsychotic, is currently approved for treatment of schizophrenia and bipolar depression. Little is known about whether lurasidone might also cause pedal edema. A 55-year-old female patient had been diagnosed with bipolar I disorder from the age of 26. She had been prescribed escitalopram and quetiapine 300 mg/day for her persistent depressive mood. Later, she took lurasidone plus escitalopram to treat depressive episode. Consequently, she developed mild bilateral swelling over the lower legs and ankles. After lurasidone was discontinued, the bilateral pedal edema was completely resolved, and no further recurrence of edema occurred. Resolution of the edema after discontinuation of lurasidone indicates that the edema may have been caused by lurasidone. Caution should be needed when prescribing lurasidone for patients, as pedal edema may affect patients' adherence to the prescription.

Exploring the influence of brilliant blue G on amyloid fibril formation of lysozyme.

Evidence suggests that amyloid fibril mitigation/inhibition is considered a promising approach toward treating amyloid diseases. In this work, we first examined how amyloid fibrillogenesis of lysozyme was affected by BBG, a safe triphenylmethane compound with nice blood-brain-barrier-permeability, and found that shorter fibrillar species were formed in the lysozyme samples treated with BBG. Next, alterations in the features including the secondary as well as tertiary structure, extent of aggregation, and molecular distribution of lysozyme triggered by the addition of BBG were examined by various spectroscopic techniques, right-angle light scattering, dynamic light scattering, and SDS-PAGE. In addition, we have investigated how BBG affected the lysozyme fibril-induced cytotoxicity in SH-SY5Y cells. We found that a large quantity of shorter fibrillar species and more lysozyme monomers were present in the samples treated with BBG. Also, the addition of BBG rescued SH-SY5Y cells from cell death induced by amyloid fibrils of lysozyme. Finally, information about the binding sites and interacting forces involved in the BBG-lysozyme interaction was further explored using synchronous fluorescence and molecular docking approaches. Molecular docking results revealed that, apart from the hydrophobic interaction(s), hydrogen bonding, electrostatic interactions, and van der Waal forces may also be involved in the binding interaction.

Alteration of mesenchymal stem cells polarity by laminar shear stimulation promoting ß-catenin nuclear localization.

Mesenchymal stem cell (MSC) is mechanosensitive and the respond to mechanical force is pattern specific. We previously reported that oscillatory shear stress at 0.5 ±â€¯4 dyne/cm2 guided MSCs polarity vertical to net flow direction before apolaric stage at 30 min resulting in phosphorylation of ß-catenin and inhibition of Wnt signaling. This time, we explored laminar shear stress (LS) at 0.5 dyne/cm2 polarized MSCs by guiding F-actin orientation parallel to the flow direction before apolarity at 30 min accompanied with activation of Wnt signaling. Time-dependent microarray analysis supported cell-cell junctional complex of MSCs was the major mechanosensor on MSCs to respond 0.5 dyne/cm2 LS. Three-dimensional immunofluorescence image confirmed LS promoting ß-catenin nuclear localization during 15 min to 1 h with a peak at 30 min. Functional analysis of proteomic study on MSC with 30 min LS stimulation indicated that upregulation of ß-catenin downstream proteins related to cardiovascular development, endothelial cell protection and angiogenesis. Conditioned medium from MSCs with 30 min LS stimulation improved the viability of human endothelial cells from oxidative damage. In conclusion, 0.5 dyne/cm2 LS on MSCs for 30 min guides MSCs lack of polarity and promotes ß-catenin nuclear translocation favoring Wnt activation and paracrine cardiovascular support.

Lysozyme amyloid fibrillization in presence of tacrine/acridone-coumarin heterodimers.

Amyloid aggregates of proteins are one of the most abundant and important naturally occurring self-associated assemblies. Formation of poly/peptide amyloid aggregates is also associated with the widely spread diseases, so called amyloidosis, which include Alzheimer's disease, diabetes mellitus and lysozyme amyloidosis. These disorders are still incurable and novel therapeutical approaches are focused on using small molecules for inhibition of amyloid aggregation. We have observed effect of three structurally distinct groups of tacrine/acridone - coumarin heterodimers on hen egg white (HEW) lysozyme fibrillization in vitro. The ability of heterodimers to interfere with lysozyme amyloid aggregation was examined using Thioflavin T fluorescence assay, atomic force microscopy and docking method. The obtained data suggest that inhibitory effect of heterodimers on lysozyme fibrillization depends on their composition. We have shown that tacrine-coumarin heterodimers with alkylenediamine linker are the most effective inhibitors of lysozyme fibrillization. The inhibitory activities were quantified through IC50 values; the most potent heterodimers interfere with lysozyme aggregation in the scale of micromolar concentrations (19.2 µM-105.4 µM). The molecular docking showed that the modes of possible interactions involved in the binding are mainly hydrophobic interactions, hydrogen bonding and van der Waals interactions. Studied heterodimers had none or weak cytotoxic effect on human neuroblastoma cells. The obtained results can be helpful for the design and development of new therapeutics for amyloid-related diseases.

Brilliant blue R dye is capable of suppressing amyloid fibril formation of lysozyme.

Amyloid fibril formation is associated with an array of degenerative diseases. While no real cure is currently available, evidence suggests that suppression of amyloid fibrillogenesis is an effective strategy toward combating these diseases. Brilliant blue R (BBR), a disulfonated triphenylmethane compound, has been shown to interact with fibril-forming proteins but exert different effects on amyloid fibrillogenesis. These inconsistent findings prompted us to further evaluate BBR's effect on the inhibition/suppresion of protein fibrillogenesis. Using 129-residue hen lysozyme, which shares high sequence homology to human lysozyme associated with hereditary non-neuropathic systemic amyloidosis, as a model, this study is aimed at thoroughly examining the influence of BBR on the in vitro protein fibrillogenesis. We first showed that BBR dose-dependently attenuated lysozyme fibril formation probably by affecting the fibril growth rate, with the value of IC50 determined to be ~4.39 µM. Next, we employed tryptophan fluorescence quenching method to determine the binding constant and number of binding site(s) associated with BBR-lysozyme binding. In addition, we further conducted molecular docking studies to gain a better understanding of the possible binding site(s) and interaction(s) between lysozyme and BBR. We believe some of the information and/or knowledge concerning the structure-function relationship associated with BBR's suppressing activity obtained here can be applied for the future work in the subject matter related with the therapeutic strategies for amyloid diseases.

Comparative Studies on Ultraviolet-Light-Derived Photoresponse Properties of ZnO, AZO, and GZO Transparent Semiconductor Thin Films.

ZnO, Al-doped ZnO (AZO), and Ga-doped ZnO (GZO) semiconductor thin films were deposited on glass substrates via a sol-gel spin-coating process for application in a photoconductive ultraviolet (UV) detector. The doping concentrations of Al and Ga were 1.0 at % in the precursor solutions. In this study, the microstructural features and the optical and electrical properties of sol-gel-derived ZnO, AZO, and GZO thin films were compared, and the performance of ZnO-based UV photodetectors under ultraviolet A (UVA) light were measured. Experimental results confirmed the synthesis of single-phase nanocrystalline ZnO-based thin films and the successful substitution of Al and Ga into Zn sites in ZnO crystals. The results also demonstrated that the optical transmittance and electrical properties of ZnO thin films could be improved by Al and Ga doping. UV photodetectors based on ZnO-based thin films, having a metal-semiconductor-metal (MSM) configuration, were fabricated with Al inter-digitated electrodes. All photodetectors showed an ohmic nature between semiconductor and electrode contacts and exhibited a sharp increase in photocurrent under illumination with UVA light. We found that the MSM UV photodetector based on the GZO semiconductor thin film exhibited the best UV response (IUVA/Idark) of 73.3 and the highest photocurrent responsivity of 46.2 A/W under UVA light (power density ~0.825 mW/cm²) at 5 V bias.

Investigating the effects of erythrosine B on amyloid fibril formation derived from lysozyme.

Formation of amyloid fibrils has been associated with at least 30 different protein aggregation diseases. The 129-residue polypeptide hen lysozyme, which is structurally homologous to human lysozyme, has been demonstrated to exhibit amyloid fibril-forming propensity in vitro. This study is aimed at exploring the influence of erythrosine B on the in vitro amyloid fibril formation of hen lysozyme at pH 2.0 and 55°C using ThT binding assay, transmission electron microscopy, far-UV circular dichroism absorption spectroscopy, 1-anilinonaphthalene-8-sulfonic acid fluorescence spectroscopy, and synchronous fluorescence study. We found that lysozyme fibrillogenesis was dose-dependently suppressed by erythrosine B. In addition, our far-UV CD and ANS fluorescence data showed that, as compared with the untreated lysozyme control, the α-to-ß transition and exposure of hydrophobic clusters in lysozyme were reduced upon treatment with erythrosine B. Moreover, it could be inferred that the binding of erythrosine B occurred in the vicinity of the tryptophan residues. Finally, molecular docking and molecular dynamics simulations were further employed to gain some insights into the possible binding site(s) and interactions between lysozyme and erythrosine B. We believe the results obtained here may contribute to the development of potential strategies/approaches for the suppression of amyloid fibrillogenesis, which is implicated in amyloid pathology.

Stromal Tissue Rigidity Promotes Mesenchymal Stem Cell-Mediated Corneal Wound Healing Through the Transforming Growth Factor ß Signaling Pathway.

The healing of a corneal epithelial defect is essential for preventing infectious corneal ulcers and subsequent blindness. We previously demonstrated that mesenchymal stem cells (MSCs) in the corneal stroma, through a paracrine mechanism, yield a more favorable therapeutic benefit for corneal wound re-epithelialization than do MSCs in the corneal epithelium. In this study, MSCs were grown on a matrix with the rigidity of the physiological human vitreous (1 kPa), corneal epithelium (8 kPa), or corneal stroma (25 kPa) for investigating the role of corneal tissue rigidity in MSC functions regarding re-epithelialization promotion. MSC growth on a 25-kPa dish significantly promoted the wound healing of human corneal epithelial (HCE-T) cells. Among growth factors contributing to corneal epithelial wound healing, corneal stromal rigidity selectively enhanced transforming growth factor-beta (TGF-ß) secretion from MSCs. Inhibitors of TGF-ß pan receptor, TGF-ß receptor 1, and Smad2 dose dependently abrogated MSC-mediated HCE-T wound healing. Furthermore, MSCs growth on a matrix with corneal stromal rigidity enhanced the ability of themselves to promote corneal re-epithelialization by activating matrix metalloproteinase (MMP) expression and integrin ß1 production in HCE-T cells through TGF-ß signaling pathway activation. Smad2 activation resulted in the upregulation of MMP-2 and -13 expression in HCE-T cells, whereas integrin ß1 production favored a Smad2-independent TGF-ß pathway. Altogether, we conclude that corneal stromal rigidity is a critical factor for MSC-induced promotion of corneal re-epithelialization. The activation of the TGF-ß signaling pathway, which maintains the balance between integrin and MMP expression, in HCE-T cells is the major pathway responsible for MSC-mediated wound healing. Stem Cells 2016;34:2525-2535.

Regulation of metastatic ability and drug resistance in pulmonary adenocarcinoma by matrix rigidity via activating c-Met and EGFR.

Lung fibrosis is a poor prognostic factor for pulmonary adenocarcinoma, and the effect of a rigid microenvironment on cancer behavior is unclear. We cultured A549 cells on matrices of 0.2, 2, and 25 kPa to mimic the rigidities of normal lung parenchyma, progressive fibrotic change, and lung fibrosis, respectively. Lung tissue from patients with pulmonary adenocarcinoma was used to confirm the in vitro findings. Increased matrix rigidity promoted cell proliferation and upregulated the epidermal growth factor receptor (EGFR), hepatocyte growth factor receptor (c-Met), and Snail expression in A549 cells. A549 cells became more resistant to the EGFR inhibitor (Erlotinib) and c-Met inhibitor (PHA-665752) when matrix rigidity increased; however, a high concentration of PHA-665752 reversed the rigidity-induced morphological pleomorphism. In human lung tissue, expression of type I collagen was more consistent with clinical fibrosis than the expression of alpha-smooth muscle antibody was. c-Met- and Snail-expressing tumor cells, rather than EGFR-experssing cells, were localized with lung parenchyma rich in type I collagen. Our findings suggest that c-Met causes the rigidity-induced biophysical reaction in pulmonary adenocarcinoma. Treatment targeting both EGFR and c-Met should be considered for patients with lung fibrosis and who are abundant type I collagen expression in the tumor mass.