Matrix Contraction Research Articles

Optimization of Accurate Spacing for Gas Extraction from Damaged Coal Seams Based on a Dual-Penetration Model

In order to develop precise hole placement technology for gas extraction, this paper combines industrial testing, the evolution law of coal rock damage permeability, and the coal double-pore double-permeability model; establishes a coupling model of the coal damage deformation field and matrix-fracture double diffusion seepage field; and determines precise hole placement spacing for a coal seam gas by using the COMSOL Multiphysics 5.6 numerical simulation software. This study shows that the effective radius of gas extraction is 4.8 m after 180 d of extraction, which is a power function of the extraction time. The permeability of the coal body is affected by matrix adsorption, and contraction and effective stress, and the influence range between the boreholes under multi-hole extraction increases with the increase in spacing; at the same time, we took into account the positive effect of the permeability change on the extraction effect and ultimately determined the optimal spacing of boreholes in Dashucun Mine to be 6 m, which was arranged in the square area. The extraction effect was good after the on-site test, and the results of this study can provide guidance for the optimal arrangement of the spacing of boreholes in the underground areas of coal mines.

Effect of Gelatin-Based Hemostats on Fibroblasts and Relevant Growth Factors in Wound Healing.

Gelatin-based hemostats have been used in various surgical fields and showed advantageous effects on central aspects of wound healing when compared to cellulose-based hemostats. Nevertheless, the influence of gelatin-based hemostats on wound healing has not been fully explored yet. Hemostats were applied to fibroblast cell cultures for 5, 30, 60 min, 24 h, 7 and 14 days and measurements were taken at 3, 6, 12, 24 h and 7 or 14 days, respectively. Cell proliferation was quantified after different exposure times and a contraction assay was conducted to measure the extent of the extracellular matrix over time. We further assessed quantitative levels of vascular endothelial growth factor and basic fibroblast growth factor using enzyme-linked immunosorbent assay. Fibroblast counts decreased significantly at 7 and 14 days independent of the application duration (p < 0.001 for 5 min application). The gelatin-based hemostat did not have a negative impact on cell matrix contraction. After application of gelatin-based hemostat, the basic fibroblast growth factor did not change; yet, the vascular endothelial growth factor significantly increased after a prolonged 24 h application time when compared to controls or to a 6 h exposure (p < 0.05). Gelatin-based hemostats did not impair contraction of the extracellular matrix or growth factor production (vascular endothelial growth factor and basic fibroblast growth factor), while cell proliferation diminished at late time points. In conclusion, the gelatin-based material seems to be compatible with central aspects of wound healing. For further clinical assessment, future animal and human studies are necessary.

Increased Matrix Metalloproteinase-1 Activation Enhances Disruption and Regression of k-RasV12–Expressing Arteriovenous Malformation-Like Vessels

This study sought to identify potential mechanisms by which k-RasV12-expressing endothelial cell (EC) tubes demonstrate an increased propensity to regress compared with controls. Activated k-Ras mutations play a role in a variety of pathological conditions, including arteriovenous malformations, which are prone to bleed, causing serious hemorrhagic complications. ECs expressing active k-RasV12 demonstrate markedly excessive lumen formation with widened and shortened tubes accompanied by reduced pericyte recruitment and basement membrane deposition, leading to deficient capillary network assembly. The current study showed that active k-Ras-expressing ECs secreted greater amounts of MMP-1 proenzyme compared with control ECs, and readily converted it to increased active MMP-1 levels through the action of plasmin or plasma kallikrein (generated from their added zymogens). Active MMP-1 degraded three-dimensional collagen matrices, leading to more rapid and extensive regression of the active k-Ras-expressing EC tubes, in conjunction with matrix contraction, compared with control ECs. Under conditions where pericytes protect control EC tubes from plasminogen- and MMP-1-dependent tube regression, this failed to occur with k-RasV12 ECs, due to reduced pericyte interactions. In summary, k-RasV12-expressing EC vessels showed an increased propensity to regress in response to serine proteinases through accentuated levels of active MMP-1, a novel pathogenic mechanism that may underlie hemorrhagic events associated with arteriovenous malformation lesions.

Variant STAT4 and Response to Ruxolitinib in an Autoinflammatory Syndrome

BackgroundDisabling pansclerotic morphea (DPM) is a rare systemic inflammatory disorder, characterized by poor wound healing, fibrosis, cytopenias, hypogammaglobulinemia, and squamous-cell carcinoma. The cause is unknown, and mortality is high.MethodsWe evaluated four patients from three unrelated families with an autosomal dominant pattern of inheritance of DPM. Genomic sequencing independently identified three heterozygous variants in a specific region of the gene that encodes signal transducer and activator of transcription 4 (STAT4). Primary skin fibroblast and cell-line assays were used to define the functional nature of the genetic defect. We also assayed gene expression using single-cell RNA sequencing of peripheral-blood mononuclear cells to identify inflammatory pathways that may be affected in DPM and that may respond to therapy.ResultsGenome sequencing revealed three novel heterozygous missense gain-of-function variants in STAT4. In vitro, primary skin fibroblasts showed enhanced interleukin-6 secretion, with impaired wound healing, contraction of the collagen matrix, and matrix secretion. Inhibition of Janus kinase (JAK)–STAT signaling with ruxolitinib led to improvement in the hyperinflammatory fibroblast phenotype in vitro and resolution of inflammatory markers and clinical symptoms in treated patients, without adverse effects. Single-cell RNA sequencing revealed expression patterns consistent with an immunodysregulatory phenotype that were appropriately modified through JAK inhibition.ConclusionsGain-of-function variants in STAT4 caused DPM in the families that we studied. The JAK inhibitor ruxolitinib attenuated the dermatologic and inflammatory phenotype in vitro and in the affected family members. (Funded by the American Academy of Allergy, Asthma, and Immunology Foundation and others.)

Left atrial appendage anatomical changes following radiofrequency-based ostial isolation

Abstract Funding Acknowledgements Type of funding sources: None. Background Left atrial appendage (LAA) electrical isolation (ei) may be achieved via radiofrequency (RF) energy applications at the level of the appendage ostium targeting the sites of earliest activation recorded by a mapping catheter. Notably, RF has long been used in vascular, orthopedic, and aesthetic surgery to promote thermal-induced collagen matrix contraction, fibrosis, and tissue retraction. LAA anatomical changes associated to RF-induced tissue retraction have never been reported. Purpose To quantify the anatomical changes of the LAA ostium following RF-based LAAei. Methods Thirty-four consecutive patients requiring AF ablation with LAAei underwent transesophageal echocardiography (TEE) within 7 days before (baseline TEE) and apx 6 months after (follow-up TEE) ablation. The diameter of LAA orifice and landing zone were measured at 4 different views (0°, 45°, 90°, 135°). Measurements were performed by two independent reviewers blinded to the patient’s identity. Results Among 43 AF patients (67±6yrs, 72.1% males), the LAA morphology was classified as chicken wing in 19 (44.2%) patients, windsock in 12 (27.9%), cactus in 8 (18.6%), and cauliflower in 4 (9.3%). At baseline TEE, the mean maximum and mean minimum ostial diameters were 25±4mm and 22±4mm, respectively. The mean maximum and mean minimum diameters of the landing zone were 26±4mm and 23±3mm, respectively. On average, LAAei was achieved after 17±6 minutes of RF at a power of 45W. Follow-up TEE was performed 294±136 days after LAAei. The median LAA contraction velocity was 0.1 m/s (IQR: 0.02-0.18) and was significantly impaired in all patients. At follow-up TEE, the mean maximum and mean minimum ostial diameters were 19±4mm and 17±3mm, respectively. The mean maximum and mean minimum diameters of the landing zone were 20±4mm and 18±4mm, respectively. The mean relative reduction of the ostium and the landing zone was -24.4% and -22.5%, respectively. Box-Whisker plots of the maximum and minimum ostial diameters before and after LAAei are reported in Fig.1. Conclusion RF led to a &amp;gt;20% reduction of the diameters of the ostium and the landing zone. These changes may have important implications for a successful percutaneous occlusion procedure and justify a staged approach of isolation and occlusion.

Effect of N-oleoyl dopamine on myofibroblast trans-differentiation of retinal pigment epithelial cells

Retinal pigment epithelial (RPE) cells contribute to several clinical conditions resulting in retinal fibrotic scars. Myofibroblast trans-differentiation of RPE cells is a critical step in the process of retinal fibrosis. In this study, we investigated the effects of N-oleoyl dopamine (OLDA), a newer endocannabinoid with a structure distinct from classic endocannabinoids, on TGF-β2-induced myofibroblast trans-differentiation of porcine RPE cells. Using an in vitro collagen matrix contraction assay, OLDA was found to inhibit TGF-β2 induced contraction of collagen matrices by porcine RPE cells. This effect was concentration-dependent, with significant inhibition of contraction observed at 3 μM and 10 μM. OLDA did not affect the proliferation of porcine RPE cells. Immunocytochemistry showed that at 3 μM, OLDA decreased incorporation of α-SMA in the stress fibers of TGF-β2-treated RPE cells. In addition, western blot analysis showed that 3 μM OLDA significantly downregulated TGF-β2-induced α-SMA protein expression. Taken together these results demonstrate that OLDA inhibits TGF-β induced myofibroblast trans-differentiation of RPE cells. It has been established that classic endocannabinoid such as anandamide, by activating the CB1 cannabinoid receptor, promote fibrosis in multiple organ systems. In contrast, this study demonstrates that OLDA, an endocannabinoid with a chemical structure distinct from classic endocannabinoids, inhibits myofibroblast trans-differentiation, an important step in fibrosis. Unlike classic endocannabinoids, OLDA has weak affinity for the CB1 receptor. Instead, OLDA acts on non-classic cannabinoid receptors such as GPR119, GPR6, and TRPV1. Therefore, our study indicates that the newer endocannabinoid OLDA and its non-classic cannabinoid receptors could potentially be novel therapeutic targets for treating ocular diseases involving retinal fibrosis and fibrotic pathologies in other organ systems.

Identification of the hub genes associated with prostate cancer tumorigenesis.

Prostate cancer (PCa) is one of the most common malignant tumors of the male urogenital system; however, the underlying mechanisms remain largely unclear. This study integrated two cohort profile datasets to elucidate the potential hub genes and mechanisms in PCa. Gene expression profiles GSE55945 and GSE6919 were filtered from the Gene Expression Omnibus (GEO) database to obtain 134 differentially expressed genes (DEGs) (14 upregulated and 120 downregulated) in PCa. Gene Ontology and pathway enrichment were performed using the Database for Annotation, Visualization, and Integrated Discovery, showing that these DEGs were mainly involved in biological functions such as cell adhesion, extracellular matrix, migration, focal adhesion, and vascular smooth muscle contraction. The STRING database and Cytoscape tools were used to analyze protein-protein interactions and identify 15 hub candidate genes. Violin plot, boxplot, and prognostic curve analyses were performed using Gene Expression Profiling Interactive Analysis, which identified seven hub genes, including upregulated expressed SPP1 and downregulated expressed MYLK, MYL9, MYH11, CALD1, ACTA2, and CNN1 in PCa compared with normal tissue. Correlation analysis was performed using the OmicStudio tools, which showed that these hub genes were moderately to strongly correlated with each other. Finally, quantitative reverse transcription PCR and western blotting were performed to validate the hub genes, showing that the abnormal expression of the seven hub genes in PCa was consistent with the analysis results of the GEO database. Taken together, MYLK, MYL9, MYH11, CALD1, ACTA2, SPP1, and CNN1 are hub genes significantly associated with PCa occurrence. These genes are abnormally expressed, leading to the formation, proliferation, invasion, and migration of PCa cells and promoting tumor neovascularization. These genes may serve as potential biomarkers and therapeutic targets in patients with PCa.

PD02-01 NUAK1 AND -2 PROMOTE CONTRACTION, PROLIFERATION AND SUPPRESSION OF CELL DEATH IN HUMAN PROSTATE STROMAL CELLS: EVIDENCE FROM ISOFORM-SPECIFIC SILENCING

You have accessJournal of UrologyCME1 Apr 2023PD02-01 NUAK1 AND -2 PROMOTE CONTRACTION, PROLIFERATION AND SUPPRESSION OF CELL DEATH IN HUMAN PROSTATE STROMAL CELLS: EVIDENCE FROM ISOFORM-SPECIFIC SILENCING Yuhan Liu, Ru Huang, Ruixiao Wang, Alexander Tamalunas, Raphaela Waidelich, Frank Strittmatter, Christian G. Stief, and Martin Hennenberg Yuhan LiuYuhan Liu More articles by this author , Ru HuangRu Huang More articles by this author , Ruixiao WangRuixiao Wang More articles by this author , Alexander TamalunasAlexander Tamalunas More articles by this author , Raphaela WaidelichRaphaela Waidelich More articles by this author , Frank StrittmatterFrank Strittmatter More articles by this author , Christian G. StiefChristian G. Stief More articles by this author , and Martin HennenbergMartin Hennenberg More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003219.01AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: In benign prostatic hyperplasia (BPH), contraction and growth in the prostate contribute to urethral obstruction and voiding symptoms. NUAK1 and -2 are serine/threonine kinases and promote myosin light chain phosphorlyation, actin organization, proliferation and suppression of cell death in non-muscle cells, which are critical for smooth muscle contraction and growth. Here, we examined effects of NUAK silencing on contraction and growth-related functions of human prostate stromal cells (WPMY-1). METHODS: Effects of NUAK1 and -2 silencing on matrix plug contraction, proliferation (EdU, Ki-67 mRNA), apoptosis and cell death (flowcytometry), viability (CCK-8) and actin organization (phalloidin) were examined in cultured WPMY-1 cells. Effects of NUAK inhibitors were examined in matrix contraction and in colony formation assays. RESULTS: Silencing of NUAK1 and -2 by transfection of WPMY-1 cells with isoform-specific siRNAs was confirmed by RT-PCR. Effects of silencing were most pronounced on proliferation and cell death. Proliferation rates were decreased by 60% and 70% by silencing of NUAK1 and NUAK2 (compared to scramble siRNA-transfected controls, 48 h after transfection), paralleled by decreases in Ki-67 mRNA by 75% and 77%. Numbers of dead cells amounted to 2.8 fold of scramble-transfected controls after silencing of NUAK1, and 4.9 fold of scramble controls after NUAK2 silencing, while no effect on apoptosis was observed. Viability was reduced by 31% and 49%, respectively, 24 h after transfection with NUAK1 and NUAK2 siRNA, compared to scramble controls. Polymerized actin was decreased by 66% and 70% by silencing of NUAK1 and -2. Time-dependent contractions in matrix contraction assays were reduced up to 45% by silencing of NUAK1, and up to 58% by silencing of NUAK2. The presumed NUAK inhibitors HTH01-015 and WZ4003 (both 10 µM) reduced contractions of WPMY-1 cells by up to 54% and 57%, and reduced the number of colonies in colony formation assays up to 78% and 64%, respectively. CONCLUSIONS: NUAK1 and -2 suppress cell death, and promote proliferation and contraction in prostate stromal cells. A role of NUAKs for BPH-relevant prostate functions driving voiding symptoms appears possible. NUAK inhibitors mimic effects of silencing on contraction, and inhibit the growth of stromal cells. Source of Funding: Deutsche Forschungsgemeinschaft (DFG), grant HE 5825/9-1; China Scholarship Council (CSC), grant 201908440478. Conflicts of Interest: Nothing to disclose © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e69 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yuhan Liu More articles by this author Ru Huang More articles by this author Ruixiao Wang More articles by this author Alexander Tamalunas More articles by this author Raphaela Waidelich More articles by this author Frank Strittmatter More articles by this author Christian G. Stief More articles by this author Martin Hennenberg More articles by this author Expand All Advertisement PDF downloadLoading ...

Platelet-Derived Extracellular Vesicles Promote Tenogenic Differentiation of Stem Cells on Bioengineered Living Fibers

Tendon mimetic scaffolds that recreate the tendon hierarchical structure and niche have increasing potential to fully restore tendon functionality. However, most scaffolds lack biofunctionality to boost the tenogenic differentiation of stem cells. In this study, we assessed the role of platelet-derived extracellular vesicles (EVs) in stem cells' tenogenic commitment using a 3D bioengineered in vitro tendon model. First, we relied on fibrous scaffolds coated with collagen hydrogels encapsulating human adipose-derived stem cells (hASCs) to bioengineer our composite living fibers. We found that the hASCs in our fibers showed high elongation and cytoskeleton anisotropic organization, typical of tenocytes. Moreover, acting as biological cues, platelet-derived EVs boosted the hASCs' tenogenic commitment, prevented phenotypic drift, enhanced the deposition of the tendon-like extracellular matrix, and induced lower collagen matrix contraction. In conclusion, our living fibers provided an in vitro system for tendon tissue engineering, allowing us to study not only the tendon microenvironment but also the influence of biochemical cues on stem cell behavior. More importantly, we showed that platelet-derived EVs are a promising biochemical tool for tissue engineering and regenerative medicine applications that are worthy of further exploration, as paracrine signaling might potentiate tendon repair and regeneration.

Toward Ab Initio Reaction Discovery Using the Artificial Force Induced Reaction Method.

Predicting the whole process of a chemical reaction while solving kinetic equations presents an opportunity to realize an on-the-fly kinetic simulation that directly discovers chemical reactions with their product yields. Such a simulation avoids the combinatorial explosion of reaction patterns to be examined by narrowing the search space based on the kinetic analysis of the reaction path network, and would open a new paradigm beyond the conventional two-step approach, which requires a reaction path network prior to performing a kinetic simulation. The authors addressed this issue and developed a practical method by combining the artificial force induced reaction method with the rate constant matrix contraction method. Two algorithms are available for this purpose: a forward mode with reactants as the input and a backward mode with products as the input. This article first numerically verifies these modes for known reactions and then demonstrates their application to the actual reaction discovery. Finally, the challenges of this method and the prospects for ab initio reaction discovery are discussed. Expected final online publication date for the Annual Review of Physical Chemistry, Volume 74 is April 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

The Influence of Cryogenic Treatment on the Microstructure and Mechanical Characteristics of Aluminum Silicon Carbide Matrix Composites

Aluminum matrix composites have been widely used in aerospace and automotive fields due to their excellent physical properties. Cryogenic treatment was successfully adopted to improve the performance of aluminum alloy components, while its effect and mechanism on the aluminum matrix composite remained unclear. In this work, the effects of cryogenic treatment on the microstructure evolution and mechanical properties of 15%SiCp/2009 aluminum matrix composites were systematically investigated by means of Thermoelectric Power (TEP), Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM). The results showed that TEP measurement can be an effective method for evaluating the precipitation characteristics of 15%SiCp/2009 aluminum matrix composites during aging. The addition of cryogenic treatment after solution and before aging treatment promoted the precipitation from the beginning stage of aging. Furthermore, the aging time for the maximum precipitation of the θ″ phase was about 4 h advanced, as the conduction of cryogenic treatment accelerates the aging kinetics. This was attributed to the great difference in the linear expansion coefficient between the aluminum alloy matrix and SiC-reinforced particles, which could induce high internal stress in their boundaries for precipitation. Moreover, the lattice contraction of the aluminum alloy matrix during cryogenic treatment led to the increase in dislocation density and micro defects near the boundaries, thus providing more nucleation sites for precipitation during the aging treatment. After undergoing artificial aging treatment for 20 h, the increase in dispersive, distributed precipitates after cryogenic treatment improved the hardness and yield strength by 4% and 16 MPa, respectively.

Anatomically resolved transcriptome and proteome landscapes reveal disease‐relevant molecular signatures and systematic changes in heart function of end‐stage dilated cardiomyopathy

AbstractDilated cardiomyopathy (DCM), as characterized by the left ventricular dilatation and contractile dysfunction, is one of the molecular mechanisms of which are largely unexplored. Here, we profiled the region‐resolved transcriptome and proteome of healthy and DCM human myocardial tissue and obtained the deep‐coverage dataset consisting 7,605 proteins and 19,880 transcripts in four chambers of the human heart. On the basis of the core proteome and transcriptome characters of the healthy hearts, chamber‐specific proteome alterations were further revealed in end‐stage DCM, among which extracellular matrix (ECM), mitochondrial function, and muscle contraction were the most dysregulated biological processes. Protein–protein interaction network demonstrated divergent functional networks of DCM atrium and ventricle. Additionally, a 4‐biomarker panel (CTSB, vWF, C9, and MFGE8) was established with promising diagnostic potential for the DCM. Collectively, our data provide a global proteomic basis of the chamber‐specific cardiac tissue, and establish a protein catalog that holds promise for better definition and diagnosis of DCM.

Dysregulation of TSP2-Rac1-WAVE2 axis in diabetic cells leads to cytoskeletal disorganization, increased cell stiffness, and dysfunction

Fibroblasts are a major cell population that perform critical functions in the wound healing process. In response to injury, they proliferate and migrate into the wound space, engaging in extracellular matrix (ECM) production, remodeling, and contraction. However, there is limited knowledge of how fibroblast functions are altered in diabetes. To address this gap, several state-of-the-art microscopy techniques were employed to investigate morphology, migration, ECM production, 2D traction, 3D contraction, and cell stiffness. Analysis of cell-derived matrix (CDM) revealed that diabetic fibroblasts produce thickened and less porous ECM that hindered migration of normal fibroblasts. In addition, diabetic fibroblasts were found to lose spindle-like shape, migrate slower, generate less traction force, exert limited 3D contractility, and have increased cell stiffness. These changes were due, in part, to a decreased level of active Rac1 and a lack of co-localization between F-actin and Waskott-Aldrich syndrome protein family verprolin homologous protein 2 (WAVE2). Interestingly, deletion of thrombospondin-2 (TSP2) in diabetic fibroblasts rescued these phenotypes and restored normal levels of active Rac1 and WAVE2-F-actin co-localization. These results provide a comprehensive view of the extent of diabetic fibroblast dysfunction, highlighting the regulatory role of the TSP2-Rac1-WAVE2-actin axis, and describing a new function of TSP2 in regulating cytoskeleton organization.

Matrix Adhesiveness Regulates Myofibroblast Differentiation from Vocal Fold Fibroblasts in a Bio-orthogonally Cross-linked Hydrogel.

Repeated mechanical and chemical insults cause an irreversible alteration of extracellular matrix (ECM) composition and properties, giving rise to vocal fold scarring that is refractory to treatment. Although it is well known that fibroblast activation to myofibroblast is the key to the development of the pathology, the lack of a physiologically relevant in vitro model of vocal folds impedes mechanistic investigations on how ECM cues promote myofibroblast differentiation. Herein, we describe a bio-orthogonally cross-linked hydrogel platform that recapitulates the alteration of matrix adhesiveness due to enhanced fibronectin deposition when vocal fold wound healing is initiated. The synthetic ECM (sECM) was established via the cycloaddition reaction of tetrazine (Tz) with slow (norbornene, Nb)- and fast (trans-cyclooctene, TCO)-reacting dienophiles. The relatively slow Tz-Nb ligation allowed the establishment of the covalent hydrogel network for 3D cell encapsulation, while the rapid and efficient Tz-TCO reaction enabled precise conjugation of the cell-adhesive RGDSP peptide in the hydrogel network. To mimic the dynamic changes of ECM composition during wound healing, RGDSP was conjugated to cell-laden hydrogel constructs via a diffusion-controlled bioorthognal ligation method 3 days post encapsulation. At a low RGDSP concentration (0.2 mM), fibroblasts residing in the hydrogel remained quiescent when maintained in transforming growth factor beta 1 (TGF-β1)-conditioned media. However, at a high concentration (2 mM), RGDSP potentiated TGF-β1-induced myofibroblast differentiation, as evidenced by the formation of an actin cytoskeleton network, including F-actin and alpha-smooth muscle actin. The RGDSP-driven fibroblast activation to myofibroblast was accompanied with an increase in the expression of wound healing-related genes, the secretion of profibrotic cytokines, and matrix contraction required for tissue remodeling. This work represents the first step toward the establishment of a 3D hydrogel-based cellular model for studying myofibroblast differentiation in a defined niche associated with vocal fold scarring.

Laser-imprinting of micro-3D printed protein hydrogels enables real-time independent modification of substrate topography and elastic modulus

Independent control over the Young's modulus and topography of a hydrogel cell culture substrate is necessary to characterize how attributes of its adherent surface affect cellular responses. Arbitrary, real-time manipulation of these parameters at the micron scale would further provide cellular biologists and bioengineers with the tools to study and control numerous highly dynamic behaviors including cellular adhesion, motility, metastasis, and differentiation. Although physical, chemical, thermal, and light-based strategies have been developed to influence Young's modulus and topography of hydrogel substrates, independent control of these physical attributes has remained elusive, spatial resolution is often limited, and features commonly must be pre-patterned. We recently reported a strategy in which biomaterials having specified three-dimensional (3D) morphologies are micro-3D printed in a two-step process: laser-scanning bioprinting of a protein-based hydrogel, followed by biocompatible hydrogel re-scanning to create microscale imprinted features at user-defined times. In this approach, a pulsed near-infrared laser beam is focused within the printed hydrogel to promote matrix contraction through multiphoton crosslinking, where scanning the laser focus projects a user-defined topographical pattern on the surface without subjecting the hydrogel-solution interface to damaging light intensities. Here, we extend this strategy, demonstrating the ability to decouple dynamic topographical changes from changes in hydrogel Young's modulus at the substrate surface by increasing the isolation distance between the surface and re-scanning planes. Using atomic force microscopy, we show that robust topographic changes can be imposed without altering the Young's modulus measured at the substrate surface by scanning at a depth of greater than ∼6 μm. Transmission electron microscopy of hydrogel thin sections reveals changes to hydrogel porosity and density distribution within scanned regions, and that such changes to the hydrogel matrix are highly localized to regions of laser exposure. These results represent valuable new capabilities for deconvolving the effects of substrate dynamic physical attributes on the behavior of adherent cells.

A Human atlas of smooth muscle cell gene expression; insights from the FANTOM consortium CAGE dataset

Abstract Background Under normal physiological conditions, smooth muscle cells (SMC's) have a role in both vessel contraction and generation of the extra-cellular matrix. It has now become apparent that SMCs throughout the vasculature play a key role in several cardiovascular conditions including hypertension, atherosclerosis, vasculopathy and myocardial infarction. However, how SMC gene expression compares in different vascular and non-vascular anatomical regions remains unknown. Purpose To create a human SMC gene expression map using data from the FANTOM consortium Cap-analysis gene expression (CAGE) dataset, a primary database of almost 600 human primary cell samples. Methods The gene level expression data was obtained by aggregating expression levels of all the promoters belonging to same gene as defined by the FANTOM CAGE dataset. The anatomical regions (n: 13) included the following vascular sites: aortic, brachiocephalic, carotid, coronary, internal thoracic &amp; pulmonary arteries. Non-vascular regions included colon, oesophagus, prostate, uterine, trachea and bronchial. Weighted correlation network analysis (WGCNA) was used as an unsupervised approach to identify clustering of gene sets. Results Hierarchical clustering (Figure 1) demonstrates a distinct clustering of vascular SMC gene sets (aortic, carotid, coronary, internal thoracic, pulmonary &amp; subclavian arteries). The same pattern of vascular SMC clustering was also seen in WGCNA (Figure 2). Gene ontology enrichment of vascular SMCs that clustered in WGCNA identified common gene sets relating to cytokine and chemokine activity. KEGG pathway analysis highlighted several pathways linked to inflammation (TNF, IL17 &amp; NF-κB signalling) as well as pathways linked to atherosclerosis and lipid metabolism. Conclusion Vascular SMCs distinctly cluster away from non-vascular SMC types in humans and the separation is primarily driven by gene sets relating to inflammation, lipid metabolism &amp; atherosclerosis. Mapping SMC gene expression at various anatomical regions in both healthy and diseased states may give us better insight into the role of SMCs in disease progression and could identify novel mechanistic targets for future therapy. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): British Heart Foundation

Fibroblast-derived CXCL12 increases vascular permeability in a 3-D microfluidic model independent of extracellular matrix contractility.

Cancer-associated fibroblasts (CAFs) play an active role in remodeling the local tumor stroma to support tumor initiation, growth, invasion, metastasis, and therapeutic resistance. The CAF-secreted chemokine, CXCL12, has been directly implicated in the tumorigenic progression of carcinomas, including breast cancer. Using a 3-D in vitro microfluidic-based microtissue model, we demonstrate that stromal CXCL12 secreted by CAFs has a potent effect on increasing the vascular permeability of local blood microvessel analogues through paracrine signaling. Moreover, genetic deletion of fibroblast-specific CXCL12 significantly reduced vessel permeability compared to CXCL12 secreting CAFs within the recapitulated tumor microenvironment (TME). We suspected that fibroblast-mediated extracellular matrix (ECM) remodeling and contraction indirectly accounted for this change in vessel permeability. To this end, we investigated the autocrine effects of CXCL12 on fibroblast contractility and determined that antagonistic blocking of CXCL12 did not have a substantial effect on ECM contraction. Our findings indicate that fibroblast-secreted CXCL12 has a significant role in promoting a leakier endothelium hospitable to angiogenesis and tumor cell intravasation; however, autocrine CXCL12 is not the primary upstream trigger of CAF contractility.

Efficient evaluation of the Breit operator in the Pauli spinor basis.

The frequency-independent Coulomb-Breit operator gives rise to the most accurate treatment of two-electron interaction in the non-quantum-electrodynamics regime. The Breit interaction in the Coulomb gauge consists of magnetic and gauge contributions. The high computational cost of the gauge term limits the application of the Breit interaction in relativistic molecular calculations. In this work, we apply the Pauli component integral-density matrix contraction scheme for gauge interaction with a maximum spin- and component separation scheme. We also present two different computational algorithms for evaluating gauge integrals. One is the generalized Obara-Saika algorithm, where the Laplace transformation is used to transform the gauge operator into Gaussian functions and the Obara-Saika recursion is used for reducing the angular momentum. The other algorithm is the second derivative of Coulomb interaction evaluated with Rys-quadrature. This work improves the efficiency of performing Dirac-Hartree-Fock with the variational treatment of Breit interaction for molecular systems. We use this formalism to examine relativistic trends in the Periodic Table and analyze the relativistic two-electron interaction contributions in heavy-element complexes.

Heterotropic roles of divalent cations in the establishment of allostery and affinity maturation of integrin αXβ2.

SUMMARYAllosteric activation and silencing of leukocyte β2-integrins transpire through cation-dependent structural changes, which mediate integrin biosynthesis and recycling, and are essential to designing leukocyte-specific drugs. Stepwise addition of Mg2+ reveals two mutually coupled events for the αXβ2 ligand-binding domain—the αX I-domain—corresponding to allostery establishment and affinity maturation. Electrostatic alterations in the Mg2+-binding site establish long-range couplings, leading to both pH− and Mg2+-occupancy-dependent biphasic stability change in the αX I-domain fold. The ligand-binding sensorgrams show composite affinity events for the αX I-domain accounting for the multiplicity of the αX I-domain conformational states existing in the solution. On cell surfaces, increasing Mg2+ concentration enhanced adhesiveness of αXβ2. This work highlights how intrinsically flexible pH− and cation-sensitive architecture endows a unique dynamic continuum to the αI-domain structure on the intact integrin, thereby revealing the importance of allostery establishment and affinity maturation in both extracellular and intracellular integrin events.

Comments on "Anatomically distinct fibroblast subsets determine skin autoimmune patterns".

Comments on "Anatomically distinct fibroblast subsets determine skin autoimmune patterns".