Fibronectin Protein Research Articles

Expression of FLRT2 in Postnatal Central Nervous System Development and After Spinal Cord Injury.

Fibronectin and leucine-rich transmembrane (FLRT) proteins are necessary for various developmental processes and in pathological conditions. FLRT2 acts as a homophilic cell adhesion molecule, a heterophilic repulsive ligand of Unc5/Netrin receptors, and a synaptogenic molecule; the last feature is mediated by binding to latrophilins. Although the function of FLRT2 in regulating cortical migration at the late gestation stage has been analyzed, little is known about the expression pattern of FLRT2 during postnatal central nervous system (CNS) development. In this study, we used Flrt2-LacZ knock-in (KI) mice to analyze FLRT2 expression during CNS development. At the early postnatal stage, FLRT2 expression was largely restricted to several regions of the striatum and deep layers of the cerebral cortex. In adulthood, FLRT2 expression was more prominent in the cerebral cortex, hippocampus, piriform cortex (PIR), nucleus of the lateral olfactory tract (NLOT), and ventral medial nucleus (VM) of the thalamus, but lower in the striatum. Notably, in the hippocampus, FLRT2 expression was confined to the CA1 region and partly localized on pre- and postsynapses whereas only few expression was observed in CA3 and dentate gyrus (DG). Finally, we observed temporally limited FLRT2 upregulation in reactive astrocytes around lesion sites 7 days after thoracic spinal cord injury. These dynamic changes in FLRT2 expression may enable multiple FLRT2 functions, including cell adhesion, repulsion, and synapse formation in different regions during CNS development and after spinal cord injury.

Oxymatrine improves renal fibrosis and inflammation in diabetic rats by modulating CHK1/2 phosphorylation

To explore the role of cell cycle checkpoint kinase 1/2 (CHK1/2) in mediating the inhibitory effect of oxymatrine (OMT) against renal inflammation and fibrosis in diabetic rats. SD rats were randomly divided into normal control group, diabetes model group (DM) and OMT treatment group (n=6). HE and Masson staining were used to observe histopathological changes of the renal tissue, and the expressions of CHK1, CHK2, p-CHK1 and p-CHK2 were localized by immunohistochemical staining. The contents of interleukin-6 (IL-6) and IL-1β in the renal tissue were detected using ELISA, and the expression levels of CHK1, CHK2, p-CHK1, p-CHK2, type Ⅲ collagen (Col-Ⅲ), type Ⅳ collagen (Col-Ⅳ), and fibronectin (FN) were determined using Western blotting. The changes in the expressions of CHK1, CHK2, p-CHK1, p-CHK2, Col-Ⅲ, Col-Ⅳ and FN proteins were also examined with Western blotting in NRK-52E cells in response to high glucose exposure, OMT treatment and siRNA-mediated CHK1/2 knockdown. In diabetic rats, OMT treatment significantly decreased the levels of blood glucose, serum creatinine and 24 h urinary protein (P < 0.05) and obviously improved inflammatory cell infiltration and fibrosis phenotype in the renal tissue (P < 0.05). CHK1 and CHK2 were mainly expressed in the cytoplasm and nuclei of renal tubule cells, and their phosphorylation levels were significantly higher in DM group than in the control group and OMT group. OMT treatment significantly decreased the protein expression levels of p-CHK1, p-CHK2, Col-Ⅲ, Col-Ⅳ and FN in the renal tissue of diabetic rats and in NRK-52E cells exposed to high glucose (P < 0.05). In NRK-52E cells, CHK1/2 knockdown resulted in significant reduction of the protein expressions of p-CHK1/2, Col-Ⅲ, Col-Ⅳ and FN (P < 0.05). The inhibitory effects of OMT against renal inflammation and fibrosis in diabetic rats are mediated probably by lowered phosphorylation levels of CHK1 and CHK2, which result in reduced release of the downstream inflammatory mediators and decreased secretion and deposition of extracellular matrix.

Peptide-modified substrate enhances cell migration and migrasome formation

Extracellular vesicles (EVs) are cell-to-cell communication tools. Migrasomes are recently discovered microscale EVs formed at the rear ends of migrating cells, and thus are suggested to be involved in communicating with neighboring cells. In cell culture, peptide scaffolds on substrates have been used to demonstrate cellular function for regenerative medicine. In this study, we evaluated peptide scaffolds, including cell penetrating, virus fusion, and integrin-binding peptides, for their potential to enable the formation of migrasome-like vesicles. Through structural and functional analyses, we confirmed that the EVs formed on these peptide-modified substrates were migrasomes. We further noted that the peptide interface comprising cell-penetrating peptides (pVEC and R9) and virus fusion peptide (SIV) have superior properties for enabling cell migration and migrasome formation than fibronectin protein, integrin-binding peptide (RGD), or bare substrate. This is the first report of migrasome formation on peptide-modified substrates. Additionally, the combination of 95% RGD and 5% pVEC peptides provided a functional interface for effective migrasome formation and desorption of cells from the substrate via a simple ethylenediaminetetraacetic acid treatment. These results provide a functional substrate for the enhancement of migrasome formation and functional analysis.

Identification of splice regulators of fibronectin-EIIIA and EIIIB by direct measurement of exon usage in a flow-cytometry based CRISPR screen

The extracellular matrix protein fibronectin (FN) is alternatively spliced in a variety of inflammatory conditions, resulting in increased inclusion of alternative exons EIIIA and EIIIB. Inclusion of these exons affects fibril formation, fibrosis, and inflammation. To define upstream regulators of alternative splicing in FN, we have developed an in vitro flow-cytometry based assay, using RNA-binding probes to determine alternative exon inclusion level in aortic endothelial cells. This approach allows us to detect exon inclusion in the primary transcripts themselves, rather than in surrogate splicing reporters. We validated this assay in cells with and without FN-EIIIA and -EIIIB expression. In a small-scale CRISPR KO screen of candidate regulatory splice factors, we successfully detected known regulators of EIIIA and EIIIB splicing, and detected several novel regulators. Finally, we show the potential in this approach to broadly interrogate upstream signaling pathways in aortic endothelial cells with a genome-wide CRISPR-KO screen, implicating the TNFalpha and RIG-I-like signaling pathways and genes involved in the regulation of fibrotic responses. Thus, we provide a novel means to screen the regulation of splicing of endogenous transcripts, and predict novel pathways in the regulation of FN-EIIIA inclusion.

Role of miR-138 on Radio Sensitivity of Gastric Carcinoma Cells and Its Molecular Mechanism

To explore FOXP4 and miR-138 in gastric carcinoma (GC) and its related mechanisms. Sixty-eight GC patients from January 2018 to January 2019 were selected as group A, and 66 healthy people as group B. GC cells and gastric mucosal epithelial cells were purchased, sh-FOXP4, si-FOXP4, NC, miR-138-inhibitor, and miR-138-mimics were transfected into MKN-45 and NCI-N87 cells. The FOXP4 and miR-138 expression levels in samples were tested by qRT-PCR, and N-cadherin, vimentin, Fibronectin, Slug, E-Cadherin and Y-catenin downstream proteins in cells were detected by WB. The proliferation was tested by MTT assay, invasion was tested by Transwell assay, and apoptosis was detected by flow cytometry assay. F0XP4 was highly expressed in GC, miR-138 was poorly expressed in GC, and AUC of FOXP4 and miR-138 was &gt; 0.8. FOXP4 and miR-138 were tied to the age, gender, tumor invasion, differentiation degree, tumor location and TNM stage of GC patients. Silent expression of FOXP4 and overexpression of miR-138 can promote apoptosis, inhibit cell growth and epithelial-mesenchymal transition (ETM). The two also can inhibit N-cadherin, vimentin, Fibronectin and Slug proteins, and promote E-Cadherin and y-catenin. Dual luciferase report confirmed that FOXP4 and miR-138 had targeted relationship. In terms of radiological parameters, the SF2 and D0 (Gy) values of transfected miR-138mimic+pcDNA3. 1-FOXP4 were dramatically higher than those of transfected miR-138mimic+pcDNA3. 1 cells, and SER was lower than those of transfected miR-138 mimic+ pcDNA3. 1 cells (P&lt;0.05), suggesting FOXP4 partially adjusted the radiosensitization role of miR-138. In conclusion, miR-138 can regulate EMT of GC cells by adjusting F0XP4 and enhance radiosensitivity of those cells.

SYST-03. A PHASE I/II STUDY TO EVALUATE SAFETY AND PRELIMINARY ACTIVITY OF THE TUMOR-TARGETING ANTIBODY-CYTOKINE FUSION PROTEIN L19TNF IN PATIENTS WITH GLIOBLASTOMA AT FIRST RELAPSE

Abstract Treatment options for recurrent glioblastoma are limited and there is a need for novel effective therapies. We previously demonstrated encouraging anti-tumor activity with the targeted delivery of tumor necrosis factor a (TNF) in preclinical orthotopic glioma models. TNF- a is a potent pro-inflammatory cytokine which may trigger strong anti-tumor immunity. However, its systemic administration at therapeutically active doses is hampered by toxic side effects. L19TNF is a fully human antibody-cytokine fusion protein, comprising TNF- a fused to the antibody L19 that binds a tumor-specific epitope of the extracellular matrix protein fibronectin. This allows a targeted delivery of therapeutically relevant doses of TNF to the tumor site upon intravenous administration while sparing healthy tissues. In this phase I/II open label, non-randomized, monocentric study, we investigated the safety and preliminary activity of L19TNF for patients with isocitrate dehydrogenase (IDH) wildtype World Health Organization (WHO) grade III / IV glioma at first relapse. Twenty patients were enrolled from 2019-2020 and treated with intravenous infusions of L19TNF. In the phase I part of the study, 6 patients were assigned to two different dose levels of L19TNF and a dose of 13 µg/kg was established as the recommended dose. In the phase II part 14 patients were treated at the recommended dose. No dose-limiting toxicities were observed and survival follow-up is ongoing. In almost all patients, we observed treatment-associated emerging tumor necrosis. For patients that had re-surgery at progression on or after treatment with L19TNF, we demonstrated increased numbers of tumor-infiltrating lymphocytes compared to the tumor tissue obtained at primary diagnosis. Translational studies to better understand the effects of L19TNF on a molecular and immunophenotypic level are ongoing. ClinicalTrials.gov Identifier: NCT 03779230

Acquired Esophageal Strictures in Children: Morphometric and Immunohistochemical Analyses.

Esophageal strictures (ES) in children are not well characterized pathologically. We report unique histopathologic analyses of resected acquired ES and control esophagi (CE). Muscle layer thicknesses were measured in intact well-oriented areas; inflammatory cells were counted in the most inflamed high power field (hpf). Sections were stained with relevant antibodies. Results were expressed as median, lower and upper quartiles. Wilcoxon Rank Sums non-parametric test was used to compare groups; P ≤ 0.05 was considered significant. All ES (N = 10) showed focal replacement of lamina propria, muscularis mucosa and submucosa by actin+ fibers emanating from muscularis propria. Compared to CE (N = 8), ES displayed significantly thickened muscularis mucosa and propria, and increased mast cells (tryptase- and chymase-positive), and eosinophils in muscle layers (all P ≤ 0.01). Matrix proteins periostin and fibronectin were identified in the muscle layers of CE, and in the extracellular matrix in areas of disrupted architecture in ES. Compared to CE, acquired ES in children show significant structural alterations, including obliterative muscularization, inflammatory cell mural infiltrates, and extracellular matrix protein deposits. Therapies targeting connective tissue expansion, mast cells, eosinophils and inflammation may be beneficial to treat ES.

Novel Insights Into Cellular Changes in HPV8-E7 Positive Keratinocytes: A Transcriptomic and Proteomic Analysis.

Human papillomavirus type 8 (HPV8) is associated with the development of non-melanoma skin cancer. In the past we already delved into the mechanisms involved in keratinocyte invasion, showing that the viral E7 oncoprotein is a key player that drives invasion of basal keratinocytes controlled by the extracellular protein fibronectin. To unravel further downstream effects in E7 expressing keratinocytes we now aimed at characterizing gene and protein/phosphoprotein alterations to narrow down on key cellular targets of HPV8-E7. We now show that gene expression of GADD34 and GDF15 are strongly activated in the presence of E7 in primary human keratinocytes. Further analyses of fibronectin-associated factors led to the identification of the Src kinase family members Fyn and Lyn being aberrantly activated in the presence of HPV8-E7. Phospho-proteomics further revealed that E7 not only targets cell polarity and cytoskeletal organization, but also deregulates the phosphorylation status of nuclear proteins involved in DNA damage repair and replication. Many of these differentially phosphorylated proteins turned out to be targets of Fyn and Lyn. Taken together, by using unbiased experimental approaches we have now arrived at a deeper understanding on how fibronectin may affect the signaling cascades in HPV8 positive keratinocytes, which may be key for skin tumorigenesis and that may also aid in the development of novel therapeutic approaches for betaHPV-mediated cancers.

TGF-Β SIGNALING REGULATES WNT/Β-CATENIN PATHWAY IN UTERINE LEIOMYOMA CELLS

Uterine leiomyomas, also known as uterine fibroids, are the most common benign gynecologic tumor of the female reproductive tract. Despite their high prevalence and heavy burden, their pathogenesis is not clear. Several signaling aberrations have been identified, including Wnt/β-catenin and TGF-β. TGF-β pathway is a crucial mediator of extracellular matrix (ECM) deposition and fibrosis and is upregulated in leiomyoma compared to the myometrium. The Wnt/β-catenin pathway is a conserved pathway implicated in proliferation, differentiation and stem cell renewal and has also been implicated in uterine leiomyoma development. Due to the importance of these two pathways in uterine leiomyoma development, we are interested in studying the interaction between these two pathways to better understand the pathogenesis of these tumors. After obtaining informed consent, primary fibroid and myometrial cells were isolated from hysterectomy surgeries at Johns Hopkins Hospital. Primary cells and immortalized fibroid (HuLM) and myometrial (UtSM) were treated with TGF-β3 (10ng/mL) for 48 hours. The effect of TGF-β3 on the Wnt/β-catenin pathway was examined using western blot through assessing the expression of total β-catenin, non-phosphorylated β-catenin, the active form of β-catenin, and Dickkopf-1 (DKK1), an inhibitor of Wnt pathway. The student’s t-test was used to determine statistically significant differences (P<0.05). As expected, TGF-β3 treatment induced the expression of ECM proteins collagen 1A and fibronectin in both primary and immortalized fibroid and myometrial cells. The expression of total β-catenin and non-phosphorylated β-catenin was higher in leiomyoma cells, and TGF-β3 treatment significantly increased the expression of total β-catenin and non-phosphorylated β-catenin in both myometrial by 66% and 59%, respectively and fibroid cells by 58% and 62%, respectively (p-value <0.05). The expression of the Wnt pathway inhibitor, DKK-1, was significantly lower in primary leiomyoma cells compared to myometrial cells, and TGF-β3 treatment resulted in a significant reduction in DKK-1 expression in both myometrial by 85% and fibroid cells by 61%(p-value <0.05). These results indicate that TGF-β3 and Wnt/β-catenin signal transduction pathways interact in uterine leiomyoma. Further analyses are needed to clarify the interaction and the role of DKK-1 in leiomyoma development.

Abstract 07: Collecting Duct (Pro) Renin Receptor(PRR) Promotes Kidney Fibrosis Via Macrophage Alternative Activation Mediated By Soluble PRR (sPRR)-Dependent Activation Of Yap/Taz Axis

Renal collecting duct (CD), an important site for fine-tuning urinary Na + and water excretion, is not traditionally thought to play a role in pathogenesis of chronic kidney disease. In the present study, we examined a profibrotic role of CD PRR in a mouse model of unilateral ureteral obstruction (UUO) and further defined the underlying mechanism involving soluble PRR (sPRR)-dependent activation of alternative macrophages activation. We subjected mice with CD-specific deletion of PRR (CD PRR KO) and their floxed controls to UUO or sham surgery. After 7 days of UUO, CD PRR KO decreased the fibronectin (28.6±6.8%) and α-SMA (39.2±7.8%) protein expression in obstructed kidneys. The Masson’s trichrome staining data also showed that CD PRR KO significantly attenuated UUO-induced collagen deposition and histological damage in the kidney. In parallel, CD PRR KO reduced TGF-β1 (56.7±3.2%) and TGF-β2 (53±1.2%) mRNA expression in obstructed kidneys. Moreover, in macrophages sorted from obstructed kidneys of CD PRR KO mice showed a significantly reduced of M2 macrophage markers such as mannose receptor ( MR ) (58.7±2.1%), arginase-1 ( Arg-1 ) (50±1.5%), chitinase-like lectins ( YM-1 ) (59.3±1.9%), inflammatory zone-1 ( Fizz1 ) (39.1±4.1%) mRNA expression and Yes-associated protein ( Yap ) (77±6.8%)/ transcriptional coactivator with PDZ-binding motif ( Taz ) (54.5±1.8%) mRNA expression compared with macrophages sorted from obstructed kidneys of floxed mice. Meanwhile, plasma sPRR was elevated in floxed mice by UUO and this elevation was blunted by CD PRR KO (23.7±0.4%). Administration of site-1 protease (S1P) inhibitor PF-429242 to C57BL/6 mice with UUO almost completely recapitulated the antifibrotic action as well as the inhibitory effect on M2 activation of CD PRR KO. In bone marrow-derived macrophages, sPRR-His treatment promoted macrophage M2 polarization, fibrosis and Yap/Taz expression. Overall, these results suggest that activation of CD PRR releases sPRR that activates M2 polarization via Yap/Taz axis, leading to renal fibrosis during UUO.

Clinical and immunological features and prognosis of different phenotypes of occupational asthma

Introduction. Occupational asthma is a complex and heterogeneous disease. Development of personalized treatment and prevention tactics becomes feasible due to phenotyping, which means identifying of markers to combine cases of occupational asthma with similar manifestations (clinical, instrumental, laboratory) and prognosis to phenotypes. The study aims to determination and comparative analysis of spirographic and immunological parameters for different phenotypes of occupational asthma. Materials and methods. In this study, we included 170 patients with different phenotypes of occupational asthma and 50 participants in control group. The spirographic examination was performed using computer spirograph with determination of the following parameters: forced vital capacity (FVC), forced expiratory volume during the first second (FEV1), Tiffeneau-Pinelli index (FEV1/FVC), peak expiratory flow (PEF), maximal expiratory flow at 75%, 50%, 25% of the forced vital capacity (MEF75%VC, MEF50%VC, MEF25%VC). Quantification of the immunoglobulins IgA, IgM, and IgG in human serum was conducted by Mancini method; levels of IgE, C-reactive protein and fibronectin were determined using a solid-phase enzyme immunoassay. Fibrinogen concentration in plasma was measured by an automatic coagulometer. Haptoglobin concentration in serum was determined by spectrophotometry. Results. According to spirometry and laboratory results obtained, there is a strong evidence, that the phenotype "occupational asthma - occupational chronic obstructive pulmonary disease" has the lowest values in pulmonary function tests and the most significant changes in immunoglobulins, fibronectin and acute phase proteins levels among other studied phenotypes of occupational asthma. Conclusions. Dynamic determination of spirometric, immunological parameters, fibronectin and proteins of the acute phase of inflammation in workers at risk for the formation of occupational bronchial asthma may be recommended to be carried out once every 6 months, followed by the isolation of disease phenotypes. This will optimize diagnostics, therapeutic and preventive tactics, as well as predict the course of this pathology.

EVALUATING THE INHIBITORY EFFECT OF ELAGOLIX & RELUGOLIX ON LEIOMYOMA GROWTH IN 2D CELL CULTURE

Elagolix and relugolix, both oral GnRH antagonists, are structurally similar; however their pharmakinetic properties differ. At the GnRH receptor elagolix’s IC50 is 1.5 nM with a half-life of 4-6 hours, while relugolix’s IC50 is 0.33 nM with a half-life of 49 hours. The goal of this study is to compare the effect elagolix and relugolix have on the expression of extracellular matrix (ECM) proteins in human leiomyoma cells in 2D cell culture. We hypothesized that both GnRH antagonists directly impact ECM protein expression relative to untreated leiomyoma cells. Leiomyoma cells were grown 2-dimentionally and exposed to elagolix and relugolix for 24 and 48 hours. Expression of various ECM proteins and GnRHR were analyzed using western blot and immunohistochemistry. At 100 nM relugolix (n=10) resulted in maximal, decreased expression of col1A1 at 24 hours (1.78 ± 0.06-fold; p<0.01) and 48 hours (1.92 ± 0.14-fold; p<0.04) and the difference between the two time points was not significant (p=0.87). Elagolix (n=11) resulted in maximal, decreased expression of col1A1 at 24 hours (1.56 ± 0.14-fold; p<0.03) but suppression was lost by 48 hours; the difference between the time points was significant (p<0.01). At 100 nM relugolix resulted in maximal, decreased expression of fibronectin at 24 hours (1.7 ± 0.07-fold; p<0.01) and 48 hours (1.8 ± 0.07-fold; p<0.01) with the difference between the two time points not being significant (p=0.52); elagolix resulted in maximal, decreased expression of fibronectin at 24 hours (1.7 ± 0.14-fold; p=0.01) and 48 hours (2.0 ± 0.09-fold; p<0.01) with no significant difference between the time points (p=0.19). An upregulation was seen in versican expression when cells were treated with relugolix for 24 hours though no concentration achieved growth to statistical significance when compared to untreated cells. A significant, concentration dependent downregulation was achieved at 48 hours (p<0.01) and at 100 nM relugolix resulted in maximal, decreased expression of versican (1.5 ± 0.07-fold; p<0.01). At 24 hours maximal, decreased expression of versican was seen at 100 nM (1.6 ± 0.10-fold; p<0.01) for cells treated with elagolix; a statistically significant upregulation was seen at 48 hours (p=0.02). In terms of the GnRH receptor, no significant difference between GnRH receptor protein expression in relugolix and elagolix was seen at 24 hours (p=0.57). At 48 hours, GnRH receptor protein expression increased in cells treated with elagolix when compared to cells treated with relugolix (p=0.037) suggesting the suppressive effect of elagolix at the receptor is lost by 48 hours. Our findings suggest that treatment with relugolix and elagolix directly regulated collagen, fibronectin, versican, and GnRH receptor protein expression in our 2D human leiomyoma cell culture model.

Unraveling the Local Relation Between Tissue Composition and Human Brain Mechanics Through Machine Learning.

The regional mechanical properties of brain tissue are not only key in the context of brain injury and its vulnerability towards mechanical loads, but also affect the behavior and functionality of brain cells. Due to the extremely soft nature of brain tissue, its mechanical characterization is challenging. The response to loading depends on length and time scales and is characterized by nonlinearity, compression-tension asymmetry, conditioning, and stress relaxation. In addition, the regional heterogeneity–both in mechanics and microstructure–complicates the comprehensive understanding of local tissue properties and its relation to the underlying microstructure. Here, we combine large-strain biomechanical tests with enzyme-linked immunosorbent assays (ELISA) and develop an extended type of constitutive artificial neural networks (CANNs) that can account for viscoelastic effects. We show that our viscoelastic constitutive artificial neural network is able to describe the tissue response in different brain regions and quantify the relevance of different cellular and extracellular components for time-independent (nonlinearity, compression-tension-asymmetry) and time-dependent (hysteresis, conditioning, stress relaxation) tissue mechanics, respectively. Our results suggest that the content of the extracellular matrix protein fibronectin is highly relevant for both the quasi-elastic behavior and viscoelastic effects of brain tissue. While the quasi-elastic response seems to be largely controlled by extracellular matrix proteins from the basement membrane, cellular components have a higher relevance for the viscoelastic response. Our findings advance our understanding of microstructure - mechanics relations in human brain tissue and are valuable to further advance predictive material models for finite element simulations or to design biomaterials for tissue engineering and 3D printing applications.

Glioblastoma invasion factor ODZ1 is induced by microenvironmental signals through activation of a Stat3-dependent transcriptional pathway

We have previously shown that the transmembrane protein ODZ1 serves for glioblastoma (GBM) cells to invade the surrounding tissue through activation of RhoA/ROCK pathway. However, the transcriptional machinery used by GBM cells to regulate the expression of ODZ1 is unknown. Here we show that interaction with tumor microenvironment elements, mainly activated monocytes through IL-6 secretion, and the extracellular matrix protein fibronectin, induces the Stat3 transcriptional pathway and upregulates ODZ1 which results in GBM cell migration. This signaling route is abrogated by blocking the IL-6 receptor, inhibiting Jak kinases or knocking down Stat3. Furthermore, we have identified a Stat3 responsive element in the ODZ1 gene promoter, about 1 kb from the transcription start site. Luciferase-reporter assays confirmed that the promoter responds to the presence of monocytic cells and this activation is greatly reduced when the Stat3 site is mutated or following treatment with a neutralizing anti-IL-6 receptor antibody or transfecting GBM cells with a dominant negative variant of Stat3. Overall, we show that monocyte-secreted IL-6 and the extracellular matrix protein fibronectin activate the axis Stat3-ODZ1 and promote migration of GBM cells. This is the first described transcriptional mechanism used by tumor cells to promote the expression of the invasion factor ODZ1.

Engineered protein-small molecule conjugates empower selective enzyme inhibition

Potent, specific ligands drive precision medicine and fundamental biology. Proteins, peptides, and small molecules constitute effective ligand classes. Yet greater molecular diversity would aid the pursuit of ligands to elicit precise biological activity against challenging targets. We demonstrate a platform to discover protein-small molecule (PriSM) hybrids to combine unique pharmacophore activities and shapes with constrained, efficiently engineerable proteins. In this platform, a fibronectin protein library is displayed on yeast with a single cysteine coupled to acetazolamide via a maleimide-poly(ethylene glycol) linker. Magnetic and flow cytometric sorts enrich specific binders to carbonic anhydrase isoforms. Isolated PriSMs exhibit potent, specific inhibition of carbonic anhydrase isoforms with efficacy superior to that of acetazolamide or protein alone, including an 80-fold specificity increase and 9-fold potency gain. PriSMs are engineered with multiple linker lengths, protein conjugation sites, and sequences against two different isoforms, which reveal platform flexibility and impacts of molecular designs. PriSMs advance the molecular diversity of efficiently engineerable ligands.

Key Roles of RGD-Recognizing Integrins During Cardiac Development, on Cardiac Cells, and After Myocardial Infarction

Cardiac cells interact with the extracellular matrix (ECM) proteins through integrin mechanoreceptors that control many cellular events such as cell survival, apoptosis, differentiation, migration, and proliferation. Integrins play a crucial role in cardiac development as well as in cardiac fibrosis and hypertrophy. Integrins recognize oligopeptides present on ECM proteins and are involved in three main types of interaction, namely with collagen, laminin, and the oligopeptide RGD (Arg-Gly-Asp) present on vitronectin and fibronectin proteins. To date, the specific role of integrins recognizing the RGD has not been addressed. In this review, we examine their role during cardiac development, their role on cardiac cells, and their upregulation during pathological processes such as heart fibrosis and hypertrophy. We also examine their role in regenerative and angiogenic processes after myocardial infarction (MI) in the peri-infarct area. Specific targeting of these integrins may be a way of controlling some of these pathological events and thereby improving medical outcomes.

Plasma Exosome Profile in ST-Elevation Myocardial Infarction Patients with and without Out-of-Hospital Cardiac Arrest.

The identification of new biomarkers allowing an early and more accurate characterization of patients with ST-segment elevation myocardial infarction (STEMI) is still needed, and exosomes represent an attractive diagnostic tool in this context. However, the characterization of their protein cargo in relation to cardiovascular clinical manifestation is still lacking. To this end, 35 STEMI patients (17 experiencing resuscitated out-of-hospital cardiac arrest (OHCA-STEMI) and 18 uncomplicated) and 32 patients with chronic coronary syndrome (CCS) were enrolled. Plasma exosomes were characterized by the nanoparticle tracking analysis and Western blotting. Exosomes from STEMI patients displayed a higher concentration and size and a greater expression of platelet (GPIIb) and vascular endothelial (VE-cadherin) markers, but a similar amount of cardiac troponin compared to CCS. In addition, a difference in exosome expression of acute-phase proteins (ceruloplasmin, transthyretin and fibronectin) between STEMI and CCS patients was found. GPIIb and brain-associated marker PLP1 accurately discriminated between OHCA and uncomplicated STEMI. In conclusion, the exosome profile of STEMI patients has peculiar features that differentiate it from that of CCS patients, reflecting the pathophysiological mechanisms involved in STEMI. Additionally, the exosome expression of brain- and platelet-specific markers might allow the identification of patients experiencing ischemic brain injury in STEMI.

Paramyxovirus replication induces the hexosamine biosynthetic pathway and mesenchymal transition via the IRE1α-XBP1s arm of the unfolded protein response.

The paramyxoviridae, respiratory syncytial virus (RSV), and murine respirovirus are enveloped, negative-sense RNA viruses that are the etiological agents of vertebrate lower respiratory tract infections (LRTIs). We observed that RSV infection in human small airway epithelial cells induced accumulation of glycosylated proteins within the endoplasmic reticulum (ER), increased glutamine-fructose-6-phosphate transaminases (GFPT1/2) and accumulation of uridine diphosphate (UDP)-N-acetylglucosamine, indicating activation of the hexosamine biosynthetic pathway (HBP). RSV infection induces rapid formation of spliced X-box binding protein 1 (XBP1s) and processing of activating transcription factor 6 (ATF6). Using pathway selective inhibitors and shRNA silencing, we find that the inositol-requiring enzyme (IRE1α)-XBP1 arm of the unfolded protein response (UPR) is required not only for activation of the HBP, but also for expression of mesenchymal transition (EMT) through the Snail family transcriptional repressor 1 (SNAI1), extracellular matrix (ECM)-remodeling proteins fibronectin (FN1), and matrix metalloproteinase 9 (MMP9). Probing RSV-induced open chromatin domains by ChIP, we find XBP1 binds and recruits RNA polymerase II to the IL6, SNAI1, and MMP9 promoters and the intragenic superenhancer of glutamine-fructose-6-phosphate transaminase 2 (GFPT2). The UPR is sustained through RSV by an autoregulatory loop where XBP1 enhances Pol II binding to its own promoter. Similarly, we investigated the effects of murine respirovirus infection on its natural host (mouse). Murine respirovirus induces mucosal growth factor response, EMT, and the indicators of ECM remodeling in an IRE1α-dependent manner, which persists after viral clearance. These data suggest that IRE1α-XBP1s arm of the UPR pathway is responsible for paramyxovirus-induced metabolic adaptation and mucosal remodeling via EMT and ECM secretion.

A novel role of snail in regulating tuberin/AMPK pathways to promote renal fibrosis in the new mouse model of type II diabetes.

Epithelial–mesenchymal transition (EMT) plays an important role in tissue fibrosis following chronic exposure to hyperglycemia. This study investigates the role of chronic diabetes in regulating tuberin/snail/AMPK to enhance EMT and increase renal fibrosis. A new mouse model of db/db/TSC2 +/− was generated by backcrossing db/db mice and TSC2 +/− mice. Wild type (WT), db/db, TSC2 +/− and dbdb/TSC2 +/− mice were sacrificed at ages 6 and 8 months old. Tuberin protein level was significantly decreased in kidneys from diabetic compared to WT mice at both ages. In addition, tuberin and E‐cadherin protein levels were significantly decreased in dbdb/TSC2 +/− compared to TSC2 +/− and db/db mice. In contrast, p‐PS6K, NFkB, snail, vimentin, fibronectin, and α‐SMA protein levels were significantly increased in dbdb/TSC2 +/− compared to db/db and TSC2 +/− mice at ages 6 and 8 months. Both downregulation of AMPK by DN‐AMPK and downregulation of tuberin by siRNA resulted in increased NFkB, snail, and fibronectin protein expression and decreased E‐cadherin protein expression in mouse primary renal proximal tubular cells. Interestingly, downregulation of snail by siRNA increased tuberin expression via feedback through activation of AMPK and reversed the expression of epithelial proteins such as E‐cadherin as well as mesenchymal proteins such as fibronectin, NF‐KB, vimentin, and α‐SMA in mouse primary renal proximal tubular cells isolated from kidneys of four mice genotypes. The data show that chronic diabetes significantly decreases tuberin expression and that provides strong evidence that tuberin is a major key protein involved in regulating EMT. These data also demonstrated a novel role for snail in regulating of AMPK/tuberin to enhance EMT and renal cell fibrosis in diabetes.

Proteomic characterization of serum proteins from Atlantic salmon (Salmo salar L.) from an outbreak with cardiomyopathy syndrome

Cardiomyopathy syndrome (CMS), caused by piscine myocarditis virus (PMCV), is a serious challenge to Atlantic salmon (Salmo salar L.) aquaculture. Regrettably, husbandry techniques are the only tool to manage CMS outbreaks, and no prophylactic measures are available at present. Early diagnosis of CMS is therefore desirable, preferably with non-lethal diagnostic methods, such as serum biomarkers. To identify candidate biomarkers for CMS, the protein content of pools of sera (4 fish/pool) from salmon with a CMS outbreak (3 pools) and from clinically healthy salmon (3 pools) was compared using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Overall, seven proteins were uniquely identified in the sera of clinically healthy fish, while 27 proteins were unique to the sera of CMS fish. Of the latter, 24 have been associated with cardiac disease in humans. These were grouped as leakage enzymes (creatine kinase, lactate dehydrogenase, glycogen phosphorylase and carbonic anhydrase); host reaction proteins (acute-phase response proteins-haptoglobin, fibrinogen, α2-macroglobulin and ceruloplasmin; and complement-related proteins); and regeneration/remodelling proteins (fibronectin, lumican and retinol). Clinical evaluation of the suitability of these proteins as biomarkers of CMS, either individually or as part of a panel, is a logical next step for the development of early diagnostic tools for CMS.