Epidermal Growth Factor Family Research Articles

The role of epidermal growth factor-like domain-related abnormalities, protein S Tokushima, and anti-protein S autoantibodies in pregnancy loss

BackgroundProtein S (PS) deficiency and autoantibodies that bind to PS (anti-PS) have been described in patients with adverse pregnancy outcomes, including pregnancy loss. PS Tokushima is a congenital abnormality of the second epidermal growth factor (EGF)-like domain, and anti-PS has been reported to recognize EGF-like domains. ObjectivesWe evaluated the role of PS Tokushima and anti-PS in patients with pregnancy loss. MethodsPatients with recurrent early pregnancy loss (n = 324; group A), those with one or more mid-to-late pregnancy loss (n = 196; group B), and infertile women having no pregnancy loss (n = 650; group C) were screened for PS type II deficiency and anti-PS. Patients who were diagnosed with PS type II deficiency underwent genetic analysis for the detection of PS Tokushima. ResultsThe incidence of patients with PS Tokushima was 1.85 %, 5.10 %, and 1.23 % in groups A, B, and C, respectively. The incidence of patients with PS Tokushima was significantly higher in group B (p = 0.0027) than in group C. The incidence of patients with anti-PS was 20.1 %, 23.0 %, and 19.2 % in groups A, B, and C, respectively. The incidence of patients with anti-PS was significantly higher in groups A (p = 0.0229), B (p = 0.0071), and C (p = 0.0288) than in previously reported healthy nonpregnant women (7.1 %, 4/56). ConclusionsOur data suggest that PS Tokushima is associated with mid-to-late pregnancy loss, while anti-PS are associated with recurrent early pregnancy loss, mid-to-late pregnancy loss, and infertility.

Tie1 contributes to the development of ovarian hyperstimulation syndrome under the regulation of EGR1 in granulosa cells

The expression of tyrosine kinase with immunoglobulin-like and epidermal growth factor-like domains 1 (Tie1), a transmembrane protein expressed almost exclusively by endothelial cells, has been reported in granulosa cells. However, its significance in ovarian hyperstimulation syndrome (OHSS), which can occur after the injection of gonadotropins in infertile women undergoing controlled ovarian stimulation, is unknown. Here, we report significantly increased Tie1 and vascular endothelial growth factor (VEGF) expression in cultured granulosa cells from OHSS patients, as well as ovaries from rats with experimentally established OHSS, compared to controls, with the levels of both proteins also increasing in granulosa and SVOG cells (a nontumorigenic human granulosa-lutein cell line) treated with an acute dose of human chorionic gonadotropin (hCG). Tie1 silencing abolished the hCG-induced VEGF level in SVOG cells and attenuated the progression of OHSS in rats, as determined by histological analysis. Further studies in SVOG cells revealed that the hCG-induced upregulation of Tie1 expression involved the phosphoinositide 3-kinase/protein kinase B signaling pathway. We also report that early growth response protein 1 (EGR1), whose expression was also upregulated by hCG, bound directly to the Tie1 promoter and activated its transcription. Taken together, our results indicate that Tie1 may be a therapeutic target in cases of moderate-to-severe OHSS. Further studies are needed to address its clinical relevance.

Long noncoding TUG1 promotes angiogenesis of HUVECs in PE via regulating the miR-29a-3p/VEGFA and Ang2/Tie2 pathways.

Preeclampsia (PE) is a pregnancy-specific disease that is associated with oxidative stress-induced endothelial dysfunction. Long noncoding RNAs (lncRNAs) are related to PE progression. The purpose is to study whether lncRNA taurine-upregulated gene 1 (TUG1) takes part in endothelial dysfunction in PE. The placenta tissues were collected from PE patients and normal subjects. Human umbilical vein endothelial cells (HUVECs) were suffered from hypoxia-reoxygenation (H/R). TUG1, miR-29a-3p and vascular endothelial growth factor A (VEGFA) were detected via qRT-PCR. soluble fms-related tyrosine kinase-1 (sFLT1) and soluble endoglin (sENG) levels were detected by ELISA. Cell proliferation, migration, invasion and angiogenesis were examined via MTT, wound healing analysis, transwell and tube formation analysis. The proteins in VEGFA and angiopoietin 2 (Ang2)/tyrosine kinase with immunoglobulin-like and EGF-like domains 2 (Tie2) signaling were measured by western blot. The binding relationship was analyzed via Starbase, Jefferson and dual-luciferase reporter analysis. TUG1 and VEGFA levels were downregulated, and levels of miR-29a-3p, sFLT1 and sENG were increased in PE patients. TUG1 abundance was reduced in H/R-stimulated HUVECs, and TUG1 overexpression increased proliferation, migration, invasion and angiogenesis, and activated the VEGFA and Ang2/Tie2 signaling in H/R-stimulated HUVECs. TUG1 sponged miR-29a-3p, and miR-29a-3p overexpression reversed the function of TUG1 on H/R-induced HUVECs dysfunction. MiR-29a-3p knockdown attenuated H/R-induced inhibition of proliferation, migration, invasion, angiogenesis and activation of the VEGFA and Ang2/Tie2 signaling in HUVECs. VEGFA and Ang2 were targeted by miR-29a-3p, and VEGFA or Ang2 silence weakened the role of miR-29a-3p knockdown in H/R-caused HUVECs dysfunction. TUG1 facilitates proliferation, migration, invasion and angiogenesis in H/R-stimulated HUVECs via activating the VEGFA and Ang2/Tie2 signaling by regulating miR-29a-3p.

Generation of Properly Folded Epidermal Growth Factor-Like (EGF) Repeats and Glycosyltransferases Enables In Vitro O-Glycosylation.

The epidermal growth factor-like (EGF) domain is an evolutionarily conserved motif found widely distributed among numerous secreted and membrane-anchored proteins, including the Notch receptors. Notch receptors include numerous EGF repeats tandemly connected in the extracellular domain. These EGF repeats must be properly folded in order for them to undergo the three different types of O-glycosylation associated with these extracellular proteins: O-fucose, O-glucose, and O-N-acetylglucosamine via glycosyltransferases POFUT1, POGLUT1, and EOGT. The O-glycosylation of the EGF repeats in the Notch receptors regulates the activation of Notch signaling and mutations in POFUT1, POGLUT1, and EOGT have been linked to specific human diseases. A large amount of EGF repeat and glycosyltransferase protein is required to construct an in vitro O-glycosylation system. Here, we describe how we prepared properly folded EGF repeats using two different bacterial expression vectors, generated recombinant glycosyltransferases, and performed in vitro O-glycosylation and subsequent product analysis by mass spectrometry. The methods described here are useful for investigating the enzymatic activities of mutated glycosyltransferases, revealing the structural basis of the O-glycosylation mechanism by co-crystallization of the glycosyltransferase-EGF repeat complexes, or identifying potential inhibitors of these glycosyltransferases.

Targeting the HER3 pseudokinase domain with small molecule inhibitors.

HER3 is a potent oncogenic growth factor receptor belonging to the human epidermal growth factor (HER/EGFR) family of receptor tyrosine kinases. In contrast to other EGFR family members, HER3 is a pseudokinase, lacking functional kinase activity. As such, efforts to develop small molecule tyrosine kinase inhibitors against this family member have been limited. In response to HER3-specific growth factors such as neuregulin (NRG, also known as heregulin or HRG), HER3 must couple with catalytically active family members, including its preferred partner HER2. Dimerization of the intracellular HER2:HER3 kinase domains is a critical part of the activation mechanism and HER3 plays a specialized role as an allosteric activator of the active HER2 kinase partner. Intriguingly, many pseudokinases retain functionally important nucleotide binding capacity, despite loss of kinase activity. We demonstrated that occupation of the nucleotide pocket of the pseudokinase HER3 retains functional importance for growth factor signaling through oncogenic HER2:HER3 heterodimers. Mutation of the HER3 nucleotide pocket both disrupts signaling and disrupts HER2:HER3 dimerization. Conversely, ATP competitive drugs which bind to HER3, but not HER2, can stabilize HER2:HER3 dimers, induce signaling and promote cell growth in breast cancer models. This indicates a nucleotide-dependent conformational role for the HER3 kinase domain. Critically, our recent proof-of-concept work demonstrated that HER3-directed small molecule inhibitors can also disrupt HER2:HER3 dimerization and signaling, supporting the prospect that HER3 can be a direct drug target despite its lack of intrinsic activity. In this chapter we will describe methods for identifying and validating small molecule inhibitors against the HER3 pseudokinase.

Decoding the byssus fabrication by spatiotemporal secretome analysis of scallop foot

Secretome is involved in almost all physiological, developmental, and pathological processes, but to date there is still a lack of highly-efficient research strategy to comprehensively study the secretome of invertebrates. Adhesive secretion is a ubiquitous and essential physiological process in aquatic invertebrates with complicated protein components and unresolved adhesion mechanisms, making it a good subject for secretome profiling studies. Here we proposed a computational pipeline for systematic profiling of byssal secretome based on spatiotemporal transcriptomes of scallop. A total of 186 byssus-related proteins (BRPs) were identified, which represented the first characterized secretome of scallop byssal adhesion. Scallop byssal secretome covered almost all of the known structural elements and functional domains of aquatic adhesives, which suggested this secretome-profiling strategy had both high efficiency and accuracy. We revealed the main components of scallop byssus (including EGF-like domain containing proteins, the Tyr-rich proteins and 4C-repeats containing proteins) and the related modification enzymes primarily contributing to the rapid byssus assembly and adhesion. Spatiotemporal expression and co-expression network analyses of BRPs suggested a simultaneous secretion pattern of scallop byssal proteins across the entire region of foot and revealed their diverse functions on byssus secretion. In contrast to the previously proposed “root-initiated secretion and extension-based assembly” model, our findings supported a novel “foot-wide simultaneous secretion and in situ assembly” model of scallop byssus secretion and adhesion. Systematic analysis of scallop byssal secretome provides important clues for understanding the aquatic adhesive secretion process, as well as a common framework for studying the secretome of non-model invertebrates.

EGF domain peptide of Developmentally regulated endothelial locus1 facilitates gene expression of extracellularly applied plasmid DNA

BackgroundThe successful development of messenger RNA vaccines for SARS-CoV-2 opened up venues for clinical nucleotide-based vaccinations. For development of DNA vaccines, we tested whether the EGF domain peptide of Developmentally regulated endothelial locus1 (E3 peptide) enhances uptake of extracellularly applied plasmid DNA. MethodsDNA plasmid encoding lacZ or GFP was applied with a conditioned culture medium containing E3 peptide to cell lines in vitro or mouse soleus muscles in vivo, respectively. After 48 h incubation, gene expression was examined by β-galactosidase (β-gal) assay and fluorescent microscope, respectively. ResultsApplication of E3 peptide-containing medium to cultured cell lines induced intense β-gal activity in a dose-dependent manner. Intra-gastrocnemius injection of E3 peptide-containing medium to mouse soleus muscle succeeded in the induction of GFP fluorescence in many cells around the injection site. ConclusionsThe administration of E3 peptide facilitates transmembrane uptake of extracellular DNA plasmid which induces sufficient extrinsic gene expression.

Von Willebrand factor D and EGF domains regulate ameloblast differentiation and enamel formation.

Cell- and tissue-specific extracellular matrix (ECM) composition plays an important role in organ development, including teeth, by regulating cell behaviors, such as cell proliferation and differentiation. Here, we demonstrate for the first time that von Willebrand factor D and epidermal growth factor (EGF) domains (Vwde), a previously uncharacterized ECM protein, is specifically expressed in teeth and regulates cell proliferation and differentiation in inner enamel epithelial cells (IEEs) and enamel formation. We identified the Vwde as a novel ECM protein through bioinformatics using the NCBI expressed sequence tag database for mice. Vwde complementary DNA encodes 1773 amino acids containing a signal peptide, a von Willebrand factor type D domain, and tandem calcium-binding EGF-like domains. Real-time polymerasechain reaction demonstrated that Vwde is highly expressed in tooth tissue but not in other tissues including the brain, lung, heart, liver, kidney, and bone. In situ hybridization revealed that the IEEs expressed Vwde messenger RNA in developing teeth. Immunostaining showed that VWDE was localized at the proximal and the distal ends of the pericellular regions of the IEEs. Vwde was induced during the differentiation of mouse dental epithelium-derived M3H1 cells. Vwde-transfected M3H1 cells secreted VWDE protein into the culture medium and inhibited cell proliferation, whereas ameloblastic differentiation was promoted. Furthermore, Vwde increased the phosphorylation of extracellular signal-regulated kinase 1/2 and protein kinase B and strongly induced the expression of the intercellular junction protein, N-cadherin (Ncad). Interestingly, the suppression of endogenous Vwde inhibited the expression of Ncad. Finally, we created Vwde-knockout mice using the CRISPR-Cas9 system. Vwde-null mice showed low mineral density, rough surface, and cracks in the enamel, indicating the enamel hypoplasia phenotype. Our findings suggest that Vwde assembling the matrix underneath the IEEs is essential for Ncad expression and enamel formation.

Meta-analysis of genome-wide association studies for litter size in sheep

Litter size and ovulation rate are important reproduction traits in sheep and have important impacts on the profitability of farm animals. To investigate the genetic architecture of litter size, we report the first meta-analysis of genome-wide association studies (GWAS) using 522 ewes and 564,377 SNPs from six sheep breeds. We identified 29 significant associations for litter size which 27 of which have not been reported in individual GWAS for each population. However, we could confirm the role of BMPR1B in prolificacy. Our gene set analysis discovered biological pathways related to cell signaling, communication, and adhesion. Functional clustering and enrichment using protein databases identified epidermal growth factor-like domain affecting litter size. Through analyzing protein-protein interaction data, we could identify hub genes like CASK, PLCB4, RPTOR, GRIA2, and PLCB1 that were enriched in most of the significant pathways. These genes have a role in cell proliferation, cell adhesion, cell growth and survival, and autophagy. Notably, identified SNPs were scattered on several different chromosomes implying different genetic mechanisms underlying variation of prolificacy in each breed. Given the different layers that make up the follicles and the need for communication and transfer of hormones and nutrients through these layers to the oocyte, the significance of pathways related to cell signaling and communication seems logical. Our results provide genetic insights into the litter size variation in different sheep breeds.

Biallelic ANGPT2 loss-of-function causes severe early-onset non-immune hydrops fetalis

BackgroundHydrops fetalis, a pathological fluid accumulation in two or more body compartments, is aetiologically heterogeneous. We investigated a consanguineous family with recurrent pregnancy loss due to severe early-onset non-immune hydrops...

Characterization of recombinant \u03b2 subunit of human MUC4 mucin (rMUC4\u03b2)

MUC4 is a transmembrane mucin expressed on various epithelial surfaces, including respiratory and gastrointestinal tracts, and helps in their lubrication and protection. MUC4 is also aberrantly overexpressed in various epithelial malignancies and functionally contributes to cancer development and progression. MUC4 is putatively cleaved at the GDPH site into a mucin-like α-subunit and a membrane-tethered growth factor-like β-subunit. Due to the presence of several functional domains, the characterization of MUC4β is critical for understanding MUC4 biology. We developed a method to produce and purify multi-milligram amounts of recombinant MUC4β (rMUC4β). Purified rMUC4β was characterized by Far-UV CD and I-TASSER-based protein structure prediction analyses, and its ability to interact with cellular proteins was determined by the affinity pull-down assay. Two of the three EGF-like domains exhibited typical β-fold, while the third EGF-like domain and vWD domain were predominantly random coils. We observed that rMUC4β physically interacts with Ezrin and EGFR family members. Overall, this study describes an efficient and simple strategy for the purification of biologically-active rMUC4β that can serve as a valuable reagent for a variety of biochemical and functional studies to elucidate MUC4 function and generating domain-specific antibodies and vaccines for cancer immunotherapy.

Neuregulin 1 (NRG1) fusion-positive high-grade spindle cell sarcoma: A distinct group of soft tissue tumors with metastatic potential.

Neuregulin 1 (NRG1) is an epidermal growth factor (EGF)-like ligand that activates receptor tyrosine kinases of the ErbB family of receptors. NRG1 gene fusions, which are rare (<1%) but recurrent events in solid tumors, are an emerging oncogenic driver that is potentially actionable using ErbB-targeted tyrosine kinase inhibitors. Largely characterized only in carcinomas, we describe three cases of NRG1-rearranged sarcomas. The patients were all female, aged 32-47 years old. Two cases were deep-seated tumors in the lower extremities (right thigh and calf); one case presented as a uterine mass. The tumors measured 9-11.5 cm in the greatest dimensions. Histologically, all three tumors were high-grade spindle cell sarcomas composed of monomorphic spindle cells arranged in interlacing fascicles. The tumor cells were set in the loose collagenous stroma with branching, curvilinear thin-walled vasculature in the background. Cytologically, the neoplastic cells displayed ovoid to fusiform nuclei with finely stippled chromatin, inconspicuous nucleoli, scant to moderate clear to eosinophilic cytoplasm, occasional cytoplasmic vacuoles, and elongated cytoplasmic processes. Mitotic activity was elevated (> 20/10 high power fields) and tumor necrosis was present. None of the tumors expressed lineage-specific immunophenotypical markers. Targeted RNA-sequencing uncovered gene fusions involving NRG1 and the 5' untranslated regions of PPHLN1, HMBOX1, or MTUS1. In all cases, the C-terminal EGF-like domain of NRG1 was preserved in the predicted chimeric protein product. All three patients developed metastatic disease within 2 years from initial presentation and were alive with disease at last follow-up (mean follow-up period=19 months). In conclusion, we present the first case series of NRG1-rearranged sarcomas characterized by high-grade fascicular spindle cell morphology, non-specific immunoprofile, and aggressive clinical behavior. Further studies are needed to determine whether this distinct subgroup of spindle cell sarcomas are amenable to targeted therapies.

The EGF Domains of MUC4 Oncomucin Mediate HER2 Binding Affinity and Promote Pancreatic Cancer Cell Tumorigenesis.

Simple SummaryA feature of pancreatic cancer (PC) is the frequent overexpression of tyrosine kinase membrane receptor HER2 along with its membrane partner the MUC4 oncomucin in the early stages of the pancreatic carcinogenesis. However, therapeutic approaches targeting HER2 in PC are not efficient. MUC4 could indeed represent an alternative therapeutic strategy to target HER2 signaling pathway, but this approach needs to characterize MUC4/HER2 interaction at the molecular level. In this study, we successfully showed the impact of the EGF domains of MUC4 on HER2 binding affinity and demonstrated their “growth factor-like” biological activities in PC cells. Moreover, homology models of the MUC4EGF/HER2 complexes allowed identification of binding hotspots mediating binding affinity with HER2 and PC cell proliferation. These results allow a better understanding of the mechanisms involved in the MUC4/HER2 complex formation and may lead to the design of potential MUC4/HER2 inhibitors.The HER2 receptor and its MUC4 mucin partner form an oncogenic complex via an extracellular region of MUC4 encompassing three EGF domains that promotes tumor progression of pancreatic cancer (PC) cells. However, the molecular mechanism of interaction remains poorly understood. Herein, we decipher at the molecular level the role and impact of the MUC4EGF domains in the mediation of the binding affinities with HER2 and the PC cell tumorigenicity. We used an integrative approach combining in vitro bioinformatic, biophysical, biochemical, and biological approaches, as well as an in vivo study on a xenograft model of PC. In this study, we specified the binding mode of MUC4EGF domains with HER2 and demonstrate their “growth factor-like” biological activities in PC cells leading to stimulation of several signaling proteins (mTOR pathway, Akt, and β-catenin) contributing to PC progression. Molecular dynamics simulations of the MUC4EGF/HER2 complexes led to 3D homology models and identification of binding hotspots mediating binding affinity with HER2 and PC cell proliferation. These results will pave the way to the design of potential MUC4/HER2 inhibitors targeting the EGF domains of MUC4. This strategy will represent a new efficient alternative to treat cancers associated with MUC4/HER2 overexpression and HER2-targeted therapy failure as a new adapted treatment to patients.

Abstract 12593: A Disordered Linker Regulates the Dynamics and Activity of the Protease Domain of the Urokinase-Type Plasminogen Activator

Introduction: The urokinase-type Plasminogen Activator (uPA) catalyzes the rate-limiting step in the fibrinolysis pathway. How the activity of uPA is regulated is not well characterized and its understanding could unravel alternative ways of tuning fibrinolysis. The uPA has three domains: an EGF-like and a Kringle domain linked through a disordered linker to a serine protease domain. The linker binds to the protease domain through a disulfide bond that is located 22.7 Å away from the catalytic triad. There is no crystal structure available for full-length uPA and it is unknown if these domains interact among themselves. We recently showed that full-length uPA is more active than the protease domain by itself. Hydrogen Deuterium Exchange Mass Spectrometry (HDXMS) revealed that the protease domain was less dynamic in the presence of the EGF and Kringle domains, however, our data did not show any interface between the domains. We hypothesize that the linker is allosterically regulating the protease domain. Methods: We mutated two residues in the linker of full-length uPA (S138D and P141A) and measured their activity using a small substrate analog and the endogenous substrate Plasminogen. In addition, changes in dynamics of the protease domain were evaluated using HDXMS. Results: The S138D mutation decreased the activity of the enzyme towards both substrates, while the P141A mutation increased the activity towards the small chromogenic substrate and did not alter the activity towards Plasminogen. HDXMS revealed how the dynamics of different loops in the protease domain were affected by the mutations. The 220s loop was less dynamic in the P141A mutant, however the 110s loop, the activation loop and the β9 strand were more dynamic on this mutant compared to the wildtype. The 110s, 170s and 140s loops together with the activation loop and the β9 strand were more dynamic in the S138D mutant. Interestingly, all these loops are between 20 to 40 Å away from the cysteine that connects the linker to the protease domain, as measured from PDB: 1C5W. Conclusions: Our results support the hypothesis that the long-range dynamic allostery in full-length uPA is mediated by the disordered linker. In addition, this allostery regulates the activity and dynamics of the protease domain of uPA.

Phenotypic Variability of MEGF10 Variants Causing Congenital Myopathy: Report of Two Unrelated Patients from a Highly Consanguineous Population

Congenital myopathies are rare neuromuscular hereditary disorders that manifest at birth or during infancy and usually appear with muscle weakness and hypotonia. One of such disorders, early-onset myopathy, areflexia, respiratory distress, and dysphagia (EMARDD, OMIM: 614399, MIM: 612453), is a rare autosomal recessive disorder caused by biallelic mutations (at homozygous or compound heterozygous status) in MEGF10 (multiple epidermal growth factor-like domains protein family). Here, we report two unrelated patients, who were born to consanguineous parents, having two novel MEGF10 deleterious variants. Interestingly, the presence of MEGF10 associated EMARDD has not been reported in Saudi Arabia, a highly consanguineous population. Moreover, both variants lead to a different phenotypic onset of mild and severe types. Our work expands phenotypic features of the disease and provides an opportunity for genetic counseling to the inflicted families.

Characterization of wall-associated kinase/wall-associated kinase-like (WAK/WAKL) family in rose (Rosa chinensis) reveals the role of RcWAK4 in Botrytis resistance

BackgroundWall-associated kinase (WAK)/WAK-like (WAKL) is one of the subfamily of receptor like kinases (RLK). Although previous studies reported that WAK/WAKL played an important role in plant cell elongation, response to biotic and abiotic stresses, there are no systematic studies on RcWAK/RcWAKL in rose.ResultsIn this study, we identified a total of 68 RcWAK/RcWAKL gene family members within rose (Rosa chinensis) genome. The RcWAKs contained the extracellular galacturonan-binding domain and calcium-binding epidermal growth factor (EGF)-like domain, as well as an intracellular kinase domains. The RcWAKLs are missing either calcium-binding EGF-like domain or the galacturonan-binding domain in their extracellular region. The phylogenetic analysis showed the RcWAK/RcWAKL gene family has been divided into five groups, and these RcWAK/RcWAKL genes were unevenly distributed on the 7 chromosomes of rose. 12 of RcWAK/RcWAKL genes were significantly up-regulated by Botrytis cinerea-inoculated rose petals, where RcWAK4 was the most strongly expressed. Virus induced gene silencing of RcWAK4 increased the rose petal sensitivity to B. cinerea. The results indicated RcWAK4 is involved in the resistance of rose petal against B. cinerea.ConclusionOur study provides useful information to further investigate the function of the RcWAK/RcWAKL gene family and breeding research for resistance to B. cinerea in rose.

Modulating Endothelial Cells with EGFL7 to Diminish aGVHD after Allogeneic Bone Marrow Transplantation in Mice

Acute graft versus host (aGVHD) is the second cause of death after allogeneic-hematopoietic stem cell transplant (allo-HSCT) underscoring the need for novel therapies. Based on previous work that endothelial cell dysfunction is present in aGVHD and that epidermal growth factor-like domain 7 (EGFL7) plays a significant role in decreasing inflammation by repressing endothelial cell activation and T cell migration, we hypothesized that increasing EGFL7 levels after allo-HSCT will diminish the severity of aGVHD. Here, we show that treatment with recombinant EGFL7 (rEGFL7) decreases aGVHD severity and improves survival in recipient mice after allogeneic transplantation with respect to controls without affecting graft versus leukemia effect. Histopathology analysis revealed higher amount of leukocyte infiltration in both large intestine and liver of PBS group compared to rEGFL7-treated mice. Furthermore, damage to the gut was reduced in EGFL7 treated mice. Finally, we showed that rEGFL7 treatment results in higher thymocytes, T, B and dendritic cells in recipient mice after allo-HSCT. This study constitutes a proof of concept of the ability of rEGFL7 therapy to reduce GHVD severity and mortality after allo-HSCT. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Sustained ErbB Activation Causes Demyelination and Hypomyelination by Driving Necroptosis of Mature Oligodendrocytes and Apoptosis of Oligodendrocyte Precursor Cells.

Oligodendrocytes are vulnerable to genetic and environmental insults and its injury leads to demyelinating diseases. The roles of ErbB receptors in maintaining the CNS myelin integrity are largely unknown. Here, we overactivate ErbB receptors that mediate signaling of either neuregulin (NRG) or epidermal growth factor (EGF) family growth factors and found their synergistic activation caused deleterious outcomes in white matter. Sustained ErbB activation induced by the tetracycline-dependent mouse tool Plp-tTA resulted in demyelination, axonal degeneration, oligodendrocyte precursor cell (OPC) proliferation, astrogliosis, and microgliosis in white matter. Moreover, there was hypermyelination before these inflammatory pathologic events. In contrast, sustained ErbB activation induced by another tetracycline-dependent mouse tool Sox10+/rtTA caused hypomyelination in the corpus callosum and optic nerve, which appeared to be a developmental deficit and did not associate with OPC regeneration, astrogliosis, or microgliosis. By tracing the differentiation states of cells expressing tetracycline-controlled transcriptional activator (tTA)/reverse tTA (rtTA)-dependent transgene or pulse-labeled reporter proteins in vitro and in vivo, we found that Plp-tTA targeted mainly mature oligodendrocytes (MOs), whereas Sox10+/rtTA targeted OPCs and newly-formed oligodendrocytes (NFOs). The distinct phenotypes of mice with ErbB overactivation induced by Plp-tTA and Sox10+/rtTA consolidated their nonoverlapping targeting preferences in the oligodendrocyte lineage, and enabled us to demonstrate that ErbB overactivation in MOs induced necroptosis that caused inflammatory demyelination, whereas in OPCs induced apoptosis that caused noninflammatory hypomyelination. Early interference with aberrant ErbB activation ceased oligodendrocyte deaths and restored myelin development in both mice. This study suggests that aberrant ErbB activation is an upstream pathogenetic mechanism of demyelinating diseases, providing a potential therapeutic target.SIGNIFICANCE STATEMENT Primary oligodendropathy is one of the etiologic mechanisms for multiple sclerosis, and oligodendrocyte necroptosis is a pathologic hallmark in the disease. Moreover, the demyelinating disease is now a broad concept that embraces schizophrenia, in which white matter lesions are an emerging feature. ErbB overactivation has been implicated in schizophrenia by genetic analysis and postmortem studies. This study suggests the etiologic implications of ErbB overactivation in myelin pathogenesis and elucidates the pathogenetic mechanisms.

Protein S EGF-like domain related problems in patients with pregnancy morbidity

Protein S EGF-like domain related problems in patients with pregnancy morbidity

In silico structural analysis of truncated 2’ fluoro-RNA aptamer: Elucidating EGF-1 and EGF-2 binding domains on factor IX protein

A stable truncated 2’ fluoro-RNA aptamer contributes to the upstream interruption of the blood coagulation pathway by selectively binding with factor IX protein (FIX). This work followed a rational method for virtually analyzing structural orientation and binding interactions between aptamer and FIX. Three possible aptamer models from the same sequence were designed and evaluated for optimum binding with FIX. Molecular docking was performed to find a prime docking domain and the best docking conformation for a stable aptamer-FIX complex. Possible interactions are observed at PRO126-U9, LYS125-U9, LYS122-A7, ARG116-U9, GLY102-G22, THR101-G22, GLN74-G6, GLY60-G5, CGU36-U11, CGU40-A12, and ASP49-A10. A molecular dynamic simulation assessed the strength of the interaction between aptamer and FIX. It was revealed that the aptamer was effectively bound with the average amino acid fluctuation of 1 Å. The complex reached equilibrium within ten ns during the initial phase of the simulation and then remained stable over 100 ns. B factor analysis showed a moderate atomic displacement from the crystal structure, except at N-terminal from atom 2247−5938. Principal component analysis of post-dynamic trajectories further revealed the stability by the arrayed orientation of amino acids dihedral angles. Overall, 2’ fluoro-RNA aptamer exhibits stronger stability in their interaction with FIX. Noteworthy, the aptamer's stability as an inhibitor to deactivate the clotting cascade by specifically binding with epidermal growth factor-like (EGF) 1 and 2 domains was evidenced.