Decoding spatiotemporal transcriptional dynamics and epithelial fibroblast crosstalk during gastroesophageal junction development through single cell analysis.

The gastroesophageal squamocolumnar junction (GE-SCJ) is a critical tissue interface between the esophagus and stomach, with significant relevance in the pathophysiology of gastrointestinal diseases. Despite this, the molecular mechanisms underlying GE-SCJ development remain unclear. Using single-cell transcriptomics, organoids, and spatial analysis, we examine the cellular heterogeneity and spatiotemporal dynamics of GE-SCJ development from embryonic to adult mice. We identify distinct transcriptional states and signaling pathways in the epithelial and mesenchymal compartments of the esophagus and stomach during development. Fibroblast-epithelial interactions are mediated by various signaling pathways, including WNT, BMP, TGF-ß, FGF, EGF, and PDGF. Our results suggest that fibroblasts predominantly send FGF and TGF-ß signals to the epithelia, while epithelial cells mainly send PDGF and EGF signals to fibroblasts. We observe differences in the ligands and receptors involved in cell-cell communication between the esophagus and stomach. Our findings provide insights into the molecular mechanisms underlying GE-SCJ development and fibroblast-epithelial crosstalk involved, paving the way to elucidate mechanisms during adaptive metaplasia development and carcinogenesis.

Effects of Banhabaekchulcheonma-Tang on Brain Injury and Cognitive Function Impairment Caused by Bilateral Common Carotid Artery Stenosis in a Mouse Model.

Vascular dementia (VD) is the second most prevalent dementia type, with no drugs approved for its treatment. Here, the effects of Banhabaekchulcheonma-Tang (BBCT) on ischemic brain injury and cognitive function impairment were investigated in a bilateral carotid artery stenosis (BCAS) mouse model. Mice were divided into sham-operated, BCAS control, L-BBCT (40 ml/kg), and H-BBCT (80 ml/kg) groups. BBCT's effects were characterized using the Y-maze test, novel object recognition test (NORT), immunofluorescence staining, RNA sequencing, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) analyses. The NORT revealed cognitive function improvement in the H-BBCT group, while the Y-maze test revealed no significant difference among the four groups. The CD68+ microglia and GFAP+ astrocyte numbers were reduced in the H-BBCT group. Furthermore, H-BBCT treatment restored the dysregulation of gene expression caused by BCAS. The major BBCT targets were predicted to be cell division cycle protein 20 (CDC20), Epidermal growth factor (EGF), and tumor necrosis factor receptor-associated factor 1 (TRAF1). BBCT regulates the neuroactive ligand-receptor interaction and neuropeptide signaling pathways, as predicted by KEGG and GO analyses, respectively. BBCT significantly improved cognitive impairment in a BCAS mouse model by inhibiting microglial and astrocyte activation and regulating the expression of CDC20, EGF, TRAF1, and key proteins in the neuroactive ligand-receptor interaction and neuropeptide signaling pathways.

JCAD deficiency delayed liver regenerative repair through the Hippo-YAP signalling pathway.

BACKGROUND AND AIMS: Liver regeneration retardation post partial hepatectomy (PH) is a common clinical problem after liver transplantation. Identification of key regulators in liver regeneration post PH may be beneficial for clinically improving the prognosis of patients after liver transplantation. This study aimed to clarify the function of junctional protein-associated with coronary artery disease (JCAD) in liver regeneration post PH and to reveal the underlying mechanisms. METHODS: JCAD knockout (JCAD-KO), liver-specific JCAD-KO (Jcad△Hep) mice and their control group were subjected to 70% PH. RNA sequencing was conducted to unravel the related signalling pathways. Primary hepatocytes from KO mice were treated with epidermal growth factor (EGF) to evaluate DNA replication. Fluorescent ubiquitination-based cell cycle indicator (FUCCI) live-imaging system was used to visualise the phases of cell cycle. RESULTS: Both global and liver-specific JCAD deficiency postponed liver regeneration after PH as indicated by reduced gene expression of cell cycle transition and DNA replication. Prolonged retention in G1 phase and failure to transition over the cell cycle checkpoint in JCAD-KO cell line was indicated by a FUCCI live-imaging system as well as pharmacologic blockage. JCAD replenishment by adenovirus reversed the impaired DNA synthesis in JCAD-KO primary hepatocyte in exposure to EGF, which was abrogated by a Yes-associated protein (YAP) inhibitor, verteporfin. Mechanistically, JCAD competed with large tumour suppressor 2 (LATS2) for WWC1 interaction, leading to LATS2 inhibition and thereafter YAP activation, and enhanced expression of cell cycle-associated genes. CONCLUSION: JCAD deficiency led to delayed regeneration after PH as a result of blockage in cell cycle progression through the Hippo-YAP signalling pathway. These findings uncovered novel functions of JCAD and suggested a potential strategy for improving graft growth and function post liver transplantation. KEY POINTS: JCAD deficiency leads to an impaired liver growth after PH due to cell division blockage. JCAD competes with LATS2 for WWC1 interaction, resulting in LATS2 inhibition, YAP activation and enhanced expression of cell cycle-associated genes. Delineation of JCADHippoYAP signalling pathway would facilitate to improve prognosis of acute liver failure and graft growth in living-donor liver transplantation.

Plumbagin as a preferential lead molecule to combat EGFR-driven matrix abundance and migration of cervical carcinoma cells.

The handshake between the complex networks of matrix components in the tumor micro-environment (TME) is considered as a crucial event in the progression of several cancers including cervical carcinoma (CC). A number of studies report a connection between epidermal growth factor (EGF) and matrix component production. Studies demonstrate that the mechano-transduction trigger by collagen, influences the tumor cells to undergo epithelial-mesenchymal transition (EMT) and block the entry of drugs. We hypothesize that the intervention to prevent EGF triggered deposition of matrix components could sensitize several therapies for CC cells. We utilized morphological assessment, MTT assay, mitored tracking, acridine orange (AO)/ ethidium bromide (EtBr) staining and bromodeoxyuridine (BrdU) assay to measure the cell viability, mitochondrial activity, cellular apoptosis, and DNA synthesis. Clonogenic assay and scratch healing assay were executed to address the stemness and migratory potential. Detection of glycosaminoglycan's (GAGs), collagen, matrix metalloproteinase (MMP)-2/9 secretion and calcium (Ca2+) ions were performed to assess the production of matrix components. Finally, the interaction between EGFR and plumbagin was evaluated by employing molecular dynamics (MD) simulation. Pre-treating the cells with plumbagin inhibited the EGF-induced EMT along with reduction in cell proliferation, migration, clonogenesis and depletion of matrix components. The actions of EGF and plumbagin were more pronounced in HPV-positive CC cells than HPV-negative CC cells. This study identified that increased matrix production triggered by EGF-rich milieu is inhibited by plumbagin in human papilloma viral (HPV) 68 positive ME180, HPV 16 positive SiHa and HPV-negative C33A cell lines. Delivery of plumbagin directly to TME would effectively accelerate the clearance of CC cells, reduce metastasis and matrix abundance by employing targeted delivery to minimize the undesired effects of plumbagin.

Regulation of STAT1 and STAT4 Expression by Growth Factor and Interferon Supplementation in Sjögren's Syndrome Cell Culture Models.

Our goal was to investigate the effects of epidermal growth factor (EGF) and interferons (IFNs) on signal transducer and activator of transcription STAT1 and STAT4 mRNA and active phosphorylated protein expression in Sjögren's syndrome cell culture models. iSGECs (immortalized salivary gland epithelial cells) and A253 cells were treated with EGF, IFN-alpha, -beta, -gamma, or mitogen-activated protein kinase p38 alpha (p38-MAPK) inhibitor for 0-24-48-72 h. STAT1 and STAT4 mRNA expression was quantified by qRT-PCR. Untreated and treated cells were compared using the delta-delta-CT method based on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) normalized relative fold changes. phospho-tyrosine-701-STAT1 and phospho-serine-721-STAT4 were detected by Western blot analysis. STAT4 mRNA expression decreased 48 h after EGF treatment in A253 cells, immortalized salivary gland epithelial cells iSGECs nSS2 (sicca patient origin), and iSGECs pSS1 (anti-SSA negative Sjögren's Syndrome patient origin). EGF and p38-MAPK inhibitor decreased A253 STAT4 mRNA levels. EGF combined with IFN-gamma increased phospho-STAT4 and phospho-STAT1 after 72 h in all cell lines, suggesting additive effects for phospho-STAT4 and a major effect from IFN-gamma for phospho-STAT1. pSS1 and nSS2 cells responded differently to type I and type II interferons, confirming unique functional characteristics between iSGEC cell lines. EGF/Interferon related pathways might be targeted to regulate STAT1 and STAT4 expression in salivary gland epithelial cells. Further investigation is required learn how to better target the Janus kinases/signal transducer and activator of transcription proteins (JAK/STAT) pathway-mediated inflammatory response in Sjögren's syndrome.

Protein Dynamic Landscape during Mouse Mammary Gland Development from Virgin to Pregnant, Lactating, and Involuting Stages.

The mammary gland undergoes significant physiological changes as it undergoes a transition from virgin to pregnancy, lactation, and involution. However, the dynamic role of proteins in regulating these processes during mouse mammary gland development has not been thoroughly explored. In this study, we collected mouse mammary gland tissues from mature virgins aged 8-10 weeks (V), day 16 of pregnancy (P16d), day 12 of lactation (L12d), day 1 of forced weaning (FW 1d), and day 3 of forced weaning (FW 3d) stages for analysis using DIA-based quantitative proteomics technology. A total of 3,312 proteins were identified, of which 843 were DAPs that were categorized into nine clusters based on their abundance changes across developmental stages. Notably, DAPs in cluster 2, which peaked at the L12d stage, were primarily associated with mammary gland development and lactation. The protein-protein interaction network revealed that the epidermal growth factor (EGF) was central to this cluster. Our study provides a comprehensive overview of the mouse mammary gland development proteome and identifies some important proteins, such as EGF, Janus kinase 1 (JAK1), and signal transducer and activator of transcription 6 (STAT6) that may serve as potential targets for future research to provide guidelines for a deeper understanding of the developmental biology of mammary glands.

NRF2 regulates EGF stability through OTUD4 in lung adenocarcinoma.

NRF2 (NFE2L2) is a transcription factor mainly for regulating cellular antioxidant response and therefore promotes tumor progression. The target genes of NRF2 also play important roles in cellular processes including glucose metabolism, de novo serine synthesis, iron metabolism, etc. Here, by modulating NRF2 expression in lung adenocarcinoma (LUAD) cells, we showed that NRF2 regulated EGF expression at protein level. Furthermore, EGF was identified as a ubiquitinated protein. We predicted three deubiquitinases of EGF, and OTUD4 had the highest correlation with NRF2 in LUAD among the three. OTUD4 expression was reduced upon NRF2 knocking-down and recovered upon NRF2 rescuing in A549 cells. Then a potential binding site for NRF2 in OTUD4 promoter was searched out. By binding with OTUD4 promoter, NRF2 transcriptionally activated OTUD4, thus promoted EGF deubiquitination and enhanced its stability. More importantly, OTUD4 and NRF2 expression was found being correlated in LUAD patients. The data collectively revealed a novel mechanism of NRF2 regulating on EGF stability through OTUD4 in LUAD.

High expression of CD9 and Epidermal Growth Factor Receptor promotes the development of tongue cancer.

Tongue cancer is distinguished by aggressive behavior, a high risk of recurrence, lymph, and distant metastases. Hypoxia-Induced Factor 1 α functions as a CD9 transcription factor. CD9 is a transmembrane protein that may be found on the cell membrane. It can modulate the expression of the Epidermal Growth Factor Receptor (EGFR) pathway. ELISA was used to measure serum CD9, p-EGFR, and p-Akt levels in 70 tongue cancer patients and 35 healthy controls. RT-PCR was used to analyze the gene expression of the related genes. The gene as well as protein expression of CD9, EGFR/p-EGFR, and Akt/p-Akt was significantly higher in case subjects when compared with the controls. The expression of CD9 was higher in case subjects who were smokers/alcoholics when to control subjects who were smokers/alcoholics. Overexpression of CD9 due to hypoxic conditions leads to the activation of EGFR-signaling pathway resulting in cancer progression, resistance to chemotherapy. Hence, CD9 could be a potential target to suppress cancer progression.

Epidermal growth factor receptor (EGFR) is a target of the tumor-suppressor E3 ligase FBXW7.

FBXW7 is an E3 ubiquitin ligase that targets proteins for proteasome-mediated degradation and is mutated in various cancer types. Here, we use CRISPR base editors to introduce different FBXW7 hotspot mutations in human colon organoids. Functionally, FBXW7 mutation reduces EGF dependency of organoid growth by ~10,000-fold. Combined transcriptomic and proteomic analyses revealed increased EGFR protein stability in FBXW7 mutants. Two distinct phosphodegron motifs reside in the cytoplasmic tail of EGFR. Mutations in these phosphodegron motifs occur in human cancer. CRISPR-mediated disruption of the phosphodegron motif at T693 reduced EGFR degradation and EGF growth factor dependency. FBXW7 mutant organoids showed reduced sensitivity to EGFR-MAPK inhibitors. These observations were further strengthened in CRC-derived organoid lines and validated in a cohort of patients treated with panitumumab. Our data imply that FBXW7 mutations reduce EGF dependency by disabling EGFR turnover.

Epidermal growth factor receptor phosphorylation contributes to levobupivacaine-induced contraction in isolated rat aorta.

Vasoconstriction induced by levobupivacaine, a local anesthetic, is mediated by increased levels of calcium, tyrosine kinase, c-Jun NH2-terminal kinase (JNK), and phospholipase D, which are associated with prolonged local anesthesia. Epidermal growth factor receptor (EGFR) phosphorylation is associated with vasoconstriction. However, its role in levobupivacaine-induced contractions remains unknown. We determined whether EGFR phosphorylation is associated with levobupivacaine-induced contractions in isolated rat thoracic aortas and identified the underlying cellular signaling pathways. The effects of various inhibitors and a calcium-free solution alone or in combination on levobupivacaine-induced contractions were then assessed. Furthermore, we examined the effects of various inhibitors on levobupivacaine-induced EGFR and JNK phosphorylation and calcium levels in vascular smooth muscle cells (VSMCs) of rat aortas. The EGFR tyrosine kinase inhibitor AG1478, matrix metalloproteinase (MMP) inhibitor GM6001, Src kinase inhibitors PP1 and PP2, and JNK inhibitor SP600125 attenuated levobupivacaine-induced contractions. Moreover, although the calcium-free solution abolished levobupivacaine-induced contractions, calcium reversed this inhibitory effect. The magnitude of the calcium-mediated reversal of abolished levobupivacaine-induced contractions was lower in the combination treatment with calcium-free solution and AG1478 than in the treatment with calcium-free solution alone. Levobupivacaine induced EGFR and JNK phosphorylation. However, AG1478, GM6001, and PP2 attenuated levobupivacaine-induced EGFR and JNK phosphorylation. Moreover, although levobupivacaine induced JNK phosphorylation in control siRNA-transfected VSMCs, EGFR siRNA inhibited levobupivacaine-induced JNK phosphorylation. Furthermore, AG1478 inhibited levobupivacaine-induced calcium increases in VSMCs. Collectively, these findings suggest that levobupivacaine-induced EGFR phosphorylation, which may occur via the Src kinase-MMP pathway, contributes to vasoconstriction via JNK phosphorylation and increased calcium levels.

Epidermal Growth Factor Suppresses the Development of GABAergic Neurons Via the Modulation of Perineuronal Net Formation in the Neocortex of Developing Rodent Brains.

Previously, we reported that epidermal growth factor (EGF) suppresses GABAergic neuronal development in the rodent cortex. Parvalbumin-positive GABAergic neurons (PV neurons) have a unique extracellular structure, perineuronal nets (PNNs). PNNs are formed during the development of PV neurons and are mainly formed from chondroitin sulfate (CS) proteoglycans (CSPGs). We examined the effect of EGF on CSPG production and PNN formation as a potential molecular mechanism for the inhibition of inhibiting GABAergic neuronal development by EGF. In EGF-overexpressing transgenic (EGF-Tg) mice, the number of PNN-positive PV neurons was decreased in the cortex compared with that in wild-type mice, as in our previous report. The amount of CS and neurocan was also lower in the cortex of EGF-Tg mice, with a similar decrease observed in EGF-treated cultured cortical neurons. PD153035, an EGF receptor (ErbB1) kinase inhibitor, prevented those mentioned above excess EGF-induced reduction in PNN. We explored the molecular mechanism underlying the effect of EGF on PNNs using fluorescent substrates for matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinases (ADAMs). EGF increased the enzyme activity of MMPs and ADAMs in cultured neurons. These enzyme activities were also increased in the EGF-Tg mice cortex. GM6001, a broad inhibitor of MMPs and ADAMs, also blocked EGF-induced PNN reductions. Therefore, EGF/EGF receptor signals may regulate PNN formation in the developing cortex.

Extracellular domain mutations of the EGF receptor differentially modulate high-affinity and low-affinity responses to EGF receptor ligands.

The EGF receptor is mutated in a number of cancers. In most cases, the mutations occur in the intracellular tyrosine kinase domain. However, in glioblastomas, many of the mutations are in the extracellular ligand binding domain. To determine what changes in receptor function are induced by such extracellular domain mutations, we analyzed the binding and biological response to the seven different EGF receptor ligands in three common glioblastoma mutants-R84K, A265V, and G574V. Our data indicate that all three mutations significantly increase the binding affinity of all seven ligands. In addition, the mutations increase the potency of all ligands for stimulating receptor autophosphorylation, phospholipase Cγ, Akt, and MAP kinase activity. In all mutants, the rank order of ligand potency seen at the wild-type receptor was retained, suggesting that the receptors still discriminate among the different ligands. However, the low-affinity ligands, EPR and EPG, did show larger than average enhancements of potency for stimulating Akt and MAPK but not receptor autophosphorylation and phospholipase Cγ activation. Relative to the wild-type receptor, these changes lead to an increase in the responsiveness of these mutants to physiological concentrations of ligands and an alteration in the ratio of activation of the different pathways. This may contribute to their oncogenic potential. In the context of recent findings, our data also suggest that so-called "high"-affinity biological responses arise from activation by isolated receptor dimers, whereas "low"-affinity biological responses require clustering of receptors which occurs at higher concentrations of ligand.

Cholesterol suppresses spontaneous activation of EGFR-mediated signal transduction.

Epidermal growth factor receptor (EGFR)-mediated signal transduction controls cell growth and proliferation. The signaling pathway is regulated so that it is activated only by external EGF stimuli, but the mechanisms that prevent EGF-independent spontaneous activation of EGFR-mediated signaling are unknown. Here we report cholesterol depletion activates EGFR-mediated signaling without EGF. We applied automated single-molecule imaging to EGFR and characterized the lateral diffusion and cluster formation on cholesterol-depleted and cholesterol-supplemented membranes. In cells in which cholesterol was depleted by methyl-ß-cyclodextrin (MßCD) treatment, EGFR exhibited a reduction in lateral diffusion, an acceleration of cluster formation, and autophosphorylation without EGF. Concurrently, extracellular signal-regulated kinase (ERK), which is regulated by EGFR-mediated signaling, exhibited phosphorylation and nuclear translocation without EGF. These cholesterol depletion-induced changes were similar, albeit less efficient, to those that occurred with EGF stimulation in normal cells without MßCD, indicating the spontaneous activation of EGFR signaling. The exogenous supplementation of cholesterol suppressed the MßCD-induced spontaneous activation of EGFR and ERK nuclear translocation. Single-molecule imaging of EGFR in a large number of cells revealed cell-to-cell heterogeneity, with a sub-population showing a high ability for spontaneous activation. These results provide evidence that EGFR-mediated signaling is properly regulated by cholesterol metabolism to prevent uncontrolled spontaneous activation.

Impact of antibody architecture and paratope valency on effector functions of bispecific NKp30 x EGFR natural killer cell engagers.

Natural killer (NK) cells emerged as a promising effector population that can be harnessed for anti-tumor therapy. In this work, we constructed NK cell engagers (NKCEs) based on NKp30-targeting single domain antibodies (sdAbs) that redirect the cytotoxic potential of NK cells toward epidermal growth factor receptor (EGFR)-expressing tumor cells. We investigated the impact of crucial parameters such as sdAb location, binding valencies, the targeted epitope on NKp30, and the overall antibody architecture on the redirection capacity. Our study exploited two NKp30-specific sdAbs, one of which binds a similar epitope on NKp30 as its natural ligand B7-H6, while the other sdAb addresses a non-competing epitope. For EGFR-positive tumor targeting, humanized antigen-binding domains of therapeutic antibody cetuximab were used. We demonstrate that NKCEs bivalently targeting EGFR and bivalently engaging NKp30 are superior to monovalent NKCEs in promoting NK cell-mediated tumor cell lysis and that the architecture of the NKCE can substantially influence killing capacities depending on the NKp30-targeting sdAb utilized. While having a pronounced impact on NK cell killing efficacy, the capabilities of triggering antibody-dependent cellular phagocytosis or complement-dependent cytotoxicity were not significantly affected comparing the bivalent IgG-like NKCEs with cetuximab. However, the fusion of sdAbs can have a slight impact on the NK cell release of immunomodulatory cytokines, as well as on the pharmacokinetic profile of the NKCE due to unfavorable spatial orientation within the molecule architecture. Ultimately, our findings reveal novel insights for the engineering of potent NKCEs triggering the NKp30 axis.

The HIF-1α/EGF/EGFR Signaling Pathway Facilitates the Proliferation of Yak Alveolar Type II Epithelial Cells in Hypoxic Conditions.

The yak is a unique creature that thrives in low-oxygen environments, showcasing its adaptability to high-altitude settings with limited oxygen availability due to its unique respiratory system. However, the impact of hypoxia on alveolar type II (AT2) epithelial cell proliferation in yaks remains unexplored. In this study, we investigated the effects of different altitudes on 6-month-old yaks and found an increase in alveolar septa thickness and AT2 cell count in a high-altitude environment characterized by hypoxia. This was accompanied by elevated levels of hypoxia-inducible factor-1α (HIF-1α) and epidermal growth factor receptor (EGFR) expression. Additionally, we observed a significant rise in Ki67-positive cells and apoptotic lung epithelial cells among yaks inhabiting higher altitudes. Our in vitro experiments demonstrated that exposure to hypoxia activated HIF-1α, EGF, and EGFR expression leading to increased proliferation rates among yak AT2 cells. Under normal oxygen conditions, activation of HIF-1α enhanced EGF/EGFR expressions which subsequently stimulated AT2 cell proliferation. Furthermore, activation of EGFR expression under normoxic conditions further promoted AT2 cell proliferation while simultaneously suppressing apoptosis. Conversely, inhibition of EGFR expression under hypoxic conditions had contrasting effects. In summary, hypoxia triggers the proliferation of yak AT2 cells via activation facilitated by the HIF-1α/EGF/EGFR signaling cascade.

Searching for EGF Fragments Recreating the Outer Sphere of the Growth Factor Involved in Receptor Interactions.

The aims of this study were to determine whether it is possible to use peptide microarrays obtained using the SPOT technique (immobilized on cellulose) and specific polyclonal antibodies to select fragments that reconstruct the outer sphere of proteins and to ascertain whether the selected peptide fragments can be useful in the study of their protein-protein and/or peptide-protein interactions. Using this approach, epidermal growth factor (EGF) fragments responsible for the interaction with the EGF receptor were searched. A library of EGF fragments immobilized on cellulose was obtained using triazine condensing reagents. Experiments on the interactions with EGFR confirmed the high affinity of the selected peptide fragments. Biological tests on cells showed the lack of cytotoxicity of the EGF fragments. Selected EGF fragments can be used in various areas of medicine.

Ferrous-sucrose complex supplementation regulates maternal plasma metabolism and the fecal microbiota composition and improves neonatal immunity and placental glucose transportation by activating the EGF/PI3K/AKT signaling pathways in sows.

Pregnancy is a dynamic state involving rapid physiological changes in metabolism, affecting the health and development of the offspring. During pregnancy, the placenta constitutes a physical and immunological barrier to provide fetal nutrition through the maternal blood and prevent the exposure of the fetus to dangerous signals. Metabolic changes in the plasma, the fecal microbiota profile, and functional regulation in the placenta were studied in sows supplied with a ferrous-sucrose complex (FeSuc) from late gestation to parturition. The results revealed that maternal FeSuc supplementation enhanced arginine and proline metabolism, glutathione metabolism, with increased glutamic acid, beta-D-glucosamine, L-proline, 1-butylamine, and succinic acid and reduced sphingosine and chenodeoxycholic acid sulfate levels in the plasma. Moreover, significantly increased abundances of Christensenellaceae_R-7_group, Prevotellaceae_NK3B31_group, and Lachnospiraceae_NK4B4_group were detected in the feces of sows from the FeSuc group (P < 0.05). Spearman's correlation analysis indicated that Prevotellaceae_NK3B31_group abundances were positively correlated with glutamic acid, indoxyl sulfate, acetyl-DL-leucine, and beta-D-glucosamine, while Christensenellaceae_R-7_group was positively correlated with beta-D-glucosamine. Furthermore, maternal FeSuc supplementation significantly increased neonatal glucose (P < 0.01) and iron (P < 0.01) in the neonatal serum, significantly increased IL-10 and TGF-ß1 levels in the neonatal liver (P < 0.01) and jejunum (P < 0.05), promoted the transcription of immune molecules in the placenta, and significantly increased the protein expressions of EGF (P < 0.05), PI3K (P < 0.01), p-PI3K (P < 0.001), p-AKT (P < 0.01), and glucose transporter 1 (GLUT1) (P < 0.001) in the placenta. The current study demonstrated that FeSuc supplementation regulated maternal metabolism processes by altering the fecal microbial composition and improved neonatal immunity and placental glucose transportation by activating the EGF/PI3K/AKT signaling pathways in sows.

Collecting duct water permeability inhibition by EGF is associated with decreased cAMP, PKA activity, and AQP2 phosphorylation at Ser269.

Prior studies showed that epidermal growth factor (EGF) inhibits vasopressin-stimulated osmotic water permeability in the renal collecting duct. Here, we investigated the underlying mechanism. Using isolated perfused rat inner medullary collecting ducts (IMCDs), we found that the addition of EGF to the peritubular bath significantly decreased 1-deamino-8-d-arginine vasopressin (dDAVP)-stimulated water permeability, confirming prior observations. The inhibitory effect of EGF on water permeability was associated with a reduction in intracellular cAMP levels and protein kinase A (PKA) activity. Using phospho-specific antibodies and immunoblotting in IMCD suspensions, we showed that EGF significantly reduces phosphorylation of AQP2 at Ser264 and Ser269. This effect was absent when 8-cpt-cAMP was used to induce AQP2 phosphorylation, suggesting that EGF's inhibitory effect was at a pre-cAMP step. Immunofluorescence labeling of microdissected IMCDs showed that EGF significantly reduced apical AQP2 abundance in the presence of dDAVP. To address what protein kinase might be responsible for Ser269 phosphorylation, we used Bayesian analysis to integrate multiple-omic datasets. Thirteen top-ranked protein kinases were subsequently tested by in vitro phosphorylation experiments for their ability to phosphorylate AQP2 peptides using a mass spectrometry readout. The results show that the PKA catalytic-α subunit increased phosphorylation at Ser256, Ser264, and Ser269. None of the other kinases tested phosphorylated Ser269. In addition, H-89 and PKI strongly inhibited dDAVP-stimulated AQP2 phosphorylation at Ser269. These results indicate that EGF decreases the water permeability of the IMCD by inhibiting cAMP production, thereby inhibiting PKA and decreasing AQP2 phosphorylation at Ser269, a site previously shown to regulate AQP2 endocytosis.NEW & NOTEWORTHY The authors used native rat collecting ducts to show that inhibition of vasopressin-stimulated water permeability by epidermal growth factor involves a reduction of aquaporin 2 phosphorylation at Ser269, a consequence of reduced cAMP production and PKA activity.

Receptor mediated targeting of EGF-conjugated alginate-PAMAM nanoparticles to lung adenocarcinoma: 2D/3D in vitro and in vivo evaluation.

Carboplatin (cis-diamine (1,1-cyclobutandicarboxylaso)­platinum (II)) is a second-generation antineoplastic drug, which is widely used for chemotherapy of lung, colon, breast, cervix, testicular and digestive system cancers. Although preferred over cisplatin due to the lower incidence of nephrotoxicity and ototoxicity, efficient carboplatin delivery remains as a major challenge. In this study, carboplatin loaded alginate- poly(amidoamine) (PAMAM) hybrid nanoparticles (CAPs) with mean sizes of 192.13 ± 4.15 nm were synthesized using a microfluidic platform, then EGF was conjugated to the surface of CAPs (EGF-CAPs) for the receptor-targeted delivery. Hence, increased FITC+ cell counts were observed in A549 spheroids after EGF-CAP treatment compared to CAP in the 3D cellular uptake study. As such, the cytotoxicity of EGF-CAP was approximately 2-fold higher with an IC50 value of 35.89 ± 10.37 µg/mL compared to the CAPs in A549 spheroids. Based on in vivo experimental animal model, anti-tumor activities of the group treated with CAP decreased by 61 %, whereas the group treated with EGF-CAP completely recovered. Additionally, EGF-CAP application was shown to induce apoptotic cell death. Our study provided a new strategy for designing a hybrid nanoparticle for EGFR targeted carboplatin delivery with improved efficacy both in vitro and in vivo applications.

Chemical mixture that targets the epidermal growth factor pathway impairs human trophoblast cell functions.

Pregnant women are exposed to complex chemical mixtures, many of which reach the placenta. Some of these chemicals interfere with epidermal growth factor receptor (EGFR) activation, a receptor tyrosine kinase that modulates several placenta cell functions. We hypothesized that a mixture of chemicals (Chem-Mix) known to reduce EGFR activation (polychlorinated biphenyl (PCB)-126, PCB-153, atrazine, trans-nonachlor, niclosamide, and bisphenol S) would interfere with EGFR-mediated trophoblast cell functions. To test this, we determined the chemicals' EGFR binding ability, EGFR and downstream effectors activation, and trophoblast functions (proliferation, invasion, and endovascular differentiation) known to be regulated by EGFR in extravillous trophoblasts (EVTs). The Chem-Mix competed with EGF for EGFR binding, however only PCB-153, niclosamide, trans-nonachlor, and BPS competed for binding as single chemicals. The effects of the Chem-Mix on EGFR phosphorylation were tested by exposing the placental EVT cell line, HTR-8/SVneo to control (0.1% DMSO), Chem-Mix (1, 10, or 100 ng/ml), EGF (30 ng/ml), or Chem-Mix + EGF. The Chem-Mix - but not the individual chemicals - reduced EGF-mediated EGFR phosphorylation in a dose dependent manner, while no effect was observed in its downstream effectors (AKT and STAT3). None of the individual chemicals affected EVT cell invasion, but the Chem-Mix reduced EVT cell invasion independent of EGF. In support of previous studies that have explored chemicals targeting a specific pathway (estrogen/androgen receptor), current findings indicate that exposure to a chemical mixture that targets the EGFR pathway can result in a greater impact compared to individual chemicals in the context of placental cell functions.