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Foods (Basel, Switzerland) Jul 2023Cell-cultured protein technology has become increasingly attractive due to its sustainability and climate benefits. The aim of this study is to determine the nutritional...
Cell-cultured protein technology has become increasingly attractive due to its sustainability and climate benefits. The aim of this study is to determine the nutritional quality of the human-induced pluripotent stem cell (hiPSC)-cultured proteins in an advanced 3D peptide hydrogel system for the highly efficient production of cell-cultured proteins. Our previous study demonstrated a PGmatrix peptide hydrogel for the 3D embedded culture of long-term hiPSC maintenance and expansion (PGmatrix-hiPSC (PG-3D)), which showed significantly superior pluripotency when compared with traditional 2D cell culture on Matrigel and/or Vitronectin and other existing 3D scaffolding systems such as Polyethylene glycol (PEG)-based hydrogels. In this study, we designed a PGmatrix 3D suspension (PG-3DSUSP) system from the PG-3D embedded system that allows scaling up a hiPSC 3D culture volume by 20 times (e.g., from 0.5 mL to 10 mL). The results indicated that the PG-3DSUSP was a competitive system compared to the well-established PG-3D embedded method in terms of cell growth performance and cell pluripotency. hiPSCs cultured in PG-3DSUSP consistently presented a 15-20-fold increase in growth and a 95-99% increase in viability across multiple passages with spheroids with a size range of 30-50 μm. The expression of pluripotency-related genes, including , , , , and , in PG-3DSUSP-cultured hiPSCs was similar to or higher than that observed in a PG-3D system, suggesting continuous pluripotent maintenance. The nutritional value of the hiPSC-generated proteins from the PG-3DSUSP system was further evaluated for amino acid composition and in vitro protein digestibility. The amino acid composition of the hiPSC-generated proteins demonstrated a significantly higher essential amino acid content (39.0%) than human skeletal muscle protein (31.8%). In vitro protein digestibility of hiPSC-generated proteins was significantly higher (78.0 ± 0.7%) than that of the commercial beef protein isolate (75.7 ± 0.6%). Taken together, this is the first study to report an advanced PG-3DSUSP culture system to produce highly efficient hiPSC-generated proteins that possess more essential amino acids and better digestibility. The hiPSC-generated proteins with superior nutrition quality may be of particular significance as novel alternative proteins in food engineering and industries for future food, beverage, and supplement applications.
PubMed: 37509805
DOI: 10.3390/foods12142713 -
Mediators of Inflammation 2023Vitronectin (VTN) has been reported to trigger cell pyroptosis to aggravate inflammation in our previous study. However, the function of VTN in inflammatory bowel...
OBJECTIVE
Vitronectin (VTN) has been reported to trigger cell pyroptosis to aggravate inflammation in our previous study. However, the function of VTN in inflammatory bowel disease (IBD) remains to be addressed.
METHODS
Real-time PCR and western blotting were performed to analyze VTN-regulated intestinal epithelial cell (IEC) differentiation through ferroptosis, and immunofluorescence (IF), luciferase, and chromatin immunoprecipitation were used to identify whether VTN-modulated ferroptosis is dependent on phosphodiesterase 4 (PDE4)/protein kinase A (PKA)/cyclic adenosine monophosphate-response element-binding protein (CREB) cascade pathway. experiment in mice and a pilot study in patients with IBD were used to confirm inhibition of PDE4-alleviated IECs ferroptosis, leading to cell differentiation during mucosal healing.
RESULTS
Herein, we found that caudal-related homeobox transcription factor 2-mediated IECs differentiation was impaired in response to VTN, which was attributed to enhanced ferroptosis characterized by decreased glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 expression. Inhibition of ferroptosis in IECs rescued the inhibitory effect of VTN on cell differentiation. Further analysis showed that VTN triggered phosphorylation of PDE4, leading to inhibit PKA/CREB activation and CREB nuclear translocation, which further reduced GPX4 transactivation. Endogenous PKA interacted with CREB, and this interaction was destroyed in response to VTN stimulation. What is more, overexpression of CREB in CaCO cells overcame the promotion of VTN on ferroptosis. Most importantly, inhibition of PDE4 by roflumilast or dipyridamole could alleviate dextran sulfate sodium-induced colitis in mice and in a pilot clinical study confirmed by IF.
CONCLUSIONS
These findings demonstrated that highly expressed VTN disrupted IECs differentiation through PDE4-mediated ferroptosis in IBD, suggesting targeting PDE4 could be a promising therapeutic strategy for patients with IBD.
Topics: Mice; Animals; Vitronectin; Cyclic Nucleotide Phosphodiesterases, Type 4; Ferroptosis; Pilot Projects; Inflammatory Bowel Diseases; Cell Differentiation
PubMed: 37501933
DOI: 10.1155/2023/6623329 -
International Journal of Bioprinting 2023This article provides an overview of the different types of blood-derived biomaterials that can be used as solvent additives in the formulation of inks/bioinks for use... (Review)
Review
This article provides an overview of the different types of blood-derived biomaterials that can be used as solvent additives in the formulation of inks/bioinks for use in solvent extrusion printing/bioprinting. We discuss the properties of various blood sub-products obtained after blood fractionation in terms of their use in tailoring ink/bioink to produce functional constructs designed to improve tissue repair. Blood-derived additives include platelets and/or their secretome, including signaling proteins and microvesicles, which can drive cell migration, inflammation, angiogenesis, and synthesis of extracellular matrix proteins. The contribution of plasma to ink/bioink functionalization relies not only on growth factors, such as hepatocyte growth factor and insulin growth factors, but also on adhesive proteins, such as fibrinogen/fibrin, vitronectin, and fibronectin. We review the current developments and progress in solvent-based extrusion printing/bioprinting with inks/bioinks functionalized with different blood-derived products, leading toward the development of more advanced patient-specific 3D constructs in multiple medical fields, including but not limited to oral tissues and cartilage, bone, skin, liver, and neural tissues. This information will assist researchers in identifying the most suitable blood-derived product for their ink/bioink formulation based on the intended regenerative functionality of the target tissue.
PubMed: 37457947
DOI: No ID Found -
Theranostics 2023: Renal infiltration of inflammatory cells including macrophages is a crucial event in kidney fibrogenesis. However, how macrophage regulates fibroblast activation in...
: Renal infiltration of inflammatory cells including macrophages is a crucial event in kidney fibrogenesis. However, how macrophage regulates fibroblast activation in the fibrotic kidney remains elusive. In this study, we show that macrophages promoted fibroblast activation by assembling a vitronectin (Vtn)-enriched, extracellular microenvironment. : We prepared decellularized kidney tissue scaffold (KTS) from normal and fibrotic kidney after unilateral ischemia-reperfusion injury (UIRI) and carried out an unbiased quantitative proteomics analysis. NRK-49F cells were seeded on macrophage-derived extracellular matrix (ECM) scaffold. Genetic Vtn knockout (Vtn-/-) mice and chronic kidney disease (CKD) model with overexpression of Vtn were used to corroborate a role of Vtn/integrin αvβ5/Src in kidney fibrosis. : Vtn was identified as one of the most upregulated proteins in the decellularized kidney tissue scaffold from fibrotic kidney by mass spectrometry. Furthermore, Vtn was upregulated in the kidney of mouse models of CKD and primarily expressed and secreted by activated macrophages. Urinary Vtn levels were elevated in CKD patients and inversely correlated with kidney function. Genetic ablation or knockdown of Vtn protected mice from developing kidney fibrosis after injury. Conversely, overexpression of Vtn exacerbated renal fibrotic lesions and aggravated renal insufficiency. We found that macrophage-derived, Vtn-enriched extracellular matrix scaffold promoted fibroblast activation and proliferation. In vitro, Vtn triggered fibroblast activation by stimulating integrin αvβ5 and Src kinase signaling. Either blockade of αvβ5 with neutralizing antibody or pharmacological inhibition of Src by Saracatinib abolished Vtn-induced fibroblast activation. Moreover, Saracatinib dose-dependently ameliorated Vtn-induced kidney fibrosis in vivo. These results demonstrate that macrophage induces fibroblast activation by assembling a Vtn-enriched extracellular microenvironment, which triggers integrin αvβ5 and Src kinase signaling. : Our findings uncover a novel mechanism by which macrophages contribute to kidney fibrosis via assembling a Vtn-enriched extracellular niche and suggest that disrupting fibrogenic microenvironment could be a therapeutic strategy for fibrotic CKD.
Topics: Mice; Animals; Vitronectin; Kidney; Renal Insufficiency, Chronic; src-Family Kinases; Macrophages; Fibroblasts; Fibrosis
PubMed: 37441594
DOI: 10.7150/thno.85250 -
Molecular & Cellular Proteomics : MCP Sep 2023The asialoglycoprotein receptor (ASGPR) and the mannose receptor C-type 1 (MRC1) are well known for their selective recognition and clearance of circulating...
The asialoglycoprotein receptor (ASGPR) and the mannose receptor C-type 1 (MRC1) are well known for their selective recognition and clearance of circulating glycoproteins. Terminal galactose and N-Acetylgalactosamine are recognized by ASGPR, while terminal mannose, fucose, and N-Acetylglucosamine are recognized by MRC1. The effects of ASGPR and MRC1 deficiency on the N-glycosylation of individual circulating proteins have been studied. However, the impact on the homeostasis of the major plasma glycoproteins is debated and their glycosylation has not been mapped with high molecular resolution in this context. Therefore, we evaluated the total plasma N-glycome and plasma proteome of ASGR1 and MRC1 deficient mice. ASGPR deficiency resulted in an increase in O-acetylation of sialic acids accompanied by higher levels of apolipoprotein D, haptoglobin, and vitronectin. MRC1 deficiency decreased fucosylation without affecting the abundance of the major circulating glycoproteins. Our findings confirm that concentrations and N-glycosylation of the major plasma proteins are tightly controlled and further suggest that glycan-binding receptors have redundancy, allowing compensation for the loss of one major clearance receptor.
Topics: Mice; Animals; Asialoglycoprotein Receptor; Mannose Receptor; Glycoproteins; Glycosylation; Protein Processing, Post-Translational; Carrier Proteins; Mannose
PubMed: 37414249
DOI: 10.1016/j.mcpro.2023.100615 -
Life (Basel, Switzerland) Jun 2023Nitrogen-containing bisphosphonates lead to the depletion of geranylgeranyl pyrophosphate involved in the mevalonate pathway. The effect of geranylgeraniol (GGOH) on...
Nitrogen-containing bisphosphonates lead to the depletion of geranylgeranyl pyrophosphate involved in the mevalonate pathway. The effect of geranylgeraniol (GGOH) on human osteoblast and osteoclast activities suppressed by zoledronate was investigated in this study. The effect of GGOH on human osteoblasts and osteoclasts subjected to treatment with zoledronate was analyzed by assessing cell viability, osteoclast differentiation, resorption ability, gene expression, and protein synthesis. Cell viability suppressed by bisphosphonates in osteoblasts and osteoprogenitor cells was restored with GGOH. Osteoclast differentiation was analyzed by vitronectin receptor immunofluorescence staining, and the addition of GGOH to zoledronate significantly increased osteoclast differentiation compared with zoledronate alone. A trend of reversal of osteoclast resorption by GGOH was observed; however, it was not significant in all groups. The expression of ALP, type 1 collagen, and RUNX2 in osteoblasts was recovered by the addition of GGOH. Only CALCR expression in osteoclasts was significantly recovered by GGOH addition in the zoledronate group. Although the activities of osteoblasts and osteoclasts were not entirely restored, the possibility that the topical application of GGOH in MRONJ patients or patients with dental problems and bisphosphonates might lessen the risk of development and recurrence of MRONJ is shown.
PubMed: 37374134
DOI: 10.3390/life13061353 -
Signal Transduction and Targeted Therapy Jun 2023The extracellular matrix (ECM) serves as signals that regulate specific cell states in tumor tissues. Increasing evidence suggests that extracellular biomechanical force...
The extracellular matrix (ECM) serves as signals that regulate specific cell states in tumor tissues. Increasing evidence suggests that extracellular biomechanical force signals are critical in tumor progression. In this study, we aimed to explore the influence of ECM-derived biomechanical force on breast cancer cell status. Experiments were conducted using 3D collagen, fibrinogen, and Matrigel matrices to investigate the role of mechanical force in tumor development. Integrin-cytoskeleton-AIRE and DDR-STAT signals were examined using RNA sequencing and western blotting. Data from 1358 patients and 86 clinical specimens were used for ECM signature-prognosis analysis. Our findings revealed that ECM-derived mechanical force regulated tumor stemness and cell quiescence in breast cancer cells. A mechanical force of ~45 Pa derived from the extracellular substrate activated integrin β1/3 receptors, stimulating stem cell signaling pathways through the cytoskeleton/AIRE axis and promoting tumorigenic potential and stem-like phenotypes. However, excessive mechanical force (450 Pa) could drive stem-like cancer cells into a quiescent state, with the removal of mechanical forces leading to vigorous proliferation in quiescent cancer stem cells. Mechanical force facilitated cell cycle arrest to induce quiescence, dependent on DDR2/STAT1/P27 signaling. Therefore, ECM-derived mechanical force governs breast cancer cell status and proliferative characteristics through stiffness alterations. We further established an ECM signature based on the fibrinogen/fibronectin/vitronectin/elastin axis, which efficiently predicts patient prognosis in breast cancer. Our findings highlight the vital role of ECM-derived mechanical force in governing breast cancer cell stemness/quiescence transition and suggest the novel use of ECM signature in predicting the clinical prognosis of breast cancer.
Topics: Integrins; Cell Line, Tumor; Extracellular Matrix; Signal Transduction; Fibrinogen; Neoplasms
PubMed: 37369642
DOI: 10.1038/s41392-023-01453-0 -
Virulence Dec 2023is an important bacterial pathogen in poultry. Pathogenic bacteria recruit host complement factors to resist the bactericidal effect of serum complement. Vitronectin...
is an important bacterial pathogen in poultry. Pathogenic bacteria recruit host complement factors to resist the bactericidal effect of serum complement. Vitronectin (Vn) is a complementary regulatory protein that inhibits the formation of the membrane attack complex (MAC). Microbes use outer membrane proteins (OMPs) to hijack Vn for complement evasion. However, the mechanism by which achieves evasion is unclear. This study aimed to characterise OMPs of which interact with duck Vn (dVn) during complement evasion. Far-western assays and comparison of wild-type and mutant strains that were treated with dVn and duck serum demonstrated particularly strong binding of OMP76 to dVn. These data were confirmed with strains expressing and not expressing OMP76. Combining tertiary structure analysis and homology modelling, truncated and knocked-out fragments of OMP76 showed that a cluster of critical amino acids in an extracellular loop of OMP76 mediate the interaction with dVn. Moreover, binding of dVn to inhibited MAC deposition on the bacterial surface thereby enhancing survival in duck serum. Virulence of the mutant strain ΔOMP76 was attenuated significantly relative to the wild-type strain. Furthermore, adhesion and invasion abilities of ΔOMP76 decreased, and histopathological changes showed that ΔOMP76 was less virulent in ducklings. Thus, OMP76 is a key virulence factor of . The identification of OMP76-mediated evasion of complement by recruitment of dVn contributes significantly to the understanding of the molecular mechanism by which escapes host innate immunity and provides a new target for the development of subunit vaccines.
Topics: Animals; Virulence; Ducks; Membrane Proteins; Vitronectin; Bacterial Proteins; Flavobacteriaceae Infections; Immunologic Factors; Complement System Proteins; Poultry Diseases
PubMed: 37326479
DOI: 10.1080/21505594.2023.2223060 -
ACS Applied Materials & Interfaces Jun 2023Peptide sequence periodicity is a simple design tool that can be used to generate functional peptide-based surface coatings. De novo-designed peptide N-PEG-VK16 is...
Peptide sequence periodicity is a simple design tool that can be used to generate functional peptide-based surface coatings. De novo-designed peptide N-PEG-VK16 is characterized by a hydrophobic periodicity of two that avidly binds to native polystyrene priming its surface for subsequent targeted functionalization via chemical ligation. The peptidic portion of N-PEG-VK16 is responsible for surface binding, converting polystyrene's hydrophobic surface into a wettable and electrostatically charged environment that facilitates cell attachment. Native polystyrene surfaces are coated by simple peptide adsorption from an aqueous buffered solution, and the resulting primed surface is easily functionalized by cycloaddition chemistry. Herein, we show that ligating a vitronectin-derived peptide to primed polystyrene surfaces enables adhesion, expansion, long-term culture, and phenotype maintenance of human induced pluripotent stem cells. To demonstrate scope, we also show that additional functional ligands can be used, for example, nerve growth factor protein, to control neurite outgrowth.
Topics: Humans; Polystyrenes; Cell Adhesion; Induced Pluripotent Stem Cells; Peptides; Vitronectin; Surface Properties
PubMed: 37276244
DOI: 10.1021/acsami.3c02606 -
Medical Mycology Journal 2023Trichosporon asahii is an invasive pathogenic yeast that infects immunocompromised hosts. Several virulence factors contribute to the fungal infection; however, the...
Trichosporon asahii is an invasive pathogenic yeast that infects immunocompromised hosts. Several virulence factors contribute to the fungal infection; however, the factors that contribute to the occurrence of T. asahii infections remain unclear. Since adhesins are typical virulence factors reported for pathogenic fungi, we looked for host proteins that interact with the T. asahii cell surface. T. asahii and Candida albicans were used for screening using a pull-down assay with fetal bovine serum. Serum albumin and elongation factor 2 were identified as the yeast-binding serum proteins. Additionally, we investigated the interactions of the cell surface-associated molecules (CSM) of T. asahii with vitronectin (VTN), fibronectin, fetuin-A, and alpha-1antitrypsin (AAT). The surface plasmon resonance (SPR) method was used to examine the interaction between CSM and human proteins. On the other hand, the pull-down assay was used to examine the interaction between human proteins and the T. asahii cell surface. Serum albumin, AAT, and VTN were found to interact with T. asahii in both SPR and pull-down assays. This study identified several proteins that interact with T. asahii, suggesting that these proteins play a role in infection mechanisms.
Topics: Humans; Trichosporon; Basidiomycota; Fungal Proteins; Serum Albumin; Virulence Factors; Antifungal Agents; Trichosporonosis
PubMed: 37258132
DOI: 10.3314/mmj.22-00020