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Journal of Biomedical Materials... Feb 2024Hydroxyapatite (HA) bioceramic is a promising substitute for bone defects, and the surface properties are major factors that influence bioactivity and osteoinductivity....
Hydroxyapatite (HA) bioceramic is a promising substitute for bone defects, and the surface properties are major factors that influence bioactivity and osteoinductivity. In this study, two kinds of HA bioceramics with nanoscale (n-HA) and microscale (m-HA) surface topography were designed to mimic the natural bone, thus enhancing the stimulation of osteogenic differentiation and revealing the potential mechanism. Compared to m-HA, n-HA owned a larger surface roughness, a stronger wettability, and reduced hardness and indentation modulus. Based on these properties, n-HA could maintain the conformation of vitronectin better than m-HA, which may contribute to higher cellular activities and a stronger promotion of osteogenic differentiation of mesenchymal stem cells (MSCs). Further RNA sequencing analysis compared the molecular expression between n-HA and m-HA. Six hundred twenty-seven differentially expressed genes were identified in MSCs, and 17 upregulated genes and 610 downregulated genes were included when n-HA compared to m-HA. The GO cluster analysis and enriched Kyoto encyclopedia of genes and genome signaling pathways revealed a close correlation with the immune process in both upregulated (chemokine signaling pathway and cytokine-cytokine receptor interaction) and downregulated pathways (osteoclasts differentiation). It suggested that the nanoscale surface topography of HA enhanced the osteoinductivity of MSCs and could not be separated from its regulation of immune function and the retention of adsorbed protein conformation.
Topics: Durapatite; Osteogenesis; Cell Differentiation; Mesenchymal Stem Cells; Ceramics
PubMed: 37680167
DOI: 10.1002/jbm.a.37606 -
FASEB Journal : Official Publication of... Oct 2023Mycoplasma spp., the smallest self-replicating and genome-reduced organisms, have raised a great concern in both the medical and veterinary fields due to their...
Mycoplasma spp., the smallest self-replicating and genome-reduced organisms, have raised a great concern in both the medical and veterinary fields due to their pathogenicity. The molecular determinants of these wall-less bacterium efficiently use their limited genes to ensure successful infection of the host remain unclear. In the present study, we used the ruminant pathogen Mycoplasma bovis as a model to identify the key factors for colonization and invasion into host cells. We constructed a nonredundant fluorescent transposon mutant library of M. bovis using a modified transposon plasmid, and identified 34 novel adhesion-related genes based on a high-throughput screening approach. Among them, the ΔLppB mutant exhibited the most apparent decrease in adhesion to embryonic bovine lung (EBL) cells. The surface-localized lipoprotein LppB, which is highly conserved in Mycoplasma species, was then confirmed as a key factor for M. bovis adhesion with great immunogenicity. LppB interacted with various components (fibronectin, vitronectin, collagen IV, and laminin) of host extracellular matrix (ECM) and promoted plasminogen activation through tPA to degrade ECM. The 439-502 amino acid region of LppB is a critical domain, and F465 and Y493 are important residues for the plasminogen activation activity. We further revealed LppB as a key factor facilitating internalization through clathrin- and lipid raft-mediated endocytosis, which helps the Mycoplasma invade the host cells. Our study indicates that LppB plays a key role in Mycoplasma infection and is a potential new therapeutic and vaccine target for Mycoplasma species.
Topics: Animals; Cattle; Mycoplasma bovis; Clathrin; Collagen Type IV; Mutagenesis; Plasminogen
PubMed: 37665592
DOI: 10.1096/fj.202300678R -
Basic Research in Cardiology Aug 2023While low concentrations of high-density lipoprotein-cholesterol (HDL-C) are widely accepted as an independent cardiovascular risk factor, HDL-C-rising therapies largely...
While low concentrations of high-density lipoprotein-cholesterol (HDL-C) are widely accepted as an independent cardiovascular risk factor, HDL-C-rising therapies largely failed, suggesting the importance of both HDL functions and individual subspecies. Indeed HDL particles are highly heterogeneous, with small, dense pre-beta-HDLs being considered highly biologically active but remaining poorly studied, largely reflecting difficulties for their purification. We developed an original experimental approach allowing the isolation of sufficient amounts of human pre-beta-HDLs and revealing the specificity of their proteomic and lipidomic profiles and biological activities. Pre-beta-HDLs were enriched in highly poly-unsaturated species of phosphatidic acid and phosphatidylserine, and in an unexpectedly high number of proteins implicated in the inflammatory response, including serum paraoxonase/arylesterase-1, vitronectin and clusterin, as well as in complement regulation and immunity, including haptoglobin-related protein, complement proteins and those of the immunoglobulin class. Interestingly, amongst proteins associated with lipid metabolism, phospholipid transfer protein, cholesteryl ester transfer protein and lecithin:cholesterol acyltransferase were strongly enriched in, or restricted to, pre-beta-HDL. Furthermore, pre-beta-HDL potently mediated cellular cholesterol efflux and displayed strong anti-inflammatory activities. A correlational network analysis between lipidome, proteome and biological activities highlighted 15 individual lipid and protein components of pre-beta-HDL relevant to cardiovascular disease, which may constitute novel diagnostic targets in a pathological context of altered lipoprotein metabolism.
Topics: Humans; Cardiovascular Diseases; Proteomics; Cholesterol, HDL; Heart Disease Risk Factors; Lipid Metabolism
PubMed: 37639039
DOI: 10.1007/s00395-023-01004-2 -
Biomolecules Jul 2023Adsorbing toxins from the blood to augment membrane-based hemodialysis is an active area of research. Films composed of β-cyclodextrin-co-(methacryloyloxy)ethyl...
Adsorbing toxins from the blood to augment membrane-based hemodialysis is an active area of research. Films composed of β-cyclodextrin-co-(methacryloyloxy)ethyl phosphorylcholine (p(PMβCD-co-MPC)) with various monomer ratios were formed on magnetic nanoparticles and characterized. Surface chemistry effects on protein denaturation were evaluated and indicated that unmodified magnetic nanoparticles greatly perturbed the structure of proteins compared to coated particles. Plasma clotting assays were conducted to investigate the stability of plasma in the presence of particles, where a 2:2 monomer ratio yielded the best results for a given total surface area of particles. Total protein adsorption results revealed that modified surfaces exhibited reduced protein adsorption compared to bare particles, and pure MPC showed the lowest adsorption. Immunoblot results showed that fibrinogen, α1-antitrypsin, vitronectin, prekallikrein, antithrombin, albumin, and C3 correlated with film composition. Hemocompatibility testing with whole blood illustrated that the 1:3 ratio of CD to MPC had a negative impact on platelets, as evidenced by the increased activation, reduced response to an agonist, and reduced platelet count. Other formulations had statistically significant effects on platelet activation, but no formulation yielded apparent adverse effects on hemostasis. For the first time, p(PMβCD-co-MPC)-coated MNP were synthesized and their general hemocompatibility assessed.
Topics: Phosphorylcholine; Magnetite Nanoparticles; Adsorption; Antithrombin III; Blood Coagulation
PubMed: 37627230
DOI: 10.3390/biom13081165 -
Computers in Biology and Medicine Sep 2023Digital pathology and artificial intelligence are promising emerging tools in precision oncology as they provide more robust and reproducible analysis of histologic,...
Digital pathology and artificial intelligence are promising emerging tools in precision oncology as they provide more robust and reproducible analysis of histologic, morphologic and topologic characteristics of tumor cells and the surrounding microenvironment. This study aims to develop digital image analysis workflows for therapeutic assessment in preclinical in vivo models. For this purpose, we generated pipelines that enable automatic detection and quantification of vitronectin and αvβ3 in heterotopic high-risk neuroblastoma xenografts, demonstrating that digital analysis workflows can be used to provide robust detection of vitronectin secretion and αvβ3 expression by malignant neuroblasts and to evaluate the possibility of combining traditional chemotherapy (etoposide) with extracellular matrix-targeted therapies (cilengitide). Digital image analysis added evidence for the relevance of territorial vitronectin as a therapeutic target in neuroblastoma, since its expression is modified after treatment, with a mean percentage of 60.44% in combined therapy tumors vs 45.08% in control ones. In addition, the present study revealed the efficacy of cilengitide for reducing αvβ3 expression, with a mean αvβ3 positivity of 34.17% in cilengitide treated material vs 66.14% in control and with less tumor growth when combined with etoposide, with a final mean volume of 0.04 cm in combined therapy vs 1.45 cm in control. The results of this work highlight the importance of extracellular matrix-focused therapies in preclinical studies to improve therapeutic assessment for high-risk neuroblastoma patients.
Topics: Humans; Etoposide; Tumor Microenvironment; Artificial Intelligence; Vitronectin; Workflow; Precision Medicine; Neuroblastoma
PubMed: 37598482
DOI: 10.1016/j.compbiomed.2023.107364 -
Biomaterials Advances Nov 2023Delivery of growth factors (GFs) is challenging for regulation of cell proliferation and differentiation due to their rapid inactivation under physiological conditions....
Delivery of growth factors (GFs) is challenging for regulation of cell proliferation and differentiation due to their rapid inactivation under physiological conditions. Here, a bioactive polyelectrolyte multilayer (PEM) is engineered by the combination of thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) and glycosaminoglycans to be used as reservoir for GF storage. PNIPAM-grafted-chitosan (PChi) with two degrees of substitution (DS) are synthesized, namely LMW* (DS 0.14) and HMW (DS 0.03), by grafting low (2 kDa) and high (10 kDa) molecular weight of PNIPAM on the backbone of chitosan (Chi) to be employed as polycations to form PEM with the polyanion heparin (Hep) at pH 4. Subsequently, PEMs are chemically crosslinked to improve their stability at physiological pH 7.4. Resulting surface and mechanical properties indicate that PEM containing HMW is responsive to temperature at 20 °C and 37 °C, while LMW is not. More importantly, Hep as terminal layer combined with HMW allows not only a better retention of the adhesive protein vitronectin but also a sustained release of FGF-2 at 37 °C. With the synergistic effect of vitronectin and matrix-bound FGF-2, significant promotion on adhesion, proliferation, and migration of 3T3 mouse embryonic fibroblasts is achieved on HMW-containing PEM compared to Chi-containing PEM and exogenously added FGF-2. Thus, PEM containing PNIPAM in combination with bioactive glycosaminoglycans like Hep represents a versatile approach to fabricate a GF delivery system for efficient cell culture, which can be potentially served as cell culture substrate for production of (stem) cells and bioactive wound dressing for tissue regeneration.
Topics: Animals; Mice; Heparin; Chitosan; Fibroblast Growth Factor 2; Vitronectin; Cell Adhesion; Fibroblasts; Glycosaminoglycans
PubMed: 37598438
DOI: 10.1016/j.bioadv.2023.213589 -
Urinary Proteomics for Discovery of Gastric Cancer Biomarkers to Enable Precision Clinical Oncology.Omics : a Journal of Integrative Biology Aug 2023For precision in clinical oncology practice, detection of tumor-derived peptides and proteins in urine offers an attractive and noninvasive alternative for diagnostic or...
For precision in clinical oncology practice, detection of tumor-derived peptides and proteins in urine offers an attractive and noninvasive alternative for diagnostic or screening purposes. In this study, we report comparative quantitative proteomic profiling of urine samples from patients with gastric cancer and healthy controls using tandem mass tags-based multiplexed mass spectrometry approach. We identified 1504 proteins, of which 246 were differentially expressed in gastric cancer cases. Notably, ephrin A1 (EFNA1), pepsinogen A3 (PGA3), sortilin 1 (SORT1), and vitronectin (VTN) were among the upregulated proteins, which are known to play crucial roles in the progression of gastric cancer. We also found other overexpressed proteins, including shisa family member 5 (SHISA5), mucin like 1 (MUCL1), and leukocyte cell derived chemotaxin 2 (LECT2), which had not previously been linked to gastric cancer. Using a novel approach for targeted proteomics, SureQuant, we validated changes in abundance of a subset of proteins discovered in this study. We confirmed the overexpression of vitronectin and sortilin 1 in an independent set of urine samples. Altogether, this study provides molecular candidates for biomarker development in gastric cancer, and the findings also support the promise of urinary proteomics for noninvasive diagnostics and personalized/precision medicine in the oncology clinic.
Topics: Humans; Biomarkers, Tumor; Stomach Neoplasms; Proteomics; Vitronectin; Proteins; Medical Oncology; Biomarkers; Mucins; Intercellular Signaling Peptides and Proteins
PubMed: 37579183
DOI: 10.1089/omi.2023.0077 -
International Journal of Molecular... Aug 2023Inflammation is associated with many pathology disorders and the malignant progression of most cancers. Therefore, targeting inflammatory pathways could provide a...
Inflammation is associated with many pathology disorders and the malignant progression of most cancers. Therefore, targeting inflammatory pathways could provide a promising strategy for disease prevention and treatment. In this study, we experimentally investigated the anti-inflammatory effect of CC5 and CC8, two disintegrin isoforms isolated from snake venom, on LPS-stimulated macrophages, both on human THP-1 and mouse RAW264.7 cell adherence and their underlying mechanisms by measuring cytokine release levels and Western blot assay. Equally, both molecules were evaluated on a carrageenan-induced edema rat model. Our findings suggest that CC5 and CC8 were able to reduce adhesion of LPS-stimulated macrophages both on human THP-1 and mouse RAW264.7 cells to fibrinogen and vitronectin through the interaction with the αvβ3 integrin receptor. Moreover, CC5 and CC8 reduced the levels of reactive oxygen species (ROS) mediated by the NF-κB, MAPK and AKT signaling pathways that lead to decreased production of the pro-inflammatory cytokines TNF-α, IL-6 and IL-8 and increased secretion of IL-10 in LPS-stimulated THP-1 and RAW264.7 cells. Interestingly, both molecules potently exhibited an anti-inflammatory effect in vivo by reducing paw swelling in rats. In light of these results, we can propose the CC5 and CC8 disintegrins as interesting tools to design potential candidates against inflammatory-related diseases.
Topics: Rats; Mice; Humans; Animals; Disintegrins; Lipopolysaccharides; Viperidae; Snake Venoms; NF-kappa B; Inflammation; Cytokines; Protein Isoforms; Anti-Inflammatory Agents; RAW 264.7 Cells
PubMed: 37569801
DOI: 10.3390/ijms241512427 -
Environmental Toxicology Feb 2024As the sixth most common type of cancer worldwide, liver hepatocellular carcinoma (LIHC) emerges as grave public health danger owing to its chemotherapy-resistant...
INTRODUCTION
As the sixth most common type of cancer worldwide, liver hepatocellular carcinoma (LIHC) emerges as grave public health danger owing to its chemotherapy-resistant feature. Disulfidoptosis is a newly discovered programmed cell death process affecting the normal actin cytoskeleton structure.
METHODS
Single-cell RNA (scRNA)-seq data were procured from GSE149614 and GSE202642 datasets. We utilized uniform manifold approximation and projection and clustering algorithm Louvian for dimensionality reduction and FindAllMarkers function for determining the differentially expressed genes (DEGs). Monocle2 and SCENIC were utilized to perform pseudo-time series and transcription factor analysis for selected subgroups. A series of in vitro experiments, including colony formation assay (CFA), flow cytometry targeting apoptosis and cell cycle, was applied to investigate how APLP2 regulated the LIHC progression. Two cell lines of LIHC cells, HepG2, and Huh7, were used for si-APLP2 transfection.
RESULTS
Tumor heterogeneity landscape of LIHC was depicted by detailed subgroup analysis. We found T and B cells were enriched with POU2F1 and HES1 activity. Inflammatory cancer-associated fibroblasts interacted with the cancer cells, uniquely through COL1A1/SDC1, COL1A2/SDC1 and LUM/ITGB1 pathways. The transformation from normal hepatocytes to malignant cells was displayed by cell trajectory analysis. State4, which was determined as malignant cells, was enriched in PI3K, hypoxia, and Epidermal growth factor receptor pathway, and enriched with Nuclear Receptor Subfamily 2 Group F Member 1 transcription factor activity. We observed an intense communication from the cancer cells to endothelial cells, mainly through the Vitronectin (VTN) to Kinase Insert Domain Receptor (KDR) pathway. A prognostic model targeting LIHC was constructed based on the disulfidoptosis-based DEGs, namely APLP2, PDIA6, YBX1, SPP1, whose accuracy was validated in multiple cohorts. Knockdown of APLP2 significantly increased the apoptosis and delayed cell cycle progression of LIHC cell line.
CONCLUSION
A prognostic model targeting LIHC was constructed based on the disulfidoptosis-related DEGs, which displayed high stability and accuracy in multiple cohorts. APLP2 played an active role in the carcinogenesis of LIHC by regulating the apoptosis and cell cycle.
Topics: Humans; Amyloid beta-Protein Precursor; Carcinoma, Hepatocellular; Endothelial Cells; Liver Neoplasms; Nerve Tissue Proteins; Transcription Factors
PubMed: 37515494
DOI: 10.1002/tox.23904 -
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