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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 -
Biomimetics (Basel, Switzerland) Apr 2023Hardystonite-based (HT) bioceramic foams were easily obtained via thermal treatment of silicone resins and reactive oxide fillers in air. By using a commercial silicone,...
Hardystonite-based (HT) bioceramic foams were easily obtained via thermal treatment of silicone resins and reactive oxide fillers in air. By using a commercial silicone, incorporating strontium oxide and magnesium oxide precursors (as well as CaO and ZnO), and treating it at 1100 °C, a complex solid solution (CaSrZnMgSiO) that has superior biocompatibility and bioactivity properties compared to pure hardystonite (CaZnSiO) can be obtained. Proteolytic-resistant adhesive peptide mapped on vitronectin (D2HVP), was selectively grafted to Sr/Mg-doped HT foams using two different strategies. Unfortunately, the first method (via protected peptide) was unsuitable for acid-sensitive materials such as Sr/Mg-doped HT, resulting in the release of cytotoxic levels of Zinc over time, with consequent negative cellular response. To overcome this unexpected result, a novel functionalization strategy requiring aqueous solution and mild conditions was designed. Sr/Mg-doped HT functionalized with this second strategy (via aldehyde peptide) showed a dramatic increase in human osteoblast proliferation at 6 days compared to only silanized or non-functionalized samples. Furthermore, we demonstrated that the functionalization treatment does not induce any cytotoxicity. Functionalized foams enhanced mRNA-specific transcript levels coding , , , and at 2 days post-seeding. In conclusion, the second functionalization strategy proved to be appropriate for this specific biomaterial and was effective at enhancing the material's bioactivity.
PubMed: 37218771
DOI: 10.3390/biomimetics8020185 -
Clinical Proteomics May 2023IgA nephropathy (IgAN) and IgA vasculitis with nephritis (IgAVN) are related glomerular diseases characterized by marked similarities in immunological and histological...
BACKGROUND
IgA nephropathy (IgAN) and IgA vasculitis with nephritis (IgAVN) are related glomerular diseases characterized by marked similarities in immunological and histological findings. We herein performed a comparative proteomic analysis of glomerular proteins in IgAN and IgAVN.
METHODS
We used renal biopsy specimens from 6 IgAN patients without nephrotic syndrome (NS) (IgAN-I subgroup), 6 IgAN patients with NS (IgAN-II subgroup), 6 IgAVN patients with 0-8.0% of glomeruli with crescent formation (IgAVN-I subgroup), 6 IgAVN patients with 21.2-44.8% of glomeruli with crescent formation (IgAVN-II subgroup), 9 IgAVN patients without NS (IgAVN-III subgroup), 3 IgAVN patients with NS (IgAN-IV subgroup), and 5 control cases. Proteins were extracted from laser microdissected glomeruli and analyzed using mass spectrometry. The relative abundance of proteins was compared between groups. An immunohistochemical validation study was also performed.
RESULTS
More than 850 proteins with high confidence were identified. A principal component analysis revealed a clear separation between IgAN and IgAVN patients and control cases. In further analyses, 546 proteins that were matched with ≥ 2 peptides were selected. The levels of immunoglobulins (IgA, IgG, and IgM), complements (C3, C4A, C5, and C9), complement factor H-related proteins (CFHR) 1 and 5, vitronectin, fibrinogen chains, and transforming growth factor-β inducible gene-h3 were higher (> 2.6 fold) in the IgAN and IgAVN subgroups than in the control group, whereas hornerin levels were lower (< 0.3 fold). Furthermore, C9 and CFHR1 levels were significantly higher in the IgAN group than in the IgAVN group. The abundance of some podocyte-associated proteins and glomerular basement membrane (GBM) proteins was significantly less in the IgAN-II subgroup than in the IgAN-I subgroup as well as in the IgAVN-IV subgroup than in the IgAVN-III subgroup. Among the IgAN and IgAVN subgroups, talin 1 was not detected in the IgAN-II subgroup. This result was supported by immunohistochemical findings.
CONCLUSIONS
The present results suggest shared molecular mechanisms for glomerular injury in IgAN and IgAVN, except for enhanced glomerular complement activation in IgAN. Differences in the protein abundance of podocyte-associated and GBM proteins between IgAN and IgAVN patients with and without NS may be associated with the severity of proteinuria.
PubMed: 37179321
DOI: 10.1186/s12014-023-09409-w -
International Journal of Molecular... Apr 2023Retinal artery occlusion (RAO) is a devastating condition with no effective treatment. The management of RAO could potentially be improved through an in-depth...
Retinal artery occlusion (RAO) is a devastating condition with no effective treatment. The management of RAO could potentially be improved through an in-depth understanding of the molecular alterations in the condition. This study combined advanced proteomic techniques and an experimental model to uncover the retinal large-scale protein profile of RAO. In 13 pigs, RAO was induced with an argon laser and confirmed by fluorescein angiography. Left eyes serving as controls received a sham laser without inducing occlusion. Retinal samples were collected after one, three, or six days and analyzed with liquid chromatography-tandem mass spectrometry. In RAO, 36 proteins were differentially regulated on day one, 86 on day three, and 557 on day six. Upregulated proteins included clusterin, vitronectin, and vimentin, with several proteins increasing over time with a maximum on day six, including clusterin, vimentin, osteopontin, annexin-A, signal transducer, and the activator of transcription 3. On day six, RAO resulted in the upregulation of proteins involved in cellular response to stress, hemostasis, innate immune response, and cytokine signaling. Downregulated proteins were involved in transmission across chemical synapses and visual phototransduction. This study identified the upregulation of multiple inflammatory proteins in RAO and the downregulation of proteins involved in visual pathways.
Topics: Animals; Swine; Vimentin; Clusterin; Proteomics; Retinal Artery Occlusion; Retina
PubMed: 37175625
DOI: 10.3390/ijms24097919 -
Journal of Molecular Recognition : JMR Jun 2023Vinculin is an integral component of integrin adhesions, where it functions as a molecular clutch coupling intracellular contraction to the extracellular matrix....
Vinculin is an integral component of integrin adhesions, where it functions as a molecular clutch coupling intracellular contraction to the extracellular matrix. Quantitating its contribution to the reinforcement of newly forming adhesions, however, requires ultrasensitive cell force assays covering short time and low force ranges. Here, we have combined atomic force microscopy-based single-cell force spectroscopy (SCFS) and optical tweezers force spectroscopy to investigate the role of vinculin in reinforcement of individual nascent adhesions during the first 5 min of cell contact with fibronectin or vitronectin. At minimal adhesion times (5-10 s), mouse embryonic fibroblast (MEF) wildtype (wt) and vinculin knock-out (vin ) cells develop comparable adhesion forces on the scale of several individual integrin-ligand bonds, confirming that vinculin is dispensable for adhesion initiation. In contrast, after 60 to 120 s, adhesion strength and traction reinforce quickly in wt cells, while remaining low in vin cells. Re-expression of full-length vinculin or a constitutively active vinculin mutant (vinT12) in MEF vin cells restored adhesion and traction with the same efficiency, while vinculin with a mutated talin-binding head region (vinA50I) or missing the actin-binding tail-domain (vin880) was ineffective. Integrating total internal reflection fluorescence imaging into the SCFS setup furthermore enabled us to correlate vinculin-green fluorescent protein (GFP) recruitment to nascent adhesion sites with the built-up of vinculin-dependent adhesion forces directly. Vinculin recruitment and cell adhesion reinforcement followed synchronous biphasic patterns, suggesting vinculin recruitment, but not activation, as the rate-limiting step for adhesion reinforcement. Combining sensitive SCFS with fluorescence microscopy thus provides insight into the temporal sequence of vinculin-dependent mechanical reinforcement in nascent integrin adhesions.
Topics: Animals; Mice; Cell Adhesion; Fibroblasts; Focal Adhesions; Integrins; Talin; Vinculin
PubMed: 36987702
DOI: 10.1002/jmr.3012 -
International Journal of Molecular... Mar 2023Endometrial and cervical cancers are the two most common gynaecological malignancies and among the leading causes of death worldwide. The extracellular matrix (ECM) is... (Review)
Review
Endometrial and cervical cancers are the two most common gynaecological malignancies and among the leading causes of death worldwide. The extracellular matrix (ECM) is an important component of the cellular microenvironment and plays an important role in developing and regulating normal tissues and homeostasis. The pathological dynamics of the ECM contribute to several different processes such as endometriosis, infertility, cancer, and metastasis. Identifying changes in components of ECM is crucial for understanding the mechanisms of cancer development and its progression. We performed a systematic analysis of publications on the topic of changes in the extracellular matrix in cervical and endometrial cancer. The findings of this systematic review show that matrix metalloproteinases (MMP) play an important role impacting tumour growth in both types of cancer. MMPs degrade various specific substrates (collagen, elastin, fibronectin, aggrecan, fibulin, laminin, tenascin, vitronectin, versican, nidogen) and play a crucial role in the basal membrane degradation and ECM components. Similar types of MMPs were found to be increased in both cancers, namely, MMP-1, MMP-2, MMP-9, and MMP-11. Elevated concentrations of MMP-2 and MMP-9 were correlated with the FIGO stage and are associated with poor prognosis in endometrial cancer, whereas in cervical cancer, elevated concentrations of MMP-9 have been associated with a better outcome. Elevated ADAMTS levels were found in cervical cancer tissues. Elevated disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) levels were also found in endometrial cancer, but their role is still unclear. Following these findings, this review reports on tissue inhibitors of ECM enzymes, MMPs, and ADAMTS. The present review demonstrates changes in the extracellular matrix in cervical and endometrial cancers and compared their effect on cancer development, progression, and patient prognosis.
Topics: Female; Humans; Uterine Cervical Neoplasms; Matrix Metalloproteinase 9; Matrix Metalloproteinase 2; Extracellular Matrix; Matrix Metalloproteinases; Endometrial Neoplasms; Tumor Microenvironment
PubMed: 36982551
DOI: 10.3390/ijms24065463 -
Tissue & Cell Jun 2023Extracellular matrix (ECM) proteins in the mammary gland provide structure and regulate its development and homeostasis. Alterations in its structure can regulate and...
Extracellular matrix (ECM) proteins in the mammary gland provide structure and regulate its development and homeostasis. Alterations in its structure can regulate and support pathogenesis, like breast tumors. Aiming to identify the health and tumoral canine mammary ECM scaffold protein profile by immunohistochemistry, the decellularization process was carried out to remove the cellular content. Additionally, it was verified the influence of health and tumoral ECM on the attachment of health and tumoral cells. The types I, III, IV, and V structural collagens were scarce in the mammary tumor, and ECM fibers were disorganized. Vimentin and CD44 were more common in mammary tumor stroma, suggesting a role in cell migration that results in tumor progression. Elastin, fibronectin, laminin, vitronectin, and osteopontin were similarly detected under healthy and tumor conditions, providing the attachment of normal cells in healthy ECM, while tumoral cells were able to attach in tumoral ECM. The protein pattern demonstrates ECM alteration in canine mammary tumorigenesis, presenting new knowledge on mammary tumor ECM microenvironment.
Topics: Animals; Dogs; Extracellular Matrix Proteins; Extracellular Matrix; Laminin; Connective Tissue; Neoplasms; Tumor Microenvironment
PubMed: 36933273
DOI: 10.1016/j.tice.2023.102050 -
Scientific Reports Mar 2023The pre-metastatic microenvironment consists of pro-metastatic and anti-metastatic immune cells in the early stages of cancer, when the primary tumor begins to...
The pre-metastatic microenvironment consists of pro-metastatic and anti-metastatic immune cells in the early stages of cancer, when the primary tumor begins to proliferate. Redundantly, pro-inflammatory immune cells predominated during tumor growth. Although it is well known that pre-metastatic innate immune cells and immune cells fighting primary tumor cells become exhausted, the mechanism by which this occurs is unknown. We discovered that anti-metastatic NK cells were mobilized from the liver to the lung during primary tumor progression and that the transcription factor CEBPδ, which was upregulated in a tumor-stimulated liver environment, inhibited NK cell attachment to the fibrinogen-rich bed in pulmonary vessels and sensitization to the environmental mRNA activator. CEBPδ-siRNA treated anti-metastatic NK cells regenerated the binding proteins that support sitting in fibrinogen-rich soil, such as vitronectin and thrombospondin, increasing fibrinogen attachment. Furthermore, CEBPδ knockdown restored an RNA-binding protein, ZC3H12D, which captured extracellular mRNA to increase tumoricidal activity. Refreshed NK cells using CEBPδ-siRNA with anti-metastatic abilities would work at metastatic risk areas in the pre-metastatic phase, resulting in a reduction in lung metastasis. Furthermore, tissue-specific siRNA-based therapy in lymphocyte exhaustion may be beneficial in the treatment of early metastases.
Topics: Humans; Killer Cells, Natural; Lung Neoplasms; Lung; Liver Neoplasms; Fibrinogen; RNA, Small Interfering; Tumor Microenvironment; Cell Line, Tumor
PubMed: 36890150
DOI: 10.1038/s41598-023-30476-4 -
Stem Cell Research & Therapy Mar 2023The vast and promising class of long non-coding RNAs (lncRNAs) has been under investigation for distinct therapeutic applications. Nevertheless, their role as molecular...
BACKGROUND
The vast and promising class of long non-coding RNAs (lncRNAs) has been under investigation for distinct therapeutic applications. Nevertheless, their role as molecular drivers of bone regeneration remains poorly studied. The lncRNA H19 mediates osteogenic differentiation of Mesenchymal Stem/Stromal Cells (MSCs) through the control of intracellular pathways. However, the effect of H19 on the extracellular matrix (ECM) components is still largely unknown. This research study was designed to decode the H19-mediated ECM regulatory network, and to reveal how the decellularized siH19-engineered matrices influence MSC proliferation and fate. This is particularly relevant for diseases in which the ECM regulation and remodeling processes are disrupted, such as osteoporosis.
METHODS
Mass spectrometry-based quantitative proteomics analysis was used to identify ECM components, after oligonucleotides delivery to osteoporosis-derived hMSCs. Moreover, qRT-PCR, immunofluorescence and proliferation, differentiation and apoptosis assays were performed. Engineered matrices were decellularized, characterized by atomic force microscopy and repopulated with hMSC and pre-adipocytes. Clinical bone samples were characterized by histomorphometry analysis.
RESULTS
Our study provides an in-depth proteome-wide and matrisome-specific analysis of the ECM proteins controlled by the lncRNA H19. Using bone marrow-isolated MSC from patients with osteoporosis, we identified fibrillin-1 (FBN1), vitronectin (VTN) and collagen triple helix repeat containing 1 (CTHRC1), among others, as having different pattern levels following H19 silencing. Decellularized siH19-engineered matrices are less dense and have a decreased collagen content compared with control matrices. Repopulation with naïve MSCs promotes a shift towards the adipogenic lineage in detriment of the osteogenic lineage and inhibits proliferation. In pre-adipocytes, these siH19-matrices enhance lipid droplets formation. Mechanistically, H19 is targeted by miR-29c, whose expression is decreased in osteoporotic bone clinical samples. Accordingly, miR-29c impacts MSC proliferation and collagen production, but does not influence ALP staining or mineralization, revealing that H19 silencing and miR-29c mimics have complementary but not overlapping functions.
CONCLUSION
Our data suggest H19 as a therapeutic target to engineer the bone ECM and to control cell behavior.
Topics: Humans; Extracellular Matrix; Extracellular Matrix Proteins; MicroRNAs; Osteogenesis; RNA, Long Noncoding
PubMed: 36882843
DOI: 10.1186/s13287-023-03250-6