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1.
Spectrochim Acta A Mol Biomol Spectrosc ; 266: 120468, 2022 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-34649124

RESUMO

The photometric method is widely used in real clinical tests due to its simple operation, low cost and convenient. Many of the reported colorimetric ALP assays so far are non- ratiometric because the detection was based on changes in absorbance at a single wavelength. The development of novel colorimetric and ratiometric assay is of importance for quantitatively measuring target with high accuracy. The challenge in the design of ratiometric photometric assay is that the chromophore must have a significant spectral shift before and after binding to the target. Here, we report a colorimetric and ratiometric photometric sequential assay of AA and ALP based on the complexation between ARS and Cu2+ and redox reaction between AA and Cu2+. The absorption band of ARS centered at 425 nm (yellow color), which could be shifted to 510 nm (red color) upon Cu2+ binding. However, as far as we know, this classic color reaction has not been used to develop a ratiometric photometric method to sequentially detect AA and ALP, although photometric methods based on the regulation of other color reagents with oxidizing metal ions have been reported. The proposed sensing system shows a limit of detection for ALP at 0.24 U L-1 and could be applied for detecting ALP in newborn calf serum. The established sensing system makes a useful contribution to the detection of ALP in complex clinical samples.


Assuntos
Fosfatase Alcalina , Colorimetria , Fosfatase Alcalina/metabolismo , Ácido Ascórbico , Humanos , Recém-Nascido , Limite de Detecção , Oxirredução , Espectrometria de Fluorescência
2.
Int J Mol Sci ; 22(19)2021 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-34638808

RESUMO

Tissue-nonspecific alkaline phosphatase (TNAP) is mainly known for its necessary role in skeletal and dental mineralization, which relies on the hydrolysis of the mineralization inhibitor inorganic pyrophosphate (PPi). Mutations in the gene encoding TNAP leading to severe hypophosphatasia result in strongly reduced mineralization and perinatal death. Fortunately, the relatively recent development of a recombinant TNAP with a bone anchor has allowed to correct the bone defects and prolong the life of affected babies and children. Researches on TNAP must however not be slowed down, because accumulating evidence indicates that TNAP activation in individuals with metabolic syndrome (MetS) is associated with enhanced cardiovascular mortality, presumably in relation with cardiovascular calcification. On the other hand, TNAP appears to be necessary to prevent the development of steatohepatitis in mice, suggesting that TNAP plays protective roles. The aim of the present review is to highlight the known or suspected functions of TNAP in energy metabolism that may be associated with the development of MetS. The location of TNAP in liver and its function in bile excretion, lipopolysaccharide (LPS) detoxification and fatty acid transport will be presented. The expression and function of TNAP in adipocyte differentiation and thermogenesis will also be discussed. Given that TNAP is a tissue- and substrate-nonspecific phosphatase, we believe that it exerts several crucial pathophysiological functions that are just beginning to be discovered.


Assuntos
Fosfatase Alcalina/metabolismo , Metabolismo Energético , Termogênese , Adipócitos/metabolismo , Fosfatase Alcalina/genética , Animais , Bile/metabolismo , Diferenciação Celular , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Humanos , Hipofosfatemia/genética , Hipofosfatemia/metabolismo , Lipopolissacarídeos/metabolismo , Síndrome Metabólica/genética , Síndrome Metabólica/metabolismo , Camundongos , Mutação
3.
Braz J Biol ; 84: e251970, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34705954

RESUMO

In order to better understand the ossification processes in anurans our study was carried out on tadpoles and adults of Lithobates catesbeianus. In this sense, we characterized the kinetic properties of alkaline phosphatase with p-nitrophenylphosphatase (pNPP) and pyrophosphate (PPi) and evaluated the activities of tartrate-resistant acid phosphatase and acid phosphatase. The enzyme extracts were obtained from tadpoles and adult femurs, which were divided into epiphysis and diaphysis. After homogenization, the samples were submitted to differential centrifugation to obtain cell membranes and, further, to phospholipase C (PIPLC) treatment, to remove membrane-bound proteins anchored by phosphatidylinositol. The average of specific activity for pNPP hydrolysis (at pH 10.5) by alkaline phosphatase released by phosphatidylinositol-specific phospholipase C (PIPLC) from Bacillus cereus among different bone regions at different animal ages was 1,142.57 U.mg-1, while for PPi hydrolysis (at pH 8.0), it was 1,433.82 U.mg-1. Among the compounds tested for enzymatic activity, the one that influenced the most was EDTA, with approximately 67% of inhibition for pNPPase activity and 77% for PPase activity. In the case of kinetic parameters, the enzyme showed a "Michaelian" behavior for pNPP and PPi hydrolysis. The Km value was around 0.6mM for pNPPase activity and ranged from 0.01 to 0.11mM for PPase activity, indicating that the enzyme has a higher affinity for this substrate. The study of pNPP and PPi hydrolysis by the enzyme revealed that the optimum pH of actuation for pNPP was 10.5, while for PPi, which is considered the true substrate of alkaline phosphatase, was 8.0, close to the physiological value. The results show that regardless of the ossification type that occurs, the same enzyme or isoenzymes act on the different bone regions and different life stages of anurans. The similarity of the results of studies with other vertebrates shows that anurans can be considered excellent animal models for the study of biological calcification.


Assuntos
Fosfatase Alcalina , Osteogênese , Fosfatase Alcalina/metabolismo , Animais , Osso e Ossos/metabolismo , Cinética , Rana catesbeiana
4.
PLoS One ; 16(10): e0258254, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34610044

RESUMO

Epidemiology and pathogenesis of cardiovascular diseases (CVD) and osteoporosis are strikingly overlapping. This study presents matrix metalloproteinase-9 (MMP-9), as a simple molecular link more consistently associated with the pathophysiology of both osteoporosis and CVD risk factors. 40 adult female rats were randomly distributed into 4 groups [control sham-operated, untreated osteoporosis, carvedilol-treated osteoporosis and alendronate-treated osteoporosis]. After 8 weeks, blood samples were collected to estimate Lipid profile (Total cholesterol, HDL, Triglycerides), inflammatory markers (IL-6, TNF alpha, CRP and NO), and Bone turnover markers (BTM) (Alkaline phosphatase, osteocalcin and pyridinoline). The tibias were dissected to estimate MMP-9 and NF-kB gene expression, OPG, RANKL levels and for histological examination. Induction of osteoporosis resulted in a significant elevation in BTM, inflammatory markers and dyslipidemia. MMP-9 was significantly elevated and positively correlated with BTM, inflammation and dyslipidemia markers. Carvedilol and alendronate exerted a bone preservative role and attenuated dyslipidaemia and inflammation in accordance with their respective effect on MMP-9.


Assuntos
Doenças Cardiovasculares/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Osteoporose Pós-Menopausa/metabolismo , Osteoprotegerina/metabolismo , Ovariectomia , Ligante RANK/metabolismo , Fosfatase Alcalina/metabolismo , Aminoácidos/metabolismo , Animais , Remodelação Óssea/genética , Colesterol/sangue , Osso Cortical/patologia , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Humanos , Inflamação/sangue , Inflamação/patologia , Lipoproteínas HDL/sangue , Metaloproteinase 9 da Matriz/genética , NF-kappa B/metabolismo , Osteocalcina/metabolismo , Osteoporose Pós-Menopausa/sangue , Osteoporose Pós-Menopausa/genética , Ratos , Fatores de Risco , Tíbia/patologia , Triglicerídeos/sangue
5.
Phys Chem Chem Phys ; 23(36): 20709-20717, 2021 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-34516596

RESUMO

It is usually assumed that enzymes retain their native structure during catalysis. However, the aggregation and fragmentation of proteins can be difficult to detect and sometimes conclusions are drawn based on the assumption that the protein is in its native form. We have examined three model enzymes, alkaline phosphatase (AkP), hexokinase (HK) and glucose oxidase (GOx). We find that these enzymes aggregate or fragment after addition of chemical species directly related to their catalysis. We used several independent techniques to study this behavior. Specifically, we found that glucose oxidase and hexokinase fragment in the presence of D-glucose but not L-glucose, while hexokinase aggregates in the presence of Mg2+ ion and either ATP or ADP at low pH. Alkaline phosphatase aggregates in the presence of Zn2+ ion and inorganic phosphate. The aggregation of hexokinase and alkaline phosphatase does not appear to attenuate their catalytic activity. Our study indicates that specific multimeric structures of native enzymes may not be retained during catalysis and suggests pathways for different enzymes to associate or separate over the course of substrate turnover.


Assuntos
Fosfatase Alcalina/química , Glucose Oxidase/química , Hexoquinase/química , Fosfatase Alcalina/metabolismo , Biocatálise , Glucose Oxidase/metabolismo , Hexoquinase/metabolismo , Modelos Moleculares , Estrutura Molecular , Agregados Proteicos
6.
Sci Rep ; 11(1): 17685, 2021 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-34480054

RESUMO

Staphylococcus aureus is the most frequent aetiology of bone and joint infections (BJI) and can cause relapsing and chronic infections. One of the main factors involved in the chronicization of staphylococcal BJIs is the internalization of S. aureus into osteoblasts, the bone-forming cells. Previous studies have shown that S. aureus triggers an impairment of osteoblasts function that could contribute to bone loss. However, these studies focused mainly on the extracellular effects of S. aureus. Our study aimed at understanding the intracellular effects of S. aureus on the early osteoblast differentiation process. In our in vitro model of osteoblast lineage infection, we first observed that internalized S. aureus 8325-4 (a reference lab strain) significantly impacted RUNX2 and COL1A1 expression compared to its non-internalized counterpart 8325-4∆fnbAB (with deletion of fnbA and fnbB). Then, in a murine model of osteomyelitis, we reported no significant effect for S. aureus 8325-4 and 8325-4∆fnbAB on bone parameters at 7 days post-infection whereas S. aureus 8325-4 significantly decreased trabecular bone thickness at 14 days post-infection compared to 8325-4∆fnbAB. When challenged with two clinical isogenic strains isolated from initial and relapse phase of the same BJI, significant impairments of bone parameters were observed for both initial and relapse strain, without differences between the two strains. Finally, in our in vitro osteoblast infection model, both clinical strains impacted alkaline phosphatase activity whereas the expression of bone differentiation genes was significantly decreased only after infection with the relapse strain. Globally, we highlighted that S. aureus internalization into osteoblasts is responsible for an impairment of the early differentiation in vitro and that S. aureus impaired bone parameters in vivo in a strain-dependent manner.


Assuntos
Osso Esponjoso/microbiologia , Osteoblastos/microbiologia , Osteogênese/fisiologia , Osteomielite/microbiologia , Fosfatase Alcalina/metabolismo , Animais , Osso Esponjoso/metabolismo , Colágeno Tipo I/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Modelos Animais de Doenças , Camundongos , Osteoblastos/metabolismo , Osteomielite/metabolismo , Staphylococcus aureus
7.
Int J Mol Sci ; 22(17)2021 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-34502544

RESUMO

Recently, composite scaffolding has found many applications in hard tissue engineering due to a number of desirable features. In this present study, hydroxyapatite/bioglass (HAp/BG) nanocomposite scaffolds were prepared in different ratios using a hydrothermal approach. The aim of this research was to evaluate the adhesion, growth, viability, and osteoblast differentiation behavior of human Wharton's-jelly-derived mesenchymal stem cells (hWJMSCs) on HAp/BG in vitro as a scaffold for application in bone tissue engineering. Particle size and morphology were investigated by TEM and bioactivity was assessed and proven using SEM analysis with hWJMSCs in contact with the HAp/BG nanocomposite. Viability was evaluated using PrestoBlueTM assay and early osteoblast differentiation and mineralization behaviors were investigated by ALP activity and EDX analysis simultaneously. TEM results showed that the prepared HAp/BG nanocomposite had dimensions of less than 40 nm. The morphology of hWJMSCs showed a fibroblast-like shape, with a clear filopodia structure. The viability of hWJMSCs was highest for the HAp/BG nanocomposite with a 70:30 ratio of HAp to BG (HAp70/BG30). The in vitro biological results confirmed that HAp/BG composite was not cytotoxic. It was also observed that the biological performance of HAp70/BG30 was higher than HAp scaffold alone. In summary, HAp/BG scaffold combined with mesenchymal stem cells showed significant potential for bone repair applications in tissue engineering.


Assuntos
Materiais Biocompatíveis/química , Cerâmica/química , Hidroxiapatitas/química , Células-Tronco Mesenquimais/fisiologia , Nanocompostos/química , Geleia de Wharton/citologia , Fosfatase Alcalina/metabolismo , Materiais Biocompatíveis/farmacologia , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Microscopia Eletrônica de Transmissão/métodos , Nanocompostos/ultraestrutura , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Engenharia Tecidual/métodos , Tecidos Suporte/química
8.
Biomed Res Int ; 2021: 5529368, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34368350

RESUMO

The aim of this study was to evaluate the biocompatibility and osteogenic potential of a Zeolite Socony Mobil-5 (ZSM-5) coating on a Ti-24 Nb-4 Zr-7.9 Sn (Ti-2448) surface. ZSM-5-modified Ti-2448 (ZSM-5/Ti-2448) and Ti-2448 (control) groups were employed. The physical and chemical properties of the two types of samples were evaluated by scanning electron microscopy, Fourier-transform infrared spectroscopy, nitrogen adsorption/desorption, and contact angle methods. The surface of the ZSM-5/Ti-2448 was rougher than that of the original Ti-2448, while the contact angle of the ZSM-5/Ti-2448 was smaller than that of Ti-2448. In addition, the ZSM-5/Ti-2448 largely increased the specific surface area and introduced silanol groups. A bone-like apatite layer could be formed on the surface of ZSM-5/Ti-2448 after 14 days of incubation in a simulated body fluid. ZSM-5/Ti-2448 was not cytotoxic. The number and alkaline phosphatase (ALP) activity of osteoblasts on ZSM-5/Ti-2448 were significantly higher than those on Ti-2448 surfaces, obtained in vitro using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide and ALP activity assays. Few inflammatory cells were observed around ZSM-5/Ti-2448 after insertion into the femurs of Japanese white rabbits after 4, 12, and 26 weeks through hematoxylin-eosin staining. The average gray scale of transforming growth factor-ß1 (TGF-ß1) on ZSM-5/Ti-2448 peaked earlier than that on Ti-2448, according to immunohistochemical staining. These results indicate that ZSM-5/Ti-2448 has a good biocompatibility and improved early osteogenic potential compared to a noncoated Ti-2448.


Assuntos
Ligas/farmacologia , Materiais Revestidos Biocompatíveis/farmacologia , Osteogênese/efeitos dos fármacos , Zeolitas/farmacologia , Fosfatase Alcalina/metabolismo , Animais , Biomineralização/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Implantes Experimentais , Masculino , Camundongos , Coelhos , Propriedades de Superfície , Fator de Crescimento Transformador beta/metabolismo
9.
Science ; 373(6553)2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34437092

RESUMO

Systematic and extensive investigation of enzymes is needed to understand their extraordinary efficiency and meet current challenges in medicine and engineering. We present HT-MEK (High-Throughput Microfluidic Enzyme Kinetics), a microfluidic platform for high-throughput expression, purification, and characterization of more than 1500 enzyme variants per experiment. For 1036 mutants of the alkaline phosphatase PafA (phosphate-irrepressible alkaline phosphatase of Flavobacterium), we performed more than 670,000 reactions and determined more than 5000 kinetic and physical constants for multiple substrates and inhibitors. We uncovered extensive kinetic partitioning to a misfolded state and isolated catalytic effects, revealing spatially contiguous regions of residues linked to particular aspects of function. Regions included active-site proximal residues but extended to the enzyme surface, providing a map of underlying architecture not possible to derive from existing approaches. HT-MEK has applications that range from understanding molecular mechanisms to medicine, engineering, and design.


Assuntos
Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Fosfatase Alcalina/antagonistas & inibidores , Fosfatase Alcalina/química , Biocatálise , Domínio Catalítico , Flavobacterium/enzimologia , Hidrólise , Cinética , Microfluídica , Modelos Moleculares , Mutação , Oxigênio/metabolismo , Fosfatos/metabolismo , Conformação Proteica , Dobramento de Proteína , Termodinâmica
10.
Phytomedicine ; 91: 153680, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34352588

RESUMO

BACKGROUND: Fragility fractures due to menopausal osteoporosis are a major cause of morbidity and mortality. Osteoporotic medications have substantial side effects that limit long term use. HYPOTHESES: Ingestion of a purified extract of Epimedium spp. (EP) is safe, can increase serum levels of prenylflavonoid metabolites, exert positive changes in bone specific alkaline phosphatase (BSAP), suppress of tumor necrosis factor receptor associated factor 6 (TRAF6) protein in osteoclast-precursor monocytes in peripheral blood and therefore have the potential to reduce post-menopausal bone loss. STUDY DESIGN & METHODS: Healthy postmenopausal women were randomized in a double-blind fashion to consume either EP prenylflavonoid extract (740 mg daily) or placebo daily for 6 weeks. The main outcome measures were safety and pharmacokinetics of EP flavonoids. Fasting blood was collected at 3- and 6-weeks, and two weeks after stopping medication for safety evaluations and measurement of BSAP. Peripheral blood monocytes were harvested for measurement of TRAF6 levels. Serum levels of the EP metabolites icariin, icariside I & II, icaritin and desmethylicaritin were measured using tandem mass spectrometry, and non-compartmental pharmacokinetic analyses performed using WinNonlin software. RESULTS: Between October 2018 and Jun 2020, 58 postmenopausal women, aged 57.9 ± 8.9 years, were randomized and completed the study. Consumption of EP prenylflavonoids was not associated with any significant adverse symptoms, with no changes in hepatic, hematological, and renal parameters observed. The main metabolites detected in sera after ingestion of EP prenylflavonoid capsules were desmethylicaritin, icaritin and icariside II. Icariin and icariside I were below detection levels. Ingestion of EP prenylflavonoids induced a median Cmax and AUC0→∞ for desmethylicaritin of 60.9 nM, and 157.9 nM ×day, respectively; and were associated with higher levels of BSAP (p < 0.05) and a trend (p = 0.068) towards lower levels of TRAF6 in peripheral blood monocytes eight weeks after commencing prenylflavonoid ingestion. Prenylflavonoid metabolites were not detected in the sera of placebo participants. CONCLUSIONS: Despite the widespread consumption of EP extracts, the safety, mechanisms of action of their bioactive compounds, and therapeutic indications in humans are unknown. Daily consumption of EP prenylflavonoids for six weeks was safe. The predominant metabolite in sera was desmethylicaritin. Rise in prenylflavonoid metabolites was associated with higher levels of the bone anabolic marker BSAP, suggesting potential therapeutic value for post-menopausal osteoporosis.


Assuntos
Fosfatase Alcalina/metabolismo , Epimedium , Flavonoides/uso terapêutico , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Osteoporose Pós-Menopausa , Extratos Vegetais/uso terapêutico , Idoso , Densidade Óssea , Método Duplo-Cego , Epimedium/química , Flavonoides/farmacocinética , Humanos , Pessoa de Meia-Idade , Osteoclastos , Osteoporose Pós-Menopausa/tratamento farmacológico , Extratos Vegetais/farmacocinética , Pós-Menopausa , Fator 6 Associado a Receptor de TNF
11.
Cells ; 10(8)2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34440843

RESUMO

Adequate vascularization is a fundamental prerequisite for bone regeneration, formation and tissue engineering applications. Endothelialization of scaffold materials is a promising strategy to support neovascularization and bone tissue formation. Besides oxygen and nutrition supply, the endothelial network plays an important role concerning osteogenic differentiation of osteoprogenitor cells and consecutive bone formation. In this study we aimed to enhance the growth stimulating, proangiogenic and osteogenic features of the ADSC and HUVEC coculture system by means of VEGFA165 and BMP2 application. We were able to show that sprouting phenomena and osteogenic differentiation were enhanced in the ADSC/HUVEC coculture. Furthermore, apoptosis was unidirectionally decreased in HUVECs, but these effects were not further enhanced upon VEGFA165 or BMP2 application. In summary, the ADSC/HUVEC coculture system per se is a powerful tool for bone tissue engineering applications.


Assuntos
Proteína Morfogenética Óssea 2/farmacologia , Diferenciação Celular/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Fator A de Crescimento do Endotélio Vascular/farmacologia , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Fosfatase Alcalina/metabolismo , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Técnicas de Cocultura , Células Endoteliais da Veia Umbilical Humana , Humanos , Proteínas Recombinantes/farmacologia , Células-Tronco/citologia , Células-Tronco/metabolismo , Engenharia Tecidual
12.
Int J Mol Sci ; 22(12)2021 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-34201124

RESUMO

BMP-7 has shown inductive potential for in vitro osteogenic differentiation of mesenchymal stem cells, which are an ideal resource for regenerative medicine. Externally applied, recombinant BMP-7 was able to induce the osteogenic differentiation of DPSCs but based on our previous results with BMP-2, we aimed to study the effect of the tetracyclin-inducible BMP-7 expression on these cells. DPSC, mock, and DPSC-BMP-7 cell lines were cultured in the presence or absence of doxycycline, then alkaline phosphatase (ALP) activity, mineralization, and mRNA levels of different osteogenic marker genes were measured. In the DPSC-BMP-7 cell line, the level of BMP-7 mRNA significantly increased in the media supplemented with doxycycline, however, the expression of Runx2 and noggin genes was upregulated only after 21 days of incubation in the osteogenic medium with doxycycline. Moreover, while the examination of ALP activity showed reduced activity in the control medium containing doxycycline, the accumulation of minerals remained unchanged in the cultures. We have found that the induced BMP-7 expression failed to induce osteogenic differentiation of DPSCs. We propose three different mechanisms that may worth investigating for the engineering of expression systems that can be used for the induction of differentiation of mesenchymal stem cells.


Assuntos
Proteína Morfogenética Óssea 7/metabolismo , Diferenciação Celular , Polpa Dentária/citologia , Doxiciclina/farmacologia , Osteogênese , Células-Tronco/citologia , Fosfatase Alcalina/metabolismo , Antibacterianos/farmacologia , Proliferação de Células , Células Cultivadas , Polpa Dentária/efeitos dos fármacos , Polpa Dentária/metabolismo , Humanos , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo
13.
Talanta ; 233: 122564, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34215060

RESUMO

Photoelectrochemical (PEC) immunoassay is a burgeoning and promising bioanalytical method. However, the practical application of PEC still exist some challenges such as the inevitable damage of biomolecules caused by the PEC system and the unsatisfactory sensitivity for biomarkers with low abundance in real sample. To solve the problems, we integrated the cosensitized structure of Ag2S/ZnO nanocomposities as photoelectrode with photogenerated hole-induced chemical redox cycling amplification (CRCA) strategy to develop a split-type PEC immunosensor for cardiac troponin I (cTnI) with high sensitivity. Initially, the immunoreaction was carried out on the 96-well plates in which alkaline phosphatase (ALP) could catalyze ascorbic acid 2-phosphate (AAP) to generate the signal-reporting species ascorbic acid (AA). Subsequently, the AA participated and the tris (2-carboxyethyl) phosphine (TCEP) mediated chemical redox cycling reaction took place on the photoelectrode, thus leading to signal amplification. Under the optimized conditions, the immunosensor demonstrated a detection limit (LOD) of 3.0 × 10-15 g mL-1 with a detection range of 1.0 × 10-14 g mL-1 to 1.0 × 10-9 g mL-1 for cTnI. Impressively, the proposed method could determine the cTnI in human serum samples with high sensitivity and satisfactory accuracy. Considering the virtues of the photoelectrode and the chemical redox cycling strategy, the method would hold great potential for highly sensitive biosensing and bioanalysis.


Assuntos
Técnicas Biossensoriais , Troponina I , Fosfatase Alcalina/metabolismo , Técnicas Eletroquímicas , Humanos , Imunoensaio , Limite de Detecção , Oxirredução
14.
Int J Mol Sci ; 22(14)2021 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-34299372

RESUMO

Many biomaterials have been evaluated using cultured cells. In particular, osteoblast-like cells are often used to evaluate the osteocompatibility, hard-tissue-regeneration, osteoconductive, and osteoinductive characteristics of biomaterials. However, the evaluation of biomaterial osteogenesis-inducing capacity using osteoblast-like cells is not standardized; instead, it is performed under laboratory-specific culture conditions with different culture media. However, the effect of different media conditions on bone formation has not been investigated. Here, we aimed to evaluate the osteogenesis of MC3T3-E1 cells, one of the most commonly used osteoblast-like cell lines for osteogenesis evaluation, and assayed cell proliferation, alkaline phosphatase activity, expression of osteoblast markers, and calcification under varying culture media conditions. Furthermore, the various media conditions were tested in uncoated plates and plates coated with collagen type I and poly-L-lysine, highly biocompatible molecules commonly used as pseudobiomaterials. We found that the type of base medium, the presence or absence of vitamin C, and the freshness of the medium may affect biomaterial regeneration. We posit that an in vitro model that recapitulates in vivo bone formation should be established before evaluating biomaterials.


Assuntos
Meios de Cultivo Condicionados/farmacologia , Osteogênese/efeitos dos fármacos , Células 3T3 , Fosfatase Alcalina/metabolismo , Animais , Ácido Ascórbico/farmacologia , Materiais Biocompatíveis/farmacologia , Biomarcadores/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Colágeno Tipo I/metabolismo , Camundongos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo
15.
Diagn Microbiol Infect Dis ; 100(4): 114105, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34279227

RESUMO

In this study, the utility and diagnostic accuracy of alkaline phosphatase on the surface membrane of neutrophils (mNAP) for bacteremia in patients with systemic inflammatory response syndrome (SIRS) was investigated and assessed. A total of 149 patients with SIRS were included. mNAP values were significantly higher in bacteremic SIRS group compared with that in non-bacteremic SIRS group (P < 0.001). The mNAP levels were significantly higher in SIRS patients with gram-negative bacteremia than those with gram-positive bacteremia. (P < 0.001). The receiver operating characteristic (ROC) curve analysis revealed the areas under ROC (AUC) of 0.806 for mNAP in differentiating SIRS patients with bacteremia from those without, similar to that for procalcitonin (PCT) (0.797). Combination of PCT and mNAP gave an AUC of 0.841. mNAP shares a similar diagnostic accuracy to PCT in predicting bacteremia in SIRS patients. The combination of mNAP and PCT provides a better prediction of bacteremia in patients with SIRS than either test alone.


Assuntos
Fosfatase Alcalina/metabolismo , Bacteriemia/diagnóstico , Neutrófilos/metabolismo , Síndrome de Resposta Inflamatória Sistêmica/diagnóstico , Adulto , Idoso , Bacteriemia/sangue , Bacteriemia/complicações , Biomarcadores/sangue , Membrana Celular/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pró-Calcitonina/sangue , Estudos Prospectivos , Curva ROC , Sensibilidade e Especificidade , Síndrome de Resposta Inflamatória Sistêmica/sangue , Síndrome de Resposta Inflamatória Sistêmica/complicações
16.
Biomed Res Int ; 2021: 6684709, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34195280

RESUMO

Adipose-derived mesenchymal stem cells (ADSCs) are a well-recognized multilineage stem cell with vital clinical feasibility for tissue regeneration. Extensive evidence indicates that oxidative stress and microRNAs (miRNAs/miRs) play an important role in the osteoinduction of adipose-derived mesenchymal stem cells. In this study, we investigated the mechanism of miR-125a-5p in regulating the osteogenesis of human adipose-derived mesenchymal stem cells (hADSCs) under oxidative stress. The expression of miR-125a-5p lessened gradually during the osteogenic differentiation of hADSCs. Relative to the negative group, the expression levels of runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), osteocalcin (OCN), and osterix in the miR-125a-5p group were marked lower than those in the miR-125a-5p inhibitor group. The levels of p16, p21, p53, miR-125a-5p, and ROS during osteoinduction of hADSCs were assessed in vitro under oxidative stress and were observed to be upregulated. Further experiments showed that oxidative stress and miR-125a-5p together suppressed the expression of VEGF during osteogenic differentiation of hADSCs and that the inhibition of miR-125a-5p reversed the effect of oxidative stress. In short, our study indicated that miR-125a-5p is induced under oxidative stress and inhibits the expression of VEGF, leading to the reduction of osteogenic differentiation of hADSCs. Our outcomes showed that miR-125a-5p could be a potential clinical target for bone repairing.


Assuntos
Adipócitos/citologia , Células-Tronco Mesenquimais/citologia , MicroRNAs/metabolismo , Estresse Oxidativo , Fosfatase Alcalina/metabolismo , Remodelação Óssea , Osso e Ossos/metabolismo , Diferenciação Celular , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Humanos , Osteoblastos/metabolismo , Osteocalcina/metabolismo , Osteogênese/fisiologia , Fator de Transcrição Sp7/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores
17.
Int J Nanomedicine ; 16: 3633-3648, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34079254

RESUMO

Purpose: The deposition of hydroxyapatite (HAp) crystals plays an important role in the development of vascular calcification (VC). This study aimed to demonstrate the effects of nanosized HAp (nHAp) on vascular smooth muscle cells (VSMCs) and VC progression. Methods: Transmission electron microscopy (TEM) was used to examine cellular uptake of nHAp. Cell viability was determined using CCK-8 assay kit. Mitochondrial impairment and reactive oxygen species were detected by TEM and fluorescence dye staining, respectively. Cell apoptosis was detected by Western blot analysis and Annexin V staining. Mouse model of VC was built via applying nHAp on the surface of abdominal aorta. Calcification was visualized by Alizarin red and von Kossa staining. Results: We found that nHAp could promote osteogenic transformation of VSMCs by elevating expression of runt-related factor 2 (Runx2), osteopontin (OPN) and alkaline phosphatase (ALP), impairing function and morphology of mitochondria and inducing apoptosis of VSMCs. More phosphorylation of c-Jun N-terminal protein kinase/c-JUN (JNK/c-JUN) in VSMCs was detected after mixing nHAp with VSMCs. HAp-induced osteogenic transformation of VSMCs was blocked by JNK inhibitor SP600125, resulted in decreased ALP activity, less Runx2 and OPN expressions. SP600125 also inhibited apoptosis of VSMCs. Application of nHAp to outside of aorta induced osteogenic transformation and apoptosis of VSMCs, and significant deposition of calcium on the vessel walls of mice, which can be effectively attenuated by SP600125. Conclusion: JNK/c-JUN signaling pathway is critical for nHAp-induced calcification, which could be a potential therapeutic target for controlling the progression of VC.


Assuntos
Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Durapatita/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Músculo Liso Vascular/citologia , Nanoestruturas/química , Osteogênese/efeitos dos fármacos , Fosfatase Alcalina/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Durapatita/química , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Osteopontina/metabolismo , Espécies Reativas de Oxigênio/metabolismo
18.
Int J Mol Sci ; 22(9)2021 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-34064452

RESUMO

Polycystic Kidney Disease (PKD) is a disorder that affects the kidneys and other organs, and its major forms are encoded by polycystin-1 (PC1) and polycystin-2 (PC2), as PKD1 and PKD2. It is located sandwiched inside and outside cell membranes and interacts with other cells. This protein is most active in kidney cells before birth, and PC1 and PC2 work together to help regulate cell proliferation, cell migration, and interactions with other cells. The molecular relationship and the function between PKD1 and cancer is well known, such as increased or decreased cell proliferation and promoting or suppressing cell migration depending on the cancer cell type specifically. However, its function in stem cells has not been revealed. Therefore, in this study, we investigated the biological function of PC1 and umbilical cord blood-derived mesenchymal stem cell (UCB-MSC). Furthermore, we assessed how it affects cell migration, proliferation, and the viability of cells when expressed in the PKD1 gene. In addition, we confirmed in an ex vivo artificial tooth model generated by the three-dimension printing technique that the ability to differentiate into osteocytes improved according to the expression level of the stemness markers when PKD1 was expressed. This study is the first report to examine the biological function of PKD1 in UCB-MSC. This gene may be capable of enhancing differentiation ability and maintaining long-term stemness for the therapeutic use of stem cells.


Assuntos
Diferenciação Celular/genética , Células-Tronco Mesenquimais/metabolismo , Osteócitos/metabolismo , Canais de Cátion TRPP/genética , Células A549 , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Linhagem Celular , Movimento Celular , Proliferação de Células , Terapia Baseada em Transplante de Células e Tecidos/métodos , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Expressão Gênica , Células HEK293 , Humanos , Células MCF-7 , Células-Tronco Mesenquimais/citologia , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Osteócitos/citologia , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Canais de Cátion TRPP/metabolismo , Transfecção , Transgenes
19.
Int J Mol Sci ; 22(10)2021 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-34067629

RESUMO

Tissue-nonspecific alkaline phosphatase (TNAP) is an ectoenzyme bound to the plasma membranes of numerous cells via a glycosylphosphatidylinositol (GPI) moiety. TNAP's function is well-recognized from earlier studies establishing its important role in bone mineralization. TNAP is also highly expressed in cerebral microvessels; however, its function in brain cerebral microvessels is poorly understood. In recent years, few studies have begun to delineate a role for TNAP in brain microvascular endothelial cells (BMECs)-a key component of cerebral microvessels. This review summarizes important information on the role of BMEC TNAP, and its implication in health and disease. Furthermore, we discuss current models and tools that may assist researchers in elucidating the function of TNAP in BMECs.


Assuntos
Fosfatase Alcalina/metabolismo , Células Endoteliais/metabolismo , Microvasos/metabolismo , Fosfatase Alcalina/fisiologia , Animais , Encéfalo/metabolismo , Sistema Nervoso Central/metabolismo , Humanos
20.
Cell Prolif ; 54(7): e13013, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34101928

RESUMO

BACKGROUND: Human dermal papilla (DP) cells and melanocytes (hMel) are central players in hair growth and pigmentation, respectively. In hair follicles (HFs), oxygen (O2 ) levels average 5%, being coupled with the production of reactive oxygen species (ROS), necessary to promote hair growth. MATERIALS AND METHODS: DP cell and hMel proliferation and phenotype were studied under physiological (5%O2 , physoxia) or atmospheric (21%O2 , normoxia) oxygen levels. hMel-DP cells interactions were studied in indirect co-culture or by directly co-culturing hMel with DP spheroids, to test whether their interaction affected the response to physoxia. RESULTS: Physoxia decreased DP cell senescence and improved their secretome and phenotype, as well as hMel proliferation, migration, and tyrosinase activity. In indirect co-cultures, physoxia affected DP cells' alkaline phosphatase (ALP) activity but their signalling did not influence hMel proliferation or tyrosinase activity. Additionally, ROS production was higher than in monocultures but a direct correlation between ROS generation and ALP activity in DP cells was not observed. In the 3D aggregates, where hMel are organized around the DP, both hMel tyrosinase and DP cells ALP activities, their main functional indicators, plus ROS production were higher in physoxia than normoxia. CONCLUSIONS: Overall, we showed that the response to physoxia differs according to hMel-DP cells interactions and that the microenvironment recreated when in direct contact favours their functions, which can be relevant for hair regeneration purposes.


Assuntos
Proliferação de Células/efeitos dos fármacos , Folículo Piloso/metabolismo , Melanócitos/metabolismo , Oxigênio/farmacologia , Citoesqueleto de Actina , Fosfatase Alcalina/metabolismo , Movimento Celular , Senescência Celular , Técnicas de Cocultura , Derme/citologia , Folículo Piloso/citologia , Folículo Piloso/efeitos dos fármacos , Humanos , Melanócitos/citologia , Melanócitos/efeitos dos fármacos , Monofenol Mono-Oxigenase/metabolismo , Espécies Reativas de Oxigênio/metabolismo
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