Alkaline Phosphatase Activity Research Articles

Effects of Eucommia ulmoides leaf extracts and caltrate on osteoblasts proliferation and differentiation

Osteoporosis is a significant and escalating public health issue. It is a skeletal disorder in which there is a decrease in bone strength, resulting in a heightened susceptibility to fractures. Eucommia ulmoides leaves (EUL) has been traditionally employed to alleviate muscle and bone weakness. In order to investigate the induction of osteoblast proliferation and differentiation by the two EUL extracts under non-stress conditions, as well as their combined effects with Caltrate, this study examined cell proliferation, alkaline phosphatase activity, mineralization, and abundance of osteogenic differentiation-related proteins. The results demonstrated that EUL extract 1 and Caltrate intervention alone could promote osteoblast proliferation and differentiation, and their combined treatment exhibited a synergistic effect. EUL extract 2, when administered alone, was capable of enhancing osteoblast proliferation and differentiation; however, it did not exhibit a strong synergistic effect when combined with Caltrate. Therefore, administering EUL extract 1 either individually or in combination with Caltrate serve as a potential dietary supplement for osteoporosis.

Antibacterial Activity of Novel Agent N-2-Hydroxypropyl Trimethyl Ammonium Chloride Chitosan against Streptococcus mutans.

Dental caries (DC) is one of the most common oral diseases and is mainly caused by Streptococcus mutans (S. mutans). The use of antibiotics against S. mutans usually has side effects, including developing resistance. N-2-Hydroxypropyl trimethyl ammonium chloride chitosan (N-2-HACC), a natural product, has great potential utility in antibacterial agents owing to its low toxicity and good biocompatibility. Thus, the purpose of the present study was to explore the antimicrobial activity of N-2-HACC against S. mutans through the permeability of the cell wall, integrity of cell membrane, protein and nucleic acid synthesis, respiratory metabolism, and biofilm formation. Our results confirmed that the MIC of N-2-HACC against S. mutans was 0.625 mg/mL with a 90.01 ± 1.54% inhibition rate. SEM observed the formation of cavities on the surface of S. mutans after 12 h N-2-HACC treatment. The level of alkaline phosphatase (AKP) activity was higher in the N-2-HACC treatment group than in the control group, indicating that N-2-HACC can improve the permeability of the cell wall. Also, N-2-HACC treatment can destroy the cell membrane of S. mutans by increasing conductivity and absorbance at 260 nm, decreasing cell metabolic activity, and enhancing the fluorescence at 488 nm. Respiratory metabolism revealed that the activities of the Na+-K+-ATP enzyme, pyruvate kinase (PK), succinate dehydrogenase (SDH), and malate dehydrogenase (MDH) were decreased after N-2-HACC treatment, revealing that N-2-HACC can inhibit glycolysis and the tricarboxylic acid cycle (TCA cycle) of S. mutans. Moreover, N-2-HACC can also decrease the contents of the nucleic acid and solution protein of S. mutans, interfere with biofilm formation, and decrease the mRNA expression level of biofilm formation-related genes. Therefore, these results verify that N-2-HACC has strong antibacterial activity against S. mutans, acting via cell membrane integrity damage, increasing the permeability of cell walls, interfering with bacterial protein and nucleic acid synthesis, perturbing glycolysis and the TCA cycle, and inhibiting biofilm formation. It is suggested that N-2-HACC may represent a new potential synthetically modified antibacterial material against S. mutans.

The potential role of exopolysaccharide from Graesiella sp (chlorophyte Microalga) as dietary supplement against Bisphenol A-induced hepatotoxicity in gilthead sea bream (Sparus aurata)

ABSTRACT Bioactive compounds derived from microalgae exhibit beneficial biological activities for human and animal health. The present study was conducted to assess the potential protective effect of exopolysaccharide EPSs extracted from Graesiella sp, against Bisphenol-A toxicity in gilthead sea bream (Sparus aurata). Fish were divided into two groups, the first group was fed a basal diet without EPSs and the second one was fed a supplemented diet (1.5% of EPSs) for 55 days. Subsequently, a random sample from each group were intraperitoneally injected with Bisphenol A (BPA) at a concentration of 50 mg/kg body weight. The antioxidant status, plasma biochemistry, tissue histopathology and flesh quality were then assessed after five days of BPA injection. Our results show that EPSs supplementation didn't induce negative effects for all tested parameters. BPA exposure caused a significant increment of hepatic lipid peroxidation and drops by more than 50% in the catalase (CAT), isocitrate dehydrogenase (ICDH) and reduced glutathione (GSH) activities. BPA induced also a significant increase in plasma aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) activities and numerous hepatic alterations. Interestingly, the BPA damage was reduced in fish fed with EPS-enriched diets suggesting that Graesiella-EPS can be supplemented to prevent BPA damage.

Development of injectable microgel-based scaffolds via enzymatic cross-linking of hyaluronic acid-tyramine/gelatin-tyramine for potential bone tissue engineering

Currently, the healing of large bone defects relies on invasive surgeries and the transplantation of autologous bone. As a less invasive treatment option, the provision of microenvironments that promote the regeneration of defective bones holds great promise. Here, we developed hyaluronic acid (HA)/gelatin (Ge) microgel-based scaffolds to guide bone regeneration. To enable the formation of microgels by enzymatic cross-linking in the presence of horseradish peroxidase (HRP) and hydrogen peroxide (H2O2), we modified the polymers with tyramine (TA). Spectrophotometry and proton nuclear magnetic resonance (1H NMR) spectroscopy analysis confirmed successful tyramine substitution on polymer backbones. To enable the formation of microgels by a water-in-oil emulsion approach, the HRP and H2O2 concentrations were tuned to achieve the gelation in a few seconds. By varying the stirring speed from 600 to 1000 rpm, spherical microgels were produced with an average size of 116 ± 8.7 and 68 ± 4.7 μm, respectively. The results showed that microgels were injectable through needles and showed good biocompatibility with the cultured human osteosarcoma cell line (MG-63). HA/Ge-TA microgels served as a promising substrate for MG-63 cells since they improved the alkaline phosphatase activity and level of calcium deposition. In summary, the developed HA/Ge-TA microgels are promising injectable microgel-based scaffolds in bone tissue engineering.

Alkaline Phosphatase Activity and Phosphatase-Active Bacteria in Lake Baikal Water Column and Major Tributaries

Phosphorus is one of the major biogenic elements. Its inflow facilitates eutrophication of lake water. In aquatic ecosystems, phosphorus is present mostly in organic compounds. Ability of aquatic microorganisms to assimilate phosphorus from organophosphorous compounds results from activity of alkaline phosphatases; activity of these enzymes may be an indicator of the state of the ecosystem, phosphate load, and water quality. In the present work, alkaline phosphatase activity (APA) and abundance of phosphatase-active bacteria (PAB) in Lake Baikal pelagic zone and in the mouths of its major tributaries was studied. In the pelagic zone, APA and PAB abundance decreased with depth, indicating that the main processes of phosphate generation occurred in the trophic layer of the lake. In the main tributaries, both APA and PAB abundance were considerably higher than in the pelagic zone. These results indicate active biochemical processes of transformation of organophosphorous compounds occur in the estuarine zones of the rivers. The degradation processes result in regeneration of phosphates, which are completely incorporated in the biological turnover, providing for phytoplankton development.

Impacts of Selenium Supplementation on Soil Mercury Speciation, Soil Properties and Mercury-Resistant Microorganisms and Resistant Genes

Soil mercury (Hg) contamination is a serious threat to local ecology and public health. Exogenous selenium (Se) supplementation can effectively reduce the toxicity of Hg. However, the mechanisms affecting the changes in soil Hg speciation, soil properties and the microbial Hg-resistant system during the Se–Hg interaction after exogenous Se supplementation are not clear. Therefore, in this study, soil culture experiments were conducted to analyze the effects of different Se additions on the transformation of Hg speciation, soil properties and Hg-resistant microorganisms and resistant genes (mer operon). The results indicated that Se supplementation facilitated the transformation of soil Hg from bioavailable (exchangeable and carbonate-bound) to stable forms (organic material-bound and residual), significantly reducing Hg bioavailability. Se supplementation notably decreased the electrical conductivity of Hg-contaminated soil, but had no significant effect on the soil pH, organic matter content, cation exchange capacity or alkaline phosphatase and catalase activities. The maximum activity levels of soil sucrase and urease were observed when 1 mg kg−1 Se was added. Se significantly inhibited soil peroxidase and ascorbate oxidase activities, thereby alleviating the oxidative stress in the soil system caused by Hg. Additionally, Se significantly activated the Hg-resistant system in soil microorganisms by either decreasing or increasing the regulatory genes merD and merR, and it significantly upregulated the cytoplasmic protein gene merP and the membrane protein genes merC, merF and merT. This further increased the abundance of the organomercury lyase gene merB and the mercuric reductase gene merA, promoting the conversion of Hg species to Hg⁰. Furthermore, the abundance of mer operon-containing microorganisms, such as Thiobacillus ferrooxidants, Pseudomonas, Streptomyces and Cryptococcus, significantly increased with Se addition, explaining the role of soil microorganisms in mitigating soil Hg stress via Se supplementation.

Magnetically responsive micro-clustered calcium phosphate-reinforced cell-laden microbead sodium alginate hydrogel for accelerated osteogenic tissue regeneration

The rising prevalence of bone injuries has increased the demand for minimally invasive treatments. Microbead hydrogels, renowned for cell encapsulation, provide a versatile substrate for bone tissue regeneration. They deliver bioactive agents, support cell growth, and promote osteogenesis, aiding bone repair and regeneration. In this study, we synthesized superparamagnetic iron oxide nanoparticles (Sp) coated with a calcium phosphate layer (m-Sp), achieving a distinctive flower-like micro-cluster morphology. Subsequently, sodium alginate (SA) microbead hydrogels containing m-Sp (McSa@m-Sp) were fabricated using a dropping gelation strategy. McSa@m-Sp is magnetically targetable, enhance cross-linking, control degradation rates, and provide strong antibacterial activity. Encapsulation studies with MC3T3-E1 cells revealed enhanced viability and proliferation. These studies also indicated significantly elevated alkaline phosphatase (ALP) activity and mineralization in MC3T3-E1 cells, as confirmed by Alizarin Red S (ARS) and Von Kossa staining, along with increased collagen production within the McSa@m-Sp microbead hydrogels. Immunocytochemistry (ICC) and gene expression studies supported the osteoinductive potential of McSa@m-Sp, showing increased expression of osteogenic markers including RUNX-2, collagen-I, osteopontin, and osteocalcin. Thus, McSa@m-Sp microbead hydrogels offer a promising strategy for multifunctional scaffolds in bone tissue engineering.

Influence of Varying Levels of Organic Manure, Phosphorus and Bio- inoculants on Biological Properties of Soil under Mungbean [Vigna radiata (L.) Wilczek

A field experiment was conducted for two consecutive years (during Kharif 2019 and 2020) at Instructional Farm, College of Agriculture, Jodhpur (Rajasthan) to assess the impact of varying levels of FYM, phosphorus and bio-inoculants on biological properties of soil under Mungbean. The experiment was laid out in factorial randomized block design with three replications. The experiment comprised of two levels of FYM (0 and 5 t/ha), four levels of phosphorus (0, 50, 75 and 100% P2O5 /ha) and three levels of bio-inoculations (No inoculation, Enterobacter cloacae and Bacillus amyloliquefaciens). Mungbean variety GM-4 was used for exprimentation. The results revealed that addition of FYM @ 5 t ha-1 recorded significantly higher soil microbial biomass carbon (217.92 mg /kg), soil microbial biomass phosphorus (15.47 mg/kg), acid phosphatase (1.019 \(\mu\)g PNP/g soil/h), alkaline phosphatase (7.13\(\mu\)g PNP/g soil/h) and dehydrogenase activity (6.10 \(\mu\)g TPF/g soil/ h) of soil at 40 DAS as compared to without FYM application. Among the phosphorus levels, application of 100% P2O5 /ha gave significantly highest soil microbial biomass carbon (219.13mg/kg), soil microbial biomass phosphorus (15.71 mg/kg), acid phosphatase (1.010 µg PNP/g soil/h), alkaline phosphatase (7.21\(\mu\)g PNP/g soil/h) and dehydrogenase activity (5.91\(\mu\)g TPF/g soil/h) over rest of P levels, however, it was found at par with 75 % P2O5 /ha. Similarly, the seed inoculation with B. amyloliquefaciens recorded significantly higher values of all above biological properties as compared to un-inoculated control but it was found at par with seed inoculation with E. cloacae. Thus, the results reaveal the positive effect of the application of FYM, Phosphorus and seed inoculation with biofertilizers (E. cloacae or B. amyloliquefaciens) on the biological properties of soil (soil microbial biomass carbon, soil microbial biomass phosphorus, acid phosphatase, alkaline phosphatase and dehydrogenase activity) for management of sustainable environment.

Nanocrystal hydroxyapatite carrying traditional Chinese medicine for osteogenic differentiation

Developing biomaterials with high osteogenic properties is crucial for achieving rapid bone repair and regeneration. This study focuses on the application of nanocrystal hydroxyapatite (nHAp) as a drug carrier to load Fu Yuan Huo Xue Decoction (FYHXD), a traditional Chinese medicine derived from Angelica sinensis, aiming to achieve improved efficacy in treating bone diseases such as osteoporosis. Through a facile physical adsorption approach, the FTIR result emerges new characteristic absorption peaks in the range of 1200–950 cm−1, proving the successful absorption of FYHXD onto the nHAp with a loading efficiency of 39.76 %. The modified nHAp exhibits a similar shape to the bone-derived hydroxyapatite nanocrystals, and their diameter increases slightly after modification. The drug release assay implies the rapid release of FYHXD in the first 10 h, followed by a continuously slow release within 70 h. The developed nHAp effectively enhances the adhesion, spreading, and proliferation of MC3T3-E1 cells in vitro, and significantly promotes their osteogenic differentiation, as indicated by increased alkaline phosphatase activity. Overall, the biocomposites hold great promise as active ingredients for integration into bone-associated biomaterials, offering the potential to stimulate spontaneous osteogenesis without requiring exogenous osteogenic factors.

Exploring the impact of high salinity and parasite infection on antioxidant and immune systems in Coris julis in the Pityusic Islands (Spain)

Climate change associated with human activities alters marine ecosystems and causes imbalances and abrupt changes in sea conditions. Scarce freshwater resources for human consumption often prompt the construction of desalination plants, which discharge significant amounts of brine into the sea, potentially elevating salinity levels. Furthermore, global trade together with higher temperature and pollution can facilitate the spread of parasites. The aim of this study was to assess the potential effects of salinity, an abiotic stressor, and Scaphanocephalus sp. parasitic infection responsible for black spot disease, a biotic stressor, on Coris julis, a common fish in the Balearic Islands (Spain). Fish were sampled from an area affected by a desalination plant, one with a high rate of parasite infection and a control area, and biomarkers were analysed in the liver, gills and epithelial mucosa. Both salinity and the parasite induced increases in catalase (CAT) and glutathione s-transferase activities in the liver, while superoxide dismutase (SOD) did not show significant changes. The effects of salinity were evident to a greater extent in the gills with an increase in the activity of all enzymes, as well as in the production of reactive species. The effects of the parasite were mainly observed in the mucus with significant increases in CAT and SOD activities. Regarding immune response markers in the mucus, both stressors induced an increase in lysozyme and alkaline phosphatase activities, and in the case of the parasite, also an increase in immunoglobulins. Malondialdehyde, as an indicator of oxidative damage, remained unchanged. In conclusion, both abiotic and abiotic stress induce a stress situation in C. julis that responds by activating its antioxidant and immune defence mechanisms but does cause oxidative damage. The differential tissue response to different stressors highlights the value of analysing multiple tissues to detect early indicators of diverse impacts on marine fauna.

Interfacial activation of alkaline phosphatase induced by hydrophilic metal—organic frameworks

Interfacial activation of alkaline phosphatase induced by hydrophilic metal—organic frameworks

Salinity threshold for phosphorus limitation in an estuary-coast continuum

River-derived nutrient imbalances can induce phosphorus (P) limitation in coastal plankton, but the salinity thresholds that govern this phenomenon have received little attention. Here, we provide a first insight into threshold responses in P-limitation along the salinity gradient in coastal ecosystems. Our seasonal sampling in the northern South China Sea showed a clear nonlinear decrease in alkaline phosphatase activity (APA) with increasing salinity. A pivotal finding was the identification of a salinity threshold at 32.83, presenting an early-warning indicator for P-limitation detection. Using a mixed-effects model, we further revealed that the robust positive correlation between phytoplankton biomass and APA gradually weakened as salinity increased toward this threshold. This implies a shift in phytoplankton’s P-limitation at this salinity threshold and the contribution of heterotrophic bacteria in APA within high-salinity seawater. These insights highlight the importance of refining monitoring strategies with salinity and APA dynamics as early-warning indicators for nutrient limitation in coastal ecosystems.

Epicatechin ameliorates glucose intolerance and hepatotoxicity in sodium arsenite-treated mice

Arsenic is a metalloid found in the environment that causes toxic effects in different organs, mainly the liver. This study aimed to investigate the protective effects of epicatechin (EC), a natural flavonol, on glucose intolerance (GI) and liver toxicity caused by sodium arsenite (SA) in mice. Our findings showed that SA exposure led to the development of GI. Liver tissue damage and decreased pancreatic Langerhans islet size were also observed in this study. Mice exposed to SA exhibited hepatic oxidative damage, indicated by reduced antioxidant markers (such as superoxide dismutase, catalase, glutathione peroxidase, and glutathione), along with elevated levels of thiobarbituric acid reactive substances. SA administration elevated the serum activities of liver enzymes alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase. Furthermore, notable increases in the levels of inflammatory and apoptotic markers (Toll-like receptor 4, nuclear factor-kappa B, tumor necrosis factor-α, nitric oxide, B-cell lymphoma-2, and cysteine aspartate-specific protease-3) were observed in the liver. Treatment of SA-exposed mice with EC considerably reversed these biochemical and histological changes. This study demonstrated the beneficial effects of EC in ameliorating SA-induced hyperglycemia and hepatotoxicity due to its ability to enhance the antioxidant system by modulating inflammation and apoptosis.

Guide DNA dephosphorylation-modulated Pyrococcus furiosus Argonaute fluorescence biosensor for the detection of alkaline phosphatase and aflatoxins B1

Foodborne hazardous factors pose a significant risk to public health, emphasizing the need for the development of sensitive and user-friendly detection strategies to effectively manage and control these risks in the food supply chain. Pyrococcus furiosus argonaute (PfAgo)-based biosensing approaches have been extensively explored due to its built-in signal amplification. However, the property that PfAgo is a DNA-guided DNA endonuclease has enabled almost all the existing PfAgo-based reports to be used for the detection of nucleic acids. To lend PfAgo toolbox to extended non-nucleic acid detection, we systematically investigated the mechanism characteristic of PfAgo′ preference for guide DNA (gDNA) and proposed a gDNA dephosphorylation-modulated PfAgo sensor for the detection of non-nucleic acid targets. Our results indicated that PfAgo exhibits preference for 5′-phosphorylated gDNA at a specific ratio of PfAgo to gDNA concentration. Leveraging this PfAgo′ preference and the dephosphorylation activity of alkaline phosphatase (ALP), ALP could be detected as low as 2.7 U/L. Furthermore, the PfAgo was coupled with immunolabelled ALP to develop a PfAgo-based fluorescence immunosensor, which achieves aflatoxins B1 detection with a detection limit of 29.89 pg/mL and exhibits satisfactory recoveries in wheat and maize samples. The developed method broadens the application scope of PfAgo toolbox, and provides a simple, sensitive, and universal detection platform for a variety targets.

Composted maize straw under fungi inoculation reduces soil N2O emissions and mitigates the microbial N limitation in a wheat upland

Enhancement of microbial assimilation of inorganic nitrogen (N) by straw addition is believed to be an effective pathway to improve farmland N cycling. However, the effectiveness of differently pretreated straws on soil N2O emissions and soil N-acquiring enzyme activities remains unclear. In this study, a pot experiment with four treatments (I, no addition, CK; II, respective addition of maize straw, S; III, composted maize straw under no fungi inoculation, SC; and IV, composted maize straw under fungi inoculation, SCPA) at the same quantity of carbon (C) input was conducted under the same amount of inorganic N fertilization. Results showed that the seasonal cumulative N2O emissions following the SCPA treatment were the lowest at 4.03 kg N ha−1, representing a significant reduction of 19 % compared with the CK treatment. The S and SC treatments had no significant effects on N2O emissions. The decrease of soil N2O emissions following the SCPA treatment was mainly attributed to the increase of microbial N assimilation and the increased abundance of functional genes related to N2O reductase. The SCPA treatment significantly decreased soil alkaline phosphatase (ALP) activity and increased leucine aminopeptidase (LAP) activity at the basal fertilization, while increased soil ALP and LAP activity, decreased soil N-Acetyl-β-D-Glucosidase (NAG) activity at harvest. Compared with the CK treatment, the S, SC, and SCPA treatment significantly increased soil β-glucosidase (β-GC) activity at harvest. The decrease in the (NAG+LAP)/ALP ratio following the SCPA treatment indicated that the composted maize straw under fungi inoculation alleviated microbial N limitation at harvest. Moreover, PICRUSt analysis also suggested that the SCPA treatment increased the abundance of bacterial genes associated with N assimilation and N2O reduction, whereas the S and SC treatment did not significantly affect the abundance of N2O reduction genes compared with the CK treatment. Our results suggest that the composted maize straw under fungi inoculation would reduce the risk of N2O emissions and effectively mitigate the microbial N limitation in dryland wheat system.

Efficient cadmium-resistant plant growth-promoting bacteria loaded on pig bone biochar has higher efficiency in reducing cadmium phytoavailability and improving maize performance than on rice husk biochar

Green agriculture faced challenges due to the shortage of efficient cadmium (Cd)-resistant plant growth-promoting bacteria (CdR-PGPB) and their low survival rate and activity during application. In this study, a diverse range of efficient CdR-PGPB were isolated from the rhizosphere soil of Desmodium elegans, especially those with high phosphate-solubilizing capabilities (272.87–450.45 mg L−1). Two highly efficient CdR-PGPB namely, XH1 and XH3 were loaded on to rice husk biochar (RHB) and pig bone biochar (PBB), labelled as RHBM and PBBM respectively. This study aimed to explore their effectiveness and mechanisms in promoting maize growth in a Cd-contaminated planting system. Results showed that PBBM performed best among all treatments. It significantly decreased soil phytoavailable Cd by 53.19 % and Cd content in maize shoot by 85.89 %. It also increased soil available phosphorus by 145.72 %, soil alkaline phosphatase activity by 76.34 %, maize shoot/root biomass by 47.06 %/67.98 %, Chlorophyll (a/b) content by 66.80 %/134.13 % and peroxidase activity by 171.96 %. These results were achieved through the synergistic action of efficient CdR-PGPB and PBB. Therefore, PBBM proved to be a promising and innovative application technique for sustainable agricultural development in Cd-contaminated farmland ecosystems.

Development of Biocompatible Coatings with PVA/Gelatin Hydrogel Films on Vancomycin-Loaded Titania Nanotubes for Controllable Drug Release.

This study investigates the utilization of poly(vinyl alcohol) (PVA)/gelatin hydrogel films cross-linked with glutaraldehyde as a novel material to coat the surface of vancomycin-loaded titania nanotubes (TNTs), with a focus on enhancing biocompatibility and achieving controlled vancomycin release. Hydrogel films have emerged as promising candidates in tissue engineering and drug-delivery systems due to their versatile properties. The development of these hydrogel films involved varying the proportions of PVA, gelatin, and glutaraldehyde to achieve the desired properties, including the gel fraction, swelling behavior, biocompatibility, and biodegradation. Among the formulations tested, the hydrogel with a PVA-to-gelatin ratio of 25:75 and 0.2% glutaraldehyde was selected to coat vancomycin-loaded TNTs. The coated TNTs demonstrated slower release of vancomycin compared with the uncoated TNTs. In addition, the coated TNTs demonstrated the ability to promote osteogenesis, as evidenced by increased alkaline phosphatase activity and calcium accumulation. The vancomycin-loaded TNTs coated with hydrogel film demonstrated effectiveness against both E. coli and S. aureus. These findings highlight the potential benefits and therapeutic applications of using hydrogel films to coat implant materials, offering efficient drug delivery and controlled release. This study contributes valuable insights into the development of alternative materials for medical applications, thereby advancing the field of biomaterials and drug delivery systems.

Toward the point-of-care testing of alkaline phosphatase in human serum

This work addresses challenges of simple but reliable determination of enzyme alkaline phosphatase (ALP) in μL-sized serum samples. The single-tube assay and plate-based immunoassay were developed by using a commercial glucose test strip (GTS) to transduce analytical signal from the ALP + glucose-6-phosphate (G6P) reaction. The glucose released from G6P contributed to the flow of anodic charge Q20s through GTS. The interferences from serum matrix including those from native glucose were eliminated by devising a signal ΔQ20s = Q20s (total) – Q20s (matrix). The ΔQ20s was proportional to ALP activity within a linear range of 7.4–720 IU L−1 for assay (R2, 0.995) and 10–300 IU L−1 for immunoassay (R2, 0.983), which far exceeded a normal clinical range for ALP in adult human serum (30–150 IU L−1). Both methods required no auxiliary enzymes or labels, and were accurate (93–99 % signal recovery) and precise (RSD ≤10%). The assay was a simpler option because it required only one 20-min incubation step to determine ALP. The immunoassay involved three steps but it was still less laborious (by ∼70%) than a traditional enzyme-linked immunosorbent assay (ELISA) of ALP due to the substitution of ELISA detection antibody with GTS. The GTS-G6P-based methods bring ALP testing closer to the point-of-care locations, which is important considering the growing role of ALP as a prophylactic, therapeutic, and anti-inflammatory agent, and a marker of human diseases.

Single-cell sequencing reveals that specnuezhenide protects against osteoporosis via activation of METTL3 in LEPR+ BMSCs

BackgroundOsteoporosis (OP) has garnered significant attention due to its substantial morbidity and mortality rates, imposing considerable health burdens on societies worldwide. However, the molecular mechanisms underlying osteoporosis pathogenesis remain largely elusive, and the available therapeutic interventions are limited. Therefore, there is an urgent need for innovative strategies in the treatment of osteoporosis. PurposeThe primary objective of this study was to elucidate the molecular mechanisms underlying osteoporosis pathogenesis using single-cell RNA sequencing (scRNA-seq), thereby proposing novel therapeutic agents. MethodsThe mice osteoporosis model was established through bilateral ovariectomy. Micro-computed tomography (μCT) and hematoxylin and eosin (H&E) staining were employed to assess the pathogenesis of osteoporosis. scRNA-seq was utilized to identify and analyze distinct molecular mechanisms and sub-clusters. Gradient dilution analysis was used to obtain specific sub-clusters, which were further validated by immunofluorescence staining and flow cytometry analysis. Molecular docking and cellular thermal shift assay (CETSA) were applied for screening potential agents in the TCMSPs database. Alkaline phosphatase (ALP) activity and alizarin red S (ARS) staining were performed to evaluate the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Osteogenic organoids analysis was employed to assess the proliferation and sphere-forming ability of BMSCs. Quantitative real-time PCR (qRT-PCR) and western blot analysis were conducted to investigate signaling pathways. Wound healing assay and tube formation analysis were employed to evaluate the angiogenesis of endothelial cells. ResultsThe scRNA-seq analysis revealed the crucial role of LEPR+ BMSCs in the pathogenesis of osteoporosis, which was confirmed by immunofluorescence staining of the epiphysis. Subsequently, the LEPR+ BMSCs were obtained by gradient dilution analysis and identified by immunofluorescence staining and flow cytometry. Accordingly, specnuezhenide (Spe) was screened and identified as a potential compound targeting METTL3 from the TCMSPs database. Spe promoted bone formation as evidenced by μ-CT, and H&E analysis. Additionally, Spe enhanced the osteogenic capacity of LEPR+ BMSCs through ALP and ARS assay. Notably, METTL3 pharmacological inhibitors S-Adenosylhomocysteine (SAH) attenuated the aforementioned osteo-protective effects of Spe. Particularly, Spe enhanced the LEPR+ BMSCs-dependent angiogenesis through the secretion of SLIT3, which was abolished by SAH in LEPR+ BMSCs. ConclusionCollectively, these findings suggest that Spe could enhance the osteogenic potential of LEPR+ BMSCs and promote LEPR+ BMSCs-dependent angiogenesis by activating METTL3 in LEPR+ BMSCs, indicating its potential as an ideal therapeutic agent for clinical treatment of osteoporosis.

Tissue scaffolds mimicking hierarchical bone morphology as biomaterials for oral maxillofacial surgery with augmentation: structure, properties, and performance evaluation for in vitro testing

In this study, tissue scaffolds mimicking hierarchical morphology are constructed and proposed for bone augmentation. The scaffolds are fabricated using lyophilization, before coating them with collagen (Col). Subsequently, the Col-coated scaffolds undergo a second lyophilization, followed by silk fibroin (SF) coating, and a third lyophilization. Thereafter, the scaffolds are divided into six groups with varying ratios of Col to SF: Col/SF = 7:3, 5:5, 3:7, 10:0, and 0:10, with an SF scaffold serving as the control group. The scaffold morphology is examined using a scanning electron microscope, while molecular and structural formations are characterized by Fourier transform infrared spectrometer and differential scanning calorimeter, respectively. Physical and mechanical properties including swelling and compression are tested. Biological functions are assessed throughin vitroosteoblast cell culturing. Biomarkers indicative of bone formation-cell viability and proliferation, alkaline phosphatase activity, and calcium content-are analyzed. Results demonstrate that scaffolds coated with Col and SF exhibit sub-porous formations within the main pore. The molecular formation reveals interactions between the hydrophilic groups of Col and SF. The scaffold structure contains bound water and SF formation gets disrupted by Col. Physical and mechanical properties are influenced by the Col/SF ratio and morphology due to coating. The biological functions of scaffolds with Col and SF coating show enhanced potential for promoting bone tissue formation, particularly the Col/SF (7:3) ratio, which is most suitable for bone augmentation in small defect areas.