Extracellular Alkaline Phosphatase Activity Research Articles

Peptide-Driven Assembly of Magnetic Beads for Potentiometric Sensing of Bacterial Enzyme at a Subcellular Level.

Bacterial enzymes with different subcellular localizations play a critical ecological role in biogeochemical processing. However, precisely quantifying enzymes localized at certain subcellular levels, such as extracellular enzymes, has not yet been fully realized due to the complexity and dynamism of the bacterial outer membrane. Here we present a magneto-controlled potentiometric sensing platform for the specific detection of extracellular enzymatic activity. Alkaline phosphatase (ALP), which is one of the crucial hydrolytic enzymes in the ocean, was selected as the target enzyme. Magnetic beads functionalized with an ALP-responsive self-assembled peptide (GGGGGFFFpYpYEEE, MBs-peptides) prevent negatively charged peptides from entering the bacterial outer membrane, thereby enabling direct potentiometric sensing of extracellular ALP both attached to the bacterial cell surface and released into the surrounding environment. The dephosphorylation-triggered assembly of peptide-coupled magnetic beads can be directly and sensitively measured by using a magneto-controlled sensor. In this study, extracellular ALP activity of Pseudomonas aeruginosa at concentrations ranging from 10 to 1.0 × 105 CFU mL-1 was specifically and sensitively monitored. Moreover, this magneto-controlled potentiometric method enabled a simple and accurate assay of ALP activity across different subcellular localizations.

Combination of Cinnamaldehyde/β-cyclodextrin inclusion complex and L-phenylalanine effectively reduces the postharvest green mold in citrus fruit

The essential oil and β-cyclodextrin inclusion complex was able to inhibit the growth of Penicillium digitatum, a damaging pathogen that causes green mold in citrus fruit. In this study, cinnamaldehyde-β-cyclodextrin inclusion complex (β-CDCA) for controlling citrus green mold was synthesized by the co-precipitation method. Characterization of β-CDCA revealed that the aromatic ring skeleton of cinnamaldehyde (CA) was successfully embedded into the cavity of β-CD to form the inclusion complex. β-CDCA inhibited P. digitatum at a minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of 4.0 g/L. FT-IR spectroscopy analysis, calcofluor white staining, extracellular alkaline phosphatase (AKP) activity and propidium iodide (PI) staining of hyphae morphology showed that β-CDCA may damage the cell ultrastructure and membrane permeability of P. digitatum. The study further demonstrated that hydrogen peroxide (H2O2), malondialdehyde (MDA), and reactive oxygen species (ROS) markedly accumulated in 1/2 MIC β-CDCA treated hyphae. This implied that β-CDCA inhibited growth of P. digitatum by the triggering oxidative stress, which may have caused cell death by altering cell membrane permeability. In addition, in vivo results showed that β-CDCA alone or combined with L-phenylalanine (L-PHe) displayed a comparable level to that of prochloraz. Therefore, β-CDCA combined with L-PHe can thus be used as an eco-friendly preservative for the control green mold in postharvest citrus fruit.

Presence of pharmaceutical contaminants of emerging concerns in two rivers of western Cuba and their relationship with the extracellular enzymatic activity of microbial communities

In recent years, the concern derived from the presence of emerging contaminants in the environment and the possible effects on the One Health trilogy has increased. This study determined the concentration of pharmaceutical contaminants of emerging concern and their relationship with the extracellular enzymatic activity of microbial communities from two rivers in western Cuba. Two sampling stations were analyzed; one in the Almendares River (urban) and the other in the San Juan River (rural), taking into account the pollution sources that arrive at these stations and previous physicochemical characterizations. Extracellular protease, acid phosphatase, alkaline phosphatase, lipase, and catalase activities in water and sediments were determined and correlated with contaminants of emerging concern determined by liquid chromatography with mass spectrometry. This study evidenced the presence of different pharmaceutical contaminants found in the categories of antihypertensives, stimulants, anti-inflammatories, and antibiotics in both rivers. Concentrations of contaminants of emerging concern were greater in the Almendares River compared to the San Juan River. In addition, through the canonical redundancy analysis, the influence of these contaminants on the extracellular enzymatic activities of microbial communities was documented, where in most cases they inhibit protease, phosphatase, and lipase activities and enhance catalase activity in response to oxidative stress. The present investigation constitutes the first report in Cuba of the presence of pharmaceutical contaminants of emerging concern and one of the few works that exist in the Latin American region.

Purification of Polysaccharides from Orostachys cartilaginous Cell Cultures by Macroporous Resin Absorption and Bioactivities of the Purified Polysaccharides.

Orostachys cartilaginous is a traditional herbal medicine and its cell cultures contain large amounts of polysaccharides. To utilize the cultured O. cartilaginous cells, this study purified the crude polysaccharides of O. cartilaginous cells by macroporous resin absorption and optimized the purification process in the experiment of orthogonal design with four factors (sample concentration and volume, and eluent concentration and volume) and three levels; the antibacterial and anti-cancer effects of the purified polysaccharides (OTP) were further examined. The results showed that polysaccharide purity reached 95 % in the optimized group, i. e., 1.6 mg/mL of sample (crude polysaccharides) concentration, 3.0 bed volume (BV) of sample volume, 65 % eluent (ethanol) concentration, and 3.0 BV of eluent volume. In the antibacterial experiment, the growth of three bacterial species, i. e., Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis was inhibited by OTP, whereas that of Escherichia coli was not affected; the largest diameter of the inhibitory zone was found on B. subtilis; the extracellular alkaline phosphatase activity and the electrical conductivity, nucleic acid, and protein levels of B. subtilis increased after OTP treatment, indicating that the inhibition of B. subtilis growth was caused by the leakage of cell contents. In the anti-cancer experiment, OTP decreased the cell viabilities of the tested human cancer cells, i. e., AGS (gastric cancer), HCT116 (colon cancer), HepG2 (liver cancer), and HeLa (cervical cancer), and the highest inhibitory effect was on HCT116. OTP promoted HCT116 apoptosis and affected the expression of apoptosis-related proteins, i. e., the expression of B-cell lymphoma-2 decreased and that of bcl-2 associated X protein, cytochrome c, caspase 9 and caspase 3 increased. The findings of the present study suggest that O. cartilaginous cell cultures have a potential application in food or drug production.

Surfactin inhibits the growth of Propionibacterium acnes by destroying the cell wall and membrane.

Propionibacterium acnes plays a major role in acne vulgaris. In the pre-experiment, the growth of P. acnes was inhibited effectively using surfactin; however, the antibacterial mechanism has not been described. Therefore, the aim of this study was to evaluate antibacterial activity and analyse the mechanism of surfactin against P. acnes. Minimum inhibitory concentration, time-killing kinetics and scanning electron microscopy were used to evaluate the activity of surfactin against P. acnes, which showed that 128 μg ml-1 effectively inhibited growth. Cell wall permeability was evaluated by detecting the extracellular alkaline phosphatase activity, which increased to 1·83- and 2·32-fold after incubating with 128 and 256 μg ml-1 of surfactin for 10 h, respectively. Propidium iodide fluorescence, leakage of nucleic acid, protein, K+ , and Ca2+ , membrane potential and the leakage of calcein from small unilamellar vesicles all increased after incubation with surfactin, indicating that its strong biological activities act mainly by altering membrane integrity. In a mouse model of acne, surfactin significantly reduced P. acnes-induced epidermal swelling and erythema. These results indicate that surfactin effectively inhibited the growth of P. acnes by destroying the cell wall and membrane, and is a potential candidate for acne treatment.

Effects of rhBMP-2 on Bone Formation Capacity of Rat Dental Stem/Progenitor Cells from Dental Follicle and Alveolar Bone Marrow.

Dental stem/progenitor cells are a promising cell sources for alveolar bone (AB) regeneration because of their same embryonic origin and superior osteogenic potential. However, their molecular processes during osteogenic differentiation remain unclear. The objective of this study was to identify the responsiveness of dental follicle cells (DFCs) and AB marrow-derived mesenchymal stem cells (ABM-MSCs) to recombinant human bone morphogenetic protein-2 (rhBMP-2). These cells expressed vimentin and MSC markers and did not express cytokeratin and hematopoietic stem cell markers and showed multilineage differentiation potential under specific culture conditions. DFCs exhibited higher proliferation and colony-forming unit-fibroblast efficiency than ABM-MSCs; rhBMP-2 induced DFCs to differentiate toward a cementoblast/osteoblast phenotype and ABM-MSCs to differentiate only toward a osteoblast phenotype; and rhBMP-2-induced DFCs exhibited higher osteogenic differentiation potential than ABM-MSCs. These cells adhered, grew, and produced extracellular matrix on nanohydroxyapatite/collagen/poly(l-lactide) (nHAC/PLA). During a 14-day culture on nHAC/PLA, the extracellular alkaline phosphatase (ALP) activity of DFCs decreased gradually and that of ABM-MSCs increased gradually; rhBMP-2 enhanced their extracellular ALP activity, intracellular osteocalcin (OCN), and osteopontin (OPN) protein expression; and DFCs exhibited higher extracellular ALP activity and intracellular OCN protein expression than ABM-MSCs. When implanted subcutaneously in severe combined immunodeficient mice for 3 months, DFCs+nHAC/PLA+rhBMP-2 obtained higher percentage of bone formation area, OCN, and cementum attachment protein expression and lower OPN expression than ABM-MSCs+nHAC/PLA+rhBMP-2. These results showed that DFCs possessed superior proliferation and osteogenic differentiation potential in vitro, and formed higher quantity and quality bones in vivo. It suggested that DFCs might exhibit a more sensitive responsiveness to rhBMP-2, so that DFCs enter a relatively mature stage of osteogenic differentiation earlier than ABM-MSCs after rhBMP-2 induction. The findings imply that these dental stem/progenitor cells are alternative sources for AB engineering in regenerative medicine, and developing dental tissue may provide better source for stem/progenitor cells.

Interactions of the Cyanobacterium Chrysosporum ovalisporum with Antibiotics in Water.

In this study, we assessed the effects of 11-day exposure of sulfadiazine (SD), sulfamethazine (SM2), norfloxacin (NOR), and enrofloxacin (ENR) on the growth, chlorophyll a (Chl. a) content, phycobiliproteins (PBPs) content, and alkaline phosphatase (ALP) activity of Chrysosporum ovalisporum, examined the removal rate of these antibiotics by C. ovalisporum, and performed acute toxicology test with Daphnia magna to determine the effect of interaction between antibiotics and cyanobacteria on aquatic animals. The results showed that the stress of SD and SM2 increased extracellular ALP activity and weakly inhibited the algal growth and the contents of Chl. a and PBPs compared with that noted in the control. ENR and NOR treatment groups exerted significant inhibition on algal growth as well as Chl. a and PBPs contents and ALP activity, although the cyanobacterium could degrade these two antibiotics more than SD and SM2. The results also revealed that the interaction between antibiotics and cyanobacteria could inhibit D. magna feeding.

Osteogenic Properties of 3D-Printed Silica-Carbon-Calcite Composite Scaffolds: Novel Approach for Personalized Bone Tissue Regeneration.

Carbon enriched bioceramic (C-Bio) scaffolds have recently shown exceptional results in terms of their biological and mechanical properties. The present study aims at assessing the ability of the C-Bio scaffolds to affect the commitment of canine adipose-derived mesenchymal stem cells (cAD-MSCs) and investigating the influence of carbon on cell proliferation and osteogenic differentiation of cAD-MSCs in vitro. The commitment of cAD-MSCs to an osteoblastic phenotype has been evaluated by expression of several osteogenic markers using real-time PCR. Biocompatibility analyses through 3-(4,5-dimethyl- thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), lactate dehydrogenase (LDH) activity, hemolysis assay, and Ames test demonstrated excellent biocompatibility of both materials. A significant increase in the extracellular alkaline phosphatase (ALP) activity and expression of runt-related transcription factor (RUNX), ALP, osterix (OSX), and receptor activator of nuclear factor kappa-Β ligand (RANKL) genes was observed in C-Bio scaffolds compared to those without carbon (Bio). Scanning electron microscopy (SEM) demonstrated excellent cell attachment on both material surfaces; however, the cellular layer on C-Bio fibers exhibited an apparent secretome activity. Based on our findings, graphene can improve cell adhesion, growth, and osteogenic differentiation of cAD-MSCs in vitro. This study proposed carbon as an additive for a novel three-dimensional (3D)-printable biocompatible scaffold which could become the key structural material for bone tissue reconstruction.

Degradation of diclofenac by new bacterial strains and its influence on the physiological status of cells

Diclofenac (DCF) is one of the most commonly utilized non-steroidal anti-inflammatory drugs (NSAIDs), which is known to pose an ecotoxicological threat. In this study, from activated sludge and contaminated soil, we isolated four new bacterial strains able to degrade DCF under mono-substrate and co-metabolic conditions with glucose supplementation. We found that the effectiveness of DCF removal is strictly strain-specific and the addition of the primary substrate is not always beneficial. To assess the multidirectional influence of DCF on bacterial cells we evaluated the alterations of increasing concentrations of this drug on membrane structure. A significant increase was observed in the content of 17:0 cyclo fatty acid, which is responsible for reduced fluidity and profound changes in membrane rigidity. The cell injury and oxidative stress were assessed with biomarkers used as endpoints of toxicity, i.e. catalase (CAT), superoxide dismutase (SOD), lipids peroxidation (LPX), and both intra- and extracellular alkaline and acid phosphatase activity. Results indicated that DCF induced oxidative stress, frequently intensified by the addition of glucose. However, the response of the microbial cells to the presence of DCF should not be generalized, since the overall picture of the particular alterations greatly varied for each of the examined strains.

Magnetic Nanoparticles Synergize with Pulsed Magnetic Fields to Stimulate Osteogenesis In Vitro.

Delayed bone healing is a major challenge in orthopedic clinical practice, highlighting a need for technologies to overcome ineffective cell growth and osteogenic differentiation. The objective of this study was to investigate the synergistic effects of the PhysioStim (PEMF) signal with iron-ion doped tri-calcium phosphate bone substitute on human mesenchymal stem cell (hMSC) osteogenesis in vitro. Intrinsically magnetic nano-bone substitutes (MNBS) were developed with single particles on the order of 100 nm, saturation magnetization of 0.425 emu/g, and remanent magnetization of 0.013 emu/g. MNBS were added to hMSC culture and cell viability, alkaline phosphatase (ALP) activity, mineralization, and osteogenic gene expression in the presence and absence of PEMF were quantified for up to 10 days. MNBS attached to the surface of and were internalized by hMSCs when cultured together for 4 days and had no impact on cell viability with PEMF exposure for up to 7 days. Although total ALP activity was significantly increased with PEMF treatment alone, with a peak at day 5, PEMF combined with MNBS significantly increased ALP activity, with a peak at day 3, compared with all other groups (p < 0.01). The shift can be explained by significantly increased extracellular ALP activity beginning at day 2 (p < 0.01). PEMF combined with MNBS demonstrated continuously increasing mineralization overtime, with significantly greater Alizarin Red S concentration compared with all other groups at day 7 (p < 0.01). Increases in ALP activity and mineral content were in agreement with osteogenic gene expression that demonstrated peak ALP gene expression at day 1, and upregulated BMP-2, BGLAP, and SPP1 gene expression at day 7 (p < 0.05). The results of this study demonstrate the synergistic effects of PEMF and MNBS on osteogenesis and suggest that PEMF and MNBS may provide a method for accelerated bone healing.

Response of extracellular and intracellular alkaline phosphatase in Microcystis aeruginosa to organic phosphorus.

Cyanobacterial blooms caused by Microcystis have become a menace to public health and water quality in the global freshwater ecosystem. Alkaline phosphatases (APases) produced by microorganisms play an important role in the mineralization of dissolved organic phosphorus (DOP) into orthophosphate (Pi) to promote cyanobacterial blooms. However, the response of extracellular and intracellular alkaline phosphatase activity (APA) of Microcystis to different DOP sources is poorly understood. In this study, we compared the growth of M. aeruginosa on two DOP substrates (β-glycerol-phosphate (β-GP) and lecithin (LEC)) and monitored the changes of P fractions and the extra- and intracellular APA under different P sources and concentrations. M. aeruginosa can utilize both β-GP and LEC to sustain its growth, and the bioavailability of LEC was greater than β-GP. For the β-GP treatment, there was no significant difference in the algal growth at different concentrations (P > 0.05), while the algal growth in the LEC treatment groups was significantly affected by concentrations (P < 0.05). The results showed that intracellular APA of M. aeruginosa could be detected in all DOP treatment groups and generally higher than extracellular APA. In addition, the intracellular APA per cell increased first and then decreased in all DOP treatment groups. Compared with the β-GP treatment, M. aeruginosa in the LEC groups could secret more extracellular APA.

The isoflavone genistein enhances osteoblastogenesis: signaling pathways involved

Phytoestrogens have been proposed as a natural therapy for prevention of bone loss. In this work, we studied the mechanism of action of genistein on osteoblast differentiation. Primary cell cultures of calvarial osteoblasts isolated from female Wistar rats were in vitro exposed to genistein. Osteoblast differentiation markers were measured. Genistein stimulated osteoblast migration (71-257% above control). An earlier upregulation of estrogen receptor alpha gene expression and an enhancement of mRNA levels of the Runt-related transcription factor 2 were detected after 3days of culture. The isoflavone significantly increased osteocalcin expression, extracellular collagen deposition, and alkaline phosphatase activity. The mechanism displayed by genistein involved estrogen receptor and nitric oxide pathway participation, since cell preincubation with the estrogen receptor antagonist ICI 182780, or the nitric oxide synthase inhibitor L-NAME, suppressed the phytoestrogen action. Evidence of MAPK and PI3K transduction systems participation on the stimulatory action of genistein on extracellular collagen deposition and alkaline phosphatase activity was also obtained. Genistein favored monocyte adhesion to osteoblasts (77% above control) in an ER; NOS; and MAPK kinase-dependent and PI3K-dependent manner. Co-cultured osteoblast-monocyte long term exposed (21days) to genistein exhibited a high number of multinucleated and tartrate-resistant acid phosphatase-positive cells added to osteoblasts, suggesting that the phytoestrogen promotes osteoclast differentiation. In conclusion, genistein promoted osteoblastogenesis through the participation of ER and NOS pathways, and the contribution of ERK or PI3K signal transduction pathways, and also stimulates osteoclast differentiation from its mononuclear progenitor.

Phosphorus strategy in bloom-forming cyanobacteria (Dolichospermum and Microcystis) and its role in their succession

Dolichospermum (formerly Anabaena) and Microcystis cause harmful cyanobacterial blooms in freshwater ecosystems worldwide. Input reduction of both nitrogen (N) and phosphorus (P) are commonly recognized as basic ways of controlling blooms, but little is known about the roles of nutrients and their using strategy among cyanobacteria in triggering the succession of diazotrophic to non-diazotrophic cyanobacteria. In this study, we investigated in situ responses of cyanobactria to ambient P status during the transition from Dolichospermum flos-aquae to Microcystis spp. in Lake Taihu and Lake Chaohu. While dominant in phytoplankton community, D. flos-aquae experienced P deficiency as evidenced by qualitative detection of extracellular phosphatase via enzyme labeled fluorescence (ELF). The percentage of ELF-labelled D. flos-aquae cells was 33% when it dominated the phytoplankton community, and was 78% when it co-dominated with Microcystis spp., indicating an increase in P deficiency. Meanwhile, no ELF-labelled Microcystis cells were observed while polyphosphate body (PPB) were present, suggesting that Microcystis spp. were not P deficient. Additionally, the percentages of Microcystis cells containing PPB showed an inverted “U-shaped” relationship with concentrations on soluble reactive phosphorus (SRP). To validate the field observation, a laboratory study of the monocultures of the dominant cyanobacteria was conducted. Extracellular alkaline phosphatase activity (APA) and PPB accumulation were regulated by P availability in monocultures of D. flos-aquae. Interestingly, no cell bound extracellular phosphatase was found on Microcystis aeruginasa even in the culture without P supply. Consistently, the expressions of phosphatase encoding gene phoX showed no differences among the treatments. The way in which PPB accumulation occurred in Microcystis spp. in response to P availability in the cultures was similar to that observed in the field, demonstrating a strategy of energy conservation over P accumulation. The competitive advantage of Microcystis spp. was displayed at low P concentrations: where it could rapidly uptake and store inorganic P, which also increased the P deficiency of the coexisting phytoplankton species. Responses of P-transport gene pstS confirmed this hypothesis. The physiological and molecular mechanisms mentioned above enable Microcystis to survive and proliferate in environment with low available P supply more efficiently. In conclusion, different cyanobacterial species have distinct ways of responding to P availability, suggesting that the control of cyanobacterial blooms by targeted nutrient reduction is largely dependent upon the dominant species. P reduction is more effective in controlling diazotrophic cyanobacteria than non-diazotrophic cyanobacteria.

Cinnamaldehyde Exerts Its Antifungal Activity by Disrupting the Cell Wall Integrity of Geotrichum citri-aurantii.

Our previous study showed that cinnamaldehyde (CA) significantly inhibited the mycelial growth of Geotrichum citri-aurantii, one of the main postharvest pathogens in citrus fruits. This study investigated the antifungal mechanism of CA against G. citri-aurantii. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images showed that CA treatment led to clear morphological changes in the cell walls and membranes of G. citri-aurantii. However, the membrane integrity, total lipids and ergosterol contents were not apparently affected by CA treatment. Notably, the extracellular alkaline phosphatase (AKP) activity was increased after CA treatment, suggesting impairment in cell wall permeability. A weakened fluorescence in the cell wall, a decrease in the chitin contents, and changes of ten genes involved in cell wall integrity were also observed. These results suggested that CA may exhibit its antifungal activity against G. citri-aurantii by interfering the build of cell wall and therefore lead to the damage of cell wall permeability and integrity.

Antibacterial Activity of Polygonum Orientale Extracts Against Clavibacter Michiganensis subsp. michiganensis, the Agent of Bacterial Canker of Tomato Disease

Abstract Clavibacter michiganensis subsp. michiganensis (Cmm), which is a Gram positive bacterium, causes the bacterial canker of tomato disease. The purpose of the study was to evaluate the antibacterial activity of Polygonum orientale extracts against Cmm. In this study, firstly, effects of three extracting parameters (extractive time, extractive temperature, and solid to liquid ratio) of orthogonal experiment design L27 (313) were conducted. Secondly, survival rate was determined and inhibition zone of Cmm rescued post-stress was monitored. Finally, extracellular OD260nm value, extracellular protein content, conformational structure of membrane protein, extracellular alkaline phosphatase (AKP) activity, and ATPase activity were measured to investigate the antibacterial mechanism. Results of orthogonal experiment revealed that extractive time and extractive temperature had highly significant (P<0.01) effects on the antibacterial activity of P. orientale extracts. The optimum conditions were as follows: 10h of extractive time, 60°C of extractive temperature, and 1:20 (g:mL) of solid to liquid ratio. This study also demonstrated that the living cells of each sample from survival rate test had almost no resistance or adaptability, and rescued Cmm cells were much easier to be inhibited by P. orientale extracts. The results of antibacterial mechanism indicated that cell membrane and cell wall of Cmm were seriously damaged by P. orientale extracts, and P. orientale extracts reduced the intracellular ATPase activity dramatically. All these findings suggested that P. orientale extracts had a strong antibacterial activity to inhibit Cmm, and could be used for the ecological management of the bacterial canker of tomato disease.

Synergistic effect of extracellularly supplemented osteopontin and osteocalcin on stem cell proliferation, osteogenic differentiation, and angiogenic properties.

A high demand for functional bone grafts is being observed worldwide, especially due to the increased life expectancy. Osteoinductive components should be incorporated into functional bone grafts, accelerating cell recruitment, cell proliferation, angiogenesis, and new bone formation at a defect site. Noncollagenous bone matrix proteins, especially osteopontin (OPN) and osteocalcin (OC), have been reported to regulate some physiological process, such as cell migration and bone mineralization. However, the effects of OPN and OC on cell proliferation, osteogenic differentiation, mineralization, and angiogenesis are still undefined. Therefore, we assessed the exogenous effect of OPN and OC supplementation on human bone marrow mesenchymal stem/stromal cells (hBM MSC) proliferation and osteogenic differentiation. OPN dose-dependently increased the proliferation of hBM MSC, as well as improved the angiogenic properties of human umbilical vein endothelial cells (HUVEC) by increasing the capillary-like tube formation in vitro. On the other hand, OC enhanced the differentiation of hBM MSC into osteoblasts and demonstrated an increase in extracellular calcium levels and alkaline phosphatase activity, as well as higher messenger RNA levels of mature osteogenic markers osteopontin and osteocalcin. In vivo assessment of OC/OPN-enhanced scaffolds in a critical-sized defect rabbit long-bone model revealed no infection, while new bone was being formed. Taken together, these results suggest that OC and OPN stimulate bone regeneration by inducing stem cell proliferation, osteogenesis and by enhancing angiogenic properties. The synergistic effect of OC and OPN observed in this study can be applied as an attractive strategy for bone regeneration therapeutics by targeting different vital cellular processes.

Insights into the life strategy of the common marine diatom Chaetoceros peruvianus Brightwell.

Chaetoceros peruvianus is a marine diatom species with circumglobal distribution. While frequently observed, it appears never to dominate the marine phytoplankton community hence it can be characterized as a rather opportunistic, generalistic species. Here we present ecological interpretations from a long-term data set on marine microphytoplankton in the northern Adriatic Sea, where the abundancies and relative contributions of C. peruvianus were observed along a set of steep ecological gradients. Limited supply of dissolved inorganic phosphate was identified as the driving ecological factor for this ecosystem. In parallel C. peruvianus was cultivated in monoclonal cultures and its morphological and physiological reaction to replete and phosphorus depleted medium was analysed. C. peruvianus reacted to phosphorus depletion by an increase in cell height and length as well as thickness and length of setae. This morphological reaction included an increase in cellular volume and calculated carbon content. Additionally, it represents the transition between two described morphological varieties, C. peruvianus and C. peruvianus var. robusta. C. peruvianus showed a significant induction of extracellular alkaline phosphatase activity if grown in phosphate depleted medium. Microscopical analysis demonstrated this activity to be located exclusively on the setae of the cells.

Growth, extracellular alkaline phosphatase activity, and kinetic characteristic responses of the bloom-forming toxic cyanobacterium, Microcystis aeruginosa, to atmospheric particulate matter (PM2.5, PM2.5-10, and PM>10).

Atmospheric particulate matter (APM), commonly seen and widely excited in environment, appears great enough to influence the biochemical processes in aquatic microorganisms and phytoplankton. Understanding the response of cyanobacteria to various factors is fundamental for eutrophication control. To clarify the response of cyanobacteria to APM, the effects of PM2.5, PM2.5-10, and PM>10 on Microcystis aeruginosa were researched. Variabilities in cell density, chlorophyll a, soluble protein, malondialdehyde, extracellular activity, and kinetic parameters of alkaline phosphatase were evaluated by lab-cultured experiments. Results showed that the PM2.5 had a slight stimulation impact on the growth and enhanced both of the 48- and 72-h extracellular alkaline phosphatase activity (APA), the affinity of alkaline phosphatase for substrate, and the 72-h maximum enzymatic reaction velocity (Vmax). Moreover, the stimulations in extracellular APA and Vmax enhanced with the increasing exposure concentrations. We also found there were no obvious distinctions on the effects of growth and alkaline phosphatase in M. aeruginosa between PM2.5-10 and PM>10 exposure groups. Obviously, inhibitory effects on growth existed in 4.0 and 8.0mg/L PM2.5-10 and 8.0mg/L PM>10 at 120h. Furthermore, PM2.5-10 and PM>10 exerted inhibitory effects on the extracellular APA during the 72-h exposure. Simultaneously, the Vmax was notably inhibited and the affinity of alkaline phosphatase for substrate was more inseparable compared with control in PM2.5-10 and PM>10 treatments. Nevertheless, the inhibitors in extracellular APA and kinetic parameters were unrelated to PM2.5-10 and PM>10 exposure concentrations. Two-way ANOVA results revealed that there were significant interactions between exposure concentration and diameter of APM on the 120-h cell density, soluble protein content, APA, and 72h APA of M. aeruginosa. These results in our study would be meaningful to further researches on relationships between APM deposition and cyanobacterial bloom.

Accelerated osteogenic differentiation of human bone-derived cells in ankylosing spondylitis.

Ankylosing spondylitis (AS) is characterized by excessive bone formation with syndesmophytes, leading to bony ankylosis. The contribution of osteoblasts to the pathogenesis of ankylosis is poorly understood. The aim of this study was to determine molecular differences between disease controls (Ct) and AS bone-derived cells (BdCs) during osteogenic differentiation with or without inflammation using AS patient serum. We confirmed osteoblastic differentiation of Ct and AS BdCs under osteogenic medium by observing morphological changes and measuring osteoblastic differentiation markers. Osteoblast differentiation was detected by alkaline phosphatase (ALP) staining and activity, and alizarin red and hydroxyapatite staining. Osteoblast-specific markers were analyzed by quantitative reverse-transcriptase-polymerase chain reaction, immunoblotting, and immunostaining. To examine the effects of inflammation, we added AS and healthy control serum to Ct and AS BdCs, and then analyzed osteoblast-specific markers. AS BdCs showed elevated basal intercellular and extracellular ALP activity compared to Ct. When osteoblast differentiation was induced, AS BdCs exhibited higher expression of osteoblast-specific marker genes and faster mineralization than Ct, indicating that these cells differentiated more rapidly into osteoblasts. ALP activity and mineralization accelerated when serum from AS patients was added to Ct and AS BdCs. Our results revealed that AS BdCs showed significantly increased osteoblastic activity and differentiation capacity by regulating osteoblast-specific transcription factors and proteins compared to Ct BdCs. Active inflammation of AS serum accelerated osteoblastic activity. Our study could provide useful basic data for understanding the molecular mechanism of ankylosis in AS.

Dried Plum Ingestion Increases the Osteoblastogenic Capacity of Human Serum.

In cell culture studies, dried plum (Prunus domestica L.) polyphenols increased osteoblast alkaline phosphatase (ALP) activity, mineralized nodule formation, and the expression of the bone marker genes runt-related transcription factor 2 (RUNX2) and osterix. The purpose of this study was to determine whether human serum collected 1 and 2 h after dried plum ingestion influenced osteoblast cell activity and gene expression. Five healthy women ingested 100 g of dried plum, and serum samples were collected at baseline (before dried plum ingestion) and 1 and 2 h postingestion of dried plum. MC3T3-E1 osteoblast cells were treated (2% of medium) with these serum samples for 3 or 9 days. Intracellular and extracellular ALP activities were significantly increased after 3 or 9 days of treatment with serum both postingestion time points, with no effect seen in baseline samples. Also, serum obtained 1 and 2 h postingestion significantly increased the mRNA expression of bone markers RUNX2 and connexin43 (CX43) after both 3 and 9 days of incubation periods. Finally, serum obtained 1 and 2 h postingestion increased the mRNA expression of β-catenin after 9 days of incubation. We conclude that osteoblast activity and function are increased by dried plum ingestion, which may, in part, explain its beneficial effects on bone health.