Intra-allantoic injection of calcium promotes hatching of chick embryos grown in shell-less culture.

The hatch rate of chick embryos cultured outside of the eggshell with 350 mg calcium l-lactate hydrate (CaL) and 3.5 mL water is fourfold greater in cultures in which the chorioallantoic membrane (CAM) surrounds the egg contents by incubation day 17.5 (E17.5) an event which occurs in ovo by E13. It was first investigated whether decreasing the volume of water added with 350 mg CaL would promote CAM expansion due to the smaller volume to enclose. When 350 mg CaL was present, the CAM did not surround the egg contents by E13. By E17.5, the CAM surrounded the egg contents in 53%-74% of cultures; however, CAM expansion was not significantly different when 0, 1, 2, or 3.5 mL water was present. The hatch rate with 2 or 3.5 mL water was greater than 50% but was not improved with less water. Second, it was investigated whether CaL or water inhibits CAM expansion. In the absence of CaL, the CAM surrounded the egg contents in up to two-thirds of cultures by E13, whether 2 mL water was present or not. Thus CaL, but not water, inhibits expansion of the CAM by E13, even though CaL promotes hatching. Finally, it was investigated whether injection of aqueous CaL into the allantoic fluid, in conjunction with not adding CaL to culture hammocks, would promote CAM expansion. Allantoic injection of CaL starting at E13 did not promote CAM expansion at E17.5 but resulted in hatch rates of approximately 30%. Allantoic injection is a novel route for supplementation of calcium in cultured chick embryos.

Combination of Dental-Capping Agents with Low Level Laser Therapy Promotes Proliferation of Stem Cells from Apical Papilla.

Background: Direct pulp capping is a vital pulp therapy, which stimulates differentiation of stem cells from apical papilla (SCAPs). SCAPs have multipotential capacity to differentiate into types of cells, contributing to the regeneration of tissues. Objective: Considering the promising effects of dental-capping materials, we aim to investigate the effect of dental dressing materials combined with laser therapy on the percentage of SCAP viability and the consequent dental regeneration capacity. Methods: We collected two immature third molar teeth and isolated SCAPs through collagenase type I enzymatic activity. Isolated SCAPs were then cultured with Dulbecco's modified Eagle's medium and α-minimum essential medium enriched with 15% and 10% fetal bovine serum, respectively. After reaching 70-80% confluency, cells were seeded in a 96-well plate and then treated with mineral trioxide aggregate (MTA), enamel matrix derivative (EMD), biodentine, and low level laser therapy (LLLT) alone and in combination for 24, 48, and 168 h. After that, cell survival rate was assessed using (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay. Results: We found that combination of MTA, EMD, and LLLT as well as that of biodentine, EMD, and LLLT could lead to significant increase of SCAP viability as compared with other treatment groups. Combination of MTA and biodentine with EMD could also show increased level of SCAP proliferation and viability. However, MTA and biodentine alone reduced SCAP survival rate in all time points. Conclusions: Our conclusion is that LLLT can serve as an enhancer of SCAP proliferation and differentiation rate when added to dental-capping agents such as MTA, EMD, and biodentine. Thus, LLLT combination with effective capping materials will serve as a promising option for dental tissue repair.

Well-Orientation Strategy Biosynthesis of Cefuroxime-Silver Nanoantibiotic for Reinforced Biodentine™ and Its Dental Application against Streptococcus mutans.

Dental caries results from the bacterial pathogen Streptococcus mutans (S. mutans) and is the maximum critical reason for caries formation. Consequently, the present study aims to evaluate the antibacterial activity of a newly synthesized nanoantibiotic-Biodentine formulation. The silver nanoparticles (ROE-AgNPs) were biosynthesized from the usage of Rosmarinus officinalis L. extract (ROE) and conjugated with cefuroxime to form Cefuroxime-ROE-AgNPs. Using Biodentine™ (BIOD), five groups of dental materials were prepared, in which Group A included conventional BIOD, Group B included BIOD with ROE-AgNPs, Groups C and D included BIOD with Cefuroxime-ROE-AgNPs at concentrations of 0.5% and 1.5% cefuroxime, respectively, and Group E included BIOD with 1.5% cefuroxime. The synthesized ROE-AgNPs or Cefuroxime-ROE-AgNPs were characterized for conjugating efficiency, morphology, particle size, and in vitro release. Minimum inhibitory concentration (MIC) of the cefuroxime, ROE-AgNPs, and Cefuroxime-ROE-AgNPs were additionally evaluated against cefuroxime resistant S. mutans, which furthered antibacterial efficacy of the five groups of dental materials. The UV-Visible spectrum showed the ROE-AgNPs or Cefuroxime-ROE-AgNPs peaks and their formation displayed through transmission electron microscopy (TEM), X-ray diffraction (XRD) pattern, and Fourier transforms infrared (FTIR) analysis. The end result of Cefuroxime-ROE-AgNPs showed conjugating efficiency up to 79%. Cefuroxime-ROE-AgNPs displayed the highest antibacterial efficacy against S. mutans as compared to cefuroxime or ROE-AgNPs alone. Moreover, the MIC of ROE-AgNPs and Cefuroxime-ROE-AgNPs was detected against S. mutans to be 25 and 8.5 µg/mL, respectively. Consequently, Cefuroxime-ROE-AgNPs displayed that a decrease in the MIC reached to more than three-fold less than MIC of ROE-AgNPs on the tested strain. Moreover, Cefuroxime-ROE-AgNPs/BIOD was employed as a novel dental material that showed maximum antimicrobial activity. Groups C and D of novel materials showed inhibitory zones of 19 and 26 mm, respectively, against S. mutans and showed high antimicrobial rates of 85.78% and 91.17%, respectively. These data reinforce the utility of conjugating cefuroxime with ROE-AgNPs to retrieve its efficiency against resistant S. mutant. Moreover, the nanoantibiotic delivered an advantageous antibacterial effect to BIOD, and this may open the door for future conjugation therapy of dental materials against bacteria that cause dental caries.

Effect of L-calcium lactate, zinc lactate, and ferric sodium EDTA on the physicochemical and functional properties of liquid whole egg.

The study aimed to evaluate the physicochemical and functional properties of liquid whole egg (LWE) with L-calcium lactate (L-Ca), zinc lactate (L-Zn), and sodium ferric EDTA (NaFeEDTA), and to compare with NaCl addition to determine the application potential of these mineral supplements. Results showed that salts addition significantly influenced the foaming, emulsifying, and gelling properties of LWE, which was possible through affecting the pH, particle size, surface hydrophobicity, apparent viscosity, and solubility. The addition of all the four salts reduced pH but increased the d4,3 diameter of LWE. Additionally, the addition of 200 mM L-Ca and 6 mM L-Zn significantly improved the emulsifying capacity by 41.73% and 13.6%, the foaming capacity by 26.57% and 10%, and the protein solubility by 13.89% and 12.70%, respectively. In the meantime, mineral supplements tend to produce lower hardness gel, especially with 25 mM L-Ca and 8 mM L-Zn, and the hardness was decreased from 2401.13 to 1138.29 and 1175.59 g, respectively. A relative decrease in hardness was desirable in gelled egg products. Moreover, the addition of NaCl and L-Ca showed a higher redness and yellowness, but the addition of NaFeEDTA showed an undesirable color in dark brown, which may be not accepted by the public. In summary, L-Ca and L-Zn had great potential for application in LWE, which was more appropriate than adding NaCl. This study provides a basis for improving the functional properties of LWE products in the future. PRACTICAL APPLICATION: The addition of L-Ca and L-Zn to liquid whole egg (LWE) could improve the foaming and emulsifying capacity of LWE as well as produce a lower hardness gel, which may be more conducive to the production of cake, custards, and meat products. Meantime, it is more in line with people's pursuit of a healthy diet.

Enhanced Near-Infrared Photocatalytic Eradication of MRSA Biofilms and Osseointegration Using Oxide Perovskite-Based P-N Heterojunction.

Methicillin-resistant Staphylococcus aureus (MRSA) biofilm infections after orthopedic implant increase the risk of failure and potentially cause amputation of limbs or life-threatening sepsis in severe cases. Additionally, satisfactory bone-implant integration is another important indicator of an ideal implant. Here, an antibiotic-free antibacterial nanofilm based on oxide perovskite-type calcium titanate (CTO)/fibrous red phosphorus (RP) on titanium implant surface (Ti-CTO/RP) in which the P-N heterojunction and internal electric field are established at the heterointerface, is designed. Near-infrared light-excited electron-hole pairs are effectively separated and transferred through the synergism of the internal electric field and band offset, which strongly boosts the photocatalytic eradication of MRSA biofilms by reactive oxygen species with an efficacy of 99.42% ± 0.22% in vivo. Additionally, the charge transfer endows the heterostructure with hyperthermia to assist biofilm eradication. Furthermore, CTO/RP nanofilm provides a superior biocompatible and osteoconductive platform that enables the proliferation and osteogenic differentiation of mesenchymal stem cells, thus contributing to the subsequent implant-to-bone osseointegration after eradicating MRSA biofilms.

Compositions and antimicrobial properties of binary ZnO-CuO nanocomposites encapsulated calcium and carbon from Calotropis gigantea targeted for skin pathogens.

Calotropis gigantea (C. gigantea) extract with an ecofriendly nanotechnology approach could provide promising antimicrobial activity against skin pathogens. This study investigates the antimicrobial capability of green synthesized binary ZnO-CuO nanocomposites from C. gigantea against non-MDR (Staphylococcus aureus and Escherichia coli) and MDR (Klebsiella pneumoniae, Pseudomonas aeruginosa and methicillin-resistant S. aureus) skin pathogens. Scanning electron microscopy and transmission electron microscopy revealed the size and shape of B3Z1C sample. Results of X-ray powder diffraction, energy-dispersive spectroscopy, FTIR and UV-Vis spectroscopy analyses confirmed the presence of mixed nanoparticles (i.e., zinc oxide, copper oxide, carbon and calcium) and the stabilising phytochemical agents of plant (i.e., phenol and carbonyl). Antimicrobial results showed that carbon and calcium decorated binary ZnO-CuO nanocomposites with compositions of 75 wt% of ZnO and 25 wt% CuO (B3Z1C) was a strong bactericidal agent with the MBC/MIC ratio of ≤ 4 and ≤ 2 for non-MDR and MDR pathogens, respectively. A significant non-MDR zone of inhibitions were observed for BZC by Kirby-Bauer disc-diffusion test. Further time-kill observation revealed significant fourfold reduction in non-MDR pathogen viable count after 12 h study period. Further molecular studies are needed to explain the biocidal mechanism underlying B3Z1C potential.

The use of photobiomodulation therapy or LED and mineral trioxide aggregate improves the repair of complete tibial fractures treated with wire osteosynthesis in rodents.

The repair of large bone defects is lengthy and complex. Both biomaterials and phototherapy have been used to improve bone repair. We aimed to describe histologically the repair of tibial fractures treated by wiring (W), irradiated or not, with laser (λ780 nm, 70 mW, CW, spot area of 0.5 cm2, 20.4 J/cm2 (4 × 5.1 J/cm2, Twin Flex Evolution®, MM Optics, Sao Carlos, SP, Brazil) per session, 300 s, 142.8 J/cm2 per treatment) or LED (λ850 ± 10 nm, 150 mW, spot area of 0.5 cm2, 20.4 J/cm2 per session, 64 s, 142.8 J/cm2 per treatment, Fisioled®, MM Optics, Sao Carlos, Sao Paulo, Brazil) and associated or not to the use of mineral trioxide aggregate (MTA, Angelus®, Londrina, PR, Brazil). Inflammation was discrete on groups W and W + LEDPT and absent on the others. Phototherapy protocols started immediately before suturing and repeated at every other day for 15 days. Collagen deposition intense on groups W + LEDPT, W + BIO-MTA + LaserPT and W + BIO-MTA + LEDPT and discrete or moderate on the other groups. Reabsorption was discrete on groups W and W + LEDPT and absent on the other groups. Neoformation varied greatly between groups. Most groups were partial and moderately filed with new-formed bone (W, W + LaserPT, W + LEDPT, W + BIO-MTA + LEDPT). On groups W + BIO-MTA and W + BIO-MTA + LaserPT bone, neoformation was intense and complete. Our results are indicative that the association of MTA and PBMT (λ = 780 nm) improves the repair of complete tibial fracture treated with wire osteosynthesis in a rodent model more efficiently than LED (λ = 850 ± 10 nm).

Preparation of Sterile Raw Material - Chicken Eggshells in the Process of their Transformation into Selected Calcium Salts.

BACKGROUND: The chicken eggshells and their subcrustal membranes are a valuable source of calcium, but they are not further processed but disposed of as waste from the food industry. Chicken eggshells have high content (>95%) of calcium carbonate. Some properties suggest that eggshells may be a promising alternative to the present calcium sources used in the pharmaceutical industry. METHODS: The effect of roasting chicken eggshells with a selected organic acid (citric or fumaric or lactic acid) on microbiological purity, including the presence of fungi and bacteria Salmonella spp., Staphylococcus aureus, Escherichia coli of obtained calcium salts, was investigated. In this study, chicken eggshells were subjected to chemical reactions with organic acids (citric, fumaric or lactic acid) at two different calcium-acid molar ratios (1:1 or 1:3) and the mixture was heat-treated for 1 or 3 hours at a temperature of 100°C or 120°C. RESULTS AND DISCUSSION: It was found that lactic acid was 100% effective against fungi, and the remaining citric and fumaric acids were -50% (regardless of the other examined conditions). The type of acid used has a significant effect on fungal growth inhibition (p<0.05). Fumaric acid and lactic acid will be nearly 100% effective against bacteria (100% fumaric acid and 97% lactic acid effectiveness), regardless of other factors. CONCLUSION: Lactic acid is the most effective against pathogenic flora - fungi and bacteria. The transformation of chicken eggshells into calcium lactate can provide us with sterile calcium salt, free of 100% fungi and 97% of all bacteria.

Natural and Experimental Persistence of Highly Pathogenic H5 Influenza Viruses in Slurry of Domestic Ducks, with or without Lime Treatment.

Infections by A/H5 and A/H7 avian influenza viruses (AIVs) can cause acute disease and are therefore notifiable in poultry and wild birds. During winter 2015-2016, several cases of infection caused by highly pathogenic (HP) AIVs belonging to the A/H5N1, A/H5N2, and A/H5N9 subtypes were detected in southwestern France. Throughout winter 2016-2017, several cases of infections caused mainly by A/H5N8 HP AIV (A/goose/GD/1/1996, clade 2.3.4.4) were detected across Europe. On both occasions, the viruses were widely detected on palmiped farms in France. This study was designed to evaluate the persistence of A/H5 HP AIV in slurry from various duck productions. This was achieved (i) in the laboratory setting by artificially spiking four AIV-free slurry samples with known amounts of A/H5N9 HP AIV and monitoring virus infectivity, with or without lime treatment to achieve pH 10 or pH 12, and (ii) by sampling slurry tanks on five naturally A/H5N8 HP-contaminated farms. Experimental results in artificially spiked slurry suggested virus survival for 4 weeks in slurry from Muscovy or Pekin duck breeders and for 2 weeks in slurry from ducks for foie gras production during the assisted-feeding period, without lime treatment. Persistence of infectious A/H5N9 HP AIV in all slurry samples after lime treatment at pH 10 or pH 12 was less than 1 week. The A/H5N8 HP AIV persisted in naturally contaminated untreated slurry for 7 weeks. The results obtained provide experimental support for the 60-day storage period without treatment or the 7-day interval after lime treatment defined in French regulations for slurry sanitization.IMPORTANCE From November 2015 to July 2017, two successive episodes of H5 highly pathogenic avian influenza viruses (HP AIVs) infections occurred on poultry farms in France, mostly in domestic ducks raised for foie gras production in southwestern France. During the two epizootics, epidemiological investigations were carried out on infected farms and control and biosafety measures were implemented in association with surveillance in order to stop the spread of the viruses. Effluents are known to be an important factor in environmental dissemination of viruses, and suitable effluent management is needed to help prevent the spread of epizootics to other farms or pathogen persistence at the farm level. The present study was therefore designed to assess how long infectious A/H5 HP AIVs can persist in naturally or experimentally contaminated fecal slurry samples from ducks, with or without sanitization by lime treatment.

Evaluation of Resin-Based Material Containing Copaiba Oleoresin (Copaifera Reticulata Ducke): Biological Effects on the Human Dental Pulp Stem Cells.

The purpose of this study was to analyze in vitro the biological effects on human dental pulp stem cells triggered in response to substances leached or dissolved from two experimental cements for dental pulp capping. The experimental materials, based on extracts from Copaifera reticulata Ducke (COP), were compared to calcium hydroxide [Ca(OH)2] and mineral trioxide aggregate (MTA), materials commonly used for direct dental pulp capping in restorative dentistry. For this, human dental pulp stem cells were exposed to COP associated or not with Ca(OH)2 or MTA. Cell cytocompatibility, migration, and differentiation (mineralized nodule formation (Alizarin red assay) and gene expression (RT-qPCR) of OCN, DSPP, and HSP-27 (genes regulated in biomineralization events)) were evaluated. The results showed that the association of COP reduced the cytotoxicity of Ca(OH)2. Upregulations of the OCN, DSPP, and HSP-27 genes were observed in response to the association of COP to MTA, and the DSPP and HSP-27 genes were upregulated in the Ca(OH)2 + COP group. In up to 24 h, cell migration was significantly enhanced in the MTA + COP and Ca(OH)2 + COP groups. In conclusion, the combination of COP with the currently used materials for dental pulp capping [Ca(OH)2 and MTA] improved the cell activities related to pulp repair (i.e., cytocompatibility, differentiation, mineralization, and migration) including a protective effect against the cytotoxicity of Ca(OH)2.

Bioshell calcium oxide (BiSCaO) for cleansing and healing Pseudomonas aeruginosa-infected wounds in hairless rats.

BACKGROUND: Scallop shell powder is called bioshell calcium oxide (BiSCaO), which is known to possess deodorizing properties and broad antimicrobial activity against various pathogenic microbes, including viruses, bacteria, spores, and fungi. OBJECTIVE: This study aims to investigate the applications of BiSCaO suspension cleansing in clinical situations, for instance for the prevention and treatment of infections in chronic wounds in healing-impaired patients, without delaying wound healing. METHODS: The bactericidal activities of 1000 ppm BiSCaO suspension; 500 ppm hypochlorous acid; 1000 ppm povidone iodine; and saline were compared to evaluate in vivo disinfection and healing of Pseudomonas aeruginosa-infected wounds in hairless rats. RESULTS: Cleansing of the infected wounds with BiSCaO suspension daily for 3 days significantly enhanced wound healing and reduced the in vivo bacterial counts, in comparison to hypochlorous acid, povidone iodine, and saline. Furthermore, histological examinations showed significantly advanced granulation tissue and capillary formation in the wounds cleansed with BiSCaO suspension than in those cleansed with the other solutions. CONCLUSIONS: This study suggested that the possibility of using BiSCaO suspension as a disinfectant for infected wounds and limiting disinfection to 3 days may be sufficient to avoid the negative effects on wound repair.

Raman spectroscopic study of the effect of the use of laser/LED phototherapy on the repair of complete tibial fracture treated with internal rigid fixation.

This study aimed to assess the repair of complete surgical tibial fractures fixed with internal rigid fixation (IRF) associated or not to the use of mineral trioxide aggregate (MTA) cement and treated or not with laser (λ = 780 nm, infrared) or LED (λ = 850 ±â€¯10 nm, infrared) lights, 142.8 J/cm2 per treatment, by means of Raman spectroscopy. Open surgical tibial fractures were created on 18 rabbits (6 groups of 3 animals per group, ∼8 months old) and fractures were fixed with IRF. Three groups were grafted with MTA. The groups of IRF and IRF + MTA that received laser or LED were irradiated every other day during 15 days. Animals were sacrificed after 30 days, being the tibia surgically removed. Raman spectra were collected via the probe at the defect site in five points, resulting in 15 spectra per group (90 spectra in the dataset). Spectra were collected at the same day to avoid changes in laser power and experimental setup. The ANOVA general linear model showed that the laser irradiation of tibial bone fractures fixed with IRF and grafted with MTA had significant influence in the content of phosphate (peak ∼960 cm-1) and carbonated (peak ∼1,070 cm-1) hydroxyapatites as well as collagen (peak 1,452 cm-1). Also, peaks of calcium carbonate (1,088 cm-1) were found in the groups grafted with MTA. Based on the Raman spectroscopic data collected in this study, MTA has been shown to improve the repair of complete tibial fractures treated with IRF, with an evident increase of collagen matrix synthesis, and development of a scaffold of hydroxyapatite-like calcium carbonate with subsequent deposition of phosphate hydroxyapatite.

The Influence of New Silicate Cement Mineral Trioxide Aggregate (MTA Repair HP) on Metalloproteinase MMP-2 and MMP-9 Expression in Cultured THP-1 Macrophages.

The aim of the present study was to investigate the new silicate cement mineral trioxide aggregate (MTA Repair HP) with respect to its effect on the inflammation process involving the tooth and periodontal tissues. The composition of MTA Repair HP was supplemented with plasticizer agents which can have a negative effect on the modulation of tooth inflammation. The silicate-based material in question is widely used in regeneration of the pulp-dentin complex, treatment of perforations of various locations in the tooth, as well as in surgical treatment of the complications of periapical tissue. The improved bioceramic restorative cement can affect the expression of metalloproteinases MMP-2 and MMP-9 in monocytes/macrophages involved in modulation of inflammation and regenerative processes of the tooth and periodontal tissues. The novel aspect of the present study lies in the application of the model of THP-1 monocyte/macrophage and applying the biomaterial in direct contact with the cells. Hence, it provides a representation of clinical conditions with respect to regenerative pulp and periodontal treatment with the use of MTA Repair HP. A lack of macrophage activation (as measured with flow cytometry) was found. Moreover, the study identified a lack of expression stimulation of the studied metalloproteinases (with the use of Western blotting and fluorescent microscopy). Similarly, no increase in MMP-2 and MMP-9 concentration was found (measured by ELISA method) in vitro when incubated with MTA Repair HP. Based on the results it can be concluded that new MTA Repair HP does not increase the inflammatory response in monocytes/macrophages associated with the activity of the described enzymes. It can also be speculated that they do not affect the process of dentin regeneration in which MMP-2 and MMP-9 play significant roles.

Engineering H2O2 Self-Supplying Nanotheranostic Platform for Targeted and Imaging-Guided Chemodynamic Therapy.

Developing highly efficient chemodynamic therapy (CDT)-based theranostic technology for cancer treatment is highly desired but still challenging. A novel nanotheranostic platform is constructed for enhanced CDT by engineering hybrid CaO2 and Fe3O4 nanoparticles with a hyaluronate acid (HA) stabilizer and NIR fluorophore label. This design not only enables the nanotheranostic agent to afford highly efficient CDT against tumor cells but also confers NIR fluorescence (NIRF) and magnetic resonance (MR) bimodal imaging for in vivo visualization of CDT. Moreover, the use of the HA stabilizer allows for the facile synthesis of the nanotheranostic agent with excellent biocompatibility and active targetability. The nanotheranaostic agent possesses a high capacity of self-supplying H2O2 and producing •OH in acidic conditions, while retaining the desired stability under physiological conditions. It also demonstrates high selectivity to tumor cells via CDT with minimized toxicity to normal cells. In vivo studies reveal that our nanotheranaostic agent exhibits efficacious tumor growth inhibition via a CDT mechanism with favorable biosafety. Moreover, in vivo visualization of the CDT progress via NIRF and MR bimodal imaging demonstrates specific targeting and treatment of tumors. The developed H2O2 self-supplying, active targeting, and bimodal imaging nanotheranostic platform holds the potential as a highly efficient strategy for CDT of cancer.

Identification of a Calcium-sensing Receptor in Human Dental Pulp Cells That Regulates Mineral Trioxide Aggregate-induced Mineralization.

INTRODUCTION: The purpose of this study was to verify the expression of the calcium-sensing receptor (CaSR) and its role in mineral trioxide aggregate (MTA)-induced odontoblastic differentiation and mineralization in human dental pulp cells (hDPCs). METHODS: The expression of CaSR in human dental pulp tissue and hDPCs was detected using immunohistochemical and immunofluorescent assays. Then, hDPCs were cultured in specific medium supplemented with defined concentrations of MTA dilute alone or in combination with calcimimetic R-568 (a positive allosteric modulator of CaSR [Tocris Bioscience, Bristol, UK]), and cell viability was monitored by Cell Counting Kit-8 (Dojindo Molecular Technologies, Kumamoto, Japan) analysis. Alkaline phosphatase activity, alizarin red S staining, quantitative real-time polymerase chain reaction, and Western blot were used to investigate the gene/protein expression of odontoblastic-associated markers and CaSR in medium supplemented with different combinations of diluted MTA, R-568, and calcilytic Calhex 231 (a negative allosteric modulator of CaSR [Sigma-Aldrich, St Louis, MO]). RESULTS: CaSR was slightly expressed in the central pulp tissue, whereas it was strongly expressed in the odontoblast layer, plasma membrane, and cytoplasm of hDPCs. Cell Counting Kit-8 assay indicated maximum cell viability in cultures treated with 1:8 diluted MTA additives. Compared with undifferentiated controls, the cells at the early stage of odontoblastic differentiation exhibited lower CaSR protein expression. The combination of 1:8 diluted MTA with 0.1 and 1.0 µmol/L R-568 led to significantly increased cell vitality but decreased alkaline phosphatase activity and mineralized deposit formation, and this negative effect could be attenuated by 1.0 µmol/L Calhex 231 supplementation. Quantitative polymerase chain reaction results showed a significant up-regulation of RUNX2, DSPP, DMP-1, and OCN gene expression in the 1 µmol/L R-568-treated hDPCs. Western blot analysis indicated that the treatment by MTA and R-568 alone or their combination gave no clear trend on the protein levels of CaSR and dentin sialophosphoprotein, whereas Calhex 231 can increase their expressions. In addition, the up-regulation of Akt phosphorylation was observed in R-568- and Calhex 231-treated hDPCs. CONCLUSIONS: Our data indicated that CaSR is expressed in human dental pulp and hDPCs and that it can negatively or positively regulate MTA-induced mineralization of hDPCs via the phosphoinositide 3-kinase/Akt pathway in a ligand-dependent manner, suggesting a therapeutic target for modulating reparative dentin formation.

Calcium acetate-induced reduction of cadmium accumulation in Oryza sativa: Expression of auto-inhibited calcium-ATPase and cadmium transporters.

Calcium (Ca) signalling has an essential role in regulating plant responses to various abiotic stresses. This study applied Ca in various forms (Ca acetate and CaCl2 ) and concentrations to reduce cadmium (Cd) concentration in rice and propose a possible mechanism through which Ca acts to control the Cd concentration in rice. The results showed that supplementation of Cd-contaminated soil with Ca acetate reduced the Cd concentration in rice after exposure for 7 days in both hydroponic and soil conditions. The possible involvement of the auto-inhibited Ca2+ -ATPase gene (ACA) might act to control the primary signal of the Cd stress response. The messages from ACA3 and ACA13 tended to up-regulate the low-affinity cation transporter (OsLCT1) and down-regulate Cd uptake and the Cd translocation transporter, including the genes, natural resistance-associated macrophage protein 5 (Nramp5) and Zn/Cd-transporting ATPase 2 (HMA2), which resulted in a reduction in the Cd concentration in rice. After cultivation for 120 days, the application of Ca acetate into Cd-contaminated soil inhibited Cd uptake of rice. Increasing the Ca acetate concentration in the soil lowered the Cd concentration in rice shoots and grains. Moreover, Ca acetate maintained rice productivity and quality whereas both aspects decreased under Cd stress.

The tea leaf microbiome shows specific responses to chemical pesticides and biocontrol applications.

The plant microbiome is known to be influenced by certain biotic as well as abiotic factors. Nevertheless, the drivers for specific changes in microbial community composition and structure are largely unknown. In the present study, the effects of chemical and biological treatments for plant protection on the indigenous microbiome of Camellia sinensis (L.) Kuntze were contrasted. Assessment of bacteria-specific ribosomal RNA gene fragment amplicons from a representative set of samples showed an increased microbial diversity in treated plants when compared to untreated samples. Moreover, distinct microbial fingerprints were found for plants subjected to a conventional pesticide treatment with lime sulfur as well as for plants that were biologically treated with a Piriformospora indica spore solution. The bacterial community of pesticide-treated plants was augmented by 11 taxa assigned to Proteobacteria and Actinobacteria. In contrast, plants from biological control treatments were augmented by 10 taxa representing a more diversified community enrichment and included members of Actionobacteria, Proteobacteria, Bacteroidetes, Planctomycetes, and Verrucomicrobia. Complementary, molecular quantification of fungi in the samples showed a significantly lower number of internal transcribed spacer copies in plants subjected to biological control treatments, indicating the highest efficiency against fungal pathogens. The overall results show that leaves that are used for tea production show distinct microbiome shifts that are elicited by common pest and pathogen management practices. These shifts in the microbial population indicate non-target effects of the applied treatments.

Effects of soil amendments on the growth response and phytoextraction capability of a willow variety (S. viminalis × S. schwerinii × S. dasyclados) grown in contaminated soils.

This study was conducted to evaluate the effects of lime and bisphosphonates (BPs) such as N10O chelate amendment on the growth, physiological and biochemical parameters, and phytoextraction potential of the willow variety Klara (Salix viminalis × S. schwerinii × S. dasyclados) grown in soils heavily contaminated with copper (Cu), nickel (Ni) and zinc (Zn). The plants were irrigated with tap or processed water (mine wastewater). The results suggest that the combined effects of the contaminated soil and processed water inhibited growth parameters, gas exchange parameters and chlorophyll fluorescence (Fv/Fm) values. In contrast, malondialdehyde (MDA) content, organic acids, total phenolic and total flavonoid contents, and the accumulation of metals/metalloids in the plant tissues were increased compared to the control. When the soil was supplemented with lime and N10O; growth, physiological, biochemical parameters, and resistance capacity were significantly higher compared to unamended soil treatments, especially in the contaminated soil treatments. The combined lime‒ and N10O‒amended soil treatment produced higher growth rates, resistance capacity, photosynthesis rates and phytoextraction efficiency levels relative to either the lime‒amended or the N10O‒amended soil treatments. This study provides practical evidence of the efficient chelate‒assisted phytoextraction capability of Klara and highlights its potential as a viable and inexpensive novel approach for in situ remediation of Cu‒, Ni‒ and Zn‒contaminated soils and mine wastewaters.

Antimicrobial effect of bioceramic cements on multispecies microcosm biofilm: a confocal laser microscopy study.

OBJECTIVES: To assess the viability of multispecies microcosm biofilm after contact with NeoMTA Plus, Biodentine, and MTA Angelus. MATERIALS AND METHODS: Fifty-four human dentin blocks (4 × 5 × 4 mm) were allocated to Hawley retainers, worn by six volunteers for 72 h. The blocks were then individually incubated in BHI broth for 21 days at 37 °C. At the end of experimental time for biofilm growth, the samples were randomly divided into four groups (n = 12): NeoMTA Plus, Biodentine, MTA Angelus, and negative control. The materials were placed in contact with the blocks. All samples were placed in cell-culture plate wells and incubated in BHI broth for 7 days at 37 °C. One sample from each volunteer (n = 6) was analyzed by SEM to describe the biofilm morphology. CLSM was performed to determine the percentage of viable biofilm biovolume. The data were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test (α = 5%). RESULTS: SEM showed biofilm formed by spherical and rod-shaped bacteria surrounded by an extracellular matrix. No material was able to kill all biofilm cells, and all groups had more than 50% of viable bacteria. NeoMTA Plus was significantly different from the negative control group (P < .05). CONCLUSIONS: All tested materials were not effective against multispecies microcosm biofilm. CLINICAL RELEVANCE: NeoMTA Plus, Biodentine, and MTA Angelus were not effective against multispecies microcosm biofilm. It is essential to understand that these bioceramic cements are indicated for infected clinical situations. Thus, complementary disinfection procedures should be conducted prior to filling with these materials.

Antibacterial Efficacy of Synthetic and Natural-Derived Novel Endodontic Irrigant Solutions.

The aim of this study was to compare the efficacy of grape seed extract (GSE), calcium hypochlorite [Ca(ClO)2], and sodium hypochlorite (NaOCl) irrigant solutions with rotary or reciprocating instrumentation for disinfection of root canals inoculated with Enterococcus faecalis. The mesiobuccal root canals of mandibular molars were prepared and inoculated with Enterococcus faecalis for 21 days. The roots were then randomly divided into the following eight experimental groups (n=11) according to the instrumentation technique and disinfection protocol: ProTaper Next or Reciproc R25 with sodium chloride (control group), 6% NaOCl, 6% Ca(ClO)2, or 50% GSE used for irrigation during instrumentation. The antimicrobial activity was determined on the basis of a reduction in colony-forming units (CFUs) counted on bacterial samples collected before and after root canal instrumentation and expressed as a percentage of reduction. Data were evaluated by two-way ANOVA followed by Tukey HSD post-hoc tests (p<0.05). No significant differences were observed in bacterial reduction between the ProTaper Next and Reciproc R25 systems (p>0.05), regardless of the irrigant solution used. Furthermore, all active solutions (6% NaOCl, 50% GSE, and 6% Ca(ClO)2) showed similar potential to reduce bacterial counts (p>0.05) and were significantly more effective than sodium chloride (control) (p<0.05). The results suggest that the GSE and Ca(ClO)2 have potential clinical application as irrigant solutions in endodontic therapy since they present bactericidal efficacy against Enterococcus faecalis.