Exploring the various effects of Cu doping in hydroxyapatite nanoparticle.

Adding foreign ions to hydroxyapatite (HAp) is a popular approach for improving its properties. This study focuses on the effects of calcium substitution with copper in HAp. Instead of calcium, copper ions were doped into the structure of hydroxyapatite nanoparticles at 1%, 3%, and 5% concentrations. XRD analysis showed that the amount of substituted copper was less than needed to generate a distinct phase, yet its lattice parameters and crystallinity slightly decreased. Further, the results of degradation tests revealed that copper doping in hydroxyapatite doubled calcium ion release in water. The incorporation of copper into the apatite structure also boosted the HAp zeta potential and FBS protein adsorption onto powders. According to antibacterial investigations, a concentration of 200 mg/ml of hydroxyapatite containing 5% copper was sufficient to effectively eradicate E. coli and S. aureus bacteria. Furthermore, copper improved hydroxyapatite biocompatibility. Alkaline phosphatase activity and alizarin red tests showed that copper in hydroxyapatite did not inhibit stem cell differentiation into osteoblasts. Also, the scratch test demonstrated that copper-containing hydroxyapatite extract increased HUVEC cell migration. Overall, our findings demonstrated the utility of incorporating copper into the structure of hydroxyapatite from several perspectives, including the induction of antibacterial characteristics, biocompatibility, and angiogenesis.

Gαq Signaling Activates ß-Catenin-Dependent Gene Transcription

Background: The canonical Wnt signal transduction or the Wnt/ß-catenin pathway plays a crucial role in both carcinogenesis and development of animals. Activation of the Gαq class of Gα proteins positively regulates Wnt/ß-catenin pathway, and expression of Gαq in human embryonic kidney 293 (HEK293T) cells or Xenopus oocytes leads to the inhibition of glycogen synthase kinase-3 beta and cellular accumulation of ß-catenin. This study investigated whether Gαq-mediated cellular accumulation of ß-catenin could affect the transcriptional activity of this protein. Methods: HEK-293T and HT-29 cells were used for cell culture and transfection. Protein localization and quantification were assessed by using immunofluorescence microscopy, cell fractionation assay, and Western blotting analysis. Gene expression at the transcription level was examined by quantitative reverse transcriptase/real-time PCR method. Results: Transcription of two cellular ß-catenin target genes (c-MYC and CCND1) and the ß-catenin/ T-cell factor reporter luciferase gene (TopFlash plasmid) significantly increased by Gαq activation. The Gαq-mediated increase in the expression level of the ß-catenin-target genes was sensitive to the expression of a minigene encoding a specific Gαq blocking peptide. The results of cell fractionation and Western blotting experiments showed that activation of Gαq signaling increased the intracellular ß-catenin protein level, but it blocked its membrane localization. Conclusion: Our results reveal that the Gαq-dependent cellular accumulation of ß-catenin can enhance ß-catenin transcriptional activity.

Synergy effects of copper and L-arginine on osteogenic, angiogenic, and antibacterial activities.

Copper (Cu) ions have been found to exert antibacterial and angiogenic effects. However, some studies have indicated that it inhibits osteogenesis at high concentrations. On the other hand, L-arginine (Arg) is a semi-essential amino acid required for various biological processes, including osteogenic and angiogenic activities. As a result, we hypothesized that combining Arg with Cu ions would reduce its inhibitory effects on osteogenesis while increasing its angiogenic and antibacterial capabilities. To assess osteogenic and angiogenic activities, we employed rat bone marrow mesenchymal stem cells (MSCs) and human umbilical vein endothelial cells (HUVECs), respectively. The gram-positive bacteria Staphylococcus epidermidis (S. epidermidis), Staphylococcus aureus (S. aureus), and the gram-negative bacterium Escherichia coli (E. coli) were used to investigate bacterial behaviors. According to ALP activity and calcium deposition outcomes, copper ions inhibited osteogenic development of MSCs at 100 µM; however, Arg supplementation somewhat mitigated the inhibitory effects. Furthermore, Copper and Arg synergistically stimulated migration and tube formation of HUVECs. According to our findings, copper ions and Arg in the range of 1-100 µM had no antibacterial effect on any examined bacteria. However, at a dose of 20 mM, copper demonstrated antibacterial activity, which was boosted by Arg. Overall, these findings suggest that a combination of copper and Arg may be more beneficial for bone regeneration than either copper or Arg alone.

Heat shock factor 5 is essential for spermatogenesis in mice: Detected by a new monoclonal antibody.

OBJECTIVES: Here, we examined the function of our produced monoclonal antibody (mAb10C3) to recognize one of the most important members of the HEAT shock factor family, Hsf5, in embryonic development and in spermatogenic cells of adult mouse testis. MATERIALS AND METHODS: The targeting effects of mAb10C3 were investigated by immunohistochemistry analysis in the different phases of the embryo and in the adult testis tissue sections. RESULTS: The results of immunohistochemistry staining on the mouse embryos by the supernatant of hybridoma clone that produced mAb10C3, in the early and late phases (E7.5 and E14.5) of embryonic development, indicated that mAb10C3 could only detect Hsf5 in E7.5 and it did not have any targeting activity in the late phase of development. Therefore, we showed that the hsf5 gene has expressed in early mouse embryonic development. On the other hand, mAb10C3 could detect Hsf5 in spermatogonia and spermatocytes of adult testis in comparison with a known anti-Hsf5 antibody (ab98939) and an anti-PCNA antibody as a marker of spermatogonia cells. CONCLUSION: Taken together, these data indicated that generated anti-testis mAb10C3 was generated against anti-testis proteins, specifically to target Hsf5, and can be useful as a scientific tool to investigate the critical genes in the development and spermatogenesis.

Optimized protocol for soluble prokaryotic expression, purification and refolding of the human inhibin α subunit, a cysteine rich peptide chain.

BACKGROUND: Inhibin A, a member of TGF-ß superfamily, consists of α and ß subunits. These subunits contain several cysteine residues in amino acid sequence that forms inter- and intra-subunits disulfide bonds. Due to the reducing environment of the bacterial cytoplasm, disulfide bonds formation in E.coli cytoplasm is not possible. Therefore, this can cause misfolding, aggregation and inclusion bodies formation during protein expression. As a result, the expression of inhibin subunits in E.coli produces inclusion bodiesOBJECTIVE: We aimed at identification of an optimized protocol for expression and recovery of inhibin α-subunit from inclusion bodies. METHODS: Two vectors, four different E.coli strains, and six solubilization conditions for were used for the optimization of inhibin α-subunit production. Then, the solubilized proteins were purified through Ni-NTA affinity chromatography, characterized by SDS-PAGE and Western blotting (WB) using anti-his tag antibody, and refolded by dilution. RESULTS: The results showed that inhibin α-subunits were successfully expressed in both vectors and the pET22b+inhibin α-subunit in ShuffleTM T7 strain had the highest expression; however, most of the expression was in an insoluble form. Among solubilization buffers examined, a buffer containing 2M urea with pH 12 was the best buffer to dissolve the insoluble protein. The high purity of protein was confirmed by SDS-PAGE and WB. Non-reducing SDS-PAGE demonstrating inhibin α-subunit refolded well. CONCLUSION: The current protocol is an efficient method for protocol for expression and recovery of inhibin α-subunit from inclusion bodies.

Selenium-L-methionine modulates radiation injury and Duox1 and Duox2 upregulation in rat's heart tissues.

Introduction: Redox interactions play a key role in radiation injury including heart diseases. In present study, we aimed to detect the possible protective role of selenium-L-methionine on infiltration of immune cells and Duox1&2 upregulation in rat's heart tissues. Methods: In this study, 20 rats were divided into 4 groups (5 rats in each) namely: irradiation; irradiation plus Selenium-L-methionine; control; and Selenium-L-methionine treatment. Irradiation (15 Gy to chest) was performed using a cobalt-60 gamma ray source while 4 mg/kg of selenium-L-methionine was administered intraperitoneally. Ten weeks after irradiation, rats were sacrificed for detection of IL-4 and IL-13 cytokines, infiltration of macrophages and lymphocytes as well as the expressions of IL4Ra1, Duox1, IL13Ra2 and Duox2. Results: Results showed an increase in the level of IL-4 as well as the expressions of IL4Ra1, Duox1 and Duox2. Similarly, there was an increase in the infiltration of lymphocytes and macrophages. There was significant attenuation of all these changes following treatment with selenium-L-methionine. Conclusion: Selenium-L-methionine has the potential to protect heart tissues against radiation injury. Downregulation of pro-oxidant genes and modulation of some cytokines such as IL-4 are involved in the radioprotective effect of selenium-L-methionine on heart tissues.

Evaluating the Radioprotective Effect of Curcumin on Rat's Heart Tissues.

BACKGROUND: Heart injury is one of the most important concerns after exposure to a high dose of radiation in chest cancer radiotherapy or whole body exposure to a radiation disaster. Studies have proposed that increased level of inflammatory and pro-fibrotic cytokines following radiotherapy or radiation events play a key role in the development of several side effects such as cardiovascular disorders. In the current study, we aimed to evaluate the expression of IL-4 and IL-13 cytokines as well as signaling pathways such as IL4Ra1, IL13Ra2, Duox1 and Duox2. In addition, we detected the possible protective effect of curcumin on the expression of these factors and infiltration of inflammatory cells. MATERIALS AND METHODS: Twenty rats were divided into 4 groups including control; curcumin treated; radiation; and radiation plus curcumin. After 10 weeks, rats were sacrificed for evaluation of mentioned parameters. RESULTS: Results showed an increase in the level of IL-4 and all evaluated genes, as well as increased infiltration of lymphocytes and macrophages. Treatment with curcumin could attenuate these changes. CONCLUSION: Curcumin could reduce radiation-induced heart injury markers in rats.

Protective Effect of Selenium-L-methionine on Radiation-induced Acute Pneumonitis and Lung Fibrosis in Rat.

BACKGROUND: In this study, we aimed to detect the changes in the level of interleukin (IL)-4 and IL-13 cytokines and their downstream genes including interleukin-13 receptor subunit alpha-2 (IL13Ra2), interleukin-4 receptor subunit alpha-1 (IL4Ra1), dual oxidase 1 (DUOX1) and dual oxidase 2 (DUOX2). The protective effects of Selenium-L-methionine on radiation-induced histopathological damages and changes in the level of these cytokines and genes were detected. METHODS: Four groups of 20 rats (5 rats in each) namely, control; Selenium-L-methionine, radiation and radiation plus Selenium-L-methionine were used in this study. 4 mg/kg of Selenium-Lmethionine was administered 1 day before irradiation and five consecutive days after irradiation. Irradiation was done using a dose of 15 Gy 60Co gamma rays at 109 cGy/min. All rats were sacrificed 10 weeks after irradiation for detecting changes in IL-4 and IL-13 cytokines, the expressions of IL13Ra2, IL4Ra1, Duox1 and Duox2 and histopathological changes. RESULTS: The level of IL-4 but not IL-13 increased after irradiation. This was associated with increased expression of IL4Ra1, Duox1 and Duox2, in addition to changes in morphological properties. Selenium-L-methionine could attenuate all injury markers following lung irradiation. CONCLUSION: Selenium-L-methionine can protect lung tissues against toxic effects of ionizing radiation. It is possible that the modulation of immune responses and redox interactions are involved in the radioprotective effect of this agent.

The cardiac niche role in cardiomyocyte differentiation of rat bone marrow-derived stromal cells: comparison between static and microfluidic cell culture methods.

Due to the restricted potential of the heart to regenerate its damaged region, stem cell therapy is a promising treatment modality for myocardial infarction. It has been shown that incubation of bone marrow-derived stromal cells (BMSCs) with initial steps of cardiac differentiation in vitro, can have a significant effect on their therapeutic potential to treat myocardial infarction. Based on these well-established principals we were encouraged to study the direct co-culture of rat BMSCs with neonatal mouse almost pure cardiomyocytes (APCs) and cardiac niche cells (CNCs) in static 2D and microfluidic cell culture systems. Our results showed that the difference regarding the beating rate in isolated APCs and CNCs in both 2D and the microfluidic system was not statistically significant for 30 days. No beat rate could be observed in induced BMSCs in all groups during experiment time. Except for BMSCs cultured alone in both experimental culture conditions, data obtained from Real-time PCR analysis showed that differentiated BMSCs in all co-cultured groups expressed GATA4, Nkx2.5, CX43, cTnI, cTnT, and ß-MHC during 4 weeks. BMSCs demonstrated a higher expression of these cardiac factors in microfluidic chips than those co-cultured in 24 well plates. Moreover, immunocytochemistry (ICC), also revealed the GATA4 expression in differentiated BMSCs in all co-cultured groups. It was found that, when combined with shear stress, co-culture with cardiomyocyte can differentiate BMSCs significantly toward cardiomyocyte rather than co-culture alone.

Comparative evaluation of probiotics effects on plasma glucose, lipid, and insulin levels in streptozotocin-induced diabetic rats.

BACKGROUND: Diabetes is a chronic disorder caused by the relative decrease in insulin production from the pancreas, tissue resistance to insulin, or both. Daily consumption of probiotics, particularly Lactobacilli, has been proposed as a new strategy for prevention or control of diabetes. METHODS: In the current study, the effects of various probiotics including Lactobacillus reuteri (L reuteri), Lactobacillus crispatus (L crispatus), and Bacillus subtilis on the levels of blood glucose, lipid, and insulin as well as haemoglobin A1c (HbA1c ) were investigated in rat models of streptozotocin (STZ)-induced diabetes and compared with metformin. In addition, GLUT-4 and PPAR-γ transcript levels have been analysed in adipose tissues. RESULTS: A significant decrease in plasma glucose and HbA1c levels and a dramatic increase in insulin levels have been detected after consumption of probiotics. Furthermore, lipid profiles have been improved. The expression of Glut-4 and Ppar-γ genes in adipose tissues also increased after treatment period. CONCLUSION: The results of this experiment showed that daily consumption of probiotics can be effective in control of STZ-induced diabetes and its complications.