Beneficial effects of bioactive peptides extracted from Spirulina platensis and Gracilaria gracilis algae on bone regeneration/osteogenic differentiation of mesenchymal stem cells.

Mesenchymal stem cells are used in the treatment of many diseases, particularly in the repair of bone injuries. Algae with various medicinal applications are considered important natural resources. There is limited research on the effects of bioactive peptides from algae extraction on mesenchymal stem cells. In this study the impact of bioactive proteins, protein lysates and peptide fractions (<3, <30 and <50 kDa) isolated from two algae species, Spirulina platensis and Gracilaria gracilis on the proliferation and osteogenic differentiation of human amniotic mesenchymal stem cells (hAMSCs) was investigated. The proteins were extracted ant hydrolyzed with trypsin enzyme to create peptides, which were then separated by ultrafiltration. hAMSCs were exposed to different concentrations of bioactive compounds (100, 300, 500 and 700 µg/ml) for varying time periods. Cell proliferation was assessed using the with 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and to evaluate differentiation into bone tissue, the amount of mineral deposition was measured with alizarin red staining, and alkaline phosphatase enzyme activity was determined using a colorimetric method. The expression of Runx2, Osteocalcin, and ß-Catenin genes expression was analyzed using RT-qPCR on days 7, 14 and 21 post-treatment. The results indicated that the <3 kDa peptide fraction of S. platensis and G. gracilis had no cytotoxic effects, increased cell proliferation at a concentration of 300 µg/ml, and enhanced the expression of osteogenic marker genes, alkaline phosphatase enzyme a activity, and calcium deposition in the extracellular matrix. In general, fractions that show positive effects on hAMSC differentiation have the potential to treat bone defects and promote osteoregeneration.

Osteogenic differentiation of human amniotic mesenchymal stem cells by phycocyanin and phycoerythrin pigments isolated from Spirulina platensis and Gracilaria gracilis algae.

Bone regeneration is a multistep and regular physiological process that occurs normally in fracture repair and bone defects. However, some factors such as aging, particular diseases and some drugs prevent or slowdown bone natural healing. Cell therapy using stem cells and differentiation activating factors is an effective treatment method for bone regeneration triggering in unusual conditions. Therefore, in the present study the effect of phycocyanin and phycoerythrin pigments which isolated from Spirulina platensis and Gracilaria gracilis algae was investigate on osteogenic differentiation potency of human Amniotic Mesenchymal Stem Cells (hAMSCs). For this purpose, hAMSCs were exposed to 300, 500, and 700 µg/ml concentrations of phycocyanin and phycoerythrin pigments and then the cells viability was measured with MTT assay in 48 and 72 h after treatment. The osteo-differentiation level of cells was studied by measuring ALP activity using calorimetric method and Alizarin red staining for calcium deposition in 7 and 21 days after treatment. Also, total RNA of cells was extracted in different time periods and then cDNA synthesized with specific primers, and relative expression of Runx2, ß-catenin and Osteocalcin genes were investigated using SYBR Green RT-qPCR technique. Osteogenic differentiation of hAMSCs that treated with pigments was confirmed by mineral deposits staining and increased level of ALP activity. Furthermore, these pigments elevated significantly the expression of osteogenic marker genes compared to control samples and caused hAMSCs to differentiate into osteoblast cells. According to these results, phycocyanin and phycoerythrin may suggest as suitable osteogenic supplements with low toxicity, low cost and high efficiency, although the molecular mechanism of its efficacy is not available yet.

Development and evaluation of bioactive 3D zein and zein/nano-hydroxyapatite scaffolds for bone tissue engineering application.

The aim of this study is to generate and investigate biodegradable and biocompatible zein and zein/nano-hydroxyapatite composite scaffolds for bone defect healing. 3D zein scaffold was successfully fabricated using the salt-leaching method and incorporated with 12.5 wt% nHA for osteogenic differentiation of murine myoblast cell line (C2C12 cells). The scaffolds were subjected to physicochemical and biomechanical characterizations using the scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), biodegradation, porosity, mechanical tests. C2C12 cells were cultured on scaffolds and incubated for 21 days. Cell proliferation was detected by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Quantitative real-time PCR was used to test the expression of osteoblastic-related genes including Runx2, ALP, and Col1A1. The scaffolds had an adequate mean pore size and a total porosity of 61.1%-70.6%. The addition of 12.5 wt% nHA to the zein scaffold increased the compressive modulus to 79.1 MPa and the ultimate strength to 2.7 MPa. The qRT-PCR analysis confirmed that mRNA transcript levels were significantly higher (p < 0.05) on the zein/nHA than on the pure zein scaffold. The results suggested that the developed scaffolds could be a potential candidate for bone tissue engineering due to their promising osteoinductivity, surface topography, mechanical behavior, biodegradability.

Effects of heat shock protein inducer on Hsp70 gene expression and immune parameters during Streptococcus iniae infection in a Persian sturgeon fry.

Heat shock proteins (HSPs) as stress-related factors play a fundamental role in innate and adaptive immune responses in fish, which can be considered as strong candidates for the development of new methods for fish disease prevention. It has been proven that Pro-Tex® as a heat shock protein inducer (HSPi) reduces harmful effects of cellular stress by increasing the Hsp70 protein production. We evaluated the effects of Pro-Tex® as an HSPi in a Persian sturgeon, (Acipenser persicus) exposed to a pathogenic bacterium. Therefore, A. persicus fries were pre-treated with 25.00, 50.00 and 100 mg L-1 of Pro-Tex® and then, injected with Streptococcus iniae ATCC29178. The Hsp70 gene expressions were determined in various organs including liver, gill and intestine and lysozyme (LYZ) activities along with supplemental levels of complement component 3 (C3) and immunoglobulin M (IgM) were also determined in sturgeon blood in days 3 and 7 after infection. Expression of Hsp70 gene was increased during the first three days of infection and then, it was found to be down-regulated during the infection process. Also, levels of LYZ activity, C3 and IgM increased in a concentration-dependent manner; but these parameters decreased after 7 days. Our data suggest that induction of Hsp70 is a promising approach in modulation of immune response in A. persicus and it might be employed to confer protection in fish against bacterial infections.

Surface modification of super paramagnetic iron oxide nanoparticles via milk casein for potential use in biomedical areas.

Super paramagnetic iron oxide nanoparticles (SPIONs) have proved that they have tremendous potential to use in various biomedical applications. But the surface of pure iron oxide nanoparticles so fast oxidized, that is a major drawback for biomedical applications. Covered SPIONs have good surface activity. Therefore, the first goal was to synthesize the naked SPIONs. Then we modified with 3-Aminopropyltriethoxysilane (APTES) and trichlorotriazine (TCT). Several techniques measurements were used for characterization the size and special features of naked SPIONs and TCT modified SPIONs. These results show that the SPIONs were synthesized. After that the SPIONs are coated with casein and indicate that there is an interaction between them. Moreover, we have investigated magnetic properties and anticancer effects of casein-coated SPIONs. Therefore, we showed casein could be used to increase the biocompatibility of the surface of SPIONs. At the end, we show that bonding of dipyridamole (DIP) to the surface of casein-coated SPIONs have good magnetite properties for targeted drug delivery. We find that the release of DIP by casein-coated SPIONs-DIP was sensitive to pH. Both release curves in pH 5.5 and 7.4 showed the release of DIP by ß-casein coated SPIONs-DIP better than α-casein coated SPIONs-DIP. The cell culture studies of the casein-coated SPIONs-DIP provide good anticancer effects against both breast and prostate cancer cell lines. Here, we propose a simple and inexpensive chemical method for preparation of highly biocompatible core-shell SPIONs and binding of drug for using in targeted drug delivery system. Communicated by Ramaswamy H. Sarma.

Association of PARP1 rs4653734, rs907187 and rs1136410 variants with breast cancer risk among Iranian women.

BACKGROUND: Breast cancer (BC) is the highest cause of mortality among female cancer patients. In some cases, BC is due to Poly [ADP-ribose] polymerase 1 (PARP1) gene dysregulation, which has been involved in various important cellular processes. Among Iranian women, the association between PARP1 polymorphisms and BC was never studied before so in this case-control study, the genetic association of three SNPs (rs1136410, rs907187 and rs4653734) was analyzed with susceptibility to BC. METHODS: The study subjects were 386 Iranian females divided into 186 patients and 200 healthy controls. The genotypes of PARP1 variants were detected using ARMS and a combined ARMS-RFLP PCR method. RESULTS: The results showed that Carriers of CG and GG genotypes of the variant rs4653734 were at higher risk of BC compared with wild-type carriers (CC) and this variant was statistically significant under a recessive model of inheritance. Moreover, rs907187 was related to increased BC risk in the CC and GG genotypes under dominant and recessive models of inheritance. The G allele frequency of rs4653734 and rs907187 was higher in breast cancer patients than in normal subjects. No association was detected between rs1136410 and susceptibility to BC among studied groups. Furthermore, A-G-C haplotype was linked to an increased BC risk, whereas A-C-C and A-C-G haplotypes were related to a decreased risk of BC. In Silico predictions suggested that rs907187 affects E2F and E2F-4 transcription factors binding site. CONCLUSIONS: The current study suggests that rs907187 and rs4653734 have remarkable associations with BC risk among Iranian women.

Zein nanoparticle as a novel BMP6 derived peptide carrier for enhanced osteogenic differentiation of C2C12 cells.

Zein nanoparticles as a carrier system for BMP6-derived peptide were prepared by liquid-liquid phase separation procedure and characterized with SEM, DLS, FTIR and thermogravimetric methods. After peptide encapsulation, nanoparticle size increased from 236.3 ± 92.2 nm to 379.4 ± 116.8 nm. The encapsulation efficiency of peptide was 72.6% and the release of peptide from Zein nanoparticles was partly sustained in trypsin containing phosphate buffered saline (pH 7.4) for up to 14 days. Peptide-loaded nanoparticles showed similar cell viability compared with blank ones. ALP activity of C2C12 cells treated with peptide-loaded nanoparticles (500 µg/mL) was evaluated 7, 14, 21 and 28 days after culture. In peptide-loaded nanoparticles, ALP activity was significantly higher (p < .05) compared with other groups at day 14. Alizarin Red S staining showed, C2C12 cells behind peptide-loaded nanoparticles had significantly (p < .05) higher calcium deposition at day 21. The results of RT-qPCR show that the BMP-6 peptide activated expression of RUNX2 as a transcription factor. In turn, RUNX2 regulates SPP1 and BGLAP gene expression, as osteogenic marker genes. The results confirm that the peptide-loaded Zein nanoparticles, as osteoinductive material, may be used to repair small area of bone defects, with low load bearing.

Analysis of the codon 72 polymorphism of TP53 and human papillomavirus infection in Iranian patients with prostate cancer.

The TP53 gene is one of the most important tumor suppressor genes controlling DNA transcription and cell regulation. Common polymorphisms in p53 gene may play a role in some cancers. Some studies have reported an association between human papillomavirus (HPV) infections and prostate cancer. The aim of this study was to investigate whether the TP53 codon 72 polymorphism and HPV infection are responsible for susceptibility to prostate cancer in Iranian men. The prostate biopsies were taken during surgery from 68 Iranian prostatic cancer patients, and 85 patients with benign prostate hyperplasia. For genotyping of the p53 polymorphism at codon 72, PCRRFLP methods were used and the PCR products were digested with BstU1. An attempt was also made to detect HPV DNA in benign prostate hyperplasia and prostate cancer specimens. Among cancer cases, the distribution of Arg/Arg, Arg/Pro and Pro/Pro genotypes were 26.5%, 45.4%, and 19.1%, respectively. Among patients with benign prostate hyperplasia, the distribution of Arg/Arg, Arg/Pro, and Pro/Pro genotypes were 27%, 53%, and 20%, respectively. The allele frequencies did not differ significantly between prostate cancer and benign prostate hyperplasia samples. Human papillomavirus was detected only in three patients (4.4%; P = 0.71). The results from this study suggest that the TP53 codon 72 polymorphism and HPV infection do not confer susceptibility to prostate cancer in the Iranian population. Larger population-based studies are needed to clarify the relation between prostate carcinoma and p53 polymorphism and HPV infection.

Increased acidic fibroblast growth factor concentrations in the serum and cerebrospinal fluid of patients with Alzheimer's disease.

Acidic fibroblast growth factor (aFGF), also called FGF-1, which influences the proliferation and differentiation of various cell types in vitro, was originally isolated from neural tissue. It is released from the ependymal cells of the cerebral third ventricle into the cerebrospinal fluid (CSF). FGF-1 promotes the survival of neurons. Reactive astrocytes express FGF-1 in the brain of patients with Alzheimer's disease (AD). By comparing the CSF proteome of patients with AD and normal controls it might be possible to identify proteins that have a role in AD. Because CSF is in contact with the extracellular space of the brain, modifications in the brain biochemistry could be reflected in the CSF. The aim of this study was to determine concentrations of serum and CSF FGF-1 in patients with AD. This study consisted of 64 CSF samples, from patients with AD (n=32) and those without (normal controls) (n=32). The level of CSF and serum FGF-1 in patients with AD was higher than in patients without AD. We conclude that FGF-1 is a constant component of human serum and CSF and that FGF-1 may be involved in the pathophysiology of AD.