Osteogenic differentiation of mesenchymal stem cells cultured on PLLA scaffold coated with Wharton's jelly

Poly-L-lactic acid (PLLA) electrospun nanofiber scaffold is one of the most commonly used synthetic polymer scaffolds for bone tissue engineering application. However, PLLA is hydrophobic in nature, hence does not maintain proper cell adhesion and tissue formation, moreover, it cannot provide the osteo-inductive environment due to inappropriate surface characteristic and the lack of surface motives participating in the first cellular events. To modify these shortcomings different approaches have been used, among those the most commonly used one is coating of the surface of the electrospun nanofiber with natural materials. In this work Wharton’s jelly (WJ), a tissue which surrounds the umbilical cord vessels, reaches in high amounts of extracellular matrix (ECM) components mainly; collagen, hyaluronic acid and several sulphated glycosaminoglycans (GAGs) were used to cover the surface of electrospun PLLA nanofiber scaffolds. The surface morphology of the nanofiber scaffold was evaluated via scanning electron microscope, and the in vitro osteogenic differentiation potential was determined by MTT assay and common osteogenic marker tests such as alkaline phosphatase (ALP) activity and calcium deposition tests. Coating of WJ could not change the surface morphology and diameter of the nanofibers. However, WJPLLA scaffolds showed higher proliferation of human mesenchymal stem cells (MSC) than tissue culture plate (TCP) and pristine PLLA scaffolds, moreover, WJ-PPLA scaffold demonstrated significant alkaline phosphatase activity and calcium mineralization than either TCP or PLLA nanofiber scaffolds

Simultaneous Coating of Electrospun Nanofibers with Bioactive Molecules for Stem Cell Osteogenesis In Vitro

Objective: Mesenchymal stem cells (MSCs) are widely recognized as a promising cell type for therapeutic applicationsdue to their ability to secrete and regenerate bioactive molecules. For effective bone healing, it is crucial to select ascaffold that can support, induce, and restore biological function. Evaluating the scaffold should involve assessing MSCsurvival, proliferation, and differentiation. The principal aim of this investigation was to formulate composite nanofibrousscaffolds apt for applications in bone tissue engineering.Materials and Methods: In this experimental study, nanofibrous scaffolds were fabricated using Poly-L-lactic acid(PLLA) polymer. The PLLA fibers’ surface was modified by integrating collagen and hydroxyapatite (HA) nanoparticles.Results: The findings demonstrated that the collagen- and nanohydroxyapatite-modified electrospun PLLA scaffoldpositively influenced the attachment, growth, and osteogenic differentiation of MSCs.Conclusion: Coating the nanofiber scaffold with collagen and nanoparticle HA significantly enhanced the osteogenicdifferentiation of MSCs on electrospun PLLA scaffolds

Development of monoclonal antibodies against axenic amastigotes of Leishmania infantum strain in Iran: Implication for diagnosis of Kala-azar

Objective(s): Leishmaniasis is endemic in 88 countries. Amastigote forms of Leishmania are experts at exploiting host cell processes to establish infection. Monoclonal antibodies are key reagents used in the diagnosis of infectious and non-infectious diseases. The aim of this study was to produce monoclonal antibodies against axenic amastigotes of the Leishmania infantum strain in Iran.Materials and Methods: First, standard strains were cultured and axenic amastigote antigens of L. infantum were obtained. Since then, BALB/c mice were immunized and antibody titers were determined. For hybridoma cell formation, lymphocytes isolated from spleen of immunized mice and myeloma cells were fused at a ratio of 10 to 1 in the presence of polyethylene glycol, followed by limiting dilution for the isolation of monoclones. Subsequently, antibody isotypes were determined by using the isotyping kit. The best clone was injected intraperitoneally to pristane-primed mice for large scale production of monoclonal antibodies. The specificity of antibody was determined with Western blotting.Results: Approximately 25 positive monoclones were obtained, of which four hybrids producing anti-amastigotes L. infantum monoclonal antibodies with high optical density (OD), selected and designated as 8D2 FVI6, 8D2 FVI3, 6G2 FV4 and 6G2 FV3. Results from isotype determination showed the IgG2b sub-class in 6G2FV2 and 8D2FVI6 monoclones. Conclusion: This study produced monoclonal antibody against amastigotes of Iranian strain of L. infantum for the first time. These antibodies have reactivity against Iranian strain of L. infantum and can be used in the diagnosis of Kala-azar

Screening of common CYP1B1 mutations in Iranian POAG patients using a microarray-based PrASE protocol

Purpose: The gene coding cytochrome P4501B1 (CYP1B1) has been shown to be a major cause of primary congenital glaucoma in the Iranian population. More recently it was shown to also be important in juvenile-onset open angle glaucoma (JOAG). We aimed to further investigate the role of CYP1B1 in a larger cohort of primary open angle glaucoma (POAG) patients which included late-onset patients. We also aimed to set up a microarray based protocol for mutation screening with an intent of using the protocol in a future population level screening program. Methods: Sixty three POAG patients, nine affected family members, and thirty three previously genotyped primary congenital glaucoma (PCG) patients were included in the study. Clinical examination included slit lamp biomicroscopy, IOP measurement, gonioscopic evaluation, fundus examination, and measurement of perimetry. G61E, R368H, R390H, and R469W were screened by a protocol that included multiplexed allele specific amplification in the presence of a protease (PrASE), use of sequence tagged primers, and hybridization to generic arrays on microarray slides. The entire coding sequences of CYP1B1 and myocilin (MYOC) genes were sequenced in all individuals assessed by the microarray assay to carry a mutation. Intragenic single nucleotide polymorphism (SNP) haplotpes were determined for mutated alleles. Results: Genotypes assessed by the array-based PrASE methodology were in 100 concordance with sequencing results. Seven mutation carrying POAG patients (11.1) were identified, and their distribution was quite skewed between the juvenile-onset individuals (5/21) as compared to late-onset cases (2/42). Four of the seven mutation carrying Iranian patients harbored two mutated alleles. CYP1B1 mutated alleles in Iranian PCG and POAG patients shared common haplotypes. MYOC mutations were not observed in any of the patients. Conclusions: The PrASE approach allowed reliable simultaneous genotyping of many individuals. It can be an appropriate tool for screening common mutations in large sample sizes. The results suggest that CYP1B1 is implicated in POAG among Iranians, notably in the juvenile-onset form. Contrary to POAG patients studied in other populations, many mutation harboring Iranian patients carry two mutated alleles. We propose an explanation for this observation. © 2008 Molecular Vision

Original Article Expression levels of microRNA machinery components Drosha, Dicer and DGCR8 in human (AGS, HepG2, and KEYSE-30) cancer cell lines

Abstract: MicroRNAs (miRNAs) have recently been shown to play fundamental roles in diverse cellular processes and linked to variety of cancers. Dicer and Drosha are two major enzymes in the miRNA maturation process. DGCR8 is the assistant of Drosha in the microprocessor complex. In this study, we evaluated the mRNA expression profiles of major miRNA processing machinery Drosha, Dicer, and DGCR8 in human gastrointestinal (AGS, KYSE30 and HepG2) cancer cell lines. Materials and Methods: The cells were cultured and harvested, and total cellular RNA was isolated from cells. Then, first-strand cDNA was synthesized from the RNA of cells. Afterward, Quantitative analysis was performed by real-time RT-PCR using the PowerSYBR Green PCR Master Mix. Results: Expression levels of Drosha in AGS and HepG2 cells were higher than the controls, whereas, Drosha's expression level in KYSE-30 cell line was lower. The Dicer expression levels in AGS and HepG2 cells were higher, while, its expression level in KYSE-30 cell was lower. The DGCR8 expression levels in all three cell lines were significantly higher than the control samples. Conclusion: Expression levels of the two most important enzymes of the miRNA machinery, Drosha and Dicer, and microprocessor complex component, DGCR8 were noticeably dysregulated when compared to healthy controls

The cucurbitacins D, E, and I from Ecballium elaterium (L.) upregulate the LC3 gene and induce cell-cycle arrest in human gastric cancer cell line AGS

Objective(s): Cucurbitacins exhibit a range of anti-cancer functions. We investigated the effects of cucurbitacins D, E, and I purified from Ecballium elaterium (L.) A. Rich fruits on some apoptotic and autophagy genes in human gastric cancer cell line AGS. Materials and Methods: Using quantitative reverse transcription PCR (qRT-PCR), the expression of LC3, VEGF, BAX, caspase-3, and c-MYC genes were quantified in AGS cells 24 hr after treatment with cucurbitacins D, E, and I at concentrations 0.3, 0.1 and 0.5 μg/ml, respectively. Cell cycle and death were analyzed by flowcytometry. Results: Purified cucurbitacins induced sub-G1 cell-cycle arrest and cell death in AGS cells and upregulated LC3mRNA effectively, but showed a very low effect on BAX, caspase-3, and c-MYC mRNA levels. Also after treatment with cucurbitacin I at concentration 0.5 μg/ml, VEGF mRNA levels were increased about 4.4 times. Pairwise comparison of the effect of cucurbitacins D, E, and I on LC3 mRNA expression showed that the cucurbitacin I effect is 1.3 and 1.1 times that of cucurbitacins E and D, respectively; cucurbitacin D effect is 1.2 times that of cucurbitacin E (P-valu

Expression levels of microRNA machinery components Drosha, dicer and DGCR8 in human (AGS, HepG2, and KEYSE-30) cancer cell lines

MicroRNAs (miRNAs) have recently been shown to play fundamental roles in diverse cellular processes and linked to variety of cancers. Dicer and Drosha are two major enzymes in the miRNA maturation process. DGCR8 is the assistant of Drosha in the microprocessor complex. In this study, we evaluated the mRNA expression profiles of major miRNA processing machinery Drosha, Dicer, and DGCR8 in human gastrointestinal (AGS, KYSE30 and HepG2) cancer cell lines. Materials and Methods: The cells were cultured and harvested, and total cellular RNA was isolated from cells. Then, first-strand cDNA was synthesized from the RNA of cells. Afterward, Quantitative analysis was performed by real-time RT-PCR using the PowerSYBR Green PCR Master Mix. Results: Expression levels of Drosha in AGS and HepG2 cells were higher than the controls, whereas, Drosha\u27s expression level in KYSE-30 cell line was lower. The Dicer expression levels in AGS and HepG2 cells were higher, while, its expression level in KYSE-30 cell was lower. The DGCR8 expression levels in all three cell lines were significantly higher than the control samples. Conclusion: Expression levels of the two most important enzymes of the miRNA machinery, Drosha and Dicer, and microprocessor complex component, DGCR8 were noticeably dysregulated when compared to healthy controls