Showing 6 results for Arashkia
Maryam Rafipour, Malihe Keramati, Mohammad Mehdi Aslani, Arash Arashkia, Farzin Roohvand,
Volume 24, Issue 1 (1-2020)
Abstract
Background: Streptokinase (SK), a heterogeneous plasminogen activator (PA) protein from groups A, C, and G streptococci (GAS, GCS, GGS, respectively) contains three structural domains (SKα, SKβ, and SKg). Based on the variable region of SKβ, GAS-SKs (ska) are clustered as SK1 and SK2 (including cluster2-streptokinase (SK2a)/SK2b), which show low and high fibrinogen (FG)-dependent plasminogen (Plg) activation properties, respectively. Despite being co-clustered as SK2a, GCS/GGS-SK (skcg) variants display properties similar to SK1. Herein, by SKβ exchange between GGS (G88) and GAS-SK2a (STAB902) variants, the potential roles of SK domains in amidolytic/proteolytic activity and FG-bound-Plg activation are represented. Methods: Two parental SKG88 and SKSTAB902 genes were cloned into the NdeI/XhoI site of pET26b expression vector. The two chimeric SKβ-exchanged constructs (SKC1: αG88-βSTAB-γG88 and SKC2; αSTAB-βG88-γSTAB) were constructed by BstEII/BsiWI digestion/cross-ligation in parental plasmids. SK were expressed in E. coli and purified by nickel-nitriloacetic acid chromatography. PA potencies of SKs were measured by colorimetric assay. Results: SDS-PAGE and Western-blot analyses confirmed the proper expression of 47-kDa SKs. Analyses indicated that the catalytic efficiency (Kcat/Km) for amidolytic and proteolytic activity were less and moderately dependent on SKβ, respectively. The increase of FG-bound-Plg activation for SKSTAB902/SKC1 containing SK2aβ was around six times, whereas for SKG88/SKC2 containing skcgβ, it was four times. Conclusion: Although SKβ has noticeable contribution in FG-bound-Plg activation activity, it had minor contribution in fibrin-independent, amidolytic activity. These data might be of interest for engineering fibrin-specific versions of SK.
Mahmoud Hassani, Fatemeh Hajari Taheri, Zahra Sharifzadeh, Arash Arashkia, Jamshid Hadjati, Wytske M. van Weerden, Shahriyar Abdoli, Mohammad Hossein Modarressi, Mohsen Abolhassani,
Volume 24, Issue 2 (3-2020)
Abstract
Background: Recently, modification of T cells with chimeric antigen receptor (CAR) has been an attractive approach for adoptive immunotherapy of cancers. Typically, CARs contain a single-chain variable domain fragment (scFv). Most often, scfvs are derived from a monoclonal antibody of murine origin and may be a trigger for host immune system that leads to the T-cell clearance. Nanobody is a specific antigen-binding fragment derived from camelid that has great homology to human VH and low immunogenic potential. Therefore, in this study, nanobody was employed instead of scFv in CAR construct. Methods: In this study, a CAR was constructed based on a nanobody against PSMA (NBPII-CAR). At first, Jurkat cells were electroporated with NBPII-CAR, and then flow cytometry was performed for NBPII-CAR expression. For functional analysis, CAR T cells were co-cultured with prostate cancer cells and analyzed for IL-2 secretion, CD25 expression, and cell proliferation. Results: Flow cytometry results confirmed the expression of NBPII-CAR on the transfected Jurkat cells. Our data showed the specificity of engineered Jurkat cells against prostate cancer cells by not only increasing the IL-2 cytokine (about 370 pg/ml) but also expressing the T-cell activation marker CD25 (about 30%). In addition, proliferation of engineered Jurkat cells increased nearly 60% when co-cultured with LNCaP (PSMA+), as compared with DU145 (PSMA-). Conclusion: Here, we describe the ability of nanobody-based CAR to recognize PSMA that leads to the activation of Jurkat cells. This construct might be used as a promising candidate for clinical applications in prostate cancer therapy.
Nastaran Sadat Savar, Thomas Vallet, Arash Arashkia, Kenneth Lundstrom, Marco Vignuzzi*, Hamid Mahmoudzadeh Niknam,
Volume 26, Issue 4 (7-2022)
Abstract
Background: Self-amplifying mRNA is the next-generation vaccine platform with the potential advantages in efficacy and speed of development against infectious diseases and cancer. The main aim was to present optimized and rapid methods for Semliki Forest virus (SFV)-PD self-amplifying mRNA (SAM) preparation, its packaging, and titer determination. These protocols are provided for producing and harvesting the high yields of virus replicon particle (VRP)-packaged SAM for vaccine studies.
Methods: pSFV-PD-EGFP plasmid was linearized and subjected to in vitro transcription. Different concentrations of SFV-PD SAM were first transfected into human embryonic kidney 293 cells (HEK-293) and baby hamster kidney cell line 21 (BHK-21) cell lines, and EGFP expression at different time points was evaluated by fluorescent microscopy. Replicon particle packaging was achieved by co-transfection of SFV-PD SAM and pSFV-Helper2 RNA into BHK-21 cells. The VRPs were concentrated using ultrafiltration with 100 kDa cut-off. The titers of replicon particles were determined by reverse transcription quantitative real-time PCR (RT-qPCR).
Results: In vitro transcribed SAM encoding EGFP was successfully transfected and expressed in HEK-293 and BHK-21 cell lines. Higher levels of EGFP expression was observed in BHK-21 compared to HEK-293 cells showing more stable protein overexpression and VRP packaging. Using ultrafiltration, the high yields of purified SFV-PD-EGFP particles were rapidly obtained with only minor loss of replicon particles. Accurate and rapid titer determination of replication-deficient particles was achieved by RT-qPCR.
Conclusion: Using optimized methods for SAM transfection, VRP packaging, and concentration, high yields of SFV-PD VRPs could be produced and purified. The RT-qPCR demonstrated to be an accurate and rapid method for titer determination of replication deficient VRPs.
Sara Shayan, Golnaz Bahramali, Arash Arashkia, Kayhan Azadmanesh,
Volume 27, Issue 1 (1-2023)
Abstract
Background: Hypoxic tumor microenvironment is one of the important impediments for conventional cancer therapy. This study aimed to computationally identify hypoxia-related messenger RNA (mRNA) signatures in nine hypoxic-conditioned cancer cell lines and investigate their role during hypoxia.
Methods: Nine RNA sequencing (RNA-Seq) expression data sets were retrieved from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) were identified in each cancer cell line. Then 23 common DEGs were selected by comparing the gene lists across the nine cancer cell lines. Reverse transcription-quantitative PCR (qRT-PCR) was performed to validate the identified DEGs.
Results: By comparing the data sets, GAPDH, LRP1, ALDOA, EFEMP2, PLOD2, CA9, EGLN3, HK, PDK1, KDM3A, UBC, and P4HA1 were identified as hub genes. In addition, miR-335-5p, miR-122-5p, miR-6807-5p, miR-1915-3p, miR-6764-5p, miR-92-3p, miR-23b-3p, miR-615-3p, miR-124-3p, miR-484, and miR-455-3p were determined as common micro RNAs. Four DEGs were selected for mRNA expression validation in cancer cells under normoxic and hypoxic conditions with qRT-PCR. The results also showed that the expression levels determined by qRT-PCR were consistent with RNA-Seq data.
Conclusion: The identified protein-protein interaction network of common DEGs could serve as potential hypoxia biomarkers and might be helpful for improving therapeutic strategies.
Razieh Taghizadeh Pirposhteh, Ehsan Arefian, Arash Arashkia, Dr Nasir Mohajel,
Volume 27, Issue 6 (11-2023)
Abstract
Background: The E6 oncoprotein of HPV plays a crucial role in promoting cell proliferation and inhibiting apoptosis, leading to tumor growth. Non-viral vectors such as nona-arginine (R9) peptides have shown to be potential as carriers for therapeutic molecules. This study aimed to investigate the efficacy of nona-arginine in delivering E6 shRNA and suppressing the E6 gene of HeLa cells in vitro.
Methods: HeLa cells carrying E6 gene were treated with a complex of nona-arginine and E6 shRNA. The complex was evaluated using gel retardation assay and FESEM microscopy. The optimal N/P ratio for R9 peptide to transfect HeLa cells with luciferase gene was determined. Relative real-time PCR was used to evaluate the efficiency of mRNA suppression efficiency for E6 shRNA, while the effect of E6 shRNA on cell viability was measured using an MTT assay.
Results: The results indicated that R9 efficiently binds to shRNA and effectively transfects E6 shRNA complexes at N/P ratios greater than 30. Transfection with R9 and polyethylenimine complexes resulted in a significant toxicity compared to the scrambled plasmid, indicating selective toxicity for HeLa cells. Real-time PCR confirmed the reduction of E6 mRNA expression levels in the cells transfected with anti-E6 shRNA.
Conclusion: The study suggests that R9 is a promising non-viral gene carrier for transfecting E6 shRNA in vitro, with significant transfection efficiency and minimal toxicity.
Monireh Gholizadeh, Zahra Sharifzadeh, Mohammad Nouri, Arash Arashkia, Shahriyar Abdoli, Farhad Riyazi Rad, Amir Ali Hamidieh,
Volume 28, Issue 0 (Supplementary 2024)
Abstract
Introduction: CD22 is a surface antigen predominantly found in cancerous B-cell lineages, making it a significant target for biopharmaceuticals to treat hematologic B-cell malignancies. Biopharmaceuticals, including vaccines, cell and gene therapies, and recombinant protein therapies, have revolutionized medical treatments. Monoclonal antibodies (mAbs)are prominent in this field but face challenges such as stability, aggregation, and post-translational modifications. To address these issues, researchers have developed single-chain variable fragments (scFvs), which are smaller and potentially more stable derivatives of mAbs.
Methods and Materials: An anti-CD22-scFv was designed and synthesized for expression in Escherichia coli. The protein was purified using immobilized metal affinity chromatography (IMAC) based on column refolding and purification and native purification. We optimized expression conditions, including post-induction temperature, post-induction time, and Isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration, to maximize the soluble protein yield. The binding capacity of the hybrid- and native-purified proteins to CD22+ Raji and CD22-K562 cells was assessed using flow cytometry. The efficacy of anti-CD22-scFv in inhibiting the proliferation of CD22+ Raji cells was evaluated using the MTT assay.
Results: The anti-CD22-scFv protein was successfully expressed in the Rosetta (DE3) strain using the pET-28a+ expression vector but not in the Rosetta-gami 2 strain. Hybrid purification followed by 12% SDS-PAGE and Coomassie brilliant blue staining confirmed the protein expression. Initial solubility tests indicated that the anti-CD22-scFv protein was insoluble. Optimizing the conditions to an IPTG concentration of 0.5 mM, a post-induction temperature of 25 °C, and an eight-hour post-induction time yielded higher solubility. Native-purified and hybrid-purified anti-CD22-scFvs showed binding affinities of 91.4% and 84% to CD22+ Raji cells, respectively. The anti-CD22-scFv significantly reduced the proliferation of CD22+ Raji cells.
Conclusion and Discussion: This study demonstrates the effectiveness of a one-step IMAC-based purification method for isolating anti-CD22-scFv protein from recombinant E. coli, combining the purification and refolding into a single, cost-effective step. Optimization of the environmental parameters, including expression strain, post-induction temperature, post-induction time, and IPTG concentration, significantly improved the protein yield and solubility. The anti-CD22-scFv shows promising potential in targeting and reducing the proliferation of CD22+ tumor cells, highlighting its therapeutic potential for B-cell malignancies.
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