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Background: Bladder cancer accounts for almost 54% of urinary system cancer and is the second most frequent cause of death in genitourinary malignancies after prostate cancer. About 70% of bladder tumors are non-muscle-invasive, and the rest are muscle-invasive. Recurrence of the tumor is the common feature of bladder cancer. Chemotherapy is a conventional treatment for MIBC, but it cannot improve the survival rate of these patients sufficiently. Therefore, researchers must develop new therapies. Antibody-based therapy is one of the most important strategies for the treatment of solid tumors. Selecting a suitable target is the most critical step for this strategy. Objective: The aim of this study is to detect therapeutic cell surface antigen targets in bladder cancer using data obtained by proteomic studies. Methods: Isobaric tag for relative and absolute quantitation (iTRAQ) analysis had identified 131 overexpressed proteins in baldder cancer tissue and reverse-phase proteomic array (RPPA) analysis had been done for 343 tumor tissues and 208 antibodies. All identified proteins from two studies (131+208 proteins) were collected and duplicates were removed (331 unique proteins). Gene ontology study was performed using gene ontology (GO) and protein analysis through evolutionary relationships (PANTHER) databases. The Human Protein Atlas database was used to search the protein class and subcellular location of membrane proteins obtained from the PANTHER analysis. Results: Membrane proteins that could be suitable therapeutic targets for bladder cancer were selected. These included: Epidermal growth factor receptor (EGFR), Her2, Kinase insert domain receptor (KDR), Heat shock protein 60 (HSP60), HSP90, Transferrin receptor (TFRC), Activin A Receptor Like Type 1 (ACVRL1), and cadherin 2 (CDH2). Monoclonal antibodies against these proteins or their inhibitors were used for the treatment of different cancers in preclinical and clinical trials. Conclusion: These monoclonal antibodies and inhibitor molecules and also their combination can be used for the treatment of bladder cancer.

Human follicle-stimulating hormone (hFSH) is a heterodimeric glycoprotein consisting of a common α subunit non-covalently attached to the hormone-specific β subunit. The therapeutic application of this hormone requires large quantities of biologically active contaminant-free hormones. In the current study, secretory expression and purification of codon-optimized recombinant α and β subunits of hFSH were reported using Iranian Lizard Leishmania(I.L.L.) expression system. Synthetic codon-optimized genes of α and β subunits of hFSH were amplified by PCR and cloned into the pLEXSY-hyg2 and pLEXSY-neo2 vectors, respectively. The resulted expression vectors were digested and electroporated into ILL. PCR analysis showed that the genes of α and β subunits of hFSH were inserted into the genome of the parasite. Subunits expression was analyzed by dot-blot and sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and then purified and detected by Western blot analysis. Expression analysis confirmed the presence of protein bands in sizes similar to those of the natural hormone subunits. The low electrophoretic mobility and the apparent higher molecular weight of the expressed subunits were due to successful glycosylation of subunits by ILLexpression system.

Supplemental Digital Content is available in the text.Mesenchymal stem cells are widely stimulated by transforming growth factor beta-3 (TGFβ3) for chondrocyte differentiation. The objective of our study was to establish a new method for differentiation of human mesenchymal stem cells toward chondrocyte by overexpression of MicroRNA-140 (miR-140), and also this method was compared with method of induction with TGFβ3 in high-cell density culture systems. Mesenchymal stem cells were harvested from bone marrow of human. We prepared vectors and then was used for recombinant Lenti virus production in HEK-293 cell. Transducted cells were cultured in monolayer culture system and were harvested after days 7, 14, and 21. Real time polymerase chain reaction (RT-PCR) was performed to evaluate the cartilage-specific genes in the mRNA levels. Also, in order to confirm our results, we have done immunocytochemistry technique. Bone marrow mesenchymal stem cells (BMSCs) were transducted with recombinant Lenti virus, and miR-140 was expressed. Immunocytochemical method confirmed the differentiation of BMSC toward chondrocyte with handling cartilage matrix genes. Also real-time PCR showed that after expression of miR-140 in transducted BMSCs significantly increased gene expression of collagen type II and aggrecan and downregulated expression of collagen type I when compared with the mRNA levels measured in nontransducted BMSCs. These results were compatible compared with TGFβ3 induction method as control positive. In this study, we described a new approach and technique that may be applied for differentiation of BMSCs to chondrocyte instead of stimulation with TGFβ3. Our data implies that miR-140 is a potent chondrogenic differentiation inducer for BMSCs, and we have shown increasing chondrogenic differentiation by using miR-140 overexpression.

CD24 (cluster of differentiation 24) is a small heavy glycosylated protein, which is overexpressed in many cancer and some cancer stem cells and is associated with the development, invasion, and metastasis of cancer cells. The exact role of CD24 in these processes is not fully understood, however, in this article, it has been tried to present a collection of cancer-related mechanisms attributed to CD24. Based on the literature, CD24 dis-regulates different signaling pathways in various cancer cells, including; Src kinases, STAT3, EGFR, Wnt/β-catenin and MAPK. Src kinases play an important role in the signaling pathways which activate p38 MAPK and STAT3 pathways. Akt and ERK are downstream effectors of CD24-activated EGFR, which promote cell proliferation, invasion and metastasis. CD24 increases the expression of HER2 by the activation of NF-ΚB transcription factor. Moreover, CD24 up-regulates the expression of miR-21 oncomir through the activation of Src kinases. Identification of the details of these pathways and also new pathways will help researchers to explore new CD24 targeted therapies.

Vascular endothelial growth factor (VEGF) is a potential target for cancer treatment because of its role in angiogenesis and its overexpression in most human cancers. Currently, anti-VEGF antibodies have been shown to be promising tools for therapeutic applications. However, large size, poor tumor penetration, immunogenicity, and production in cost- and labor-intensive conditions are major drawbacks of such agents. The antigen-binding regions of camelid single-chain antibodies (VHHs), due to their unique biophysical characteristics, offer an alternative to conventional antibodies for tumor-targeting purposes. The present study was undertaken to generate and characterize anti-VEGF VHHs from an immune VHH library using phage display. Four rounds of panning were performed, and selected VHHs were characterized using various immunological techniques. Assessment of the antigenic profile of VHHs was done using competition enzyme-linked immunosorbent assay (ELISA). Selected VHHs reacted strongly to VEGF in indirect ELISA and cross-reactivity ELISA tests. The binding affinity of three VHHs, ZFR-1, ZFR-2, and ZFR-5, ranged from 2.5 to 80 nM, and among them, ZFR-5, which was selected for proliferation assay, significantly inhibited the endothelial cell growth in a dose-dependent manner. Taken together, our results indicate that ZFR-5 and other VHHs may be promising tools in cancer research and treatment.

Streptokinase is widely used as an anticoagulant drug for the treatment of heart attacks. Because of antibody production against injected drug in individuals consuming streptokinase and causing allergic reactions, streptokinase treatment effects become neutral. Recombinant mutant type of streptokinase was prepared by removing of 42 amino acids from the C terminal region. ELISA plates were coated by natural and mutant streptokinase as antigen. Ninety-six normal serum samples as well as 27 streptokinase consumer serum samples (patients with acute myocardial infraction) were analyzed. The results showed that serum antibodies against natural streptokinase were three times more than those against the mutated streptokinase. In case of preserving thrombolytic activity, mutated streptokinase can be used as an alternative of the natural form.

Warfarin is widely used anticoagulant drug for the prophylaxis and treatment of venous and arterial thromboembolic disorders and exerts its anticoagulant effect by inhibiting the vitamin K epoxide reductase. To determine the impact of genetic variants of the vitamin K epoxide reductase complex subunit 1 gene (VKORC1) on the anticoagulant response to warfarin, polymorphisms in exon 1, exon 3, and 3’‐untranslated region (3’ UTR) were assessed. Results: Sixty patients (34 males and 26 females) with stable INR (2–3) were selected from cardiology and anticoagulant clinic. Three VKORC1 frameshift mutations were detected. The first frameshift mutation was nucleotide deletion (91delCC) in exon 3 (1 patient). The second variation was nucleotide addition (51addCT) in exon 3 (2 patients). All the 3 patients reported bleeding during warfarin use, while no other bleeding was reported during the study period. Warfarin maintenance dose was significantly different between 3 patients with mutations and patients without mutations. The use of a fixed‐dose warfarin for all patients and in range INR may not be sufficient for warfarin monitoring. Many factors including unknown ones may also play an important role in highly variable response among patients. Our data for the first time, suggested a new possible call for screening to reduce the risk of bleeding and guide for dosing.

Here, we report a family with gigantiform cementomas, bone fractures, and autosomal dominant inheritance. Lesions are composed of benign, lobulated, calcified masses resembling cementum. Identification of a COL1A2 mutation in one patient was a polymorphism of no pathological significance. The subject of gigantiform cementomas and the associated bone disorder is both confusing and complex. Reported familial instances indicate genetic heterogeneity with (1) osteopenia and bone fractures, (2) one form of osteogenesis imperfecta, and (3) a polyostotic diaphyseal bone disorder. © 2008 Wiley‐Liss, Inc.

The variety of recombinant protein expression systems have been developed as a resource of FVII gene expression. In the current study, the authors used a novel protein expression system based on the Iranian Lizard Leishmania, a trypanosomatid protozoan as a host for expression of FVII. Plasmid containing cDNA encoding full-length human FVII was introduced into Lizard Leishmania and positive transfectants were analyzed by SDS-PAGE and Western blot analysis. Furthermore, biological activity of purified protein was detected by PT assay. The recombinant strain harboring a construct was analyzed for expression of FVII at the mRNA and protein level. Purified rFVII was obtained and in order to confirm the purified compound was in fact rFVII. Western blot analysis was carried out. Clotting time in PT assay was reduced about 30 seconds with the purified rFVII. In Conclusion, this study has demonstrated, for the first time, that Leishmania cells can be used as an expression system for producing recombinant FVII.

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