Iranian Biomedical Journal最新文献

Programmable nucleases are powerful genomic tools for precise genome editing. These tools precisely recognize, remove, or change DNA at a defined site, thereby, stimulating cellular DNA repair pathways that can cause mutations or accurate replacement or deletion/insertion of a sequence. CRISPR-Cas9 system is the most potent and useful genome editing technique adapted from the defense immune system of certain bacteria and archaea against viruses and phages. In the past decade, this technology made notable progress, and at present, it has largely been used in genome manipulation to make precise gene editing in plants, animals, and human cells. In this review, we aim to explain the basic principle, mechanisms of action, and applications of this system in different areas of medicine, with emphasizing on the detection and treatment of parasitic diseases.

Background: Adenoid cystic carcinoma is a slow-growing malignancy that most often occurs in the salivary glands. Currently, no FDA-approved therapeutic target or diagnostic biomarker has been identified for this cancer. The aim of this study was to find new therapeutic and diagnostic targets using bioinformatics methods.

Methods: We extracted the gene expression information from two GEO datasets (including GSE59701 and GSE88804). Different expression genes between adenoid cystic carcinoma (ACC) and normal samples were extracted using R software. The biochemical pathways involved in ACC were obtained by using the Enrichr database. PPI network was drawn by STRING, and important genes were extracted by Cytoscape. Real-time PCR and immunohistochemistry were used for biomarker verification.

Results: After analyzing the PPI network, 20 hub genes were introduced to have potential as diagnostic and therapeutic targets. Among these genes, PLCG1 was presented as new biomarker in ACC. Furthermore, by studying the function of the hub genes in the enriched biochemical pathways, we found that insulin-like growth factor type 1 receptor and PPARG pathways most likely play a critical role in tumorigenesis and drug resistance in ACC and have a high potential for selection as therapeutic targets in future studies.

Conclusion: In this study, we achieved the recognition of the pathways involving in ACC pathogenesis and also found potential targets for treatment and diagnosis of ACC. Further experimental studies are required to confirm the results of this study.

Background: In the present study, a novel bioink was suggested based on the oxidized alginate (OAlg), gelatin (GL), and silk fibroin (SF) hydrogels.

Methods: The composition of the bioink was optimized by the rheological and printability measurements, and the extrusion-based 3D bioprinting process was performed by applying the optimum OAlg-based bioink.

Results: The results demonstrated that the viscosity of bioink was continuously decreased by increasing the SF/GL ratio, and the bioink displayed a maximum achievable printability (92 ± 2%) at 2% (w/v) of SF and 4% (w/v) of GL. Moreover, the cellular behavior of the scaffolds investigated by MTT assay and live/dead staining confirmed the biocompatibility of the prepared bioink.

Conclusion: The bioprinted OAlg-GL-SF scaffold could have the potential for using in skin tissue engineering applications, which needs further exploration.

Background: Mannoproteins, mannose-glycosylated proteins, play an important role in biological processes and have various applications in industries. Several methods have been already used for the extraction of mannoproteins from yeast cell-wall. The aim of this study was to evaluate the extraction and deproteinization of mannan oligosaccharide from the Kluyveromyces (K.) marxianus mannoprotein.

Methods: To acquire crude mannan oligosaccharides, K. marxianus mannoproteins were deproteinized by the Sevage, trichloroacetic acid, and hydrochloric acid (HCL) methods. Total nitrogen, crude protein content, fat, carbohydrate and ash content were measured according to the monograph prepared by the meeting of the Joint FAO/WHO Expert Committee and standard. Mannan oligosaccharide loss, percentage of deproteinization, and chemical composition of the product were assessed to check the proficiency of different methods.

Results: Highly purified (95.4%) mannan oligosaccharide with the highest deproteinization (97.33 ± 0.4%) and mannan oligosaccharide loss (25.1 ± 0.6%) were obtained following HCl method.

Conclusion: HCl, was the most appropriate deproteinization method for the removal of impurities. This preliminary data will support future studies to design scale-up procedures.

Background: CD20 is a differentiation-related antigen exclusively expressed on the membrane of B lymphocytes. CD20 amplification is observed in numerous immune-related disorders, making it an ideal target for immunotherapy of hematological malignancies and autoimmune diseases. MAb-based therapies targeting CD20 have a principal role in the treatment of several immune-related disordes and cancers, including CLL. Fc gamma receptors mediate CD20 internalization in hematopoietic cells; therefore, this study aimed to establish non-hematopoietic stable cell lines overexpressing full-length human CD20 antigen as an in vitro model for CD20-related studies.

Methods: CD20 gene was cloned into the transfer vector. The lentivirus system was transfected to packaging HEK 293T cells, and the supernatants were harvested. CHO-K1 cells were transduced using recombinant viruses, and a stable cell pool was developed by the antibiotic selection. CD20 expression was confirmed at the mRNA and protein levels.

Results: Simultaneous expression of GFP protein facilitated the detection of CD20-expressing cells. Immunophenotyping analysis of stable clones demonstrated expression of CD20 antigen. In addition, the mean fluorescence intensity was significantly higher in the CD20-CHO-K1 clones than the wild-type CHO-K1 cells.

Conclusion: This study is the first report on using second-generation lentiviral vectors for the establishment of a non-hematopoietic cell-based system, which stably expresses full-length human CD20 antigen. Results of stable CHO cell lines with different levels of CD20 antigen are well suited to be used for CD20-based investigations, including binding and functional assays.

Background: Many anogenital cancers are caused by high-risk HPV. The most common subtype is HPV16, which is prevalent in the world, including Pakistan. Various amino acid residues in HPV16 E5 are associated with high cell cycle progression and proliferation. Lack of studies on HPV16E5 in Pakistan prompted the current study. This is the first report on the occurrence of pathogenic E5 variant of HPV16 in tissue sections obtained from invasive cervical cancerous patients in Pakistan.

Methods: A subset of 11 samples from HPV-positive biopsies were subjected to E5 gene amplification using PCR and analyzed using bioinformatics programs. The bioinformatics analysis detected mutations causing structural variations, which potentially contribute to the oncogenic properties of proteins.

Results: The two-point mutations, C3979A and G4042A, observed in isolate 11 caused the substitution of isoleucine for leucine and valine at positions 44 and 65 in E5 protein. The rest of the isolates had Leu44Val65 amino acids. Intratypic variations and phylogenetic analysis revealed that the majority of the isolates were closely clustered with European-Asian lineage. Moreover, C3979A and G4042A contributed to higher degree of interactions with host receptors, i.e. EGFR.

Conclusion: This is the first study reporting HPV16 variants in a Pakistani population based on variations in the E5 region. Our findings indicate that isolate 11 has a strong interaction with the intracellular domain of EGFR, which may enhance the generation of downstream signals. Since this was a pilot study to explore E5 gene mutation, future studies with large samples are absolutely needed.

Background: Renal transplantation plays an essential role in the quality of life of patients with end-stage renal disease. At least 12% of the renal patients receiving transplantations show graft rejection. One of the methods used to diagnose renal transplantation rejection is renal allograft biopsy. This procedure is associated with some risks such as bleeding and arteriovenous fistula formation. In this study, we applied a bioinformatics approach to identify serum markers for graft rejection in patients receiving a renal transplantation.

Methods: Transcriptomic data were first retrieved from the blood of renal transplantation rejection patients using the GEO database. The data were then used to construct the protein-protein interaction and gene regulatory networks using Cytoscape software. Next, network analysis was performed to identify hub-bottlenecks, and key blood markers involved in renal graft rejection. Lastly, the gene ontology and functional pathways related to hub-bottlenecks were detected using PANTHER and DAVID servers.

Results: In PPIN and GRN, SYNCRIP, SQSTM1, GRAMD1A, FAM104A, ND2, TPGS2, ZNF652, RORA, and MALAT1 were the identified critical genes. In GRN, miR-155, miR17, miR146b, miR-200 family, and GATA2 were the factors that regulated critical genes. The MAPK, neurotrophin, and TNF signaling pathways, IL-17, and human cytomegalovirus infection, human papillomavirus infection, and shigellosis were identified as significant pathways involved in graft rejection.

Concusion: The above-mentioned genes can be used as diagnostic and therapeutic serum markers of transplantation rejection in renal patients. The newly predicted biomarkers and pathways require further studies.

Background: This study investigated the role of the immune-checkpoint receptor (ICR), CD244, and its adapter molecules, in CD8+ T cells in acute leukemia.

Methods: Blood samples were obtained from 21 acute lymphoblastic leukemia (ALL) and 6 acute myeloid leukemia (AML) patients and 20 control subjects. Relative gene expression of CD244, immune receptor tyrosine-based switch motif-associated protein (SA), EWS/FLI1-activated transcript 2 (EAT-2), and LncRNA-GSTT1-AS1 were evaluated using quantitative reverse transcription polymerase chain reaction.

Results: Expression of CD244, SAP, and EAT-2 were significantly lower in CD8+ T cells from ALL patients than those from control subjects. Interestingly, the expression of SAP was much lower than that of CD244, indicating a lower ratio of SAP to CD244. Also, SAP expression was significantly lower in AML patients compared to the control group. Expression of LncRNA-GSTT1-AS1 showed no significant difference in ALL and AML patients compared to control subjects.

Conclusion: The low SAP/CD244 expression ratio in CD8+ T cells in ALL suggests an inhibitory role for CD244 in ALL.

Background: T-cell immunoglobulin domain and mucin domain-3 (TIM-3) is an inhibitory receptor expressed in a variety of cells, including dendritic cells, T-helper 1 lymphocytes, and natural killer cells. Binding of this protein to its ligand, carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), causes T-cell exhaustion, a specific condition in which effector T cells lose their ability to proliferate and produce cytokines. Blocking this inhibitory receptor is known to be an effective strategy for treating cancer and other related diseases. Therefore, in this study, in order to block the inhibitory receptor of TIM-3, we designed and produced recombinantly a protein with a high binding affinity to this receptor.

Methods: The extracellular domain of CEACAM1 involved in binding to TIM-3 was mutated using R script to obtain a variant with the increased binding affinity to TIM-3. The binding energy of the mutant protein was calculated using the FoldX module. Finally, after recombinant production of the most appropriate CEACAM1 variant (variant 39) in E. coli, its secondary structure was determined by CD spectroscopy.

Results: The binding free energy between variant 39 and TIM-3 decreased from -5.63 to -14.49 kcal/mol, indicating an increased binding affinity to the receptor. Analysis of the secondary structure of this variant also showed that the mutation did not significantly alter the structure of the protein.

Conclusion: Our findings suggest that variant 39 could bind to TIM-3 with a higher binding affinity than the wild-type, making it a proper therapeutic candidate for blocking TIM-3.