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Background: A lizard Leishmania has been isolated from a lizard (Agama agilis) in Iran. Its genome sequence has not been determined, so far. Methods: The study was done at Shahid Beheshti University of Medical Sciences, Tehran, Iran in 2017-2023. Leishmania promastigotes were cultured in RPMI1640 culture medium and collected at logarithmic phase by centrigugation. Parasite RNA was extracted by the Qiagene standard kit and its quantity and quality was determined and sequenced by NGS method with Illumina PE machine at BGI Company (China). Results: The number of 8316 mRNA, 83 tRNA, 63 rRNA, 83 ncRNA, 5 snRNA, 1039 snoRNA, 36 region, and 3 repeat regions, 8343 CDS, 9597 Exon and 9292 Genes were identified in promastigote of Iranian lizard Leishmania. Conclusion: Genomic elements of Iranian lizards Leishmania (with unique characteristics) were determined and identified by NGS system. [ABSTRACT FROM AUTHOR]

We investigated the transcriptional profile of whole blood in early and metastatic stages of pancreatic cancer (PaC) patients to identify potential diagnostic factors for early diagnosis. Blood samples from 18 participants (6 healthy individuals, 6 patients in early stage (I/II) PaC, and 6 patients in metastatic PaC) were analyzed by RNA‐sequencing. The expression levels of identified genes were subsequently compared with their expression in pancreatic tumor tissues based on TCGA data reported in UALCAN and GEPIA2 databases. Overall, 331 and 724 genes were identified as differentially expressed genes in early and metastatic stages, respectively. Of these, 146 genes were shared by early and metastatic stages. Upregulation of PTCD3 and UBA52 genes and downregulation of A2M and ARID1B genes in PaC patients were observed from early stage to metastasis. TCGA database showed increasing trend in expression levels of these genes from stage I to IV in pancreatic tumor tissue. Finally, we found that low expression of PTCD3, A2M, and ARID1B genes and high expression of UBA52 gene were positively correlated with PaC patients survival. We identified a four‐gene set (PTCD3, UBA52, A2M, and ARID1B) expressed in peripheral blood of early stage and metastatic PaC patients that may be useful for PaC early diagnosis. [ABSTRACT FROM AUTHOR]

Background: Extracellular vesicles (EVs) may accelerate cell death during the course of infection. Mycobacteria could invade the host's immune system and survive in the host by modulation of miRNAs. MiRNAs' differential expressions can serve as biomarkers. This study evaluates THP-1 monocyte cell death by EVs from serum of patients with mycobacteria and assesses serumderived exosomal miRNAs to increase or decrease THP-1 monocyte cell death. Materials and Methods: EVs were purified from serum of patients with mycobacteria and cultured with THP-1 monocyte. The cell death was determined via annexin V-FITC and PI staining. The microRNA was isolated from serum-derived EVs of the patients. Expression level of Hsa-miR-20a-5p, Hsa-miR-29a, Hsa-miR-let7e, and Hsa-miR-155 was assessed using qRT-PCR. Results: Cell death was accelerated in 10 and 5 µg/ml concentrations of the EVs (p<0.05). Minimum cell death was seen in 2.5 and 1.2 µg/ml concentrations (p<0.05). In tuberculosis (TB) patients, expression of miR-20a-5p, miR-29a, and miR-let7e were significantly enhanced (p=0.0001), but miR-155 expression reduced. ROC analysis showed diagnostic biomarkers of miRNAs with an AUC=0.6933 for miR-20, AUC=0.6011 for miR-29a, AUC=0.7322 for miR-let7e, and AUC=0.7456 for miR-155 for active tuberculosis. Expression of miR-let7e, 20a, and 29a in M. avium vs. M. tuberculosis was overexpressed (P=0.01, P=0.0001, and P=0.0001, respectively). Also miRs let7e and 20a expression was accelerated in M. abscessus vs. M. tuberculosis (P=0.0001 and P=0.002, respectively). Conclusion: EVs accelerates cell death and may not be ideally considered for drug delivery and vaccine developments. Circulating exosomal microRNA MiR-20, miR-let7e, and miR-155 facilitate development of potential biomarkers of pulmonary tuberculosis and non-tuberculosis. [ABSTRACT FROM AUTHOR]

Background: Leishmania is a eukaryotic protozoan parasite belonging to the Trypanosomatidae family. The Iranian Lizard Leishmania (I.L.L.), which is nonpathogenic to mammals, shows great promise to be used as an expression system for recombinant protein production. Unlike other Leishmania strains, the ideal culture medium for I.L.L. has not been established, although it is commonly cultured in the RPMI1640 medium. Methods: We investigated the growth rate of the wild and recombinant I.L.L. in BHI, RPMI1640, LB, and M199 media with and without FBS, hemin, or lyophilized rabbit serum. Subsequently, the expression rate of the recombinant protein in these media was compared. Results: The growth rate of I.L.L. in RPMI1640 medium and LB broth was similar and supplementation with 10% FBS did not affect the growth rate. The amount of protein expression in the LB medium was higher than in the other three media. Conclusion: The LB broth is an appropriate medium for I.L.L. culture and recombinant protein production. [ABSTRACT FROM AUTHOR]

Introduction: Migration of fibroblast cells in wound areas is a critical aspect of the wound healing process. Employment of enhanced green fluorescent protein (EGFP) labeled fibroblast cells facilitates real-time monitoring and functional evaluation of these cells in both in vitro and in vivo settings. Plasma rich in growth factor (PRGF) is a potent accelerator of wound healing; therefore, in this study, a novel method to fabricate an electrospun bioactive scaffold containing PRGF was employed to induce in vitro cell proliferation and migration. Methods: First, the EGFP reporter gene was integrated into the AAVS1 locus of fibroblast cells using CRISPR/Cas9 system. Then, PRGF was obtained from platelet-rich plasma, and a multi-layered scaffold was fabricated using polyurethane-cellulose acetate (PU-CA) fibers as the outer layers and PRGF-containing gelatin fibers were located in the internal layer like a central strip. Scanning electron microscopy (SEM), tensile, water contact angle, and FTIR tests were performed to assess the characteristics of the scaffolds. The EGFP targeted cells were cultured on scaffolds with or without PRGF to investigate their viability, toxicity, and migration pattern in response to the release profile. Results: Fluorescence images showed that the number of migrating cells on scaffold containing PRGF was more significant than PU-CA scaffold up to day 6. Increased expression of SGPL1 , DDR2 , and VEGF genes was also observed on the scaffold containing PRGF compared to PU-CA using real-time polymerase chain reaction (PCR) analysis with around 3-, 2-, and 2-fold enhancement, respectively. Conclusion: The current scaffold provides the appropriate template for cell attachment and migration. In addition, the present results highlight the potential of reporter gene targeting for the in vitro analysis of biological processes such as migration. [ABSTRACT FROM AUTHOR]

Ecarin is one of the most widely used drug compounds in blood clotting experiments and is used to monitor and treat many diseases such as cancer, liver, lupus, and cardiovascular disease. The metalloproteinase domain is known as the active site of ecarin. In this study, an ecarin metalloproteinase cassette was designed and synthesized in the pUC57 vector. The gene fragment was released and cloned into the pET‐28a vector and expressed in Escherichia coli. The recombinant protein was confirmed by western blotting. Enzyme activity was estimated by a laboratory coagulation test, and prothrombin time and tertiary structure were determined by using the Iterative Threading ASSEmbly Refinement (I‐TASSER) server. Data from blood clotting tests for the produced ecarin activity were analyzed using an independent t test. As per I‐TASSER server prediction, model 1 with the highest confidence score 0.95, template modeling score (0.84 ± 0.08), and root mean square deviation (3.5 ± 2.4 Å) was considered as the best model, and the 2e3xA enzyme was more similar to the target protein. The predictive results helped to better understand the relationship between the structure and function of the ecarin metalloproteinase domain. Also, the production of this active site in the prokaryotic expression system, which is simpler and more cost‐effective than the production of the eukaryotic system, showed that this recombinant ecarin could be used as a substitute for the raw snake venom of Echis carinatus because it converts prothrombin into thrombin, and its activity, as estimated using the prothrombin time test, was found to be faster than normal ecarin. [ABSTRACT FROM AUTHOR]