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Background: Iranian Lizard Leishmania (I.L.L) is a nonpathogenic Leishmania strain. Due to its advantages, several recombinant proteins have been produced in this host. However, I.L.L shows a lower yield of recombinant protein expression compared to other commercial hosts. Considering the role of protease enzymes in protein digestion, we selected cysteine protease B (CPB) to investigate its impact on recombinant protein yield in I.L.L. Methods: we generated gene knockouts by utilizing homologous recombination (HR) and CRISPR methods. To assess the efficacy of the designed construct, we compared the yield of recombinant human factor VII (rhFVII) production between cells transfected with the pLEXSY-hyg2-FVII vector and the CMVpromoter-based construct (pF7cmvneo). Results: The knockout of a single CPB gene allele through the HR method or the complete knockout of all alleles through the CRISPR method led to cell death. This outcome suggests that even the deletion of a single CPB gene allele diminishes the protein to a level insufficient for the survival of I.L.L, indicating a critical dependency on the presence of this protein for the organism's viability. rhFVII exhibited a greater expression yield with the pLEXSY construct compared to the pF7cmvneo construct in I.L.L. The lower expression rate of pF7cmvneo may be influenced by epigenetic factors related to the CPC gene or the RNA polymerase used for the expression of that promoter. Conclusion: Therefore, considering alternative integration targets for CMVpromoter-based constructs and incorporating UTR sequences of I.L.L highexpression proteins in the vector may enhance recombinant protein expression rates. [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]

Background: Transgenic animals are genetically modified animals to create a specific trait that imitates an indication of pathogenesis in humans. Toll-like receptors (TLRs) are implicated in immune regulation of the female reproductive tract and, subsequently, infertility rate. This study produced Toll-like receptor 4 (Tlr4) knockout blastocysts with single-guide RNA targeting for Tlr4 by CRISPER/Cas9 technique. Materials and Methods: Web CRISPER design tools designed single-guide RNAs (sgRNAs) targeting Tlr4 gene were designed by web CRISPER design tools. Then, two strands of sgRNAs were cloned into a linearized vector for producing a gRNA-expressing eCAS9-GFP vector. The vector was then injected into the male pronucleus in the fertilized oocytes in vitro fertilization (IVF) and do polymerase chain reaction (PCR) and sequencing. Results: Gene deletion with acceptable efficiency (38%, p<0.05) successfully was confirmed by sequencing and PCR analysis. Conclusion: Our result showed that the CRISPER/Cas9 technique is an effective knockout method in mouse zygotes, potentially producing disease animal models. [ABSTRACT FROM AUTHOR]

Objectives: Choriocarcinoma spheroids, as acceptable three-dimensional (3D) models of the embryo, vary in the number of incorporated cells based on the procedures of production. Since the secretion of emitted signals from spheroids can modulate endometrial confrontation responses, improving a high throughput standard assay is precisely demanding for normalizing the characteristics of spheroids based on cellular enumeration. Materials and Methods: Spheroids derived from the suspension culture of the BeWo cell line were dimensionally categorized into three groups with sizes of 50, 100, and 200 µm in diameters. The volume of each spheroid was determined according to 4/3pr3 formula and MTT and neutral red uptake (NRU) assays were applied as conventional and modified procedures. Furthermore, the categorized groups of spheroids were enzymatically dissociated and stained by the Trypan blue dye to precisely enumerate the number of incorporated cells based on volume alterations. Results: The absorbance of spheroids in modified MTT and NRU procedures with respect to the conventional ones resulted in higher correlation coefficients with volume alterations. Similarly, modified MTT and NRU procedures with respect to the conventional ones resulted in higher correlation coefficients between the cells numeration and volume alterations. Finally, the sensitivity of modified MTT assay was higher than modified NRU assay regarding accurately estimating the number of incorporated cells per spheroid. Conclusion: In general, the modification of MTT and NRU procedures can amplify the potential of both assays to enumerate the cell number of 3D aggregates. Exclusively, the modification of MTT assay brought it as an ideal high throughput assay with high sensitivity to screen the cell number of spheroids with a diameter of 200 µm and less. [ABSTRACT FROM AUTHOR]

Objective(s): To design a multivalent DNA vaccine encoding the most immunogenic regions of the Leishmania major antigens including TSA (Thiol-specific antioxidant protein), LmSTI1 (Leishmania major stress-inducible protein1), LACK (Leishmania homologue of receptors for activated C Kinase), and KMP11 (kinetoplastid membrane protein-11) on BALB/c mice. Materials and Methods: The chimeric construct was generated including the most immunogenic epitopes containing a combination of domains and oligopeptides of the aforementioned genes. The construct was cloned into pcDNA 3.1 plasmid and named “pleish-dom.” Following intramuscular injection of mice, the capability of the vector pleish-dom alone and with pIL-12 (expressing murine IL-12) to raise protective cytokines and parasite burden was evaluated in the BALB/c mice as a susceptible animal model against L. major. Results: The immunized mice with pleish-dom/pIL-12 showed the highest and the lowest levels of interferon-gamma (IFN-γ) and interleukin-10 (IL-10), as well as the lowest leishmanin skin test (LST) reactions, were found through 8 weeks post-infection. Conclusion: Although the obtained DNA vaccine from the immunogenic chimeric protein of L. major antigens was able to induce a high level of IFN-γ, it partially protected mice against L. major. However co-administration with pIL-12 led to shift immune response to Th1 phenotype, granuloma formation, and lowered parasite burden, and consequently distinct protection was found. [ABSTRACT FROM AUTHOR]

Introduction: Today the use of electromagnetic waves has dramatically increased in modern industrial societies. This study aimed to investigate the effect of prenatal exposure to 2.4 GHz wireless frequency on forelimb development in an NMRI mouse in vivo. Methods: A total of 21 female mice weighing 25-30 g were included in the present study. They were randomly assigned to 3 groups, namely control (n=7), sham (n=7), and experimental (n=7). After mating, the experimental group was exposed to 2.4 GHz radio frequency at a distance of 20-30 cm from the device, 4 h/d until the delivery. The sham group was placed at a distance of 20-30 cm from the device every day without exposure to electromagnetic waves, and the control group had a pregnancy period without any stress and electromagnetic wave exposure. After giving birth, the forelimbs were isolated from the infants and examined by stereological studies and RT-PCR for the evaluation of osteocalcin and RUNX2 gene expression. Results: Although, at first glance, there was no macroscopic teratogen effect in forelimbs in all groups, via a stereological method, we showed that bone and cartilage volume decreased in the experimental group compared to the other groups. We also found that the experimental group had lower expression of the osteocalcin and RUNX2 gene than the control and sham groups did. However, there were no significant differences between the control and sham groups in terms of bone and cartilage volume and gene expression. Conclusion: Although teratogen effect of prenatal exposure to 2.4 GHz radio frequency on forelimbs was not demonstrated macroscopically, further studies showed negative effects on the forelimb bone, cartilage volume, and gene expression. [ABSTRACT FROM AUTHOR]

Background: Gene therapy is one of the treatment method for diabetes mellitus. An insulin gene, under control of heat shock inducer promoter was used in the study. Materials and Methods: Six mice used in this study. Streptozotocin (STZ) induced diabetes mellitus in BALB/C mice and rats. Recombinant plasmid was injected to each animal. Animal's blood sugar (BS) was measured. Warming was done at injection site with a hair dryer to induced gene expression. Results: Immediately after warming blood sugar was increased in mouse 1 and decreased after one hour. However, blood sugar increased again in mice 2 and 4. Blood sugar increased for two hours after warming in mouse 3. Blood sugar increased to 150 mg/dl after STZ injection and immediately after warming reached to 160 mg/dl, after one hour BS dropped to 116 mg/dl and on the second hour was 126 mg/dl, in mouse 5 BS had an increasing trend, BS in mouse 6 had a similar pattern to mouse 2. Conclusion: With fixing the defects of the project will be used in gene therapy in future. [ABSTRACT FROM AUTHOR]

Background: Iranian Lizard Leishmania (I.L.L) is a nonpathogenic Leishmania strain. Due to its advantages, several recombinant proteins have been produced in this host. However, I.L.L shows a lower yield of recombinant protein expression compared to other commercial hosts. Considering the role of protease enzymes in protein digestion, we selected cysteine protease B (CPB) to investigate its impact on recombinant protein yield in I.L.L., Methods: we generated gene knockouts by utilizing homologous recombination (HR) and CRISPR methods. To assess the efficacy of the designed construct, we compared the yield of recombinant human factor VII (rhFVII) production between cells transfected with the pLEXSY-hyg2-FVII vector and the CMV-promoter-based construct (pF7cmvneo)., Results: The knockout of a single CPB gene allele through the HR method or the complete knockout of all alleles through the CRISPR method led to cell death. This outcome suggests that even the deletion of a single CPB gene allele diminishes the protein to a level insufficient for the survival of I.L.L, indicating a critical dependency on the presence of this protein for the organism's viability. rhFVII exhibited a greater expression yield with the pLEXSY construct compared to the pF7cmvneo construct in I.L.L. The lower expression rate of pF7cmvneo may be influenced by epigenetic factors related to the CPC gene or the RNA polymerase used for the expression of that promoter., Conclusion: Therefore, considering alternative integration targets for CMV-promoter-based constructs and incorporating UTR sequences of I.L.L high-expression proteins in the vector may enhance recombinant protein expression rates., Competing Interests: Conflicts of Interest The authors declare no conflict of interest., (© 2024 Abdi Ghavidel et al. Published by Tehran University of Medical Sciences.)

Importance: This study is the first of its kind that suggests exosomes as a nano-carrier loaded with atovaquone (ATQ), which could be considered as a new strategy for improving the effectiveness of ATQ against acute and chronic phases of Toxoplasma gondii ., Competing Interests: The authors declare no conflict of interest.

Protein complexes are involved in many vital biological processes. Therefore, researchers need these protein complexes for biochemical and biophysical studies. Several methods exist for expressing multi-subunit proteins in eukaryotic cells, such as 2A sequences, IRES, or intein. Nevertheless, each of these elements has several disadvantages that limit their usage. In this article, we suggest a new system for expressing multi-subunit proteins, which have several advantages over existing methods meanwhile it, lacks most of their disadvantages. Leishmania is a unicellular eukaryote and member of the Trypanosomatidae family. In the expression system of Leishmania , pre-long RNAs that contain several protein sequences transcribe. Then these long RNAs separate into mature mRNAs in the process named trans splicing. For producing multi-subunit protein, Leishmania transformed with a vector containing the sequences of all subunits. Therefore, those subunits translate and form the complex under eukaryotic cell conditions. The sequence of each protein must separate by the spatial sequence needed for trans splicing. Based on a Leishmania expression pattern, not only is it possible to produce the complexes with the correct structures and post-translational modifications, but also it is possible to overcome previous method problems., Competing Interests: Conflicts of interest The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper, (Copyright © 2022 Abdi Ghavidel et al. Published by Tehran University of Medical Sciences.)