Recent Advances in CRISPR/Cas9-Mediated Genome Editing in Leishmania Strains.

BACKGROUND: Genome manipulation of Leishmania species and the creation of modified strains are widely employed strategies for various purposes, including gene function studies, the development of live attenuated vaccines, and the engineering of host cells for protein production. OBJECTIVE: Despite the introduction of novel manipulation approaches like CRISPR/Cas9 technology with significant advancements in recent years, the development of a reliable protocol for efficiently and precisely altering the genes of Leishmania strains remains a challenging endeavor. Following the successful adaptation of the CRISPR/Cas9 system for higher eukaryotic cells, several research groups have endeavored to apply this system to manipulate the genome of Leishmania. RESULTS: Despite the substantial differences between Leishmania and higher eukaryotes, the CRISPR/Cas9 system has been effectively tested and applied in Leishmania.  CONCLUSION: This comprehensive review summarizes all the CRISPR/Cas9 systems that have been employed in Leishmania, providing details on their methods and the expression systems for Cas9 and gRNA. The review also explores the various applications of the CRISPR system in Leishmania, including the deletion of multicopy gene families, the development of the Leishmania vaccine, complete gene deletions, investigations into chromosomal translocations, protein tagging, gene replacement, large-scale gene knockout, genome editing through cytosine base replacement, and its innovative use in the detection of Leishmania. In addition, the review offers an up-to-date overview of all double-strand break repair mechanisms in Leishmania.

Polycistronic Expression of Multi-Subunit Complexes in the Eukaryotic Environment: A Narrative Review.

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.

The engineered probiotics for the treatment of chronic diseases: A systematic review.

Engineered probiotics (EPs) are a group of probiotics whose proteome is manipulated by biotechnological techniques. EPs have attracted a lot of attention in recent researches for preventing and treating chronic diseases. The current study has been conducted to provide an overview regarding the EPs application in the treatment of chronic disease by a comprehensive systematic review of the published articles up to January 2022. To retrieve the related publications, three databases (PubMed/MEDLINE, Web of Sciences, and Scopus) were searched systematically. Finally, all human (n = 2) and animal (n = 37) studies were included. The included articles evaluated the effects of EPs on treatment of arthritis (n = 3), cancer (n = 2), autoimmune encephalomyelitis (EAE; n = 6), Parkinson disease (PD; n = 1), Alzheimer diseases (AD; n = 1), colitis (n = 11), celiac disease (n = 1), diabetes (n = 8) and cardiovascular disease (CVD; n = 6). Induction of oral tolerance (OT) is the most important mechanism of EPs action in the treatment of chronic disease. Providing oral vaccine and bioactive compounds are the other mechanisms of EPs action. PRACTICAL APPLICATIONS: The current systematic review gathered evidence about the application of EPs in the treatment of chronic diseases. Evidence suggests that EPs have very broad and potent effects in the treatment of chronic and even genetic diseases.

The Influence of Different Culture Media on the Growth and Recombinant Protein Production of Iranian Lizard Leishmania Promastigote.

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.

Effectiveness of Coronavirus Vaccines against Syndrome Coronavirus 2 (SARS-CoV-2) and Its New Variants.

The widespread outbreak of coronavirus disease 2019 in late 2019 caused many people worldwide to die or suffer from certain clinical complications even after the recovery. The virus has many social and economic adverse effects. Studies on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have specified that spike, surface glycoprotein antigen, is considered as a major target to stimulate the immune system. This glycoprotein binds to the angiotensin-converting enzyme 2 on the surface of human cells especially lung epithelial cells and facilitates the virus entry. Therefore, the immune response stimulated by vaccination targeting this antigen may cause immunity against the whole virus. Currently, many companies are working on SARS-CoV-2 vaccines. They include 'traditional' vaccines like attenuated or inactivated virus platforms as well as the brand-new generations of vaccines such as viral vector-based, subunit, nucleic acid-based, and virus-like particle vaccines. Certainly, each vaccine platform presents several advantages and disadvantages affecting its efficacy and safety which is the main topic of this paper.

The expression of DNMTs is dramatically decreased in peripheral blood mononuclear cells of male patients with juvenile idiopathic arthritis

INTRODUCTION: Juvenile idiopathic arthritis (JIA) is an autoimmune rheumatic disease, which affects primarily the joints in children under 16 years old. The etiology of JIA is yet unknown but research has shown that JIA is a multifactorial disease implicating several genes and environmental factors. Environmental factors affect immune cells via epigenetic mechanisms. One of the most important epigenetic mechanisms is DNA methylation catalyzed by DNA methyltransferases (DNMTs) and usually associated with gene silencing. In this study, we analyzed the expression of three DNA methyltransferases namely DNMT1, DNMT3a and DNMT3b in peripheral blood mononuclear cells (PBMCs) of patients with JIA and compared it with the expression of these genes in healthy young individuals. MATERIALS AND METHODS: Peripheral blood mononuclear cells of 28 JIA patients and 28 healthy controls were isolated. Total RNA was extracted, cDNA was synthesized and the transcript levels of DNMTs were analyzed by quantitative PCR. RESULTS: Analysis of DNMT1, DNMT3a and DNMT3b relative gene expression in PBMCs of JIA patients and control individuals shows that the expression of DNMT1 and DNMT3a is reduced significantly by 7 folds and 5.5 folds, respectively, in JIA patients compared to healthy controls. Furthermore, the expression of all three DNMTs were significantly and drastically reduced in young affected males compared to healthy males. CONCLUSION: This study shows that the expression of DNMTs is reduced in JIA patients and this reduction is severe in male JIA patients

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