Antisense Oligonucleotide (AS-ODN) Technology: Principle, Mechanism and Challenges.

Recently, there is a hopefully tremendous interest in antisense therapeutics for clinical purposes. Single-stranded synthetic antisense oligonucleotides (As-ODNs) with monomers of chemically modified 18-21 deoxynucleotides complement the mRNA sequence in target gene. The target gene expression can be blocked because of created cleavage or disability of the mRNA by binding the As-ODNs to cognate mRNA sequences via sequence-specific hybridization. The idea of antisense therapy has become particular concerning that any sequence longer than a minimal number of nucleotides (17 for DNA and 13 for RNA) can be observed only once within the human genome. The mRNA is omnipresent more probably to manipulate compared to DNA, which results in multiple in vitro and in vivo applications for As-ODNs in the field of regulatory mechanisms of biological processes, cancer, viral infections and hereditary impairments. Although, there are uncertain clinical outcomes on the ability of this approach in treatment procedures despite achieving promising findings based on previous investigations. Accordingly, the efficacy, off-target effects, delivery are issues that should be investigated to obtain satisfactory results. In this review, we will explain the mechanism of action of As-ODNs and various types of modifications and their therapeutic purposes.

Retinoic acid and/or progesterone differentiate mouse induced pluripotent stem cells into male germ cells in vitro.

Numerous reagents were employed for differentiating induced pluripotent stem cells (iPSCs) into male germ cells; however, the induction procedure was ineffective. The aim of this study was to improve the in vitro differentiation of mice iPSCs (miPSCs) into male germ cells with retinoic acid (RA) and progesterone (P). miPSCs were differentiated to embryoid bodies (EBs) in suspension with RA with or without progesterone for 0, 4, and 7 days. Then, the expression of certain genes at different stages of male germ cell development including Ddx4 (pre meiosis), Stra8 (meiosis), AKAP3 (post meiosis), and Mvh protein was examined in RNA and/or protein levels by real-time polymerase chain reaction or flow cytometry, respectively. The Stra8 gene expression increased in the RA groups on all days. But, expression of this gene declined in RA + P groups. In addition, an increased expression of Ddx4 gene was observed on day 0 in the P group. Also, a significant upregulation was observed in the expression of AKAP3 gene in the RA + P group on days 0 and 4. However, gene expression decreased in P and RA groups on day 7. The expression of Mvh protein significantly increased in the RA group on day 7. The Mvh expression was also enhanced in the P group on day 4, but it decreased on day 7, while this protein upregulated on day 0 and 7 in the RA + P group. The miPSCs have the capacity for in vitro differentiation into male germ cells by RA and/or progesterone. However, the effects of these inducers depend on the type of combination and an effective time.

In silico identification and characterization of antineoplastic asparaginase enzyme from endophytic bacteria.

It is generally accepted that L-asparagine is an important amino acid required for the fast growth of cells. Cancerous cells receive this amino acid from extracellular sources. The depletion of L-asparagine from its surrounding environments by asparaginase enzyme can be used as a therapeutic strategy in cancer patients. This therapeutic enzyme is produced commercially mainly from bacteria such as Escherichia coli and Erwinia chrysanthemi. The side effects of such drugs have persuaded scientists to find new enzyme sources. In this study, in silico approach was applied to investigate L-asparaginase producing endophytic bacteria that produce more compatible enzymes within the body. Protein-protein basic local alignment search tool with E. coli and E. chrysanthemi asparaginase enzyme sequences against 262 endophytic bacteria were performed. The results with identity more than 35%, coverage more than 80%, and E-value less than 10-4 were selected. Then, some of bioinformatics tools were used to characterize them. A total of nine sequences consisting of seven known and two hypothetical proteins were identified in six bacterial species. The results showed that some of the asparaginase enzymes produced by endophytic bacteria possess more suitable immunological indices compared with asparaginase enzymes of E. coli and E. chrysanthemi. Herbaspirillum rubrisubalbicans was predicted to produce a nonallergen and nonantigen asparaginase enzyme. The number of antigenic determinants was predicted to be lower in asparaginase enzymes produced by Bacillus amyloliquefaciens, H. rubrisubalbicans, and H. seropedicae. Moreover, the number of high-scored B-cell epitopes was lower in enzyme sequences related to the mentioned bacteria and Paenibacillus polymyxa. The number of discontinuous epitopes and the number of T-cell epitopes were lower in B. amyloliquefaciens produced enzymes. Therefore, the therapeutic use of these enzymes is possible.

The atherogenic role of immune cells in familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is an autosomal dominant disorder of lipoprotein metabolism that mainly occurs due to mutations in the low-density lipoprotein receptor gene and is characterized by increased levels of low-density lipoprotein cholesterol, leading to accelerated atherogenesis and premature coronary heart disease. Both innate and adaptive immune responses, which mainly include monocytes, macrophages, neutrophils, T lymphocytes, and B lymphocytes, have been shown to play a key role for the initiation and progression of atherogenesis in the general population. In FH patients, these immune cells have been suggested to play specific pro-atherosclerotic activities, from the initial leukocyte recruitment to plaque rupture. In fact, the accumulation of cholesterol crystals and oxLDL in the vessels in FH patients is particularly high, with consequent abnormal mobilization of immune cells and secretion of various pro-inflammatory and chemokines. In addition, cholesterol accumulation in immune cells is exaggerated with chronic exposure to relevant pro-atherosclerotic triggers. The topics considered in this review may provide a more specific focus on the immune system alterations in FH and open new insights toward immune cells as potential therapeutic targets in FH.

The pivotal role of CD69 in autoimmunity.

Autoimmune disorders are outcomes of impaired activity of the immune system regarding the maintenance of tolerance, which results in tissue damage secondary to an excess in the inflammatory response. Under normal conditions, the cells in the adaptive immune system are highly controlled to remain unresponsive against self-antigens (self-Ags) through various mechanisms and during different stages of maturation. CD69 (cluster of differentiation 69), a C-type lectin disulfide-linked homodimer, is expressed on different leukocytes, including newly-activated lymphocytes, certain subtypes of memory T-cells, infiltrating lymphocytes isolated from patients with chronic inflammatory disorders, and regulatory T-cells (Tregs). Cumulative evidence from in vitro and in vivo studies has revealed an immunoregulatory role for CD69. This marker has been reported to play a controversial role in chronic human inflammatory disorders. Many investigations have linked the absence of CD69 with a predisposition to inflammatory and/or autoimmune conditions, which indicates an immunoregulatory function for CD69 by mechanisms such as controlling the balance between differentiation of Th/Treg cells and enhancing the suppressive activity of Tregs. However, some reports from human studies have indicated that CD69 may exert a stimulatory effect on the inflammatory response. In this review, we first present a brief summary of the concept of 'immune tolerance' and, subsequently, review previous studies to uncover the details that underlie the immunoregulatory effects of CD69.

Role of long non-coding RNAs (LncRNAs) in multiple sclerosis: a brief review.

Multiple sclerosis (MS) as chronic autoimmune inflammatory neurological disease of the central nervous system (CNS) occurs due to several environmental and genetic factors, whose pathogenesis is associated with genes with regulatory role in the immune system. Long non-coding RNAs (LncRNAs) are able to reportedly regulate responses of immune systems and expression of genes, and show the tissue specificity and complexity of biofunctions. Various studies have suggested that the aberrant LncRNA expression is an underlying factor involved in the incidence of MS and that the analysis of the expression profile of these molecules can be a specific biomarker of MS for preventing the course of the disease or responding to treatment. The purpose of this research was to review the recent studies for exploring the functions of LncRNAs in the processes leading to MS disease.

Retinoic acid and 17ß-estradiol improve male germ cell differentiation from mouse-induced pluripotent stem cells.

This research aimed to explore the impacts of retinoic acid (RA)/17ß-estradiol (E) induction and embryoid body formation to enhance differentiation of mouse-induced pluripotent stem cells (miPSCs) into male germ cells in vitro. Flow cytometry and qPCR were conducted to describe miPSCs differentiation process. Various temporal expression profiles of germ cell-related genes were traced. Stra8 gene expression increased in the RA group on the 4th day compared to other groups. The RA group experienced a more significant increase than E group. The expression of Sycp3 increased in RA + E group on 4th day compared with other groups. Expression of AKAP3 enhanced in the RA + E group than other groups on day 4. Moreover, miPSCs showed that this gene expression in the RA + E group was increased in comparison to RA and E groups on day 7. AKAP3 gene expression on day 7 of miPSCs decreased in RA and E groups. Flow cytometry data indicated that 3%-8% of the cells in sub-G1 stage were haploid after RA and E induction compared to other groups on day 4. This study showed that miPSCs possess the power for differentiating into male germ cells in vitro via formation of embryoid body by RA with/or E induction.

Nanovaccine: A novel approach in immunization.

Despite great advances in the field of vaccination, there are still needs for novel and effective vaccines because still no effective vaccines have been produced for some diseases such as malaria, acquired immune deficiency syndrome (AIDS), and tuberculosis. Furthermore, many of the existing vaccines have disadvantages such as failure to stimulate completely the immune system, in vivo instability, high toxicity, the need for cold chain, and multiple administrations. Nanotechnology has been raised as a powerful tool for solving these problems in this regard. Generally, nanovaccines are a new generation of vaccines using nanoparticles (NPs) as carriers and/or adjuvants. Due to the similar scale (size) between the NPs and pathogens, the immune system can be stimulated well, resulting in triggered cellular and humoral immunity responses. Other benefits of the nanovaccines include their better stability in blood flow to increase the shelf life in blood, enhanced immune system stimulation, no need for booster doses, no need to maintain the cold chain, and ability to create active targeting. In addition, nanovaccines have raised the hope to treat diseases such as rheumatoid arthritis, AIDS, malaria, and chronic autoimmune, and so forth.

Involvement of aberrant regulation of epigenetic mechanisms in the pathogenesis of Parkinson's disease and epigenetic-based therapies.

Parkinson's disease (PD) is known as a progressive neurodegenerative disorder associated with the reduction of dopamine-secreting neurons and the formation of Lewy bodies in the substantia nigra and basal ganglia routes. Aging, as well as environmental and genetic factors, are considered as disease risk factors that can make PD as a complex one. Epigenetics means studying heritable changes in gene expression or function, without altering the underlying DNA sequence. Multiple studies have shown the association of epigenetic variations with onset or progression of various types of diseases. DNA methylation, posttranslational modifications of histones and presence of microRNA (miRNA) are among epigenetic processes involved in regulating pathways related to the development of PD. Unlike genetic mutations, most epigenetic variations may be reversible or preventable. Therefore, the return of aberrant epigenetic events in different cells is a growing therapeutic approach to treatment or prevention. Currently, there are several methods for treating PD patients, the most important of which are drug therapies. However, detection of genes and epigenetic mechanisms involved in the disease can develop appropriate diagnosis and treatment of the disease before the onset of disabilities and resulting complications. The main purpose of this study was to review the most important epigenetic molecular mechanisms, epigenetic variations in PD, and epigenetic-based therapies.

CRISPR-Cas9 in genome editing: Its function and medical applications.

The targeted genome modification using RNA-guided nucleases is associated with several advantages such as a rapid, easy, and efficient method that not only provides the manipulation and alteration of genes and functional studies for researchers, but also increases their awareness of the molecular basis of the disease and development of new and targeted therapeutic approaches. Different techniques have been emerged so far as the molecular scissors mediating targeted genome editing including zinc finger nuclease, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9). CRISPR-Cas9 is a bacterial immune system against viruses in which the single-strand RNA-guided Cas9 nuclease is linked to the targeted complementary sequences to apply changes. The advances made in the transfer, modification, and emergence of specific solutions have led to the creation of different classes of CRISPR-Cas9. Since this robust tool is capable of direct correction of disease-causing mutations, its ability to treat genetic disorders has attracted the tremendous attention of researchers. Considering the reported cases of nonspecific targeting of Cas9 proteins, many studies focused on enhancing the Cas9 features. In this regard, significant advances have been made in choosing guide RNA, new enzymes and methods for identifying misplaced targeting. Here, we highlighted the history and various direct aspects of CRISPR-Cas9, such as precision in genomic targeting, system transfer and its control over correction events with its applications in future biological studies, and modern treatment of diseases.

The role of microRNAs in embryonic stem cell and induced pluripotent stem cell differentiation in male germ cells.

New perspectives have been opened by advances in stem cell research for reproductive and regenerative medicine. Several different cell types can be differentiated from stem cells (SCs) under suitable in vitro and in vivo conditions. The differentiation of SCs into male germ cells has been reported by many groups. Due to their unlimited pluripotency and self-renewal, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can be used as valuable tools for drug delivery, disease modeling, developmental studies, and cell-based therapies in regenerative medicine. The unique features of SCs are controlled by a dynamic interplay between extrinsic signaling pathways, and regulations at epigenetic, transcriptional and posttranscriptional levels. In recent years, significant progress has been made toward better understanding of the functions and expression of specific microRNAs (miRNAs) in the maintenance of SC pluripotency. miRNAs are short noncoding molecules, which play a functional role in the regulation of gene expression. In addition, the important regulatory role of miRNAs in differentiation and dedifferentiation has been recently demonstrated. A balance between differentiation and pluripotency is maintained by miRNAs in the embryo and stem cells. This review summarizes the recent findings about the role of miRNAs in the regulation of self-renewal and pluripotency of iPSCs and ESCs, as well as their impact on cellular reprogramming and stem cell differentiation into male germ cells.

MicroRNA in leukemia: Tumor suppressors and oncogenes with prognostic potential.

Leukemia is known as a progressive malignant disease, which destroys the blood-forming organs and results in adverse effects on the proliferation and development of leukocytes and their precursors in the blood and bone marrow. There are four main classes of leukemia including acute leukemia, chronic leukemia, myelogenous leukemia, and lymphocytic leukemia. Given that a variety of internal and external factors could be associated with the initiation and progression of different types of leukemia. One of the important factors is epigenetic regulators such as microRNAs (miRNAs) and long noncoding RNAs (ncRNA). MiRNAs are short ncRNAs which act as tumor suppressor (i.e., miR-15, miR-16, let-7, and miR-127) or oncogene (i.e., miR-155, miR-17-92, miR-21, miR-125b, miR-93, miR-143-p3, miR-196b, and miR-223) in leukemia. It has been shown that deregulation of these molecules are associated with the initiation and progression of leukemia. Hence, miRNAs could be used as potential therapeutic candidates in the treatment of patients with leukemia. Moreover, increasing evidence revealed that miRNAs could be used as diagnostic and prognostic biomarkers in monitoring patients in early stages of disease or after received chemotherapy regimen. It seems that identification and development of new miRNAs could pave to the way to the development new therapeutic platforms for patients with leukemia. Here, we summarized various miRNAs as tumor suppressor and oncogene which could be introduced as therapeutic targets in treatment of leukemia.

The protective effects of curcumin on cytotoxic and teratogenic activity of retinoic acid in mouse embryonic liver.

BACKGROUND: Retinoic acid (RA) is a synthetic vitamin derivative. It exerts toxic and teratogenic effects on the development of embryonic organs in dose- and time-dependent manners in mice. Curcumin is a compound obtained from rhizomes of turmeric (Curcuma longa) and has protective effects on teratogenic agents. The current study examined the effects of curcumin on embryos treated with RA. METHODS: A total of 24 female NMRI mice (8-week-old pregnant mice) were investigated in the current study. All of them were treated for 10 days during days 15 to 50 of pregnancy. In the first group, the animals were fed with normal diets (control); in the second group, with 60 mg/kg all- trans RA; in the third group, with 10 mg/kg curcumin; and in the fourth group, with RA and curcumin in their diets. The animals were killed by cervical dislocation at the 18th day of pregnancy and embryos were separated from the uteruses. The embryo weight and crown rump (CR) length were measured, and the SPSS software was used to analyze data. RESULTS: There was a significant increase in the lengths of CR and weights of embryos after using curcumin, but RA had no effect on the length of CR and weight of embryos at a dose of 60 mg/kg. Morphometric assay of liver tissue was performed, and data analysis indicated that there were significant differences between groups in terms of morphometric parameters of liver tissue. Therefore, RA increased the cell number and sinusoid diameter and decreased the cell areas in the embryonic liver tissue. However, curcumin decreased these side effects of RA on the embryonic liver tissue. CONCLUSION: The results indicated that curcumin could decrease the toxic and teratogenic effects of RA in mouse embryos.

Pancreatic ß-cell regeneration: From molecular mechanisms to therapy.

Pancreatic ß cells are a type of cells that are present in the islets of Langerhans. These cells are highly specialized for the secretion of insulin in response to low increasing of blood glucose levels. Hence, pancreatic ß cells could contribute to maintaining systemic glucose homeostasis. Increasing evidence has revealed that a variety of internal (ie, genetic and epigenetic factors) and external factors (ie, radical-oxidative stress) are involved in the protection and/or regeneration of pancreatic ß cells. The pathways regulating ß-cell replication have been intensely investigated. Glucose has an important role in cell cycle entry of quiescent ß cells, which exerts its effect via glucose metabolism and unfolded proteins. A variety of growth factors, hormones, and signaling pathways (ie, calcium-calcineurin nuclear factor of activated T cells) are others factors that could affect ß-cell replication under different conditions. Therefore, a greater understanding of the underlying pathways involved in the regeneration and protection of pancreatic ß cells could lead to finding and developing new therapeutic approaches. Utilization of stem cells and various phytochemical agents have provided new aspects for preventing ß-cell degeneration and stimulating the endogenous regeneration of islets. Thus, these therapeutic platforms could be used as potential therapies in the treatment of insulin-dependent diabetes mellitus. Here, we summarized the various mechanisms involved in pancreatic ß-cell regeneration. Moreover, we highlighted different therapeutic approaches which could be used for the regeneration of pancreatic ß cells.

Efferocytosis: molecular mechanisms and pathophysiological perspectives.

During the life of a human being, several tons of apoptotic cells and debris are produced. These apoptotic particles should be cleared quickly and accurately from the body, as they may lose their membrane integrity with the probability of leakage of cytotoxic materials and other intracellular antigens into the environment. The action of removing apoptotic particles occurs by a process called efferocytosis. Efferocytosis is a highly regulated balance among a set of find-me, eat-me and don't-eat-me signals. Efferocytosis is accompanied by a suppression of the immune system that can explain its negative role in cancer. Additionally, defects in this process can lead to different diseases. In this review, we aim to describe the mechanism of efferocytosis and evaluate its association with the development of autoimmune diseases, airway inflammation, atherosclerosis and cancer to open a new window for the treatment of these diseases.

Electrochemical-based biosensors for detection of Mycobacterium tuberculosis and tuberculosis biomarkers.

Early detection of tuberculosis (TB) reduces the interval between infection and the beginning of treatment. However, commercially available tests cannot discriminate between BCG-vaccinated healthy persons and patients. Also, they are not suitable to be used for immunocompromised persons. In recent years, biosensors have attracted great attention due to their simple utility, accessibility, and real-time outputs. These sensors are increasingly being considered as pioneering tools for point-of-care diagnostics in communities with a high burden of TB and limited accessibility to reference laboratories. Among other types of biosensors, the electrochemical sensors have the advantages of low-cost operation, fast processing, simultaneous multi-analyte analyzing, operating with turbid samples, comparable sensitivity and readily available miniaturization. Electrochemical biosensors are sub-divided into several categories including: amperometric, impedimetric, potentiometric, and conductometric biosensors. The biorecognition element in electrochemical biosensors is usually based on antibodies (immunosensors), DNAs or PNAs (genosensors), and aptamers (aptasensors). In either case, whether an interaction of the antigen-antibody/aptamer or the hybridization of probe with target mycobacterial DNA is detected, a change in the electrical current occurs that is recorded and displayed as a plot. Therefore, impedimetric-based methods evaluate resistance to electron transfer toward an electrode by a Nyquist plot and amperometric/voltammetric-based methods weigh the electrical current by means of cyclic voltammetry, square wave voltammetry, and differential pulse voltammetry. Electrochemical biosensors provide a promising scope for the new era of diagnostics. As a consequence, they can improve detection of Mycobacterium tuberculosis traces even in attomolar scales.

Application of induced pluripotent stem cell and embryonic stem cell technology to the study of male infertility.

Stem cells (SCs) are classes of undifferentiated biological cells existing only at the embryonic, fetal, and adult stages that can divide to produce specialized cell types during fetal development and remain in our bodies throughout life. The progression of regenerative and reproductive medicine owes the advancement of respective in vitro and in vivo biological science on the stem cell nature under appropriate conditions. The SCs are promising therapeutic tools to treat currently of infertility because of wide sources and high potency to differentiate. Nevertheless, no effective remedies are available to deal with severe infertility due to congenital or gonadotoxic stem cell deficiency in prepubertal childhood. Some recent solutions have been developed to address the severe fertility problems, including in vitro formation of germ cells from stem cells, induction of pluripotency from somatic cells, and production of patient-specific pluripotent stem cells. There is a possibility of fertility restoration using the in vitro formation of germ cells from somatic cells. Accordingly, the present review aimed at studying the literature published on the medical application of stem cells in reproductive concerns.

Distribution of Human Papillomavirus Genotypes among Women in Mashhad, Iran.

OBJECTIVE: Cervical cancer is the third most common malignancy in females. Since the human papillomavirus (HPV) genotype can vary geographically, it is essential to define its genotypic distribution before establishing health care policies and vaccination programs in any given area. The aim of this study was to determine the frequency of HPV types in women in Mashhad, Iran. METHODS: We used nested PCR and reverse dot-blot hybridization for genotyping. The study included 143 cervical cytology samples from Mashhad with confirmed papillomavirus infection by molecular methods. RESULTS: We found that 74.1% of HPV types were in high-risk groups, including genotypes 16, 18, 39, 52, 58, 66, 68, and 73. Coinfection was detected in 56.4% of the cases. The low-risk group, comprising 25.9%, included genotypes 6, 11, 42, and 44/55. CONCLUSIONS: Prevention, early diagnosis, and early treatment have been proven to reduce the mortality rate of cervical cancer. Therefore, an accurate diagnosis of the genotype of the virus in infected patients is very important.

Construction and immunogenic properties of a chimeric protein comprising CfaE, CfaB and LTB against Enterotoxigenic Escherichia coli.

ETEC (Enterotoxigenic Escherichia coli) is a major cause of diarrhea in developing countries and children. ETEC has two virulence factors including colonization factors antigen (CFA) and labile enterotoxins (LTs). CFA/I consists the major pilin subunit CfaB and a minor adhesive subunit, CfaE. In this study a tripartite fusion protein containing CfaB, CfaE and LTB was designed. In silico analysis of the tertiary structure of the chimeric protein showed a protein with three main domains linked together with linkers. Linear and conformational B-cell epitopes were identified. A chimera consisting cfaB, cfaE and ltB(BET)was then synthesized with E. coli codon bias in pUC57 and sub cloned into pET32 vector. Recombinant protein was expressed and purified by affinity chromatography and confirmed by western blotting. Mice were immunized with recombinant protein and the antibody titer and specificity of the sera were analyzed by ELISA. The efficiency of the immune sera against ETEC was evaluated by binding assay and GM1-ELISA. VaxiJen analysis of the protein showed high antigenicity. Post-immune sera contained high titers of anti-BET IgG. Pretreatment of ETEC cells with sera from immunized mice decreased their ability to adhere to cells of the human colon adenocarcinoma cell line HT29.