Protective immunization against Enterohemorrhagic Escherichia coli and Shigella dysenteriae Type 1 by chitosan nanoparticle loaded with recombinant chimeric antigens comprising EIT and STX1B-IpaD.

Increasing evidence demonstrated that Enterohemorrhagic Escherichia coli (EHEC) and Shigella dysenteriae type 1 (S. dysenteriae1) are considered pathogens, that are connected with diarrhea and are still the greatest cause of death in children under the age of five years, worldwide. EHEC and S. dysenteriae 1 infections can be prevented and managed using a vaccination strategy against pathogen attachment stages. In this study, the chitosan nanostructures were loaded with recombinant EIT and STX1B-IpaD polypeptides. The immunogenic properties of this nano-vaccine candidate were investigated. The EIT and STX1B-IpaD recombinant proteins were heterologous expressed, purified, and confirmed by western blotting. The chitosan nanoparticles, were used to encapsulate the purified proteins. The immunogenicity of recombinant nano vaccine candidate, was examined in three groups of BalB/c mice by injection, oral delivery, and combination of oral-injection. ELISA and antibody titer, evaluated the humoral immune response. Finally, all three mice groups were challenged by two pathogens to test the ability of the nano-vaccine candidate to protect against bacterial infection. The Sereny test in guinea pigs was used to confirm the neutralizing effect of immune sera in controlling S. dysenteriae 1, infections. SDS-PAGE and western blotting, confirmed the presence and specificity of 63 and 27 kDa recombinant EIT and STX1B-IpaD, respectively. The results show that the nanoparticles containing recombinant proteins could stimulate the systemic and mucosal immune systems by producing IgG and IgA, respectively. The challenge test showed that, the candidate nano-vaccine could protect the animal model from bacterial infection. The combination of multiple recombinant proteins, carrying several epitopes and natural nanoparticles could evocate remarkable humoral and mucosal responses and improve the protection properties of synthetic antigens. Furthermore, compared with other available antigen delivery methods, using oral delivery as immune priming and injection as a booster method, could act as combinatorial methods to achieve a higher level of immunity. This approach could present an appropriate vaccine candidate against both EHEC and S. dysenteriae 1.

Regulation of long non-coding RNAs XIST and ROR induced by homeodomain protein TGIF2LX in colorectal cancer.

Background: Homeodomain protein transforming growth factor beta-induced factor 2 like, X-linked (TGIF2 LX) has been demonstrated to act as a transcription factor and regulate cancer cell proliferation. Long non-coding RNAs (lncRNAs) are well known as molecular regulators of colorectal cancer (CRC). Our aim was to evaluate the clinical and biological significance of TGIF2 LX and its effect on lncRNAs regulator of reprogramming (ROR) and X-inactive specific transcript (XIST) expression in CRC cells. Materials and Methods: Thirty-six CRC tissues and 22 adjacent normal colorectal tissues were subjected to RNA extraction and analysis of TGIF2 LX gene expression by quantitative real-time polymerase chain reaction (qRT-PCR). The human SW1116 cell line was transfected with cDNA for the TGIF2 LX gene. Microscopic analysis, reverse transcriptase PCR, and western blotting were used for confirming at transcriptional and translational levels. Methyl thiazolyl tetrazolium and colony formation assays were applied for evaluating the in vitro cell viability and colony-forming ability, respectively. LncRNA expression analysis was carried out using qRT-PCR. Results: The results showed that the expression levels of TGIF2 LX were significantly downregulated in CRC tissues compared to adjacent normal tissues (P = 0.032). Furthermore, the overexpression of TGIF2 LX could reduce the CRC cell line proliferation. The gene expression analysis revealed a significantly reduced level of lncRNA ROR and lncRNA XIST in TGIF2 LX-transfected SW1116 cells compared to nontransfected cells. Conclusion: Our findings provided evidence of molecular mechanisms by which TGIF2 LX may interact with lncRNAs ROR and XSIST to regulate CRC development by acting as a tumor suppressor. Thus, this protein may potentially be a promising option for CRC gene-based therapeutic strategies.

EVALUATION OF BCL2 AND ITS REGULATORY MIRS, MIR-15-B AND MIR-16 EXPRESSION CHANGES UNDER THE EXPOSURE OF EXTREMELY LOW-FREQUENCY ELECTROMAGNETIC FIELDS ON HUMAN GASTRIC CANCER CELL LINE.

In this research, changes in the expression of B-cell lymphoma 2 (BCL2), miR-15-b and miR-16 in human adenocarcinoma gastric cancer cell line (AGS) following the exposure to magnetic flux densities (MFDs) of 0.2 and 2 mT continuously and discontinuously (1.5 h on/1.5 h off) for 18 h were investigated. Changes in the cell viability were evaluated by the MTT assay. Real-time PCR was used to evaluate the expression changes of BCL2, miR-15-b and miR-16. The results showed that extremely low frequency electromagnetic field (ELF-EMF) could significantly reduce the viability of AGS cells in the continuous MFD of 2 mT. The BCL2 expression was significantly decreased following the exposure to continuous MFDs of 0.2 and 2 mT and discontinuous MFD of 2 mT. The expressions of miR-15-b and miR-16 were significantly increased in continuous and discontinuous MFD of 2 mT. According to the results, weak and moderate extremely low-frequency electromagnetic fields can change the expressions of BCL2, miR-15-b and miR-16.

An Updated Review of Various Medicinal Applications of p-Co umaric Acid: From Antioxidative and Anti-Inflammatory Properties to Effects on Cell Cycle and Proliferation.

P-Coumaric acid (p-CA) is a hydroxycinnamic acid, an organic compound that is a hydroxyl derivative of cinnamic acid. P-CA is the most abundant isomer in nature and can be found in a wide variety of edible plants such as fungi, peanuts, navy beans, tomatoes, carrots, basil, and garlic. Recently, the therapeutic properties of p-CA have received a great deal of attention from scientific society. Here, we described the medicinal effects of p-CA on various pathological conditions. This review was performed via evaluating PubMed reported studies from January 2010 to January 2020. Also, reference lists were checked to find additional studies. All intermediation or complementarity of animal models, case-control and cohort studies, in vitro studies, and controlled trials (CTs) on p-CA were acceptable. However plant extract studies without indication of main active substances were excluded due to the considerable diversities and heterogeneities. According to recent evidence regarding the beneficial effects of p-CA, numerous diseases such as nephropathies, cardiovascular diseases, neuroinflammatory diseases, liver diseases, cancers, and some metabolic disorders could potentially be controlled by this natural herb. Interestingly, autophagy is a novel molecular mechanism involved in the crosstalk between classic effects of p-CA and introduces alternative therapeutic pathways for this compound. Much work remains in clarifying the main therapeutic properties among the various p-CA effects; these will be the subject of forthcoming work, resulting in presenting further mechanism of action.

Cell death analysis of recombinant mature epsilon toxin on the kidney cell line.

BACKGROUND AND OBJECTIVES: Epsilon toxin is the third hazardous bacterial toxin causing ABS enterotoxaemia in domestic animal. In addition, epsilon toxin is known as a biological warfare agent. The aim of this study was to produce the recombinant mature epsilon toxin to evaluate cell death impact on the kidney cell line. MATERIALS AND METHODS: For this purpose, the sequence of mature epsilon toxin (46-328 aa) in pET28a was cloned and expressed in Escherichia coli BL21 (DE3) and purified by nickel-nitrilotriacetic acid (Ni-NTA) column and confirmed by western blot analysis using HRP conjugated anti-His antibody. Then, to assess the anti-proliferative effects of different concentrations of recombinant epsilon toxin, the MTT assay was done on the HEK293 cell line. The annexin V/PI staining was done to investigate the apoptotic and necrotic cell populations after exposure to epsilon toxin. RESULTS: Induction by 1 mM IPTG for 4 h at 37°C was an optimized condition for expressing mature epsilon toxin in E. coli strain BL21 (DE3). Electrophoresis on SDS-PAGE 12% gel showed the desired band approximately at 38 KDa. Our results showed that recombinant epsilon toxin is mainly expressed as an inclusion body. Furthermore, 100, 150, and 200 µg/mL of mature epsilon toxin are significantly reduced the cell viability (P≤0.05). The considerable increase of necrotic cell percentage was shown after exposing to 100, 150, and 200 µg/mL of mature epsilon toxin (P≤0.05). CONCLUSION: The recombinant mature epsilon toxin had cytotoxic effects and could induce necrosis.

Efflux pump inhibitory activity of biologically synthesized silver nanoparticles against multidrug-resistant Acinetobacter baumannii clinical isolates.

This study was carried out to investigate the possible efflux pump inhibitory activity of biologically synthesized silver nanoparticles (AgNPs) against multidrug-resistant (MDR) Acinetobacter baumannii isolates. In this study, the physicochemical characteristics of synthesized AgNPs were investigated using scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), and Fourier transform infrared spectrophotometer (FTIR) methods. Subsequently, MDR A. baumannii isolates were recovered from clinical samples and the phenotypic cartwheel efflux assay and polymerase chain reaction (PCR) were used to elucidate the possible presence of efflux pump in MDR strains. After treatment of MDR strains with sub-minimum inhibitory concentration (MIC) concentration of AgNPs, the expression level of efflux pump genes was evaluated using a quantitative real-time PCR technique. The synthesized AgNPs had a spherical nanostructure, with mean size 38.89 nm, according to SEM and TEM data. XRD and FTIR results confirmed the synthesis of AgNPs. The results of PCR revealed that among 50 strains, 12 A. baumannii strains had efflux pump genes and the expression level of AdeA, AdeC, AdeS, AdeR, AdeI, AdeJ, and AdeK efflux pump genes was downregulated significantly after the treatment with AgNPs. In addition, the inhibitory effect of AgNPs on efflux pumps can be detected when the MIC of ethidium bromide (EtBr) with AgNPs is lower than that of EtBr alone. According to the results, the biologically synthesized AgNPs exhibit efflux pumps inhibitory activity, which may be one of the possible mechanisms of their antibacterial activity against MDR A. baumannii strains.

Folate conjugated hyaluronic acid coated alginate nanogels encapsulated oxaliplatin enhance antitumor and apoptosis efficacy on colorectal cancer cells (HT29 cell line).

Oxaliplatin (OXA) has been widely used for treatment of colorectal cancer. In this study, to enhance antitumor and apoptosis efficacy, OXA was encapsulated in a novel folate conjugated hyaluronic acid coated alginate nanogels (F/HA/AL/OXA). The F/HA/AL/OXA nanogels were prepared by cross-linking process. The physico-chemical properties of F/HA/AL/OXA nanogels were characterized using scanning electron microscopy, transmission electron microscopy, fourier transform infrared spectroscopy, dynamic light scattering, and fluorescent spectrophotometry. The in-vitro antitumor activity of free OXA, AL, HA/AL, HA/AL/OXA and F/HA/AL/OXA nanogels were assessed using MTT assay against colorectal cancer cells (HT29 cell line). Finally, the effect of F/HA/AL/OXA nanogels on genes expression of Bax and Bcl2 was evaluated using quantitative real-time polymerase chain reaction (qRT-PCR) technique. The F/HA/AL/OXA nanogels were 200.3 nm in diameter and had a zeta potential of -22.0 mv. Antitumor activity of F/HA/AL/OXA nanogels on HT29 cell line indicated the highest antitumor activity as compared to free OXA and the empty nanogels. Compared to free OXA and the empty nanogels, the expression of Bax in HT29 cells treated with F/HA/AL/OXA nanogels was significantly increased along with suppression of Bcl-2 (p < .01). In general, the present F/HA/AL/OXA nanogels are a promising carrier candidate for OXA to improve the anti-tumor activity.

Evaluation of point mutations in dystrophin gene in Iranian Duchenne and Becker muscular dystrophy patients: introducing three novel variants.

Duchenne and Becker muscular dystrophies (DMD and BMD) are X-linked neuromuscular diseases characterized by progressive muscular weakness and degeneration of skeletal muscles. Approximately two-thirds of the patients have large deletions or duplications in the dystrophin gene and the remaining one-third have point mutations. This study was performed to evaluate point mutations in Iranian DMD/BMD male patients. A total of 29 DNA samples from patients who did not show any large deletion/duplication mutations following multiplex polymerase chain reaction (PCR) and multiplex ligation-dependent probe amplification (MLPA) screening were sequenced for detection of point mutations in exons 50-79. Also exon 44 was sequenced in one sample in which a false positive deletion was detected by MLPA method. Cycle sequencing revealed four nonsense, one frameshift and two splice site mutations as well as two missense variants.

Molecular analysis of factor IX gene in an Iranian female with severe hemophilia B.

Hemophilia B, a recessive X-linked coagulopathy, is rare in females, and only a few cases have been reported so far. In this report, we describe a 9-year-old female, offspring of a consanguineous marriage, with a clinically severe course of hemophilia B and a normal 46,XX karyotype. Polymerase chain reaction and conformation sensitive gel electrophoresis techniques have been applied to the important regions of the factor IX gene,and an abnormal conformation sensitive gel electrophoresis profile was identified in exon 5 of the gene. After sequencing, the mutation was found to be C17761T (R116X) in homozygous form. Then, polymerase chain reaction-restriction fragment length polymorphism using the EcoRV restriction enzyme was applied for confirmation of the homozygous mutation in the proband and for carrier testing in the relatives. In addition, haplotype analysis was informative at the HhaI polymorphic site for the female patient.