Screening and identification of DNA nucleic acid aptamers against F1 protein of Yersinia pestis using SELEX method.

BACKGROUND: Yersinia pestis is a bacterium that causes the disease plague. It has caused the deaths of many people throughout history. The bacterium possesses several virulence factors (pPla, pFra, and PYV). PFra plasmid encodes fraction 1 (F1) capsular antigen. F1 protein protects the bacterium against host immune cells through phagocytosis process. This protein is specific for Y. pestis. Many diagnostic techniques are based on molecular and serological detection and quantification of F1 protein in different food and clinical samples. Aptamers are small nucleic acid sequences that can act as specific ligands for many targets.This study, aimed to isolate the high-affinity ssDNA aptamers against F1 protein. METHODS AND RESULTS: In this study, SELEX was used as the main strategy in screening aptamers. Moreover, enzyme-linked aptamer sorbent assay (ELASA) and surface plasmon resonance (SPR) were used to determine the affinity and specificity of obtained aptamers to F1 protein. The analysis showed that among the obtained aptamers, the three aptamers of Yer 21, Yer 24, and Yer 25 were selected with a KD value of 1.344E - 7, 2.004E - 8, and 1.68E - 8 M, respectively. The limit of detection (LoD) was found to be 0.05, 0.076, and 0.033 µg/ml for Yer 21, Yer 24, and Yer 25, respectively. CONCLUSION: This study demonstrated that the synthesized aptamers could serve as effective tools for detecting and analyzing the F1 protein, indicating their potential value in future diagnostic applications.

Unraveling the potential of CD8, CD68, and VISTA as diagnostic and prognostic markers in patients with pancreatic ductal adenocarcinoma.

Introduction: Pancreatic cancer is a truculent disease with limited treatment options and a grim prognosis. Immunotherapy has shown promise in treating various types of cancer, but its effectiveness in pancreatic cancer has been lacking. As a result, it is crucial to identify markers associated with immunological pathways in order to improve the treatment outcomes for this deadly cancer. The purpose of this study was to investigate the diagnostic and prognostic significance of three markers, CD8, CD68, and VISTA, in pancreatic ductal adenocarcinoma (PDAC), the most common subtype of pancreatic cancer. Methods: We analyzed gene expression data from Gene Expression Omnibus (GEO) database using bioinformatics tools. We also utilized the STRING online tool and Funrich software to study the protein-protein interactions and transcription factors associated with CD8, CD68, and VISTA. In addition, tissue microarray (TMA) and immunohistochemistry (IHC) staining were performed on 228 samples of PDAC tissue and 10 samples of normal pancreatic tissue to assess the expression levels of the markers. We then correlated these expression levels with the clinicopathological characteristics of the patients and evaluated their survival rates. Results: The analysis of the GEO data revealed slightly elevated levels of VISTA in PDAC samples compared to normal tissues. However, there was a significant increase in CD68 expression and a notable reduction in CD8A expression in pancreatic cancer. Further investigation identified potential protein-protein interactions and transcription factors associated with these markers. The IHC staining of PDAC tissue samples showed an increased expression of VISTA, CD68, and CD8A in pancreatic cancer tissues. Moreover, we found correlations between the expression levels of these markers and certain clinicopathological features of the patients. Additionally, the survival analysis revealed that high expression of CD8 was associated with better disease-specific survival and progression-free survival in PDAC patients. Conclusion: These findings highlight the potential of CD8, CD68, and VISTA as diagnostic and prognostic indicators in PDAC.

Evaluation of anti-biofilm activity of Lactobacillus rhamnosus GG and Nisin on the expression of aap, ica-A and ica-D as biofilm-associated genes of Staphylococcus epidermidis.

Background and Objectives: In the present study, the anti-biofilm activity of Lactobacillus rhamnosus GG and Nisin was investigated on biofilm-forming abilities of Staphylococcus epidermidis strains and the expression of the biofilm-associated genes. Materials and Methods: In this study, the standard strain of L. rhamnosus GG (ATCC 53103) and Nisin were used to assess their anti-microbial and anti-biofilm effects on S. epidermidis (RP62A). Results: The MIC and MBC analysis showed that Nisin at 256 µg/mL and 512 µg/mL, and L. rhamnosus GG at 1×107 CFU/mL and 1×108 CFU/mL have anti-microbial activity compared to the negative control respectively. L. rhamnosus GG bacteria and Nisin inhibited the biofilm formation of S. epidermidis based on optical density of at 570 nm (P <0.001). The relative mRNA expression of aap, icaA, and icaD genes was significantly reduced compared to the negative control after treating S. epidermidis with sub-MIC of Nisin (0.44, 0.25 and 0.6 fold, respectively) (P>0.05). In addition, the relative expression of aap and icaA genes, but not icaD (P>0.05), was significantly lower than the negative control (0.62 and 0.7 fold, respectively) (P>0.05), after exposure to the sub MIC of L. rhamnosus GG. Conclusion: Nisin and L. rhamnosus GG exhibit potent activity against biofilm-forming abilities of S. epidermidis and these agents could be utilized as an anti-biofilm agents against S. epidermidis infections.

Dielectric barrier discharge plasma catalysis as an alternative approach for the synthesis of ammonia: a review.

Numerous researchers have attempted to provide mild reactions and environmentally-friendly methods for NH3 synthesis. Research on non-thermal plasma-assisted ammonia synthesis, notably the atmospheric-pressure nonthermal plasma synthesis of ammonia over catalysts, has recently gained attention in the academic literature. Since non-thermal plasma technology circumvents the existing crises and harsh conditions of the Haber-Bosch process, it can be considered as a promising alternative for clean synthesis of ammonia. Non-thermal dielectric barrier discharge (DBD) plasma has been extensively employed in the synthesis of ammonia due to its particular advantages such as the simple construction of DBD reactors, atmospheric operation at ambient temperature, and low cost. The combination of this plasma and catalytic materials can remarkably affect ammonia formation, energy efficiency, and the generation of by-products. The present article reviews plasma-catalysis ammonia synthesis in a dielectric barrier discharge reactor and the parameters affecting this synthesis system. The proposed mechanisms of ammonia production by this plasma catalysis system are discussed as well.

Antimicrobial properties of green synthesized novel TiO2 nanoparticles using Iranian propolis extracts.

The oral antimicrobial and cytotoxic properties of green synthesized novel titanium dioxide nanoparticles (TiO2 NPs) using Iranian propolis extracts were investigated on oral bacteria and fibroblast cells. In this study, propolis was sampled, and alcoholic extracts were prepared. The TiO2 NPs were biosynthesized using propolis extracts. The synthesized TiO2 NPs were characterized by scanning electron microscope (SEM), X-ray diffraction analysis, energy-dispersive X-ray (EDX), Fourier transform infrared spectroscopy (FTIR), dynamic light scattering, ultraviolet-visible (UV-Vis), transmission electron microscope, Brunauer-Emmett-Teller, and zeta potential. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide), minimal inhibitory concentration, minimum bactericidal concentration, minimum fungicidal concentration, biofilm formation, and degradation tests were studied to clarify the oral antimicrobial properties of green synthesized TiO2  NPs. According to the FTIR analysis, the propolis extract contained flavonoids and phenolic compounds in addition to TiO2 NPs. Additionally, UV-Vis revealed that intense bands had formed NPs. EDX spectra and SEM images revealed that the stabilizing agent was in perfect quasi-spherical shapes around 21 nm. An EDX spectrum was used to verify the presence of titanium and oxygen. There were no significant cytotoxicity effects. The antibacterial results showed that Pro1TiO2 (Khalkhal sample) had better effects than Pro2TiO2 (Gilan sample) and TiO2 NPs. The present study presents a new process for synthesizing TiO2 NPs from propolis extracts with less toxic effects and user-friendly, eco-friendly, and economical materials. Pro1TiO2 NPs may be considered the best candidate for clinical application.

The Anti-Adhesion Effect of Nisin as a Robust Lantibiotic on the Colorectal Cancer Cells.

Background: Bacteriocins are a type of antimicrobial peptide that are produced by probiotics. They have been studied as possible therapeutic drugs and have been used to suppress bacterial development in foods. Nisin is a potent bacteriocin having the anti-microbial and anti-cancer characteristics produced by Lactococcus lactis. The aim of the present paper is to evaluate the influence of Nisin on cell adhesion and its two related genes, mmp-2 and mmp-9, in the colorectal cancer cell line. Materials and Methods: For this purpose, HT-29 cells were treated with various concentrations of Nisin and the cell cytotoxicity, cell adhesion, and gene expression were evaluated using the MTT assay, cell adhesion assay, and real-time PCR. Results: Our findings showed that 32 to 1024 µg/ml of Nisin resulted in a significant reduction in cell viability (P < 0.05). Furthermore, 128 and 256 µg/ml of Nisin significantly reduced the cell adhesion, and mmp-2 and mmp-9 gene expressions (P < 0.05). Conclusion: Our findings suggested that Nisin could prevent metastasis and cancer progression.

Study of the Apoptotic Impacts of Hazelnut Oil on the Colorectal Cancer Cell Line.

Background: Hazelnut oil has a unique structure with a high oleic acid content, tocopherol, tocotrienols, and other bioactive compounds, such as phytosterols. These biochemical compounds have been widely studied because of their potential health properties. Understanding the process of apoptosis is the basis of new therapies contributing to cancer cells' death. Recently, the potential role of the evolutionary-reserved bcl-2 protein family in tumor progression and prognosis of some malignancies has been addressed in several studies. The present study is aimed at evaluating the effect of apoptotic properties of hazelnut oil on colorectal cancer cells through the major members of this family (bax and bcl-2). Materials and Methods: MTT assay, apoptotic cell staining (using Annexin V and propidium iodide), flow cytometry, and real-time PCR were used to evaluate the toxicity, percentage of apoptotic cells, and bax and bcl-2 genes' expression after exposing HT29 cells to hazelnut oil. Results: After hazelnut treatment, significant decreases in cell viability, and the gene expression of bax and bcl-2 were observed compared to the control group (P < 0.05). In addition, the total percentage of apoptotic cells after hazelnut oil treatment showed a significant increase in comparison with the negative control group (P < 0.05). Conclusion: Hazelnut oil appears to cause the death of cancerous cells through an apoptotic mechanism.

The effect of oral capsule of Ocimum basilicum leaf extract on sleep quality and insomnia severity in menopausal women: A randomized clinical trial.

One of the complications of menopause is sleep disorders, which affect women's health. Ocimum basilicum contains compounds that may affect sleep. The aim of this study was to determine the effect of an oral capsule of O. basilicum leaf extract on sleep quality and the severity of insomnia in menopausal women. This triple-blind, randomized clinical trial study was performed on 60 Iranian menopausal women aged 40 to 65 years. Subjects were randomly assigned into two groups of intervention (each capsule containing 250 mg of O. basilicum extract and 250 mg Avicel) per day for 1 month and placebo. The Pittsburgh Sleep Quality and Insomnia Intensity Index were used to assess sleep quality and severity of insomnia before, 2 weeks after and 1 month after the intervention. There was no statistically significant difference in the baseline variables between the intervention and placebo groups (p > .05). The total sleep quality scores in the two groups of intervention and placebo were 6.2 ± 0.3 versus 9.3 ± 0.3 (p < .001) and 3.7 ± 0.3 versus 9.1 ± 0.3 (p = .015) 2 weeks and 1 month after the intervention, respectively. The total insomnia severity scores in the two groups of intervention and placebo were 9.0 ± 0.3 versus 12.1 ± 0.3 (p < .001) and 5.6 ± 0.5 versus 11.0 ± 0.5 (p < .001) 2 weeks and 1 month after the intervention, respectively. Consumption of O. basilicum capsules improved sleep quality and insomnia in menopausal women. This study was approved (code IR.MUMS.NURSE.REC.1398.070) by the Ethic committee of Mashhad University of Medical Sciences and registered at the Iranian Registry of Clinical Trials, with the No. IRCT20200104046001N1 in January 2020.

Exercise Training and Probiotic Lacticaseibacillus rhamnosus GG Reduce Tetracycline-Induced Liver Oxidative Stress and Inflammation in Rats with Hepatic Steatosis.

Lifestyle modification with regular exercise can improve metabolic diseases by reducing lipid profile and inflammation. Probiotics have been recently recommended not only for gastrointestinal diseases but also for metabolic and even degenerative diseases. Therefore, in the present study, the effect of high-intensity interval training (HIIT) and Lacticaseibacillus rhamnosus strain GG (LGG) as a probiotic on tetracycline-induced hepatic steatosis in an animal model was evaluated. Eighty male Wistar rats were randomly divided into eight groups (n = 10 in each group): control, LGG, HIIT, LGG + HIIT, tetracycline-induced (TTC), TTC + LGG, TTC + HIIT, and TTC + LGG + HIIT. The rats are treated by intraperitoneal injection (IP) with 140 mg kg-1 tetracycline, an antibiotic previously known to induce steatosis. The exercise training groups performed HIIT 5 days/week for 5 weeks, and 107 CFU/ml of Lacticaseibacillus rhamnosus GG was gavaged for the LGG groups 5 days/week for 5 weeks. Fatty droplets in the hepatocyte were considered with Oil Red staining. TTC-receiving rats have more lipid accumulation and larger lipid droplets in the liver compared to healthy animals. The two-way ANOVA showed that the interaction of LGG and HIIT significantly decreased LDL, cholesterol, and triglyceride in the healthy rats (p < 0.05). In TTC-receiving rats, the interaction of LGG and HIIT significantly increased HDL and SOD and significantly decreased triglyceride, ALP, AST, and ALT (p < 0.05). The consumption of probiotic LGG, along with HIIT with control of lipid profile and liver enzymes and improvement of the oxidative capacity, neutralizes the damage of TTC to liver tissue. Therefore, this protocol can be recommended for people with hepatic steatosis.

Screening and Identification of DNA Nanostructure Aptamer Using the SELEX Method for Detection of Epsilon Toxin.

Background: Epsilon toxin (ETX), produced by Clostridium perfringens, is one of the most potent toxins known, with a lethal potency approaching that of botulinum neurotoxins. Epsilon toxin is responsible for enteritis. Therefore, the development of rapid and simple methods to detect ETX is imperative. Aptamers are single-stranded oligonucleotides that can bind tightly to specific target molecules with an affinity comparable to that of monoclonal antibodies (mAbs). DNA aptamers can serve as tools for the molecular identification of organisms, such as pathogen subspecies. Objectives: This study aimed to isolate high-affinity single-stranded DNA (ssDNA) aptamers against ETX. Methods: This study identified aptamers using the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method, enzyme-linked apta-sorbent assay (ELASA), and surface plasmon resonance (SPR) to determine the affinity and specificity of the newly obtained aptamers targeting ETX. Results: Several aptamers obtained through the SELEX process were studied. Among them, 2 aptamers, ETX clone 3 (ETX3; dissociation constant (Kd = 8.4 ± 2.4E-9M) and ETX11 (Kd = 6.3 ± 1.3E-9M) had favorable specificity for ETX. The limits of detection were 0.21 and 0.08 µg/mL for ETX3 and ETX11, respectively. . Conclusions: The discovered aptamers can be used in various aptamer-based rapid diagnostic tests for the detection of ETX.

Ameliorative effects of high intensity interval training and Lactobacillus rhamnosus GG Protect against tetracycline-induced fatty liver in rats: a gene expression profiling comparative study.

Exercise training and probiotics have been suggested as a treatment for the prevention of chronic liver damage such as non-alcoholic fatty liver disease (NAFLD). Lactobacillus rhamnosus Gorbach - Goldin (LGG) is one of the most widely used probiotic strains that decreases liver damage. Thus, this study aims to consider the ameliorative effects of high intensity interval training (HIIT) and LGG against tetracycline-induced fatty liver in rats. Eighty male Wistar rats were randomly divided into 8 groups of (n=10 each group): control, LGG, HIIT, LGG+HIIT, NAFLD, NAFLD+LGG, NAFLD+HIIT, and NAFLD+LGG+HIIT. The rats are treated by intraperitoneal injection with 140 mg/kg-1 tetracycline, an antibiotic previously known to induce steatosis. The exercise training groups performed HIIT 5 days/week for 5 weeks. 107 colony-forming units (cfu) of LGG were gavaged for LGG groups 5 days/week for 5 weeks. Probiotic supplementation in combination with interval training significantly decreased tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) mRNA and matrix metalloproteinase-2 (MMP-2) mRNA in the liver (p<0.05), while the levels of lysosomal acid lipase (LIPA) mRNA was significantly increased compared to NAFLD group. Also, compared with NAFLD group, NAFLD+LGG, NAFLD+HIIT and NAFLD+LGG+HIIT groups showed a significant decrease in hepatic monocyte chemoattractant protein-1 (MCP-1). Compared to LGG and LGG+HIIT groups, all NAFLD groups showed a significant decrease in apolipoprotein C3 (apoc3) in liver tissue (p<0.05). The results suggested that interval exercise with LGG supplementation minimizes cell destruction and inflammation in liver tissue due to NAFLD by improving gene expression profiles.

Green Synthesis of Silver Nanoparticles Using Nisin and its Antibacterial Activity against Pseudomonas aeruginosa.

Background: Green synthesized silver nanoparticles (AgNPs) have been used in a wide range of biological applications, including their use as antimicrobial agents. The aim of this study was to evaluate the antibacterial activity of green synthesis AgNPs using nisin against Pseudomonas aeruginosa (P. aeruginosa). Materials and Methods: In order to synthesize Ag-nisin, a 1 mg/ml nisin solution was mixed with a 1-mM silver nitrate solution and incubated. The Fourier transform infrared spectroscopy (FTIR) analysis was employed to determine the presence of various biomolecules around AgNPs. The AgNPs were morphologically observed and characterized using field emission scanning electron microscopy assessment, dynamic light scattering (DLS), and zeta potential analysis. The microdilution broth method based on CLSI principles was used for the assessment of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of nisin on P. aeruginosa isolates. Results: Field emission scanning electron microscope showed spherical shaped nanoparticles. DLS revealed that the average size of nanoparticles was 37.2 nm. The zeta potential of AgNPs was - 13.3 mV. FTIR findings revealed that nitrogen atoms of nisin's amine and amide groups are responsible for the capping and stability of the nanoparticles. The MIC and MBC showed that Ag/nisin nanoparticles had higher antimicrobial activity than nisin or AgNPs alone. Conclusion: The findings of this study show that the antibacterial activity of nisin can be increased by assembling it into the AgNP interface using a green chemical synthesis method. As a result, the technique may be used to develop an antibacterial formulation to enhance the effectiveness of nisin.

Design of two immunotoxins based rovalpituzumab antibody against DLL3 receptor; a promising potential opportunity.

Background and purpose: The lack of a new effective treatment for small cell lung cancer (SCLC) is an unresolved problem. Due to the new identification of delta-like ligand 3 (DLL3) and its high expression in SCLC patients, the use of DLL3 in target therapy can be effective. The use of bacterial toxins belonging to the ADP-ribosyl transferase toxins family and human enzymes to remove cancerous cells has been effective in the structure of immunotoxins. In this study, single-chain fragment variable of rovalpituzumab antibody fused to granzyme B (Rova-GrB) and PltA of typhoid toxin (Rova-Typh) as immunotoxins were designed, and bioinformatics analysis was done. Experimental approach: In silico analysis including the physicochemical properties, evaluation of the secondary and tertiary structure, refinement and validation of 3D models, and docking were performed. Immunotoxin genes were cloned and expressed in the Escherichia coli BL21 (DE3) host, purified, subsequently confirmed by western blotting and their secondary structure was evaluated by the circular dichroism method. Findings/Results: The bioinformatics analysis showed that Rova-GrB and Rova-Typh had hydrophilic properties, their codon optimization parameters were standard, validation parameters were improved after immunotoxin refinement, and docking analysis showed that the binding domain of immunotoxins could bind the N-terminal region of DLL3. immunotoxins had high expression and after purification under denaturing condition by Ni-NTA column, the immunotoxins were dialyzed against PBS buffer. Conclusion and implications: The immunotoxins had the right structure and can be produced in a prokaryotic host. The recombinant immunotoxins against DLL3 can be promising therapeutic agents for SCLC cancer.

Effect of extracellular vesicles of Lactobacillus rhamnosus GG on the expression of CEA gene and protein released by colorectal cancer cells.

Background and Objectives: Almost all living cells secret nano-sized structures enclosed by the lipid bilayer called extracellular vesicles (EVs) into their extracellular milieu. These EVs play important roles in several physiological processes as a cargo delivery system. In probiotics, EVs are the main communication tool with the host. The present study aimed to assess the effect of EVs originated from Lactobacillus rhamnosus GG on the Carcinoembryonic antigen (cea) gene expression and protein (CEA) synthesis in the SW480 and HT-29 cell lines. Materials and Methods: Different concentrations of Lactobacillus rhamnosus GG EVs were applied on the SW480 and HT-29 cell lines. The MTT assay, Real-Time PCR, and ELISA analysis methods were exploited to explore the cell viability and the expression level of the cea gene in comparison with the ß-actin gene as the control. Results: The two concentrations of 80 and 100 µg/ml of Lactobacillus rhamnosus GG EVs considerably affected the anti-proliferation and increased the amount of both CEA mRNA and protein (p < 0.05). Conclusion: Our findings showed that EVs of Lactobacillus rhamnosus GG could induce the gene expression and protein synthesis of CEA. Also, they reduced the cell proliferation of HT29 and SW480. Thus, probiotics such as EVs of Lactobacillus rhamnosus GG could be useful for preventing colorectal cancer.

Chemical Characterization and Cytotoxic/Antibacterial Effects of Nine Iranian Propolis Extracts on Human Fibroblast Cells and Oral Bacteria.

Multimicrobial infections caused by pathobionts are called dysbiotic multimicrobial illnesses. Commercial mouthwashes, such as chlorhexidine, have negative side effects that can prevent tooth decay and infection. The present study aimed to determine the antifungal, antibacterial, and cytotoxicity characteristics of the propolis extracts from different areas (Iran). The ethanolic extract of propolis was prepared. GC/MS carried out the characterization to determine the thymol, carvacrol, and menthol extracts, and also, total phenol and flavonoid were assed for all samples. The antimicrobial and antibiofilm effects were evaluated against S. mutans, S. mitis, S. salivarius, L. acidophilus, E. coli, S. aureus, and C. albicans. The cytotoxic effect of extracts was measured on human fibroblast cells by MTT test. The MIC values in mg mL-1 were ranged as follows: S. salivarius (0.003 to 0.048), S. mutans (0.003 to 0.029), S. mitis (0.007 to 0.058), L. acidophilus (0.007 to 0.117), C. albicans (0.014 to 0.234), E. coli (0.007 to 0.058), and S. aureus (0.007 to 0.058), while MBC were, respectively, S. mutans (0.007 to 0.058), S. salivarius (0.007 to 0.117), S. mitis (0.007 to 0.117), L. acidophilus (0.014 to 0.234), C. albicans (0.029 to 0.468), E. coli (0.014 to 0.234), and S. aureus (0.007 to 0.117). Cariogenic bacteria and Candida albicans were demonstrated to be resistant to propolis extracts. Therefore, propolis extracts may make good mouthwashes.

Targeting the EGFR in cancer cells by fusion protein consisting of arazyme and third loop of TGF-alpha: an in silico study.

The anticancer effects of arazyme, a bacterial metalloprotease, have been revealed in previous studies. Because of the overexpression of epidermal growth factor receptor (EGFR) in tumor cells, targeting this receptor is one of the approaches to cancer therapy. In the present study, we designed fusion protein by using a non-mitogenic binding sequence of TGFα, arazyme, and a suitable linker. The I-TASSER and Robetta web servers were employed to predict the territory structures of the Arazyme-linker-TGFαL3, and TGFαL3-linker-Arazyme. Then, models were validated by using PROCHECK, ERRAT, ProSA, and MolProbity web servers. After docking to EGFR, Arazyme-linker-TGFαL3 showed a higher binding affinity and was selected to be optimized through 100 ns Molecular dynamic (MD) simulation. Next, the stability of ligand-bound receptor was examined utilizing MD simulation for 100 ns. Furthermore, the binding free energy calculation and free energy decomposition were carried out employing MM-PBSA and MM-GBSA methods, respectively. The root mean square deviation and fluctuation (RMSD, RMSF), the radius of gyration, H-bond, and binding free energy analysis revealed the stability of the complex during simulation time. Finally, linear and conformational epitopes of B cells, as well as MHC class I and MHC class II were predicted by using different web servers. Meanwhile, the potential B cell and T cell epitopes were identified throughout arazyme protein sequence. Collectively, this study suggests a novel chimera protein candidate prevent cancer cells potentially by inducing an immune response and inhibiting cell proliferation. Communicated by Ramaswamy H. Sarma.

ARA-linker-TGFαL3: a novel chimera protein to target breast cancer cells.

Targeted cancer therapies based on overexpressed receptors and the fractions containing immunotoxins and bacterial metabolites are one of the well-known methods to overcome the chemotherapy resistance of cancer cells. In this paper, we designed ARA-linker-TGFαL3, using Arazyme, a Serratia proteamaculans metabolite, and a third loop segment of TGFα to target EGFR-expressing breast cancer cells. After cloning in pET28a (+), the expression of recombinant protein was optimized in Escherichia coli strain BL21 (DE3). MDA-MB-468 (EGFR positive) and MDA-MB-453 (EGFR negative) breast cancer cell lines were employed. Also, the chemotherapeutic drug, Taxotere (Docetaxel), was employed to compare cytotoxicity effects. Cell ELISA assessed the binding affinity of recombinant proteins to the receptor, and the cytotoxicity was detected by MTT and lactate dehydrogenase release assays. The interfacing with cancer cell adhesion was evaluated. Furthermore, the induction of apoptosis was examined utilizing flow cytometric analysis, and caspase-3 activity assay. Moreover, RT-PCR was conducted to study the expression of apoptosis (bax, bcl2, and casp3), angiogenesis (vegfr2), and metastasis (mmp2 and mmp9) genes. ARA-linker-TGFαL3 revealed a higher binding affinity, cytotoxicity, and early apoptosis induction in MDA-MB-468 cells compared to the effects of Arazyme while both recombinant proteins showed similar effects on MDA-MB-453. In addition, the Taxotere caused the highest cytotoxicity on cancer cells through induction of late apoptosis. Meanwhile, the expression of angiogenesis and metastasis genes was decreased in both cell lines after treatment with either ARA-linker-TGFαL3 or Arazyme. Our in vitro results indicated the therapeutic effect of ARA-linker-TGFαL3 on breast cancer cells.

Design, Dimerization, and Recombinant Production of MCh-AMP1-Derived Peptide in Escherichia coli and Evaluation of Its Antifungal Activity and Cytotoxicity.

Fungal species resistant to current antifungal agents are considered as a serious threat to human health, the dilemma that has dragged attentions toward other sources of antifungals such as antimicrobial peptides (AMPs). In order to improve biological activity of a recently described antifungal peptide MCh-AMP1 from Matricaria chamomilla flowers, MCh-AMP1dimer (DiMCh-AMP1), containing 61 amino acid residues connected by flexible linker (GPDGSGPDESGPDES), was designed and expressed in Escherichia coli, and its structure was analyzed using bioinformatics tools. DiMCh-AMP1 synthetic gene was cloned into pET-28a expression vector, which was then used to transform E. coli BL21 (DE3) strain. His-tag purification was achieved using metal-chelate affinity chromatography. Because there is no methionine residue in the DiMCh-AMP1 sequence, cyanogen bromide was successfully used to separate the target product from the tag. Reverse-phase high-performance liquid chromatography was used as the final step of purification. Results showed that recombinant peptide was produced in considerable amounts (0.9 mg/L) with improved antifungal activity toward both yeasts and molds compared to its monomeric counterpart. The minimum inhibition concentration and minimum fungicidal concentration values of DiMCh-AMP1 against Candida and Aspergillus species were reported in the range of 1.67-6.66 µM and 3.33-26.64 µM, respectively. Our results showed that while antifungal activity of dimerized peptide was improved considerably, its cytotoxicity was decreased, implying that DiMCh-AMP1 could be a potential candidate to design an effective antifungal agent against pathogenic yeasts and molds.

Positive Association of Matrix Proteins Alteration with TAZ and The Progression of High-Grade Bladder Cancer.

OBJECTIVE: Bladder cancer is the 9th leading cause of human urologic malignancy and the 13th cause of death worldwide. Increased collagen cross-linking, NIDOGEN1 expression and consequently stiffness of extracellular matrix (ECM) may be responsible for the mechanotransduction and regulation of transcriptional co-activator with PDZ-binding motif (TAZ) and transforming growth factor ß1 (TGF-ß1) signaling pathways, resulting in progression of tumorigenesis. The present study aimed to assess whether type 1 collagen expression is associated with TAZ nuclear localization. MATERIALS AND METHODS: In this case-control study, real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemical analysis were performed to evaluate the activation of the TAZ pathway in patients with bladder cancer (n=40) and healthy individuals (n=20). The ELISA method was also conducted to measure the serum concentrations of TGF-ß1. Masson's trichrome staining was carried out to histologically evaluate the density of type 1 collagen. RESULTS: Our findings that the expression levels of COL1A1, COL1A2, NIDOGEN1, TAZ, and TGF-ß1 genes were overexpressed in patients with bladder cancer, and their expression levels were positively associated with the grade of bladder cancer. The immunohistochemical analysis demonstrated that the nuclear localization of TAZ was markedly correlated with high-grade bladder cancer. We also found that TAZ nuclear localization was substantially higher in cancerous tissues as compared with normal bladder tissues. Masson's trichrome staining showed that the tissue density of type I collagen was considerably increased in patients with bladder cancer as compared with healthy subjects. CONCLUSION: According to our findings, it seems the alterations in the expression of type I collagen and NIDOGEN1, as well as TAZ nuclear localization influence the progression of bladder cancer. The significance of TGF-ß1 and TAZ expression in tumorigenesis and progression to high-grade bladder cancer was also highlighted. However, a possible relationship between TGF-ß1 expression and the Hippo pathway needs further investigations.

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.