MicroRNA-561-3p indirectly regulates the PD-L1 expression by targeting ZEB1, HIF1A, and MYC genes in breast cancer.

Globally, breast cancer is the second most common cause of cancer-related deaths among women. In breast cancer, microRNAs (miRNAs) are essential for both the initiation and development of tumors. It has been suggested that the tumor suppressor microRNA-561-3p (miR-561-3p) is crucial in arresting the growth of cancer cells. Further research is necessary to fully understand the role and molecular mechanism of miR-561 in human BC. The aim of this study was to investigate the inhibitory effect of miR-561-3p on ZEB1, HIF1A, and MYC expression as oncogenes that have the most impact on PD-L1 overexpression and cellular processes such as proliferation, apoptosis, and cell cycle in breast cancer (BC) cell lines. The expression of ZEB1, HIF1A, and MYC genes and miR-561-3p were measured in BC clinical samples and cell lines via qRT-PCR. The luciferase assay, MTT, Annexin-PI staining, and cell cycle experiments were used to assess the effect of miR-561-3p on candidate gene expression, proliferation, apoptosis, and cell cycle progression. Flow cytometry was used to investigate the effects of miR-561 on PD-L1 suppression in the BC cell line. The luciferase assay showed that miRNA-561-3p targets the 3'-UTRs of ZEB1, HIF1A and MYC genes significantly. In BC tissues, the qRT-PCR results demonstrated that miR-561-3p expression was downregulated and the expression of ZEB1, HIF1A and MYC genes was up-regulated. It was shown that overexpression of miR-561-3p decreased PD-L1 expression and BC cell proliferation, and induced apoptosis and cell cycle arrest through downregulation of candidate oncogenes. Furthermore, inhibition of candidate genes by miR-561-3p reduced PD-L1 at both mRNA and protein levels. Our research investigated the impact of miR-561-3p on the expression of ZEB1, HIF1A and MYC in breast cancer cells for the first time. Our findings may help clarify the role of miR-561-3p in PD-L1 regulation and point to this miR as a potential biomarker and novel therapeutic target for cancer immunotherapy.

Activation of AMPK promotes cardiac differentiation by stimulating the autophagy pathway.

Autophagy, a critical catabolic process for cell survival against different types of stress, has a role in the differentiation of various cells, such as cardiomyocytes. Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) is an energy-sensing protein kinase involved in the regulation of autophagy. In addition to its direct role in regulating autophagy, AMPK can also influence other cellular processes by regulating mitochondrial function, posttranslational acetylation, cardiomyocyte metabolism, mitochondrial autophagy, endoplasmic reticulum stress, and apoptosis. As AMPK is involved in the control of various cellular processes, it can influence the health and survival of cardiomyocytes. This study investigated the effects of an AMPK inducer (Metformin) and an autophagy inhibitor (Hydroxychloroquine) on the differentiation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). The results showed that autophagy was upregulated during cardiac differentiation. Furthermore, AMPK activation increased the expression of CM-specific markers in hPSC-CMs. Additionally, autophagy inhibition impaired cardiomyocyte differentiation by targeting autophagosome-lysosome fusion. These results indicate the significance of autophagy in cardiomyocyte differentiation. In conclusion, AMPK might be a promising target for the regulation of cardiomyocyte generation by in vitro differentiation of pluripotent stem cells.

A comparative study of the arazyme-based fusion proteins with various ligands for more effective targeting cancer therapy: an in-silico analysis.

Background and purpose: Recently, the use of immunotoxins for targeted cancer therapy has been proposed, to find new anticancer drugs with high efficacy on tumor cells with minimal side effects on normal cells. we designed and compared several arazyme (AraA)-based fusion proteins with different ligands to choose the best-targeted therapy for interleukin 13 receptor alpha 2 (IL13Rα2)-overexpressed cancer cells. For this purpose, IL13Rα2 was selected as a receptor and IL13 and IL13.E13K were evaluated as native and mutant ligands, respectively. In addition, Pep-1 and A2b11 were chosen as the peptide ligands for targeted cancer therapy. Experimental approach: Several bioinformatics servers were used for designing constructs and optimization. The structures of the chimeric proteins were predicted and verified by I-TASSER, Q-Mean, ProSA, Ramachandran plot, and Verify3D program. Physicochemical properties, toxicity, and antigenicity were predicted by ProtParam, ToxinPred, and VaxiJen. HawkDock, LigPlot+, and GROMACS software were used for docking and molecular dynamics simulation of the ligand-receptor interaction. Findings/Results: The in silico results showed AraA-A2b11 has higher values of confidence score and Q-mean score was obtained for high-resolution crystal structures. All chimeric proteins were stable, non-toxic, and non-antigenic. AraA-(A(EAAAK)4ALEA(EAAAK)4A)2-IL13 retained its natural structure and based on ligand-receptor docking and molecular dynamic analysis, the binding ability of AraA-(A(EAAAK)4ALEA(EAAAK)4A)2-IL13 to IL13Rα2 was sufficiently strong. Conclusion and implications: Based on the bioinformatics result AraA-(A(EAAAK)4ALEA(EAAAK)4A)2-IL13 was a stable fusion protein with two separate domains and high affinity with the IL13Rα2 receptor. Therefore, AraA-(A(EAAAK)4ALEA(EAAAK)4A)2-IL13 fusion protein could be a new potent candidate for target cancer therapy.

Aberrant promoter hypermethylation of miR-335 and miR-145 is involved in breast cancer PD-L1 overexpression.

PD-L1 is one of the most important immune checkpoint molecules in breast cancer that plays an important role in suppressing the immune system when confronted with tumor cells and is regulated by various microRNAs. Among them, microRNA-335-3p and microRNA-145-5p, regulated by DNA methylation, have tumor suppressor activities. We studied the role of miR-335 and -145 on PD-L1 suppression in breast cancer. The expression of miR-355 and miR-145 was significantly downregulated in BC tissues and cell lines compared to their controls, and their downregulation was negatively correlated with PD-L1 overexpression. In-silico and luciferase reporter systems confirmed that miR-335 and -145 target PD-L1. In BC tissues and cell lines, cancer-specific methylation was found in CpG-rich areas upstream of miR-335 and-145, and up-regulation of PD-L1 expression was connected with hypermethylation (r = 0.4089, P = 0.0147, and r = 0.3373, P = 0.0475, respectively). The higher levels of miR-355 and -145 in BC cells induced apoptosis, arrested the cell cycle, and reduced proliferation significantly. In summary, we found that miR-335 and -145 are novel tumor suppressors inactivated in BC, and these miRs may serve as potential therapeutic targets for breast cancer treatment.

Effect of Carvacrol on histological analysis and expression of genes involved in an animal model of multiple sclerosis.

BACKGROUND: The most common non-traumatic neurological disease in young- and middle-aged adults is multiple sclerosis (MS), leading to central nervous system (CNS) atrophy and neurological disorders with loss of myelin and axonal degeneration. Due to the inadequate efficiency of common treatments, some natural products with antioxidant properties such as Carvacrol have been considered. OBJECTIVE: the present study aimed to investigate carvacrol's anti-inflammatory and therapeutic effects on MS symptoms in healthy and experimental autoimmune encephalomyelitis (EAE) induced female Lewis rats. METHODS: The study was performed in three groups of Lewis rats: control group, EAE model, and EAE treated with carvacrol (carvacrol-treated group). The treatment group received 25 mg/kg of carvacrol intraperitoneally daily. Histologic examination and expression analysis of pro-inflammatory genes (Interleukin-1 and 17 (IL-1 and IL-17), Nuclear Factor Kappa B Cells (NF-κB) and Tumor Necrosis Factor-α (TNF-α)), myelin repair, and also regeneration genes (Myelin basic protein (MBP), Oligodendrocyte Transcription Factor 2 (OLIG2) and Platelet-Derived Growth Factor Receptor α (PDGFR-α)) were carried out. Gene studies, Hematoxylin and Eosin (H&E), and Luxol fast blue stain were performed in the lumbar region of the spinal cord. RESULTS: The EAE clinical scores in the carvacrol-treated group were lower than in untreated rats (P < 0.001). The expression of two genes, IL-17 and MBP, was confirmed using fluorescence immunohistochemistry (FIHC). A significant decrease was observed in NF-κB and IL-17, and IL-1 gene expression. The MBP and OLIG2 gene expression was increased in the carvacrol-treated group (p < 0.001). In EAE, PDGFR-α expression increased about four times. However, carvacrol administration did not affect PDGFR-α and TNF-α gene expression. In this treatment, H&E staining of spinal cord regions showed a significant decrease in inflammatory cell infiltration. Moreover, immunostaining analysis demonstrated a considerable increase in MBP and a reduction in IL-17 secretion. CONCLUSION: The results showed that carvacrol administration reduces the entry of inflammatory cells into the CNS by stimulating myelination-related processes employing increasing the expression of genes involved in myelin repair and reducing the expression of inflammatory genes. Our findings confirm that carvacrol improves the clinical and pathological symptoms of EAE through its therapeutic and modification properties as a potential adjunctive therapy and needs to be studied more.

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.

Silibinin exhibits anti-tumor effects in a breast cancer stem cell model by targeting stemness and induction of differentiation and apoptosis.

Introduction: Malignant breast cancer (BC) frequently contains a rare population of cells called cancer stem cells which underlie tumor relapse and metastasis, and targeting these cells may improve treatment options and outcomes for patients with BC. The aim of the present study was to determine the effect of silibinin on the self-renewal capacity, tumorgenicity, and metastatic potential of mammospheres. Methods: The effect of silibinin on viability and proliferation of MCF-7, MDA-MB-231 mammospheres, and MDA-MB-468 cell aggregation was determined after 72-120 hours of treatment. Colony and sphere formation ability, and the expression of stemness, differentiation, and epithelial-mesenchymal-transition (EMT)-associated genes were assessed by reverse transcription-quantitative polymerase chain reaction (qRT-PCR) in mammospheres treated with an IC50 dose of silibinin. Additionally, the antitumor capacity of silibinin was assessed in vivo, in mice. Results: The results of the present study showed that silibinin decreased the viability of all mammospheres derived from MCF-7, MDA-MB-231, and MDA-MB-468 cell aggregation in a dose-dependent manner. Colony and sphere-forming ability, as well as the expression of genes associated with EMT were reduced in mammospheres treated with silibinin. Additionally, the expression of genes associated with stemness and metastasis was also decreased and the expression of genes associated with differentiation were increased. Intra-tumoral injection of 2 mg/kg silibinin decreased tumor volumes in mice by 2.8 fold. Conclusion: The present study demonstrated that silibinin may have exerted its anti-tumor effects in BC by targeting the BC stem cells, reducing the tumorgenicity and metastasis. Therefore, silibinin may be a potential adjuvant for treatment of BC.

Melatonin Sensitizes OVCAR-3 Cells to Cisplatin through Suppression of PI3K/Akt Pathway.

This study examined the effect of melatonin on oxidative stress, expression of pro-apoptotic protein, anti-apoptotic proteins, and the activity of the PI3K/Akt signaling pathway in the human ovarian cancer cell line (OVCAR3). OVCAR3 cells were treated with cisplatin, melatonin, cisplatin + melatonin, and siRNA Akt. Reactive oxygen species levels were assessed. The expression of the proteins was determined by Western blot. Melatonin administration significantly increased intracellular ROS generation, the cleavage of caspase 3 and decreased phosphorylation of Akt. Combination therapy of cisplatin and melatonin increases apoptosis in the OVCAR-3 cells by inhibiting of PI3K/Akt signaling pathway and exacerbating oxidative stress.

Prokaryotic expression, evaluation, and prediction of the structure and function of the ecarin metalloproteinase domain.

Ecarin is one of the most widely used drug compounds in blood clotting experiments and is used to monitor and treat many diseases such as cancer, liver, lupus, and cardiovascular disease. The metalloproteinase domain is known as the active site of ecarin. In this study, an ecarin metalloproteinase cassette was designed and synthesized in the pUC57 vector. The gene fragment was released and cloned into the pET-28a vector and expressed in Escherichia coli. The recombinant protein was confirmed by western blotting. Enzyme activity was estimated by a laboratory coagulation test, and prothrombin time and tertiary structure were determined by using the Iterative Threading ASSEmbly Refinement (I-TASSER) server. Data from blood clotting tests for the produced ecarin activity were analyzed using an independent t test. As per I-TASSER server prediction, model 1 with the highest confidence score 0.95, template modeling score (0.84 ± 0.08), and root mean square deviation (3.5 ± 2.4 Å) was considered as the best model, and the 2e3xA enzyme was more similar to the target protein. The predictive results helped to better understand the relationship between the structure and function of the ecarin metalloproteinase domain. Also, the production of this active site in the prokaryotic expression system, which is simpler and more cost-effective than the production of the eukaryotic system, showed that this recombinant ecarin could be used as a substitute for the raw snake venom of Echis carinatus because it converts prothrombin into thrombin, and its activity, as estimated using the prothrombin time test, was found to be faster than normal ecarin.

Physicochemical properties, anticancer and antimicrobial activities of metallic nanoparticles green synthesized by Aspergillus kambarensis.

In the present study, metal and metal oxide nanoparticles were successfully synthesized using Aspergillus kambarensis. UV-Vis spectroscopy showed maximum absorbance of 417 nm for silver (AgNPs), 542 nm for gold (AuNPs), 582 nm for copper (CuNPs) and 367 nm for zinc oxide (ZnONPs) nanoparticles. Fourier transform infrared spectroscopy indicated the presence of various mycochemicals with diverse functional groups in the fungal cell-free filtrate. Transmission electron microscopy revealed mono and poly dispersed particles with an estimate size of 50 nm and different shapes for synthesized manufacture metallic nanoparticles (MNPs. Dynamic light scattering confirmed that MNPs were dispersed in the size range less than 50 nm. Zeta potential analysis showed values of -41.32 mV (AgNPs), -41.26 mV (AuNPs), -34.74 mV (CuNPs) and 33.72 mV (ZnONPs). X-ray diffraction analysis demonstrated crystalline nature for MNPs. All the synthesized MNPs except AuNPs showed strong antifungal and antibacterial activity in disc diffusion assay with growth inhibition zones of 13.1-44.2 mm as well as anticancer activity against HepG-2 cancer cell line with IC50 in the range of 62.01-77.03 µg/ml. Taken together, the results show that biologically active MNPs synthesized by A. kambarensis for the first time could be considered as promising antimicrobial and anticancer agents for biomedical applications.

The immunomodulatory potential of murine adipose-derived mesenchymal stem cells is enhanced following culture on chitosan film.

INTRODUCTION: Recent studies show that the paracrine immunomodulatory effects of mesenchymal stem cells (MSCs) are mediated by the secretion of interleukin-10 (IL-10), transforming growth factor-beta (TGF ß), and nitric oxide (NO). The preconditioning of MSCs improves their immunomodulatory characteristics. Chitosan is a biopolymer with low toxicity and biodegradability, used as a membrane for MSCs three-dimensional culture. The present study aimed to evaluate the levels of immunomodulatory mediators of mesenchymal cells cultured on the chitosan film. MATERIALS & METHODS: MSCs were isolated from abdominal adipose tissue of BALB/c mice. Flow cytometry and differential culture medium were used to confirm the identity of isolated mesenchymal stem cells. The MSCs were divided into three groups; The first group was treated with 10 ng/mL LPS. The second group was seeded in the flasks coated with the chitosan film (3% w/v). The last group was cultured in the flasks without any preconditioning. After 72 h, IL-10, TGF-ß, and NO concentrations were measured in the conditioned media. In addition, the arginase activity in mesenchymal stem cells was measured using a colorimetric method. RESULTS: The proliferative spindle-shaped MSCs formed several three-dimensional spheroids on the chitosan film. It was shown that the level of TGF-ß and IL-10 were increased significantly after treatment with LPS (P = 0.02) and spheroid formation (P = 0.01). In addition, the arginase activity was enormously augmented in spheroids compared to controls (7.13-fold increase; 1.71 ± 0.08 and 0.24 ± 0.01 respectively; P = 0.021). On the other hand, the LPS treatment but not the culture on chitosan film increased the NO level significantly (P = 0.02 and P = 0.14, respectively). CONCLUSION: Using chitosan film as a three-dimensional culture strategy significantly affects the production of immunosuppressive factors by MSCs in vitro through increased secretion of TGF-ß and IL-10 and arginase activity.

Synergistic effects of exosomal crocin or curcumin compounds and HPV L1-E7 polypeptide vaccine construct on tumor eradication in C57BL/6 mouse model.

Cervical cancer is the most common malignant tumor in females worldwide. Human papillomavirus (HPV) infection is associated with the occurrence of cervical cancer. Thus, developing an effective and low-cost vaccine against HPV infection, especially in developing countries is an important issue. In this study, a novel HPV L1-E7 fusion multiepitope construct designed by immunoinformatics tools was expressed in bacterial system. HEK-293T cells-derived exosomes were generated and characterized to use as a carrier for crocin and curcumin compounds. The exosomes loaded with crocin and curcumin compounds as a chemotherapeutic agent (ExoCrocin and ExoCurcumin) were used along with the L1-E7 polypeptide for evaluation of immunological and anti-tumor effects in C57BL/6 mouse model. In vitro studies showed that ExoCrocin and ExoCurcumin were not cytotoxic at a certain dose, and they could enter tumor cells. In vivo studies indicated that combination of the L1-E7 polypeptide with ExoCrocin or ExoCurcumin could produce a significant level of immunity directed toward Th1 response and CTL activity. These regimens showed the protective and therapeutic effects against tumor cells (the percentage of tumor-free mice: ~100%). In addition, both ExoCrocin and ExoCurcumin represented similar immunological and anti-tumor effects. Generally, the use of exosomal crocin or curcumin forms along with the L1-E7 polypeptide could significantly induce T-cell immune responses and eradicate tumor cells.

Immunomodulatory Activity of Human Bone Marrow and Adipose-Derived Mesenchymal Stem Cells Prolongs Allogenic Skin Graft Survival in Nonhuman Primates.

OBJECTIVE: In the present study, we examined the tolerance-inducing effects of human adipose-derived mesenchymal stem cells (hAD-MSCs) and bone marrow-derived MSCs (hBM-MSCs) on a nonhuman primate model of skin transplantation. MATERIALS AND METHODS: In this experimental study, allogenic and xenogeneic of immunomodulatory properties of human AD-MSCs and BM-MSCs were evaluated by mixed lymphocyte reaction (MLR) assays. Human MSCs were obtained from BM or AD tissues (from individuals of either sex with an age range of 35 to 65 years) and intravenously injected (2×106 MSCs/kg) after allogeneic skin grafting in a nonhuman primate model. The skin sections were evaluated by H and E staining for histopathological evaluations, particularly inflammation and rejection reaction of grafts after 96 hours of cell injection. At the mRNA and protein levels, cellular mediators of inflammation, such as CD4+IL-17+ (T helper 17; Th17) and CD4+INF-γ+ (T helper 1, Th1) cells, along with CD4+FoxP3+ cells (Treg), as the mediators of immunomodulation, were measured by RT-PCR and flow cytometry analyses. RESULTS: A significant Treg cells expansion was observed in MSCs-treated animals which reached the zenith at 24 hours and remained at a high concentration for 96 hours; however, Th1 and Th17 cells were significantly decreased. Our results showed that human MSCs significantly decrease Th1 and Th17 cell proliferation by decreasing interleukin-17 (IL-17) and interferon-γ (INF-γ) production and significantly increase Treg cell proliferation by increasing FoxP3 production. They also extend the allogenic skin graft survival in nonhuman primates. Histological evaluations showed no obvious presence of inflammatory cells or skin redness or even bulging after MSCs injection up to 96 hours, compared to the group without MSCs. There were no significant differences between hBM-MSCs and hAD-MSCs in terms of histopathological scores and inflammatory responses (P<0.05). CONCLUSION: It seems that MSCs could be regarded as a valuable immunomodulatory tool to reduce the use of immunosuppressive agents.

Effect of ß-carotene on the differentiation potential of ciliary epithelium-derived MSCs isolated from mouse eyes on alginate-based scaffolds.

Retinal degenerative diseases are considered a major challenge all over the world, and stem cell therapy is a promising approach to restore degenerative cells due to RD. MSCs are multipotent stem cells found in a variety of tissues. They are capable of differentiating into various retinal cell types, so it can be a good candidate for various degenerative disorders like retinal degenerations. ß-carotene is an antioxidant that could accelerate the stem cell differentiation while using the proper scaffold. In this study, we evaluated the effect of ß-carotene on the differentiation potential of ciliary epithelium-derived MSCs isolated from mouse eyes on alginate-based scaffolds. MSCs were isolated from mouse ciliary epithelium, cultured in DMEM medium supplemented with 10% FBS, and identified by detecting their surface antigens. Three 3D culture systems, alginate, alginate/gelatin, and gelatin hydrogels were prepared, and their structures were checked via SEM. MSCs were cultured on 3D and 2D culture system scaffolds following treated with differentiation medium containing 50 µM ß-mercaptoethanol, 1 × minimum essential medium-nonessential amino acids and 20% of knockout serum replacement and ß-carotene. MSCs viability and differentiation ability were examined by MTT and ICC, respectively. The expression changes of several retinal specific genes (Nestin, RPE65, and Rhodopsin) were also evaluated by qPCR. Over 80% of cells isolated from mouse ciliary epithelium were positive for MSC-specific markers. The viability rates of MSCs grown on all alginate-based scaffolds were above 70%. MSCs cultured on alginate-based scaffold in the differentiation medium containing ß-carotene expressed higher levels of rhodopsin protein compared to a 2D culture. Also, the expressions of Nestin, Rhodopsin, and RPE65 genes were upregulated in ß-carotene-treated MSCs grown on alginate-based scaffolds. Our results indicate that the addition of ß-carotene to the differentiation medium, along with applying alginate-based scaffolds, could induce higher differentiation in mouse ciliary epithelium-derived MSCs into specialized retinal cells.

EL4-derived Exosomes Carry Functional TNF-related Apoptosis-inducing Ligand that are Able to Induce Apoptosis and Necrosis in the Target Cells.

Exosomes released by tumor cells play critical roles in tumor progression, immune cell suppression, and cancer metastasis. The aim of the present study was to investigate whether the exosomes released by EL4 cells carry a functional TNF-related apoptosis-inducing ligand (TRAIL) molecule. Exosomes were harvested from the supernatants of EL4 cell culture, and the shape, size, and identity of EL4-derived exosomes were evaluated by utilizing scanning electron microscopy, dynamic light scattering, and dot-blot method. The expression of mRNA and TRAIL protein in EL4 cells and EL4-exosomes were investigated using real-time PCR method and dot-blot analysis. Moreover, the effects of EL4-derived exosomes on cell death in a TRAIL-sensitive cell line (4T1) were studied by using flow cytometry (annexin V/propidium iodide (PI) staining) and fluorescent microscopy analyses (acridine orange/ethidium bromide staining). The results showed that EL4 cells continuously and without the need for stimulation, produce exosomes that carry TRAIL protein. In addition, EL4-derived exosomes were capable to induce apoptosis as well as necrosis in 4T1 cells. It was ultimately revealed that EL4 cells express TRAIL protein and release exosomes containing functional TRAIL. Moreover, the released exosomes were able to induce apoptosis and necrosis in a TRAIL-sensitive cell line. Further studies are needed to reveal the potential roles of tumor-derived exosomes in the pathogenesis of cancers.

Intestinal Microbiota in Elderly Inpatients with Clostridioides difficile Infection.

PURPOSE: The incidence of Clostridioides difficile infection (CDI) has been reported as 10-fold higher among the elderly population than in young adults. The aim of this study was to compare the targeted bacteria population in the fecal microbiota in two groups of hospitalized elderly, categorized according to CDI and non-CDI. PATIENT AND METHODS: In this case-control study, 84 fecal samples of the 28 patients with CDI and 56 non-CDI patients (>65 years) were studied. C. difficile isolates were characterized by anaerobic culture and multiplex PCR. Quantitative PCR was used to analyze the bacterial elements. RESULTS: CDI group differed significantly for a prolonged hospital stay, previous surgery, residence in nursing home and exposure to a range of antibiotics including quinolone, clindamycin and cephalosporin. CDI group had significantly fewer members of Bacteroides spp., Clostridium cluster IV, Bifidobacterium spp., Faecalibacterium prausnitzii, and Prevotella spp. in their fecal microbiota than the control group (P < 0.05). The abundances of Akkermansia muciniphila, Lactobacillus spp., Escherichia coli and Klebsiella spp. were higher in group CDI compared with the control group (P < 0.05). CONCLUSION: CDI status is associated with the abundance of some bacterial populations. In this study, an increase in Akkermansia muciniphila, Lactobacillus spp., and Enterobacteriaceae genus was highlighted in CDI patients. A reduction in butyrate-producing bacteria was found in CDI patients. The differences in the composition of fecal microbiota can help to understand how antimicrobial agents impact on gut homeostasis and lead to loss of colonization resistance to C. difficile.

Study of the application of gold nanoparticles for early detection of prostate cancer.

Studies on the blood of patients with prostate cancer using Dynamic Light Scattering (DLS) and corona protein size changes have shown that this test is highly specific and sensitive, but this method has not been studied in Iran, and therefore this study intends to perform this procedure using gold nanoparticles in prostate cancer detection. Blood samples of 60 male subjects aged 40-90 years were collected from 20 healthy, 20 benign and 20 prostate cancer patients. Optical scattering changes were measured by the level of gold nanoparticles mixed with these sera, and the responses were compared with the PSA index (Prostate Specific Antigen) of the subjects. Results of D2/D1 ratio analysis were performed using SPSS statistical software R. No significant differences were found in the size of the corona protein structure between the three groups of males with cancer, males with benign tumor, and healthy males. No correlation was found between the light scattering concentration and PSA serum level Due to changes in ambient temperature, prolonged test duration or high IgG levels in apparently healthy individuals, this test is not feasible in Iran. Performing this test requires advanced equipment to maintain the same temperature that do not exist in Iran. DLS also has major limitations for prostate cancer detection, so it cannot be a simple and accurate method for the early detection of prostate cancer, and it is suggested that other methods be used to diagnose.

Production, purification, and in vivo evaluation of a novel multiepitope peptide vaccine consisted of immunodominant epitopes of SYCP1 and ACRBP antigens as a prophylactic melanoma vaccine.

Melanoma cells are significantly resistance to the current treatments. Therefore, the best option for high-risk populations is prevention. Recently, many preventive cancer vaccines have been developed. In our previous study, several bioinformatic tools were employed for selection of the most immunodominant epitopes of acrosin binding protein (ACRBP) and synaptonemal complex protein 1 (SYCP1) antigens to design multiepitope DNA and peptide cancer vaccines. In the current study, the final construct of the multiepitope DNA vaccine was placed into a pcDNA3.1 vector and then, subcloned into a pET-28a (+) expression vector for transfecting BL21 E. coli strain. The recombinant multiepitope peptide vaccine, weighing 6.35 kDa, was purified by Fast protein liquid chromatography technique (FPLC) and detected by western blotting. Subsequently, C57BL/6 mice were immunized by a mixture of the peptide vaccine and incomplete Freund's adjuvant (IFA) (four vaccinations with one-week intervals). Two weeks after the last vaccination, the serum levels of the peptide-specific IgG total, IgG2a, and IgG1 were measured by enzyme-linked immunosorbent assays (ELISA). Also, the immunized mice splenocytes efficacy for producing interleukin-4 (IL-4) and interferon-γ (IFN-γ) after stimulation with the peptide vaccine was evaluated. At last, the prophylactic effect of the peptide vaccine immunization was evaluated in B16-F10 murine melanoma model. The peptide vaccine immunization caused a significant increase in the serum levels of IgG1, IgG2a, and IgG2a. Also, the immunized mice splenocytes exhibited significantly higher ability to produce IL-4 (10-fold) and IFN-γ (16-fold) after stimulation with the peptide vaccine, in comparison with the PBS and IFA groups. The peptide immunized mice exhibited 50.2% and 43% decrease in the mean tumors' volume in comparison with PBS and IFA groups. Also, the mean survival time for the peptide immunized mice was 33 ±â€¯1.3 days which was 5 and 6 days more than the PBS and IFA groups, respectively. The obtained results exhibit high efficacy of the designed multiepitope peptide vaccine for the immune system activation and anti-tumor prophylactic effects in the murine melanoma model.

Cytotoxic and Anticancer Effects of ICD-85 (Venom Derived Peptides) in Human Breast Adenocarcinoma and Normal Human Dermal Fibroblasts.

ICD-85 (venom derived peptides) has anti-proliferative effect and anti- angiogenesis activity on cancer cells. This study was performed to test the effect of ICD-85, on Human breast adenocarcinoma (MCF-7) and normal Human Dermal Fibroblasts (HDF) cell lines. In this experimental study, Mitochondrial activity, Neutral red uptake, Lactate dehydrogenase (cell necrosis), and cell morphology were assessed under unexposed and ICD-85 exposed conditions. Caspase-9 colorimetric assay kit was used to determine caspase protease activity. Morphological changes in MCF-7 cells on treatment with ICD-85 compared with untreated MCF-7 cells are consistent with characterizing the features of apoptosis such as granulation and cell rounding which finally results in the generation of apoptotic bodies. In contrast, this difference was not observed in normal cells. In MTT assay, ICD-85 induced dose dependent manner cytotoxic effects on MCF-7 cells which were confirmed by neutral red assay. The results showed that inhibitory concentration 50% (IC50) value of ICD-85 for MCF-7 cells at 24 h was 36.45 ± 0.38 µg/mL. However, when HDF cells were exposed to ICD-85, no significant elevation of LDH release were observed at concentrations below 20 µg/mL. The apoptosis-induction of ICD-85 on MCF-7 cell was found to be through activation of caspase-9 which was 13 fold greater than unexposed cell. This study showed that ICD-85 induced apoptosis in MCF-7 cell line through caspase activation and hence it can be considered for further investigation to use ICD-85 as a potential therapy for breast cancer.

Prestimulation of Microglia Through TLR4 Pathway Promotes Interferon Beta Expression in a Rat Model of Alzheimer's Disease.

Soluble amyloid beta (Aß) oligomers are the most common forms of Aß in the early stage of Alzheimer's disease (AD). They are highly toxic to the neurons but their capability to activate microglia remains controversial. Microglia develop two distinct phenotypes, classic (M1) and alternative (M2). Tuning of microglia to the alternative (anti-inflammatory) state is of major interest in treatment of neuroinflammatory disease. This study aimed to assess tuning the microglia to produce interferon beta (IFN-ß) as an anti-inflammatory cytokine through TLR4 pathway in a rat model of AD. Microglial BV-2 cells were treated with 1 µg/ml lipopolysaccharides (LPS), Monophosphoryl lipid A (MPL), or vehicles for 24 h, and then incubated with Aß oligomer. After 24 h, cell pellets were harvested and TIR-domain-containing adapter-inducing interferon-ß (TRIF), interferon regulatory factor 3 (IRF3), and IFN-ß levels were measured. The ligands/vehicle were microinjected into the right ventricle of male Wistar rats every 3 days. Two weeks later, an osmotic pump filled with oligomeric Aß/vehicle was implanted in the left ventricle. After 2 weeks, TRIF, IRF3, and IFN-ß levels were measured in the hippocampal tissue. TNF-α and IFN-ß levels were assessed in the hippocampus using immunohistochemistry. The oligomeric Aß did not change TRIF, IRF3, and IFN-ß levels in both cell culture and hippocampal tissue. However, pretreatment with LPS or MPL increased the level of these proteins. BV-2 cells morphologically express M1 state in presence of higher dose of Aß oligomer (10 µM). Pretreatment with LPS or MPL decreased the TNF-α and increased the number of IFN-ß positive cells in the hippocampus of Aß-treated rats. In conclusion, pretreatment with low dose TLR4 agonists could induce microglia to produce neuroprotective cytokines including IFN-ß which may be considered as a potential strategy to combat neuronal degeneration in AD.