Exploring the potential of structural modeling and molecular docking for efficient siRNA screening: A promising approach to Combat viral mutants, with a focus on HIV-1.

RNA interference (RNAi) holds immense potential for sequence-specific downregulation of disease-related genes. Small interfering RNA (siRNA) therapy has made remarkable strides, with FDA approval for treating specific human diseases, showcasing its promising future in disease treatment. Designing highly efficient siRNAs is a critical step in this process. Previous studies have introduced various algorithms and parameters for siRNA design and scoring. However, these attempts have often fallen short of meeting all essential criteria or required modifications, resulting in variable and unclear effectiveness of screened siRNAs, particularly against viral mutants with non-conserved short sequences. In this study, we present a fully optimized siRNA screening system considering all necessary parameters. Notably, we highlight the critical role of molecular docking simulations between siRNA and two functional domains of the Argonaute protein (PAZ and PIWI) in identifying the most efficient siRNAs, since the appropriate interaction between the guide siRNA strand and the RISC complex is crucial. Through our stringent method, we designed approximately 50 potential siRNAs targeting the HIV-1 vpr gene. Evaluation through XTT, qRT-PCR, and flow cytometry analysis on RAW 264.7 macrophage stable cells revealed negligible cytotoxicity and exceptional gene-silencing efficiency at both the transcriptional and translational levels for the top-ranked screened siRNAs. Given the growing interest in siRNA-based therapeutics, we anticipate that the insights from this study will contribute to improving treatment strategies against mutant viruses, particularly HIV-1.

The Combinatorial Effect of Ad-IL-24 and Ad-HSV-tk/GCV on Tumor Size, Autophagy, and UPR Mechanisms in Multiple Myeloma Mouse Model.

Multiple myeloma is a type of malignant neoplasia whose treatment has changed over the past decade. This study aimed to investigate the effects of combination of Adenovector-carrying interleukin-24 and herpes simplex virus 1 thymidine kinase/ganciclovir on tumor growth, autophagy, and unfolded protein response mechanisms in mouse model of multiple myeloma. Six groups of mice, including Ad-HSV-tk/GCV, Ad-IL-24, Ad-HSV-tk/IL-24, Ad-GFP, and positive and negative controls, were investigated, and each group was injected every 72 h. The tumor size was measured several times. The expression of LC3B evaluated through western blotting and ASK-1, CHOP, Caspase-3, and ATF-6 genes in the UPR and apoptosis pathways were also analyzed by the quantitative polymerase chain reaction (qPCR) method. The present results showed that the injection of Ad-HSV-tk/GCV, Ad-HSV-tk/IL-24, and metformin reduced the tumor size. The expression of LC3B was significantly higher in the treatment groups and positive control groups compared to the negative control group. The expression of CHOP, caspase-3, and ATF-6 genes was significantly higher in the Ad-IL-24 group compared to the other treatment groups. Besides, the ASK-1 expression was significantly lower in the Ad-IL-24 group as compared to the other groups. Overall, the results indicated that the presence of the HSV-tk gene in the adenovectors reduced the size of tumors and induced autophagy by triggering the expression of LC3B protein. The presence of the IL-24 might affect tumor growth but not as much the therapeutic effect of HSV-tk. Furthermore, the results indicated that co-administration of IL-24 and HSV-tk had no synergistic effect on tumor size control.

Xanthohumol hinders invasion and cell cycle progression in cancer cells through targeting MMP2, MMP9, FAK and P53 genes in three-dimensional breast and lung cancer cells culture.

BACKGROUND: Despite recent advances in the treatment of lung and breast cancer, the mortality with these two types of cancer is high. Xanthohumol (XN) is known as a bioactive compound that shows an anticancer effect on cancer cells. Here, we intended to investigate the anticancer effects of XN on the breast and lung cancer cell lines, using the three-dimensional (3D) cell culture. METHODS: XN was isolated from Humulus lupulus using Preparative-Thin Layer Chromatography (P-TLC) method and its authenticity was documented through Fourier Transform Infrared spectroscopy (FT-IR) and Hydrogen Nuclear Magnetic Resonance (H-NMR) methods. The spheroids of the breast (MCF-7) and lung (A549) cancer cell lines were prepared by the Hanging Drop (HD) method. Subsequently, the IC50s of XN were determined using the MTT assay in 2D and 3D cultures. Apoptosis was evaluated by Annexin V/PI flow cytometry and NFκB1/2, BAX, BCL2, and SURVIVIN expressions. Cell cycle progression was determined by P21, and P53 expressions as well as PI flow cytometry assays. Multidrug resistance was investigated through examining the expression of MDR1 and ABCG2. The invasion was examined by MMP2, MMP9, and FAK expression and F-actin labeling with Phalloidin-iFluor. RESULTS: While the IC50s for the XN treatment were 1.9 µM and 4.74 µM in 2D cultures, these values were 12.37 µM and 31.17 µM in 3D cultures of MCF-7 and A549 cells, respectively. XN induced apoptosis in MCF-7 and A549 cell lines. Furthermore, XN treatment reduced cell cycle progression, multidrug resistance, and invasion at the molecular and/or cellular levels. CONCLUSIONS: According to our results of XN treatment in 3D conditions, this bioactive compound can be introduced as an adjuvant anti-cancer agent for breast and lung cancer.

Significance of microRNA-targeted ErbB signaling pathway genes in cardiomyocyte differentiation.

OBJECTIVE(S): Cardiomyocyte differentiation is a complex process that follows the progression of gene expression alterations. The ErbB signaling pathway is necessary for various stages of cardiac development. We aimed to identify potential microRNAs targeting the ErbB signaling pathway genes by in silico approaches. METHODS: Small RNA-sequencing data were obtained from GSE108021 for cardiomyocyte differentiation. Differentially expressed miRNAs were acquired via the DESeq2 package. Signaling pathways and gene ontology processes for the identified miRNAs were determined and the targeted genes of those miRNAs affecting the ErbB signaling pathway were determined. RESULTS: Results revealed highly differentially expressed miRNAs were common between the differentiation stages and they targeted the genes involved in the ErbB signaling pathway as follows: let-7g-5p targets both CDKN1A and NRAS, while let-7c-5p and let-7d-5p hit CDKN1A and NRAS exclusively. let-7 family members targeted MAPK8 and ABL2. GSK3B was targeted by miR-199a-5p and miR-214-3p, and ERBB4 was targeted by miR-199b-3p and miR-653-5p. miR-214-3p, miR-199b-3p, miR-1277-5p, miR-21-5p, and miR-21-3p targeted CBL, mTOR, Jun, JNKK, and GRB1, respectively. MAPK8 was targeted by miR-214-3p, and ABL2 was targeted by miR-125b-5p and miR-1277-5p, too. CONCLUSION: We determined miRNAs and their target genes in the ErbB signaling pathway in cardiomyocyte development and consequently heart pathophysiology progression.

Induction of apoptosis and cell cycle arrest in colorectal cancer cells by novel anticancer metabolites of Streptomyces sp. 801.

BACKGROUND: Colorectal cancer is the third and most significant cause of death and fourth most common cancer in the world. Chemotherapy can be introduced in the cases of locally or distantly invasive colorectal cancer. In recent years Actinomycetes, especially the genus Streptomyces, contain numerous bioactive compounds, some of which are known as important anti-tumor chemotherapy drugs. In this research, we aimed to explore the anti-cancer mode of action of Streptomyces sp. 801 on colorectal cancer cells in vitro conditions. METHODS: Fermented supernatant of strain Streptomyces sp. 801 isolated from soil showed maximum growth inhibition on human colorectal cancer cells. The cytotoxic effects of various concentrations of EtOAc extract from bacterial culture supernatant on HT-29, HCT 116 and SW480 cancer cells were surveyed using the MTT assay. Moreover, flow cytometry assays and Bax, Bcl-2, Cyclin D1 and P21 gene expressions were carried out to assess the apoptotic and cell cycle effects. Also, the scratch assay was performed to measure migration. Finally, Ethyl acetate (EtOAc) extract was analyzed by LC-MS to identify anti-cancer compounds. RESULTS: The cell viability of all three cell lines were decreased in a dose-dependent manner. The successful induction of apoptosis and cell cycle arrest at IC50 values, were confirmed by flow cytometry as well as by the mRNA expression levels of the genes involved in these processes. Scratch assays indicated the inhibition of cell migration in the cancer cell lines treated by Streptomyces sp. 801. Nine anti-cancer compounds of Streptomyces sp. 801 were detected by liquid chromatography-mass spectrometry (LC-MS) analysis. CONCLUSIONS: These findings suggest that Streptomyces sp. 801 can be a source of promising anticancer metabolites.

Synergistic effect of cell and molecule - imprinted substrates for bone tissue engineering.

BACKGROUND: The purpose of this research was to investigate the in vitro osteogenic induction of MG-63 cells using topography and collagen protein printed on polydimethyl siloxane (PDMS). METHODS: ALKALINE PHOSPHATASE (ALP) assay, calcium content, alizarin red staining, immunocytochemistry (ICC), and real-time polymerase chain reaction (PCR) were used to evaluate the osteo-differentiation of human adipose stem cells on the MG-63 cell pattern, MG-63 cells/collagen pattern, and collagen pattern. Also, the differentiated cell shape was studied by crystal violet staining and scanning electron microscopy (SEM). RESULTS: Our results showed that calcium content and ALP activity increased significantly on the MG-63 cells /collagen pattern (P < 0.05). The gene expression analysis (ALKALINPHOSPHATASE, COLLAGEN1 and OSTEOCALCIN) and bone marker protein expression (OSTEOCALCIN) confirmed the osteo differentiation of adipose stem cells (ADSCs) seeded on the imprinting substrate. DISCUSSION: Cell and molecular printing enhanced osteogenic development of adipose stem cells, according to our findings.

Neuronal differentiation of mesenchymal stem cells by polyvinyl alcohol/Gelatin/crocin and beta-carotene.

BACKGROUND: Nerve tissues are important in coordinating the motions and movements of the body. Nerve tissue repair and regeneration is a slow process that might take a long time and cost a lot of money. As a result, tissue engineering was employed to treat nerve tissue lesions. The aim of this study was to investigate the proliferation of C6 cells and human mesenchymal stem cells derived bone marrow (hBMMSCs) differentiate into neuronal-like cells on the polyvinyl alcohol/gelatin/crocin (PVA/Gel/Cro) nanofiber scaffolds in vitro. METHODS: PVA/Gel scaffolds containing crocin in three concentrations (1%, 3%, and 5%) were prepared by the electrospinning method. The human bone marrow-derived mesenchymal stem cells (hBMSCs) differentiation on the PVA/Gel/Cro 5% that induced by beta-carotene (ßC), was analyzed during 10 days. Morphology of differentiated cells on the scaffolds was taken by scanning electron microscope (SEM). The expression of the neural cell markers was studied by quantitative reverse transcription- polymerase chain reaction (qRT-PCR) and immunocytochemistry (ICC). RESULTS: MTT results of C6 cells culture on the scaffolds showed that proliferation and metabolic activity on PVA/Gel scaffold containing crocin 5% (PVA/Gel/Cro 5%) are significantly more than the other concentrations (P = 0.01). MSC differentiation to nerve-like cells was approved by MAP-2 expression at the mRNA level and NESTIN and MAP-2 at the protein level. CONCLUSIONS: These results suggested that PVA/Gel/Cro 5% and ßC could lead to hBMSCs differentiation to neural cells.

Mesenchymal stem cells encapsulation in chitosan and carboxymethyl chitosan hydrogels to enhance osteo-differentiation.

BACKGROUND: Recently biomaterials utilized for designing scaffolds in tissue engineering are not cost-effective and eco-friendly. As a result, we design and develop biocompatible and bioactive hydrogels for osteo-tissue regeneration based on the natural polysaccharide chitosan. Three distinct hydrogel components were used for this. METHODS: Hydrogels networks were created using chitosan 2% (CTS 2%), carboxymethyl chitosan 2% (CMC 2%), and 50:50 mixtures of CTS and CMC (CTS/CMC 50:50). Furthermore, scanning electron microscopy (SEM), Fourier transforms infrared spectroscopy (FTIR), degradation, and swelling behavior of design hydrogels were studied. Also, the cytocompatibility and osteo-differentiation potency were examined by encapsulating mesenchymal stem cells derived from adipose tissue (AMSCs) on the designed hydrogels. RESULTS: According to the findings, our results showed an acceptable pore structure, functional groups, and degradation rate of the designed hydrogels for in vitro evaluation. In addition, employing CMC instead of CTS or adding 50% CMC to the hydrogel component could improve the hydrogel's osteo-bioactivity without the use of external osteogenic differentiation agents. CONCLUSION: The CMC-containing hydrogel not only caused early osteogenesis but also accelerated differentiation to the maturity phase of osteoblasts.

Downregulation of Talin-1 is associated with the increased expression of miR-182-5p and miR-9-5p in coronary artery disease.

BACKGROUND: Evidence indicates that the dysregulation of extracellular matrix (ECM) components can lead to cardiovascular diseases. The Talin-1 (TLN1) gene is a major component of the ECM, and it mediates integrin adhesion to the ECM. In this study, we aimed to determine microRNAs (miRs) that regulate the expression of TLN1 and determine expression alterations in TLN1 and its targeting miRs in coronary artery disease (CAD). METHODS: Data sets of CAD and normal samples of blood exosomes were downloaded, and TLN1 was chosen as one of the genes with differential expressions in an in silico analysis. Next, miR-182-5p and miR-9-5p, which have a binding site on 3´-UTR of TLN1, were selected using bioinformatics tools. Then, the miR target site was cloned in the psiCHECK-2 vector, and direct interaction between the miR target site and the TLN1 3'-UTR putative target site was investigated by luciferase assay. The expression of miR-182-5p, miR-9-5p, and TLN1 in the serum samples of CAD and non-CAD individuals was assessed via a real-time quantitative polymerase chain reaction. RESULTS: Our data revealed that miR-182-5p directly regulated the expression of TLN1. Moreover, miR-182-5p and miR-9-5p were significantly upregulated in the CAD group. Hence, both bioinformatics and experimental analyses determined the downregulated expression of TLN1 in the CAD samples. CONCLUSIONS: Our findings demonstrated that miR-182-5p and miR-9-5p could play significant roles in TLN1 regulation and participate in CAD development by targeting TLN1. These findings introduce novel biomarkers with a potential role in CAD pathogenesis.

Cold atmospheric plasma induced genotoxicity and cytotoxicity in esophageal cancer cells.

In this paper, we studied the functional effects of cold atmospheric plasma (CAP) on the esophageal cancer cell line (KYSE-30) by direct and indirect treatment and fibroblast cell lines as normal cells. KYSE-30 cells were treated with CAP at different time points of 60, 90, 120 and, 240 s for direct exposure and 90, 180, 240 and, 360 s for indirect exposure. Cell viability was studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and apoptosis induction in the treated cells was measured by Annexin-V/PI using flow cytometry. The expression of apoptotic related genes (BAX/BCL-2) was analyzed by real-time polymerase chain reaction. Moreover, the genotoxicity was analyzed by comet assay. Cell viability results showed that direct CAP treatment has a markedly cytotoxic impact on the reduction of KYSE-30 cells at 60 s (p = 0.000), while indirect exposure was less impactful (p > 0.05). The results of the Annexin-V/PI staining confirmed this analysis. Subsequently, the genotoxicity study of the direct CAP treatment demonstrated a longer tail-DNA length and caused increase in DNA damage in the cells (p < 0.00001) as well as shift BAX/BCL-2 toward apoptosis. The concentration of H2O2 and NO2- in direct CAP treatment was significantly higher than indirect (p > 0.05). Treatment with direct CAP showed genotoxicity in cancer cells. Collectively, our results pave a deeper understanding of CAP functions and the way for further investigations in the field of esophageal cancer treatment.

A nanobody-derived mimotope against VEGF inhibits cancer angiogenesis.

Vascular Endothelial Growth Factor (VEGF) promotes angiogenesis in tumours of various cancers. Monoclonal antibodies and nanobodies are one of the potent agents in the treatment of cancer. Due to their high costs, researchers are considering to design and produce peptides as a substitute approach in recent years. The aim of the current study was designing a mimotope against VEGF and evaluate its effects on cell proliferation and tube formation in the HUVEC cell line. For this, a peptide was designed against VEGF and chemically produced. The effects of synthetic peptide and nanobody on the inhibition of proliferation of HUVEC cells were examined using MTT and tube formation assays. The data indicate that the peptide was able to significantly inhibit both HUVEC cell proliferation and tube formation through inhibition of VEGF, highlighting the potential of peptides as a 'novel' class of candidate drugs to inhibit angiogenesis.

Carboxymethyl dextran-trimethyl chitosan coated superparamagnetic iron oxide nanoparticles: An effective siRNA delivery system for HIV-1 Nef.

Gene therapy, including small interfering RNA (siRNA) technology, is one of the leading strategies that help to improve the outcomes of the current therapeutic systems against HIV-1 infection. The successful therapeutic application of siRNAs requires their safe and efficient delivery to specific cells. Here, we introduce a superparamagnetic iron oxide nanoparticle (SPION) for delivering siRNA against HIV-1 nef (anti-nef siRNA) into two cell lines, HEK293 and macrophage RAW 264.7. SPIONs were coated with trimethyl chitosan (TMC), and thereafter, different concentrations of SPION-TMC were coated with different ratios of a carboxymethyl dextran (CMD) to modify the physicochemical properties and improve the biological properties of the nanocarriers. The nanoparticles exhibited a spherical shape with an average size of 112 nm. The obtained results showed that the designed delivery route enhanced the uptake of siRNA into both HEK293 and RAW 264.7 cells compared with control groups. Moreover, CMD-TMC-SPIONs containing anti-nef siRNA significantly reduced the expression of HIV-1 nef in HEK293 stable cells. The modified siRNA-loaded SPIONs also displayed no toxicity or apoptosis-inducing effects on the cells. The CMD-TMC-SPIONs are suggested as potential nanocarriers for siRNA delivery in gene therapy of HIV-1 infection.

ANRIL and ANRASSF1 long noncoding RNAs are upregulated in gastric cancer.

Gastric cancer (GC) is the fifth most frequent cancer and the third-leading cause of cancer-related death worldwide. It is a highly heterogeneous disease regarding the morphological and molecular viewpoints. Since it is curable in primary stages, early detection could improve the survival rate. Long noncoding RNAs contribute to a variety of cellular mechanisms, and their dysregulation is reported in various diseases such as cancer. Thus, they have a great potential to be used as diagnostic and prognostic biomarkers and therapeutic targets as well. In the current study, ANRIL and ANRASSF1 expression levels were compared between GC tumors and the adjacent normal tissues collected from 39 Iranian patients using the quantitative real-time polymerase chain reaction method. Correlation between ANRIL and ANRASSF1 expression levels and other clinical parameters was also evaluated. ANRIL and ANRASSF1 were significantly overexpressed in GC tumors compared with adjacent tissues ( P < 0.0001 and P = 0.001, respectively). No significant correlation between ANRIL and ANRASSF1 expression levels and demographic information was found. This study suggests that ANRIL and ANRASSF1 may play a critical role in GC progression and can be considered as a potential diagnostic or therapeutics biomarkers.

The effect of miR-579 on the PI3K/AKT pathway in human glioblastoma PTEN mutant cell lines.

Glioblastoma multiform (GBM) is a type of aggressive brain cancer with limited success in standard treatment. MicroRNAs are one of the most beneficial tools for diagnosis, prognosis, and treatment of cancer. This study aimed to investigate the effect of miR-579 on cellular behaviors and expression of PI3K/AKT signaling pathway in GBM cell lines. In the present study, miR-579 was overexpressed in U251 and A-172 cell lines by using lentil vector, and its effect on cellular behavior such as proliferation and migration was investigated by the cell cycle, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Annexin V, colony formation, Transwell and wound healing assays. MiR-579 predicted target genes (AKT1, Rheb, PDK1, and a few others) were also evaluated by real-time polymerase chain reaction or luciferase assay and Western blot analysis. Our results represented that overexpression of miR-579 could inhibit proliferation, migration, cell cycle and also promoted the apoptosis of GBM cell lines. The luciferase reporter assay showed miR-579 directly targets the 3 UTR of mTOR, Rheb, and PDK1 and repressed their expressions. Furthermore, the Western blot analysis showed that miR-579 could downregulate the AKT1 and Rheb protein expression. Overall, our findings propose that miR-579 functions as a novel tumor suppressor gene in GBM by regulating the PI3K/AKT signaling pathway and may serve as a therapeutic target for clinical therapy of glioblastoma multiform.

Occult hepatitis C virus infection in patients with beta-thalassemia major: Is it a neglected and unexplained phenomenon?

Occult hepatitis C virus (HCV) infection (OCI) is described as the presence of viral genome in both hepatocytes and peripheral blood mononuclear cells (PBMCs) despite constant negative results on serum HCV RNA tests. Beta-thalassemia major (BTM) describes a group of inherited blood diseases. Patients with BTM require repeated blood transfusions, increasing the risk of exposure to infectious agents. We aimed to assess the prevalence of OCI in Iranian BTM patients and to identify the role of host factors in OCI positivity. A total of 181 BTM patients with HCV negative markers were selected. HCV RNA was tested in PBMCs using nested polymerase chain reaction assay. The positive samples were then genotyped via restriction fragment-length polymorphism (RFLP) and 5'-untranslated region sequencing. Six (3.3%) out of 181 BTM patients had viral HCV genomes in PBMC samples. Three (50.0%), two (33.3%), and one (16.7%) out of these six patients were infected with HCV-1b, HCV-1a, and HCV-3a, respectively. OCI positivity was significantly associated with the serum level of uric acid (P = 0.045) and ABO blood group (P = 0.032). Also, OCI patients had unfavorable IFNL3 rs12979860 TT, IFNL3 rs8099917 GG, IFNL3 rs12980275 GG, and IFNL4 ss469415590 ∆G/∆G genotypes. In conclusion, we indicated the low frequency of OCI in BTM patients. Nevertheless, more attention is warranted considering the importance of this infection. Also, further studies are necessary to determine the actual prevalence of OCI among BTM patients in Iran.

Gene and protein delivery using four cell penetrating peptides for HIV-1 vaccine development.

Cell penetrating peptides (CPPs) can potently transport therapeutic molecules to target cells for treatment of a variety of diseases. Thus, their use is critical to improve therapeutic vaccines. Histidine-rich nona-arginine (HR9) and primary amphipathic peptide (MPG) showed the ability to transfer DNA into the cells. Moreover, the peptide derived from the C-terminal of the tumor suppressor protein p14ARF (M918) and arginine-rich peptide (penetratin) were utilized to deliver polypeptides and proteins into the living cells. In this study, the immunostimulatory properties of HIV-1 Nef DNA and protein constructs were evaluated using small heat shock protein 20 (sHsp20) and Freund's emulsion as an adjuvant, and four CPPs (HR9, MPG, M918, and penetratin) as a gene or protein carrier in BALB/c mice. Our data indicated that the HR9/DNA, MPG/DNA, M918/protein, and penetratin/protein complexes formed the stable nanoparticles that were effectively delivered in HEK-293T cell line at certain ratios. Moreover, a heterologous Hsp20-Nef DNA + MPG prime/rHsp20-Nef protein+M918 boost regimen significantly elicited higher levels of IgG2a, IgG2b, IFN-gamma, and Granzyme B directed toward Th1 responses in a long period (3 months) after the last immunization compared to other groups. Furthermore, the effective role of Hsp20 was detected as a natural adjuvant in enhancing immune responses against HIV-1 Nef antigen. These findings demonstrated that the simultaneous use of M918 and MPG CPPs as protein and gene carriers improves HIV-1 Nef-specific B- and T-cell immune responses as a promising approach for development of HIV-1 monovalent vaccine.

Design and in vitro delivery of HIV-1 multi-epitope DNA and peptide constructs using novel cell-penetrating peptides.

OBJECTIVES: Developing an effective HIV vaccine that stimulates the humoral and cellular immune responses is still challenging because of the diversity of HIV-1 virus, polymorphism of human HLA and lack of a suitable delivery system. RESULTS: Using bioinformatics tools, we designed a DNA construct encoding multiple epitopes. These epitopes were highly conserved within prevalent HIV-1 subtypes and interacted with prevalent class I and II HLAs in Iran and the world. The designed DNA construct included Nef60-84, Nef126-144, Vpr34-47, Vpr60-75, Gp16030-53, Gp160308-323 and P248-151 epitopes (i.e., nef-vpr-gp160-p24 DNA) which was cloned into pET-24a(+) and pEGFP-N1 vectors. The recombinant polyepitope peptide (rNef-Vpr-Gp160-P24; ~ 32 kDa) was successfully generated in E. coli expression system. The pEGFP-nef-vpr-gp160-p24 and rNef-Vpr-Gp160-P24 polyepitope peptide were delivered into HEK-293 T cells using cell-penetrating peptides (CPPs). The MPG and HR9 CPPs, as well as the novel LDP-NLS and CyLoP-1 CPPs, were utilized for DNA and peptide delivery into the cells, respectively. SEM results confirmed the formation of stable MPG/pEGFP-N1-nef-vpr-gp160-p24, HR9/pEGFP-N1-nef-vpr-gp160-p24, LDP-NLS/rNef-Vpr-Gp160-P24 and CyLoP-1/rNef-Vpr-Gp160-P24 nanoparticles with a diameter of < 200 nm through non-covalent bonds. MTT assay results indicated that these nanoparticles did not have any major toxicity in vitro. Fluorescence microscopy, flow cytometry and western blot data demonstrated that these CPPs could significantly deliver the DNA and peptide constructs into HEK-293 T cells. CONCLUSION: The use of these CPPs can be considered as an approach in HIV vaccine development for in vitro and in vivo delivery of DNA and peptide constructs into mammalian cells.

Interleukin 12B mRNA level and rs3212227 genotyping in peripheral blood mononuclear cells of inflammatory bowel disease patients

Background/aim: Inflammatory bowel disease (IBD) is a multifactorial disorder. Single nucleotide polymorphisms (SNPs) in the IL-12 gene, which are the main factors to regulate the immune reaction, play an important role in the production of IL-12 molecules. The aim of this study was to evaluate the correlation between the SNP on position +1188 of the 3 ' UTR region of the IL-12 p40 subunit gene and expression of the IL-12 p40 gene. Materials and methods: This case-control study was performed with 102 patients with IBD and 107 healthy people. PCR-RFLP and comparative real-time PCR were performed to assess the association between genotype and IL-12 gene expression. Results: The frequency of AA, CA, and CC genotypes of this gene at position +1188 was calculated to be 58.8%, 32.4%, and 8.8% in patients and 61.7%, 26.2%, and 12.1% in the control group, respectively, with no significant difference between the two groups (IL- 12B rs3212227: AA (Reference 1), CA (P = 0.407); OR (95% CI) 0.771 (0.418­1.424), CC (P = 0.561); OR (95% CI) 1.313 (0.524­3.292)). Also, the IL-12B mRNA expression level was compared between IBD patients and healthy controls and demonstrated a significant association (R 2 0.136, 95% CI 1.892­3.872, P < 0.0001). Conclusion: Our results show that IL-12B expression in IBD may be associated with altered immune and inflammatory responses.

IL28B rs12980275 and HLA rs4273729 genotypes as a powerful predictor factor for rapid, early, and sustained virologic response in patients with chronic hepatitis C.

Single-nucleotide polymorphisms (SNPs) in the Interleukin-28B (IL28B) gene and rs4273729 in the human leukocyte antigen (HLA) gene in chronic hepatitis C (CHC) virus infection are important for predicting treatment outcome. In this study, the distribution of IL28B SNPs (rs12979860 and rs12980275) and HLA rs4273729 in rapid virologic response (RVR), complete early virologic response (cEVR) and sustained virologic response (SVR) in HCV Iranian patients with CHC virus infection was assessed. IL28B genotyping and rs4273729 were performed using the amplification refractory mutation system (ARMS)-PCR and direct sequencing in 190 CHC virus infections, respectively. RVR, cEVR, and SVR were 53.2 %, 78.9 %, and 65.8 %, respectively. Multivariate regression analysis demonstrated that the responses significantly predicted SVR in patients with age <40 years (p = 0.008), HCV genotypes (p = 0.032), IL28B rs12979860 CC genotype (p < 0.001), rs12980275 AA genotype (p < 0.001), rs4273729 GG genotype (p < 0.001), RVR (p < 0.001) and cEVR (p = 0.024). Three critical predictor factors based on RVR response were rs12979860 CC genotype (p = 0.033), rs12980275 AA genotype (p < 0.001) and rs4273729 GG genotype (p < 0.001), while rs12980275 AA (p = 0.003) and rs4273729 GG genotypes (p < 0.001) predicted cEVR. For the first time in Iran, these results revealed that the rs12980275 and HLA rs4273729 are important for the treatment of CHC infection. These findings may help predict responses to CHC infection treatment and reduce the cost and side effects of therapy.

Deregulation of miR-93 and miR-143 in human esophageal cancer.

Esophageal squamous cell carcinoma (ESCC) is the second and third most common malignancy in Iranian males and females, respectively. Treatment of ESCC is largely ineffective due to lack of detection at early stages of the disease. In recent years, miRNA, a small RNA molecule, has drawn much attention to researchers as a potential biomarker for esophageal cancer. miR-93 and miR-143 are two miRNA molecules reported to be frequently deregulated in various cancers, including prostate, stomach, cervix, and etc. The purpose of this study was to investigate the expression levels of these miRNAs and evaluate their diagnostic and therapeutic potential in esophageal squamous cell carcinoma. In this study, total RNA was extracted from 30 tumor tissues and 30 nontumor tissues of esophageal tumor margins, using RNX-plus solution. After validating the quality and quantity of total RNA, cDNAs of interest were synthesized using microRNA-specific cDNA Synthesis Kit. The expression level of miR-93 and miR-143 was evaluated using quantitative real-time PCR with miRNA-specific primers. Finally, the obtained data was analyzed by SPSS ver.20 software and paired t test was performed to observe the significance of difference between groups. The expression level of miR-93 was significantly increased and of miR-143 was significantly decreased in most of the examined tumor tissues, compared to nontumor tissues. Also, our findings did not detect correlation between mir-93 and mir-143 expressions in regard to stage and grade of the samples. These findings suggest that the deregulation of these miRNAs may play an important role in esophageal squamous cell carcinoma. Both miR-93 and miR-143 might be used as potential biomarkers in esophageal squamous cell carcinoma. However, more studies with large population of samples are necessary.