The Association between Histidine-Rich Glycoprotein rs10770 Genotype and Recurrent Miscarriage in Iranian Women.

Purpose: Recurrent miscarriage (RM) is a significant reproductive concern affecting numerous women globally. Genetic factors are believed to play a crucial role in RM, making the histidine-rich glycoprotein (HRG) gene, a topic of interest due to its potential involvement in angiogenesis. This study is aimed at investigating the association between the HRG rs10770 genotype and RM. Method: Blood samples were collected from a total of 200 women at the beginning of the study. Subsequently, a comparative analysis was conducted between the blood samples of 100 women with a history of RM (case group) and the blood samples of another 100 healthy women (control group). HRG rs10770 genotyping was performed through polymerase chain reaction restriction-fragment length polymorphism (PCR-RFLP), followed by statistical analysis to evaluate the relationship between HRG rs10770 genotype and RM. Results: The results indicated a significant statistical difference between the C/C genotype (OR = 3.32, CI: 1.22-9.04, p = 0.01) and the C/T genotype (OR = 1.24, CI: 0.67-2.30, p = 0.47) in both the case and control groups. Additionally, a significant correlation was observed in the C allelic frequency among RM participants compared to the control group (OR = 1.65, CI: 1.06-2.58, p = 0.02). Conclusion: The study highlights the importance of HRG rs10770 in understanding RM, shedding light on its implications for reproductive health. Furthermore, it became evident that women carrying the homozygous C/C genotype exhibited increased susceptibility to the risk of RM.

Molecular identification of Phlebotomus kandelakii apyrase and assessment of the immunogenicity of its recombinant protein in BALB/c mice.

Sand fly salivary proteins have immunomodulatory and anti-inflammatory features; hence, they are proven to perform important roles in the early establishment of Leishmania parasite in the vertebrate host. Among them, salivary apyrase with anti-hemostatic properties has a crucial role during the blood meal process. In the present study, a Genome-Walking method was used to characterize a full-length nucleotide sequence of Phlebotomus (P.) kandelakii apyrase (Pkapy). Bioinformatics analyses revealed that Pkapy is a ~ 36 kDa stable and hydrophilic protein that belongs to the Cimex family of apyrases. Moreover, recombinant proteins of Pkapy and P. papatasi apyrase (Ppapy) were over-expressed in Escherichia coli BL2 (DE3) and their antigenicity in BALB/c mice was evaluated. Dot-blot and ELISA results indicated that both recombinant apyrases could induce antibodies in BALB/c. Moreover, a partial cross-reactivity between Pkapy and Ppapy was found. In vitro stimulation of splenocytes from immunized mice with the recombinant proteins indicated cross-reactive T cell proliferative responses. Cytokine analysis revealed significant production of IFN-γ (p < 0.001) and IL-10 (p < 0.01) in response to Pkapy. In conclusion, the full-length nucleotide sequence and molecular characteristics of Pkapy were identified for the first time. Immunologic analyses indicated that Pkapy and Ppapy are immunogenic in BALB/c mice and show partial cross-reactive responses. The immunity to Pkapy was found to be a Th1-dominant response that highlights its potential as a component for an anti-Leishmania vaccine.

Targeted integration in CHO cells using CRIS-PITCh/Bxb1 recombinase-mediated cassette exchange hybrid system.

Recombinant Chinese hamster ovary (CHO) cell line development for complex biotherapeutic production is conventionally based on the random integration (RI) approach. Due to the lack of control over the integration site and copy number, RI-generated cell pools are always coupled with rigorous screening to find clones that satisfy requirements for production titers, quality, and stability. Targeted integration into a well-defined genomic site has been suggested as a possible strategy to mitigate the drawbacks associated with RI. In this work, we employed the CRISPR-mediated precise integration into target chromosome (CRIS-PITCh) system in combination with the Bxb1 recombinase-mediated cassette exchange (RMCE) system to generate an isogenic transgene-expressing cell line. We successfully utilized the CRIS-PITCh system to target a 2.6 kb Bxb1 landing pad with homology arms as short as 30 bp into the upstream region of the S100A gene cluster, achieving a targeting efficiency of 10.4%. The platform cell line (PCL) with a single copy of the landing pad was then employed for the Bxb1-mediated landing pad exchange with an EGFP encoding cassette to prove its functionality. Finally, to accomplish the main goal of our cell line development method, the PCL was applied for the expression of a secretory glycoprotein, human recombinant soluble angiotensin-converting enzyme 2 (hrsACE2). Taken together, on-target, single-copy, and stable expression of the transgene over long-term cultivation demonstrated our CRIS-PITCh/RMCE hybrid approach might possibly improve the cell line development process in terms of timeline, specificity, and stability. KEY POINTS: • CRIS-PITCh system is an efficient method for single copy targeted integration of the landing pad and generation of platform cell line • Upstream region of the S100A gene cluster of CHO-K1 is retargetable by recombinase-mediated cassette exchange (RMCE) approach and provides a stable expression of the transgene • CRIS-PITCh/Bxb1 RMCE hybrid system has the potential to overcome some limitations of the random integration approach and accelerate the cell line development timeline.

Quality by Design in Downstream Process Development of Romiplostim

Background: Background: Downstream processing of therapeutic recombinant proteins expressed as the inclusion bodies (IBs) in E. coli is quite challenging. This study aimed to use the quality by design approach for developing the multi-step downstream process of a structurally complex therapeutic Fc-Peptide fusion protein, romiplostim. Methods: Methods: For development of a successful downstream process, risk analysis and experimental designs were used to characterize the most critical quality attributes (CQAs) and effects of process parameters on these quality attributes. Results: Results: The solubilization of IBs was optimized by design of experiment on three parameters with a focus on solubility yield, which resulted in >75% increase of the target protein solubilization. The pH of sample was identified as CQA in anion exchange chromatography that might have an impact on achieving >85% host cell proteins removal and >90% host cell DNA reduction. In the refolding step, process parameters were screened. Cystine/cysteine ratio, pH, and incubation time identified as CPPs were further optimized using Box-Behnken analysis, which >85% of the target protein was refolded. The design space for further purification step by HIC was mapped with a focus on high molecular weight impurities. After polishing by gel filtration, the final product's biological activity showed no statistically significant differences among the groups received romiplostim and Nplate®, as the reference product. Conclusions: Conclusion: This research presents a precise and exhaustive model for mapping the design space in order to describe and anticipate the link between the yield and quality of romiplostim and its downstream process parameters.

Immunoinformatics Evaluation of a Fusion Protein Composed of Leishmania infantum LiHyV and Phlebotomus kandelakii Apyrase as a Vaccine Candidate against Visceral Leishmaniasis.

Background: Visceral leishmaniasis (VL) is a lethal parasitic disease, transmitted by sand fly vectors. Immunomodulatory properties of sand fly saliva proteins and their protective effects against Leishmania infection in pre-exposed animals suggest that a combination of an antigenic salivary protein along with a Leishmania antigen can be considered for designing a vaccine against leishmaniasis. Methods: Three different fusion forms of L. infantum hypothetical protein (LiHyV) in combination with Phlebotomus kandelakii salivary apyrase (PkanAp) were subjected to insilico analyses. Major Histocompatibility Complex (MHC) class I and II epitopes in both humans and BALB/c mice were predicted. Antigenicity, immunogenicity, epitope conservancy, toxicity, and population coverage were also evaluated. Results: Highly antigenic promiscuous epitopes consisting of truncated LiHyV (10-285) and full-length PkanAp (21-329) were identified in human and was named Model 1. This model contained 25 MHC-I and 141 MHC-II antigenic peptides which among them, MPANSDIRI and AQSLFDFSGLALDSN were fully conserved. LALDSNATV, RCSSALVSI, ALVSINVPL, SAVESGALF of MHC-I epitopes, and 28 MHC-II binding epitopes showed 60% conservancy among various clades. A population coverage with a rate of >75% in the Iranian population and >70% in the whole world was also identified. Conclusion: Based on this in-silico approach, the predicted Model 1 could potentially be used as a vaccine candidate against VL.

Co-delivery of PLGA nanoparticles loaded with rSAG1 antigen and TLR ligands: An efficient vaccine against chronic toxoplasmosis.

Although vaccination is a promising approach for the control of toxoplasmosis, there is currently no commercially available human vaccine. Adjuvants such as delivery vehicles and immunomodulators are critical components of vaccine formulations. In this study, Poly (D, l-lactide-co-glycolide) (PLGA) nanoparticles were applied to serve as delivery system for both surface antigen-1 (SAG1), a candidate vaccine against toxoplasmosis and two TLR ligands, monophosphoryl lipid A (MPL) and imiquimod (IMQ), respectively. Compared to rSAG1 alone, CBA/J mice immunized with rSAG1-PLGA produced higher anti-SAG1 IgG antibodies titers. This response was increased by the co-administration of IMQ-PLGA (p < 0.01). Compared to IMQ-PLGA co-administration, MPL-PLGA co-administration further increased the humoral response (p < 0.01) and potentiated the Th1 humoral response. Compared to rSAG1 alone, rSAG1-PLGA, or rSAG1-PLGA mixed with IMQ-PLGA or MPL-PLGA similarly enhanced the cellular response characterized by the production of IFN-γ, IL-2, TNF-α and low levels of IL-5, indicating a Th1-biased immunity. The induced immune responses, led to significant brain cyst reductions (p < 0.01) after oral challenge with T. gondii cysts in mice immunized with either rSAG1-PLGA, rSAG1-PLGA + IMQ-PLGA, rSAG1-PLGA + MPL-PLGA formulations. Taken together the results indicated that PLGA nanoparticles could serve as a platform for dual-delivery of antigens and immunomodulators to provide efficacious vaccines against toxoplasmosis.

An overview of the sand fly salivary proteins in vaccine development against leishmaniases.

Leishmaniases are a group of vector-borne parasitic diseases transmitted through the infected sand flies. Leishmania parasites are inoculated into the host skin along with sand fly saliva. The sand fly saliva consists of biologically active molecules with anticoagulant, anti-inflammatory, and immunomodulatory properties. Such properties help the parasite circumvent the host's immune responses. The salivary compounds support the survival and multiplication of the parasite and facilitate the disease progression. It is documented that frequent exposure to uninfected sand fly bites produces neutralizing antibodies against specific salivary proteins and further activates the cellular mechanisms to prevent the establishment of the disease. The immune responses due to sand fly saliva are highly specific and depend on the composition of the salivary molecules. Hence, thorough knowledge of these compounds in different sand fly species and information about their antigenicity are paramount to designing an effective vaccine. Herein, we review the composition of the sand fly saliva, immunomodulatory properties of some of its components, immune responses to its proteins, and potential vaccine candidates against leishmaniases.

Computational Engineering of Protein L to Achieve an Optimal Affinity Chromatography Resin for Purification of Antibody Fragments.

Protein L affinity chromatography is a useful method for the purification of antibody fragments containing kappa light chains. In affinity chromatography, increasing the binding affinity leads to increased product purity, recovery, and dynamic binding capacity (DBC). In this study, molecular docking and molecular dynamics simulation techniques were used to design the engineered Protein L with higher affinity to the kappa light chain. Each engineered ligand was produced as a recombinant protein and coupled to a solid matrix. The purity, recovery, and DBC of the engineered resins were evaluated and then compared to those of a commercially available resin. The results showed important parameters for engineering more efficient Protein L ligands for affinity chromatography.

High resolution melting assay in discrimination of the main etiologic agents of leishmaniasis in Iran.

BACKGROUND AND OBJECTIVES: The three old world Leishmania species i.e., L. major, L. tropica, and L. infantum are considered as potential etiological agents of the various clinical forms of leishmaniasis in Iran. Different species co-exist in some areas. Accurate differentiation between the species is essential for choosing an appropriate therapy. Conventional and gold standard methods for the detection and characterization of parasites are time-consuming, laborious, and have low sensitivity. A polymerase chain reaction followed by high resolution melting (PCR-HRM) analysis has been employed for detection and species identification. Most of the studies suffer from the use of multiple targets and/or requiring more than one reaction to identify a single sample. The present study aimed to design a PCR method based on the amplification of kinetoplast DNA minicircles (kDNA) and HRM analysis of the amplicons for rapid discrimination of the three mentioned species. MATERIALS AND METHODS: DNA from reference strains including L. major, L. tropica, and L. infantum and fifty-eight strains subjected to PCR-HRM analysis targeting kDNA. All the samples were also analyzed by conventional kDNA-PCR. RESULTS: The PCR-HRM analysis allowed discrimination between the three Old World species. The normalized HRM curves for the amplicons of kDNA indicated a unique and repeatable melting plot for each species, even in combination with human and mouse genomic DNA. Conventional kDNA-PCR could not properly discriminate L. tropica from L. infantum. CONCLUSION: PCR-HRM analysis of kDNA proved to be fast and accurate for discrimination of L. major, L. tropica, and L. infantum.

Development of a Quantitative Real-Time PCR Assay for Detection of Toxoplasma gondii in Brain Samples.

BACKGROUND: Toxoplasmosis is a worldwide-distributed infection that can cause serious diseases, mainly in congenitally infected and immunodeficient individuals. PCR assays play an indispensable role in the detection of Toxoplasma gondii in different biological samples. METHODS: This study was conducted in the Parasitology Department at Pasteur Institute of Iran (Tehran) during 2016-2018. We designed a highly sensitive quantitative real-time PCR (RT-qPCR) targeted REP-529, a noncoding repetitive DNA. We cloned the amplicon in a plasmid (pTZREP-529) and used it to generate the standard curve. The Toxoplasma RT-qPCR characteristics, i.e., detection limit, specificity, linear dynamic range, linearity, intra-, and inter-assay precisions, were determined. The detection limit of the assay was one plasmid copy number (PCN) per reaction (about 0.004 T. gondii genome), and the linear dynamic range was equal to 6 logs (1× 101 to 1× 107 PCN per reaction). RESULTS: The assay showed no signal when genomic DNA of Plasmodium falciparum, Leishmania major, and Trichomonas vaginallis were used. The standard curve was drawn using dilutions of pTZREP-529 plasmid spiked with genomic DNA from a mouse brain, and test characteristics were shown unaffected. Applying the Toxoplasma RT-qPCR, we showed brain cysts were significantly decreased in mice vaccinated with GRA2 antigen of Toxoplasma formulated in Monophosphoryl Lipid A (MPL) adjuvant. CONCLUSION: We have developed a quantitative, specific, and highly sensitive PCR for detecting T. gondii in biological samples.

Circulating miR-20a and miR-26a as Biomarkers in Prostate Cancer

Objective: Circulating microRNAs (miRNAs), present in body fluids, have been considering importance as cancer biomarkers. The primary aim of this study was to assess whether circulatory miR-20a and miR-26a can be used as diagnostic biomarkers in prostate cancer (PCa). Methods: Relative expression miR-20a and miR-26a has been assessed in 40 patients with PCa and 40 non-cancerous volunteer. Sample Collection of patients was performed before and one week after prostatectomy. Total RNA was extracted from serum and miR-20a and miR-26a expressions were quantified by using Real-Time PCR method. Results: miR-20a was significantly up-regulated in pre-operation serum samples of PCa patients compared to the serum samples of non-cancerous controls, however, in post-operation samples no significant differences was showed. miR-26a level was not significantly decreased in pre and post-operation serum samples compared to the serum samples of controls. However, the expression level ratios of both miR-20a and miR-26a were insignificantly decreased when post-operation serum samples compared to pre-operation ones. Conclusion: Decrement of circulating miR-20a and miR-26a in patients after surgery may reflect the tumoral origin of those microRNAs and the results may use for tumor remnant monitoring after prostatectomy.

Soluble expression of IGF1 fused to DsbA in SHuffle™ T7 strain: optimization of expression and purification by Box-Behnken design.

Production of insulin-like growth factor 1 (IGF1) in Escherichia coli mostly results in the formation of inclusion bodies. In the present study, IGF1 was fused to disulfide bond oxidoreductase A (DsbA) and expressed in SHuffle™ T7 strain, in order to obtain correctly folded protein. Soluble expression and IMAC purification of DsbA-IGF1 were optimized by applying the Box-Behnken design of response surface methodology. The optimization greatly increased concentration of soluble protein from 317 to 2600 mg/L, and IMAC yield from 400 to 1900 mg/L. Results of ANOVA showed induction OD600 and temperature had significant effects on the soluble protein expression while isopropyl-ß-d thiogalactoside, in the concentrations tested, displayed no significant effect. Moreover, the three parameters of the binding buffer including, pH, concentration of NaCl, and imidazole displayed significant effects on the IMAC yield. Then, purified DsbA-IGF1 was cleaved by human rhinovirus 3C protease, and authentic IGF1 was obtained in flow through of a subtractive IMAC. Final polishing of the protein by reversed-phase HPLC yielded IGF1 with purity of 96%. The quality attributes of purified IGF1 such as purity, identity, molecular size, molecular weight, secondary structure, and biological activity were assessed and showed to be comparable to the standard IGF1. The final yield of purified IGF1 was estimated to be 120 ± 18 mg from 1 L of the culture. Our results demonstrated a simple and easily scalable strategy for production of large amounts of bioactive IGF1 by rational designing soluble protein expression, and further optimization of expression and purification methods.

Circulating miR-26a and miR-21 as biomarkers for glioblastoma multiform.

Glioblastoma multiform is the most common and lethal primary central nervous system tumor. Circulating microRNAs (miRNAs), present in cell-free bodily fluids, have been gaining importance as cancer biomarkers. The primary aim of this study was to assess whether circulating miRNA-128, -21, and -26a in glioblastoma patients can be used as diagnostic biomarkers. Venous blood samples were collected from 11 noncancerous volunteers and 15 glioblastoma patients pre- and post operation. Also, tissue tumor samples were obtained intra-operationally to assay consistency of miRNA levels in serum and tissue samples. Serum and tissue levels of miRNAs were determined by quantitative reverse transcription PCR. miR-21 and miR-26a were both significantly upregulated in pre- and postoperation serum samples of glioblastoma patients compared with the serum samples of noncancerous controls. We found that all three miR-128, -21, and -26a expression levels were reduced in postoperative serum samples compared with pre-operative serum samples, though this decrease was only significant for miR-26a. The serum miR-26a and miR-21 upregulation in glioblastoma patients compared to noncancerous controls and their downregulation in postoperative serum from glioblastoma patients suggest that these miRNAs could be used as serum-derived miRNA biomarkers for glioblastoma.

miR-1266-5p and miR-185-5p Promote Cell Apoptosis in Human Prostate Cancer Cell Lines

Objective: Small non-coding RNA molecules are dysregulated in prostate cancer (PCa). In our previous study, downregulation of miR-1266 and miR-185 was demonstrated in PCa tissues and cell lines. The aim of the present study was to investigate whether miR-1266 and miR-185 are involved in the regulation of B-cell lymphoma (BCL) 2 and BCL2L1, respectively, and whether transfection of PCa cell lines with miR-1266 and miR-185 mimics can alter tumorigenic phenotypes. Methods: In order to investigate the regulation of BCL2 and BCL2L1 mRNA levels by miR-1266 and miR-185, respectively, a luciferase reporter assay was used. Real-time PCR was also used to analyze changes in the levels of BCL2 and BCL2L1 mRNAs in PCa cell lines following transfection with synthetic miR-1266 and miR-185. Cell apoptosis was determined by Annexin V protein expression analysis via flow cytometry. In addition to the MTT assay, a cell proliferation assay was performed. Result: A luciferase assay confirmed that the BCL2 and BCL2L1 genes may be targeted by miR-1266 and miR-185, respectively, through binding to their 3'UTR regions. Transfection of PC3 and DU145 cells with miR-1266 and miR-185 induced apoptosis and reduced proliferation, which also revealed an inverse correlation with BCL2 and BCL2L1 gene expression in the treated cells. Conclusion: Our data suggests that miR-1266 and miR-185 may be novel candidates for further research in PCa treatment through the anti-apoptotic pathway.

Downregulation of miR-1266-5P, miR-185-5P and miR-30c-2 in prostatic cancer tissue and cell lines.

Over the latest decade, the role of microRNAs (miRNAs/miRs) has received more attention. miRNAs are small non-coding RNAs that may serve a role as oncogenes or tumor suppressor genes. Certain miRNAs regulate the apoptosis pathway by influencing pro- or anti-apoptotic genes. We hypothesized that increases in the expression of B cell lymphoma 2 (BCL2) and BCL2-like 1 (BCL2L1) genes, which have been reported in various types of cancer tissues, may be due to the downregulation of certain miRNAs. The present study aimed to identify miRNAs that target BCL2 and BCL2L1 anti-apoptotic genes in prostate cancer (PCa) clinical tissue samples. Certain candidate miRNAs were selected bioinformatically and their expression in PCa samples was analyzed and compared with that in benign prostatic hyperplasia (BPH) tissue samples. The candidate miRNAs that targeted BCL2 and BCL2L1 genes were searched in online databases (miRWalk, microRNA.org, miRDB and TargetScan). A total of 12 miRNAs that target the 3'-untranslated region of the aforementioned genes and/or for which downregulation of their expression has previously been reported in cancer tissues. A total of 30 tumor tissue samples from patients with PCa and 30 samples tissues from patients with BPH were obtained and were subjected to reverse transcription-quantitative polymerase chain reaction for expression analysis of 12 candidate miRNAs, and the BCL2 and BCL2L1 genes. Additionally, expression of 3 finally selected miRNAs and genes was evaluated in prostate cancer PC3 and DU145 cell lines and human umbilical vein endothelial cells. Among 12 miRNA candidates, the expression of miR-1266, miR-185 and miR-30c-2 was markedly downregulated in PCa tumor tissues and cell lines. Furthermore, downregulation of these miRNAs was associated with upregulation of the BCL2 and BCL2L1 genes. An inverse association between three miRNAs (miR-1266, miR-185 and miR-30c-2) and two anti-apoptotic genes (BCL2 and BCL2L1) may be considered for interventional miRNA therapy of PCa.

SHuffle™ T7 strain is capable of producing high amount of recombinant human fibroblast growth factor-1 (rhFGF-1) with proper physicochemical and biological properties.

BACKGROUND: Human fibroblast growth factor-1 (FGF-1) has powerful mitogenic activities in a variety of cell types and plays significant roles in many physiological processes e.g. angiogenesis and wound healing. There is increasing demand for large scale production of recombinant human FGF-1 (rhFGF-1), in order to investigate the potential medical use. In the present study, we explored SHuffle™ T7 strain for production of rhFGF-1. METHODS: A synthetic gene encoding Met-140 amino acid form of human FGF-1 was utilized for expression of the protein in three different E. coli hosts (BL21 (DE3), Rosetta-gami™ 2(DE3), SHuffle™ T7). Total expressions and soluble/insoluble expression ratios of rhFGF-1 in different hosts were analyzed and compared. Soluble rhFGF-1 produced in SHuffle™ T7 cells was purified using one-step heparin-Sepharose affinity chromatography and characterized by a variety of methods for physicochemical and biological properties. RESULTS: The highest level of rhFGF-1 expression and maximum soluble/insoluble ratio were achieved in SHuffle™ T7 strain. Using a single-step heparin-Sepharose chromatography, about 1500mg of purified rhFGF-1 was obtained from one liter of the culture, representing purification yield of ∼70%. The purified protein was reactive toward anti-FGF-1 ployclonal antibody in immunoblotting. Mass spectrometry confirmed the protein had expected amino acid sequence and molecular weight. In reverse-phase high-performance liquid chromatography (RP-HPLC), the protein displayed the same retention time with the human FGF-1 standard, and purity of 94%. Less than 0.3% of the purified protein was comprised of oligomers and/or aggregates as judged by high-performance size-exclusion chromatography (HP-SEC). Secondary and tertiary structures of the protein, investigated by circular dichroism and intrinsic fluorescence spectroscopy methods, respectively, represented native folding of the protein. The purified rhFGF-1 was bioactive and stimulated proliferation of NIH 3T3 cells with EC50 of 0.84ng/mL. CONCLUSION: Although SHuffle™ T7 has been introduced for production of disulfide-bonded proteins in cytoplasm, we herein successfully recruited it for high yield production of soluble and bioactive rhFGF-1, a protein with 3 free cysteine and no disulfide bond. To our knowledge, this is the highest-level of rhFGF-1 expression in E. coli reported so far. Extensive physicochemical and biological analysis showed the protein had similar characteristic to authentic FGF-1.

MiR-377 reverses cancerous phenotypes of pancreatic cells via suppressing DNMT1 and demethylating tumor suppressor genes.

AIM: The aim was to investigate the effect of miR-377 on DNMT1 expression and cancer phenotype in pancreatic cancer cells. MATERIALS & METHODS: Real-time PCR, luciferase assay, MTT and Annexin-PI staining were used. RESULTS: Decreased miR-377 and increased DNMT1 (verified as a target for mir-377) levels in pancreatic cancer tissues and cell lines in comparison with normal tissues was confirmed to be influenced by promoter methylation. Also hypermethylation of BNIP3, SPARC, TFPI2 and PENK promoters was observed in tumor samples but not in normal tissues which negatively correlated with their expression. Restoration of miR-377 resulted in a reduction of the expression of DNMT1 and reactivation of BNIP3 and SPARC genes via promoter demethylation. Furthermore, enhanced expression of miR-377 could significantly inhibit cell proliferation and induce apoptosis. CONCLUSION: Our findings showed that miR-377 through targeting DNMT1 could reduce DNA methylation of some tumor suppressor genes and restore their expression in pancreatic cancer cells.

Contribution of dopamine neurotransmission in proconvulsant effect of Toxoplasma gondii infection in male mice.

Epilepsy is one of the most common neurologic disorders worldwide with no distinguishable cause in 60% of patients. One-third of world's population is infected with Toxoplasma gondii (T. gondii). This intracellular parasite has high tendency to excitable cells including neurons. We assessed seizure susceptibility and involvement of dopaminergic system in male mice with acute and chronic T. gondii infection. Mice were infected by intraperitoneal injection of T. gondii cysts. Acute and chronic stages of infection were determined by quantification of SAG1/BAG1 transcripts and level of repetitive REP-529 sequence in the brain of mice by real-time PCR. Threshold of clonic seizures was measured by tail vein infusion of pentylenetetrazole. The infected mice were pretreated with D1 and D2 dopamine receptor antagonists, and seizure threshold was measured. Moreover, seizure threshold was determined after treatment of toxoplasmosis by sulfamethoxazole and trimethoprim. SAG1 level reached the maximum at week 2 after infection and then declined. The maximum level of BAG1 was observed at the week 3 and preserved till the week 8. REP-529 was detected at first week after infection, reached maximum at the week 3 and kept at this level till the eighth week. Threshold of seizures significantly decreased in both acute and chronic phases of infection. D1 and D2 receptors antagonists inhibited proconvulsant effect of toxoplasmosis. Chemotherapy inhibited parasite growth and multiplication, and returned seizure susceptibility to the level of non-infected mice. Dopaminergic neurotransmission participates in proconvulsant effect of T. gondii. The effect of parasite is eliminated by antibiotic therapy. © 2017 Wiley Periodicals, Inc.

Association of the Asp1312Gly Thyroglobulin Gene Polymorphism with Susceptibility to Differentiated Thyroid Cancer in an Iranian Population

Background and aim: While the causes of thyroid cancer in most patients remain largely unknown, it has recently been reported that there may be links to particular chromosome regions. In particular, polymorphisms (SNPs) in the thyroglobulin (TG) gene could be susceptibility factors. Methods: In this case-control study, any association of the Asp1312Gly single nucleotide polymorphism (SNP) in the TG gene (rs2069556) with susceptibility to differentiated thyroid cancer (DTC) was investigated among 103 Iranian patients and 100 controls who had no history of any type of cancer. Genomic DNA was extracted from the whole blood by salting out procedure. High Resolution Melting (HRM) technique was used to detect this SNP. Results: Data were analyzed with SPSS software and the results showed that the recessive GG genotype was associated with an increased risk of differentiated thyroid carcinoma when compared to the AA+AG genotypes (OR: 2.06; CI: 1.09-3.89; P-value: 0.025). Conclusion: Although our study demonstrated that differentiated thyroid cancer is significantly associated with this polymorphism, further studies with larger populations are required to confirm our findings.

MiR-339 and especially miR-766 reactivate the expression of tumor suppressor genes in colorectal cancer cell lines through DNA methyltransferase 3B gene inhibition.

It is observed that upregulation of DNMT3B enzyme in some cancers, including colon cancer, could lead to silencing of tumor suppressor genes. MiR-339 and miR-766 have been predicted to target 3'UTR of DNMT3B gene. Luciferase reporter assay validated that individual and co-transfection of miR-766 and miR-339 into the HEK293T cell reduced luciferase activity to 26% ± 0.41%, 43% ± 0.42 and 64% ± 0.52%, respectively, compared to the control (P < 0.05). Furthermore, transduction of miR-339 and miR-766 expressing viruses into colon cancer cell lines (SW480 and HCT116) decreased DNMT3B expression (1.5, 3-fold) and (3, 4-fold), respectively. In addition, DNA methylation of some tumor suppressor genes decreased. Expression of these genes such as SFRP1 (2 and 1.6-fold), SFRP2 (0.07 and 4-fold), WIF1 (0.05 and 4-fold), and DKK2 (2 and 4-fold) increased in SW-339 and SW-766 cell lines; besides, expression increments for these genes in HCT-339 and HCT-766 cell lines were (2.8, 4-fold), (0.005, 1.5-fold), (1.7 and 3-fold) and (0.04, 1.7-fold), respectively. Also, while in SW-766, cell proliferation reduced to 2.8% and 21.7% after 24 and 48 hours, respectively, SW-339 showed no reduced proliferation. Meanwhile, HCT-766 and HCT-339 showed (3.5%, 12.8%) and (18.8%, 33.9%) reduced proliferation after 24 and 48 hours, respectively. Finally, targeting DNMT3B by these miRs, decreased methylation of tumor suppressor genes such as SFRP1, SFRP2, WIF1 and DKK2 in the mentioned cell lines, and returned the expression of these tumor suppressor genes which can contribute to lethal effect on colon cancer cells and reducing tumorigenicity of these cells.