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Background and objectives This study aimed to determine whether the selected neuregulin1 (NRG1) gene variants (rs2439272 and rs6988339) are associated with schizophrenia, and Cognitive Impairments Associated with Schizophrenia (CIAS). Methods Participants with schizophrenia (N = 276) and healthy controls (N = 193) were enrolled. Cognitive assessment using the Wisconsin card sorting test (WCST) and Wechsler adult intelligence scale and genotyping for selected variants of NRG1 were performed. Results For both the rs2439272 and rs6988339, compared to healthy controls, we found significant higher frequencies of the G allele and the GG genotype in all and male participants with schizophrenia, and with all Wechsler adult intelligence score (WAIS) and Wisconsin card sorting test (WCST) scores (except for non-perseverative score) in all, male and female participants. Conclusions There was an association between rs2439272, rs6988339 of NRG1 variants and schizophrenia and CIAS (intelligence and executive functions).

Background: The CRISPR-Cas9 system, a powerful tool, has revolutionized genome engineering in eukaryotic cells and living organisms. However, this approach poses unique concerns and limitations when used by conventional transfection methods, including limited packaging size and low delivery efficiency. Here, we aim at assessing the transfection efficiency of DNA encoding for the CRISPR-Cas9 system by PEI coated Magnetic NanoParticle (MNPs) to improve the delivery of CRISPR/Cas9 constructs into eukaryotic cells. Results: Superparamagnetic iron oxide nanoparticles (SPIONs) coated with polyethylenimine (PEI) and then complexed with pCXLE-dCas9VPH-T2A-GFP-shP53 plasmid DNA. We used HEK-293 (human embryonic kidney) and Human foreskin fibroblasts (HFF) cells to express GFP after transfection to evaluate delivery efficiency with MNPs and Lipofection methods. PEI-coated nanoparticles with magnetic iron oxide core were synthesized by co-precipitation technique resulting in an average size of ~ 20 nm in diameter. Characterization of Magnetic Nano Particle (MNPs) revealed that particles have narrow size distribution sufficient colloidal stability. The result showed that the magnetofection method with an efficiency around 85.7% for HEK-293 and 28.2% for HFF. Also, transfection efficiency by lipofection method was 83.2% and 7.89% for HEK-293 and HFF respectively. Conclusion: The magnetofection was revealed to be more efficient than classic Lipofectamine transfection as measured by GFP expression. We show that PEI-MNPs enable effective delivery and improved safety of plasmids encoding CRISPR/Cas9 into eukaryote cells.

Schizophrenia (SCZ) is a debilitating, destructive, and chronic mental disorder and affects approximately one percent of the human population. Diagnosis in psychiatry is based on the patient's descriptions of his/her symptoms, interviewer's observations, history of disorder over time, and response to treatment. All of these data measure phenotype-based functions. But it appears that accurate diagnosis of such a complex disorder must be based on valid and reliable factors. In the present study, gene selection was based on the possible role of γ-aminobutyric acid (GABA) in psychopathology of SCZ and expression in blood. We evaluated the association of Na

Multiple sclerosis is a disease that affects male and female patients differently. Several studies have been performed to explain the gender differences in MS susceptibility, but the genetic causes underlying gender differences remain unknown. The association between multiple sclerosis and the HLA-DRB1*15:01 haplotype has been confirmed to be female-specific. We hypothesized other immunological components such as lnc-DC may be gender-specific among multiple sclerosis patients, especially when MS patients are negative for the HLA-DRB1*15:01 allele. Therefore, the current study, considering the results of previous studies, aimed to evaluate the expression level of the lnc-DC gene in HLA-DRB1*15:01-negative female patients with relapsing remitting MS (RRMS). A total of 50 MS female patients and 50 female healthy controls were enrolled in this observational case-control study. HLA-DRB1*15:01, as a critical risk factor for MS, was ruled out in all patients. The peripheral blood mononuclear cells were obtained from all patients and total RNA was isolated and cDNA synthesis was carried out. The gene expression of lnc-DC was evaluated by real-time quantitative PCR. Our results have shown that lnc-DC expression level was significantly higher in total MS female patients compared with female controls (P = 0.0044). In addition, the correlation between lnc-DC with disease duration, EDSS, and age at onset did not reach a statistical significance in our study (r = 0.0336, P = 0.817; r = 0.0914, P = 0.5278 and r = 0.0743, P = 0.6083, respectively). Our results give further evidence that lnc-DC may play a gender-dependent role in MS pathogenesis.

Hirudin is an inhibitor of thrombin and used as an effective anticoagulant, but has a potential to develop unacceptable immune responses. In this study, two computational tools were used to predict T-cell epitopes within Hirudin variant III (HVIII) sequence, and design mutations that would lessen its antigenicity. Homology models of native and mutant HVIII proteins (T4K, S9G, V21G, and V21K) were generated, and further used to assess their interactions with thrombin. The docking experiment showed that all mutants had a suitable pattern of interactions, with similar or lower interaction energies compared with the native protein. These complexes were subsequently subjected to molecular dynamics simulation. All mutants complexes had overall stable structures over simulation time, with RMSD, gyration radius, hydrogen bonds numbers, and accessible surface areas patterns that were comparable with the native HVIII over time. Interestingly, in all mutants, a shorter length was observed for the two salt bridges Arg73-Asp55 and Arg77-Glu57, which are suggested to be important in Hirudin-thrombin complex formation. Best selected mutants expressed in Escherichia coli BL21(DE3), subsequently SDS-PAGE and Western blot analysis confirmed the successful same expression of Hirudin and mutants. In conclusion, we believe that this computational approach could identify potentially safer proteins with preserved or even improved functionality.

International audience; Angiogenesis targeting is an attractive approach for cancer treatment. Vascular endothelial growth factor receptor 2 (VEGFR2) is such an important target that is overexpressed in tumor vasculature compared to the endothelium cells of resting blood vessels and blocking of its signaling inhibits neovascularization and tumor metastasis. Immunotoxins represent a promising group of targeted therapeutics to combat tumors. They consist of an antibody linked to a toxin and are designed to kill specifically the tumor cells. In this study, we fused a VEGFR2-specific Nanobody, the antigen-binding single-domain fragment derived from functional Heavy-chain antibody of Camelidae, to the truncated form of Pseudomonas exotoxin A and evaluated its ability to bind the VEGFR2 molecule on the cell surface. We demonstrate that this immunotoxin inhibits the proliferation of VEGFR2-expressing cells in vitro. This finding is considered to be a significant achievement in tumor therapy and it forms a basis for further studies in animal models.

International audience; In the present study, 193 Aspergillus strains were isolated from a total of 100 soil samples of pistachio orchards, which all of them were identified as Aspergillus flavus as the most abundant species of Aspergillus section Flavi existing in the environment. Approximately 59%, 81%, and 61% of the isolates were capable of producing aflatoxins (AFs), cyclopiazonic acid (CPA), and sclerotia, respectively. The isolates were classified into four chemotypes (I to IV) based on the ability to produce AFs and CPA. The resulting dendrogram of random amplified polymorphic DNA (RAPD) analysis of 24 selected A. flavus isolates demonstrated the formation of two separate clusters. Cluster 1 contained both aflatoxigenic and non-aflatoxigenic isolates (17 isolates), whereas cluster 2 comprised only aflatoxigenic isolates (7 isolates). All the isolates of cluster 2 produced significantly higher levels of AFs than those of cluster 1 and the isolates that produced both AFB(1) and AFB(2) were found only in cluster 2. RAPD genotyping allowed the differentiation of A. flavus from Aspergillus parasiticus as a closely related species within section Flavi. The present study has provided for the first time the relevant information on distribution and genetic diversity of different A. flavus populations from nontoxigenic to highly toxigenic enable to produce hazardous amounts of AFB(1) and CPA in soils of pistachio orchards. These fungi, either toxigenic or not-toxigenic, should be considered as potential threats for agriculture and public health.

International audience; The expression of four aflatoxin (AF) biosynthetic pathway genes (aflD, aflO, aflP and aflQ) was evaluated in 24 Aspergillus flavus strains isolated from soils of pistachio orchards, with the aim of rapidly and accurately differentiating toxigenic from non-toxigenic strains. The amounts of AFB1 produced by 20 aflatoxigenic strains varied from 1.25 to 321.56 ng/mg fungal dry weights in YES medium. RT-PCR results showed that transcription of the four genes was not always correlated with AF production. The expression pattern of aflO and aflQ, however, was found to be well correlated with the amounts of AFB1 produced when strains were arbitrarily classified into two types: type I, comprised of strains producing ≥30 ng/mg; and type II, low (≤30 ng/mg) and non-AF producers. The present study suggests that, under specific growth conditions, the expression pattern of aflatoxin biosynthetic pathway genes such as aflO and aflQ can be used to infer the AF-producing capability of A. flavus strains.

Thalassemia, which may be due to point mutations, translocations, and deletions involving the α or βglobin gene, is the most prevalent single gene disorder in Iran.This study aims to calculate the α/β ratio in normal cases, α- and β-thalassemia carriers by RT-PCR, real-time PCR, and in vitro globin chain synthesis (GCS) in order to establish the most accurate technique to distinguish between α- and β-thalassemia carriers in suspicious cases.The α/β ratios were calculated in all samples by RT-PCR, real-time RT-PCR, and in vitro GCS.Using RT-PCR, the ratios were 1.09 ± 0.07 in normal samples, 1.2 ± 0.17 in β-thalassemia, 1.08 ± 0.19 in mild α-thalassemia, and 0.96 ± 0.19 in severe α-thalassemia carriers. In real-time RT-PCR, the ratios were 2.21 ± 1.36 in normal samples, 5.12 ± 1.83 in β-thalassemia, 2.88 ± 0.81 in mild α-thalassemia, and 1.18 ± 0.52 in severe α-thalassemia carriers. With GCS, the ratios were 1.03 ± 0.1 in normal samples, 1.9 ± 0.37 in β-thalassemia, 0.8 ± 0.13 in mild α-thalassemia, and 0.59 ± 0.12 in severe α-thalassemia carriers.To determine the most accurate technique, we statistically analyzed the α/β ratios obtained from the three standard methods. The ratio obtained by GCS and real-time PCR were helpful in distinguishing between α and β carriers in suspicious patients in whom the mutation detection was limited and the risk for offspring was not clear. The use of this technique is more obvious when time is restricted (i.e. during the pregnancy period).

Angiogenesis targeting is an attractive approach for cancer treatment. Vascular endothelial growth factor receptor 2 (VEGFR2) is such an important target that is overexpressed in tumor vasculature compared to the endothelium cells of resting blood vessels and blocking of its signaling inhibits neovascularization and tumor metastasis. Immunotoxins represent a promising group of targeted therapeutics to combat tumors. They consist of an antibody linked to a toxin and are designed to kill specifically the tumor cells. In this study, we fused a VEGFR2-specific Nanobody, the antigen-binding single-domain fragment derived from functional Heavy-chain antibody of Camelidae, to the truncated form of Pseudomonas exotoxin A and evaluated its ability to bind the VEGFR2 molecule on the cell surface. We demonstrate that this immunotoxin inhibits the proliferation of VEGFR2-expressing cells in vitro. This finding is considered to be a significant achievement in tumor therapy and it forms a basis for further studies in animal models.

Purpose: To develop a hirudin therapeutic protein that eliminates unwanted immune response.Methods: Molecular dynamic simulation was performed on immunodominant hirudin epitopes 1-15 and 13-27 and its analog, modified peptide ligands (MPLs), namely, [Lys4] Hir1-15 and [Gly9] Hir1-15, [Gly21] Hir13-27 and [Lys21] Hir13-27. The selected epitopes were modeled and 20 ns of molecular dynamics simulation was performed on peptide-HLA1 0101 and MPLs-HLA1 0101 complexes to gain a better understanding of molecular recognition mechanisms of MHC peptide binding. Characterization of the process was done by evaluation of root mean square deviation (RMSD) and total energy of binding.Result: All complexes of MPLs-HLA-DRB1 0101 showed thermodynamically unstable structure in comparison with native epitopes-HLA-DRB1 0101. The findings indicate that these analogs have different orientation in HLA grooves and are not available for suitable interaction with HLA-DRB1 0101.Conclusion: Altogether, the results show the potentials of predictive methods and molecular modeling in molecular mimicry of peptide-MHC interaction and provide insights into the binding characteristics of antigen presentation mechanism.Keywords: Modified peptide ligand, Epitopes, MHC peptide binding, Hirudin, Modified peptide ligands, Molecular dynamic simulation, Binding free energy

International audience; Vascular endothelial growth factor receptor-2 (VEGFR2) is an important tumor-associated receptor and blockade of the VEGF receptor signaling can lead to the inhibition of neovascularization and tumor metastasis. Nanobodies are the smallest intact antigen binding fragments derived from heavy chain-only antibodies occurring in camelids. Here, we describe the identification of a VEGFR2-specific Nanobody, named 3VGR19, from dromedaries immunized with a cell line expressing high levels of VEGFR2. We demonstrate by FACS, that 3VGR19 Nanobody specifically binds VEGFR2 on the surface of 293KDR and HUVECs cells. Furthermore, the 3VGR19 Nanobody potently inhibits formation of capillary-like structures. These data show the potential of Nanobodies for the blockade of VEGFR2 signaling and provide a basis for the development of novel cancer therapeutics.

Background. Thyroid peroxidase gene (TPO) mutations are one of the most common causes of thyroid dyshormonogenesis in patients with congenital hypothyroidism (CH). In this study, the prevalence of TPO gene mutations in patients with thyroid dyshormonogenesis in Isfahan was investigated.Methods. In this cross-sectional study, genomic DNA of 41 patients with permanent CH due to thyroid dyshormonogenesis was extracted using the salting out method. The 17 exonic regions of the TPO gene were amplified. SSCP technique was performed for scanning of the exonic regions of the TPO gene, except exon 8. DNA sequencing was performed for those with different migration patterns in SSCP by chain termination method. Exon 8 was sequenced directly in all patients. In 4 patients, all fragments were also sequenced.Results. One missense mutationc.2669G>A(NM_000547.5) at exon 15 (14th coding exon) in one patient in homozygous form and seven different single nucleotide polymorphisms (SNPs) in exons 1, 7, 8, 11, and 15 of TPO gene.Conclusion. The TPO gene mutations among CH patients with dyshormonogenesis in Isfahan were less frequent in comparison with other similar studies. It may be due to the presence of other unknown gene mutations which could not be detected by SSCP and sequencing methods.

Antibodies and their derivative fragments have long been used as tools in a variety of applications, in fundamental research work, biotechnology, diagnosis, and therapy. Camels produce single heavy-chain antibodies (VHH) in addition to usual antibodies. These minimal-sized binders are very robust and bind the antigen with high affinity in a monomeric state. Vascular endothelial growth factor recepror-2 (VEGFR2) is an important tumor-associated receptor that blockade of its signaling can lead to the inhibition of neovascularization and tumor metastasis. Here, we describe the construction, expression, and purification VEGFR2-specific Diabody. Two variable fragments of a same camel anti-VEGFR2 antibody were linked together by the upper hinge segment of antibody to make a diabody. We showed the ability of diabody to recognition of VEGFR2 on the cell surface by FACS. Diabodies can be produced in the low-cost prokaryotic expression system, so they are suitable molecules for diagnostic and therapeutic issues.

Genetic markers are used for identity testing and paternity analysis depends on knowing the allele frequencies in the population. Minisatellites show allelic variability in the number of repeat units. We have studied the allele frequencies and heterozygosity of two VNTRs (ApoB and D1S80) in Iranian populations. A total of 96 and 82 chromosomes were analyzed by PCR and gel electrophoresis for ApoB and D1S80 respectively. In the ApoB system, allele 37 was the most common followed by allele 35 whereas allele 23 was the most common followed by allele18 at the D1S80 locus. Observed heterozygosity was relatively low in ApoB than D1S80 locus, however, no significant differences were found between observed and expected heterozygosity.