Iranian Biomedical Journal
Volume:23 Issue: 6, Nov 2019

Acute myelocytic leukemia (AML) is a clonal malignancy resulting from the accumulation of genetic abnormalities in the cells. Human baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5), encodes survivin, is one of only a handful of genes that is differentially over-expressed in numerous malignant diseases including AML.

The BIRC5 was silenced permanently in two AML cell lines, HL‑60 and KG-1, via the CRISPR/Cas9n system. After transfection of CRISPR constructs, genomic DNA was extracted and amplified to assess mutation detection. To evaluate BIRC5 gene expression, quantitative real-time PCR was performed. Also, MTT cell viability and Annexin‑V/propidium iodide flowcytometric staining were performed, and the data were analyzed using the Kolmogorov-Smirnov, Levene's, and ANOVA tests.

The results indicated that Cas9n and its sgRNAs successfully triggered site-specific cleavage and mutation in the BIRC5 gene locus. Moreover, suppression of BIRC5 resulted in the reduction of cell viability, and induction of apoptosis and necrosis in HL60 and KG1 suggested that the permanent suppression of BIRC5 remarkably dropped the gene expression and cells viability.

This study reinforces the idea that BIRC5 disruption via Cas9n:sgRNAs has favorable effects
on the AML clinical outcome. It thereby can be a promising candidate in a variety of leukemia treatments.

Crimean-Congo hemorrhagic fever (CCHF) is an acute viral zoonotic disease, with a mortality rate of 30-50%. There is no approved vaccine or any specific antiviral treatment for CCHF; therefore, the rapid diagnosis seems to be crucial for both efficient supportive therapy and control of infection spread. In this study, the potency of recombinant nucleoprotein of virus expressed in prokaryotic system was investigated for diagnosis of the infection.

The DNA sequence of complete nucleoprotein ORF was codon optimized based on E. coli codon usage and synthesized commercially. The gene was subcloned in pCA4 vector and expressed in E. coli BL21 (DE3). Refolding and simultaneous purification of nucleoprotein were performed using protein folding liquid chromatography method. The recombinant nucleoprotein was analyzed by Western blotting, ELISA, immunofluorescence assay, and circular dichroism. Forty eight human samples, in three IgM positive and three negative control groups, were evaluated using recombinant nucleoprotein in a capture ELISA setting. Serum from healthy individuals, those suspected to viral hemorrhagic fevers, and positive samples of Chikungunya and Dengue were considered as negative controls.

The existence and structure of recombinant nucleoprotein were verified and confirmed. Capture IgM ELISA detected all positive samples (sensitivity of 100%), but none of the 25 negative samples was detected as positive (specificity of 100%). The test also detected all the included genotypes of virus.

Our recombinant nucleoprotein can be used in IgM capture ELISA for easy and efficient detection of CCHF in any lab in endemic regions.

There is a growing interest in development of an effective adjuvant system for improving DNA vaccines. Recent findings have confirmed an important role for autophagy in both innate and adaptive immunity. The current study was undertaken to determine the efficacy of autophagy induction with Beclin-1, as a novel adjuvant system, in mice immunized with human papilloma virus (HPV) DNA vaccine.

To determine whether autophagy induction with Beclin-1 enhances the efficacy of HPV DNA vaccine, female C57BL/6 mice were challenged with TC-1 tumor cells and were immunized three times at one-week intervals. Two weeks after the final immunization, the mice were sacrificed, and the antitumor effects were assessed by measurement of lymphocyte proliferation, cytotoxicity, cytokine production, and tumor regression.

Beclin-1 in combination with HPV-16 DNA vaccine encoding the E7 antigen induced a higher level of lymphocyte proliferation and cytotoxicity than the DNA vaccine alone. The novel combination increased the production of IFN-γ and highly inhibited tumor progression in comparison with DNA vaccine alone.

Administration of Beclin-1, as an autophagy inducer, with HPV DNA vaccine produces antitumor effects, providing an effective adjuvant for the induction of a strong antitumor immune response.

It is believed that the loading value of anticancer drug conjugated to the monoclonal antibody, called drug-to-antibody ratio (DAR), is the main quality feature of antibody-drug conjugates.

In this study, matrix assisted laser desorption/ionization mass spectrometry was used to determine the average molecular weight of trastuzumab and its three conjugated forms. The differences in the measured masses for each conjugate and unconjugated trastuzumab were compared to the expected mass change through the conjugation of one mole of related drug-linker, in order to measure DAR.

There was a consistency between the loading results of mass spectrometry and the measurements of UV spectrometry in most cases.

According to our findings, the MALDI-MS method for determining the loading values can be used rapidly and reliably to estimate the covalently bound drugs conjugated to antibodies when ESI-TOF-MS is unavailable.

Hair follicle stem cells (HFSCs) located in the bulge area has shown to be highly proliferative and could differentiate into neurons, glia, smooth muscle cell, and melanocytes in vitro. Simvastatin is an HMG-CoA reductase inhibitor that exerts pleiotropic effects beyond simple low-density lipoprotein lowering and has a similar impact on the differentiation of bone marrow stromal cells and peripheral blood mononuclear cells. The present study examined the hypothesis that the application of simvastatin would induce the HFSCs differentiation into keratinocyte.

The bulge of the hair follicle was anatomized, and HFSCs were cultivated. The flow cytometry and immunocytochemical staining for detection of nestin, CD34, and Kr15 biomarkers were performed before differentiation. In order to hasten the HFSCs differentiation to keratinocyte, HFSCs were treated with 1 µM, 2 µM, and 5 µM of simvastatin daily for a week. After differentiation, the flow cytometry and immunocytochemical staining were performed with Kr15 and Kr10 biomarkers, and the MTT assay was carried out as an index of cell viability and cell growth.

Our results showed that bulge of HFSCs were nestin and CD34 positive and Kr15 negative. Simvastatin significantly increased the viability of HFSCs (p < 0.05) at the concentration of 5 µM. In addition, the percentages of keratinocyte-differentiated cells treated with 5 µM of simvastatin showed a significant increase compared to all other treated groups (p < 0.05).

 Our findings demonstrate that 5 µM of simvastatin could induce HFSCs differentiation into keratinocyte.

MUTYH DNA glycosylase germline mutations are linked to the recessive inheritance of multiple adenoma. Studies have revealed that germline mutations in this gene are ethnicity related. This study aimed to identify the germline mutations in MUTYH gene and determine their prevalence among Jordanian patients with colorectal adenoma.

In this study, 150 colorectal adenoma patients and 150 cancer-free individuals with no previous history of polyps were recruited. Sanger DNA sequencing of the MUTYH gene (accession number NG_008189.1) was carried out using 3130xL Genetic Analyzer. Sequencing results were analyzed by ChromasPro, and mutational effects were predicted by online bioinformatics tools.

Two novel variants, g.87C>T and c.1264G>C, were identified. g.87C>T was also found in 60 (40%) patients and 10 (6.7%) controls. However, c.1264G>C was detected in 90 (60%) patients and 7 (4.7%) controls. Thus, a significant association was observed between these two variants and colorectal adenoma (p value for both variants was <0.0001). Moreover, the newly identified germline variant, c.1264G>C, was found to be significantly associated with colorectal adenoma transformation into malignancy (p < 0.0001).

The data showed high prevalence of two germline mutations in MUTYH gene among Jordanians with colorectal adenoma, which may make them as potential early biomarkers for diagnosis of colorectal adenoma.

Hypercoagulability and hypofibrinolysis are among the symptoms exhibited by diabetic patients. Our study aimed to address the polymorphic nature of Alu DNA fragment in the human tissue plasminogen activator gene within diabetes mellitus (DM) Jordanian patients.

Genomic DNA was isolated from 76 DM patients and 60 non-diabetic Jordanian individuals, and the Alu fragment was amplified using PCR.

The results showed that 80% of the non-diabetic Jordanian subjects were homozygotes for the deletion of the Alu fragment (Alu-/-), 16.7% were homozygotes for its insertion (Alu+/+), and 3.3% were heterozygotes (Alu+/-). Besides, 36.8% of the diabetic patients exhibited the Alu-/- or Alu+/- genotype, and 26.3% were Alu+/+. The Alu-/- genotype occurred less frequently in the diabetic individuals.

The high frequency of the Alu-/- genotype constitutes a protective deletion with respect to DM within the normal subjects.

Hemoglobin (Hb) Alesha is a rare and very unstable Hb variant, resulting in disruption of the heme pocket and producing severe hemolysis in heterozygous statues. In this study, we describe the first report of this variant in an Iranian boy originated from south of Iran with severe hemolytic anemia and mild splenomegaly.

A six-year-old boy from Khuzestan Province and his parents were studied. Gap-PCR and direct sequencing were performed to detect the a-globin gene deletions and β-globin gene mutations, respectively.

The subject had a sporadic mutation GTG to ATG (Val [valine]>Met [methionine]) at codon 67 in heterozygous form on β-globin gene, which was not detected in his parents.

Since both parents proved to be normal, this Hb variant could be considered as a de novo mutation, which is highly useful for prenatal diagnosis.