فهرست مطالب

Reports of Biochemistry & Molecular Biology - Volume:8 Issue: 4, 2020
  • Volume:8 Issue: 4, 2020
  • تاریخ انتشار: 1399/03/17
  • تعداد عناوین: 15
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  • Adish Zhansaya*, Mukantayev Kanatbek, Tursunov Kanat, Ingirbay Bakhytkali, Kanayev Darkhan, Kulyyassov Arman, Tarlykov Pavel, Mukanov Kasym, Ramankulov Yerlan Pages 347-357
    Background

    The programmed cell deathprotein 1 (PD-1), which is a member of the CD28 receptor family, can negatively regulate antitumor immune responses by interacting with its ligands, PD-L1 or PD-L2. The PD-1–PD-L1 signaling pathway is a checkpoint mechanism that plays essential roles in downregulating immune responses in cancerous tissues. Thus, blocking this signaling pathway leads to enhanced antitumor immunity, potentially preventing tumor progression.

    Methods

    We synthesized the extracellular domain of the PD-1 receptor (rPD-1) de novoby using a two-step polymerase chain reaction and the Phusion® DNA polymerase. The synthesized gene was cloned into the pET28 expression plasmid and transformed into competent Escherichia coli. Purification of rPD-1 was performed by metal-affinity chromatography, using a HisTrap column.Purified rPD-1 was characterized by western blotting and mass spectrometry using the SwissProt database and the Mascot program.

    Results

    Designed and synthesized construct of rPD-1 was 500 bpin size. Analysis of the electrophoresis data of purified rPD-1 showed the presence of a protein with a molecular mass of 21 kDa. Mass spectrometry data using the SwissProt database and the Mascot program outputted the highest-scoring sequence to correspond to rPD-1.

    Conclusions

    Synthesized de novo rPD-1 may have potential therapeutic applications in enhancing antitumor immune responses

    Keywords: Cancer, PD-1 ligands, PD-1 receptor, Tumor immunotherapy
  • MohammadAli Esmaili, Ahmad Kazemi, Farhad Zaker, Mohammad Faranoush, MohammadReza Rezvany* Pages 358-365
    Background

    The current study aims to investigate the relationship of miR-24 expression with plasma methotrexate (MTX) levels, therapy-related toxicities, and event-free survival (EFS) in Iranian pediatric acute lymphoblastic leukemia (ALL) patients.

    Methods

    The study included 74 ALL patients in consolidation phase and 41 healthy children. RNA was extracted from plasma, polyadenylated, and reverse transcribed. miR-24 expression was determined by quantitative polymerase chain reaction (qPCR). Plasma MTX concentrations were measured by high performance liquid chromatography (HPLC) 48 h after high-dose methotrexate (HD-MTX) injection. The diagnosis of ALL was further subclassified as B-ALL or T-ALL via flow cytometry.

    Results

    miR-24 expression was less in pediatric ALL patients than in the control group (p = 0.0038). Furthermore, downregulation of miR-24 was correlated with intermediate-to high-grade HD-MTX therapy toxicities (p = 0.025). Nevertheless, no statistically significant associations were seen between miR-24 levels and plasma MTX levels 48 h after HD-MTX administration (p > 0.05) or EFS in pediatric ALL patients (p > 0.05).

    Conclusions

    miR-24 expression may contribute to interindividual variability in response to intermediate-to high-grade HD-MTX therapy toxicities under Berlin Frankfurt Munster (BFM) treatment.

    Keywords: Acute Lymphoblastic Leukemia, Event Free Survival, Methotrexate, Mir, Toxicity
  • Jagadish Babu Dasari, Bini Chhetri Soren, Alessio Ottaviani, Cinzia Tesauro, Simona Marino, Beatrice Messina, Paola Fiorani* Pages 366-375
    Background

    DNA topoisomerases 1B are a class of ubiquitous enzyme that solves the topological problems associated with biological processes such as replication, transcription and recombination. Numerous sequence alignment of topoisomerase 1B from different species shows that the lengths of different domains as well as their amino acids sequences are quite different. In the present study a hybrid enzyme, generated by swapping the N-terminal of Plasmodium falciparum into the corresponding domain of the human, has been characterized.

    Methods

    The chimeric enzyme was generated using different sets of PCR. The in vitro characterization was carried out using different DNA substrate including radio-labelled oligonucleotides.

    Results

    The chimeric enzyme displayed slower relaxation activity, cleavage and re-ligation kinetics strongly perturbed when compared to the human enzyme.

    Conclusions

    These results indicate that the N-terminal domain has a crucial role in modulating topoisomerase activity in different species.

    Keywords: N-terminal domain, Plasmodium falciparum topoisomerase 1B, Topoisomerase 1B
  • Nasser Beikzadeh*, AbdolReza Varasteh Pages 376-382
    Background

    Pathogenesis-related (PR) proteins are induced in response to biotic and abiotic stresses. Some plant proteins, including Mal d 1, Mal d 2, and Mal d 3 in apple, are allergens. In this study, the effects of Erwinia amylovora infection of two apple cultivars, Red and Golden Delicious, on the expression of PR proteins homologous to Mal d 1, 2, and 3 were investigated.

    Methods

    In natural conditions trees with or without disease symptoms were sampled. In addition, seeds of the cultivars were grown in a greenhouse and seedlings were examined in three groups: 1) those inoculated by E. amylovora, 2) those inoculated by sterilized distilled water, and 3) uninoculated. Real-time PCR was used to determine expression of the Mal d 1, 2, and 3 genes (Mal d 1, 2, and 3) in infected and uninfected samples. Statistical analyses were performed using SPSS and graphs were produced by Excel. P values < 0.05 were considered significant.

    Results

     The analysis of variance showed that in natural conditions the effect of infection on the mean relative expression of Mal d 2 and 3 was significant, and more so in Red than in Golden Delicious. The analysis of variance of the greenhouse samples showed that the effect of infection on the mean relative expression of Mal d 1, 2, and 3 in both cultivars was significant.

    Conclusions

    Our results suggest that Mal d 2 is more related to plant defense than Mal d 1 or Mal d 3, and is more highly expressed in E. amylovora-resistant than in E. amylovora-sensitive cultivars.

    Keywords: Allergens, Erwinia amylovora, Homologous, Pathogenesis-related proteins
  • Reza Kamali Kakhki, Ehsan Aryan, Zahra Meshkat, Mojtaba Sankian* Pages 383-393
    Background

    The line probe assay (LPA) is one of the most accurate diagnostic tools for detection of different Mycobacterium species. Several commercial kits based on the LPA for detection of Mycobacterium species are currently available. Because of their high cost, especially for underdeveloped and developing countries, and the discrepancy of non-tuberculous mycobacteria (NTM) prevalence across geographic regions, it would be reasonable to consider the development of an in-house LPA. The aim of this study was to develop an LPA to detect and differentiate mycobacterial species and to evaluate the usefulness of PCR-LPA for direct application on clinical samples.

    Methods

    One pair of biotinylated primers and 15 designed DNA oligonucleotide probes were used based on multiple aligned internal transcribed spacer (ITS) sequences. Specific binding of the PCR-amplified products to the probes immobilized on nitrocellulose membrane strips was evaluated by the hybridization method. Experiments were performed three times on separate days to evaluate the assay’s repeatability. The PCR-LPA was evaluated directly on nine clinical samples and their cultivated isolates.

    Results

    All 15 probes used in this study hybridized specifically to ITS sequences of the corresponding standard species. Results were reproducible for all the strains on different days. Mycobacterium species of the nine clinical specimens and their cultivated isolates were correctly identified by PCR-LPA and confirmed by sequencing.

    Conclusions

    In this study, we describe a PCR-LPA that is readily applicable in the clinical laboratory. The assay is fast, cost-effective, highly specific, and requires no radioactive materials.

    Keywords: Diagnosis, Line Probe Assay (LPA), Mycobacterium Infection, Tuberculosis
  • Nazila Ariaee, Mojtaba Sankian, Abdolreza Varasteh, Mitra Hosseinpour, Farahzad Jabbari* Pages 394-400
    Background

    The diagnosis and treatment of allergic diseases require high quality pollen allergen extracts for reliable test results and effective treatments. The quality of the pollen allergen extracts is influenced by pharmacologically inert ingredients, such as stabilizers which are added to prevent the degradation of the allergenic activity. This study was conducted to develop a stabilizer formulation in order to protect the allergenic activity of the pollen’s extracts.

    Methods

    Pine and orchard grass pollen allergen extracts were incubated for 40 days at 37 °C. The effects of chemicals were examined via inhibition ELISA on days 7, 14, 21, 28, and 40 to evaluate the ability of the pollen allergen extracts to inhibit specific IgE in the sera of sensitized patients.

    Results

    Our findings showed that the pine pollen and orchard grass allergen extracts treated with Lys/Glu had the best stabilizing effect resulting in a 97% IgE inhibition following the 40 days of incubation. In the non-treatment group, the IgE inhibition decreased to 23% at the end of the 40 days. The orchard grass pollen allergen extracts receiving no treatment decreased to 12% IgE inhibition following the 40-day incubation.

    Conclusions

    Amino acids are able to act as an effective stabilizer for pollen allergen extracts and prevent the degradation of their activity over time. Particularly applying Lys/ Glu in pollen allergenic extracts can protect allergenic activity and potency of the pollen extracts to inhibit specific IgE in human sera.

    Keywords: Amino acids, Pollen, Skin Prick Test, Stabilizing
  • Morteza Bagheri*, Khadijeh Makhdoomi, Ali Taghizadeh Afshari, AhmadAli Nikibakhsh, Isa Abdi Rad Pages 401-406
    Background

    Autosomal dominant polycystic kidney disease (ADPKD) is a delayed-onset renal disorder that results from a mutation in the PKD1 or PKD2 genes. Autosomal dominant polycystic kidney disease results in end-stage renal disease due to renal cystic dysplasia. The aim of this study was to evaluate, by exon sequencing, the disease-causing variants of PKD2 (exons 4, 6, and 8) in Iranian ADPKD patients.

    Methods

    Genomic DNA was extracted from 3-5 ml of peripheral blood by the salting-out method. PKD2 exons 4, 6, and 8 were PCR-amplified and sequenced.

    Results

    Three disease-causing PKD2 variants were identified; all three were missense mutations in exon 4. The mutations were AGC → ACC (c.893G>C, cDNA.959G>C, S298T), TAC → TTC (c.1043A>T, cDNA.1109 A>T, Y348F), and GAA → GAT (c.1059A>T, cDNA.1125 A>T, E353D. These novel pathogenic variants may cause loss of the normal protein function.

    Conclusions

    Our results suggest that AGC → ACC (c.893G>C, cDNA.959G>C, S298T), TAC → TTC (c.1043A>T, cDNA.1109 A>T, Y348F), and GAA → GAT (c.1059A>T, cDNA.1125 A>T, E353D variants are common in Iranian ADPKD patients. These mutations modify the transmembrane domain and likely influence PC2 function.

    Keywords: Pathogenic Variants, PKD2, Autosomal Dominant Polycystic Kidney Disease
  • Merat Mahmoodi, Farnaz Sedghy*, Abdollah Jafarzadeh Pages 407-412
    Background

    Thymus vulgaris, or thyme belongs to the Lamiaceae family of aromatic plant species and has established antioxidant and anti-inflammatory properties. We examined the association between thyme extract treatment to recovered urinary levels of melatonin, a hormone with neuroprotective effects, in mice induced with EAE.

    Methods

    Eight B6 mice induced with EAE were randomized into two groups and exposed to either 50 mg/kg of thyme extract or PBS. After EAE induction, mice were injected i.p every other day from day 0 to 21. Four B6 mice without EAE were considered the healthy control group. Urine samples were collected consecutively for two 24 h periods on day 19 and 20. We examined whether thyme extract treatment modified urinary melatonin sulfate concentration (ng/mL) in EAE-induced mice using an ELISA.

    Results

    The clinical score and body weight in thyme-treated EAE group were significantly lower in comparison to the EAE control group at indicated time points. The urinary melatonin concentration was significantly lower in the EAE control group compared to the healthy mice. There was no significant difference between thyme-treated and EAE groups regarding the urine melatonin concentration.

    Conclusions

    Our results show that exposing EAE mice to thyme extract improved their clinical symptoms, however, there was no significant effect on urinary melatonin concentration.

    Keywords: Enzyme-linked immunosorbent assay (ELISA), Experimental autoimmune encephalomyelitis (EAE), Melatonin, Thymus vulgaris (Thyme), Urine
  • Rasoul Kaviani, Iman Pouladi, Mohammad Niakan*, Reza Mirnejad Pages 413-418
    Background

    Acinetobacter baumannii (A. baumannii) is one of the most important bacteria causing nosocomial infections worldwide. Over the past few years, several strains of A. baumannii have shown antibiotic resistance, which may be due to the activity of efflux pumps. This study was aimed to detect AdeFG efflux pump genes and their contribution to antibiotic resistance in A. baumannii clinical isolates.

    Methods

    A total of 200 A. baumannii clinical isolates were collected from clinical specimens of ulcers, pus, sputum, and blood. All isolates were identified using standard biochemical tests. After identifying and cleaving the genome by boiling, PCR was performed on samples using specific primers. The antimicrobial susceptibility patterns were determined by disk diffusion, with and without CCCP efflux pump inhibitor were determined according to CLSI guidelines.

    Results

    We identified 60 clinical isolates of A. baumannii using biochemical differential tests. Identification of all A. baumannii isolates was confirmed by blaOXA-51-like PCR. According to the results of our study, 98.37% of A. baumannii isolates were resistant to ciprofloxacin, norfloxacin, and levofloxacin. PCR results indicated that all 60 A. baumannii isolates contained the AdeF and 76.66% contained AdeG.

    Conclusions

    the results of this study demonstrated that most of the A. baumannii isolates contained AdeF and AdeG efflux pump genes, and more than 98% of the isolates were resistant to ciprofloxacin, norfloxacin, and levofloxacin. This reflected the significant contribution of efflux pumps to the development of resistance to these antibiotics.

    Keywords: Acinetobacter baumannii, AdeFG, Antibiotic Resistance, Efflux pump, Molecular detection
  • Hasan Mahmud Reza*, Razwa Saleh, Preeti Jain, Ghazi Muhammad Sayedur Rahman, Asim Kumar Bepari Pages 419-428
    Background

    c-MAF, a transcription factor that belongs to the b-Zip Maf transcription factor family, was found to be critical for lens development in vertebrates. It is a well-known fact that the adult human ocular surface expresses c-MAF, however, its role in the limbus, cornea and conjunctiva remains unknown. Thus, the present study aimed to investigate c-MAF expression within the human ocular surface, and its potential role in pterygium pathogenesis.

    Methods

    We performed immunohistochemical staining to detect c-MAF expression in frozen adult human tissue sections, including the limbus, cornea and conjunctiva, and cultured cells from eye cadavers. We then compared c-MAF expression to the expression of a known protein, P63. Lastly, we performed RT-PCR, and immunohistochemistry for c-MAF expression in healthy adult human conjunctiva and pterygium.

    Results

    We found differential c-MAF expression between adult human limbus, cornea and conjunctiva tissues. Further, we observed that c-MAF is downregulated in the pterygium compared to healthy conjunctiva.

    Conclusions

    Overall, our results suggest that c-MAF may play a context-specific role in maintaining limbal, corneal and conjunctival homeostasis, and may be critical for preventing pterygium development in humans.

    Keywords: Conjunctiva, C-MAF Expression, Human Ocular Surface, Pterygium
  • Atefeh Sohan Forooshan Moghadam, Mitra Ataei, Ghazaleh Arabzadeh, Kowsar Falahati, Fatemeh Roshani, MohammadHossein Sanati* Pages 429-437
    Background

    In multiple sclerosis (MS), the immune system acts against myelin lesions of the central nervous system, destroying neuronal fibers resulting in signal transmission disturbances in the nervous system. MicroRNAs play important roles in the post-transcriptional regulation of gene expression and in the regulation of disease activity and its response to treatment. The goal of this study was to determine the role of miR-18a-5p by comparing its expression in MS patients and healthy subjects.

    Methods

    RNA was isolated from blood samples of 32 MS patients and 32 healthy individuals, and miR-18a-5p expression was determined by real-time polymerase chain reaction (real-time PCR).

    Results

    miR-18a-5p expression was significantly less in MS patients than in healthy subjects.

    Conclusions

    The reduction of miR-18a-5p expression may be via pathway signaling. Altered signaling plays an important role in MS pathogenesis and the miR-18a-5p expression profile in blood cells can be described as a prognostic biomarker and identifier of high-risk individuals in MS.

    Keywords: MicroRNA (miRNA), MiRNA-18a-5p, Multiple Sclerosis (MS)
  • Elaheh Seyed Abutorabi, Shiva Irani, Marjan Yaghmaie, Seyed Hamid Ghaffari* Pages 438-445
    Background

    Thyroid cancer is the most prevalent endocrine malignancies globally. Anaplastic thyroid carcinoma (ATC) accounts for 1-3% of all Thyroid cancer. The evidence showed that ATC is a highly invasive solid tumor with poor prognosis. Despite conventional chemotherapy treatments, a considerable number of patients show developing resistance to therapeutic agents and tumor relapse. The aim of this study was the investigation anti-tumor effect of Abemaciclib (novel targeted cancer therapy drug) on Anaplastic Thyroid carcinoma SW1736 and C643 cell lines.

    Methods

    SW1736 and C643 cell lines were treated by desire concentrations of Abemaciclib (0, 1, 2.5, 5, 10, and 20 μM) and cell viability was measured by MTT assay. Also, Anoikis resistance assay was conducted for non-adherent the cells in the exposure of Abemaciclib. The gene expression of apoptotic and anti-apoptotic genes was conducted by quantitative Real-time PCR.

    Results

    Abemaciclib at the concentration of 10 and 20 μM effectively reduced cell proliferation and growth of the ATC cells compared to the control (p=0.000). Furthermore, we showed that 10 and 20 μM doses of the Abemaciclib inhibited the non-adherent ATC cells which were resistant to Anoikis death significantly (p=0.001). Moreover, we demonstrated this targeted therapy significantly reduced anti-apoptotic gene expression levels (BCL2 and CMYC) (p<0.05) and increased apoptotic gene expressions such as P21 and BAX (p<0.05).

    Conclusions

    Our data suggested that Abemaciclib can be utilized as a novel therapeutic agent in ATC cancer. Further in vivo and in vitro investigations are needed to evaluate molecular and clinical mechanisms of Abemaciclib.

    Keywords: Abemaciclib, Anaplastic Thyroid Carcinoma, CDK4, 6 inhibitor
  • Sajjad Biglari, Koorosh Kamali, Sousan Banihashemi, Arash Faal Sezavari, Seyed Mohsen Aghajanpour Mir, Farkhondeh Behjati* Pages 446-453
    Background

    Alzheimer’s disease is one of the most common neurodegenerative and dementia disorders in people between the ages of 30 and 65. When symptoms appear in this age group, the disease is referred to as early-onset Alzheimer’s disease (EOAD). Unfortunately, the symptoms are progressive and no current treatments are effective.

    Methods

    In this research, 13 patients, aged 37 to 65 years with symptoms of early-onset Alzheimer’s disease, were studied. First, patient lymphocytes were isolated and cultured in RPMI 1640 medium using a special micronucleus (MN) culture method. Next, the lymphocytes were harvested and prepared on slides. The slides were then examined by fluorescent microscopy using a unique FISH protocol specific for MNs. The patients were divided into groups aged 30-39, 40-49, and 50-65.

    Results

    We found that 19.76% of the MNs from our EOAD patients originated in chromosome 21. Micronuclei originated in chromosome 21 in 21.20 and 16.52% of patients without and with family histories of Alzheimer’s, respectively. This difference was not significant. Also, the percentage of micronuclei originating in chromosome 21 was not dependent on the patient age at the time of the study, or symptom onset age or duration.

    Conclusions

    This study shows that the rate of micronuclei with the origin of chromosome 21 is high in these patients. However, the micronucleus increased has no significant relationship with age and duration of disease or family history of it.

    Keywords: Early-onset Alzheimer’s disease (EOAD), Chromosomal instability, Fluorescence in Situ Hybridization (FISH), Neurodegenerative diseases, Micronucleus (MN)
  • Sanaz Zebardast, Mehdi Sahmani, Saeed Mohammadi, Farshad Foroughi, Ali Dehghani Fard, Zahra Mohammadi, Sahar Khojastepour, Mehdi Azad* Pages 454-457
    Background

    DNA methylation is an epigenetic modification that has the ability to alter gene expression and function. These epigenetic changes have been associated with the development of cancer. Previous research has found that DNA methylation patterns can predict disease prognosis for patients with Acute Promyelocytic Leukemia (APL). The role of DNMT1 and CDH1 in regulating the extension of cells are studied in this study.

    Methods

    DNA was extracted from peripheral blood samples of APL patients and treated with bisulfite. DNMT1 and CDH1 gene promoter methylation was subsequently analyzed using methylation-specific PCR (MSP). Real-time PCR was used to measure the expression level of DNMT1 and CDH1 genes.

    Results

    Partial methylation of the CDH1 gene promoter was detected in 20% of APL patients and an unmethylated status was detected in 80% of patient samples. Additionally, an unmethylated status in the DNMT1 gene promoter was detected in 100% of APL patient samples.

    Conclusions

    Our study found the CDH1 gene promoter to be unmethylated in almost all APL patients, while the DNMT1 promoter was unmethylated in all APL patients. Furthermore, we observed an increase in both CDH1 and DNMT1 gene expression in APL patients compared to healthy controls. These findings suggest that DNMT1 may not have a specific role in inhibiting CDH1 gene expression in APL. Applying higher resolution techniques would help to better uncover the DNA methylation patterns in patients with APL. Further research is required to determine the role of DNA methylation and CDH1 and DNMT1 gene expression in APL.

    Keywords: Acute Promyelocytic Leukemia, CDH1, DNMT, Promoter Methylation
  • Omid Salemi, Zahra Noormohammadi, Fariborz Bahrami, Seyed Davar Siadat, Soheila Ajdary* Pages 465-472
    Background

    It is estimated that one third of the world's population is infected with Mycobacterium tuberculosis (Mtb), the causative agent of Tuberculosis (TB). The BCG vaccine is widely used to fight against TB; however, many question its ability to provide complete protection from Mtb. Recently, the "Region of Difference 1" (RD1) set of genes were shown to be involved in the pathogenesis of Mtb. Downstream of RD1 transcription region, two proteins are encoded, known as EspB and EspC, which were found to contribute to Mtb virulence. In this study these two proteins are targeted as potential vaccine candidates against TB.

    Methods

    The EspB and EspC Mtb genes were codon-optimized for expression and synthesis in Escherichia coli (E. coli). The amplicons were cloned into a pET21a expression vector and transformed into E. coli BL21(DE3). The expression and purity of the expressed proteins (i.e. rEspC, rEspB and rEspC/EspB) were confirmed by SDS-PAGE and Western blotting. Moreover, BALB/c mice were immunized against Mtb using the recombinant proteins. Finally, the mice sera were analyzed via Western blotting.

    Results

    EspC, EspB, and EspC/EspB fusion genes were cloned and expressed in E. coli. Both SDS-PAGE and Western blots confirmed the presence and successful purification of the desired proteins. Moreover, antisera produced against the purified recombinant proteins reacted with Mtb proteins.

    Conclusions

    rEspC, rEspB, and rEspC/EspB could be expressed and purified using an E. coli expression system. The recombinant proteins induced the production of antibodies in BALB/c mice that reacted with Mtb proteins.

    Keywords: EspB, EspC, ESX-1, Mycobacterium tuberculosis