STAT protein family and cardiovascular diseases: overview of pathological mechanisms and therapeutic implications.

Globally, cardiovascular diseases (CVD) are one of the significant causes of death and are considered a major concern of human society. One of the most crucial objectives of scientists is to reveal the mechanisms associated with the pathogenesis of CVD, which has attracted the attention of many scientists. Accumulating evidence showed that the signal transducer and activator of transcription (STAT) signaling pathway is involved in various physiological and pathological processes. According to research on the molecular mechanisms of CVDs, the STAT family of proteins is one of the most crucial players in these diseases. Numerous studies have demonstrated the undeniable relevance of STAT family proteins in various CVDs. The aim of this review is to shed light on how STAT signaling pathways are related to CVD and the potential for using these signaling pathways as therapeutic targets.

Global and Regional DNA methylation silencing of PPARγ Associated with Glioblastoma Multiforme Pathogenesis.

BACKGROUND: The relationship between peroxisome proliferator-activated receptor gamma (PPARγ) expression level and epigenetic modifications occurring in glioblastoma multiforme (GBM) pathogenesis is largely unknown. Herein, we examine the association of PPARγ expression with its promoter and genomic global DNA methylation status, as well as DNA methyltransferases (DNMTs) gene expression in GBM patients. METHODS: We examined the patterns of promoter methylation and PPARγ expression in 26 GBM tissues and 13 adjacent non-tumor tissues by methylation-specific PCR (MSP), real-time PCR, and ELISA, respectively. Also, we examined the genomic global 5-methyl cytosine levels and DNMTs gene expression using ELISA and real-time PCR methods, respectively. RESULTS: We found that hypermethylation on a specific region of the PPARγ promoter is significantly associated with the downregulation of the PPARγ gene and protein level in GBM patients. Interestingly, the amount of 5-methyl cytosine level was significantly reduced in GBM patients and positively correlated with PPARγ protein expression. Furthermore, the expression level of DNMT1, DNMT3A, and 3B were upregulated in GBM patients and the average expression level of all three DNMTs was positively correlated with tumor area. Also, we found that tumors from cortical regions exhibited a higher global DNA hypomethylation and PPARγ hypermethylation was related to the increase in GBM risk. CONCLUSION: Our study demonstrated that global DNA methylation and PPARγ epigenetic silencing is associated with the GBM risk. Our data provide a novel molecular mechanistic insight into epigenetic silencing of PPARγ in GBM patients that may be relevant as a key tumor marker for GBM pathogenesis.

Overexpression of SIRT6 alleviates apoptosis and enhances cell viability and monoclonal antibody expression in CHO-K1 cells.

BACKGROUND: Chinese hamster ovary (CHO) cells are the most predominantly utilized host for the production of monoclonal antibodies (mAbs) and other complex glycoproteins. A major challenge in the process of CHO cell culture is the occurrence of cell death following different stressful conditions, which hinders the production yield. Engineering genes involved in pathways related to cell death is a remarkable strategy to delay apoptosis, improve cell viability and enhance productivity. SIRT6 is a stress-responsive protein that regulates DNA repair, maintains genome integrity, and is critical for longevity and cell survival in organisms. METHODS AND RESULTS: In this study, SIRT6 was stably overexpressed in CHO-K1 cells and the impact of its expression on apoptosis related gene expression profile, viability, apoptosis, and mAb productivity was investigated. While a significant increase was observed in Bcl-2 mRNA level, caspase-3 and Bax mRNA levels were decreased in the SIRT6 engineered cells compared to the parental CHO-K1 cells. Moreover, improved cell viability and decreased rate of apoptotic progression was observed in a SIRT6-derived clone in comparision to the CHO-K1 cells during 5 days of batch culture. anti-CD52 IgG1 mAb titers were improved up to 1.7- and 2.8-fold in SIRT6-derived clone during transient and stable expression, respectively. CONCLUSIONS: This study indicates the positive effects of SIRT6 overexpression on cell viability and anti-CD52 IgG1 mAb expression in CHO-K1 cells. Further studies are needed to examine the potential of SIRT6-engineered host cells for the production of recombinant biotherapeutics in industrial settings.

Evaluation of in vitro coculture of keratinocytes derived from foreskin and adipose-derived mesenchymal stem cells (AMSCs) on a multilayer oxygen-releasing electrospun scaffold based on PU/PCL.Sodium percarbonate (SPC)-gelatine/PU.

Despite significant advancements in tissue engineering and regenerative medicine during the last two decades, the fabrication of proper scaffolds with appropriate cells can still be considered a critical achievement in this field. Hypoxia is a major stumbling block to chronic wound healing, which restrains tissue engineering plans because a lack of oxygen may cause cell death. This study evaluated the cocultured human keratinocytes and human adipose-derived mesenchymal stem cells (AMSCs) on a multilayer oxygen-releasing electrospun scaffold based on PU/PCL.Sodium percarbonate (SPC)-gelatin/PU. The scaffold was characterized using Fourier transform infrared (FTIR) and scanning electron microscopy (SEM) methods. Flow cytometry confirmed mesenchymal stem cells, and then the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay and DAPI staining were used to assess the in vitro biocompatibility of the scaffold. The experimental results showed that the multilayer electrospun scaffold containing 2.5% SPC could efficiently produce oxygen. Furthermore, according to cell viability results, this structure makes a suitable substrate for the coculture of keratinocytes and AMSCs. Gene expression analysis of various markers such as Involucrin, Cytokeratin 10, and Cytokeratin 14 after 14 days confirmed that keratinocytes and AMSCs coculture on PU/PCL.SPC-gelatin/PU electrospun scaffold promotes dermal differentiation and epithelial proliferation compared to keratinocytes single-cell culture. Therefore, our study supports using oxygen-releasing scaffolds as a potential strategy to hasten skin tissue regeneration. Based on the results, this structure is suggested as a promising candidate for cell-based skin tissue engineering. Given that the developed oxygen-generating polymeric electrospun scaffolds could be used as part of a future strategy for skin tissue engineering, the PU/PCL.SPC-gelatin/PU hybrid electrospun multilayer scaffold in combination with keratinocyte/AMSC coculture is proposed as an effective substrate for skin tissue engineering and regenerative medicine platforms.

Construction and evaluation of wild and mutant ofatumumab scFvs against the human CD20 antigen.

Several monoclonal antibodies targeting the CD20 have been produced and Ofatumumab is a case in point. Although whole antibodies target cancer cells effectively, their applications are restricted in some ways. Single-chain fragment variable antibodies, rather than employing the entire structure of antibodies, have proven a practical approach for creating completely functional antigen-binding fragments. In current research, the DNA coding sequence of VL and VH of the wild and mutant forms of ofatumumab were joined with a flexible linker (GGGGS)3 separately. Using the E. coli BL21 (DE3) expression system, the VL-linker-VH genes were cloned into the pET-28 a (+), and the associated recombinant proteins were produced. Purified and refolded scFvs (scFv-C and scFv-V3) represented a concentration of around 0.7 mg/ml from 1 L of initial E. coli culture with a molecular weight of about 27 kDa. Affinity measurement disclosed anti-CD20 scFv-V3 possesses a higher affinity constant compared to anti-CD20 scFv-C. The recombinant scFvs exclusively attach to Raji cells but not to Jurkat cells, according to a cell-ELISA analysis. The MTT test signified anti-CD20 scFvs could affect cell viability in Raji cells but had no impact on Jurkat cells and also, Raji cells viability was affected more significantly by anti-CD20 scFv-V3.

Long non-coding RNA MIR4435-2HG: a key molecule in progression of cancer and non-cancerous disorders.

MIR4435-2HG (LINC00978) is a long non-coding RNA (lncRNA) that acts as an oncogene in almost all cancers. This lncRNA participates in the molecular cascades involved in other disorders such as coronary artery diseases, osteonecrosis, osteoarthritis, osteoporosis, and periodontitis. MIR4435-2HG exerts its functions via the spectrum of different mechanisms, including inhibition of apoptosis, sponging microRNAs (miRNAs), promoting cell proliferation, increasing cell invasion and migration, and enhancing epithelial to mesenchymal transition (EMT). MIR4435-2HG can regulate several signaling pathways, including Wnt, TGF-ß/SMAD, Nrf2/HO-1, PI3K/AKT, MAPK/ERK, and FAK/AKT/ß­catenin signaling pathways; therefore, it can lead to tumor progression. In the present review, we aimed to discuss the potential roles of lncRNA MIR4435-2HG in developing cancerous and non-cancerous conditions. Due to its pivotal role in different disorders, this lncRNA can serve as a potential biomarker in future investigations. Moreover, it may serve as a potential therapeutic target for the treatment of various diseases.

An overview of current drugs and prophylactic vaccines for coronavirus disease 2019 (COVID-19).

Designing and producing an effective vaccine is the best possible way to reduce the burden and spread of a disease. During the coronavirus disease 2019 (COVID-19) pandemic, many large pharmaceutical and biotechnology companies invested a great deal of time and money in trying to control and combat the disease. In this regard, due to the urgent need, many vaccines are now available earlier than scheduled. Based on their manufacturing technology, the vaccines available for COVID-19 (severe acute respiratory syndrome coronavirus 2 (SAR-CoV2)) infection can be classified into four platforms: RNA vaccines, adenovirus vector vaccines, subunit (protein-based) vaccines, and inactivated virus vaccines. Moreover, various drugs have been deemed to negatively affect the progression of the infection via various actions. However, adaptive variants of the SARS-CoV-2 genome can alter the pathogenic potential of the virus and increase the difficulty of both drug and vaccine development. In this review, along with drugs used in COVID-19 treatment, currently authorized COVID-19 vaccines as well as variants of the virus are described and evaluated, considering all platforms.

Long noncoding RNA LINC00978 acts as a potential diagnostic biomarker in patients with colorectal cancer.

Colorectal cancer (CRC) as a lethal malignancy has been associated with dysregulation of several genes and pathways. Long noncoding RNAs (lncRNAs) play an important role in gene expression regulation. In the current research, we aim to evaluate the expression of LINC00978 in CRC samples and adjacent tissues. Using Quantitative Real-Time PCR (qRT-PCR) method, we assessed the expression levels of LINC00978 and ß-catenin in 70 pairs of CRC and adjacent tissues. Moreover, the association between clinicopathological features and the LINC00978 expression levels was investigated. To assess the diagnostic power of LINC00978 expression in CRC, receiver operating characteristic (ROC) curve was plotted. The relationship between LINC00978 and ß-catenin expression levels was evaluated using correlation analysis. A markedly increased level of LINC00978 and ß-catenin expression levels was observed in CRC samples compared with adjacent tissues (P < 0.0001). No significant association was detected between LINC00978 expression level and the patient's clinicopathological features. The results of Pearson's correlation coefficient highlighted a positive correlation between LINC00978 and ß-catenin expression (r2 = 0.4695, P < 0.0001). According to the area under curve (AUC) value, LINC00978 expression differentiates CRC samples from the adjacent tissues (AUC = 0.81, P < 0.0001). The present results suggest that LINC00978 may play a critical role in CRC progression via Wnt pathway. The potential role of LINC00978 as a diagnostic biomarker needs to be further investigated in future studies.

The utility of dermal fibroblasts in treatment of skin disorders: A paradigm of recessive dystrophic epidermolysis bullosa.

Dermal fibroblasts are the most accessible cells in the skin that have gained significant attention in cell therapy. Applying dermal fibroblasts' regenerative capacity can introduce new patterns to develop cell-based therapies to treat skin disorders. Dermal fibroblasts originate from mesenchymal cells and are located within the dermis. These cells are mainly responsible for synthesizing glycosaminoglycans, collagens, and components of extracellular matrix supporting skin's structural integrity. Preclinical studies suggested that allogeneic and autologous dermal fibroblasts provide widespread and beneficial applications for wound healing, burn ulcers, and inherited skin disorders. In this regard, generating induced pluripotent stem cells (iPSCs) from fibroblasts and gene-edited fibroblasts are promising approaches for treating skin disorders. Here, we aimed to review literature about ongoing and completed clinical trials that applied fibroblasts and bioengineered fibroblasts as therapeutic agents for various skin disorders. This review explores cell therapy protocols from the earliest phase of allogeneic and autologous fibroblasts development in different benches to translating them into bedside-level treatment for skin disorders, particularly recessive dystrophic epidermolysis bullosa.

The cooperative effects of micro-grooved topography and TGF-ß1 on the vascular smooth muscle cell contractile protein expression of the mesenchymal stem cells.

Cell morphological changes induced by micro-grooved topography have been shown to be an important regulator of smooth muscle (SM) differentiation of mesenchymal stem cells (MSCs). In addition to the micro-grooved topography, transforming growth factor-ß1 (TGF-ß1) can also modulate MSCs differentiation towards smooth muscle cells (SMCs) through alterations in cell morphological characteristics. Thus, it can be hypothesized that substrate topography and TGF-ß1 may interact to facilitate differentiation of MSCs into SMCs. In this study, we investigated the time-course cooperative effects of substrate topography and TGF-ß1 in the regulation of SM differentiation of human MSCs. Western blotting, followed by image analysis, was performed to assess the protein expression of α-actin, h1-calponin and gelsolin. Three-way analysis of variance was employed to investigate the main effect of each independent variable, i.e. TGF-ß1 conditioning, substrate topography and culture time, along with the interactions of these variables. Each of TGF-ß1, substrate topography and culture time significantly affected the protein expression of α-actin, h1-calponin and gelsolin. Overall, TGF-ß1 conditioning of the cells and culturing the cells on the micro-grooved substrate resulted in greater protein expression of α-actin and h1-calponin, and lesser protein expression of gelsolin. In addition to the isolated effects of the variables, treatment type interacted with substrate topography and culture time to regulate the expression of the above-mentioned proteins. This study indicated the feasibility of promoting SM differentiation of human MSCs by simultaneous recruitment of micro-grooved topography and TGF-ß1. The findings could be of assistance when effective utilization of chemo-physical cues is needed to achieve functional SMC-like MSCs in vitro.

Conformational change and GTPase activity of human tubulin: A comparative study on Alzheimer's disease and healthy brain.

In Alzheimer's disease (AD), the most common form of dementia, microtubules (MTs) play a pivotal role through their highly dynamic structure and instability. They mediate axonal transport that is crucial to synaptic viability. MT assembly, dynamic instability and stabilization are modulated by tau proteins, whose detachment initiates MT disintegration. Albeit extensive research, the role of GTPase activity in molecular mechanism of stability remains controversial. We hypothesized that GTPase activity is altered in AD leading to microtubule dynamic dysfunction and ultimately to neuronal death. In this paper, fresh tubulin was purified by chromatography from normal young adult, normal aged, and Alzheimer's brain tissues. Polymerization pattern, assembly kinetics and dynamics, critical concentration, GTPase activity, interaction with tau, intermolecular geometry, and conformational changes were explored via Förster Resonance Energy Transfer (FRET) and various spectroscopy methods. Results showed slower MT assembly process in samples from the brains of people with AD compared with normal young and aged brains. This observation was characterized by prolonged lag phase and increased critical and inactive concentration of tubulin. In addition, the GTPase activity in samples from AD brains was significantly higher than in both normal young and normal aged samples, concurrent with profound conformational changes and contracted intermolecular MT-tau distances as revealed by FRET. These alterations were partially restored in the presence of a microtubule stabilizer, paclitaxel. We proposed that alterations of both tubulin function and GTPase activity may be involved in the molecular neuropathogenesis of AD, thus providing new avenues for therapeutic approaches.

Affinity maturation and characterization of the ofatumumab monoclonal antibody.

CD20 molecule, a phosphoprotein with 297 amino acids and four transmembrane domains, is a member of MS4A protein family. Anti-CD20 antibodies such as ofatumumab, which have been developed for cancer treatment and has demonstrated efficacy in relapsed/refractory chronic lymphocytic leukemia, are among the most successful therapies to date. Rational engineering methods can be applied with reasonable success to improve functional characteristics of antibodies. Considering the importance of this issue, we have used in silico modeling approach for the improvement of ofatumumab monoclonal antibody. Four mutated variants of ofatumumab were developed and expressed in Chinese hamster ovary (CHO) cells along with the unmodified antibody. Analysis of affinity of the purified antibodies with CD20 showed significant improvement in antigen-binding characteristics of one of the variants compared with the control antibody. This study represents the first step toward development of the second generation ofatumumab antibody with improved affinity.

Development of an improved lentiviral based vector system for the stable expression of monoclonal antibody in CHO cells.

Therapeutic monoclonal antibodies (mAbs) have become the dominant products in biopharmaceutical industry. Mammalian cell expression systems including Chinese hamster ovary (CHO) cells are the most commonly used hosts for the production of complex recombinant proteins. However, development of stable, high producing CHO cell lines suffers from the low expression level and instability of the transgene. The increasing efforts in the development of novel therapeutic antibodies and the advent of biosimilars have revealed the necessity for the development of improved platforms for rapid production of products for initial characterization and testing. In line with this premise, vector design and engineering has been applied to improve the expression level and stability of the transgene. This study reports the application of an improved lentiviral vector system containing the human interferon-ß scaffold attachment region (IFN-SAR) for the development of antibody producing stable CHO cells. mAb expressing clones producing 1100 µg/L of IgG1 monoclonal antibody were isolated without extensive screening of a large number of clones. Our results here indicate the positive effects of IFN-SAR on stable mAb expression using lentiviral based expression vectors. We also observed that although IFN-SAR can improve light chain (LC) and heavy chain (HC) gene copy numbers in stable cell pools, mAb expression in single cell clones was not affected by the transgene copy number.

Design, expression and evaluation of novel chimeric protein constructed from colorectal tumor-associated antigen.

There were 134,000 new diagnosis and 49,000 deaths in 2016 due to colorectal cancer. Similar to most cancers, early diagnosis increases the chance of successful treatment. Detection of tumor-associated antigens or the immune response against such markers is one of the most common methods of diagnosis. In that regard, we aimed to design and express a chimeric protein from the most common tumor-associated antigens in colorectal cancer and assess its ability to detect the immune response in comparison with the parental tumor-associated antigens in patient's sera. Through bioinformatics approaches a chimeric protein from carcinoembryonic antigen (CEA) and carbohydrate antigen 19.9 (CA19-9) was designed and expressed in E. coli (BL21DE3). Proper folding, expression levels and immune reactivity were assessed by western blot, ELISA and immunohistochemistry. Recombinant proteins functionality and immune reactivity were confirmed by ELISA and Western blot. Results showed that recombinant CEA, recombinant CA19.9 and chimeric protein of CEA- CA19.9 have strong reactivity with antibodies in the sera of colorectal cancer patients, whereas no reactivity was seen with the sera of healthy volunteers. Significantly stronger immune reactivity was seen with the chimeric protein than each of the CEA or CA19.9 alone. Overall, it was concluded that the designed recombinant proteins in this study could be used to detect autoantibodies produced against the colorectal tumor-associated antigens. The chimeric CEA-CA19.9 protein shows a stronger reactivity with the sera antibodies of colorectal cancer patients that CEA or CA19.9 alone.

Development of a novel anti-HER2 scFv by ribosome display and in silico evaluation of its 3D structure and interaction with HER2, alone and after fusion to LAMP2B.

HER2 is a member of epidermal factor receptor (EGFR) family which is overexpressed in breast cancer, ovarian cancer and gastric cancer. Development of new binders for cancer cell surface receptors and expressing them at the surface of exosomes would be a great approach in targeted cancer therapy. We found a high affinity scFv against HER2 using ribosome display with the approach of applying it as a targeting moiety at the surface of exosomes by fusion to lysosomal associated membrane protein 2B (LAMP2B). We also provide some structural information about the ribosome display selected scFv (scFv HFS2) through modeling the 3D structure of scFv HFS2 using RosettaAntibody and docked it at the extracellular domain of HER2. We also evaluated the structure of scFv HFS2 and its binding to HER2 after fusion to LAMP2B. Our results showed no significant change in 3D structure of scFv HFS2 when fused to LAMP2B (RMSD 1.3) and interaction analysis represented that scFv HFS2 binds HER2 domain III before and after fusion to LAMP2B. Although binding domain of scFv HFS2 on HER2 was the same at both state, residues involved in their interactions showed significant differences as it was probably due to the spatial hindrance of scFv HFS2 when fused to LAMP2B through a short linker and it should be considered before proceeding to experiment.

Application of immuno-PCR assay for the detection of serum IgE specific to Bermuda allergen.

In vivo and in vitro tests are the two major ways of identifying the triggering allergens in sensitized individuals with allergic symptoms. Both methods are equally significant in terms of sensitivity and specificity. However, in certain circumstances, in vitro methods are highly preferred because they circumvent the use of sensitizing drugs in patients. In current study, we described a highly sensitive immuno-PCR (iPCR) assay for serum IgE specific to Bermuda allergens. Using oligonucleotide-labelled antibody, we used iPCR for the sensitive detection of serum IgE. The nucleotide sequence was amplified using conventional PCR and the bands were visualized on 2.5% agarose gel. Results demonstrated a 100-fold enhancement in sensitivity of iPCR over commercially available enzyme-linked immunosorbent assay (ELISA) kit. Our iPCR method was highly sensitive for Bermuda-specific serum IgE and could be beneficial in allergy clinics.

A novel recombinant anti-epidermal growth factor receptor peptide vaccine capable of active immunization and reduction of tumor volume in a mouse model.

Over-expression of epidermal growth factor receptor (EGFR) has been reported in a number of human malignancies. Strong expression of this receptor has been associated with poor survival in many such patients. Active immunizations that elicit antibodies of the desired type could be an appealing alternative to conventional passive immunization. In this regard, a novel recombinant peptide vaccine capable of prophylactic and therapeutic effects was constructed. A novel fusion recombinant peptide base vaccine consisting of L2 domain of murine extra-cellular domain-EGFR and EGFR mimotope (EM-L2) was constructed and its prophylactic and therapeutic effects in a Lewis lung carcinoma mouse (C57/BL6) model evaluated. Constructed recombinant peptide vaccine is capable of reacting with anti-EGFR antibodies. Immunization of mice with EM-L2 peptide resulted in antibody production against EM-L2. The constructed recombinant peptide vaccine reduced tumor growth and increased the survival rate. Designing effective peptide vaccines could be an encouraging strategy in contemporary cancer immunotherapy. Investigating the efficacy of such cancer immunotherapy approaches may open exciting possibilities concerning hyperimmunization, leading to more promising effects on tumor regression and proliferation.

Humanizing glycosylation pathways in eukaryotic expression systems.

Glycosylation represents the most widespread posttranslational modifications, found in a broad spectrum of natural and therapeutic recombinant proteins. It highly affects bioactivity, site-specificity, stability, solubility, immunogenicity, and serum half-life of glycoproteins. Numerous expression hosts including yeasts, insect cells, transgenic plants, and mammalian cells have been explored for synthesizing therapeutic glycoproteins. However, glycosylation profile of eukaryotic expression systems differs from human. Glycosylation strategies have been proposed for humanizing the glycosylation pathways in expression hosts which is the main theme of this review. Besides, we also highlighted the glycosylation potential of protozoan parasites by emphasizing on the mammalian-like glycosylation potential of Leishmania tarentolae known as Leishmania expression system.

Camelid-derived heavy-chain nanobody against Clostridium botulinum neurotoxin E in Pichia pastoris.

Botulinum neurotoxins (BoNTs) result in severe and often fatal disease, botulism. Common remedial measures such as equine antitoxin and human botulism immunoglobulin in turn are problematic and time-consuming. Therefore, diagnosis and therapy of BoNTs are vital. The variable domain of heavy-chain antibodies (VHH) has unique features, such as the ability to identify and bind specifically to target epitopes and ease of production in bacteria and yeast. The Pichia pastoris is suitable for expression of recombinant antibody fragments. Disulfide bond formation and correct folds of protein with a high yield are some of the advantages of this eukaryotic host. In this study, we have expressed and purified the camelid VHH against BoNT/E in P. pastoris. The final yield of P. pastoris-expressed antibody was estimated to be 16 mg/l, which is higher than that expressed by Escherichia coli. The nanobody expressed in P. pastoris neutralized 4LD50 of the BoNT/E upon i.p. injection in 25% of mice. The nanobody expressed in E. coli extended the mice's survival to 1.5-fold compared to the control. This experiment indicated that the quality of expressed protein in the yeast is superior to that of the bacterial expression. Favorable protein folding by P. pastoris seems to play a role in its better toxin-binding property.

Antifungal effect of Echinophora platyloba on expression of CDR1 and CDR2 genes in fluconazole-resistant Candida albicans.

BACKGROUND: Several studies examined the effect of the Echinophora platyloba extract in treatment of azole-resistant Candida albicans clinical isolates. OBJECTIVE: We investigated the effect of E. platyloba extract on expression of CDR1 and CDR2 genes in fluconazole-resistant clinical isolates of C. albicans using real-time PCR. MATERIALS AND METHODS: The crude extract of E. platyloba was obtained using percolation method. Using serial dilution method, different concentrations of extract were achieved. Two hundred microlitres of fungal suspension (10(6) CFU/ml) was added to the media and cultured with different concentrations and then incubated at 37 °C for 48 h. The concentration of extract in the first tube, which inhibited the growth of C. albicans, was recorded as the Minimal Inhibitory Concentration (MIC). In order to analyse the expression of CDR1 and CDR2 genes, RNA was extracted from C. albicans isolates before and after treatment with MIC of E. platyloba using glass beads and the denaturing buffer agents in an RNase-free environment and then the cDNA was synthesised and used for real-time PCR assay. RESULTS: Twenty of total of 148 isolates were resistant to fluconazole. The MIC and MFC for the alcoholic extract of E. Platyloba were 64 mg/ml and 128 mg/ml, respectively. Real-time PCR results revealed that the mRNA levels of CDR1 and CDR2 genes significantly declined after incubation with E. Platyloba (both p values < 0.001). CONCLUSION: E. Platyloba is effective in reducing CDR1 and CDR2 expression which in turn plays an important role in fluconazole resistance in Candida species.