Publications by authors named "Laleh Shariati"

27 Publications

  • Page 1 of 1

Organoid Technology: Current Standing and Future Perspectives.

Stem Cells 2021 Mar 30. Epub 2021 Mar 30.

Department of Mechanical Engineering, Australian College of Kuwait, Mishref, Safat, Kuwait.

Organoids are powerful systems to facilitate the study of individuals' disorders and personalized treatments. Likewise, emerging this technology has improved the chance of translatability of drugs for pre-clinical therapies and mimicking the complexity of organs, while it proposes numerous approaches for human disease modeling, tissue engineering, drug development, diagnosis, and regenerative medicine. In this review, we outline the past/present organoid technology and summarize its faithful applications, then, we discuss the challenges and limitations encountered by 3D organoids. In the end, we offer the human organoids as basic mechanistic infrastructure for "human modelling" systems to prescribe personalized medicines. © AlphaMed Press 2021 SIGNIFICANCE STATEMENT: This concise review concerns about organoids, available methods for in vitro organoid formation and different types of human organoid models. We, then, summarize biological approaches to improve 3D organoids complexity and therapeutic potentials of organoids. Despite the existing incomprehensive review articles in literature that examine partial aspects of the organoid technology, the present review article comprehensively and critically presents this technology from different aspects. It effectively provides a systematic overview on the past and current applications of organoids and discusses the future perspectives and suggestions to improve this technology and its applications.
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http://dx.doi.org/10.1002/stem.3379DOI Listing
March 2021

Gold Nano/Micro-Islands Overcome the Molecularly Imprinted Polymer Limitations to Achieve Ultrasensitive Protein Detection.

ACS Sens 2021 03 19;6(3):797-807. Epub 2021 Jan 19.

Department of Bioengineering, McGill University, Montreal, Quebec H3A 0E9, Canada.

Here, we report on an electrochemical biosensor based on core-shell structure of gold nano/micro-islands (NMIs) and electropolymerized imprinted -phenylenediamine (o-PD) for detection of heart-fatty acid binding protein (H-FABP). The shape and distribution of NMIs (the core) were tuned by controlled electrodeposition of gold on a thin layer of electrochemically reduced graphene oxide (ERGO). NMIs feature a large active surface area to achieve a low detection limit (2.29 fg mL, a sensitivity of 1.34 × 10 μA mM) and a wide linear range of detection (1 fg mL to 100 ng mL) in PBS. Facile template H-FABP removal from the layer (the shell) in less than 1 min, high specificity against interference from myoglobin and troponin T, great stability at ambient temperature, and rapidity in detection of H-FABP (approximately 30 s) are other advantages of this biomimetic biosensor. The electrochemical measurements in human serum, human plasma, and bovine serum showed acceptable recovery (between 91.1 ± 1.7 and 112.9 ± 2.1%) in comparison with the ELISA method. Moreover, the performance of the biosensor in clinical serum showed lower detection time and limit of detection against lateral flow assay (LFA) rapid test kits, as a reference method. Ultimately, the proposed biosensor based on the core-shell structure of gold NMIs and MIP opens interesting avenues in the detection of proteins with low cost, high sensitivity and significantstability for clinical applications.
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http://dx.doi.org/10.1021/acssensors.0c01701DOI Listing
March 2021

Leukemia inhibitory factor: A main controller of breast cancer.

J Biosci 2020;45

Department of Cell and Molecular Biology, School of Biology, Kish International Campus, University of Tehran, Kish, Iran.

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January 2020

Generation of HBsAg DNA aptamer using modified cell-based SELEX strategy.

Mol Biol Rep 2021 Jan 5;48(1):139-146. Epub 2021 Jan 5.

Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Islamic Republic of Iran.

Aptamers as potential alternatives for antibodies could be employed against hepatitis B surface antigen (HBsAg), the great hallmark and first serological marker in HBV, for further theragnostic applications. Therefore, isolation HBsAg specific aptamer was performed in this study with a modified Cell-SELEX method. HEK293T overexpressing HBsAg and HEK293T as target and control cells respectively, were incubated with single-stranded rounds of DNA library during six SELEX and Counter SELEX rounds. Here, we introduced the new modified Cell-SELEX using deoxyribonuclease I digestion to separate single stranded DNA aptamers against the HBsAg. Characterization and evaluation of selected sequences were performed using flow cytometry analysis. The results led to isolation of 15 different ssDNA clones in six rounds of selection which were categorized to four clusters based on common structural motifs. The evaluation of SELEX progress showed growth in aptamer affinity with increasing in the cycle number. Taken together, the application of modified cell-SELEX demonstrated the isolation of HBsAg-specific ssDNA aptamers with proper affinity. Modified cell-SELEX as an efficient method can shorten the selection procedure and increase the success rate while the benefits of cell-based SELEX will be retained. Selected aptamers could be applied in purification columns, diagnostic kits, and drug delivery system against HBV-related liver cancer.
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http://dx.doi.org/10.1007/s11033-020-05995-2DOI Listing
January 2021

Targeting of cholera toxin A () gene by zinc finger nuclease: pitfalls of using gene editing tools in prokaryotes.

Res Pharm Sci 2020 Apr 11;15(2):182-190. Epub 2020 May 11.

Department of Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, I.R. Iran.

Background And Purpose: The study was launched to use zinc finger nuclease (ZFN) technology to disrupt the cholera toxin gene () for inhibiting CT toxin production in .

Experimental Approach: An engineered ZFN was designed to target the catalytic site of the gene. The coding sequence of ZFN was cloned to pKD46, pTZ57R T/A vector, and plasmid and transformed to Top10 and . The efficiency of ZFN was evaluated by colony counting.

Findings/results: No expression was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting in transformed . The gene sequencing did not show any mutation. Polymerase chain reaction on pKD46-ZFN plasmid had negative results. Transformation of Top10 with T/A vectors containing whole ZFN sequence led to 7 colonies all of which contained bacteria with self-ligated vector. Transformation with left array ZFN led to 24 colonies of which 6 contained bacteria with self-ligated vector and 18 of them contained bacteria with vector/left array. Transformation of with vectors containing whole ZFN did not produce any colonies. Transformation with left array vectors led to 17 colonies containing bacteria with vector/left array. Left array protein band was captured using western blot assay.

Conclusions And Implications: ZFN might have off target on bacterial genome causing lethal double-strand DNA break due to lack of non-homologous end joining (NHEJ) mechanism. It is recommended to develop ZFNs against bacterial genes, engineered packaging host with NHEJ repair system is essential.
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http://dx.doi.org/10.4103/1735-5362.283818DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7306252PMC
April 2020

Protective effects of doxepin cream on radiation dermatitis in breast cancer: A single arm double-blind randomized clinical trial.

Br J Clin Pharmacol 2020 09 25;86(9):1875-1881. Epub 2020 Feb 25.

Isfahan Cardiovascular Research Center, Cardiovascular Research Institute, Isfahan University of Medical Sciences, Isfahan, Iran.

Aims: Breast cancer is the most frequently occurring cancer in women. Lumpectomy followed by radiotherapy is suggested to be as effective as a total mastectomy. Radiation-induced dermatitis often occurs as a result of breast radiotherapy. Recent studies suggest that doxepin has promising anti-inflammatory properties. This study was undertaken to evaluate the effects of doxepin therapy on radiation dermatitis.

Methods: A double-blind randomized clinical trial was launched from 2016 to 2017, with a total of 48 patients who had undergone breast-conserving surgery and received postoperative radiation therapy. Radiotherapy was applied 5 days per week for 5 weeks. Adverse dermatological effects were evaluated by a physician at the beginning of the fifth week of radiotherapy and the patients were then randomly assigned (1:1 ratio) to receive either doxepin (5%) or placebo cream for 7 days.

Results: There were no significant differences in the dermatitis grade between doxepin and placebo groups at baseline (P > .5). The occurrence of acute dermatitis (grade 2 or higher) was significantly lower with the use of doxepin than with placebo (P ≤ .0001, Z = 1.96 at 95% confidence interval).

Conclusion: Doxepin cream prevents dermatitis grade 2 or higher during post-operative breast irradiation. Doxepin cream is easy to use, affordable and prevents pain and irritation.
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http://dx.doi.org/10.1111/bcp.14238DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7444764PMC
September 2020

MicroRNAs as the actors in the atherosclerosis scenario.

J Physiol Biochem 2020 Feb 5;76(1):1-12. Epub 2019 Dec 5.

Isfahan Cardiovascular Research Center, Cardiovascular Research Institute, Isfahan University of Medical Sciences, Isfahan, Iran.

Atherosclerosis is considered as the most common cardiovascular disease and a leading cause of global mortality, which develops through consecutive steps. Various cellular and molecular biomarkers such as microRNAs are identified to be involved in atherosclerosis progression. MicroRNAs are a group of endogenous, short, non-coding RNAs, which are able to bind to specific sequences on target messenger RNAs and thereby modulate gene expression post-transcriptionally. MicroRNAs are key players in wide range of biological processes; thus, their expression level is regulated in pathophysiological conditions. Ample evidences including in vitro and in vivo studies approved a critical role of microRNAs in epigenetic and the sequential processes of atherosclerosis from risk factors to plaque formation, progression, and rupture. Based on these findings, miRNAs seems to be promising candidates for therapeutic approach. This review summarizes the role of miRNAs in atherosclerosis development, epigenetic, and therapy. Moreover, the application of exosomes in miRNA delivery, and/or their prognostic and diagnostic values are also discussed.
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http://dx.doi.org/10.1007/s13105-019-00710-7DOI Listing
February 2020

Evidence for expression of promoterless GFP cassette: Is GFP an ideal reporter gene in biotechnology science?

Res Pharm Sci 2019 Aug;14(4):351-358

Isfahan Cardiovascular Research Center, Cardiovascular Research Institute, Isfahan University of Medical Sciences, Isfahan, I.R. Iran.

Green fluorescent protein (GFP) has played an important role in biochemistry and cell biology as a reporter gene. It has been used to assess the potency of promoters for recombinant protein production. This investigation reveals evidences suggesting that the gene () could be expressed without the promoter. In a study, a pLenti-F/GFP vector was constructed with the purpose to allow GFP expression in transduced cells but not in packaging cells; however, after transfecting the HEK293T cell line, was expressed, compared to pLOX/CW-transfected cells showed expression lag, lower levels and reduced percentage of GFP expression in the cells. This unexpected result could be due to auto transduction in packaging cell, possible retrotransposon activity in the cell line, possible contamination of pLenti-F/GFP with the pLOX/CWgfp and possible presence of a promoter within backbone of the vector. All the possibilities were ruled out. To exclude the possibility that a sequence within the region might act as a promoter, the fragment to be transfected was minimized to a region containing "from the start of the gene to 5'LTR R". The gene was again expressed. Therefore, our findings suggest the does not need promoter for expression. This should appeal to the researchers designing GFP based assays to evaluate the potency of promoters, since possible aberrant expression may have a potential to influence on the results of a planned experiment.
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http://dx.doi.org/10.4103/1735-5362.263559DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6714119PMC
August 2019

Development of α4 integrin DNA aptamer as a potential therapeutic tool for multiple sclerosis.

J Cell Biochem 2019 09 20;120(9):16264-16272. Epub 2019 May 20.

Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Islamic Republic of Iran.

One of the most important molecules for multiple sclerosis pathogenesis is α4 integrin, which is responsible for autoreactive leukocytes migration into the brain. The monoclonal antibody, natalizumab, was introduced to market for blocking the extravasation of autoreactive leukocytes via inhibition of α4 integrin. However, the disadvantages of antibodies provided a suitable background for other agents to be replaced with antibodies. Considering the profound advantages of aptamers over antibodies, aptamer isolation against α4 integrin was intended in the current study. The α4 integrin-specific aptamers were selected using cell-systematic evolution of ligands by exponential enrichment (SELEX) method with human embryonic kidney (HEK)-293T overexpressing α4 integrin and HEK-293T as target and control cells, respectively. Evaluation of selected aptamer was performed through flow cytometric analysis. The selected clones were then sequenced and analyzed for any possible secondary structure and affinity. The results of this study led to isolation of 13 different single-stranded DNA clones in 11 rounds of selection which were categorized to three clusters based on common structural motifs and the equilibrium dissociation constant (K ) of the most stable structure was calculated. The evaluation of SELEX progress showed growth in aptamer affinity with increasing of the number of cycles. Taken together, the findings of this study demonstrated the isolation of α4-specific single-stranded DNA aptamers with suitable affinity for ligand, which can further be replaced with natalizumab.
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http://dx.doi.org/10.1002/jcb.28907DOI Listing
September 2019

Engineered zinc-finger nuclease to generate site-directed modification in the KLF1 gene for fetal hemoglobin induction.

J Cell Biochem 2018 Dec 16. Epub 2018 Dec 16.

Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.

Elevation of hemoglobin F (HbF) ameliorates symptoms of β-thalassemia, as a common autosomal recessive disorder. In this study, the ability of an engineered zinc-finger nuclease (ZFN) system was assesed to disrupt the KLF1 gene to inhibit the γ to β hemoglobin switching in K562 cells. This study was performed using a second generation integration-deficient lentiviral vector assigned to transient gene targeting. The sequences coding for zinc finger protein arrays were designed and subcloned in TDH plus as a transfer vector. Transduction of K562 cells was performed with the integrase minus lentivirus containing ZFN. The indel percentage of the transducted cells with lentivirus containing ZFN was about 29%. Differentiation of K562 cell line into erythroid cell lineage was induced with cisplatin concentration of 15 µg/mL. After differentiation, γ-globin and HbF expression were evaluated using real-time reverse-transcription polymerase chain reaction and hemoglobin electrophoresis methods. The levels of γ-globin messenger RNA were nine-fold higher in the ZFN treated cells compared with untreated cells 5 days after differentiation. Hemoglobin electrophoresis method showed the same results for HbF level measurement. Application of the ZFN tool to induce KLF1 gene mutation in adult erythroid progenitors might be a candidate to stimulate HbF expression in β-thalassemia patients.
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http://dx.doi.org/10.1002/jcb.28130DOI Listing
December 2018

Induction of Apoptosis in Infected Hela Cells by Cisplatin and Sodium Azide and Isolation of Apoptotic Bodies and Potential Use for Vaccination against .

Iran J Parasitol 2018 Jul-Sep;13(3):406-415

Dept. of Parasitology and Mycology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.

Background: can infect a wide range of mammalians, especially humans. It controls several intracellular signals for the inhibition of apoptosis. This study aimed to investigate the apoptogenic effect of cisplatin and sodium azide on infected HeLa cells and isolate apoptotic bodies (blebs) as a potent stimulator of the immune system.

Methods: he cytotoxic properties of cisplatin and sodium azide (NaN3) on HeLa cells were evaluated by MTT assay. Moreover, the apoptogenic activity of cisplatin and NaN3 was studied using flow cytometry (Annexin V/PI double staining) and scanning electron microscopy (SEM). Finally, apoptotic bodies were separated by centrifugation.

Results: MTT assay data showed that the survival rate of cells treated with different concentration of NaN3 was significantly reduced, compared to negative control groups. Concerning cisplatin, only concentration of 20 μM had not a significant impact on the cell viability; however, the other concentration of cisplatin significantly reduced cell viability, compared to negative control groups. The level of early apoptosis in uninfected HeLa cells was higher compared to infected HeLa cells treated with cisplatin and NaN3. Finally, apoptotic bodies were separated from T. gondii infected HeLa cells treated with cisplatin.

Conclusion: Apoptosis was induced in both uninfected and infected HeLa cells with T. gondii and apoptotic bodies were isolated from infected cells. Therefore, further studies on apoptotic bodies are required in order to find a proper candidate for vaccine preparation against infections.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6243158PMC
November 2018

Construction and characterization of human embryonic kidney-(HEK)-293T cell overexpressing truncated α4 integrin.

Res Pharm Sci 2018 Aug;13(4):353-359

Isfahan Neurosciences Research Center, Alzahra Research Institute, Isfahan University of Medical Sciences, Isfahan, I.R. Iran.

Blockade of α4 integrin by antibodies could be an appropriate treatment strategy in multiple sclerosis and Crohn's disease. Considering disadvantages of antibodies, other elements (e.g. aptamers) have been proposed for antibodies replacement. Isolation of aptamers through cell-SELEX (systematic evolution of ligands by exponential enrichment) method requires positive and negative expressing α4 integrin cell lines. For a better isolation, we intended to construct a negative cell line lacking of specific ligand binding site of α4 integrin. strain top 10 was used for truncated integrin subunit α4 () expression vector. Human embryonic kidney (HEK)-293T cell was transfected with linearized plasmid and subsequently screened for stable truncated expressing cells. Chromosomal DNA of truncated -transfected cells was extracted and the presence of truncated gene in HEK-293T genome was confirmed by polymerase chain reaction (PCR). The expression level of truncated on HEK-293T cells was also analysed by real-time PCR and flow cytometry. Real-time PCR and flow cytometric analysis showed significant difference of truncated expression between untransfected HEK-293T cells compared to transfected cells. The results suggest that we have successfully constructed the truncated integrin α4 expressing HEK-293T cell, which will facilitate further research into the production of antibody, nanobody, and aptamer against α4 integrin.
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http://dx.doi.org/10.4103/1735-5362.235162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6040166PMC
August 2018

Disruption of SOX6 gene using CRISPR/Cas9 technology for gamma-globin reactivation: An approach towards gene therapy of β-thalassemia.

J Cell Biochem 2018 11 16;119(11):9357-9363. Epub 2018 Jul 16.

Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.

Elevation of Hemoglobin F ameliorates symptoms of β-thalassemia, a common autosomal recessive disorder. The transcription factor SOX6 plays a key role in the γ to β-globin gene switching. In the current investigation, a mutation was induced using the CRISPR/Cas9 technology in the binding domain region of SOX6 to reactivate γ-globin expression. Three CRISPR/Cas9 cassettes were provided, whose single-guide RNAs targeted different regions in the SOX6 gene-binding domain. After transfection of K562 cells with CRISPR a, b and c, and subsequent erythroid differentiation, the indel percentage of the cells was about 30%, 25%, and 24%, respectively. Relative quantification showed that the γ-globin mRNA level increased to 1.3-, 2.1-, and 1.1-fold in the cells treated with CRISPR/Cas9 a, b, and c, respectively, compared with untreated cells. Our results show that mutation induction in the binding site of the SOX6 gene leads to γ-globin reactivation. These findings support the idea that CRISPR interrupts the SOX6 binding site, and, as a result, SOX6 is incapable of binding the γ-globin promoter. In conclusion, SOX6 disruption could be considered as a therapeutic approach for β-thalassemia treatment. CRISPR/Cas9 was selected for this purpose as it is the most rapidly evolving technology.
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http://dx.doi.org/10.1002/jcb.27253DOI Listing
November 2018

A Novel Prokaryotic Green Fluorescent Protein Expression System for Testing Gene Editing Tools Activity Like Zinc Finger Nuclease.

Adv Biomed Res 2017 30;6:155. Epub 2017 Nov 30.

Isfahan Cardiovascular Research Center, Cardiovascular Research Institute, Isfahan University of Medical Sciences, Isfahan, Iran.

Background: Gene editing technology has created a revolution in the field of genome editing. The three of the most famous tools in gene editing technology are zinc finger nucleases (ZFNs), transcription activator-like effector nucleases, clustered regularly interspaced short palindromic repeats (CRISPR), and CRISPR-associated systems. As their predictable nature, it is necessary to assess their efficiency. There are some methods for this purpose, but most of them are time labor and complicated. Here, we introduce a new prokaryotic reporter system, which makes it possible to evaluate the efficiency of gene editing tools faster, cheaper, and simpler than previous methods.

Materials And Methods: At first, the target sites of a custom ZFN, which is designed against a segment of ampicillin resistance gene, were cloned on both sides of green fluorescent protein (GFP) gene to construct pPRO-GFP. Then pPRO-GFP was transformed into TOP10F' that contains pZFN (contains expression cassette of a ZFN against ampicillin resistant gene), or p15A-KanaR as a negative control. The transformed bacteria were cultured on three separate media that contained ampicillin, kanamycin, and ampicillin + kanamycin; then the resulted colonies were assessed by flow cytometry.

Results: The results of flow cytometry showed a significant difference between the case (bacteria contain pZFN) and control (bacteria contain p15A, KanaR) in MFI (Mean Fluorescence Intensity) ( < 0.0001).

Conclusion: According to ZFN efficiency, it can bind and cut the target sites, the bilateral cutting can affect the intensity of GFP fluorescence. Our flow cytometry results showed that this ZFN could reduce the intensity of GFP color and colony count of bacteria in media containing amp + kana versus control sample.
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http://dx.doi.org/10.4103/2277-9175.219420DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5735555PMC
November 2017

The silencing effect of miR-30a on gene expression : an approach for gene therapy.

Res Pharm Sci 2017 Dec;12(6):456-464

Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, I.R. Iran.

Integrins are adhesion molecules which play crucial roles in cell-cell and cell-extracellular matrix interactions. Very late antigen-4 or α4β1 and lymphocyte Peyer's patch adhesion molecule-1 or α4β7, are key factors in the invasion of tumor cells and metastasis. Based on the previous reports, integrin α4 () is overexpressed in some immune disorders and cancers. Thus, inhibition of could be a therapeutic strategy. In the present study, miR-30a was selected in order to suppress expression. The 3' UTR was amplified, cloned in the Z2827-M67-() plasmid and named as Z2827-M67/3'UTR. HeLa cells were divided into five groups; (1) untreated without any transfection, (2) mock with Z2827-M67/3'UTR transfection and X-tremeGENE reagent, (3) negative control with Z2827-M67/3'UTR transfection alone, (4) test with miR-30a mimic and Z2827-M67/3'UTR transfection and (5) scramble with miR-30a scramble and Z2827-M67/3'UTR transfection. The MTT assay was performed to evaluate cell survival and cytotoxicity in each group. Real-time RT-PCR was applied for the expression analysis. The findings of this study showed that miR-30a downregulated expression and had no effect on the cell survival. Due to the silencing effect of miR-30a on the gene expression, this agent could be considered as a potential tool for cancer and immune disorders therapy.
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http://dx.doi.org/10.4103/1735-5362.217426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5691572PMC
December 2017

Inducing indel mutation in the SOX6 gene by zinc finger nuclease for gamma reactivation: An approach towards gene therapy of beta thalassemia.

J Cell Biochem 2018 03 30;119(3):2512-2519. Epub 2017 Nov 30.

Department of Genetics and Molecular biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.

β-thalassemia is a common autosomal recessive disorder characterized by a deficiency in the synthesis of β-chains. Evidences show that increased HbF levels improve the symptoms in patients with β-thalassemia or sickle cell anemia. In this study, ZFN technology was applied to induce a mutation in the binding domain region of SOX6 to reactivate γ-globin expression. The sequences coding for ZFP arrays were designed and sub cloned in TDH plus as a transfer vector. The ZFN expression was confirmed using Western blot analysis. In the next step, using the site-directed mutagenesis strategy through the overlap PCR, a missense mutation (D64V) was induced in the catalytic domain of the integrase gene in the packaging plasmid and verified using DNA sequencing. Then, the integrase minus lentivirus containing ZFN cassette was packaged. Transduction of K562 cells with this virus was performed. Mutation detection assay was performed. The indel percentage of the cells transducted with lenti virus containing ZFN was 31%. After 5 days of erythroid differentiation with 15 μg/mL cisplatin, the levels of γ-globin mRNA were sixfold in the cells treated with ZFN compared to untreated cells. In the meantime, the measurement of HbF expression levels was carried out using hemoglobin electrophoresis and showed the same results. Integrase minus lentivirus can provide a useful tool for efficient transient gene expression and helps avoid disadvantages of gene targeting using the native virus. The ZFN strategy applied here to induce indel on SOX6 gene in adult erythroid progenitors may provide a method to activate fetal hemoglobin expression in individuals with β-thalassemia.
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http://dx.doi.org/10.1002/jcb.26412DOI Listing
March 2018

A novel pathogenic variant in an Iranian Ataxia telangiectasia family revealed by next-generation sequencing followed by in silico analysis.

J Neurol Sci 2017 Aug 12;379:212-216. Epub 2017 Jun 12.

Ahvaz Noor Genetics Laboratory, Ahvaz, Iran; Department of Medical Genetics, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. Electronic address:

Ataxia telangiectasia (A-T) is a neurodegenerative autosomal recessive disorder with the main characteristics of progressive cerebellar degeneration, sensitivity to ionizing radiation, immunodeficiency, telangiectasia, premature aging, recurrent sinopulmonary infections, and increased risk of malignancy, especially of lymphoid origin. Ataxia Telangiectasia Mutated gene, ATM, as a causative gene for the A-T disorder, encodes the ATM protein, which plays an important role in the activation of cell-cycle checkpoints and initiation of DNA repair in response to DNA damage. Targeted next-generation sequencing (NGS) was performed on an Iranian 5-year-old boy presented with truncal and limb ataxia, telangiectasia of the eye, Hodgkin lymphoma, hyper pigmentation, total alopecia, hepatomegaly, and dysarthria. Sanger sequencing was used to confirm the candidate pathogenic variants. Computational docking was done using the HEX software to examine how this change affects the interactions of ATM with the upstream and downstream proteins. Three different variants were identified comprising two homozygous SNPs and one novel homozygous frameshift variant (c.80468047delTA, p.Thr2682ThrfsX5), which creates a stop codon in exon 57 leaving the protein truncated at its C-terminal portion. Therefore, the activation and phosphorylation of target proteins are lost. Moreover, the HEX software confirmed that the mutated protein lost its interaction with upstream and downstream proteins. The variant was classified as pathogenic based on the American College of Medical Genetics and Genomics guideline. This study expands the spectrum of ATM pathogenic variants in Iran and demonstrates the utility of targeted NGS in genetic diagnostics.
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http://dx.doi.org/10.1016/j.jns.2017.06.012DOI Listing
August 2017

Genetic study of the BRAF gene reveals new variants and high frequency of the V600E mutation among Iranian ameloblastoma patients.

J Oral Pathol Med 2018 Jan 7;47(1):86-90. Epub 2017 Nov 7.

Department of Oral and Maxillofacial Pathology, School of Dentistry, Isfahan University of Medical Sciences, Isfahan, Iran.

Background: Ameloblastoma is a benign, slow-growing and locally invasive tumor. It is one of the most prevalent odontogenic tumors, with an incidence rate of 1% of all oral tumors and approximately 18% of odontogenic tumors. A group of genes have been investigated in patients with ameloblastoma. The BRAF V600E mutation has been implicated as the most common mutation in ameloblastoma. The presence or absence of this mutation has been associated with several clinicopathological properties, including location, age at diagnosis, histology, and prognosis. Although some populations have been investigated so far, little data are available on the Iranian population. The current research was launched to study the BRAF V600E mutation among a cohort of Iranian patients with ameloblastoma.

Methods: In this clinicopathological and molecular biology study, a total of 19 formalin-fixed, paraffin-embedded tissues were studied. DNA extraction was performed, followed by PCR-sequencing of exons 10 and 15 of the BRAF gene to identify mutations. In silico analysis was performed for the identified variants. Results were analyzed by T test, Chi-square, and Fisher's exact test.

Results: Totally, 12 of 19 samples (63%) harbored the p. V600E hotspot mutation. In addition, we identified several variants, two of which were novel. The c.1769T>G (p. V590G) and c.1751C>T (p.L584F) as the novel variants showed a possible damaging effect by in silico analysis. No variant was found within exon 10.

Conclusions: Our study confirms the role of BRAF mutations in ameloblastoma in the Iranian patients studied.
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http://dx.doi.org/10.1111/jop.12610DOI Listing
January 2018

Aptamers Against Pro- and Anti-Inflammatory Cytokines: A Review.

Inflammation 2017 Feb;40(1):340-349

Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.

Inflammatory disorders result from continuous inflammation in injured sites. Many molecules are involved in this process; the inhibition of which could prevent the inflammation. Chemokines are a group of these biological mediators which are categorized into pro-, anti-, and pro-/anti-inflammatory. Thus, targeting these essential molecules can be an effective way for prevention and control of inflammatory diseases. Various therapeutic agents have been developed for primary and secondary prevention of these disorders, but each of them has its own limitations. Aptamers, as novel therapeutic agents, are a new generation of drugs which could replace other medications even antibodies. Aptamer can bind to its target molecule to trap it and prohibit its function. Among large group of inflammatory cytokines, only 11 aptamers have been selected either against cytokines or their related receptors. These cytokines include interleukin (IL)-2, IL-6, IL-10, IL-11, IL-17, IL-32, TGF-β, TNF-α, IFN-γ, CCL2, and IP-10. Most of the isolated aptamers are against pro-inflammatory or dual function cytokines, and it seems that they could be used for diagnosis, prevention, and treatment of the related inflammatory diseases. Most of the aptamers have been tested in vitro, but so far, none of them has been approved for in vivo use. Given a vast number of inflammatory cytokines, more aptamers against this group of biological molecules will be selected in the near future. The available aptamers will also be tested in clinical trials. Therefore, a significant improvement is expected for the prevention and control of inflammatory disorders.
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http://dx.doi.org/10.1007/s10753-016-0477-1DOI Listing
February 2017

Gamma reactivation using the spongy effect of KLF1-binding site sequence: an approach in gene therapy for beta-thalassemia.

Iran J Basic Med Sci 2016 Oct;19(10):1063-1069

Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.

Objectives: β-thalassemia is one of the most common genetic disorders in the world. As one of the promising treatment strategies, fetal hemoglobin (Hb F) can be induced. The present study was an attempt to reactivate the γ-globin gene by introducing a gene construct containing KLF1 binding sites to the K562 cell line.

Materials And Methods: A plasmid containing a 192 bp sequence with two repeats of KLF1 binding sites on β-globin and BCL11A promoters was constructed and used to transfect the K562 cell line. Positive selection was performed under treatment with 150 μg/ml hygromycin B. The remaining cells were expanded and harvested on day 28, and genomic DNA was extracted. The PCR was carried out to verify insertion of DNA fragment to the genome of K562 cells. The cells were differentiated with 15 μg/ml cisplatin. Flowcytometry was performed to identify erythroid differentiation by detection of CD235a+ cells. Real-time RT-PCR was performed to evaluate γ-globin expression in the transfected cells.

Results: A 1700 bp fragment was observed on agarose gel as expected and insertion of DNA fragment to the genome of K562 cells was verified. Totally, 84% of cells were differentiated. The transfected cells significantly increased γ-globin expression after differentiation compared to untransfected ones.

Conclusion: The findings demonstrate that the spongy effect of KLF1-binding site on BCL11A and β-globin promoters can induce γ-globin expression in K562 cells. This novel strategy can be promising for the treatment of β-thalassemia and sickle cell disease.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5110654PMC
October 2016

Genetic disruption of the KLF1 gene to overexpress the γ-globin gene using the CRISPR/Cas9 system.

J Gene Med 2016 Oct;18(10):294-301

Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.

Background: β-thalassemia comprises a major group of human genetic disorders involving a decrease in or an end to the normal synthesis of the β-globin chains of hemoglobin. KLF1 is a key regulatory molecule involved in the γ- to β-globin gene switching process directly inducing the expression of the β-globin gene and indirectly repressing γ-globin. The present study aimed to investigate the ability of an engineered CRISPR/Cas9 system with respect to disrupting the KLF1 gene to inhibit the γ- to β-hemoglobin switching process in K562 cells.

Methods: We targeted three sites on the KLF1 gene, two of which are upstream of codon 288 in exon 2 and the other site being in exon 3.

Results: The average indel percentage in the cells transfected with CRISPR a, b and c was approximately 24%. Relative quantification was performed for the assessment of γ-globin expression. The levels of γ-globin mRNA on day 5 of differentiation were 8.1-, 7.7- and 1.8-fold in the cells treated with CRISPR/Cas9 a, b and c, respectively,compared to untreated cells. The measurement of HbF expression levels confirmed the same results.

Conclusions: The findings obtained in the present study support the induction of an indel mutation in the KLF1 gene leading to a null allele. As a result, the effect of KLF1 on the expression of BCL11A is decreased and its inhibitory effect on γ-globin gene expression is removed. Application of CRISPR technology to induce an indel in the KLF1 gene in adult erythroid progenitors may provide a method for activating fetal hemoglobin expression in individuals with β-thalassemia or sickle cell disease.
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http://dx.doi.org/10.1002/jgm.2928DOI Listing
October 2016

Zinc Finger Nuclease: A New Approach to Overcome Beta-Lactam Antibiotic Resistance.

Jundishapur J Microbiol 2016 Jan 2;9(1):e29384. Epub 2016 Jan 2.

Department of Immunology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, IR Iran.

Background: The evolution of antibiotic-resistant bacteria (ARB) and antibiotic-resistance genes (ARGs) has been accelerated recently by the indiscriminate application of antibiotics. Antibiotic resistance has challenged the success of medical interventions and therefore is considered a hazardous threat to human health.

Objectives: The present study aimed to describe the use of zinc finger nuclease (ZFN) technology to target and disrupt a plasmid-encoded β-lactamase, which prevents horizontal gene transfer-mediated evolution of ARBs.

Materials And Methods: An engineered ZFN was designed to target a specific sequence in the ampicillin resistance gene (amp(R)) of the pTZ57R plasmid. The Escherichia coli bacteria already contained the pZFN kanamycin-resistant (kana(R)) plasmid as the case or the pP15A, kana(R) empty vector as the control, were transformed with the pTZ57R; the ability of the designed ZFN to disrupt the β-lactamase gene was evaluated with the subsequent disturbed ability of the bacteria to grow on ampicillin (amp) and ampicillin-kanamycin (amp-kana)-containing media. The effect of mild hypothermia on the ZFN gene targeting efficiency was also evaluated.

Results: The growth of bacteria in the case group on the amp and amp-kana-containing media was significantly lower compared with the control group at 37°C (P < 0.001). Despite being more efficient in hypothermic conditions at 30°C (P < 0.001), there were no significant associations between the incubation temperature and the ZFN gene targeting efficiency.

Conclusions: Our findings revealed that the ZFN technology could be employed to overcome ampicillin resistance by the targeted disruption of the ampicillin resistance gene, which leads to inactivation of β-lactam synthesis. Therefore, ZFN technology could be engaged to decrease the antibiotic resistance issue with the construction of a ZFN archive against different ARGs. To tackle the resistance issue at the environmental level, recombinant phages expressing ZFNs against different ARGs could be constructed and released into both hospital and urban wastewater systems.
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http://dx.doi.org/10.5812/jjm.29384DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4833962PMC
January 2016

Staphylococcus aureus Isolates Carrying Panton-Valentine Leucocidin Genes: Their Frequency, Antimicrobial Patterns, and Association With Infectious Disease in Shahrekord City, Southwest Iran.

Jundishapur J Microbiol 2016 Jan 2;9(1):e28291. Epub 2016 Jan 2.

Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, IR Iran; Pediatric Inherited Diseases Research Center, Research Institute for Primordial Prevention of Non-Communicable Disease, Isfahan University of Medical Sciences, Isfahan, IR Iran.

Background: A diversity of virulence factors work together to create the pathogenicity of Staphylococcus aureus. These factors include cell surface components that promote adherence to surfaces as well as exoproteins such as Panton-Valentine leukocidin (PVL), encoded by the luk-PV genes, that invade or bypass the immune system and are toxic to the host, thereby enhancing the severity of infections caused by methicillin-resistant Staphylococcus aureus (MRSA).

Objectives: The aim of this study was to determine the frequency of PVL-positive MRSA strains by real-time PCR and their antibiotic susceptibility patterns by phenotypic test.

Materials And Methods: In total, 284 Staphylococcus isolates, identified by phenotypic methods from clinical samples of Shahrekord University Hospitals, Shahrekord, Iran, were tested for nuc, mecA, and PVL genes by TaqMan real-time PCR. The antibiotic susceptibility patterns of PVL-containing MRSA strains were determined via the disk diffusion method.

Results: In total, 196 isolates (69%) were nuc positive (i.e., S. aureus); of those isolates, 96 (49%) were mecA positive (MRSA). Eighteen (18.8%) of the 96 MRSA positive and 3 (3%) of the 100 methicillin-susceptible Staphylococcus aureus (MSSA) strains were PVL positive. PVL-positive MRSA strains were mostly recovered from tracheal specimens. Eight PVL-positive MRSA strains were resistant to all the tested antibiotics except vancomycin. A significant correlation (P = 0.001) was found between the mecA positivity and the presence of luk-PV genes.

Conclusions: Community acquired (CA)-MRSA is becoming a public health concern in many parts of the world, including Asian countries. The variable prevalence of luk-PV-positive MRSA isolates in different regions and their rather high frequency in pneumonia necessitate the application of rapid diagnostic methods such as real-time PCR to improve treatment effectiveness.
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http://dx.doi.org/10.5812/jjm.28291DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4834141PMC
January 2016

Comparison of different methods for erythroid differentiation in the K562 cell line.

Biotechnol Lett 2016 Aug 13;38(8):1243-50. Epub 2016 Apr 13.

Department of Molecular Medicine, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran.

Objective: To compare methods for erythroid differentiation of K562 cells that will be promising in the treatment of beta-thalassemia by inducing γ-globin synthesis.

Results: Cells were treated separately with: RPMI 1640 medium without glutamine, RPMI 1640 medium without glutamine supplemented with 1 mM sodium butyrate, RPMI 1640 medium supplemented with 1 mM sodium butyrate, 25 µg cisplatin/ml, 0.1 µg cytosine arabinoside/ml. The highest differentiation (84 %) with minimum toxicity was obtained with cisplatin at 15 µg /ml. Real-time RT-PCR showed that expression of the γ-globin gene was significantly higher in the cells differentiated with cisplatin compared to undifferentiated cells (P < 0.001).

Conclusions: Cisplatin is useful in the experimental therapy of ß-globin gene defects and can be considered for examining the basic mechanism of γ-reactivation.
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http://dx.doi.org/10.1007/s10529-016-2101-8DOI Listing
August 2016

Comparison of different methods for erythroid differentiation in the K562 cell line.

Biotechnol Lett 2016 Aug 13;38(8):1243-50. Epub 2016 Apr 13.

Department of Molecular Medicine, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran.

Objective: To compare methods for erythroid differentiation of K562 cells that will be promising in the treatment of beta-thalassemia by inducing γ-globin synthesis.

Results: Cells were treated separately with: RPMI 1640 medium without glutamine, RPMI 1640 medium without glutamine supplemented with 1 mM sodium butyrate, RPMI 1640 medium supplemented with 1 mM sodium butyrate, 25 µg cisplatin/ml, 0.1 µg cytosine arabinoside/ml. The highest differentiation (84 %) with minimum toxicity was obtained with cisplatin at 15 µg /ml. Real-time RT-PCR showed that expression of the γ-globin gene was significantly higher in the cells differentiated with cisplatin compared to undifferentiated cells (P < 0.001).

Conclusions: Cisplatin is useful in the experimental therapy of ß-globin gene defects and can be considered for examining the basic mechanism of γ-reactivation.
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http://dx.doi.org/10.1007/s10529-016-2101-8DOI Listing
August 2016

Comparison of real-time PCR with disk diffusion, agar screen and E-test methods for detection of methicillin-resistant Staphylococcus aureus.

Curr Microbiol 2010 Dec 20;61(6):520-4. Epub 2010 Apr 20.

Department of Microbiology and Immunology, Cellular and Molecular Research Center, Shahrekord University of Medical Sciences, Shahrekord, Iran.

Methicillin-resistant Staphylococcus aureus (MRSA) is a nosocomial pathogen. Our main objective was to compare oxacillin disk test, oxacillin E-test, and oxacillin agar screen for detection of methicillin resistance in S. aureus, using real-time PCR for mecA as the "gold standard" comparison assay. 196 S. aureus isolates were identified out of 284 Staphylococcus isolates. These isolates were screened for MRSA with several methods: disk diffusion, agar screen (6.0 μg/ml), oxacillin E-test, and real-time PCR for detection of mecA gene. Of the 196 S. aureus isolates tested, 96 isolates (49%) were mecA-positive and 100 isolates (51%) mecA-negative. All methods tested had a statistically significant agreement with real-time PCR. E-test was 100% sensitive and specific for mecA presence. The sensitivity and specificity of oxacillin agar screen method were 98 and 99%, respectively and sensitivity and specificity of oxacillin disk diffusion method were 95 and 93%, respectively. In the present study, oxacillin E-test is proposed as the best phenotypic method. For economic reasons, the oxacillin agar screen method (6.0 μg/ml), which is suitable for the detection of MRSA, is recommended due to its accuracy and low cost.
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http://dx.doi.org/10.1007/s00284-010-9647-9DOI Listing
December 2010