Publications by authors named "Farzaneh Aboualizadeh"

13 Publications

  • Page 1 of 1

A homogeneous split-luciferase assay for rapid and sensitive detection of anti-SARS CoV-2 antibodies.

Nat Commun 2021 03 22;12(1):1806. Epub 2021 Mar 22.

Donnelly Centre, University of Toronto, Toronto, ON, Canada.

Better diagnostic tools are needed to combat the ongoing COVID-19 pandemic. Here, to meet this urgent demand, we report a homogeneous immunoassay to detect IgG antibodies against SARS-CoV-2. This serological assay, called SATiN, is based on a tri-part Nanoluciferase (tNLuc) approach, in which the spike protein of SARS-CoV-2 and protein G, fused respectively to two different tNLuc tags, are used as antibody probes. Target engagement of the probes allows reconstitution of a functional luciferase in the presence of the third tNLuc component. The assay is performed directly in the liquid phase of patient sera and enables rapid, quantitative and low-cost detection. We show that SATiN has a similar sensitivity to ELISA, and its readouts are consistent with various neutralizing antibody assays. This proof-of-principle study suggests potential applications in diagnostics, as well as disease and vaccination management.
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http://dx.doi.org/10.1038/s41467-021-22102-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7985487PMC
March 2021

Split Intein-Mediated Protein Ligation for detecting protein-protein interactions and their inhibition.

Nat Commun 2020 05 15;11(1):2440. Epub 2020 May 15.

Donnelly Centre, University of Toronto, Toronto, ON, Canada.

Here, to overcome many limitations accompanying current available methods to detect protein-protein interactions (PPIs), we develop a live cell method called Split Intein-Mediated Protein Ligation (SIMPL). In this approach, bait and prey proteins are respectively fused to an intein N-terminal fragment (IN) and C-terminal fragment (IC) derived from a re-engineered split intein GP41-1. The bait/prey binding reconstitutes the intein, which splices the bait and prey peptides into a single intact protein that can be detected by regular protein detection methods such as Western blot analysis and ELISA, serving as readouts of PPIs. The method is robust and can be applied not only in mammalian cell lines but in animal models such as C. elegans. SIMPL demonstrates high sensitivity and specificity, and enables exploration of PPIs in different cellular compartments and tracking of kinetic interactions. Additionally, we establish a SIMPL ELISA platform that enables high-throughput screening of PPIs and their inhibitors.
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http://dx.doi.org/10.1038/s41467-020-16299-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7229206PMC
May 2020

A drug discovery platform to identify compounds that inhibit EGFR triple mutants.

Nat Chem Biol 2020 05 24;16(5):577-586. Epub 2020 Feb 24.

Department for Lung Diseases Jordanovac, Clinical Hospital Centre Zagreb, University of Zagreb, Zagreb, Croatia.

Receptor tyrosine kinases (RTKs) are transmembrane receptors of great clinical interest due to their role in disease. Historically, therapeutics targeting RTKs have been identified using in vitro kinase assays. Due to frequent development of drug resistance, however, there is a need to identify more diverse compounds that inhibit mutated but not wild-type RTKs. Here, we describe MaMTH-DS (mammalian membrane two-hybrid drug screening), a live-cell platform for high-throughput identification of small molecules targeting functional protein-protein interactions of RTKs. We applied MaMTH-DS to an oncogenic epidermal growth factor receptor (EGFR) mutant resistant to the latest generation of clinically approved tyrosine kinase inhibitors (TKIs). We identified four mutant-specific compounds, including two that would not have been detected by conventional in vitro kinase assays. One of these targets mutant EGFR via a new mechanism of action, distinct from classical TKI inhibition. Our results demonstrate how MaMTH-DS is a powerful complement to traditional drug screening approaches.
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http://dx.doi.org/10.1038/s41589-020-0484-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8123931PMC
May 2020

Fetal RHD Genotyping Using Real-Time Polymerase Chain Reaction Analysis of Cell-Free Fetal DNA in Pregnancy of RhD Negative Women in South of Iran.

Int J Fertil Steril 2016 Apr-Jun;10(1):62-70. Epub 2016 Apr 5.

Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.

Background: Maternal-fetal RhD antigen incompatibility causes approximately 50% of clinically significant alloimmunization cases. The routine use of prophylactic anti-D immunoglobulin has dramatically reduced hemolytic disease of the fetus and newborn. Recently, fetal RHD genotyping in RhD negative pregnant women has been suggested for appropriate use of anti-D immunoglobulin antenatal prophylaxis and decrease unnecessary prenatal interventions.

Materials And Methods: In this prospective cohort study, in order to develop a reliable and non-invasive method for fetal RHD genotyping, cell free fetal DNA (cffD- NA) was extracted from maternal plasma. Real-time quantitative polymerase chain reaction (qPCR) for detection of RHD exons 7, 5, 10 and intron 4 was performed and the results were compared to the serological results of cord blood cells as the gold standard method. SRY gene and hypermethylated Ras-association domain family member 1 (RASSF1A) gene were used to confirm the presence of fetal DNA in male and female fetuses, respectively.

Results: Out of 48 fetuses between 8 and 32 weeks (wks) of gestational age (GA), we correctly diagnosed 45 cases (93.75%) of RHD positive fetuses and 2 cases (4.16%) of the RHD negative one. Exon 7 was amplified in one sample, while three other RHD gene sequences were not detected; the sample was classified as inconclusive, and the RhD serology result after birth showed that the fetus was RhD-negative.

Conclusion: Our results showed high accuracy of the qPCR method using cffDNA for fetal RHD genotyping and implicate on the efficiency of this technique to predict the competence of anti-D immunoglobulin administration.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4845531PMC
http://dx.doi.org/10.22074/ijfs.2016.4770DOI Listing
April 2016

Development of an In-House TaqMan Real Time RT-PCR Assay to Quantify Hepatitis C Virus RNA in Serum and Peripheral Blood Mononuclear Cells in Patients With Chronic Hepatitis C Virus Infection.

Hepat Mon 2015 Aug 31;15(8):e28895. Epub 2015 Aug 31.

Diagnosis Laboratory Sciences and Technology Research Center, School of Paramedical, Sciences Shiraz University of Medical Sciences, Shiraz, IR Iran.

Background: Viral load measurements are commonly used to monitor HCV infection in patients with chronic diseases or determining the number of HCV-genomes in serum samples of patients after sustained virological response. However, in some patients, HCV viral load in serum samples is too low to be detected by PCR, especially after treatment.

Objectives: The aim of this study was to develop a highly specific, sensitive, and reproducible in-house quantitative PCR using specific primers and probe cited in highly conservative region of HCV genome that allows simultaneous detection of HCV genotypes 1 - 4.

Materials And Methods: In this study, three sets of primer pairs and a TaqMan probe for amplification and detection of selected region within 5'-non-coding (5'NCR) of four HCV genotypes were used. Using plasmid containing 5'NCR region of HCV, standard curve, threshold, and threshold cycle (CT) values were determined. Real-time and nested PCR were performed on HCV genotypes 1 - 4 extracted from plasma and peripheral blood mononuclear cells (PBMCs) samples collected from patients with chronic HCV infection.

Results: The lower limit detection of this in-house HCV real-time RT-PCR was determined as 100 RNA copies/mL. Inter- and intra-assay coefficient of variation (CV) of this in-house HCV real-time RT-PCR ranged from 0.9% to 1.8% and 1.76% to 3.94%, respectively. The viral load of the genotyped samples ranged from 2.0 × 10(6) ± 0.31 to 2.7 × 10(5) ± 0.46 copies/mL in serum samples and 5 × 10(2) ± 0.36 to 4.0 × 10(3) ± 0.51 copies/10(6) cells/mL of PBMCs.

Conclusions: The quite sensitive in-house TaqMan real time RT-PCR assay was able to detect and quantify all four main HCV genotypes prevailing around all geographical regions of Iran.
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http://dx.doi.org/10.5812/hepatmon.28895DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4584366PMC
August 2015

Evaluation of a Modified DNA Extraction Method for Isolation of Cell-Free Fetal DNA from Maternal Serum.

Avicenna J Med Biotechnol 2015 Apr-Jun;7(2):85-8

Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.

Background: Discovery of short cell free fetal DNA (cffDNA) fragments in maternal plasma has created major changes in the field of prenatal diagnosis. The use of cffDNA to set up noninvasive prenatal test is limited due to the low concentration of fetal DNA in maternal plasma therefore, employing a high efficiency extraction method leads to more accurate results. The aim of this study was to evaluate the efficiency of Triton/Heat/Phenol (THP) protocol in comparison with the QIAamp DNA Blood mini Kit for cffDNA purification.

Methods: In order to evaluate the efficiency of THP protocol, DNA of Rhesus D (RhD) negative pregnant women's plasma was collected, then real-time PCR for RHD exon 7 was performed. The Ct value data of real time PCR obtained by two different methods were compared and after delivery serology test on cord blood was done to validate the real time PCR results.

Results: The results indicated significant differences between two extraction methods (p=0.001). The mean±SD of Ct-value using THP protocol was 33.8±1.6 and 36.1±2.47 using QIAamp DNA Blood mini Kit.

Conclusion: Our finding demonstrated that THP protocol was more effective than the QIAamp DNA Blood mini Kits for cffDNA extraction and lead to decrease the false negative results.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4483320PMC
July 2015

The association of circulating adiponectin and + 45 T/G polymorphism of adiponectin gene with gestational diabetes mellitus in Iranian population.

J Diabetes Metab Disord 2015 16;14:30. Epub 2015 Apr 16.

Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.

Background: Adiponectin is an adipokine with insulin-sensitizing effects. We investigated the relationship between the single nucleotide polymorphism (SNP) +45 T > G ( rs 2241766 ;Gly15Gly) in the adiponectin gene, serum adiponectin levels, insulin resistance and risk of gestational diabetes (GDM) in Iranian population.

Methods: 65 GDM patients and 70 healthy pregnant women were enrolled in this study. Genotyping for SNP +45 T > G in the adiponectin gene ( rs 2241766 ) was performed by the polymerase chain reaction-restriction fragment length polymorphism method. The level of fasting serum adiponectin, insulin, glucose, and lipid levels were measured. Insulin resistance was estimated using homeostasis model of assessment for insulin resistance (HOMA-IR).

Results: The G allele and TG/GG genotype of rs 2241766 were more frequent than the T allele and TT genotype in GDM patients compared to the controls (p < 0.05). Multiple logistic regression analysis revealed that the risk of GDM was significantly higher in subjects with the TG/GG genotype to those with TT genotype [odds ratio = 2.38, 95% CI 1.09-5.22, p = 0.030]. No significant association was observed between genotypes of rs 2241766 and circulating concentrations of adiponectin. Multiple regression analysis showed that serum adiponectin levels was negatively associated with HOMA-IR in GDM patients (β = -0.385, p <0.01).

Conclusion: The findings demonstrated that TG/GG genotype of rs 2241766 was an independent risk factor of GDM in our population. Furthermore, circulating adiponectin level was negatively correlated with insulin resistance in GDM patients.
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http://dx.doi.org/10.1186/s40200-015-0156-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4407396PMC
April 2015

G22A Polymorphism of Adenosine Deaminase and its Association with Biochemical Characteristics of Gestational Diabetes Mellitus in an Iranian Population.

Iran J Med Sci 2015 Mar;40(2):170-4

Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.

Adenosine deaminase (ADA) is an important regulator of insulin action. The single nucleotide polymorphism (SNP) G22A in the ADA gene decreases enzymatic activity of ADA. The aim of this study was to investigate the relationship between the SNP G22A and blood glycemic control, insulin resistance, and obesity of gestational diabetes mellitus (GDM) patients in an Iranian population. SNP G22A was determined in women with GDM (N=70) and healthy pregnant women (control, N=70) using polymerase chain reaction-restriction fragment length polymorphism. Fasting plasma glucose (FPG), hemoglobin A1C (HbA1c), plasma insulin levels and plasma lipids were measured using commercial kits. Homeostasis model of assessment for insulin resistance (HOMA-IR) was calculated. The distribution of genotypes and alleles among GDM patients was similar to that of the control group. FPG and HbA1c were significantly higher in GDM patients with GG genotype compared with GDM patients with GA+AA genotype and non-GDM patients. The frequency of GG genotype was significantly higher in obese GDM patients compared to lean GDM patients. The SNP G22A in the ADA gene was not associated with the risk of GDM in our population. GG genotype was associated with poor glycemic control and obesity in GDM patients.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4359938PMC
March 2015

A Comprehensive Membrane Interactome Mapping of Sho1p Reveals Fps1p as a Novel Key Player in the Regulation of the HOG Pathway in S. cerevisiae.

J Mol Biol 2015 Jun 30;427(11):2088-103. Epub 2015 Jan 30.

Donnelly Centre, University of Toronto, 160 College Street, Toronto, ON M5S 3E1, Canada; Department of Biochemistry, University of Toronto, Medical Science Building, 1 King's College Circle, Toronto, ON M5S 1A8, Canada; Department of Molecular Genetics, University of Toronto, Medical Sciences Building, 1 King's College Circle, Toronto, ON M5S 1A8, Canada. Electronic address:

Sho1p, an integral membrane protein, plays a vital role in the high-osmolarity glycerol (HOG) mitogen-activated protein kinase pathway in the yeast Saccharomyces cerevisiae. Activated under conditions of high osmotic stress, it interacts with other HOG pathway proteins to mediate cell signaling events, ensuring that yeast cells can adapt and remain viable. In an attempt to further understand how the function of Sho1p is regulated through its protein-protein interactions (PPIs), we identified 49 unique Sho1p PPIs through the use of membrane yeast two-hybrid (MYTH), an assay specifically suited to identify PPIs of full-length integral membrane proteins in their native membrane environment. Secondary validation by literature search, or two complementary PPI assays, confirmed 80% of these interactions, resulting in a high-quality Sho1p interactome. This set of putative PPIs included both previously characterized interactors, along with a large subset of interactors that have not been previously identified as binding to Sho1p. The SH3 domain of Sho1p was found to be important for binding to many of these interactors. One particular novel interactor of interest is the glycerol transporter Fps1p, which was shown to require the SH3 domain of Sho1p for binding via its N-terminal soluble regulatory domain. Furthermore, we found that Fps1p is involved in the positive regulation of Sho1p function and plays a role in the phosphorylation of the downstream kinase Hog1p. This study represents the largest membrane interactome analysis of Sho1p to date and complements past studies on the HOG pathway by increasing our understanding of Sho1p regulation.
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http://dx.doi.org/10.1016/j.jmb.2015.01.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5331858PMC
June 2015

Relationship between AHSP gene expression, β/α globin mRNA ratio, and clinical severity of the β-thalassemia patients.

Ann Clin Lab Sci 2014 ;44(2):189-93

PhD.; Deputy Paramedical Education, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran; phone:+987112270240;

Background: Alpha hemoglobin stabilizing protein (AHSP) is a chaperone-like molecule specialized for erythroid series which binds to free α-globin chain. According to this characteristic, AHSP can be considered an important factor which reduces beta thalassemia symptoms.

Materials And Methods: Reticulocytes RNA extraction and a subsequent cDNA synthesis were performed, followed by Relative qRT-PCR for AHSP, alpha, and beta globin chain genes. The beta actin gene was used as an endogenous reference as well. The relationship between AHSP gene expression, disease severity, and the β/α globin mRNA ratio was studied among different homozygote β-thalassemia patients (mild, moderate and severe) and compared with minor thalassemia and the normal population.

Results: Analysis of the β-globin/α-globin mRNA ratio has shown that disease severity enhanced with a decrease in this proportion. Evaluation of the correlation between AHSP gene expression and the average of the β-globin/α-globin expression ratio indicated a significant but indirect relationship in considered groups. Our results demonstrated that the AHSP gene expression increases in accordance with augmentation of clinical symptoms.

Conclusions: Although one of the main reasons for reduced clinical severity in homozygote β-thalassemia can be the high level of AHSP gene expression as a chaperon molecule, our findings indicated that AHSP gene expression decreased in a mild category as compared to that in severe and moderate groups.
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December 2014

Evaluation of red cell membrane cytoskeletal disorders using a flow cytometric method in South iran.

Turk J Haematol 2014 Mar 5;31(1):25-31. Epub 2014 Mar 5.

Diagnostic Laboratory, Sciences and Research Technology Center, Shiraz University of Medical Sciences, Shiraz, Iran ; School of Para Medical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.

Objective: The diagnosis of hereditary red blood cell (RBC) membrane disorders, and in particular hereditary spherocytosis (HS) and Southeast Asian ovalocytosis (SAO), is based on clinical history, RBC morphology, and other conventional tests such as osmotic fragility. However, there are some milder cases of these disorders that are difficult to diagnose. The application of eosin-5'-maleimide (EMA) was evaluated for screening of RBC membrane defects along with some other anemias. We used EMA dye, which binds mostly to band 3 protein and to a lesser extent some other membrane proteins, for screening of some membrane defects such as HS.

Materials And Methods: Fresh RBCs from hematologically normal controls and patients with HS, SAO, hereditary elliptocytosis, hereditary spherocytosis with pincered cells, severe iron deficiency, thalassemia minor, and autoimmune hemolytic anemia were stained with EMA dye and analyzed for mean fluorescent intensity (MFI) using a flow cytometer.

Results: RBCs from patients with HS and iron deficiency showed a significant reduction in MFI compared to those from normal controls (p<0.0001 and p<0.001, respectively), while macrocytic RBCs showed a significant increase in MFI (p<0.01). A significant correlation was shown between mean corpuscular volume and MFI, with the exceptions of HS and thalassemia minor.

Conclusion: Our results showed that the flow cytometric method could be a reliable diagnostic method for screening and confirmation, with higher sensitivity and specificity (95% and 93%, respectively) than conventional routine tests for HS patients prior to further specific membrane protein molecular tests.
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http://dx.doi.org/10.4274/Tjh.2012.0146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3996639PMC
March 2014

Assessment of different permeabilization methods of minimizing damage to the adherent cells for detection of intracellular RNA by flow cytometry.

Avicenna J Med Biotechnol 2014 Jan;6(1):38-46

Shiraz Transplant Research center, Shiraz University of Medical Sciences, Shiraz, Iran.

Background: Various fixation and permeabilization techniques have been developed for detection of intracellular antigens by flow cytometry; however, there are few studies using flow cytometry to detect the frequency of intracellular nucleic acids, particularly RNA. We tested six different permeabilization methods in order to gain access to a high quality method with minimal damage to intracellular components focusing on 18S rRNA in HeLa cells.

Methods: HeLa cells were fixed in 2% paraformaldehyde. A variety of detergents and enzymes including saponin, TritonX-100, Tween-20, NP40, Proteinase K, and streptolysin O were used to optimize a protocol of permeabilization for the flow cytometric enumeration of intracellular 18S rRNA. Treated cells were subjected to standard protocol of flow cytometric in situ hybridization in the presence of FITC-labeled sense and antisense probes to detect 18S ribosomal RNAs. Samples were then analyzed on a FACSCalibur flow cytometer. To evaluate cell morphology, following hybridization the cells were fixed on glass slide, covered with DAPI, and evaluated on a fluorescent microscope with appropriate filter sets.

Results: In comparison with other methods, maximum cell frequency in percentage and fluorescent intensity (M1 = 2.1%, M2 = 97.9%) were obtained when the cells were treated with 0.2% Tween-20 and incubated for 30 min (p = 0.001).

Conclusion: Our study indicated that the highest levels of mean fluorescence could be obtained when the cells were treated with Tween-20. However, it should be taken into consideration that for a successful flow cytometric result, other interfering factors such as hybridization conditions should also be optimized.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3895578PMC
January 2014

Analysis of β/α globin ratio by using relative qRT-PCR for diagnosis of beta-thalassemia carriers.

J Clin Lab Anal 2013 Jul;27(4):267-71

Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.

Background: Current routine tests for premarital screening of β-thalassemia carriers are not applicable for diagnosis of rare atypical minor β-thalassemia cases. A more specialized laboratory evaluation for them is the measurement of β/α chain synthesis ratio with the assistance of radioactive amino acids. This method is also no longer routinely accessible. Consequently it is required to establish a rapid, trouble-free, and reliable method that encompasses all the cases of β-thalassemia carriers. Therefore we have determined β/α-globin mRNA ratio by applying relative qRT-PCR in various β-thalassemia patients.

Methods: Reticulocytes RNA extraction and subsequent cDNA synthesis were performed, followed by relative qRT-PCR for α- and β-globin chain genes and β-actin gene as an endogenous reference. β/α-Globin gene ratio was then evaluated with the Pfaffl method.

Results: The mean of β/α ratio was 0.99, 0.81, 0.69, and 0.69 for normal population, minor, intermediate, and major β-thalassemia, respectively. Approximately 6% of cases with minor thalassemia RBC index and normal HbA2 and having a decreased β/α ratio were located in the minor β-thalassemia group. The mean of β/α mRNA ratio in normal individuals and minor β-thalassemia was significantly different with all other groups (P-value < 0.05). Nevertheless, there was no such association between β/α mRNA ratio in major and intermediate β-thalassemia.

Conclusion: According to the significant differences achieved, no overlapping between minor β-thalassemia and normal group, capability of diagnosing atypical minor β-thalassemia, and accessibility of this technique, we can declare that this method could be suggested as a routine premarital screening test for β-thalassemia carriers.
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http://dx.doi.org/10.1002/jcla.21594DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6807357PMC
July 2013