Maryam Kay - Academia.edu (original) (raw)

Papers by Maryam Kay

Research paper thumbnail of Increase of Clavulanic acid production by using recombinant Streptomyces clavuligerus strain including claR gene

Biological Journal of Microorganism, Jul 15, 2014

Research paper thumbnail of Targeted mutations in Val101 and Arg27 interferon beta protein increase its transcriptional and translational activities

Cytokine, 2016

Interferon β (IFNβ) is the most prescribed drug that has been used frequently for the treatment o... more Interferon β (IFNβ) is the most prescribed drug that has been used frequently for the treatment of multiple sclerosis (MS) patients. The aim of this study is to improve the production of IFNβ by induction of site directed mutagenesis. Accordingly, recombinant constructs were designed in order to enhance the expression of IFNβ mRNA and protein. The recombinant plasmids were transfected to the CHO cell line, following RNA extractions and cDNA synthesis. The effects of recombinant constructs were analyzed by real time PCR, ELISA and MTT assay. Transfected samples with either IFNβ101 or IFNβ101+27 have shown 11.55 and 2.26 fold elevation and over-expression compare to the wild type construct respectively. Our data also indicated that the IFNβ101 and IFNβ101+27 constructs increase IFNβ protein expression more than 2.2 and 4.5 fold, respectively compared to the control group. It could be concluded that the substitution of Phe in the codon 101 position, which may increase the binding activity of IFNβ with its receptors and introduction of an additional N glycosylation site (Asn-X-Thr) in the position 27 of IFNβ protein may cause such an effect. The proliferative activity of transfected cells by a recombinant IFNβ101 decreases in comparison to the wild type, although it was not statistically significant. Over-expression of IFNβ in such a level is promising not only for the patients but also for the pharmaceutical industries.

Research paper thumbnail of The molecular study of IFNβ pleiotropic roles in MS treatment

Iranian Journal of Neurology, 2013

Multiple sclerosis (MS) is one of the most important autoimmune diseases recognized by demyelinat... more Multiple sclerosis (MS) is one of the most important autoimmune diseases recognized by demyelination and axonal lesion. It is the most common cause of disability in the young population. Various immunomodulatory and immunosuppressive therapies, including different formulations of interferon beta (IFNβ), glatiramer acetate (GA), mitoxantrone, and natalizumab are available for this disease. However, interferon has been the best prescribed. Although the precise mechanism of IFNβ is unclear, many studies indicate some potential mechanism including blocking T cells activation, controlling pro-and antiinflammatory cytokine secretion, preventing activated immune cell migration through BBB, and inducing repair activity of damaged nerve cells by differentiating neural stem cells into oligodendrocytes. These molecular mechanisms have significant roles in IFNβ therapy. More researches are required in order for us to comprehend the mechanism of action of IFNβ, and improve and develop drugs for more efficient MS treatment.

Research paper thumbnail of Mechanism of Action of Interferon Beta in Treatment of Multiple Sclerosis

Research paper thumbnail of Molecular dynamics study of biodegradation of azo dyes via their interactions with AzrC azoreductase

Journal of Biomolecular Structure and Dynamics, 2015

Azo dyes are one of the most important class of dyes, which have been widely used in industries. ... more Azo dyes are one of the most important class of dyes, which have been widely used in industries. Because of the environmental pollution of azo dyes, many studies have been performed to study their biodegradation using bacterial systems. In present work, the AzrC of mesophilic gram-positive Bacillus sp. B29 has been considered to study its interaction with five common azo dyes (orange G, acid red 88, Sudan I, orange I, and methyl red). The molecular dynamics simulations have been employed to study the interaction between AzrC and azo dyes. The trajectory was confirmed using root mean square deviation and the root mean square fluctuation analyses. Then, the hydrogen bond and alanine scanning analyses were performed to reveal active site residues. Phe105 (A), Phe125 (B), Phe172 (B), and Pro132 (B) have been found as the most important hydrophobic residues whereas Asn104 (A), Tyr127 (B), and Asn187 (A) have key role in making hydrogen bond. The results of molecular mechanics Poisson-Boltzmann surface area and molecular mechanics generalized Born surface area calculations proved that the hydrophobic azo dyes like Acid red 88 binds more tightly to the AzrC protein. The calculated data suggested MR A 121 (B) I as a potential candidate for improving the AzrC-MR interactions.

Research paper thumbnail of A novel recombinant AzrC protein proposed by molecular docking and in silico analyses to improve azo dye's binding affinity

Gene, 2015

Azo dyes are broadly used in different industries through their chemical stability and ease of sy... more Azo dyes are broadly used in different industries through their chemical stability and ease of synthesis. These dyes are usually identified as critical environmental pollutants and many attentions were performed to degradation of azo dyes using biological systems. In this study, the interactions of an azoreductase from mesophilic gram-positive Bacillus sp. B29, AzrC, with four common azo dyes (orange I, orange II, orange G and acid red 88) were investigated. Fifteen points, double, triple and quadruple mutant forms of AzrC were made using Molegro Virtual Docker 6.0 in order to improve the binding affinity of azo dyes to AzrC. The impact of 15 different mutations on azo dye affinity potency of AzrC was computationally analyzed using AzrC-azo dye molecular docking, and each interaction was scored based on AutoDock 4.2 free binding energy. Our results have indicated that Asn 104 (A), Asn 187 (B), and Tyr 151 (A) make stable hydrogen bond between AzrC and azo dyes. The hydrophobic amino acids like Phe105 (A), Phe 125 (B), and Phe 172 (B) in wild type form make hydrophobic interactions. In addition, the presence of more hydrophobic residues F60 (B), I119 (B), I121 (B) and F132 (B) in mutant forms made more powerful hydrophobic pocket in the active site. In conclusion, recombinant AzrC with quadruple mutations was suggested in order to increase the biodegradation capacity of AzrC through improving its affinity to four studied azo dyes. This study would be promising for future experimental analyses in order to produce recombinant form of AzrC.

Research paper thumbnail of New Insights into VEGF-A Alternative Splicing: Key Regulatory Switching in the Pathological Process

Avicenna journal of medical biotechnology, 2014

Vascular endothelial growth factor (VEGF-A) is one of the most important regulatory factors in pa... more Vascular endothelial growth factor (VEGF-A) is one of the most important regulatory factors in pathological and physiological angiogenesis. Alternative splicing is a complicated molecular process in VEGF-A gene expression which adds complexity to VEGF-A biology. Among all VEGF-A exons, alternative splicing of exon 8 is the key determinant of isoform switching from pro-angio-genic VEGF-xxx to anti-angiogenic VEGF-xxxb. This is known as a key molecular switching in many pathological situations. In fact, the balance between VEGF-xxx and VEGF-xxxb isoforms is a critical controlling switch in both conditions of health and disease. Here, the properties of VEGF-xxx and VEGF-xxxb isoforms were discussed and their regulatory mechanism and their roles in certain pathological processes were evaluated. In summary, it was suggested that C-terminal VEGF-A alternative splicing can provide a new treatment opportunity in angiogenic diseases.

Research paper thumbnail of The molecular study of IFNβ pleiotropic roles in MS treatment

Iranian journal of neurology, 2013

Multiple sclerosis (MS) is one of the most important autoimmune diseases recognized by demyelinat... more Multiple sclerosis (MS) is one of the most important autoimmune diseases recognized by demyelination and axonal lesion. It is the most common cause of disability in the young population. Various immunomodulatory and immunosuppressive therapies, including different formulations of interferon beta (IFNβ), glatiramer acetate (GA), mitoxantrone, and natalizumab are available for this disease. However, interferon has been the best prescribed. Although the precise mechanism of IFNβ is unclear, many studies indicate some potential mechanism including blocking T cells activation, controlling pro- and anti-inflammatory cytokine secretion, preventing activated immune cell migration through BBB, and inducing repair activity of damaged nerve cells by differentiating neural stem cells into oligodendrocytes. These molecular mechanisms have significant roles in IFNβ therapy. More researches are required in order for us to comprehend the mechanism of action of IFNβ, and improve and develop drugs fo...

Research paper thumbnail of Effects of disruption of the nucleotide pattern in CRID element and Kozak sequence of interferon β on mRNA stability and protein production

Autoimmunity, 2015

Interferon β (IFNβ) is the most important drug that has been used frequently for multiple scleros... more Interferon β (IFNβ) is the most important drug that has been used frequently for multiple sclerosis treatment. This study has tried to improve the IFNβ production by introducing mutations in the coding region of IFNβ, while its amino acid sequence is intact. Two recombinant vectors IFNβK and IFNβK+CRID were designed by site-directed mutagenesis. The IFNβK and IFNβK+CRID have two substitutions in Kozak sequence and four substitutions in CRID sequence, respectively. The Chinese hamster ovary (CHO) cell codon usage optimization was also performed for both of them. They were transiently transfected to CHO-dhfr(-) cell line using Lipofectamine kit (Invitrogen, Grand Island, NY). The amount of mRNA and protein was determined by real time PCR and ELISA. The results of this study indicate that the amount of IFNβ protein produced by CHO cells containing IFNβK has been elevated up to 3.5-fold. On the other hand, enormous amounts of IFNβ mRNA and protein were produced by cells containing IFNβK+CRID construct; more than 4.6-fold and 6-fold, respectively. It could be concluded that disruption of AT pattern in CRID element increase RNA and protein production, improve IFNβ mRNA stability and, may also enhance mRNA half-life. In a similar way, more proteins are produced by modification of Kozak sequence.

Research paper thumbnail of Experimental evidences for hsa-miR-497-5p as a negative regulator of SMAD3 gene expression

Gene, 2016

The SMAD family comprises of transcription factors that function as signal transducers of transfo... more The SMAD family comprises of transcription factors that function as signal transducers of transforming growth factor (TGFβ) superfamily members. MiRNAs are a class of small noncoding RNAs that may play a major role in post transcriptional regulation of SMAD genes. Here, we intended to investigate if hsa-miR-497-5p is capable of regulating SMAD3 gene expression. Hsa-miR-497-5p was bioinformatically predicted as a candidate regulator of SMAD3 gene expression and then, hsa-miR-497-5p expression status was analyzed in different cell lines using RT-qPCR. Overexpression of hsa-miR-497-5p in HEK293t cells resulted in downregulation of SMAD3 which was detected by RT-qPCR and western analysis. Further, dual luciferase assay results supported direct interaction of hsa-miR-497-5p with 3'-UTR sequences of SMAD3 transcript. Overexpression of hsa-miR-497-5p in HEK293t cells resulted in cell cycle arrest in G0/G1 phase, detected by flow cytometry. Overall, accumulative results indicated that hsa-miR-497-5p by targeting SMAD3 is potentially one of the regulators of the TGFβ signaling pathway.

Research paper thumbnail of Increase of Clavulanic acid production by using recombinant Streptomyces clavuligerus strain including claR gene

Biological Journal of Microorganism, Jul 15, 2014

Research paper thumbnail of Targeted mutations in Val101 and Arg27 interferon beta protein increase its transcriptional and translational activities

Cytokine, 2016

Interferon β (IFNβ) is the most prescribed drug that has been used frequently for the treatment o... more Interferon β (IFNβ) is the most prescribed drug that has been used frequently for the treatment of multiple sclerosis (MS) patients. The aim of this study is to improve the production of IFNβ by induction of site directed mutagenesis. Accordingly, recombinant constructs were designed in order to enhance the expression of IFNβ mRNA and protein. The recombinant plasmids were transfected to the CHO cell line, following RNA extractions and cDNA synthesis. The effects of recombinant constructs were analyzed by real time PCR, ELISA and MTT assay. Transfected samples with either IFNβ101 or IFNβ101+27 have shown 11.55 and 2.26 fold elevation and over-expression compare to the wild type construct respectively. Our data also indicated that the IFNβ101 and IFNβ101+27 constructs increase IFNβ protein expression more than 2.2 and 4.5 fold, respectively compared to the control group. It could be concluded that the substitution of Phe in the codon 101 position, which may increase the binding activity of IFNβ with its receptors and introduction of an additional N glycosylation site (Asn-X-Thr) in the position 27 of IFNβ protein may cause such an effect. The proliferative activity of transfected cells by a recombinant IFNβ101 decreases in comparison to the wild type, although it was not statistically significant. Over-expression of IFNβ in such a level is promising not only for the patients but also for the pharmaceutical industries.

Research paper thumbnail of The molecular study of IFNβ pleiotropic roles in MS treatment

Iranian Journal of Neurology, 2013

Multiple sclerosis (MS) is one of the most important autoimmune diseases recognized by demyelinat... more Multiple sclerosis (MS) is one of the most important autoimmune diseases recognized by demyelination and axonal lesion. It is the most common cause of disability in the young population. Various immunomodulatory and immunosuppressive therapies, including different formulations of interferon beta (IFNβ), glatiramer acetate (GA), mitoxantrone, and natalizumab are available for this disease. However, interferon has been the best prescribed. Although the precise mechanism of IFNβ is unclear, many studies indicate some potential mechanism including blocking T cells activation, controlling pro-and antiinflammatory cytokine secretion, preventing activated immune cell migration through BBB, and inducing repair activity of damaged nerve cells by differentiating neural stem cells into oligodendrocytes. These molecular mechanisms have significant roles in IFNβ therapy. More researches are required in order for us to comprehend the mechanism of action of IFNβ, and improve and develop drugs for more efficient MS treatment.

Research paper thumbnail of Mechanism of Action of Interferon Beta in Treatment of Multiple Sclerosis

Research paper thumbnail of Molecular dynamics study of biodegradation of azo dyes via their interactions with AzrC azoreductase

Journal of Biomolecular Structure and Dynamics, 2015

Azo dyes are one of the most important class of dyes, which have been widely used in industries. ... more Azo dyes are one of the most important class of dyes, which have been widely used in industries. Because of the environmental pollution of azo dyes, many studies have been performed to study their biodegradation using bacterial systems. In present work, the AzrC of mesophilic gram-positive Bacillus sp. B29 has been considered to study its interaction with five common azo dyes (orange G, acid red 88, Sudan I, orange I, and methyl red). The molecular dynamics simulations have been employed to study the interaction between AzrC and azo dyes. The trajectory was confirmed using root mean square deviation and the root mean square fluctuation analyses. Then, the hydrogen bond and alanine scanning analyses were performed to reveal active site residues. Phe105 (A), Phe125 (B), Phe172 (B), and Pro132 (B) have been found as the most important hydrophobic residues whereas Asn104 (A), Tyr127 (B), and Asn187 (A) have key role in making hydrogen bond. The results of molecular mechanics Poisson-Boltzmann surface area and molecular mechanics generalized Born surface area calculations proved that the hydrophobic azo dyes like Acid red 88 binds more tightly to the AzrC protein. The calculated data suggested MR A 121 (B) I as a potential candidate for improving the AzrC-MR interactions.

Research paper thumbnail of A novel recombinant AzrC protein proposed by molecular docking and in silico analyses to improve azo dye's binding affinity

Gene, 2015

Azo dyes are broadly used in different industries through their chemical stability and ease of sy... more Azo dyes are broadly used in different industries through their chemical stability and ease of synthesis. These dyes are usually identified as critical environmental pollutants and many attentions were performed to degradation of azo dyes using biological systems. In this study, the interactions of an azoreductase from mesophilic gram-positive Bacillus sp. B29, AzrC, with four common azo dyes (orange I, orange II, orange G and acid red 88) were investigated. Fifteen points, double, triple and quadruple mutant forms of AzrC were made using Molegro Virtual Docker 6.0 in order to improve the binding affinity of azo dyes to AzrC. The impact of 15 different mutations on azo dye affinity potency of AzrC was computationally analyzed using AzrC-azo dye molecular docking, and each interaction was scored based on AutoDock 4.2 free binding energy. Our results have indicated that Asn 104 (A), Asn 187 (B), and Tyr 151 (A) make stable hydrogen bond between AzrC and azo dyes. The hydrophobic amino acids like Phe105 (A), Phe 125 (B), and Phe 172 (B) in wild type form make hydrophobic interactions. In addition, the presence of more hydrophobic residues F60 (B), I119 (B), I121 (B) and F132 (B) in mutant forms made more powerful hydrophobic pocket in the active site. In conclusion, recombinant AzrC with quadruple mutations was suggested in order to increase the biodegradation capacity of AzrC through improving its affinity to four studied azo dyes. This study would be promising for future experimental analyses in order to produce recombinant form of AzrC.

Research paper thumbnail of New Insights into VEGF-A Alternative Splicing: Key Regulatory Switching in the Pathological Process

Avicenna journal of medical biotechnology, 2014

Vascular endothelial growth factor (VEGF-A) is one of the most important regulatory factors in pa... more Vascular endothelial growth factor (VEGF-A) is one of the most important regulatory factors in pathological and physiological angiogenesis. Alternative splicing is a complicated molecular process in VEGF-A gene expression which adds complexity to VEGF-A biology. Among all VEGF-A exons, alternative splicing of exon 8 is the key determinant of isoform switching from pro-angio-genic VEGF-xxx to anti-angiogenic VEGF-xxxb. This is known as a key molecular switching in many pathological situations. In fact, the balance between VEGF-xxx and VEGF-xxxb isoforms is a critical controlling switch in both conditions of health and disease. Here, the properties of VEGF-xxx and VEGF-xxxb isoforms were discussed and their regulatory mechanism and their roles in certain pathological processes were evaluated. In summary, it was suggested that C-terminal VEGF-A alternative splicing can provide a new treatment opportunity in angiogenic diseases.

Research paper thumbnail of The molecular study of IFNβ pleiotropic roles in MS treatment

Iranian journal of neurology, 2013

Multiple sclerosis (MS) is one of the most important autoimmune diseases recognized by demyelinat... more Multiple sclerosis (MS) is one of the most important autoimmune diseases recognized by demyelination and axonal lesion. It is the most common cause of disability in the young population. Various immunomodulatory and immunosuppressive therapies, including different formulations of interferon beta (IFNβ), glatiramer acetate (GA), mitoxantrone, and natalizumab are available for this disease. However, interferon has been the best prescribed. Although the precise mechanism of IFNβ is unclear, many studies indicate some potential mechanism including blocking T cells activation, controlling pro- and anti-inflammatory cytokine secretion, preventing activated immune cell migration through BBB, and inducing repair activity of damaged nerve cells by differentiating neural stem cells into oligodendrocytes. These molecular mechanisms have significant roles in IFNβ therapy. More researches are required in order for us to comprehend the mechanism of action of IFNβ, and improve and develop drugs fo...

Research paper thumbnail of Effects of disruption of the nucleotide pattern in CRID element and Kozak sequence of interferon β on mRNA stability and protein production

Autoimmunity, 2015

Interferon β (IFNβ) is the most important drug that has been used frequently for multiple scleros... more Interferon β (IFNβ) is the most important drug that has been used frequently for multiple sclerosis treatment. This study has tried to improve the IFNβ production by introducing mutations in the coding region of IFNβ, while its amino acid sequence is intact. Two recombinant vectors IFNβK and IFNβK+CRID were designed by site-directed mutagenesis. The IFNβK and IFNβK+CRID have two substitutions in Kozak sequence and four substitutions in CRID sequence, respectively. The Chinese hamster ovary (CHO) cell codon usage optimization was also performed for both of them. They were transiently transfected to CHO-dhfr(-) cell line using Lipofectamine kit (Invitrogen, Grand Island, NY). The amount of mRNA and protein was determined by real time PCR and ELISA. The results of this study indicate that the amount of IFNβ protein produced by CHO cells containing IFNβK has been elevated up to 3.5-fold. On the other hand, enormous amounts of IFNβ mRNA and protein were produced by cells containing IFNβK+CRID construct; more than 4.6-fold and 6-fold, respectively. It could be concluded that disruption of AT pattern in CRID element increase RNA and protein production, improve IFNβ mRNA stability and, may also enhance mRNA half-life. In a similar way, more proteins are produced by modification of Kozak sequence.

Research paper thumbnail of Experimental evidences for hsa-miR-497-5p as a negative regulator of SMAD3 gene expression

Gene, 2016

The SMAD family comprises of transcription factors that function as signal transducers of transfo... more The SMAD family comprises of transcription factors that function as signal transducers of transforming growth factor (TGFβ) superfamily members. MiRNAs are a class of small noncoding RNAs that may play a major role in post transcriptional regulation of SMAD genes. Here, we intended to investigate if hsa-miR-497-5p is capable of regulating SMAD3 gene expression. Hsa-miR-497-5p was bioinformatically predicted as a candidate regulator of SMAD3 gene expression and then, hsa-miR-497-5p expression status was analyzed in different cell lines using RT-qPCR. Overexpression of hsa-miR-497-5p in HEK293t cells resulted in downregulation of SMAD3 which was detected by RT-qPCR and western analysis. Further, dual luciferase assay results supported direct interaction of hsa-miR-497-5p with 3'-UTR sequences of SMAD3 transcript. Overexpression of hsa-miR-497-5p in HEK293t cells resulted in cell cycle arrest in G0/G1 phase, detected by flow cytometry. Overall, accumulative results indicated that hsa-miR-497-5p by targeting SMAD3 is potentially one of the regulators of the TGFβ signaling pathway.