Biomechanical properties of cells and tissues not only regulate their shape and function but are ... more Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in elasticity can propagate or trigger the onset of major diseases like cancer or osteoarthritis (OA). Atomic force microscopy (AFM) has emerged as a strong tool to qualitatively and quantitatively characterize the biomechanical properties of specific biological target structures on a microscopic scale, measuring forces in a range from as small as the piconewton to the micronewton. Biomechanical properties are of special importance in musculoskeletal tissues, which are subjected to high levels of strain. OA as a degenerative disease of the cartilage results in the disruption of the pericellular matrix (PCM) and the spatial rearrangement of the chondrocytes embedded in their extracellular matrix (ECM). Disruption in PCM and ECM has been associated with changes in the biomechanical properties of cartilage. In the present study we used AFM to quantify these changes in relation to the specific spatial pattern changes of the chondrocytes. With each pattern change, significant changes in elasticity were observed for both the PCM and ECM. Measuring the local elasticity thus allows for drawing direct conclusions about the degree of local tissue degeneration in OA.
Modern wheat is a worldwide staple food. Gluten is an immune-dominant component of wheat. Consump... more Modern wheat is a worldwide staple food. Gluten is an immune-dominant component of wheat. Consumption of wheat, in enetically predisposed individuals, triggers immunological reactions causing gluten-related disorders (GRDs) including celiac disease (CD). A gluten-free diet currently is the sole effective treatment option for GRDs and CD. A low-immunogenic wheat variety can be beneficial for such individuals. In recent years, various biotechnology-based approaches have been applied to develop a hypoimmunogenic wheat variety. Such safe wheat options have been developed using genetically modified organisms (GMO) technologies. However, due to specific limitations, such varieties have not been accepted for domestic consumption. Recent studies suggest that genome editing (GE) techniques such as clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) modified non-transgenic wheat variety could be a safer option for GRDs and CD. This review article discusses how CRISPR/Cas 9...
International Journal of Functional Nutrition, 2022
celiac disease (cD) is a chronic enteropathy caused by the ingestion of gluten in genetically sus... more celiac disease (cD) is a chronic enteropathy caused by the ingestion of gluten in genetically susceptible individuals. cD is a common food-related disorder with a prevalence of ~1% worldwide. Failure to follow the only available treatment, i.e., a standard gluten-free diet (GFD), increases the risk of adverse outcomes, such as refractory cD. Studies have reported that the long-term avoidance of a GFD may lead to the development of certain types of cancer in patients with cD. An increased risk of gastrointestinal cancers and intestinal lymphomas are associated with cD. On the other hand, recent studies have demonstrated that the risk of colon cancer, ovarian and breast cancer is low in patients with cD. It has also been demonstrated that a strict GFD exerts a positive effect in reducing the cancer risk. However, only a limited number of studies have been conducted in this area, and the outcomes of these studies warrant further verifications. The present review article summarises and discusses the possible links between cD and cancer, and the probable reasons behind their association. In addition, the present review also discusses whether a strict GFD reduces the risk of developing certain types of cancer in patients with cD.
KRAS-mutated colorectal cancers (CRCs) are resistant to cetuximab treatment. The multifunctional ... more KRAS-mutated colorectal cancers (CRCs) are resistant to cetuximab treatment. The multifunctional Y-box binding protein 1 (YB-1) is overexpressed in CRC and is associated with chemoresistance. In this study, the effects of oncogenic mutated KRAS(G12V) and KRAS(G13D) on YB-1 phosphorylation were investigated in CRC cells. The effects of the inhibition of p90 ribosomal S6 kinase (RSK) on YB-1 phosphorylation, cell proliferation and survival were tested with and without treatment with 5-fluorouracil using pharmacological inhibitors and siRNA. YB-1 phosphorylation status and subcellular distribution in CRC patient tissues were determined by immunofluorescence staining and confocal microscopy. Endogenous expression of mutated KRAS(G13D) and conditional expression of KRAS(G12V) significantly stimulated YB-1 phosphorylation via RSK and were associated with cetuximab resistance. Inhibition of YB-1 by targeting RSK stimulated the Akt signaling pathway, and this stimulation occurred independen...
Tumor microenvironment (TME) is a specialized ecosystem of host components, designed by tumor cel... more Tumor microenvironment (TME) is a specialized ecosystem of host components, designed by tumor cells for successful development and metastasis of tumor. With the advent of 3D culture and advanced bioinformatic methodologies, it is now possible to study TME’s individual components and their interplay at higher resolution. Deeper understanding of the immune cell’s diversity, stromal constituents, repertoire profiling, neoantigen prediction of TMEs has provided the opportunity to explore the spatial and temporal regulation of immune therapeutic interventions. The variation of TME composition among patients plays an important role in determining responders and non-responders towards cancer immunotherapy. Therefore, there could be a possibility of reprogramming of TME components to overcome the widely prevailing issue of immunotherapeutic resistance. The focus of the present review is to understand the complexity of TME and comprehending future perspective of its components as potential t...
Malignant melanoma is the most lethal form of skin cancer; its incidence has increased over the l... more Malignant melanoma is the most lethal form of skin cancer; its incidence has increased over the last five decades. Y-box binding protein 1 (YB-1) plays a prominent role in mediating metastatic behavior by promoting epithelial-to-mesenchymal transition (EMT) processes. Migratory melanoma cells exhibit two major phenotypes: elongated mesenchymal or rounded amoeboid. The actomyosin cytoskeleton is key in both phenotypes, but intermediate filaments also undergo a significant rearrangement process, switching from cytokeratin-rich to vimentin and nestin-rich network. In this study, we aimed to investigate to what extent YB-1 impacts the biomechanical (cell stiffness) and biochemical aspects of melanoma cells and their cytoskeleton. To this end, we subjected A375 YB-1 knock-out and parental cells to atomic force microscopy investigations (stiffness determination), immunolabelling, and proteome analysis. We found that YB-1 expressing cells were significantly stiffer compared to the correspo...
Wheat gluten contains epitopes that trigger celiac disease (CD). A life-long strict gluten-free d... more Wheat gluten contains epitopes that trigger celiac disease (CD). A life-long strict gluten-free diet is the only treatment accepted for CD. However, very low-gluten wheat may provide an alternative treatment to CD. Conventional plant breeding methods have not been to produce celiac-safe wheat. RNA interference technology, to some extent, has succeeded in the development of safer wheat varieties. However, these varieties have multiple challenges in terms of their implementation. Clustered Regularly Interspaced Short Palindromic Repeats-associated nuclease 9 (CRISPR/Cas9) is a versatile gene-editing tool that has the ability to edit immunogenic gluten genes. So far, only a few studies have applied CRISPR/Cas9 to modify the wheat genome. In this article, we reviewed the published literature that applied CRISPR/Cas9 in wheat genome editing to investigate the current status of the CRISPR/Cas9 system to produce a low-immunogenic wheat variety. We found that in recent years, the CRISPR/Cas...
The multifunctional protein Y-box binding protein-1 (YB-1) regulates all the so far described can... more The multifunctional protein Y-box binding protein-1 (YB-1) regulates all the so far described cancer hallmarks including cell proliferation and survival. The MAPK/ERK and PI3K/Akt pathways are also the major pathways involved in cell growth, proliferation, and survival, and are the frequently hyperactivated pathways in human cancers. A gain of function mutation in KRAS mainly leads to the constitutive activation of the MAPK pathway, while the activation of the PI3K/Akt pathway occurs either through the loss of PTEN or a gain of function mutation of the catalytic subunit alpha of PI3K (PIK3CA). In this study, we investigated the underlying signaling pathway involved in YB-1 phosphorylation at serine 102 (S102) in KRAS(G13D)-mutated triple-negative breast cancer (TNBC) MDA-MB-231 cells versus PIK3CA(H1047R)/PTEN(E307K) mutated TNBC MDA-MB-453 cells. Our data demonstrate that S102 phosphorylation of YB-1 in KRAS-mutated cells is mainly dependent on the MAPK/ERK pathway, while in PIK3CA...
Gluten, a wheat protein, contains epitopes that trigger celiac disease (CD). So far, there is no ... more Gluten, a wheat protein, contains epitopes that trigger celiac disease (CD). So far, there is no treatment available for CD other than following a life-long, strict gluten-free diet (GFD). A very low-gluten or gluten-free wheat could provide an alternative treatment to CD. Till date, conventional plant breeding methods are not sufficient to produce celiac-safe wheat. Clustered Regularly Interspaced Short Palindromic Repeats-associated protein 9 (CRISPR/Cas9) is a versatile gene-editing system may efficiently edit the immunogenic gluten protein thereby producing a celiac-safe wheat variety. However, CRISPR/Cas9-mediated genome editing system has not been widely investigated to edit the wheat genome. Published literature available on various scientific platforms, that applied the CRISPR/Cas9 system to edit the wheat genome were explored. Only original research articles were included. Review articles, protocols, scientific presentations, and Ph.D. thesis were excluded. CRISPR/Cas9 is a...
Ionizing radiation (IR) and epidermal growth factor (EGF) stimulate Y-box binding protein-1 (YB-1... more Ionizing radiation (IR) and epidermal growth factor (EGF) stimulate Y-box binding protein-1 (YB-1) phosphorylation at Ser-102 in KRAS wild-type (KRASwt) cells, whereas in KRAS mutated (KRASmut) cells, YB-1 is constitutively phosphorylated, independent of IR or EGF. YB-1 activity stimulates the repair of IR-induced DNA double-strand breaks (DSBs) in the nucleus. Thus far, the YB-1 nuclear translocation pattern after cell exposure to various cellular stressors is not clear. In the present study, we investigated the pattern of YB-1 phosphorylation and its possible translocation to the nucleus in KRASwt cells after exposure to IR, EGF treatment, and conditional expression of mutated KRAS(G12V). IR, EGF, and conditional KRAS(G12V) expression induced YB-1 phosphorylation in both the cytoplasmic and nuclear fractions of KRASwt cells. None of the stimuli induced YB-1 nuclear translocation, while p90 ribosomal s6 kinase (RSK) translocation was enhanced in KRASwt cells after any of the stimul...
Background: The inbred mouse strain BTBR T+ tf/J (BTBR) exhibits behavioral deficits that mimic t... more Background: The inbred mouse strain BTBR T+ tf/J (BTBR) exhibits behavioral deficits that mimic the core deficits of autism. Neuroanatomically, the BTBR strain is also characterized by a complete absence of the corpus callosum. The goal of this study was to identify novel molecular and cellular changes in the BTBR mouse, focusing on neuronal, synaptic, glial and plasticity markers in the limbic system as a model for identifying putative molecular and cellular substrates associated with autistic behaviors. Methods: Forebrains of 8 to 10-week-old male BTBR and age-matched C57Bl/6J control mice were evaluated by immunohistochemistry using free-floating and paraffin embedded sections. Twenty antibodies directed against antigens specific to neurons, synapses and glia were used. Nissl, Timm and acetylcholinesterase (AchE) stains were performed to assess cytoarchitecture, mossy fibers and cholinergic fiber density, respectively. In the hippocampus, quantitative stereological estimates for the mitotic marker bromodeoxyuridine (BrdU) were performed to determine hippocampal progenitor proliferation, survival and differentiation, and brain-derived neurotrophic factor (BDNF) mRNA was quantified by in situ hybridization. Quantitative image analysis was performed for NG2, doublecortin (DCX), NeuroD, GAD67 and Poly-Sialic Acid Neural Cell Adhesion Molecule (PSA-NCAM).
Gluten, a wheat protein, contains epitopes that trigger celiac disease (CD). So far, there is no ... more Gluten, a wheat protein, contains epitopes that trigger celiac disease (CD). So far, there is no treatment available for CD other than following a life-long, strict gluten-free diet (GFD). A very low-gluten or gluten-free wheat could provide an alternative treatment to CD. Till date, conventional plant breeding methods are not sufficient to produce celiac-safe wheat. Clustered Regularly Interspaced Short Palindromic Repeats-associated protein 9 (CRISPR/Cas9) is a versatile gene-editing system may efficiently edit the immunogenic gluten protein thereby producing a celiac-safe wheat variety. However, CRISPR/Cas9-mediated genome editing system has not been widely investigated to edit the wheat genome. Published literature available on various scientific platforms, that applied the CRISPR/Cas9 system to edit the wheat genome were explored. Only original research articles were included. Review articles, protocols, scientific presentations, and Ph.D. thesis were excluded. CRISPR/Cas9 is a...
Biomechanical properties of cells and tissues not only regulate their shape and function but are ... more Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in elasticity can propagate or trigger the onset of major diseases like cancer or osteoarthritis (OA). Atomic force microscopy (AFM) has emerged as a strong tool to qualitatively and quantitatively characterize the biomechanical properties of specific biological target structures on a microscopic scale, measuring forces in a range from as small as the piconewton to the micronewton. Biomechanical properties are of special importance in musculoskeletal tissues, which are subjected to high levels of strain. OA as a degenerative disease of the cartilage results in the disruption of the pericellular matrix (PCM) and the spatial rearrangement of the chondrocytes embedded in their extracellular matrix (ECM). Disruption in PCM and ECM has been associated with changes in the biomechanical properties of cartilage. In the present study we used AFM to quantify these changes in relation to the specific spatial pattern changes of the chondrocytes. With each pattern change, significant changes in elasticity were observed for both the PCM and ECM. Measuring the local elasticity thus allows for drawing direct conclusions about the degree of local tissue degeneration in OA.
Modern wheat is a worldwide staple food. Gluten is an immune-dominant component of wheat. Consump... more Modern wheat is a worldwide staple food. Gluten is an immune-dominant component of wheat. Consumption of wheat, in enetically predisposed individuals, triggers immunological reactions causing gluten-related disorders (GRDs) including celiac disease (CD). A gluten-free diet currently is the sole effective treatment option for GRDs and CD. A low-immunogenic wheat variety can be beneficial for such individuals. In recent years, various biotechnology-based approaches have been applied to develop a hypoimmunogenic wheat variety. Such safe wheat options have been developed using genetically modified organisms (GMO) technologies. However, due to specific limitations, such varieties have not been accepted for domestic consumption. Recent studies suggest that genome editing (GE) techniques such as clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) modified non-transgenic wheat variety could be a safer option for GRDs and CD. This review article discusses how CRISPR/Cas 9...
International Journal of Functional Nutrition, 2022
celiac disease (cD) is a chronic enteropathy caused by the ingestion of gluten in genetically sus... more celiac disease (cD) is a chronic enteropathy caused by the ingestion of gluten in genetically susceptible individuals. cD is a common food-related disorder with a prevalence of ~1% worldwide. Failure to follow the only available treatment, i.e., a standard gluten-free diet (GFD), increases the risk of adverse outcomes, such as refractory cD. Studies have reported that the long-term avoidance of a GFD may lead to the development of certain types of cancer in patients with cD. An increased risk of gastrointestinal cancers and intestinal lymphomas are associated with cD. On the other hand, recent studies have demonstrated that the risk of colon cancer, ovarian and breast cancer is low in patients with cD. It has also been demonstrated that a strict GFD exerts a positive effect in reducing the cancer risk. However, only a limited number of studies have been conducted in this area, and the outcomes of these studies warrant further verifications. The present review article summarises and discusses the possible links between cD and cancer, and the probable reasons behind their association. In addition, the present review also discusses whether a strict GFD reduces the risk of developing certain types of cancer in patients with cD.
KRAS-mutated colorectal cancers (CRCs) are resistant to cetuximab treatment. The multifunctional ... more KRAS-mutated colorectal cancers (CRCs) are resistant to cetuximab treatment. The multifunctional Y-box binding protein 1 (YB-1) is overexpressed in CRC and is associated with chemoresistance. In this study, the effects of oncogenic mutated KRAS(G12V) and KRAS(G13D) on YB-1 phosphorylation were investigated in CRC cells. The effects of the inhibition of p90 ribosomal S6 kinase (RSK) on YB-1 phosphorylation, cell proliferation and survival were tested with and without treatment with 5-fluorouracil using pharmacological inhibitors and siRNA. YB-1 phosphorylation status and subcellular distribution in CRC patient tissues were determined by immunofluorescence staining and confocal microscopy. Endogenous expression of mutated KRAS(G13D) and conditional expression of KRAS(G12V) significantly stimulated YB-1 phosphorylation via RSK and were associated with cetuximab resistance. Inhibition of YB-1 by targeting RSK stimulated the Akt signaling pathway, and this stimulation occurred independen...
Tumor microenvironment (TME) is a specialized ecosystem of host components, designed by tumor cel... more Tumor microenvironment (TME) is a specialized ecosystem of host components, designed by tumor cells for successful development and metastasis of tumor. With the advent of 3D culture and advanced bioinformatic methodologies, it is now possible to study TME’s individual components and their interplay at higher resolution. Deeper understanding of the immune cell’s diversity, stromal constituents, repertoire profiling, neoantigen prediction of TMEs has provided the opportunity to explore the spatial and temporal regulation of immune therapeutic interventions. The variation of TME composition among patients plays an important role in determining responders and non-responders towards cancer immunotherapy. Therefore, there could be a possibility of reprogramming of TME components to overcome the widely prevailing issue of immunotherapeutic resistance. The focus of the present review is to understand the complexity of TME and comprehending future perspective of its components as potential t...
Malignant melanoma is the most lethal form of skin cancer; its incidence has increased over the l... more Malignant melanoma is the most lethal form of skin cancer; its incidence has increased over the last five decades. Y-box binding protein 1 (YB-1) plays a prominent role in mediating metastatic behavior by promoting epithelial-to-mesenchymal transition (EMT) processes. Migratory melanoma cells exhibit two major phenotypes: elongated mesenchymal or rounded amoeboid. The actomyosin cytoskeleton is key in both phenotypes, but intermediate filaments also undergo a significant rearrangement process, switching from cytokeratin-rich to vimentin and nestin-rich network. In this study, we aimed to investigate to what extent YB-1 impacts the biomechanical (cell stiffness) and biochemical aspects of melanoma cells and their cytoskeleton. To this end, we subjected A375 YB-1 knock-out and parental cells to atomic force microscopy investigations (stiffness determination), immunolabelling, and proteome analysis. We found that YB-1 expressing cells were significantly stiffer compared to the correspo...
Wheat gluten contains epitopes that trigger celiac disease (CD). A life-long strict gluten-free d... more Wheat gluten contains epitopes that trigger celiac disease (CD). A life-long strict gluten-free diet is the only treatment accepted for CD. However, very low-gluten wheat may provide an alternative treatment to CD. Conventional plant breeding methods have not been to produce celiac-safe wheat. RNA interference technology, to some extent, has succeeded in the development of safer wheat varieties. However, these varieties have multiple challenges in terms of their implementation. Clustered Regularly Interspaced Short Palindromic Repeats-associated nuclease 9 (CRISPR/Cas9) is a versatile gene-editing tool that has the ability to edit immunogenic gluten genes. So far, only a few studies have applied CRISPR/Cas9 to modify the wheat genome. In this article, we reviewed the published literature that applied CRISPR/Cas9 in wheat genome editing to investigate the current status of the CRISPR/Cas9 system to produce a low-immunogenic wheat variety. We found that in recent years, the CRISPR/Cas...
The multifunctional protein Y-box binding protein-1 (YB-1) regulates all the so far described can... more The multifunctional protein Y-box binding protein-1 (YB-1) regulates all the so far described cancer hallmarks including cell proliferation and survival. The MAPK/ERK and PI3K/Akt pathways are also the major pathways involved in cell growth, proliferation, and survival, and are the frequently hyperactivated pathways in human cancers. A gain of function mutation in KRAS mainly leads to the constitutive activation of the MAPK pathway, while the activation of the PI3K/Akt pathway occurs either through the loss of PTEN or a gain of function mutation of the catalytic subunit alpha of PI3K (PIK3CA). In this study, we investigated the underlying signaling pathway involved in YB-1 phosphorylation at serine 102 (S102) in KRAS(G13D)-mutated triple-negative breast cancer (TNBC) MDA-MB-231 cells versus PIK3CA(H1047R)/PTEN(E307K) mutated TNBC MDA-MB-453 cells. Our data demonstrate that S102 phosphorylation of YB-1 in KRAS-mutated cells is mainly dependent on the MAPK/ERK pathway, while in PIK3CA...
Gluten, a wheat protein, contains epitopes that trigger celiac disease (CD). So far, there is no ... more Gluten, a wheat protein, contains epitopes that trigger celiac disease (CD). So far, there is no treatment available for CD other than following a life-long, strict gluten-free diet (GFD). A very low-gluten or gluten-free wheat could provide an alternative treatment to CD. Till date, conventional plant breeding methods are not sufficient to produce celiac-safe wheat. Clustered Regularly Interspaced Short Palindromic Repeats-associated protein 9 (CRISPR/Cas9) is a versatile gene-editing system may efficiently edit the immunogenic gluten protein thereby producing a celiac-safe wheat variety. However, CRISPR/Cas9-mediated genome editing system has not been widely investigated to edit the wheat genome. Published literature available on various scientific platforms, that applied the CRISPR/Cas9 system to edit the wheat genome were explored. Only original research articles were included. Review articles, protocols, scientific presentations, and Ph.D. thesis were excluded. CRISPR/Cas9 is a...
Ionizing radiation (IR) and epidermal growth factor (EGF) stimulate Y-box binding protein-1 (YB-1... more Ionizing radiation (IR) and epidermal growth factor (EGF) stimulate Y-box binding protein-1 (YB-1) phosphorylation at Ser-102 in KRAS wild-type (KRASwt) cells, whereas in KRAS mutated (KRASmut) cells, YB-1 is constitutively phosphorylated, independent of IR or EGF. YB-1 activity stimulates the repair of IR-induced DNA double-strand breaks (DSBs) in the nucleus. Thus far, the YB-1 nuclear translocation pattern after cell exposure to various cellular stressors is not clear. In the present study, we investigated the pattern of YB-1 phosphorylation and its possible translocation to the nucleus in KRASwt cells after exposure to IR, EGF treatment, and conditional expression of mutated KRAS(G12V). IR, EGF, and conditional KRAS(G12V) expression induced YB-1 phosphorylation in both the cytoplasmic and nuclear fractions of KRASwt cells. None of the stimuli induced YB-1 nuclear translocation, while p90 ribosomal s6 kinase (RSK) translocation was enhanced in KRASwt cells after any of the stimul...
Background: The inbred mouse strain BTBR T+ tf/J (BTBR) exhibits behavioral deficits that mimic t... more Background: The inbred mouse strain BTBR T+ tf/J (BTBR) exhibits behavioral deficits that mimic the core deficits of autism. Neuroanatomically, the BTBR strain is also characterized by a complete absence of the corpus callosum. The goal of this study was to identify novel molecular and cellular changes in the BTBR mouse, focusing on neuronal, synaptic, glial and plasticity markers in the limbic system as a model for identifying putative molecular and cellular substrates associated with autistic behaviors. Methods: Forebrains of 8 to 10-week-old male BTBR and age-matched C57Bl/6J control mice were evaluated by immunohistochemistry using free-floating and paraffin embedded sections. Twenty antibodies directed against antigens specific to neurons, synapses and glia were used. Nissl, Timm and acetylcholinesterase (AchE) stains were performed to assess cytoarchitecture, mossy fibers and cholinergic fiber density, respectively. In the hippocampus, quantitative stereological estimates for the mitotic marker bromodeoxyuridine (BrdU) were performed to determine hippocampal progenitor proliferation, survival and differentiation, and brain-derived neurotrophic factor (BDNF) mRNA was quantified by in situ hybridization. Quantitative image analysis was performed for NG2, doublecortin (DCX), NeuroD, GAD67 and Poly-Sialic Acid Neural Cell Adhesion Molecule (PSA-NCAM).
Gluten, a wheat protein, contains epitopes that trigger celiac disease (CD). So far, there is no ... more Gluten, a wheat protein, contains epitopes that trigger celiac disease (CD). So far, there is no treatment available for CD other than following a life-long, strict gluten-free diet (GFD). A very low-gluten or gluten-free wheat could provide an alternative treatment to CD. Till date, conventional plant breeding methods are not sufficient to produce celiac-safe wheat. Clustered Regularly Interspaced Short Palindromic Repeats-associated protein 9 (CRISPR/Cas9) is a versatile gene-editing system may efficiently edit the immunogenic gluten protein thereby producing a celiac-safe wheat variety. However, CRISPR/Cas9-mediated genome editing system has not been widely investigated to edit the wheat genome. Published literature available on various scientific platforms, that applied the CRISPR/Cas9 system to edit the wheat genome were explored. Only original research articles were included. Review articles, protocols, scientific presentations, and Ph.D. thesis were excluded. CRISPR/Cas9 is a...
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