Kanishka De silva | University of Sri Jayewardenepura (original) (raw)

Papers by Kanishka De silva

Research paper thumbnail of Evaluation of non‑coding region sequence variants and mitochondrial haplogroups as potential biomarkers of sporadic breast cancer in individuals of Sri Lankan Sinhalese ethnicity

Biomedical Reports, 2020

Mitochondrial DNA (mtDNA) mutations have been reported to be associated with various diseases, in... more Mitochondrial DNA (mtDNA) mutations have been reported to be associated with various diseases, including cancer. The present study investigated the mtDNA non-coding region mutations and mitochondrial haplogroups as potential biomarkers of sporadic breast cancer in Sri Lankan Sinhalese women. Mitochondrial macro-haplogroups were determined using PCR-restriction fragment length polymorphism, whereas non-coding region sequences were determined using Sanger sequencing. The sequence of the non-coding region was also used to confirm haplogroup status. Neither the mutations in the non-coding region nor the mitochondrial haplogroups that were reported as risk factors in other populations, were determined to be potential risk factors for sporadic breast cancer in the present study. Furthermore, several novel mutations were identified in the present matched pairs case-controlled study. The M65a haplogroup with an additional mutation at position 16311 (P=0.0771) and mutations at the ori-b site (P=0.05) were considered a weak risk factor and protective factor, respectively, for sporadic breast cancer in Sinhalese women. Previous studies have indicated the use of mtDNA mutations as a biomarker; however, the present study showed that such biomarkers need to be validated for individual ethnic groups before they can be recommended for use in the prediction of disease.

Research paper thumbnail of Pattern of nucleotide variants of TP53 and their correlation with the expression of p53 and its downstream proteins in a Sri Lankan cohort of breast and colorectal cancer patients

Background Breast cancer (BC) is known to be the most common malignancy in females whereas colore... more Background Breast cancer (BC) is known to be the most common malignancy in females whereas colorectal cancer (CRC) incidence also higher in both genders in Sri Lanka. TP53 is an important tumour suppressor gene and its somatic mutations are reported in approximately 27% of BC and 43% of CRC cases. Analysis of TP53 gene variants not only provides clues for the aetiology of the tumour formation, but also has an impact on treatment efficacy. The current study was conducted to investigate the pattern of TP53 variants in patients with BC and CRC from Sri Lanka. Methods 30 patients with BC, 21 patients with CRC and an equal number of healthy controls were screened for mutational status of TP53 by polymerase chain reaction (PCR) followed by direct sequencing. In addition, a subset of these samples were analysed for the protein expression of p53 and comparison made with the mutational status of TP53. We also analysed the protein expression of p21 and MDM2 as potential indicators of p53 func...

Research paper thumbnail of Nucleotide variants and protein expression of TP53 in a Sri Lankan cohort of patients with head and neck cancer

Molecular Medicine Reports, 2019

Head and neck cancer (HNC) is the leading cancer in Sri Lankan males and second most common cance... more Head and neck cancer (HNC) is the leading cancer in Sri Lankan males and second most common cancer among Sri Lankan females. This is the first study, to the best of our knowledge, that has focused on investigating the association between TP53 somatic DNA variants, with p53 protein expression and risk factors in a cohort of Sri Lankan patients with HNC. A total of 44 patients with cancer and 20 healthy controls were studied. In total, 36 genomic DNA sequence variants were found, including several novel variants (two deletions in exons 4 and 6, two in the 3' untranslated region and several intronic variants). A total of 14 tumour samples carried pathogenic TP53 mutations. A random selection of 24 samples was analysed immunohistochemically for p53 protein expression. All the samples with point missense variants were strongly immuno-positive, whereas, samples with nonsense and frameshift TP53 variants were immuno-negative for p53 immunohistochemical staining. Although, the human papilloma virus is a known risk factor for HNC, results from the present study identified an absence or lower level of infection in the Sri Lankan cohort.

Research paper thumbnail of Circulating leptin, soluble leptin receptor, free leptin index, visfatin and selected leptin and leptin receptor gene polymorphisms in sporadic breast cancer

Endocrine journal, Jan 11, 2017

Leptin and visfatin are implicated in breast cancer risk but studies accounting for bioavailabili... more Leptin and visfatin are implicated in breast cancer risk but studies accounting for bioavailability of leptin are sparse. Reports on the association of leptin gene (LEP) and leptin receptor gene (LEPR) polymorphisms with breast cancer are also inconsistent. Only a very few studies have examined biochemical and genetic variables concomitantly in the same cohort. A matched pairs study was carried out to ascertain whether plasma leptin, soluble leptin receptor, free leptin index (leptin/soluble leptin receptor), serum visfatin and selected LEP and LEPR polymorphisms are risk factors for sporadic breast cancer. Newly diagnosed sporadic breast cancer patients (N=80) were matched for age, body mass index (BMI) and menopausal status with healthy controls. Plasma leptin, soluble leptin receptor and serum visfatin were measured by enzyme-immunoassay. LEP -2548 A/G and LEPR K109R, LEPR Q223R polymorphisms were determined by genotyping. Leptin (p=0.0234), leptin/BMI (p=0.0468), free leptin ind...

Research paper thumbnail of Novel and reported pathogenic variants in exon 11 of BRCA2 gene in a cohort of Sri Lankan young breast cancer patients

Familial Cancer, 2016

Women with breast carcinoma diagnosed before 40 years of age with a strong familial risk have a g... more Women with breast carcinoma diagnosed before 40 years of age with a strong familial risk have a greater prevalence of germline BRCA1 or BRCA2 variants than late onset breast cancer. Previously germline variants in BRCA1 and BRCA2 genes were characterized in a cohort of Sri Lankan breast cancer patients unselected for age of onset. This study focused on young breast cancer patients who were screened for previously identified hotspot regions in BRCA2 gene. A total of 48 young breast cancer patients with family history of cancer and 25 healthy controls were studied. Direct sequencing was used to detect pathogenic and other sequence variants in the hotspot regions of BRCA2 gene. Thirty-six sequence variants including seven pathogenic (c.2411_2412delAA/p.Glu804Valfs*2, c.2500_2501insG/p.Leu834Cysfs*4, c.3881T>G/p.Leu1294*, c.4768A>T/p.Lys1590*, c.5645C>G/p.Ser1882*, c.5747delC/p.His1916Phefs*3, c.6728C>T/p.Ser2243Phe) and two likely pathogenic (c.1922C>T and c.3378A>T) variants, two intronic variants of unknown significance (c.1910-74T>C, c.1910-51G>T), two variants of uncertain significance (c.2324C>T c.5104C>T) and 23 benign variants were detected. Among them, seven were novel (pathogenic 5 and likely pathogenic 2). Prevalence of pathogenic and likely pathogenic variants in the hotspots regions of BRCA2 was 23 and 6.3 % respectively in this cohort. This justifies BRCA2 variant testing in young breast cancer patients with family history of cancer in Sri Lanka.

Research paper thumbnail of Murine muscle stem cell response to perturbations of the neuromuscular junction are attenuated with aging

eLife, 2021

During aging and neuromuscular diseases, there is a progressive loss of skeletal muscle volume an... more During aging and neuromuscular diseases, there is a progressive loss of skeletal muscle volume and function impacting mobility and quality of life. Muscle loss is often associated with denervation and a loss of resident muscle stem cells (satellite cells or MuSCs); however, the relationship between MuSCs and innervation has not been established. Herein, we administered severe neuromuscular trauma to a transgenic murine model that permits MuSC lineage tracing. We show that a subset of MuSCs specifically engraft in a position proximal to the neuromuscular junction (NMJ), the synapse between myofibers and motor neurons, in healthy young adult muscles. In aging and in a mouse model of neuromuscular degeneration (Cu/Zn superoxide dismutase knockout – Sod1-/-), this localized engraftment behavior was reduced. Genetic rescue of motor neurons in Sod1-/- mice reestablished integrity of the NMJ in a manner akin to young muscle and partially restored MuSC ability to engraft into positions prox...

Research paper thumbnail of Surface Chemistry of Carbon Nanotubes Impacts the Growth and Expression of Water Channel Protein in Tomato Plants

Small, 2012

Specific properties of carbon nanotubes, such as their level of agglomeration in the medium and t... more Specific properties of carbon nanotubes, such as their level of agglomeration in the medium and their surface characteristics, can be critical for the physiological response of plants upon application of carbon nanotubes. The correlations among the level of aggregation, the type of functional group on the surface of the carbon nanotubes, and the growth performance of tomato plants are documented.

Research paper thumbnail of Bioresponse to Nanotubes: Surface Chemistry of Carbon Nanotubes Impacts the Growth and Expression of Water Channel Protein in Tomato Plants (Small 15/2012)

Research paper thumbnail of Complex genetic, photothermal, and photoacoustic analysis of nanoparticle-plant interactions

Proceedings of the National Academy of Sciences, 2010

Understanding the nature of interactions between engineered nanomaterials and plants is crucial i... more Understanding the nature of interactions between engineered nanomaterials and plants is crucial in comprehending the impact of nanotechnology on the environment and agriculture with a focus on toxicity concerns, plant disease treatment, and genetic engineering. To date, little progress has been made in studying nanoparticle-plant interactions at single nanoparticle and genetic levels. Here, we introduce an advanced platform integrating genetic, Raman, photothermal, and photoacoustic methods. Using this approach, we discovered that multiwall carbon nanotubes induce previously unknown changes in gene expression in tomato leaves and roots, particularly, up-regulation of the stress-related genes, including those induced by pathogens and the water-channel LeAqp2 gene. A nano-bubble amplified photothermal/photoacoustic imaging, spectroscopy, and burning technique demonstrated the detection of multiwall carbon nanotubes in roots, leaves, and fruits down to the single nanoparticle and cell ...

Research paper thumbnail of Arabidopsis thaliana calcium-dependent lipid-binding protein (AtCLB): a novel repressor of abiotic stress response

Journal of Experimental Botany, 2011

Ca 2+ is an important second messenger in plant signal transduction pathways regulating stress-in... more Ca 2+ is an important second messenger in plant signal transduction pathways regulating stress-induced gene expression. Functional analysis of plant proteins containing Ca 2+-binding domains (C2 domains) will help us understand the mechanisms behind the role of transcriptional regulators in the Ca 2+ signalling pathway and open new perspectives for crop genetic improvement. We identified a novel transcriptional regulator, a Ca 2+-dependent lipid-binding protein (AtCLB) containing a C2 domain. AtCLB binds specifically to the promoter of the Arabidopsis thalianol synthase gene (AtTHAS1), whose expression is induced by gravity and light. Here we describe the role of the Atclb gene encoding the AtCLB protein. Expression of the Atclb gene was documented in all analysed tissues of Arabidopsis (leaf, root, stem, flower, and silique) by real-time PCR analysis. Immunofluorescence analysis revealed that AtCLB protein is localized in the nucleus of cells in Arabidopsis root tips. We demonstrated that the AtCLB protein was capable of binding to the membrane lipid ceramide. The role of the Atclb gene in negatively regulating responses to abiotic stress in Arabidopsis thaliana was identified. The loss of the Atclb gene function confers an enhanced drought and salt tolerance and a modified gravitropic response in T-DNA insertion knockout mutant lines. Expression of AtTHAS1 in Atclb knockout mutant lines was increased compared with wild type and a 35S-Atclb overexpression line suggesting AtCLB as a transcriptional repressor of AtTHAS1.

Research paper thumbnail of Reduction of inositol (1,4,5)–trisphosphate affects the overall phosphoinositol pathway and leads to modifications in light signalling and secondary metabolism in tomato plants

Journal of Experimental Botany, 2011

The phosphoinositol pathway is one of the major eukaryotic signalling pathways. The metabolite of... more The phosphoinositol pathway is one of the major eukaryotic signalling pathways. The metabolite of the phosphoinositol pathway, inositol-(1,4,5) trisphosphate (InsP 3), is a regulator of plant responses to a wide variety of stresses, including light, drought, cold, and salinity. It was found that the expression of InsP 5-ptase, the enzyme that hydrolyses InsP 3 , also dramatically affects the levels of inositol phosphate metabolites and the secondary metabolites in transgenic tomato plants. Tomato plants expressing InsP 5-ptase exhibited a reduction in the levels of several important inositol phosphates, including InsP 1 , InsP 2 , InsP 3 , and InsP 4. Reduced levels of inositol phosphates accompanied an increase in the accumulation of phenylpropanoids (rutin, chlorogenic acid) and ascorbic acid (vitamin C) in the transgenic fruits of tomato plants. The enhanced accumulation of these metabolites in transgenic tomato plants was in direct correspondence with the observed up-regulation of the genes that express the key enzymes of ascorbic acid metabolism (myo-inositol oxygenase, MIOX; L-galactono-g-lactone dehydrogenase, GLDH) and phenylpropanoid metabolism (chalcone synthase, CHS1; cinnamoyl-CoA shikimate/quinate transferase, HCT). To understand the molecular links between the activation of different branches of plant metabolism and InsP 3 reduction in tomato fruits, the expression of transcription factors known to be involved in light signalling was analysed by real-time RT-PCR. The expression of LeHY5, SIMYB12, and LeELIP was found to be higher in fruits expressing InsP 5-ptase. These results suggest possible interconnections between phosphoinositol metabolism, light signalling, and secondary metabolism in plants. Our study also revealed the biotechnological potential for the genetic improvement of crop plants by the manipulation of the phosphoinositol pathway.

Research paper thumbnail of Carbon Nanotubes Induce Growth Enhancement of Tobacco Cells

ACS Nano, 2012

Carbon nanotubes have shown promise as regulators of seed germination and plant growth. Here, we ... more Carbon nanotubes have shown promise as regulators of seed germination and plant growth. Here, we demonstrate that multiwalled carbon nanotubes (MWCNTs) have the ability to enhance the growth of tobacco cell culture (55À64% increase over control) in a wide range of concentrations (5À500 μg/mL). Activated carbon (AC) stimulated cell growth (16% increase) only at low concentrations (5 μg/mL) while dramatically inhibited the cellular growth at higher concentrations (100À500 μg/mL). We found a correlation between the activation of cells growth exposed to MWCNTs and the upregulation of genes involved in cell division/cell wall formation and water transport. The expression of the tobacco aquaporin (NtPIP1) gene, as well as production of the NtPIP1 protein, significantly increased in cells exposed to MWCNTs compared to control cells or those exposed to AC. The expression of marker genes for cell division (CycB) and cell wall extension (NtLRX1) was also up-regulated in cells exposed to MWCNTs compared to control cells or those exposed to activated carbon only.

Research paper thumbnail of Author response: Murine muscle stem cell response to perturbations of the neuromuscular junction are attenuated with aging

During aging and neuromuscular diseases, there is a progressive loss of skeletal muscle volume an... more During aging and neuromuscular diseases, there is a progressive loss of skeletal muscle volume and function impacting mobility and quality of life. Muscle loss is often associated with denervation and a loss of resident muscle stem cells (satellite cells or MuSCs); however, the relationship between MuSCs and innervation has not been established. Herein, we administered severe neuromuscular trauma to a transgenic murine model that permits MuSC lineage tracing. We show that a subset of MuSCs specifically engraft in a position proximal to the neuromuscular junction (NMJ), the synapse between myofibers and motor neurons, in healthy young adult muscles. In aging and in a mouse model of neuromuscular degeneration (Cu/Zn superoxide dismutase knockout-Sod1-/-), this localized engraftment behavior was reduced. Genetic rescue of motor neurons in Sod1-/mice reestablished integrity of the NMJ in a manner akin to young muscle

Research paper thumbnail of Evaluation of non‑coding region sequence variants and mitochondrial haplogroups as potential biomarkers of sporadic breast cancer in individuals of Sri Lankan Sinhalese ethnicity

Biomedical Reports, 2020

Mitochondrial DNA (mtDNA) mutations have been reported to be associated with various diseases, in... more Mitochondrial DNA (mtDNA) mutations have been reported to be associated with various diseases, including cancer. The present study investigated the mtDNA non-coding region mutations and mitochondrial haplogroups as potential biomarkers of sporadic breast cancer in Sri Lankan Sinhalese women. Mitochondrial macro-haplogroups were determined using PCR-restriction fragment length polymorphism, whereas non-coding region sequences were determined using Sanger sequencing. The sequence of the non-coding region was also used to confirm haplogroup status. Neither the mutations in the non-coding region nor the mitochondrial haplogroups that were reported as risk factors in other populations, were determined to be potential risk factors for sporadic breast cancer in the present study. Furthermore, several novel mutations were identified in the present matched pairs case-controlled study. The M65a haplogroup with an additional mutation at position 16311 (P=0.0771) and mutations at the ori-b site (P=0.05) were considered a weak risk factor and protective factor, respectively, for sporadic breast cancer in Sinhalese women. Previous studies have indicated the use of mtDNA mutations as a biomarker; however, the present study showed that such biomarkers need to be validated for individual ethnic groups before they can be recommended for use in the prediction of disease.

Research paper thumbnail of Pattern of nucleotide variants of TP53 and their correlation with the expression of p53 and its downstream proteins in a Sri Lankan cohort of breast and colorectal cancer patients

Background Breast cancer (BC) is known to be the most common malignancy in females whereas colore... more Background Breast cancer (BC) is known to be the most common malignancy in females whereas colorectal cancer (CRC) incidence also higher in both genders in Sri Lanka. TP53 is an important tumour suppressor gene and its somatic mutations are reported in approximately 27% of BC and 43% of CRC cases. Analysis of TP53 gene variants not only provides clues for the aetiology of the tumour formation, but also has an impact on treatment efficacy. The current study was conducted to investigate the pattern of TP53 variants in patients with BC and CRC from Sri Lanka. Methods 30 patients with BC, 21 patients with CRC and an equal number of healthy controls were screened for mutational status of TP53 by polymerase chain reaction (PCR) followed by direct sequencing. In addition, a subset of these samples were analysed for the protein expression of p53 and comparison made with the mutational status of TP53. We also analysed the protein expression of p21 and MDM2 as potential indicators of p53 func...

Research paper thumbnail of Nucleotide variants and protein expression of TP53 in a Sri Lankan cohort of patients with head and neck cancer

Molecular Medicine Reports, 2019

Head and neck cancer (HNC) is the leading cancer in Sri Lankan males and second most common cance... more Head and neck cancer (HNC) is the leading cancer in Sri Lankan males and second most common cancer among Sri Lankan females. This is the first study, to the best of our knowledge, that has focused on investigating the association between TP53 somatic DNA variants, with p53 protein expression and risk factors in a cohort of Sri Lankan patients with HNC. A total of 44 patients with cancer and 20 healthy controls were studied. In total, 36 genomic DNA sequence variants were found, including several novel variants (two deletions in exons 4 and 6, two in the 3' untranslated region and several intronic variants). A total of 14 tumour samples carried pathogenic TP53 mutations. A random selection of 24 samples was analysed immunohistochemically for p53 protein expression. All the samples with point missense variants were strongly immuno-positive, whereas, samples with nonsense and frameshift TP53 variants were immuno-negative for p53 immunohistochemical staining. Although, the human papilloma virus is a known risk factor for HNC, results from the present study identified an absence or lower level of infection in the Sri Lankan cohort.

Research paper thumbnail of Circulating leptin, soluble leptin receptor, free leptin index, visfatin and selected leptin and leptin receptor gene polymorphisms in sporadic breast cancer

Endocrine journal, Jan 11, 2017

Leptin and visfatin are implicated in breast cancer risk but studies accounting for bioavailabili... more Leptin and visfatin are implicated in breast cancer risk but studies accounting for bioavailability of leptin are sparse. Reports on the association of leptin gene (LEP) and leptin receptor gene (LEPR) polymorphisms with breast cancer are also inconsistent. Only a very few studies have examined biochemical and genetic variables concomitantly in the same cohort. A matched pairs study was carried out to ascertain whether plasma leptin, soluble leptin receptor, free leptin index (leptin/soluble leptin receptor), serum visfatin and selected LEP and LEPR polymorphisms are risk factors for sporadic breast cancer. Newly diagnosed sporadic breast cancer patients (N=80) were matched for age, body mass index (BMI) and menopausal status with healthy controls. Plasma leptin, soluble leptin receptor and serum visfatin were measured by enzyme-immunoassay. LEP -2548 A/G and LEPR K109R, LEPR Q223R polymorphisms were determined by genotyping. Leptin (p=0.0234), leptin/BMI (p=0.0468), free leptin ind...

Research paper thumbnail of Novel and reported pathogenic variants in exon 11 of BRCA2 gene in a cohort of Sri Lankan young breast cancer patients

Familial Cancer, 2016

Women with breast carcinoma diagnosed before 40 years of age with a strong familial risk have a g... more Women with breast carcinoma diagnosed before 40 years of age with a strong familial risk have a greater prevalence of germline BRCA1 or BRCA2 variants than late onset breast cancer. Previously germline variants in BRCA1 and BRCA2 genes were characterized in a cohort of Sri Lankan breast cancer patients unselected for age of onset. This study focused on young breast cancer patients who were screened for previously identified hotspot regions in BRCA2 gene. A total of 48 young breast cancer patients with family history of cancer and 25 healthy controls were studied. Direct sequencing was used to detect pathogenic and other sequence variants in the hotspot regions of BRCA2 gene. Thirty-six sequence variants including seven pathogenic (c.2411_2412delAA/p.Glu804Valfs*2, c.2500_2501insG/p.Leu834Cysfs*4, c.3881T>G/p.Leu1294*, c.4768A>T/p.Lys1590*, c.5645C>G/p.Ser1882*, c.5747delC/p.His1916Phefs*3, c.6728C>T/p.Ser2243Phe) and two likely pathogenic (c.1922C>T and c.3378A>T) variants, two intronic variants of unknown significance (c.1910-74T>C, c.1910-51G>T), two variants of uncertain significance (c.2324C>T c.5104C>T) and 23 benign variants were detected. Among them, seven were novel (pathogenic 5 and likely pathogenic 2). Prevalence of pathogenic and likely pathogenic variants in the hotspots regions of BRCA2 was 23 and 6.3 % respectively in this cohort. This justifies BRCA2 variant testing in young breast cancer patients with family history of cancer in Sri Lanka.

Research paper thumbnail of Murine muscle stem cell response to perturbations of the neuromuscular junction are attenuated with aging

eLife, 2021

During aging and neuromuscular diseases, there is a progressive loss of skeletal muscle volume an... more During aging and neuromuscular diseases, there is a progressive loss of skeletal muscle volume and function impacting mobility and quality of life. Muscle loss is often associated with denervation and a loss of resident muscle stem cells (satellite cells or MuSCs); however, the relationship between MuSCs and innervation has not been established. Herein, we administered severe neuromuscular trauma to a transgenic murine model that permits MuSC lineage tracing. We show that a subset of MuSCs specifically engraft in a position proximal to the neuromuscular junction (NMJ), the synapse between myofibers and motor neurons, in healthy young adult muscles. In aging and in a mouse model of neuromuscular degeneration (Cu/Zn superoxide dismutase knockout – Sod1-/-), this localized engraftment behavior was reduced. Genetic rescue of motor neurons in Sod1-/- mice reestablished integrity of the NMJ in a manner akin to young muscle and partially restored MuSC ability to engraft into positions prox...

Research paper thumbnail of Surface Chemistry of Carbon Nanotubes Impacts the Growth and Expression of Water Channel Protein in Tomato Plants

Small, 2012

Specific properties of carbon nanotubes, such as their level of agglomeration in the medium and t... more Specific properties of carbon nanotubes, such as their level of agglomeration in the medium and their surface characteristics, can be critical for the physiological response of plants upon application of carbon nanotubes. The correlations among the level of aggregation, the type of functional group on the surface of the carbon nanotubes, and the growth performance of tomato plants are documented.

Research paper thumbnail of Bioresponse to Nanotubes: Surface Chemistry of Carbon Nanotubes Impacts the Growth and Expression of Water Channel Protein in Tomato Plants (Small 15/2012)

Research paper thumbnail of Complex genetic, photothermal, and photoacoustic analysis of nanoparticle-plant interactions

Proceedings of the National Academy of Sciences, 2010

Understanding the nature of interactions between engineered nanomaterials and plants is crucial i... more Understanding the nature of interactions between engineered nanomaterials and plants is crucial in comprehending the impact of nanotechnology on the environment and agriculture with a focus on toxicity concerns, plant disease treatment, and genetic engineering. To date, little progress has been made in studying nanoparticle-plant interactions at single nanoparticle and genetic levels. Here, we introduce an advanced platform integrating genetic, Raman, photothermal, and photoacoustic methods. Using this approach, we discovered that multiwall carbon nanotubes induce previously unknown changes in gene expression in tomato leaves and roots, particularly, up-regulation of the stress-related genes, including those induced by pathogens and the water-channel LeAqp2 gene. A nano-bubble amplified photothermal/photoacoustic imaging, spectroscopy, and burning technique demonstrated the detection of multiwall carbon nanotubes in roots, leaves, and fruits down to the single nanoparticle and cell ...

Research paper thumbnail of Arabidopsis thaliana calcium-dependent lipid-binding protein (AtCLB): a novel repressor of abiotic stress response

Journal of Experimental Botany, 2011

Ca 2+ is an important second messenger in plant signal transduction pathways regulating stress-in... more Ca 2+ is an important second messenger in plant signal transduction pathways regulating stress-induced gene expression. Functional analysis of plant proteins containing Ca 2+-binding domains (C2 domains) will help us understand the mechanisms behind the role of transcriptional regulators in the Ca 2+ signalling pathway and open new perspectives for crop genetic improvement. We identified a novel transcriptional regulator, a Ca 2+-dependent lipid-binding protein (AtCLB) containing a C2 domain. AtCLB binds specifically to the promoter of the Arabidopsis thalianol synthase gene (AtTHAS1), whose expression is induced by gravity and light. Here we describe the role of the Atclb gene encoding the AtCLB protein. Expression of the Atclb gene was documented in all analysed tissues of Arabidopsis (leaf, root, stem, flower, and silique) by real-time PCR analysis. Immunofluorescence analysis revealed that AtCLB protein is localized in the nucleus of cells in Arabidopsis root tips. We demonstrated that the AtCLB protein was capable of binding to the membrane lipid ceramide. The role of the Atclb gene in negatively regulating responses to abiotic stress in Arabidopsis thaliana was identified. The loss of the Atclb gene function confers an enhanced drought and salt tolerance and a modified gravitropic response in T-DNA insertion knockout mutant lines. Expression of AtTHAS1 in Atclb knockout mutant lines was increased compared with wild type and a 35S-Atclb overexpression line suggesting AtCLB as a transcriptional repressor of AtTHAS1.

Research paper thumbnail of Reduction of inositol (1,4,5)–trisphosphate affects the overall phosphoinositol pathway and leads to modifications in light signalling and secondary metabolism in tomato plants

Journal of Experimental Botany, 2011

The phosphoinositol pathway is one of the major eukaryotic signalling pathways. The metabolite of... more The phosphoinositol pathway is one of the major eukaryotic signalling pathways. The metabolite of the phosphoinositol pathway, inositol-(1,4,5) trisphosphate (InsP 3), is a regulator of plant responses to a wide variety of stresses, including light, drought, cold, and salinity. It was found that the expression of InsP 5-ptase, the enzyme that hydrolyses InsP 3 , also dramatically affects the levels of inositol phosphate metabolites and the secondary metabolites in transgenic tomato plants. Tomato plants expressing InsP 5-ptase exhibited a reduction in the levels of several important inositol phosphates, including InsP 1 , InsP 2 , InsP 3 , and InsP 4. Reduced levels of inositol phosphates accompanied an increase in the accumulation of phenylpropanoids (rutin, chlorogenic acid) and ascorbic acid (vitamin C) in the transgenic fruits of tomato plants. The enhanced accumulation of these metabolites in transgenic tomato plants was in direct correspondence with the observed up-regulation of the genes that express the key enzymes of ascorbic acid metabolism (myo-inositol oxygenase, MIOX; L-galactono-g-lactone dehydrogenase, GLDH) and phenylpropanoid metabolism (chalcone synthase, CHS1; cinnamoyl-CoA shikimate/quinate transferase, HCT). To understand the molecular links between the activation of different branches of plant metabolism and InsP 3 reduction in tomato fruits, the expression of transcription factors known to be involved in light signalling was analysed by real-time RT-PCR. The expression of LeHY5, SIMYB12, and LeELIP was found to be higher in fruits expressing InsP 5-ptase. These results suggest possible interconnections between phosphoinositol metabolism, light signalling, and secondary metabolism in plants. Our study also revealed the biotechnological potential for the genetic improvement of crop plants by the manipulation of the phosphoinositol pathway.

Research paper thumbnail of Carbon Nanotubes Induce Growth Enhancement of Tobacco Cells

ACS Nano, 2012

Carbon nanotubes have shown promise as regulators of seed germination and plant growth. Here, we ... more Carbon nanotubes have shown promise as regulators of seed germination and plant growth. Here, we demonstrate that multiwalled carbon nanotubes (MWCNTs) have the ability to enhance the growth of tobacco cell culture (55À64% increase over control) in a wide range of concentrations (5À500 μg/mL). Activated carbon (AC) stimulated cell growth (16% increase) only at low concentrations (5 μg/mL) while dramatically inhibited the cellular growth at higher concentrations (100À500 μg/mL). We found a correlation between the activation of cells growth exposed to MWCNTs and the upregulation of genes involved in cell division/cell wall formation and water transport. The expression of the tobacco aquaporin (NtPIP1) gene, as well as production of the NtPIP1 protein, significantly increased in cells exposed to MWCNTs compared to control cells or those exposed to AC. The expression of marker genes for cell division (CycB) and cell wall extension (NtLRX1) was also up-regulated in cells exposed to MWCNTs compared to control cells or those exposed to activated carbon only.

Research paper thumbnail of Author response: Murine muscle stem cell response to perturbations of the neuromuscular junction are attenuated with aging

During aging and neuromuscular diseases, there is a progressive loss of skeletal muscle volume an... more During aging and neuromuscular diseases, there is a progressive loss of skeletal muscle volume and function impacting mobility and quality of life. Muscle loss is often associated with denervation and a loss of resident muscle stem cells (satellite cells or MuSCs); however, the relationship between MuSCs and innervation has not been established. Herein, we administered severe neuromuscular trauma to a transgenic murine model that permits MuSC lineage tracing. We show that a subset of MuSCs specifically engraft in a position proximal to the neuromuscular junction (NMJ), the synapse between myofibers and motor neurons, in healthy young adult muscles. In aging and in a mouse model of neuromuscular degeneration (Cu/Zn superoxide dismutase knockout-Sod1-/-), this localized engraftment behavior was reduced. Genetic rescue of motor neurons in Sod1-/mice reestablished integrity of the NMJ in a manner akin to young muscle