Dimanthi Uduwela | University Of Peradeniya,Sri Lanka (original) (raw)

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Papers by Dimanthi Uduwela

Ceylon Journal of Science, 2020

Dendrophthoe falcata (L.f.) Ettingsh is a hemiparasitic plant, used in Sri Lankan and Indian indi... more Dendrophthoe falcata (L.f.) Ettingsh is a hemiparasitic plant, used in Sri Lankan and Indian indigenous medicine to treat various diseases such as asthma, cancerous tumors, diabetes and wounds. Despite the hemiparasite on the host Limonia acidissima is used to treat various diseases, any bioactivity study of Dendrophthoe falcata grown on the particular host has not been conducted yet. This study aimed to investigate several bioactivities of the hemiparasite grown on Limonia acidissima (La) and compare with that grown on Mangifera indica (Mi). Sequential extracts by Soxhlet method from hexane (La-HE and Mi-HE), ethyl acetate (La-EAE and Mi-EAE) and methanol (La-ME and MiME) were investigated for bioactivities. Antioxidant activity was determined using DPPH radical scavenging assay in which MiME and Mi-EAE showed the highest activity, that is five times more than that of α-tocopherol. Brine shrimp lethality assay was conducted as a preliminary toxicity assay, where La-EAE showed the highest activity that is approximately four times that of K 2 Cr 2 O 7. The total polyphenolic content was determined by Folin-Ciocalteu method in which both methanol extracts showed the highest polyphenolic content of which MiME was approximately four times more than that of LaME. These results suggest that the bioactivity of the hemiparasite vary considerably with the host. Further, extracts of the hemiparasite on Limonia acidissima showing high antioxidant activity coupled with high toxicity justifies its applicability in indigenous medicine.

Representative structures from RMSD-clustering of MD trajectories

PDB files contain representative structures of RMSD-based clusters obtained from the simulation o... more PDB files contain representative structures of RMSD-based clusters obtained from the simulation of each variant using the combined last 50ns of all three replicas of the simulation perfomed for each variant. The clustering was performed on all heavy atoms of the side chains of the following residues: 13, 14, 50, 51, 72, 73, 74, 75, 113, 115, 138, 139, 155, 156, 157, 158, 159, 160, 211, 212, 317, 318, 321, 325, 330, 331, 375, as well as heavy atoms of the substrate. In each case the structures in each trajectory were aligned using backbone heavy atoms of the entire protein prior to clustering. Clustering was performed with cpptraj (Amber Tools) using average linkage algorithm. Summary of the clustering for each system can be found in *summary.out files, which contain the list of all clusters determined through the clustering analysis, with number of frames in each cluster, total fraction of the simulation these frames consittute, average distance between points in the cluster (AvgDist), standard deviation of points in the cluster (Stdev), centroid frame (Centroid) and average distance of a given cluster to every other cluster (AvgCDist). PDB files containing the representative structures of each cluster are labelled with the number corresponding to the number of a given cluster as listed in *summary.out file

Frontiers in Molecular Biosciences

The study of urinary phase II sulfate metabolites is central to understanding the role and fate o... more The study of urinary phase II sulfate metabolites is central to understanding the role and fate of endogenous and exogenous compounds in biological systems. This study describes a new workflow for the untargeted metabolic profiling of sulfated metabolites in a urine matrix. Analysis was performed using ultra-high-performance liquid chromatography-high resolution tandem mass spectrometry (UHPLC-HRMS/MS) with data dependent acquisition (DDA) coupled to an automated script-based data processing pipeline and differential metabolite level analysis. Sulfates were identified through k-means clustering analysis of sulfate ester derived MS/MS fragmentation intensities. The utility of the method was highlighted in two applications. Firstly, the urinary metabolome of a thoroughbred horse was examined before and after administration of the anabolic androgenic steroid (AAS) testosterone propionate. The analysis detected elevated levels of ten sulfated steroid metabolites, three of which were ide...

Data from: Enhancing the steroid sulfatase activity of the arylsulfatase from Pseudomonas aeruginosa

Steroidal sulfate esters play a central role in many physiological processes. They serve as the r... more Steroidal sulfate esters play a central role in many physiological processes. They serve as the reservoir for endogenous sex hormones and form a significant fraction of the steroid metabolite pool. The analysis of steroid sulfates is thus essential in fields such as medical science and sports drug testing. Although the direct detection of steroid sulfates can be readily achieved using liquid chromatography-mass spectrometry, many analytical approaches, including gas chromatography-mass spectrometry, are hampered due to the lack of suitable enzymatic or chemical methods for sulfate ester hydrolysis prior to analysis. Enhanced methods of steroid sulfate hydrolysis would expand analytical possibilities for the study of these widely occurring metabolites. The arylsulfatase from Pseudomonas aeruginosa (PaS) is a purified enzyme capable of hydrolysing steroid sulfates. However, this enzyme requires improvement to hydrolytic activity and substrate scope in order to be useful in analytical applications. These improvements were sought by applying semi-rational design to mutate amino acid residues neighbouring the enzyme active site. Mutagenesis was implemented on both single and multiple residue sites. Screening by UPLC-MS was performed to test the steroid sulfate hydrolysis activity of these mutant libraries against testosterone sulfate. This approach revealed the steroid sulfate binding pocket and resulted in three mutants that showed an improvement in catalytic efficiency (Vmax/KM) of more than 150 times that of wild-type PaS. The substrate scope of PaS was expanded and a modest increase in thermostability was observed. Finally, molecular dynamics simulations of enzyme-substrate complexes were used to provide qualitative insight into the structural origin of the observed effects

Impact of environmental regions in Sri Lanka on the bioactivity of Dendrophthoe falcata grown on the host Limonia acidissima

Ceylon Journal of Science, 2021

Dendrophthoe falcata (L.f.) Ettingsh (Loranthaceae family) is a hemiparasite which has many medic... more Dendrophthoe falcata (L.f.) Ettingsh (Loranthaceae family) is a hemiparasite which has many medicinal applications and grows on a variety of hosts. Bioactivity of the hemiparasite shows a great host dependence, where it contains high antioxidant activity coupled with high toxicity when grown on the host Limonia acidissima. This study aimed to investigate the impact of environmental conditions on the bioactivity of the hemiparasite grown on L. acidissima (Rutaceae family), as the environmental stress may play a determining role in the production of secondary metabolites in a plant. The hemiparasite grown in Hambantota (Hamb – dry zone), Kurunegala (Kuru – intermediate zone) and Ambalantota (Amba – dry zone), were selected for this study. Sequential extracts by Soxhlet method from hexane (HE), ethyl acetate (EAE) and methanol (ME) were compared for antioxidant activity, polyphenolic content, toxicity and alkaloid content determined by DPPH, Folin-Ciocalteu, brine shrimp lethality and acid-base assays respectively. Antioxidant activity was approximately 40% higher for Kuru-ME compared to Hamb-ME and Amba-ME. A significant 180 - 200% higher polyphenolic content was observed for Hamb-ME compared to the Amba-ME and Kuru-ME. Toxicity studies revealed that Hamb-EAE is 43% and 29% more toxic than Kuru-EAE and Amba-EAE. The alkaloid content of Hamb-ME showed the highest percentage with a less significant difference between the extracts of other two locations. On average, among the three locations, extracts of Kurunegala, Ambalantota and Hambantota showed the least, intermediate and highest bioactivities respectively experiencing the least, intermediate and most environmental stressed conditions. Hence, it can be concluded that the bioactivity is influenced by the environmental stress due to the impact of governing the secondary metabolites produced by Dendrophthoe falcata.

ACS Catalysis

Steroidal sulfate esters play a central role in many physiological processes. They serve as the r... more Steroidal sulfate esters play a central role in many physiological processes. They serve as the reservoir for endogenous sex hormones and form a significant fraction of the steroid metabolite pool. The analysis of steroid sulfates is thus essential in fields such as medical science and sports drug testing. Although the direct detection of steroid sulfates can be readily achieved using liquid chromatographymass spectrometry, many analytical approaches, including gas chromatography-mass spectrometry, are hampered due to the lack of suitable enzymatic or chemical methods for sulfate ester hydrolysis prior to analysis. Enhanced methods of steroid sulfate hydrolysis would expand analytical possibilities for the study of these widely occurring metabolites. The arylsulfatase from Pseudomonas aeruginosa (PaS) is a purified enzyme capable of hydrolysing steroid sulfates. However, this enzyme requires improvement to hydrolytic activity and substrate scope in order to be useful in analytical applications. These improvements were sought by applying semi-rational design to mutate amino acid residues neighbouring the enzyme active site. Mutagenesis was implemented on both single and multiple residue sites. Screening by UPLC-MS was performed to test the steroid sulfate hydrolysis activity of these mutant libraries against testosterone sulfate. This approach revealed the steroid sulfate binding pocket and resulted in three mutants that showed an improvement in catalytic efficiency (Vmax/KM) of more than 150 times that of wild-type PaS. The substrate scope of PaS was expanded and a modest increase in thermostability was observed. Finally, molecular dynamics simulations of enzymesubstrate complexes were used to provide qualitative insight into the structural origin of the observed effects.

Drug Testing and Analysis

In the course of investigations into the metabolism of testosterone (T) by means of deuterated T ... more In the course of investigations into the metabolism of testosterone (T) by means of deuterated T and hydrogen isotope ratio mass spectrometry, a pronounced influence of the oral administration of T on sulfoconjugated steroid metabolites was observed. Especially in case of epiandrosterone sulfate (EPIA_S), the contribution of exogenous T to the urinary metabolite was traceable up to 8 days after a single oral dose of 40 mg of T. These findings initiated follow-up studies on the capability of EPIA_S to extend the detection of T and T analogue misuse by carbon isotope ratio (CIR) mass spectrometry in sports drug testing. Excretion study urine samples obtained after transdermal application of T and after oral administration of 4-androstenedione, dihydrotestosterone, and EPIA were investigated regarding urinary concentrations and CIR. With each administered steroid, EPIA_S was significantly depleted and prolonged the detectability when compared to routinely used steroidal target compounds by a factor of 2 to 5. In order to simplify the sample preparation procedure for sulfoconjugated compounds, enzymatic cleavage by Pseudomonas aeruginosa arylsulfatase was tested and implemented into CIR measurements for the first time. Further simplification was achieved by employing multidimensional gas chromatography to ensure the required peak purity for CIR determinations, instead of sample purification strategies using liquid chromatographic fractionation. Taking into account these results that demonstrate the unique and broad applicability of EPIA_S for the detection of illicit administrations of T or T-related steroids, careful consideration of how this steroid can be implemented into routine doping control analysis appears warranted.

Ceylon Journal of Science, 2020

Dendrophthoe falcata (L.f.) Ettingsh is a hemiparasitic plant, used in Sri Lankan and Indian indi... more Dendrophthoe falcata (L.f.) Ettingsh is a hemiparasitic plant, used in Sri Lankan and Indian indigenous medicine to treat various diseases such as asthma, cancerous tumors, diabetes and wounds. Despite the hemiparasite on the host Limonia acidissima is used to treat various diseases, any bioactivity study of Dendrophthoe falcata grown on the particular host has not been conducted yet. This study aimed to investigate several bioactivities of the hemiparasite grown on Limonia acidissima (La) and compare with that grown on Mangifera indica (Mi). Sequential extracts by Soxhlet method from hexane (La-HE and Mi-HE), ethyl acetate (La-EAE and Mi-EAE) and methanol (La-ME and MiME) were investigated for bioactivities. Antioxidant activity was determined using DPPH radical scavenging assay in which MiME and Mi-EAE showed the highest activity, that is five times more than that of α-tocopherol. Brine shrimp lethality assay was conducted as a preliminary toxicity assay, where La-EAE showed the highest activity that is approximately four times that of K 2 Cr 2 O 7. The total polyphenolic content was determined by Folin-Ciocalteu method in which both methanol extracts showed the highest polyphenolic content of which MiME was approximately four times more than that of LaME. These results suggest that the bioactivity of the hemiparasite vary considerably with the host. Further, extracts of the hemiparasite on Limonia acidissima showing high antioxidant activity coupled with high toxicity justifies its applicability in indigenous medicine.

Representative structures from RMSD-clustering of MD trajectories

PDB files contain representative structures of RMSD-based clusters obtained from the simulation o... more PDB files contain representative structures of RMSD-based clusters obtained from the simulation of each variant using the combined last 50ns of all three replicas of the simulation perfomed for each variant. The clustering was performed on all heavy atoms of the side chains of the following residues: 13, 14, 50, 51, 72, 73, 74, 75, 113, 115, 138, 139, 155, 156, 157, 158, 159, 160, 211, 212, 317, 318, 321, 325, 330, 331, 375, as well as heavy atoms of the substrate. In each case the structures in each trajectory were aligned using backbone heavy atoms of the entire protein prior to clustering. Clustering was performed with cpptraj (Amber Tools) using average linkage algorithm. Summary of the clustering for each system can be found in *summary.out files, which contain the list of all clusters determined through the clustering analysis, with number of frames in each cluster, total fraction of the simulation these frames consittute, average distance between points in the cluster (AvgDist), standard deviation of points in the cluster (Stdev), centroid frame (Centroid) and average distance of a given cluster to every other cluster (AvgCDist). PDB files containing the representative structures of each cluster are labelled with the number corresponding to the number of a given cluster as listed in *summary.out file

Frontiers in Molecular Biosciences

The study of urinary phase II sulfate metabolites is central to understanding the role and fate o... more The study of urinary phase II sulfate metabolites is central to understanding the role and fate of endogenous and exogenous compounds in biological systems. This study describes a new workflow for the untargeted metabolic profiling of sulfated metabolites in a urine matrix. Analysis was performed using ultra-high-performance liquid chromatography-high resolution tandem mass spectrometry (UHPLC-HRMS/MS) with data dependent acquisition (DDA) coupled to an automated script-based data processing pipeline and differential metabolite level analysis. Sulfates were identified through k-means clustering analysis of sulfate ester derived MS/MS fragmentation intensities. The utility of the method was highlighted in two applications. Firstly, the urinary metabolome of a thoroughbred horse was examined before and after administration of the anabolic androgenic steroid (AAS) testosterone propionate. The analysis detected elevated levels of ten sulfated steroid metabolites, three of which were ide...

Data from: Enhancing the steroid sulfatase activity of the arylsulfatase from Pseudomonas aeruginosa

Steroidal sulfate esters play a central role in many physiological processes. They serve as the r... more Steroidal sulfate esters play a central role in many physiological processes. They serve as the reservoir for endogenous sex hormones and form a significant fraction of the steroid metabolite pool. The analysis of steroid sulfates is thus essential in fields such as medical science and sports drug testing. Although the direct detection of steroid sulfates can be readily achieved using liquid chromatography-mass spectrometry, many analytical approaches, including gas chromatography-mass spectrometry, are hampered due to the lack of suitable enzymatic or chemical methods for sulfate ester hydrolysis prior to analysis. Enhanced methods of steroid sulfate hydrolysis would expand analytical possibilities for the study of these widely occurring metabolites. The arylsulfatase from Pseudomonas aeruginosa (PaS) is a purified enzyme capable of hydrolysing steroid sulfates. However, this enzyme requires improvement to hydrolytic activity and substrate scope in order to be useful in analytical applications. These improvements were sought by applying semi-rational design to mutate amino acid residues neighbouring the enzyme active site. Mutagenesis was implemented on both single and multiple residue sites. Screening by UPLC-MS was performed to test the steroid sulfate hydrolysis activity of these mutant libraries against testosterone sulfate. This approach revealed the steroid sulfate binding pocket and resulted in three mutants that showed an improvement in catalytic efficiency (Vmax/KM) of more than 150 times that of wild-type PaS. The substrate scope of PaS was expanded and a modest increase in thermostability was observed. Finally, molecular dynamics simulations of enzyme-substrate complexes were used to provide qualitative insight into the structural origin of the observed effects

Impact of environmental regions in Sri Lanka on the bioactivity of Dendrophthoe falcata grown on the host Limonia acidissima

Ceylon Journal of Science, 2021

Dendrophthoe falcata (L.f.) Ettingsh (Loranthaceae family) is a hemiparasite which has many medic... more Dendrophthoe falcata (L.f.) Ettingsh (Loranthaceae family) is a hemiparasite which has many medicinal applications and grows on a variety of hosts. Bioactivity of the hemiparasite shows a great host dependence, where it contains high antioxidant activity coupled with high toxicity when grown on the host Limonia acidissima. This study aimed to investigate the impact of environmental conditions on the bioactivity of the hemiparasite grown on L. acidissima (Rutaceae family), as the environmental stress may play a determining role in the production of secondary metabolites in a plant. The hemiparasite grown in Hambantota (Hamb – dry zone), Kurunegala (Kuru – intermediate zone) and Ambalantota (Amba – dry zone), were selected for this study. Sequential extracts by Soxhlet method from hexane (HE), ethyl acetate (EAE) and methanol (ME) were compared for antioxidant activity, polyphenolic content, toxicity and alkaloid content determined by DPPH, Folin-Ciocalteu, brine shrimp lethality and acid-base assays respectively. Antioxidant activity was approximately 40% higher for Kuru-ME compared to Hamb-ME and Amba-ME. A significant 180 - 200% higher polyphenolic content was observed for Hamb-ME compared to the Amba-ME and Kuru-ME. Toxicity studies revealed that Hamb-EAE is 43% and 29% more toxic than Kuru-EAE and Amba-EAE. The alkaloid content of Hamb-ME showed the highest percentage with a less significant difference between the extracts of other two locations. On average, among the three locations, extracts of Kurunegala, Ambalantota and Hambantota showed the least, intermediate and highest bioactivities respectively experiencing the least, intermediate and most environmental stressed conditions. Hence, it can be concluded that the bioactivity is influenced by the environmental stress due to the impact of governing the secondary metabolites produced by Dendrophthoe falcata.

ACS Catalysis

Steroidal sulfate esters play a central role in many physiological processes. They serve as the r... more Steroidal sulfate esters play a central role in many physiological processes. They serve as the reservoir for endogenous sex hormones and form a significant fraction of the steroid metabolite pool. The analysis of steroid sulfates is thus essential in fields such as medical science and sports drug testing. Although the direct detection of steroid sulfates can be readily achieved using liquid chromatographymass spectrometry, many analytical approaches, including gas chromatography-mass spectrometry, are hampered due to the lack of suitable enzymatic or chemical methods for sulfate ester hydrolysis prior to analysis. Enhanced methods of steroid sulfate hydrolysis would expand analytical possibilities for the study of these widely occurring metabolites. The arylsulfatase from Pseudomonas aeruginosa (PaS) is a purified enzyme capable of hydrolysing steroid sulfates. However, this enzyme requires improvement to hydrolytic activity and substrate scope in order to be useful in analytical applications. These improvements were sought by applying semi-rational design to mutate amino acid residues neighbouring the enzyme active site. Mutagenesis was implemented on both single and multiple residue sites. Screening by UPLC-MS was performed to test the steroid sulfate hydrolysis activity of these mutant libraries against testosterone sulfate. This approach revealed the steroid sulfate binding pocket and resulted in three mutants that showed an improvement in catalytic efficiency (Vmax/KM) of more than 150 times that of wild-type PaS. The substrate scope of PaS was expanded and a modest increase in thermostability was observed. Finally, molecular dynamics simulations of enzymesubstrate complexes were used to provide qualitative insight into the structural origin of the observed effects.

Drug Testing and Analysis

In the course of investigations into the metabolism of testosterone (T) by means of deuterated T ... more In the course of investigations into the metabolism of testosterone (T) by means of deuterated T and hydrogen isotope ratio mass spectrometry, a pronounced influence of the oral administration of T on sulfoconjugated steroid metabolites was observed. Especially in case of epiandrosterone sulfate (EPIA_S), the contribution of exogenous T to the urinary metabolite was traceable up to 8 days after a single oral dose of 40 mg of T. These findings initiated follow-up studies on the capability of EPIA_S to extend the detection of T and T analogue misuse by carbon isotope ratio (CIR) mass spectrometry in sports drug testing. Excretion study urine samples obtained after transdermal application of T and after oral administration of 4-androstenedione, dihydrotestosterone, and EPIA were investigated regarding urinary concentrations and CIR. With each administered steroid, EPIA_S was significantly depleted and prolonged the detectability when compared to routinely used steroidal target compounds by a factor of 2 to 5. In order to simplify the sample preparation procedure for sulfoconjugated compounds, enzymatic cleavage by Pseudomonas aeruginosa arylsulfatase was tested and implemented into CIR measurements for the first time. Further simplification was achieved by employing multidimensional gas chromatography to ensure the required peak purity for CIR determinations, instead of sample purification strategies using liquid chromatographic fractionation. Taking into account these results that demonstrate the unique and broad applicability of EPIA_S for the detection of illicit administrations of T or T-related steroids, careful consideration of how this steroid can be implemented into routine doping control analysis appears warranted.