185 research outputs found
Author and Owner Intersection in Sound Recordings in The Copyright Act of India
No full text245-250The present work focuses on the intersection of author and owner concerning sound recordings. The interpretation of copyright law on the author and owner intersection by the Court's are rather varied. It may be because the restricted issues at its hand lead the courts. More particularly, interpretation of provisos (b) and (c) of Section 17 of The Copyright Act, 1957 leads to differing interpretations by the Courts. The present analysis is made by studying three recent judgments to understand the author and owner conflicts of sound recordings
Treatment of Denture Stomatitis Using Modified Tissue Conditioners: A Systematic Review
No full textThe most common sequela of wearing removable dentures is denture stomatitis. This review article uses a Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) format to collect data regarding articles that report on the treatment of denture stomatitis using tissue conditioners modified with antifungal drugs, inorganic compound, and phytomedicines. Their advantages, disadvantages, and mechanism of action are discussed
Regulation of the divalent metal ion transporter via membrane budding
No full textData source: Supplementary information, https://doi.org/10.1038/celldisc.2016.11The release of extracellular vesicles (EVs) is important for both normal physiology and disease. However, a basic understanding of the targeting of EV cargoes, composition and mechanism of release is lacking. Here we present evidence that the divalent metal ion transporter (DMT1) is unexpectedly regulated through release in EVs. This process involves the Nedd4-2 ubiquitin ligase, and the adaptor proteins Arrdc1 and Arrdc4 via different budding mechanisms. We show that mouse gut explants release endogenous DMT1 in EVs. Although we observed no change in the relative amount of DMT1 released in EVs from gut explants in Arrdc1 or Arrdc4 deficient mice, the extent of EVs released was significantly reduced indicating an adaptor role in biogenesis. Furthermore, using Arrdc1 or Arrdc4 knockout mouse embryonic fibroblasts, we show that both Arrdc1 and Arrdc4 are non-redundant positive regulators of EV release. Our results suggest that DMT1 release from the plasma membrane into EVs may represent a novel mechanism for the maintenance of iron homeostasis, which may also be important for the regulation of other membrane proteins.Kimberly D Mackenzie, Natalie J Foot, Sushma Anand, Hazel E Dalton, Natasha Chaudhary, Brett M Collins, Suresh Mathivanan and Sharad Kuma
Structural and Functional Studies on Salmonella typhimurium Propionate Kinase and Photorhabdus luminescens Oxalate Decarboxylase
No full textAcetate and propionate are low molecular mass carbon compounds found abundantly in the soil. Although these compounds have been extensively used as food preservatives because of their ability to inhibit microbial growth, surprisingly, bacteria such as Escherichia coli and Salmonella typhimurium are able to grow on propionate as their sole carbon and energy source. Only in the presence of glucose, acetate and other short chain fatty acids inhibit microbial growth.
Propionate is produced during the β-oxidation of odd-numbered carbon-chain fatty acids, fermentation of carbohydrates, oxidative degradation of branched-chain amino acids such as valine and isoleucine. They are also produced during the catabolism of threonine, methionine, thymine and cholesterol. In Escherichia coli and Salmonella typhimurium, enzymes involved in the degradation of L-serine and L-threonine to acetate and propionate, respectively, are encoded by the anaerobically regulated tdc operon. L-threonine is anaerobically degraded to propionate in four consecutive reaction steps catalyzed by biodegradative threonine deaminase (TdcB), 2-ketobutyrate formate lyase (TdcE), phosphotransacetylase (Pta) and propionate kinase (TdcD). Detailed studies on the structure and function of two of these enzymes have earlier been carried out in our laboratory. However, these studies did not reveal the precise substrate binding site in Salmonella typhimurium TdcD (StTdcD). It was also not possible to provide a satisfactory explanation of the structural basis of substrate specificity. The present studies were therefore aimed at locating the substrate binding site, elucidating the structural basis of substrate specificity and mechanism of catalysis of StTdcD.
Oxalic acid is toxic to almost all organisms and its excessive occurrence leads to a variety of pathological conditions. In humans and other vertebrates, secretion of oxalic acid leads to formation of low soluble calcium oxalate, which precipitates as kidney stones. Formation of kidney stones is aggravated by lack of enzymes that catabolize oxalate. Oxalate oxidase, oxalate decarboxylase and oxalyl-CoA decarboxylase constitute three distinct categories of oxalate degrading enzymes. Photorhabdus luminescens is a Gram-negative, symbiotic bacterium associated with the entomopathogenic nematodes of the family Heterorhabditidae. Novel insecticidal genes from these symbiotic bacteria are now being examined for their potential in generating pest resistant transgenic plants. As part of this project, the three-dimensional X-ray crystal structure of an oxalate oxidase (OXDC) enzyme from Photorhabdus luminescens (PlOXDC) was determined.
The introductory chapter (Chapter I) of the thesis presents the earlier investigations carried out in the laboratory on the structure and function of StTdcD. It also provides a summary of the earlier literature pertaining to propionate metabolism in S. typhimurium. The crystal structure of StTdcD in the apo form as well as in complex with ADP and the non-hydrolysable nucleotide analog AMPPNP were determined by earlier Dr. Simanshu ( Simanshu et al., 2005 , 2008 ). Subsequently, Dr Chittori determined the structures of the enzyme in complex with various other nucleotides ( Chittori et al., 2013 ). These studies along with enzyme assays performed by Chittori revealed that StTdcD possesses broad specificity and it could be activated by various nucleotides and metal ions and catalyzes phosphorylation of both propionate and acetate ( Chittori et al., 2013 ). In spite of these extensive studies, the precise mode of binding of the substrate propionate to StTdcD could not be elucidated. The chapter also presents a summary of the literature on oxalate, its toxic effects and enzymes that degrade oxalate. The importance of structural and functional studies on oxalate degrading enzymes and other enzymes encoded by Photorhabdus luminescens is also briefly discussed.
All the experimental protocols and computational methods applicable for most of the investigations reported in Chapters 4, 5 and 6 are presented in Chapter II. The experimental procedures described include cloning, overexpression, purification, enzymatic assays, crystallization and X-ray diffraction data collection. Computational methods covered include summary of crystallographic theory and details of various programs used during data processing, structure solution, refinement, model building, validation and analysis. The databases that were used in the course of these investigations are also cited.
The experience gained during attempts to determine the structure of StTdcD by single wavelength anomalous dispersion (SAD) method is described in Chapter III. The impetus for this work was the urge to examine the power of SAD technique making use of a newly acquired rotating anode X-ray generator equipped with a chromium anode. As expected, the structure determined by SAD was very close to the earlier determined structure of StTdcD. The structure contained a citrate, which was part of the crystallization cocktail at the active site. This is in contrast with acetate kinase, where it was found that citrate binds at the dimeric interface. The present studies demonstrated that the identification of a plausible regulatory site at the interface of dimeric structure in acetokinases based on the structure of acetate kinase (Chittori et al., 2013) is not valid for propionate kinase.
Extensive efforts carried out to obtain structures of StTdcD and its mutants StTdcD A88V and StTdcD G207A complexed with either the substrate or substrate analogues provided several crystal structures. In most of these structures, the ligand was bound at a position distinct from the substrate binding site. These structures and their analysis are described Chapter IV. Asn206 was transformed from a disallowed region to an allowed region of the Ramachandran map in these structures whenever an anion was bound at the position corresponding to the γ- phosphate of the nucleotide substrate. This structural transformation might enhance the affinity of the enzyme for the substrate. In the structure of StTdcD A88V in complex with AMPPNP, AMPPNP was found to be cleaved to AMP and PNP either due to catalytic activity of the enzyme or due to radiation damage. The released PNP probably reacted with propionate forming propionyl-pyrophosphate. These structures also demonstrate that the nucleotide site readily accommodates the substrate or substrate analogues in the absence of a bound nucleotide.
StTdcD catalyzes the Mg2+ ion dependent inter-conversion of propionate and ATP to propionyl phosphate and ADP. Two distinct catalytic mechanisms have been proposed for the phosphoryl transfer reaction catalyzed by acetokinase family enzymes: 1) direct-in-line transfer mechanism and 2) triple displacement mechanism (Anthony and Spector, 1972; Matte et al., 1998). In both, the configuration of the transferred phosphate undergoes an inversion, which has been experimentally demonstrated. Structural studies carried out with the view of elucidating the catalytic mechanism of StTdcD is described in Chapter V. Fortunately, it was possible to obtain the crystal structures of StTdcD and its mutants with propionate and AMPPNP bound at the active site. The structure supported an associative SN2 type direct in-line transfer mechanism of catalysis. The studies also revealed that Arg236 and His175 are catalytically important residues. As suggested earlier, Ala88 has a major role in specificity determination. However, Ala88 is not the sole determinant of specificity. Active site volume determining residues, Arg86, His118, Asp143 and the segment Pro116-Leu117-His118 are also important for substrate specificity. The catalytic mechanism proposed in this chapter may also be applicable to other acetokinase family members.
The final Chapter VI describes three different crystal structures of PlOXDC. As expected from sequence similarity with B. subtilis and T. maritima OXDCs, PlOXDC polypeptide was found to possess a bicupin structure. However, the functional unit was a trimer in contrast to BsOXDC which functions as a hexamer. The difference is shown to be due to the disorder in the amino terminal segment of PlOXDC. The polypeptide was truncated during purification by a non-specific cleavage at residue Lys26 either by thrombin used for cleaving the covalently attached GST tag or by some other protease. However, in the crystal structure, the amino terminal 90 residues were disordered. The observed trimeric form of PlOXDC may represent its inherent nature or a result of the missing N-terminal residues. There is some controversy in the literature on whether both or only one cupin domain of the protomer is catalytically active. The structures presented in this chapter provided significant information on the mode of ligand binding to PlOXDC. In one of the structures, EDO was bound to both the cupin domains and was involved in similar interactions with protein atoms. This may imply that the substrate binds at both the sites and both cupin domains may have catalytic function.
The thesis ends with a short note on future perspectives. It is clear that substantial work has been carried out on acetokinases. These studies have provided significant understanding of their structure and function. In the future, appropriate site-specific mutations of the substrate specificity determining residues may be made and their effect on enzyme specificity could be studied. Similarly, mutagenesis experiments could be performed to inter-convert acetate, propionate and butyrate kinases. These studies will provide deeper insights on intricacies of enzyme function. In contrast to the work on short chain fatty acid kinases, work on OXDC should be considered preliminary and further biochemical and structural studies are needed to illustrate the catalytic mechanism and examine if the protein is a suitable candidate for generating transgenic crops resistant to insect pests.
The following manuscripts have been published or will be communicated for publication based on the results presented in the thesis
Abstract 3932: Dual role of p120ctn in cancer: epithelial vs mesenchymal
No full textAbstract
Neuroblastoma, a paediatric cancer, accounts for 15% of childhood cancer mortality. Even though neuroblastoma is an aggressive cancer, the exact mechanisms by which the cells resist treatment is poorly understood. Here, we hypothesise that neuroblastoma cells have high expression of mesenchymal markers and hence could attribute to the aggressive phenotype. P120ctn is downregulated in epithelial cancers and is known to play a major role in EMT and aggressiveness. In this study, immunohistochemical staining of neuroblastoma patient tissues suggested that p120ctn is highly abundant. Hence, the role of p120ctn and N-Myc in neuroblastoma aggressiveness was investigated by using RNA interference. Amplification of N-Myc oncogene occurs in 20% of neuroblastoma patients and is considered high risk as it correlates with aggressiveness and poor prognosis. Interestingly, knockdown of p120ctn down regulated N-Myc both at mRNA and protein levels. Upon knockdown of p120ctn and N-Myc, the proliferation, invasion and migration of neuroblastoma cells were significantly reduced. Quantitative proteomic and qPCR analysis of the wild type and knockdown cells revealed that p120ctn knockdown cells underwent mesenchymal-to-epithelial transition. Confocal microscopy and Western blotting analysis of subcellular fractionation showed nuclear accumulation of β-catenin upon p120ctn knockdown. Once in the nucleus, β-catenin activated Wnt signalling pathway and up regulated Wnt target genes including C-Myc. Interestingly, down regulation of p120ctn sensitised the neuroblastoma cells to doxorubicin. Currently, there is no published study that explores the role of p120ctn in neuroblastoma. However, these findings are contradictory to scientific literature in the context of the functional role of p120ctn in epithelial cancer. Hence to validate our findings, we established knockdown of p120ctn in epithelial colorectal cancer cells. Consistent with the literature, knockdown of p120ctn induced EMT, proliferation and migration. These results suggest that the role of p120ctn is cell type dependent. Overall, the findings from this study suggest that p120ctn plays a pivotal role in progression of neuroblastoma.
Citation Format: Pamali Fonseka, Suresh Mathivanan, Michael Liem, Ishara Atukorala. Dual role of p120ctn in cancer: epithelial vs mesenchymal [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3932. doi:10.1158/1538-7445.AM2017-3932</jats:p
Exosomes from N-Myc amplified neuroblastoma cells induce migration and confer chemoresistance to non-N-Myc amplified cells: implications of intra-tumour heterogeneity
No full textNeuroblastoma accounts for 15% of childhood cancer mortality. Amplification of the oncogene N-Myc is a well-established poor prognostic marker for neuroblastoma. Whilst N-Myc amplification status strongly correlates with higher tumour aggression and resistance to treatment, the role of N-Myc in the aggressiveness of the disease is poorly understood. Exosomes are released by many cell types including cancer cells and are implicated as key mediators in cell-cell communication via the transfer of molecular cargo. Hence, characterising the exosomal protein components from N-Myc amplified and non-amplified neuroblastoma cells will improve our understanding on their role in the progression of neuroblastoma. In this study, a comparative proteomic analysis of exosomes isolated from cells with varying N-Myc amplification status was performed. Label-free quantitative proteomic profiling revealed 968 proteins that are differentially abundant in exosomes released by the neuroblastoma cells. Gene ontology-based analysis highlighted the enrichment of proteins involved in cell communication and signal transduction in N-Myc amplified exosomes. Treatment of SH-SY5Y cells with N-Myc amplified SK-N-BE2 cell-derived exosomes increased the migratory potential, colony forming abilities and conferred resistance to doxorubicin induced apoptosis. Incubation of exosomes from N-Myc knocked down SK-N-BE2 cells abolished the transfer of resistance to doxorubicin induced apoptosis. These findings suggest that exosomes could play a pivotal role in N-Myc-driven aggressive neuroblastoma and transfer of chemoresistance between cells. Abbreviations: RNA = ribonucleic acid; DNA = deoxyribonucleic acid; FCS = foetal calf serum; NTA = nanoparticle tracking analysis; LC-MS = liquid chromatography–mass spectrometry; KD = knockdown; LTQ = linear trap quadropole; TEM = transmission electron microscop
Dry sliding wear behaviour of Ta/NbC filled glass-epoxy composites at elevated temperatures
No full textIn this work an attempt was made to evaluate wear loss, specific wear rate and coefficient of friction of Glass-Epoxy (G-E) composites with and without Tantalum Niobium Carbide (Ta/NbC) filler. A vacuum assisted resin transfer moulding (VARTM) technique was employed to fabricate the composite specimens. The fabricated wear specimens were tested by using pin-on-disk test rig at various temperatures viz., 30, 60, 90 and 120° C at normal applied loads of 10 N and 20 N. Sliding velocity of the disc of 1.5 m/s was maintained and test was continued for each sample up to a sliding distance of 5000 m. The wear loss in both the composites increases with increase in temperature and applied normal load. However, Ta/NbC particulate filler incorporated G-E composite exhibits lower wear rate and higher coefficient of friction as compared to unfilled G-E composite. The features of worn surfaces of the specimens were examined under scanning electron microscopy (SEM) and findings are analysed
Efficient Data Assumption and Optimization of Analog Response Communication Systems
No full textThis paper is focused on the examination of connection between the premier remote reason exactitude of transmission information and qualities for driving edge correspondence structures and straightforward data cesium (AFCS) identified with transmission of signs from fundamental sources. It's displayed that the mean sq. oversight of transmission picks information properties of AFCS. Varieties between the cutoff purposes of AFCS thought of as summed up correspondence channel and their forward channel square measure investigated. The new impacts appearing in incredible AFCS and in DCS working near Shannon's cutoff square measure thought of also. Amid this works advanced and essential correspondence frameworks were thought of with none inclinations and no confirmation that the use of simple cesium is frequently less able than modernized correspondence structures had been given start inside the no in this way far off past. The secured comes about unambiguously showed the capacity of simple data cesium to transmit the signs while not committal to composing with to a little degree rate up to the uttermost ranges of the forward control
Reduce the complexity of the E-learning authoring process
No full textFor every problem, there is one solution which is simple, neat, and wrong. The production of E-Learning contents is today the largest cost factor in the E-Learning and also the major issue of insecurity. This is an obstacle with the further propagation of the E-Learning. At present there are hardly visible numbers of tools to the production of E-Leaning contents. Besides the partial very high prices for this software they have the deficiency that they are usable only after a training course phase by the E-Learning author due to their complexity and its extent. The new challenge for designers and the researchers is to develop software tools for effective E-Learning. This Master thesis proposes an E-learning authoring tool which automatically uploads the file (OpenOffice document) which is selected by the enduser to the LMS/server. It also narrates how the content and the metadata are transformed as a SCORM package as well as its simplicity comparing to the other tools
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