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Structural and Related Studies on Mycobacterial Lectins
Get PDFThis thesis is concerned with the first ever X-ray crystallographic and complimentary solution studies on mycobacterial lectins. Lectins, described as multivalent carbohydrate binding proteins of non-immune origin, are found in all kingdoms of life. As explained in the introductory chapter, those from plants and animals are the best characterized in terms of structure and function. Although not that extensive, important studies have been carried out on viral, fungal and parasite lectins as well. Bacterial lectins studied so far can be classified in to fimbrial, surface and secretory (or toxic). Applications of lectins include blood typing, cell separation and purification of glycoconjugates, mitogenic stimulation of lymphocytes, mapping of neuronal pathways and drug targeting and delivery. The work reported in the thesis lies at the intersection of two major long range programs in this laboratory, one on lectins and the other on mycobacterial proteins. Three putative lectins Rv1419 and Rv2813 from M. tuberculosis and MSMEG_3662 from M. smegmatis were chosen for exploratory studies on the basis of preliminary genomic searches.
Exploratory studies on Rv1419, Rv2813 and MSMEG_3662 are described in the second chapter. MSMEG_3662 contains two domains, a LysM domain and a lectin domain (MSL) connected by a long polypeptide chain. The two M. tuberculosis proteins, full length MSMEG_3662 and MSL were cloned, expressed, purified and characterized. Rv2813 did not show any appreciable agglutination activity. It showed ATPase activity. Clearly the protein was not a lectin. Rv1419, full length MSMEG_3662 and MSL exhibited lectin characteristics. Among them, Rv1419 and MSL could be crystallized. Preliminary X-ray diffraction studies on them were carried out.
Rv1419 could be successfully expressed only once. However, that was enough for the determination of crystal structure and the glycan array analysis of the lectin (Chapter 3). The monomeric lectin has a β-trefoil fold. It has high affinity for LacNAc and its Neu5Ac derivatives. Modeling studies using complexes of homologous structures, led to the identification of two carbohydrate binding sites on the lectins. Sequence comparisons of Rv1419 with homologous proteins with known structures and phylogenetic analysis involving them provide interesting insights into the relationship among trefoil lectins from different sources.
X-ray crystal structure analysis of MSL and its complexes with mannose and methyl-α-mannose, the first comprehensive effort of its kind on a mycobacterial lectin, reveals a structure very similar to β-prism II fold lectins from plant sources, but with extensive unprecedented domain swapping in dimer formation (Chapter 4). The two subunits in a dimer often show small differences in structure, but the two domains, not always related by 2-fold symmetry, have the same structure. Each domain carries three sugar-binding sites, similar to those in plant lectins, one on each Greek key motif. The occurrence of β-prism II fold lectins in bacteria, with characteristics similar to those from plants, indicates that this family of lectins is of ancient origin and had evolved into a mature system before bacteria and plants diverged. In plants, the number of binding sites per domain varies between one and three, whereas the number is two in the recently reported lectin domains from Pseudomonas putida and Pseudomonas aeruginosa. An analysis of the sequences of the lectins and the lectin domains shows that the level of sequence similarity among the three Greek keys in each domain has a correlation with the number of binding sites in it. Furthermore, sequence conservation among the lectins from different species is the highest for that Greek key which carries a binding site in all of them. Thus, it would appear that carbohydrate binding influences the course of the evolution of the lectin.
LysM domains have been recognized in bacteria and eukaryotes as carbohydrate-binding protein modules, but the mechanism of their binding to chitooligosaccharides is underexplored. Binding of a full length MSMEG_3662 containing LysM and lectin (MSL) domains to chitooligosaccharides has been studied using isothermal titration calorimetry and fluorescence titration (Chapter 5). This investigation demonstrates the presence of two binding sites of non-identical affinities per dimeric MSL-LysM molecule. Affinity of the molecule for chitooligosaccharides correlates with the length of the carbohydrate chain. Its binding to chitooligosaccharides is characterized by negative cooperativity in the interactions of the two domains. Apparently, the flexibility of the long linker that connects the LysM and MSL domains plays a facilitating role in this recognition. The LysM domain in MSL-LysM, like other bacterial domains but unlike plant LysM domains, recognizes equally well peptidoglycan fragments as well as chitin polymers. Interestingly, in the present case two LysM domains are enough for binding to peptidoglycan in contrast to the three reportedly required by the LysM domains of Bacillus subtilis and Lactococcus lactis. Also, the affinity of MSL-LysM for chitooligosaccharides is higher than that of LysM-chitooligosaccharide interactions reported so far.
A part of the work presented in this thesis has been reported in the following publications:
• Patra D, Mishra P, Surolia A, Vijayan M. 2014. Structure, interactions and evolutionary implications of a domain-swapped lectin dimer from Mycobacterium smegmatis. Glycobiology, 24:956-965.
• Patra D, Sharma A, Chandran D, Vijayan M. 2011. Cloning, expression, purification, crystallization and preliminary X-ray studies of the mannose-binding lectin domain of MSMEG_3662 from Mycobacterium smegmatis. Acta Crystallogr Sect F Struct Biol Cryst Commun, 67:596-599.
• Patra D, Srikalaivani R, Misra A, Singh DD, Selvaraj M, Vijayan M. 2010. Cloning, expression, purification, crystallization and preliminary X-ray studies of a secreted lectin (Rv1419) from Mycobacterium tuberculosis. Acta Crystallogr Sect F Struct Biol Cryst Commun, 66:1662-1665
Structural Studies Of Mycobacterial Uracil-DNA Glycosylase (Ung) And Single-Stranded DNA Binding Protein (SSB)
Get PDFFor survival and successful propagation, every organism has to maintain the genomic integrity of the cell. The information content, in the form of nucleotide bases, is constantly threatened by endogenous agents and environmental pollutants. In particular, pathogenic mycobacteria are constantly exposed to DNA-damaging assaults such as reactive oxygen species (ROS) and reactive nitrogen intermediate (RNI), in their habitat which is inside host macrophage. In addition, the genome of Mycobacterium tuberculosis makes it more susceptible for guanine oxidation and cytosine deamination as it is G-C rich. Therefore DNA repair mechanisms are extremely important for the mycobacterium. An important enzyme involved in DNA repair is uracil-DNA glycosylase (Ung). To access the genomic information, during repair as well as DNA replication and recombination, dsDNA must unwind to form single stranded (ss) intermediates. ssDNA is more prone to chemical and nuclease attacks that can produce breaks or lesions and can also inappropriately self associate. In order to preserve ssDNA intermediates, cells have evolved a specialized class of ssDNA-binding proteins (SSB) that associate with ssDNA with high affinity. As part of a major programme on mycobacterial proteins in this laboratory, structural studies on mycobacterial uracil-DNA glycosylase (Ung) and single-stranded DNA binding protein (SSB) have been carried out.
The structures were solved using the well-established techniques of protein X-ray crystallography. The hanging drop vapour diffusion and microbatch methods were used for crystallization in all cases. X-ray intensity data were collected on a MAR Research imaging plate mounted on a Rigaku RU200 X-ray generator. The data were processed using the HKL program suite. The structures were solved by the molecular replacement method using the program PHASER and AMoRe. Structure refinements were carried out using the programs CNS and REFMAC. Model building was carried out using COOT. PROCHECK, ALIGN, INSIGHT and NACCESS were used for structure validation and analysis of the refined structures. MD simulations were performed using the software package GROMACS v 3.3.1.
Uracil-DNA glycosylase (UNG), a repair enzyme involved in the excision of uracil from DNA, from mycobacteria differs from UNGs from other sources, particularly in the sequence in the catalytically important loops. The structure of the enzyme from Mycobacterium tuberculosis (MtUng) in complex with a proteinaceous inhibitor (Ugi) has been determined by X-ray analysis of a crystal containing seven crystallographically independent copies of the complex. This structure provides the first geometric characterization of a mycobacterial UNG. A comparison of the structure with those of other UNG proteins of known structure shows that a central core region of the molecule is relatively invariant in structure and sequence, while the N- and C-terminal tails exhibit high variability. The tails are probably important in folding and stability. The mycobacterial enzyme exhibits differences in UNG-Ugi interactions compared with those involving UNG from other sources. The MtUng-DNA complex modelled on the basis of the known structure of the complex involving the human enzyme indicates a domain closure in the enzyme when binding to DNA. The binding involves a larger burial of surface area than is observed in binding by human UNG. The DNA-binding site of MtUng is characterized by the presence of a higher proportion of arginyl residues than is found in the binding site of any other UNG of known structure. In addition to the electrostatic effects produced by the arginyl residues, the hydrogen bonds in which they are involved compensate for the loss of some interactions arising from changes in amino-acid residues, particularly in the catalytic loops. The results arising from the present investigation represent unique features of the structure and interaction of mycobacterial Ungs.
To gain further insights, the structure of Mycobacterium tuberculosis Ung (MtUng) in its free form was also determined. Comparison with appropriate structures indicate that the two domain enzyme slightly closes up when binding to DNA while it slightly opens up when binding to its proteinaceous inhibitor Ugi. The structural changes on complexation in the catalytic loops reflect the special features of their structure in the mycobacterial protein. A comparative analysis of available sequences of the enzyme from different sources indicates high conservation of amino acid residues in the catalytic loops. The uracil binding pocket in the structure is occupied by a citrate ion. The interactions of the citrate ion with the protein mimic those of uracil in addition to providing insights into other possible interactions that inhibitors could be involved in.
SSB is an essential accessory protein required during DNA replication, repair and recombination, and various other DNA transactions. Eubacteral single stranded DNA binding (SSB) proteins constitute an extensively studied family of proteins. The variability in the quaternary association in these tetrameric proteins was first demonstrated through the X-ray analysis of the crystal structure of Mycobacterium tuberculosis SSB (MtSSB) and Mycobacterium smegmatis (MsSSB) in this laboratory. Subsequent studies on these proteins elsewhere have further explored this variability, but attention was solely concentrated on the variability in the relative orientation of the two dimers that constitute the tetramer. Furthermore, the effect of this variability on the properties of the tetrameric molecule was not adequately addressed. In order to further explore this variability and strengthen structural information on mycobacterial SSBs in particular, and on SSB proteins in general, the crystal structures of two forms of Mycobacterium leprae single stranded DNA-binding protein (MlSSB) has been determined. Comparison of the structures with other eubacterial SSB structures indicates considerable variation in their quaternary association although the DNA binding domains in all of them exhibit the same OB-fold. This variation has no linear correlation with sequence variation, but it appears to correlate well with variation in protein stability. Molecular dynamics simulations have been carried out on tetrameric molecules derived from the two forms and the prototype E. coli SSB and the individual subunits of both the proteins. The X-ray studies and molecular dynamics simulations together yield information on the relatively rigid and flexible regions of the molecule and the effect of oligomerization on flexibility. The simulations provide insights into the changes in the subunit structure on oligomerization. They also provide insights into the stability and time evolution of the hydrogen bonds/water-bridges that connect two pairs of monomers in the tetramer.
In continuation of our effort to understand structure-function relationships of mycobacterial SSBs, the structure of MsSSB complexed with a 31-mer polydeoxy-cytidine single stranded DNA (ssDNA) was determined. The mode of ssDNA binding in the MsSSB is different from the modes in the known structures of similar complexes of the proteins from E. coli (EcSSB) and Helicobacter pylori (HpSSB). The modes in the EcSSB and HpSSB also exhibit considerable differences between them. A comparison of the three structures reveals the promiscuity of DNA-binding to SSBs from different species in terms of symmetry and the path followed by the bound DNA chain. It also reveals commonalities within the diversity. The regions of the protein molecule involved in DNA-binding and the nature of the residues which interact with the DNA, exhibit substantial similarities. The regions which exhibit similarities are on the central core of the subunit which is unaffected by tetramerisation. The variable features of DNA binding are associated with the periphery of the subunit, which is involved in oligomerization. Thus, there is some correlation between variability in DNA-binding and the known variability in tetrameric association in SSBs.
In addition to the work on Ung and SSB, the author was involved in X-ray studies on crystals of horse methemoglobin at different levels of hydration, which is described in the Appendix of the thesis. The crystal structure of high-salt horse methaemoglobin has been determined at environmental relative humidities (r.h.) of 88, 79, 75 and 66%. The molecule is in the R state in the native and the r.h. 88% crystals. At r.h.79% the molecule appears to move towards the R2 state. The crystal structure at r.h.66% is similar, but not identical, to that at r.h.75%. Thus variation in hydration leads to variation in the quaternary structure. Furthermore, partial dehydration appears to shift the structure from the R state to the R2 state. This observation is in agreement with the earlier conclusion that the changes in protein structure that accompany partial dehydration are similar to those that occur during protein action.
A part of the work presented in the thesis has been reported in the following publications.
1. Singh, P., Talawar, R.K., Krishna, P.D., Varshney, U. & Vijayan, M. (2006). Overexpression, purification, crystallization and preliminary X-ray analysis of uracil N-glycosylase from Mycobacterium tuberculosis in complex with a proteinaceous inhibitor. Acta Crystallogr. F62, 1231-1234.
2. Kaushal, P.S., Talawar, R.K., Krishna, P.D., Varshney, U. & Vijayan, M. (2008). Unique features of the structure and interactions of mycobacterial uracil-DNA glycosylase: structure of a complex of the Mycobacterium tuberculosis enzyme in comparison with those from other sources. Acta Crystallogr. D64, 551-560.
3. Kaushal, P.S., Sankaranarayanan, R. & Vijayan, M. (2008). Water-mediated variability in the structure of relaxed-state haemoglobin. Acta Crystallogr. F64, 463-469
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Structural Studies On Three-Fold Symmetric Plant Lectins
Get PDFLectins, multivalent carbohydrate-binding proteins of non-immune origin, have the unique ability to decode the information contained in complex carbohydrate structures of glycoproteins and glycolipids by stereo-specifically recognizing and binding to carbohydrates and carbohydrate linkages. The ubiquitous distribution of lectins in all forms of life and viruses along with their involvement in various biological processes such as cell-cell communication, host-pathogen interaction, cancer metastasis, embryogenesis, tissue development and mitogenic stimulation further emphasizes the importance of lectins in biological systems. Although not much is known about the endogenous roles of plant lectins, they constitute the most thoroughly studied class of lectins. On the basis of their subunit folds plant lectins have been divided in six major classes. They include jelly roll fold lectins (or legume lectins), hevein domain lectins (or cereal lectins), β-trefoil fold lectins, β-prism II fold lectins (or bulb lectins), β-prism I fold lectins and the most recently discovered lectin homologous to cyanovirin-N (http://www.cermav.cnrs.fr/lectines). Interestingly, of these, lectin subunits harbor an approximate three-fold symmetry in three cases and each subunit is believed to have evolved through successive gene duplication, fusion and divergent evolution. One of the major research activities in this laboratory involves structural studies on plant lectins. Decades of extensive studies in the laboratory have shed light on various structural and functional aspects of lectins such as variability in quaternary association, lectin-carbohydrate interactions, strategies for generating ligand specificity and multivalency. Furthermore, the β-prism I fold was first identified as a lectin fold in this laboratory through the X-ray analysis of the methyl-α-galactose complex of jacalin, one of the two lectins from the seeds of Artocarpus integrifolia. Subsequently, many other lectins with the same fold have been structurally characterized here and else where (http://www.cermav.cnrs.fr/lectines). They include mannose specific tetrameric artocarpin and dimeric banana lectin studied in this laboratory. Also investigated here is the structure of first dimeric β-prism II fold lectin, namely, garlic lectin. The subsequent work, carried out by the author, on the structure and dynamics of three-fold symmetric lectins form the subject matter of this thesis.
Different web-servers available at NCBI and EXPASY web sites were used for sequence annotation studies. MRBAYES and MEGA were used for phylogenetic analysis. Molecular dynamics (MD) simulations were carried out using the simulation package GROMACS v.3.3.1. OPLS-AA/L and GLYCAM-06 force fields were used for proteins and carbohydrates respectively. Simulations were performed in explicit water system with TIP4P water model under NPT conditions with unit dielectric constant. The hanging drop method was used for crystallizing banana lectin and its complexes. Intensity data were collected on a MAR 345 image plate mounted on a Rigaku RU200 rotating-anode X-ray generator. The Oxford cryosystem was used when collecting data at low temperature. The data were processed using DENZO and SCALEPACK of HKL suite of programs. The structure factors from the processed data were calculated using TRUNCATE of CCP4 suite of programs. The molecular replacement program MOLREP was used for structure solution. Structure refinements were carried out using the CNS software package and REFMAC of CCP4. Model building was done using the molecular graphics program COOT. INSIGHT II, ALIGN, CONTACT, MUSTANG and SC of CCP4 were used for analysis of structural features. PROCHECK and web-server MOLPROBITY were used for the validation of the refined structures.
The β-prism II fold lectins of known structure, all from monocots, invariably have three carbohydrate-binding sites in each subunit / domain. Until recently, β-prism I fold lectins of known structure were all from dicots and they exhibited one carbohydrate-binding site per subunit / domain. However, the recently determined structure of the β-prism I fold lectin from banana, a monocot, has two very similar carbohydrate-binding sites. This prompted a detailed analysis of all the sequences appropriate for the two lectin folds and which carry one or more relevant carbohydrate-binding motifs. The recent observation of a β-prism I fold lectin, griffithsin, with three binding sites in each domain further confirmed the need for such an analysis. The detailed sequence and phylogenetic analysis of all the β-prism I fold lectin or lectin-like sequences, available then, with particular attention to their carbohydrate-binding sites in them, in conjunction with the analysis of available three-dimensional structures demonstrate substantial diversity in the number of binding sites, unrelated to the taxonomical position of the plant source. However, the number of binding sites and the symmetry within the sequence exhibit reasonable correlation. The distribution of the two families of β-prism fold lectins among plants and the number of binding sites in them, appear to suggest that both of them arose through successive gene duplication, fusion and divergent evolution of the same primitive carbohydrate-binding motif involving a Greek key. Analysis with sequences in individual Greek keys as independent units lends further support to this conclusion. It would seem that the prepondence of three carbohydrate-binding sites per domain in monocot lectins, particularly those with the β-prism II fold, is related to the role of plant lectins in defence.
Jacalin is the most thoroughly studied β-prism I fold lectin. A wealth of structural and thermodynamic data, mostly from this laboratory, led to a thorough characterization of carbohydrate-recognition in the case of jacalin. One aspect of jacalin that has not been investigated so far was its dynamics. The issue was addressed through reasonably long MD simulations, in explicit solvent system using all atom force field, of all the jacalin-carbohydrate complexes of known structure, models of unliganded molecules derived from the complexes and also models of relevant complexes where X-ray structures are not available. Results of the simulations and the available crystal structures involving jacalin permit delineation of the relatively rigid and flexible regions of the molecule and the dynamical variability of the hydrogen bonds involved in stabilizing the structure. Local flexibility appears to be related to solvent accessibility. Hydrogen bonds involving side chains and water bridges involving buried water molecules appear to be important in the stabilization of loop structures. The lectin-carbohydrate interactions observed in crystal structures, the average parameters pertaining to them derived from simulations, energetic contribution of the stacking residue estimated from quantum mechanical calculations and the scatter of the locations of carbohydrate and carbohydrate-binding residues, are consistent with the known thermodynamic parameters of jacalin-carbohydrate interactions. The simulations, along with X-ray results, provide a fuller picture of carbohydrate binding by jacalin than provided by crystallographic analysis alone. The simulations confirm that in the unliganded structures water molecules tend to occupy the positions occupied by carbohydrate oxygens in the lectin-carbohydrate complexes. Population distributions in simulations of the free lectin, the ligands and the complexes indicate a combination of conformational selection and induced fit.
Mannose-specific β-prism I fold lectins, like lectins belonging to other plant families, exhibit interesting variability in their quaternary association. Mannose specific artocarpin and MornigaM are tetrameric, heltuba is octameric in the crystal structure and banana lectin and calsepa are dimeric. The modes of the dimerization in the last two are however, entirely different. This variability was explored through modelling and molecular dynamics simulations based on the known three-dimensional structures. This study, which combines computational approaches and results of X-ray analyses, provides valuable insights into the origin of the variability in quaternary association. MD simulations on individual subunits and the oligomers provide insights into the changes in the structure brought about in the protomers on oligomerization, including swapping of the N-terminal stretch in one instance. The regions which undergo changes also tend to exhibit dynamic flexibility during MD simulations. The internal symmetries of individual oligomers are substantially retained during the calculations. Simulations were also carried out on models using all possible oligomers employing the four different protomers. The unique dimerization pattern observed in calsepa could be traced to unique substitutions in a peptide stretch involved in dimerization. The impossibility of a specific mode of oligomerization involving a particular protomer is often expressed in terms of unacceptable steric contacts or dissociation of the oligomer during simulations. The calculations also lead to a rationale for the observation of a heltuba tetramer in solution although the lectin exists as an octamer in the crystal, in addition to providing insights into relations among evolution, oligomerization and ligand binding.
The known crystal structures of banana lectin in its native and ligand bound forms revealed interesting features including the presence of two functional carbohydrate-binding sites per subunit. However, some confusion remained on the role of glycosidic linkage in carbohydrate-binding. The three crystal structures reported in this thesis provide information on details of the interactions of mannose and mannosylα-1,3-mannose with banana lectin and evidence for the binding of glucosyl-α-1,2glucose to the lectin. The known structures involving the lectin include a complex with glucosyl-β-1,3-glucose. Modelling studies on the three disaccharide complexes with the reducing end and the non-reducing end at the primary binding site are also presented here. The results of the X-ray and modelling studies show that the disaccharides with an α-1,3 linkage prefers to have the non-reducing end at the primary binding site while the reducing end is preferred at the site when the linkage is β-1,3 in mannose/glucose specific β-prism I fold lectins. In the corresponding galactose-specific lectins, however, α-1,3 linked disaccharides cannot bind the lectin with the non-reducing end at the primary binding site on account of steric clashes with an aromatic residue which occurs only when the lectin is galactose-specific. MD simulations based on the known structures involving banana lectin enrich the information on lectin-carbohydrate interactions obtained from crystal structures. They demonstrate that conformational selection as well as induced fit operate when carbohydrates bind to banana lectin.
Snake gourd seed lectin (SGSL) isolated from Trichosanthes anguina is a glycosylated, galactose-specific, non-toxic lectin similar to type II ribosome inactivating proteins (RIPs) with a molecular weight of ~53kDa. It was established through preliminary X-ray studies that chain A with molecular weight of ~23kDa adopts the same fold as that of type I RIPs and the toxic chain of type II RIPs. Chain B with molecular weight ~32kDa has two β-trefoil fold domains and is responsible for the lectin activity of the protein. The two chains are connected with a disulphide bond. The sequence of the protein could not be determined using conventional methods despite extensive effort. It was derived from X-ray data at 2.4 Å resolution, which was used for structure analysis. The non-toxicity of SGSL appears to result from a combination of changes in the catalytic site in chain A and sugar-binding site in chain B. Detailed analysis of the sequences of type II RIPs of known structure and their homologues with unknown structure, provide valuable insights into the evolution of this class of proteins. It also indicates some variability in carbohydrate-binding sites, which appears to contribute to different levels of toxicity exhibited by lectins from various sources.
In addition to the work on plant lectins, the author was also involved in studies on the crystal structures of the adipic acid complexes of L- and DL-Lysine. This investigation is presented in an appendix.
A part of the work presented in the thesis has been reported in the following
publications.
Sharma, A., Thamotharan, S., Roy, S., & Vijayan, M. (2006). X-ray studies of
crystalline complexes involving amino acids and peptides. XLIII. Adipic acid
complexes of L- and DL-lysine. Acta Cryst, C62, o148-o152.
Sharma, A., Chandran, D., Singh, D.D., & Vijayan, M. (2007). Multiplicity of
carbohydrate-binding sites in beta-prism fold lectins: occurrence and possible
evolutionary implications. J Biosci, 32, 1089-1110.
Sharma, A., Sekar, K., & Vijayan, M. (2009). Structure, dynamics, and interactions of
jacalin. Insights from molecular dynamics simulations examined in conjunction
with results of X-ray studies. Proteins, 77, 760-777
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
Unveiling the hydrogen bonding network in liquid crystalline natural-based glycosides containing polymeric complexes : experimental and theoretical assessment
Get PDFThis work was supported by the Ministry of Education of Malaysia [FRGS/1/2019/TK05/UITM/02/9, 2019]; Royal Academy of Engineering, U.K., and Academy of Science, Malaysia [NRCP1516/4/61, 2016]; University of Aberdeen [SF10192, 2018] and University Malaya [UMRG grant RP038B-17AFR]. CRediT authorship contribution statement Nurul Fadhilah Kamalul Aripin: Conceptualization, Data curation, Writing - original draft, Writing - review & editing, Funding acquisition. Jonathan Heap: Data curation, Formal analysis. Rafael Piñol: Data curation, Methodology. Vijayan M. Achari: Data curation, Formal analysis, Writing - original draft. Alfonso Martinez-Felipe: Conceptualization, Investigation, Writing - original draft, Writing - review & editing, Funding acquisition.Peer reviewe
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