1,721,177 research outputs found
Structure of the capsular polysaccharide of the KPC-2-producing Klebsiella pneumoniae strain KK207-2 and assignment of the glycosyltransferases functions
Full text linkKlebsiella pneumoniae strain KK207-2 was isolated in 2010 froma bloodstreaminfection of an inpatient at an Italian hospital. It was previously found to produce the KPC-2 carbapenemase and to belong to clade 1 of sequence type 258. Genotyping of the conserved wzi and wzc genes from strain KK207-2 yielded contrasting results: the wzc-based method assigned the cps207–2 to a new K-type, while the wzi-based method assigned it to the known K41 K-type. In order to resolve this contradiction, the capsular polysaccharide of K. pneumoniae KK207-2 was purified and its structure determined by using GLC-MS of appropriate carbohydrate derivatives, ESI-MS of both partial hydrolysis and Smith degradation derived oligosaccharides, andNMR spectroscopy of oligosaccharides, and the lithium degraded, native and de-O-acetylated polysaccharide. All the collected data demonstrated
the following repeating unit for the K. pneumoniae KK207-2 capsular polysaccharide:
OAc
6
[3)-β-D-Gal-(1-4)-β-D-Glc-(1-]n
4
I
1
β-D-Glcp-(1-6)-α-D-Glcp-(1-4)-β-D-GlcpA-(1-6)-α-D-Glcp
The polysaccharide contains about 0.60 acetyl groups per repeating unit on C6 of the Gal residue. The reactions catalysed by each glycosyltransferase in the cpsKK207-2 gene cluster were assigned on the basis of structural homology with other Klebsiella K antigens
The development of physico-chemical quality control methods for Haemophilus influenzae type b vaccine production
No full textThe development of a low cost Haemophilus influenzaetype b (Hib) manufacturing platform at The Biovac Institute (TBI) required analytical method development in parallel with the production process development. Technology transfer enabled TBI to develop Hib vaccine production which could lead to the development of vaccine manufacturing capacity in subSaharan Africa. Initial studies were conducted in the Research and Development (R&D) department from where the process was transferred to the Good Manufacturing Process (GMP) environments of the Production and Quality Control departments respectively. Scaling of the development process to a process commercially viable required the development of additional quality control test methods. The quality control of Hib is performed by characterisation of the manufactured batch using physico-chemical analysis. The data generated are compared against that of a successful clinical trial batch. Animal based models for the potency and safety tests of Hib are ineffective. Chromatographic methods of analysis are often used in the pharmaceutical and biotechnological industry. Gas chromatography with flame ionisation detection (GC-FID) is a conventional technique used for the analysis of volatile analytes. The analysis of process residuals ethanol and ethylene glycol were performed using headspace or direct injection GC-FID analysis. Ethylene glycol, a non-volatile solvent, was chemically dried after which it was derivatised with a trimethylsilylating reagent. In addition, a method was developed to determine polyribosylribitolphosphate. Samples were dried by means of lyophilisation and then subjected to methanolysis to yield methyl glycosides. A trimethylsilylating reagent was used to volatilise the analyte and analysis was performed using GC-FID with direct injection. The use of internal standards throughout the sample preparation processes minimised errors due to sample handling, processing or injector reproducibility. Analytical method validation parameters were applied to the developed methods
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe 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
Physicochemical studies of a novel adjuvant and conjugate vaccines
No full textIncludes abstract.Includes bibliographical references.South African children currently receive vaccines against Diphtheria, Tetanus, Pertussis (administered as DTP), Hepatitis B (HBV) and Haemophilus influenzae type b (Hib) at 6, 10 and 14 weeks. The use of combination vaccines provides a means of avoiding the logistical problems and costs associated with multiple injections of these different vaccines. A local vaccine manufacturer is in the process of developing a combined tetravalent DTP-HBV as well as a liquid pentavalent DTP-HBV-Hib vaccine. The Hib vaccine is a glycoconjugate in which Haemophilus influenzae type b capsular polysaccharide (polyribosylribitolphosphate or PRP) is conjugated to a carrier protein. The conjugate is immunogenic in infants who have a higher risk of infection while the polysaccharide vaccine is not. The compatibility of the Hib antigenic component in the presence of the other antigens and adjuvant presents a challenge. Aluminium containing adjuvants have been the most widely used adjuvants in human vaccines. Aluminium hydroxide has been found to catalyse the hydrolysis of PRP when added to Hib conjugate vaccines. Although this can be circumvented by the use of aluminium phosphate, there is a need for new adjuvants that elicit broader immune responses. This thesis presents a study of the size, structure and composition of a locally developed experimental adjuvant called Pheroid™ by use of NMR spectroscopy and Coulter Counter. NMR analysis of Pheroid™ formulations provided a structural fingerprint for the formulations and indicated the relative proportions of the major components present, whereas their particle size distribution was profiled using a Coulter Counter. The average size distribution was similar in the formulations tested including those that had been activated using nitrous oxide. The primary focus of this thesis was the application of appropriate physicochemical procedures for evaluation of the locally manufactured Hib vaccine alone and when in combination with DTP-HBV-Hib. Investigating the stability and integrity of Hib conjugate vaccines requires determination of the total saccharide and unbound or free saccharide which is expressed as the percentage of free saccharide present. In the absence of a suitable Hib conjugate, model compounds such as human serum albumin (HSA) , meningococcal group A polysaccharide (PsA) and the derived conjugate (Mn A-IT) were used to investigate three methods of free saccharide separation: solid phase extraction (SPE), acid precipitation using deoxycholate (DOC/HCI) and ultrafiltration (UF). At physiological pH, the binding capacity was low and so the SPE method was not investigated further. For Mn A-IT the DOC/HCI method generally gave lower free saccharide values than the UF method; this was attributed to entrapment and coprecipitation of free saccharide by DOC/HCI. In contrast, washing steps in the UF method ensured good free saccharide recovery. A colorimetric assay for phosphorus and high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) were investigated for saccharide quantification in Mn A-TT vaccines. The HPAEC-PAD method for the monomer (mannosamine-6-phosphate from acid hydrolysis) permitted higher specificity and sensitivity for the free saccharide analysis compared to the phosphate assay. The DOC/HCI and UF methods were compared by their application to Mn A-TT samples subjected to an accelerated stability study
Modelling and validation of bacterial O-antigen conformations: ring puckering in Shigella flexneri 7a and 7b O-antigens as a case study
No full textThis work forms part of a larger project investigating the conformation and dynamics of Shigella flexneri O-antigens. S. flexneri is a leading cause of diarrhoeal related diseases, especially in Sub-Saharan Africa and Southeast Asia. There are over 30 S. flexneri serotypes and, with the exception of serotype 6, all share a common O-antigen backbone (serotype Y), with variations in glucosylation, phosphorylation, and O-acetylation. O-antigens are carbohydrate polymers on the outer membrane of gram-negative bacteria. In S. flexneri, O-antigens are a primary antigenic component and are a target for conjugate vaccines currently in development. Analysis of O-antigen conformation for different S. flexneri serotypes may inform the vaccine development process. However, determining molecular conformation experimentally is challenging. Systematic molecular modelling protocols have proven useful in elucidating conformations of polysaccharide antigens, especially when experimental methods, such as nuclear magnetic resonance, are used for verification of modelling results. Here we use a combination of molecular modelling and 1H nuclear magnetic resonance spectroscopy experiments to probe the O-antigen conformations of S. flexneri 7a and 7b. Simulations of six repeating units of both O-antigens show that they are highly flexible, similar to the S. flexneri Y O-antigen. However, we found frequent puckering of the β-D-GlcpNAc ring away from the canonical 4C1 conformer, which has not been previously observed in studies of Shigella or other bacterial O-antigens. To provide further insight, molecular dynamics and metadynamics simulations of a range of 3,4-disubstituted β-D-GlcNAc trisaccharides with two carbohydrate force fields (CHARMM36 and GLYCAM06) were performed. The simulations reveal that 3,4-α-linked β-D-GlcpNAc puckers to a similar extent as the S. flexneri 7a and 7b O-antigens. Moreover, for both force fields the range of β-D-GlcNAc puckering is dependant on the anomeric configuration of both the 3- and 4- linkage, with non-4C1 conformations dominant in 3,4-α-linked β-D-GlcpNAc trisaccharides. 1H nuclear Overhauser effect spectroscopy experiments were used to calculate 1H-1H distances in S. flexneri 7a and 7b O-antigens. These experimentally derived distances match those calculated from the repeating unit simulations when a mix of 4C1 and boat/skew states of β-DGlcpNAc are considered at a ratio of 85:15. The results also suggest that puckering of β-DGlcpNAc does occur in S. flexneri 7a and 7b O-antigens but that it may be over represented in the simulations. Two general observations can be drawn from this study: (1) the 3,4-disubstitution of β-DGlcpNAc with two bulky substituents leads to the ring puckering out of the 4C1 conformer; and (2) the conformation and dynamic behaviour of β-D-GlcpNAc puckering is not accurately modelled by current methods, identifying the need for further enhancements to existing carbohydrate force fields
Investigation of the potential beneficial effects of supplemental polyunsaturated fatty acids and glycosaminoglycans on the risk factors for calcium oxalate kidney stone formation using theoretical, experimental and human models
No full textIncludes bibliographical references.Introduction: Two hypotheses with regard to calcium oxalate (CaOx) renal stone formation were tested in this thesis. The first hypothesis is that fatty acid (FA) supplementation (n-6 and n-3) and chondroitin sulphate (CS) supplementation may reduce the plasma (FA) and urinary (FA and CS) risk factors for CaOx renal stone formation, and ultimately serve as therapeutic agents in the management of this disease. The notion that FAs may reduce plasma risk factors is based on previous studies which have shown that n-6 and n-3 FA supplementation reduces the concentrations of arachidonic acid, while the notion of an effect on urinary risk factors is based on reports of these supplements decreasing urinary calcium and/or oxalate excretion in animal and human studies. The notion of CS playing a role in reducing CaOx stone risk is based on its chemical structure which presents potential binding sites for calcium and magnesium. The second hypothesis is that black and white healthy South African subjects may respond differently to these dietary supplements and that these differences may provide insights which could account for the lower stone incidence in the former group compared to the latter. This hypothesis is based on the observation in many previous studies of different renal responses in the two race groups, to different dietary and supplemental challenges. FA’s and CS have not been previously investigated in this regard. Methods: These hypotheses were tested simultaneously by administering n-6 and n-3 FA supplements individually and in combination, and supplemental CS, to different groups of black and white healthy male subjects. For the FA studies, blood samples were analyzed for serum biomarkers (25-hydroxyvitamin 03 and triglycerides) for CaOx stone formation and FA profiles in plasma total phospholipids since arachidonic acid regulates calcium excretion. Urine samples were analyzed for individual CaOx stone risk factors, risk indices (Tiselius risk index and supersaturation (SS) of calcium oxalate, brushite and uric acid); crystallization experiments (metastable limit and crystal growth kinetics) were also conducted. For the CS studies, thermodynamic binding constants for calcium-CS and magnesium-CS complexes were determined by isothermal titration calorimetry. These constants were then used to model speciation in different urines using the computer program Joint Expert Speciation System (JESS), and to calculate supersaturation values under different urinary conditions. This was followed by in vitro crystallization experiments in which the effects of exogeneous CS on the CaOx metastable limit and CaOx crystallization kinetics were investigated in artificial and real urine samples. Finally, human studies were performed in which CS supplements were administered to subjects to test their efficacy on reducing the urinary risk factors for CaOx stone formation. Urines were analyzed and crystallization experiments were performed as described for the FA supplementation studies. Results: In the FA studies, favourable changes in the plasma CaOx stone risk factors were achieved by the supplementation of n-3 FA alone. Post-supplementation, the concentration of arachidonic acid in plasma total phospholipids was significantly reduced in both groups, thereby implying a reduction in urinary calcium excretion. However, FA supplementation had no positive effect on the urinary risk factors or on CaOx metastable limits and CaOx crystallization kinetics. In the CS studies, theoretical modelling showed that the reduction of ionized calcium concentrations can only be attainable at 100 times physiological concentrations of urinary CS and that the formation of the calcium-CS complex does not influence the urinary supersaturation of CaOx. The formation of the magnesium-CS complexes was unfavourable because it resulted in an increase in the concentrations of ionized oxalate, a risk factor for CaOx stone formation. The in vitro crystallization experiments showed that exogeneous CS at physiological and above physiological concentrations had no favourable effect on the metastable limit and crystal growth kinetics in all the tested urine samples. Finally, the human study showed that CS supplementation had no effect on urine chemistry and crystallization kinetics. Speciation calculations also showed that the SS values of CaOx and the concentrations of ionized calcium were not significantly changed by supplementation. Within groups, the effects of FA supplementation on the urinary risk factors were different. n-6 FA supplementation significantly increased magnesium and significantly decreased urate in the black group whereas in whites citrate, oxalate and potassium were significantly increased while ionized calcium was significantly reduced. In the n-3 FA study, magnesium was significantly increased and SS value of brushite was significantly decreased in whites. In the black group, there was no significant difference in the urinary risk factors after supplementation compared to baseline values. With regards to n-6 & n-3 FA supplementation, citrate was significantly increased while oxalate and SS CaOx were significantly decreased in the black group whereas in the white group, magnesium was significant increased. With regards to the CS study, SS values for brushite, tribasic calcium phosphate, hydroxylapatite and octacalcium phosphate decreased significantly in black subjects after CS supplementation, whereas the SS value for hydroxylapatite increased significantly in the white group. These effects could not be attributed to complexation of CS with calcium or magnesium. However, they are noteworthy because they are different in the two groups. Discussion: The results of these studies do not support the hypothesis that supplemental fatty acids or chondroitin sulfate have significant beneficial effects for reducing blood and urinary risk factors for calcium oxalate stone formation. Although the response to FA and CS supplementation was different between the two race groups, these findings did not provide new information to explain the difference in the incidence of calcium oxalate stone disease in the two population groups. The work described in this thesis provides a foundation for future studies in which CaOx stone patients, rather than healthy individuals are investigated
Variations on the Author
Full text link“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
Full text linkWe 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
An investigation towards a conjugate vaccine against Streptococcus pneumoniae serotype 19A
Full text linkStreptococcus pneumoniae is a human pathogen that causes invasive pneumococcal diseases (IPD) such as pneumonia, otitis media and sepsis particularly in children, the elderly, and patients with HIV, and other immunosuppressive conditions. Conjugate vaccines comprised of the bacterial surface polysaccharide conjugated to a carrier protein are very effective in protecting young children against disease by inducing immunological memory and reducing carriage of the bacteria. A pneumococcal conjugate vaccine against seven serotypes (PCV7) was licensed in 2000, which resulted in a dramatic reduction of IPD. However; there was a gradual increase in the number of cases due to non-vaccine serotypes (serotype replacement). Serotype 19A, not included in PCV7 as the structurally similar serotype 19F was assumed to crossprotect against 19A disease, emerged as the most prevalent serotype in several studies with significant presence in Sub-Saharan Africa and South-East Asia and is associated with multidrug resistance. As a result a 13-valent conjugate vaccine that includes serotype 19A was developed and licensed which provided broader coverage. The aim of the work presented in this thesis was to develop processes for the manufacture of 19A capsular polysaccharide (CPS) for the production of a conjugate vaccine based on work performed on the model Pn1. The cultivation process included clonal selection for the growth and isolation of a serotype 19A clone producing high levels of CPS and cultivation using disposable bag technology as an alternative to the traditional fermentor. The culture was inactivated at low temperatures using cold phenol to prevent CPS degradation and to improve the release of CPS from the bacteria. The culture was clarified using a scalable flow-through centrifugation process. The Pn19A polysaccharide was purified using a single step process utilizing differential filtration with ethanol. Analytical tests including identity, purity (from nucleic acid and protein) and size analysis were optimized and performed on Pn19A CPS lots. All purified batches of polysaccharide met World Health Organisation (WHO) specifications as defined in the Technical Report Series. Structural studies were performed on closely related CPS namely; 19F and 19A CPS, both of which contain a labile phosphodiester linkage. The composition of the polysaccharides determined by colorimetric assays was confirmed by hydrolysis and monomeric analysis using gas chromatography/mass spectroscopy (GC/MS) of the methyl glycoside derivatives. Use of 1H, 13C and 31P nuclear magnetic resonance (NMR) experiments confirmed the structure of the 19F and 19A CPS repeating units and permitted determination of the extent of the cell wall polysaccharide contamination. CPS was size-reduced by microfluidization prior to conjugation experiments using cyanylating chemistry and a model carrier protein bovine serum albumin (BSA) and tetanus toxoid (TT). Pn19F and Pn19A conjugates prepared using TT were subjected to thermal stability studies and demonstrated similar stability based on the free saccharide generated. This proof of concept study established small-scale processes that can be further optimized for the manufacture of a conjugate vaccine against Pn19A disease
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