The relationship between structure and thermostability of a nitrile hydratase from Goebacillus pallidus RAPc8

Philosophiae Doctor - PhDNitrile hydratases (NHases) are very important biocatalysts for the enzymatic conversion of nitriles to industrially important amides such as acrylamide and nicotinamide. An “ideal” NHase should fulfil several essential criteria including, high substrate conversion rates, being able to tolerate high substrate and product concentrations as well as being highly thermostable. The NHase used in the present study was isolated from Geobacillus pallidus RAPc8, a moderate thermophile. The primary aims of this study were to use random mutagenesis to engineer the G. pallidus RAPc8 NHase towards improved thermostability and then to use X-ray crystallography to investigate the molecular mechanism(s) involved in the enhanced thermostability. Two randomly mutated libraries were constructed using MnCl2 mediated errorprone PCR. The PCR reaction was performed using 0.05 mM and 0.10 mM MnCl2 and a biased dNTP concentration. The hydroxamic acid assay was used to screen the randomly mutated libraries for NHase mutants with enhanced thermostability. Six mutants that exhibited thermostability-enhancing mutations were isolated from the randomly mutated libraries. The thermostabilised mutants contained between 3 and 7 nucleotide changes per NHase operon. The wild-type and four thermostabilised mutant NHases (7D, 8C, 9C, 9E) were over-expressed, purified, crystallised and subjected to X-ray crystallography. The resolution of the diffraction data for the all the mutant NHases were better than the 2.4Å previously obtained for the wild-type G. pallidus NHase. The best quality data was collected for mutant 9E, which diffracted to a resolution of 1.15Å. The high quality crystal structures allowed each thermostability-enhancing mutation to be viewed in detail. As most of the NHase mutants contained multiple mutations, the crystal structures were important in correlating the observed thermostabilisation with the structural effect of the mutations. Analysis of the X-ray crystal structures illustrated the importance of electrostatic interactions, particularly salt bridges and hydrogen bonds in enhancing the thermostability of the mutant NHases. The difference in the free energy of activation of thermal unfolding (DDG) was used to compare the wild-type and mutant NHases thermostability. The most improved NHase, mutant 9C, was stabilised by both a buried inter-subunit salt bridge between aR169 and bD218 and an inter-helical hydrogen bond between bK43 and bK50. The stabilisation provided by these electrostatic interactions was 7.62 kJ/mol. Mutant 8C was primarily stabilised by the introduction an electrostatic network consisting of a salt bridge between bE96 and aR28 and a hydrogen bond between bE96 and bE92. Also, an intra-helical salt bridge between aE192 and aK195 stabilised the helix consisting of a190-196 in mutant 8C by shielding the helix backbone from solvation and preventing co-operative unfolding of the a helix. However, mutant 8C was also destabilised by a mutation that disrupted a water-mediated hydrogen bond between bD167 and bK168 at the heterotetramer interface of the enzyme. Consequently, the net stabilisation energy provided as a result of stabilising and destabilising interactions was 6.16 kJ/mol. Mutant 7D was the only NHase mutant with only one possible thermostabilising mutation. This mutant was stabilised by 3.40 kJ/mol as the result of a water-mediated hydrogen bond between aS47 and bE33. Similarly, a water-mediated hydrogen bond between aS23 and bS103 provided a stabilisation energy of 4.27 kJ/mol to mutant 9E. This project has shown that moderate-frequency randomly mutated libraries can yield mutants with multiple thermostabilising interactions. Also, the importance of utilising X-ray crystallography to investigate structure-function relationships in proteins has been illustrated.South Afric

Optimising the growth of Cryptococcus species SS1, a potential probiotic for farmed abalone

Includes bibliographical references.Farmed abalone is a reliable and good quality source of abalone. Cryptococcus species SS1 was isolated from the gut of the South African abalone, Haliotis midae, and has been identified as a potential probiotic for farmed abalone. The implementation of strain SS1 as a probiotic for aquacultured abalone required the design of a fermentation system to produce high concentrations of the yeast strain in order to supply the probiotic to commercial abalone producers. The aim of this project was to assist in the recommendation of a commercial fermentation process that is economically feasible for the production of strain SS1. This involved evaluation of all the main factors that will contribute to the cost of the fermentation; i.e. cultivation medium, fermentation space and time, and productivity

Human Hair as a Testing Substrate in the Era of Precision Medicine: Potential Role of ‘Omics-Based Approaches

Minimally and noninvasive investigation of pathology and treatment monitoring is highly attractive in medicine. The use of human hair samples as a non-invasive testing substrate is potentially poised to improve diagnostic and forensic medicine. Hair has the unique ability to capture long-term information about health and disease in an individual as compared to urine and blood. Testing long hair offers a potential means of long-term monitoring of drug compliance, drug abuse, chronic alcohol abuse, and diagnostic biomarker discovery. Even though human hair is mostly composed of keratin and keratin-associated proteins, very little literature has been published on human hair proteomics. Emerging high throughput omics based techniques such as proteomics are increasingly improving our depth of knowledge about the diagnosis, prognosis and prediction of diseases globally. Although many aspects of the use of these novel molecular aids to improve disease diagnosis and patient management remains elusive; it is evident that these techniques have improved precision medicine tremendously. This chapter aims to discuss current plausible application of human hair omics-based approaches to the field of pathology, diagnostics and precision/individualized medicine

Signal transduction and activator of transcription-3 (STAT3) in patients with colorectal cancer: associations with the phenotypic features of the tumour and host

Purpose: In patients with colorectal cancer (CRC), a high-density local inflammatory infiltrate response is associated with improved survival, whereas elevated systemic inflammatory responses are associated with poor survival. One potential unifying mechanism is the IL-6/JAK/STAT3 pathway. The present study examines the relationship between tumour total STAT3 and phosphorylated STAT3Tyr705 (pSTAT3) expression, host inflammatory responses and survival in patients undergoing resection of stage I-III CRC. Experimental Design: Immunohistochemical assessment of STAT3/pSTAT3 expression was performed using a tissue microarray and tumour cell expression divided into tertiles using the weighted histoscore. The relationship between STAT3/pSTAT3 expression and local inflammatory (CD3+, CD8+, CD45R0+, FOXP3+ T-cell density and Klintrup-Mäkinen grade) and systemic inflammatory responses and cancer-specific survival were examined. Results: 196 patients were included in the analysis. Cytoplasmic and nuclear STAT3 expression strongly correlated (r=0.363, P<0.001); nuclear STAT3 and pSTAT3 expression weakly correlated (r=0.130, P=0.068). Cytoplasmic STAT3 was inversely associated with the density of CD3+ (P=0.012), CD8+ (P=0.003) and FOXP3+ T-lymphocytes (P=0.002) within the cancer cell nests and was associated with an elevated systemic inflammatory response as measured by modified Glasgow Prognostic Score (mGPS2: 19% vs. 4%, P=0.004). The combination of nuclear STAT3/pSTAT3 stratified five-year survival from 81% to 62% (P=0.012), however was not associated with survival independent of venous invasion, tumour perforation or tumour budding. Conclusion In patients undergoing CRC resection, STAT3 expression was associated with adverse host inflammatory responses and reduced survival. Up-regulation of tumour STAT3 may be an important mechanism whereby the tumour deregulates local and systemic inflammatory responses

Systems Approach to Human Hair Fibers: Interdependence Between Physical, Mechanical, Biochemical and Geometric Properties of Natural Healthy Hair

Contextual interpretation of hair fiber data is often blind to the effects of the dynamic complexity between different fiber properties. This intrinsic complexity requires systems thinking to decipher hair fiber accurately. Hair research, studied by various disciplines, follows a reductionist research approach, where elements of interest are studied from a local context with a certain amount of detachment from other elements or contexts. Following a systems approach, the authors are currently developing a cross-disciplinary taxonomy to provide a holistic view of fiber constituents and their interactions within large-scale dynamics. Based on the development process, this paper presents a review that explores the associated features, interrelationships and interactive complexities between physical, mechanical, biochemical and geometric features of natural, healthy hair fibers. Through the review, the importance of an appropriate taxonomy for interpreting hair fiber data across different disciplines is revealed. The review also demonstrates how seemingly unrelated fiber constituents are indeed interdependent and that these interdependencies may affect the behavior of the fiber. Finally, the review highlights how a non-integrative approach may have a negative impact on the reliability of hair data interpretation

Microevolution of Serial Clinical Isolates of Cryptococcus neoformans var. grubii and C. gattii

We thank the Broad Institute Sequencing Platform for generating the Illumina sequences. We thank Chen-Hsin Yu for helping on the data processing of the phenotypic tests. We acknowledge the South African National Institute for Communicable Diseases’ GERMS-SA surveillance network through which these isolates were originally collected. This project has been funded in whole or in part by the following U.S. Health and Human Services grants from the National Institute of Allergy and Infectious Diseases: U19 AI110818 (Broad Institute), R01 AI93257 (J.R.P.), R01 AI73896 (J.R.P.), and R01 AI025783 (T.G.M.). R.A.F. was supported by the Wellcome Trust. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The content is solely our responsibility and does not necessarily represent the official views of the funders. The use of product names in this manuscript does not imply their endorsement by the U.S. Department of Health and Human Services. The findings and conclusions in this article are those of the authors and do not necessarily represent the views of the CDC.Peer reviewedPublisher PD

A prospective cohort study of school-going children investigating reproductive and neurobehavioral health effects due to environmental pesticide exposure in the Western Cape, South Africa: study protocol

Abstract Background Research on reproductive health effects on children from low-level, long-term exposure to pesticides currently used in the agricultural industry is limited and those on neurobehavioral effects have produced conflicting evidence. We aim at investigating the association between pesticide exposure on the reproductive health and neurobehavior of children in South Africa, by including potential relevant co-exposures from the use of electronic media and maternal alcohol consumption. Methods The design entails a prospective cohort study with a follow-up duration of 2 years starting in 2017, including 1000 school going children between the ages of 9 to 16 years old. Children are enrolled with equal distribution in sex and residence on farms and non-farms in three different agricultural areas (mainly apple, table grapes and wheat farming systems) in the Western Cape, South Africa. The neurobehavior primary health outcome of cognitive functioning was measured through the iPad-based CAmbridge Neuropsychological Test Automated Battery (CANTAB) including domains for attention, memory, and processing speed. The reproductive health outcomes include testicular size in boys and breast size in girls assessed in a physical examination, and blood samples to detect hormone levels and anthropometric measurements. Information on pesticide exposure, co-exposures and relevant confounders are obtained through structured questionnaire interviews with the children and their guardians. Environmental occurrence of pesticides will be determined while using a structured interview with farm owners and review of spraying records and collection of passive water and air samples in all three areas. Pesticide metabolites will be analysed in urine and hair samples collected from the study subjects every 4 months starting at baseline. Discussion The inclusion of three different agricultural areas will yield a wide range of pesticide exposure situations. The prospective longitudinal design is a further strength of this study to evaluate the reproductive and neurobehavioural effects of different pesticides on children. This research will inform relevant policies and regulatory bodies to improve the health, safety and learning environments for children and families in agricultural settings