Evolution of New Catalytic Mechanisms for Xenobiotic Hydrolysis

Several different evolutionary processes allow enzymes to gain new catalytic activity, or enhance existing properties. Xenobiotic-degrading enzymes are often used to study the molecular basis for the evolution of new catalytic activity, as the substrates are often synthetic and comparatively new to the biosphere, the directionality of the evolutionary process is generally known and the evolved protein can often be traced back to a specific ancestral enzyme. However, the molecular basis behind the evolution of novel catalytic activities in different xenobiotic degrading enzymes is not well understood. In this work several different xenobiotic hydrolysing metalloenzymes were investigated to better understand new enzymatic reactions can evolve: the bacterial phosphotriesterases, triazine chlorohydrolases, and linuron/molinate hydrolases. These three enzyme families were studied using a combination of protein crystallography, enzyme kinetics, metal-ion dependence analysis, thermostability and solubility quantification, bioinformatics analyses, and critical analysis relevant literature. The conclusions that were drawn from these investigations cover fundamental concepts relating to enzymatic catalysis, including (1) the role of metal-ion cofactors in catalysis and the evolution of new function, (2) the desolvation of charged catalytic residues, the energetic cost of this desolvation and how this contributes to catalysis, and (3) the epistatic restrictions imposed on the evolution of new enzyme activity. Various smaller milestones were accomplished as a result of these investigations. The molecular structures of hydrolases for the herbicides molinate and linuron (molinate hydrolase and phenylurea hydrolase B) have been solved for the first time and, in combination with extensive kinetic and metal analysis, a catalytic mechanism has been proposed. This information will be useful for optimization of both enzymes for the bioremediation of herbicide-contaminated sites. Detailed information outlining the structural and catalytic basis of triazine hydrolase activity and solubility was also obtained. Work towards understanding the structural basis underlying how triazine hydrolase can accumulate in a soluble, active, form and catalyse the hydrolysis of triazine based herbicides will aid the future protein engineering of more stable or active variants. The activity of phosphotriesterase was improved 5000-fold with the most widely used pesticide in the US, malathion. This phosphotriesterase variant has great potential for bioremediation applications and also provides a useful case study into the role that epistasis plays in the engineering of improved enzyme function. Finally, future research ideas are proposed, using results described in this work

Evolutionary expansion of the amidohydrolase superfamily in bacteria in response to the synthetic compounds molinate and diuron

The amidohydrolase superfamily has remarkable functional diversity, with considerable structural and functional annotation of known sequences. In microbes, the recent evolution of several members of this family to catalyze the breakdown of environmental xenobiotics is not well understood. An evolutionary transition from binuclear to mononuclear metal ion coordination at the active sites of these enzymes could produce large functional changes such as those observed in nature, but there are few clear examples available to support this hypothesis. To investigate the role of binuclear-mononuclear active-site transitions in the evolution of new function in this superfamily, we have characterized two recently evolved enzymes that catalyze the hydrolysis of the synthetic herbicides molinate (MolA) and phenylurea (PuhB). In this work, the crystal structures, mutagenesis, metal ion analysis, and enzyme kinetics of both MolA and PuhB establish that these enzymes utilize a mononuclear active site. However, bioinformatics and structural comparisons reveal that the closest putative ancestor of these enzymes had a binuclear active site, indicating that a binuclear-mononuclear transition has occurred. These proteins may represent examples of evolution modifying the characteristics of existing catalysts to satisfy new requirements, specifically, metal ion rearrangement leading to large leaps in activity that would not otherwise be possible

The lid domain is important, but not essential, for catalysis of Escherichia coli pyruvate kinase

Pyruvate kinase catalyses the final step of the glycolytic pathway in central energy metabolism. The monomeric structure comprises three domains: a catalytic TIM-barrel, a regulatory domain involved in allosteric activation, and a lid domain that encloses the substrates. The lid domain is thought to close over the TIM-barrel domain forming contacts with the substrates to promote catalysis and may be involved in stabilising the activated state when the allosteric activator is bound. However, it remains unknown whether the lid domain is essential for pyruvate kinase catalytic or regulatory function. To address this, we removed the lid domain of Escherichia coli pyruvate kinase type 1 (PKTIM+Reg) using protein engineering. Biochemical analyses demonstrate that, despite the absence of key catalytic residues in the lid domain, PKTIM+Reg retains a low level of catalytic activity and has a reduced binding affinity for the substrate phosphoenolpyruvate. The enzyme retains allosteric activation, but the regulatory profile of the enzyme is changed relative to the wild-type enzyme. Analytical ultracentrifugation and small-angle X-ray scattering data show that, beyond the loss of the lid domain, the PKTIM+Reg structure is not significantly altered and is consistent with the wild-type tetramer that is assembled through interactions at the TIM and regulatory domains. Our results highlight the contribution of the lid domain for facilitating pyruvate kinase catalysis and regulation, which could aid in the development of small molecule inhibitors for pyruvate kinase and related lid-regulated enzymes

TRIM69 inhibits vesicular stomatitis Indiana virus (VSIV)

Vesicular stomatitis Indiana virus (VSIV), formerly known as vesicular stomatitis virus (VSV) Indiana (VSVIND), is a model virus that is exceptionally sensitive to the inhibitory action of interferons (IFNs). Interferons induce an antiviral state by stimulating the expression of hundreds of interferon-stimulated genes (ISGs). These ISGs can constrain viral replication, limit tissue tropism, reduce pathogenicity, and inhibit viral transmission. Since VSIV is used as a backbone for multiple oncolytic and vaccine strategies, understanding how ISGs restrict VSIV not only helps in understanding VSIV-induced pathogenesis but also helps us evaluate and understand the safety and efficacy of VSIV-based therapies. Thus, there is a need to identify and characterize the ISGs that possess anti-VSIV activity. Using arrayed ISG expression screening, we identified TRIM69 as an ISG that potently inhibits VSIV. This inhibition was highly specific as multiple viruses, including influenza A virus, HIV-1, Rift Valley fever virus, and dengue virus, were unaffected by TRIM69. Indeed, just one amino acid substitution in VSIV can govern sensitivity/resistance to TRIM69. Furthermore, TRIM69 is highly divergent in human populations and exhibits signatures of positive selection that are consistent with this gene playing a key role in antiviral immunity. We propose that TRIM69 is an IFN-induced inhibitor of VSIV and speculate that TRIM69 could be important in limiting VSIV pathogenesis and might influence the specificity and/or efficacy of vesiculovirus-based therapies

Selected marketing and human resources variables influencing sponsorship initiatives within corporate businesses :a South African perspective

In an ever changing world, consumers are less and less responsive to traditional advertising, which creates a challenge for marketers, as they need to constantly develop new marketing communication tools to make a lasting impression on current and potential consumers. Consumers of the twenty first century are described as emotionally acting individuals, and has led to a increase in creative and emotional marketing communication tools, for example sponsorship, which is an increasingly important element of an integrated marketing communication strategy. Because of the value sponsorship can have for a business, and the high costs involved in this marketing initiative, it is imperative to examine variables that might be influential, in order to apply this promotional activity to the business’ advantage (Belch & Belch 2007:12; Hartland, Skinner & Griffiths 2005:164-173; Nicholis, Roslow & Dublish 1999:365-387; Pham & Vanheule 1997:407-417). Because of the important effect of sponsorship, marketing managers seek clarity in respect of which events to support and to link their business’ brand with or not. There is however little guidance in literature available on which events to sponsor and how to exploit resources efficiently and effectively (Crimmins & Horn 1996; Speed & Thompson 2000). Therefore, the objective of this study is to identify factors that might influence sponsorship initiatives within corporate businesses in South Africa. The variables that was identified for the study at hand was marketing related variables namely branding, marketing ethics and green marketing, and human resources related variables namely, corporate culture, values and employee empowerment. In order to determine the influence of the predetermined variables on sponsorship, an empirical investigation was conducted. The positivistic (phenomenological) approach was used in this study as the aim was to determine whether a relationship exists between selected independent variables and the dependent variable, via the intervening variables, using statistical analysis. In order to gather primary data, selfadministered questionnaires were issued to 182 respondents by means of convenience and snowball sampling of which the results were analysed to arrive at conclusions regarding the research in question. The empirical analysis of the data followed the following statistical steps: exploratory factor analysis to test the validity of the measuring instrument, Cronbach Alpha correlation coefficients to confirm the reliability of the questionnaire, SEM goodness-of-fit, multiple regression analysis to test the hypothesised relationships between the independent, intervening and dependent variables, ANOVA and descriptive statistics. The main findings of this study suggest that branding and green marketing have a significantly positive relationship with sponsorship, indicating the importance of a business’ brand and environmental awareness in terms of its emotional marketing initiatives. These relationships iIn an ever changing world, consumers are less and less responsive to traditional advertising, which creates a challenge for marketers, as they need to constantly develop new marketing communication tools to make a lasting impression on current and potential consumers. Consumers of the twenty first century are described as emotionally acting individuals, and has led to a increase in creative and emotional marketing communication tools, for example sponsorship, which is an increasingly important element of an integrated marketing communication strategy. Because of the value sponsorship can have for a business, and the high costs involved in this marketing initiative, it is imperative to examine variables that might be influential, in order to apply this promotional activity to the business’ advantage (Belch & Belch 2007:12; Hartland, Skinner & Griffiths 2005:164-173; Nicholis, Roslow & Dublish 1999:365-387; Pham & Vanheule 1997:407-417). Because of the important effect of sponsorship, marketing managers seek clarity in respect of which events to support and to link their business’ brand with or not. There is however little guidance in literature available on which events to sponsor and how to exploit resources efficiently and effectively (Crimmins & Horn 1996; Speed & Thompson 2000). Therefore, the objective of this study is to identify factors that might influence sponsorship initiatives within corporate businesses in South Africa. The variables that was identified for the study at hand was marketing related variables namely branding, marketing ethics and green marketing, and human resources related variables namely, corporate culture, values and employee empowerment. In order to determine the influence of the predetermined variables on sponsorship, an empirical investigation was conducted. The positivistic (phenomenological) approach was used in this study as the aim was to determine whether a relationship exists between selected independent variables and the dependent variable, via the intervening variables, using statistical analysis. In order to gather primary data, selfadministered questionnaires were issued to 182 respondents by means of convenience and snowball sampling of which the results were analysed to arrive at conclusions regarding the research in question. The empirical analysis of the data followed the following statistical steps: exploratory factor analysis to test the validity of the measuring instrument, Cronbach Alpha correlation coefficients to confirm the reliability of the questionnaire, SEM goodness-of-fit, multiple regression analysis to test the hypothesised relationships between the independent, intervening and dependent variables, ANOVA and descriptive statistics. The main findings of this study suggest that branding and green marketing have a significantly positive relationship with sponsorship, indicating the importance of a business’ brand and environmental awareness in terms of its emotional marketing initiatives. These relationships imply that, according to respondents, if these two aspects improve within the business, so could its sponsorship initiatives. Interestingly, it was established that ethics was insignificant in terms of a business’sponsorship initiatives. Corporate culture and values had a positive relationship with sponsorship, and employee empowerment proved to be negatively related to Sport and Broadcast sponsorship, with no significant relationship with Education and Community sponsorship. Additionally, the empirical investigation revealed that the ethnicity of respondents exerted an influence on the perception employees have regarding ethics, employee empowerment and Sport and Broadcast sponsorship within the businesses they are employed at. As this study assisted in the development of sponsorship strategies for businesses to implement, it will have a direct benefit to marketers and businesses in general so that sponsorship initiatives can be directed in such a way to maximise the return on investment for a business. The implications of this study will be of great value to marketing managers as sponsorship is such an important marketing strategy and communications tool which impacts on the overall business objectives.mply that, according to respondents, if these two aspects improve within the business, so could its sponsorship initiatives. Interestingly, it was established that ethics was insignificant in terms of a business’ sponsorship initiatives. Corporate culture and values had a positive relationship with sponsorship, and employee empowerment proved to be negatively related to Sport and Broadcast sponsorship, with no significant relationship with Education and Community sponsorship. Additionally, the empirical investigation revealed that the ethnicity of respondents exerted an influence on the perception employees have regarding ethics, employee empowerment and Sport and Broadcast sponsorship within the businesses they are employed at. As this study assisted in the development of sponsorship strategies for businesses to implement, it will have a direct benefit to marketers and businesses in general so that sponsorship initiatives can be directed in such a way to maximise the return on investment for a business. The implications of this study will be of great value to marketing managers as sponsorship is such an important marketing strategy and communications tool which impacts on the overall business objectives

Resurrection of 2′-5′-oligoadenylate synthetase 1 (OAS1) from the ancestor of modern horseshoe bats blocks SARS-CoV-2 replication

The prenylated form of the human 2′-5′-oligoadenylate synthetase 1 (OAS1) protein has been shown to potently inhibit the replication of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the virus responsible for the Coronavirus Disease 2019 (COVID-19) pandemic. However, the OAS1 orthologue in the horseshoe bats (superfamily Rhinolophoidea), the reservoir host of SARS-related coronaviruses (SARSr-CoVs), has lost the prenylation signal required for this antiviral activity. Herein, we used an ancestral state reconstruction approach to predict and reconstitute in vitro, the most likely OAS1 protein sequence expressed by the Rhinolophoidea common ancestor prior to its prenylation loss (RhinoCA OAS1). We exogenously expressed the ancient bat protein in vitro to show that, unlike its non-prenylated horseshoe bat descendants, RhinoCA OAS1 successfully blocks SARS-CoV-2 replication. Using protein structure predictions in combination with evolutionary hypothesis testing methods, we highlight sites under unique diversifying selection specific to OAS1’s evolution in the Rhinolophoidea. These sites are located near the RNA-binding region and the C-terminal end of the protein where the prenylation signal would have been. Our results confirm that OAS1 prenylation loss at the base of the Rhinolophoidea clade ablated the ability of OAS1 to restrict SARSr-CoV replication and that subsequent evolution of the gene in these bats likely favoured an alternative function. These findings can advance our understanding of the tightly linked association between SARSr-CoVs and horseshoe bats

The apparent interferon resistance of transmitted HIV-1 is possibly a consequence of enhanced replicative fitness

HIV-1 transmission via sexual exposure is an inefficient process. When transmission does occur, newly infected individuals are colonized by the descendants of either a single virion or a very small number of establishing virions. These transmitted founder (TF) viruses are more interferon (IFN)-resistant than chronic control (CC) viruses present 6 months after transmission. To identify the specific molecular defences that make CC viruses more susceptible to the IFN-induced ‘antiviral state’, we established a single pair of fluorescent TF and CC viruses and used arrayed interferon-stimulated gene (ISG) expression screening to identify candidate antiviral effectors. However, we observed a relatively uniform ISG resistance of transmitted HIV-1, and this directed us to investigate possible underlying mechanisms. Simple simulations, where we varied a single parameter, illustrated that reduced growth rate could possibly underly apparent interferon sensitivity. To examine this possibility, we closely monitored in vitro propagation of a model TF/CC pair (closely matched in replicative fitness) over a targeted range of IFN concentrations. Fitting standard four-parameter logistic growth models, in which experimental variables were regressed against growth rate and carrying capacity, to our in vitro growth curves, further highlighted that small differences in replicative growth rates could recapitulate our in vitro observations. We reasoned that if growth rate underlies apparent interferon resistance, transmitted HIV-1 would be similarly resistant to any growth rate inhibitor. Accordingly, we show that two transmitted founder HIV-1 viruses are relatively resistant to antiretroviral drugs, while their matched chronic control viruses were more sensitive. We propose that, when present, the apparent IFN resistance of transmitted HIV-1 could possibly be explained by enhanced replicative fitness, as opposed to specific resistance to individual IFN-induced defences. However, further work is required to establish how generalisable this mechanism of relative IFN resistance might be

Intraoperative surgical site infection control and prevention : a position paper and future addendum to WSES intra-abdominal infections guidelines

Correction: Volume: 16 Issue: 1, Article Number: 18 DOI: 10.1186/s13017-021-00361-4Background Surgical site infections (SSI) represent a considerable burden for healthcare systems. They are largely preventable and multiple interventions have been proposed over past years in an attempt to prevent SSI. We aim to provide a position paper on Operative Room (OR) prevention of SSI in patients presenting with intra-abdominal infection to be considered a future addendum to the well-known World Society of Emergency Surgery (WSES) Guidelines on the management of intra-abdominal infections. Methods The literature was searched for focused publications on SSI until March 2019. Critical analysis and grading of the literature has been performed by a working group of experts; the literature review and the statements were evaluated by a Steering Committee of the WSES. Results Wound protectors and antibacterial sutures seem to have effective roles to prevent SSI in intra-abdominal infections. The application of negative-pressure wound therapy in preventing SSI can be useful in reducing postoperative wound complications. It is important to pursue normothermia with the available resources in the intraoperative period to decrease SSI rate. The optimal knowledge of the pharmacokinetic/pharmacodynamic characteristics of antibiotics helps to decide when additional intraoperative antibiotic doses should be administered in patients with intra-abdominal infections undergoing emergency surgery to prevent SSI. Conclusions The current position paper offers an extensive overview of the available evidence regarding surgical site infection control and prevention in patients having intra-abdominal infections.Peer reviewe

A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research

The recent emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the underlying cause of Coronavirus Disease 2019 (COVID-19), has led to a worldwide pandemic causing substantial morbidity, mortality, and economic devastation. In response, many laboratories have redirected attention to SARS-CoV-2, meaning there is an urgent need for tools that can be used in laboratories unaccustomed to working with coronaviruses. Here we report a range of tools for SARS-CoV-2 research. First, we describe a facile single plasmid SARS-CoV-2 reverse genetics system that is simple to genetically manipulate and can be used to rescue infectious virus through transient transfection (without in vitro transcription or additional expression plasmids). The rescue system is accompanied by our panel of SARS-CoV-2 antibodies (against nearly every viral protein), SARS-CoV-2 clinical isolates, and SARS-CoV-2 permissive cell lines, which are all openly available to the scientific community. Using these tools, we demonstrate here that the controversial ORF10 protein is expressed in infected cells. Furthermore, we show that the promising repurposed antiviral activity of apilimod is dependent on TMPRSS2 expression. Altogether, our SARS-CoV-2 toolkit, which can be directly accessed via our website at https://mrcppu-covid.bio/, constitutes a resource with considerable potential to advance COVID-19 vaccine design, drug testing, and discovery science