A cellular automaton model of crystalline cellulose hydrolysis by cellulases

<p>Abstract</p> <p>Background</p> <p>Cellulose from plant biomass is an abundant, renewable material which could be a major feedstock for low emissions transport fuels such as cellulosic ethanol. Cellulase enzymes that break down cellulose into fermentable sugars are composed of different types - cellobiohydrolases I and II, endoglucanase and β-glucosidase - with separate functions. They form a complex interacting network between themselves, soluble hydrolysis product molecules, solution and solid phase substrates and inhibitors. There have been many models proposed for enzymatic saccharification however none have yet employed a cellular automaton approach, which allows important phenomena, such as enzyme crowding on the surface of solid substrates, denaturation and substrate inhibition, to be considered in the model.</p> <p>Results</p> <p>The Cellulase 4D model was developed <it>de novo </it>taking into account the size and composition of the substrate and surface-acting enzymes were ascribed behaviors based on their movements, catalytic activities and rates, affinity for, and potential for crowding of, the cellulose surface, substrates and inhibitors, and denaturation rates. A basic case modeled on literature-derived parameters obtained from <it>Trichoderma reesei </it>cellulases resulted in cellulose hydrolysis curves that closely matched curves obtained from published experimental data. Scenarios were tested in the model, which included variation of enzyme loadings, adsorption strengths of surface acting enzymes and reaction periods, and the effect on saccharide production over time was assessed. The model simulations indicated an optimal enzyme loading of between 0.5 and 2 of the base case concentrations where a balance was obtained between enzyme crowding on the cellulose crystal, and that the affinities of enzymes for the cellulose surface had a large effect on cellulose hydrolysis. In addition, improvements to the cellobiohydrolase I activity period substantially improved overall glucose production.</p> <p>Conclusions</p> <p>Cellulase 4D simulates the enzymatic hydrolysis of cellulose to glucose by surface and solution phase-acting enzymes and accounts for complex phenomena that have previously not been included in cellulose hydrolysis models. The model is intended as a tool for industry, researchers and educators alike to explore options for enzyme engineering and process development and to test hypotheses regarding cellulase mechanisms.</p

Progressive skeletal benefits of physical activity when young as assessed at the midshaft humerus in male baseball players

Physical activity benefits the skeleton, but there is contrasting evidence regarding whether benefits differ at different stages of growth. The current study demonstrates that physical activity should be encouraged at the earliest age possible and be continued into early adulthood to gain most skeletal benefits. INTRODUCTION: The current study explored physical activity-induced bone adaptation at different stages of somatic maturity by comparing side-to-side differences in midshaft humerus properties between male throwing athletes and controls. Throwers present an internally controlled model, while inclusion of control subjects removes normal arm dominance influences. METHODS: Throwing athletes (n = 90) and controls (n = 51) were categorized into maturity groups (pre, peri, post-early, post-mid, and post-late) based on estimated years from peak height velocity (10 years). Side-to-side percent differences in midshaft humerus cortical volumetric bone mineral density (Ct.vBMD) and bone mineral content (Ct.BMC); total (Tt.Ar), medullary (Me.Ar), and cortical (Ct.Ar) areas; average cortical thickness (Ct.Th); and polar Strength Strain Index (SSIP) were assessed. RESULTS: Significant interactions between physical activity and maturity on side-to-side differences in Ct.BMC, Tt.Ar, Ct.Ar, Me.Ar, Ct.Th, and SSIP resulted from the following: (1) greater throwing-to-nonthrowing arm differences than dominant-to-nondominant arm differences in controls (all p < 0.05) and (2) throwing-to-nonthrowing arm differences in throwers being progressively greater across maturity groups (all p < 0.05). Regional analyses revealed greatest adaptation in medial and lateral sectors, particularly in the three post-maturity groups. Years throwing predicted 59% of the variance of the variance in throwing-to-nonthrowing arm difference in SSIP (p < 0.001). CONCLUSION: These data suggest that physical activity has skeletal benefits beginning prior to and continuing beyond somatic maturation and that a longer duration of exposure to physical activity has cumulative skeletal benefits. Thus, physical activity should be encouraged at the earliest age possible and be continued into early adulthood to optimize skeletal benefits

The Redox Cofactor F-420 Protects Mycobacteria from Diverse Antimicrobial Compounds and Mediates a Reductive Detoxification System

A defining feature of mycobacterial redox metabolism is the use of an unusual deazaflavin cofactor, F420. This cofactor enhances the persistence of environmental and pathogenic mycobacteria, including after antimicrobial treatment, although the molecular basis for this remains to be understood. In this work, we explored our hypothesis that F420 enhances persistence by serving as a cofactor in antimicrobial-detoxifying enzymes. To test this, we performed a series of phenotypic, biochemical, and analytical chemistry studies in relation to the model soil bacterium Mycobacterium smegmatis. Mutant strains unable to synthesize or reduce F420 were found to be more susceptible to a wide range of antibiotic and xenobiotic compounds. Compounds from three classes of antimicrobial compounds traditionally resisted by mycobacteria inhibited the growth of F420 mutant strains at subnanomolar concentrations, namely, furanocoumarins (e.g., methoxsalen), arylmethanes (e.g., malachite green), and quinone analogues (e.g., menadione). We demonstrated that promiscuous F420H2-dependent reductases directly reduce these compounds by a mechanism consistent with hydride transfer. Moreover, M. smegmatis strains unable to make F420H2 lost the capacity to reduce and detoxify representatives of the furanocoumarin and arylmethane compound classes in whole-cell assays. In contrast, mutant strains were only slightly more susceptible to clinical antimycobacterials, and this appeared to be due to indirect effects of F420 loss of function (e.g., redox imbalance) rather than loss of a detoxification system. Together, these data show that F420 enhances antimicrobial resistance in mycobacteria and suggest that one function of the F420H2-dependent reductases is to broaden the range of natural products that mycobacteria and possibly other environmental actinobacteria can reductively detoxify

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

Cofactor F420-dependent enzymes: An under-explored resource for asymmetric redox biocatalysis

The asymmetric reduction of enoates, imines and ketones are among the most important reactions in biocatalysis. These reactions are routinely conducted using enzymes that use nicotinamide cofactors as reductants. The deazaflavin cofactor F420 also has electrochemical properties that make it suitable as an alternative to nicotinamide cofactors for use in asymmetric reduction reactions. However, cofactor F420-dependent enzymes remain under-explored as a resource for biocatalysis. This review considers the cofactor F420-dependent enzyme families with the greatest potential for the discovery of new biocatalysts: the flavin/deazaflavin-dependent oxidoreductases (FDORs) and the luciferase-like hydride transferases (LLHTs). The characterized F420-dependent reductions that have the potential for adaptation for biocatalysis are discussed, and the enzymes best suited for use in the reduction of oxidized cofactor F420 to allow cofactor recycling in situ are considered. Further discussed are the recent advances in the production of cofactor F420 and its functional analog FO-5′-phosphate, which remains an impediment to the adoption of this family of enzymes for industrial biocatalytic processes. Finally, the prospects for the use of this cofactor and dependent enzymes as a resource for industrial biocatalysis are discussed

Mycobacterial F420H2-dependent reductases promiscuously reduce diverse compounds through a common mechanism

An unusual aspect of actinobacterial metabolism is the use of the redox cofactor F420. Studies have shown that actinobacterial F420H2-dependent reductases promiscuously hydrogenate diverse organic compounds in biodegradative and biosynthetic processes. These enzymes therefore represent promising candidates for next-generation industrial biocatalysts. In this work, we undertook the first broad survey of these enzymes as potential industrial biocatalysts by exploring the extent, as well as mechanistic and structural bases, of their substrate promiscuity. We expressed and purified 11 enzymes from seven subgroups of the flavin/deazaflavin oxidoreductase (FDOR) superfamily (A1, A2, A3, B1, B2, B3, B4) from the model soil actinobacterium Mycobacterium smegmatis. These enzymes reduced compounds from six chemical classes, including fundamental monocycles such as a cyclohexenone, a dihydropyran, and pyrones, as well as more complex quinone, coumarin, and arylmethane compounds. Substrate range and reduction rates varied between the enzymes, with the A1, A3, and B1 groups exhibiting greatest promiscuity. Molecular docking studies suggested that structurally diverse compounds are accommodated in the large substrate-binding pocket of the most promiscuous FDOR through hydrophobic interactions with conserved aromatic residues and the isoalloxazine headgroup of F420H2. Liquid chromatography-mass spectrometry (LC/MS) and gas chromatography-mass spectrometry (GC/MS) analysis of derivatized reaction products showed reduction occurred through a common mechanism involving hydride transfer from F420H- to the electron-deficient alkene groups of substrates. Reduction occurs when the hydride donor (C5 of F420H-) is proximal to the acceptor (electrophilic alkene of the substrate). These findings suggest that engineered actinobacterial F420H2-dependent reductases are promising novel biocatalysts for the facile transformation of a wide range of α,β-unsaturated compounds.This work was supported by a CSIRO Office of the Chief Executive Postdoctoral Fellowship and an ARC DECRA DE120102673

Cofactor Tail Length Modulates Catalysis of Bacterial F420-Dependent Oxidoreductases

F420 is a microbial cofactor that mediates a wide range of physiologically important and industrially relevant redox reactions, including in methanogenesis and tetracycline biosynthesis. This deazaflavin comprises a redox-active isoalloxazine headgroup conjugated to a lactyloligoglutamyl tail. Here we studied the catalytic significance of the oligoglutamate chain, which differs in length between bacteria and archaea. We purified short-chain F420 (two glutamates) from a methanogen isolate and long-chain F420 (five to eight glutamates) from a recombinant mycobacterium, confirming their different chain lengths by HPLC and LC/MS analysis. F420 purified from both sources was catalytically compatible with purified enzymes from the three major bacterial families of F420-dependent oxidoreductases. However, long-chain F420 bound to these enzymes with a six- to ten-fold higher affinity than short-chain F420. The cofactor side chain also significantly modulated the kinetics of the enzymes, with long-chain F420 increasing the substrate affinity (lower Km) but reducing the turnover rate (lower kcat) of the enzymes. Molecular dynamics simulations and comparative structural analysis suggest that the oligoglutamate chain of F420 makes dynamic electrostatic interactions with conserved surface residues of the oxidoreductases while the headgroup binds the catalytic site. In conjunction with the kinetic data, this suggests that electrostatic interactions made by the oligoglutamate tail result in higher-affinity, lower-turnover catalysis. Physiologically, we propose that bacteria have selected for long-chain F420 to better control cellular redox reactions despite tradeoffs in catalytic rate. Conversely, this suggests that industrial use of shorter-length F420 will greatly increase the rates of bioremediation and biocatalysis processes relying on purified F420-dependent oxidoreductases

F420H2-Dependent Degradation of Aflatoxin and other Furanocoumarins Is Widespread throughout the Actinomycetales

Two classes of F420-dependent reductases (FDR-A and FDR-B) that can reduce aflatoxins and thereby degrade them have previously been isolated from Mycobacterium smegmatis. One class, the FDR-A enzymes, has up to 100 times more activity than the other. F420 is a cofactor with a low reduction potential that is largely confined to the Actinomycetales and some Archaea and Proteobacteria. We have heterologously expressed ten FDR-A enzymes from diverse Actinomycetales, finding that nine can also use F420H2 to reduce aflatoxin. Thus FDR-As may be responsible for the previously observed degradation of aflatoxin in other Actinomycetales. The one FDR-A enzyme that we found not to reduce aflatoxin belonged to a distinct clade (herein denoted FDR-AA), and our subsequent expression and analysis of seven other FDR-AAs from M. smegmatis found that none could reduce aflatoxin. Certain FDR-A and FDR-B enzymes that could reduce aflatoxin also showed activity with coumarin and three furanocoumarins (angelicin, 8-methoxysporalen and imperatorin), but none of the FDR-AAs tested showed any of these activities. The shared feature of the compounds that were substrates was an α,β-unsaturated lactone moiety. This moiety occurs in a wide variety of otherwise recalcitrant xenobiotics and antibiotics, so the FDR-As and FDR-Bs may have evolved to harness the reducing power of F420 to metabolise such compounds. Mass spectrometry on the products of the FDR-catalyzed reduction of coumarin and the other furanocoumarins shows their spontaneous hydrolysis to multiple products

Mendelian adult-onset leukodystrophy genes in Alzheimer´s disease. Critical influence of CSF1R and NOTCH3

Mendelian adult-onset leukodystrophies are a spectrum of rare inherited progressive neurodegenerative disorders affecting the white matter of the central nervous system. Among these, Cerebral Autosomal Dominant and Recessive Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL and CARASIL), Cerebroretinal vasculopathy (CRV), Metachromatic leukodystrophy (MLD), Hereditary diffuse Leukoencephalopathy with spheroids (HDLS), Vanishing white matter disease (VWM) present with rapidly progressive dementia as dominant feature and are caused by mutations in NOTCH3, HTRA1, TREX1, ARSA, CSF1R, EIF2B1, EIF2B2, EIF2B3, EIF2B4, EIF2B5, respectively. Given the rare incidence of these disorders and the lack of unequivocally diagnostic features, leukodystrophies are frequently misdiagnosed with common sporadic dementing diseases such as Alzheimer’s disease (AD), raising the question of whether these overlapping phenotypes may be explained by shared genetic risk factors. To investigate this intriguing hypothesis, we have combined gene expression analysis 1) in 6 different AD mouse strains (APPPS1, HOTASTPM, HETASTPM, TPM, TAS10 and TAU), at 5 different developmental stages (Embryo [E15], 2 months, 4 months, 8 months and 18 months), 2) in APPPS1 primary cortical neurons under stress conditions (oxygen-glucose deprivation) and single-variant and single-gene (c-alpha and SKAT tests) based genetic screening in a cohort composed of 332 Caucasian late-onset AD patients and 676 Caucasian elderly controls. Csf1r was significantly overexpressed (Log2FC>1, adj. p-val<0.05) in the cortex and hippocampus of aged HOTASTPM mice with extensive Aβ core dense plaque pathology. We identified 3 likely pathogenic mutations in CSF1R TK domain (p.L868R, p.Q691H and p.H703Y) in our discovery and validation cohort, composed of 465 AD and MCI Caucasian patients from the UK. Moreover, NOTCH3 was a significant hit in the c-alpha test (adj p-val = 0.01). Adult onset Mendelian leukodystrophy genes are not common factors implicated in AD. Nevertheless, our study suggests a potential pathogenic link between NOTCH3, CSF1R and sporadic LOAD, that warrants further investigation