Analysis of the galactomannan binding ability of ß-mannosidases, BtMan2A and CmMan5A, regarding their activity and synergism with a ß-mannanase.

Both ß-mannanases and ß-mannosidases are required for mannan-backbone degradation into mannose. In this study, two ß-mannosidases of glycoside hydrolase (GH) families 2 (BtMan2A) and 5 (CmMan5A) were evaluated for their substrate specificities and galactomannan binding ability. BtMan2A preferred short manno-oligomers, while CmMan5A preferred longer ones; DP >2, and galactomannans. BtMan2A displayed irreversible galactomannan binding, which was pH-dependent, with higher binding observed at low pH, while CmMan5A had limited binding. Docking and molecular dynamics (MD) simulations showed that BtMan2A galactomannan binding was stronger under acidic conditions (-8.4 kcal/mol) than in a neutral environment (-7.6 kcal/mol), and the galactomannan ligand was more unstable under neutral conditions than acidic conditions. Qualitative surface plasmon resonance (SPR) experimentally confirmed the reduced binding capacity of BtMan2A at pH 7. Finally, synergistic ß-mannanase to ß-mannosidase (BtMan2A or CmMan5A) ratios required for maximal galactomannan hydrolysis were determined. All CcManA to CmMan5A combinations were synergistic (≈1.2-fold), while combinations of CcManA with BtMan2A (≈1.0-fold) yielded no hydrolysis improvement. In conclusion, the low specific activity of BtMan2A towards long and galactose-containing oligomers and its non-catalytic galactomannan binding ability led to no synergy with the mannanase, making GH2 mannosidases ineffective for use in cocktails for mannan degradation.

Identification of a subgroup of black South Africans with type 1 diabetes who are older at diagnosis but have lower levels of glutamic acid decarboxylase and islet antigen 2 autoantibodies.

AIMS: To compare the age at diagnosis and prevalence of islet autoantibody [glutamic acid decarboxylase (65 kDa) 65 and islet antigen 2] positivity in black and white participants with type 1 diabetes in South Africa, and to analyse the relationship between age at diagnosis and the presence of autoantibodies. METHODS: Participants were recruited from diabetes outpatient departments and autoantibodies to glutamic acid decarboxylase (65 kDa) and islet antigen 2 were measured by enzyme-linked immunosorbent assay. RESULTS: We recruited 472 (353 black and 119 white) participants with type 1 diabetes. Age at diagnosis of diabetes was later in black (19.7 ± 10.5) than in white participants (12.7 ± 10.8 years; P < 0.001) with a median (interquartile range) disease duration of 5.0 (2.0-10.0) and 8.5 (4.0-20.0) years (P < 0.001), respectively. An older age at diagnosis (≥ 21 years) was more frequent in black (152 of 340, 45%) than in white participants (24 of 116, 21%; P < 0.001). The prevalence of islet antigen 2 autoantibodies was 19% (66/352) in black and 41% in white participants (48/118; P < 0.001). There was no significant difference in glutamic acid decarboxylase (65 kDa) autoantibody positivity between black (212/353, 60%) and white participants (77/117, 66%; P = 0.269). In black, but not white, participants the prevalence of both glutamic acid decarboxylase (65 kDa) and islet antigen 2 autoantibody positivity was significantly lower in participants diagnosed at age ≥ 21 years (P < 0.001 for both comparisons). CONCLUSIONS: The older age at diagnosis, lower prevalence of islet antigen 2 autoantibodies and a distinct subgroup of participants with type 1 diabetes with age at diagnosis of > 20 years in the black compared to white population suggest a difference in the immunological aetiology of type 1 diabetes in these two population groups.

Protein and fat meal content increase insulin requirement in children with type 1 diabetes - Role of duration of diabetes.

BACKGROUND AND OBJECTIVE: Hyperglycaemia remains a challenge in type 1 diabetes since current regimes used to determine meal insulin requirements prove to be ineffective. This is particularly problematic for meals containing high amounts of protein and fat. We aimed to determine the post-prandial glycaemic response and total insulin need for mixed meals, using sensor-augmented insulin pumps in children with type 1 diabetes. METHODS: Twenty-two children with type 1 diabetes, aged 4-17 years on insulin pump therapy completed this home-based, cross-over, randomised controlled trial. Two meals with identical carbohydrate content - one with low fat and protein (LFLP) and one with high fat and protein (HFHP) contents - were consumed using normal insulin boluses. Blood glucose monitoring was done for 10 h post-meal, with correction bolus insulin given two-hourly if required. RESULTS: The HFHP meal required significantly more total insulin (3.48 vs. 2.7 units) as a result of increased post-meal correction insulin requirement (1.2 vs. 0.15 units) spread over a longer duration (6 vs. 3 h). The HFHP meals significantly increased the time spent above target glucose level. Duration of diabetes and total daily insulin use significantly influenced the post-prandial blood glucose response to the two meals. CONCLUSION: When consuming carbohydrate-based mixed meals, children with type 1 diabetes on insulin pump therapy, required significantly more insulin over a longer period of time than the insulin requirement calculated using current regimes. This additional amount required is influenced by the duration of diabetes and total daily insulin use.

Formulation of an optimized synergistic enzyme cocktail, HoloMix, for effective degradation of various pre-treated hardwoods.

In this study, two selected hardwoods were subjected to sodium chlorite delignification and steam explosion, and the impact of pre-treatments on synergistic enzymatic saccharification evaluated. A cellulolytic core-set, CelMix, and a xylanolytic core-set, XynMix, optimised for glucose and xylose release, respectively, were used to formulate HoloMix cocktail for optimal saccharification of various pre-treated hardwoods. For delignified biomass, the optimized HoloMix consisted of 75%:25% protein dosage, CelMix: XynMix, while for untreated and steam exploded biomass the HoloMix consisted of 93.75%:6.25% protein dosage. Saccharification by HoloMix (27.5mgprotein/gbiomass) for 24h achieved 70-100% sugar yields. Pre-treatment of the hardwoods (especially those with a higher proportion of lignin) with a laccase, improved saccharification by HoloMix. This study provided insights into enzymatic hydrolysis of various pre-treated hardwood substrates and showed the same lignocellulolytic cocktail comparable to/if not better than commercial enzyme preparations can be used to efficiently hydrolyse different hardwood species.

Using an artificial neural network to predict the optimal conditions for enzymatic hydrolysis of apple pomace.

The enzymatic degradation of lignocellulosic biomass such as apple pomace is a complex process influenced by a number of hydrolysis conditions. Predicting optimal conditions, including enzyme and substrate concentration, temperature and pH can improve conversion efficiency. In this study, the production of sugar monomers from apple pomace using commercial enzyme preparations, Celluclast 1.5L, Viscozyme L and Novozyme 188 was investigated. A limited number of experiments were carried out and then analysed using an artificial neural network (ANN) to model the enzymatic hydrolysis process. The ANN was used to simulate the enzymatic hydrolysis process for a range of input variables and the optimal conditions were successfully selected as was indicated by the R 2 value of 0.99 and a small MSE value. The inputs for the ANN were substrate loading, enzyme loading, temperature, initial pH and a combination of these parameters, while release profiles of glucose and reducing sugars were the outputs. Enzyme loadings of 0.5 and 0.2 mg/g substrate and a substrate loading of 30% were optimal for glucose and reducing sugar release from apple pomace, respectively, resulting in concentrations of 6.5 g/L glucose and 28.9 g/L reducing sugars. Apple pomace hydrolysis can be successfully carried out based on the predicted optimal conditions from the ANN.

Time dependence of enzyme synergism during the degradation of model and natural lignocellulosic substrates.

Cellulosic ethanol production relies on the biochemical (enzymatic) conversion of lignocellulose to fermentable sugars and ultimately to bioethanol. However, the cost of lignocellulolytic enzymes is a limiting factor in the commercialisation of this technology. This therefore necessitates the optimisation of lignocellulolytic enzyme cocktails through the elucidation of synergistic interactions between enzymes so as to improve lignocellulose hydrolysis and also lower protein loadings in these reactions. However, many factors affect the synergism that occurs between these lignocellulolytic enzymes, such as enzyme ratios, substrate characteristics, substrate loadings, enzyme loadings and time. This review examines the effect of time on the synergistic dynamics between lignocellulolytic enzymes during the hydrolysis of both complex (true) lignocellulosic substrates and model substrates. The effect of sequential and simultaneous application of the lignocellulolytic enzymes on the synergistic dynamics during the hydrolysis of these substrates is also explored in this review. Finally, approaches are further proposed for efficient and synergistic hydrolysis of both complex lignocellulosic substrates and model substrates. With respect to the synergistic enzymatic hydrolysis of lignocellulosic biomass, this review exposed knowledge gaps that should be covered in future work in order to fully understand how enzyme synergism works: e.g. elucidating protein to protein interactions that exist between these enzymes in establishing synergy; and the effect of lignocellulose degradation products of one enzyme on the behaviour of the other enzyme and ultimately their synergistic relationship.

Analysis of Global Radiotherapy Needs and Costs by Geographic Region and Income Level.

Recent years have seen various reviews on the lack of access to radiotherapy often based on geographic regions of the world such as Africa, Asia Pacific, Europe, Latin America and North America. Countries are often defined by their national income per capita levels based on World Bank definitions of high income, upper middle income, lower middle income and low income. Within the world regions, there are significant variations in gross national income (GNI) per capita among the different countries, and even within similar income levels, large variations exist. This report presents the actual status of radiotherapy and analyses the current needs and costs to provide full access in the different regions of the world. Actual coverage of the needs ranges from 34% in Africa to over 92% in Europe to about double the needs in North America. In line with this, proportional additional investments and operational costs are as high as more than 200% in Africa to almost none in North America. Two world regions face substantial challenges: Africa, based on the important demands to build new capacity and subsequently to maintain operational capability; and Asia Pacific, due to its high population density, translating into large absolute needs in radiotherapy treatments and resources, and hence in associated costs. With the data highlighting a large variability of GNI/capita even within similar income levels in the various world regions, it is expected that additional investment in resources and costs may be more dependent on income level of the country than on the GNI group or the geographic region of the world.

Mobilising Expertise and Resources to Close the Radiotherapy Gap in Cancer Care.

Closing the gap in cancer care within low- and middle-income countries and in indigenous and geographically isolated populations in high-income countries requires investment and innovation. This is particularly true for radiotherapy, for which the global disparity is one of the largest in healthcare today. New models and paradigms and non-traditional collaborations have been proposed to improve global equity in cancer control. We describe recent initiatives from within the radiation oncology community to increase access to treatment, build the low- and middle-income countries' radiation oncology workforce, mobilise more professionals from within high-income countries and raise awareness of the global need for equitable cancer care.

Use of naloxone to minimize extubation failure after premedication for INSURE procedure in preterm neonates.

OBJECTIVES: A new guideline for the early respiratory management of preterm infants that included early nCPAP and INSURE was recently introduced in our NICU. This case series describes the clinical courses of a group of preterm infants managed according to this guideline, and reports the rates of successful extubation within 30 minutes of surfactant administration with and without the use of naloxone and adverse events encountered. STUDY DESIGN: Descriptive case series of all preterm babies admitted to our unit who were candidates for INSURE procedure with premedication from August 2012 to August 2013. RESULTS: A total of 31 infants were included with a mean birth weight of 1178 grams and a mean gestational age of 28.4 weeks. Twelve out of thirteen (92%) infants in the naloxone group were extubated within 30 minutes of surfactant administration while only 12/18 (67%) in the non-naloxone group were extubated within the same time frame. No adverse reactions were noted with naloxone usage in this context. CONCLUSION: Naloxone can be effective in reversing the respiratory depressive effect of analgesic premedication and in turn facilitates expeditious extubation in some preterm infants intubated for INSURE procedure.

A review of the enzymatic hydrolysis of mannans and synergistic interactions between ß-mannanase, ß-mannosidase and α-galactosidase.

Mannan is an important polysaccharide found in softwoods and many other plant sources. Mannans from various sources display large differences in composition, structure and complexity. To hydrolyse mannan into its monomer sugars requires a number of enzymes working in synergy. This review examines mannan structure and the enzymes required for its hydrolysis. Several studies have investigated the effect of supplementing ß-mannanases with ß-mannosidases and α-galactosidases in binary and ternary combinations. Synergistic enhancement of hydrolysis has been found in some, but not all cases. In the case of mannosidases, they sometimes display an anti-synergistic effect with mannanases, most likely due to competition for binding sites. Most importantly, in the case of α-galactosidases, the same enzyme from different families display differences in synergistic interactions due to different specificities. An improved understanding of enzyme interactions will aid in achieving enhanced hydrolysis of mannans and higher sugar yields. This review highlights areas which require further research in order to gain a better understanding of mannan hydrolysis and utilisation. Such knowledge is very important as this can be used in the optimisation of commercial or purified enzyme mixtures to improve the economic viability of the conversion of high mannan-containing biomass such as softwoods into fermentable sugars for bioethanol production.

The addition of accessory enzymes enhances the hydrolytic performance of cellulase enzymes at high solid loadings.

The pretreatment process used and the nature of the biomass feedstock will influence the role that accessory enzymes can play in synergistically interacting with cellulases to effectively deconstruct the substrate. The work reported here assessed the possible boosting effects of the xylanase and lytic polysaccharide monooxygenase (AA9, formerly known as GH61) on the hydrolytic potential of cellulase enzyme mixtures during hydrolysis of steam pretreated poplar and corn stover at high (10-20% w/v) substrate concentrations. A higher proportion of xylanase was required when the substrate had a relatively high xylan content and at high substrate concentrations. In contrast, a relatively small amount of AA9 (about 2 mg/g cellulose) was enough, regardless of the nature or concentration of the substrate. The overall protein loading required to achieve effective hydrolysis of high concentrations of pretreated biomass substrates could be substantially reduced by optimizing the ratio of enzymes in the "cellulase" mixture.

The accessible cellulose surface influences cellulase synergism during the hydrolysis of lignocellulosic substrates.

Effective enzymatic hydrolysis of insoluble cellulose requires the synergistic action of a suite of cellulase components. Most previous studies have only assessed cellulase synergism on model cellulosic substrates. When the actions of individual and combinations of cellulases (Cel7A, Cel6A, Cel7B, Cel5A) were assessed on various pretreated lignocellulosic substrates, Cel7A was shown to be the major contributor to overall cellulose hydrolysis, with the other enzymes synergistically enhancing its hydrolytic efficiency, at least partially, by facilitating Cel7A desorption (assessed by a double-sandwich enzyme-linked immunosorbent assay). When the influences of various substrate physicochemical characteristics on the effectiveness of enzyme synergism were assessed, a strong relationship was observed between cellulose accessibility (as determined by the cellulose binding module technique) and the degree of synergism, with greater synergy observed on the more disorganized/accessible cellulose surface.

Optimisation of enzymatic hydrolysis of apple pomace for production of biofuel and biorefinery chemicals using commercial enzymes.

Apple pomace, a waste product from the apple juice industry is a potential feedstock for biofuel and biorefinery chemical production. Optimisation of hydrolysis conditions for apple pomace hydrolysis using Viscozyme L and Celluclast 1.5L was investigated using 1 L reaction volumes. The effects of temperature, pH, ß-glucosidase supplementation and substrate feeding regimes were determined. Hydrolysis at room temperature using an unbuffered system gave optimal performance. Reactors in batch mode resulted in a better performance (4.2 g/L glucose and 16.8 g/L reducing sugar, 75 % yield for both) than fed-batch (3.2 g/L glucose and 14.6 g/L reducing sugar, 65.5 and 73.1 % yield respectively) in 72 h. The addition of Novozyme 188 to the core mixture of Viscozyme L and Celluclast 1.5L resulted in the doubling of glucose released. The main products (yield %) released from apple pomace hydrolysis were galacturonic acid (78 %), glucose (75 %), arabinose (90 %) and galactose (87 %). These products are potential raw materials for biofuel and biorefinery chemical production.

Organochlorine pesticide levels in Clarias gariepinus from polluted freshwater impoundments in South Africa and associated human health risks.

There are increasing concerns regarding the safe human consumption of fish from polluted, freshwater impoundments. The aim of this study was to analyse the muscle tissue of the sharptooth catfish Clarias gariepinus for selected organo-chlorine pesticides (OCPs) and to perform a human health risk assessment using a standard protocol described by the United States Environmental Protection Agency (US EPA). Fish were collected from the polluted Roodeplaat-(RDPD), Rietvlei-(RVD) and Hartbeespoort (HBPD) Dam impoundments located in the north-eastern regions of South Africa. GC-MS analyses showed levels of various OCPs in fish muscle samples from all three impoundments. For fish collected from the RDPD, p,p'-DDE, endosulfan, lindane and ß- and δ-HCH were the most prevalent OCPs detected, while p,p'-DDE and endosulfan were the most predominant in fish from the RVD. Lindane and ß- and δ-HCH were the main OCPs detected in fish from the HBPD. Dieldrin was the only OCP detected at concentrations for which a cancer risk and a hazard index above the acceptable risk levels were estimated. This was the case for fish from both the RDPD and RVD impoundments. No toxic risk was estimated should fish from the HBPD be consumed.

The effect of mixtures of organophosphate and carbamate pesticides on acetylcholinesterase and application of chemometrics to identify pesticides in mixtures.

Organophosphate (OP) and carbamate (CP) pesticides act by the inhibition of acetylcholinesterase (AChE). This enables the use of this enzyme for the detection of these pesticides in the environment. While many studies have looked at the effect of single pesticides on AChE, the effect of mixtures of pesticides still requires extensive investigation. This is important to evaluate the cumulative risk in the case of simultaneous exposure to multiple pesticides. Therefore we examined the effect of five different pesticides (carbaryl, carbofuran, parathion, demeton-S-methyl, and aldicarb) on AChE activity to determine whether combinations had an additive, synergistic, or antagonistic inhibitory effect. Results indicated that the mixtures had an additive inhibitory effect on AChE activity. The data from the assays of the mixtures were used to develop and train an artificial neural network (ANN) which was then utilised successfully for the identification of pesticides and their concentrations in mixtures. This study is significant because it evaluated mixtures of OPs and CPs where previous studies focused on either OPs or CPs. Previous studies have only examined up to three pesticides while this study evaluated mixtures of five pesticides simultaneously. This is also the first study where an ANN was able to utilise data from the inhibition of a single enzyme to differentiate five different pesticides and their concentrations from mixtures.