In vitro expansion of U87-MG human glioblastoma cells under hypoxic conditions affects glucose metabolism and subsequent in vivo growth

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Degradação de Ocratoxina a: estudo de processo e toxicidade

A ocratoxina A (OTA), micotoxina encontrada em diferentes níveis e em diversas matrizes, apresenta efeitos carcinogênicos, nefrotóxicos e teratogênicos. O desenvolvimento de métodos capazes de diminuir esta contaminação a níveis permitidos pela legislação é incentivado e os processos biológicos utilizados envolvem o uso de enzimas e/ou microrganismos para degradação da OTA e são preferenciais pela especificidade, bem como pelas condições brandas para a detoxificação. O objetivo do trabalho foi estudar a ação de carboxipeptidase A nos níveis e na toxicidade de OTA, visando aplicar a técnica para detoxificar farinhas de trigo. Primeiramente foi estimado o risco de exposição à ocratoxina A pelo consumo de farinhas de trigo. Para isso foram estabelecidas condições de determinação de OTA em farinhas de trigo, empregando técnicas de estatística multivariada para definir os principais interferentes na extração de OTA pelo método de QuEChERS e detecção em CLAE-FL. O método validado permitiu a avaliação da ocorrência natural em 20 amostras de farinha de trigo, estando estas contaminadas na faixa de 0,22 a 0,85 µg.kg-1 , apresentando um valor de ingestão diária de 0,08 ngOTA.dia-1 .kgmassacorpórea -1 e uma disponibilidade de 94,4%. Em seguida foi realizada a padronização da extração de carboxipeptidase A em biomassa de Rhizopus oryzae que consistiu em agitação ultrassônica durante 30 minutos numa potencia fixa de 150 W e 40 kHz e a triagem de agentes biológicos para degradação de OTA. Para o estudo da degradação in vitro de OTA, método de extração e detecção de OTA e OTα em CLAEFL foi validado e o processo de degradação foi realizado com Rhizopus oryzae e Trichoderma reesei, obtendo-se uma redução máxima de 63,5% e 57,7%, respectivamente. A degradação apresentou uma correlação alta (R>0,9) e significativa (p<0,05) com a produção de Otα, indicando que ocorreu a produção de enzimas capazes de hidrolisar a micotoxina, por exemplo, a carboxipeptidase A. O estudo da toxicidade de OTA e seu metabólito OTα foi realizado em neutrófilos humanos, onde foi observado a ausência de efeito tóxico de OTα. Também foi determinado o mecanismo de toxicidade de OTA pelo aumento de Ca2+ intracelular pela liberação a partir das reservas internas. Esta liberação, subsequentemente, provoca uma cascata de eventos, nomeadamente: a produção de espécies reativas, depleção de ATP, perda de ΔΨm, levando à morte por necrose. Para reduzir o risco de exposição à micotoxina pela ingestão de matéria prima contaminada, carboxipeptidase A extraída de diferentes fontes foi aplicada na hidrólise de OTA em farinha de trigo para posterior determinação do conteúdo residual de OTA e OTα, empregando método validado. O estudo mostrou uma redução de OTA entre 16,8 e 78,5% e produção de OTα entre 2 a 8,2 ng.g-1 . As carboxipeptidases mais promissoras para degradação foram as provenientes de Rhizopus e Trichoderma e a carboxipeptidase comercial. Ficou demonstrado que se pode recomendar a aplicação de enzimas proteolíticas, tipo carboxipeptidase, para reduzir o risco de exposição à micotoxina quando utilizada matéria prima contaminada, por exemplo, farinha de trigo para diferentes processos. A transformação de OTA para OTα e seus efeitos na redução da toxicidade da micotoxina corroboram com esta afirmação.

Post consumer pet depolymerization by acid hydrolysis

The reaction of post-consumer poly(ethylene terephthalate) with aqueous solutions of sulfuric acid 7.5M was investigated in terms of temperature, time and particle size. The reaction extent reached 80% in four days at 100 degrees C and 90% in 5 hours at 135 degrees C. TPA obtained was purified and considered in the same level of quality of the commercial one after tests of elemental analysis, particle size and color. It was concluded that the hydrolysis occurred preferentially at the chain ends and superficially, having as controller mechanism the acid diffusion into the polymer structure. The shrinking-core model can explain the reaction kinetics.

Valorization of the Macroalgae Sargassum Muticum by Enzymatic Hydrolysis, Interest of Surfactants to Improve the Extraction of Phlorotannins and Polysaccharides

The use of surfactants to improve enzymatic hydrolysis of the macroalgae Sargassum muticum has been investigated. Visible absorption spectroscopy has been used to quantify the solubilization of both polysaccharides and phlorotannins in the hydrolysates.   After total extraction, results showed that Sargassum muticum contained 2.74% (expressed in percent of the dry weight of the algae) of phlorotannins whose 32 % were in the cell wall. This result shows that it is important to access to the parietal phlorotannins. To reach this objective, we chose the enzymatic approach for destructurating the cell wall of the algae. The use of 5% dry weight (DW - 5% by weight of hydrolyzed algae) of an enzymatic mix containing a commercial beta-glucanase, a commercial protease and an alginate lyase extracted from Pseudomonas alginovora led after 3 hours of hydrolysis to the solubilization of 2.43% DW polysaccharides and 0.52% DW phlorotannins. The use of 0.5% volume of the surfactant Triton® X-100 with 10% DW of the enzymatic mix has allowed to reaching the value of 2.63% DW of solubilized phlorotannins, that is 96% of the total phenolic content.   The use of non-ionic surfactant, combined to enzymatic hydrolysis, showed an increased efficiency in disrupting cell wall and solubilizing phlorotannins in Sargassum muticum.

Enzyme Optimization for Lignocellulose Hydrolysis using Mechanistic Modeling

A novel mechanistic model for the saccharification of cellulose and hemicellulose is utilized to predict the products of hydrolysis over a range of enzyme loadings and times. The mechanistic model considers the morphology of the substrate and the kinetics of enzymes to optimize enzyme concentrations for the enzymatic hydrolysis of cellulose and hemicellulose simultaneously. Substrates are modeled based on their fraction of accessible sites, glucan content, xylan content, and degree of polymerizations. This enzyme optimization model takes into account the kinetics of six core enzymes for lignocellulose hydrolysis: endoglucanase I (EG1), cellobiohydrolase I (CBH1), cellobiohydrolase II (CBH2), and endo-xylanase (EX) from Trichoderma reesei; β-glucosidase (BG), and β-xylosidase (BX) from Aspergillus niger. The model employs the synergistic action of these enzymes to predict optimum enzyme concentrations for hydrolysis of Avicel and ammonia fiber explosion (AFEX) pretreated corn stover. Glucan, glucan + xylan, glucose and glucose + xylose conversion predictions are given over a range of mass fractions of enzymes, and a range of enzyme loadings. Simulation results are compared with optimizations using statistically designed experiments. BG and BX are modeled in solution at later time points to predict the effect on glucose conversion and xylose conversion.

Defining fluid restriction in the management of infants following cardiac surgery and understanding the subsequent impact on nutrient delivery and growth outcomes

Adequacy of nutritional intake during the postoperative period, as measured by a change in weight-for-age z-scores from surgery to the time of discharge, was evaluated in infants (n = 58) diagnosed with a congenital heart defect and admitted for surgical intervention at Miami Children’s Hospital using a prospective observational study design. Parental consent was obtained for all infants who participated in the study. ^ Forty patients had a weight available at hospital discharge. The mean preoperative weight-for-age z-score was -1.3 ±1.43 and the mean weight-for-age z-score at hospital discharge was -1.89 ±1.35 with a mean difference of 0.58 ±0.5 (P = 0.2).^ Nutritional intake during the postoperative period was inadequate based on a decrease in weight-for-age z-scores from the time of surgery until discharged home. Our findings suggested that limited fluid volume for nutrition likely contributes to suboptimal nutritional delivery during the postoperative period; however, inadequate nutrition prescription may also be an important contributing factor. Development of a nutrition protocol for initiation and advancement of nutrition support may reduce the delay in achieving patient’s nutritional goals and may attenuate the observed decrease in z-scores during the postoperative period.^

Commentary on "Stress ulcer prophylaxis in hospitalized patients, subsequent use in primary care and physicians' opinions about acid-suppressive therapy"

SIN FINANCIACIÓN

Fabrication of heterogeneous biocatalyst tethering artificial prosthetic groups to obtain omega-3-fatty acids by selective hydrolysis of fish oils

The active site of lipase from Bacillus thermocathenolatus was selectively modified with allyl and naphthyl chains at different positions. Lipase immobilization and selective tethering of a naphthyl side chain to its position 320 improve both the hydrolysis rate of fish oils and the selectivity towards the eicosapentaenoic acid acyl chains. © The Royal Society of Chemistry 2016.

An investigation of the possible health-promoting modes of action of regular- and super- doses of phytase in the broiler chicken

The overall objective of this thesis was to study the effects of regular and high (super-) doses of phytase in the gut of broilers, with the aim of documenting the mechanism of their action leading to improvements in animal health. Phytase is often supplemented to commercial broiler diets to facilitate the hydrolysis of plant phytate and release of phosphorus for utilisation. Although not the original intention of its addition, phytase supplementation leads to improvements in growth performance parameters and enhanced nutrient utilisation. Further benefits have also been observed following the addition of super-doses of phytase which are not explained by an increase in phosphorus release, and thus have been termed ‘extra-phosphoric effects’. Using diets formulated to be adequate or marginally deficient in available phosphorus (aP; forming the negative control, NC), phytase was supplemented at 1,500 and 3,000 FTU/kg phytase in the first study (both super-doses) and the partitioning of nutrients within the body was investigated. It appeared that there were some metabolic changes between 1,500 and 3,000 FTU/kg, switching between protein and fat accretion, potentially as a consequence of nutrient availability, although these changes were not reflected by changes in growth performance parameters. However, the loss of the NC treatment without phytase on day 12 limits the comparison of the phytase within the NC treatment, but does allow for comparison of each dose at adequate or low dietary aP levels. As expected, a greater degree of phytate hydrolysis was achieved with 3,000 than with 1,500 FTU/kg phytase, but changes in carcass accretion characteristics were greater with 1,500 than 3,000 FTU/kg. Using these findings and the observation that there were no further changes in the parameters measured by increasing phytase from 1,500 to 3,000 FTU/kg (aside from phytate hydrolysis), 1,500 FTU/kg phytase was selected as the super-dose to be used in subsequent studies. The next study considered the influence of regular (500 FTU/kg) and super doses (1,500 FTU/kg) of phytase from within the gut. Overall, it was observed that changes were occurring to the gut environment, which ultimately would influence the absorptive capacity and conditions for further phytate hydrolysis. Dietary treatment influenced gut conditions such as pH, intestinal morphology and bacterial populations which can subsequently influence nutrient utilisation and potential for growth. The subsequent study was designed to investigate the effects within the gut in more detail. The release of nutrients from phytate hydrolysis and their bioavailability within the digesta can influence conditions within intestine, facilitating enhanced absorption. One of the parameters investigated was the expression of genes involved in the transport of nutrients in the intestine. Overall, there were few significant dietary treatment influences on gene expression in the intestine, however there was a dose-dependent response of phytase on the expression of the jejunual divalent mineral transporter. This indicates a change in divalent mineral bioavailability in the intestine, with correlations with inositol phosphate esters (IPs) being identified. This is likely explained by the IPs produced by phytase hydrolysis and accumulating in the digesta, differing between regular and high doses of phytase. It became apparent that interactions between the products of phytate hydrolysis (IP3, IP4) and minerals in the digesta had the potential to influence the gut environment and subsequent nutrient bioavailability and overall phytase action. The final study was designed to increase the content of the IPs, and investigate the influence of phytase under these conditions. As the complete hydrolysis of phytate to myo-inositol has been reported to be beneficial due to its proposed insulin mimetic effects, myo-inositol was also supplemented to one of the diets to see if any further benefits would be observed when supplemented alongside super-doses of phytase. Neither increased concentrations of the higher IP esters (IP6, IP5 and IP4) nor myo-inositol (myo-) had any effect on broiler growth performance, however there were still apparent beneficial influences of phytase supplementation. The results suggest considerable and important interactions between minerals and IP esters within the digesta, which ultimately have the potential to influence gut conditions and thus nutrient utilisation and growth performance. Reduced concentrations of blood glucose in the high IP ester diet with additional phytase supplementation suggest some insulin-like effects of myo- production. Additionally, the lack of effect of myo- supplementation on blood glucose and insulin concentrations suggests a difference between the structure of phytase-produced myo- and supplemented myo-. Although there were no improvements in growth performance by increasing phytase from 500 to 1,500 FTU/kg, there were changes occurring at the level of the gut and expression of genes in the intestine, influencing nutrient utilisation and the partitioning of nutrients within the body. There are many factors to be considered when supplementing phytase, with dietary nutrient content and nutrient release and IP production during phytate hydrolysis having an influence on phytase action, nutrient absorption and conditions within the gut. Super-doses of phytase may be beneficial for maintaining optimal gut conditions, clearing IP esters from the digesta, reducing their potential to form complexes with minerals and other nutrients, ultimately influencing the efficiency of production.

Near-infrared spectroscopic study of basic aluminum sulfate and nitrate

The tridecameric Al-polymer [AlO4Al12(OH)24(H2O)12]7+ was prepared by forced hydrolysis of Al3+ up to an OH/Al molar ratio of 2.2. Under slow evaporation crystals were formed of Al13-nitrate. Upon addition of sulfate the tridecamer crystallised as the monoclinic Al13-sulfate. These crystals have been studied using near-infrared spectroscopy and compared to Al2(SO4)3.16H2O. Although the near-infrared spectra of the Al13-sulfate and nitrate are very similar indicating similar crystal structures, there are minor differences related to the strength with which the crystal water molecules are bonded to the salt groups. The interaction between crystal water and nitrate is stronger than with the sulfate as reflected by the shift of the crystal water band positions from 6213, 4874 and 4553 cm–1 for the Al13 sulfate towards 5925, 4848 and 4532 cm–1 for the nitrate. A reversed shift from 5079 and 5037 cm–1 for the sulfate towards 5238 and 5040 cm–1 for the nitrate for the water molecules in the Al13 indicate that the nitrate-Al13 bond is weakened due to the influence of the crystal water on the nitrate. The Al-OH bond in the Al13 complex is not influenced by changing the salt group due to the shielding by the water molecules of the Al13 complex.

The ILC's Articles on Responsibility of States for Internationally Wrongful Acts: Completion of the Second Reading

In 2001 the International Law Commission finally adopted on second reading the Draft Articles on Responsibility of States for Internationally Wrongful Acts with commentaries, bringing to an end nearly 50 years of ILC work on the subject. This article reviews the final group of changes to the text, focusing on the definitions of ‘injury’ and ‘damage’, assurances of non‐repetition in the light of the LaGrand case, procedural aspects of countermeasures and the controversy over measures taken in response to a breach by states which are not individually injured. The focus of debate now turns to the UNGA Sixth Committee, which will have to decide what to make of the Draft Articles. The ILC itself recommended an initial resolution taking note of the Articles, with subsequent consideration (after a period of years) of a possible diplomatic conference with a view to concluding a convention. This modest proposal allows for further reflection on the text and may help to avoid possibly divisive and inconclusive debate in the Sixth Committee. At the same time it allows time for better understanding of the many changes made as compared with the first reading text (1996).

The ageing of alumina hydrolysates synthesized from sec-butoxyaluminium(III)

A combination of X-ray diffraction, thermal analysis and Raman spectroscopy was employed to characterise the ageing of alumina hydrolysates synthesised from the hydrolysis of anhydrous tri-sec-butoxyaluminium(III). X-Ray diffraction showed that the alumino-oxy(hydroxy) hydrolysates were pseudoboehmite. For boehmite the lamellar spacings are in the b direction and multiple d(020) peaks are observed for the un-aged hydrolysate. After 4 h of ageing, a single d(020) peak is observed at 6.53 Å. Thermal analysis showed five endotherms at 70, 140, 238, 351 and 445°C. These endotherms are attributed to the dehydration and dehydroxylation of the boehmite-like hydrolysate. Raman spectroscopy shows the presence of bands for the washed hydrolysates at 333, 355, 414, 455, 475, 495, 530 and 675 cm–1. These bands are attributed to pseudoboehmite. Ageing of the hydrolysates results in an increase in the crystallite size of the pseudoboehmite.

Mechanochemical treatment of kaolinite

Kaolinite surfaces were modified by mechanochemical treatment for periods of time up to 10 h. X-ray diffraction shows a steady decrease in intensity of the d(001) spacing with mechanochemical treatment, resulting in the delamination of the kaolinite and a subsequent decrease in crystallite size with grinding time. Thermogravimetric analyses show the dehydroxylation patterns of kaolinite are significantly modified. Changes in the molecular structure of the kaolinite surface hydroxyls were followed by infrared spectroscopy. Hydroxyls were lost after 10 h of grinding as evidenced by a decrease in intensity of the OH stretching vibrations at 3695 and 3619 cm−1 and the deformation modes at 937 and 915 cm−1. Concomitantly an increase in the hydroxyl stretching vibrations of water is found. The water-bending mode was observed at 1650 cm−1, indicating that water is coordinating to the modified kaolinite surface. Changes in the surface structure of the OSiO units were reflected in the SiO stretching and OSiO bending vibrations. The decrease in intensity of the 1056 and 1034 cm−1 bands attributed to kaolinite SiO stretching vibrations were concomitantly matched by the increase in intensity of additional bands at 1113 and 520 cm−1 ascribed to the new mechanically synthesized kaolinite surface. Mechanochemical treatment of the kaolinite results in a new surface structure.

Longitudinal Student Evaluations of a Postgraduate Unit Using Importance-Performance Analysis

The Australian tourism tertiary education sector operates in a competitive and dynamic environment, which necessitates a market orientation to be successful. Academic staff and management in the sector must regularly assess the perceptions of prospective and current students, and monitor the satisfaction levels of current students. This study is concerned with the setting and monitoring of satisfaction levels of current students, reporting the results of three longitudinal investigations of student satisfaction in a postgraduate unit. The study also addresses a limitation of a university’s generic teaching evaluation instrument. Importance-performance analysis (IPA) has been recommended as a simple but effective tool for overcoming the deficiencies of many student evaluation studies, which have generally measured only attribute importance or importance at the end of a semester. IPA was used to compare student expectations of the unit at the beginning of semester with their perceptions of performance ten weeks later. The first stage documented key benchmarks for which amendments to the unit based on student feedback could be evaluated during subsequent teaching periods.

The science of assessment : identifying and predicting environmental impacts

The historical challenge of environmental impact assessment (EIA) has been to predict project-based impacts accurately. Both EIA legislation and the practice of EIA have evolved over the last three decades in Canada, and the development of the discipline and science of environmental assessment has improved how we apply environmental assessment to complex projects. The practice of environmental assessment integrates the social and natural sciences and relies on an eclectic knowledge base from a wide range of sources. EIA methods and tools provide a means to structure and integrate knowledge in order to evaluate and predict environmental impacts.----- This Chapter will provide a brief overview of how impacts are identified and predicted. How do we determine what aspect of the natural and social environment will be affected when a mine is excavated? How does the practitioner determine the range of potential impacts, assess whether they are significant, and predict the consequences? There are no standard answers to these questions, but there are established methods to provide a foundation for scoping and predicting the potential impacts of a project.----- Of course, the community and publics play an important role in this process, and this will be discussed in subsequent chapters. In the first part of this chapter, we will deal with impact identification, which involves appplying scoping to critical issues and determining impact significance, baseline ecosystem evaluation techniques, and how to communicate environmental impacts. In the second part of the chapter, we discuss the prediction of impacts in relation to the complexity of the environment, ecological risk assessment, and modelling.