Disruption of a putative mitochondrial oxaloacetate shuttle protein in Aspergillus carbonarius results in secretion of malic acid at the expense of citric acid production

FWN – Publicaties zonder aanstelling Universiteit Leide

The production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi

In this review we will focus on the current status and views concerning the production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi. We will focus on single-chain antibody fragment production (scFv and V(HH)) by these lower eukaryotes and the possible applications of these proteins. Also the coupling of fragments to relevant enzymes or other components will be discussed. As an example of the fusion protein strategy, the 'magic bullet' approach for industrial applications, will be highlighted

Metabolic engineering with ATP-citrate lyase and nitrogen source supplementation improves itaconic acid production in Aspergillus niger

FWN – Publicaties zonder aanstelling Universiteit Leide

Humeral shaft fractures: Retrospective results of non-operative and operative treatment of 186 patients

Background: Humeral shaft fractures account for 1-3% of all fractures and 20% of the fractures involving the humerus. The aim of the current study was to compare the outcome after operative and non-operative treatment of humeral shaft fractures, by comparing the time to radiological union and the rates of delayed union and complications. Methods: All patients aged 16 years or over treated for a humeral shaft fracture during a 5-year period were included in this retrospective analysis; periprosthetic and pathological fractures were excluded. Radiographs and medical charts were retrieved and reviewed in order to collect data on fracture classification, time to radiographic consolidation and the occurrence of adverse events. Results: A total of 186 patients were included; 91 were treated non-operatively and 95 were treated operatively. Mean age was 58.7 ± 1.5 years and 57.0% were female. In 83.3% of the patients, only the humerus was affected. A fall from standing height was the most common cause of the fracture (72.0%). Consolidation time varied from a median of 11-28 weeks. The rate of radial nerve palsy in both groups was similar: 8.8% versus 9.5%. In 5.3% of the operatively treated patients, the palsy resulted from the operation. Likewise, delayed union rates were similar in both groups: 18.7% following non-operative trea

Комбинированная кинезотерапия в лечении больных коксартрозом

Вивчені результати лікування 54 пацієнтів з коксартрозом. Включення комплексного лікування даного контингенту пацієнтів комбінованої кінезотерапії методикою Євмінова дозволяє підвищити ефективність проведеної терапії.This article studied results of treatment of 54 patients with coxarthrosis. Upon inclusion of the combined kinesitherapy under Evminov method into the complex treatment of the mentioned group of patients it became possible to increase the effectiveness of the whole therapy

Expression of human α1-proteinase inhibitor in Aspergillus niger

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In vivo validation of DNA adduct formation by estragole in rats predicted by physiologically based biodynamic modelling

Estragole is a naturally occurring food-borne genotoxic compound found in a variety of food sources, including spices and herbs. This results in human exposure to estragole via the regular diet. The objective of this study was to quantify the dose-dependent estragole-DNA adduct formation in rat liver and the urinary excretion of 1'-hydroxyestragole glucuronide in order to validate our recently developed physiologically based biodynamic (PBBD) model. Groups of male outbred Sprague Dawley rats (n = 10, per group) were administered estragole once by oral gavage at dose levels of 0 (vehicle control), 5, 30, 75, 150, and 300mg estragole/kg bw and sacrificed after 48h. Liver, kidney and lungs were analysed for DNA adducts by LC-MS/MS. Results obtained revealed a dose-dependent increase in DNA adduct formation in the liver. In lungs and kidneys DNA adducts were detected at lower levels than in the liver confirming the occurrence of DNA adducts preferably in the target organ, the liver. The results obtained showed that the PBBD model predictions for both urinary excretion of 1'-hydroxyestragole glucuronide and the guanosine adduct formation in the liver were comparable within less than an order of magnitude to the values actually observed in vivo. The PBBD model was refined using liver zonation to investigate whether its predictive potential could be further improved. The results obtained provide the first data set available on estragole-DNA adduct formation in rats and confirm their occurrence in metabolically active tissues, i.e. liver, lung and kidney, while the significantly higher levels found in liver are in accordance with the liver as the target organ for carcinogenicity. This opens the way towards future modelling of dose-dependent estragole liver DNA adduct formation in huma

Identifying inhibitory compounds in lignocellulosic biomass hydrolysates using an exometabolomics approach

BACKGROUND: Inhibitors are formed that reduce the fermentation performance of fermenting yeast during the pretreatment process of lignocellulosic biomass. An exometabolomics approach was applied to systematically identify inhibitors in lignocellulosic biomass hydrolysates. RESULTS: We studied the composition and fermentability of 24 different biomass hydrolysates. To create diversity, the 24 hydrolysates were prepared from six different biomass types, namely sugar cane bagasse, corn stover, wheat straw, barley straw, willow wood chips and oak sawdust, and with four different pretreatment methods, i.e. dilute acid, mild alkaline, alkaline/peracetic acid and concentrated acid. Their composition and that of fermentation samples generated with these hydrolysates were analyzed with two GC-MS methods. Either ethyl acetate extraction or ethyl chloroformate derivatization was used before conducting GC-MS to prevent sugars are overloaded in the chromatograms, which obscure the detection of less abundant compounds. Using multivariate PLS-2CV and nPLS-2CV data analysis models, potential inhibitors were identified through establishing relationship between fermentability and composition of the hydrolysates. These identified compounds were tested for their effects on the growth of the model yeast, Saccharomyces. cerevisiae CEN.PK 113-7D, confirming that the majority of the identified compounds were indeed inhibitors. CONCLUSION: Inhibitory compounds in lignocellulosic biomass hydrolysates were successfully identified using a non-targeted systematic approach: metabolomics. The identified inhibitors include both known ones, such as furfural, HMF and vanillin, and novel inhibitors, namely sorbic acid and phenylacetaldehyde

Evaluation of Human Interindividual Variation in Bioactivation of Estragole Using Physiologically Based Biokinetic Modeling

The present study investigates interindividual variation in liver levels of the proximate carcinogenic metabolite of estragole, 1′-hydroxyestragole, due to variation in two key metabolic reactions involved in the formation and detoxification of this metabolite, namely 1′-hydroxylation of estragole and oxidation of 1′-hydroxyestragole. Formation of 1′-hydroxyestragole is predominantly catalyzed by P450 1A2, 2A6, and 2E1, and results of the present study support that oxidation of 1′-hydroxyestragole is catalyzed by 17β-hydroxysteroid dehydrogenase type 2 (17β-HSD2). In a first approach, the study defines physiologically based biokinetic (PBBK) models for 14 individual human subjects, revealing a 1.8-fold interindividual variation in the area under the liver concentration-time curve (AUC) for 1′-hydroxyestragole within this group of human subjects. Variation in oxidation of 1′-hydroxyestragole by 17β-HSD2 was shown to result in larger effects than those caused by variation in P450 enzyme activity. In a second approach, a Monte Carlo simulation was performed to evaluate the extent of variation in liver levels of 1′-hydroxyestragole that could occur in the population as a whole. This analysis could be used to derive a chemical-specific adjustment factor (CSAF), which is defined as the 99th percentile divided by the 50th percentile of the predicted distribution of the AUC of 1′-hydroxyestragole in the liver. The CSAF was estimated to range between 1.6 and 4.0, depending on the level of variation that was taken into account for oxidation of 1′-hydroxyestragole. Comparison of the CSAF to the default uncertainty factor of 3.16 for human variability in biokinetics reveals that the default uncertainty factor adequately protects 99% of the populatio

Use of Physiologically Based Biokinetic (PBBK) Modeling to Study Estragole Bioactivation and Detoxification in Humans as Compared with Male Rats

The extent of bioactivation of the herbal constituent estragole to its ultimate carcinogenic metabolite 1′-sulfooxyestragole depends on the relative levels of bioactivation and detoxification pathways. The present study investigated the kinetics of the metabolic reactions of both estragole and its proximate carcinogenic metabolite 1′-hydroxyestragole in humans in incubations with relevant tissue fractions. Based on the kinetic data obtained a physiologically based biokinetic (PBBK) model for estragole in human was defined to predict the relative extent of bioactivation and detoxification at different dose levels of estragole. The outcomes of the model were subsequently compared with those previously predicted by a PBBK model for estragole in male rat to evaluate the occurrence of species differences in metabolic activation. The results obtained reveal that formation of 1′-oxoestragole, which represents a minor metabolic route for 1′-hydroxyestragole in rat, is the main detoxification pathway of 1′-hydroxyestragole in humans. Due to a high level of this 1′-hydroxyestragole oxidation pathway in human liver, the predicted species differences in formation of 1′-sulfooxyestragole remain relatively low, with the predicted formation of 1′-sulfooxyestragole being twofold higher in human compared with male rat, even though the formation of its precursor 1′-hydroxyestragole was predicted to be fourfold higher in human. Overall, it is concluded that in spite of significant differences in the relative extent of different metabolic pathways between human and male rat there is a minor influence of species differences on the ultimate overall bioactivation of estragole to 1′-sulfooxyestragol