The influence of nutrient deprivation on nucleus pulposus cells in vivo and in vitro

Intervertebral disc degeneration is one of the causes of low back pain with a multifactorial etiology of which lack of nutrition for the nucleus pulposus cells has been proposed as one of the causes. During ageing, the cartilaginous endplates of the disc calcify and become less permeable to nutrients. Since the disc is a large avascular structure the cells inside the disc depend on diffusion for solute transport. For the nucleus pulposus the most important route is the route through the endplate. In this research project we want to see if partial blockage of this endplate route (mimicking endplate calcification in humans) results in nutrient deprivation, and if this nutrient deprivation could cause disc degeneration. This study has an in vivo and an in vitro part. In the in vivo part we test the endplate perfusion inhibition, and how the cells respond in a short term to the nutrient deprivation caused by this perfusion inhibition. In the in vitro part we test the sensitivity of isolated nucleus pulposus cells to low glucose conditions with or without serum and with or without cyclic hydrostatic loading. Also we test the capability of bone marrow derived stromal cells (BMSCs) to survive in hydrogels which is no problem to nucleus pulposus cells and articular chondrocytes, this with respect to their potential for disc and cartilage repair for future development of biological treatments. In vivo: Under general anesthesia cuts were made in sheep vertebrae, close and parallel to the endplate to disrupt the blood supply to the disc. Immediately or 4 weeks later, diffusion of N2O into the disc was measured amperometrically. Post-mortem a fluorescent dye (procion red) was infused into the lumbar vasculature to visualize the vascular buds in histological section. In the 4 week study gene expression, DNA quantification and cell viability assessment were added as outcome measures. The results of the acute study confirmed that disrupting the blood supply decreased the perfusion of the endplate and inhibited diffusion of N2O into the disc. There was a significant correlation between the amount of perfused vascular buds and the amount of diffused N2O inside the disc. In the 4 week study there were problems with the diffusion measurements, but the cell viability was lower and Collagen I and MMP13 gene expression were significantly up-regulated in the experimental discs compared to the control discs. In vitro : Isolated nucleus pulposus cells were cultured in 3D cell pellets under high (4.5 g/l, only serum group) low (1.0 g/l) and ultra low (0.1 g/l) glucose conditions with and without cyclic hydrostatic load (sine wave, 0.5 - 1.5 MPa, 1Hz, 4 hours per day) and with or without serum. At days 0, 7 and 14 pellets were harvested for DNA, GAG, gene expression analysis and histology (only d14). In all serum groups DNA content per pellet increased over the 14 day culture period. In the serum groups, GAG increased over time in the ultra low glucose group. In the low and high glucose groups GAG content increased till d7, but had dropped again at d14. A similar trend was found in aggrecan and collagen II gene expression. In the serum free groups GAG content was low at d7, but had increased at d14. Histology also showed more GAG in the ultra low glucose groups and the serum free groups than in the low and high glucose groups when pellets were cultured with serum. In none of the groups an effect of load on any of the outcome measures was found. BMSCs, articular chondrocytes and nucleus pulposus cells cast in 1.2% alginate or 2% agarose were cultured for 21 days in serum containing media or (only BMSCs) in medium with 1% ITS+ and 10ng/ml TGF??1. By day 21, nucleus pulposus cells and articular chondrocytes proliferated, maintained up-regulation of aggrecan and collagen type II, produced GAGs and stained positively for collagen type II in both scaffolds. In contrast, number of living BMSCs and DNA content of their constructs decreased in both scaffolds. Addition of TGF??1 resulted in cell survival and behavior more similar (gene expression, GAG production and collagen type II synthesis) to articular chondrocytes and nucleus pulposus cells. In conclusion: Nucleus pulposus cells are very tough cells that survived very low glucose conditions. But when perfusion has been inhibited in vivo, they have problems surviving and maintaining their gene expression. When considering BMSCs as a strategy for disc (or cartilage) repair, chondrogenic differentiation is advised in order to maintain their viability. This research shows that glucose deprivation alone or combined with load does not result in degenerative changes in nucleus pulposus cells and that in order to study disc de- and regeneration in vitro oxygen concentration and pH should also be included

Review of the geomorphological, benthic ecological and biogeomorphological effects of nourishments on the shoreface and surf zone of the Dutch coast

Most knowledge on the morphodynamic behaviour of shoreface and beach nourishments originates from data analysis studies. Numerical modeling tools have been used successfully in hindcasting behaviour of nourishments, but do not yet have the predictive power for reliably forecasting

Production of D-malate by maleate hydratase from Pseudomonas pseudoalcaligenes

The biological activity of a chiral compound with respect to its pharmaceutical and agrochemical activity, flavour and taste can vary dramatically for the different enantiomers. Especially when using chiral compounds for pharmaceutical or agrochemical applications, the presence of the "wrong" stereoisomer can have severe effects on patients or may cause an additional environmental load. The availability of (cheap) optically pure compounds is, therefore, of prime importance, especially for the pharmaceuticals industry.There are several ways in which optically active compounds can be produced. One way is by using biocatalysts. During the last decade, the importance of biocatalysis for the production of optically pure fine-chemicaIs has increased, and it is expected to increase even further in the near future. Advantages of enzymes as catalysts are the fact that they work under mild reaction conditions and their reaction-, regio-, and stereospecificity. These last three aspects also imply that fewer side- products are produced which is positive in view of the increasing environmental concern. Enzymes that can transform a substrate for 100% into a 100% optically pure product, and which do not require cofactor recycling are, both from an economical and an environmental point of view, most suitable as biocatalysts.D-Malate is an optically active compound which can be used as a synthon in organic synthesis, as a resolving agent and as a ligand in asymmetric catalysis (Chapter 1).D-Malate can be produced in several ways, but the approach using the lyase maleate hydratase (malease; EC 4.2.1.31), which catalyzes the hydration of maleate toD-malate, seems to be the most promising approach for commercialization (Chapter 1).Lyases are enzymes that catalyze the cleavage of C-C, C-N, C-O and other bonds by elimination to produce double bonds or, conversely, catalyze the addition of groups to double bonds. These enzymes do not require cofactor recycling, show an absolute stereospecificity (100% e.e.) and can give a theoretical yield of 100%. Lyases are attracting increasing interest as biocatalysts for the production of optically active compounds, and have already found application in several large commercial processes (Chapter 2).We have screened more than 300 microorganisms for the presence of malease activity (Chapter 3). Many strains (n = 128) could convert maleate toD-malate with an enantiomeric purity of more than 97%. Accumulation of fumarate during incubation of permeabilized cells with maleate was shown to be indicative for the presence of the unwanted maleate cis-trans -isomerase activity, which ultimately results in the formation of the unwantedLenantiomer of malate. The ratio in which fumarate and malate accumulated could be used to estimate the enantiomeric composition of the malate formed (Chapter 3).Pseudomonas pseudoalcaligenes NCIMB 9867 was selected for more detailed studies, because it contained one of the highest malease activities, theD-malate formed had an enantiomeric purity of more than 99.97%, it did not degradeD-malate and because it did not show any side-product formation (Chapter 3).The highest malease activity in P.pseudoalcaligenes was observed when it was grown on 3-hydroxybenzoate (Chapter 4). Growth on gentisate also resulted in an enhanced malease activity. Both compounds are degraded via maleate in this microorganism. The specific malease activity of cells grown on 3-hydroxybenzoate was constant during the logarithmic phase, but dropped rapidly as soon as growth ceased.Malease from P. pseudoalcaligenes was purified (Chapter 5). The purified enzyme (89 kDa) consisted of two subunits (57 and 24 kDa). No cofactor was required for full activity of this colorless enzyme. The stability of the enzyme was dependent on the protein concentration and the presence of dicarboxylic acids. Maximum enzyme activity was measured at pH 8 and 45°C. The purified enzyme also catalyzes the hydration of citraconate (2-methylmaleate) formingD-(+)-citramalate and of 2,3- dimethylmaleate at, respectively, 54 and 0.8% of the rate of maleate hydration (30°C). The KM of malease for maleate was 0.35 mM, and for citraconate 0.20 mM. The productsD-malate andD-citramalate and the substrate analog 2,2-dimethylsuccinate were strong competitive inhibitors of malease (Chapter 5).Hydratases catalyze equilibrium reactions. To optimize the reaction conditions, data concerning the equilibrium constant are necessary to determine the maximal obtainable yield. In literature no data were available concerning the equilibrium of the malease catalyzed reactions. Therefore, we determined the equilibrium constants ( Kapp ) for the malease catalyzed hydration reactions. The Kapp for the maleate, citraconate, and 2,3-dimethylmaleate hydration reactions were 2050, 104 and 11.2, respectively, under standard biochemical conditions (25°C, pH 7.0, I =0.1) (Chapter 5 and 6). The equilibrium constants for the maleate and citraconate hydration reactions make yields of more than 99% possible.As especially maleate has a high p K a 2 , the influence of the pH (6.0-8.5) on the Kapp was determined to describe the influence of the presence of the different forms of maleate (i.e. dianionic, monoanionic and diprotonated) on the equilibrium constant (Chapter 6). Also the influence of the temperature (10°C - 40°C) on Kapp was determined. From these experiments the Gibbs-free-energy change (ΔG°'), the enthalpy change (ΔH°'), and entropy change (ΔS°') under standard biochemical conditions for the maleate 2-and citraconate 2-hydration reactions were calculated (Chapter 6). Also the effect of the temperature on the maleate and citraconate hydration rates of the purified enzyme was determined to calculate the activation energy. At low temperatures the hydration rate of citraconate was higher, while at temperatures above 18°C the maleate hydration reaction was faster (Chapter 6).P. pseudoalcaligenes is not able to grow on maleate, probably because it lacks an uptake system for maleate (Chapter 3). Intact cells of P.pseudoalcaligenes do not show any accumulation ofD-malate from maleate but Triton X-100 treated cells showed accumulation ofD-malate from maleate, indicating that these cells were permeabilized or lysed (Chapter 7). Incubation of cells with Triton X-100 also resulted in an increase in the protein concentration of the supernatant, indicating the occurrence of lysis. Permeabilization and lysis were time-dependent: longer incubations resulted in higher malease activities and more protein in the supernatant. The permeabilization and lysis rates were also dependent on the Triton X-100 and biomass concentration (Chapter 7).Malease activity of permeabilized cells of P.pseudoalcaligenes decreased strongly when Na 2 - maleate concentrations higher than 0.6 M were used as the substrate (Chapter 8). When other counter- ions than Na +were used, in some instances the malease activity was found to be affected much less by high substrate concentrations. When for instance Mg 2+was used as the counter-ion, a much larger percentage of maleate is present as metal-substrate complex, thereby reducing the "real" substrate concentration resulting in less substrate inhibition. When Ca 2+and Ba 2+were used as the counter- ion, the malease activity was not at all affected by increasing substrate concentrations. The use of these metal-ions resulted in the formation of a crystal-liquid two-phase system, due to the low solubility of the metalsubstrate complex. In this situation, the "real" substrate concentration was independent on the total amount of substrate present. Ca 2+was the best counter-ion for the conversion of maleate intoD-malate. The use of this metal-ion resulted in the highest malease activities and the absence of substrate inhibition at high substrate concentrations (Chapter 8). In this way, high concentrations (up to 160 g/l) of either maleate or citraconate were converted by malease of P.pseudoalcaligenes intoD-malate andD-citramalate, respectively, with yields of more than 99%.To determine the potential of the crystal-liquid two-phase system for the large scale conversion of maleate intoD-malate, the conversion of one kilogram of maleate was studied (Chapter 9). At a substrate concentration of 200 g/l, permeabilized cells of P.pseudoalcaligenes (1 g/l protein) converted maleate within two days into 1. 15 kg ofD-malate with a yield of 99.4%.During our studies on theD-malate production from maleate with malease from P.pseudoalcaligenes, also three Japanese groups published about their studies on this conversion, using other biocatalysts. P.pseudbalcaligenes was the best malease containing biocatalyst (Chapter 10). It producedD-malate at the highest product concentrations, with the highest yield and the highest specific activity. Comparing these data with the data for the already commercialized process forL-malate production catalyzed by fumarase, suggests that commercialization of the malease process is very promising (Chapter 10)

Genetic algorithm based two-mode clustering of metabolomics data

Metabolomics and other omics tools are generally characterized by large data sets with many variables obtained under different environmental conditions. Clustering methods and more specifically two-mode clustering methods are excellent tools for analyzing this type of data. Two-mode clustering methods allow for analysis of the behavior of subsets of metabolites under different experimental conditions. In addition, the results are easily visualized. In this paper we introduce a two-mode clustering method based on a genetic algorithm that uses a criterion that searches for homogeneous clusters. Furthermore we introduce a cluster stability criterion to validate the clusters and we provide an extended knee plot to select the optimal number of clusters in both experimental and metabolite modes. The genetic algorithm-based two-mode clustering gave biological relevant results when it was applied to two real life metabolomics data sets. It was, for instance, able to identify a catabolic pathway for growth on several of the carbon sources

Diagnosis of urinary schistosomiasis: a novel approach to compare bladder pathology measured by ultrasound and three methods for hematuria detection

We aggregated published data from field studies documenting prevalence of Schistosoma haematobium infection and bladder pathology determined by ultrasonography or hematuria detected by reagent strip, questionnaire, or visual examination. A mathematical expression was used to describe the associations between prevalence of pathology/morbidity and infection. This allows for indirect comparison of these methods, which are rarely used simultaneously. All four methods showed a similar, marked association with infection. Surprisingly, ultrasound revealed higher prevalences of pathology in schools than in communities with the same prevalence of infection, implying a need for age-related cut-off values. Reagent strip testing yielded a higher prevalence than questionnaire, which in turn was higher than by visual examination. After correction for morbidity due to other causes, a consistent ratio in prevalence of hematuria of 3:2:1 resulted for the three respective methods. The simple questionnaire approach is not markedly inferior to the other techniques, making it the best option for field use

Prevalence of inappropriate tuberculosis treatment regimens: a systematic review

A potential threat to the success of new tuberculosis (TB) drugs is the development of resistance. Using drugs in appropriate regimens, such as those recommended in the World Health Organization (WHO) treatment guidelines, prevents the development of resistance. We performed a systematic review to assess the prevalence of inappropriate prescription of TB drugs for the treatment of TB

Metabolism of carveol and dihydrocarveol in Rhodococcus erythropolis DCL14

Rhodococcus erythropolis DCL14 assimilates all stereoisomers of carveol and dihydrocarveol as sole source of carbon and energy. Induction experiments with carveol- or dihydrocarveol-grown cells showed high oxygen consumption rates with these two compounds and with carvone and dihydrocarvone. (Dihydro)carveol-grown cells of R. erythropolis DCL14 contained the following enzymic activities involved in the carveol and dihydrocarveol degradation pathways of this micro-organism: (dihydro)carveol dehydrogenase (both NAD - and dichlorophenolindophenol-dependent activities), an unknown cofactor-dependent carvone reductase, (iso-)dihydrocarvone isomerase activity, NADPH-dependent dihydrocarvone monooxygenase (Baeyer–Villiger monooxygenase), -lactone hydrolase and an NAD -dependent 6-hydroxy-3-isopropenylheptanoate dehydrogenase. Product accumulation studies identified (4R)-carvone, (1R,4R)-dihydrocarvone, (4R,7R)-4-isopropenyl-7-methyl-2-oxo-oxepanone, (3R)-6-hydroxy-3-isopropenylheptanoate, (3R)-3-isopropenyl-6-oxoheptanoate, (3S,6R)-6-isopropenyl-3-methyl-2-oxo-oxepanone and (5R)-6-hydroxy-5-isopropenyl-2-methylhexanoate as intermediates in the (4R)-carveol degradation pathway. The opposite stereoisomers of these compounds were identified in the (4S)-carveol degradation pathway. With dihydrocarveol, the same intermediates are involved except that carvone was absent. These results show that R. erythropolis DCL14 metabolizes all four diastereomers of carveol via oxidation to carvone, which is subsequently stereospecifically reduced to (1R)-(iso-)dihydrocarvone. At this point also dihydrocarveol enters the pathway, and this compound is directly oxidized to (iso-)dihydrocarvone. Cell extracts contained both (1R)-(iso-)dihydrocarvone 1,2-monooxygenase and (1S)-(iso)-dihydrocarvone 2,3-monooxygenase activity, resulting in a branch point of the degradation pathway; (1R)-(iso-)dihydrocarvone was converted to 4-isopropenyl-7-methyl-2-oxo-oxepanone, while (1S)-(iso)-dihydrocarvone, which in vivo is isomerized to (1R)-(iso-)dihydrocarvone, was converted to 6-isopropenyl-3-methyl-2-oxo-oxepanone. 4-Isopropenyl-7-methyl-2-oxo-oxepanone is hydrolysed to 6-hydroxy-3-isopropenylheptanoate, which is subsequently oxidized to 3-isopropenyl-6-oxoheptanoate, thereby linking the (dihydro)carveol degradation pathways to the limonene degradation pathway of this micro-organism. 6-Isopropenyl-3-methyl-2-oxo-oxepanone is, in vitro, hydrolysed to 6-hydroxy-5-isopropenyl-2-methylhexanoate, which is thought to be a dead-end metabolite