Activated sludge encapsulation in gellan gum microbeads for gasoline biodegradation

A two-phase dispersion technique, termed emulsification–internal gelation, is proposed for encapsulation of activated sludge in gellan gum microbeads. The influence of emulsion parameters on size distribution of microbeads was investigated. Mean diameter of microbeads varied within a range of 34–265 µm as a descending function of emulsion stirring rate (1,000–5,000 rpm), emulsification time (10–40 min), and emulsifier concentration (0–0.1% w/w), and as an ascending function of disperse phase volume fraction (0.08–0.25). Encapsulated sludge expressed a high biodegradation activity compared with non-encapsulated sludge cultures even at 4.4 times lower level of overall biomass loading. Over 90% of gasoline at an initial concentration of 35 and 70 mg l^–1 was removed by both encapsulated and non-encapsulated sludge cultures in sealed serum bottles within 7 days. Encapsulation of activated sludge in gellan gum microbeads enhanced the biological activity of microbial populations in the removal of gasoline hydrocarbons. The results of this study demonstrated the feasibility of the production of size-controlled gellan gum-encapsulated sludge microbeads and their use in the biodegradation of gasoline.     

Local backbone structure prediction of proteins

A statistical analysis of the PDB structures has led us to define a new set of small 3D structural prototypes called Protein Blocks (PBs). This structural alphabet includes 16 PBs, each one is defined by the (phi, psi) dihedral angles of 5 consecutive residues. The amino acid distributions observed in sequence windows encompassing these PBs are used to predict by a Bayesian approach the local 3D structure of proteins from the sole knowledge of their sequences. LocPred is a software which allows the users to submit a protein sequence and performs a prediction in terms of PBs. The prediction results are given both textually and graphically.     

Influence of microarrays experiments missing values on the stability of gene groups by hierarchical clustering

Background

Microarray technologies produced large amount of data. The hierarchical clustering is commonly used to identify clusters of co-expressed genes. However, microarray datasets often contain missing values (MVs) representing a major drawback for the use of the clustering methods. Usually the MVs are not treated, or replaced by zero or estimated by the k-Nearest Neighbor (kNN) approach. The topic of the paper is to study the stability of gene clusters, defined by various hierarchical clustering algorithms, of microarrays experiments including or not MVs.

Results

In this study, we show that the MVs have important effects on the stability of the gene clusters. Moreover, the magnitude of the gene misallocations is depending on the aggregation algorithm. The most appropriate aggregation methods (e.g. complete-linkage and Ward) are highly sensitive to MVs, and surprisingly, for a very tiny proportion of MVs (e.g. 1%). In most of the case, the MVs must be replaced by expected values. The MVs replacement by the kNN approach clearly improves the identification of co-expressed gene clusters. Nevertheless, we observe that kNN approach is less suitable for the extreme values of gene expression.

Conclusion

The presence of MVs (even at a low rate) is a major factor of gene cluster instability. In addition, the impact depends on the hierarchical clustering algorithm used. Some methods should be used carefully. Nevertheless, the kNN approach constitutes one efficient method for restoring the missing expression gene values, with a low error level. Our study highlights the need of statistical treatments in microarray data to avoid misinterpretation.

     

Circadian phase and period responses to light stimuli in two nocturnal rodents

We report period response curves (τRC) for two nocturnal Murid species from India, Mus booduga and Mus platythrix. We further discuss the method of phase shift estimation in the presence of τ-changes, because such changes pose a serious methodological problem in the estimation of phase shifts. Although the τRC indicates that most of the phase shifts are associated with small changes in τ, the period changes across all the phases showed a significant positive correlation with the phase shifts. We conclude that τRCs are a reality even in nocturnal mammals, although their amplitude is less than what is usually found in diurnal mammals, and requires a larger data set to be distinguished from noise.     

Rafts promote assembly and atypical targeting of a nonenveloped virus, rotavirus, in Caco-2 cells

Rotavirus follows an atypical pathway to the apical membrane of intestinal cells that bypasses the Golgi. The involvement of rafts in this process was explored here. VP4 is the most peripheral protein of the triple-layered structure of this nonenveloped virus. High proportions of VP4 associated with rafts within the cell as early as 3 h postinfection. In the meantime a significant part of VP4 was targeted to the Triton X-100-resistant microdomains of the apical membrane, suggesting that this protein possesses an autonomous signal for its targeting. At a later stage the other structural rotavirus proteins were also found in rafts within the cells together with NSP4, a nonstructural protein required for the final stage of virus assembly. Rafts purified from infected cells were shown to contain infectious particles. Finally purified VP4 and mature virus were shown to interact with cholesterol- and sphingolipid-enriched model lipid membranes that changed their phase preference from inverted hexagonal to lamellar structures. Together these results indicate that a direct interaction of VP4 with rafts promotes assembly and atypical targeting of rotavirus in intestinal cells.     

The identification of intestinal scavenger receptor class B, type I (SR-BI) by expression cloning and its role in cholesterol absorption

The molecular mechanisms of cholesterol absorption in the intestine are poorly understood. With the goal of defining candidate genes involved in these processes a fluorescence-activated cell sorter-based, retroviral-mediated expression cloning strategy has been devised. SCH354909, a fluorescent derivative of ezetimibe, a compound which blocks intestinal cholesterol absorption but whose mechanism of action is unknown, was synthesized and shown to block intestinal cholesterol absorption in rats. Pools of cDNAs prepared from rat intestinal cells enriched in enterocytes were introduced into BW5147 cells and screened for SCH354909 binding. Several independent clones were isolated and all found to encode the scavenger receptor class B, type I (SR-BI), a protein suggested by others to play a role in cholesterol absorption. SCH354909 bound to Chinese hamster ovary (CHO) cells expressing SR-BI in specific and saturable fashion and with high affinity (K(d) approximately 18 nM). Overexpression of SR-BI in CHO cells resulted in increased cholesterol uptake that was blocked by micromolar concentrations of ezetimibe. Analysis of rat intestinal sections by in situ hybridization demonstrated that SR-BI expression was restricted to enterocytes. Cholesterol absorption was determined in SR-B1 knockout mice using both an acute, 2-h, assay and a more chronic fecal dual isotope ratio method. The level of intestinal cholesterol uptake and absorption was similar to that seen in wild-type mice. When assayed in the SR-B1 knockout mice, the dose of ezetimibe required to inhibit hepatic cholesterol accumulation induced by a cholesterol-containing 'western' diet was similar to wild-type mice. Thus, the binding of ezetimibe to cells expressing SR-B1 and the functional blockade of SR-B1-mediated cholesterol absorption in vitro suggest that SR-B1 plays a role in intestinal cholesterol metabolism and the inhibitory activity of ezetimibe. In contrast studies with SR-B1 knockout mice suggest that SR-B1 is not essential for intestinal cholesterol absorption or the activity of ezetimibe.     

First observation of solution structures of bradykinin-penta-O-galloyl-D-glucopyranose complexes as determined by NMR and simulated annealing

Polyphenols (tannins) are known for their high propensity to precipitate proteins. They bind most strongly to proteins with a high proline content. Understanding the mechanism of this association is of prime interest because this interaction might induce protein conformational changes that may modify their biological activity. To investigate the interaction, an NMR study was carried out on the binding of a representative polyphenol, penta-O-galloyl-D-glucopyranose, to a nonapeptide hormone, bradykinin (BDK), where proline accounts for 30% of residues. Series of 1D and 2D-NMR experiments were performed. For the first time, a three-dimensional structure of complexes was determined using 2D-NMR experiments and molecular modeling. These structure calculations are a potent tool to understand how the association arises. They clearly show that the interaction is a complex phenomenon where several parameters are involved. The PGG/BDK complexes are formed by multiple weak interactions between peptide side chains and galloyl rings. Proline and arginine are good anchoring points and the glycine gives a certain flexibility in the peptide backbone that allows the polyphenol to approach and interact. Therefore, it is not only the hydrophobic stackings between galloyl rings and proline and hydrogen bonding involving arginine and aromatic rings which are important. The residue sequence and the side chain steric bulk also intervene.     

Genetic model organisms in the study of N-glycans

Recently the genomic sequences of three multicellular eukaryotes, Caenorhabditis elegans, Drosophila melanogaster and Arabidopsis thaliana, have been elucidated. A number of cDNAs encoding glycosyltransferases demonstrated to have a role in N-linked glycosylation have already been cloned from these organisms, e.g., GlcNAc transferases and alpha 1,3-fucosyltransferases. However, many more homologues of glycosyltransferases and other glycan modifying enzymes have been predicted by analysis of the genome sequences, but the predictions of full length open reading frames appear to be particularly poor in Caenorhabditis. The use of these organisms as models in glycobiology may be hampered since they all have N-linked glycosylation repertoires unlike those of mammals. Arabidopsis and Drosophila have glycosylation similar to that of other plants or insects, while our new data from MALDI-TOF analysis of PNGase A-released neutral N-glycans of Caenorhabditis indicate that there exists a range of pauci- and oligomannosidic structures, with up to four fucose residues and up to two O-methyl groups. With all these three 'genetic model organisms', however, much more work is required for a full understanding of their glycobiology.     

Analysis of Asn-linked glycans from vegetable foodstuffs: widespread occurrence of Lewis a, core alpha1,3-linked fucose and xylose substitutions

The N-glycans from 27 "plant" foodstuffs, including one from a gymnospermic plant and one from a fungus, were prepared by a new procedure and examined by means of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). For several samples, glycan structures were additionally investigated by size-fractionation and reverse-phase high-performance liquid chromatography in conjunction with exoglycosidase digests and finally also (1)H-nuclear magnetic resonance spectroscopy. The glycans found ranged from the typical vacuolar "horseradish peroxidase" type and oligomannose to complex Le(a)-carrying structures. Though the common mushroom exclusively contained N-glycans of the oligomannosidic type, all plant foods contained mixtures of the above-mentioned types. Apple, asparagus, avocado, banana, carrot, celery, hazelnut, kiwi, onion, orange, pear, pignoli, strawberry, and walnut were particularly rich in Le(a)-carrying N-glycans. Although traces of Le(a)-containing structures were also present in almond, pistachio, potato, and tomato, no such glycans could be found in cauliflower. Coconut exhibited almost exclusively N-glycans containing only xylose but no fucose. Oligomannosidic N-glycans dominated in buckwheat and especially in the legume seeds mung bean, pea, peanut, and soybean. Papaya presented a unique set of hybrid type structures partially containing the Le(a) determinant. These results are not only compatible with the hypothesis that the carbohydrate structures are another potential source of immunological cross-reaction between different plant allergens, but they also demonstrate that the Le(a)-type structure is very widespread among plants.