Characterization of the substitution pattern of cellulose derivatives using carbohydrate-binding modules

Background: Derivatized celluloses, such as methylcellulose (MC) and hydroxypropyl methylcellulose (HPMC), are of pharmaceutical importance and extensively employed in tablet matrices. Each batch of derivatized cellulose is thoroughly characterized before utilized in tablet formulations as batch-to-batch differences can affect drug release. The substitution pattern of the derivatized cellulose polymers, i.e. the mode on which the substituent groups are dispersed along the cellulose backbone, can vary from batch-to-batch and is a factor that can influence drug release. Results: In the present study an analytical approach for the characterization of the substitution pattern of derivatized celluloses is presented, which is based on the use of carbohydrate-binding modules (CBMs) and affinity electrophoresis. CBM4-2 from Rhodothermus marinus xylanase 10A is capable of distinguishing between batches of derivatized cellulose with different substitution patterns. This is demonstrated by a higher migration retardation of the CBM in acrylamide gels containing batches of MC and HPMC with a more heterogeneous distribution pattern. Conclusions: We conclude that CBMs have the potential to characterize the substitution pattern of cellulose derivatives and anticipate that with use of CBMs with a very selective recognition capacity it will be possible to more extensively characterize and standardize important carbohydrates used for instance in tablet formulation

Affinity maturation generates greatly improved xyloglucan-specific carbohydrate binding modules

<p>Abstract</p> <p>Background</p> <p>Molecular evolution of carbohydrate binding modules (CBM) is a new approach for the generation of glycan-specific molecular probes. To date, the possibility of performing affinity maturation on CBM has not been investigated. In this study we show that binding characteristics such as affinity can be improved for CBM generated from the CBM4-2 scaffold by using random mutagenesis in combination with phage display technology.</p> <p>Results</p> <p>Two modified proteins with greatly improved affinity for xyloglucan, a key polysaccharide abundant in the plant kingdom crucial for providing plant support, were generated. Both improved modules differ from other existing xyloglucan probes by binding to galactose-decorated subunits of xyloglucan. The usefulness of the evolved binders was verified by staining of plant sections, where they performed better than the xyloglucan-binding module from which they had been derived. They discriminated non-fucosylated from fucosylated xyloglucan as shown by their ability to stain only the endosperm, rich in non-fucosylated xyloglucan, but not the integument rich in fucosylated xyloglucan, on tamarind seed sections.</p> <p>Conclusion</p> <p>We conclude that affinity maturation of CBM selected from molecular libraries based on the CBM4-2 scaffold is possible and has the potential to generate new analytical tools for detection of plant carbohydrates.</p

Comparison between an African town and a neighbouring village shows delayed, but not decreased, sleep during the early stages of urbanisation

The well-established negative health outcomes of sleep deprivation, and the suggestion that availability of electricity may enable later bed times without compensating sleep extension in the morning, have stimulated interest in studying communities whose sleep pattern may resemble a pre-industrial state. Here, we describe sleep and activity in two neighbouring communities, one urban (Milange) and one rural (Tengua), in a region of Mozambique where urbanisation is an ongoing process. The two communities differ in the amount and timing of daily activity and of light exposure, with later bedtimes (≈1 h) associated with more evening and less daytime light exposure seen in the town of Milange. In contrast to previous reports comparing communities with and without electricity, sleep duration did not differ between Milange (7.28 h) and Tengua (7.23 h). Notably, calculated sleep quality was significantly poorer in rural Tengua than in Milange, and poor sleep quality was associated with a number of attributes more characteristic of rural areas, including more intense physical labour and less comfortable sleeping arrangements. Thus, whilst our data support the hypothesis that access to electricity delays sleep timing, the higher sleep quality in the urban population also suggests that some aspects of industrialisation are beneficial to sleep

Sex, War, and Disease: The Role of Parasite Infection on Weapon Development and Mating Success in a Horned Beetle (Gnatocerus cornutus)

While parasites and immunity are widely believed to play important roles in the evolution of male ornaments, their potential influence on systems where male weaponry is the object of sexual selection is poorly understood. We experimentally infect larval broad-horned flour beetles with a tapeworm and study the consequent effects on: 1) adult male morphology 2) male-male contests for mating opportunities, and 3) induction of the innate immune system. We find that infection significantly reduces adult male size in ways that are expected to reduce mating opportunities in nature. The sum of our morphological, competition, and immunological data indicate that during a life history stage where no new resources are acquired, males allocate their finite resources in a way that increases future mating potential

Engineering protein-carbohydrate interactions - lessons from natural and evolved carbohydrate binding modules

Protein-carbohydrate interactions are of central role in all living organism. In the studies presented in this thesis several strategies for engineering such interactions have been investigated. In the first study the binding affinity of a carbohydrate binding module (CBM) was improved a 100-fold through affinity maturation. Also, in that study it was discovered that all the selected, matured mutants carried a mutation in common (glutamic acid), which was shown to be responsible for the increased affinity. Furthermore, the binding interaction of two different CBM was analyzed with x-ray crystallography. One of the CBM can only bind xylans, while the other binds also beta-glucans and xyloglucan. The crystal structures revealed that the binding cleft of the multi-specific CBM is flexible, permitting reorientation of side-chains to avoid steric clashes. Also the multi-specific CBM harbored an important phenylalanine that due to its chemical composition has a pi-electron surface and can interact with hydrogen atoms on the different ligands it recognizes. The specific CBM on the other hand harbor a leucine in this position and can only interact with xylans. In the last study, the capacity of CBM for use as analytical tools in the characterization of the distribution pattern of cellulose derivatives was investigated. The study showed that some CBM are able to distinguish between cellulose derivatives with different substitution distribution. This is a potentially new application for CBM. In all, the thesis demonstrates strategies in which carbohydrate binding proteins can be generated, improved and utilized

Bioinspired assemblies of plant cell wall polymers unravel the affinity properties of carbohydrate-binding modules

Lignocellulose-acting enzymes play a central role in the biorefinery of plant biomass to make fuels, chemicals and materials. These enzymes are often appended to carbohydrate binding modules (CBMs) that promote substrate targeting. When used in plant materials, which are complex assemblies of polymers, the binding properties of CBMs can be difficult to understand and predict, thus limiting the efficiency of enzymes. In order to gain more information on the binding properties of CBMs, some bioinspired model assemblies that contain some of the polymers and covalent interactions found in the plant cell walls have been designed. The mobility of three engineered CBMs has been investigated by FRAP in these assemblies, while varying the parameters related to the polymer concentration, the physical state of assemblies and the oligomerization state of CBMs. The features controlling the mobility of the CBMs in the assemblies have been quantified and hierarchized. We demonstrate that the parameters can have additional or opposite effects on mobility, depending on the CBM tested. We also find evidence of a relationship between the mobility of CBMs and their binding strength. Overall, bioinspired assemblies are able to reveal the unique features of affinity of CBMs. In particular, the results show that oligomerization of CBMs and the presence of ferulic acid motifs in the assemblies play an important role in the binding affinity of CBMs. Thus we propose that these features should be finely tuned when CBMs are used in plant cell walls to optimise bioprocesses

New insights into mobility and affinity of engineered family 4 CBMs in bioinspired lignocellulosic assemblies

Lignocellulose-acting enzymes are often appended to one or more carbohydrate binding modules (CBMs) which promote substrate targeting due to their variable substrate specificity and affinity. They thus play a central role in the enzymatic deconstruction of plant cell wall biomass, but their fine characterization is generally performed with pure isolated polymers which do not reflect the complex plant cell walls. In order to gain more information on the behavior and mobility of CBMs in complex 3D substrates, we have devised and used bioinspired model assemblies that contain some of the polymers and covalent interactions found in plant cell walls. These assemblies contain feruloylated arabinoxylans (FAXs) and cellulose nano-crystals (CNCs) at various concentrations in solution (free polymers) or in gels (cross-linked polymers). The three CBMs studied herein belong to family 4 and have been engineered so that they have different affinities and specificities toward FAX and CNC: CBM4-2 binds FAX and CNC, CBM X-2 binds only FAX and CBM G-4 binds neither FAX nor CNC. These CBMs have been produced as monomers and as dimers and then been grafted to a fluorophore so that their mobility in the bioinspired assemblies can be followed by measuring the fluorescence recovery after photobleaching. By varying three parameters related to the assemblies (CNC concentration, solution or gel state) and to the CBMs (oligomerisation state), we have been able to calculate an apparent affinity and to model the mobility of the CBMs. Overall, our results reveal that apparent affinity is drastically modified in a 3D polymer network in comparison to their behavior in solution, and parameters varied influence differently each CBM. This demonstrates that bioinspired assemblies may provide important information relevant for studies of carbohydrate active proteins such as the selection of appropriate CBM constructs to be further exploited in applications targeting complex carbohydrate substrates

Microdistribution of xyloglucan in differentiating poplar cells

Recent studies on the ultrastructure and composition of the gelatinous layer (g-layer) in poplar have reported findings of xyloglucan. Using correlated fluorescence, scanning- and transmission electron micros-copy, we found evidence for xyloglucan present in and surrounding the g-layer, using the fucosylated xyloglucan specific CCRC-M1 antibody and the carbohydrate binding module FXG-14b. However, labeling of isolated gelatinous layer remained negative