Search

Enzyme engineering is a powerful tool for improving or altering the properties of biocatalysts for industrial, research, and therapeutic applications. Fast and accurate screening of variant libraries is often the bottleneck of enzyme engineering and may be overcome by growth-based screening strategies with simple processes to enable high throughput. The currently available growth-based screening strategies have been widely employed for enzymes but not yet for catalytically potent and oxygen-sensitive metalloenzymes. Here, we present a screening system that couples the activity of an oxygen-sensitive formate dehydrogenase to the growth of Escherichia coli . This system relies on the complementation of the E. coli formate hydrogenlyase (FHL) complex by Mo-dependent formate dehydrogenase H ( Ec FDH-H). Using an Ec FDH-H-deficient strain, we demonstrate that growth inhibition by acidic glucose fermentation products can be alleviated by FHL complementation. This allows the identification of catalytically active Ec FDH-H variants at a readily measurable cell density readout, reduced handling efforts, and a low risk of oxygen contamination. Furthermore, a good correlation between cell density and formate oxidation activity was established using Ec FDH-H variants with variable catalytic activities. As proof of concept, the growth assay was employed to screen a library of 1,032 Ec FDH-H variants and reduced the library size to 96 clones. During the subsequent colorimetric screening of these clones, the variant A12G exhibiting an 82.4% enhanced formate oxidation rate was identified. Since many metal-dependent formate dehydrogenases and hydrogenases form functional complexes resembling E. coli FHL, the demonstrated growth-based screening strategy may be adapted to components of such electron-transferring complexes.IMPORTANCEOxygen-sensitive metalloenzymes are highly potent catalysts that allow the reduction of chemically inert substrates such as CO 2 and N 2 at ambient pressure and temperature and have, therefore, been considered for the sustainable production of biofuels and commodity chemicals such as ammonia, formic acid, and glycine. A proven method to optimize natural enzymes for such applications is enzyme engineering using high-throughput variant library screening. However, most screening methods are incompatible with the oxygen sensitivity of these metalloenzymes and thereby limit their relevance for the development of biosynthetic production processes. A microtiter plate-based assay was developed for the screening of metal-dependent formate dehydrogenase that links the activity of the tested enzyme variant to the growth of the anaerobically grown host cell. The presented work extends the application range of growth-based screening to metalloenzymes and is thereby expected to advance their adoption to biosynthesis applications., Competing Interests: The authors declare no conflict of interest.

One of the most important properties of membranes is their permeability to water and other small molecules. A targeted change in permeability allows the passage of molecules to be controlled. Vesicles made of membranes with low water permeability are preferable for drug delivery, for example, because they are more stable and maintain the drug concentration inside. This study reports on the very low water permeability of pure protein membranes composed of a bilayer of the amphiphilic protein hydrophobin HFBI. Using a droplet interface bilayer setup, we demonstrate that HFBI bilayers are essentially impermeable to water. HFBI bilayers withstand far larger osmotic pressures than lipid membranes. Only by disturbing the packing of the proteins in the HFBI bilayer is a measurable water permeability induced. To investigate possible molecular mechanisms causing the near-zero permeability, we used all-atom molecular dynamics simulations of various HFBI bilayer models. The simulations suggest that the experimental HFBI bilayer permeability is compatible neither with a lateral honeycomb structure, as found for HFBI monolayers, nor with a residual oil layer within the bilayer or with a disordered lateral packing similar to the packing in lipid bilayers. These results suggest that the low permeabilities of HFBI and lipid bilayers rely on different mechanisms. With their extremely low but adaptable permeability and high stability, HFBI membranes could be used as an osmotic pressure-insensitive barrier in situations where lipid membranes fail such as desalination membranes.

Hydrogen oxidizing autotrophic bacteria are promising hosts for conversion of CO 2 into chemicals. In this work, we engineered the metabolically versatile lithoautotrophic bacterium R. opacus strain DSM 43205 for synthesis of polymer precursors. Aspartate decarboxylase (panD) or lactate dehydrogenase (ldh) were expressed for beta-alanine or L-lactic acid production, respectively. The heterotrophic cultivations on glucose produced 25 mg L -1 beta-alanine and 742 mg L -1 L-lactic acid, while autotrophic cultivations with CO 2 , H 2 , and O 2 resulted in the production of 1.8 mg L -1 beta-alanine and 146 mg L -1 L-lactic acid. Beta-alanine was also produced at 345 μg L -1 from CO 2 in electrobioreactors, where H 2 and O 2 were provided by water electrolysis. This work demonstrates that R. opacus DSM 43205 can be engineered to produce chemicals from CO 2 and provides a base for its further metabolic engineering., Competing Interests: Authors LS, LO, GP, ML, and MT were employed by the company VTT Technical Research Centre of Finland Ltd. and author J-PP was employed by the company Solar Foods Ltd. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Salusjärvi, Ojala, Peddinti, Lienemann, Jouhten, Pitkänen and Toivari.)

This study evaluates the techno-economic feasibility of five solar-powered concepts for the production of autotrophic microorganisms for food and feed production; the main focus is on three concepts based on hydrogen-oxidizing bacteria (HOB), which are further compared to two microalgae-related concepts. Two locations with markedly different solar conditions are considered (Finland and Morocco), in which Morocco was found to be the most economically competitive for the cultivation of microalgae in open ponds and closed systems (1.4 and 1.9 € kg -1 , respectively). Biomass production by combined water electrolysis and HOB cultivation results in higher costs for all three considered concepts. Among these, the lowest production cost of 5.3 € kg -1 is associated with grid-assisted electricity use in Finland, while the highest production cost of >9.1 € kg -1 is determined for concepts using solely photovoltaics and/or photoelectrochemical technology for on-site electricity production and solar-energy conversion to H 2 by water electrolysis. All assessed concepts are capital intensive. Furthermore, a sensitivity analysis suggests that the production costs of HOB biomass can be lowered down to 2.1 € kg -1 by optimization of the process parameters among which volumetric productivity, electricity strategy, and electricity costs have the highest cost-saving potentials. The study reveals that continuously available electricity and H 2 supply are essential for the development of a viable HOB concept due to the capital intensity of the needed technologies. In addition, volumetric productivity is the key parameter that needs to be optimized to increase the economic competitiveness of HOB production., Competing Interests: The authors declare no competing financial interest., (© 2020 The Authors. Published by American Chemical Society.)

Recent advances in enzymatic electrosynthesis of desired chemicals in biological-inorganic hybrid systems has generated interest because it can use renewable energy inputs and employs highly specific catalysts that are active at ambient conditions. However, the development of such innovative processes is currently limited by a deficient understanding of the molecular mechanisms involved in electrode-based electron transfer and biocatalysis. Mechanistic studies of non-electrosynthetic electron transferring proteins have provided a fundamental understanding of the processes that take place during enzymatic electrosynthesis. Thus, they may help explain how redox proteins stringently control the reduction potential of the transferred electron and efficiently transfer it to a specific electron acceptor. The redox sites at which electron donor oxidation and electron acceptor reduction take place are typically located in distant regions of the redox protein complex and are electrically connected by an array of closely spaced cofactors. These groups function as electron relay centers and are shielded from the surrounding environment by the electrically insulating apoporotein. In this matrix, electrons travel via electron tunneling, i.e. hopping between neighboring cofactors, over impressive distances of upto several nanometers and, as in the case of the Shewanella oneidensis Mtr electron conduit, traverse the bacterial cell wall to extracellular electron acceptors such as solid ferrihydrite. Here, the biochemical strategies of protein-based electron transfer are presented in order to provide a basis for future studies on the basis of which a more comprehensive understanding of the structural biology of enzymatic electrosynthesis may be attained., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 The Author(s).)

Background: The HIV epidemic in Cambodia is strongly and disproportionately concentrated among key populations. One important hard-to-reach key population is the expanding community of female entertainment workers (FEWs). HIV as well as other sexual and reproductive health (SRH) outcomes including sexually transmitted infections (STIs), contraception, and gynecologic health are also substandard among FEWs. To address these concerns, a mobile health intervention (mHealth) using short message service (SMS) and voice message (VM) services-the Mobile Link project-was constructed. This paper aims to describe the development of this mHealth intervention that used participatory methodologies and to illustrate how these findings can be useful in future mHealth projects., Methods: This intervention development process used an iterative, participatory approach. Twenty-seven focus group discussions (FGDs) covering SRH topics were designed and conducted and implemented across four provinces in Cambodia. Additionally, six in-depth interviews (IDIs) were conducted with FEWs living with HIV in Siem Reap and Phnom Penh. Data from the FGDs and IDIs were analyzed using content and matrix analysis methods to identify prioritized themes for messages. Two data validation workshops were organized to present the prioritized themes to FEWs and outreach workers (outreach workers) for validation. The workshops included activities stimulating participation such as listening to sample messages in order to determine health priorities as well as message tone and style., Results: The findings from the qualitative research provided guidance on how to tailor the intervention to the FEW community in terms of the tone, timing, content and delivery mode of the messages. Participants preferred a friendly, professional female voice for VM. Participants revealed that health priorities such as gynecologic issues (vaginal infections/irritation) and cervical and breast cancer, were emphasized more than HIV and family planning. Participants also reported a number of misconceptions about contraception, particularly around oral contraceptives and intrauterine devices, and STI transmission. Participants expressed the need to build trust in outreach workers and linkages, affirming the emphasis on the link within the Mobile Link project. Lastly, from the IDIs, FEWs living with HIV highlighted wanting supportive/messages to address depressive feelings that may stem from their perceived stigma., Conclusions: Utilizing participatory methodologies was demonstrated to be useful in intervention content creation and program implementation. As a result of this intervention development process, the research team gleaned lessons that may be applicable to future mHealth projects including the idea of adding some layers of choice to mHealth interventions for further tailoring at the individual level and the importance of human contact and trusting relationship., Competing Interests: Conflicts of Interest: The authors have no conflicts of interest to declare.

Class II hydrophobins are amphiphilic proteins produced by filamentous fungi. One of their typical features is the tendency to accumulate at the interface between an aqueous phase and a hydrophobic phase, such as the air-water interface. The kinetics of the interfacial self-assembly of wild-type hydrophobins HFBI and HFBII and some of their engineered variants at the air-water interface were measured by monitoring the accumulated mass at the interface via nondestructive ellipsometry measurements. The resulting mass vs time curves revealed unusual kinetics for a monolayer formation that did not follow a typical Langmuir-type of behavior but had a rather coverage-independent rate instead. Typically, the full surface coverage was obtained at masses corresponding to a monolayer. The formation of multilayers was not observed. Atomic force microscopy revealed formation and growth of non-fusing protein clusters at the interface. The mechanism of the adsorption was studied by varying the structure or charges of the protein or the ionic strength of the subphase, revealing that the lateral interactions between the hydrophobins play a role in their interfacial assembly. Additionally, a theoretical model was introduced to identify the underlying mechanism of the unconventional adsorption kinetics.

Hydrogen-oxidizing bacteria (HOB) have been shown to be promising micro-organisms for the reduction of carbon dioxide to a wide range of value-added products in bioelectrochemical systems with in situ water electrolysis of the cultivation medium, also known as a hybrid biological-inorganic systems (HBI). However, scaling up of this process requires overcoming the inherent constraints of the low energy efficiency partly associated with the pH-neutral electrolyte with low conductivity. Most of the research in the field is concentrated on the bacterial cultivation, whereas the analysis and evaluation of the electrode material performance have received little attention in the literature so far. Therefore, in the present work, in situ electrolysis of a pH-neutral medium for HOB cultivation was performed with different combinations of electrode materials. Besides conventional electrode types, electrodes with coatings made of earth-abundant cobalt and a nickel-iron alloy, known for their catalytic activity for the kinetically sluggish oxygen evolution reaction (OER), were prepared and tested as potential substitutes for catalysts made of precious metals. The cultivation of HOB with in situ water electrolysis has been successfully tested in a small scale electrobioreactor in order to support the experimental results. A simplified water electrolysis model was developed and applied to evaluate the current-voltage characteristics of an bioelectrochemical system prototype. Application of the developed model allows quantitative evaluation and comparison of reversible, ohmic, and activation overvoltages of different electrode sets. The modeling results were found to agree well with the experimental data. The developed model and the data gathered can be applied to further investigation, simulation, and optimization of HBI systems.

Biosensors detect signals using biological sensing components such as redox enzymes and biological cells. Although cellular versatility can be beneficial for different applications, limited stability and efficiency in signal transduction at electrode surfaces represent a challenge. Recent studies have shown that the Mtr electron conduit from Shewanella oneidensis MR-1 can be produced in Escherichia coli to generate an exoelectrogenic model system with well-characterized genetic tools. However, means to specifically immobilize this organism at solid substrates as electroactive biofilms have not been tested previously. Here, we show that mannose-binding Fim pili can be produced in exoelectrogenic E. coli and can be used to selectively attach cells to a mannose-coated material. Importantly, cells expressing fim genes retained current production by the heterologous Mtr electron conduit. Our results demonstrate the versatility of the exoelectrogenic E. coli system and motivate future work that aims to produce patterned biofilms for bioelectronic devices that can respond to various biochemical signals., (© 2018 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Hydrophobins are a family of small-sized proteins featuring a distinct hydrophobic patch on the protein's surface, rendering them amphiphilic. This particularity allows hydrophobins to self-assemble into monolayers at any hydrophilic/hydrophobic interface. Moreover, stable pure protein bilayers can be created from two interfacial hydrophobin monolayers by contacting either their hydrophobic or their hydrophilic sides. In this study, this is achieved via a microfluidic approach, in which also the bilayers' adhesion energy can be determined. This enables us to study the origin of the adhesion of hydrophobic and hydrophilic core bilayers made from the class II hydrophobins HFBI and HFBII. Using different fluid media in this setup and introducing genetically modified variants of the HFBI molecule, the different force contributions to the adhesion of the bilayer sheets are studied. It was found that in the hydrophilic contact situation, the adhesive interaction was higher than that in the hydrophobic contact situation and could be even enhanced by reducing the contributions of electrostatic interactions. This effect indicates that the van der Waals interaction is the dominant contribution that explains the stability of the observed bilayers.

Electrosynthesis of formate is a promising technology to convert CO 2 and electricity from renewable sources into a biocompatible, soluble, non-flammable, and easily storable compound. In the model methanogen Methanococcus maripaludis, uptake of cathodic electrons was shown to proceed indirectly via formation of formate or H 2 by undefined, cell-derived enzymes. Here, we identified that the multi-enzyme heterodisulfide reductase supercomplex (Hdr-SC) of M. maripaludis is capable of direct electron uptake and catalyzes rapid H 2 and formate formation in electrochemical reactors (-800 mV vs Ag/AgCl) and in Fe(0) corrosion assays. In Fe(0) corrosion assays and electrochemical reactors, purified Hdr-SC primarily catalyzed CO 2 reduction to formate with a coulombic efficiency of 90% in the electrochemical cells for 5 days. Thus, this report identified the first enzyme that stably catalyzes the mediator-free electrochemical reduction of CO 2 to formate, which can serve as the basis of an enzyme electrode for sustained electrochemical production of formate., (Copyright © 2018. Published by Elsevier Ltd.)

Pure protein bilayers and vesicles are formed using the native, fungal hydrophobin HFBI. Bilayers with hydrophobic (red) and hydrophilic (blue) core are produced and, depending on the type of core, vesicles in water, oily media, and even in air can be created using microfluidic jetting. Vesicles in water are even able to incorporate functional gramicidin A pores., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Hydrophobins are extracellular proteins produced by filamentous fungi. They show a variety of functions at interfaces that help fungi to adapt to their environment by, for example, adhesion, formation of coatings, and lowering the surface tension of water. Hydrophobins fold into a globular structure and have a distinct hydrophobic patch on their surface that makes these proteins amphiphilic. Their amphiphilicity implies interfacial assembly, but observations indicate that intermolecular interactions also contribute to their functional properties. Here, we used the class II hydrophobin HFBI from Trichoderma reesei as a model to understand the structural basis for the function of hydrophobins. Four different variants were made in which charged residues were mutated. The residues were chosen to probe the role of different regions of the hydrophilic part of the proteins. Effects of the mutations were studied by analyzing the formation and structure of self-assembled layers, multimerization in solution, surface adhesion, binding of secondary layers of proteins on hydrophobins, and the viscoelastic behavior of the air-water interface during formation of protein films; the comparison showed clear differences between variants only in the last two analyses. Surface viscoelasticity behavior suggests that the formation of surface layers is regulated by specific interactions that lead to docking of proteins to each other. One set of mutations led to assemblies with a remarkably high elasticity at the air-water interface (1.44 N/m). The variation of binding of secondary layers of protein on surface-adsorbed hydrophobins suggest a mechanism for a proposed function of hydrophobins, namely, that hydrophobins can act as a specific adhesive layer for the binding of macromolecules to interfaces.

HFBI (derived from Trichoderma sp.) is a unique structural protein, which forms a self-organized monolayer at both air/water interface and water/solid interfaces in accurate two-dimensional ordered structures. We have taken advantage of the unique functionality of HFBI as a molecular carrier for preparation of ordered molecular phase on solid substrate surfaces. The HFBI molecular carrier can easily form ordered structures; however, the dense molecular layers form an electrochemical barrier between the electrode and solution phase. In this study, the electrochemical properties of HFBI self-organized membrane-covered electrodes were investigated. Wild-type HFBI has balanced positive and negative charges on its surface. Highly oriented pyrolytic graphite (HOPG) electrodes coated with HFBI molecules were investigated electrochemically. To improve the electrochemical properties of this HFBI-coated electrode, the two types of HFBI variants, with oppositely charged surfaces, were prepared genetically. All three types of HFBI-coated HOPG electrode perform electron transfer between the electrode and solution phase through the dense HFBI molecular layer. This is because the HFBI self-organized membrane has a honeycomb-like structure, with penetrating holes. In the cases of HFBI variants, the oppositely charged HFBI membrane phases shown opposite electrochemical behaviors in electrochemical impedance spectroscopy. HFBI is a molecule with a unique structure, and can easily form honeycomb-like structures on solid material surfaces such as electrodes. The molecular membrane phase can be used for electrochemical molecular interfaces., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The strategic surface immobilization of a protein can add new functionality to a solid substrate; however, protein activity, e.g., enzymatic activity, can be drastically decreased on immobilization onto a solid surface. The concept of a designed and optimized "molecular interface" is herein introduced in order to address this problem. In this study, molecular interface was designed and constructed with the aim of attaining high enzymatic activity of a solid-surface-immobilized a using the hydrophobin HFBI protein in conjunction with a fusion protein of HFBI attached to glucose oxidase (GOx). The ability of HFBI to form a self-organized membrane on a solid surface in addition to its adhesion properties makes it an ideal candidate for immobilization. The developed fusion protein was also able to form an organized membrane, and its structure and immobilized state on a solid surface were investigated using QCM-D measurements. This method of immobilization showed retention of high enzymatic activity and the ability to control the density of the immobilized enzyme. In this study, we demonstrated the importance of the design and construction of molecular interface for numerous purposes. This method of protein immobilization could be utilized for preparation of high throughput products requiring structurally ordered molecular interfaces, in addition to many other applications., (Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.)

Hydrophobins are small fungal proteins that are amphiphilic and have a strong tendency to assemble at interfaces. By taking advantage of this property, hydrophobins have been used for a number of applications: as affinity tags in protein purification, for protein immobilization, such as in foam stabilizers, and as dispersion agents for insoluble drug molecules. Here, we used site-directed mutagenesis to gain an understanding of the molecular basis of their properties. We especially focused on the role of charged amino acids in the structure of hydrophobins. For this purpose, fusion proteins consisting of Trichoderma reesei hydrophobin I (HFBI) and the green fluorescent protein (GFP) that contained various combinations of substitutions of charged amino acids (D30, K32, D40, D43, R45, K50) in the HFBI structure were produced. The effects of the introduced mutations on binding, oligomerization, and partitioning were characterized in an aqueous two-phase system. It was found that some substitutions caused better surface binding and reduced oligomerization, while some showed the opposite effects. However, all mutations decreased partitioning in surfactant systems, indicating that the different functions are not directly correlated and that partitioning is dependent on finely tuned properties of hydrophobins. This work shows that not all functions in self-assembly are connected in a predictable way and that a simple surfactant model for hydrophobin function is insufficient.

Hydrophobins are structural proteins produced by filamentous fungi that are amphiphilic and function through self-assembling into structures such as membranes. They have diverse roles in the growth and development of fungi, for example in adhesion to substrates, for reducing surface tension to allow aerial growth, in forming protective coatings on spores and other structures. Hydrophobin membranes at the air-water interface and on hydrophobic solids are well studied, but understanding how hydrophobins can bind to a polar surface to make it more hydrophobic has remained unresolved. Here we have studied different class II hydrophobins for their ability to bind to polar surfaces that were immersed in buffer solution. We show here that the binding under some conditions results in a significant increase of water contact angle (WCA) on some surfaces. The highest contact angles were obtained on cationic surfaces where the hydrophobin HFBI has an average WCA of 62.6° at pH 9.0, HFBII an average of 69.0° at pH 8.0, and HFBIII had an average WCA of 61.9° at pH 8.0. The binding of the hydrophobins to the positively charged surface was shown to depend on both pH and ionic strength. The results are significant for understanding the mechanism for formation of structures such as the surface of mycelia or fungal spore coatings as well as for possible technical applications.

Cross-linking of β-casein by Trichoderma reesei tyrosinase (TrTyr) and Streptoverticillium mobaraense transglutaminase (Tgase) was analyzed by (31)P nuclear magnetic resonance (NMR) spectroscopy in ionic liquid (IL). According to (31)P NMR, 91% of the tyrosine side chains were cross-linked by TrTyr at high dosages. When Tgase was used, no changes were observed because a different cross-linking mechanism was operational. However, this verified the success of the phosphitylation of phenolics within the protein matrix in the IL. Atomic force microscopy (AFM) in solid state showed that disk-shaped nanoparticles were formed in the reactions with average diameters of 80 and 20 nm for TrTyr and Tgase, respectively. These data further advance the current understanding of the action of tyrosinases on proteins on molecular and chemical bond levels. Quantitative (31)P NMR in IL was shown to be a simple and efficient method for the study of protein modification.

Hydrophobins are adhesive proteins produced by filamentous fungi. They are in many cases secreted into the medium and adsorb readily to a number of different surfaces. They fulfill many different tasks such as the formation of various coatings and mediating adhesion of fungi to surfaces. The mechanism of how hydrophobins adhere and how they mediate fungal adhesion is of interest both from the point of view of fungal biology and for various biotechnical immobilization applications. It has been shown that hydrophobins typically form a monomolecular layer on solid substrates. We are especially interested in how a surface layer of hydrophobin can mediate the adhesion of a second layer of another protein. In this work we systematically studied how proteins adsorb onto hydrophobins that are bound as monomolecular layers on nonpolar surfaces. We found that several types of proteins readily adsorb onto hydrophobins, but only under defined conditions of pH and ionic strength. The binding conditions were also highly dependent on the adhering protein. By studying solution conditions such as pH and ionic strength, we conclude that the surface adhesion is due to selective Coulombic charge interactions. We conclude that hydrophobins can transform a nonpolar surface into one that efficiently recruits other proteins by charge interactions.

Insufficient accumulation levels of recombinant proteins in plants and the lack of efficient purification methods for recovering these valuable proteins have hindered the development of plant biotechnology applications. Hydrophobins are small and surface-active proteins derived from filamentous fungi that can be easily purified by a surfactant-based aqueous two-phase system. In this study, the hydrophobin HFBI sequence from Trichoderma reesei was fused to green fluorescent protein (GFP) and transiently expressed in Nicotiana benthamiana plants by Agrobacterium tumefaciens infiltration. The HFBI fusion significantly enhanced the accumulation of GFP, with the concentration of the fusion protein reaching 51% of total soluble protein, while also delaying necrosis of the infiltrated leaves. Furthermore, the endoplasmic reticulum-targeted GFP-HFBI fusion induced the formation of large novel protein bodies. A simple and scalable surfactant-based aqueous two-phase system was optimized to recover the HFBI fusion proteins from leaf extracts. The single-step phase separation was able to selectively recover up to 91% of the GFP-HFBI up to concentrations of 10 mg mL(-1). HFBI fusions increased the expression levels of plant-made recombinant proteins while also providing a simple means for their subsequent purification. This hydrophobin fusion technology, when combined with the speed and posttranslational modification capabilities of plants, enhances the value of transient plant-based expression systems.

Carbohydrate-protein interactions govern many crucial life processes involved in cell recognition events, but are often difficult to study because the interactions are weak, and multivalent exposure appears to be crucial for their biological function. We have used self-assembled monolayers (SAMs) of neoglycoconjugates as a model system to probe the specific interactions between the lectin wheat germ agglutinin (WGA) and monosaccharides by surface plasmon resonance (SPR) and atomic force microscopy (AFM) force measurements. SAMs presenting N-acetyl-D-glucosamine (GlcNAc) as a neoglycoconjugate were produced on gold surfaces, where the SAM formation was monitored using a quartz crystal microbalance (QCM) and shown to be a very rapid process. In the AFM force measurements WGA was covalently coupled to flexible polyethylene glycol (PEG) molecules at a probe surface using amine coupling. GlcNAc-specific binding events were detected with a WGA-modified probe on the GlcNAc-neoglycoconjugate SAM at bond rupture forces of 47 +/- 15 pN. Additionally, less frequent GlcNAc-specific unbinding events were detected at higher forces (120 +/- 20 pN) which are believed to originate from simultaneous detachment of multiple binding sites from the SAM surface. SPR measurements confirmed that WGA has higher affinity toward the immobilized GlcNAc-SAM than toward the soluble free monosaccharide. The binding constants obtained for soluble chitinoligosaccharides suggested up to three subsites within one carbohydrate-binding site of the WGA molecule and also provided further evidence of the multivalent binding character of the WGA dimer.

Detailed neuropsychological assessment was performed in 86 women (48 patients with stable relapsing-remitting multiple sclerosis (MS) and 38 matched healthy controls (HC)). Patients were categorized into patients without (EDSS < or =1, n = 26) and with physical disability (EDSS > or =2, n = 22). Patients with EDSS > or =2 scored significantly (P < 0.05) higher in Beck's depression inventory (BDI) and depression scores (DS) compared to HC and patients with EDSS < or =1. No significant differences were found with respect to the use of specific coping strategies between the patient groups, who preferred active (EDSS < or =1) or distracting (EDSS > or =2) strategies. Cognitive deficits were significantly increased in MS with EDSS > or =2 with regard to visuo-construction and visual memory, in particular with respect to geometric figures, compared to MS with EDSS < or =1. Significant positive correlations of depression variables (BDI, DS and BL) and depressive as well as denying coping strategies were found. Our results showed increased depression scores and increased cognitive deficits in advanced physically disabled patients, without selection of specific coping strategies. This supports an individual MS-specific neuropsychological therapeutic approach in order to improve disease related deficits together with social functioning.

Cognitive and emotional capabilities were evaluated in 73 female patients with stable relapsing-remitting definite, and/or laboratory-supported multiple sclerosis (MS) and were compared with 32 matched healthy controls. Patients were categorized according to their score in the expanded disability status scale (EDSS) to either no (EDSS 0, n = 33) or few clinical signs (EDSS 1-2, n = 40) of MS without physical disability. Patients with EDSS > 0 were characterized by significantly (p < 0.001) higher scores on "von Zerssen's" depression scale, compared to controls. Patients with higher EDSS scores (1-2) showed significantly decreased performance with respect to the total score of Kimura's Recurring-Figures-Test (p < 0.001), in addition. Regarding visuo-constructive functioning, patients with EDSS=0 performed to a significantly lower level (p < 0.001), compared to controls. These results indicate that depression may present as an early sign in MS followed by cognitive impairment, in particular visuo-spatial short-term memory, before physical disability appears. Neuropsychological tests as mentioned here could serve as early diagnostic tools to detect subtle disease progression and to initiate and monitor disease modifying therapies.