Structural and Functional Role of the Disulfide Bridges in the Hydrophobin SC3

Hydrophobins function in fungal development by self-assembly at hydrophobic-hydrophilic interfaces such as the interface between the fungal cell wall and the air or a hydrophobic solid. These proteins contain eight conserved cysteine residues that form four disulfide bonds. To study the effect of the disulfide bridges on the self-assembly, the disulfides of the SC3 hydrophobin were reduced with 1,4-dithiothreitol. The free thiols were then blocked with either iodoacetic acid (IAA) or iodoacetamide (IAM), introducing eight or zero negative charges, respectively. Circular dichroism and infrared spectroscopy showed that after opening of the disulfide bridges SC3 is initially unfolded. IAA-SC3 did not self-assemble at the air-water interface upon shaking an aqueous solution. Remarkably, after drying down IAA-SC3 or after exposing it to Teflon, it refolded into a structure similar to that observed for native SC3 at these interfaces. Iodoacetamide-SC3 on the other hand, which does not contain extra charges, spontaneously refolded in water in the amyloid-like β-sheet conformation, characteristic for SC3 assembled at the water-air interface. From this we conclude that the disulfide bridges of SC3 are not directly involved in self-assembly but keep hydrophobin monomers soluble in the fungal cell or its aqueous environment, preventing premature self-assembly.

Use of hydrophobins in formulation of water insoluble drugs for oral administration

The poor water solubility of many drugs requires a specific formulation to achieve a sufficient bioavailability after oral administration. Suspensions of small drug particles can be used to improve the bioavailability. We here show that the fungal hydrophobin SC3 can be used to make suspensions of water insoluble drugs. Bioavailability of two of these drugs, nifedipine and cyclosporine A (CyA), was tested when administered as a SC3-based suspension. SC3 (in a 1:2 (w/w) drug:SC3 ratio) or 100% PEG400 increased the bioavailability of nifedipine to a similar degree (6±2- and 4±3-fold, respectively) compared to nifedipine powder without additives. Moreover, SC3 (in a 7:1 (w/w) drug:hydrophobin ratio) was as effective as a 20-fold diluted Neoral® formulation by increasing bioavailability of CyA 2.3±0.3-fold compared to CyA in water. Interestingly, using SC3 in the CyA formulation resulted in a slower uptake (p < 0.001 in Tmax) of the drug, with a lower peak concentration (Cmax 1.8 mg ml−1) at a later time point (Tmax 9±2 h) compared to Neoral® (Cmax 2.2 mg ml−1; Tmax 3.2±0.2). Consequently, SC3 will result in a more constant, longer lasting drug level in the body. Taken together, hydrophobins are attractive candidates to formulate hydrophobic drugs.

Influences between online-exclusive, conjoint and offline-exclusive friendship networks: The moderating role of shyness

Prior research has indicated that shy adolescents are more motivated to form friendships online than to form friendships offline. Little is known about whether having friendships found exclusively online may impact self-esteem and forming offline friendships for these adolescents. This study therefore aimed to provide insight into the moderating role of shyness in the longitudinal interplay between friendships in online and offline contexts in early adolescence. Adolescents and their friends (193 girls, 196 boys; Mage = 13.29) were followed with three consecutive measurements with intervals of eight months. Results showed that particularly for shy adolescents, having friends exclusively online predicted increases in self-esteem. Self-esteem, in turn, was found to predict forming more friendships found both offline and online and forming more friendships found exclusively offline. Thus, findings supported the social compensation perspective that shy adolescents may benefit from having friends exclusively online, as these friendships may increase self-esteem, thereby facilitating the formation of friendships found partially and completely offline

Field Studies

One way to implement and evaluate the effectiveness of recommendation systems in software engineering is to conduct field studies. Field studies are important as they are the extension of laboratory experiments into real-life situations of organizations and/or society. They bring greater realism to the phenomena that are under study. However, field studies require following a rigorous research approach with many challenges attached, such as difficulties in implementing the research design, achieving sufficient control, replication, validity, and reliability. In practice, another challenge is to find organizations who are prepared to be studied. In this chapter, we provide a step-by-step process for the construction and deployment of recommendation systems in software engineering in the field. We also emphasize three main challenges (organizational, data, design) encountered during field studies, both in general and specifically with respect to software organizations

Self-assembly of the hydrophobin SC3 proceeds via two structural intermediates

Hydrophobins self assemble into amphipathic films at hydrophobic–hydrophilic interfaces. These proteins are involved in a broad range of processes in fungal development. We have studied the conformational changes that accompany the self-assembly of the hydrophobin SC3 with polarization-modulation infrared reflection absorption spectroscopy, attenuated total reflection Fourier transform infrared spectroscopy, and circular dichroism, and related them to changes in morphology as observed by electron microcopy. Three states of SC3 have been spectroscopically identified previously as follows: the monomeric state, the α-helical state that is formed upon binding to a hydrophobic solid, and the β-sheet state, which is formed at the air–water interface. Here, we show that the formation of the β-sheet state of SC3 proceeds via two intermediates. The first intermediate has an infrared spectrum indistinguishable from that of the α-helical state of SC3. The second intermediate is rich in β-sheet structure and has a featureless appearance under the electron microscope. The end state has the same secondary structure, but is characterized by the familiar 10-nm-wide rodlets

Assembly of the Fungal SC3 Hydrophobin into Functional Amyloid Fibrils Depends on Its Concentration and Is Promoted by Cell Wall Polysaccharides

Class I hydrophobins function in fungal growth and development by self-assembling at hydrophobic-hydrophilic interfaces into amyloid-like fibrils. SC3 of the mushroom-forming fungus Schizophyllum commune is the best studied class I hydrophobin. This protein spontaneously adopts the amyloid state at the water-air interface. In contrast, SC3 is arrested in an intermediate conformation at the interface between water and a hydrophobic solid such as polytetrafluoroethylene (PTFE; Teflon). This finding prompted us to study conditions that promote assembly of SC3 into amyloid fibrils. Here, we show that SC3 adopts the amyloid state at the water-PTFE interface at high concentration (300 μg ml−1) and prolonged incubation (16 h). Moreover, we show that amyloid formation at both the water-air and water-PTFE interfaces is promoted by the cell wall components schizophyllan (β(1–3),β(1–6)-glucan) and β(1–3)-glucan. Hydrophobin concentration and cell wall polysaccharides thus contribute to the role of SC3 in formation of aerial hyphae and in hyphal attachment