Production of Hydrophobins from fungi

Hydrophobins (HPs) are industrially important surface active, amphipathic proteins produced by fungi. There are many applications reported for HPs in the literature notably as, agents for enhancing bioavailability of water insoluble drugs, food stabilizers, antifouling agents for biomedical devices like catheters, fusion partner for recombinant proteins for purification, low friction coatings on biomaterials, immobilizing enzymes in biosensors, etc. However, there are limitations for industrial scale production of HPs. Various methods have been reported for their production e.g. use of wild fungi from natural hydrophobic environments, use of modified bioreactors for submerged and solid state fermentation and recombinant homologous as well as heterologous microbes. Knowing the industrial importance of HPs many reviews have been published focusing on technical and medical applications of these proteins; however there is no comprehensive overview of HP production in the literature. This review summarizes the efforts made to improve yields of HPs by various bioprocesses and also highlights the strategies designed to overcome problems of low yield of HPs.     

Lactate dehydrogenase in the cyanobacterium Microcystis PCC7806

Abstract The cyanobacterium Microcystis PCC7806 was found to possess an NAD-dependent lactate dehydrogenase (EC 1.1.1.27) which catalyzes the reduction of pyruvate to l-lactate. The enzyme required fructose 1,6-bisphosphate for activity and displayed positive cooperativity towards pyruvate. Lactate was not formed during fermentation by cell suspensions, possibly due to low intracellular concentrations of fructose 1,6-bisphosphate and/or pyruvate.     

iPSC technology-Powerful hand for disease modeling and therapeutic screen

Cardiovascular and neurodegenerative diseases are major health threats in many developed countries. Recently, target tissues derived from human embryonic stem (hES) cells and induced pluripotent stem cells (iPSCs), such as cardiomyocytes (CMs) or neurons, have been actively mobilized for drug screening. Knowledge of drug toxicity and efficacy obtained using stem cell-derived tissues could parallel that obtained from human trials. Furthermore, iPSC disease models could be advantageous in the development of personalized medicine in various parts of disease sectors. To obtain the maximum benefit from iPSCs in disease modeling, researchers are now focusing on aging, maturation, and metabolism to recapitulate the pathological features seen in patients. Compared to pediatric disease modeling, adult-onset disease modeling with iPSCs requires proper maturation for full manifestation of pathological features. Herein, the success of iPSC technology, focusing on patient-specific drug treatment, maturation-based disease modeling, and alternative approaches to compensate for the current limitations of patient iPSC modeling, will be further discussed. [BMB Reports 2015; 48(5): 256-265]     

Population density,roads and altitude influences on spatial distribution of hares positive to EBHSV

Understanding of the ecology of infected animals facilitates disease risk assessment and is also crucial for wildlife conservation. Relatively little is known about the spatial distribution of infected wild mammals in relation to environmental factors. In neighboring Mediterranean ecosystems 250 European brown hares (Lepus europaeus) were collected and examined with RT-PCR to detect European Brown Hare Syndrome Virus (EBHSV). Multivariate statistics and Geographical Information System (GIS) analysis were applied to estimate spatial patterns of biotic and abiotic factors and human activities as determinants of EBHSV positivity. Hare population abundance was estimated using faeces counts and belt drive censuses. The study showed that EBHSV infected hares had widespread distribution even in isolated areas. However, EBHSV infection prevalence was higher in areas with higher hare abundance, closer to paved road networks and at lower altitudes. The risk map revealed the potential distribution of EBHSV-infected hares. This study shows that host abundance and landscape influence the ecology of the disease, a finding that should be taken into account in future studies. The management of harvest and restocking of hares is also discussed for population conservation.     

Citric acid and polyols production by Aspergillus niger at high glucose concentration in solid state fermentation on inert support

Summary Aspergillus niger cultures at high initial glucose concentration (up to 400 g/1) on Amberlite as inert support were carried out. Citric acid was accumulated in the support showing high concentration (94.54 g/l) and productivity (1.35 g/l h) without inhibition related to the presence of metals (Mn²⁺, Zn²⁺, Co²⁺, Cu²⁺, and Ca²⁺) at high concentrations. Citric acid accumulation was clearly associated with both, glycerol production and to the age of the culture. Glycerol and erythritol, the major osmoregulator metabolites, were also produced (8.16 and 24.57 g/l respectively) at 400 g/l of glucose.     

Nanomechanics of Drug-target Interactions and Antibacterial Resistance Detection

The cantilever sensor, which acts as a transducer of reactions between model bacterial cell wall matrix immobilized on its surface and antibiotic drugs in solution, has shown considerable potential in biochemical sensing applications with unprecedented sensitivity and specificity^1-5. The drug-target interactions generate surface stress, causing the cantilever to bend, and the signal can be analyzed optically when it is illuminated by a laser. The change in surface stress measured with nano-scale precision allows disruptions of the biomechanics of model bacterial cell wall targets to be tracked in real time. Despite offering considerable advantages, multiple cantilever sensor arrays have never been applied in quantifying drug-target binding interactions.Here, we report on the use of silicon multiple cantilever arrays coated with alkanethiol self-assembled monolayers mimicking bacterial cell wall matrix to quantitatively study antibiotic binding interactions. To understand the impact of vancomycin on the mechanics of bacterial cell wall structures^1,6,7. We developed a new model¹ which proposes that cantilever bending can be described by two independent factors; i) namely a chemical factor, which is given by a classical Langmuir adsorption isotherm, from which we calculate the thermodynamic equilibrium dissociation constant (K_d) and ii) a geometrical factor, essentially a measure of how bacterial peptide receptors are distributed on the cantilever surface. The surface distribution of peptide receptors (p) is used to investigate the dependence of geometry and ligand loading. It is shown that a threshold value of p ~10% is critical to sensing applications. Below which there is no detectable bending signal while above this value, the bending signal increases almost linearly, revealing that stress is a product of a local chemical binding factor and a geometrical factor combined by the mechanical connectivity of reacted regions and provides a new paradigm for design of powerful agents to combat superbug infections.     

Ezrin/Radixin/Moesin Are Required for the Purinergic P2X7 Receptor (P2X7R)-dependent Processing of the Amyloid Precursor Protein

The amyloid precursor protein (APP) can be cleaved by α-secretases in neural cells to produce the soluble APP ectodomain (sAPPα), which is neuroprotective. We have shown previously that activation of the purinergic P2X7 receptor (P2X7R) triggers sAPPα shedding from neural cells. Here, we demonstrate that the activation of ezrin, radixin, and moesin (ERM) proteins is required for the P2X7R-dependent proteolytic processing of APP leading to sAPPα release. Indeed, the down-regulation of ERM by siRNA blocked the P2X7R-dependent shedding of sAPPα. We also show that P2X7R stimulation triggered the phosphorylation of ERM. Thus, ezrin translocates to the plasma membrane to interact with P2X7R. Using specific pharmacological inhibitors, we established the order in which several enzymes trigger the P2X7R-dependent release of sAPPα. Thus, a Rho kinase and the MAPK modules ERK1/2 and JNK act upstream of ERM, whereas a PI3K activity is triggered downstream. For the first time, this work identifies ERM as major partners in the regulated non-amyloidogenic processing of APP.     

ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber diameter, composition and organization. In addition to providing structural support, the physical and chemical properties of the ECM play an important role in multiple cellular processes including adhesion, differentiation, and apoptosis. In vivo, the ECM is assembled by exposing cryptic self-assembly (fibrillogenesis) sites within proteins. This process varies for different proteins, but fibronectin (FN) fibrillogenesis is well-characterized and serves as a model system for cell-mediated ECM assembly. Specifically, cells use integrin receptors on the cell membrane to bind FN dimers and actomyosin-generated contractile forces to unfold and expose binding sites for assembly into insoluble fibers. This receptor-mediated process enables cells to assemble and organize the ECM from the cellular to tissue scales. Here, we present a method termed surface-initiated assembly (SIA), which recapitulates cell-mediated matrix assembly using protein-surface interactions to unfold ECM proteins and assemble them into insoluble fibers. First, ECM proteins are adsorbed onto a hydrophobic polydimethylsiloxane (PDMS) surface where they partially denature (unfold) and expose cryptic binding domains. The unfolded proteins are then transferred in well-defined micro- and nanopatterns through microcontact printing onto a thermally responsive poly(N-isopropylacrylamide) (PIPAAm) surface. Thermally-triggered dissolution of the PIPAAm leads to final assembly and release of insoluble ECM protein nanofibers and nanostructures with well-defined geometries. Complex architectures are possible by engineering defined patterns on the PDMS stamps used for microcontact printing. In addition to FN, the SIA process can be used with laminin, fibrinogen and collagens type I and IV to create multi-component ECM nanostructures. Thus, SIA can be used to engineer ECM protein-based materials with precise control over the protein composition, fiber geometry and scaffold architecture in order to recapitulate the structure and composition of the ECM in vivo.     

Spectral imaging for precise surgical intervention in Hirschsprung's Disease

We used advanced spectral imaging for intrasurgical decision making in a preclinical study, on a mouse model of Hirschsprung's Disease. Our imaging device sampled areas from normal and abnormal (aganglionic) colon in these animals. Spectral segmentation and classification of the resulting images showed a clear distinction between the normal and aganglionic regions, as confirmed by pathological analysis and use of mutant mice. We developed a simple algorithm that could distinguish normal from aganglionic colon with high spatial resolution and reproducibility, and the following statistics: sensitivity = 97%, specificity = 94%, positive predictive value = 92%, negative predictive value = 98%. These studies showed translational proof of concept that spectral imaging could be used during operations, in real time, to help surgeons precisely distinguish normal from abnormal tissue without requiring traditional biopsy. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)