To tread or not to tread: comparison between water treading and conventional flapping wing kinematics.

Hovering insects are limited by their physiology and need to rotate their wings at the end of each back-and-forth motion to keep the wing's leading edge ahead of its trailing edge. The wing rotation at the end of each half-stroke pushes the leading edge vortex away from the wing which leads to a loss in the lift. Unlike biological fliers, human-engineered flapping wing micro air vehicles have different design limitations. They can be designed to avoid the end of stroke wing rotation and use so-called water-treading flapping kinematics. Flapping wings using conventional flapping kinematics have a designated leading and trailing edge. In the water-treading mode, the role of the leading and trailing edges are continuously alternated throughout the stroke. Here, we compare velocity field and force measurements for a rectangular flapping wing conducting normal hovering and water-treading kinematics to study the difference in fluid dynamic performance between the two types of flapping kinematics. We show that for similar power consumption, the water-treading mode produces more lift than the conventional hovering mode and is 50% more efficient for symmetric pitching kinematics. In the water-treading mode, the leading edge vortex from the previous stroke is not pushed away but is captured and keeps the newly formed leading edge vortex closer to the wing, leading to a more rapid increase of the lift coefficient which is sustained for longer. This makes the water-treading mode a promising alternative for human-engineered flapping wing vehicles.

Aeroelastic characterisation of a bio-inspired flapping membrane wing.

Natural fliers like bats exploit the complex fluid-structure interaction between their flexible membrane wings and the air with great ease. Yet, replicating and scaling the balance between the structural and fluid-dynamical parameters of unsteady membrane wings for engineering applications remains challenging. In this study, we introduce a novel bio-inspired membrane wing design and systematically investigate the fluid-structure interactions of flapping membrane wings. The membrane wing can passively camber, and its leading and trailing edges rotate with respect to the stroke plane. We find optimal combinations of the membrane properties and flapping kinematics that out-perform their rigid counterparts both in terms of increased stroke-average lift and efficiency, but the improvements are not persistent over the entire input parameter space. The lift and efficiency optima occur at different angles of attack and effective membrane stiffnesses which we characterise with the aeroelastic number. At optimal aeroelastic numbers, the membrane has a moderate camber between 15% and 20% and its leading and trailing edges align favourably with the flow. Higher camber at lower aeroelastic numbers leads to reduced aerodynamic performance due to negative angles of attack at the leading edge and an over-rotation of the trailing edge. Most of the performance gain of the membrane wings with respect to rigid wings is achieved in the second half of the stroke when the wing is decelerating. The stroke-maximum camber is reached around mid-stroke but is sustained during most of the remainder of the stroke which leads to an increase in lift and a reduction in power. Our results show that combining the effect of variable stiffness and angle of attack variation can significantly enhance the aerodynamic performance of membrane wings and has the potential to improve the control capabilities of micro air vehicles.

Phenomenology and scaling of optimal flapping wing kinematics.

Biological flapping wing fliers operate efficiently and robustly in a wide range of flight conditions and are a great source of inspiration to engineers. The unsteady aerodynamics of flapping wing flight are dominated by large-scale vortical structures that augment the aerodynamic performance but are sensitive to minor changes in the wing actuation. We experimentally optimise the pitch angle kinematics of a flapping wing system in hover to maximise the stroke average lift and hovering efficiency with the help of an evolutionary algorithm andin situforce and torque measurements at the wing root. Additional flow field measurements are conducted to link the vortical flow structures to the aerodynamic performance for the Pareto-optimal kinematics. The optimised pitch angle profiles yielding maximum stroke-average lift coefficients have trapezoidal shapes and high average angles of attack. These kinematics create strong leading-edge vortices early in the cycle which enhance the force production on the wing. The most efficient pitch angle kinematics resemble sinusoidal evolutions and have lower average angles of attack. The leading-edge vortex grows slower and stays close-bound to the wing throughout the majority of the stroke-cycle. This requires less aerodynamic power and increases the hovering efficiency by 93% but sacrifices 43% of the maximum lift in the process. In all cases, a leading-edge vortex is fed by vorticity through the leading edge shear layer which makes the shear layer velocity a good indicator for the growth of the vortex and its impact on the aerodynamic forces. We estimate the shear layer velocity at the leading edge solely from the input kinematics and use it to scale the average and the time-resolved evolution of the circulation and the aerodynamic forces. The experimental data agree well with the shear layer velocity prediction, making it a promising metric to quantify and predict the aerodynamic performance of the flapping wing hovering motion.

Heptahelical receptors GprC and GprD of Aspergillus fumigatus Are essential regulators of colony growth, hyphal morphogenesis, and virulence.

The filamentous fungus Aspergillus fumigatus normally grows on compost or hay but is also able to colonize environments such as the human lung. In order to survive, this organism needs to react to a multitude of external stimuli. Although extensive work has been carried out to investigate intracellular signal transduction in A. fumigatus, little is known about the specific stimuli and the corresponding receptors activating these signaling cascades. Here, two putative G-protein-coupled receptors, GprC and GprD, were characterized with respect to their cellular functions. Deletion of the corresponding genes resulted in drastic growth defects as hyphal extension was reduced, germination was retarded, and hyphae showed elevated levels of branching. The growth defect was found to be temperature dependent. The higher the temperature the more pronounced was the growth defect. Furthermore, compared with the wild type, the sensitivity of the mutant strains toward environmental stress caused by reactive oxygen intermediates was increased and the mutants displayed an attenuation of virulence in a murine infection model. Both mutants, especially the DeltagprC strain, exhibited increased tolerance toward cyclosporine, an inhibitor of the calcineurin signal transduction pathway. Transcriptome analyses indicated that in both the gprC and gprD deletion mutants, transcripts of primary metabolism genes were less abundant, whereas transcription of several secondary metabolism gene clusters was upregulated. Taken together, our data suggest the receptors are involved in integrating and processing stress signals via modulation of the calcineurin pathway.

Protein kinase A regulates growth, sporulation, and pigment formation in Aspergillus fumigatus.

Aspergillus fumigatus is an opportunistic human pathogenic fungus causing severe infections in immunocompromised patients. Cyclic AMP (cAMP) signal transduction plays an important role in virulence. A central component of this signaling cascade is protein kinase A (PKA), which regulates cellular processes by phosphorylation of specific target proteins. Here we describe the generation and analysis of A. fumigatus mutants expressing the gene encoding the catalytic subunit of PKA, pkaC1, under control of an inducible promoter. Strains overexpressing pkaC1 showed high PKA activity, reduced growth, sporulation deficiency, and formation of a dark pigment in the mycelium. These data indicate that cAMP-PKA signaling is involved in the regulation of important processes, such as growth, asexual reproduction, and biosynthesis of secondary metabolites. Furthermore, elevated PKA activity led to increased expression of the pksP gene. The polyketide synthase PksP is an essential enzyme for production of dihydroxynaphthalene-melanin in A. fumigatus and contributes to virulence. Our results suggest that increased pksP expression is responsible for pigment formation in the mycelium. Comparative proteome analysis of the pkaC1-overexpressing strain and the wild-type strain led to the identification of proteins regulated by the cAMP-PKA signal transduction pathway. We showed that elevated PKA activity resulted in activation of stress-associated proteins and of enzymes involved in protein biosynthesis and glucose catabolism. In contrast, proteins which were involved in nucleotide and amino acid biosynthesis were downregulated, as were enzymes involved in catabolism of carbon sources other than glucose.

The opportunistic human pathogenic fungus Aspergillus fumigatus evades the host complement system.

The opportunistic human pathogenic fungus Aspergillus fumigatus causes severe systemic infections and is a major cause of fungal infections in immunocompromised patients. A. fumigatus conidia activate the alternative pathway of the complement system. In order to assess the mechanisms by which A. fumigatus evades the activated complement system, we analyzed the binding of host complement regulators to A. fumigatus. The binding of factor H and factor H-like protein 1 (FHL-1) from human sera to A. fumigatus conidia was shown by adsorption assays and immunostaining. In addition, factor H-related protein 1 (FHR-1) bound to conidia. Adsorption assays with recombinant factor H mutants were used to localize the binding domains. One binding region was identified within N-terminal short consensus repeats (SCRs) 1 to 7 and a second one within C-terminal SCR 20. Plasminogen was identified as the fourth host regulatory molecule that binds to A. fumigatus conidia. In contrast to conidia, other developmental stages of A. fumigatus, like swollen conidia or hyphae, did not bind to factor H, FHR-1, FHL-1, and plasminogen, thus indicating the developmentally regulated expression of A. fumigatus surface ligands. Both factor H and plasminogen maintained regulating activity when they were bound to the conidial surface. Bound factor H acted as a cofactor to the factor I-mediated cleavage of C3b. Plasminogen showed proteolytic activity when activated to plasmin by urokinase-type plasminogen activator. These data show that A. fumigatus conidia bind to complement regulators, and these bound host regulators may contribute to evasion of a host complement attack.

The CCAAT-binding complex of eukaryotes: evolution of a second NLS in the HapB subunit of the filamentous fungus Aspergillus nidulans despite functional conservation at the molecular level between yeast, A.nidulans and human.

The heterotrimeric CCAAT-binding complex is evolutionarily conserved in eukaryotic organisms, including fungi, plants and mammals. In the filamentous fungus Aspergillus nidulans, the corresponding complex was designated AnCF (A.nidulans CCAAT-binding factor). AnCF consists of the subunits HapB, HapC and HapE. All three subunits are necessary for DNA binding. HapB contains two putative nuclear localisation signal sequences (NLSs) designated NLS1 and NLS2. Previously, it was shown that only NLS2 was required for nuclear localisation of HapB. Furthermore, HapC and HapE are transported to the nucleus only in complex with HapB via a piggy back mechanism. Here, by using various GFP constructs and by establishing a novel marker gene for transformation of A.nidulans, i.e. the pabaA gene encoding p-aminobenzoic acid synthase, it was shown that the HapB homologous proteins of both Saccharomyces cerevisiae (Hap2p) and human (NF-YA) use an NLS homologous to HapB NLS1 for nuclear localisation in S.cerevisiae. Interestingly, for A.nidulans HapB, NLS1 was sufficient for nuclear localisation in S.cerevisiae. In A.nidulans, HapB NLS1 was also functional when present in a different protein context. However, in A.nidulans, both S.cerevisiae Hap2p and human NF-YA entered the nucleus only when HapB NLS2 was present in the respective proteins. In that case, both proteins Hap2p and NF-YA complemented, at least in part, the hap phenotype of A.nidulans with respect to lack of growth on acetamide. Similarly, A.nidulans HapB and human NF-YA complemented a hap2 mutant of S.cerevisiae. In summary, HapB, Hap2p and NF-YA are interchangeable. Because the A.nidulans hapB mutant was complemented, at least in part, by both the human NF-YA and S.cerevisiae Hap2p this finding suggests that the piggy-back mechanism of nuclear transport found for A.nidulans is conserved in yeast and human.

Regulation of penicillin biosynthesis in filamentous fungi.

The beta-lactam antibiotic penicillin is one of the mainly used antibiotics for the therapy of infectious diseases. It is produced as end product by some filamentous fungi only, most notably by Aspergillus (Emericella) nidulans and Penicillium chrysogenum. The penicillin biosynthesis is catalysed by three enzymes which are encoded by the following three genes: acvA (pcbAB), ipnA (pcbC) and aatA (penDE). The genes are organised into a gene cluster. Although the production of secondary metabolites as penicillin is not essential for the direct survival of the producing organisms, several studies indicated that the penicillin biosynthesis genes are controlled by a complex regulatory network, e.g. by the ambient pH, carbon source, amino acids, nitrogen etc. A comparison with the regulatory mechanisms (regulatory proteins and DNA elements) involved in the regulation of genes of primary metabolism in lower eukaryotes is thus of great interest. This has already led to the elucidation of new regulatory mechanisms. Positively acting regulators have been identified such as the pH dependent transcriptional regulator PACC, the CCAAT-binding complex AnCF and seem also to be represented by recessive trans-acting mutations of A. nidulans (prgA1, prgB1, npeE1) and R chrysogenum (carried by mutants Npe2 and Npe3). In addition, repressors like AnBH1 and VeA are involved in the regulation. Furthermore, such investigations have contributed to the elucidation of signals leading to the production of penicillin and can be expected to have a major impact on rational strain improvement programs.