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Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited condition that can cause fatal cardiac arrhythmia. Human mutations in the Ca2+ sensor calmodulin (CaM) have been associated with CPVT susceptibility, suggesting that CaM dysfunction is a key driver of the disease. However, the detailed molecular mechanism remains unclear. Focusing on the interaction with the cardiac ryanodine receptor (RyR2), we determined the effect of CPVT-associated variants N53I and A102V on the structural characteristics of CaM and on Ca2+ fluxes in live cells. We provide novel data showing that interaction of both Ca2+/CaM-N53I and Ca2+/CaM-A102V with the RyR2 binding domain is decreased. Ca2+/CaM–RyR23583-3603 high-resolution crystal structures highlight subtle conformational changes for the N53I variant, with A102V being similar to wild type (WT). We show that co-expression of CaM-N53I or CaM-A102V with RyR2 in HEK293 cells significantly increased the duration of Ca2+ events; CaM-A102V exhibited a lower frequency of Ca2+ oscillations. In addition, we show that CaMKIIδ (also known as CAMK2D) phosphorylation activity is increased for A102V, compared to CaM-WT. This paper provides novel insight into the molecular mechanisms of CPVT-associated CaM variants and will facilitate the development of strategies for future therapies.

Significance Many filamentous fungi colonizing animal or plant tissue, waste matter, or soil must find optimal paths through the constraining geometries of their microenvironment. Imaging of live fungal growth in custom-built microfluidics structures revealed the intracellular mechanisms responsible for this remarkable efficiency. In meandering channels, the Spitzenkörper (an assembly of vesicles at the filament tip) acted like a natural gyroscope, conserving the directional memory of growth, while the fungal cytoskeleton organized along the shortest growth path. However, if an obstacle could not be negotiated, the directional memory was lost due to the disappearance of the Spitzenkörper gyroscope. This study can impact diverse environmental, industrial, and medical applications, from fungal pathogenicity in plants and animals to biology-inspired computation., Filamentous fungi that colonize microenvironments, such as animal or plant tissue or soil, must find optimal paths through their habitat, but the biological basis for negotiating growth in constrained environments is unknown. We used time-lapse live-cell imaging of Neurospora crassa in microfluidic environments to show how constraining geometries determine the intracellular processes responsible for fungal growth. We found that, if a hypha made contact with obstacles at acute angles, the Spitzenkörper (an assembly of vesicles) moved from the center of the apical dome closer to the obstacle, thus functioning as an internal gyroscope, which preserved the information regarding the initial growth direction. Additionally, the off-axis trajectory of the Spitzenkörper was tracked by microtubules exhibiting “cutting corner” patterns. By contrast, if a hypha made contact with an obstacle at near-orthogonal incidence, the directional memory was lost, due to the temporary collapse of the Spitzenkörper–microtubule system, followed by the formation of two “daughter” hyphae growing in opposite directions along the contour of the obstacle. Finally, a hypha passing a lateral opening in constraining channels continued to grow unperturbed, but a daughter hypha gradually branched into the opening and formed its own Spitzenkörper–microtubule system. These observations suggest that the Spitzenkörper–microtubule system is responsible for efficient space partitioning in microenvironments, but, in its absence during constraint-induced apical splitting and lateral branching, the directional memory is lost, and growth is driven solely by the isotropic turgor pressure. These results further our understanding of fungal growth in microenvironments relevant to environmental, industrial, and medical applications.

Screening cells for their differentiation potential requires a combination of tissue culture models and imaging methods that allow for long-term tracking of the location and function of cells. Embryonic kidney re-aggregationin vitroassays have been established which allow for the monitoring of organotypic cell behaviour in re-aggregated and chimeric renal organoids. However, evaluation of cell integration is hampered by the high photonic load of standard fluorescence microscopy which poses challenges for imaging three-dimensional systems in real-time over a time course. Therefore, we employed light sheet microscopy, a technique that vastly reduces photobleaching and phototoxic effects. We have also developed a new method for culturing the re-aggregates which involves immersed culture, generating organoids which more closely reflect developmentin vivo. To facilitate imaging from various angles, we embedded the organoids in a freely rotatable hydrogel cylinder. Endpoint fixing and staining were performed to provide additional biomolecular information. We succeeded in imaging labelled cells within re-aggregated kidney organoids over 15 hours and tracking their fate while simultaneously monitoring the development of organotypic morphological structures. Our results show that Wt1-expressing embryonic kidney cells obtained from transgenic mice could integrate into re-aggregated chimeric kidney organoids and contribute to developing nephrons. Furthermore, the nascent proximal tubules that formed in the re-aggregated tissues using the new culture method displayed secretory function, as evidenced by their ability to secrete an organic anion mimic into the tubular lumen.

Gold nanorods are excellent contrast agents for imaging technologies which rely on near-infrared absorption such as photoacoustic imaging. For cell tracking applications, the cells of interest are labeled with the contrast agent prior to injection. However, after uptake into cells by endocytosis, the confinement and high concentration in endosomes leads to plasmon band broadening and reduced absorbance. This would limit the potential of multispectral optoacoustic tomography in terms of spectral processing and, consequently, sensitivity. Here, we show that steric hindrance provided by silica coating of the nanorods leads to the preservation of their spectral properties and improved photoacoustic sensitivity. This strategy allowed the detection and monitoring of as few as 2 × 10(4) mesenchymal stem cells in mice over a period of 15 days with a high spatial resolution. Importantly, the silica-coated nanorods did not affect the viability or differentiation potential of the transplanted mesenchymal stem cells.

Despite occupying physically and chemically heterogeneous natural environments, the growth dynamics of filamentous fungi is typically studied on the surface of homogeneous laboratory media. Fungal exploration and exploitation of complex natural environments requires optimal survival and growth strategies at the colony, hyphal, and intra hyphal level, with hyphal space-searching strategies playing a central role. We describe a new methodology for the characterisation and analysis of hyphal space-searching strategies, which uses purposefully designed three-dimensional microfluidics structures mimicking some of the characteristics of natural environments of the fungi. We also demonstrate this new methodology by running a comparative examination of two Neurospora crassa strains, i.e., the wild type of N. crassa -- a commonly used model organism for the study of filamentous fungi -- and the N. crassa ro-1 mutant strain -- which is deficient in hyphal and mycelial growth. Continuous live imaging showed that both strains responded actively to the geometrically confined microstructured environments without any detectable temporal delay or spatial adjustment. While both strains navigated the test structures exhibiting similar geometry-induced space-searching mechanisms, they presented fundamentally different growth patterns that could not be observed on geometrically unconfined, flat agar surfaces.

Although filamentous fungi live in physically and chemically complex natural environments that require optimal survival strategies, both at colony and individual cell level, their growth dynamics are usually studied on homogenous media. This study proposes a new research methodology based on the purposeful design, fabrication and operation of microfluidics structures to examine the temporal and spatial responses of filamentous fungi. Two model fungal strains, the wild type of Neurospora crassa - a commonly used model organisms - and the ro-1 mutant strain of this species impaired in hyphal growth and morphology, have been chosen to demonstrate the potential of this new methodology. Time-lapse observations of both species show that filamentous fungi respond rapidly to the physically microstructured environment without any detectable temporal or spatial adjustment period. Despite their genetic differences, and consequently different growth behaviour, both strains present efficient space-search strategies enabling them to solve the microsized networks successfully and in similar periods, thus demonstrating that the space-searching algorithms are robust and mutation-independent. Additionally, the use of the proposed methodology could put in evidence new biological mechanisms responsible for the apical extension of filamentous fungi, beyond the classical theory based on the central role of Spitzenkorper.

Microorganisms live in environments that are geometrically, physically and chemically heterogeneous at the micro-scale. Consequently, they have to optimise their behaviour at the cellular level within limited time frames by responding to stimuli that are spatially distributed at the micro-scale. Microorganisms achieve this by using a range of different sensing mechanisms as a means to gain biologically-critical and dynamic information about their physical (e.g., available space) and chemical (e.g., distribution of nutrients) environment. Despite this micro-scale dependent behaviour, the response of microorganisms is usually studied in and on homogeneous media, which have little resemblance to their natural environment. Semiconductor fabrication offers large possibilities for the design and creation of physically- and chemically-microstructured environments, which either mimic the natural ones, or represent a multitude of possible environments to be used in combinatorial experiments. In order to test the dynamic behaviour of microorganisms we propose here the design and fabrication of test microstructured arrays and demonstrate their applications using representative fungal (Neurospora crassa) and bacterial (Escherichia coli) species.

The optical properties of metal nanoparticles depend strongly on their composition and size. The plasmon absorption band can be shifted between the blue range and the red range of the optical spectrum, if silver and gold are mixed and aggregation of particles is also induced. The formation of different nanoparticles and nanoparticle aggregates was studied in a two-step flow-through process using a glass/Si/glass static micromixer. The educt solution was pumped through the mixer chips by programmable syringe pumps. The process was monitored online by a compact spectrometer connected to the outlet tube by glass fibers. The product solution was collected and investigated by a UV/VIS spectrophotometer. Selected samples were investigated by Scanning Electron Microscopy (SEM) to evaluate the particle morphology. The typical Ag nanoparticle absorption was found, when an excess of Ag ions was introduced into the system. The intensity of the peak decreased when the flow rate of AgNO3 solution was reduced in a reaction system, which included HAuCl4 solution and polyvinyl alcohol, as well as the reducing agent (ascorbic acid). The experiments show that the educt concentration ratios as well as the total flow rates influence the shape and composition of the nanoparticles. Therefore, microreactors with fast mixing and well reproducible mixing conditions are interesting options for the synthesis of nanomaterials.

From the 1950s onwards, our understanding of the formation and intracellular trafficking of membrane vesicles was informed by experiments in which cells were exposed to gold nanoparticles and their uptake and localisation, studied by electron microscopy. In the last decade, building on progress in the synthesis of gold nanoparticles and their controlled functionalisation with a large variety of biomolecules (DNA, peptides, polysaccharides), new applications have been proposed, including the imaging and sensing of intracellular events. Yet, as already demonstrated in the 1950s, uptake of nanoparticles results in confinement within an intracellular vesicle which in principle should preclude sensing of cytosolic events. To study this apparent paradox, we focus on a commercially available nanoparticle probe that detects mRNA through the release of a fluorescently-labelled oligonucleotide (unquenching the fluorescence) in the presence of the target mRNA. Using electron, fluorescence and photothermal microscopy, we show that the probes remain in endocytic compartments and that they do not report on mRNA level. We suggest that the validation of any nanoparticle-based probes for intracellular sensing should include a quantitative and thorough demonstration that the probes can reach the cytosolic compartment.

From the 1950s onwards, our understanding of the formation and intracellular trafficking of membrane vesicles was informed by experiments in which cells were exposed to gold nanoparticles and their uptake and localisation, studied by electron microscopy. In the last decade, building on progress in the synthesis of gold nanoparticles and their controlled functionalisation with a large variety of biomolecules (DNA, peptides, polysaccharides), new applications have been proposed, including the imaging and sensing of intracellular events. Yet, as already demonstrated in the 1950s, uptake of nanoparticles results in confinement within an intracellular vesicle which in principle should preclude sensing of cytosolic events. To study this apparent paradox, we focus on a commercially available nanoparticle probe that detects mRNA through the release of a fluorescently labelled oligonucleotide (unquenching the fluorescence) in the presence of the target mRNA. Using electron, fluorescence and photothermal microscopy, we show that the probes remain in endocytic compartments and that they do not report on mRNA level. We suggest that the validation of any nanoparticle-based probes for intracellular sensing should include a quantitative and thorough demonstration that the probes can reach the cytosolic compartment.

Filamentous fungi include serious plant and animal pathogens that explore their environment efficiently in order to penetrate the host. This environment is physically and chemi cally heterogeneous and the fu ngi rely on specific physical and chemical signals to find the optimal point/s of attack. This study presents a methodology to introduce distinct structures with dimensions similar to the hyphal diameter and specific chemical surface groups into a controllable environment in order to study the fungal response. We introduced 3.3 µm polystyrene beads covered with Epoxy surface groups into microfluidic channels made from PDMS by rapid replica molding. The experimental setup resulted in different areas with low and high densities of beads as well as densely packed patches. The observations of the fungus exploring the areas long-term showed that the growth parameters were altered significantly, compared with the values measured on agar. The fungus responded to both, the physical and chemical parameters of the beads, including temporary directional changes, increased branching angles, decreased branching distances, decreased apical extension velocities and occasional cell wall lysis. The wealth and magnitude of the observed responses indicates that the microfluidic structures provid e a powerful platform for the investigation of micron-sized features on filamentous fungi. Keywords: Neurospora crassa , hypha, micro bead, Epoxy , microfluidics, PDMS

Microtubules are a crucial part of the fungal cytoskeleton and facilitate long-distance vesicle transport to the growing apex that constitutes one of the main driving forces of polarized growth. This study observed the spatial and temporal distribution of microtubules in growing Neurospora crassa hyphae in two and three dimensions. The fungal strain used expressed a normal growth pattern combined with the expression of green fluorescent protein (GFP) along the microtubules enabling their direct study with fluorescence and confocal microscopy. Time-lapse imaging revealed that the microtubules were dynamic, being assembled and disassembled at high rates as well as moving with the cytoplasmic flow. In the apical compartment, the filaments were arranged mostly parallel but not helical to the growth axis and appeared to determine the growth direction in close interaction with the Spitzenkorper. The microtubule distribution in subapical compartments was more random and their motility appeared to be driven by the cytoplasmic flow. However, this flow is affected by hyphal septa that act as partition walls with small connecting pores. Three-dimensional imaging showed that in order to pass through a septum, the filaments had to align parallel to the growth axis. The aim of this study was to attempt to reveal deeper insight into the role of microtubules in fungal growth thus confirming and challenging some suggestions proposed in previous literature.

All multi-cellular living organisms are very complex microfluidics systems, which are assembled 'inside-out', as the result of a complex 'tug-of-war' process comprising both feed-forward modules, in particular the program embedded in the species' DNA, and feed-back processes, in particular the response to external environmental conditions. Living organisms have to solve a perennial problem: for an environment with limited resources (finite and spatial distribution of nutrients, geometrical limitations, competition from other species, etc.), finite growth rate and penalties applied to nonoptimal behaviour, what is the best strategy that will maximise growth and survival of the individual and the species? Identifying how organisms resolve this problem is very difficult due to the high complexity involved. However, filamentous fungi and other simple organisms, provide a means to address these issues due to the availability of microscopic imaging and microfabricated geometries in which the behaviour can be tested. Semiconductor fabrication (photolithography, RIE etching, and soft lithography techniques) was used here to produce artificial micro-networks, which are physically and chemically structured 3D microenvironments. Continuous imaging of fungal behaviour in microfluidics networks required the use of PDMS, which is transparent and O2-permeable. Using these structures, the dynamic growth behaviour of two fungal species Neurospora crassa and Armillaria mellea was observed microscopically in real-time. Growth parameters such as the tip extension velocities, branching angles and branching distances on plain surfaces differed vastly between the species. Features in the micronetworks of a size similar to the hyphal diameter induced the largest change in growth parameters such as, the largest decrease in branching distances in N. crassa and a near complete suppression for A. mellea. Despite these fundamental differences, both species negotiated the structures successfully. These results open the way to asking more fundamental questions, such as which species use algorithms that are most efficient for solving a particular type of mathematical problem coded in a network? Can these natural algorithms be used for either better control of fungal growth and metabolism for both medical and biotechnology applications; or even for new computation paradigms?

Accurate quantification of the hydrophobic/hydrophilic properties of protein surfaces requires detailed knowledge of the hydrophobicity of amino acids at the atomic level. As discussed previously in various published papers, molecular modeling can be used with effect to acquire such knowledge. In this study, molecular dynamics methods have been employed to examine the role of the distance between an amino acid atom and its nearest water molecule in relation to its intrinsic atom hydrophobicity. This distance is the radius of the water-excluding-region around the atom; therefore, it can provide information on the solvent accessibility and steric hindrance that may influence the atom hydrophobicity. Molecular models of tripeptide in the form of GXG, and pentapeptides in the form of AcWLXLL-NH 2 and AcGGXGGNH 2 for 20 natural amino acids in the X position were constructed and allowed to dynamically interact with surrounding water for a sufficient period of time. The distance value for each atom in all natural amino acids were calculated and analyzed against the atom/amino acid's other parameters such as radial distribution function, solvent-accessible surface area, and hydrogen bonding. It was observed that, when the dynamic factor is taken into account, peptide molecular conformation is modified noticeably with residue type. For protein surface identification purposes, preliminary results are consistent with those reported in the literature on the need to include the amino acid structural properties as well as the effects of its neighboring residues. Further investigation is envisaged in order to verify these observations.

Filamentous fungi like Neurospora crassa are a large and evolutionary successful group of organisms that can efficiently colonise microconfined networks like soil, wood, leaf litter, plant and animal tissues. Growth of the fungus Neurospora crassa was monitored for three-dimensional interactions with artificial profiled surface and two-dimensional interactions with a patterned surface. Specific growth parameters that included branching angles and branching distances were used to measure the responses of growing hyphae to the confining features. In three dimensional microfluidic and mazelike structures, changes in the growth parameters were observed and revealed an exceptional directional memory by growing hyphae that was maintained over long distances. Comparison with data from a previous study using another species revealed that different fungal species exhibit surprisingly specific sets of growth parameters. A second set of experiments showed that Neurospora crassa had a distinct affinity to edges and tips. On a surface covered with microscale pyramids, hyphae balanced on and bridged between tips.

The hydrophobic force is one of the main driving forces in protein folding and binding. However, its nature is not yet well understood and consequently there are more than 80 different scales published trying to quantify it. Most of the hydrophobicity scales are amino acid-based, but the interaction between the molecular surface of the proteins (and DNA) and surfaces they are immobilized on, e.g., on biomedical micro/nanodevices, occurs on fractions of, rather than whole amino acids. This fragmented structure of the biomolecular surface requires the derivation of atom-level hydrophobicity. Most attempts for the evaluation of atomic hydrophobicities are derived from amino acid-based values, which ignore dynamic and steric factors. This contribution reports on the Molecular Dynamics simulations that aim to overcome this simplification. The calculations examine various tripeptides in an aqueous solution and the analysis focuses on the distance of the nearest water molecules to the individual atoms in the peptides. Different environments result in a variation of average distances for similar atoms in different tripeptides. Comparison with the atomic hydrophobicities derived from the amino acid-based hydrophobicity obtained from peptide partition in water-octanol (Dgoct) and transport through the membrane interface (Dgwif) shows a similar trend to the calculated distances. The variations are likely due to the steric differences of similar types of atoms in different geometric contexts. Therefore, Molecular Dynamics simulations proved convenient for the evaluation of atomic hydrophobicities and open new research avenues. The atomic hydrophobicities can be used to design surfaces that mimic the biomolecular surfaces and therefore elicit an expected biomolecular activity from the immobilized biomolecules.

The airway epithelium is exposed to a variety of harmful agents during breathing and appropriate cellular responses are essential to maintain tissue homeostasis. Recent evidence has highlighted the contribution of epithelial barrier dysfunction in the development of many chronic respiratory diseases. Despite intense research efforts, the responses of the airway barrier to environmental agents are not fully understood, mainly due to lack of suitable in vitro models that recapitulate the complex in vivo situation accurately. Using an interdisciplinary approach, we describe a novel dynamic 3D in vitro model of the airway epithelium, incorporating fully differentiated primary human airway epithelial cells at the air-liquid interface and a basolateral microfluidic supply of nutrients simulating the interstitial flow observed in vivo. Through combination of the microfluidic culture system with an automated fraction collector the kinetics of cellular responses by the airway epithelium to environmental agents can be analysed at the early phases for the first time and with much higher sensitivity compared to common static in vitro models. Following exposure of primary differentiated epithelial cells to pollen we show that CXCL8/IL-8 release is detectable within the first 2h and peaks at 4-6h under microfluidic conditions, a response which was not observed in conventional static culture conditions. Such a microfluidic culture model is likely to have utility for high resolution temporal profiling of toxicological and pharmacological responses of the airway epithelial barrier, as well as for studies of disease mechanisms.

Filamentous fungi are very successful in colonising various microconfined environments, but their behaviour is usually tested on flat surfaces. This contribution presents the design, the fabrication and the use of microstructures, made of a biocompatible polymer (poly(dimethylsiloxane), PDMS) for studying the dynamic micro-scale behaviour of the filamentous fungus Neurospora crassa. The proposed methodology is simple to implement and uses low cost fabrication methods. The observations of the fungus growing through a variety of fabricated micro-environments revealed distinct structure-dependent and structure-induced responses. Generalising the proposed methodology we propose a tool for high-throughput studies of numerous fungal species.