The chemotrophic behaviour of Aspergillus niger: Mapping hyphal filaments during chemo-sensing; the first step towards directed materials formation.

In the development of fungal based materials for applications in construction through to biomedical materials and fashion, understanding how to regulate and direct growth is key for gaining control over the form of material generated. Here, we show how simple 'chemical food' cues can be used to manipulate the growth of fungal networks by taking Aspergillus niger as an exemplar species. Chemotrophic responses towards a range of nitrogen and carbon containing biomolecules including amino acids, sugars and sugar alcohols were quantified in terms of chemotrophic index (CI) under a range of basal media compositions (low and high concentrations of N and C sources). Growth of filamentous networks was followed using fluorescence microscopy at single time points and during growth by an AI analytical approach to explore chemo sensing behaviour of the fungus when exposed to pairs (C-C, C-N, N-N) of biomolecules simultaneously. Data suggests that the directive growth of A. niger can be controlled towards simple biomolecules with CI values giving a good approximation for expected growth under a range of growth conditions. This is a first step towards identifying conditions for researcher-led directed growth of hyphae to make mycelial mats with tuneable morphological, physicochemical, and mechanical characteristics.

Structural Flexibility and Disassembly Kinetics of Single Ferritin Molecules Using Optical Nanotweezers.

Ferritin, a spherical protein shell assembled from 24 subunits, functions as an efficient iron storage and release system through its channels. Understanding how various chemicals affect the structural behavior of ferritin is crucial for unravelling the origins of iron-related diseases in living organisms including humans. In particular, the influence of chemicals on ferritin's dynamics and iron release is barely explored at the single-protein level. Here, by employing optical nanotweezers using double-nanohole (DNH) structures, we examined the effect of ascorbic acid (reducing reagent) and pH on individual ferritin's conformational dynamics. The dynamics of ferritin increased as the concentration of ascorbic acid approached saturation. At pH 2.0, ferritin exhibited significant structural fluctuations and eventually underwent a stepwise disassembly into fragments. This work demonstrated the disassembly pathway and kinetics of a single ferritin molecule in solution. We identified four critical fragments during its disassembly pathway, which are 22-mer, 12-mer, tetramer, and dimer subunits. Moreover, we present single-molecule evidence of the cooperative disassembly of ferritin. Interrogating ferritin's structural change in response to different chemicals holds importance for understanding their roles in iron metabolism, hence facilitating further development of medical treatments for its associated diseases.

Tidying up the conformational ensemble of a disordered peptide by computational prediction of spectroscopic fingerprints.

The most advanced structure prediction methods are powerless in exploring the conformational ensemble of disordered peptides and proteins and for this reason the "protein folding problem" remains unsolved. We present a novel methodology that enables the accurate prediction of spectroscopic fingerprints (circular dichroism, infrared, Raman, and Raman optical activity), and by this allows for "tidying up" the conformational ensembles of disordered peptides and disordered regions in proteins. This concept is elaborated for and applied to a dodecapeptide, whose spectroscopic fingerprint is measured and theoretically predicted by means of enhanced-sampling molecular dynamics coupled with quantum mechanical calculations. Following this approach, we demonstrate that peptides lacking a clear propensity for ordered secondary-structure motifs are not randomly, but only conditionally disordered. This means that their conformational landscape, or phase-space, can be well represented by a basis-set of conformers including about 10 to 100 structures. The implications of this finding have profound consequences both for the interpretation of experimental electronic and vibrational spectral features of peptides in solution and for the theoretical prediction of these features using accurate and computationally expensive techniques. The here-derived methods and conclusions are expected to fundamentally impact the rationalization of so-far elusive structure-spectra relationships for disordered peptides and proteins, towards improved and versatile structure prediction methods.

Quantitative Raman microscopy to describe structural organisation in hollow microcrystals built from silicon catecholate and amines.

Macroscopic scale hollow microcrystals are a promising group of materials for gas and liquid uptake as well as sensing. In this contribution we describe the structure of hollow hexagonal cross-section crystals formulated as salts of a silicon catecholate anion and a tetramethylenediamine (TEMED) cation. Using a combination of X-ray single crystal diffraction, Raman spectroscopy and quantum chemistry we explore the structural properties of the hollow microcrystals. With the X-ray structural data as a starting point and assisted with quantum chemistry we compute Raman tensors to fit polarisation sensitive spectral responses and predict the orientation and packing of unit cells in respect to the long and short axis of the synthesised microcrystals. Using these newly developed methods for predicting molecular Raman responses in space with dependence on local orientation, we present the quantitative analysis of experimental Raman images of both hexagonal and tetragonal cross section hollow microcrystals formed from silicon catecholate anions using different amines as counterions. We describe the distributions of chemical components at the surfaces and edges of microcrystals, address the effect of catcholate hydrophobicity on water uptake and discuss possible strategies in chemical and post-assembly modifications to widen the functional properties of this group of environmentally friendly silicon organic framework (SOF) materials.

Raman microscopy tracks maturity of melanin intermediates in Botrytis cinerea, a plant pathogen.

We use Raman microscopy to describe the structure and chemical composition of both conidiophore and hyphae of Botrytis cinerea, a common plant pathogen. To interpret experimental data, we use density functional theory (DFT) to compute Raman tensors specific to an important fungal glycopeptide, a segment of α-chitin, and several naphthalene-based precursors of increasing complexity, which we propose play a role in the melanin synthesis pathway. Using spectral interpretations based on quantum chemical validation, we review microscopy images reconstructed for specific Raman activities and describe differences in distributions of structural components, photo-protective secondary naphthalene-based pigments, and proteins in both spores and hyphal filaments. Comparison of our results with literature data on other fungi suggests an example of convergent evolution expressed at the level of secondary metabolites specific to plant pathogenic fungi. Our results indicate that pre-resonant Raman monitoring of melanin precursors may help assessment of local Botrytis population biology to aid agricultural production.

A ratiometric, fluorometric approach for surface charge mapping of biosilica features.

We describe a surface charge imaging method for heterogeneous biosilicas based on relationships between zeta (ζ) potential, feature size of nanoparticles, and PDMPO fluorescence and apply it to silicified structures from plants and diatoms. The methodology provides the first opportunity to map the surface charge of large heterogeneous biosilica materials and indicates that local surface charge is related to morphology below the diffraction limit (ca. 20-130 nm) with sharper features showing less negative zeta potential equivalent surface charge suggesting that the zeta potential of silica structures can be adjusted by engineering surface morphology. We show that the approach can be used to study living silicified biological tissues without recourse to sectioning and fixation. Further, the approach could be used for the study of other metal oxides possessing hydroxylated moieties. The method has potential to open up opportunities for the engineering of materials with defined charge characteristics for the solution of biomedical engineering problems including materials for tissue replacement.

Cu(Proline)2 Complex: A Model of Bio-Copper Structural Ambivalence.

Complexes of Cu2+(d9) with proline may be considered a simple model to address the structural flexibility and electronic properties of copper metalloproteins. To discuss optical electronic spectra and infrared spectral responses, we use quantum chemistry applied to model systems prepared under different geometries and degree of hydration. A comparison of experimental data with calculations indicates that first explicit neighbor water clustering next to the Cu2+(d9) complex is critical for a correct description of the electronic properties of this system. We deduce that the moderately hydrated trans conformer is the main structural form of the complex in water. Further, we suggest that the antisymmetric stretching mode of the carbonyl moieties of the conformer is dominant in the spectrally broadened infrared resonance at 1605 cm-1, where inhomogeneity of the transition at the blue side can be ascribed to a continuum of less optimal interactions with the solvent. Extracted structural properties and hydration features provide information on the structural flexibility/plasticity specific to Cu2+(d9) systems in correlation with the electronic behavior upon photoexcitation. We discuss the role and the nature of the axial ligand in bio-copper structural ambivalence and reactivity.

Performance of GFN1-xTB for periodic optimization of metal organic frameworks.

Tight-binding approaches bridge the gap between force field methods and Density Functional Theory (DFT). Density Functional Tight Binding (DFTB) has been employed for a wide range of systems including proteins, clays and 2D and 3D materials. DFTB is 2-3 orders of magnitude faster than DFT, allowing calculations containing up to ca. 5000 atoms. The efficiency of DFTB comes via pre-computed integrals, which are parameterized for each pair of atoms, and the requirement for this parameterization has previously prevented widespread use of DFTB for Metal-Organic Frameworks. The GFN-xTB (Geometries, Frequencies, and Non-covalent interactions Tight Binding) method provides parameters for elements up to Z ≤ 86. We have therefore employed GFN-xTB to periodic optimizations of the Computation Ready Experimental (CoRE) database of MOF structures. We find that 75% of all cell parameters remain within 5% of the reference (experimental) value and that bonds containing metal atoms are typically well conserved with a mean average deviation of 0.187 Å. Therefore GFN-xTB provides the ability to calculate MOF structures more accurately than force fields, and ca. 2 orders of magnitude faster than DFT. We therefore propose that GFN-xTB is a suitable method for screening of hypothetical MOFs (Z ≤ 86), with the advantage of accurate binding energies for adsorption applications.

Anchoring of a hydrophobic heptapeptide (AFILPTG) on silica facilitates peptide unfolding at the abiotic-biotic interface.

A hydrophobic heptapeptide, with sequence AFILPTG, as part of a phage capsid protein binds effectively to silica particles carrying negative charge. Here, we explore the silica binding activity of the sequence as a short polypeptide with polar N and C terminals. To describe the structural changes that occur on binding, we fit experimental infrared, Raman and circular dichroism data for a number of structures simulated in the full configuration space of the hepta-peptide using replica exchange molecular dynamics. Quantum chemistry was used to compute normal modes of infrared and Raman spectra and establish a relationship to structures from MD data. To interpret the circular dichroism data, instead of empirical factoring of optical activity into helical/sheet/random components, we exploit natural transition orbital theory and specify the contributions of backbone amide units, side chain functional groups, water, sodium ions and silica to the observed transitions. Computed optical responses suggest a less folded backbone and importance of the N-terminal when close to silica. We further discuss the thermodynamics of the interplay of charged and hydrophobic moieties of the polypeptide on association with the silica surface. The outcomes of this study may assist in the engineering of novel artificial bio-silica heterostructures.

Lessons from a Challenging System: Accurate Adsorption Free Energies at the Amino Acid/ZnO Interface.

We undertake steps to overcome four challenges that have hindered the understanding of ZnO/biomolecule interfaces at the atomic scale: parametrization of a classical force field, ZnO surface termination and amino acid protonation state in methanol, and convergence of enhanced sampling molecular dynamics simulations. We predict adsorption free energies for histidine, serine, cysteine, and tryptophan in remarkable agreement with experimental measurements obtained via a novel indicator-displacement assay. Adsorption is driven by direct surface/amino-acid interactions mediated by terminal hydroxyl groups and stabilized by strongly structured methanol solvation shells.

Mapping blood biochemistry by Raman spectroscopy at the cellular level.

We report how Raman difference imaging provides insight on cellular biochemistry in vivo as a function of sub-cellular dimensions and the cellular environment. We show that this approach offers a sensitive diagnostic to address blood biochemistry at the cellular level. We examine Raman microscopic images of the distribution of the different hemoglobins in both healthy (discocyte) and unhealthy (echinocyte) blood cells and interpret these images using pre-calculated, accurate pre-resonant Raman tensors for scattering intensities specific to hemoglobins. These tensors are developed from theoretical calculations of models of the oxy, deoxy and met forms of heme benchmarked against the experimental visible spectra of the corresponding hemoglobins. The calculations also enable assignments of the electronic transitions responsible for the colour of blood: these are mainly ligand to metal charge transfer transitions.

Gamma estimator of Jarzynski equality for recovering binding energies from noisy dynamic data sets.

A fundamental problem in thermodynamics is the recovery of macroscopic equilibrated interaction energies from experimentally measured single-molecular interactions. The Jarzynski equality forms a theoretical basis in recovering the free energy difference between two states from exponentially averaged work performed to switch the states. In practice, the exponentially averaged work value is estimated as the mean of finite samples. Numerical simulations have shown that samples having thousands of measurements are not large enough for the mean to converge when the fluctuation of external work is above 4 kBT, which is easily observable in biomolecular interactions. We report the first example of a statistical gamma work distribution applied to single molecule pulling experiments. The Gibbs free energy of surface adsorption can be accurately evaluated even for a small sample size. The values obtained are comparable to those derived from multi-parametric surface plasmon resonance measurements and molecular dynamics simulations.

Polariton condensation and surface enhanced Raman in spherical ZnO microcrystals.

Preparation and characterization of polariton Bose-Einstein condensates in micro-cavities of high quality are at the frontier of contemporary solid state physics. Here, we report on three-dimensional polariton condensation and confinement in pseudo-spherical ZnO microcrystals. The boundary of micro-spherical ZnO resembles a stable cavity that enables sufficient coupling of radiation with material response. Exciting under tight focusing at the low frequency side of the bandgap, we detect efficiency and spectral nonlinear dependencies, as well as signatures of spatial delocalization of the excited states which are characteristics of dynamics in polariton droplets. Expansion of the photon component of the condensate boosts the leaky field beyond the boundary of the ZnO microcrystals. Using this, we observe surface polariton field enhanced Raman responses at the interface of ZnO microspheres. The results demonstrate how readily available spherical semiconductor microstructures facilitate engineering of polariton based electronic states and sensing elements for diagnostics at interfaces.

Correction: Azamacrocycles and tertiary amines can be used to form size tuneable hollow structures or monodisperse oxide nanoparticles depending on the 'M' source.

Correction for 'Azamacrocycles and tertiary amines can be used to form size tuneable hollow structures or monodisperse oxide nanoparticles depending on the 'M' source' by Graham E. Tilburey, et al., Dalton Trans., 2019, 48, 15470-15479.

Materials-Based Approach for Interrogating Human Prostate Cancer Cell Adhesion and Migratory Potential Using a Fluoroalkylsilica Culture Surface.

OPCT-1 is a heterogeneous prostate cancer cell line derived from primary (rather than metastatic) disease which contains epithelial, mesenchymal, and CD44high/CD24low cancer stem cell (CSC) subpopulations and from which we have previously generated and characterized stable mesenchymal (P4B6B) and epithelial (P5B3) cell subpopulations. In this contribution, we explore the effect of tissue culture surface chemistry (standard tissue culture plastic (TCP) and a fluoroalkylsilica (FS) culture surface with inherently low surface energy) on the phenotype and adherent capacity of mesenchymal and epithelial cell populations. We demonstrate that OPCT-1 cells adherent to FS surfaces comprise both epithelial- and mesenchymal-like populations; a mesenchymal subpopulation derived from OPCT1 (P4B6B) poorly adheres to FS and formed spheroids, whereas an epithelial subpopulation derived from OPCT1 (P5B3) forms an adherent monolayer. In contrast, P4B6B cells do adhere to FS when cocultured with P5B3 cells. Taken together, these findings demonstrate that EMT/cell differentiation status dictates cell adhesive capacity and provide a novel insight into the relationship between epithelial and mesenchymal cell populations in metastasis. Importantly, the differences in adherence capacity between P4B6B and P5B3 are not apparent using standard TCP-based culture, thereby highlighting the value of using alternative culture surfaces for studying cell surface interaction/adhesion phenomena and interrogating mechanisms involved in adhesion and detachment of metastatic tumor cells.

Platform for Screening Abiotic/Biotic Interactions Using Indicator Displacement Assays.

This paper describes novel adaptations of optically sectioned planar format assays to screen compounds for their affinities to materials surfaces. The novel platform, which we name optically sectioned indicator displacement assays (O-IDA), makes use of displaceable dyes in a format adaptable to high-throughput multiwell plate technologies. We describe two approaches: the first being where the dye exhibits fluorescence in both the surface bound and unbound state and the second, where fluorescence is lost upon displacement of the dye from the surface. Half maximal inhibitory concentration (IC50), binding affinity (Ki), and binding free energy (ΔGads) values can be extracted from the raw data. Representative biomolecules were tested for interactions with silica in an aqueous environment and ZnO(0001)-Zn and (10-10) facets in a nonaqueous environment. We provide the first experimental values for both the binding of small molecules to silica and the facet-dependent ZnO binding affinity of key amino acids associated with ZnO-specific oligopeptides. The specific data will be invaluable to those studying interactions at interfaces both experimentally and computationally. O-IDA provides a general framework for the high-throughput screening of molecule binding to materials surfaces, which has important applications in drug delivery, (bio-) catalysis, biosensing, and biomaterial engineering.

C-H Olefination of Tryptophan Residues in Peptides: Control of Residue Selectivity and Peptide-Amino Acid Cross-linking.

There is high demand for new methods to modify peptides, for application in drug discovery and biomedicine. A C-H functionalization protocol for the olefination of tryptophan residues in peptides is described. The modification is successful for Trp residues at any position in the peptide, has broad scope in the styrene coupling partner, and offers opportunities for conjugating peptides with other biomolecules. For peptides containing both Trp and Phe, directing group manipulation enables full control of residue selectivity.

Azamacrocycles and tertiary amines can be used to form size tuneable hollow structures or monodisperse oxide nanoparticles depending on the 'M' source.

We show that the azamacrocycle 'cyclam' (1,4,8,11-tetraazacyclodecane) in conjunction with a silicon catecholate ion generates novel hollow tetragonal tube-like crystalline materials [(C6H4O2)3Si][C10H26N4]·H2O, whose dimensions can be tuned according to the pH of the reaction medium. The synthesis approach was successful for both silicon and germanium and we hypothesise that a range of other catecholate precursors of elements such as iron could be used to generate a large array of inorganic materials with interesting morphologies. The synthesis approach can be extended to tertiary diamines with functional group spacing playing an important role in the efficacy of complexation. Of the molecules explored to date, a C2 spacing (N,N,N',N'-tetramethylethylenediamine (4MEDAE)), leads to the most efficient structure control with hollow hexagonal tube-like structures being formed. In addition, we show that azamacrocycles, in the presence of unbuffered tetramethoxysilane (TMOS) solutions can be used to manipulate silica formation and provide a fast (ca. 10 minutes) synthesis route to particles whose diameter can be tuned from ca. 20 nm to several hundreds of nm under reaction conditions (no extremes of pH) that make the sols suitable for direct use in biotechnological applications.

Distributions of Silica and Biopolymer Structural Components in the Spore Elater of Equisetum arvense, an Ancient Silicifying Plant.

Equisetum species are primitive vascular plants that benefit from the biogenesis of silica bio-organic inclusions in their tissues and participate in the annual biosilica turnover in local eco-systems. As means of Equisetum reproduction and propagation, spores are expected to reflect the evolutionary adaptation of the plants to the climatic conditions at different times of the year. Combining methods of Raman and scanning electron microscopy and assisted with density functional theory, we conducted material spatial-spectral correlations to characterize the distribution of biopolymers and silica based structural elements that contribute to the bio-mineral content of the elater. The elater tip has underlying skeletal-like structural elements where cellulose fibers provide strength and flexibility, both of which are necessary for locomotion. The surface of the elater tips is rich with less ordered pectin like polysaccharide and shows a ridged, folded character. At the surface we observe silica of amorphous, colloidal form in nearly spherical structures where the silica is only a few layers thick. We propose the observed expansion of elater tips upon germination and the form of silica including encapsulated biopolymers are designed for ready dispersion, release of the polysaccharide-arginine rich content and to facilitate silica uptake to the developing plant. This behavior would help to condition local soil chemistry to facilitate competitive rooting potential and stem propagation.

From phage display to structure: an interplay of enthalpy and entropy in the binding of the LDHSLHS polypeptide to silica.

Polypeptide based biosilica composites show promise as next generation multi-functional nano-platforms for diagnostics and bio-catalytic applications. Following the identification of a strong silica binder (LDHSLHS) by phage display, we conduct structural analysis of the polypeptide at the interface with amorphous silica nanoparticles in an aqueous environment. Our approach relies on modelling infrared and Raman spectral responses using predictions of molecular dynamics simulations and quantum studies of the normal modes for several potential structures. By simultaneously fitting both infrared and Raman responses in the amide spectral region, we show that the main structural conformer has a beta-like central region and helix-twisted terminals. Classical simulations, as conducted previously (Chem. Mater., 2014, 26, 5725), predict that the association of the main structure with the interface is stimulated by electrostatic interactions though surface binding also requires spatially distributed sodium ions to compensate for negatively charged acidic silanol groups. Accordingly, diffusion of sodium ions would contribute to a stochastic character of the peptide association with the surface. Consistent with the described dynamics at the interface, the results obtained from isothermal titration calorimetry (ITC) confirm a significant enhancement of polypeptide binding to silica at higher concentrations of Na+. The results of this study suggest that the tertiary structure of a phage capsid protein plays a significant role in regulating the conformation of peptide LDHSLHS, increasing its binding to silica during the phage display process. The results presented here support design-led engineering of polypeptide-silica nanocomposites for bio-technological applications.