The potential role of Rac signalling and the planar cell polarity pathway in wiring of the enteric nervous system

The functional development of the enteric nervous system (ENS) requires newly generated neurons and their progenitors to migrate to their appropriate sites, extend neurites, guide axons and dendrites to suitable locations and establish synaptic connections with the appropriate targets. Very little is known about the molecular mechanism underlying these processes. Recent studies have suggested a potential role of Rho GTPases as intracellular regulators of several ENS developmental processes. However, the relative participation of specific members of the family in migration, neurogenesis and axonal guidance of enteric progenitors has not been addressed yet. Here, we investigate the in vivo role and genetic interaction of two members of the Rho-GTPase family, Rac1 and Rac3 in enteric neurogenesis. Taking advantage of the Cre/loxP recombination system and a Rac1 conditional inactivation mouse strain (Rac1flox/flox), we generated a Sox10Cre; Rac1flox/flox;R26ReYFP mouse line, where Rac1 gene is specifically ablated in the neural crest population which is also labeled by the expression of Yellow Fluorescent Protein. Secondly, we generated double Rac1;Rac3 mutant animals by crossing the Sox10Cre;Rac1flox;R26ReYFP mouse line to a constitutive Rac3 KO strain (Rac3-/-). In vivo and in vitro studies on Rac-deficient enteric neural crest cells and neurons showed distinctive roles for Rac1 and Rac3 in migration of enteric neural crest cells (ENCCs), in development of enteric neurons and in control of cell polarity within the developing ENS. In addition, we also undertook a candidate gene approach to investigate the involvement of Wnt-signaling genes in enteric axon guidance and circuit formation. We found that two of the core components of the Planar Cell Polarity pathway, the Wnt receptor Frizzled 3 (Fzd3) and the Cadherin EGF LAG seven-pass G-type receptor 3 (Celsr3) are expressed specifically in ENCCs during embryonic development. Here we show, by using a combination of in vivo approaches that in mice deficient in either protein, enteric neurons had characteristic defects in neuronal tract formation and in patterning of individual axonal projections evident from early stages of ENS development. Furthermore, preliminary data show that these specific defects in ENS wiring might be the cause of impaired intestinal function and, therefore, provide the basis for understanding the aetiopathology of several idiopathic enteric neuropathies in humans

CHARMM force field parameterization protocol for self-assembling peptide amphiphiles : the Fmoc moiety

Aromatic peptide amphiphiles are known to self-assemble into nanostructures but the molecular level structure and the mechanism of formation of these nanostructures is not yet understood in detail. Molecular dynamic simulations using the CHARMM force field have been applied to a wide variety of peptide-based systems to obtain molecular level details of processes that are inaccessible with experimental techniques. However, this force field does not include parameters for the aromatic moieties which dictate the self-assembly of these systems. The standard CHARMM force field parameterization protocol uses hydrophilic interactions for the non-bonding parameters evaluation. However, to effectively reproduce the self-assembling behaviour of these molecules, the balance between the hydrophilic and hydrophobic nature of the molecule is essential. In this work, a modified parameterization protocol for the CHARMM force field for these aromatic moieties is presented. This protocol is applied for the specific case of the Fmoc moiety. The resulting set of parameters satisfies the conformational and interactions analysis and is able to reproduce experimental results such as the Fmoc-S-OMe water/octanol partition free energy and the self-assembly of Fmoc-S-OH and Fmoc-Y-OH into spherical micelles and fibres, respectively, while also providing detailed information on the mechanism of these processes. The effectiveness of the parameters for the Fmoc moiety validates the protocol as a robust approach to paramterise this class of compounds

Molecular dynamics simulations reveal disruptive self-assembly in dynamic peptide libraries

There is a significant interest in the use of unmodified self-assembling peptides as building blocks for functional, supramolecular biomaterials. Recently, dynamic peptide libraries (DPLs) have been proposed to select self-assembling materials from dynamically exchanging mixture of dipeptide inputs in the presence of a nonspecific protease enzyme, where peptide sequences are selected and amplified based on their self-assembling tendencies. It was shown that the results of DPL of mixed sequences (e.g. starting from a mixture of dileucine, L2 and diphenylalanine, F2) did not give the same outcome as the separate L2 and F2 libraries (which give rise to formation of F6 and L6), implying that interaction between these sequences could disrupt the self-assembly. In this study, coarse grained molecular dynamic (CG-MD) simulations are used to understand the DPL results for F2, L2 and mixed libraries. CG-MD simulations demonstrate that interactions between precursors can cause the low formation yield of hexapeptides in mixtures of dipeptides and show that this ability to disrupt is influenced by the concentration of the different species in the DPL. The disrupting self-assembly effect between the species in DPL is an important effect to take into account in dynamic combinatorial chemistry as it affects the possible discovery of new materials. The work shows that combined computational and experimental screening can be used complementary and in combination provide a powerful means to discover new supramolecular peptide nanostructures

Supramolecular fibers in gels can be at thermodynamic equilibrium : a simple packing model reveals preferential fibril formation versus crystallization

Low molecular weight gelators are able to form nanostructures, typically fibers, which entangle to form gel-phase materials. These materials have wide-ranging applications in biomedicine and nanotechnology. While it is known that supramolecular gels often represent metastable structures due to the restricted molecular dynamics in the gel state, the thermodynamic nature of the nanofibrous structure is not well understood. Clearly, 3D extended structures will be able to form more interactions than 1D structures. However, self-assembling molecules are typically amphiphilic, thus giving rise to a combination of solvophobic and solvophilic moieties where a level of solvent exposure at the nanostructure surface is favorable. In this study, we introduce a simple packing model, based on prisms with faces of different nature (solvophobic and solvophilic) and variable interaction parameters, to represent amphiphile self-assembly. This model demonstrates that by tuning shape and "self" or "solvent" interaction parameters either the 1D fiber or 3D crystal may represent the thermodynamic minimum. The model depends on parameters that relate to features of experimentally known systems: The number of faces exposed to the solvent or buried in the fiber; the overall shape of the prism; and the free energy penalties associated with the interactions can be adjusted to match their chemical nature. The model is applied to describe the pH-dependent gelation/precipitation of well-known gelator Fmoc-FF. We conclude that, despite the fact that most experimentally produced gels probably represent metastable states, one-dimensional fibers can represent thermodynamic equilibrium. This conclusion has critical implications for the theoretical treatment of gels

Metastable hydrogels from aromatic dipeptides

We demonstrate that the well-known self-assembling dipeptide diphenylalanine (FF) and its amidated derivative (FF-NH2) can form metastable hydrogels upon sonication of the dipeptide solutions. The hydrogels show instantaneous syneresis upon mechanical contact resulting in rapid expulsion of water and collapse into a semi-solid gel

Enzymatically activated emulsions stabilised by interfacial nanofibre networks

We report on-demand formation of emulsions stabilised by interfacial nanoscale networks. These are formed through biocatalytic dephosphorylation and self-assembly of Fmoc(9-fluorenylmethoxycarbonyl)-dipeptide amphiphiles in aqueous/organic mixtures. This is achieved by using alkaline phosphatase which transforms surfactant-like phosphorylated precursors into self-assembling aromatic peptide amphiphiles (Fmoc-tyrosine-leucine, Fmoc-YL) that form nanofibrous networks. In biphasic organic/aqueous systems, these networks form preferentially at the interface thus providing a means of emulsion stabilisation. We demonstrate on-demand emulsification by enzyme addition, even after storage of the biphasic mixture for several weeks. Experimental (Fluorescence, FTIR spectroscopy, fluorescence microscopy, electron microscopy, atomic force microscopy) and computational techniques (atomistic molecular dynamics) are used to characterise the interfacial self-assembly process

Spontaneous aminolytic cyclization and self-assembly of dipeptide methyl esters in water

Dipeptides are known to spontaneously cyclize to diketopiperazines, and in some cases these cyclic dipeptides have been shown to self-assemble to form supramolecular nanostructures. Herein, we demonstrate the in situ cyclization of dipeptide methyl esters in aqueous buffer by intramolecular aminolysis, leading to the formation of diverse supramolecular nanostructures. The chemical nature of the amino acid side chains dictates the supramolecular arrangement and resulting nanoscale architectures. For c[LF], supramolecular gels are formed, and the concentration of starting materials influences the mechanical properties of hydrogels. Moreover, by adding metalloporphyrin to the starting dipeptide ester solution, these become incorporated through cooperative assembly, resulting in the formation of nanofibers able to catalyse the oxidation of organic phenol in water. The approach taken here, which combines the chemically activated assembly with the versatility of short peptides might pave the way for achieving the spontaneous formation of supramolecular order and function using simple building blocks

Enteric Neurospheres Are Not Specific to Neural Crest Cultures: Implications for Neural Stem Cell Therapies

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Assessing the utility of infrared spectroscopy as a structural diagnostic tool for β-sheets in self-assembling aromatic peptide amphiphiles

beta-Sheets are a commonly found structural motif in self-assembling aromatic peptide amphiphiles, and their characteristic "amide I" infrared (IR) absorption bands are routinely used to support the formation of supramolecular structure. In this paper, we assess the utility of IR spectroscopy as a structural diagnostic tool for this class of self-assembling systems. Using 9-fluorene-methyloxycarbonyl dialanine (Fmoc-AA) and the analogous 9-fluorene-methylcarbonyl dialanine (Fmc-AA) as examples, we show that the origin of the band around 1680-1695 cm(-1) in Fourier transform infrared (FTIR) spectra, which was previously assigned to an antiparallel beta-sheet conformation, is in fact absorption of the stacked carbamate group in Fmoc-peptides. IR spectra from C-13-labeled samples support our conclusions. In addition, DFT frequency calculations on small stacks of aromatic peptides help to rationalize these results in terms of the individual vibrational modes