Boron clusters as broadband membrane carriers.

The membrane translocation of hydrophilic substances constitutes a challenge for their application as therapeutic compounds and labelling probes1-4. To remedy this, charged amphiphilic molecules have been classically used as carriers3,5. However, such amphiphilic carriers may cause aggregation and non-specific membrane lysis6,7. Here we show that globular dodecaborate clusters, and prominently B12Br122-, can function as anionic inorganic membrane carriers for a broad range of hydrophilic cargo molecules (with molecular mass of 146-4,500 Da). We show that cationic and neutral peptides, amino acids, neurotransmitters, vitamins, antibiotics and drugs can be carried across liposomal membranes. Mechanistic transport studies reveal that the carrier activity is related to the superchaotropic nature of these cluster anions8-12. We demonstrate that B12Br122- affects cytosolic uptake of different small bioactive molecules, including the antineoplastic monomethyl auristatin F, the proteolysis targeting chimera dBET1 and the phalloidin toxin, which has been successfully delivered in living cells for cytoskeleton labelling. We anticipate the broad and distinct delivery spectrum of our superchaotropic carriers to be the starting point of conceptually distinct cell-biological, neurobiological, physiological and pharmaceutical studies.

Size and Polarizability of Boron Cluster Carriers Modulate Chaotropic Membrane Transport.

Perhalogenated closo-borates represent a new class of membrane carriers. They owe this activity to their chaotropicity, which enables the transport of hydrophilic molecules across model membranes and into living cells. The transport efficiency of this new class of cluster carriers depends on a careful balance between their affinity to membranes and cargo, which varies with chaotropicity. However, the structure-activity parameters that define chaotropic transport remain to be elucidated. Here, we have studied the modulation of chaotropic transport by decoupling the halogen composition from the boron core size. The binding affinity between perhalogenated decaborate and dodecaborate clusters carriers was quantified with different hydrophilic model cargos, namely a neutral and a cationic peptide, phalloidin and (KLAKLAK)2. The transport efficiency, membrane-lytic properties, and cellular toxicity, as obtained from different vesicle and cell assays, increased with the size and polarizability of the clusters. These results validate the chaotropic effect as the driving force behind the membrane transport propensity of boron clusters. This work advances our understanding of the structural features of boron cluster carriers and establishes the first set of rational design principles for chaotropic membrane transporters.

Photoswitchable Calixarene Activators for Controlled Peptide Transport across Lipid Membranes.

Supramolecular synthetic transporters are crucial to understand and activate the passage across lipid membranes of hydrophilic effector molecules. Herein, we introduce photoswitchable calixarenes for the light-controlled transport activation of cationic peptide cargos across model lipid bilayers and inside living cells. Our approach was based on rationally designed p-sulfonatocalix[4]arene receptors equipped with a hydrophobic azobenzene arm, which recognize cationic peptide sequences at the nM range. Activation of membrane peptide transport is confirmed, in synthetic vesicles and living cells, for calixarene activators featuring the azobenzene arm in the E configuration. Therefore, this method allows the modulation of the transmembrane transport of peptide cargos upon Z-E photoisomerization of functionalized calixarenes using 500 nm visible light. These results showcase the potential of photoswitchable counterion activators for the light-triggered delivery of hydrophilic biomolecules and pave the way for potential applications in remotely controlled membrane transport and photopharmacology applications of hydrophilic functional biomolecules.

Metallacarborane Cluster Anions of the Cobalt Bisdicarbollide-Type as Chaotropic Carriers for Transmembrane and Intracellular Delivery of Cationic Peptides.

Cobalt bisdicarbollides (COSANs) are inorganic boron-based anions that have been previously reported to permeate by themselves through lipid bilayer membranes, a propensity that is related to their superchaotropic character. We now introduce their use as selective and efficient molecular carriers of otherwise impermeable hydrophilic oligopeptides through both artificial and cellular membranes, without causing membrane lysis or poration at low micromolar carrier concentrations. COSANs transport not only arginine-rich but also lysine-rich peptides, whereas low-molecular-weight analytes such as amino acids as well as neutral and anionic cargos (phalloidin and BSA) are not transported. In addition to the unsubstituted isomers (known as ortho- and meta-COSAN), four derivatives bearing organic substituents or halogen atoms have been evaluated, and all six of them surpass established carriers such as pyrenebutyrate in terms of activity. U-tube experiments and black lipid membrane conductance measurements establish that the transport across model membranes is mediated by a molecular carrier mechanism. Transport experiments in living cells showed that a fluorescent peptide cargo, FITC-Arg8, is delivered into the cytosol.

Ghost introgression in ricefishes of the genus Adrianichthys in an ancient Wallacean lake.

Because speciation might have been promoted by ancient introgression from an extinct lineage, it is important to detect the existence of 'ghost introgression' in focal taxa and examine its contribution to their diversification. In this study, we examined possible ghost introgression and its contributions to the diversification of ricefishes of the genus Adrianichthys in Lake Poso, an ancient lake on Sulawesi Island, in which some extinctions are known to have occurred. Population-genomic analysis revealed that two extant Adrianichthys species, A. oophorus and A. poptae are reproductively isolated from each other. Comparisons of demographic models demonstrated that introgression from a ghost population, which diverged from the common ancestor of A. oophorus and A. poptae, is essential for reconstructing the demographic history of Adrianichthys. The best model estimated that the divergence of the ghost population greatly predated the divergence between A. oophorus and A. poptae, and that the ghost population secondarily contacted the two extant species within Lake Poso more recently. Genome scans and simulations detected a greatly divergent locus, which cannot be explained without ghost introgression. This locus was also completely segregated between A. oophorus and A. poptae. These findings suggest that variants that came from a ghost population have contributed to the divergence between A. oophorus and A. poptae, but the large time-lag between their divergence and ghost introgression indicates that the contribution of introgression may be restricted.

Genetic basis for the evolution of pelvic-fin brooding, a new mode of reproduction, in a Sulawesian fish.

Modes of reproduction in animals are diverse, with different modes having evolved independently in multiple lineages across a variety of taxa. However, an understanding of the genomic change driving the transition between different modes of reproduction is limited. Several ricefishes (Adrianichthyidae) on the island of Sulawesi have a unique mode of reproduction called "pelvic-fin brooding," wherein females carry externally fertilized eggs until hatching using their pelvic fins. Phylogenomic analysis demonstrated pelvic-fin brooders to have evolved at least twice in two distant clades of the Adrianichthyidae. We investigated the genetic architecture of the evolution of this unique mode of reproduction. Morphological analyses and laboratory observations revealed that females of pelvic-fin brooders have longer pelvic fins and a deeper abdominal concavity, and that they can carry an egg clutch for longer than nonbrooding adrianichthyids, suggesting that these traits play important roles in this reproductive mode. Quantitative trait locus mapping using a cross between a pelvic-fin brooder Oryzias eversi and a nonbrooding O. dopingdopingensis reveals different traits involved in pelvic-fin brooding to be controlled by different loci on different chromosomes. Genomic analyses of admixture detected no signatures of introgression between two lineages with pelvic-fin brooders, indicating that introgression is unlikely to be responsible for repeated evolution of pelvic-fin brooding. These findings suggest that multiple independent mutations may have contributed to the convergent evolution of this novel mode of reproduction.

Diversity of sex chromosomes in Sulawesian medaka fishes.

Recent genetic and genomic studies have revealed tremendous diversity in sex chromosomes across diverse taxa. Closely related species with different sex chromosomes provide us excellent opportunities to investigate the driving forces and the consequences of sex chromosome turnover. In the present study, we investigated the diversity of sex chromosomes of 13 Oryzias species from Sulawesi, Indonesia, which diversified during the last 4.86 million years. Using pooled sequencing, we found sex chromosomes in nine species that all had XY systems, with a species being possibly modified by multiple loci. Seven species (O. woworae, O. asinua, O. wolasi, O. matanensis, O. celebensis, O. hadiatyae, and O. dopingdopingensis) share linkage group (LG) 24 as sex chromosomes; however, they differed in the length and magnitude of sequence divergence between the X and Y chromosomes. The sex chromosome of O. eversi was LG4, which has not been reported as a sex chromosome in any other medaka species. In O. sarasinorum, LG16 and LG22 are associated with sex. Although LG16 was found to be sex-linked in another medaka species previously examined, the sex-determining regions did not overlap. No significant signatures for sex chromosomes were identified in the other four species (O. marmoratus, O. nigrimas, O. nebulosus, and O. orthognathus). Frequent turnovers and the great diversity of the sex chromosomes will make Sulawesian medaka species a model system for investigating the driving forces and consequences of sex chromosome turnover.

A 3D Peptide/[60]Fullerene Hybrid for Multivalent Recognition.

Fully substituted peptide/[60]fullerene hexakis-adducts offer an excellent opportunity for multivalent protein recognition. In contrast to monofunctionalized fullerene hybrids, peptide/[60]fullerene hexakis-adducts display multiple copies of a peptide in close spatial proximity and in the three dimensions of space. High affinity peptide binders for almost any target can be currently identified by in vitro evolution techniques, often providing synthetically simpler alternatives to natural ligands. However, despite the potential of peptide/[60]fullerene hexakis-adducts, these promising conjugates have not been reported to date. Here we present a synthetic strategy for the construction of 3D multivalent hybrids that are able to bind with high affinity the E-selectin. The here synthesized fully substituted peptide/[60]fullerene hybrids and their multivalent recognition of natural receptors constitute a proof of principle for their future application as functional biocompatible materials.

Stronger Together: Multivalent Phage Capsids Inhibit Virus Entry.

Antivirals are now more important than ever. To efficiently inhibit virus replication, antiviral multivalent strategies need sufficient affinity to overcome the excellent matching between the virus and its receptor. This report highlights a phage capsid scaffold strategy that can be used to precisely position sialic acid moieties to inhibit influenza A virus replication.

Species divergence and repeated ancient hybridization in a Sulawesian lake system.

An increasing volume of empirical studies demonstrated that hybridization between distant lineages may have promoted speciation in various taxa. However, the timing, extent and direction of introgressive hybridization remain unknown in many cases. Here, we report a possible case in which repeated hybridization promoted divergence of Oryzias ricefishes (Adrianichthyidae) on Sulawesi, an island of Wallacea. Four Oryzias species are endemic to the Malili Lake system in central Sulawesi, which is composed of five tectonic lakes; of these, one lake is inhabited by two species. Morphological and population genomic analyses of genome-wide single-nucleotide polymorphisms revealed that these two sympatric species are phylogenetically sister to but substantially reproductively isolated from each other. Analyses of admixture and comparison of demographic models revealed that the two sympatric species experienced several substantial introgressions from outgroup populations that probably occurred soon after they had secondary contact with each other in the lake. However, the ratio of migrants from the outgroups was estimated to be different between the two species, which is consistent with the hypothesis that these introgressions aided their divergence or prevented them from forming a hybrid swarm. Repeated lake fragmentations and fusions may have promoted diversification of this freshwater fish species complex that is endemic to this ancient lake system.

Mesozoic origin and 'out-of-India' radiation of ricefishes (Adrianichthyidae).

The Indian subcontinent has an origin geologically different from Eurasia, but many terrestrial animal and plant species on it have congeneric or sister species in other parts of Asia, especially in the Southeast. This faunal and floral similarity between India and Southeast Asia is explained by either of the two biogeographic scenarios, 'into-India' or 'out-of-India'. Phylogenies based on complete mitochondrial genomes and five nuclear genes were undertaken for ricefishes (Adrianichthyidae) to examine which of these two biogeographic scenarios fits better. We found that Oryzias setnai, the only adrianichthyid distributed in and endemic to the Western Ghats, a mountain range running parallel to the western coast of the Indian subcontinent, is sister to all other adrianichthyids from eastern India and Southeast-East Asia. Divergence time estimates and ancestral area reconstructions reveal that this western Indian species diverged in the late Mesozoic during the northward drift of the Indian subcontinent. These findings indicate that adrianichthyids dispersed eastward 'out-of-India' after the collision of the Indian subcontinent with Eurasia, and subsequently diversified in Southeast-East Asia. A review of geographic distributions of 'out-of-India' taxa reveals that they may have largely fuelled or modified the biodiversity of Eurasia.

1D to 2D Self Assembly of Cyclic Peptides.

Despite recent developments in two-dimensional self-assembly, most supramolecular 2D materials are assembled by tedious methodologies, with complex surface chemistry and small sizes. We here report d/l-alternating cyclic peptides that undergo one-dimensional self-assembly into amphiphilic nanotubes, which subsequently arrange as tubular bilayers to form giant nanosheets in the mesoscale. Reversible transitions between the assembled, dispersed, and aggregated states of these nanosheets can be triggered by external stimuli. The characteristic flexibility, defined chemical topology, and length scale of these nanosheets set a clear distinction between this new supramolecular architecture and previously reported 2D nanostructures. The sequential 1D-to-2D self-assembly of peptides described here provides a conceptually new approach to achieve two-dimensional materials with hierarchical organization. These giant nanosheets represent one of the largest 2D supramolecular materials ever made, with potential application as long-range molecular transporters, responsive surfaces, and (bio)sensors.

An Adhesive Peptide from the C-Terminal Domain of α-Synuclein for Single-Layer Adsorption of Nanoparticles onto Substrates.

The two-dimensional (2D) homogeneous assembly of nanoparticle monolayer arrays onto a broad range of substrates constitutes an important challenge for chemistry, nanotechnology, and material science. α-Synuclein (αS) is an intrinsically disordered protein associated with neuronal protein complexes and has a high degree of structural plasticity and chaperone activity. The C-terminal domain of αS has been linked to the noncovalent interactions of this protein with biological targets and the activity of αS in presynaptic connections. Herein, we have systematically studied peptide fragments of the chaperone-active C-terminal sequence of αS and identified a 17-residue peptide that preserves the versatile binding nature of αS. Attachment of this short peptide to gold nanoparticles afforded colloidally stable nanoparticle suspensions that allowed the homogeneous 2D adhesion of the conjugates onto a wide variety of surfaces, including the formation of crystalline nanoparticle superlattices. The peptide sequence and the strategy reported here describe a new adhesive molecule for the controlled monolayer adhesion of metal nanoparticles and sets a stepping-stone toward the potential application of the adhesive properties of αS.

Sequence Decoding of 1D to 2D Self-Assembling Cyclic Peptides.

The inherent ability of peptides to self-assemble with directional and rationally predictable interactions has fostered a plethora of synthetic two-dimensional (2D) supramolecular biomaterials. However, the design of peptides with hierarchical assembly in different dimensions across mesoscopic lengths remains a challenging task. We here describe the structural exploration of a d/l-alternating cyclic octapeptide capable of assembling one-dimensional (1D) nanotubes in water, which subsequently pack laterally to form giant 2D nanosheets up to 500 µm long with a constant 3.2 nm thickness. Specific amino acid mutations allowed the mapping of structure-assembly relationships that determine 2D self-assembly. Nine peptide modifications were studied, revealing key features in the peptide sequence that nanosheets tolerated, while a total of three peptide variants included modifications that compromised their 2D arrangement. These lessons will serve as guide and inspiration for new 2D supramolecular peptide designs.

Synthesis and Supramolecular Functional Assemblies of Ratiometric pH Probes.

Tracking pH with spatiotemporal resolution is a critical challenge for synthetic chemistry, chemical biology and beyond. Over the last decade, different small probes and supramolecular systems have emerged for in cellulo or in vivo pH tracking. However, pH reporting still presents critical limitations, such as background reduction, improved sensor stability, cell targeting, endosomal escape, near- and far-infrared ratiometric pH tracking and adaption to new imaging techniques (i.e., super-resolution). These challenges will require the combined efforts of synthetic and supramolecular chemistry working together to develop the next generation of smart materials that will resolve current limitations. Herein, recent advances in the synthesis of small fluorescent probes, together with new supramolecular functional systems employed for pH tracking, are described with an emphasis on ratiometric probes. The combination of organic synthesis and stimuli-responsive supramolecular functional materials will be essential to solve future challenges of pH tracking, such as improved signal to noise ratio, on target activation and microenvironment reporting.

Spatially Controlled Supramolecular Polymerization of Peptide Nanotubes by Microfluidics.

Despite the importance of spatially resolved self-assembly for molecular machines, the spatial control of supramolecular polymerization with synthetic monomers had not been experimentally established. Now, a microfluidic-regulated tandem process of supramolecular polymerization and droplet encapsulation is used to control the position of self-assembled microfibrillar bundles of cyclic peptide nanotubes in water droplets. This method allows the precise preferential localization of fibers either at the interface or into the core of the droplets. UV absorbance, circular dichroism and fluorescence microscopy indicated that the microfluidic control of the stimuli (changes in pH or ionic strength) can be employed to adjust the packing degree and the spatial position of microfibrillar bundles of cyclic peptide nanotubes. Additionally, this spatially organized supramolecular polymerization of peptide nanotubes was applied in the assembly of highly ordered two-dimensional droplet networks.

Monitoring the Formation of Amyloid Oligomers Using Photoluminescence Anisotropy.

The formation of oligomeric soluble aggregates is related to the toxicity of amyloid peptides and proteins. In this manuscript, we report the use of a ruthenium polypyridyl complex ([Ru(bpy)2(dpqp)]2+) to track the formation of amyloid oligomers at different times using photoluminescence anisotropy. This technique is sensitive to the rotational correlation time of the molecule under study, which is consequently related to the size of the molecule. [Ru(bpy)2(dpqp)]2+ presents anisotropy values of zero when free in solution (due to its rapid rotation and long lifetime) but larger values as the size and concentration of amyloid-ß (Aß) oligomers increase. Our assays show that Aß forms oligomers immediately after the assay is started, reaching a steady state at ∼48 h. SDS-PAGE, DLS, and TEM were used to confirm and characterize the formation of oligomers. Our experiments show that the rate of formation for Aß oligomers is temperature dependent, with faster rates as the temperature of the assay is increased. The probe was also effective in monitoring the formation of α-synuclein oligomers at different times.

Where in the Cell Is our Cargo? Methods Currently Used To Study Intracellular Cytosolic Localisation.

The internalisation and delivery of active substances into cells is a field of growing interest for chemical biology and therapeutics. As we move from small-molecule-based drugs towards bigger cargos, such as antibodies, enzymes, nucleases or nucleic acids, the development of efficient delivery systems becomes critical for their practical application. Different strategies and synthetic carriers have been developed; these include cationic lipids, gold nanoparticles, polymers, cell-penetrating peptides (CPPs), protein surface modification etc. However, all of these methodologies still present limitations relating to the precise targeting of the different intracellular compartments and, in particular, difficulties in access to the cellular cytosol. Additionally, the precise quantification of the cellular uptake of a compound is not enough to demonstrate delivery and/or functional activity. Therefore, methods to determine cellular distributions of cargos and carriers are of critical importance for identifying the barriers that are blocking the activity. Herein we survey the different techniques that can currently be used to track and to monitor the subcellular localisation of the synthetic compounds that we deliver inside cells.

Glycosylated Cell-Penetrating Peptides (GCPPs).

The cell membrane regulates the exchange of molecules and information with the external environment. However, this control barrier hinders the delivery of exogenous bioactive molecules that can be applied to correct cellular malfunctions. Therefore, the traffic of macromolecules across the cell membrane represents a great challenge for the development of the next generation of therapies and diagnostic methods. Cell-penetrating peptides are short peptide sequences capable of delivering a broad range of biomacromolecules across the cellular membrane. However, penetrating peptides still suffer from limitations, mainly related to their lack of specificity and potential toxicity. Glycosylation has emerged as a potential promising strategy for the biological improvement of synthetic materials. In this work we have developed a new convergent strategy for the synthesis of penetrating peptides functionalized with glycan residues by an oxime bond connection. The uptake efficiency and intracellular distribution of these glycopeptides have been systematically characterized by means of flow cytometry and confocal microscopy and in zebrafish animal models. The incorporation of these glycan residues into the peptide structure influenced the internalization efficiency and cellular toxicity of the resulting glycopeptide hybrids in the different cell lines tested. The results reported herein highlight the potential of the glycosylation of penetrating peptides to modulate their activity.

Self-assembled micro-fibres by oxime connection of linear peptide amphiphiles.

Linear peptide amphiphiles are excellent biocompatible scaffolds for the hierarchical self-assembly of one-dimensional nano-structures in aqueous media. However, their structural exploration and screening of self-assembling properties are often limited by time-consuming synthesis and purification steps. We here describe the application of an oxime bond as a powerful synthetic tool towards the conjugation of peptide heads bearing a hydroxylamine group with hydrophobic aldehyde tails. This methodology allowed the quick preparation of a small library of oxime-connected peptide amphiphiles, whose supramolecular screening revealed nano-to-micro-fibrillation with dependency on their chemical structure. These results demonstrate the simplicity and the synthetic potential of the oxime conjugation for the preparation of peptide amphiphiles with improved self-assembling capabilities.