Modification of Ammonia Decomposition Activity of Ruthenium Nanoparticles by N-Doping of CNT Supports

The use of ammonia as a hydrogen vector has the potential to unlock the hydrogen economy. In this context, this paper presents novel insights into improving the ammonia decomposition activity of ruthenium nanoparticles supported on carbon nanotubes (CNT) by nitrogen doping. Our results can be applied to develop more active systems capable of delivering hydrogen on demand, with a view to move towards the low temperature target of less than 150?°C. Herein we demonstrate that nitrogen doping of the CNT support enhances the activity of ruthenium nanoparticles for the low temperature ammonia decomposition with turnover frequency numbers at 400?°C of 6200 LH₂ mol_Ru ^?1 h^?1, higher than the corresponding value of unmodified CNT supports under the same conditions (4400 LH₂ mol_Ru ^?1 h^??1), despite presenting similar ruthenium particle sizes. However, when the nitrogen doping process is carried out with cetyltrimethylammonium bromide (CTAB) to enhance the dispersion of CNTs, the catalyst becomes virtually inactive despite the small ruthenium particle size, likely due to interference of CTAB, weakening the metal–support interaction. Our results demonstrate that the low temperature ammonia decomposition activity of ruthenium can be enhanced by nitrogen doping of the CNT support due to simultaneously increasing the support’s conductivity and basicity, electronically modifying the ruthenium active sites and promoting a strong metal–support interaction.     

Biodistribution of single and aggregated gold nanoparticles exposed to the human lung epithelial tissue barrier at the air-liquid interface

Background

The lung represents the primary entry route for airborne particles into the human body. Most studies addressed possible adverse effects using single (nano)particles, but aerosolic nanoparticles (NPs) tend to aggregate and form structures of several hundreds nm in diameter, changing the physico-chemical properties and interaction with cells. Our aim was to investigate how aggregation might affect the biodistribution; cellular uptake and translocation over time of aerosolized NPs at the air-blood barrier interface using a multicellular lung system.

Results

Model gold nanoparticles (AuNPs) were engineered and well characterized to compare single NPs with aggregated NPs with hydrodynamic diameter of 32 and 106 nm, respectively. Exposures were performed by aerosolization of the particles onto the air-liquid interface of a three dimensional (3D) lung model. Particle deposition, cellular uptake and translocation kinetics of single and aggregated AuNPs were determined for various concentrations, (30, 60, 150 and 300 ng/cm²) and time points (4, 24 and 48 h) using transmission electron microscopy and inductively coupled plasma optical emission spectroscopy. No apparent harmful effect for single and aggregated AuNPs was observed by lactate dehydrogenase assay, nor pro-inflammation response by tumor necrosis factor α assessment. The cell layer integrity was also not impaired. The bio-distribution revealed that majority of the AuNPs, single or aggregated, were inside the cells, and only a minor fraction, less than 5%, was found on the basolateral side. No significant difference was observed in the translocation rate. However, aggregated AuNPs showed a significantly faster cellular uptake than single AuNPs at the first time point, i.e. 4 h.

Conclusions

Our studies revealed that aggregated AuNPs showed significantly faster cellular uptake than single AuNPs at the first time point, i.e. 4 h, but the uptake rate was similar at later time points. In addition, aggregation did not affect translocation rate across the lung barrier model since similar translocation rates were observed for single as well as aggregated AuNPs.

     

PBAT/organoclay composite films: preparation and properties

Environmental problems caused by the increased waste associated with short-term use of plastic materials, particularly by the food packaging industry, prompted the search for biodegradable alternatives. This contribution studied one of these alternatives, poly(butylene adipate-co-terephthalate)—PBAT, a polymer that is fully biodegradable in common landfills, compounded with a small amount of Cloisite 20A organoclay. Materials were mixed in a laboratory internal mixer and films prepared in a chill roll extruder. Results show that the presence of organoclay does not increase degradation of the polymer matrix during processing, nor affects its crystallization characteristics. However, organoclay addition significantly diminished oxygen and carbon dioxide permeability of the films, making them a very interesting alternative for the food packaging industry.     

Expanding the Genetic Code in Xenopus laevis Oocytes

Heterologous expression of ligand‐gated ion channels (LGICs) in Xenopus laevis oocytes combined with site‐directed mutagenesis has been demonstrated to be a powerful approach to study structure–function relationships. In particular, introducing unnatural amino acids (UAAs) has enabled modifications that are not found in natural proteins. However, the current strategy relies on the technically demanding in vitro synthesis of aminoacylated suppressor tRNA. We report here a general method that circumvents this limitation by utilizing orthogonal aminoacyl‐tRNA synthetase (aaRS)/suppressor tRNA_CUA pairs to genetically encode UAAs in Xenopus oocytes. We show that UAAs inserted in the N‐terminal domain of N‐methyl‐D ‐aspartate receptors (NMDARs) serve as photo‐crosslinkers that lock the receptor in a discrete conformational state in response to UV photo treatment. Our method should be generally applicable to studies of other LGICs in Xenopus oocytes.     

Using a Fragment‐Based Approach To Target Protein–Protein Interactions

The ability to identify inhibitors of protein–protein interactions represents a major challenge in modern drug discovery and in the development of tools for chemical biology. In recent years, fragment‐based approaches have emerged as a new methodology in drug discovery; however, few examples of small molecules that are active against chemotherapeutic targets have been published. Herein, we describe the fragment‐based approach of targeting the interaction between the tumour suppressor BRCA2 and the recombination enzyme RAD51; it makes use of a screening pipeline of biophysical techniques that we expect to be more generally applicable to similar targets. Disruption of this interaction in vivo is hypothesised to give rise to cellular hypersensitivity to radiation and genotoxic drugs. We have used protein engineering to create a monomeric form of RAD51 by humanising a thermostable archaeal orthologue, RadA, and used this protein for fragment screening. The initial fragment hits were thoroughly validated biophysically by isothermal titration calorimetry (ITC) and NMR techniques and observed by X‐ray crystallography to bind in a shallow surface pocket that is occupied in the native complex by the side chain of a phenylalanine from the conserved FxxA interaction motif found in BRCA2. This represents the first report of fragments or any small molecule binding at this protein–protein interaction site.     

Macrocyclization of Organo‐Peptide Hybrids through a Dual Bio‐orthogonal Ligation: Insights from Structure–Reactivity Studies

Macrocycles constitute an attractive structural class of molecules for targeting biomolecular interfaces with high affinity and specificity. Here, we report systematic studies aimed at exploring the scope and mechanism of a novel chemo‐biosynthetic strategy for generating macrocyclic organo‐peptide hybrids (MOrPHs) through a dual oxime‐/intein‐mediated ligation reaction between a recombinant precursor protein and bifunctional, oxyamino/1,3‐amino‐thiol compounds. An efficient synthetic route was developed to access structurally different synthetic precursors incorporating a 2‐amino‐ mercaptomethyl‐aryl (AMA) moiety previously found to be important for macrocyclization. With these compounds, the impact of the synthetic precursor scaffold and of designed mutations within the genetically encoded precursor peptide sequence on macrocyclization efficiency was investigated. Importantly, the desired MOrPHs were obtained as the only product from all the different synthetic precursors probed in this study and across peptide sequences comprising four to 15 amino acids. Systematic mutagenesis of the “i?1” site at the junction between the target peptide sequence and the intein moiety revealed that the majority of the 20 amino acids are compatible with MOrPH formation; this enables the identification of the most and the least favorable residues for this critical position. Furthermore, interesting trends with respect to the positional effect of conformationally constrained (Pro) and flexible (Gly) residues on the reactivity of randomized hexamer peptide sequences were observed. Finally, mechanistic investigations enabled the relative contributions of the two distinct pathways (side‐chain→C‐end ligation versus C‐end→side‐chain ligation) to the macrocyclization process to be dissected. Altogether, these studies demonstrate the versatility and robustness of the methodology to enable the synthesis and diversification of a new class of organo‐peptide macrocycles and provide valuable structure–reactivity insights to inform the construction of macrocycle libraries through this chemo‐biosynthetic strategy.     

Isolation and Characterisation of a Ferrirhodin Synthetase Gene from the Sugarcane Pathogen Fusarium sacchari

FSN1, a gene isolated from the sugar‐cane pathogen Fusarium sacchari, encodes a 4707‐residue nonribosomal peptide synthetase consisting of three complete adenylation, thiolation and condensation modules followed by two additional thiolation and condensation domain repeats. This structure is similar to that of ferricrocin synthetase, which makes a siderophore that is involved in intracellular iron storage in other filamentous fungi. Heterologous expression of FSN1 in Aspergillus oryzae resulted in the accumulation of a secreted metabolite that was identified as ferrirhodin. This siderophore was found to be present in both mycelium and culture filtrates of F. sacchari, whereas ferricrocin is found only in the mycelium, thus suggesting that ferricrocin is an intracellular storage siderophore in F. sacchari, whereas ferrirhodin is used for iron acquisition. To our knowledge, this is the first report to characterise a ferrirhodin synthetase gene functionally.     

Oxidative Folding of Peptides with Cystine‐Knot Architectures: Kinetic Studies and Optimization of Folding Conditions

Bioactive peptides often contain several disulfide bonds that provide the main contribution to conformational rigidity and structural, thermal, or biological stability. Among them, cystine‐knot peptides—commonly named “knottins”—make up a subclass with several thousand natural members. Hence, they are considered promising frameworks for peptide‐based pharmaceuticals. Although cystine‐knot peptides are available through chemical and recombinant synthetic routes, oxidative folding to afford the bioactive isomers still remains a crucial step. We therefore investigated the oxidative folding of ten protease‐inhibiting peptides from two knottin families, as well as that of an HIV entry inhibitor and of aprotinin, under two conventional sets of folding conditions and by a newly developed procedure. Kinetic studies identified folding conditions that resulted in correctly folded miniproteins with high rates of conversion even for highly hydrophobic and aggregation‐prone peptides in concentrated solutions.     

Fluorescent Probes for G‐Quadruplex Structures

Mounting evidence supports the presence of biologically relevant G‐quadruplexes in single‐cell organisms, but the existence of endogenous G‐quadruplex structures in mammalian cells remains highly controversial. This is due, in part, to the common misconception that DNA and RNA molecules are passive information carriers with relatively little structural or functional complexity. For those working in the field, however, the lack of available tools for characterizing DNA structures in vivo remains a major limitation to addressing fundamental questions about structure–function relationships of nucleic acids. In this review, we present progress towards the direct detection of G‐quadruplex structures by using small molecules and modified oligonucleotides as fluorescent probes. While most development has focused on cell‐permeable probes that selectively bind to G‐quadruplex structures with high affinity, these same probes can induce G‐quadruplex folding, thereby making the native conformation of the DNA or RNA molecule (i.e., in the absence of probe) uncertain. For this reason, modified oligonucleotides and fluorescent base analogues that serve as “internal” fluorescent probes are presented as an orthogonal means for detecting conformational changes, without necessarily perturbing the equilibria between G‐quadruplex, single‐stranded, and duplex DNA. The major challenges and motivation for the development of fluorescent probes for G‐quadruplex structures are presented, along with a summary of the key photophysical, biophysical, and biological properties of reported examples.     

Spectroscopic and Theoretical Study on Electronically Modified Chromophores in LOV Domains: 8‐Bromo‐ and 8‐Trifluoromethyl‐Substituted Flavins

Two chemically synthesized flavin derivatives, 8‐trifluoromethyl‐ and 8‐bromoriboflavin (8‐CF₃RF and 8‐BrRF), were photochemically characterized in H₂O and studied spectroscopically after incorporation into the LOV domain of the blue light photoreceptor YtvA from Bacillus subtilis. The spectroscopic studies were paralleled by high‐level quantum chemical calculations. In solution, 8‐BrRF showed a remarkably high triplet quantum yield (0.97, parent compound riboflavin, RF: 0.6) and a small fluorescence quantum yield (0.07, RF: 0.27). For 8‐CF₃RF, the triplet yield was 0.12, and the fluorescence quantum yield was 0.7. The high triplet yield of 8‐BrRF is due to the bromine heavy atom effect causing a stronger spin–orbit coupling. Theoretical calculations reveal that the decreased triplet yield of 8‐CF₃RF is due to a smaller charge transfer and a less favorable energetic position of T₂, required for intersystem crossing from S₁ to T₁, as an effect of the electron‐withdrawing CF₃ group. The reconstitution of the LOV domain with the new flavins resulted in the typical LOV photochemistry, consisting of triplet state formation and covalent binding of the chromophore, followed by a thermal recovery of the parent state, albeit with different kinetics and photophysical properties.