Bacteria use a process of chemical communication called quorum sensing to assess their population density and to change their behavior in response to fluctuations in the cell number and species composition of the community. In this work, we identified the quorum-sensing-regulated proteome in the model organism Vibrio harveyi by bio-orthogonal non-canonical amino acid tagging (BONCAT). BONCAT enables measurement of proteome dynamics with temporal resolution on the order of minutes. We deployed BONCAT to characterize the time-dependent transition of V. harveyi from individual- to group-behaviors. We identified 176 quorum-sensing-regulated proteins at early, intermediate, and late stages of the transition, and we mapped the temporal changes in quorum-sensing proteins controlled by both transcriptional and post-transcriptional mechanisms. Analysis of the identified proteins revealed 86 known and 90 new quorum-sensing-regulated proteins with diverse functions, including transcription factors, chemotaxis proteins, transport proteins, and proteins involved in iron homeostasis.

Hydrogenases are complex metalloenzymes that catalyze the reversible splitting of molecular hydrogen into protons and electrons essentially without overpotential. The NAD+-reducing soluble hydrogenase (SH) from Ralstonia eutropha is capable of H2 conversion even in the presence of usually toxic dioxygen. The molecular details of the underlying reactions are largely unknown, mainly because of limited knowledge of the structure and function the various metal cofactors present in the enzyme. Here all iron-containing cofactors of the SH were investigated by 57Fe specific nuclear resonance vibrational spectroscopy (NRVS). Our data provide experimental evidence for one [2Fe2S] center and four [4Fe4S] clusters, which is consistent with amino acid sequence composition. Only the [2Fe2S] cluster and one of the four [4Fe4S] clusters were reduced upon incubation of the SH with NADH. This finding explains the discrepancy between the large number of FeS clusters and the small amount of FeS cluster-related signals as detected by electron paramagnetic resonance spectroscopic analysis of several NAD+-reducing hydrogenases. For the first time, Fe-CO and Fe-CN modes derived from the [NiFe] active site could be distinguished by NRVS through selective 13C labeling of the CO ligand. This strategy also revealed the molecular coordinates that dominate the individual Fe-CO modes. The present approach explores the complex vibrational signature of the Fe-S clusters and the hydrogenase active site, thereby showing that NRVS represents a powerful tool for the elucidation of complex biocatalysts containing multiple cofactors.

Propranolol is shown to undergo lipidation reactions in three types of lipid membrane: (1) synthetic single-component glycerophospholipid liposomes; (2) liposomes formed from complex lipid mixtures extracted from E. coli or liver cells; and (3) in cellulo in Hep G2 cells. Fourteen different lipidated propranolol homologues were identified in extracts from Hep G2 cells cultured in a medium supplemented with propranolol. This isolation of lipidated drug molecules from liver cells demonstrates a new drug reactivity in living systems. Acyl transfer from lipids to the alcoholic group of propranolol was favoured over transfer to the secondary amine. Migration of acyl groups from the alcohol to the amine was diminished. Other drugs that were examined did not form detectable levels of lipidation products, but many of these drugs did affect the lysolipid levels in model membranes. The propensity for a compound to induce lysolipid formation in a model system was found to be a predictor for phospholipidosis activity in cellulo.

Although a number of advances have been made in RNA sequencing and structural characterization, the lack of a method for directly determining the sequence and structure of single RNA molecules has limited our ability to probe heterogeneity in gene expression at the level of single cells. Here we present a method for direct nucleotide identification and structural label mapping of single RNA molecules via Quantum Molecular Sequencing (QMSeq). The method combines non-perturbative quantum tunneling spectroscopy to probe the molecular orbitals of ribonucleotides, new experimental biophysical parameters that fingerprint these molecular orbitals, and a machine learning classification algorithm to distinguish between the ribonucleotides. The algorithm uses tunneling spectroscopy measurements on an unknown ribonucleotide to determine its chemical identity and the presence of local chemical modifications. Combining this with structure-dependent chemical labeling presents the possibility of mapping both the sequence and local structure of individual RNA molecules. By optimizing the base-calling algorithm, we show a high accuracy for both ribonucleotide discrimination (>99.8%) and chemical label identification (>98%) with a relatively modest molecular coverage (35 repeat measurements). This lays the groundwork for simultaneous sequencing and structural mapping of single unknown RNA molecules, and paves the way for probing the sequence-structure-function relationship within the transcriptome at an unprecedented level of detail.

Lysosomal pHs are maintained at low values by the cooperative action of a proton pump and a chloride channel to maintain electroneutrality. Owing to the biological significance of lysosomal chloride ions, measurements of their levels are of great importance to understand lysosome-associated biological events. However, appropriate probes to selectively detect Cl- ions within acidic lysosomes have not been developed to date. In this study, we prepared MQAE-MP, a lysosomal Cl--selective fluorescent probe, and applied it to gain information about biological processes associated with lysosomes. The fluorescence of MQAE-MP is pH-insensitive over physiological pH ranges and is quenched by Cl- with a Stern-Volmer constant of 204 M-1. Because MQAE-MP detects lysosomal Cl- selectively, it was employed to assess the effects of eleven substances on lysosomal Cl- concentrations. The results show that lysosomal Cl- concentrations decrease in cells treated with substances that inhibit proteins responsible for lysosomal membrane stabilization, induce lysosomal membrane permeabilization, and transport lysosomal Cl- to the cytosol. In addition, we investigated the effect of lysosomal chloride ions on the fusion of autophagosomes with lysosomes to generate autolysosomes during autophagy inhibition promoted by substances. It was found that changes in lysosomal Cl- concentrations did not affect the fusion of autophagosomes with lysosomes but an increase in the cytosolic Ca2+ concentration blocked the fusion process. We demonstrate from the current study that MQAE-MP has great potential as a lysosomal Cl--selective fluorescent probe for studies of biological events associated with lysosomes.

In an effort to better define the nature of the nuclear coordinate associated with excited state dynamics in first-row transition metal-based chromophores, variable-temperature ultrafast time-resolved absorption spectroscopy has been used to determine activation parameters associated with ground state recovery dynamics in a series of low-spin Fe(ii) polypyridyl complexes. Our results establish that high-spin (5T2) to low-spin (1A1) conversion in complexes of the form [Fe(4,4'-di-R-2,2'-bpy')3]2+ (R = H, CH3, or tert-butyl) is characterized by a small but nevertheless non-zero barrier in the range of 300-350 cm-1 in fluid CH3CN solution, a value that more than doubles to ∼750 cm-1 for [Fe(terpy)2]2+ (terpy = 2,2':6',2''-terpyridine). The data were analyzed in the context of semi-classical Marcus theory. Changes in the ratio of the electronic coupling to reorganization energy (specifically, H ab 4/λ) reveal an approximately two-fold difference between the [Fe(bpy')3]2+ complexes (∼1/30) and [Fe(terpy)2]2+ (∼1/14), suggesting a change in the nature of the nuclear coordinate associated with ground state recovery between these two types of complexes. These experimentally-determined ratios, along with estimates for the 5T2/1A1 energy gap, yield electronic coupling values between these two states for the [Fe(bpy')3]2+ series and [Fe(terpy)2]2+ of 4.3 ± 0.3 cm-1 and 6 ± 1 cm-1, respectively, values that are qualitatively consistent with the second-order nature of high-spin/low-spin coupling in a d6 ion. In addition to providing useful quantitative information on these prototypical Fe(ii) complexes, these results underscore the utility of variable-temperature spectroscopic measurements for characterizing ultrafast excited state dynamics in this class of compounds.

Identifying a target ligand binding site is an important step for structure-based rational drug design as shown here for G protein-coupled receptors (GPCRs), which are among the most popular drug targets. We applied long-time scale molecular dynamics simulations, coupled with mutagenesis studies, to two prototypical GPCRs, the M3 and M4 muscarinic acetylcholine receptors. Our results indicate that unlike synthetic antagonists, which bind to the classic orthosteric site, the endogenous agonist acetylcholine is able to diffuse into a much deeper binding pocket. We also discovered that the most recently resolved crystal structure of the LTB4 receptor comprised a bound inverse agonist, which extended its benzamidine moiety to the same binding pocket discovered in this work. Analysis on all resolved GPCR crystal structures indicated that this new pocket could exist in most receptors. Our findings provide new opportunities for GPCR drug discovery.

Luminogens with aggregation-induced emission (AIEgens) characteristics have been well developed and applied in various areas such as bio-imaging, theranostics, organic photoelectronics and chemo/bio sensors. However, most of the reported AIEgens suffer from the disadvantages of complex organic synthesis and high cost, as well as being environmentally unfriendly and hard to degrade, which have largely limited their real applications. In this work, we discovered berberine chloride, a natural isoquinoline alkaloid isolated from Chinese herbal plants, as an unconventional rotor-free AIEgen with bright solid-state emission and water-soluble characteristics. Single crystal structure analysis and optical property, viscosity, and host-guest interaction studies suggested that intramolecular vibration and twisted intramolecular charge transfer were responsible for the AIE phenomenon of berberine chloride. Moreover, berberine chloride was biocompatible and could specifically target lipid droplets in a fluorescence turn-on and wash-free manner, demonstrating the great potential of natural products as promising AIE probes.

A family of Al-rich ZSM-5 zeolites with Si/Al = 8 ± 0.5 was prepared according to a biomass-mediated supramolecular approach. A combination of advanced characterisation techniques and periodic density functional theory (DFT) calculations unraveled the purity and stability of un-expected Al-enriched ZSM-5 structures, hence allowing to cross the frontier of Si/Al lower limit. In addition, these Al-rich ZSM-5 zeolites demonstrated high catalytic activity in n-hexane cracking and methanol conversion into hydrocarbons, being in line with the presence of numerous Brønsted acid sites.

Development of tumor microenvironment responsive and modulating theranostic nano-systems is of great importance for specific and efficient cancer therapy. Herein, we report a redox-sensitive nanoagent combining manganese dioxide (MnO2) and gold nanoshell coated silicon nanoparticles for synergistic chemo-photothermal therapy of hypoxia solid tumors. In highly reducing tumor tissues, the outer MnO2 nanosheet with the loaded drug would be dissociated by intracellular glutathione (GSH), resulting in on-demand drug release, as well as generating Mn2+ ions which provided high contrast magnetic resonance imaging (MRI), and fluorescence imaging (FI) in vitro and in vivo. While upon near-infrared (NIR) light irradiation, the gold nanoshell modulated the hypoxic tumor microenvironment via increasing blood flow, achieving enhanced photothermal therapy (PTT) and chemotherapy. After tail vein injection into tumor-bearing mice and monitoring in real time, the intelligent redox-activated nanoagent exhibited high tumor accumulation and powerful synergistic chemo-photothermal therapy efficiency. The proposed work developed a noninvasive strategy to modulate the tumor microenvironment and enhance the anticancer therapeutic effect. We believe that this single nano-platform exhibits promising potential as a comprehensive theranostic agent to enhance the efficacies of synergistic cancer therapy.

Drop-casting and inkjet printing are virtually the most versatile and cost-effective methods for depositing active materials on surfaces. However, drawbacks associated with the coffee-ring effect, as well as uncontrolled aggregation of the coating materials, have impeded the use of these methods for applications requiring high control of film properties. We now report on a simple method based on covalent cross-linking of monodisperse materials that enables the formation of thin films with homogeneous thicknesses and macroscale cohesion. The coffee-ring effect is impeded by triggering gelation of the coating materials via a thioacetate-disulfide transition which counterbalances the capillary forces induced by evaporation. Aggregates are prevented by monodisperse building blocks that ensure that the resulting gel resists sedimentation until complete droplet drying. This combined strategy yields an unprecedented level of homogeneity in the resulting film thickness in the 100 nm to 10 μm range. Moreover, macroscale cohesion is preserved as evidenced by the long-range charge transfer within the matrix. We highlight the impact of this method with bioelectrocatalysts for H2 and NADPH oxidation. Peak catalytic performances are reached at about 10-fold lower catalyst loading compared to conventional approaches owing to the high control on film cohesion and thickness homogeneity, thus setting new benchmarks in catalyst utilization.

Protein-protein interactions (PPIs) play pivotal roles in the majority of biological processes. Therefore, improved approaches to target and disrupt PPIs would provide tools for chemical biology and leads for therapeutic development. PPIs with α-helical components are appealing targets given that the secondary structure is well understood and can be mimicked or stabilised to render small-molecule and constrained-peptide-based inhibitors. Here we present a strategy to target α-helix-mediated PPIs that exploits de novo coiled-coil assemblies and test this using the MCL-1/NOXA-B PPI. First, computational alanine scanning is used to identify key α-helical residues from NOXA-B that contribute to the interface. Next, these residues are grafted onto the exposed surfaces of de novo designed homodimeric or heterodimeric coiled-coil peptides. The resulting synthetic peptides selectively inhibit a cognate MCL-1/BID complex in the mid-nM range. Furthermore, the heterodimeric system affords control as inhibition occurs only when both the grafted peptide and its designed partner are present. This establishes proof of concept for exploiting peptides stabilised in de novo coiled coils as inhibitors of PPIs. This dependence on supramolecular assembly introduces new possibilities for regulation and control.

Nanoscale engineering of surfaces is becoming an indispensable technique to modify membranes and, thus cellular behaviour. Here, such membrane engineering related was explored on the surface of a living animal using DNA nanotechnology. We demonstrate the immobilization of oligonucleotides functionalized with a membrane anchor on 2 day old zebrafish. The protruding single-stranded DNA on the skin of zebrafish served as a handle for complementary DNAs, which allowed the attachment of small molecule cargo, liposomes and dynamic relabeling by DNA hybridization protocols. Robust anchoring of the oligonucleotides was proven as DNA-based amplification processes were successfully performed on the outer membrane of the zebrafish enabling the multiplication of surface functionalities from a single DNA-anchoring unit and the dramatic improvement of fluorescent labeling of these animals. As zebrafish are becoming an alternative to animal models in drug development, toxicology and nanoparticles characterization, we believe the platform presented here allows amalgamation of DNA nanotechnology tools with live animals and this opens up yet unexplored avenues like efficient bio-barcoding as well as in vivo tracking.

Phosphorus doped tin(iv) oxide (P:SnO2) films have been synthesised by an aerosol assisted chemical vapour deposition route. Triethyl phosphate was used as the phosphorus dopant source. The phosphorus concentration in solution was found to be key to electrical properties, with concentrations between 0.25-0.5 mol% phosphorus giving the lowest resistivities of the deposited films. The conductivity of the films synthesised improved on doping SnO2 with phosphorus, with resistivity values of 7.27 × 10-4 Ω cm and sheet resistance values of 18.2 Ω □-1 achieved for the most conductive films. Phosphorus doping up to 1.0 mol% was shown to improve visible light transmission of the deposited films. The phosphorus doping also had a significant effect on film morphology, with varying microstructures achieved. The films were characterised by X-ray diffraction, scanning electron microscopy, UV/vis spectroscopy, Hall effect measurements and X-ray photoelectron spectroscopy. The data generated was used to build computational models of phosphorus as a dopant for SnO2, showing that the phosphorus acts as a shallow one-electron n-type donor allowing for good conductivities. Phosphorus does not suffer from self-compensation issues associated with other dopants, such as fluorine.

The ionic assembly of oppositely charged polyelectrolyte-surfactant complexes (PESCs) is often done with the aim of constructing more functional colloids, for instance as advanced delivery systems. However, PESCs are often not easily loaded with a solubilisate due to intrinsic restrictions of such complexes. This question was addressed from a different starting point: by employing microemulsion droplets as heavily loaded surfactant systems and thereby avoiding potential solubilisation limitations from the beginning. We investigated mixtures of cationic oil-in-water (O/W) microemulsion droplets and oppositely charged sodium polyacrylate (NaPA) and determined structure and phase behaviour as a function of the mixing ratio for different droplet sizes and different M w (NaPA). Around an equimolar charge ratio an extended precipitate region is present, which becomes wider for larger droplets and with increasing M w of the NaPA. Static and dynamic light scattering (SLS and DLS) and small-angle neutron scattering (SANS) show the formation of one-dimensional arrangements of microemulsion droplets for polyelectrolyte excess, which become more elongated with increasing M w (NaPA) and less so with increasing NaPA excess. What is interesting is a marked sensitivity to ionic strength, where already a modest increase to ∼20 mM leads to a dissolution of the complexes. This work shows that polyelectrolyte/microemulsion complexes (PEMECs) are structurally very versatile hybrid systems, combining the high solubilisate loading of microemulsions with the larger-scale structuring induced by the polymer, thereby markedly extending the concept of conventional PESCs. This type of system has not been described before and is highly promising for future applications where high payloads are to be formulated.

Mature microRNAs (miRNAs) produced from precursor microRNAs (pre-miRNAs) by the RNase Dicer have showed significant potential for cancer diagnosis and prognosis due to their key regulatory roles in various pathological processes. However, discriminatory detection of low-abundance miRNAs and pre-miRNAs remains a key challenge since the mature sequence is also present in the pre-miRNA forms. Herein, we report a novel cascade reaction to sensitively distinguish miRNAs versus pre-miRNAs in living cells based on two pairs of programmable hairpin oligonucleotide probes with a simple sequence design. The programmable hairpin probes can metastably coexist until the introduction of miRNAs or pre-miRNAs, which can trigger a specific hybridization chain reaction (HCR), respectively, leading to the self-assembly of nicked DNA duplex structures and a remarkable specific fluorescence intensity increase. The system can readily and sensitively assess the miRNA or pre-miRNA abundance in a homogeneous solution. The intracellular miRNA and pre-miRNA expression level assessment in different living cells is realized. Thus, we provide a novel investigation tool for discriminatorily and accurately assessing miRNA and pre-miRNA abundance, which could be useful for the biomedical application of miRNAs.

Here, we present a highly specific, sensitive and cost-effective system to quantify microRNA (miRNA) expression based on two-step RT-qPCR with EvaGreen detection chemistry, called linear-hairpin variable primer RT-qPCR. It takes advantage of the novel designed variable primer, which is initially designed to be linear, extending to form a hairpin structure and replacing the target miRNA for cyclic RT. Then the RT product is quantified by conventional EvaGreen based qPCR. The results show that this method has a dynamic range of 8 logs and the sensitivity is sufficient to directly detect down to 4 target miRNA molecules with a total analysis time of less than 2 hours. It is capable of discriminating between similar miRNAs, leading to an accurate representation of the mature miRNA content in a sample. The RT step can be multiplexed and the 8 miRNA profiles measured in 7 mouse tissues by this method show an excellent correlation with the commercial standard TaqMan RT-qPCR assays (r 2 = 0.9881).

Conjugation with poly(ethylene glycol) (PEG) or PEGylation is a widely used tool to overcome the shortcomings of native proteins, such as poor stability, inadequate pharmacokinetic (PK) profiles, and immunogenicity. However, PEGylation is often accompanied by an unwanted detrimental effect on bioactivity, particularly, resulting from the amphiphilic nature of PEG. This is especially true for PEGylated proteins with large binding targets. Pegasys, a PEGylated interferon alpha-2a (IFN-α2a) bearing a 40 kDa branched PEG, is a typical example that displays only 7% in vitro activity of the unmodified IFN-α2a. In this work, by employing IFN-α2a as a model protein, we demonstrated that a protein conjugated with zwitterionic polymers (or zwitterlation) could significantly mitigate the antiproliferative bioactivity loss in vitro after polymer conjugation. The retained antiproliferative activity of zwitterlated IFN-α2a is 4.4-fold higher than that of the PEGylated IFN-α2a with the same polymer molecular weight, or 3-fold higher than that of the PEGylated IFN-α2a with a similar hydrodynamic size. It is hypothesized that nonspecific interactions between zwitterionic polymers and IFN-α2a/IFN-α2a receptors can be mitigated due to the super-hydrophilic nature of zwitterionic polymers. This, in turn, reduces the 'nonspecific blocking' between IFN-α2a and IFN-α2a receptors. In addition, we demonstrated that zwitterlated IFN-α2a showed a prolonged circulation time and a mitigated accelerated blood clearance after repeated injections in rats.

Target engagement is a key concept in drug discovery and its direct measurement can provide a quantitative understanding of drug efficacy and/or toxicity. Failure to demonstrate target occupancy in relevant cells and tissues has been recognised as a contributing factor to the low success rate of clinical drug development. Several techniques are emerging to quantify target engagement in cells; however, in situ measurements remain challenging, mainly due to technical limitations. Here, we report the development of a non-covalent clickable probe, based on SCH772984, a slow off-rate ERK1/2 inhibitor, which enabled efficient pull down of ERK1/2 protein via click reaction with tetrazine tagged agarose beads. This was used in a competition setting to measure relative target occupancy by selected ERK1/2 inhibitors. As a reference we used the cellular thermal shift assay, a label-free biophysical assay relying solely on ligand-induced thermodynamic stabilization of proteins. To validate the EC50 values measured by both methods, the results were compared with IC50 data for the phosphorylation of RSK, a downstream substrate of ERK1/2 used as a functional biomarker of ERK1/2 inhibition. We showed that a slow off-rate reversible probe can be used to efficiently pull down cellular proteins, significantly extending the potential of the approach beyond the need for covalent or photoaffinity warheads.

The peroxidase activity of cytochrome c (cyt c) plays a key role during apoptosis. Peroxidase catalysis requires a vacant Fe coordination site, i.e., cyt c must undergo an activation process involving structural changes that rupture the native Met80-Fe contact. A common strategy for dissociating this bond is the conversion of Met80 to sulfoxide (MetO). It is widely believed that this MetO formation in itself is sufficient for cyt c activation. This notion originates from studies on chloramine-T-treated cyt c (CT-cyt c) which represents a standard model for the peroxidase activated state. CT-cyt c is considered to be a "clean" species that has undergone selective MetO formation, without any other modifications. Using optical, chromatographic, and mass spectrometry techniques, the current work demonstrates that CT-induced activation of cyt c is more complicated than previously thought. MetO formation alone results in only marginal peroxidase activity, because dissociation of the Met80-Fe bond triggers alternative ligation scenarios where Lys residues interfere with access to the heme. We found that CT causes not only MetO formation, but also carbonylation of several Lys residues. Carbonylation is associated with -1 Da mass shifts that have gone undetected in the CT-cyt c literature. Proteoforms possessing both MetO and Lys carbonylation exhibit almost fourfold higher peroxidase activity than those with MetO alone. Carbonylation abrogates the capability of Lys to coordinate the heme, thereby freeing up the distal site as required for an active peroxidase. Previous studies on CT-cyt c may have inadvertently examined carbonylated proteoforms, potentially misattributing effects of carbonylation to solely MetO formation.