Make It Shine: Labelling the ER for Light and Fluorescence Microscopy.

The ER is a highly dynamic network of tubules and membrane cisternae. Hence, imaging this organelle in its native and mobile state is of great importance. Here we describe methods of labelling the native plant ER using fluorescent proteins and lipid dyes as well as methods for immunolabelling on plant tissue.

Juggling Optoelectronics and Catalysis: The Dual Talents of Bench Stable 1,4-Azaborinines.

Boron- and nitrogen-doped polycyclic aromatic hydrocarbons (B-PAHs) have established a strong foothold in the realm of organic electronics. However, their catalytic potential remains largely untapped. In this study, we synthesise and characterise two bench stable B,N-doped PAH derivatives based on a 1,4-azaborinine motif. Most importantly, the anthracene derived structure is an efficient catalyst in the reduction of various carbonyls and imines. These results underscore the potential of B,N-PAHs in catalytic transformations, setting the stage for deeper exploration in this chemical space.

Synthesis of C6-modified mannose 1-phosphates and evaluation of derived sugar nucleotides against GDP-mannose dehydrogenase.

Sufferers of cystic fibrosis are at significant risk of contracting chronic bacterial lung infections. The dominant pathogen in these cases is mucoid Pseudomonas aeruginosa. Such infections are characterised by overproduction of the exopolysaccharide alginate. We present herein the design and chemoenzymatic synthesis of sugar nucleotide tools to probe a critical enzyme within alginate biosynthesis, GDP-mannose dehydrogenase (GMD). We first synthesise C6-modified glycosyl 1-phosphates, incorporating 6-amino, 6-chloro and 6-sulfhydryl groups, followed by their evaluation as substrates for enzymatic pyrophosphorylative coupling. The development of this methodology enables access to GDP 6-chloro-6-deoxy-ᴅ-mannose and its evaluation against GMD.

A Reversibly Porous Supramolecular Peptide Framework.

The ability to use bio-inspired building blocks in the assembly of novel supramolecular frameworks is at the forefront of an exciting research field. Herein, we present the first polyproline helix to self-assemble into a reversibly porous, crystalline, supramolecular peptide framework (SPF). This framework is assembled from a short oligoproline, adopting the polyproline II conformation, driven by hydrogen-bonding and dispersion interactions. Thermal activation, guest-induced dynamic porosity and enantioselective guest inclusion have been demonstrated for this novel system. The principles of the self-assembly associated with this SPF will be used as a blueprint allowing for the further development of helical peptide linkers in the rational design of SPFs and metal-peptide frameworks.

Chemical synthesis of 4'-thio and 4'-sulfinyl pyrimidine nucleoside analogues.

Analogues of the canonical nucleosides required for nucleic acid synthesis have a longstanding presence and proven capability within antiviral and anticancer research. 4'-Thionucleosides, that incorporate bioisosteric replacement of furanose oxygen with sulfur, represent an important chemotype within this field. Established herein is synthetic capability towards a common 4-thioribose building block that enables access to thio-ribo and thio-arabino pyrimidine nucleosides, alongside their 4'-sulfinyl derivatives. In addition, this building block methodology is templated to deliver 4'-thio and 4'-sulfinyl analogues of the established anticancer drug gemcitabine. Cytotoxic capability of these new analogues is evaluated against human pancreatic cancer and human primary glioblastoma cell lines, with observed activities ranging from low µM to >200 µM; explanation for this reduced activity, compared to established nucleoside analogues, is yet unclear. Access to these chemotypes, with thiohemiaminal linkages, will enable a wider exploration of purine and triphosphate analogues and the application of such materials for potential resistance towards relevant hydrolytic enzymes within nucleic acid biochemistries.

Ligand isomerism fine-tunes structure and stability in zinc complexes of fused pyrazolopyridines.

Fused-ring pyrazoles offer a versatile platform for derivitization to give finely tuned and functional ligands in coordination assemblies. Here, we explore the pyrazolo[4,3-b]pyridine (HL1) and pyrazolo[3,4-c]pyridine (HL2) backbones and their N-substituted derivatives, using their coordination chemistry with zinc(II) in the solid state and in solution to examine the steric and electronic effects of varying their substitution pattern. The parent heterocycles HL1 and HL2 both generate robust and permanently porous isomeric MOFs on reaction with zinc and a dicarboxylate co-ligand. The subtle geometric change offered by the position of the backbone pyridyl nitrogen atom leads to substantial changes in the pore size and total pore volume, which is reflected in both their surface areas and CO2 uptake performance. Both materials are also unusually resilient to atmospheric water vapour by virtue of the strong metal-azolate bonding. The isomeric chelating ligands L3-L6, generated by N-arylation of the parent heterocycles with a 2-pyridyl group, each coordinate to zinc to give either mononuclear or polymeric coordination compounds depending on the involvement of the backbone pyridine nitrogen atom. While crystal packing influences based on the steric preferences of the ligands are dominant in the crystalline phase, fluorescence spectroscopy is used to show that the 2H isomers L4 and L6 show distinct coordination behaviour to the 1H isomers L3 and L5, forming competing [ML] and [ML2] species in soution. The first stability constant for L6 with zinc(II) is an order of magnitude larger than for the other three ligands, suggesting an improved binding strength based on the electron configuration in this isomer. These results show that careful control of remote substitution on fused pyrazole ligands can lead to substantial improvements in the stability of the resulting complexes, with consequences for the design of stable coordination assemblies containining labile metal ions.

Squaramide-Based Self-Associating Amphiphiles for Anion Recognition.

The synthesis and characterisation of two novel self-assembled amphiphiles (SSAs) SQS-1 and SQS-2 are reported. Both compounds, based on the squaramide motif, were fully soluble in a range of solvents and were shown to undergo self-assembly through a range of physical techniques. Self-assembly was shown to favour the formation of crystalline domains on the nanoscale but also fibrillar film formation, as suggested by SEM analysis. Moreover, both SQS-1 and SQS-2 were capable of anion recognition in DMSO solution as demonstrated using 1 H NMR and UV/Vis absorption spectroscopy, but displayed lower binding affinities for various anions when compared against other squaramide based receptors. In more competitive solvent mixtures SQS-1 gave rise to a colourimetric response in the presence of HPO42- that was clearly visible to the naked eye. We anticipate that the observed response is due to the basic nature of the HPO42- anion when compared against other biologically relevant anions.

Coordination sphere hydrogen bonding as a structural element in metal-organic Frameworks.

In the design of new metal-organic frameworks, the constant challenges of framework stability and structural predictability continue to influence ligand choice in favour of well-studied dicarboxylates and similar ligands. However, a small subset of known MOF ligands contains suitable functionality for coordination sphere hydrogen bonding which can provide new opportunities in ligand design. Such interactions may serve to support and rigidity the coordination geometry of mononuclear coordination spheres, as well as providing extra thermodynamic and kinetic stabilisation to meet the challenge of hydrolytic stability in these materials. In this perspective, a collection of pyrazole, amine, amide and carboxylic acid containing species are examined through the lens of (primarily) inner-sphere hydrogen bonding. The influence of these interactions is then related to the overall structure, stability and function of these materials, to provide starting points for harnessing these interactions in future materials design.

Functional characterization of Schistosoma mansoni fucosyltransferases in Nicotiana benthamiana plants.

Helminth parasites secrete a wide variety of immunomodulatory proteins and lipids to dampen host immune responses. Many of these immunomodulatory compounds are modified with complex sugar structures (or glycans), which play an important role at the host-parasite interface. As an example, the human blood fluke Schistosoma mansoni produces highly fucosylated glycan structures on glycoproteins and glycolipids. Up to 20 different S. mansoni fucosyltransferase (SmFucT) genes can be found in genome databases, but thus far only one enzyme has been functionally characterized. To unravel the synthesis of highly fucosylated N-glycans by S. mansoni, we examined the ability of ten selected SmFucTs to modify N-glycans upon transient expression in Nicotiana benthamiana plants. All enzymes were localized in the plant Golgi apparatus, which allowed us to identify the SmFucTs involved in core fucosylation and the synthesis of complex antennary glycan motifs. This knowledge provides a starting point for investigations into the role of specific fucosylated glycan motifs of schistosomes in parasite-host interactions. The functionally characterized SmFucTs can also be applied to synthesize complex N-glycan structures on recombinant proteins to study their contribution to immunomodulation. Furthermore, this plant expression system will fuel the development of helminth glycoproteins for pharmaceutical applications or novel anti-helminth vaccines.

p24 Family Proteins Are Involved in Transport to the Plasma Membrane of GPI-Anchored Proteins in Plants.

p24 proteins are a family of type-I membrane proteins that cycle between the endoplasmic reticulum (ER) and the Golgi apparatus via Coat Protein I (COPI)- and COPII-coated vesicles. These proteins have been proposed to function as cargo receptors, but the identity of putative cargos in plants is still elusive. We previously generated an Arabidopsis (Arabidopsis thaliana) quadruple loss-of-function mutant affecting p24 genes from the δ-1 subclass of the p24 delta subfamily (p24δ3δ4δ5δ6 mutant). This mutant also had reduced protein levels of other p24 family proteins and was found to be sensitive to salt stress. Here, we used this mutant to test the possible involvement of p24 proteins in the transport to the plasma membrane of glycosylphosphatidylinositol (GPI)-anchored proteins. We found that GPI-anchored proteins mostly localized to the ER in p24δ3δ4δ5δ6 mutant cells, in contrast to plasma membrane proteins with other types of membrane attachment. The plasma membrane localization of GPI-anchored proteins was restored in the p24δ3δ4δ5δ6 mutant upon transient expression of a single member of the p24 δ-1 subclass, RFP-p24δ5, which was dependent on the coiled-coil domain in p24δ5. The coiled-coil domain was also important for a direct interaction between p24δ5 and the GPI-anchored protein arabinogalactan protein4 (AGP4). These results suggest that Arabidopsis p24 proteins are involved in ER export and transport to the plasma membrane of GPI-anchored proteins.

A Series of Manganese(III) Salen Complexes as a Result of Team-Based Inquiry in a Transnational Education Programme.

The development of a team-based approach to research-led transnational practical chemistry teaching is described in which a team of five Chinese students on an articulated transnational degree programme, supported by a team of academic and technical staff, carried out a study examining the structural chemistry of a series of manganese(III) salen complexes. A series of four crystallographically characterized manganese(III) salen complexes with ancillary carboxylate ligands are reported here. The carboxylate coordination modes range from the bridging syn-anti µ2 -κO : κO' mode observed in the predominant cyclohexanoate and isobutyrate species, to a capping terminal monodentate mode for the adamantanoate species, and an unusual mixture of bridging and terminal coordination modes observed in a second minor phase of the cyclohexanoate species. The variation on extended structures based on the weakly interacting aliphatic backbones may provide a useful basis for further structural studies.

Differences in intracellular localisation of ANKH mutants that relate to mechanisms of calcium pyrophosphate deposition disease and craniometaphyseal dysplasia.

ANKH mutations are associated with calcium pyrophosphate deposition disease and craniometaphyseal dysplasia. This study investigated the effects of these ANKH mutants on cellular localisation and associated biochemistry. We generated four ANKH overexpression-plasmids containing either calcium pyrophosphate deposition disease or craniometaphyseal dysplasia linked mutations: P5L, E490del and S375del, G389R. They were transfected into CH-8 articular chondrocytes and HEK293 cells. The ANKH mutants dynamic differential localisations were imaged and we investigated the interactions with the autophagy marker LC3. Extracellular inorganic pyrophosphate, mineralization, ENPP1 activity expression of ENPP1, TNAP and PIT-1 were measured. P5L delayed cell membrane localisation but once recruited into the membrane it increased extracellular inorganic pyrophosphate, mineralization, and ENPP1 activity. E490del remained mostly cytoplasmic, forming punctate co-localisations with LC3, increased mineralization, ENPP1 and ENPP1 activity with an initial but unsustained increase in TNAP and PIT-1. S375del trended to decrease extracellular inorganic pyrophosphate, increase mineralization. G389R delayed cell membrane localisation, trended to decrease extracellular inorganic pyrophosphate, increased mineralization and co-localised with LC3. Our results demonstrate a link between pathological localisation of ANKH mutants with different degrees in mineralization. Furthermore, mutant ANKH functions are related to synthesis of defective proteins, inorganic pyrophosphate transport, ENPP1 activity and expression of ENPP1, TNAP and PIT-1.

Cyclic Aliphatic Hydrocarbons as Linkers in Metal-Organic Frameworks: New Frontiers for Ligand Design.

In this Minireview we outline the development of cyclic aliphatic moieties as ligands in metal-organic frameworks (MOFs), with a focus on the relationship between ligand design and synthesis and the properties of the subsequent materials. Aliphatic ligands have received considerably less attention than aromatic analogues in MOF chemistry but offer advantages in their unique combinations of geometric and electronic properties which are unattainable from conventional ligands. Here, we focus on rigid and semi-rigid backbone moieties derived from monocyclic and fused polycyclic aliphatic backbones, including cyclohexane and adamantane, cubane and bicyclo[2.2.2]octane, and discuss the synthetic chemistry of these species along with their potential importance as the next generation of building blocks for microporous materials.

Balancing connectivity with function in silver(i) networks of pyridyltriazole (tzpa) ligands results in the formation of a metallogel.

A new flexible and divergent 1,2,3-triazol-4-yl-picolinamide (tzpa) ligand 2 and the half-equivalent model ligand 1, both functionalised with pendant 3-pyridyl groups, are reported and their coordination behaviour with silver(i) ions is explored, both in the crystalline phase and through the formation of a supramolecular metallogel. The self-assembly of tzpa ligand 1 with AgCF3SO3 resulted in the formation of a 1D coordination polymer, binding in a bidentate fashion through the pyridyl and triazole nitrogen atoms of the tzpa binding site and a pendant pyridyl nitrogen atom of an adjacent ligand. Doubling the number of metal binding sites in ligand 2, while retaining the same metal binding domain, gives rise to the formation of a supramolecular metallogel on reaction with AgBF4 at 5 wt% in MeCN, possessing self-healing properties.

Fluorescent supramolecular hierarchical self-assemblies from glycosylated 4-amino- and 4-bromo-1,8-naphthalimides.

An investigation into the self-assembly of two 4-amino- and a 4-bromo-1,8-naphthalimide (Nap) based structures (1-3) possessing an appended glycan unit, from protic polar media, is presented. The results demonstrate the formation of complex hierarchical luminescent aggregates, wherein the morphologies, sizes and spherical structures were highly dependent on both the media and the Nap structure. Upon cleaving the native glycosidic bond, using an enzyme, the structure/morphology of the self-assembly of 3 in buffered solution was significantly transformed.

A signal motif retains Arabidopsis ER-α-mannosidase I in the cis-Golgi and prevents enhanced glycoprotein ERAD.

The Arabidopsis ER-α-mannosidase I (MNS3) generates an oligomannosidic N-glycan structure that is characteristically found on ER-resident glycoproteins. The enzyme itself has so far not been detected in the ER. Here, we provide evidence that in plants MNS3 exclusively resides in the Golgi apparatus at steady-state. Notably, MNS3 remains on dispersed punctate structures when subjected to different approaches that commonly result in the relocation of Golgi enzymes to the ER. Responsible for this rare behavior is an amino acid signal motif (LPYS) within the cytoplasmic tail of MNS3 that acts as a specific Golgi retention signal. This retention is a means to spatially separate MNS3 from ER-localized mannose trimming steps that generate the glycan signal required for flagging terminally misfolded glycoproteins for ERAD. The physiological importance of the very specific MNS3 localization is demonstrated here by means of a structurally impaired variant of the brassinosteroid receptor BRASSINOSTEROID INSENSITIVE 1.

Unexpected linkage isomerism in chiral tetranuclear bis-tridentate (1,2,3-triazol-4-yl)-picolinamide (tzpa) grids.

The synthesis of a chiral bis-tridentate (1,2,3-triazol-4-yl)-picolinamide (tzpa) ligand is described and its coordination chemistry with Cu(NO3)2 and [Cu(MeCN)4]PF6 is explored in the crystalline phase as well as in solution. Chiral [2 × 2] tetranuclear square grid complexes [Cu4(H21)4](NO3)8 and [Cu4(H1)4](PF6)4 were observed, and crystallographically analysed, these being linkage isomers with N4O2 and N5O coordination spheres, respectively. These come about by an unusual in situ amide deprotonation and coordination, which accompanies a CuI → CuII oxidation process.

Plants Neither Possess nor Require Consciousness.

In claiming that plants have consciousness, 'plant neurobiologists' have consistently glossed over the remarkable degree of structural and functional complexity that the brain had to evolve for consciousness to emerge. Here, we outline a new hypothesis proposed by Feinberg and Mallat for the evolution of consciousness in animals. Based on a survey of the brain anatomy, functional complexity, and behaviors of a broad spectrum of animals, criteria were established for the emergence of consciousness. The only animals that satisfied these criteria were the vertebrates (including fish), arthropods (e.g., insects, crabs), and cephalopods (e.g., octopuses, squids). In light of Feinberg and Mallat's analysis, we consider the likelihood that plants, with their relative organizational simplicity and lack of neurons and brains, have consciousness to be effectively nil.

Direct imaging of the recruitment and phosphorylation of S6K1 in the mTORC1 pathway in living cells.

Knowledge of protein signalling pathways in the working cell is seen as a primary route to identifying and developing targeted medicines. In recent years there has been a growing awareness of the importance of the mTOR pathway, making it an attractive target for therapeutic intervention in several diseases. Within this pathway we have focused on S6 kinase 1 (S6K1), the downstream phosphorylation substrate of mTORC1, and specifically identify its juxtaposition with mTORC1. When S6K1 is co-expressed with raptor we show that S6K1 is translocated from the nucleus to the cytoplasm. By developing a novel biosensor we demonstrate in real-time, that phosphorylation and de-phosphorylation of S6K1 occurs mainly in the cytoplasm of living cells. Furthermore, we show that the scaffold protein raptor, that typically recruits mTOR substrates, is not always involved in S6K1 phosphorylation. Overall, we demonstrate how FRET-FLIM imaging technology can be used to show localisation of S6K1 phosphorylation in living cells and hence a key site of action of inhibitors targeting mTOR phosphorylation.