Nitrogen-vacancy colour centres in diamond can undergo strong, spin-sensitive optical transitions under ambient conditions, which makes them attractive for applications in quantum optics 1 , nanoscale magnetometry 2,3 and biolabelling 4 . Although nitrogen-vacancy centres have been observed in aggregated detonation nanodiamonds 5 and milled nanodiamonds 6 , they have not been observed in very small isolated nanodiamonds 7 . Here, we report the first direct observation of nitrogen-vacancy centres in discrete 5-nm nanodiamonds at room temperature, including evidence for intermittency in the luminescence (blinking) from the nanodiamonds. We also show that it is possible to control this blinking by modifying the surface of the nanodiamonds.Detonation nanodiamond is routinely produced on an industrial scale, and the raw material can be disintegrated into a stable 5-nm monodisperse colloid 8 . The combination of inert core and chemically reactive surface, which can host a variety of moieties, is appealing for chemists, biologists and material scientists 9,10 . Quantum magnetometry 2,3 is an example of an emerging technology that will directly benefit from the availability of nanocrystals with welldefined sizes in the 5-nm range, because the sensitivity to single spins is inversely proportional to the cube of the distance between the sensor (that is, the nitrogen-vacancy (NV) centre) and the spin being detected.Producing and detecting NV colour centres in isolated 5-nm detonation nanodiamond has been controversial, and there has been some scepticism regarding their stability as a useful emitter in a discrete crystal. For example, theoretical calculations of the crystal energy budget favour the location of nitrogen on the surface rather than in the core, which seems to explain the limited observation of NV centres in chemical vapour deposition and high-pressure high-temperature grains of less than 40 nm in size 11,12 , and favours the prediction that nanodiamonds smaller than 10 nm in size do not contain NV centres 7,13 . Although sub-10-nm nanodiamonds with NV centres have been produced using a top-down approach (milling luminescent high-pressure hightemperature microdiamonds into 7-nm particles 6,14 ), the question of NV stability in isolated detonation nanodiamonds persists.In aggregated detonation nanodiamonds (agglomerates and agglutinates 8 ), high-sensitivity, time-gated luminescence and electronic paramagnetic resonance spectroscopy have been used to extract a weak NV signal from a strong luminescence background 5 . The experiments highlight the eclipsing nature of the graphitic surface layers in nanodiamond aggregates-NV centres were simply not visible through the broadband luminescence from the surface and grain boundary material. To distinguish the NV spectral signature from the large grain boundary luminescence overhead, diamond synthesis yielding discrete sub-10-nm detonation nanodiamonds is vital. Here, we use a robust deaggregation and dispersion method, which diminishes the crystal-crystal interaction to...

Most proteins adopt a well defined three-dimensional structure; however, it is increasingly recognized that some proteins can exist with at least two stable conformations. Recently, a class of intracellular chloride ion channel proteins (CLICs) has been shown to exist in both soluble and integral membrane forms. The structure of the soluble form of CLIC1 is typical of a soluble glutathione S-transferase superfamily protein but contains a glutaredoxin-like active site. In this study we show that on oxidation CLIC1 undergoes a reversible transition from a monomeric to a non-covalent dimeric state due to the formation of an intramolecular disulfide bond (Cys-24 -Cys-59). We have determined the crystal structure of this oxidized state and show that a major structural transition has occurred, exposing a large hydrophobic surface, which forms the dimer interface. The oxidized CLIC1 dimer maintains its ability to form chloride ion channels in artificial bilayers and vesicles, whereas a reducing environment prevents the formation of ion channels by CLIC1. Mutational studies show that both Cys-24 and Cys-59 are required for channel activity.Chloride ion channels control a variety of cellular processes that are central to normal function and disease states (1). The CLIC 1 family is a recently identified class of Cl Ϫ channel proteins that consists of seven members (p64, parchorin, CLIC1-5) (2, 3). A conserved C-terminal CLIC module of ϳ240 amino acids is present in each member of the family with several members containing additional, unrelated Nterminal domains. Most CLICs are localized to intracellular membranes and have been linked to functions including apoptosis, pH, and cell cycle regulation (4 -6). The CLIC ion channels are unusual in that they possess both soluble and integral membrane forms (2). In this regard they are similar to some bacterial toxins and several classes of intracellular proteins including Bcl-x L and the annexins (7). Our understanding of how such dual natured proteins enter the membrane is limited by the dearth of high resolution structures for key states in this process.We have recently determined the crystal structure of a soluble monomeric form of CLIC1 (8) and found that it is a structural homologue of the GST superfamily of proteins (9). This soluble form of CLIC1 consists of two domains, the N-domain possessing a thioredoxin fold closely resembling glutaredoxin and an all ␣-helical C-domain, which is typical of the GST superfamily. CLIC1 contains an intact glutathione-binding site that was shown to covalently bind glutathione via a conserved CLIC cysteine residue, Cys-24. This led to the suggestion that CLIC1 function may be under redox control, possibly via reactive oxygen or nitrogen species.The structure and stoichiometry of the integral membrane form of the CLIC proteins is still unclear. Electrophysiology of purified, soluble (Escherichia coli-expressed) recombinant CLIC1 in reconstituted artificial bilayers shows that CLIC1 alone is sufficient for chloride ion channel formation (8, 1...

Inflammatory monocyte-derived effector cells play an important role in the pathogenesis of numerous inflammatory diseases. However, no treatment option exists that is capable of modulating these cells specifically. We show that infused negatively charged, immune-modifying microparticles (IMPs), derived from polystyrene, microdiamonds, or biodegradable poly(lactic-co-glycolic) acid, were taken up by inflammatory monocytes, in an opsonin-independent fashion, via the macrophage receptor with collagenous structure (MARCO). Subsequently, these monocytes no longer trafficked to sites of inflammation; rather, IMP infusion caused their sequestration in the spleen through apoptotic cell clearance mechanisms and, ultimately, caspase-3–mediated apoptosis. Administration of IMPs in mouse models of myocardial infarction, experimental autoimmune encephalomyelitis, dextran sodium sulfate–induced colitis, thioglycollate-induced peritonitis, and lethal flavivirus encephalitis markedly reduced monocyte accumulation at inflammatory foci, reduced disease symptoms, and promoted tissue repair. Together, these data highlight the intricate interplay between scavenger receptors, the spleen, and inflammatory monocyte function and support the translation of IMPs for therapeutic use in diseases caused or potentiated by inflammatory monocytes.

Background In the last stage of the C. elegans equivalent of epiboly, an open pocket in the epidermis is closed by marginal epidermal P/pocket cells that express and require VAB-1/Eph and PLX-2/plexin receptors for migration toward and alignment with contralateral partners at the ventral midline. Cellular mechanisms affected by these signaling proteins remain unknown. Results A cellular bridge comprising four neuron cell bodies that spans the open pocket serves as a substratum for migration of contra-lateral P cell pair P9/10 to the midline which can facilitate similar migration of neighboring P cells. This bridge is formed by a stereotypical rearrangement of five sister pairs of PLX-2 and VAB-1 expressing cells, of which three pairs serve as a scaffold for bridge assembly and two pairs form the bridge. Bridge formation requires VAB-1 kinase-dependent extension of presumptive bridge cell protrusions toward the ventral midline. An unassembled mutant bridge obstructs but does not block P cell progression toward the midline, however, cell type-specific rescue experiments show that VAB-1 or a nearly complete cytoplasmic deletion of VAB-1 expressed by scaffold and bridge cells or by P9/10 can facilitate P cell progression to the midline. MAB-20/semaphorin and VAB-1 also exhibit complex redundancies to regulate adhesion and prevent gaps between sister bridge and scaffold forming cells that would otherwise completely block P cell migration. Conclusions The Eph receptor functions to mediate cell extensions required for bridge formation, independently facilitates P cell migration to the ventral midline, and acts redundantly with PLX-2/plexin to prevent gaps between sister plexin band cells that normally serve as a substratum for P9/10 cell migration.