Nanometre-scale thermometry in a living cell.

Sensitive probing of temperature variations on nanometre scales is an outstanding challenge in many areas of modern science and technology. In particular, a thermometer capable of subdegree temperature resolution over a large range of temperatures as well as integration within a living system could provide a powerful new tool in many areas of biological, physical and chemical research. Possibilities range from the temperature-induced control of gene expression and tumour metabolism to the cell-selective treatment of disease and the study of heat dissipation in integrated circuits. By combining local light-induced heat sources with sensitive nanoscale thermometry, it may also be possible to engineer biological processes at the subcellular level. Here we demonstrate a new approach to nanoscale thermometry that uses coherent manipulation of the electronic spin associated with nitrogen-vacancy colour centres in diamond. Our technique makes it possible to detect temperature variations as small as 1.8 mK (a sensitivity of 9 mK Hz(-1/2)) in an ultrapure bulk diamond sample. Using nitrogen-vacancy centres in diamond nanocrystals (nanodiamonds), we directly measure the local thermal environment on length scales as short as 200 nanometres. Finally, by introducing both nanodiamonds and gold nanoparticles into a single human embryonic fibroblast, we demonstrate temperature-gradient control and mapping at the subcellular level, enabling unique potential applications in life sciences.

Cyanine fluorophores for cellular protection against ROS in stimulated macrophages and two-photon ROS detection.

We report the first example of a novel two-photon active, biocompatible, and macrophage cell-membrane permeable carbazole-based cyanine fluorophore for the detection of three biologically important ROS, namely, ˙OH, O2(-) and OCl(-) in solution. This versatile probe shows cellular protection not only in stimulated macrophages from phorbol-12-myristate-13-acetate-induced morphological changes but also lipopolysaccharide-induced cytotoxicity by quenching with the O2(-) and OCl(-) production, respectively. Such protection could be visualized by a distinct change in the fluorescence intensity of the probe.

All-optical sensing of a single-molecule electron spin.

We demonstrate an all-optical method for magnetic sensing of individual molecules in ambient conditions at room temperature. Our approach is based on shallow nitrogen-vacancy (NV) centers near the surface of a diamond crystal, which we use to detect single paramagnetic molecules covalently attached to the diamond surface. The manipulation and readout of the NV centers is all-optical and provides a sensitive probe of the magnetic field fluctuations stemming from the dynamics of the electronic spins of the attached molecules. As a specific example, we demonstrate detection of a single paramagnetic molecule containing a gadolinium (Gd(3+)) ion. We confirm single-molecule resolution using optical fluorescence and atomic force microscopy to colocalize one NV center and one Gd(3+)-containing molecule. Possible applications include nanoscale and in vivo magnetic spectroscopy and imaging of individual molecules.

An iron-catalysed C-C bond-forming spirocyclization cascade providing sustainable access to new 3D heterocyclic frameworks.

Heterocyclic architectures offer powerful creative possibilities to a range of chemistry end-users. This is particularly true of heterocycles containing a high proportion of sp3-carbon atoms, which confer precise spatial definition upon chemical probes, drug substances, chiral monomers and the like. Nonetheless, simple catalytic routes to new heterocyclic cores are infrequently reported, and methods making use of biomass-accessible starting materials are also rare. Here, we demonstrate a new method allowing rapid entry to spirocyclic bis-heterocycles, in which inexpensive iron(III) catalysts mediate a highly stereoselective C-C bond-forming cyclization cascade reaction using (2-halo)aryl ethers and amines constructed using feedstock chemicals readily available from plant sources. Fe(acac)3 mediates the deiodinative cyclization of (2-halo)aryloxy furfuranyl ethers, followed by capture of the intermediate metal species by Grignard reagents, to deliver spirocycles containing two asymmetric centres. The reactions offer potential entry to key structural motifs present in bioactive natural products.

Identification of a novel mouse p53 target gene DDA3.

We have identified a novel p53 regulated gene designated DDA3 through differential mRNA display on IW32 erythroleukemia cells containing a temperature sensitive p53 allele, tsp53val-135. DDA3 mRNA induction could be observed in all sublines expressing tsp53val-135 cultured at permissive temperature as well as in NIH3T3 cells undergoing DNA damage. Upregulation of DDA3 could be detected within 2 h after down-shifting the temperature to 32.5 degrees C; upon shifting back to 38.5 degrees C, DDA3 mRNA rapidly degraded with a half-life of less than 2 h. Actinomycin D, but not cycloheximide, inhibited the p53 dependent DDA3 induction, suggesting that the activation is through transcriptional regulation and does not require de novo protein synthesis. DDA3 was expressed in multiple mouse tissues including brain, spleen, lung, kidney and testis. Full-length DDA3 cDNA was cloned and it contained an open reading frame predicted to encode a proline rich protein of 329 amino acids. Overexpression of DDA3 in H1299 lung carcinoma cells suppressed colony formation. These results suggest that DDA3 is a p53-regulated gene that might participate in the p53-mediated growth suppression.

Pharmacokinetic studies of ivermectin: effects of formulation.

Studies are reported which describe the effects of formulation, animal species, and route of administration on the pharmacokinetics of ivermectin. Biological half-life t1/2 increases in the order: swine (0.5 day) less than dogs (1.8 day) less than cattle approximately equal to sheep (2.8 day). Formulation modifications, based upon the solubility properties of the drug, have been directed towards the development of a nonaqueous injectable formulation for cattle and an aqueous vehicle for horses. Bioavailability following subcutaneous injection in cattle can be regulated by control of injection solvent composition: a vehicle composed of a mixed aqueous-organic solvent exhibits pharmacokinetic properties (i.e., Cp, t1/2, AUC, and F) intermediate between those furnished by an aqueous formulation and via a purely nonaqueous solvent. The longer apparent biological half-life from this latter vehicle (t1/2 = 8.3 days) confirms that a slow absorption process dominates the pharmacokinetics in the nonaqueous injectable product to produce an effective controlled-release formulation. These bioavailability results illustrate the increase in the concentration of an organic solvent and a concomitant decrease in surfactant concentration in a micellar aqueous system for prolonged drug delivery via injection.

CD49f and CD61 identify Her2/neu-induced mammary tumor-initiating cells that are potentially derived from luminal progenitors and maintained by the integrin-TGFß signaling.

Human epidermal growth factor receptor 2 (HER2)/Neu is overexpressed in 20-30% of breast cancers and associated with aggressive phenotypes and poor prognosis. For deciphering the role of HER2/Neu in breast cancer, mouse mammary tumor virus (MMTV)-Her2/neu transgenic mice that develop mammary tumors resembling human HER2-subtype breast cancer have been established. Several recent studies have revealed that HER2/Neu is overexpressed in and regulates self renewal of breast tumor-initiating cells (TICs). However, in the MMTV-Her2/neu transgenic mouse model, the identity of TICs remains elusive, despite previous studies showing supportive evidence for existence of TICs in Her2/neu-induced mammary tumors. Through systematic screening and characterization, we identified that surface markers CD49f, CD61 and ESA were aberrantly overexpressed in Her2-overexpressing mammary tumor cells. Analysis of these markers and CD24 detected anomalous expansion of the luminal progenitor population in preneoplastic mammary glands of Her2/neu transgenic mice, indicating that aberrant luminal progenitors originated in Her2-induced mammary tumors. The combined markers, CD49f and CD61, further delineated the CD49f(high)CD61(high)-sorted fraction as a TIC-enriched population, which displayed increased tumorsphere formation ability, enhanced tumorigenicity both in vitro and in vivo and drug resistance to pacitaxel and doxorubicin. Moreover, the TIC-enriched population manifested increased transforming growth factor-ß (TGFß) signaling and exhibited gene expression signatures of stemness, TGFß signaling and epithelial-to-mesenchymal transition. Our findings that self-renewal and clonogenicity of TICs were suppressed by pharmacologically inhibiting the TGFß signaling further indicate that the TGFß pathway is vital for maintenance of the TIC population. Finally, we showed that the integrin-ß3 (CD61) signaling pathway was required for sustaining active TGFß signaling and self-renewal of TICs. We for the first time developed a technique to highly enrich TICs from mammary tumors of Her2/neu transgenic mice, unraveled their properties and identified the cooperative integrin-ß3-TGFß signaling axis as a potential therapeutic target for HER2-induced TICs.

Correlative light and electron microscopy using cathodoluminescence from nanoparticles with distinguishable colours.

Correlative light and electron microscopy promises to combine molecular specificity with nanoscale imaging resolution. However, there are substantial technical challenges including reliable co-registration of optical and electron images, and rapid optical signal degradation under electron beam irradiation. Here, we introduce a new approach to solve these problems: imaging of stable optical cathodoluminescence emitted in a scanning electron microscope by nanoparticles with controllable surface chemistry. We demonstrate well-correlated cathodoluminescence and secondary electron images using three species of semiconductor nanoparticles that contain defects providing stable, spectrally-distinguishable cathodoluminescence. We also demonstrate reliable surface functionalization of the particles. The results pave the way for the use of such nanoparticles for targeted labeling of surfaces to provide nanoscale mapping of molecular composition, indicated by cathodoluminescence colour, simultaneously acquired with structural electron images in a single instrument.

Identification of a mouse thiamine transporter gene as a direct transcriptional target for p53.

p53 tumor suppressor is a transcription factor that functions, in part, through many of its downstream target genes. We have identified a p53-inducible gene by performing mRNA differential display on IW32 murine erythroleukemia cells containing a temperature-sensitive p53 mutant allele, tsp53(Val-135). Sequence analysis of the full-length cDNA revealed its identity as the mouse homologue of the human thiamine transporter 1 (THTR-1). Induction of the mouse THTR-1 (mTHTR-1) mRNA was detectable as early as 1 h at 32.5 degrees C; upon shifting back to 38.5 degrees C, mTHTR-1 transcript was rapidly degraded with a half-life of less than 2 h. Elevation of mTHTR-1 expression was found in DNA damage-induced normal mouse embryonic fibroblast cells, but not in p53(-/-) mouse embryonic fibroblast cells, suggesting that mTHTR-1 induction was p53-dependent. A region within the first intron of the mTHTR-1 gene bound to p53 and conferred the p53-mediated transactivation. Furthermore, increased thiamine transporter activities were found in cells overexpressing mTHTR-1 and under conditions of DNA damage or p53 activation. Our findings indicate that p53 may be involved in maintaining thiamine homeostasis through transactivation of THTR-1.