During surface-enhanced Raman scattering (SERS), molecules exhibit a significant increase in their Raman signals when attached, or in very close vicinity, to gold or silver nanostructures. This effect is exploited as the basis of a new class of optical labels. Here we demonstrate robust and sensitive SERS labels as probes for imaging live cells. These hybrid labels consist of gold nanoparticles with Rose Bengal or Crystal Violet attached as reporter molecules. These new labels are stable and nontoxic, do not suffer from photobleaching, and can be excited at any excitation wavelength, even in the near infrared. SERS labels can be detected and imaged through the specific Raman signatures of the reporters. In addition, surface-enhanced Raman spectroscopy in the local optical fields of the gold nanoparticles also provides sensitive information on the immediate molecular environment of the label in the cell and allows imaging of the native constituents of the cell. This is demonstrated by images based on a characteristic Raman line of the reporter as well as by displaying lipids based on the SERS signal of the C-H deformation/bending modes at ∼1470 cm −1 .

Keloids are reactive or spontaneous fibroproliferative dermal tumors characterized by the exaggerated and uncontrolled accumulation of extracellular collagen. Current approaches to mitigate keloidogenesis are largely procedural in nature. However, a better understanding of its biological drivers may lead to novel targeted treatments for keloids. Through whole-genome expression analysis, we found that an HIF-1a transcriptional footprint is preferentially upregulated (activation score ¼ 2.024; P ¼ 1.05EÀ19) in keloid fibroblasts compared with normal dermal fibroblasts. We verified that HIF-1a protein is more strongly expressed in keloid specimens compared with normal skin (P ¼ 0.035) and that hypoxia (1% O 2 ) leads to increased collagen, especially in the extracellular compartment. Collagen levels were reduced uniformly by selective HIF-1a inhibitor CAY10585.Our results indicate that collagen secretion may be intimately linked to a hypoxic microenvironment within keloid tumors and that HIF-1a blockade could be a novel avenue of treatment for these tumors.

Two growth inhibitory hurdles that must be overcome by the evolving cancer cell include pathways regulated by RB and p53. In human melanoma cells, inactivation of a single locus, CDKN2A, can lead to abrogation of both RB and p53 functionality through loss of the two CDKN2A cognate transcripts-p16 and p14ARF, respectively. We thus set out to assess how recurrent patterns of genetic injury at three critical human melanoma loci-CDKN2A, TP53, and CDK4-cooperate to disrupt both RB and p53 pathways. Overall, 77.8% of the melanoma cell lines analyzed showed genetic evidence of dual RB and p53 pathway compromise; this percentage is even higher if protein expression loss is considered. Although homozygous deletion of all three critical CDKN2A exons (exons 1 beta, 1 alpha, and 2) represent the most common mechanism, concurrent loss of CDKN2A(Exon1 alpha) and CDKN2A(Exon1 beta) and simultaneous point mutagenesis of CDK4 and TP53 reflect alternative cassettes of dual inactivation. In cell lines with isolated CDKN2A(Exon2) mutations, coincident alterations in TP53 or deletion of CDKN2A(Exon1 beta) suggest that p16 transcript may be preferentially targeted over the p14ARF transcript as additional p53 pathway lesions are recruited. Moreover, predictive modeling of CDKN2A(Exon2) missense mutations further suggests that the amino acid substitutions in this region negatively impact p16 to a greater extent than p14ARF. Taken together, our data point to a clear need in human melanoma cell lines, as in its murine counterpart, to disrupt both RB and p53 pathways and recurrent mechanisms may play into the unique genetic vulnerabilities of this tumor type.

Long wavelength solar UVA radiation stimulates formation of reactive oxygen species (ROS) and prostaglandin E 2 (PGE 2 ), which are involved in skin photosensitivity and tumor promotion. High levels of 7-dehydrocholesterol (7-DHC), the precursor to cholesterol, cause exaggerated photosensitivity to UVA in patients with Smith-Lemli-Opitz syndrome (SLOS). Partially replacing cholesterol with 7-DHC in keratinocytes rapidly (<5 min) increased UVA-induced ROS, intracellular calcium, phospholipase A 2 activity, PGE 2 , and NADPH oxidase activity. UVA-induced ROS and PGE 2 production were inhibited in these cells by depleting the Nox1 subunit of NADPH oxidase using siRNA or using a mitochondrial radical quencher, MitoQ. Partial replacement of cholesterol with 7-DHC also disrupted membrane lipid raft domains, although depletion of cholesterol, which also disrupts lipid rafts, did not affect UVA-induced increases in ROS and PGE 2 . Phospholipid liposomes containing 7-DHC were more rapidly oxidized by a free radical mechanism than those containing cholesterol. These results indicate that 7-DHC enhances rapid UVA-induced ROS and PGE 2 formation by enhancing free radical-mediated membrane lipid oxidation and suggests that this mechanism might underlie the UVA-photosensitivity in SLOS.