Raman spectroscopy differentiates squamous cell carcinoma (SCC) from normal skin following treatment with a high-powered CO2 laser.

BACKGROUND AND OBJECTIVES: The number of cases of non-melanoma skin cancer (NMSC), which include squamous cell carcinoma (SCC) and basal cell carcinoma (BCC), continues to rise as the aging population grows. Mohs micrographic surgery has become the treatment of choice in many cases but is not always necessary or feasible. Ablation with a high-powered CO2 laser offers the advantage of highly precise, hemostatic tissue removal. However, confirmation of complete cancer removal following ablation is difficult. In this study we tested for the first time the feasibility of using Raman spectroscopy as an in situ diagnostic method to differentiate NMSC from normal tissue following partial ablation with a high-powered CO2 laser. MATERIALS AND METHODS: Twenty-five tissue samples were obtained from eleven patients undergoing Mohs micrographic surgery to remove NMSC tumors. Laser treatment was performed with a SmartXide DOT Fractional CO2 Laser (DEKA Laser Technologies, Inc.) emitting a wavelength of 10.6 µm. Treatment levels ranged from 20 mJ to 1200 mJ total energy delivered per laser treatment spot (350 µm spot size). Raman spectra were collected from both untreated and CO2 laser-treated samples using a 785 nm diode laser. Principal Component Analysis (PCA) and Binary Logistic Regression (LR) were used to classify spectra as originating from either normal or NMSC tissue, and from treated or untreated tissue. RESULTS: Partial laser ablation did not adversely affect the ability of Raman spectroscopy to differentiate normal from cancerous residual tissue, with the spectral classification model correctly identifying SCC tissue with 95% sensitivity and 100% specificity following partial laser ablation, compared with 92% sensitivity and 60% selectivity for untreated NMSC tissue. The main biochemical difference identified between normal and NMSC tissue was high levels of collagen in the normal tissue, which was lacking in the NMSC tissue. CONCLUSION: The feasibility of a combined high-powered CO2 laser ablation, Raman diagnostic procedure for the treatment of NMSC is demonstrated since CO2 laser treatment does not hinder the ability of Raman spectroscopy to differentiate normal from diseased tissue. This combined approach could be employed clinically to greatly enhance the speed and effectiveness of NMSC treatment in many cases.

Determination of penetratin secondary structure in live cells with Raman microscopy.

Cell penetrating peptides (CPPs) have attracted recent interest as drug delivery tools, although the mechanisms by which CPPs are internalized by cells are not well-defined. Here, we report a new experimental approach for the detection and secondary structure determination of CPPs in live cells using Raman microscopy with heavy isotope labeling of the peptide. As a first demonstration of principle, penetratin, a 16-residue CPP derived from the Antennapedia homeodomain protein of Drosophila, was measured in single, living melanoma cells. Carbon-13 labeling of the Phe residue of penetratin was used to shift the intense aromatic ring-breathing vibrational mode from 1003 to 967 cm(-1), thereby enabling the peptide to be traced in cells. Difference spectroscopy and principal components analysis (PCA) were used independently to resolve the Raman spectrum of the peptide from the background cellular Raman signals. On the basis of the position of the amide I vibrational band in the Raman spectra, the secondary structure of the peptide was found to be mainly random coil and beta-strand in the cytoplasm, and possibly assembling as beta-sheets in the nucleus. The rapid entry and almost uniform cellular distribution of the peptide, as well as the lack of correlation between peptide and lipid Raman signatures, indicated that the mechanism of internalization under the conditions of study was probably nonendocytotic. This experimental approach can be used to study a wide variety of CPPs as well as other classes of peptides in living cells.

Whole genome sequencing of mutation accumulation lines reveals a low mutation rate in the social amoeba Dictyostelium discoideum.

Spontaneous mutations play a central role in evolution. Despite their importance, mutation rates are some of the most elusive parameters to measure in evolutionary biology. The combination of mutation accumulation (MA) experiments and whole-genome sequencing now makes it possible to estimate mutation rates by directly observing new mutations at the molecular level across the whole genome. We performed an MA experiment with the social amoeba Dictyostelium discoideum and sequenced the genomes of three randomly chosen lines using high-throughput sequencing to estimate the spontaneous mutation rate in this model organism. The mitochondrial mutation rate of 6.76×10(-9), with a Poisson confidence interval of 4.1×10(-9) - 9.5×10(-9), per nucleotide per generation is slightly lower than estimates for other taxa. The mutation rate estimate for the nuclear DNA of 2.9×10(-11), with a Poisson confidence interval ranging from 7.4×10(-13) to 1.6×10(-10), is the lowest reported for any eukaryote. These results are consistent with low microsatellite mutation rates previously observed in D. discoideum and low levels of genetic variation observed in wild D. discoideum populations. In addition, D. discoideum has been shown to be quite resistant to DNA damage, which suggests an efficient DNA-repair mechanism that could be an adaptation to life in soil and frequent exposure to intracellular and extracellular mutagenic compounds. The social aspect of the life cycle of D. discoideum and a large portion of the genome under relaxed selection during vegetative growth could also select for a low mutation rate. This hypothesis is supported by a significantly lower mutation rate per cell division in multicellular eukaryotes compared with unicellular eukaryotes.

High relatedness is necessary and sufficient to maintain multicellularity in Dictyostelium.

Most complex multicellular organisms develop clonally from a single cell. This should limit conflicts between cell lineages that could threaten the extensive cooperation of cells within multicellular bodies. Cellular composition can be manipulated in the social amoeba Dictyostelium discoideum, which allows us to test and confirm the two key predictions of this theory. Experimental evolution at low relatedness favored cheating mutants that could destroy multicellular development. However, under high relatedness, the forces of mutation and within-individual selection are too small for these destructive cheaters to spread, as shown by a mutation accumulation experiment. Thus, we conclude that the single-cell bottleneck is a powerful stabilizer of cellular cooperation in multicellular organisms.