Accurate identification of breast cancer margins in microenvironments of ex-vivo basal and luminal breast cancer tissues using Raman spectroscopy.

Better knowledge of the breast tumor microenvironment is required for surgical resection and understanding the processes of tumor development. Raman spectroscopy is a promising tool that can assist in uncovering the molecular basis of disease and provide quantifiable molecular information for diagnosis and treatment evaluation. In this work, eighty-eight frozen breast tissue sections, including forty-four normal and forty-four tumor sections, were mapped in their entirety using a 250-µm-square measurement grid. Two or more smaller regions of interest within each tissue were additionally mapped using a 25 µm-square step size. A deep learning algorithm, convolutional neural network (CNN), was developed to distinguish histopathologic features with-in individual and across multiple tissue sections. Cancerous breast tissue were discriminated from normal breast tissue with 90 % accuracy, 88.8 % sensitivity and 90.8 % specificity with an excellent Area Under the Receiver Operator Curve (AUROC) of 0.96. Features that contributed significantly to the model were identified and used to generate RGB images of the tissue sections. For each grid point (pixel) on a Raman map, color was assigned to intensities at frequencies of 1002 cm-1 (Phenylalanine), 869 cm-1 (Proline, CC stretching of hydroxyproline-collagen assignment, single bond stretching vibrations for the amino acids proline, valine and polysaccharides) and 1309 cm-1 (CH3/CH2 twisting or bending mode of lipids). The Raman images clearly associate with hematoxylin and eosin stained tissue sections and allow clear visualization of boundaries between normal adipose, connective tissue and tumor. We demonstrated that this simple imaging technique allows high-resolution, straightforward molecular interpretation of Raman images. Raman spectroscopy provides rapid, label-free imaging of microscopic features with high accuracy. This method has application as laboratory tool and can assist with intraoperative tissue assessment during Breast Conserving surgery.

Applications of Raman spectroscopy in cancer diagnosis.

Novel approaches toward understanding the evolution of disease can lead to the discovery of biomarkers that will enable better management of disease progression and improve prognostic evaluation. Raman spectroscopy is a promising investigative and diagnostic tool that can assist in uncovering the molecular basis of disease and provide objective, quantifiable molecular information for diagnosis and treatment evaluation. This technique probes molecular vibrations/rotations associated with chemical bonds in a sample to obtain information on molecular structure, composition, and intermolecular interactions. Raman scattering occurs when light interacts with a molecular vibration/rotation and a change in polarizability takes place during molecular motion. This results in light being scattered at an optical frequency shifted (up or down) from the incident light. By monitoring the intensity profile of the inelastically scattered light as a function of frequency, the unique spectroscopic fingerprint of a tissue sample is obtained. Since each sample has a unique composition, the spectroscopic profile arising from Raman-active functional groups of nucleic acids, proteins, lipids, and carbohydrates allows for the evaluation, characterization, and discrimination of tissue type. This review provides an overview of the theory of Raman spectroscopy, instrumentation used for measurement, and variation of Raman spectroscopic techniques for clinical applications in cancer, including detection of brain, ovarian, breast, prostate, and pancreatic cancers and circulating tumor cells.

Ceramide changes in abdominal subcutaneous and visceral adipose tissue among diabetic and nondiabetic patients.

BACKGROUND: This study profiles ceramides extracted from visceral and subcutaneous adipose tissue of human subjects by liquid chromatography-mass spectrometry to determine a correlation with status of diabetes and gender. METHODS: Samples of visceral and abdominal wall subcutaneous adipose tissue (n = 36 and n = 31, respectively) were taken during laparoscopic surgery from 36 patients (14 nondiabetic, 22 diabetic and prediabetic) undergoing bariatric surgery with a body mass index (BMI) >35 kg/m2 with ≥1 existing comorbidity or BMI ≥40 kg/m2 . Sphingolipids were extracted and analyzed using liquid chromatography-mass spectrometry. RESULTS: After logarithm 2 conversion, paired analysis of visceral to subcutaneous tissue showed differential accumulation of Cer(d18:1/16:0), Cer(d18:1/18:0), and Cer(d18:1/24:1) in visceral tissue of prediabetic/diabetic female subjects, but not in males. Within-tissue analysis showed higher mean levels of ceramide species linked to insulin resistance, such as Cer(d18:1/18:0) and Cer(d18:1/16:0), in visceral tissue of prediabetic/diabetic patients compared with nondiabetic subjects and higher content of Cer(d18:1/14:0) in subcutaneous tissue of insulin-resistant female patients compared with prediabetic/diabetic males. Statistically significant differences in mean levels of ceramide species between insulin-resistant African American and insulin-resistant Caucasian patients were not evident in visceral or subcutaneous tissue. CONCLUSIONS: Analysis of ceramides is important for developing a better understanding of biological processes underlying type 2 diabetes, metabolic syndrome, and obesity. Knowledge of the accumulated ceramides/dihydroceramides may reflect on the prelipolytic state that leads the lipotoxic phase of insulin resistance and may shed light on the predisposition to insulin resistance by gender.

roX RNAs and Genome Regulation in Drosophila Melanogaster.

Organisms with dimorphic sex chromosomes suffer a potentially lethal imbalance in gene expression in one sex. Addressing this fundamental problem can be considered the first, and most essential, aspect of sexual differentiation. In the model organisms Drosophila, Caenorhabditis elegans, and mouse, expression from X-linked genes is modulated by selective recruitment of chromatin-modifying complexes to X chromatin. In both flies and mammals, large noncoding RNAs have a central role in recruitment and activity of these complexes. This review will summarize current knowledge of the function of the noncoding roX genes in this process in Drosophila. Identification of an autosomal function for the roX RNAs raises intriguing questions about the origin of the modern dosage compensation system in flies.

Coordinated regulation of heterochromatic genes in Drosophila melanogaster males.

Dosage compensation modifies the chromatin of X-linked genes to assure equivalent expression in sexes with unequal X chromosome dosage. In Drosophila dosage compensation is achieved by increasing expression from the male X chromosome. The ribonucleoprotein dosage compensation complex (DCC) binds hundreds of sites along the X chromosome and modifies chromatin to facilitate transcription. Loss of roX RNA, an essential component of the DCC, reduces expression from X-linked genes. Surprisingly, loss of roX RNA also reduces expression from genes situated in proximal heterochromatin and on the small, heterochromatic fourth chromosome. Mutation of some, but not all, of the genes encoding DCC proteins produces a similar effect. Reduction of roX function suppresses position effect variegation (PEV), revealing functional alteration in heterochromatin. The effects of roX mutations on heterochromatic gene expression and PEV are limited to males. A sex-limited role for the roX RNAs in autosomal gene expression was unexpected. We propose that this reflects a difference in the heterochromatin of males and females, which serves to accommodate the heterochromatic Y chromosome present in the male nucleus. roX transcripts may thus participate in two distinct regulatory systems that have evolved in response to highly differentiated sex chromosomes: compensation of X-linked gene dosage and modulation of heterochromatin.

Modulation of Heterochromatin by Male Specific Lethal Proteins and roX RNA in Drosophila melanogaster Males.

The ribonucleoprotein Male Specific Lethal (MSL) complex is required for X chromosome dosage compensation in Drosophila melanogaster males. Beginning at 3 h of development the MSL complex binds transcribed X-linked genes and modifies chromatin. A subset of MSL complex proteins, including MSL1 and MSL3, is also necessary for full expression of autosomal heterochromatic genes in males, but not females. Loss of the non-coding roX RNAs, essential components of the MSL complex, lowers the expression of heterochromatic genes and suppresses position effect variegation (PEV) only in males, revealing a sex-limited disruption of heterochromatin. To explore the molecular basis of this observation we examined additional proteins that participate in compensation and found that MLE, but not Jil-1 kinase, contributes to heterochromatic gene expression. To determine if identical regions of roX RNA are required for dosage compensation and heterochromatic silencing, we tested a panel of roX1 transgenes and deletions and find that the X chromosome and heterochromatin functions are separable by some mutations. Chromatin immunoprecipitation of staged embryos revealed widespread autosomal binding of MSL3 before and after localization of the MSL complex to the X chromosome at 3 h AEL. Autosomal MSL3 binding was dependent on MSL1, supporting the idea that a subset of MSL proteins associates with chromatin throughout the genome during early development. The broad localization of these proteins early in embryogenesis supports the idea of direct action at autosomal sites. We postulate that this may contribute to the sex-specific differences in heterochromatin that we, and others, have noted.