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.

Conjugated estrogen plus medroxyprogesterone does not impair blood rheological properties in hypertensive postmenopausal women.

OBJECTIVES: Hypertension and estrogens are both prothrombotic. We used the microchannel method to investigate whether hormone replacement therapy (HRT) with conjugated equine estrogen (CEE) plus medroxyprogesterone acetate (MPA) affects blood flow through the microchannels in hypertensive postmenopausal women being treated with antihypertensive drugs and in normotensive postmenopausal women. METHODS: Sixty-two consecutive postmenopausal women were randomly assigned to a hypertensive HRT group (n=16), hypertensive control group (n=15), normotensive HRT group (n=16) and normotensive control group (n=15). Each HRT group received CEE 0.625 mg plus MPA 2.5 mg daily orally for 12 months. Both hypertensive groups were being treated with antihypertensive drugs before the study. Microvascular blood flow was assessed on the basis of blood passage time, the time required for 100 microl of whole blood to pass through a cylinder, was determined before and 12 months after the start of HRT by the microchannel method (micro channel array flow analyzer). RESULTS: CEE plus MPA therapy did not change blood passage time in any of the groups. Microscopic observation showed that the whole blood passed smoothly through the microchannels in every group. CONCLUSIONS: CEE plus MPA therapy may not impair blood flow through the microchannels in hypertensive postmenopausal women receiving antihypertensive drugs or in normotensive postmenopausal women. However, administration of CEE plus MPA to postmenopausal women with hypertension warrants caution against the occurrence of thromboembolic events.

Mass drug administration against lymphatic filariasis: experiences from Kozhikode district of Kerala State.

The mass DEC drug administration to eliminate lymphatic filariasis in Kozhikode district was monitored from 2001 to 2003 to assess the drug distribution coverage, compliance, reasons for non-compliance, side reactions, mf prevalence and intensity, infection and infectivity rates in the vector. The drug distribution coverage and compliance were much below the required level. "No disease so not necessary" (42.5%) and "fear of side reactions" (25.2%) were the two major reasons for non-compliance. The adverse reactions were minimal. No appreciable changes were found in the mf prevalence and intensity. For the successful implementation of the MDA programme, proper planning, intense and timely efforts to motivate the community and innovative drug delivery strategies are required.

Effect of antihypertensive therapy on blood rheology in patients with essential hypertension.

Hypertension is an important risk factor for cardiovascular disease, and antihypertensive drugs can decrease the occurrence of such events in hypertensive patients. This study compared the rheological properties of blood in 22 untreated hypertensive patients, 42 patients taking antihypertensive drugs and 74 normotensive subjects. Using a microchannel method, the whole blood passage time was measured and blood movement was observed with a microscope connected to an image display unit. The blood passage time in untreated hypertensive patients was significantly higher than in treated hypertensive patients or normotensive subjects, but was similar in the latter two groups. Microscopic observations showed that platelet aggregation and leucocyte adhesion were increased in untreated hypertensive patients, resulting in poor flow, while blood samples from treated hypertensive patients and normotensive subjects passed smoothly through the microchannels. These rheological differences could contribute to the decrease in cardiovascular disease seen when hypertensive patients are treated effectively.

Decline of brugian filariasis in Cherthala taluk, Alappuzha district, Kerala.

A total of 4492 persons from 5 panchayats and 1 town were investigated from the Brugia malayi most endemic taluk of Cherthala, Alappuzha district of Kerala state. The urban area in Cherthala taluk only revealed mf carriers; mf rate was 0.13%. Rural areas in Cherthala taluk were free from infection. Microfilaria rate had declined by 99.5% and disease rate by 90.7% in Cherthala compared to 1934 prevalence. Shedding of sheath by B. malayi microfilariae was recorded for the first time in India. The youngest person with microfilaria and disease manifestation was 4 1/2 and 9 years respectively. All the 3 major vectors, Mansonia annulifera, Ma.uniformis and Culex quinquefasciatus were prevalent throughout. Complete disappearance of brugian filariasis from this taluk is a distinct possibility. The reasons for the drastic decline are discussed.

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.

Dynamic patterns in the locomotion and feeding behaviors by the placozoan Trichoplax adhaerence.

The placozoan Trichoplax adhaerence is one of the most primitive multi-cellular organisms, and moves about accompanying perpetual changes in its shape. Changes in position, locomotion velocity and the outer shape of the organism were monitored quantitatively with use of a computer image analysis, and their dynamic patterns in free locomotion and upon feeding were analyzed in terms of non-linear dynamics. The organism changed its behavioral patterns discontinuously in response to various concentrations of yeast extracts (food). (1) At low concentrations, the organism moved fast with perpetual random changes in shape. Both locomotion velocity and shape changes exhibited 1/f fluctuations. (2) At high concentrations, the shape of the organism as well as the locomotion exhibited oscillations with periods of about 8 min. These limit cycle oscillations bifurcated into the period 2 at the highest concentration tested. The organism flattened more strongly and the locomotion was more reduced on the whole at higher concentrations. (3) At the intermediate concentrations, two patterns as monitored above appeared: one pattern continued for a while and switched to the other abruptly. (4) The average square displacement of the organism increased linearly with time in all cases, indicating that the locomotion is a Brownian movement. In this way, the feeding behaviors by the placozoan are organized as successive co-operative transitions among non-linear dynamic states.

[Synthesis of ethyl 1-(Difluoro-1,3,5,-triazinyl)-2-methylindolizine-3-carboxylate as a fluorescent derivatization reagent and its reactivity].

Ethyl 1-(difluoro-1,3,5-triazinyl)-2-methylindolizine-3-carboxylate was found to be a selective fluorescence derivatization reagent for primary and secondary amines. The reagent reacted with amines in aqueous acetonitrile in the presence of sodium hydroxide to give the corresponding fluorescent products, which could be separated on a TSK gel ODS-80TM reversed-phase column with aqueous methanol as eluent. 2-Phenethylamine and spermidine were used to investigate the derivatization conditions. The detection limits (signal-to-noise ratio = 3) of each compound were 3 and 2 pmol per 10 microliters injection volume, respectively. Alcohols, thiols and aromatic amines did not give any fluorescent products under these derivatization conditions.

[Studies on the constituents of Pinus densiflora Sieb. et Zucc. leaves. II. On the ethanol-extractable amino acids compared with Pinus thunbergii Parl. leaves].

In order to identify the constituents of ethanol-extractable amino acids (free amino acids) from Pinus densiflora Sieb. et Zucc. and Pinus thunbergii Parl. leaves, analytical studies were performed by the use of high performance liquid chromatography. According to the results obtained, 29 amino acids in Pinus densiflora (the tree aged 3), 27 amino acids in Pinus thunbergii (the tree aged 3) and 25 amino acids in both pine (the tree aged 25) were confirmed. The total amounts of free amino acids in Pinus densiflora were found to be more rich than in Pinus thunbergii. The peaks of many free amino acids in Pinus densiflora (the tree aged 3) were found in February, but in Pinus thunbergii (the tree aged 3) the peaks of free amino acids were observed in May and from February to April. The mode of occurrence of asparagine at germination of buds explains clearly the distinction between Pinus densiflora and Pinus thunbergii.

Early development of acute filariasis in a migrant from non-endemic to hyper endemic area: a case report.

A case report of a European woman who contracted filariasis after staying for a few weeks in a filaria endemic area of south India is presented in this paper.

Plasma triglyceride-decreasing components of pine needles.

An ultrafiltrate (M.W. < 10000) of 0.1 M acetic acid extract from Japanese red pine needles was treated with Sep-Pak Vac C18, and an absorbable fraction (pine aqueous components fraction, PAC) was obtained. Since the intraperitoneal administration of PAC to rats decreased plasma triglyceride in a screening test for bioactivity, we tried to further isolate the active substances with respect to this triglyceride-decreasing action. Active substances were precipitated by acid, and we then divided them into fractions using reversed phase HPLC. The active fractions were hydrolyzed, then the hydrolysate was re-separated by the same HPLC system. Negative ion mode FAB-LC/MS of the bioactive fractions of this hydrolysate revealed an [M-H] molecular ion peak at m/z 169 and 321. The 13C-NMR spectra were consistent with those of authentic gallic acid and galloyl gallic acid, a dimer of gallic acid. Authentic gallic acid also showed a triglyceride-decreasing action, and galloyl gallic acid even more markedly decreased triglyceride. These findings suggest that the triglyceride-decreasing action of Japanese red pine needles is due to these polyphenol compounds. In addition, amino acids were also detected in the hydrolysate of PAC, indicating that the components in Japanese red pine needles that decrease triglycerides are complexes of a hydrolyzable tannin, which contains gallic acid and galloyl gallic acid as components and peptides.