Optical diagnosis of cervical cancer by fluorescence spectroscopy technique

In the present work, we examine normal and malignant stage IIIB cervical tissue by laser induced fluorescence, with 2 different objectives. (i) Development of the fluorescence spectroscopy technique as a standard optical method for discrimination of normal and malignant tissue samples and, (ii) Optimization of the technique by the method of matching of a sample spectrum with calibration sets of spectra of pathologically certified samples. Laser-induced fluorescence spectra were measured using samples from 62 subjects at different excitation wavelengths. Principal component analysis (PCA) of spectra and intensity ratios of curve-resolved fluorescence peaks were tested for discrimination. It was found that PCA of total fluorescence at 325 nm excitation gives specificity and sensitivity over 95%. Use of calibration sets of spectra of histo-pathologically certified samples combined with PCA for matching and pass/fail classification of test samples is shown to have high sensitivity/specificity for routine diagnostic purposes as well as for possible staging of the disease. Further, the multi-component origin of the fluorescence spectra is illustrated by curve resolution and fluorescence spectra of separated proteins of tissue homogenates

Strong impact of TGF-β1 gene polymorphisms on breast cancer risk in Indian women: a case-control and population-based study

Introduction: TGF-β1 is a multi-functional cytokine that plays an important role in breast carcinogenesis. Critical role of TGF-β1 signaling in breast cancer progression is well documented. Some TGF-β1 polymorphisms influence its expression; however, their impact on breast cancer risk is not clear. Methods: We analyzed 1222 samples in a candidate gene-based genetic association study on two distantly located and ethnically divergent case-control groups of Indian women, followed by a population-based genetic epidemiology study analyzing these polymorphisms in other Indian populations. The c.29C>T (Pro10Leu, rs1982073 or rs1800470) and c.74G>C (Arg25Pro, rs1800471) polymorphisms in the TGF-β1 gene were analyzed using direct DNA sequencing, and peripheral level of TGF-β1 were measured by ELISA. Results: c.29C>T substitution increased breast cancer risk, irrespective of ethnicity and menopausal status. On the other hand, c.74G>C substitution reduced breast cancer risk significantly in the north Indian group (p  =  0.0005) and only in the pre-menopausal women. The protective effect of c.74G>C polymorphism may be ethnicity-specific, as no association was seen in south Indian group. The polymorphic status of c.29C>T was comparable among Indo-Europeans, Dravidians and Tibeto-Burmans. Interestingly, we found that Tibeto-Burmans lack polymorphism at c.74G>C locus as true for the Chinese populations. However, the Brahmins of Nepal (Indo-Europeans) showed polymorphism in 2.08% of alleles. Mean TGF-β1 was significantly elevated in patients in comparison to controls (p<0.001). Conclusion: c.29C>T and c.74G>C polymorphisms in the TGF-β1 gene significantly affect breast cancer risk, which correlates with elevated TGF-β1 level in the patients. The c.29C>T locus is polymorphic across ethnically different populations, but c.74G>C locus is monomorphic in Tibeto-Burmans and polymorphic in other Indian populations

Inflammation induced insulin resistance is associated with DNA methylation changes in vascular endothelial cells

Vascular insulin resistance manifests in decreased production of nitric oxide subsequently leading to vasoconstriction and atherosclerosis. Inflammatory molecules such as IL-6 are known to induce insulin resistance in vascular endothelial cells. Epigenetic mechanisms including promoter DNA methylation have been demonstrated in development and progression of metabolic disorders and atherosclerosis. However, precise and underlying epigenetic mechanisms governing vascular insulin resistance are not known. Human endothelial cells treated with a) IL-6 and insulin together, b) pretreated with IL-6 and c) hyperinsulinemic conditions induced vascular insulin resistance leading to decreased Akt/eNOS activation and subsequently stabilized STAT3 phosphorylation. In 3D spheroid and matrigel assays, IL-6 abrogated insulin effects on angiogenesis. IL-6 induced insulin resistance was associated with down regulation of DNMT1 and DNMT3B, but not DNMT3A and resulted in decreased enzyme activity leading to global DNA hypomethylation. Protein levels of DNMT1 and DNMT3B were inversely correlated with S-phase of cell cycle. CpG microarray analysis revealed hypomethylation of promoters associated with 199 genes and promoter hypermethylation of 98 genes. Further, methylation status of promoters of genes associated with insulin signaling and angiogenesis such as RPS6KA2, PI3KR2, FoxD3, Exoc7, MAP3K8, ITPKB, EPHA6, IGF1R and FOXC2 were validated by bisulfite sequencing. Differentially methylated CpG sites of four out of five hypomethylated genes harboured putative binding site for HMGB1, a potent inflammatory mediator, suggesting HMGB1 might serve as epigenetic switch facilitating a pro-inflammatory milieu in response to IL-6 in endothelial cells. Our data indicates causal link between IL-6 induced DNMT1 changes and altered gene expression involved in insulin signaling and angiogenesis

Advances in development and application of human organoids

Innumerable studies associated with cellular differentiation, tissue response and disease modeling have been conducted in two-dimensional (2D) culture systems or animal models. This has been invaluable in deciphering the normal and disease states in cell biology; the key shortcomings of it being suitability for translational or clinical correlations. The past decade has seen several major advances in organoid culture technologies and this has enhanced our understanding of mimicking organ reconstruction. The term organoid has generally been used to describe cellular aggregates derived from primary tissues or stem cells that can self-organize into organotypic structures. Organoids mimic the cellular microenvironment of tissues better than 2D cell culture systems and represent the tissue physiology. Human organoids of brain, thyroid, gastrointestinal, lung, cardiac, liver, pancreatic and kidney have been established from various diseases, healthy tissues and from pluripotent stem cells (PSCs). Advances in patient-derived organoid culture further provides a unique perspective from which treatment modalities can be personalized. In this review article, we have discussed the current strategies for establishing various types of organoids of ectodermal, endodermal and mesodermal origin. We have also discussed their applications in modeling human health and diseases (such as cancer, genetic, neurodegenerative and infectious diseases), applications in regenerative medicine and evolutionary studies