FTIR microspectroscopy and multivariate analysis facilitate identification of dynamic changes in epithelial-to-mesenchymal transition induced by TGF-ß in prostate cancer cells.

The standard diagnosis of prostate cancer is accomplished by the identification of cytomorphological deviations in biopsied tissues while immunohistochemistry is used to resolve the equivocal cases. Accumulating evidence favors the concept that epithelial-to-mesenchymal transition (EMT) is a stochastic process composed of multiple intermediate states instead of a single binary switch. Despite its significant role in promoting cancer aggressiveness, the current tissue-based risk stratification tools do not include any of the EMT phenotypes as a metric. As a proof-of-concept, the present study analyzes the temporal progression of EMT in transforming growth factor-beta (TGF-ß) treated PC3 cells encompassing multifarious characteristics such as morphology, migration and invasion, gene expression, biochemical fingerprint, and metabolic activity. Our multimodal approach reinstates EMT plasticity in TGF-ß treated PC3 cells. Further, it highlights that mesenchymal transition is accompanied by discernible changes in cellular morphometry and molecular signatures particularly in the range of 1800-1600 cm-1 and 3100-2800 cm-1 of Fourier-transformed infrared (FTIR) spectra signifying Amide III and lipid, respectively. Investigation of attenuated total reflectance (ATR)-FTIR spectra of extracted lipids from PC3 cell populations undergoing EMT identifies changes in stretching vibration at FTIR peaks at 2852, 2870, 2920, 2931, 2954, and 3010 cm-1 characteristics of fatty acids and cholesterol. Chemometric analysis of these spectra indicates that the level of unsaturation and acyl chain length of fatty acid coregister with differential epithelial/mesenchymal states of TGF-ß treated PC3 cells. Observed changes in lipids also correlate with cellular nicotinamide adenine dinucleotide hydrogen (NADH) and flavin adenine dinucleotide dihydrogen (FADH2) levels and mitochondrial oxygen consumption rate. In summary, our study establishes that morphological and phenotypic traits of epithelial/mesenchymal variants of PC3 cells concur with their respective biochemical and metabolic properties. It also underscores that spectroscopic histopathology has a definitive potential to refine the diagnosis of prostate cancer reckoning its molecular and biochemical heterogeneities.

Clinico-pathological significance of Drp1 dysregulation and its correlation to apoptosis in oral cancer patients.

Dysregulation in mitochondrial dynamics has been associated with several diseases including cancer. Present study assessed the alteration in mitochondrial fission protein (Drp1) in oral epithelial cells collected from clinically confirmed pre-cancer and cancer patients and further correlates it with the cellular apoptosis signaling. Results indicate the ROS accumulation in OSCC patients is accompanied by several changes including increase in mitochondrial mass, expression of mitochondrial fission protein (Drp1) and alteration in apoptotic signaling. The positive co-relation has been observed between the expressions of anti-apoptotic Bcl-2proteinswith mitochondrial fission protein Drp1. Higher mitochondrial fission in oral cancer cells was also correlated with the increased expression of cell cycle marker CyclinD1 indicating highly proliferative stage of oral cancer cells. The clinical correlation can be extended to develop biomarker for diagram and program in oral cancer management.

Epithelial cell functionality on electroconductive Fe/Sr co-doped biphasic calcium phosphate.

In the perspective of dental restorative applications, co-doped bioceramics have not been explored much. From the clinical perspective, a successful dental implant is expected to interact with peri-prosthetic bones, gingival tissue, and surrounding connective tissues. The interaction of implant and implant coating materials with bone tissue is well studied. However, their interaction with surrounding epithelial components needs scientific validation. In this context, the present study aims at quantitative evaluation of the electrical properties of Fe/Sr co-doped biphasic calcium phosphate (BCP) samples and assessment of their cytocompatibility with epithelial (vero) cells. Sr/Fe co-doped BCPs were prepared by sol-gel synthesis technique, with different dopant concentration. Impact of co-doping on conductivity was assessed and interestingly an increase in conductivity with dopant amount was recorded in different co-doped BCPs. Cellular study showed the significant ( p = 0.01) increase in both cellular viability and functionality with increasing conductivity of samples. Higher epithelial cell adhesion indicates that (Sr/Fe) co-doped BCP would be favorable for faster epithelial sealing and also would reduce the chances of infection. Real-time PCR and immunofluorescence studies indicated that the expression of the epithelial marker (E-cadherin) significantly ( p = 0.01) increased in 10, 30 and 40 mol% co-doped samples in comparison to undoped BCP. In contrast to E-cadherin, fold change of ß-catenin remains unchanged amongst the co-doped ceramics, implying the absence of tumorigenic potential of (Sr/Fe) co-doped BCP. In addition, immune-fluorescence signatures for cellular polarity are established from enhanced expression PARD3 protein, which has major relevance for cellular morphogenesis and cell division. Summarizing, the present study establishes the efficacy of Sr/Fe co-doped BCPs as a dental implant coating material and its ability to modulate vero cell functionality.

Chemometric analysis of integrated FTIR and Raman spectra obtained by non-invasive exfoliative cytology for the screening of oral cancer.

FTIR spectroscopy and Raman spectroscopy of biological analytes are increasingly explored as screening tools for early detection of cancer. In the present study, an integrated analysis of the FTIR and Raman spectra obtained from exfoliated cells is adopted to improve discrimination of normal, pre-cancerous and cancerous conditions. Multiple spectra were obtained from 13 normal, 13 pre-cancer and 10 cancer patients in both modes. Compared to normal patients, significant differences were observed at 1550, 1580, 1640, 2370, 2330, 2950-3000 and 3650-3750 cm-1 (FTIR) and 520, 640, 785, 827, 850, 935, 1003, 1175, 1311 cm-1 and 1606 cm-1 (Raman) vibrations of the other two. The increase in DNA, protein and lipid content with malignancy was more clearly elucidated by examining both spectra. Principal component analysis (PCA)-linear discriminant analysis (LDA) with 10-fold cross validation of the FTIR and Raman spectral data sets showed efficient discrimination between normal and pathological conditions while overlapping was seen between the two pathologies. The PCA-LDA model of the dual spectra yielded a classification accuracy of 98% in comparison with either FTIR (85%) or Raman (82%) in a spectrum-wise comparison. In the patient-wise approach (mean of all spectra from a patient), the overall classification efficiency was 73%, 80% and 87% for FTIR, Raman and integrated spectral approaches respectively. Moreover, the efficiency of the integrated FTIR-Raman PCA-LDA model as a prediction tool was tested to screen susceptible individuals (11 cigarette smokers) using the dual spectra acquired from these individuals. The study presents proof-of-concept for adopting a large-scale, follow-up trial of the approach for mass screening purposes.

Electrospun chitosan/polycaprolactone-hyaluronic acid bilayered scaffold for potential wound healing applications.

Fabrication of mechanically stable, biocompatible bilayered polymeric scaffold consisting of chitosan(CS)/polycaprolactone(PCL) and hyaluronic acid(HA) using less toxic solvent system is presented in this study. Electrospinning technique to make the scaffold was used followed by morphological, physiochemical and mechanical characterizations. Average fiber diameter of CS/PCL-HA bilayered scaffold was found 362.2 ±â€¯236 nm which is in the range of collagen fiber found in the extracellular matrices. Enhanced swelling, degradation, hydrophilicity and water vapour transmission rate were found for the bilayered scaffold compared to that of the PCL and CS-PCL scaffolds. Antimicrobial property evaluation revealed reduction in bacterial adhesion on bilayered scaffolds. Invitro studies with vero cells [kidney epithelial cell, extracted from African Green Monkey (Chlorocebus sp.)] confirm enhanced proliferation, growth and migration of vero cell on the bilayered CS/PCL-HA scaffold to that of PCL and CS/PCL scaffolds. Novelty of this study includes the use of HA for mechanically stabilized scaffold with acceptable biological properties for wound healing applications.

Repositing honey incorporated electrospun nanofiber membranes to provide anti-oxidant, anti-bacterial and anti-inflammatory microenvironment for wound regeneration.

Topical application of honey for tissue regeneration, has recently regained attention in clinical practice with controlled studies affirming its efficacy and indicating its role in regeneration over repair. Parallely, to overcome difficulties of applying raw honey, several product development studies like nanofibrous matrices have been reported. However, one approach concentrated on achieving highest possible honey loading in the nanofiber membranes while other studies have found that only specific honey dilutions result in differential cellular responses on wound healing and re-epithelization. From these results, it can be suggested that high honey loading provides optimum external microenvironment, low-loaded membranes could provide a more conducive internal microenvironment for tissue regeneration. With this hypothesis, this paper sought to evaluate ability of low-honey loaded nanofibers to modulate the anti-oxidant, anti-biofilm and anti-inflammatory properties which are important to be maintained in wound micro-environment. A loading-dependent reduction of biofilm formation and anti-oxidant activity was noted in different concentration ranges investigated. After scratch assay, a certain honey loading (0.5%) afforded the maximum re-epithelization. Since there is lack of methods to determine anti-inflammatory properties of nanofiber membranes during epithelial healing process, we performed anti-inflammatory assessment of nano-fibers by evaluating the expressions of pro-inflammatory markers-Cycloxygenase-2 (COX-2) and Interleukin-6 (IL-6) and to confirm the optimized concentration. Considering the role of COX-2 and IL-6, the novel methodology used in this study can also be developed as an assay for anti-inflammatory matrices for wound healing.

(Fe/Sr) Codoped Biphasic Calcium Phosphate with Tailored Osteoblast Cell Functionality.

Although doped bioceramics have been widely investigated for biomedical applications, the codoped bioceramics remain mostly unexplored for bone regeneration applications. For example, the impact of codoping of Sr2+ and Fe3+ ions on the phase stability and cytocompatibility is not explored so far. In this perspective, the objective of the present study is to quantitatively understand this aspect in case of Fe/Sr codoped biphasic calcium phosphate (BCP). Following sol-gel synthesis, codoped BCP samples with Sr/Fe dopant concentrations of 2, 10, 20, 30, and 40 mol % as well as doped BCPs with single dopant (Sr or Fe) with similar compositions were calcined at 800 °C in air. Using extensive Rietveld analysis, the dopant content dependent crystallographic properties (e.g lattice parameters) and phase stability of HA/TCP are quantitatively assessed. In vitro cytocompatibility of codoped samples has been assessed using mouse osteoblast cells. An important observation is that, while singular dopant of Sr/Fe at 20 mol % or higher amount reduces cell viability significantly, osteoblast viability is not compromised to any significant extent on Sr/Fe codoped BCP, compared to undoped BCP. Our results indicate that one can tailor osteoblast functionality by controlling the codopant content. More importantly, all the codoped BCPs support cell proliferation, when single doped BCP exhibits significant reductionin cell viability, at dopant content of 10 mol % or higher. Cell morphological analysis supports extensive cell spreading on codoped BCPs. An attempt has been made to correlate the variation in cellular response with HA/TCP ratio and ion dissolution behavior. Taken together, the present work establishes unique advantage of Sr/Fe codoping approach toward realizing their bone replacement application.