Characterization and classification of pathogenic bacteria using native fluorescence and spectral deconvolution.

Identification and classification of pathogenic bacterial strains is of current interest for the early treatment of diseases. In this work, protein fluorescence from eight different pathogenic bacterial strains were characterized using steady state and time resolved fluorescence spectroscopy. The spectral deconvolution method was also employed to decompose the emission contribution from different intrinsic fluorophores and extracted various key parameters, such as intensity, emission maxima, emission line width of the fluorophores, and optical redox ratio. The change in average lifetime values across different bacterial strains exhibits good statistical significance (p ≤ 0.01). The variations in the photophysical characteristics of bacterial strains are due to the different conformational states of the proteins. The stepwise multiple linear discriminate analysis of fluorescence emission spectra at 280 nm excitation across eight different bacterial strains classifies the original groups and cross validated group with 100% and 99.5% accuracy, respectively.

Significance of Fluorescent Spectroscopy in Screening Oral Potentially Malignant Disorders and Oral Cancer by Characterization of Salivary DNA Using Ethidium Bromide-A Comparative Study.

Sangeetha RamamoorthyBackground Optical diagnosis is one of the upcoming methods in screening and diagnosing oral cancer at the earlier stage. Currently, DNA-based diagnosis is used along with light-based imaging methods to diagnose oral cancer rapidly. Aim The aim of this study was to discriminate oral cancer and oral potentially malignant disorders from normal patient with fluorescence spectroscopic characterization of salivary DNA using ethidium bromide dye. Materials and Methods A total of 40 patients with average age of 20 to 60 years in both the genders were selected and divided into three groups. Group A included clinically proven cases of oral cancer, group B1 included clinically diagnosed cases of leukoplakia, group B2 included clinically diagnosed cases of oral submucous fibrosis, and group C included controls. Salivary DNA fluorescence spectrum obtained after adding ethidium bromide was analyzed using FluoroLog spectrophotometer at 480 nm wavelength. Results The discriminant analysis of fluorescent emission of salivary DNA shows predictive accuracy of 90% between group C and group A, 95% between group C and group B1, and 65% observed between group C and group B2. Conclusion From this study, screening of oral cancer can be done at the earliest with the help of fluorescence spectroscopic characterization of salivary DNA. This method can be done rapidly and noninvasively.

Comparison and estimation of photoneutron dose produce between 10 MV flattened and unflattened beam in Elekta Versa HD™ medical linac.

BACKGROUND: In a high-energy medical linear accelerator (linac), if the interaction of photon energy is higher than the neutron binding energy of high atomic material, it emits a neutron field through photonuclear (γ, n) reaction. AIM: The current study, evaluates the photoneutron dose equivalent (PNDE) produced between the 10 MV flattened and unflattened beams as a function of field sizes in the Elekta Versa HD™ linac. MATERIALS AND METHODS: The PNDE produced from Versa linac was recorded along the patient plane using the bubble detector personal neutron dosimeter and from the measured PNDE values, the theoretical PNDE values were simulated for various field sizes using nonlinear least-squares curve-fitting as a function of a polynomial. The percentage of deviation (PoD) and Chi-square (χ2) tests were performed between the measured and simulated PNDE values to study the reliability and validity. RESULTS: The results show that the mean PoD between the measured and simulated PNDE values for respective positions of a field size of FF beam was found to be -1.99% for 0.3×0.3, -4.39% for 5×5, -3.868% for 10×10, 0.590% for 15×15, 9.18% for 20×20, -4.133% for 25×25, and 0.467% for 30×30 cm2. Similarly, the mean PoD between the measured and simulated PNDE values for flattening filter-free (FFF) beam was found to be 1.36% for 0.3×0.3, -1.39% for 5×5, -5.38% for 10×10, 4.41% for 15×15, 3.84% for 20×20, 5.69% for 25×25, and -1.75% for 30×30 cm2. The maximum deviation between the measured and simulated PNDE values lies within the range ± 5%. CONCLUSIONS: From the study, it is observed that the FFF beam produces lesser neutron contamination than the FF beam.

Support vector machine based methodology for classification of thermal images pertaining to breast cancer.

Breast cancer has been and continues to be a cause of major health concern for women. It is more prevalent in old age, but its incidence has increased in recent years in groups below 50 years old, as in India. According to the Indian Council of Medical Research (ICMR) 2020, 50% of all the cases are in the age group of 25-50 where the numbers are staggering and constantly rising. The increase in incidence over the years indicates an urging need for innovative approaches to enhance breast cancer detection early. Thermography is non-contact imaging modalities and has potential to detect breast cancer at an early stage. Though thermography has capable of detecting breast cancer early, the challenge lies in the interpretation of the breast thermograms with respect to features and subsequent analysis. The present work discusses image acquisition, image processing related pre-processing, segmentation, and feature extraction. The extracted features were analyzed using ANOVA (Analysis of variance) statistical analysis. Statistical analyses were done in order to find the appropriate feature on the whole and quadrant breast. Statistical analysis results clearly reveal existence of thermal symmetry for the healthy subjects (p value > .05) in both whole and quadrant breast regions. In the case of abnormal subjects, whole breast analyses revealed the significance (p value < .05) for features like mean, variance, standard deviation, kurtosis, skewness, entropy, energy, homogeneity and contrast whereas upper outer quadrant analyses showed significance for all above features except contrast. The well correlated features of upper outer quadrant and whole breast were given as input for the Support Vector Machine - Radial Basis Function (SVM - RBF) classifier with grid search method. The results revealed that whole breast analysis has achieved 92.86% accuracy and upper outer quadrant breast analysis has achieved 85.71% accuracy. The results clearly indicate the involvement of upper outer quadrant and whole breast in early detection of breast cancer using thermal imaging.

Evaluation of photoneutron dose equivalent in 10 MV and 15 MV beams for wedge and open fields in the Elekta Versa HD linac.

In a high-energy medical linear accelerator (linac), if the interaction of photon energy is higher than the neutron binding energy of high atomic material, it emits a neutron field through a photonuclear reaction. The objective of this current study is to measure the photoneutron dose equivalent produces in a motorized wedge field and open field of 10 MV and 15 MV photon beams in Elekta Versa HD™ linac. The PNDE values were recorded at various positions along the patient plane using the Bubble Detector-Personal Neutron Dosimeter (BD-PND). The results revealed that the PNDE values are higher in 20 × 20 cm2 than 10 × 10 cm2 field sizes for both the 60° wedge and open fields of 10 MV and 15 MV beams. In addition, the 60° wedge fields generate higher photoneutron contamination when compared with the 45°, 30° wedge fields and open field sizes. Hence, on average PNDE values produced by the 15 MV beam were higher by a factor of 1.98 and 2.11 times for open and 60° wedge fields than the 10 MV beam, respectively.

Pulsed laser deposition of nanostructured bioactive glass and hydroxyapatite coatings: Microstructural and electrochemical characterization.

Bioactive coatings on metallic implants promote osseointegration between bone and implant interfaces. A suitable coating enhances the life span of the implant and reduces the requirement of revision surgery. The coating process needs to be optimized such that it does not alter the bioactivity of the material. To understand this, the biocompatibility of nanostructured bioactive glass and hydroxyapatite-coated Titanium substrate by pulsed laser deposition method is evaluated. Raman and IR spectroscopic techniques based on silica and phosphate functional groups mapping have confirmed homogeneity in coatings by pulse laser deposition method. Comparative studies on nanostructured bioactive glass and hydroxyapatite on titanium surface elaborated the significance of bioactivity, hemocompatibility, and cytocompatibility of the coated surface. Notably, both hydroxyapatite and bioactive glass show good hemocompatibility in powder form. Hemocompatibility and cytocompatibility results validate the enhanced sustenance for hydroxyapatite coating. These results signify the importance of the choice of coating methodology of bioceramics towards implant applications.

Green synthesis of white light emitting carbon quantum dots: Fabrication of white fluorescent film and optical sensor applications.

In this work, we have reported on the facile synthesis of white light-emitting carbon quantum dots (CQD) from corncob by hydrothermal method. This CQD has a broad emission from 380 nm to 650 nm with high photoluminescence intensity even after three months of shelf-life and stable at variable pH conditions. The presence of Si and N impurities in the biomass gives a greater advantage in producing white light emission with high quantum yield (54%) and enhanced lifetime at ambient conditions. The CQD is highly sensitive towards DNA, paracetamol, Pb2+, Cu2+, Fe3+, and Cr3+ fluorescence sensing and signifies its application as a multi-modal fluorescence sensor. The results of optical sensitivity calculated from the linear range of 1-10 ng/mL, 0.10-0.30 mg/mL, 2.5446 ng/mL, 0.0694 mg/mL, 0.3103-1.5515 µM/mL, 0.4299-4.7293 µM/mL, 1.3010 µM/mL and 0.05-2.5 µM/mL. The limit of detection is 2.5446 ng/mL, 0.0694 mg/mL, 0.8641 µM/mL, 1.2454 µM/mL, 1.3010 µM/m, 0.8550 µM/mL and 2.8562 µM/mL, respectively. And also, the relative standard deviation values of 2.30%, 4.46%, 1.79%, 1.84%, 0.26%, 1.23% and 0.35% are evidences its possibility of development towards potential optical sensor applications. Flexible white light-emitting sheets were fabricated from the CQD, illuminates uniform brightness, and has good color reproducibility and higher stability under various UV light excitation.

In vitro and In silico Analysis of the Anti-diabetic and Anti-microbial Activity of Cichorium intybus Leaf extracts.

OBJECTIVE: To screen the selected phytochemicals against diabetes by docking studies in comparison with experimental analysis. METHODS: Ethanol crude extract was obtained from the leaves of C.intybus and its chemical compounds were identified using GC- MS. Docking studies were carried out for selected phytochemicals to find the binding affinity and H-bond interaction using Schrodinger suite. Dynamic simulations were carried out for protein-ligand complex up to 50ns using desmond OPLS AA forcefield and α- Amylase and α- Glucosidase assay were carried for the ethanolic extract to infer its inhibition. RESULTS: Four compounds were chosen for induced fit docking based on the docking score and glide energy obtained from GLIDE-XP docking. The compounds were docked with the protein target human aldose reductase (PDB ID: 2FZD) for checking the anti-diabetic nature. The molecular dynamics simulations were carried out for the most favorable compounds and stability was checked during the simulations. The ethanol extract exhibits significant α-amylase and α-glucosidase inhibitory activities with an IC50 value of 38µg and 88µg dry extract, respectively, and well compared with standard acarbose drug. The antimicrobial activity was also carried out for various extracts (Chloroform, Ethyl acetate, and Ethanol) of the same (C. intybus) screened against four selected human pathogens. Compared to other solvent extracts, ethanol and chloroform extracts show better inhibition and their minimal inhibitory concentration (MIC) value has been calculated. CONCLUSION: In silico studies and in vitro studies reveals that C. intybus plant compounds have more potent for treating diabetes.

Polarization gating technique extracts depth resolved fluorescence redox ratio in oral cancer diagnostics.

Mortality of oral cancer is often due to late diagnosis. Effective non-invasive diagnostic techniques may increase the survival rate based on an earlier diagnosis.. We report on the application of the polarization gating technique for isolating weakly scattered and highly scattered components of fluorescence emission from the superficial and deeper layers of tissue due to intrinsic fluorophores NADH and FAD. The fluorescence polarization spectra were collected from 21 normal and 67 oral squamous cell carcinoma biopsy tissues. The tissues were excited at 350 nm and the fluorescence emission had two peaks corresponding to NADH, and FAD respectively. The spectra were analyzed using the polarization gating technique along with the spectral deconvolution method to derive the optical redox ratio from different layers of tissue. The fractional change in redox ratio between superficial and deeper layers of tissue exhibits excellent statistical significance (p<10-3) which may be due to a shift in the metabolic pathway from oxidative phosphorylation to glycolysis in the cancer cell. Further, variation in collagen intensity in deeper layers of tissue is observed which may be attributed to the breakdown of collagen fibers in the stroma. Linear discriminant analysis showed that oral cancer tissue is discriminated with a better accuracy using polarization gating technique than that of conventional fluorescence spectroscopy.

Chitosan mediated 5-Fluorouracil functionalized silica nanoparticle from rice husk for anticancer activity.

Herein, we have reported a cost-effective method of synthesizing highly efficient silica nanomaterial from rice husks for its application as a chemotherapeutic agent. Silica particle with two different sizes ~20 nm and ~40 nm were achieved from silica precursor obtained from rice husk. 5-Fluorouracil was functionalized onto the surface of silica nanoparticles by direct conjugation and chitosan mediated conjugation. Particle size analysis, zeta potential and functional analyzes were performed in systematic methodology to confirm chitosan coating and 5-Fluorouracil conjugation on silica nanoparticles. The drug loading percentage with respect to the particle size shows that ~20 nm particles have higher loading capacity. The chitosan mediated conjugation of drug shows sustained release in acidic pH and hence suitable for cancer cell-targeted delivery. The in vitro cell culture studies performed on MC3T3 fibroblast cell lines, MCF-7 and A549 cancer cell lines depicts that, compared to direct conjugation of 5-Fluorouracil, chitosan mediated drug conjugation on the surface of silica nanoparticles shows lesser toxic to fibroblast cell lines and higher toxicity towards cancer cell lines. The results of this toxicity were also confirmed from the nucleic acid spectral signature of Raman spectra treated with drug conjugated silica nanoparticles.

Synchronous Luminescence Spectroscopy as a Tool in the Discrimination and Characterization of Oral Cancer Tissue.

High incidence of oral cancer is primarily due to ongoing tobacco epidemic. In this work, synchronous luminescence spectroscopy (SLS) has been used to characterize and discriminate oral cancer tissue. Spectral deconvolution method is employed to compute the fluorescence intensity, peak wavelength, and full width half maxima for different endogenous fluorophores. The fluorescence measurements were made on 21 normal and 88 oral squamous cell carcinoma biopsy tissues. Besides, variations in relative concentration of collagen, NADH, and FAD, peak shifts and broadening of peaks are observed for tryptophan, NADH, and FAD, in oral cancer tissues indicating both biochemical and micro environmental changes at cellular level. Linear discriminant analysis showed that oral cancer tissue is discriminated with a sensitivity and specificity of 100% and 95.2% respectively.

Near-infrared Raman spectroscopy for estimating biochemical changes associated with different pathological conditions of cervix.

The molecular level changes associated with oncogenesis precede the morphological changes in cells and tissues. Hence molecular level diagnosis would promote early diagnosis of the disease. Raman spectroscopy is capable of providing specific spectral signature of various biomolecules present in the cells and tissues under various pathological conditions. The aim of this work is to develop a non-linear multi-class statistical methodology for discrimination of normal, neoplastic and malignant cells/tissues. The tissues were classified as normal, pre-malignant and malignant by employing Principal Component Analysis followed by Artificial Neural Network (PC-ANN). The overall accuracy achieved was 99%. Further, to get an insight into the quantitative biochemical composition of the normal, neoplastic and malignant tissues, a linear combination of the major biochemicals by non-negative least squares technique was fit to the measured Raman spectra of the tissues. This technique confirms the changes in the major biomolecules such as lipids, nucleic acids, actin, glycogen and collagen associated with the different pathological conditions. To study the efficacy of this technique in comparison with histopathology, we have utilized Principal Component followed by Linear Discriminant Analysis (PC-LDA) to discriminate the well differentiated, moderately differentiated and poorly differentiated squamous cell carcinoma with an accuracy of 94.0%. And the results demonstrated that Raman spectroscopy has the potential to complement the good old technique of histopathology.

An in vitro diagnosis of oral premalignant lesion using time-resolved fluorescence spectroscopy under UV excitation-a pilot study.

In spite of rapid advancement in cancer treatment, early diagnosis of cancer and medicable precursors are still the finest approach towards the assurance of patient lives and enhancement in the quality of their life. In this regard, the present study deals with the time-resolved fluorescence spectroscopy of normal and premalignant oral tissues under UV excitations (280nm and 310nm). The decay kinetics at 350nm emission of normal tissues exhibit higher fluorescence lifetime than that of premalignant tissues and subsequent statistical analysis shows that the data were statistically significant. Further, the decay kinetics at 450nm emission for normal and premalignant oral tissues was obtained. Subsequently, statistical analysis revealed that except fast component, rest of the component lifetimes and fractional amplitudes were not statistically significant. An attempt has also been made to explore the better statistical tool to discriminate premalignant tissues from normal ones at 350nm emission. Among stepwise linear discriminant analysis (SLDA) and receiver operator characteristics (ROC), the former discriminates premalignant from normal tissues with 86.7% specificity and 93.3% sensitivity. Hence, fluorescence lifetime spectroscopy at 350nm emission opens a new avenue for early detection of oral cancer.

Polarized Raman spectroscopy unravels the biomolecular structural changes in cervical cancer.

Polarized Raman spectroscopy has emerged as a promising technique giving a wealth of information about the orientation and symmetry of bond vibrations in addition to the general chemical information from the conventional Raman spectroscopy. In this regard, polarized Raman Spectroscopic technique was employed to study the changes in the orientation of biomolecules in normal and cancerous conditions. This technique was compared to the conventional Raman spectroscopic technique and was found to yield additional information about the orientation of tyrosine, collagen and DNA. The statistically analyzed depolarization ratios by Linear Discriminant Analysis yielded better accuracy than the statistical results of conventional Raman spectroscopy. Thus, this study reveals that polarized Raman spectroscopy has better diagnostic potential than the conventional Raman spectroscopic technique.