Cancer diagnosis continuously evolves due to the better understanding of tumorigenic processes. DNA-methylation is consolidated as an effective biomarker for cancer prognosis and diagnostic even in tumors of unknown origin. The reversibility of this epigenetic mechanism also places it as a high-profile tool for the development of more sophisticated and personalized therapies. Current methodologies, such as bisulfite conversion or PCR amplification, rely on complex procedures that make difficult the standardization of epigenetics analyses. Here we present an optical biosensor methodology based on Surface Plasmon Resonance that employs poly-purine reverse-Hoogsten hairpin probes capable of interacting directly with ds-DNA fragments by triple helix formation. The direct interaction with the material of interest can greatly enhance the reliability of the analysis providing a more accurate and precise diagnosis. We have demonstrated the capabilities of our methodology for the direct capture of ds-DNA fragments and specific methyl-cytosine quantification. Our poly-purine hairpin probe demonstrated the specific capture of ds-DNA fragments while the standard duplex-forming probes failed to do so. In addition, the biosensor methodology showed a strong correlation with the different DNA methylation status between the sequences with a low signal variation (≤ 8%CV) along 35 hybridization/regeneration cycles. Through its straightforward procedure and versatility of detecting different DNA modifications related to the DNA methylation process, we anticipate that our strategy will enable a greater level of accuracy and precision in cancer diagnostics making a strong impact on the development of personalized therapies.
Recent advances in optical techniques have created a great range of possibilities for diagnosis and therapeutics in liver related diseases. With the uses of efficient light sources like lasers and LEDs (Light Emitting Diodes) it is possible to employ the light-tissue interaction to promote hepatic tissue regeneration after partial hepatectomy, to detect hepatocarcinoma and steatosis by utilizing optical fluorescence, to evaluate the metabolism of the liver during hepatic transplantation as well as to treat liver tumors. We present here an overview of the technique presently in development at the Ribeirâo Preto Faculty of Medicine - USP in cooperation with the Physics Institute of São Carlos -USP. The results obtained so far have been the subject of a list of publications and are here presented as an overview. A new perspective for modern application of optical techniques in different medical practices related to the liver is presented.
The evaluation of microleakage is a method conventionally used to evaluate the composite restoration in cavities prepared by laser or conventional drill. The samples were five sound molar human extracted teeth with cavity preparation Class V in each surface (buccal, lingual, mesial and distal) diVided in: GIcavity preparation with Er:YAG laser, adhesive agent Single Bond (3M), composite Z100 (3M) color Al; Gilcavity preparation laser, 35%phosphoric acid etching, adhesive, composite; Gil!cavity preparation with a drill, adhesive, composite; GIVcavity preparation with a drill, acid etching, adhesive, composite. The samples were sealed with a coating of nail varnish except on the area of the restoration and immersed in 50% aqueous solution of silver nitrate for 24 hours while kept in darkness. Samples were rinsed, soaked in photorevealing solution and exposed to fluorescent light for 6 hours. After, they were embedded in epoxy resin and sectioned longitudinally using the diamond saw microtone under numing water. The specimens were observed under optical microscopy and the results were analyzed under Kruskall Wallis resulting in difference statistically between GI X GIV at the 5% level; and under Mann-Whitney resulting non-difference staüstically between groups divided in cervical or occiusal walls.
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Significance:
Accurate early diagnosis of malignant skin lesions is critical in providing adequate and timely treatment; unfortunately, initial clinical evaluation of similar-looking benign and malignant skin lesions can result in missed diagnosis of malignant lesions and unnecessary biopsy of benign ones.
Aim:
To develop and validate a label-free and objective image-guided strategy for the clinical evaluation of suspicious pigmented skin lesions based on multispectral autofluorescence lifetime imaging (maFLIM) dermoscopy.
Approach:
We tested the hypothesis that maFLIM-derived autofluorescence global features can be used in machine-learning (ML) models to discriminate malignant from benign pigmented skin lesions. Clinical widefield maFLIM dermoscopy imaging of 41 benign and 19 malignant pigmented skin lesions from 30 patients were acquired prior to tissue biopsy sampling. Three different pools of global image-level maFLIM features were extracted: multispectral intensity, time-domain biexponential, and frequency-domain phasor features. The classification potential of each feature pool to discriminate benign versus malignant pigmented skin lesions was evaluated by training quadratic discriminant analysis (QDA) classification models and applying a leave-one-patient-out cross-validation strategy.
Results:
Classification performance estimates obtained after unbiased feature selection were as follows: 68% sensitivity and 80% specificity with the phasor feature pool, 84% sensitivity, and 71% specificity with the biexponential feature pool, and 84% sensitivity and 32% specificity with the intensity feature pool. Ensemble combinations of QDA models trained with phasor and biexponential features yielded sensitivity of 84% and specificity of 90%, outperforming all other models considered.
Conclusions:
Simple classification ML models based on time-resolved (biexponential and phasor) autofluorescence global features extracted from maFLIM dermoscopy images have the potential to provide objective discrimination of malignant from benign pigmented lesions. ML-assisted maFLIM dermoscopy could potentially assist with the clinical evaluation of suspicious lesions and the identification of those patients benefiting the most from biopsy examination.
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