Molecular effects of photodynamic therapy with curcumin on Leishmania major promastigotes.

Leishmaniasis is a neglected disease mainly affecting low-income populations. Conventional treatment involves several side effects, is expensive, and, in addition, protozoa can develop resistance. Photodynamic therapy (PDT) is a promising alternative in treating the disease. PDT involves applying light at a specific wavelength to activate a photosensitive compound (photosensitizer, PS), to produce reactive oxygen species (ROS). Curcumin and its photochemical characteristics make it a good candidate for photodynamic therapy. Studies evaluating gene expression can help to understand the molecular events involved in the cell death caused by PDT. In the present study, RNA was extracted from promastigotes from the control and treated groups after applying PDT. RT-qPCR was performed to verify the expression of the putative ATPase beta subunit (ATPS), ATP synthase subunit A (F0F1), argininosuccinate synthase 1 (ASS), ATP-binding cassette subfamily G member 2 (ABCG2), glycoprotein 63 (GP63), superoxide dismutase (FeSODA), and glucose-6-phosphate dehydrogenase (G6PDH) genes (QR). The results suggest that PDT altered the expression of genes that participate in oxidative stress and cell death pathways, such as ATPS, FeSODA, and G6PD. The ATP-F0F1, ASS, and GP63 genes did not have their expression altered. However, it is essential to highlight that other genes may be involved in the molecular mechanisms of oxidative stress and, consequently, in the death of parasites.

PCR-RFLP and FTIR-based detection of high-risk human papilloma virus for cervical cancer screening and prevention.

BACKGROUND: Approximately 70% of cervical carcinoma cases show the presence of high-risk Human Papilloma Virus (HPV), especially HPV-16 and HPV-18, and can be used to stratify high risk patients from low risk and healthy. Currently, molecular biology techniques such as polymerase chain reaction (PCR) are used to identify the presence of virus in patient samples. While the methodology is highly sensitive, it is labor intensive and time-consuming. Alternative techniques, such as vibrational spectroscopy, has been suggested as a possible rapid alternative. Therefore, in this study, we evaluate the efficiency of cervical fluid Fourier Transform Infrared spectroscopy (FTIR) in patient risk stratification informed by PCR. METHODS: Cervical fluid samples (n = 91) were obtained from patients who have undergone routine Papanicolaou (Pap) test. Viral genome was identified and classified as high/low-risk by PCR-Restriction Fragment Length Polymorphism (PCR-RFLP). FTIR spectra were acquired from samples identified by PCR-RFLP as No-HPV (n = 10), high-risk HPV (n = 7), and low-risk HPV (n = 7). RESULTS: Of the 91 samples, was detected the viral genome by PCR in 36 samples. Of these 36 samples, nine samples were identified to contain high-risk HPV (HR-HPV) and nine samples were found to have low-risk HPV (LR-HPV). The FTIR spectra acquired from No-HPV, LR-HPV, and HR-HPV showed differences in 1069, 1437, 1555, 1647, 2840, 2919, and 3287 cm-1 bands. Principal Component Analysis (PCA) showed distinct clusters for No-HPV and HR-HPV and No-HPV and LR-HPV, but there was significant overlap in the clusters of HR-HPV and LR-HPV. PCA-Linear Discriminant Analysis (PC-LDA) after Leave One Out Cross Validation (LOOCV) classified No-HPV from HR-HPV and No-HPV from LR-HPV with 100% efficiency in the 1400-1800 cm-1 spectral range. LOOCV classifications for LR-HPV and HR-HPV from each other were 71 and 75%, respectively, in the 2800-3400 cm-1 spectral range. CONCLUSIONS: The results highlight the high sensitivity of PCR-RFLP in HPV identification and show that FTIR can classify samples identified as healthy, low, and high-risk samples by PCR-RFLP. GENERAL SIGNIFICANCE: We show the possibility of using FTIR for initial cervical cancer risk stratification followed by detailed PCR-RFLP investigations for suspect cases.

Molecular detection of HPV and FT-IR spectroscopy analysis in women with normal cervical cytology.

BACKGROUND: The increase in the incidence of Cervical Cancer in the female population worldwide has been an issue that deserves further attention from the scientific community. Several studies have already proven the relationship of its development with the molecular mechanisms that Human Papillomavirus (HPV) induces in cervical cells. The gene amplification provided by molecular biology techniques has been used as the gold standard diagnostic method of this virus because of its high specificity and sensitivity.However, the high investments associated with the acquisition of reagents, equipment and labor demonstrate the need for the development of more accessible techniques that present the same accuracy. FT-IR spectroscopy has been studied as an inexpensive and easily accessible technology that can provide the differentiation of malignant and benign cells. This study aimed to demonstrate the effectiveness and sensitivity of molecular analysis by PCR in relation to cytological analysis and to evaluate the sensitivity of FT-IR spectroscopy in the diagnosis of HPV for cervical cancer prevention. METHODS: Cervical fluid samples obtained from 50 patients with absence of cellular lesion by cytological analysis were analyzed by molecular and spectroscopic analyzes. Oncotic colpocitology analysis was performed by the Papanicolaou staining, amplification of the L1 viral gene by PCR was performed using primers MY09 and MY11 and biochemical analysis of the fluids by FT-IR was performed using the Spectrum 400 system equipped with a microscope. RESULTS: Of the 50 patients without evident morphological alteration of the cells, seven were diagnosed by molecular analysis as positive for presence of HPV. Principal component analysis of spectroscopy was not able to separate the negative samples from the HPV positive samples and, therefore, did not present as an effective diagnostic technique. CONCLUSIONS: We highlight the efficacy, sensitivity and specificity of molecular biology by PCR in the identification of the virus and we emphasize that more studies should be used for the application of FT-IR spectroscopy in the diagnosis of this infection and its application in the prevention of cervical cancer.

ATR-FTIR spectroscopy and CDKN1C gene expression in the prediction of lymph nodes metastases in papillary thyroid carcinoma.

Thyroid cancer has become in recent years the most common endocrine malignancy. Among its different types, papillary thyroid carcinoma (PTC) has the highest incidence. PTC is slow growing, but shows a high rate of lymph node metastasis. Tissue biochemical characterization and identification of molecular markers can facilitate stratification of patients into those requiring surgical assessment of lymph nodes and patients for whom this surgical procedure is unnecessary; thus, leading to a more accurate prognosis. To this end, the study aimed to predict lymph node metastasis by Attenuated Total Reflectance - Fourier transform infrared (ATR-FTIR) spectroscopy of primary PTC tumors. Another objective of the study was to determine whether CCNA1, CDKN1C, FOS, HSPA5, JUN, KSR1, MAP2K6, MAPK8IP2 and SFN gene expression in primary PTC tumors could be used as predictive markers of lymph node metastasis. Three PTC with lymph node involvement (PTC+), six PTC without lymph node involvement (PTC-), and five normal (N) thyroid tissues were used for FTIR spectroscopy analysis; while 18 PTC+, 17 PTC-, and 6 N samples were used for molecular analysis by real-time quantitative PCR (RT-qPCR). FTIR spectral analysis revealed changes in phosphate groups possibly associated with nucleic acid (1236 cm-1), and protein/lipids (1452, 2924, 3821 cm-1) in PTC + compared to PTC-, and multivariate analysis could distinguish the two groups. Molecular analysis showed significant increase in CDKN1C gene expression in PTC + compared to PTC-. Being a cell growth regulator, increased CDKN1C provides some supporting evidence to the FTIR spectroscopy based finding of increased nucleic acids in PTC+. Thus, the study suggests the possibility of using FTIR spectroscopy and CDKN1C expression for predicting metastasis using primary tumor alone.

Human papillomavirus detection using PCR and ATR-FTIR for cervical cancer screening.

The human papillomavirus (HPV) genital infection is considered one of the most common sexually transmitted diseases worldwide, and has been associated with cervical cancer. The objective of this study was to investigate the efficacy of the diagnostic methods: polymerase chain reaction (PCR) and Fourier transform infrared (FTIR) equipped with an ATR (Attenuated Total Reflectance) unit (Pike Tech) spectroscopy, to diagnose HPV infection in women undergoing gynecological examination. Seventeen patients (41.46%) of the 41 patients analyzed were diagnosed with exophytic/condyloma acuminate lesions by clinical analysis, 29 patients (70.7%) (G1 group) of the 41 patients, showed positive result for HPV cell injury by oncotic colpocitology and 12 patients (29.3%) (G2 group), presented negative result for cellular lesion and absence of clinical HPV lesion. Four samples were obtained per patient, which were submitted oncotic colpocitology analysis (Papanicolau staining, two samples), PCR (one sample) and ATR-FTIR analysis (one sample). L1 gene was amplified by PCR technique with specific GP5+/GP6+ and MY09/MY11 primers. PCR results were uniformly positive for presence of HPV in all analyzed samples. Multivariate analysis of ATR-FTIR spectra suggests no significant biochemical changes between groups and no clustering formed, concurring with results of PCR. This study suggests that PCR and ATR-FTIR are highly sensitive technique for HPV detection.

Analysis of molecular markers as predictive factors of lymph node involvement in breast carcinoma.

Nodal status is the most significant independent prognostic factor in breast cancer. Identification of molecular markers would allow stratification of patients who require surgical assessment of lymph nodes from the large numbers of patients for whom this surgical procedure is unnecessary, thus leading to a more accurate prognosis. However, up to now, the reported studies are preliminary and controversial, and although hundreds of markers have been assessed, few of them have been used in clinical practice for treatment or prognosis in breast cancer. The purpose of the present study was to determine whether protein phosphatase Mg2+/Mn2+ dependent 1D, ß-1,3-N-acetylglucosaminyltransferase, neural precursor cell expressed, developmentally down-regulated 9, prohibitin, phosphoinositide-3-kinase regulatory subunit 5 (PIK3R5), phosphatidylinositol-5-phosphate 4-kinase type IIα, TRF1-interacting ankyrin-related ADP-ribose polymerase 2, BCL2 associated agonist of cell death, G2 and S-phase expressed 1 and PAX interacting protein 1 genes, described as prognostic markers in breast cancer in a previous microarray study, are also predictors of lymph node involvement in breast carcinoma Reverse transcription-quantitative polymerase chain reaction analysis was performed on primary breast tumor tissues from women with negative lymph node involvement (n=27) compared with primary tumor tissues from women with positive lymph node involvement (n=23), and was also performed on primary tumors and paired lymph node metastases (n=11). For all genes analyzed, only the PIK3R5 gene exhibited differential expression in samples of primary tumors with positive lymph node involvement compared with primary tumors with negative lymph node involvement (P=0.0347). These results demonstrate that the PIK3R5 gene may be considered predictive of lymph node involvement in breast carcinoma. Although the other genes evaluated in the present study have been previously characterized to be involved with the development of distant metastases, they did not have predictive potential.

Micro-Raman spectroscopic study of thyroid tissues.

Thyroid carcinomas are the most common endocrine malignancy. Inconclusive results for the analysis of malignancies are an issue in the diagnosis of thyroid carcinomas; 20% of thyroid cancer diagnoses are indeterminate or suspicious, resulting in a surgical procedure without immediate need. The use of Raman spectroscopy may help improve the diagnosis of thyroid carcinoma. In this study, 30 thyroid samples, including normal thyroid, goiter and thyroid cancer, were analyzed by confocal Raman spectroscopy. Principal component analysis (PCA), linear discriminant analysis (LDA) with cross validation and binary logistic regression (BLR) analysis were applied to discriminate among tissues. Significant discrimination was observed, with a consistent rate of concordant pairs of 89.2% for normal thyroid versus cancer, 85.7% for goiter versus cancer and 80.6% for normal thyroid versus goiter using just the amide III region. Raman spectroscopy was thus proven to be an important and fast tool for the diagnosis of thyroid tissues. The spectral region of 1200-1400cm-1 discriminated normal versus goiter tissues despite the great similarity of these tissues.

Caracterização molecular e bioquímica de lesões de tireoide / Biochemical and Molecular characteristics of thyroid lesions

Introdução: O diagnóstico óptico através da espectroscopiaRaman é amplamente utilizado na indústria para estudo decomposição de materiais e recentemente foi introduzido emanálises de tecidos biológicos. É possível a caracterizaçãode lesões através de alterações bioquímicas identificadasprecocemente às alterações morfológicas. Objetivo: caracterizare diferenciar tecidos normais de tumorais da tireoide pelatécnica de espectroscopia Raman confocal. Método: Um totalde 21 espectros de tecido de tireoide, sendo 8 normais e 13tumorais (carcinoma papilífero), foram obtidos pela técnica deespectroscopia Raman confocal e processados pelos softwaresMatlab 6.1, Origin 8.6 e OPUS®. A análise estatística para aseparação das amostras baseados nas características bioquímicasde cada grupo foi realizada pelo programa Minitab®. Resultados:um importante aumento de intensidade de alguns modosvibracionais foi verificado nos espectros de câncer em relação aonormal. A análise multivariada possibilitou diferenciação de 100%para tecidos normais e de 84,6% para tumorais, o que equivalea uma proporção discriminante de 90,5%. Conclusão: A técnicade espectroscopia Raman se mostrou eficiente na detecção dealterações bioquímicas das lesões de tireoide e na diferenciaçãoentre os tecidos.