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The use of Fourier transform infrared (FTIR) and Raman spectroscopy (RS) for the analysis of lymphocytes in clinical applications is increasing in the field of biomedicine. The pre-analytical phase, which is the most vulnerable stage of the testing process, is where most errors and sample variance occur; however, it is unclear how pre-analytical variables affect the FTIR and Raman spectra of lymphocytes. In this study, we evaluated how pre-analytical procedures undertaken before spectroscopic analysis influence the spectral integrity of lymphocytes purified from the peripheral blood of male volunteers (n = 3). Pre-analytical variables investigated were associated with (i) sample preparation, (blood collection systems, anticoagulant, needle gauges), (ii) sample storage (fresh or frozen), and (iii) sample processing (inter-operator variability, time to lymphocyte isolation). Although many of these procedural pre-analytical variables did not alter the spectral signature of the lymphocytes, evidence of spectral effects due to the freeze-thaw cycle, in vitro culture inter-operator variability and the time to lymphocyte isolation was observed. Although FTIR and RS possess clinical potential, their translation into a clinical environment is impeded by a lack of standardisation and harmonisation of protocols related to the preparation, storage, and processing of samples, which hinders uniform, accurate, and reproducible analysis. Therefore, further development of protocols is required to successfully integrate these techniques into current clinical workflows.
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This review focuses on recent advances and future perspectives in the use of Raman spectroscopy for cervical cancer, a global women's health issue. Cervical cancer is the fourth most common women's cancer in the world, and unfortunately mainly affects younger women. However, when detected at the early precancer stage, it is highly treatable. High-quality cervical screening programmes and the introduction of the human papillomavirus (HPV) vaccine are reducing the incidence of cervical cancer in many countries, but screening is still essential for all women. Current gold standard methods include HPV testing and cytology for screening, followed by colposcopy and histopathology for diagnosis. However, these methods are limited in terms of sensitivity/specificity, cost, and time. New methods are required to aid clinicians in the early detection of cervical precancer. Over the past 20 years, the potential of Raman spectroscopy together with multivariate statistical analysis has been shown for the detection of cervical cancer. This review discusses the research to date on Raman spectroscopic approaches for cervical cancer using exfoliated cells, biofluid samples, and tissue ex vivo and in vivo.
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Infecções por Papillomavirus , Neoplasias do Colo do Útero , Feminino , Humanos , Neoplasias do Colo do Útero/patologia , Detecção Precoce de Câncer , Análise Espectral Raman/métodos , Infecções por Papillomavirus/diagnóstico , Saúde da MulherRESUMO
Irradiation of the tumour site during treatment for cancer with external-beam ionising radiation results in a complex and dynamic series of effects in both the tumour itself and the normal tissue which surrounds it. The development of a spectral model of the effect of each exposure and interaction mode between these tissues would enable label free assessment of the effect of radiotherapeutic treatment in practice. In this study Fourier transform Infrared microspectroscopic imaging was employed to analyse an in-vitro model of radiotherapeutic treatment for prostate cancer, in which a normal cell line (PNT1A) was exposed to low-dose X-ray radiation from the scattered treatment beam, and also to irradiated cell culture medium (ICCM) from a cancer cell line exposed to a treatment relevant dose (2 Gy). Various exposure modes were studied and reference was made to previously acquired data on cellular survival and DNA double strand break damage. Spectral analysis with manifold methods, linear spectral fitting, non-linear classification and non-linear regression approaches were found to accurately segregate spectra on irradiation type and provide a comprehensive set of spectral markers which differentiate on irradiation mode and cell fate. The study demonstrates that high dose irradiation, low-dose scatter irradiation and radiation-induced bystander exposure (RIBE) signalling each produce differential effects on the cell which are observable through spectroscopic analysis.
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Efeito Espectador , Lesões por Radiação , Masculino , Humanos , Efeito Espectador/efeitos da radiação , Quebras de DNA de Cadeia Dupla , Sobrevivência Celular/efeitos da radiação , Linhagem CelularRESUMO
Cell communication via exosomes is capable of influencing cell fate in stress situations such as exposure to ionizing radiation. In vitro and in vivo studies have shown that exosomes might play a role in out-of-target radiation effects by carrying molecular signaling mediators of radiation damage, as well as opposite protective functions resulting in resistance to radiotherapy. However, a global understanding of exosomes and their radiation-induced regulation, especially within the context of an intact mammalian organism, has been lacking. In this in vivo study, we demonstrate that, compared to sham-irradiated (SI) mice, a distinct pattern of proteins and miRNAs is found packaged into circulating plasma exosomes after whole-body and partial-body irradiation (WBI and PBI) with 2 Gy X-rays. A high number of deregulated proteins (59% of WBI and 67% of PBI) was found in the exosomes of irradiated mice. In total, 57 and 13 miRNAs were deregulated in WBI and PBI groups, respectively, suggesting that the miRNA cargo is influenced by the tissue volume exposed to radiation. In addition, five miRNAs (miR-99b-3p, miR-200a-3p, miR-200a, miR-182-5p, miR-182) were commonly overexpressed in the exosomes from the WBI and PBI groups. In this study, particular emphasis was also given to the determination of the in vivo effect of exosome transfer by intracranial injection in the highly radiosensitive neonatal cerebellum at postnatal day 3. In accordance with a major overall anti-apoptotic function of the commonly deregulated miRNAs, here, we report that exosomes from the plasma of irradiated mice, especially in the case of WBI, prevent radiation-induced apoptosis, thus holding promise for exosome-based future therapeutic applications against radiation injury.
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Exossomos , MicroRNAs , Lesões por Radiação , Animais , Apoptose , Cerebelo/metabolismo , Exossomos/metabolismo , Mamíferos/metabolismo , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Proteômica , Lesões por Radiação/metabolismoRESUMO
The quest for the discovery and validation of radiosensitivity biomarkers is ongoing and while conventional bioassays are well established as biomarkers, molecular advances have unveiled new emerging biomarkers. Herein, we present the validation of a new 4-gene signature panel of CDKN1, FDXR, SESN1 and PCNA previously reported to be radiation-responsive genes, using the conventional G2 chromosomal radiosensitivity assay. Radiation-induced G2 chromosomal radiosensitivity at 0.05 Gy and 0.5 Gy IR is presented for a healthy control (n = 45) and a prostate cancer (n = 14) donor cohort. For the prostate cancer cohort, data from two sampling time points (baseline and Androgen Deprivation Therapy (ADT)) is provided, and a significant difference (p > 0.001) between 0.05 Gy and 0.5 Gy was evident for all donor cohorts. Selected donor samples from each cohort also exposed to 0.05 Gy and 0.5 Gy IR were analysed for relative gene expression of the 4-gene signature. In the healthy donor cohort, there was a significant difference in gene expression between IR dose for CDKN1, FXDR and SESN1 but not PCNA and no significant difference found between all prostate cancer donors, unless they were classified as radiation-induced G2 chromosomal radiosensitive. Interestingly, ADT had an effect on radiation response for some donors highlighting intra-individual heterogeneity of prostate cancer donors.
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Inibidor de Quinase Dependente de Ciclina p21/genética , Proteínas de Choque Térmico/genética , Proteínas Mitocondriais/genética , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Antígeno Nuclear de Célula em Proliferação/genética , Neoplasias da Próstata/genética , Tolerância a Radiação/genética , Transcriptoma , Adulto , Idoso , Idoso de 80 Anos ou mais , Antagonistas de Androgênios/farmacologia , Antagonistas de Androgênios/uso terapêutico , Estudos de Casos e Controles , Cromossomos/efeitos da radiação , Estudos de Coortes , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Multiplex/métodos , Prognóstico , Neoplasias da Próstata/sangue , Neoplasias da Próstata/tratamento farmacológico , Doses de Radiação , Tolerância a Radiação/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Adulto JovemRESUMO
The brain undergoes ionizing radiation exposure in many clinical situations, particularly during radiotherapy for brain tumors. The critical role of the hippocampus in the pathogenesis of radiation-induced neurocognitive dysfunction is well recognized. The goal of this study is to test the potential contribution of non-targeted effects in the detrimental response of the hippocampus to irradiation and to elucidate the mechanisms involved. C57Bl/6 mice were whole body (WBI) or partial body (PBI) irradiated with 0.1 or 2.0 Gy of X-rays or sham irradiated. PBI consisted of the exposure of the lower third of the mouse body, whilst the upper two thirds were shielded. Hippocampi were collected 15 days or 6 months post-irradiation and a multi-omics approach was adopted to assess the molecular changes in non-coding RNAs, proteins and metabolic levels, as well as histological changes in the rate of hippocampal neurogenesis. Notably, at 2.0 Gy the pattern of early molecular and histopathological changes induced in the hippocampus at 15 days following PBI were similar in quality and quantity to the effects induced by WBI, thus providing a proof of principle of the existence of out-of-target radiation response in the hippocampus of conventional mice. We detected major alterations in DAG/IP3 and TGF-ß signaling pathways as well as in the expression of proteins involved in the regulation of long-term neuronal synaptic plasticity and synapse organization, coupled with defects in neural stem cells self-renewal in the hippocampal dentate gyrus. However, compared to the persistence of the WBI effects, most of the PBI effects were only transient and tended to decrease at 6 months post-irradiation, indicating important mechanistic difference. On the contrary, at low dose we identified a progressive accumulation of molecular defects that tended to manifest at later post-irradiation times. These data, indicating that both targeted and non-targeted radiation effects might contribute to the pathogenesis of hippocampal radiation-damage, have general implications for human health.
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Irradiação Craniana , Hipocampo/metabolismo , Hipocampo/efeitos da radiação , Metaboloma , Neurogênese/genética , Neurogênese/efeitos da radiação , Proteoma , Transcriptoma , Animais , Biologia Computacional/métodos , Irradiação Craniana/efeitos adversos , Feminino , Regulação da Expressão Gênica , Imuno-Histoquímica , Camundongos , Doses de Radiação , Transdução de SinaisRESUMO
Surface-enhanced Raman scattering (SERS) is a powerful and sensitive technique for the detection of fingerprint signals of molecules and for the investigation of a series of surface chemical reactions. Many studies introduced quantitative applications of SERS in various fields, and several SERS methods have been implemented for each specific application, ranging in performance characteristics, analytes used, instruments, and analytical matrices. In general, very few methods have been validated according to international guidelines. As a consequence, the application of SERS in highly regulated environments is still considered risky, and the perception of a poorly reproducible and insufficiently robust analytical technique has persistently retarded its routine implementation. Collaborative trials are a type of interlaboratory study (ILS) frequently performed to ascertain the quality of a single analytical method. The idea of an ILS of quantification with SERS arose within the framework of Working Group 1 (WG1) of the EU COST Action BM1401 Raman4Clinics in an effort to overcome the problematic perception of quantitative SERS methods. Here, we report the first interlaboratory SERS study ever conducted, involving 15 laboratories and 44 researchers. In this study, we tried to define a methodology to assess the reproducibility and trueness of a quantitative SERS method and to compare different methods. In our opinion, this is a first important step toward a "standardization" process of SERS protocols, not proposed by a single laboratory but by a larger community.
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OBJECTIVE: In 2016, there were an estimated 56 870 new cases of thyroid cancer (TC) in the USA. Fine needle aspiration cytology (FNAC) is the most safe, accurate and cost-effective method for the initial investigation of thyroid nodules. FNAC is limited by the inability to diagnose malignancy in follicular-patterned lesions accurately and, as a result, 20%-30% of cases under investigation for TC are classified as cytologically indeterminate, illustrating a problem with current FNAC procedure. Raman spectroscopy has shown promising results for the detection of many cancers; however, to date there has been no report on the performance of Raman spectroscopy on thyroid cytological samples. The aim of this study was to examine whether Raman spectroscopy could be used to correctly classify cell lines representing benign thyroid cells and various subtypes of TC. METHODS: A benign thyroid cell line and seven TC cell lines were prepared as ThinPrep® cytology slides and analysed with Raman spectroscopy. Principal components analysis and linear discriminant analysis were implemented to develop effective diagnostic algorithms for classification of Raman spectra of different TC subtypes. RESULTS: The spectral differences separating benign and TC cell lines were assigned to differences in the composition of nucleic acids, lipids, carbohydrates and protein in the benign and cancer cells. Good sensitivities (74%-85%), specificities (65%-93%) and diagnostic accuracies (71%-88%) were achieved for the identification of TC. CONCLUSION: These findings suggest that Raman spectroscopy has potential for preoperative TC diagnosis on FNAC samples.
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Citodiagnóstico/métodos , Análise Espectral Raman/métodos , Neoplasias da Glândula Tireoide/diagnóstico , Nódulo da Glândula Tireoide/diagnóstico , Biópsia por Agulha Fina , Feminino , Humanos , Masculino , Período Pré-Operatório , Glândula Tireoide/diagnóstico por imagem , Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/diagnóstico por imagem , Neoplasias da Glândula Tireoide/patologia , Nódulo da Glândula Tireoide/diagnóstico por imagem , Nódulo da Glândula Tireoide/patologiaRESUMO
Raman spectroscopy can provide a molecular-level signature of the biochemical composition and structure of cells with submicrometer spatial resolution and could be useful to monitor changes in composition for early stage and non-invasive cancer diagnosis, both ex-vivo and in vivo. In particular, the fingerprint spectral region (400-1800cm-1) has been shown to be very promising for optical biopsy purposes. However, limitations for discrimination of dysplastic and inflammatory processes based on the fingerprint region have been demonstrated. In addition, the Raman spectral signal of dysplastic cells is one important source of misdiagnosis of normal versus pathological tissues. The high wavenumber region (2800-3600cm-1) provides more specific information based on NH, OH and CH vibrations and can be used to identify the subtle changes which could be important for discrimination of samples. In this study, we demonstrate the potential of the high-wavenumber spectral region in this context by collecting Raman spectra of nucleolus, nucleus and cytoplasm from oral epithelial cancer (SCC-4) and dysplastic (DOK) cell lines and from normal oral epithelial primary cells, in vitro, in water immersion, which were then analyzed by principal components analysis as a method to discriminate the spectra. Analysis was performed before and after digital subtraction of the bulk water signal. In the normal cell line, the three subcellular regions are well differentiated before water subtraction, although the discrimination of the two nuclear regions is less well defined after water subtraction. Comparing the respective subcellular regions of the three cell lines, before water subtraction, the cell lines can be discriminated using sequential PCA and Feature Discriminant Analysis with up to ~100% sensitivity and 97% specificity for the cytoplasm, which is improved to 100% sensitivity and 99% specificity for the nucleus. The results are discussed in terms of discrimination comparing the CH vibrational modes of nucleic acids, proteins and lipids. The potential role of the OH vibrations, considering free water and confined water, in the discrimination of cell cultures and pathological processes are also discussed.
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Transformação Celular Neoplásica/patologia , Detecção Precoce de Câncer , Neoplasias Bucais/diagnóstico , Análise Espectral Raman , Linhagem Celular Tumoral , Núcleo Celular/patologia , Citoplasma/patologia , Células Epiteliais/patologia , Humanos , Neoplasias Bucais/patologiaRESUMO
Extensive research has been undertaken on the examination of tissue biopsies using vibrational spectroscopic techniques. However, fewer studies have focused on less invasive and commonly acquired blood samples. Recent studies have shown the ability of Raman and Fourier transform infrared (FTIR) spectroscopy to discriminate between non-cancer controls and cancer cases using blood serum or plasma. Even though many studies have proposed Raman spectroscopy as a potential diagnostic tool in various cancers, the Raman spectroscopic technique has not been introduced as a routine clinical technology. This is due to multiple drawbacks with the application of the technique, including sample preparation, the requirement for expensive substrates and long acquisition times. The current study aims to overcome these limitations and focuses on the translation of Raman spectroscopy into a high throughput clinical diagnostic tool for prostate cancer. In this study, the effect of different instrumental and sample preparation parameters were investigated, with the aim of identifying a combination that would reduce the overall acquisition time for spectra from peripheral blood plasma, reduce the complexity of sample preparation and retain the classification accuracy from Raman spectroscopic diagnostics. A high throughput (HT) system was developed and Raman spectroscopic measurements were performed on plasma samples from 10 prostate cancer patients and 10 healthy volunteers. The spectra were pre-processed and classified by principal component analysis - linear discriminant analysis (PCA-LDA) in the R environment. Statistically significant differences were observed between Raman spectra of prostate cancer patients and non-cancer controls. The (HT) classification resulted in a sensitivity and specificity of 96.5% and 95% respectively. Overall, this study has overcome some of the limitations associated with clinical translation of Raman spectroscopy. The HT-Raman spectroscopy method developed in this study can be used for rapid and accurate diagnosis of prostate cancer using liquid plasma samples.
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Detecção Precoce de Câncer , Neoplasias da Próstata/sangue , Neoplasias da Próstata/diagnóstico , Análise Espectral Raman , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Análise Discriminante , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Componente Principal , Adulto JovemRESUMO
Modern models of radiobiological effects include mechanisms of damage initiation, sensing and repair, for those cells that directly absorb ionizing radiation as well as those that experience molecular signals from directly irradiated cells. In the former case, the effects are termed targeted effects while, in the latter, non-targeted effects. It has emerged that phenomena occur at low doses below 1 Gy in directly irradiated cells that are associated with cell-cycle-dependent mechanisms of DNA damage sensing and repair. Likewise in non-targeted bystander-irradiated cells the effect saturates at 0.5 Gy. Both effects at these doses challenge the limits of detection of vibrational spectroscopy. In this paper, a study of the sensing of both targeted and non-targeted effects in HaCaT human keratinocytes irradiated with gamma ray photons is conducted with vibrational spectroscopy. In the case of directly irradiated cells, it is shown that the HaCaT cell line does exhibit both hyperradiosensitivity and increased radioresistance at low doses, a transition between the two effects occurring at a dose of 200 mGy, and that cell survival and other physiological effects as a function of dose follow the induced repair model. Both Raman and FTIR signatures are shown to follow a similar model, suggesting that the spectra include signatures of DNA damage sensing and repair. In bystander-irradiated cells, pro- and anti-apoptotic signalling and mechanisms of ROS damage were inhibited in the mitogen-activated protein kinase (MAPK) transduction pathway. It is shown that Raman spectral profiles of bystander-irradiated cells are correlated with markers of bystander signalling and molecular transduction. This work demonstrates for the first time that both targeted and non-targeted effects of ionizing radiation damage are detected by vibrational spectroscopy in vitro.
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Efeito Espectador/efeitos da radiação , Raios gama , Queratinócitos/efeitos da radiação , Análise Espectral , Vibração , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA , Relação Dose-Resposta à Radiação , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Raman spectroscopy can provide a molecular-level fingerprint of the biochemical composition and structure of cells with excellent spatial resolution and could be useful to monitor changes in composition for dysplasia and early, non-invasive cancer diagnosis (carcinoma in situ), both ex-vivo and in vivo. In this study, we demonstrate this potential by collecting Raman spectra of the nucleoli, nuclei and cytoplasm from oral epithelial cancer (SCC-4) and dysplastic (pre-cancerous, DOK) cell lines and from normal oral epithelial primary cell cultures, in vitro, which were then analysed by principal component analysis (PCA) as a multivariate statistical method to discriminate the spectra. Results show significant discrimination between cancer and normal cell lines. Furthermore, the dysplastic and cancer cell lines could be discriminated based on the spectral profiles of the cytoplasmic regions. The principal component loading plot, which elucidates the biochemical features responsible for the discrimination, showed significant contributions of nucleic acid and proteins for nucleolar and nuclear sites and variation in features of lipids for the cytoplasmic area. This technique may provide a rapid screening method and have potential use in the diagnosis of dysplasia and early, non-invasive oral cancer, the treatment of which involves much less extensive and complex surgery and a reduction in associated co-morbidity for the patient.
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Carcinoma de Células Escamosas/diagnóstico , Detecção Precoce de Câncer/métodos , Neoplasias Bucais/diagnóstico , Análise Espectral Raman/métodos , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Células Cultivadas , Humanos , Neoplasias Bucais/metabolismoRESUMO
Cervical cancer is the fourth most common cancer in women worldwide and mainly affects younger women. The mortality associated with cervical cancer can be reduced if the disease is detected at the pre-cancer stage. Current best-practice methods include cytopathology, HPV testing, and histopathology, but these methods are limited in terms of subjectivity, cost, and time. There is an unmet clinical need for new methods to aid clinicians in the early detection of cervical pre-cancer. These methods should be objective and rapid and require minimal sample preparation. Raman spectroscopy is a vibrational spectroscopic technique by which incident radiation is used to induce vibrations in the molecules of a sample and the scattered radiation may be used to characterise the sample in a rapid and non-destructive manner. Raman spectroscopy is sensitive to subtle biochemical changes occurring at the molecular level, enabling spectral variations corresponding to disease onset to be detected. Over the past 15 years, there have been numerous reports revealing the potential of Raman spectroscopy together with multivariate statistical analysis for the detection of a variety of cancers. This paper discusses the recent advances and challenges for cervical-cancer screening and diagnosis and offers some perspectives for the future.
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Colo do Útero/patologia , Análise Espectral Raman/métodos , Neoplasias do Colo do Útero/diagnóstico , Alphapapillomavirus/isolamento & purificação , Colo do Útero/virologia , Feminino , Humanos , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/patologia , Neoplasias do Colo do Útero/virologiaRESUMO
Cervical cancer is the third most common cancer affecting women worldwide. The mortality associated with cervical cancer can, however, be significantly reduced if the disease is detected at the pre-malignant stage. The aim of this study was to evaluate the potential of Raman microspectroscopy for elucidation of the biochemical changes associated with the pre-malignant stages of cervical cancer. Formalin fixed paraffin preserved tissue sections from cervical biopsies classified as negative for intraepithelial lesion and malignancy (NILM), low grade squamous intraepithelial lesion (LSIL) or high grade squamous intraepithelial lesion (HSIL) were analysed by Raman spectral mapping. Raman mapping, with K-means cluster analysis (KMCA), was able to differentiate the NILM cervical tissue into three layers including stroma, basal/para-basal and superficial layers, characterised by spectral features of collagen, DNA bases and glycogen respectively. In the LSIL and HSIL samples, KMCA clustered regions of the superficial layer with the basal layer. Using principal components analysis (PCA), biochemical changes associated with disease were also observed in normal areas of the abnormal samples, where morphological changes were not apparent. This study has shown that Raman microspectroscopy could be useful for the early detection of pre-malignant changes in cervical tissue.
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Análise Espectral Raman/métodos , Lesões Intraepiteliais Escamosas Cervicais/diagnóstico , Displasia do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/diagnóstico , Análise por Conglomerados , Feminino , Humanos , Análise de Componente PrincipalRESUMO
In vitro and in vivo observations accumulated over several decades have firmly shown that the biological effects of ionizing radiation can spread from irradiated cells/tissues to non-targeted cells/tissues. Redox-modulated intercellular communication mechanisms that include a role for secreted factors and gap junctions, can mediate these non-targeted effects. Clearly, the expression of such effects and their transmission to progeny cells has implications for issues related to radiation protection. Their elucidation is also relevant towards enhancing the efficacy of cancer radiotherapy and reducing its impact on the development of normal tissue toxicities. In addition, the study of non-targeted effects is pertinent to our basic understanding of intercellular communications under conditions of oxidative stress. This review will trace the history of non-targeted effects of radiation starting with early reports of abscopal effects which described radiation induced effects in tissues distant from the site of radiation exposure. A related effect involved the production of clastogenic factors in plasma following irradiation which can induce chromosome damage in unirradiated cells. Despite these early reports suggesting non-targeted effects of radiation, the classical paradigm that a direct deposition of energy in the nucleus was required still dominated. This paradigm was challenged by papers describing radiation induced bystander effects. This review will cover mechanisms of radiation-induced bystander effects and the potential impacts on radiation protection and radiation therapy.
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Efeito Espectador , Efeito Espectador/efeitos da radiação , Humanos , Animais , Comunicação Celular/efeitos da radiação , Estresse Oxidativo/efeitos da radiaçãoRESUMO
Understanding the interaction of anticancer drugs with model cell lines is important to elucidate the mode of action of these drugs as well as to develop cost effective and rapid screening methods. Raman spectroscopy has been demonstrated to be a valuable technique for high throughput, noninvasive analysis. The interaction of vincristine with a human lung adenocarcinoma cell line (A549) was investigated using Raman micro spectroscopy. The results were correlated with parallel measurements from the MTT cytotoxicity assay, which yielded an IC50 value of 0.10 ± 0.03 µM. The Raman spectral data acquired from vincristine treated A549 cells was analysed to understand its interaction with the nucleus in the cell and elucidate DNA intercalation. The dose dependent spectral changes in the nucleus are analysed by PLS-Jack knifing for the identification of the more significant changes associated with the mode of action of the drug. Results are correlated with a similar dose dependent expression analysis of the bcl-2 protein, an anti-apoptotic protein associated with DNA damage, in the vincristine treated A549 cells using flow cytometry. The results indicate the co-existence of two modes of action, microtubule binding at low doses and DNA intercalation at high doses.
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Regulação Neoplásica da Expressão Gênica , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Análise Espectral Raman/métodos , Vincristina/análise , Vincristina/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Humanos , Ligação Proteica/fisiologiaRESUMO
It is known that ionising radiation (IR) induces a complex signalling apoptotic cascade post-exposure to low doses ultimately to remove damaged cells from a population, specifically via the intrinsic pathway. Therefore, it was hypothesised that bystander reporter cells may initiate a similar apoptotic response if exposed to low doses of IR (0.05Gy and 0.5Gy) and compared to directly irradiated cells. Key apoptotic genes were selected according to their role in the apoptotic cascade; tumour suppressor gene TP53, pro-apoptotic Bax and anti-apoptotic Bcl2, pro-apoptotic JNK and anti-apoptotic ERK, initiator caspase 2 and 9 and effector caspase 3, 6 and 7. The data generated consolidated the role of apoptosis following direct IR exposure for all doses and time points as pro-apoptotic genes such as Bax and JNK as well as initiator caspase 7 and effector caspase 3 and 9 were up-regulated. However, the gene expression profile for the bystander response was quite different and more complex in comparison to the direct response. The 0.05Gy dose point had a more significant apoptosis gene expression profile compared to the 0.5Gy dose point and genes were not always expressed within 1h but were sometimes expressed 24h later. The bystander data clearly demonstrates initiation of the apoptotic cascade by the up-regulation of TP53, Bax, Bcl-2, initiator caspase 2 and effector caspase 6. The effector caspases 3 and 7 of the bystander samples demonstrated down-regulation in their gene expression levels at 0.05Gy and 0.5Gy at both time points therefore not fully executing the apoptotic pathway. Extensive analysis of the mean-fold gene expression changes of bystander data demonstrated that the apoptosis is initiated in the up-regulation of pro-apoptotic and initiator genes but may not very well be executed to final stages of cell death due to down-regulation of effector genes.
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Apoptose/efeitos da radiação , Biomarcadores/metabolismo , Efeito Espectador/efeitos da radiação , Perfilação da Expressão Gênica , Queratinócitos/efeitos da radiação , Radiação Ionizante , Transdução de Sinais/efeitos da radiação , Caspases/metabolismo , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Fibrinogen assays are commonly used as part of clinical screening tests to investigate haemorrhagic states, for detection of disseminated intravascular coagulation and as a predictor of a variety of cardiovascular events. The Clauss assay, which measures thrombin clotting time, is the most commonly used method for measuring fibrinogen levels. Nevertheless, inconsistencies are present in inter-manufacturer reagent sources, calibration standards and methodologies. Automated coagulation analysers, which measure changes in optical density during the prothrombin time (PT-Fg), have found use in many hospitals. However, the PT-Fg method is found to give falsely elevated values due to varying choices of calibrants, reagents and analysers. As an alternative, Raman spectroscopy has previously been applied to the analysis of blood and its various constituents to determine various analyte concentrations such as glucose, urea, triglycerides and cholesterol. In this study, Raman spectroscopy was investigated for its ability to accurately quantify fibrinogen concentration in blood plasma. Samples collected from 34 patients were analysed by Raman spectroscopy and the resultant spectra were fitted with a Partial Least Squares Regression model using target values obtained through a pre-calibrated Clauss fibrinogen assay. Various spectral pre-processing methods were utilised to prepare data to be entered into a calibration model. A root mean square error of prediction of 0.72 ± 0.05 g/L was achieved with as few as 25 spectra. In this pilot study, Raman spectroscopy has been demonstrated to be a robust technique providing rapid and reagent-free quantification of fibrinogen levels in blood plasma and a potential alternative to the Clauss assay.
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Fibrinogênio/análise , Plasma , Análise Espectral Raman/métodos , Calibragem , Humanos , Indicadores e Reagentes , Análise de Regressão , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
OBJECTIVES: To describe the contribution of women radiobiologists in Ireland to the development of the discipline internationally and at home and to discuss the history of radiobiology in Ireland to date. This parallels the history of the evolution of a small radiobiology group in Kevin Street, Dublin Institute of Technology (DIT) which was formerly part of the City of Dublin Vocational Education Committee. There followed years of development first as a radiobiological research center which evolved in the FOCAS Research Institute now embedded within Technological University Dublin (TU Dublin). CONCLUSIONS: Over the last 45 years, the women of the Radiation and Environmental Science Centre (RESC) contributed to the major paradigm shift in low dose radiobiology contributing exciting new research concerning non-targeted effects, including discovery of lethal mutations, medium transfer bystander mechanisms, and signaling pathways. They also developed translational research using human explant culture systems with unique immunocytochemical methods and more recently evolved to molecular and spectroscopic analysis of clinical samples. The RESC also developed unique in vitro research methods into effects of radiation on non-human species of concern in ecosystems.
Assuntos
Ecossistema , Radiobiologia , Academias e Institutos , Meios de Cultura , Feminino , Humanos , IrlandaRESUMO
Recent reports have shown a link between radiation exposure and non-cancer diseases such as radiation-induced heart disease (RIHD). Radiation exposures are often inhomogeneous, and out-of-target effects have been studied in terms of cancer risk, but very few studies have been carried out for non-cancer diseases. Here, the role of miRNAs in the pathogenesis of RIHD was investigated. C57Bl/6J female mice were whole- (WBI) or partial-body-irradiated (PBI) with 2 Gy of X-rays or sham-irradiated (SI). In PBI exposure, the lower third of the mouse body was irradiated, while the upper two-thirds were shielded. From all groups, hearts were collected 15 days or 6 months post-irradiation. The MiRNome analysis at 15 days post-irradiation showed that miRNAs, belonging to the myomiR family, were highly differentially expressed in WBI and PBI mouse hearts compared with SI hearts. Raman spectral data collected 15 days and 6 months post-irradiation showed biochemical differences among SI, WBI and PBI mouse hearts. Fibrosis in WBI and PBI mouse hearts, indicated by the increased deposition of collagen and the overexpression of genes involved in myofibroblast activation, was found 6 months post-irradiation. Using an in vitro co-culture system, involving directly irradiated skeletal muscle and unirradiated ventricular cardiac human cells, we propose the role of miR-1/133a as mediators of the abscopal response, suggesting that miRNA-based strategies could be relevant for limiting tissue-dependent reactions in non-directly irradiated tissues.