RESUMEN
Simple and standardized approaches for genome analysis of human papillomavirus (HPV) by next-generation sequencing are needed. The aim of the study was to develop a protocol for direct deep sequencing of high-risk (hr) HPV strains, based on the widely used commercial Hybrid Capture 2 (QIAGEN) test, without any additional probe design. This protocol was applied to 15 HPV-positive and two HPV-negative cervical samples or cell lines and validated at the genotype level by comparing the sequencing results to those obtained using a commercial genotyping kit. The performance of our protocol, presented in this proof-of-principle study, supports its use for accurate characterization of genetic variants of hrHPV.
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Alphapapillomavirus , Infecciones por Papillomavirus , Neoplasias del Cuello Uterino , Cuello del Útero , ADN Viral , Femenino , Genotipo , Humanos , Papillomaviridae/genética , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Despite the availability of safe and effective HPV vaccines in France, more than 80% of girls remain unvaccinated. SETTING: A regional university hospital referral center in France. OBJECTIVE: To estimate the overall prevalence and distribution of HPV in vaccinated, sexually active young French women who were screened for cervical cancer by cytology and HPV testing. METHODS: High-risk HPV (HR-HPV) prevalence, genotype-specific prevalence and extent of multiple infections were assessed in 125 cervical samples from females with available vaccine data using hc2 assay and INNO-LiPA assay. HPV status was analyzed in accordance with cytological data. RESULTS: In our series, mean age was 23 years, overall prevalence of HR-HPV was 52% and was correlated with the lesion grade. The diversity of HPV genotypes was broad. Single HR-HPV infections were identified in 11%, 21% and 47% of women with NILM, ASC-US/-H and LSIL respectively. Multiple infections with HR-HPV were detected in 28% of the specimens. Only 24.5% of women with NILM presented infections with 2 genotypes or more, vs 28% of women with ASC-US/-H and 35% of women with LSIL. The overall prevalence of genotypes covered by the quadrivalent vaccine was low (5.9%); with 4.2%, 0%, 0.8% and 0.8% for HPV 16, HPV 18, HPV 6 and HPV 11 respectively. CONCLUSION: Among HPV-vaccinated young women, HR-HPV are detected at a high rate, and an association with the grade of cytological abnormalities was observed. However, HPV 16 and 18, both targeted by the vaccines, are remarkably rare among young French women since program implementation.
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Papillomavirus Humano 11 , Papillomavirus Humano 16 , Papillomavirus Humano 18 , Papillomavirus Humano 6 , Infecciones por Papillomavirus/virología , Vacunas contra Papillomavirus/uso terapéutico , Neoplasias del Cuello Uterino/prevención & control , Adulto , Femenino , Francia/epidemiología , Humanos , Infecciones por Papillomavirus/epidemiología , Prevalencia , Adulto JovenRESUMEN
Prescription of an HPV test in practice will enable the clinician to optimize the monitoring and the management of patients, especially in the context of cervical cancer screening. Numerous HPV tests are available that present different analytical and clinical sensitivity and specificity. International recommendations on clinical performance of HPV tests used for cervical cancer screening have been published by a group of experts, and tests that meet these performance criteria should be used. Apart from the HPV detection kit, the whole circuit from sampling to report of the results must be considered. This implies that the pre-analytical (sampling, quality of sample collection medium, storage condition and sample transportation ) and post-analytical steps (quality of result reporting, providing expert advices ) are also standardized. For this purpose, medical-biology laboratories are subjected to a COFRAC certification, as defined by the international standard ISO 15189 providing quality criteria for any clinical laboratory test and HPV test in particular.
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Sondas de ADN de HPV/normas , Infecciones por Papillomavirus/diagnóstico , Manejo de Especímenes/normas , Femenino , HumanosRESUMEN
OBJECTIVES: To assess opinions, practices and difficulties of general practitioners (GP) of Besançon concerning human papillomavirus (HPV) vaccination. MATERIALS AND METHODS: A survey among the 140 GP of Besançon, France, was conducted in 2015. RESULTS: A percentage of 77.1 reported being favourable to HPV vaccination and 72.9% practices HPV vaccination. The 2 main concerns about HPV vaccination for GP are the fear of side effects (for 40.6% of GP) and the doubt on efficacy. According to GP, the hepatitis B vaccination controversy, the fear of side effects, the limited clinical efficacy experience and the lack of confidence in health authorities are concerns about HPV vaccination for 77.1%, 76%, 74% and 49% of patients, respectively. CONCLUSION: Courses for GP on HPV vaccination must be pursued and reinforced. A school-based program could be developed to facilitate communication between GP and patients to improve HPV vaccination coverage.
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Actitud del Personal de Salud , Médicos Generales/estadística & datos numéricos , Conocimientos, Actitudes y Práctica en Salud , Infecciones por Papillomavirus/prevención & control , Vacunas contra Papillomavirus/uso terapéutico , Vacunación/estadística & datos numéricos , Adulto , Anciano , Femenino , Francia , Humanos , Masculino , Persona de Mediana EdadAsunto(s)
ADN Viral/análisis , Cejas/química , Poliomavirus/aislamiento & purificación , Psoriasis/tratamiento farmacológico , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Remoción del Cabello , Humanos , Inmunosupresores/uso terapéutico , Poliomavirus de Células de Merkel/aislamiento & purificación , Metotrexato/uso terapéutico , Poliomavirus/genética , Estudios ProspectivosRESUMEN
OBJECTIVES: High risk human papilloma-viruses (HR HPV) are associated with risk of cervical dysplasia and carcinoma. The risk is increased in patients with immune deficiency or auto-immune disease as systemic lupus erythematosus. Currently, no data are available about the human papillomavirus status in women with systemic sclerosis (SSc). METHODS: Thirty-one women with SSc were evaluated for cervical HPV infection and dysplasia, and compared to fifty age-matched control. Cervical swabs were tested by the INNO-LiPA assay®. Serum antibodies against HPV 16 and 18 were assessed using enzyme-linked immunosorbent assay in the SSc group. RESULTS: The overall HPV frequency was comparable between SSc and controls (32% vs. 38%), as well as the HR HPV frequency (28% vs. 34%), but infection by ≥2 HPV was two times more frequent in the SSc group (50% vs. 26% of the HPV positive samples). The most prevalent genotype was 52 in the SSc group (12%), and 52/53 in the control group (8% for both). Pap smears were within the normal range. Seropositivity for HPV 16 and 18 was 13% and 6.5%, respectively. A diffuse systemic sclerosis and a younger age at first intercourse were more frequent in cases of overall HPV positivity. Current smoking and a higher number of sexual partners were only observed in cases of seropositivity. CONCLUSIONS: This is the first study to evaluate HPV status in women with SSc. HR HPV52 was the most common genotype with a greater multi-HPV infection rate. This result needs to be confirmed in a larger study.
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ADN Viral/genética , Infecciones por Papillomavirus/epidemiología , Esclerodermia Sistémica/epidemiología , Neoplasias del Cuello Uterino/epidemiología , Anciano , Anticuerpos Antivirales/inmunología , Estudios de Casos y Controles , Detección Precoz del Cáncer , Femenino , Genotipo , Papillomavirus Humano 16/inmunología , Papillomavirus Humano 18/inmunología , Humanos , Persona de Mediana Edad , Prueba de Papanicolaou , Papillomaviridae/genética , Infecciones por Papillomavirus/diagnóstico , Infecciones por Papillomavirus/inmunología , Factores de Riesgo , Estudios Seroepidemiológicos , Parejas Sexuales , Fumar/epidemiología , Neoplasias del Cuello Uterino/diagnóstico , Neoplasias del Cuello Uterino/inmunología , Frotis VaginalRESUMEN
BACKGROUND: Tumour necrosis factor alpha (TNF-α) inhibitors are associated with an increased risk of infections and with a still debatable risk of skin cancer. Furthermore, cutaneous human papillomavirus (HPV) infection may be involved in skin cancer. OBJECTIVES: Our primary objective was to assess the HPV DNA prevalence in psoriasis patients treated with TNF inhibitors and the secondary objective was to assess the same parameter before and during treatment. METHODS: Plucked eyebrow hairs were collected from 151 consecutive patients with moderate to severe chronic plaque psoriasis, including 48 patients treated with anti-TNF-α agents, 21 patients treated with methotrexate (MTX) and 82 patients with no previous systemic treatment. Among them, 38 patients were subsequently treated with either MTX or anti-TNF-α agents. HPV genotyping was performed using the HPV type-specific E7 PCR bead-based multiplex allowing the detection of 27 genus-α types, 25 genus-ß types, 16 genus-γ types and one single genus-µ type. Follow-up provided a total of 972.7 person-months of overall exposure for patients treated with TNF inhibitors and 326.9 person-months for patients treated with MTX. RESULTS: Our data confirm the high prevalence of ß-HPV infection in healthy skin of psoriasis patients (68.9%), with no significant difference between untreated patients (64.6%), patients treated with MTX (76.2%) and patients treated with anti-TNF-α agents (72.9%). The mean number of different HPV types and the distribution of HPV types were similar in different groups of patients. Moreover, in prospectively treated patients, we did not observe any change in the HPV DNA prevalence in the distribution of HPV types and the number of HPV types after a mean duration of treatment of 332 ± 39.8 days. CONCLUSION: Despite the small number of patients in our cohort, our results are quite encouraging in view of the increased use of anti-TNF-α agents in different auto inflammatory immune diseases.
Asunto(s)
ADN Viral/análisis , Cejas , Cabello/química , Papillomaviridae/genética , Psoriasis/tratamiento farmacológico , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Humanos , Psoriasis/fisiopatologíaRESUMEN
OBJECTIVE: To assess the expected impact in France of a quadrivalent HPV 6/11/16/18 vaccine on the occurrence of genital HPV-induced lesions in women. METHODS: A Markov model based on a quadrivalent vaccination of 14-year-old girls as recommended in France was performed to assess the number of subjects needed to vaccinate to prevent an HPV-related event during their lifetime and the expected annual number of cases which could be prevented by vaccination. This model was based on prevalence data reported in four large French studies (EDiTH I-IV) reporting an HPV 6/11/16/18 prevalence of 82% (95% CI: 78.5-85.1) in cervical cancer (CC), 64% (95% CI: 59.7-68.1) in CIN2/3, 34% (95% CI: 28.9-38.1) in low-grade squamous intraepithelial lesions (LSIL) and 83% (95% CI 77.6-87.8) in female external acuminata condylomata (EAC) cases. RESULTS: Using a theoretical vaccine efficacy of 100%, 130 young women need to be vaccinated to prevent a case of CC, 17 for a case of CIN2/3 and 13 for a case of EAC. Immunization of 80% of 14-year-old girls could prevent 2495 CC (72%), 17,985 CIN2/3 (54%), 8004 CIN1 (27%), and 22,531 EAC female cases (65%) in France annually. CONCLUSION: A good adhesion to the preferentially recommended HPV quadrivalent vaccination would thus substantially reduce the burden of female genital lesions in France.
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Condiloma Acuminado/prevención & control , Infecciones por Papillomavirus/prevención & control , Vacunas contra Papillomavirus , Displasia del Cuello del Útero/prevención & control , Neoplasias del Cuello Uterino/prevención & control , Adolescente , Condiloma Acuminado/epidemiología , Condiloma Acuminado/virología , Femenino , Francia/epidemiología , Humanos , Cadenas de Markov , Modelos Teóricos , Infecciones por Papillomavirus/epidemiología , Vacunas contra Papillomavirus/administración & dosificación , Neoplasias del Cuello Uterino/epidemiología , Neoplasias del Cuello Uterino/virología , Displasia del Cuello del Útero/epidemiología , Displasia del Cuello del Útero/virologíaRESUMEN
BACKGROUND: Human immunodeficiency virus (HIV)+ patients have an increased risk of anogenital warts. High-risk (HR) human papillomaviruses (HPVs), especially types 16 and 18, are major risk factors for precancerous and cancerous lesions of the anogenital tract, while low-risk (LR) HPVs are associated with benign lesions. Cure of genital warts with ablative techniques, surgical excision, podophyllotoxin or trichloroacetic acid is frequently difficult. Treatment with imiquimod cream showed a total clearance of external genital or perianal warts in about 50% of immunocompetent subjects. However, total clearance was reduced in HIV+ subjects not treated with highly active antiretroviral therapy (HAART). OBJECTIVES: To assess clinically and by monitoring HPV content the efficacy of 5% topical imiquimod to treat anogenital warts in HIV+ subjects with at least partially restored immune functions. METHODS: Fifty HIV+ patients successfully treated with HAART (total CD4+ cells > or = 200 cells mm(-3) and plasma HIV RNA load < 10(4) copies mL(-1)) with anogenital warts were included. Imiquimod 5% cream was applied on external genital or perianal warts three times weekly for up to 16 weeks. Warts were tested at entry and after treatment for human LR- and HR-HPV DNA. RESULTS: Total wart clearance was observed in 16 of 50 (32%) patients at week 16. At enrolment, HPV DNA was present in more than 90% of lesions with a majority of lesions co-infected by HR- and LR-HPV. At study end, the HPV load decreased or became undetectable in 40% of cases studied. CONCLUSIONS: Imiquimod 5% cream did not show safety concerns and is suitable for use in HIV+ subjects with anogenital warts and successful HAART treatment.
Asunto(s)
Infecciones Oportunistas Relacionadas con el SIDA/tratamiento farmacológico , Adyuvantes Inmunológicos/administración & dosificación , Aminoquinolinas/administración & dosificación , Enfermedades del Ano/tratamiento farmacológico , Condiloma Acuminado/tratamiento farmacológico , Infecciones por Papillomavirus/tratamiento farmacológico , Infecciones Oportunistas Relacionadas con el SIDA/virología , Administración Cutánea , Adolescente , Adulto , Terapia Antirretroviral Altamente Activa , Enfermedades del Ano/virología , Condiloma Acuminado/virología , Esquema de Medicación , Femenino , Humanos , Imiquimod , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Infecciones por Papillomavirus/virología , Resultado del Tratamiento , Adulto JovenRESUMEN
The ligninolytic fungus Irpex lacteus was shown as an efficient degrader of oligocyclic aromatic hydrocarbons (PAHs; 'polycyclic aromatic hydrocarbons') possessing 3-6 aromatic rings in complex liquid media. The strain produced mainly Mn-dependent peroxidase in media without pollutants. Activity of ligninolytic enzymes was higher in a N-limited medium. However, after contamination with PAHs (especially pyrene) the values increased and significant activity of Mn-independent peroxidase appeared in the complex medium. Other factors (such as the increase in nitrogen concentration or the presence of solvent(s) for dissolution of PAHs) had no effect. Cytochrome P-450 was detected in the microsomal fraction of biomass grown in the complex medium. The rate of PAH degradation was also affected by the presence of various combinations of PAHs. However, independently of the enzyme activities, anthracene was shown to have a positive influence on degradation of pyrene and fluoranthene.
Asunto(s)
Basidiomycota/enzimología , Basidiomycota/metabolismo , Proteínas Fúngicas/metabolismo , Lignina/metabolismo , Peroxidasas/metabolismo , Hidrocarburos Policíclicos Aromáticos/metabolismo , Basidiomycota/genética , Biodegradación Ambiental , Proteínas Fúngicas/genética , Peroxidasas/genética , Hidrocarburos Policíclicos Aromáticos/químicaRESUMEN
INTRODUCTION: Cervical intraepithelial neoplasia (CIN) 2 and CIN3 lesions clearly represent precancerous states even if some of them would heal spontaneously. Management is based on surgical excision of part of the uterine cervix because such lesions can potentially progress into carcinomas. In most cases, this treatment leads to the cure of intraepithelial lesions. However, even after such an efficient treatment, theses patients are still at a higher risk of developing an invasive cervical cancer. That is why guidelines recommend a specific follow-up in order to screen for residual disease (incomplete excision) or for recurrences (after a complete excision). The actual problem in the follow-up strategy lies in the screening tools in use - cervical smears and colposcopy - whose sensitivities are low and hence, not quite sufficient when applied to a high risk population. These intraepithelial lesions are due to high risk human papillomaviruses (HPV) and there cannot be any lesion progression without HPV. Consequently, a viral testing would help in identifying a high risk subpopulation of women after cone loop cervical excision. MATERIAL AND METHODS: We studied, retrospectively, the contribution of HPV testing (Hybrid Capture 2((R))) in the follow-up after CIN2-3 treatment in 386 cone loop cervical excisions performed at a single centre during 80 months. RESULTS: Between three to six months follow-up after surgery, HPV remained present in 22.5% cases. The sensitivity of HPV testing in the screening for residual lesions or for recurrences was 100%, that of cervical smears cytology was 72%, whereas that of the pathological analysis of margins reached only 67%. The negative predictive value of a negative HPV detection associated with a normal cytology was 100%. DISCUSSION: Owing to its clinical relevance, HPV testing optimises postoperative follow-up and leads to the rapid and efficient selection of a subgroup, representing less than one upon three patients who are really at risk of an invasive lesion and to wholly reassure the others. Indeed, a negative HPV testing, associated with a normal cervical cytology, obtained after surgery correspond to a negative predictive value of almost 100% and this allows us to increase the time-interval between two screenings and to rapidly place the patient in a routine follow-up.
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Tamizaje Masivo , Papillomaviridae/aislamiento & purificación , Displasia del Cuello del Útero/virología , Neoplasias del Cuello Uterino/virología , Adulto , Femenino , Estudios de Seguimiento , Humanos , Recurrencia Local de Neoplasia/diagnóstico , Neoplasia Residual , Infecciones por Papillomavirus/diagnóstico , Valor Predictivo de las Pruebas , Estudios Retrospectivos , Sensibilidad y Especificidad , Neoplasias del Cuello Uterino/cirugía , Displasia del Cuello del Útero/cirugíaRESUMEN
Cervical cancer, the second most common cancer in young women in France, is still today imperfectly screened even with the advent of primary prevention for this cancer in the form of prophylactic HPV vaccination. Indeed, the cervical Pap smear and its cytologic analysis, both operator and reader dependent, have limited sensitivities requiring repeated samplings and above all, producing a high rate of falsely negative tests. Although most cancers occur in women who are either not or insufficiently screened, the problem with cervical smears is the fact that cancers are also often diagnosed in young women having follow-ups in accordance with professional guidelines. The absence of an organized screening in France results in an inadequate female population coverage. Nowadays, it is unanimously recognized that high-risk papillomaviruses (HR HPV) represent the only independent risk factor for cervical cancer and that there cannot be any disease without this virus. It is therefore this strong association between a viral agent and the cervical cancer which opened the door firstly, to the notion of prophylactic vaccination and secondly, to the integration of HR HPV testing in the screening for precancerous lesions. Molecular biological techniques based on the HR HPV genome detection within the female genital tract have shown a very high sensitivity without any inter and intraobserver variability and an excellent negative predictive value. Their integration in the primary screening for cervical cancer would improve the relevance of the latter and would suit the need for a wider population coverage and even for an organized screening thanks to the possibility for self-sampling. The specificity of these tests is inferior to that of the cervical smear, but the management of the falsely positive HPV tests has proved to be efficient by sorting residual cells obtained from liquid-based cytology. What is urgent in France is the need for an organized screening programme in order to improve population coverage and, this does not go against neither a vaccination promotion nor the integration of new technologies. Moreover, the last three randomized trials published in October 2007 have shown that it was quite safely possible to extend the time interval between two consecutive viral testing and thus improving the cost-effectiveness of cervical cancer screening. The aim of this work was to analyze publications on the subject in order to conclude, according to proof levels obtained by different studies, on its usefulness in the secondary prevention of cervical cancer.
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Papillomaviridae/aislamiento & purificación , Neoplasias del Cuello Uterino/prevención & control , Neoplasias del Cuello Uterino/virología , ADN Viral/análisis , Reacciones Falso Negativas , Femenino , Francia/epidemiología , Humanos , Tamizaje Masivo , Prueba de Papanicolaou , Papillomaviridae/genética , Vacunas contra Papillomavirus , Factores de Riesgo , Sensibilidad y Especificidad , Neoplasias del Cuello Uterino/epidemiología , Frotis VaginalRESUMEN
The study was aimed to evaluate the feasibility of detecting human papillomavirus (HPV) in women with normal or abnormal cervical smears using the Roche Amplicor MWP HPV Test. We compared by AMPLICOR Test and Hybrid Capture II (HCII) Test, the prevalence of HR-HPV in 470 cervical samples including 55 samples with WNL cytology, 208 ASC-US, 193 LGSIL and 14 HGSIL. Samples with discordant results were retested with INNO-LiPA Genotyping HPV Test v2. The HR-HPV positivity in WNL cytology samples was similar (21.8%) by AMPLICOR and HCII. In ASC-US, the HPV positivity was 42.3% by both tests. In LGSIL, HPV positivity was 66.3% and 66.8% by AMPLICOR and HCII, respectively. In HGSIL, 92.8% of samples were positive by AMPLICOR and 85.7% by HCII. The agreement of both tests was 96.2% with a Kappa value of 0.92. Eighteen cases were discordant: 9 HCII positive/AMPLICOR negative and 9 HCII negative/AMPLICOR positive. The INNO-LiPA test revealed HPV positivity in every case. Interestingly, all HCII+/AMPLICOR- samples were found to harbour HPV53. As for the HCII-/AMPLICOR+ samples, 8 demonstrated a multiple infection with HR 16- and/or 18- and/or 56-phylogenetically related HPV types. Moreover, two of these samples were co-infected with HPV6 and two other with HPV54. By using consensus HR-HPV as our reference HPV positivity, the sensitivity (96.6%) and specificity (100%) of AMPLICOR was similar to that of HCII Test. The AMPLICOR HPV Test is sensitive, specific, feasible and appropriate for routine HPV detection.
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Papillomaviridae/genética , Papillomaviridae/aislamiento & purificación , Infecciones por Papillomavirus/virología , Juego de Reactivos para Diagnóstico , Displasia del Cuello del Útero/virología , Neoplasias del Cuello Uterino/virología , Adolescente , Adulto , Anciano , Cuello del Útero , Femenino , Genotipo , Humanos , Persona de Mediana Edad , Técnicas de Amplificación de Ácido Nucleico , Hibridación de Ácido Nucleico , Papillomaviridae/clasificación , Infecciones por Papillomavirus/patología , Reacción en Cadena de la Polimerasa , Sensibilidad y Especificidad , Neoplasias del Cuello Uterino/patología , Frotis Vaginal , Displasia del Cuello del Útero/patologíaRESUMEN
AIMS: To investigate human papillomavirus (HPV) genotype-specific prevalence in the high-risk Kazakh ethnic group with esophageal squamous cell carcinoma (ESCC). METHODS: Sixty-seven Kazakh patients with primary ESCC were studied. From each patient, two tissue samples were collected: one sample of the tumor and one sample of normal esophageal tissue from an area away from the tumor. Tissues were analyzed by INNO-LiPA HPV Genotyping test v2 assay allowing the detection of at least 24 different HPV genotypes. RESULTS: Twenty cancer patients (30%) had HPV DNA detected in collected specimens. Interestingly, 14 patients (21%) had HPV only in the tumor and six (9%) had HPV only in the normal esophageal tissue. Overall, HPV16 was the viral type most frequently detected being present in eight out of 20 positive cases (40%). No correlation between the presence of HPVs and the gender or ESCC grade was observed. CONCLUSION: If the causative factors of esophageal carcinogenesis remain to be firmly established in the Kazakh population, HPV found in 30% of patients might play a role in the etiology of esophageal SCC.
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Carcinoma de Células Escamosas/virología , Neoplasias Esofágicas/virología , Infecciones por Papillomavirus/etnología , Infecciones por Papillomavirus/virología , Adulto , Anciano , Carcinoma de Células Escamosas/etnología , Carcinoma de Células Escamosas/patología , China/epidemiología , Neoplasias Esofágicas/etnología , Neoplasias Esofágicas/patología , Femenino , Genotipo , Papillomavirus Humano 16 , Humanos , Masculino , Persona de Mediana Edad , Infecciones por Papillomavirus/clasificación , PrevalenciaRESUMEN
This article introduces smart sample holders for optical microscopy. Their purpose is to allow the absolute determination of the position of the observed zone with respect to the sample holder itself and with a high accuracy. It becomes then straightforward to find a given zone of interest by positioning coarsely the microscope slide to the same position coordinates. Furthermore images recorded during different observation sessions; i.e. for slightly different positions; can be processed numerically in order to superimpose them with a high accuracy. Thus the slight deviations of the microscope slide position and orientation due to the different observations are compensated numerically and a perfect superimposition of the recorded images is performed. Then accurate site-by-site image comparisons become possible even for images recorded during different observation sessions and over a long period of time. The subpixel capability of the proposed method is demonstrated and those smart microscope slides constitute a new tool for live cell experiment. In practise, an encoded geometrical pattern used as position reference is inserted in a plane parallel to the surface receiving the tissue section or sample. Then the transition of the focus position from the tissue section to the position reference requires only a vertical adjustment and does not affect the lateral coordinates of observation. The numeric processing of the image of the position reference pattern allows the retrieval of the lateral coordinates that are also used for the tissue section image. Thus each image is recorded with a set of position coordinates that defines accurately the position of the observed area with respect to the sample holder itself.
RESUMEN
Hydroxyphenylureas are the first main metabolites formed in the environment from pesticide and biocide urea compounds. Because fungi release potent exocellular oxidases, we studied the ability of laccases produced by the white rot fungus, T. versicolor, to catalyze in vitro the transformation of five hydroxyphenylureas, to identify transformation pathways and mechanisms. Our results establish that the pH of the reaction has a strong influence on both the kinetics of the reaction and the nature of the transformation products. Structural characterization by spectroscopic methods (NMR, mass spectrometry) of eleven transformation products shows that laccase oxidizes the substrates to quinones or to polyaromatic oligomers. Slightly acidic conditions favor the formation of quinones as final transformation products. In contrast, at pH 5-6, the quinones further react with the remaining substrate in solution to give hetero-oligomers via carbon-carbon or carbon-oxygen bond formation. A reaction pathway is proposed for each of the identified products. These results demonstrate that fungal laccases could assist the transformation of hydroxyphenylureas.
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Compuestos de Fenilurea/metabolismo , Compuestos de Cloro/metabolismo , Concentración de Iones de Hidrógeno , Hidroxilación , Cinética , Lacasa/metabolismo , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Oxidación-ReducciónRESUMEN
We recently argued for a major role of p53 in staurosporine(ST)-induced apoptosis of immortalized epithelial cells, depending on their p53 status. Here, we studied the effects of PRIMA-1 (p53 reactivation and induction of massive apoptosis) and Pifithrin-alpha (p fifty-three inhibitor) in combination with ST to reinforce our previous results by respectively restoring or inhibiting the p53 transcriptional activity in different cell lines.PRIMA-1 does modify neither expression of apoptosis-related proteins nor the percentage of wild-type p53 HeLa and CaSki cells with [symbol: see text]delta psi m and DNA cleavage, whilst it increases by 45% Bax expression and apoptosis of mutated p53 C33A cells. Pifithrin-alpha, does modify neither Bax expression nor apoptosis level of C33A cells, but readily inhibits both [symbol: see text]delta psi m and DNA fragmentation of p53wt cells with decreasing Bax expression. These data support the evidence that PRIMA-1 could be a good candidate, as an anti-cancer drug targeting mutant p53, in order to increase ST efficiency. Moreover, Pifithrin-alpha could be used in combination with ST and PRIMA-1 to prevent side effects of anti-tumor therapies in cells expressing mutant P53.
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Apoptosis/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Proteínas de la Membrana/farmacología , Proteínas del Tejido Nervioso/farmacología , Tiazoles/farmacología , Tolueno/análogos & derivados , Proteína p53 Supresora de Tumor/genética , Benzotiazoles , Técnicas de Cultivo de Célula , Línea Celular Transformada , Línea Celular Tumoral , Transformación Celular Viral , ADN de Neoplasias/análisis , ADN de Neoplasias/metabolismo , Combinación de Medicamentos , Inhibidores Enzimáticos/farmacología , Femenino , Células HeLa , Humanos , Potenciales de la Membrana/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mutación , Papillomaviridae/fisiología , Estaurosporina/farmacología , Tolueno/farmacología , Transcripción Genética/efectos de los fármacos , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Laccases are oxidizing enzymes of interest because of their potential environmental and industrial applications. We performed site-directed mutagenesis of a laccase produced by Trametes versicolor in order to improve its catalytic properties. Considering a strong interaction of the Asp residue in position 206 with the substrate xylidine, we replaced it with Glu, Ala or Asn, expressed the mutant enzymes in the yeast Yarrowia lipolytica and assayed the transformation of phenolic and non-phenolic substrates. The transformation rates remain within the same range whatever the mutation of the laccase and the type of substrate: at most a 3-fold factor increase was obtained for k(cat) between the wild-type and the most efficient mutant Asp206Ala with 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic) acid as a substrate. Nevertheless, the Asn mutation led to a significant shift of the pH (DeltapH = 1.4) for optimal activity against 2,6-dimethoxyphenol. This study also provides a new insight into the binding of the reducing substrate into the active T1 site and induced modifications in catalytic properties of the enzyme.