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Aim: Cervical cancer is the most common gynecological cancer. Recent studies have revealed that the F-box and WD repeat domain containing 7 (FBXW7) gene, which encodes a subunit of Skp1-Cul1-F-box protein (SCF) ubiquitin ligase, is frequently mutated in cervical squamous cell carcinomas. In this study, we investigated whether Chinese cervical cancer cells also harbor these mutations. Methods: Using PCR and sequencing assays, a total of 190 specimens from Han Chinese patients with cervical cancer were analyzed for FBXW7 mutations. Results: Two FBXW7 mutations (p.R479P and p.L443H), were identified from a study of 145 (1.4%) cervical squamous cell carcinomas. The p.L443H somatic mutation has not been previously reported. Functional assays showed that both of these FBXW7 mutations could promote cell proliferation, migration, and invasion. Conclusion: A low frequency (1.4%) of cervical squamous cell carcinomas were identified with FBXW7 mutations. We did, however, identify a novel FBXW7 mutation. Our results also demonstrated that the identified FBXW7 mutations could promote cell proliferation, migration, and invasion in cervical cancer cells.

Endometriosis is characterized by the ectopic implant of endometrial tissue outside the uterine cavity and found in ˜35-50% of subfertile women. Previous studies have found that endometriosis had frequent defects in zona pellucida (ZP), and mutations in ZP genes could lead to ZP defects, raising the possibility that mutations in ZP genes might exist in endometriosis. We analyzed a total of 152 Han Chinese samples with ovarian endometriosis for the presence of mutations in the ZP1, ZP2, ZP3 and ZP4 genes. Two novel nonsynonymous ZP4 mutations were identified in three out of 152 (2.0%) samples: a p.M1?/(c.3 G > C) mutation in a 27- and 35-year-old sample, respectively, and a p.A433 V (c.1298C > T) mutation in a 31-year-old patient. No mutations were detected in ZP1, ZP2 or ZP3 genes; furthermore, no mutations in ZP genes were identified in 85 female control samples without endometriosis. The p.M1?/(c.3 G > C) mutation could lead to the usage of a downstream translation initiation site, while the evolutionary conservation and protein structural modeling analyses suggested that the p.A433 V mutation might be functionally important. However, there were strikingly different fertility outcomes among the three samples with ZP4 mutations: the p.A433V-mutated sample had no problem in fertility; while the p.M1?-mutated samples presented with paradoxical effects on fertility: the 35-year-old patient had a child while the 27-year-old patient was infertile, who underwent two spontaneous abortions and an implantation failure after IVF treatment. These results suggested that the potential role of ZP4 mutations on human fertility might be more complex than we thought, and other genetic and environment factors might play a role. In conclusion, we identified two novel mutations in the ZP4 gene in 2.0% of Han Chinese patients with ovarian endometriosis for the first time, our results suggested that mutations in ZP4, but not ZP1, ZP2 and ZP3, might play active roles in the pathogenesis of ovarian endometriosis, despite the mutation-carriers present with complex fertility outcomes.

Adenomyosis is one of the most common gynecological disorders that the molecular events underlying its pathogenesis remain not fully understood. Prior studies have shown that endometrial stromal cells (ESCs) played crucial roles in the pathogenesis of adenomyosis. In this study, we utilized two-dimensional gel electrophoresis combined with protein identification by mass spectrometry (2D/MS) proteomics analysis to compare the differential protein expression profile between the paired eutopic and ectopic ESCs (EuESCs and EcESCs) in adenomyosis, and a total of 32 significantly altered protein spots were identified. Among which, the expression of LIM and SH3 protein 1 (LASP1) was increased significantly in EcESCs compared to EuESCs. Immunohistochemical assay showed that LASP1 was overexpressed in the stromal cells of ectopic endometriums compared to eutopic endometriums; further functional analyses revealed that LASP1 overexpression could enhance cell proliferation, migration and invasion of EcESCs. Furthermore, we also showed that the dysregulated expression of LASP1 in EcESCs was associated with DNA hypermethylation in the promoter region of the LASP1 gene. However, the detailed molecular mechanisms of enhancing cell proliferation, invasion and migration caused by upregulated LASP1 in adenomyosis needs further study. For the first time, our data suggested that LASP1 plays important roles in the pathogenesis of adenomyosis, and could serve as a prognostic biomarker of adenomyosis.

Endometriosis is a common chronic gynecological disease greatly affecting women health. Prior studies have implicated that dysferlin (DYSF) aberration might be involved in the pathogenesis of ovarian endometriosis. In the present study, we explore the potential presence of DYSF mutations in a total of 152 Han Chinese samples with ovarian endometriosis. We analyze the potential presence of DYSF mutations by direct DNA sequencing. A total of seven rare variants/mutations in the DYSF gene in 10 out of 152 samples (6.6%) were identified, including 5 rare variants and 2 novel mutations. For the 5 rare variants, p.R334W and p.G941S existed in 2 samples, p.R865W, p.R1173H and p.G1531S existed in single sample, respectively; for the two novel mutations, p.W352* and p.I1642F, they were identified in three patients. These rare variants/mutations were absent or existed at extremely low frequency either in our 1006 local control women without endometriosis, or in the China Metabolic Analytics Project (ChinaMAP) and Genome Aggregation Database (gnomAD) databases. Evolutionary conservation analysis results suggested that all of these rare variants/mutations were evolutionarily conserved among 11 vertebrate species from Human to Fox. Furthermore, in silico analysis results suggested these rare variants/mutations were disease-causing. Nevertheless, we find no significant association between DYSF rare variants/mutations and the clinical features in our patients. To our knowledge, this is the first report revealing frequent DYSF mutations in ovarian endometriosis. We identified a high frequency of DYSF rare variants/mutations in ovarian endometriosis for the first time. This study suggests a new correlation between DYSF rare variants/mutations and ovarian endometriosis, implicating DYSF rare variants/mutations might be positively involved in the pathogenesis of ovarian endometriosis.

Endometriosis is a complex and heterogeneous pre-malignant inflammatory disease harboring multiple gene mutations. Previous studies have suggested that caspase recruitment domain family member (CARD)10 and CARD11 mutations may exist in endometriosis. In the present study, a collection of endometriotic lesions and paired peripheral blood from 101 patients with ovarian endometriosis were obtained, and the entire coding sequences of the CARD10 and CARD11 genes were sequenced. Evolutionary conservation analysis and online prediction programs were applied to analyze the disease-causing potential of the identified mutations. A total of 4 novel somatic mutations were identified in 4 out of the 101 (4.0%) samples: 2 in-frame deletions in CARD10 (c.785_790delAGGAGA, p.K272_E273delKE; c.785_802delAGGAGAAGGAGAAGGAGA, p.K272_V277delKEPDNV) and 2 heterozygous missense mutations in CARD11 (c.49G>T, p.D17Y; c.160G>C, p.E54Q). The sample with CARD10 p.K272_E273delKE deletion was obtained from a 47-year-old patient who was also diagnosed with uterine leiomyoma, while the CARD10 p.K272_V277delKEPDNV-mutated sample was from a 43-year-old patient exhibiting a decreased blood eosinophil granulocyte ratio (0.3%) and an elevated serum creatine kinase level (314 U/l). The patient with the CARD11 p.D17Y mutation was 38 years old and exhibited an increased level of cancer antigen 125 (45.4 U/ml), while the patient with the CARD11 p.E54Q mutation was 46 years old and exhibited no other gynecological conditions. Evolutionary conservation analysis and online prediction programs suggested that these mutations may be disease-causing. In summary, 4 novel somatic mutations in the CARD10 and CARD11 genes were identified from amongst 101 cases of ovarian endometriosis for the first time, these mutations may serve active roles in the development of ovarian endometriosis.

The aim of this study was to assess the ability of Raman spectroscopy to discriminate and quantify five antineoplastic drugs in an aqueous matrix at low concentrations before patient administration. Five antineoplastic drugs were studied at therapeutic concentrations in aqueous 0.9% sodium chloride: 5-fluorouracil (5FU), gemcitabine (GEM), cyclophophamide (CYCLO), ifosfamide (IFOS) and doxorubicin (DOXO). All samples were packaged in glass vials and analyzed using Raman spectrometry from 400 to 4000cm-1. Discriminant analyses were performed using Partial Least Squares Discriminant Analysis (PLS-DA) and quantitative analyses using PLS regression. The best discrimination model was obtained using hierarchical PLS-DA models including three successive models for concentrations higher than the lower limit of quantification (0% of fitting and cross-validation error rate with an excellent accuracy of 100%). According to these hierarchical discriminative models, 90.8% (n=433) of external validation samples were correctly predicted, 2.5% (n=12) were misclassified and 6.7% (n=32) of the external validation set were not assigned. The quantitative analysis was characterized by the RMSEP that ranged from 0.23mg/mL for DOXO to 3.05mg/mL for 5FU. The determination coefficient (R2) was higher than 0.9994 for all drugs evaluated except for 5FU (R2=0.9986). This study provides additional information about the potential value of Raman spectroscopy for real-time quality control of cytotoxic drugs in hospitals. In some situations, this technique therefore constitutes a powerful alternative to usual methods with ultraviolet (UV) detection to ensure the correct drug and the correct dose in solutions before administration to patients and to limit exposure of healthcare workers during the analytical control process.

Endometriosis is a potential premalignant disorder. The underlying molecular aberrations, however, are not fully understood. A recent exome sequencing study found that 25% (10/39) of deep infiltrating endometriosis harbored cancer driver gene mutations. However, it is unclear whether these mutations also exist in ovarian endometriosis. Here, a total of 101 ovarian endometriosis samples were analyzed for the presence of these gene mutations, including KRAS, PPP2R1A, PIK3CA and ARID1A. In addition, 6 other cancer-associated genes (BRAF, NRAS, HRAS, ERK1, ERK2 and PTEN) were also analyzed. In total, four somatic mutations were identified in three out of 101 ovarian endometriotic lesions (4%, 4/101), including a KRAS p.G12V, a PPP2R1A p.S256F and two ARID1A nonsense mutations (p.Q403* and p.G1926*); while no mutations were identified in the remaining 7 genes (BRAF, NRAS, HRAS, ERK1, ERK2, PTEN and PIK3CA). Note that the KRAS G12V and ARID1A Q403* mutations co-occurred in a 36-year-old sample who had a high serum CA125 (308.4 U/mL) and a late menarche age (18-year-old). Additionally, no mutations in any of the 10 genes were identified in either the healthy eutopic endometrial tissues from 85 control individuals without endometriosis, or in 62 healthy ovarian tissues from ovarian cysts samples (without endometriosis). Our study revealed, for the first time, the presence of classical cancer driver gene mutations in ovarian endometriosis. Furthermore, the co-occurrence of KRAS and ARID1A mutations was identified in a single individual for the first time. The observations of cancer driver gene mutations in our ovarian endometriosis samples, together with several prior observations, further support the notion that endometriosis is a premalignant disorder.

Polycyclic aromatic hydrocarbons (PAHs) are environmental contaminants with carcinogenic effect raising worldwide concerns. Hydroxylated PAHs (OH-PAHs) could be formed in the body as metabolites of PAHs in human urine samples and thus considered as biomarkers of PAH exposure. In this study, five OH-PAHs including 3-phenanthrol, 1-naphthol, 2-hydroxy fluorene, 1-hydroxprene and 6-hydroxy chrysene in human urine samples were selectively enriched by C18 solid-phase microextraction (SPME), then SPME fiber was connected high voltage and then was inserted into a glass-capillary to elute and ionize the analytes for mass spectrometric (MS) detection. The coupling of SPME-MS showed excellent analytical performance for detection of urinary OH-PAHs under optimal conditions, providing an easy operation for rapid detection of a single sample within minutes. By use of internal standard (i.e., 2-hydroxy fluorene-d9), the limit of detection (LOD) and limit of quantitation (LOQ) of OH-PAHs were found to be less than 0.05 ng L−1 level (S/N > 3) and less than 0.1 ng L−1 level (S/N > 10), respectively. The dynamic ranges of five OH-PAHs were found to be a range at 0.1–5.0 ng L−1 with excellent coefficient (R2 > 0.99). This method also showed good precisions (intra-day: 3.4–5.5%, inter-day: 7.0–9.8%, n = 5) and good accuracy (85.3–95.3%, n = 5). Moreover, ion suppression and matrix effect in detection of OH-PAHs in urine were also investigated. Human urine samples collected from 12 volunteers including 6 non-smokers and 6 smokers have been successfully analyzed, it was found that individual OH-PAHs in smokers were higher than in non-smokers. This study demonstrated that SPME coupled with glass-capillary nanoESI-MS is a sensitive method for rapid detection of urinary OH-PAHs for health risk assessment of PAHs exposure.

Introduction Le controle analytique des chimiotherapies permet d’assurer la qualite physico-chimique des preparations. La spectroscopie vibrationnelle Raman (SR) est une technique rapide, non destructive et non invasive largement utilisee dans l’industrie pharmaceutique. L’objectif de cette etude a ete de montrer la faisabilite de la SR pour la discrimination et de la quantification de plusieurs molecules anticancereuses, et de comparer ces performances aux techniques separatives de routine avec detection UV. Materiels et methode Cinq molecules ont ete analysees apres dilution dans du NaCl 0,9 % a des concentrations couvrant la gamme therapeutique : gemcitabine (GEM) de 1 a 40 mg/mL, doxorubicine (DOXO) de 0,01 a 2 mg/mL, 5-fluorouracile (5-FU) de 1 a 50 mg/mL, cyclophosphamide (CYCLO) de 0,5 a 20 mg/mL et ifosphamide (IFOS) de 1 a 40 mg/mL. Les solutions conditionnees dans des flacons en verre, ont ete analysees avec un spectrometre Raman Labram HR Evolution (3 mesures/echantillon). Les echantillons ont ete repartis en 2 sets de calibration pour le developpement des modeles (n = 450) et de validation externe pour l’evaluation des performances predictives des modeles (n = 477). Les modeles de discrimination et de quantification ont ete developpes par analyse multivariee, Partial Least Square discrimination analysis (PLS-DA) et regression (PLS-R). Plusieurs pretraitement ont ete evalues (derivee premiere, seconde et normalisation vectorielle). Les echantillons de validation externe ont ete analyses egalement par injection directe dans le flux (FIA/UV) ou separation chromatographique (HPLC-UV). Resultats et discussion Au total, 144 modeles de discrimination et 96 de quantification ont ete developpes. Deux approches d’analyse discriminante ont ete testees globale et hierarchique. La meilleure discrimination a ete obtenue par analyse hierarchique reposant sur 3 discriminations successives pour des concentrations superieures a la limite de quantification (LLOQ). Sur les echantillons de validation externe, 90,8 % etaient bien assignes, 2,5 % mal classes et 6,7 % non assignes. Les modeles de quantification les plus performants presentaient des erreurs de prediction (RMSEP) comprises entre 0,23 mg/mL pour la DOXO et 3,05 mg/mL pour le 5FU avec des coefficients de determination (r2) superieurs a 0,9989. Les LLOQ variaient de 0,17 mg/mL pour la DOXO a 2,83 mg/mL pour GEM. Selon l’approche de Bland-Altman comparant la SR et les methodes UV, seules trois methodes (GEM, IFOS et 5FU pour des concentrations > 10 mg/mL) sont transposables avec un biais et des limites de confidence (+1,96SD) compris dans les limites fixees respectivement de ±2 % et de ±20 %. Conclusion Cette etude confirme la potentialite de la SR pour l’analyse quantitative et discriminante des molecules anticancereuses. Malgre des limites de sensibilite pour les faibles concentrations, cette technique permettant des analyses in situ sans echantillonnage constitue une perspective interessante pour les seringues ou les diffuseurs jusque-la non echantillonnable. Cette methode non invasive permet toutefois de reduire les risques potentiels lies a l’exposition aux cytotoxiques.

Uterine leiomyomas represents a major public health problem. Despite their prevalence, the causation and pathogenesis of leiomyomas are poorly understood. A broad range of organisms and tissues contain 14-3-3 proteins which have been associated with the pathogenesis of many diseases through participating in signal transduction pathways. This study was designed to evaluate which 14-3-3 isoforms might be optimal targets in leiomyomas, and to further explore their relationship with estrogen and progesterone receptor (ER and PR).Paired samples of leiomyoma and adjacent myometrium were obtained from 80 subjects who had surgical excision of uterine leiomyomas. The expression of 14-3-3 isoforms was detected by Western bolt and RT-PCR, and their relationship with ER and PR was analysed by immunohistochemistry.The expressions of 14-3-3σ had decreased significantly in leiomyoma compared with that in normal myometrium and was negatively correlated with ER and PR by immunohistochemistry.The down-regulation of 14-3-3σ in leiomyoma suggests that 14-3-3σ may play a role in tumorigenesis, and that its mechanism may be involved in the up-regulation of ER and PR.

Implementation of ICH guideline for validation of analytical methods was tested in the case of two enzymatic assays of determination of superoxide dismutase (SOD) activity. Analytical figures of merits of two tested methods (NBT reference method and the so called WST-1 method) demonstrate the feasibility of such approach. Nevertheless specification usually admitted for physicochemical method (e.g., HPLC), needs to be clearly enlarged, for instance, up to 10% for repeatability and 20% for reproducibility. On the other hand, the cross-validation performed between the two enzymatic activity determination techniques, though based on the same principle, clearly shows that the quality of results depends on small variation in the experimental conditions. This shows that an enzymatic activity determination should be strictly related to the technique used, especially in the pharmaceutical control quality field, and confirms that analytical figures of merits are strongly function of the technique used. Finally, it was demonstrated that both NBT reference method and WST-1 method give strictly similar results.

A general approach, still few exploited so far and never associated with microbore-LC, consisting of detection of various lipid classes (i.e. phospholipids, triglycerides, ceramides and glycosphingolipids) by non-covalent association with 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence probe is developed. This mode of detection was coupled with non-aqueous reversed-phase microbore-LC (C18) by using classical post-column fluorescence detection. The classical LC system was first adapted to microbore-chromatography (internal diameter 1 mm) without apparatus miniaturization of the solvent delivery system and the detection cell. For this purpose, the detection parameters (probe concentration, post-column flow rate, post-column reactor length and post-column system temperature) were optimized by a central composite design (CCD) using a mixture of phosphatidylcholine (PC) species as a lipid model and DPH (lambda(ex) = 350 nm, lambda(em) = 430 nm) as a fluorescence probe. The optimal conditions of detection for the various molecular species of PC were determined for a DPH concentration of 3.35 micromol/L, a post-column flow rate of 0.5 mL/min, a reactor length of 1.4 m and a temperature of 35 degrees C. The fluorescence response was linear over a wide range of PC species from 5 microg/mL to 100 microg/mL and the lower limit of detection (signal/noise = 3) was about 1 microg/mL, that is equivalent to evaporative light scattering detection (ELSD). Others molecular species of various classes of lipids, i.e. triglycerides, ceramides and glycosphingolipids were also easily detected. Thus, this study demonstrated the versatility of the proposed system of detection which was shown to be sensitive, easy to perform, non-destructive and allowed, in contrast to ELSD, for a linear response with various polarity lipid classes.

Ceramide analysis was developed with gradient elution in non-aqueous reversed-phase liquid chromatography with evaporative light scattering detection (ELSD) or postcolumn fluorescence detection. Fluorescence detection (excitation, 360 nm; emission, 425 nm) after postcolumn formation of mixed assemblies between eluted ceramides and 1,6-diphenyl-1,3,5-hexatriene was developed. In comparison with ELSD, fluorescence detection allows a better detection of the minor species ceramide from ceramide type III (commercial mixture of non-hydroxy fatty acid-sphingosine) and appears to be more sensitive for quantitation of ceramides at low concentrations. The fluorescence response is linear over a wide range of injected amount of ceramide III (expressed as stearoyl-phytosphingosine): 10 ng to 1000 ng. The response of ELSD is non linear but can be linearized in double logarithmic coordinates for calculations over a narrow range, e.g. between 10 to 350 ng ceramide III injected. The lower quantitation limits of these two detectors are similar: 5 ng ceramide III was injected.

Une etude portant sur 51 sujets, dont 15 hommeshemizygotes 12 femmes heterozygotes et 24 temoins, decrit une methode de depistage par dosage de l’α-galactosidase A sur goutte de sang recueillie sur papier filtre, et son interet dans les populations a risque de maladie de Fabry

A precise and sensitive method is described for the determination of N-nitrosodiethanolamine (NDELA) at the 1 μl l -1 level. NDELA was found to be extracted from aqueous solution by ethyl acetate at pH 1.0. The technique is based on the alkaline denitrosation of NDELA and reaction of the nitrite liberated with Coumarin 120 (4-methyl-7-aminocoumarin) in sulphuric acid medium to give the corresponding 7-diazo compound. After hydrolysis at 100°C, the corresponding 7-hydroxycoumarin (4-methylumbelliferone) is detected fluorimetrically at 380 nm after excitation at 325 nm. HPLC is essential to separate excess Coumarin 120 from 4-methylumbelliferone. Linearity was obtained from 1 to 30 μl l -1 (r = 0.997). The limit of detection was 0.8 μg l -1 for a signal-to-noise ratio of 3. The repeatability expressed as R.S.D. was 6.8% at 1 μl l -1 and 5.5% at 30 μg l -1 . The reproducibility, established by two operators on three consecutive days at 5 μl l -1 (n = 6), was 9.2% (R.S.D.). Under the conditions adopted, detection of trace levels of NDELA in ethanolamine derivatives is possible.

La prevalence de la maladie de Fabry est tres faible dans la population generale. Toutefois, la determination de l’activite de l’α-galactosidase A presente un interet dans les populations a risque.