H-terminated polycrystalline boron doped diamond electrode for geochemical sensing into underground components of nuclear repositories

Nuclear waste repositories are being installed in deep excavated rock formations in some places in Europe to isolate and store radioactive waste. In France, Callovo-Oxfordian formation (COx) is potential candidate for nuclear waste repository. It is thus necessary to measure in situ the state of a structure's health during its entire life. The monitoring of the near-field rock and the knowledge of the geochemical transformations can be carried out by a set of sensors for a sustainable management of long-term safety, reversibility and retrievability. Among the chemical parameters, the most significant are pH, conductivity and redox potential. Wide band gap semiconductors are favored materials for chemical sensing because of their high stability to many chemical agents. Among the wide band gap materials, Chemical Vapor Deposition (CVD) boron doped diamond (BDD) benefits from a large band gap (5.45 eV), which gives rise to a wide electrochemical potential window (~3 V/Saturated Calomel Electrode(SCE)) (Angus et al. 1999). It is moreover described as a radiation, corrosion and bio-corrosion resistant. These remarkable properties, in addition to a low double layer capacity and a low residual current, make BDD a promising material for geochemical sensor elaboration. This work aimed to investigate BDD- based electrodes coated with p-type polycrystalline BDD- hydrogen-terminated surfaces (1 cm2) for pH and/or redox measurements into the underground components of nuclear repositories. The boron-doped p-type channel was grown in a microwave plasma reactor (BJS 150) (Silva et al. 2009). The boron-doped channel was hydrogen terminated by a hydrogen plasma treatment in the CVD reactor, resulting in full saturation of the surface carbon bonds with hydrogen atoms. Figure 1 shows the Scanning Electron Microscopy (SEM) of the polycrystalline BDD coating with a Bore/Carbon ratio of 500 ppm and its Raman spectrum. SEM micrograph illustrates the typical columnar growth of the polycrystalline CVD diamond. A homogeneous surface was observed concerning the crystallite size which average was 1.5 microns. On the Raman spectrum of a single crystal diamond intrinsic film (undoped), the diamond peak is usually observed at 1332 cm-1. In Figure 1, the intense peak at 1327 cm-1 corresponding to diamond is shifted due to the "Fano" effect according to doping, which is observed through a broad peak at 910 cm-1. Its intensity shows that the investigated sample was highly doped. Gheeraert et al. (1993) suggested that the peaks at 500 and 1230 cm-1 appears when the boron concentration reaches the critical value of 3×1020 at.cm-3 corresponding to a metallic conductivity. The lack of peak around 1350 cm-1 and 1570 cm-1, which corresponds respectively to D and G graphite peak of impurity phases of non-diamond carbon (sp2), attests to the crystalline quality of the deposit. The slight width at half maximum of the characteristic peak of diamond compared to that of natural diamond reflects the degree of organization and structural perfection of this phase indicating that the coating was of high quality. Electrodes made in this way have been used for 8 month without any surface treatments or conditioning. The electrochemical behavior of Hydrogen-terminated BDD was studied by cyclic voltammetry. Electrodes showed a wide potential range of about 2 V/SCE. They also showed and a rapid reversible charge transfer in the presence of redox probes such FeCN63-/4- and Ru(NH)63+/2+. Performances, reliability and robustness for pH or redox monitoring were examined by potentiometric measurements at 25°C under anaerobic conditions (oxygen-free atmosphere, 100 % nitrogen) in a glove box. Investigation has been limited in pH, ranging from 5.5 to 13.5, close to those encountered in the environment of the nuclear repositories. The feasibility of measuring pH with BDD electrodes was first tested in NH4Cl/NH3-NaCl (0.1mol L-1) buffer solutions, leading to electrode calibration over the widest range of pH, from around neutral to basic pH. Experiments were also conducted in NaHCO3/Na2CO3 buffer samples, similar to conditions prevailing in the COx formation. For redox measurements, [Fe3+]/[Fe2+] ratios were analysed at different pH and/or ionic strengths (supporting electrolytes concentration ranged from 0.05 to 1 mol.L-1). The same measurements were also done using a 10-mm disk platinum electrode with a surface of 78.54 mm². No pH sensitivity was observed, thus the energy level of the state was not moved. However, for redox measurements the potential acquired by Hydrogen-terminated BDD and Platinum electrode converged to a value of the same order of magnitude, independently of the sample. This fact demonstrates that, under the same experimental conditions, the redox couples fixe identically the potential of the electrodes. Investigations with reference to ionic strength in thermodynamically equilibrated Fe(III)/Fe(II) samples were highly interesting. Independently of the electrode, the voltage measurement was not or little affected, whereas both the solution conductivity as well as the speciation were affected, due to the increase in salinity. This means that the term [Fe3+]/[Fe2+] is practically unaffected. This implies that assuming the ratio of the activity coefficients, γFe3+/ γFe2+ as equal to 1 has a minor effect on the measured redox potential. H-terminated BDD electrode appears well suited for redox monitoring. Work is in progress to demonstrate the robustness of the H-terminated BDD electrode for redox monitoring into COx over a long period

Mapping lung cancer epithelial-mesenchymal transition states and trajectories with single-cell resolution.

Elucidating the spectrum of epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) states in clinical samples promises insights on cancer progression and drug resistance. Using mass cytometry time-course analysis, we resolve lung cancer EMT states through TGFβ-treatment and identify, through TGFβ-withdrawal, a distinct MET state. We demonstrate significant differences between EMT and MET trajectories using a computational tool (TRACER) for reconstructing trajectories between cell states. In addition, we construct a lung cancer reference map of EMT and MET states referred to as the EMT-MET PHENOtypic STAte MaP (PHENOSTAMP). Using a neural net algorithm, we project clinical samples onto the EMT-MET PHENOSTAMP to characterize their phenotypic profile with single-cell resolution in terms of our in vitro EMT-MET analysis. In summary, we provide a framework to phenotypically characterize clinical samples in the context of in vitro EMT-MET findings which could help assess clinical relevance of EMT in cancer in future studies

The use of a combined bipedicled axial perforator based fasciocutaneous flap for the treatment of a traumatic diabetic foot wound: a case report

The axial and perforator vascularised fasciocutaneous flaps are reliable and effective treatment methods for covering lower limb post-traumatic, septic, Charcot, and diabetic foot wounds. The authors describe the unique utilisation of a hybrid flap as an axial-perforator flap combination for the treatment of a traumatic diabetic foot wound

Reproductive potential and performance of fertility preservation strategies in BRCA-mutated breast cancer patients

Background: Preclinical evidence suggests a possible negative impact of deleterious BRCA mutations on female fertility. However, limited and rather conflicting clinical data are available. This study assessed the reproductive potential and performance of fertility preservation strategies in BRCA-mutated breast cancer patients. Patients and methods: This was a retrospective analysis of two prospective studies investigating oocyte cryopreservation and ovarian tissue cryopreservation in newly diagnosed early breast cancer patients. In the current analysis, baseline anti-Mullerian hormone (AMH) and performance of cryopreservation strategies were compared between patients with or without germline deleterious BRCA mutations. Results: Out of 156 patients included, 101 had known BRCA status of whom 29 (18.6%) were BRCA-mutated and 72 (46.1%) had no mutation. Median age in the entire cohort was 31 years [interquartile range (IQR) 28-33). Median AMH levels were 1.8 lg/l (IQR 1.0-2.7) and 2.6 \u3bcg/l (IQR 1.5-4.1) in the BRCA-positive and BRCA-negative cohorts, respectively (P=0.109). Among patients who underwent oocyte cryopreservation (N=29), women in the BRCA-positive cohort tended to retrieve (6.5 versus 9; P=0.145) and to cryopreserve (3.5 versus 6; P=0.121) less oocytes than those in the BRCA-negative cohort. Poor response rate (i.e. retrieval of 644 oocytes) was 40.0% and 11.1% in the BRCA-positive and BRCA-negative cohorts, respectively (P=0.147). Among patients who underwent ovarian tissue cryopreservation (N=72), women in the BRCA-positive cohort tended to have a numerically lower number of oocytes per fragment (0.08 versus 0.14; P=0.193) and per square millimeter (0.33 versus 0.78; P=0.153) than those in the BRCA-negative cohort. Two BRCA-mutated patients were transplanted after chemotherapy and one delivered at term a healthy baby. No difference between BRCA1- and BRCA2-mutated patients was observed in any of the above-mentioned outcomes. Conclusion: A consistent trend for reduced reproductive potential and performance of cryopreservation strategies was observed in BRCA-mutated breast cancer patients. Independent validation of these results is needed

Gene expression profile of circulating tumor cells in breast cancer by RT-qPCR

<p>Abstract</p> <p>Background</p> <p>Circulating tumor cells (CTCs) have been associated with prognosis especially in breast cancer and have been proposed as a liquid biopsy for repeated follow up examinations. Molecular characterization of CTCs is difficult to address since they are very rare and the amount of available sample is very limited.</p> <p>Methods</p> <p>We quantified by RT-qPCR <it>CK-19, MAGE-A3, HER-2, TWIST1, hTERT α+β+</it>, and <it>mammaglobin </it>gene transcripts in immunomagnetically positively selected CTCs from 92 breast cancer patients, and 28 healthy individuals. We also compared our results with the CellSearch system in 33 of these patients with early breast cancer.</p> <p>Results</p> <p>RT-qPCR is highly sensitive and specific and can detect the expression of each individual gene at the one cell level. None of the genes tested was detected in the group of healthy donors. In 66 operable breast cancer patients, <it>CK-19 </it>was detected in 42.4%, <it>HER-2 </it>in 13.6%, <it>MAGE-A3 </it>in 21.2%, <it>hMAM </it>in 13.6%, <it>TWIST-1 </it>in 42.4%, and <it>hTERT α+β+ </it>in 10.2%. In 26 patients with verified metastasis, <it>CK-19 </it>was detected in 53.8%, <it>HER-2 </it>in 19.2%, <it>MAGE-A3 </it>in 15.4%, <it>hMAM </it>in 30.8%, <it>TWIST-1 </it>in 38.5% and <it>hTERT </it>α⁺β⁺in 19.2%. Our preliminary data on the comparison between RT-qPCR and CellSearch in 33 early breast cancer patients showed that RT-qPCR gives more positive results in respect to CellSearch.</p> <p>Conclusions</p> <p>Molecular characterization of CTCs has revealed a remarkable heterogeneity of gene expression between breast cancer patients. In a small percentage of patients, CTCs were positive for all six genes tested, while in some patients only one of these genes was expressed. The clinical significance of these findings in early breast cancer remains to be elucidated when the clinical outcome for these patients is known.</p

Tumor-infiltrating lymphocytes in patients receiving trastuzumab/pertuzumab-based chemotherapy : a TRYPHAENA Substudy

Background: There is an urgent requirement to identify biomarkers to tailor treatment in human epidermal growth factor receptor 2 (HER2)-amplified early breast cancer treated with trastuzumab/pertuzumab-based chemotherapy. Methods: Among the 225 patients randomly assigned to trastuzumab/pertuzumab concurrently or sequentially with an anthracycline-containing regimen or concurrently with an anthracycline-free regimen in the Tryphaena trial, we determined the percentage of tumor-infiltrating lymphocytes (TILs) at baseline in 213 patients, of which 126 demonstrated a pathological complete response (pCR; ypT0/is ypN0), with 28 demonstrating event-free survival (EFS) events. We investigated associations between baseline TIL percentage and either pCR or EFS after adjusting for clinicopathological characteristics using logistic and Cox regression models, respectively. To understand TIL biology, we evaluated associations between baseline TILs and baseline tumor gene expression data (800 gene set by NanoString) in a subset of 173 patients. All statistical tests were two-sided. Results: Among the patients with measurable TILs at baseline, the median level was 14.1% (interquartile range = 7.1%-32.4%). After adjusting for clinicopathological characteristics, baseline percentage TIL was not associated with pCR (adjusted odds ratio [aOR] for every 10-percentage unit increase in TILs = 1.12, 95% confidence interval [CI] = 0.95 to 1.31, P = .17). At a median follow-up of 4.7 years, for every increase in baseline TILs of 10%, there was a 25% reduction in the hazard for an EFS event (aOR = 0.75, 95% CI = 0.56 to 1.00, P = .05) after adjusting for baseline clinicopathological characteristics and pCR. Additionally, genes associated with epithelial-mesenchymal transition, angiogenesis, and T-cell inhibition such as SNAIL1, ZEB1, NOTCH3, and B7-H3 were statistically significantly inversely correlated with percentage TIL. Conclusions: Baseline TIL percentage provides independent prognostic information in patients treated with trastuzumab/pertuzumab-based neoadjuvant chemotherapy. However, further validation is required

Semiautomated isolation and molecular characterisation of single or highly purified tumour cells from CellSearch enriched blood samples using dielectrophoretic cell sorting

Background: Molecular characterisation of single circulating tumour cells (CTCs) holds considerable promise for predictive biomarker assessment and to explore CTC heterogeneity. We evaluate a new method, the DEPArray system, that allows the dielectrophoretic manipulation and isolation of single and 100% purified groups of CTCs from pre-enriched blood samples and explore the feasibility of their molecular characterisation.Methods:Samples containing known numbers of two cell populations were used to assess cell loss during sample loading. Cultured breast cancer cells were isolated from spiked blood samples using CellSearch CTC and Profile kits. Single tumour cells and groups of up to 10 tumour cells were recovered with the DEPArray system and subjected to transcriptional and mutation analysis.Results:On average, 40% cell loss was observed when loading samples to the DEPArray system. Expected mutations in clinically relevant markers could be obtained for 60% of single recovered tumour cells and all groups of tumour cells. Reliable gene expression profiles were obtained from single cells and groups of up to 10 cells for 2 out of 3 spiked breast cancer cell lines.Conclusion:We describe a semiautomated workflow for the isolation of small groups of 1 to 10 tumour cells from whole blood samples and provide proof of principle for the feasibility of their comprehensive molecular characterisation

Anti-Epithelial Cell Adhesion Molecule Antibodies and the Detection of Circulating Normal-Like Breast Tumor Cells

Identification of specific subtypes of circulating tumor cells in peripheral blood of cancer patients can provide information about the biology of metastasis and improve patient management. However, to be effective, the method used to identify circulating tumor cells must detect all tumor cell types. We investigated whether the five subtypes of human breast cancer cells that have been defined by global gene expression profiling—normal-like, basal, HER2-positive, and luminal A and B—were identified by CellSearch, a US Food and Drug Administration–approved test that uses antibodies against the cell surface–expressed epithelial cell adhesion molecule (EpCAM) to isolate circulating tumor cells. We used global gene expression profiling to determine the subtypes of a well-defined panel of 34 human breast cancer cell lines (15 luminal, nine normal-like, five basal-like, and five Her2-positive). We mixed 50-150 cells from 10 of these cell lines with 7.5 mL of blood from a single healthy human donor, and the mixtures were subjected to the CellSearch test to isolate the breast cancer cells. We found that the CellSearch isolation method, which uses EpCAM on the surface of circulating tumor cells for cell isolation, did not recognize, in particular, normal-like breast cancer cells, which in general have aggressive features. New tests that include antibodies that specifically recognize normal-like breast tumor cells but not cells of hematopoietic origin are needed

Minimal residual disease and circulating tumor cells in breast cancer

Tumor cell dissemination in bone marrow or other organs is thought to represent an important step in the metastatic process. The detection of bone marrow disseminated tumor cells is associated with worse outcome in early breast cancer. Moreover, the detection of peripheral blood circulating tumor cells is an adverse prognostic factor in metastatic breast cancer, and emerging data suggest that this is also true for early disease. Beyond enumeration, the characterization of these cells has the potential to improve risk assessment, treatment selection and monitoring, and the development of novel therapeutic agents, and to advance our understanding of the biology of metastasis