Absorption/Attenuation Spectral Description of ESKAPEE Bacteria: Application to Seeder-Free Culture Monitoring, Mammalian T-Cell and Bacteria Mixture Analysis and Contamination Description.

Despite numerous innovations, measuring bacteria concentrations on a routine basis is still time consuming and ensuring accurate measurements requires careful handling. Furthermore, it often requires sampling small volumes of bacteria suspensions which might be poorly representative of the real bacteria concentration. In this paper, we propose a spectroscopy measurement method based on a description of the absorption/attenuation spectra of ESKAPEE bacteria. Concentrations were measured with accuracies less than 2%. In addition, mixing the mathematical description of the absorption/attenuation spectra of mammalian T-cells and bacteria allows for the simultaneous measurements of both species' concentrations. This method allows real-time, sampling-free and seeder-free measurement and can be easily integrated into a closed-system environment.

Absorption Spectra Description for T-Cell Concentrations Determination and Simultaneous Measurements of Species during Co-Cultures.

Advanced Therapy Medicinal Products are promising drugs for patients in therapeutic impasses. Their complex fabrication process implies regular quality controls to monitor cell concentration. Among the different methods available, optical techniques offer several advantages. Our study aims to measure cell concentration in real time in a potential closed-loop environment using white light spectroscopy and to test the possibility of simultaneously measuring concentrations of several species. By analyzing the shapes of the absorption spectra, this system allowed the quantification of T-cells with an accuracy of about 3% during 30 h of cultivation monitoring and 26 h of doubling time, coherent with what is expected for normal cell culture. Moreover, our system permitted concentration measurements for two species in reconstructed co-cultures of T-cells and Candida albicans yeasts. This method can now be applied to any single or co-culture, it allows real-time monitoring, and can be easily integrated into a closed system.

AB186 Inhibits Migration of Triple-Negative Breast Cancer Cells and Interacts with α-Tubulin.

Breast cancer is one of the leading causes of cancer-related death among females worldwide. A major challenge is to develop innovative therapy in order to treat breast cancer subtypes resistant to current treatment. In the present study, we examined the effects of two Troglitazone derivatives Δ2-TGZ and AB186. Previous studies showed that both compounds induce apoptosis, nevertheless AB186 was a more potent agent. The kinetic of cellular events was investigated by real-time cell analysis system (RTCA) in MCF-7 (hormone dependent) and MDA-MB-231 (triple negative) breast cancer (TNBC) cells, followed by cell morphology analysis by immuno-localization. Both compounds induced a rapid modification of both impedance-based signals and cellular morphology. This process was associated with an inhibition of cell migration measured by wound healing and transwell assays in TNBC MDA-MB-231 and Hs578T cells. In order to identify cytoplasmic targets of AB186, we performed surface plasmon resonance (SPR) and pull-down analyses. Subsequently, 6 cytoskeleton components were identified as potential targets. We further validated α-tubulin as one of the direct targets of AB186. In conclusion, our results suggested that AB186 could be promising to develop novel therapeutic strategies to treat aggressive forms of breast cancer such as TNBC.

Immuno-MALDI-MS in human plasma and on-chip biomarker characterizations at the femtomole level.

Immuno-SPR-MS is the combination of immuno-sensors in biochip format with mass spectrometry. This association of instrumentation allows the detection and the quantification of proteins of interest by SPR and their molecular characterization by additional MS analysis. However, two major bottlenecks must be overcome for a wide diffusion of the SPR-MS analytical platform: (i) To warrant all the potentialities of MS, an enzymatic digestion step must be developed taking into account the spot formats on the biochip and (ii) the biological relevancy of such an analytical solution requires that biosensing must be performed in complex media. In this study, we developed a procedure for the detection and the characterization at ~1 µg/mL of the LAG3 protein spiked in human plasma. The analytical performances of this new method was established, particularly its specificity (S/N > 9) and sensitivity (100% of LAG3 identification with high significant mascot score >68 at the femtomole level). The collective and automated on-chip MALDI-MS imaging and analysis based on peptidic fragments opens numerous applications in the fields of proteomics and diagnosis.

Epidemiological Impact of GII.17 Human Noroviruses Associated With Attachment to Enterocytes.

For the last 30 years, molecular surveys have shown that human norovirus (HuNoV), predominantly the GII.4 genotype, is one of the main causative agents of gastroenteritis. However, epidemiological surveys have revealed the worldwide emergence of GII.17 HuNoVs. Genetic analysis confirmed that GII.17 strains are distributed into three variants (i.e., Kawasaki 308, Kawasaki 323, and CS-E1). Here, virus-like particles (VLPs) were baculovirus-expressed from these variants to study putative interactions with HBGA. Qualitative analysis of the HBGA binding profile of each variant showed that the most recent and predominant GII.17 variant, Kawasaki 308, possesses a larger binding spectrum. The retrospective study of GII.17 strains documented before the emergence of the dominant Kawasaki 308 variant showed that the emergence of a new GII.17 variant could be related to an increased binding capacity toward HBGA. The use of duodenal histological sections confirmed that recognition of enterocytes involved HBGA for the three GII.17 variants. Finally, we observed that the relative affinity of recent GII.17 VLPs for HBGA remains lower than that of the GII.4-2012 variant. These observations suggest a model whereby a combination of virological factors, such as polymerase fidelity and increased affinity for HBGA, and immunological factors was responsible for the incomplete and non-persistent replacement of GII.4 by new GII.17 variants.

Seismic microzonation of a region with complex surficial geology based on different site classification approaches.

A seismic microzonation study was conducted to refine the seismic hazard model for the city of Saguenay, Canada. The Quaternary geology underlying Saguenay shows complex glacial and post-glacial stratigraphy with a number of buried valleys filled with fluvioglacial and glaciomarine sediments. High impedance contrast between rock formations and surficial sediments is prone to seismic amplification. To evaluate their applicability, advantages and limitations in capturing the geological specificity of the study area, four site classification methods were applied: the current National Building Code of Canada (NBCC) and Eurocode 8, both mainly based on the average shear-wave velocity for the surficial sediments (V S,avg ) and for the top 30 m (V S,30 ); a method based on the fundamental site period (T 0 ); and a hybrid method based on the combination of V S,30 , T 0 and V S,avg . The study specifically aimed to evaluate the importance of the site classification parameters on the resulting microzonation maps. V S,30 is capable to present the geological and geotechnical site conditions, however, the results may be further improved by considering V s,avg in shallow and T 0 in thick layers of soil sediments as secondary parameters. The T 0 method gives also satisfactory results with T 0 showing a better correlation to V s,30 than to V s,avg . The versatile hybrid method may be challenging to apply in certain cases with its nine different site categories and parameters.

Geomechanical characterization of a heterogenous rock mass using geological and laboratory test results: a case study of the Niobec Mine, Quebec (Canada).

ABSTRACT: To conduct a successful geomechanical characterization of rock masses, an appropriate interpretation of lithological heterogeneity should be attained by considering both the geological and geomechanical data. In order to clarify the reliability and applicability of geological surveys for rock mechanics purposes, a geomechanical characterization study is conducted on the heterogeneous rock mass of Niobec Mine (Quebec, Canada), by considering the characteristics of its various identified lithological units. The results of previous field and laboratory test campaigns were used to quantify the variability associated to intact rock geomechanical parameters for the different present lithological units. The interpretation of geomechanical similarities between the lithological units resulted in determination of three main rock units (carbonatite, syenite, and carbonatite-syenite units). Geomechanical parameters of these rock units and their associated variabilities are utilized for stochastic estimation of geomechanical parameters of the heterogeneous rock mass using the Monte Carlo Simulation method. A comparison is also made between the results of probabilistic and deterministic analyses to highlight the presence of intrinsic variability associated with the heterogeneous rock mass properties. The results indicated that, for the case of Niobec Mine, the carbonatite-syenite rock unit could be considered as a valid representative of the entire rock mass geology since it offers an appropriate geomechanical approximation of all the present lithological units at the mine site, in terms of both the magnitude and dispersion of the strength and deformability parameters. ARTICLE HIGHLIGHTS: Evaluating the reliability and applicability of geological survey outcomes for rock mechanics purposes.A geomechanical characterization study is conducted on the heterogeneous rock mass by considering the various identified rock lithotypes.The geomechanical parameters of intact units and their associated variabilities are used to stochastically estimate the geomechanical parameters of the heterogeneous rock mass by employing the Monte Carlo Simulation.A comparison is also made between the results of probabilistic and deterministic geomechanical analyses.The results indicate that, in the case of Niobec Mine, the combined syenite-carbonatite rock unit could be considered as a valid representative of the entire rock mass.

Assessment of Shear-Dependent Kinetics of Primary Haemostasis With a Microfluidic Acoustic Biosensor.

Primary haemostasis is a complex dynamic process, which involves in-flow interactions between platelets and sub-endothelial matrix at the area of the damaged vessel wall. It results in a first haemostatic plug, which stops bleeding, before coagulation ensues and consolidates it. The diagnosis of primary haemostasis defect would benefit from evaluation of the whole sequence of mechanisms involved in platelet plug formation in flow. This work proposes a new approach that is based on characterization of the shear-dependent kinetics that enables the evaluation of the early stages of primary haemostasis. We used a label-free method with a quartz crystal microbalance (QCM) biosensor to measure the platelet deposits over time onto covalently immobilized type I fibrillar collagen. We defined three metrics: total frequency shift, lag time, and growth rate. The measurement was completed at four predefined shear rates prevailing in small vessels (500, 770, 1000 and 1500 s-1) during five minutes of perfusion with anticoagulated normal whole blood. The rate of the frequency shift over the first five minutes was strongly influenced by shear rate conditions, presenting a maximum around 770 s-1, and varying by a factor larger than three in the studied shear rate range. To validate the biosensor signal, the total frequency shift was compared to results obtained by atomic force microscopy (AFM) on final platelet deposits. The results show that shear-dependent kinetic assays are promising as an advanced method for screening of primary haemostasis.

Detecting a Defective Casing Seal at the Top of a Bedrock Aquifer.

An improperly sealed casing can produce a direct hydraulic connection between two or more originally isolated aquifers with important consequences regarding groundwater quantity and quality. A recent study by Richard et al. (2014) investigated a monitoring well installed in a fractured rock aquifer with a defective casing seal at the soil-bedrock interface. A hydraulic short circuit was detected that produced some leakage between the rock and the overlying deposits. A falling-head permeability test performed in this well showed that the usual method of data interpretation is not valid in this particular case due to the presence of a piezometric error. This error is the direct result of the preferential flow originating from the hydraulic short circuit and the subsequent re-equilibration of the piezometric levels of both aquifers in the vicinity of the inlet and the outlet of the defective seal. Numerical simulations of groundwater circulation around the well support the observed impact of the hydraulic short circuit on the results of the falling-head permeability test. These observations demonstrate that a properly designed falling-head permeability test may be useful in the detection of defective casing seals.

Naturally occurring thallium: a hidden geoenvironmental health hazard?

This paper illustrates a real environmental concern and draws attention to the fact that natural processes can mobilize thallium (Tl), a highly toxic metal, which may enter the food chain as a "hidden health killer" with severe health impacts on local human population. Natural processes may be exacerbated by human activities such as mining and farming, and may cause enrichment of Tl in the environment. In geochemically anomalous areas with concentrated levels of Tl in the surface environment (bedrocks, waters, soils, and crops), such as the Lanmuchang area in southwestern Guizhou Province, China, it is essential to establish base-level values and to pay heed to the geological context of "natural contamination," as high concentrations of Tl in bedrocks/ores (6-35,000 mg/kg) can lead to enrichment of Tl in the aquatic system (0.005-1100 microg/l in groundwaters and 0.07-31 microg/l in surface waters) and soil layers (1.5-124 mg/kg). In sensitive areas such as the Yanshang area of southwestern Guizhou, elevated natural levels of Tl from bedrocks may also cause higher concentrations of Tl in the surface environment, and thus more attention must be paid to geoenvironmental management of human activities if socio-economic catastrophes are to be avoided. Due to high uptake of Tl by crops, Tl can be transferred from soils to crops and remarkably concentrated in food crops. Concentrations of 1-500 mg/kg Tl based on dry weight (DW) were determined in many food crops growing on Tl-contaminated arable soils from the Lanmuchang area. The daily intake of 1.9 mg of Tl from consumed food crops was estimated for the local adult inhabitant of Lanmuchang. Thus, Tl is regarded as a latent health hazard with potential risk of toxicity in humans within areas of "natural" contamination by Tl.

Calibration and validation of an integrated nitrate transport model within a well capture zone.

Groundwater contamination by nitrate was investigated in an agricultural area in southern Quebec, Canada, where a municipal well is the local source of drinking water. A network of 38 piezometers was installed within the capture zone of the municipal well to monitor water table levels and nitrate concentrations in the aquifer. Nitrate concentrations were also measured in the municipal well. A Water flow and Nitrate transport Global Model (WNGM) was developed to simulate the impact of agricultural activities on nitrate concentrations in both the aquifer and municipal well. The WNGM first uses the Agriflux model to simulate vertical water and nitrate fluxes below the root zone for each of the seventy agricultural fields located within the capture zone of the municipal well. The WNGM then uses the HydroGeoSphere model to simulate three-dimensional variably-saturated groundwater flow and nitrate transport in the aquifer using water and nitrate fluxes computed with the Agriflux model as the top boundary conditions. The WNGM model was calibrated by reproducing water levels measured from 2005 to 2007 in the network of piezometers and nitrate concentrations measured in the municipal well from 1997 to 2007. The nitrate concentrations measured in the network of piezometers, however, showed greater variability than in the municipal well and could not be reproduced by the calibrated model. After calibration, the model was validated by successfully reproducing the decrease of nitrate concentrations observed in the municipal well in 2006 and 2007. Although it cannot predict nitrate concentrations in individual piezometers, the calibrated and validated WNGM can be used to assess the impact of changes in agricultural practices on global nitrate concentrations in the aquifer and in the municipal well.

Neuropilin-2 expression promotes TGF-ß1-mediated epithelial to mesenchymal transition in colorectal cancer cells.

Neuropilins, initially characterized as neuronal receptors, act as co-receptors for cancer related growth factors and were recently involved in several signaling pathways leading to cytoskeletal organization, angiogenesis and cancer progression. Then, we sought to investigate the ability of neuropilin-2 to orchestrate epithelial-mesenchymal transition in colorectal cancer cells. Using specific siRNA to target neuropilin-2 expression, or gene transfer, we first observed that neuropilin-2 expression endows HT29 and Colo320 for xenograft formation. Moreover, neuropilin-2 conferred a fibroblastic-like shape to cancer cells, suggesting an involvement of neuropilin-2 in epithelial-mesenchymal transition. Indeed, the presence of neuropilin-2 in colorectal carcinoma cell lines was correlated with loss of epithelial markers such as cytokeratin-20 and E-cadherin and with acquisition of mesenchymal molecules such as vimentin. Furthermore, we showed by surface plasmon resonance experiments that neuropilin-2 is a receptor for transforming-growth factor-ß1. The expression of neuropilin-2 on colon cancer cell lines was indeed shown to promote transforming-growth factor-ß1 signaling, leading to a constitutive phosphorylation of the Smad2/3 complex. Treatment with specific TGFß-type1 receptor kinase inhibitors restored E-cadherin levels and inhibited in part neuropilin-2-induced vimentin expression, suggesting that neuropilin-2 cooperates with TGFß-type1 receptor to promote epithelial-mesenchymal transition in colorectal cancer cells. Our results suggest a direct role of NRP2 in epithelial-mesenchymal transition and highlight a cross-talk between neuropilin-2 and TGF-ß1 signaling to promote cancer progression. These results suggest that neuropilin-2 fulfills all the criteria of a therapeutic target to disrupt multiple oncogenic functions in solid tumors.

Membrane-associated Hsp72 from tumor-derived exosomes mediates STAT3-dependent immunosuppressive function of mouse and human myeloid-derived suppressor cells.

Myeloid-derived suppressor cells (MDSCs) have been identified in humans and mice as a population of immature myeloid cells with the ability to suppress T cell activation. They accumulate in tumor-bearing mice and humans and have been shown to contribute to cancer development. Here, we have isolated tumor-derived exosomes (TDEs) from mouse cell lines and shown that an interaction between TDE-associated Hsp72 and MDSCs determines the suppressive activity of the MDSCs via activation of Stat3. In addition, tumor-derived soluble factors triggered MDSC expansion via activation of Erk. TDE-associated Hsp72 triggered Stat3 activation in MDSCs in a TLR2/MyD88-dependent manner through autocrine production of IL-6. Importantly, decreasing exosome production using dimethyl amiloride enhanced the in vivo antitumor efficacy of the chemotherapeutic drug cyclophosphamide in 3 different mouse tumor models. We also demonstrated that this mechanism is relevant in cancer patients, as TDEs from a human tumor cell line activated human MDSCs and triggered their suppressive function in an Hsp72/TLR2-dependent manner. Further, MDSCs from cancer patients treated with amiloride, a drug used to treat high blood pressure that also inhibits exosome formation, exhibited reduced suppressor functions. Collectively, our findings show in both mice and humans that Hsp72 expressed at the surface of TDEs restrains tumor immune surveillance by promoting MDSC suppressive functions.