Circulating microRNA profiling in patients with advanced non-squamous NSCLC receiving bevacizumab/erlotinib followed by platinum-based chemotherapy at progression (SAKK 19/05).

OBJECTIVES Molecular subclassification of non small-cell lung cancer (NSCLC) is essential to improve clinical outcome. This study assessed the prognostic and predictive value of circulating micro-RNA (miRNA) in patients with non-squamous NSCLC enrolled in the phase II SAKK (Swiss Group for Clinical Cancer Research) trial 19/05, receiving uniform treatment with first-line bevacizumab and erlotinib followed by platinum-based chemotherapy at progression. MATERIALS AND METHODS Fifty patients with baseline and 24 h blood samples were included from SAKK 19/05. The primary study endpoint was to identify prognostic (overall survival, OS) miRNA's. Patient samples were analyzed with Agilent human miRNA 8x60K microarrays, each glass slide formatted with eight high-definition 60K arrays. Each array contained 40 probes targeting each of the 1347 miRNA. Data preprocessing included quantile normalization using robust multi-array average (RMA) algorithm. Prognostic and predictive miRNA expression profiles were identified by Spearman's rank correlation test (percentage tumor shrinkage) or log-rank testing (for time-to-event endpoints). RESULTS Data preprocessing kept 49 patients and 424 miRNA for further analysis. Ten miRNA's were significantly associated with OS, with hsa-miR-29a being the strongest prognostic marker (HR=6.44, 95%-CI 2.39-17.33). Patients with high has-miR-29a expression had a significantly lower survival at 10 months compared to patients with a low expression (54% versus 83%). Six out of the 10 miRNA's (hsa-miRN-29a, hsa-miR-542-5p, hsa-miR-502-3p, hsa-miR-376a, hsa-miR-500a, hsa-miR-424) were insensitive to perturbations according to jackknife cross-validation on their HR for OS. The respective principal component analysis (PCA) defined a meta-miRNA signature including the same 6 miRNA's, resulting in a HR of 0.66 (95%-CI 0.53-0.82). CONCLUSION Cell-free circulating miRNA-profiling successfully identified a highly prognostic 6-gene signature in patients with advanced non-squamous NSCLC. Circulating miRNA profiling should further be validated in external cohorts for the selection and monitoring of systemic treatment in patients with advanced NSCLC.

Chondrocytes expressing intracellular collagen type II enter the cell cycle and co-express collagen type I in monolayer culture.

For autologous chondrocyte transplantation, articular chondrocytes are harvested from cartilage tissue and expanded in vitro in monolayer culture. We aimed to characterize with a cellular resolution the synthesis of collagen type II (COL2) and collagen type I (COL1) during expansion in order to further understand why these cells lose the potential to form cartilage tissue when re-introduced into a microenvironment that supports chondrogenesis. During expansion for six passages, levels of transcripts encoding COL2 decreased to <0.1%, whereas transcript levels encoding COL1 increased 370-fold as compared to primary chondrocytes. Flow cytometry for intracellular proteins revealed that chondrocytes acquired a COL2/COL1-double positive phenotype during expansion, and the COL2 positive cells were able to enter the cell cycle. While the fraction of COL2 positive cells decreased from 70% to <2% in primary chondrocytes to passage six cells, the fraction of COL1 positive cells increased from <1% to >95%. In parallel to the decrease of the fraction of COL2 positive cells, the cells' potential to form cartilage-like tissue in pellet cultures steadily decreased. Intracellular staining for COL2 enables for characterization of chondrocyte lineage cells in more detail with a cellular resolution, and it may allow predicting the effectiveness of expanded chondrocytes to form cartilage-like tissue.

Controlled assembly and single electron charging of monolayer protected Au 144 clusters: an electrochemistry and scanning tunneling spectroscopy study

Single gold particles may serve as room temperature single electron memory units because of their size dependent electronic level spacing. Here, we present a proof-of-concept study by electrochemically controlled scanning probe experiments performed on tailor-made Au particles of narrow dispersity. In particular, the charge transport characteristics through chemically synthesized hexane-1-thiol and 4-pyridylbenzene-1-thiol mixed monolayer protected Au144 clusters (MPCs) by differential pulse voltammetry (DPV) and electrochemical scanning tunneling spectroscopy (EC-STS) are reported. The pyridyl groups exposed by the Au-MPCs enable their immobilization on Pt(111) substrates. By varying the humidity during their deposition, samples coated by stacks of compact monolayers of Au-MPCs or decorated with individual, laterally separated Au-MPCs are obtained. DPV experiments with stacked monolayers of Au144-MPCs and EC-STS experiments with laterally separated individual Au144-MPCs are performed both in aqueous and ionic liquid electrolytes. Lower capacitance values were observed for individual clusters compared to ensemble clusters. This trend remains the same irrespective of the composition of the electrolyte surrounding the Au144-MPC. However, the resolution of the energy level spacing of the single clusters is strongly affected by the proximity of neighboring particles.

CO2 Electroreduction on Cu-Modified Platinum Single Crystal Electrodes in Aprotic Media

Techniques of electrode modification by copper deposits are developed that allow obtaining compact bulk quasi-epitaxial deposits on basal Pt(hkl) single crystal faces. The issues of the deposit roughness and characterization are discussed. Problems of drying and transferring electrodes with copper deposits into other solutions are considered. The obtained deposits are used for CO2 electroreduction in propylene carbonate and acetonitrile solutions of 0.1 M TBAPF6, and the relationship between the electrode surface structure and its electrocatalytic activity in CO2 electroreduction is discussed. We also demonstrate that the restructuring of Cu deposits occurs upon CO2 electroreduction. Complementary reactivity studies are presented for bare Pt(hkl) and Cu(hkl) single crystal electrodes. Cu-modified Pt(hkl) electrodes display the highest activity as compared to bare Pt(hkl) and Cu(hkl). Particularly, the Cu/Pt(110) electrode shows the highest activity among the electrodes under study. Such high activity of Cu/Pt(hkl) electrodes can be explained not only by the increasing actual surface area but also by structural effects, namely by the presence of a large amount of specific defect sites (steps, kinks) on Cu crystallites.

The Interplay between Melting, Refertilization and Carbonatite Metasomatism in Off-Cratonic Lithospheric Mantle under Zealandia: an Integrated Major, Trace and Platinum Group Element Study

It is widely accepted that stabilization of the continental crust requires the presence of sub-continental lithospheric mantle. However, the degree of melt depletion required to stabilize the lithosphere and whether widespread refertilization is a significant process remain unresolved. Here, major and trace element, including platinum group elements (PGE), characterization of 40 mantle xenoliths from 13 localities is used to constrain the melt depletion, refertilization and metasomatic history of lithospheric mantle underneath the micro-continent Zealandia. Our previously published Re–Os isotopic data for a subset of these xenoliths indicate Phanerozoic to Paleoproterozoic ages and, reinterpreted with the new major and trace element data presented here, demonstrate that a large volume (>2 million km3) of lithospheric mantle with an age of 1·99 ± 0·21 Ga is present below the much younger crust of Zealandia. A peritectic melting model using moderately incompatible trace elements (e.g. Yb) in bulk-rocks demonstrates that these peridotites experienced a significant range of degrees of partial melting, between 3 and 28%. During subsolidus equilibration clinopyroxene gains significant rare earth elements (REE), which then leads to the underestimation of the degree of partial melting by ≤12% in fertile xenoliths. A new approach taking into account the effects of subsolidus re-equilibration on clinopyroxene composition effectively removes discrepancies in the calculated degree of melting and provides consistent estimates of between 4 and 29%. The estimated amount of melting is independent of the Re–Os model ages of the samples. The PGE patterns record simple melt depletion histories and the retention of primary base metal sulfides in the majority of the xenoliths. A rapid decrease in Pt/IrN observed at c. 1·0 wt % Al2O3 is a direct result of the exhaustion of sulfide in the mantle residue at c. 20–25% partial melting and the inability of Pt to form a stable alloy phase. Major elements preserve evidence for refertilization by a basaltic component that resulted in the formation of secondary clinopyroxene and low-forsterite olivine. The majority of xenoliths show the effects of cryptic metasomatic overprinting, ranging from minor to strong light REE enrichments in bulk-rocks (La/YbN = 0·16–15·9). Metasomatism is heterogeneous, with samples varying from those with weak REE enrichment and notable positive Sr and U–Th anomalies and negative Nb–Ta anomalies in clinopyroxene to those that have extremely high concentrations of REE, Th–U and Nb. Chemical compositions are consistent with a carbonatitic component contributing to the metasomatism of the lithosphere under Zealandia. Notably, the intense metasomatism of the samples did not affect the PGE budget of the peridotites as this was controlled by residual sulfides.

Establishment of a confluent monolayer model with human primary trophoblast cells: novel insights into placental glucose transport

STUDY HYPOTHESIS Using optimized conditions, primary trophoblast cells isolated from human term placenta can develop a confluent monolayer in vitro, which morphologically and functionally resembles the microvilli structure found in vivo. STUDY FINDING We report the successful establishment of a confluent human primary trophoblast monolayer using pre-coated polycarbonate inserts, where the integrity and functionality was validated by cell morphology, biophysical features, cellular marker expression and secretion, and asymmetric glucose transport. WHAT IS KNOWN ALREADY Human trophoblast cells form the initial barrier between maternal and fetal blood to regulate materno-fetal exchange processes. Although the method for isolating pure human cytotrophoblast cells was developed almost 30 years ago, a functional in vitro model with primary trophoblasts forming a confluent monolayer is still lacking. STUDY DESIGN, SAMPLES/MATERIALS, METHODS Human term cytotrophoblasts were isolated by enzymatic digestion and density gradient separation. The purity of the primary cells was evaluated by flow cytometry using the trophoblast-specific marker cytokeratin 7, and vimentin as an indicator for potentially contaminating cells. We screened different coating matrices for high cell viability to optimize the growth conditions for primary trophoblasts on polycarbonate inserts. During culture, cell confluency and polarity were monitored daily by determining transepithelial electrical resistance (TEER) and permeability properties of florescent dyes. The time course of syncytia-related gene expression and hCG secretion during syncytialization were assessed by quantitative RT-PCR and enzyme-linked immunosorbent assay, respectively. The morphology of cultured trophoblasts after 5 days was determined by light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Membrane makers were visualized using confocal microscopy. Additionally, glucose transport studies were performed on the polarized trophoblasts in the same system. MAIN RESULTS AND THE ROLE OF CHANCE During 5-day culture, the highly pure trophoblasts were cultured on inserts coated with reconstituted basement membrane matrix . They exhibited a confluent polarized monolayer, with a modest TEER and a size-dependent apparent permeability coefficient (Papp) to fluorescently labeled compounds (MW ∼400-70 000 Da). The syncytialization progress was characterized by gradually increasing mRNA levels of fusogen genes and elevating hCG secretion. SEM analyses confirmed a confluent trophoblast layer with numerous microvilli, and TEM revealed a monolayer with tight junctions. Immunocytochemistry on the confluent trophoblasts showed positivity for the cell-cell adhesion molecule E-cadherin, the tight junction protein 1 (ZO-1) and the membrane proteins ATP-binding cassette transporter A1 (ABCA1) and glucose transporter 1 (GLUT1). Applying this model to study the bidirectional transport of a non-metabolizable glucose derivative indicated a carrier-mediated placental glucose transport mechanism with asymmetric kinetics. LIMITATIONS, REASONS FOR CAUTION The current study is only focused on primary trophoblast cells isolated from healthy placentas delivered at term. It remains to be evaluated whether this system can be extended to pathological trophoblasts isolated from diverse gestational diseases. WIDER IMPLICATIONS OF THE FINDINGS These findings confirmed the physiological properties of the newly developed human trophoblast barrier, which can be applied to study the exchange of endobiotics and xenobiotics between the maternal and fetal compartment, as well as intracellular metabolism, paracellular contributions and regulatory mechanisms influencing the vectorial transport of molecules. LARGE-SCALE DATA Not applicable. STUDY FUNDING AND COMPETING INTERESTS This study was supported by the Swiss National Center of Competence in Research, NCCR TransCure, University of Bern, Switzerland, and the Swiss National Science Foundation (grant no. 310030_149958, C.A.). All authors declare that their participation in the study did not involve factual or potential conflicts of interests.