Determination of Intra- and Extracellular Metabolic Adaptations of 3D Cell Cultures upon Challenges in Real-Time by NMR

NMR flow devices provide longitudinal real-time quantitative metabolome characterisation of living cells. However, discrimination of intra- and extracellular contributions to the spectra represents a major challenge in metabolomic NMR studies. The present NMR study demonstrates the possibility to quantitatively measure both metabolic intracellular fingerprints and extracellular footprints on human control fibroblasts by using a commercially available flow tube system with a standard 5 mm NMR probe. We performed a comprehensive 3D cell culture system characterisation. Diffusion NMR was employed for intra- and extracellular metabolites separation. In addition, complementary extracellular footprints were determined. The implemented perfused NMR bioreactor system allowed the determination of 35 metabolites and intra- and extracellular separation of 19 metabolites based on diffusion rate differences. We show the reliability and sensitivity of NMR diffusion measurements to detect metabolite concentration changes in both intra- and extracellular compartments during perfusion with different selective culture media, and upon complex I inhibition with rotenone. We also demonstrate the sensitivity of extracellular footprints to determine metabolic variations at different flow rates. The current method is of potential use for the metabolomic characterisation of defect fibroblasts and for improving physiological comprehension.     

Cloning and Functional Characterization of Dog OCT1 and OCT2: Another Step in Exploring Species Differences in Organic Cation Transporters

OCT1 and OCT2 are polyspecific membrane transporters that are involved in hepatic and renal drug clearance in humans and mice. In this study, we cloned dog OCT1 and OCT2 and compared their function to the human and mouse orthologs. We used liver and kidney RNA to clone dog OCT1 and OCT2. The cloned and the publicly available RNA-Seq sequences differed from the annotated exon-intron structure of OCT1 in the dog genome CanFam3.1. An additional exon between exons 2 and 3 was identified and confirmed by sequencing in six additional dog breeds. Next, dog OCT1 and OCT2 were stably overexpressed in HEK293 cells and the transport kinetics of five drugs were analyzed. We observed strong differences in the transport kinetics between dog and human orthologs. Dog OCT1 transported fenoterol with 12.9-fold higher capacity but 14.3-fold lower affinity (higher K_M) than human OCT1. Human OCT1 transported ipratropium with 5.2-fold higher capacity but 8.4-fold lower affinity than dog OCT1. Compared to human OCT2, dog OCT2 showed 10-fold lower transport of fenoterol and butylscopolamine. In conclusion, the functional characterization of dog OCT1 and OCT2 reported here may have implications when using dogs as pre-clinical models as well as for drug therapy in dogs.     

High-temperature performance of InGaAs-InGaAlAs-InP 1.79-μmdiode lasers grown in solid-source molecular-beam epitaxy

1.79-μm InGaAs-InGaAlAs strained-layer quantum-well diode lasers have been fabricated. A characteristic temperature of 72 K has been achieved. At a temperature as high as 100°C, a continuous-wave output power of more than 6.5 mW per facet has been demonstrated with lasers using as-cleaved facets as mirrors     

A Si bipolar monolithic RF bandpass amplifier

The application of monolithic inductors to the realization of Si bipolar monolithic RF amplifiers is investigated. As a test vehicle, a bipolar monolithic bandpass amplifier was fabricated and characterized. A 4-nH silicon integrated inductor was used to achieve a peak S ₂₁ gain of 8 dB, a simulated noise figure of 6.4 dB, and a matched input impedance of 50 Ω in the frequency range of 1-2 GHz     

A 100-MHz macropipelined VAX microprocessor

A macropipelined CISC microprocessor was implemented in a 0.75-μm CMOS 3.3-V technology. The 1.3-million-transistor custom chip measures 1.62×1.46 cm² and dissipates 16.3 W. The 100-MHz parts were benchmarked at 50 SPEC marks. The on-chip clocking system and several high-performance logic and circuit techniques are described. Macroinstruction handling, micropipeline management, and control store structures highlight the design architecture. The hierarchical array organization and fast tag comparison technique of the primary cache are discussed. Power estimation procedures are outlined, and the results are compared to measurements. Physical design and verification methods, and CAD tools are also described. After extensive functional verification efforts are described, chip and system test results are presented     

Characterization of the CH4/H2/Ar high density plasma etching process for HgCdTe

High density plasma etching of mercury cadmium telluride using CH₄/H₂/Ar plasma chemistries is investigated. Mass spectrometry is used to identify and monitor etch products evolving from the surface during plasma etching. The identifiable primary etch products are elemental Hg, TeH₂, and Cd(CH₃)₂. Their relative concentrations are monitored as ion and neutral fluxes (both in intensity and composition), ion energy and substrate temperature are varied. General insights are made into surface chemistry mechanisms of the etch process. These insights are evaluated by examining etch anisotropy and damage to the remaining semiconductor material. Regions of process parameter space best suited to moderate rate, anisotropic, low damage etching of HgCdTe are identified.     

In situ Characterization of SiO2 Nanoparticle Biointeractions Using BrightSilica

By adding a gold core to silica nanoparticles (BrightSilica), silica‐like nanoparticles are generated that, unlike unmodified silica nanoparticles, provide three types of complementary information to investigate the silica nano‐biointeraction inside eukaryotic cells in situ. Firstly, organic molecules in proximity of and penetrating into the silica shell in live cells are monitored by surface‐enhanced Raman scattering (SERS). The SERS data show interaction of the hybrid silica particles with tyrosine, cysteine and phenylalanine side chains of adsorbed proteins. Composition of the biomolecular corona of BrightSilica nanoparticles differs in fibroblast and macrophage cells. Secondly, quantification of the BrightSilica nanoparticles using laser ablation inductively coupled plasma mass spectrometry (LA‐ICP‐MS) micromapping indicates a different interaction of silica nanoparticles compared to gold nanoparticles under the same experimental conditions. Thirdly, the metal cores allow the investigation of particle distribution and interaction in the cellular ultrastructure by cryo nanoscale X‐ray tomography (cryo‐XT). In 3D reconstructions the assumption is confirmed that BrightSilica nanoparticles enter cells by an endocytotic mechanism. The high SERS intensities are explained by the beneficial plasmonic properties due to agglomeration of BrightSilica. The results have implications for the development of multi‐modal qualitative and quantitative characterization in comparative nanotoxicology and bionanotechnology.     

Mechanism of O2-anneal induced Vfb shifts of Ru gated stacks

A systematic study of the flat-band voltage (V_fb) shift of Ru gated metal-oxide-semiconductor (MOS) capacitors subjected to thermal treatment in O₂ has been performed. The dependence of the V_fb shift on the thickness of Ru, anneal temperature and time is studied. The V_fb shift is ascribed to the shift of metal gates’ work function (WF), and is not significantly dependent on the type of dielectric (HfO₂ or SiO₂). From time-of-flight secondary ion mass spectrometry (TOF-SIMS) measurement, it was found that after thermal treatment in ¹⁸O₂, ¹⁸O penetrated through Ru and was incorporated in the Ru/dielectric interface region. We believe that the formation of the thin interfacial RuO_x layer is responsible for the V_fb shift.