Real-time,label-free characterization of oligosaccharide-binding proteins using carbohydrate microarrays and an ellipsometry-based biosensor

Carbohydrates present on cell surfaces mediate cell behavior through interactions with other biomolecules. Due to their structural complexity, diversity, and heterogeneity, it is difficult to fully characterize a variety of carbohydrates and their binding partners. As a result, novel technologies for glycomics applications have been developed, including carbohydrate microarrays and label-free detection methods. In this paper, we report using the combination of oligosaccharide microarrays and the label-free oblique-incidence reflectivity difference microscopy for real-time characterization of oligosaccharide-binding proteins. Aminated human milk oligosaccharides were immobilized on epoxy-coated glass substrates as microarrays for reactions with Family 1 of solute-binding proteins from Bifidobacterium longum subsp. infantis (B. infantis). Binding affinities of these protein–oligosaccharide interactions showed preferences of Family 1 of solute-binding proteins to host glycans, which helps in characterizing the complex process of human milk oligosaccharides foraging by B. infantis.     

Cellular Dielectric Spectroscopy: A Label-Free Technology for Drug Discovery

CDS is a technology that enables comprehensive pharmacological evaluation of cell surface receptors in real time. It is a universal assay that allows measurement of multiple types of receptors, including G protein-coupled receptors (GPCRs), tyrosine kinase receptors (TKRs), and steroid receptors using the same platform, without the need for transfections or special reagents. The CDS-based platform is sensitive enough to consistently monitor ligand-mediated activation of endogenously expressed receptors, and for this reason, has the ability to generate more physiologically relevant data than obtained with genetically and chemically manipulated cells.The label-free CDS measurement is a measure of cell function as opposed to binding events that are typical of many current label-free approaches: classical (BIAcore's SPR technology) and more recent (Corning's EPIC, SRU Biosystems' BIND, CSEM's WIOS, and Akubio's RAP technology). A detailed comparison of these and other impedance-based measurement systems is available in a recent review of label-free technologies.⁵⁸To illustrate the power of the CDS technology, data has been presented demonstrating the use of this label-free approach in pharmacological evaluation of ligands for a number of receptors in a variety of cell types, including primary cells. Specifically, the CDS technology has been applied to hit confirmation, receptor selectivity analysis, ligand potency, and Schild analysis of receptor-selective antagonists. CDS measurements quantitatively align with results from other cell-based assays in determining the potency and ranking of agonists and antagonists. The robust and reproducible nature of CDS assays is reflected in low coefficients of variation, high signal-to-background, and excellent Z'-scores of the data. The data generated using CDS technology are unique in that the response profiles reflect receptor-mediated signaling pathways and are characteristic of main subsets of GPCRs within a cell line. Clustering analysis has been performed using response profiles from receptors that lead to potential novel applications including identification of the G-protein coupling of unknown GPCRs and the cataloging of active endogenous receptors in cell lines. Because the CDS assay requires no stable or transiently transfected cells or special reagents, assay development and data acquisition are simple and fast. These studies demonstrate that the CDS platform provides an easy-to-use, label-free, universal assay with applications in secondary screening, hit identification and lead optimization areas of drug discovery. The CellKey™ system, based on CDS technology, will be commercially available in late 2005.²⁹     

Multiple Imputation Approaches Applied to the Missing Value Problem in Bottom-Up Proteomics

Analysis of differential abundance in proteomics data sets requires careful application of missing value imputation. Missing abundance values widely vary when performing comparisons across different sample treatments. For example, one would expect a consistent rate of “missing at random” (MAR) across batches of samples and varying rates of “missing not at random” (MNAR) depending on the inherent difference in sample treatments within the study. The missing value imputation strategy must thus be selected that best accounts for both MAR and MNAR simultaneously. Several important issues must be considered when deciding the appropriate missing value imputation strategy: (1) when it is appropriate to impute data; (2) how to choose a method that reflects the combinatorial manner of MAR and MNAR that occurs in an experiment. This paper provides an evaluation of missing value imputation strategies used in proteomics and presents a case for the use of hybrid left-censored missing value imputation approaches that can handle the MNAR problem common to proteomics data.     

MPP+-Induced Changes in Cellular Impedance as a Measure for Organic Cation Transporter (SLC22A1-3) Activity and Inhibition

The organic cation transporters OCT1-3 (SLC22A1-3) facilitate the transport of cationic endo- and xenobiotics and are important mediators of drug distribution and elimination. Their polyspecific nature makes OCTs highly susceptible to drug–drug interactions (DDIs). Currently, screening of OCT inhibitors depends on uptake assays that require labeled substrates to detect transport activity. However, these uptake assays have several limitations. Hence, there is a need to develop novel assays to study OCT activity in a physiological relevant environment without the need to label the substrate. Here, a label-free impedance-based transport assay is established that detects OCT-mediated transport activity and inhibition utilizing the neurotoxin MPP⁺. Uptake of MPP⁺ by OCTs induced concentration-dependent changes in cellular impedance that were inhibited by decynium-22, corticosterone, and Tyrosine Kinase inhibitors. OCT-mediated MPP⁺ transport activity and inhibition were quantified on both OCT1-3 overexpressing cells and HeLa cells endogenously expressing OCT3. Moreover, the method presented here is a valuable tool to identify novel inhibitors and potential DDI partners for MPP⁺ transporting solute carrier proteins (SLCs) in general.     

Oxide-on-graphene field effect bio-ready sensors

Electrical detection schemes using nanoscale devices offer fast and label-free alternatives to biosensing techniques based on chemical and optical interactions. Here we report on the design, fabrication, and operation of oxide-on-graphene ion-sensitive field effect sensor arrays using large-area graphene sheets synthesized by chemical vapor deposition. In this scheme, HfO2 and SiO2 thin films are deposited atop the graphene sheet and play the dual role of the sensing interface, as well as the passivation layer protecting the channel and electrodes underneath from direct contact with the electrolyte. We further demonstrate the functionalization of the SiO2 surface with 3-aminopropyltrimethoxysilane （APTMS）. The oxide-on-graphene sensors operate in solution with high stability and a high average mobility of 5,000 cm2/（V＇s）. As a proof of principle, we demonstrate pH sensing using the bare or the APTMS-functionalized SiO2 as the sensing surface. The measured sensitivities, 46 mV/pH and 43 mV/pH, respectively, agree well with existing studies. We further show that by applying the solution gate voltage in pulse, the hysteresis in the transfer curve of the graphene transducer can be eliminated, greatly improving the ionic potential resolution of the sensor. These experiments demonstrate the potential of oxide-on-graphene ion-sensitive field effect sensors in on-chip, label-free and real-time biosensing applications.     

Molecular Interaction of a New Antibacterial Polymer with a Supported Lipid Bilayer Measured by an in situ Label-Free Optical Technique

The interaction of the antibacterial polymer–branched poly(ethylene imine) substituted with quaternary ammonium groups, PEO and alkyl chains, PEI₂₅QI₅J₅A8₁₅–with a solid supported lipid bilayer was investigated using surface sensitive optical waveguide spectroscopy. The analysis of the optogeometrical parameters was extended developing a new composite layer model in which the structural and optical anisotropy of the molecular layers was taken into consideration. Following in situ the change of optical birefringence we were able to determine the composition of the lipid/polymer surface layer as well as the displacement of lipid bilayer by the antibacterial polymer without using additional labeling. Comparative assessment of the data of layer thickness and optical anisotropy helps to reveal the molecular mechanism of antibacterial effect of the polymer investigated.     

Multimodal Label-Free Monitoring of Adipogenic Stem Cell Differentiation Using Endogenous Optical Biomarkers

Stem cell-based therapies carry significant promise for treating human diseases. However, clinical translation of stem cell transplants for effective treatment requires precise non-destructive evaluation of the purity of stem cells with high sensitivity (<0.001% of the number of cells). Here, a novel methodology using hyperspectral imaging (HSI) combined with spectral angle mapping-based machine learning analysis is reported to distinguish differentiating human adipose-derived stem cells (hASCs) from control stem cells. The spectral signature of adipogenesis generated by the HSI method enables identifying differentiated cells at single-cell resolution. The label-free HSI method is compared with the standard techniques such as Oil Red O staining, fluorescence microscopy, and qPCR that are routinely used to evaluate adipogenic differentiation of hASCs. HSI is successfully used to assess the abundance of adipocytes derived from transplanted cells in a transgenic mice model. Further, Raman microscopy and multiphoton-based metabolic imaging is performed to provide complementary information for the functional imaging of the hASCs. Finally, the HSI method is validated using matrix-assisted laser desorption/ionization-mass spectrometry imaging of the stem cells. The study presented here demonstrates that multimodal imaging methods enable label-free identification of stem cell differentiation with high spatial and chemical resolution.     

受激拉曼散射显微技术用于快速无标记病理成像

对病变组织进行快速成像以获取足够的诊断信息,可以帮助医生在术中做出重要决策。受激拉曼散射显微技术是一种新兴的无标记成像技术,利用生物分子自身的光散射性质,在不需要对组织进行任何处理的情况下即可获取类比于传统病理的成像结果。越来越多的研究表明受激拉曼散射显微技术作为一种虚拟组织成像工具,可以迅速区分病变与正常组织。这篇综述主要阐述了受激拉曼散射显微成像的基本原理、发展现状及其在组织成像上的应用。     

Proteomic Landscape of Adeno-Associated Virus (AAV)-Producing HEK293 Cells

Adeno-associated viral (AAV) vectors are widely used for gene therapy, providing treatment for diseases caused by absent or defective genes. Despite the success of gene therapy, AAV manufacturing is still challenging, with production yields being limited. With increased patient demand, improvements in host cell productivity through various engineering strategies will be necessary. Here, we study the host cell proteome of AAV5-producing HEK293 cells using reversed phase nano-liquid chromatography and tandem mass spectrometry (RPLC-MS/MS). Relative label-free quantitation (LFQ) was performed, allowing a comparison of transfected vs. untransfected cells. Gene ontology enrichment and pathway analysis revealed differential expression of proteins involved in fundamental cellular processes such as metabolism, proliferation, and cell death. Furthermore, changes in expression of proteins involved in endocytosis and lysosomal degradation were observed. Our data provides highly valuable insights into cellular mechanisms involved during recombinant AAV production by HEK293 cells, thus potentially enabling further improvements of gene therapy product manufacturing.