Simplified Protocol for Detection of Protein−Ligand Interactions via Surface-Enhanced Resonance Raman Scattering and Surface-Enhanced Fluorescence

simple and effective protocol for detections of protein−protein and protein−small molecule interactions has been developed. After interactions between proteins and their corresponding ligands, we employed colloidal silver staining for producing active substrates for surface-enhanced Raman scattering (SERS) and surface-enhanced fluorescence (SEF). Tetramethylrhodamine isothiocyanate (TRITC) and Atto610 were used for both Raman and fluorescent probes. We detected interactions between human IgG and TRITC−anti-human IgG, and those between avidin and Atto610-biotin by surface-enhanced resonance Raman scattering (SERRS) and SEF. The detection limits of the proposed SERRS-based method are comparable to those of the proposed SEF-based one, 0.9 pg/mL for anti-human IgG and 0.1 pg/mL for biotin. This protocol exploits several advantages of simplicity over other SERS and SEF-based related methods because of the protein staining-based strategy for silver nanoparticle assembling, high sensitivity from SERRS and SEF, and high stability in photostability comparing to fluorescence-based protein detections. Therefore, the proposed method for detection of protein−ligand interactions has great potential in high-sensitivity and high-throughput chip-based protein function determination.

http://pubs.acs.org/cgi-bin/abstract.cgi/ancham/asap/abs/ac800642g.html

Surface-structure-regulated cell-membrane penetration by monolayer-protected nanoparticles

Nanoscale objects are typically internalized by cells into membrane-bounded endosomes and fail to access the cytosolic cell machinery. Whereas some biomacromolecules may penetrate or fuse with cell membranes without overt membrane disruption, no synthetic material of comparable size has shown this property yet. Cationic nano-objects pass through cell membranes by generating transient holes, a process associated with cytotoxicity. Studies aimed at generating cell-penetrating nanomaterials have focused on the effect of size, shape and composition. Here, we compare membrane penetration by two nanoparticle ‘isomers’ with similar composition (same hydrophobic content), one coated with subnanometre striations of alternating anionic and hydrophobic groups, and the other coated with the same moieties but in a random distribution. We show that the former particles penetrate the plasma membrane without bilayer disruption, whereas the latter are mostly trapped in endosomes. Our results offer a paradigmfor analysing the fundamental problemof cell-membrane-penetrating bio- and macro-molecules.

http://www.nature.com/nmat/journal/v7/n7/full/nmat2202.html

Development of aptamer-based affinity assays using temperature gradient focusing: Minimization of the limit of detection

A method is described for an aptamer-based affinity assay using a combination of two nonconventional techniques, temperature gradient focusing (TGF) and field-amplified continuous sample injection TGF (FACSI-TGF), with fluorescence detection. Human immunodeficiency virus reverse transcriptase (HIVRT) is used as the protein target for the assay. The TGF and FACSI-TGF assays are compared to similar results obtained with conventional CE. A range of starting aptamer concentrations are used to determine the optimal LOD for human immunodeficiency virus reverse transcriptase (HIVRT) using each approach. The results indicate that the LODs for HIVRT obtained with TGF and FACSI-TGF are comparable to or even lower than the LODs obtained with conventional CE in spite of the inferior detector used for the TGF and FACSI-TGF assays (arc lamp and low-cost CCD for TGF versus LIF with PMT for CE). It is hypothesized that this is due to the greater reproducibility of the TGF and FACSI-TGF techniques since they do not employ a defined sample injection. The lowest LOD achieved with the new aptamer assay approach is more than an order of magnitude lower than that reported for a similar CE-based aptamer assay for the same target.

On-Chip, Cell-Based Microarray Immunofluorescence Assay for High-Throughput Analysis of Target Proteins

We have developed an immunofluorescence-based assay for high-throughput analysis of target proteins on a three-dimensional cellular microarray platform. This process integrates the use of three-dimensional cellular microarrays, which should better mimic the cellular microenvironment, with sensitive immunofluorescence detection and provides quantitative information on cell function. To demonstrate this assay platform, we examined the accumulation of the α subunit of the hypoxia-inducible factor (HIF-1α) after chemical stimulation of human pancreatic tumor cells encapsulated in 3D alginate spots in volumes as low as 60 nL. We also tested the effect of the known dysregulator of HIF-1α, 2-methoxyestradiol (2ME2), on the levels of HIF-1α using a dual microarray stamping technique. This chip-based in situ Western immunoassay protocol was able to provide quantitative information on cell function, namely, the cellular response to hypoxia mimicking conditions and the reduction of HIF-1α levels after cell treatment with 2ME2. This system is the first to enable high-content screening of cellular protein levels on a 3D human cell microarray platform.

http://pubs.acs.org/cgi-bin/abstract.cgi/ancham/asap/abs/ac800848j.html

Toward a Fast, Easy, and Versatile Immobilization of Biomolecules into Carbon Nanotube/Polysulfone-Based Biosensors for the Detection of hCG Hormone

The aim of this study was the fabrication and characterization of biomembranes by the phase inversion (PI) method followed by their subsequent casting onto screen-printed electrodes (SPE) for biomedical applications. The combination of multiwalled carbon nanotubes (MWCNT) as a transducer with polysulfone (PSf) polymer enables easy incorporation of biological moieties (hormones or antibodies), providing a 3D composite with high electrochemical response to corresponding analytes. Antibody/MWCNT/PSf biosensors were characterized by confocal scanning laser microscopy (CSLM), scanning electron microscopy (SEM), and electrochemical methods. For biomedical purposes, human chorionic gonadotropin (hCG) hormone was tested by competitive immunoassay. The detection limit was determined to be 14.6 mIU/mL with a linear range up to 600 mIU/mL. We concluded that the easy and fast incorporation of biomolecules by the PI method, as well as their stability and distribution throughout the 3D polysulfone composite, are testament to the utility for the versatile fabrication of biosensors for clinical diagnosis.

Since our group has recently involved in CNT research this may be another interesting one.

http://pubs.acs.org/cgi-bin/abstract.cgi/ancham/asap/abs/ac7025282.html

Ligand-Induced Structural Changes in Maltose Binding Proteins Measured by Atomic Force Microscopy

We use atomic force microscopy (AFM) based force-compression measurements to probe the ligand-induced functional conformational changes in surface-immobilized dicysteine-terminated maltose binding proteins (dicys-MBPs). The proteins are immobilized at well-defined locations directly on Au substrates using the previously reported technique of nanografting. By measuring the difference between the ligand-free and ligand-bound mechanical work performed by the AFM-tip during the protein compression, we determine the open-closed transition energy for dicys-MBPs to be ΔE0 ) (8 ( 4) Kcal/mol. We also compare the binding kinetics of two different ligands (maltose and maltotriose) to dicys-MBPs by performing AFM-friction measurements. We show that our results are consistent with a simple model for the surface-immobilized dicys-MBPs: the protein consists of two rigid lateral lobes connected by a hinge-loaded spring.

http://pubs.acs.org/cgi-bin/asap.cgi/nalefd/asap/html/nl801553h.html

Matuzumab Binding to EGFR Prevents the Conformational Rearrangement Required for Dimerization

Cancer Cell, 13, 2008, 365

This article talks about EGFR monoclonal antibody therapy.

An increasing number of therapeutic antibodies targeting tumors that express the epidermal growth factor receptor (EGFR) are in clinical use or late stages of clinical development. Here we investigate the molecular basis for inhibition of EGFR activation by the therapeutic antibody matuzumab (EMD72000). We describe the X-ray crystal structure of the Fab fragment of matuzumab (Fab72000) in complex with isolated domain III from the extracellular region of EGFR. Fab72000 interacts with an epitope on EGFR that is distinct from the ligand-binding region on domain III and from the cetuximab/Erbitux epitope. Matuzumab blocks ligand-induced receptor activation indirectly by sterically preventing the domain rearrangement and local conformational changes that must occur for high-affinity ligand binding and receptor dimerization.

Highly Efficient, Functional Engraftment of Skeletal Muscle Stem Cells in Dystrophic Muscles

Cell, 134, 2008, 37

Interesting for tissue regrowth.

Satellite cells reside beneath the basal lamina of skeletal muscle fibers and include cells that act as precursors for muscle growth and repair. Although they share a common anatomical localization and typically are considered a homogeneous population, satellite cells actually exhibit substantial heterogeneity. We used cell-surface marker expression to purify from the satellite cell pool a distinct population of skeletal muscle precursors (SMPs) that function as muscle stem cells. When engrafted into muscle of dystrophin-deficient mdx mice, purified SMPs contributed to up to 94% of myofibers, restoring dystrophin expression and significantly improving muscle histology and contractile function. Transplanted SMPs also entered the satellite cell compartment, renewing the endogenous stem cell pool and participating in subsequent rounds of injury repair. Together, these studies indicate the presence in adult skeletal muscle of prospectively isolatable muscle-forming stem cells and directly demonstrate the efficacy of myogenic stem cell transplant for treating muscle degenerative disease.

New Insights into Tumor Microstructure Using Temporal Diffusion Spectroscopy

Cancer Research, 68, 2008, 5941

Magnetic resonance images (MRI) that depict rates of water diffusion in tissues can be used to characterize the cellularity of tumors and are valuable in assessing their early response to treatment. Water diffusion rates are sensitive to the cellular and molecular content of tissues and are affected by local microstructural changes associated with tumor development. However, conventional maps of water diffusion reflect the integrated effects of restrictionsto free diffusion at multiple scales up to a specific limiting spatial dimension, typically several micrometers. Such measurements cannot distinguish effects caused by structural variations at a smaller scale. Variations in diffusion rates then largely reflect variations in the density of cells, and no information is available about changes on a subcellular scale. We report here our experiences using a new approach based on Oscillating Gradient Spin-Echo (OGSE) MRI methods that can differentiate the influence on water diffusion of structural changes on scales much smaller than the diameter of a single cell. MRIs of glioblastomas in rat brain in vivo show an increased contrast and spatial heterogeneity when diffusion measurements are selectively sensitized to shorter distance scales. These results show the benefit of OGSE methods for revealing microscopic variations in tumors in vivo and confirm that diffusion measurements depend on factors other than cellularity.

Cell Lineage Analysis of a Mouse Tumor

Cancer Research, 68, 2008, 5924

Revealing the lineage relations among cancer cells can shed light on tumor growth patterns and metastasis formation, yet cell lineages have been difficult to come by in the absence of a suitable method. We previously developed a method for reconstructing cell lineage trees from genomic variability caused by somatic mutations. Here, we apply the method to cancer and reconstruct, for the first time, a lineage tree of neoplastic and adjacent normal cells obtained by laser microdissection from tissue sections of a mouse lymphoma. Analysis of the reconstructed tree reveals that the tumor initiated from a single founder cell, 5 months before diagnosis, that the tumor grew in a physically coherent manner, and that the average number of cell divisions accumulated in cancerous cells was almost twice than in adjacent normal lung epithelial cells but slightly less than the expected figure for normal B lymphocytes. The cells were also genotyped at the TP53 locus, and neoplastic cells were found to share a common mutation, which was most likely present in a heterozygous state. Our work shows that the ability to obtain data regarding the physical appearance, precise anatomic position, genotypic profile, and lineage position of single cells may be useful for investigating cancer development, progression, and interaction with the microenvironment.