Quantitative proteomics for identification of cancer biomarkers

Quantitative proteomics can be used for the identification of cancer biomarkers that could be used for early detection, serve as therapeutic targets, or monitor response to treatment. Several quantitative proteomics tools are currently available to study differential expression of proteins in samples ranging from cancer cell lines to tissues to body fluids. 2-DE, which was classically used for proteomic profiling, has been coupled to fluorescence labeling for differential proteomics. Isotope labeling methods such as stable isotope labeling with amino acids in cell culture (SILAC), isotope-coded affinity tagging (ICAT), isobaric tags for relative and absolute quantitation (iTRAQ), and (18) O labeling have all been used in quantitative approaches for identification of cancer biomarkers. In addition, heavy isotope labeled peptides can be used to obtain absolute quantitative data. Most recently, label-free methods for quantitative proteomics, which have the potential of replacing isotope-labeling strategies, are becoming popular. Other emerging technologies such as protein microarrays have the potential for providing additional opportunities for biomarker identification. This review highlights commonly used methods for quantitative proteomic analysis and their advantages and limitations for cancer biomarker analysis.     

Monoclonal antibody cocktail as an enrichment tool for acetylome analysis

The availability and robustness of methods to analyze phosphorylated proteins has greatly expanded our knowledge of phosphorylation based cell signaling. A key ingredient to the success of these studies is the ability to enrich phosphopeptides using antibodies or other chemical approaches. Most other post-translational modifications, such as lysine acetylation, are still poorly characterized because of the lack of availability of such enrichment methods. Recently, some groups have reported identification of acetylation sites in a global fashion by enriching acetylated peptides with a polyclonal antibody from a single source that was raised against pan-acetylated lysine. Instead of the use of this polyclonal antibody, we used a cocktail of monoclonal antibodies where each was directed against acetylated lysine in different contexts. Using high resolution Fourier transform mass spectrometry, we observed that the majority of acetylated lysine residues identified using the monoclonal antibody cocktail were distinct from those enriched by the polyclonal antibody used by the other groups. Our study demonstrates that immunoaffinity enrichment of acetylated peptides is somewhat limited by substrate specificity and that an optimal yield of enrichment can be achieved by employing a broader array of affinity reagents.     

Production and mechanical properties of SiC<Subscript>p</Subscript> particle-reinforced 2618 aluminum alloy composites

In this study, 2618 aluminum alloy metal matrix composites (MMCs) reinforced with two different sizes and weight fractions of SiC_p particles upto 10% weight were fabricated by stir cast method and subsequent forging operation. The effects of SiC_p particle content and size of the particles on the mechanical properties of the composites such as hardness, tensile strength, hot tensile strength (at 120 °C), and impact strength were investigated. The density measurements showed that the samples contained little porosity with increasing weight fraction. Optical microscopic observations of the microstructures revealed uniform distribution of particles and at some locations agglomeration of particles and porosity. The results show that hardness and tensile strength of the composites increased, with decreasing size and increasing weight fraction of the particles. The hardness and tensile strength of the forged composites were higher than those of the cast samples.     

Enhanced accumulation of phosphate by Lolium multiflorum cultivars grown in phosphate-enriched medium

Agricultural and animal husbandry practices combined with soil composition have caused phosphate overloading of farmlands in different parts of the U.S. and Europe. Movement of soluble phosphates (Pi) from phosphorus enriched soils results in degradation of natural aquatic systems, triggering serious environmental problems. Remediation of such sites using plants that tolerate and accumulate high concentrations of Pi in their aerial parts may be an attractive remediation technology. In the present study, Pi transport and accumulation potential of Marshall and Gulf ryegrass (Lolium multiflorum cultivars) were determined using a solution culture of seedlings. Ryegrass seedlings accumulated phosphorus (P) in excess of 2% of dry weight in their aerial parts when supplied with 5 g/L KH2PO4 in medium. Phosphorus accumulation was positively correlated with the concentration of phosphate (0-5 g/L KH2PO4) in medium. Plants grew well on medium containing 5 g/L KH2PO4, but concentrations above 5 g/L caused symptoms of toxicity. Scanning electron microscopy and energy-dispersive X-ray spectroscopy confirmed high P accumulation in different cell types of grass roots and shoots. Phosphate starvation and replenishment experiments point to the unique ability of these grasses to concentrate phosphate in the above-ground parts. It is hypothesized that the unique ability of these ryegrass cultivars may be due to the presence of efficient phosphate transport and sequestration mechanisms.     

Electrical Conductivity,Selective Adhesion,and Biocompatibility in Bacteria‐Inspired Peptide–Metal Self‐Supporting Nanocomposites

Bacterial type IV pili (T4P) are polymeric protein nanofibers that have diverse biological roles. Their unique physicochemical properties mark them as a candidate biomaterial for various applications, yet difficulties in producing native T4P hinder their utilization. Recent effort to mimic the T4P of the metal‐reducing Geobacter sulfurreducens bacterium led to the design of synthetic peptide building blocks, which self‐assemble into T4P‐like nanofibers. Here, it is reported that the T4P‐like peptide nanofibers efficiently bind metal oxide particles and reduce Au ions analogously to their native counterparts, and thus give rise to versatile and multifunctional peptide–metal nanocomposites. Focusing on the interaction with Au ions, a combination of experimental and computational methods provides mechanistic insight into the formation of an exceptionally dense Au nanoparticle (AuNP) decoration of the nanofibers. Characterization of the thus‐formed peptide–AuNPs nanocomposite reveals enhanced thermal stability, electrical conductivity from the single‐fiber level up, and substrate‐selective adhesion. Exploring its potential applications, it is demonstrated that the peptide–AuNPs nanocomposite can act as a reusable catalytic coating or form self‐supporting immersible films of desired shapes. The films scaffold the assembly of cardiac cells into synchronized patches, and present static charge detection capabilities at the macroscale. The study presents a novel T4P‐inspired biometallic material.     

Tissue-specific activation of the osmotin gene by ABA, C2H4 and NaCl involves the same promoter region

The gene encoding the antifungal protein osmotin is induced by several hormonal and environmental signals. In this study, tissue-specific and inducer-mediated expression of the reporter gene beta-glucuronidase (uidA) fused to different fragment lengths of the osmotin promoter was evaluated in transgenic tobacco (Nicotiana tabacum). The region of the promoter between -248 to -108 (Fragment A) was found to be essential and sufficient for inducer (abscisic acid (ABA), C2H4 and NaCl)-mediated expression of the reporter gene. Expression of the reporter gene was developmentally regulated and increased with maturity of leaves, stem and flowers. Expression also was tissue-specific being most highly expressed in epidermis and vascular parenchyma of the stem. The regulators ABA, C2H4 and NaCl exhibited tissue-specific induction of this promoter. The promoter was specifically responsive to C2H4 in flowers at virtually all stages of development, but not responsive in these tissues to ABA or NaCl. Conversely, ABA and NaCl were able to induce reporter gene activity using promoter Fragment A in specific tissues of root where C2H4 was unable to induce activity. Further dissection of the promoter Fragment A into fragments containing either the conserved GCC element (PR); PR/AT; or G/AT sequences, and subsequent testing of these fragments fused to GUS in transgenic plants was performed. These experiments revealed that the promoter fragment containing PR element alone, although required, was barely able to allow responsiveness to C2H4. However, significant C2H4-induced activity was obtained with a promoter fragment containing the AT and PR elements together.