Antibody phage display libraries: contributions to oncology

Since the advent of phage display technology, dating back to 1985, antibody libraries displayed on filamentous phage surfaces have been used to identify specific binders for many different purposes, including the recognition of tumors. Phage display represents a high-throughput technique for screening billions of random fusion antibodies against virtually any target on the surface or inside cancer cells, or even soluble markers found in patient serum. Many phage display derived binders targeting important tumor markers have been identified. Selection directed to tumoral cells' surfaces lead to the identification of unknown tumoral markers. Also the improvement of methods that require smaller amounts of cells has opened the possibility to use this approach on patient samples. Robust techniques combining an antibody library displayed on the phage surface and protein microarray allowed the identification of auto antibodies recognized by patient sera. Many Ab molecules directly or indirectly targeting angiogenesis have been identified, and one of them, ramucirumab, has been tested in 27 phase I-III clinical trials in a broad array of cancers. Examples of such antibodies will be discussed here with emphasis on those used as probes for molecular imaging and other clinical trials.     

Increasing the X-ray Diffraction Power of Protein Crystals by Dehydration: The Case of Bovine Serum Albumin and a Survey of Literature Data

Serum albumin is one of the most widely studied proteins. It is the most abundant protein in plasma with a typical concentration of 5 g/100 mL and the principal transporter of fatty acids in plasma. While the crystal structures of human serum albumin (HSA) free and in complex with fatty acids, hemin, and local anesthetics have been characterized, no crystallographic models are available on bovine serum albumin (BSA), presumably because of the poor diffraction power of existing hexagonal BSA crystals. Here, the crystallization and diffraction data of a new BSA crystal form, obtained by the hanging drop method using MPEG 5K as precipitating agent, are presented. The crystals belong to space group C2, with unit-cell parameters a = 216.45 Å, b = 44.72 Å, c = 140.18 Å, β = 114.5°. Dehydration was found to increase the diffraction limit of BSA crystals from ~8 Å to 3.2 Å, probably by improving the packing of protein molecules in the crystal lattice. These results, together with a survey of more than 60 successful cases of protein crystal dehydration, confirm that it can be a useful procedure to be used in initial screening as a method of improving the diffraction limits of existing crystals.     

High Resolution Crystal Structures of the Cerebratulus lacteus Mini-Hb in the Unligated and Carbomonoxy States

The nerve tissue mini-hemoglobin from Cerebratulus lacteus (CerHb) displays an essential globin fold hosting a protein matrix tunnel held to allow traffic of small ligands to and from the heme. CerHb heme pocket hosts the distal TyrB10/GlnE7 pair, normally linked to low rates of O(2) dissociation and ultra-high O(2) affinity. However, CerHb affinity for O(2) is similar to that of mammalian myoglobins, due to a dynamic equilibrium between high and low affinity states driven by the ability of ThrE11 to orient the TyrB10 OH group relative to the heme ligand. We present here the high resolution crystal structures of CerHb in the unligated and carbomonoxy states. Although CO binds to the heme with an orientation different from the O(2) ligand, the overall binding schemes for CO and O(2) are essentially the same, both ligands being stabilized through a network of hydrogen bonds based on TyrB10, GlnE7, and ThrE11. No dramatic protein structural changes are needed to support binding of the ligands, which can freely reach the heme distal site through the apolar tunnel. A lack of main conformational changes between the heme-unligated and -ligated states grants stability to the folded mini-Hb and is a prerequisite for fast ligand diffusion to/from the heme.     

Genotoxic and mutagenic effects of permethrin in mice: Micronuclei analysis in peripheral blood erythrocytes

Pyrethroids such as permethrin are synthetic compounds widely used in the agriculture of many countries to combat plagues and in domestic products, such as acaricides. Not so long ago these chemicals were characterized as non‐toxic for non‐target organisms; however, recent studies have showed that these compounds could present toxic potential for many organisms. In this sense, this study presents genotoxic and mutagenic potential of permethrin administered intraperitoneally in mice under artificial conditions by the use of micronucleus assay in the peripheral blood of these animals. The mice were divided into five groups: group I = negative control (distilled water), group II = positive control (cyclophosphamide), group III = 30% of permethrin LD₅₀ (96 mg/kg), group IV = 50% of permethrin LD₅₀ (160 mg/kg), and group V = 80% of permethrin LD₅₀ (256 mg/kg). The peripheral blood was collected 24, 48, and 72 h after treatment. Results showed that all the tested permethrin dosages presented genotoxic and mutagenic effects 24 h after treatment, which would contradict the classification of this chemical product as moderately toxic, i.e., unable to cause damages to the cell DNA. Microsc. Res. Tech. © 2012 Wiley Periodicals, Inc.     

Effect of physical properties on the stability of Lactobacillus bulgaricus in a freeze-dried galacto-oligosaccharides matrix

The ability of galacto-oligosaccharides (GOS) to protect Lactobacillus delbrueckii subsp. bulgaricus upon freeze drying was analyzed on the basis of their capacity to form glassy structures. Glass transition temperatures (T(g)) of a GOS matrix at various relative humidities (RH) were determined by DSC. Survival of L. bulgaricus in a glassy GOS matrix was investigated after freezing, freeze drying, equilibration at different RHs and storage at different temperatures. At 32 °C, a drastic viability loss was observed. At 20 °C, the survival was affected by the water content, having the samples stored at lower RHs, the highest survival percentages. At 4°C, no decay in the cells count was observed after 45 days of storage. The correlation between molecular mobility [as measured by Proton nuclear magnetic resonance (1H NMR)] and loss of viability explained the efficiency of GOS as cryoprotectants. The preservation of microorganisms was improved at low molecular mobility and this condition was obtained at low water contents and low storage temperatures. These results are important in the developing of new functional foods containing pre and probiotics.     

The genome of wine yeast Dekkera bruxellensis provides a tool to explore its food-related properties

The yeast Dekkera/Brettanomyces bruxellensis can cause enormous economic losses in wine industry due to production of phenolic off-flavor compounds. D. bruxellensis is a distant relative of baker's yeast Saccharomyces cerevisiae. Nevertheless, these two yeasts are often found in the same habitats and share several food-related traits, such as production of high ethanol levels and ability to grow without oxygen. In some food products, like lambic beer, D. bruxellensis can importantly contribute to flavor development. We determined the 13.4 Mb genome sequence of the D. bruxellensis strain Y879 (CBS2499) and deduced the genetic background of several "food-relevant" properties and evolutionary history of this yeast. Surprisingly, we find that this yeast is phylogenetically distant to other food-related yeasts and most related to Pichia (Komagataella) pastoris, which is an aerobic poor ethanol producer. We further show that the D. bruxellensis genome does not contain an excess of lineage specific duplicated genes nor a horizontally transferred URA1 gene, two crucial events that promoted the evolution of the food relevant traits in the S. cerevisiae lineage. However, D. bruxellensis has several independently duplicated ADH and ADH-like genes, which are likely responsible for metabolism of alcohols, including ethanol, and also a range of aromatic compounds.     

Classification and adulteration control of vegetable oils based on microwave reflectometry analysis

Olive oil production represents a big part of the Mediterranean economy, and as such it must be protected from frauds. For this reason, it is necessary to develop alternative low-cost techniques, applicable on large scale, for checking the quality of the product and for detecting adulteration. On such bases, the present work deals with the possibility of adopting microwave reflectometry for obtaining a ‘spectral signature’ of vegetable oils. For this purpose, time domain reflectometry (TDR) measurements, in combination with specific data processing, are first used for the dielectric characterization of several oil types. Successively, the acquired data are processed through the principal component analysis (for identifying clusters of oil types that exhibit common features) and through the partial least square analysis (for identifying a predictive model for detecting oil adulteration). Results confirm that the proposed procedure holds considerable potential for quality and anti-adulteration control purposes, especially in view of practical applications.     

Effect of electron beam irradiation on quality indicators of peanut butter over a storage period

Electron beam (e-beam) irradiation is an effective non-thermal processing step for the reduction of Salmonella in peanut butter. The objective of this study was to evaluate the effects of e-beam irradiation on quality indicators of peanut butter. Peanut butter samples were exposed to a range of e-beam doses and examined over a 14-day period at 22 °C. Colour analysis (L^∗, a^∗, b^∗), spreadability, peroxide values (PV) and thiobarbituric acid reactive substances test (TBARS) for lipid oxidation were monitored, fatty acid and amino acid profiles were verified and protein degradation was determined by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). Changes in colour, PV, and TBARS were observed as e-beam dose increased. No significant changes in spreadability were observed (P > 0.05). When applied to peanut butter, e-beam irradiation will produce significant changes in quality indicators; future studies should include sensory evaluation and consumer acceptance studies.