Second harmonic atomic force microscopy imaging of live and fixed mammalian cells

Higher harmonic contributions in the movement of an oscillating atomic force microscopy (AFM) cantilever are generated by nonlinear tip–sample interactions, yielding additional information on structure and physical properties such as sample stiffness. Higher harmonic amplitudes are strongly enhanced in liquid compared to the operation in air, and were previously reported to result in better structural resolution in highly organized lattices of proteins in bacterial S-layers and viral capsids [J. Preiner, J. Tang, V. Pastushenko, P. Hinterdorfer, Phys. Rev. Lett. 99 (2007) 046102]. We compared first and second harmonics AFM imaging of live and fixed human lung epithelial cells, and microvascular endothelial cells from mouse myocardium (MyEnd). Phase–distance cycles revealed that the second harmonic phase is 8 times more sensitive than the first harmonic phase with respect to variations in the distance between cantilever and sample surface. Frequency spectra were acquired at different positions on living and fixed cells with second harmonic amplitude values correlating with the sample stiffness. We conclude that variations in sample stiffness and corresponding changes in the cantilever–sample distance, latter effect caused by the finite feedback response, result in second harmonic images with improved contrast and information that is not attainable in the fundamental frequency of an oscillating cantilever.     

Volatile compounds in fresh meats subjected to high pressure processing: Effect of the packaging material

The effect of high pressure treatment (400MPa, 10min at 12°C) on the volatile profile of minced beef and chicken breast, packaged with or without aluminum foil in a multilayer polymeric bag, was investigated. The analysis of the volatile fraction was carried out by dynamic headspace extraction coupled to gas chromatography-mass spectrometry. Pressurization produced significant changes in the levels of some volatile compounds presumably coming from microbial activity. Some alcohols and aldehydes decreased, while other compounds, such as 2,3-butanedione and 2-butanone, were more abundant in high pressure processed meats. A significant migration of compounds from the plastic material was observed, mainly branched-chain alkanes and benzene compounds. Two functions built by the principal component analysis explained a high percentage of the variance and could be used to separate the samples into four distinct groups, according to high pressure treatment and packaging material.     

Construction and optimization of a CC49-based scFv-beta-lactamase fusion protein for ADEPT

CC49 is a clinically validated antibody with specificity for TAG-72, a carbohydrate epitope that is over-expressed and exposed on a large fraction of solid malignancies. We constructed a single chain fragment (scFv) based on CC49 and fused it to beta-lactamase. The first generation fusion protein, TAB2.4, was expressed at low levels in Escherichia coli and significant degradation was observed during production. We optimized the scFv domain of TAB2.4 by Combinatorial Consensus Mutagenesis (CCM). An improved variant TAB2.5 was identified that resulted in an almost 4-fold improved expression and 2.5 degrees higher thermostability relative to its parent molecule. Soluble TAB2.5 can be manufactured in low-density E.coli cultures at 120 mg/l. Our studies suggest that CCM is a rapid and efficient method to generate antibody fragments with improved stability and expression. The fusion protein TAB2.5 can be used for antibody directed enzyme prodrug therapy (ADEPT).     

Progress and utilization of small nuclear reactors

In this study, we encompass different designs of small reactors, making a review since the beginning of the concept in the military and academic research, to the actual designs; and the prospective of this kind of reactors in the development of the nuclear industry. Small reactors were developed many years ago and have been in operation since 1950, but now, there is an increasing interest in developing small reactors for different applications or co-generation including a large variety of reactor designs with features ranging in coolant, cycle type, fuel, and energy applications; and enhancing features such as nuclear inherent safety, passive safety, and proliferation resistance. The modularity of some of these reactors makes them also a possible choice for nuclear development of some countries with units manufactured off-site and possible scaling into multimodular plants. Different lines of small reactors and current status are reviewed in this work.     

Observation of shear band formation in nanocrystalline Pd–Au alloy during in situ SEM compression testing

Understanding the deformation behavior of nanocrystalline (nc) materials is important because of their possible application as structural materials. The investigation of macroscopic nc samples during in situ mechanical tests can shed light on operating deformation mechanisms. For example, observations during a compression test in a scanning electron microscope (SEM) or results obtained by more localized methods like transmission electron microscopy can extend our knowledge on this subject. In this study, we present the results of in situ SEM compression tests on nc Pd–10 at.% Au alloy with a mean grain size of 23 nm produced using the combination of inert gas condensation with subsequent high-pressure torsion. We show that plastic flow in this material is very inhomogeneous on both the macro and the mesoscale as it is localized in one area of the compression sample and within shear bands. The formation of shear bands is accompanied by strain softening. We propose that such behavior can be explained by the activation of grain boundary mediated deformation mechanisms instead of dislocation-based plasticity. This conclusion was supported by in situ synchrotron XRD measurements which revealed that no preferential grain orientation is forming during the compression of identically prepared samples of the same material.     

Cyanidin suppresses neoplastic cell transformation by directly targeting phosphatidylinositol 3-kinase

Cyanidin, an anthocyanidin found in fruits and vegetables, has been reported to possess anti-cancer effects. However, there is no study on the chemopreventive effect of cyanidin against neoplastic cell transformation and its molecular mechanisms. In the present study, we compare the anti-carcinogenic effects of cyanidin and cyanidin-3-glucoside (C3G) and investigate their underlying mechanisms. The inhibitory effect of cyanidin on EGF-induced cell transformation was higher than those of C3G in JB6 P+ mouse epidermal (JB6 P+) cells. Both cyanidin and C3G showed dose-dependent radical scavenging activities. It is indicated that the divergent inhibitory effects of cyanidin and C3G are not due to their antioxidant activities. We found that cyanidin, but not C3G, inhibited the EGF-induced Akt/p70S6K phosphorylation. Moreover, cyanidin directly suppressed the activity of PI3K by binding to PI3K directly in an ATP-competitive manner, which indicates that PI3K is one of the molecular targets of cyanidin.     

Glycation compounds in peanuts

In the present study, the extent of the Maillard reaction in peanuts was investigated, using N-ε-fructosyllysine (ε-Fru-Lys, determined via furosine) as an indicator for the early stage, and pyrraline and N-ε-carboxymethyllysine (CML) as representatives for advanced glycation. In commercial samples, ε-Fru-Lys ranged between 1.5 and 13.3 mmol/mol lysine. Pyrraline was found in amounts between not detectable (below 0.3 mmol/mol lysine) and 9.0 mmol/mol lysine, and CML between 0.8 and 2.7 mmol/mol lysine. Lysine modification by glycation products was very low in cooked and fried peanuts (below 1%). In laboratory-scale roasting experiments, the amount of ε-Fru-Lys reached maximum values of 12.0 mmol/mol lysine after 20 min at 160 °C, whereas pyrraline increased up to 38.5 mmol/mol lysine after 25 min at 170 °C. Amount of CML was up to 1.8 mmol/mol lysine in peanuts roasted for 25 min at 170 °C. Such high amounts of pyrraline have not yet been described for any other food item. Only about one tenth of the totally observed lysine modification of up to 50% can be explained by the three glycation compounds, indicating that currently unknown reactions occur during peanut roasting. Reactions between proteins and carbonyl compounds, most likely originating from oxidative degradation of lipids, may play an important role for lysine derivatization in peanuts and should be analyzed more in detail.     

Effect of α‐tocopherol and α‐tocotrienol on the performance of Chilean hazelnut oil (Gevuina avellana Mol) at high temperature

The high temperature antioxidant efficiency of α‐tocopherol, α‐tocotrienol and a mixture of both on hazelnut oil were evaluated. Crude hazelnut oil (HZO), crude hazelnut oil treated with alumina (THZO), as well as three samples of THZO to which 150 mg kg^?1 of α‐tocopherol, 140 mg kg^?1 of α‐tocotrienol or a mixture containing 70 mg kg^?1 of α‐tocopherol and 70 mg kg^?1 of α‐tocotrienol, were added and submitted to thermal treatment at 180°C for 18 h. The addition of tocols to THZO decreased the formation of polar compounds and increased its oxidative stability in all the systems studied. However, α‐tocopherol showed a higher antioxidant capacity than α‐tocotrienol at high temperature. In addition, α‐tocotrienol showed a more rapid degradation rate than α‐tocopherol under the conditions studied. Copyright © 2004 Society of Chemical Industry     

Outgrowth inhibition of Clostridium beijerinckii spores by a bacteriocin-producing lactic culture in ovine milk cheese

In the manufacture of model cheeses, ovine milk was deliberately contaminated with spores of Clostridium beijerinckii INIA 63, a wild isolate from Manchego cheese with late blowing defect, and inoculated with nisin- and lacticin 481-producing Lactococcus lactis subsp. lactis INIA 415 as starter, to test its potential to prevent the late blowing defect, or with L. lactis subsp. lactis INIA 415-2, a spontaneous mutant not producing bacteriocins. Cheeses made individually with the lactococcal strains, without clostridial spores, served as controls. Cheese made with clostridial spores and L. lactis subsp. lactis INIA 415-2 showed late blowing defect after 120 days of ripening. Spoilt cheese also showed lower concentrations of lactic acid, and higher levels of acetic, propionic and butyric acids, and of other volatile compounds such as 2-propanol and 1-butanol, than control cheese. In addition, cheese made with the bacteriocin producer did not show any late blowing symptoms, despite its spore counts similar to those of blown cheese, pointing to outgrowth inhibition of C. beijerinckii spores by bacteriocins. Besides, cheese made with the bacteriocin producer showed similar concentrations of lactic acid and volatile compounds than control cheese. Inclusion of L. lactis subsp. lactis INIA 415 in starter cultures seems a feasible method to prevent late blowing defect in cheese without altering its sensory characteristics.     

Using High-Pressure Processing for Reduction of Proteolysis and Prevention of Over-ripening of Raw Milk Cheese

High-pressure-processing (HPP) at 400 or 600 MPa was applied to cheeses made from ewe raw milk, on days 21 or 35 after manufacturing, to reduce proteolysis and prevent over-ripening. The characteristics of HPP and non-pressurized (control) cheeses were compared during ripening at 8 °C until day 60 and further storage at 4 °C until day 240. HPP and control cheeses showed similar pH values throughout ripening, but on day 240 pH values remained 0.4–0.6 units lower for HPP cheeses than for the control cheeses. Casein degradation was significantly retarded in the 600 MPa cheeses. Their α-casein concentration was 48–52 % higher on day 60 and 30–33 % higher on day 240 than in the control cheeses while β-casein concentration was 25–26 % higher on day 60 and 100–103 % higher on day 240. No significant differences in para-κ-casein concentration between cheeses were found on day 60, but on day 240, it was 22–35 % higher in the 600 MPa cheeses than in the control cheese. Hydrophilic peptides, hydrophobic peptides and total free amino acids evolved similarly in HPP and control cheeses during the 60-day ripening period. However, on day 240 hydrophilic peptides were at 34–39 % lower levels in the 600 MPa cheeses than in the control cheeses, hydrophobic peptides at 7–16 % lower levels and total free amino acids at 25–29 % lower levels. Flavour intensity scores increased at a slower rate in HPP cheeses than in the control cheese. Flavour quality declined markedly in the control cheeses during refrigerated storage while it did not vary significantly in 600 MPa cheeses.