Tailoring Proteins to Re-Evolve Nature: A Short Review

Proteins are key biomolecules for most biological processes, their function is related to their conformation that is also dictated by their sequence of amino acids. Through evolution, nature has produced an immense variety of enzymatic tools of high efficiency and selectivity, and thanks to the understanding of the molecular basis of life and the technological advances, scientists have learned to introduce mutations and select mutant enzymes, to optimize and control their molecular fitness characteristics mainly for industrial, medical and environmental applications. The relationship between protein structure and enzymatic functionality is essential, and there are various experimental and instrumental techniques for unravelling the molecular changes, activities and specificities. Protein engineering applies computational tools, in hand with experimental tools for mutations, like directed evolution and rational design, along with screening methods to obtain protein variations with the desired properties under a short time frame. With innovations in technology, it is possible to fine tune properties in proteins and reach new frontiers in their applications. The present review will briefly discuss these points and methods, with a glimpse on their strengths and pitfalls, while giving an overview of the versatility of synthetic proteins and their huge potential for biotechnological and biomedical fields.     

Vancomycin-Resistant Enterococcus spp. Isolated from Wastewater and Chicken Feces in the United States

Vancomycin-resistant Enterococcus spp. (VRE) were isolated from sewage and chicken feces but not from other animal fecal sources (dog, cow, and pig) or from surface waters tested. VRE from hospital wastewater were resistant to ≥20 μg of vancomycin/ml and possessed the vanA gene. VRE from residential wastewater and chicken feces were resistant to 3 to 5 μg of vancomycin/ml and possessed the vanC gene.     

Cathepsins F and S block HDL3-induced cholesterol efflux from macrophage foam cells

In atherosclerosis, accumulation of cholesterol in macrophages may partially depend on its defective removal by high-density lipoproteins (HDL). We studied the proteolytic effect of cathepsins F, S, and K on HDL(3) and on lipid-free apoA-I, and its consequence on their function as inductors of cholesterol efflux from cholesterol-filled mouse peritoneal macrophages in vitro. Incubation of HDL(3) with cathepsin F or S, but not with cathepsin K, led to rapid loss of prebeta-HDL, and reduced cholesterol efflux by 50% in only 1min. Cathepsins F or K partially degraded lipid-free apoA-I and reduced its ability to induce cholesterol efflux, whereas cathepsin S totally degraded apoA-I, leading to complete loss of apoA-I cholesterol acceptor function. These results suggest that cathepsin-secreting cells induce rapid depletion of lipid-poor (prebeta-HDL) and lipid-free apoA-I and inhibit cellular cholesterol efflux, so tending to promote the formation and maintenance of foam cells in atherosclerotic lesions.     

Sleep patterns of day-working, evening high-schooled adolescents of São Paulo, Brazil

Children who grow up in developing countries of the world must work to help financially support their families, and they must also attend school. We investigated the impact of work on the sleep of working vs. nonworking high school students. Twenty-seven S?o Paulo, Brazil, public high school students (eight male and eight female working students plus six nonworking female and five nonworking male students) 14-18 yrs of age who attended school Monday-Friday between 19:00 to 22:30h participated. A comprehensive questionnaire about work and living conditions, health status, and diseases and their symptoms was also answered. The activity level and rest pattern (sleep at night and napping during the day) were continuously assessed by wrist actigraphy (Ambulatory Monitoring, USA). The main variables were analyzed by a two-factor ANOVA with application of the Tukey HSD test for multiple comparisons, and the length of sleep during weekdays vs. weekends was compared by Student t-test. Working students went to sleep earlier weekends [F(1,23)=6.1; p=0.02] and woke up earlier work days than nonworking students [F(1,23) = 17.3; p = 0.001]. The length of nighttime sleep during weekdays was shorter among all the working [F(1,23)= 16.7; p <0.001] than all the nonworking students. The sleep duration of boys was shorter than of girls during weekends [F(1,23)= 10.8; p <0.001]. During weekdays, the duration of napping by working and nonworking male students was shorter than nonworking female students. During weekdays working girls took the shortest naps [F(1,23)= 5.6; p = 0.03]. The most commonly reported sleep complaint during weekdays was difficulty waking up in the morning [F(1,23) = 6.5; p = 0.02]. During weekdays, the self-perceived sleep quality of working students was worse than nonworking students [F(1,23) = 6.2; p = 0.02]. The findings of this study show that work has negative effects on the sleep of adolescents, with the possible build-up of a chronic sleep debt with potential consequent impact on quality of life and school learning.     

Hydrophobic complementarity in protein-protein docking

Formation of hydrophobic contacts across a newly formed interface is energetically favorable. Based on this observation we developed a geometric-hydrophobic docking algorithm that estimates quantitatively the hydrophobic complementarity at protein-protein interfaces. Each molecule to be docked is represented as a grid of complex numbers, storing information regarding the shape of the molecule in the real part and information regarding the hydropathy of the surface in the imaginary part. The grid representations are correlated using fast Fourier transformations. The algorithm is used to compare the extent of hydrophobic complementarity in oligomers (represented by D2 tetramers) and in hetero-dimers of soluble proteins (complexes). We also test the implication of hydrophobic complementarity in distinguishing correct from false docking solutions. We find that hydrophobic complementarity at the interface exists in oligomers and in complexes, and in both groups the extent of such complementarity depends on the size of the interface. Thus, the non-polar portions of large interfaces are more often juxtaposed than non-polar portions of small interfaces. Next we find that hydrophobic complementarity helps to point out correct docking solutions. In oligomers it significantly improves the ranks of nearly correct reassembled and modeled tetramers. Combining geometric, electrostatic and hydrophobic complementarity for complexes gives excellent results, ranking a nearly correct solution < 10 for 5 of 23 tested systems, < 100 for 8 systems and < 1000 for 19 systems.     

In vitro exposure of human lymphocytes to 900 MHz CW and GSM modulated radiofrequency: studies of proliferation, apoptosis and mitochondrial membrane potential

The aim of this study was to investigate the nonthermal effects of radiofrequency (RF) fields on human immune cells exposed to a Global System for Mobile Communication (GSM) signal generated by a commercial cellular phone and by a sinusoidal non-modulated signal. To assess whether mobile phone RF-field exposure affects human immune cell functions, peripheral blood mononuclear cells (PBMCs) from healthy donors were exposed in vitro to a 900 MHz GSM or continuous-wave (CW) RF field 1 h/day for 3 days in a transverse electromagnetic mode (TEM) cell system (70-76 mW/kg average specific absorption rate, SAR). The cells were cultured for 48 or 72 h, and the following end points were studied: (1) mitogen-induced proliferation; (2) cell cycle progression; (3) spontaneous and 2-deoxy-D-ribose (dRib)-induced apoptosis; (4) mitochondrial membrane potential modifications during spontaneous and dRib-induced-apoptosis. Data obtained from cells exposed to a GSM-modulated RF field showed a slight decrease in cell proliferation when PBMCs were stimulated with the lowest mitogen concentration and a slight increase in the number of cells with altered distribution of phosphatidylserine across the membrane. On the other hand, cell cycle phases, mitochondrial membrane potential and susceptibility to apoptosis were found to be unaffected by the RF field. When cells were exposed to a CW RF field, no significant modifications were observed in comparison with sham-exposed cells for all the end points investigated.     

Effects of ischemia on epicardial deformation in the passive rabbit heart

BACKGROUND: Surgically induced ischemia in the arrested heart can result in changes in the mechanical properties of the myocardium. Regions of ischemia may be characterized based on the amount of epicardial deformation for a given load. Computer aided speckle interferometry (CASI), which tracks the movement of clusters of particles, is developed as a technique for measuring epicardial deformation, thereby determining the perfusion status of the passive heart. MATERIALS AND METHODS: Silicone carbide particles and retroreflective beads were dispersed randomly onto the epicardial surface of 11 isolated rabbit hearts to form speckle images. The hearts were arrested with hyperkalemic Krebs-Henseleit buffered solution. Each heart was then exposed to a series of intracavitary pressures, and at each pressure speckle images were acquired with a charge-coupled device (CCD) camera. Nine hearts were exposed to global ischemia, and two hearts were exposed to regional ischemia by occluding the second diagonal branch of the left anterior descending artery (LAD). The hearts were again loaded and the speckle images were acquired. CASI was used to determine the distribution of deformation field. RESULTS: CASI was able to determine displacements with a spatial resolution of about 50 microns. Global ischemia resulted in a significant increase in the maximum principle strain and the first invariant of the 2-D strain tensor. In the regionally ischemic heart, a large difference in deformation between the ischemic and perfused regions was clearly observed. CONCLUSION: Based on epicardial deformation, CASI is able to distinguish between perfused and ischemic myocardium, with a spatial resolution of 50 microns.