Laboratory evaluation of certain natural compounds against the melon ladybird beetle,Epilachna chrysomelina F. attacking cucurbit plants

Neem Azal-T/S, Neudosan and Spruzit flüssig at different concentrations were evaluated against the second and fourth instar larvae of E. chrysomelina under laboratory conditions. In case of acute effects, the highest mortality was recorded when larvae were offered squash leaves treated with the highest concentration (2%) of Neem Azal-T/S, and the lowest mortality was, however recorded after treating them with the lowest concentration (0.25%). On the other hand, the second instar larvae were more susceptible to any compound at any concentration than the fourth instar one. One per cent Neudosan formulation showed the highest toxicity to both the 2nd and 4th instar larvae. As the concentration of this compound decreased the mortality percentage of treated larvae decreased to reach the minimum at the lowest concentration (0.25%). The same trend could be applied for Spruzit flüssig compound. When Neem Azal-T/S was compared with Neudosan, the later at any concentration was more toxic than the former. Generally, Neudosan and Spruzit flüssig seemed to be active against E. chrysomelina during the first three days after treatment, then, deterioration occurred among this compound. In case of latent effects, the duration of the treated fourth instar larvae of E. chrysomelina with any tested compound, was prolonged. An increase in the concentration caused prolongation of this period to reach the longest at the highest concentration. This prolongation, was more pronounced after Neem Azal-T/S treatment followed by Neudosan and Spruzit flüssig. The same trend could be applied for the duration of the pupal stage as being affected by the different concentrations of the tested compounds which were applied to the fourth instar larvae. However, adults of E. Chrysomelina treated as fourth instar larvae with high concentrations of Neem Azal-T/S showed some adult malformations. The sex ratios of adults produced from fourth instar larvae treated with different concentrations of the tested natural compounds were about 1?:?1, except for two cases; 1% Neudosan where the number of females were double the number of males and 0.5% Spruzit flüssig where the number of males were double the number of females. An increase in the concentration of any tested compound caused an obvious decrease in the percentage of emerged adults. The least emergence percentage was recorded in the case of Spruzit flüssig, followed by Neudosan and Neem Azal-T/S. Treating the fourth instar larvae with the different concentrations of the tested natural compounds significantly shortened the longevity of produced adults and decreased the fecundity.     

Can the presence of curved forms of the diatom Aulacoseira ambigua in the Nile (Egypt) and Vaal (South Africa) Rivers be ascribed to similar water quality conditions?

Spiral colonies of the diatom Aulacoseira ambigua were assigned the rank of forma (Aulacoseira ambigua f. japonica). This spiral-shaped, colonial, centric diatom has limited geographical distribution and is currently reported to occur in only a few countries in the world. In Africa this species was described for the first time from the Vaal River, South Africa, but recent research on the Nile River also revealed its presence in Egypt. Physical and chemical data on water quality in these two river systems were compared to determine whether the presence of this uncommon ‘phenoecodeme’ could be ascribed to similar environmental conditions. Results indicate that the rivers are dissimilar with regard to many variables, but both rivers provide turbid, warm and eutrophic waters with medium to high mineral content and it was concluded that these factors favour and sustain the growth of this curved form.     

Development of “Plug and Play” Fiducial Marks for Structural Studies of GPCR Signaling Complexes by Single-Particle Cryo-EM

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Exploring the capacity of minimalist protein interfaces: interface energetics and affinity maturation to picomolar KD of a single-domain antibody with a flat paratope

A major architectural class in engineered binding proteins ("antibody mimics") involves the presentation of recognition loops off a single-domain scaffold. This class of binding proteins, both natural and synthetic, has a strong tendency to bind a preformed cleft using a convex binding interface (paratope). To explore their capacity to produce high-affinity interfaces with diverse shape and topography, we examined the interface energetics and explored the affinity limit achievable with a flat paratope. We chose a minimalist paratope limited to two loops found in a natural camelid heavy-chain antibody (VHH) that binds to ribonuclease A. Ala scanning of the VHH revealed only three "hot spot" side chains and additional four residues important for supporting backbone-mediated interactions. The small number of critical residues suggested that this is not an optimized paratope. Using selection from synthetic combinatorial libraries, we enhanced its affinity by >100-fold, resulting in variants with Kd as low as 180 pM with no detectable loss of binding specificity. High-resolution crystal structures revealed that the mutations induced only subtle structural changes but extended the network of interactions. This resulted in an expanded hot spot region including four additional residues located at the periphery of the paratope with a concomitant loss of the so-called "O-ring" arrangement of energetically inert residues. These results suggest that this class of simple, single-domain scaffolds is capable of generating high-performance binding interfaces with diverse shape. More generally, they suggest that highly functional interfaces can be designed without closely mimicking natural interfaces.     

Optimizing Production of Antigens and Fabs in the Context of Generating Recombinant Antibodies to Human Proteins

We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that in vivo biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 in vivo biotinylated antigen domains. High-affinity Fabs (<20nM) were obtained for 196. We constructed and optimized a new expression vector to produce in vivo biotinylated Fabs in E. coli. This increased average yields up to 10-fold, with an average yield of 4 mg/L. For 118 antigens, we identified Fabs that could immunoprecipitate their full-length endogenous targets from mammalian cell lysates. One Fab for each antigen was converted to a recombinant IgG and produced in mammalian cells, with an average yield of 15 mg/L. In summary, we have optimized each step of the pipeline to produce recombinant antibodies, significantly increasing both efficiency and yield, and also showed that these Fabs and IgGs can be generally useful for chromatin immunoprecipitation (ChIP) protocols.     

Weight loss is not mandatory for exercise-induced effects on health indices in females with metabolic syndrome

The aim of this study was to investigate the impact of moderate aerobic training on functional, anthropometric, biochemical, and health-related quality of life (HRQOL) parameters on women with metabolic syndrome (MS). Fifteen untrained women with MS performed moderate aerobic training for 15 weeks, without modifications of dietary behaviours. Functional, anthropometric, biochemical, control diet record and HRQOL parameters were assessed before and after the training. Despite body weight maintenance, the patients presented decreases in waist circumference (P = 0.001), number of MS components (P = 0.014), total cholesterol (P = 0.049), HDL cholesterol (P = 0.004), LDL cholesterol (P = 0.027), myeloperoxidase activity (P = 0.002) and thiobarbituric acid-reactive substances levels (P = 0.006). There were no differences in total energy, carbohydrate, protein and lipid intake pre- and post-training. Furthermore, improvements in the HRQOL subscales of physical functioning (P = 0.03), role-physical (P = 0.039), bodily pain (P = 0.048), general health (P = 0.046) and social functioning scoring (P = 0.011) were reported. Despite the absence of weight loss, aerobic training induced beneficial effects on functional, anthropometric, biochemical and HRQOL parameters in women with MS.     

Estimation of the membrane potential of cultured macrophages from the fast potential transient upon microelectrode entry

Analysis of membrane potential recordings upon microelectrode impalement of four types of macrophages (cell lines P388D1 and PU5-1.8, cultured mouse peritoneal macrophages, and cultured human monocytes) reveals that these cells have membrane potentials at least two times more negative than sustained potential values (E(s)) frequently reported. Upon microelectrode entry into the cell (P388D1), the recorded potential drops to a peak value (E(p)) (mean -37 mV for 50 cells, range -15 to -70 mV) within 2 ms, after which it decays to a depolarized potential (E(n)) (mean -12 mV) in about 20 ms. Thereafter, the membrane develops one or a series of slow hyperpolarizations before a final sustained membrane potential (E(s)) (mean -14 mV, range -5 to -40) is established. The mean value of the peak of the first hyperpolarization (E(h)) is -30 mV (range -10 to -55 mV). The initial fast peak transient, measured upon microelectrode entry, was first described and analyzed by Lassen et al. (Lassen, U.V., A.M. T. Nielson, L. Pape, and L. O. Simonsen, 1971, J. Membr. Biol. 6:269-288 for other change in the membrane potential from its real value before impalement to a sustained depolarized value. This was shown to be true for macrophages by two-electrode impalements of single cells. Values of E(p), E(n), E(h), E(s), and membrane resistance (R(m)) measured for the other macrophages were similar to those of P388D1. From these results we conclude that E(p) is a better estimate of the true membrane potential of macrophages than E(s), and that the slow hyperpolarizations upon impalement should be regarded as transient repolarizations back to the original membrane potentials. Thus, analysis of the initial fast impalement transient can be a valuable aid in the estimation of the membrane potential of various sorts of small isolated cells by microelectrodes.     

Solvent structure in crystals of trypsin determined by X-ray and neutron diffraction.

The solvent structure in orthorhombic crystals of bovine trypsin has been independently determined by X-ray diffraction to 1.35 A resolution and by neutron diffraction to 2.1 A resolution. A consensus model of the water molecule positions was obtained using oxygen positions identified in the electron density map determined by X-ray diffraction, which were verified by comparison to D2O-H2O difference neutron scattering density. Six of 184 water molecules in the X-ray structure, all with B-factors greater than 50 A2, were found to be spurious after comparison with neutron results. Roughly two-thirds of the water of hydration expected from thermodynamic data for proteins was localized by neutron diffraction; approximately one-half of the water of hydration was located by X-ray diffraction. Polar regions of the protein are well hydrated, and significant D2O-H2O difference density is seen for a small number of water molecules in a second shell of hydration. Hydrogen bond lengths and angles calculated from unconstrained refinement of water positions are distributed about values typically seen in small molecule structures. Solvent models found in seven other bovine trypsin and trypsinogen and rat trypsin structures determined by X-ray diffraction were compared. Internal water molecules are well conserved in all trypsin structures including anionic rat trypsin, which is 65% homologous to bovine trypsin. Of the 22 conserved waters in trypsin, 19 were also found in trypsinogen, suggesting that they are located in regions of the apoprotein that are structurally conserved in the transition to the mature protein. Seven waters were displaced upon activation of trypsinogen. Water structure at crystal contacts is not generally conserved in different crystal forms. Three groups of integral structural water molecules are highly conserved in all solvent structures, including a spline of water molecules inserted between two beta-strands, which may resemble an intermediate in the formation of beta sheets during the folding of a protein.