Variation in sensitivity of the cardiac glycoside receptor characteristics of (Na+ + K+)-ATPase to lipolysis and temperature.

1. The rate of binding of [3H]ouabain to untreated membrane preparations of [Na+ +K+]-ATPase is a timperature--dependent process displaying a thermal transition close to 25degreesC. The apparent energies of activation which can be calculated above and below this transition are similar to, but not identical with, those previously reported for activation of the enzyme by cations. 2. Treatment of the enzyme preparation with detergents or lipolysis with phospholipase A eliminates the thermal transition resulting in linear Arrhenius plots. 3. The number of sites available for [3H]ouabain binding is not temperature dependent as the amount of [3H]ouabain bound at equillbrium is not changed between 10 and 37 degrees C. 4. Treatment of the enzyme with phospholipase A results in time-dependent changes in the number of binding sites for [3H]ouabain at equilibrium. 5. Treatment of the membrane enzyme preparations with detergents reveals additional [3H]ouabain binding sites which are extremely sensitive to lipolysis with phospholipase A. 6. There are a number of [3H]ouabain binding sites which remain resistant to lipolysis by phospholipase A in either untreated or detergent-treated membrane preparations. 7. It is suggested that [3H]ouabain binding sites exist in the membrane in at least two different environments, one of which is resistant the other sensitive to attack by phopholipase A.     

Network-Level Structural Abnormalities of Cerebral Cortex in Type 1 Diabetes Mellitus

Type 1 diabetes mellitus (T1DM) usually begins in childhood and adolescence and causes lifelong damage to several major organs including the brain. Despite increasing evidence of T1DM-induced structural deficits in cortical regions implicated in higher cognitive and emotional functions, little is known whether and how the structural connectivity between these regions is altered in the T1DM brain. Using inter-regional covariance of cortical thickness measurements from high-resolution T1-weighted magnetic resonance data, we examined the topological organizations of cortical structural networks in 81 T1DM patients and 38 healthy subjects. We found a relative absence of hierarchically high-level hubs in the prefrontal lobe of T1DM patients, which suggests ineffective top-down control of the prefrontal cortex in T1DM. Furthermore, inter-network connections between the strategic/executive control system and systems subserving other cortical functions including language and mnemonic/emotional processing were also less integrated in T1DM patients than in healthy individuals. The current results provide structural evidence for T1DM-related dysfunctional cortical organization, which specifically underlie the top-down cognitive control of language, memory, and emotion.     

Implementation Fidelity of the National Malaria Control Program in Burkina Faso

Background

Every year 40,000 people die of malaria in Burkina Faso. In 2010, the Burkinabè authorities implemented a national malaria control program that provides for the distribution of mosquito nets and the home-based treatment of children with fever by community health workers. The objective of this study was to measure the implementation fidelity of this program.

Methods

We conducted a case study in two comparable districts (Kaya and Zorgho). Data were collected one year after the program’s implementation through field observations (10 weeks), documentary analysis, and individual interviews with stakeholders (n = 48) working at different levels of the program. The analysis framework looked at the fidelity of (i) the intervention’s content, (ii) its coverage, and (iii) its schedule.

Results

The program’s implementation was relatively faithful to what was originally planned and was comparable in the two districts. It encountered certain obstacles in terms of the provision of supplies. Coverage fidelity was better in Kaya than in Zorgho, where many community health workers (CHW) experienced problems with the restocking of artemisinin-based combination therapy and with remuneration for periods of training. In both districts, the community was rarely involved in the process of selecting CHWs. The components affected by scheduling all experienced successive implementation delays that pushed nets distribution and the initial provision of artemisinin-based combination therapies to the CHWs past the 2010 malaria season.

Conclusions

The activities intended by the program were mostly implemented with good fidelity. However, the implementation was plagued by delays that probably postponed the expected beneficial effects.     

Redesigning the stereospecificity of tyrosyl‐tRNA synthetase

d ‐Amino acids are largely excluded from protein synthesis, yet they are of great interest in biotechnology. Unnatural amino acids have been introduced into proteins using engineered aminoacyl‐tRNA synthetases (aaRSs), and this strategy might be applicable to d ‐amino acids. Several aaRSs can aminoacylate their tRNA with a d ‐amino acid; of these, tyrosyl‐tRNA synthetase (TyrRS) has the weakest stereospecificity. We use computational protein design to suggest active site mutations in Escherichia coli TyrRS that could increase its d ‐Tyr binding further, relative to l ‐Tyr. The mutations selected all modify one or more sidechain charges in the Tyr binding pocket. We test their effect by probing the aminoacyl‐adenylation reaction through pyrophosphate exchange experiments. We also perform extensive alchemical free energy simulations to obtain l ‐Tyr/d ‐Tyr binding free energy differences. Agreement with experiment is good, validating the structural models and detailed thermodynamic predictions the simulations provide. The TyrRS stereospecificity proves hard to engineer through charge‐altering mutations in the first and second coordination shells of the Tyr ammonium group. Of six mutants tested, two are active towards d ‐Tyr; one of these has an inverted stereospecificity, with a large preference for d ‐Tyr. However, its activity is low. Evidently, the TyrRS stereospecificity is robust towards charge rearrangements near the ligand. Future design may have to consider more distant and/or electrically neutral target mutations, and possibly design for binding of the transition state, whose structure however can only be modeled. Proteins 2016; 84:240–253. © 2015 Wiley Periodicals, Inc.     

Protein side chain conformation predictions with an MMGBSA energy function

The prediction of protein side chain conformations from backbone coordinates is an important task in structural biology, with applications in structure prediction and protein design. It is a difficult problem due to its combinatorial nature. We study the performance of an “MMGBSA” energy function, implemented in our protein design program Proteus, which combines molecular mechanics terms, a Generalized Born and Surface Area (GBSA) solvent model, with approximations that make the model pairwise additive. Proteus is not a competitor to specialized side chain prediction programs due to its cost, but it allows protein design applications, where side chain prediction is an important step and MMGBSA an effective energy model. We predict the side chain conformations for 18 proteins. The side chains are first predicted individually, with the rest of the protein in its crystallographic conformation. Next, all side chains are predicted together. The contributions of individual energy terms are evaluated and various parameterizations are compared. We find that the GB and SA terms, with an appropriate choice of the dielectric constant and surface energy coefficients, are beneficial for single side chain predictions. For the prediction of all side chains, however, errors due to the pairwise additive approximation overcome the improvement brought by these terms. We also show the crucial contribution of side chain minimization to alleviate the rigid rotamer approximation. Even without GB and SA terms, we obtain accuracies comparable to SCWRL4, a specialized side chain prediction program. In particular, we obtain a better RMSD than SCWRL4 for core residues (at a higher cost), despite our simpler rotamer library. Proteins 2016; 84:803–819. © 2016 Wiley Periodicals, Inc.     

Oviposition behaviour and host colony size discrimination in Episyrphus balteatus (Diptera: Syrphidae)

Three bioassays were used to examine the oviposition behaviour of the hoverfly, Episyrphus balteatus (Degeer) in which various stimuli purported to influence host assessment and choice were tested. Episyrphus balteatus failed to exhibit enhanced behavioural responses, in terms of approaches and landings, to artificial leaves with the highest numbers of aphids, suggesting that females are merely responding to the colour of the artificial leaves. A density-dependent oviposition response was reported in a second bioassay using whole bean plants, although there was an asymptotic relationship. Syrphid responses, which were measured by oviposition over a five-day period, were greatest towards those plants with the highest number of aphids. In a final series of trials, gravid female E. balteatus showed a behavioural preference for filter papers treated with the greatest honeydew concentrations (0.26 mg microl(-1) honeydew). More time was spent in the treated areas and syrphids demonstrated more proboscis and ovipositor extensions in these treatments. These bioassays provided an opportunity to isolate some of the individual components of decision-making by female E. balteatus during egg-laying behaviour. A greater understanding of natural enemy behaviour is essential before enhanced control of pest populations in the field can be established.     

Protein-protein recognition: an experimental and computational study of the R89K mutation in Raf and its effect on Ras binding

Binding of the protein Raf to the active form of Ras promotes activation of the MAP kinase signaling pathway, triggering cell growth and differentiation. Raf/Arg89 in the center of the binding interface plays an important role determining Ras-Raf binding affinity. We have investigated experimentally and computationally the Raf-R89K mutation, which abolishes signaling in vivo. The binding to [gamma-35S]GTP-Ras of a fusion protein between the Raf-binding domain (RBD) of Raf and GST was reduced at least 175-fold by the mutation, corresponding to a standard binding free energy decrease of at least 3.0 kcal/mol. To compute this free energy and obtain insights into the microscopic interactions favoring binding, we performed alchemical simulations of the RBD, both complexed to Ras and free in solution, in which residue 89 is gradually mutated from Arg into Lys. The simulations give a standard binding free energy decrease of 2.9+/-1.9 kcal/mol, in agreement with experiment. The use of numerous runs with three different force fields allows insights into the sources of uncertainty in the free energy and its components. The binding decreases partly because of a 7 kcal/mol higher cost to desolvate Lys upon binding, compared to Arg, due to better solvent interactions with the more concentrated Lys charge in the unbound state. This effect is expected to be general, contributing to the lower propensity of Lys to participate in protein-protein interfaces. Large contributions to the free energy change also arise from electrostatic interactions with groups up to 8 A away, namely residues 37-41 in the conserved effector domain of Ras (including 4 kcal/mol from Ser39 which loses a bifurcated hydrogen bond to Arg89), the conserved Lys84 and Lys87 of Raf, and 2-3 specific water molecules. This analysis will provide insights into the large experimental database of Ras-Raf mutations.     

Geneflow from GM plants--towards a more quantitative risk assessment

Assessing the risks associated with geneflow from GM crops to wild relatives is a significant scientific challenge. Most researchers have focused on assessing the frequency of gene flow, too often on a localized scale, and ignoring the hazards caused by geneflow. To quantify risk, multi-disciplinary research teams need to unite and scale up their studies.     

Leukocytosis occurs in response to resistance exercise in men

The purpose of this study was to determine the effects of a single bout of resistance exercise on immune cell numbers of moderately active men. Subjects were 16 male volunteers (mean +/- standard deviation [SD] age 30 +/- 7 years, height 180.1 +/- 7.0 cm, mass 83.97 +/- 10.33 kg); 8 were randomly assigned to treatment and 8 to control groups. Treatment was a common resistance training routine (3 sets of 8-10 repetitions at 75% of 1 repetition maximum) of 8 large muscle mass exercises using resistance machines. Blood samples were drawn before exercise and at 0 minutes (P0), 15 minutes (P15), and 30 minutes (P30) postexercise. Control subjects sat quietly in the training facility; blood was drawn at the same intervals as treatment. Leukocyte and lymphocyte (LY) subpopulation numbers were determined. Statistical analysis was analysis of variance (ANOVA) (repeated measures, p < or = 0.050) and multiple comparisons (Dunn method) to isolate variability. All leukocyte subpopulations, except basophils (BA) and eosinophils (EO), increased and counts declined by P15 and P30. Only neutrophils (NE) did not return to preexercise levels by P30. The majority of resistance exercise induced leukocytosis was due to an increase in circulating LY (natural killer cells increased most, CD4+/CD8+ ratio unchanged) and monocytes (MO). The transient, inconsequential immune cell population responses to resistance exercise are similar to those during aerobic activity. The lack of large alterations in and rapid recovery from cell number changes suggests that resistance exercise is not immunosuppressive.     

Crystallization and preliminary X-ray structural studies of hemoglobin A2 and hemoglobin E,isolated from the blood samples of beta-thalassemic patients

Hemoglobin A(2) (alpha(2)delta(2)), a minor (2-3%) component of circulating red blood cells, acts as an anti-sickling agent and its elevated concentration in beta-thalassemia is a useful clinical diagnostic. In beta-thalassemia major, where there is a failure of beta-chain production, HbA(2) acts as the predominant oxygen delivery mechanism. Hemoglobin E, is another common abnormal hemoglobin, caused by splice site mutation in exon 1 of beta globin gene, when combines with beta-thalassemia, causes severe microcytic anemia. The purification, crystallization, and preliminary structural studies of HbA(2) and HbE are reported here. HbA(2) and HbE are purified by cation exchange column chromatography in presence of KCN from the blood samples of individuals suffering from beta-thalassemia minor and E beta-thalassemia. X-ray diffraction data of HbA(2) and HbE were collected upto 2.1 and 1.73 A, respectively. HbA(2) crystallized in space group P2(1) with unit cell parameters a=54.33 A, b=83.73 A, c=62.87 A, and beta=99.80 degrees whereas HbE crystallized in space group P2(1)2(1)2(1) with unit cell parameters a=60.89 A, b=95.81 A, and c=99.08 A. Asymmetric unit in each case contains one Hb tetramer in R(2) state.