Intradomain confinement of disulfides in the folding of two consecutive modules of the LDL receptor

© 2015 Martínez-Oliván et al. The LDL receptor internalizes circulating LDL and VLDL particles for degradation. Its extracellular binding domain contains ten (seven LA and three EGF) cysteine-rich modules, each bearing three disulfide bonds. Despite the enormous number of disulfide combinations possible, LDLR oxidative folding leads to a single native species with 30 unique intradomain disulfides. Previous folding studies of the LDLR have shown that non native disulfides are initially formed that lead to compact species. Accordingly, the folding of the LDLR has been described as a >coordinated nonvectorial> reaction, and it has been proposed that early compaction funnels the reaction toward the native structure. Here we analyze the oxidative folding of LA4 and LA5, the modules critical for ApoE binding, isolated and in the LA45 tandem. Compared to LA5, LA4 folding is slow and inefficient, resembling that of LA5 disease-linked mutants. Without Ca++, it leads to a mixture of many two-disulfide scrambled species and, with Ca++, to the native form plus two three-disulfide intermediates. The folding of the LA45 tandem seems to recapitulate that of the individual repeats. Importantly, although the folding of the LA45 tandem takes place through formation of scrambled isomers, no interdomain disulfides are detected, i.e. the two adjacent modules fold independently without the assistance of interdomain covalent interactions. Reduction of incredibly large disulfide combinatorial spaces, such as that in the LDLR, by intradomain confinement of disulfide bond formation might be also essential for the efficient folding of other homologous disulfide-rich receptors.Peer Reviewe

Thermal Analysis of High Pressure Micro Plasma Discharge

High pressure micro plasma discharge has been at the center of interest in recent years, because of their vast applications, ease of access and cost efficiency. This attributes to atmospheric discharges that are generated in ambient conditions and therefore can be readily applicable to everyday use. The absence of vacuum makes these high pressure discharges to be inexpensive to operate. Despite the ease of operation, the high pressure is a source of enhanced gas heating as the gas temperature cannot be controlled by diffusion alone. Gas heating is therefore an important factor when it comes to the simulation of high pressure micro plasma discharge, unlike their low pressure counterpart where the heat generation is almost negligible. Low pressure discharge due to their low degree of collisionality generates ionic species and electrons at small concentrations, whereas high pressure discharge due to their higher gas density produces ions and electrons at higher concentrations which is a direct consequence of increase collision. The higher gas density and consequential large concentration of ionic species and electron contributes directly to higher heat generation rates. . In this thesis the gas temperature transport In this thesis the gas temperature transport of high pressure micro plasma discharge has been studied with a special focus on the heat source terms, temperature boundary conditions, temperature distribution in the solid phase electrodes and the gas phase and their overall influence on the plasma characteristics. For this purpose a multi-physics mathematical model has been developed that comprised of a plasma module, neutral gas temperature module, external circuit module and conjugate heat transfer module. The plasma module consisted of conservation of the different ionic, electronically excited species, radicals, neutrals and electrons, conservation of the electron temperature, and electric field. The external circuit module resolved the coupled driving circuit comprised of a voltage source, ballast resistor and capacitance. A detailed gas phase chemical kinetic model was also implemented. One-dimensional simulation has been performed to study the effects of the neutral gas temperature on a micro plasma discharge operating in the “abnormal” glow mode. In addition, two dimensional simulation has been conducted to simulate the “normal” glow regime of a micro plasma discharge that has multi-dimensional spatial dependence. The effects of conjugate heat transfer on the gas temperature distribution and the overall plasma characteristics i.e. the voltage-current curve and electron number density has been investigated. The conjugate heat transfer is found to significantly affect the plasma behavior. Finally a temporally varying temperature boundary condition has been proposed that reduces the computational overhead but resolves the conjugate heat transfer effect with reasonable accuracy

Nonuniform sampling and maximum entropy reconstruction in multidimensional NMR

NMR spectroscopy is one of the most powerful and versatile analytic tools available to chemists. The discrete Fourier transform (DFT) played a seminal role in the development of modern NMR, including the multidimensional methods that are essential for characterizing complex biomolecules. However, it suffers from well-known limitations: chiefly the difficulty in obtaining high-resolution spectral estimates from short data records. Because the time required to perform an experiment is proportional to the number of data samples, this problem imposes a sampling burden for multidimensional NMR experiments. At high magnetic field, where spectral dispersion is greatest, the problem becomes particularly acute. Consequently multidimensional NMR experiments that rely on the DFT must either sacrifice resolution in order to be completed in reasonable time or use inordinate amounts of time to achieve the potential resolution afforded by high-field magnets.Maximum entropy (MaxEnt) reconstruction is a non-Fourier method of spectrum analysis that can provide high-resolution spectral estimates from short data records. It can also be used with nonuniformly sampled data sets. Since resolution is substantially determined by the largest evolution time sampled, nonuniform sampling enables high resolution while avoiding the need to uniformly sample at large numbers of evolution times. The Nyquist sampling theorem does not apply to nonuniformly sampled data, and artifacts that occur with the use of nonuniform sampling can be viewed as frequency-aliased signals. Strategies for suppressing nonuniform sampling artifacts include the careful design of the sampling scheme and special methods for computing the spectrum. Researchers now routinely report that they can complete an N-dimensional NMR experiment 3 times faster (a 3D experiment in one ninth of the time). As a result, high-resolution three- and four-dimensional experiments that were prohibitively time consuming are now practical. Conversely, tailored sampling in the indirect dimensions has led to improved sensitivity.Further advances in nonuniform sampling strategies could enable further reductions in sampling requirements for high resolution NMR spectra, and the combination of these strategies with robust non-Fourier methods of spectrum analysis (such as MaxEnt) represent a profound change in the way researchers conduct multidimensional experiments. The potential benefits will enable more advanced applications of multidimensional NMR spectroscopy to study biological macromolecules, metabolomics, natural products, dynamic systems, and other areas where resolution, sensitivity, or experiment time are limiting. Just as the development of multidimensional NMR methods presaged multidimensional methods in other areas of spectroscopy, we anticipate that nonuniform sampling approaches will find applications in other forms of spectroscopy

RELATIONSHIP BETWEEN FLORAL MORPHOLOGY, FRUIT SETTING BEHAVIOR AND FINAL YIELD IN SOME EGGPLANT (Solanum melongena) GENOTYPES FROM IRAN

Heterostyly in eggplant flowers is a common trait that may affect the fruit production. The objective of this study was to evaluate the impact of heterostyly on fruit setting and yield of 13 eggplant genotypes from Iran. Flowers capable of setting fruit, including long- (LGs) and medium-styled flowers (MEs), accounted for the largest number of flowers (43.60-75.62%), while total short-styled flowers (SRTs) constituted a smaller percentage (20.47-45.51%) in different genotypes. However, SRTs represented a considerable proportion of the total number of eggplant flowers. Different eggplant genotypes produced 42.59-77.25% fruits from LGs+MEs, and only 0.0-3.77% from SRTs. In general, although the percentage of fruit setting of LGs and MEs was much greater compared to values of SRTs, there was no significant correlation between final yield and style length. Therefore, in order to increase eggplant yield, protecting fruits formed from LGs and MEs might be more effective than increasing the proportion of LGs and MEs to SRTs

Effects of sorghum residue in presence of pre-emergence herbicides on emergence and biomass of Echinochloa colona and Chloris virgata

In conservation agriculture systems, farmers gain many advantages from retaining crop residue on the soil surface, but crop residue retention in these systems may intervene with the activity of pre-emergence herbicides. A pot study was conducted to evaluate the effect of different rates of pre-emergence herbicides [imazethapyr (100 and 150 g a. i. ha-1), isoxaflutole (100 and 200 g a. i. ha-1), metolachlor (1.5 and 2.25 kg a. i. ha-1), pendimethalin (2.25 and 3.38 kg a. i. ha-1) and prosulfocarb + metolachlor (2.5 and 3.75 kg a. i. ha-1)] on seedling emergence and biomass of Echinochloa colona and Chloris virgata when applied in the presence of sorghum residue at rates equivalent to (0, 3 and 6 t ha-1). When seeds of E. colona and C. virgata were not covered with sorghum residue, the seedling emergence and biomass of both weeds was inhibited by 93–100% and 56–100%, respectively, with the application (both rates) of isoxaflutole, metolachlor, pendimethalin and prosulfocarb + metolachlor. Using sorghum residue resulted in lower herbicide efficacy on both weeds. At 3 t ha-1 sorghum residue, E. colona emergence and biomass reduced by 38–100% and 30–100%, respectively, with application of isoxaflutole, metolachlor and pendimethalin (both rates) in comparison with the no-herbicide treatment. Similarly, the emergence and biomass of C. virgata was also reduced by 92–100% and 25–100%, respectively. The results of this study suggest that crop residue may influence efficacy of commonly used pre-emergence herbicides and that the amount of crop residue on the soil surface should be adjusted according to the nature of the pre-emergence herbicides to achieve adequate weed control

Selenoether oxytocin analogues have analgesic properties in a mouse model of chronic abdominal pain

Poor oral availability and susceptibility to reduction and protease degradation is a major hurdle in peptide drug development. However, drugable receptors in the gut present an attractive niche for peptide therapeutics. Here we demonstrate, in a mouse model of chronic abdominal pain, that oxytocin receptors are significantly upregulated in nociceptors innervating the colon. Correspondingly, we develop chemical strategies to engineer non-reducible and therefore more stable oxytocin analogues. Chemoselective selenide macrocyclization yields stabilized analogues equipotent to native oxytocin. Ultra-high-field nuclear magnetic resonance structural analysis of native oxytocin and the seleno-oxytocin derivatives reveals that oxytocin has a pre-organized structure in solution, in marked contrast to earlier X-ray crystallography studies. Finally, we show that these seleno-oxytocin analogues potently inhibit colonic nociceptors both in vitro and in vivo in mice with chronic visceral hypersensitivity. Our findings have potentially important implications for clinical use of oxytocin analogues and disulphide-rich peptides in general

Reducing seed dependent variability of non-uniformly sampled multidimensional NMR data

The application of NMR spectroscopy to study the structure, dynamics and function of macromolecules requires the acquisition of several multidimensional spectra. The one-dimensional NMR time-response from the spectrometer is extended to additional dimensions by introducing incremented delays in the experiment that cause oscillation of the signal along “indirect” dimensions. For a given dimension the delay is incremented at twice the rate of the maximum frequency (Nyquist rate). To achieve high-resolution requires acquisition of long data records sampled at the Nyquist rate. This is typically a prohibitive step due to time constraints, resulting in sub-optimal data records to the detriment of subsequent analyses. The multidimensional NMR spectrum itself is typically sparse, and it has been shown that in such cases it is possible to use non-Fourier methods to reconstruct a high-resolution multidimensional spectrum from a random subset of non-uniformly sampled (NUS) data. For a given acquisition time, NUS has the potential to improve the sensitivity and resolution of a multidimensional spectrum, compared to traditional uniform sampling. The improvements in sensitivity and/or resolution achieved by NUS are heavily dependent on the distribution of points in the random subset acquired. Typically, random points are selected from a probability density function (PDF) weighted according to the NMR signal envelope. In extreme cases as little as 1% of the data is subsampled. The heavy under-sampling can result in poor reproducibility, i.e. when two experiments are carried out where the same number of random samples is selected from the same PDF but using different random seeds. Here, a jittered sampling approach is introduced that is shown to improve random seed dependent reproducibility of multidimensional spectra generated from NUS data, compared to commonly applied NUS methods. It is shown that this is achieved due to the low variability of the inherent sensitivity of the random subset chosen from a given PDF. Finally, it is demonstrated that metrics used to find optimal NUS distributions are heavily dependent on the inherent sensitivity of the random subset, and such optimisation is therefore less critical when using the proposed sampling scheme