Using a FRET Library with Multiple Probe Pairs To Drive Monte Carlo Simulations of α-Synuclein

We describe a strategy for experimentally-constraining computational simulations of intrinsically disordered proteins (IDPs), using α-synuclein, an IDP with a central role in Parkinson’s disease pathology, as an example. Previously, data from single-molecule Förster Resonance Energy Transfer (FRET) experiments have been effectively utilized to generate experimentally constrained computational models of IDPs. However, the fluorophores required for single-molecule FRET experiments are not amenable to the study of short-range (<30 Å) interactions. Using ensemble FRET measurements allows one to acquire data from probes with multiple distance ranges, which can be used to constrain Monte Carlo simulations in PyRosetta. To appropriately employ ensemble FRET data as constraints, we optimized the shape and weight of constraining potentials to afford ensembles of structures that are consistent with experimental data. We also used this approach to examine the structure of α-synuclein in the presence of the compacting osmolyte trimethylamine-N-oxide. Despite significant compaction imparted by 2 M trimethylamine-N-oxide, the underlying ensemble of α-synuclein remains largely disordered and capable of aggregation, also in agreement with experimental data. These proof-of-concept experiments demonstrate that our modeling protocol enables one to efficiently generate experimentally constrained models of IDPs that incorporate atomic-scale detail, allowing one to study an IDP under a variety of conditions.     

Purification of Xylitol from Fermented Hemicellulosic Hydrolyzate Using Liquid–Liquid Extraction and Precipitation Techniques

Xylitol was produced by Candida guilliermondii by fermentation of sugarcane bagasse hemicellulosic hydrolysate. Undesirable impurities were extracted from the broth using either ethyl acetate, chloroform or dichloromethane. The best results on clarification of the broth without xylitol loss were obtained with ethyl acetate. When ethanol, acetone or tetrahydrofuran were used for precipitation of impurities, only tetrahydrofuran clarified the fermented broth, but a high xylitol loss (~30%) was observed.     

The Extrapolation of Vapour–liquid Equilibrium Curves of Pure Fluids in Alternative Gibbs Ensemble Monte Carlo Implementations

Extrapolation schemes based on Taylor series expansion to determine the vapour–liquid equilibrium (VLE) curves of pure molecular fluids are presented for the NpH and μVL versions of the Gibbs ensemble Monte Carlo (GEMC) simulations. The coexistence curves of the various configurational quantities can be expressed as Taylor series around the simulated equilibrium point as a function of pressure in the NpH version and chemical potential in the μVL version. The coefficients of the Taylor series are calculated from single GEMC simulations using Clapeyron-like equations and fluctuation formulas. A Padè approximant is used to widen the range where the extrapolation is accurate. These methods are demonstrated on atomic Lennard-Jones fluid. The procedure is found to be an accurate and useful tool to calculate wide sections of the VLE curves. With this procedure the saturation heat capacity can be directly determined using the calculated derivatives.     

Monte Carlo simulations of vapour–liquid phase equilibrium and microstructure for the system containing azeotropes

Abstract

Phase equilibrium data of the mixtures including alcohols, esters and organic acids are of first interest particularly to design and optimise biodiesel production and reactive distillation processes. In this work, vapour–liquid phase equilibrium of these systems was simulated at low pressure using Gibbs ensemble Monte Carlo method. All Lennard–Jones parameters of pseudo-atoms involved in the systems were derived from previous parametrisations of TraPPE-UA force field. The Fourier coefficients of dihedrals encountered in ethyl acetate molecule have been obtained from the quantum calculations. Using this force field, temperature-composition diagrams are well reproduced for ethyl acetate + ethanol, ethyl acetate + methanol at 70.00 kPa and ethyl acetate + acetic acid mixtures at 77.33 kPa. The transferability of this force field to mixtures in these systems is noticeable. Analysis of the microstructure for the ethyl acetate + ethanol and ethyl acetate + acetic acid mixtures was presented. We found that the hydrogen bond networks consist of autoassociation and cross-association and autoassociation occupies the main position as compared with cross association in the ethyl acetate + ethanol mixture. OC_HAc–H_HAc and OC_EtOAc–H_HAc hydrogen bond interactions play a significant role in the phase behaviours or structures of ethyl acetate + acetic acid mixture.     

Theoretical Calculation of the Liquid—Vapor Coexistence Curve of Water,Chloroform and Methanol with the Cavity-Biased Monte Carlo Method in the Gibbs Ensemble

Abstract

The Gibbs ensemble computer simulation method of Panagiotopoulos is combined by the cavity-biased sampling technique used previously in the grand-canonical ensemble. The combined technique is applied to the determination of the liquid—vapor coexistence curve of the Lennard—Jones fluid as a test case, two water models (SPC and TIP4P) as well as methanol and chloroform, both described with the OPLS model. The application of the virial-based sampling technique, used earlier in the isobaric ensemble is also discussed.     

Molecular Cloning and Expression of an α-Amylase Inhibitor from Rye with Potential for Controlling Insect Pests

Alpha-amylase inhibitors have important roles in plant defense mechanisms, particularly against insects, and several of these inhibitors have been expressed in different crops to increase their resistance to particular insects. In this work, we report the cloning and expression of a gene encoding for a new -amylase inhibitor (BIII) from rye (Secale cereale) seeds. The BIII gene contains 354 nucleotides that encode for 118 amino acids sequence. A 313 bp fragment of the gene was expressed in Escherichia coli and resulted in a functional inhibitor that reduced the activity of -amylases of larvae of the coleopteran pests Acanthoscelides obtectus, Zabrotess subfasciatus and Anthonomus grandis. In contrast, the inhibitor did not inhibit the activity of porcine pancreatic -amylase. Although the amino acid sequence of BIII showed high identity with those of bifunctional inhibitors, the recombinant protein was unable to inhibit trypsin-like serine proteinases. The effects of recombinant BIII were evaluated in vivo against A. grandis. When first instar larvae were reared on an artificial diet containing four different concentrations of BIII, a reduction in larval weight and a mortality of 83% were observed at the highest concentration.     

Liquid–liquid extraction of an extracellular alkaline protease from fermentation broth using aqueous two-phase and reversed micelles systems

Summary The study of recovery of an extracellular alkaline protease from fermentation broth produced by Norcadiopsis sp, was carried out with liquid–liquid extraction through sodium di-(2-ethylhexyl) sulphosuccinate/isooctane reversed micelles systems and aqueous two-phase systems (polyethylene glycol/potassium phosphate). The best conditions for extraction and back-extraction with the reversed micelles system was obtained at pH 9.0 and pH 5.0, respectively, showing a yield of protein of 6.16%, a specific activity of 4.10 U/ml and a purification factor of 1.80. The studies using aqueous two-phase systems of polyethylene glycol/potassium phosphate at pH 10.0 showed purification factors of 2 and 5, and protein yield of 11 and 4%, respectively, for polyethylene glycol 550/potassium phosphate and polyethylene glycol 8000/potassium phosphate. The results indicate that the aqueous two-phase systems are more attractive as a first step in the isolation and purification processes.     

Isoprene Emissions from Downy Oak under Water Limitation during an Entire Growing Season: What Cost for Growth?

Increases in the production of terpene- and phenolic-like compounds in plant species under abiotic stress conditions have been interpreted in physiological studies as a supplementary defense system due to their capacity to limit cell oxidation. From an ecological perspective however, these increases are only expected to confer competitive advantages if they do not imply a significant cost for the plant, that is, growth reduction. We investigated shifts of isoprene emissions, and to a lesser extent phenolic compound concentration, of Quercus pubescens Willd. from early leaf development to leaf senescence under optimal watering (control: C), mild and severe water stress (MS, SS). The impact of water stress was concomitantly assessed on plant physiological (chlorophyll fluorescence, stomatal conductance, net photosynthesis, water potential) functional (relative leaf water content, leaf mass per area ratio) and growth (aerial and root biomass) traits. Growth changes allowed to estimate the eventual costs related to the production of isoprene and phenolics. The total phenolic content was not modified under water stress whereas isoprene emissions were promoted under MS over the entire growing cycle despite the decline of P_n by 35%. Under SS, isoprene emissions remained similar to C all over the study despite the decline of Pn by 47% and were thereby clearly uncoupled to Pn leading to an overestimation of the isoprene emission factor by 44%. Under SS, maintenance of isoprene emissions and phenolic compound concentration resulted in very significant costs for the plants as growth rates were very significantly reduced. Under MS, increases of isoprene emission and maintenance of phenolic compound concentration resulted in moderate growth reduction. Hence, it is likely that investment in isoprene emissions confers Q. pubescens an important competitive advantage during moderate but not severe periods of water scarcity. Consequences of this response for air quality in North Mediterranean areas are also discussed.     

A Nonlinear Stability Analysis of Vegetative Turing Pattern Formation for an Interaction–Diffusion Plant-Surface Water Model System in an Arid Flat Environment

The development of spontaneous stationary vegetative patterns in an arid flat environment is investigated by means of a weakly nonlinear diffusive instability analysis applied to the appropriate model system for this phenomenon. In particular, that process can be modeled by a partial differential interaction–diffusion equation system for the plant biomass density and the surface water content defined on an unbounded flat spatial domain. The main results of this analysis can be represented by closed-form plots in the rate of precipitation versus the specific rate of plant density loss parameter space. From these plots, regions corresponding to bare ground and vegetative patterns consisting of parallel stripes, labyrinth-like mazes, hexagonal arrays of gaps, irregular mosaics, and homogeneous distributions of vegetation, respectively, may be identified in this parameter space. Then those theoretical predictions are compared with both relevant observational evidence involving tiger and pearled bush patterns and existing numerical simulations of similar model systems as well as placed in the context of the results from some recent nonlinear vegetative pattern formation studies.     

Comparison between an event-by-event Monte Carlo code,NOREC, and ETRAN for electron scaled point kernels between 20 keV and 1 MeV

An event-by-event Monte Carlo code called NOREC, a substantially improved version of the Oak Ridge electron transport code (OREC), was released in 2003, after a number of modifications to OREC. In spite of some earlier work, the characteristics of the code have not been clearly shown so far, especially for a wide range of electron energies. Therefore, NOREC was used in this study to generate one of the popular dosimetric quantities, the scaled point kernel, for a number of electron energies between 0.02 and 1.0 MeV. Calculated kernels were compared with the most well-known published kernels based on a condensed history Monte Carlo code, ETRAN, to show not only general agreement between the codes for the electron energy range considered but also possible differences between an event-by-event code and a condensed history code. There was general agreement between the kernels within about 5% up to 0.7 r/r ₀ for 100 keV and 1 MeV electrons. Note that r/r ₀ denotes the scaled distance, where r is the radial distance from the source to the dose point and r ₀ is the continuous slowing down approximation (CSDA) range of a mono-energetic electron. For the same range of scaled distances, the discrepancies for 20 and 500 keV electrons were up to 6 and 12%, respectively. Especially, there was more pronounced disagreement for 500 keV electrons than for 20 keV electrons. The degree of disagreement for 500 keV electrons decreased when NOREC results were compared with published EGS4/PRESTA results, producing similar agreement to other electron energies.     

Electrodiffusion Models of Neurons and Extracellular Space Using the Poisson-Nernst-Planck Equations—Numerical Simulation of the Intra- and?Extracellular Potential for an Axon Model

In neurophysiology, extracellular signals—as measured by local field potentials (LFP) or electroencephalography—are of great significance. Their exact biophysical basis is, however, still not fully understood. We present a three-dimensional model exploiting the cylinder symmetry of a single axon in extracellular fluid based on the Poisson-Nernst-Planck equations of electrodiffusion. The propagation of an action potential along the axonal membrane is investigated by means of numerical simulations. Special attention is paid to the Debye layer, the region with strong concentration gradients close to the membrane, which is explicitly resolved by the computational mesh. We focus on the evolution of the extracellular electric potential. A characteristic up-down-up LFP waveform in the far-field is found. Close to the membrane, the potential shows a more intricate shape. A comparison with the widely used line source approximation reveals similarities and demonstrates the strong influence of membrane currents. However, the electrodiffusion model shows another signal component stemming directly from the intracellular electric field, called the action potential echo. Depending on the neuronal configuration, this might have a significant effect on the LFP. In these situations, electrodiffusion models should be used for quantitative comparisons with experimental data.     

A Practical Tool to Identify Water Bodies with Potential for Mosquito Habitatunder Mangrove Canopy: Large-scale Airborne Scanning in the Thermal Band 8–13 μm

We address the practical issue of using thermal image data without adjustment or calibration for projects which do not require actual temperatures per se. Large scale airborne scanning in the thermal band at 8.5–13 μm was obtained for a mangrove and salt marsh in subtropical eastern Australia. For open sites, the raw image values were strongly positively correlated with ground level temperatures. For sites under mangrove canopy cover, image values indicated temperatures 2–4°C lower than those measured on the ground. The raw image was useful in identifying water bodies under canopy and has the potential for locating channel lines of deeper water. This could facilitate modification to increase flushing in the system, thereby reducing mosquito larval survival.     

Human papilloma virus–specific T cells can be generated from naïve T cells for use as an immunotherapeutic strategy for immunocompromised patients

Human papilloma virus (HPV) is a known cause of cervical cancer, squamous cell carcinoma and laryngeal cancer. Although treatments exist for HPV-associated malignancies, patients unresponsive to these therapies have a poor prognosis. Recent findings from vaccine studies suggest that T-cell immunity is essential for disease control. Because Epstein-Barr Virus (EBV)-specific T cells have been highly successful in treating or preventing EBV-associated tumors, we hypothesized that the development of a manufacturing platform for HPV-specific T cells from healthy donors could be used in a third-party setting to treat patients with high-risk/relapsed HPV-associated cancers. Most protocols for generating virus-specific T cells require prior exposure of the donor to the targeted virus and, because the seroprevalence of high-risk HPV types varies greatly by age and ethnicity, manufacturing of donor-derived HPV-specific T cells has proven challenging. We, therefore, made systematic changes to our current Good Manufacturing Practice (GMP)-compliant protocols to improve antigen presentation, priming and expansion for the manufacture of high-efficacy HPV-specific T cells. Like others, we found that current methodologies fail to expand HPV-specific T cells from most healthy donors. By optimizing dendritic cell maturation and function with lipopolysaccharide (LPS) and interferon (IFN)γ, adding interleukin (IL)-21 during priming and depleting memory T cells, we achieved reliable expansion of T cells specific for oncoproteins E6 and E7 to clinically relevant amounts (mean, 578-fold expansion; n?=?10), which were polyfunctional based on cytokine multiplex analysis. In the third-party setting, such HPV-specific T-cell products might serve as a potent salvage therapy for patients with HPV-associated diseases.     

Application of the phase-space distribution approach of Monte Carlo for radiation contamination dose estimation from the (n,γ), (γ,n) nuclear reactions and linac leakage photons in the megavoltage radiotherapy facility

AimThe aim of this study was to characterize the radiation contamination inside and outside the megavoltage radiotherapy room.BackgroundRadiation contamination components in the 18 MV linac room are the secondary neutron, prompt gamma ray, electron and linac leakage radiation.Materials and MethodsAn 18 MV linac modeled in a typical bunker employing the MCNPX code of Monte Carlo. For fast calculation, phase-space distribution (PSD) file modeling was applied and the calculations were conducted for the radiation contamination components dose and spectra at 6 locations inside and outside the bunker.ResultsThe results showed that the difference of measured and calculated percent depth-dose (PDD) and photo beam-profile (PBP) datasets were lower than acceptable values. At isocenter, the obtained photon dose and neutron fluence were 2.4 × 10⁻¹⁴ Gy/initial e° and 2.22 × 10^-8 n°/cm², respectively. Then, neutron apparent source strength (Q_N) value was found as 1.34 × 10¹² n°/Gy X at isocenter and the model verified to photon and neutron calculations. A surface at 2 cm below the flattening filter was modeled as phase-space (PS) file for PDD and PBP calculations. Then by use of a spherical cell in the center of the linac target as a PS surface, contaminant radiations dose, fluence and spectra were estimated at 6 locations in a considerably short time, using the registered history of all particles and photons in the 13GB PSD file as primary source in the second step.ConclusionDesigning the PSD file in MC modeling helps user to solve the problems with complex geometry and physics precisely in a shorter run-time.     

Epidemiology and Evolution of Rotaviruses and Noroviruses from an Archival WHO Global Study in Children (1976–79) with Implications for Vaccine Design

Prompted by the discovery of new gastrointestinal viruses, the NIH, NIAID and WHO investigated the etiology of acute diarrhea that occurred from 1976–1979 in a global cohort of infants and young children. Rotaviruses were found to be major pathogens worldwide, whereas the Norwalk virus could not be detected using a radioimmunoassay. The aim of this study is to re-evaluate the role and diversity of rotaviruses and noroviruses in the original cohort using more sensitive current technologies. Stools collected from Asia, Africa, and South America (n = 485) were evaluated for viral genotypes by RT-PCR and sequencing. Rotaviruses were detected in 28.9% and noroviruses in 9.7% of the specimens, with G1 rotaviruses and GII noroviruses accounting for the majority of each respective virus. Various strains in this study predated the currently assigned dates of discovery for their particular genotype, and in addition, two noroviruses (KL45 and T091) could not be assigned to current genotypes. Phylogenetic analyses demonstrated a relative constancy in circulating rotavirus genotypes over time, with several genotypes from this study becoming established in the current repertoire of viral species. Similarly, GII noroviruses have maintained dominance, with GII.4 noroviruses continuing as a predominant genotype over time. Taken together, the complex molecular epidemiology of rotaviruses and noroviruses circulating in the 1970’s is consistent with current patterns, an important consideration in the design of multivalent vaccines to control these viruses.     

Generation of Zika virus–specific T cells from seropositive and virus-naïve donors for potential use as an autologous or “off-the-shelf” immunotherapeutic

BackgroundZika virus (ZIKV) infection can cause severe birth defects in newborns with no effective currently available treatment. Adoptive transfer of virus-specific T cells has proven to be safe and effective for the prevention or treatment of many viral infections, and could represent a novel treatment approach for patients with ZIKV infection. However, extending this strategy to the ZIKV setting has been hampered by limited data on immunogenic T-cell antigens within ZIKV. Hence, we have generated ZIKV-specific T cells and characterized the cellular immune responses against ZIKV antigens.MethodsT-cell products were generated from peripheral blood of ZIKV-exposed donors, ZIKV-naive adult donors and umbilical cord blood by stimulation with pentadecamer (15mer) overlapping peptide libraries spanning four ZIKV polyproteins (C, M, E and NS1) using a Good Manufacturing Practice–compliant protocol.ResultsWe successfully generated T cells targeting ZIKV antigens with clinically relevant numbers. The ex vivo–expanded T cells comprised both CD4⁺ and CD8⁺ T cells that were able to produce Th1-polarized effector cytokines and kill ZIKV-infected HLA-matched monocytes, confirming functionality of this unique T-cell product as a potential “off-the-shelf” therapeutic. Epitope mapping using peptide arrays identified several novel HLA class I and class II–restricted epitopes within NS1 antigen, which is essential for viral replication and immune evasion.DiscussionOur findings demonstrate that it is feasible to generate potent ZIKV-specific T cells from a variety of cell sources including virus naïve donors for future clinical use in an “off-the-shelf” setting.     

Detecting Long-term Global Forest Change Using Continuous Fields of Tree-Cover Maps from 8-km Advanced Very High Resolution Radiometer (AVHRR) Data for the Years 1982–99

This paper describes global changes in forest cover from 1982 to 1999 based on the 8-km Pathfinder Advanced Very High Resolution Radiometer (AVHRR) data set. The procedure involves the use of a regression tree in predicting percent tree cover for the years 1982–99. Training data are created from high-resolution imagery and used with phenological metrics derived from the annual AVHRR time series. Using the 18 years of estimated tree cover, and based on a thresholding approach, we identified locations where change in tree cover has occurred. The change sites were then compared to a set of high-resolution deforestation analyses to yield area estimates of deforestation and regrowth. Percent tree cover was found to have decreased globally, from the 1980s to 1990s, in contrast to United Nations Food and Agriculture Organization (FAO) reports of a global increase in forest cover. Latin America and tropical Asia are the two dominant deforestation regions. Paraguay shows the highest rate of forest clearing over the time series, while Indonesia had the greatest increase in deforestation from the 1980s to 1990s. We also suggest that the percent tree-cover maps can be used in standardizing national forest statistics, as an objective means of identifying hot spots of change, and for facilitating ecosystem monitoring.