Effects of Hydroxylated Carbon Nanotubes on the Aggregation of Aβ16–22 Peptides: A Combined Simulation and Experimental Study

The pathogenesis of Alzheimer’s disease (AD) is associated with the aggregation of amyloid-β (Aβ) peptides into toxic aggregates with β-sheet character. In a previous computational study, we showed that pristine single-walled carbon nanotubes (SWCNTs) can inhibit the formation of β-sheet-rich oligomers in the central hydrophobic core fragment of Aβ (Aβ_16–22). However, the poor solubility of SWCNTs in water hinders their use in biomedical applications and nanomedicine. Here, we investigate the influence of hydroxylated SWCNT, a water-soluble SWCNT derivative, on the aggregation of Aβ_16–22 peptides using all-atom explicit-water replica exchange molecular dynamics simulations. Our results show that hydroxylated SWCNTs can significantly inhibit β-sheet formation and shift the conformations of Aβ_16–22 oligomers from ordered β-sheet-rich structures toward disordered coil aggregates. Detailed analyses of the SWCNT-Aβ interaction reveal that the inhibition of β-sheet formation by hydroxylated SWCNTs mainly results from strong electrostatic interactions between the hydroxyl groups of SWCNTs and the positively charged residue K16 of Aβ_16–22 and hydrophobic and aromatic stacking interactions between SWCNTs and F19 and F20. In addition, our atomic force microscopy and thioflavin T fluorescence experiments confirm the inhibitory effect of both pristine and hydroxylated SWCNTs on Aβ_16–22 fibrillization, in support of our previous and present replica exchange molecular dynamics simulation results. These results demonstrate that hydroxylated SWCNTs efficiently inhibit the aggregation of Aβ_16–22; in addition, they offer molecular insight into the inhibition mechanism, thus providing new clues for the design of therapeutic drugs against amyloidosis.     

Effects of Hydrophobic Macromolecular Crowders on Amyloid β (16–22) Aggregation

In Alzheimer’s disease (AD), the amyloid β (Aβ) peptide aggregates in the brain to form progressively larger oligomers, fibrils, and plaques. The aggregation process is strongly influenced by the presence of other macromolecular species, called crowders, that can exert forces on the proteins. One very common attribute of macromolecular crowders is their hydrophobicity. We examined the effect of hydrophobic crowders on protein aggregation by using discontinuous molecular dynamics (DMD) simulations in combination with an intermediate resolution protein model, PRIME20. The systems considered contained 48 Aβ (16–22) peptides and crowders with diameters of 5 Å, 20 Å, and 40 Å, represented by hard spheres or spheres with square-well/square-shoulder interactions, at a crowder volume fraction of ϕ = 0.10. Results show that low levels of crowder hydrophobicity are capable of increasing the fibrillation lag time and high levels of crowder hydrophobicity can fully prevent the formation of fibrils. The types of structures that remain during the final stages of the simulations are summarized in a global phase diagram that shows fibril, disordered oligomer, or β-sheet phases in the space spanned by crowder size and crowder hydrophobicity. In particular, at high levels of hydrophobicity, simulations with 5 Å crowders result in only disordered oligomers and simulations with 40 Å crowders result in only β-sheets. The presence of hydrophobic crowders reduces the antiparallel β-sheet content of fibrils, whereas hard sphere crowders increase it. Finally, strong hydrophobic crowders alter the secondary structure of the Aβ (16–22) monomers, bending them into a shape that is incapable of forming ordered β-sheets or fibrils. These results qualitatively agree with previous theoretical and experimental work.     

Mechanisms of the Effects of Crocin on Aggregation and Deposition of Aβ1–40 Fibrils in Alzheimer’s Disease

Alzheimer??s disease (AD) is among the most important health-care problems in the world. The two pathological hallmarks of AD are extracellular neuritic amyloid plaques and intracellular neurofibrillary tangles. The aggregation of A?? and ??-sheet formation are considered to be the critical events which render these peptides neurotoxic. AD is affecting a large percentage of the elderly around the world. Many studies have been done on drugs to cure or at least slow Alzheimer??s disease. Most drugs produced for this disease aim at compensating for the performance of specific cell groups affected by the disease or restoring the function of these cells.This study examined the interaction of crocin, the main pigment of saffron, with the amyloid-?? peptides 1?+?40 (A?? 40) to determine the effects on peptide conformation and fibril formation using fluorescence spectroscopy, CD spectroscopy and electron microscopy. ThT data demonstrated the appearance of well-defined amyloid fibrils indicating an enhanced nucleation of A??40. Incubation of pre-formed A??40 fibrils with crocin resulted in extensive lateral aggregation and precipitation of the fibrils. Consistent with this, electron microscopy data indicated that crocin decreased the number of fibrils formed and significantly reduced the average fibril length of A??40 as assessed by low levels of thioflavin T binding data. The mechanism by which, crocin prevented fibril formation was demonstrated by ANS binding assay and CD spectroscopy. In summary, crocin interacts with A?? peptides and prevents amyloid formation. This means that it has the potential to be an important therapeutic drug against AD.     

The Structure of Aβ42 C-Terminal Fragments Probed by a Combined Experimental and Theoretical Study

The C-terminus of amyloid β-protein (Aβ) 42 plays an important role in this protein's oligomerization and may therefore be a good therapeutic target for the treatment of Alzheimer's disease. Certain C-terminal fragments (CTFs) of Aβ42 have been shown to disrupt oligomerization and to strongly inhibit Aβ42-induced neurotoxicity. Here we study the structures of selected CTFs [Aβ(x-42); x = 29-31, 39] using replica exchange molecular dynamics simulations and ion mobility mass spectrometry. Our simulations in explicit solvent reveal that the CTFs adopt a metastable β-structure: β-hairpin for Aβ(x-42) (x = 29-31) and extended β-strand for Aβ(39-42). The β-hairpin of Aβ(30-42) is converted into a turn-coil conformation when the last two hydrophobic residues are removed, suggesting that I41 and A42 are critical in stabilizing the β-hairpin in Aβ42-derived CTFs. The importance of solvent in determining the structure of the CTFs is further highlighted in ion mobility mass spectrometry experiments and solvent-free replica exchange molecular dynamics simulations. A comparison between structures with solvent and structures without solvent reveals that hydrophobic interactions are critical for the formation of β-hairpin. The possible role played by the CTFs in disrupting oligomerization is discussed.     

Effects of oxidation on redox and cytotoxic properties of copper complex of Aβ1–16 peptide

Abstract

The effect of oxidation on redox and cytotoxic properties of copper complex of amyloid beta (Aβ) peptide was studied by gamma radiolysis. The oxidation of Aβ1–16 and Aβ1–16/Cu(II) complex was carried out using hydroxyl (^?OH) radicals produced by gamma radiolysis and the products were analyzed using mass spectrometry. The presence of Cu(II) was found to enhance the oxidation of Aβ1–16 peptide. The oxidation of residues Asp1, His6, and His13 was enhanced due to their involvement in copper binding. The oxidation of His residues of Aβ1–16 peptide, which are chiefly responsible for copper binding, resulted in altered redox properties and subsequently in higher cytotoxicity of the Aβ1–16 peptide in SH-SY5Y cells.     

Differential Axonal Conduction Patterns of Mechano-Sensitive and Mechano-Insensitive Nociceptors – A Combined Experimental and Modelling Study

Cutaneous pain sensations are mediated largely by C-nociceptors consisting of both mechano-sensitive (CM) and mechano-insensitive (CMi) fibres that can be distinguished from one another according to their characteristic axonal properties. In healthy skin and relative to CMi fibres, CM fibres show a higher initial conduction velocity, less activity-dependent conduction velocity slowing, and less prominent post-spike supernormality. However, after sensitization with nerve growth factor, the electrical signature of CMi fibres changes towards a profile similar to that of CM fibres. Here we take a combined experimental and modelling approach to examine the molecular basis of such alterations to the excitation thresholds. Changes in electrical activation thresholds and activity-dependent slowing were examined in vivo using single-fibre recordings of CM and CMi fibres in domestic pigs following NGF application. Using computational modelling, we investigated which axonal mechanisms contribute most to the electrophysiological differences between the fibre classes. Simulations of axonal conduction suggest that the differences between CMi and CM fibres are strongly influenced by the densities of the delayed rectifier potassium channel (K_dr), the voltage-gated sodium channels Na_V1.7 and Na_V1.8, and the Na⁺/K⁺-ATPase. Specifically, the CM fibre profile required less K_dr and Na_V1.8 in combination with more Na_V1.7 and Na⁺/K⁺-ATPase. The difference between CM and CMi fibres is thus likely to reflect a relative rather than an absolute difference in protein expression. In support of this, it was possible to replicate the experimental reduction of the ADS pattern of CMi nociceptors towards a CM-like pattern following intradermal injection of nerve growth factor by decreasing the contribution of K_dr (by 50%), increasing the Na⁺/K⁺-ATPase (by 10%), and reducing the branch length from 2 cm to 1 cm. The findings highlight key molecules that potentially contribute to the NGF-induced switch in nociceptors phenotype, in particular Na_V1.7 which has already been identified clinically as a principal contributor to chronic pain states such as inherited erythromelalgia.     

Exploring the Influence of Carbon Nanoparticles on the Formation of β-Sheet-Rich Oligomers of IAPP22–28 Peptide by Molecular Dynamics Simulation

Recent advances in nanotechnologies have led to wide use of nanomaterials in biomedical field. However, nanoparticles are found to interfere with protein misfolding and aggregation associated with many human diseases. It is still a controversial issue whether nanoparticles inhibit or promote protein aggregation. In this study, we used molecular dynamics simulations to explore the effects of three kinds of carbon nanomaterials including graphene, carbon nanotube and C₆₀ on the aggregation behavior of islet amyloid polypeptide fragment 22–28 (IAPP_22–28). The diverse behaviors of IAPP_22–28 peptides on the surfaces of carbon nanomaterials were studied. The results suggest these nanomaterials can prevent β-sheet formation in differing degrees and further affect the aggregation of IAPP_22–28. The π–π stacking and hydrophobic interactions are different in the interactions between peptides and different nanoparticles. The subtle differences in the interaction are due to the difference in surface curvature and area. The results demonstrate the adsorption interaction has competitive advantages over the interactions between peptides. Therefore, the fibrillation of IAPP_22–28 may be inhibited at its early stage by graphene or SWCNT. Our study can not only enhance the understanding about potential effects of nanomaterials to amyloid formation, but also provide valuable information to develop potential β-sheet formation inhibitors against type II diabetes.     

‘A Dispassionate and Objective Effort:’ Negotiating the First Study on the Biological Effects of Atomic Radiation

The National Academy of Science’s 1956 study on the Biological Effects of Atomic Radiation (BEAR) was designed to provide an objective analysis to assess conflicting statements by leading geneticists and by officials in the Atomic Energy Commission. Largely because of its status as a detached, non-governmental evaluation by eminent scientists, no studies have had a broader impact on the development of biological thinking in regard to nuclear policies. This paper demonstrates that despite the first BEAR study’s reputation as an objective and independent study, it was the product of careful negotiation between Academy scientists, the Atomic Energy Commission, and Britain’s Medical Research Council. This paper also reveals the fragility of the consensus that produced the Academy’s report, the range of political uses of the report, and the subsequent disaffection of the scientists who took part in it.     

Impact of Routing Flexibility on the Performance of an FMS—A Simulation Study

The evolving manufacturing environment is characterized by a drive toward increasing flexibility. One possible manifestation of flexibility within an FMS is in the form of routing flexibility. Providing this typically is an expensive proposition, and system designers therefore aim to provide only the required levels commensurate with a given set of operating conditions. This paper presents a framework based on a Taguchi experimental design for studying the nature of the impact of varying levels of routing flexibility on the performance of an FMS. Simulation results indicate that increases in routing flexibility, when made available at the cost of an associated penalty on operation processing time, is not always beneficial. There is an optimal flexibility level, beyond which system performance deteriorates, as judged by the makespan measure of performance. It is suggested that the proposed methodology can be used in practice for not only setting priorities on specific design and control factors but also for highlighting likely factor level combinations that could yield near-optimal shop performance.     

A Lipidomic Study of the Effects of N-methyl-N′-nitro-N-nitrosoguanidine on Sphingomyelin Metabolism

Systems biology is a new and rapidly developing research area in which,by quantitativelydescribing the interaction among all the individual components of a cell,a systems-level understanding of abiological response can be achieved.Therefore,it requires high-throughput measurement technologies forbiological molecules,such as genomic and proteomic approaches for DNA/RNA and protein,respectively.Recently,a new concept,lipidomics,which utilizes the mass spectrometry(MS)method for lipid analysis,has been proposed.Using this lipidomic approach,the effects of N-methyl-N'-nitro-N-nitrosoguanidine(MNNG)on sphingomyelin metabolism,a major class of sphingolipids,were evaluated.Sphingomyelin moleculeswere extracted from cells and analyzed by matrix-assisted laser desorption ionization-time of flight MS.Itwas found that MNNG induced profound changes in sphingomyelin metabolism,including the appearance ofsome new sphingomyelin species and the disappearance of some others,and the concentrations of severalsphmgomyelin species also changed.This was accompanied by the redistribution of acid sphingomyelinase(ASM),a key player in sphingomyelin metabolism.On the other hand,imipramine,an inhibitor of ASM,caused the accumulation of sphingomyelin.It also prevented some of the effects of MNNG,as well as theredistribution of ASM.Taken together,these data suggested that the lipidomic approach is highly effectivefor the systematic analysis of cellular lipids metabolism.     

Effects of oxidation on copper-binding properties of Aβ1-16 peptide: A pulse radiolysis study

Abstract

The reaction of hydroxyl radicals (^?OH) with Aβ1-16 peptide was carried out using pulse radiolysis to understand the effect of oxidation of peptide on its copper-binding properties. This reaction produced oxidized, dimeric and trimeric Aβ1-16 peptide species. The formation of these products was established with the help of fluorescence spectroscopy and mass spectrometry. The mass spectral data indicate that the major site of oxidation is at His6, while the site for dimerization is at Tyr10. Diethyl pyrocarbonate-treated Aβ1-16 peptide did not produce any trimeric species upon oxidation with ^?OH. The quantitative chemical modification studies indicated that one of the three histidine residues is covalently modified during pulse radiolysis. The copper-binding studies of the oxidized peptide revealed that it has similar copper-binding properties as the unoxidized peptide. Further, the cytotoxicity studies point out that both oxidized and unoxidized Aβ1-16 peptide are equally efficient in producing free radicals in presence of copper and ascorbate that resulted in comparable cell death.     

A Lipidomic Study of the Effects of N-methyl-N＇-nitro-N-nitrosoguanidine on Sphingomyelin Metabolism

Systems biology is a new and rapidly developing research area in which, by quantitatively describing the interaction among all the individual components of a cell, a systems-level understanding of a biological response can be achieved. Therefore, it requires high-throughput measurement technologies for biological molecules, such as genomic and proteomic approaches for DNA/RNA and protein, respectively.Recently, a new concept, lipidomics, which utilizes the mass spectrometry （MS） method for lipid analysis,has been proposed. Using this lipidomic approach, the effects of N-methyl-N＇-nitro-N-nitrosoguanidine （MNNG） on sphingomyelin metabolism, a major class of sphingolipids, were evaluated. Sphingomyelin molecules were extracted from cells and analyzed by matrix-assisted laser desorption ionization-time of flight MS. It was found that MNNG induced profound changes in sphingomyelin metabolism, including the appearance of some new sphingomyelin species and the disappearance of some others, and the concentrations of several sphingomyelin species also changed. This was accompanied by the redistribution of acid sphingomyelinase （ASM）, a key player in sphingomyelin metabolism. On the other hand, imipramine, an inhibitor of ASM,caused the accumulation of sphingomyelin. It also prevented some of the effects of MNNG, as well as the redistribution of ASM. Taken together, these data suggested that the lipidomic approach is highly effective for the systematic analysis of cellular lipids metabolism.     

Effects of Familial Mutations on the Monomer Structure of Aβ42

Amyloid beta (Aβ) peptide plays an important role in Alzheimer’s disease. A number of mutations in the Aβ sequence lead to familial Alzheimer’s disease, congophilic amyloid angiopathy, or hereditary cerebral hemorrhage with amyloid. Using molecular dynamics simulations of ∼200 μs for each system, we characterize and contrast the consequences of four pathogenic mutations (Italian, Dutch, Arctic, and Iowa) for the structural ensemble of the Aβ monomer. The four familial mutations are found to have distinct consequences for the monomer structure.Amyloid beta (Aβ) peptides have long been thought to play a central role in Alzheimer’s disease (AD). Usually 40 or 42 residues in length, Aβ peptides are proteolytic products of the Aβ precursor protein and they aggregate to form the fibrillar plaques in AD patients’ brains. Besides fibrillar plaques, Aβ oligomers are also neurotoxic. The significance and nature of Aβ oligomerization has recently become a focus of intensive research studies and debates (1,2). Notably, numerous pathogenic mutations have been identified in the Aβ precursor protein sequence and in the enzymes involved in Aβ processing (3). These mutations generally lead to early onset of AD or cerebral amyloid angiopathy. Understanding how the pathogenic mutations alter Aβ oligomerization/aggregation is essential to our understanding of the disease mechanism.Four of these pathogenic mutations (Italian E22K, Dutch E22Q, Arctic E22G, and Iowa D23N) cluster in the region of E22 and D23 in the Aβ sequence (distal from proteolytic cleavage sites) and they have higher neurotoxicity compared to wild-type (WT) Aβ (4). These mutations are thought to modify the physicochemistry of the peptide. For example, kinetic studies (4) show that the E22K and E22Q mutations lead to faster peptide aggregation, whereas the E22G and D23N mutations result in slightly slower aggregation than WT Aβ₄₂ (although the E22G mutation shows increased protofibril formation (5)). Recent solid-state NMR studies also suggest that rather than the in-register β-sheet conformation adopted by WT Aβ, the Iowa D23N mutant forms amyloid fibrils with antiparallel β-sheet structure (6).To understand how the mutations modify the peptide oligomerization/aggregation it is critical to characterize the starting point of the process, the monomers. Unfortunately, investigating the early phase of the oligomerization process experimentally is a challenging task due to the high aggregation propensity of Aβ and its intrinsic disorder. Therefore, a number of computational approaches have been adopted to investigate the consequences of mutations for the monomer structure (7–16). However, due to the high computational demands of explicit-solvent molecular dynamics (MD) simulations to simulate full-length Aβ peptides, most of these computational studies are either on Aβ fragments (to decrease the system size) using explicit-solvent simulations (8–12) or on full-length Aβ using implicit-solvent simulations (which are less computationally demanding and enable longer simulation times, but lack explicit water molecules in the simulations to fully describe water-peptide interactions) (13–15). In a very recent report, explicit-solvent simulations were used to study the effects of the E22Q mutation on full-length Aβ; however, rather limited data (<10 μs) were collected (16). Thus, characterizing full-length Aβ monomers remains quite a daunting task even with simulations.To characterize the effects of mutations on full-length Aβ monomer using explicit-solvent MD simulations, we employed distributed computing (17) to simulate the WT Aβ₄₂, Aβ₄₂-E22K, Aβ₄₂-E22Q, Aβ₄₂-E22G, and Aβ₄₂-D23N monomers. MD simulations of >200 μs were performed for each system and AMBER ff99sb (18) and the tip3p water model (19) were used for force field parameters. Peptide configurations in the MD trajectories were clustered with the root mean-square deviation metric to identify representative conformations (i.e., states) and transitions between these states were counted. Markov state model analysis was then performed where the master equations were solved and the equilibrium population of each state deduced (20). Details of the MD simulation procedures and Markov state model analysis can be found in the Supporting Material.Each of the five Aβ monomer systems exhibits great structural diversity and can only be characterized in an ensemble fashion (rather than described by a handful of representative configurations). This is in accord with the notion that full-length Aβ peptides are intrinsically disordered (21,22). Using the Dictionary of Secondary Structure of Proteins program (23) to assign secondary structure, it is clear that the five Aβ monomer systems are found overall not well structured, although small β-hairpins and α-helices are observed. In Fig. 1 we plot the residue-dependent extended β propensity and α-helix propensity, in the top and bottom panels, respectively, for each Aβ monomer system. Although we are reasonably confident of the convergence behavior of the α-helix propensity, we note that the convergence of the extended β-propensity might be more challenging and demand a much longer sampling time than the current aggregate simulation time of ∼200 μs (24).Open in a separate windowFigure 1Ensemble-averaged %population of β-strand (top) and α-helix (bottom) propensity for all five monomer systems. The sequence of the WT Aβ₄₂ is given on the x axis.We observe in Fig. 1 that all five Aβ monomer systems share a rather similar residue-dependent tendency to form an extended β-structure, although minor differences are present. On the other hand, these pathogenic mutations alter the α-helix propensity quite significantly. The E22K and E22Q mutations increase the α-helix propensity in the region of residues 20–23. All four mutations (E22K, E22Q, E22G, and D23N) decrease the α-helix propensity in the region of residues 33–36.Notably, we find that in all five systems only short stretches of α-helices are formed. That is, when a residue is involved in α-helix formation, it participates in forming mostly short helical segments (consisting of only four helical residues). To provide more insight into the changes of α-helix propensity due to the mutations, in Fig. S1 we plot the tendency of forming short α-helices along the sequence for all five systems. Each data point in Fig. S1 represents the propensity to form an α-helix of four residues in length, ending at the specific residue. For example, in the structural ensemble adopted by the WT peptide, ∼5.5% of the conformations have a short α-helix of size four, involving residues 15–18. We see from Fig. S1 that the E22K and E22Q mutations induce the formation of two short helices in residues 19–22 and 20–23. The higher α-helix propensity in this region for the E22K mutant compared to the WT was previously attributed to the elimination of the electrostatic repulsion between E22 and D23 in the WT by the mutation and the longer aliphatic chain of K22 in the mutant compared to E22 in the WT (9,22). This is consistent with the observation that the E22Q mutation also induces helix formation in this region (by eliminating the electrostatic repulsion between E22 and D23 in the WT) but to a lesser extent, possibly due to the shorter aliphatic chain of Q22 compared to K22.In the E22G mutant, although the mutation eliminates the electrostatic repulsion between E22 and D23 in the WT peptide, glycine is known to be a helix breaker (25), leading to diminished α-helix propensity in the region around residue G22 seen in Fig. S1.In the D23N mutant, although the mutation eliminates the electrostatic repulsion between E22 and D23 in the WT peptide, it does not induce (or rather even slightly decreases) helix formation around residue 23. This may be due to the short aliphatic chain of N23 but it is possible that the mutation induces some nonlocal effects on the peptide structure, disfavoring helix formation in this region.It is worth noting that all four mutations (E22K, E22Q, E22G, and D23N) virtually eliminate the α-helix propensity in the region of residues 33–36. This region is rather far away from the mutation sites in sequence but its α-helix propensity is nonetheless affected. The origin of such a nonlocal effect is less straightforward to explain and further analysis will aid untangling this behavior. Nonetheless, the diminished α-helix propensity in the region of residues 33–36 appears to be a consistent feature across all four mutants.The four mutations studied here (E22K, E22Q, E22G, and D23N) have been thought to modify the physicochemistry of the peptide and alter the oligomerization/aggregation process, leading to higher neurotoxicity. In predicting intrinsic aggregation propensities using peptide sequences, all four mutants are suggested to be more aggregation prone (26). On the other hand, kinetic studies show that only the E22K and E22Q mutants aggregate more quickly, whereas the E22G and D23N mutations result in slightly slower aggregation than WT Aβ₄₂ (4). Our simulation results suggest these pathogenic mutations have complicated effects on the monomer structure—all four mutations decrease helix propensity in residues 33–36, whereas only the E22K and E22Q mutations increase helix propensity in residues 20–23. It is interesting to note that α-helix propensity is generally thought to anticorrelate with aggregation propensity; however, recent studies have suggested an important role of α-helical intermediates in amyloid oligomerization (27–29). Our studies suggest that it would be of great value to investigate how the distinct patterns of α-helix propensity in these five systems may propagate to give rise to different oligomerization kinetics or even mechanisms. The pathogenic mutations studied here have complex effects on the oligomerization of the peptide. The characterization of the monomer structural ensembles reported here should aid understanding of such an important and complicated process.     

Stress Reduction by Technology? An Experimental Study into the Effects of Brainmachines on Burnout and State Anxiety

Stress and burnout are widely acknowledged as major causes of societal and individual problems in the Western world. In order to reduce material and immaterial expenses, increased efforts are made to enhance relaxation and stress reduction. Based on neuropsychological findings, alternative ways have been explored, one of them being the application of so-called brain wave synchronizers, which are said to induce a relaxation response by entraining alpha brain-wave activity (8–13 Hz) through audiovisual stimulation. A double blind, quasi-experiment was conducted among employees at a Dutch addiction care center to investigate the possible effects of two distinct brainmachine programs on burnout and anxiety. Subjects in both conditions showed a significant, immediate decrease in state anxiety as assessed by Spielberger's State-Trait Anxiety Inventory (STAI) and reported a range of subjective effects. However, a long-term effect on burnout, as measured with Maslach's Burnout Inventory (MBI-NL), could not be established. A long-term effect on anxiety (STAI), as investigated by interrupted time-series measurement, could not be established either. These and other findings suggest that the major claims with respect to these machines cannot hold over time, although pleasant short-term effects do occur. Individual differences in baseline responsivity, the stable character of burnout dimensions, or the ill-defined nature of relaxation, or a combination of these, may account for these results.     

Effects of 17β-estradiol and bisphenol A on the formation of reproductive organs in planarians

Planarians have a remarkable capacity for regeneration after ablation, and they reproduce asexually by fission. However, some planarians can also reproduce and maintain their sexual organs. During the regenerative process, their existing sexual organs degenerate and new ones develop. However, little is known about hormonal regulation during the development of reproductive organs in planarians. In this study, we investigated the effects of 17β-estradiol (a steroid) and bisphenol A (an endocrine disrupter) on the formation of sexual organs in the hermaphroditic planarian Dugesia ryukyuensis. Under control conditions, all worm tissues regenerated into sexual planarians with sexual organs within 4 weeks after ablation. However, in the presence of bisphenol A or 17β-estradiol, although they apparently regenerated into sexual planarians, the yolk glands, which are one of the female sexual organs, failed to regenerate even 7 weeks after ablation. These data suggest that planarians have a steroid hormone system, which plays a key role in the formation and maturation of sexual organs.     

Effects of Experimental Choices and Analysis Noise on Surveys of the “Rare Biosphere”

When planning a survey of 16S rRNA genes from a complex environment, investigators face many choices including which primers to use and how to taxonomically classify sequences. In this study, we explored how these choices affected a survey of microbial diversity in a sample taken from the aerobic basin of the activated sludge of a North Carolina wastewater treatment plant. We performed pyrosequencing reactions on PCR products generated from primers targeting the V1-V2, V6, and V6-V7 variable regions of the 16S rRNA gene. We compared these sequences to 16S rRNA gene sequences found in a whole-genome shotgun pyrosequencing run performed on the same sample. We found that sequences generated from primers targeting the V1-V2 variable region had the best match to the whole-genome shotgun reaction across a range of taxonomic classifications from phylum to family. Pronounced differences between primer sets, however, occurred in the “rare biosphere” involving taxa that we observed in fewer than 11 sequences. We also examined the results of analysis strategies comparing a classification scheme using a nearest-neighbor approach to directly classifying sequences with a naïve Bayesian algorithm. Again, we observed pronounced differences between these analysis schemes in infrequently observed taxa. We conclude that if a study is meant to probe the rare biosphere, both the experimental conditions and analysis choices will have a profound impact on the observed results.For nearly 3 decades, investigations of the distribution of microbes in complex environments have focused on the use of rRNA genes (1, 2, 4, 11, 16, 18, 19, 22, 24). Because the full-length 16S rRNA sequence can be obtained with paired-end reads via traditional Sanger sequencing, until recently most studies of the 16S rRNA gene captured most or nearly most of the 16S sequence length. New pyrosequencing technologies, however, have recently been introduced that greatly reduce the per base cost of sequencing but with shorter read lengths than traditional Sanger sequencing (17). This new approach has proven powerful, yielding a previously unobtainable view of rare taxa (7, 12-14, 25).The shorter reads produced by pyrosequencing require the choice of a particular region of the 16S rRNA gene to target for pyrosequencing as well as the choice of an algorithm to classify the taxonomy of the shorter reads. In their initial surveys of microbial diversity with pyrosequencing (12, 14, 25), Sogin and colleagues targeted the V6 variable region, in part because it is was small enough to be captured with the 100-bp reads of the pyrosequencing technology available at the time. Recently, the read length of 454 pyrosequencing machines has been increased to an average of ∼250 bp. This allows for more flexibility in primer design and opens up the possibility of targeting regions of the 16S rRNA gene other than V6. In recent work, Huse et al. took advantage of this new capability to compare the classifications made for the human gut microbiome with the V6 and longer V3 regions (13). Plotting the taxonomic abundance of these two sequence sets against each other yielded an excellent correlation (r² = 0.99), suggesting that the choice of which variable region to target makes little difference. In this report, we introduce a data set examining the performance of sets of primers targeting the V1-V2, V6, and V6-V7 regions. By using a sample for which we have also generated a whole-genome shotgun sequencing run with 250 bp reads, we were able to compare the observed 16S rRNA genes in samples with and without an initial PCR step targeting the 16S rRNA gene. Our results demonstrate that experimental choices such as which region of the 16S rRNA gene to sequence and which algorithm to use to classify taxa are much more likely to affect observations of the “rare biosphere” than more commonly observed taxa.     

Determining the Mode of Action Involved in the Antimicrobial Activity of?Synthetic Peptides: A Solid-State NMR and FTIR Study

We have previously shown that leucine to lysine substitution(s) in neutral synthetic crown ether containing 14-mer peptide affect the peptide structure and its ability to permeabilize bilayers. Depending on the substitution position, the peptides adopt mainly either a α-helical structure able to permeabilize dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) vesicles (nonselective peptides) or an intermolecular β-sheet structure only able to permeabilize DMPG vesicles (selective peptides). In this study, we have used a combination of solid-state NMR and Fourier transform infrared spectroscopy to investigate the effects of nonselective α-helical and selective intermolecular β-sheet peptides on both types of bilayers. ³¹P NMR results indicate that both types of peptides interact with the headgroups of DMPC and DMPG bilayers. ²H NMR and Fourier transform infrared results reveal an ordering of the hydrophobic core of bilayers when leakage is noted, i.e., for DMPG vesicles in the presence of both types of peptides and DMPC vesicles in the presence of nonselective peptides. However, selective peptides have no significant effect on the ordering of DMPC acyl chains. The ability of these 14-mer peptides to permeabilize lipid vesicles therefore appears to be related to their ability to increase the order of the bilayer hydrophobic core.     

Effects of hydroxypropyl-β-cyclodextrin on the growth and morphology of Absidia coerulea

Cyclodextrin has been found to be an attractive novel solubilizer due to its unique material properties. Absidia coerulea is widely used in steroid bioconversion. The effects of hydroxypropyl-β-cyclodextrin (HP-β-CD) on the growth, morphology, and steroid-converting activity of A. coerulea CICC 40302 were systematically studied. HP-β-CD affected A. coerulea growth, resulting in changes in its spore morphology and mycelial morphology. It induced an increase in the spore germination rate and a decrease in cell biomass at the stationary phase. Optical microscopy revealed that HP-β-CD altered the mycelial morphology and reduced the pellet compactness of A. coerulea. A convenient and feasible computing method was used to measure pellet compactness, and it demonstrated that the compactness degree of the pellet decreased as HP-β-CD increased, which could be attributed to the modification of the physical properties of the fermentation medium. Moreover, the changing of mycelial morphology influenced steroid-converting activity. The results showed that HP-β-CD had multiple concentration-dependent effects on A. coerulea cells. HP-β-CD in the proper concentration range holds great potential as a biocompatible solubilizer.     

Effect of Alanine Replacement of L17 and F19 on the Aggregation and Neurotoxicity of Arctic-Type Aβ40

Alzheimer’s disease is the most common form of neurodegenerative disease. Beta-amyloid peptides (Aβ) are responsible for neuronal death both in vitro and in vivo. Previously, L17 and F19 residues were identified as playing key roles in the stabilization of the Aβ₄₀ conformation and in the reduction of its neurotoxicity. In this study, the effects of L17A/F19A mutations on the neurotoxicity of Aβ genetic mutant Arctic-type Aβ₄₀(E22G) were tested. The results showed that compared to Aβ₄₀(E22G), Aβ₄₀(L17A/F19A/E22G) reduced the rate of conformation conversion, aggregation, and cytotoxicity, suggesting that L17 and F19 are critical residues responsible for conformational changes which may trigger the neurotoxic cascade of Aβ. Aβ₄₀(L17A/F19A/E22G) also had decreased damage due to reactive oxygen species. The results are consistent with the discordant helix hypothesis, and confirm that residues 17–25 are in the discordant helix region. Compared to Aβ₄₀(L17A/F19A), reduction in aggregation of Aβ₄₀(L17A/F19A/E22G) was less significantly decreased. This observation provides an explanation based on the discordant helix hypothesis that the mutation of E22 to G22 of Aβ₄₀(E22G) alters the propensity of the discordant helix. Arctic-type Aβ₄₀(E22G) aggregates more severely than wild-type Aβ₄₀, with a consequential increase in toxicity.