Effects of turn residues in directing the formation of the beta-sheet and in the stability of the beta-sheet

The designed peptide (denoted 20-mer, sequence VFITS(D)PGKTYTEV(D)PGOKILQ) has been shown to form a three-strand antiparallel beta-sheet. It is generally believed that the (D)Pro-Gly segment has the propensity to adopt a type II' beta-turn, thereby promoting the formation of this beta-sheet. Here, we replaced (D)Pro-Gly with Asp-Gly, which should favor a type I' turn, to examine the influence of different type of turns on the stability of the beta-sheet. Contrary to our expectation, the mutant peptide, denoted P6D, forms a five-residue type I turn plus a beta-bulge between the first two strands due to a one amino-acid frameshift in the hydrogen bonding network and side-chain inversion of the first beta-strand. In contrast, the same kind of substitution at (D)Pro-14 in the double mutant, denoted P6DP14D, does not yield the same effect. These observations suggest that the SDGK sequence disfavors the type I' conformation while the VDGO sequence favors a type I' turn, and that the frameshift in the first strand provides a way for the peptide to accommodate a disfavored turn sequence by protruding a bulge in the formation of the beta-hairpin. Thus, different types of turns can affect the stability of a beta-structure.     

Complete genome sequence of the halophilic bacterium Spirochaeta africana type strain (Z-7692T) from the alkaline Lake Magadi in the East African Rift

Spirochaeta africana Zhilina et al. 1996 is an anaerobic, aerotolerant, spiral-shaped bacterium that is motile via periplasmic flagella. The type strain of the species, Z-7692^T, was isolated in 1993 or earlier from a bacterial bloom in the brine under the trona layer in a shallow lagoon of the alkaline equatorial Lake Magadi in Kenya. Here we describe the features of this organism, together with the complete genome sequence, and annotation. Considering the pending reclassification of S. caldaria to the genus Treponema, S. africana is only the second ''true'' member of the genus Spirochaeta with a genome-sequenced type strain to be published. The 3,285,855 bp long genome of strain Z-7692^T with its 2,817 protein-coding and 57 RNA genes is a part of the G enomic E ncyclopedia of B acteria and A rchaea project.     

红火蚁巢穴土壤中生物碱成分分析

【目的】红火蚁Solenopsis invicta栖息在真菌和细菌较丰富的土壤环境中,容易受多种真菌和细菌性病原体的侵染,因红火蚁的毒液具有很好的防御和抗菌作用,致使红火蚁能生存于此类土壤环境中。为了探索这一奥秘,本研究旨在建立红火蚁巢穴土壤中的毒液生物碱的最佳提取方法,并对毒液生物碱成分进行定量分析。【方法】采集蚁巢周边3 m处的土壤,进行添加、回收红火蚁毒液实验。采用抽滤法提取土壤中的毒液生物碱,利用GC-FID对毒液生物碱成分进行定量分析,筛选最佳的提取溶剂,并且确定添加三乙胺的最佳体积比。然后用最优方法提取蚁巢土壤中的毒液生物碱,并进行定量分析。【结果】正己烷、二氯甲烷、乙酸乙酯、丙酮和甲醇5种提取溶剂中,正己烷处理的生物碱回收率略优。当添加的三乙胺的体积为1 mL及以上时,提取效果最佳。红火蚁巢穴土壤中的毒液生物碱成分中,trans-C15∶1的含量最高,trans-C13∶1的含量次之。巢穴土壤中总生物碱含量约为22μg/g。【结论】三乙胺有助于提取蚂蚁巢穴土壤中的毒液生物碱。红火蚁巢穴土壤中的生物碱浓度较高,有可能对巢穴土壤微生物群落产生重要影响。     

Effect of the N3 residue on the stability of the alpha-helix

N3 is the third position from the N terminus in the alpha-helix with helical backbone dihedral angles. All 20 amino acids have been placed in the N3 position of a synthetic helical peptide (CH(3)CO-[AAX AAAAKAAAAKAGY]-NH(2)) and the helix content measured by circular dichroism spectroscopy at 273 K. The dependence of peptide helicity on N3 residue identity has been used to determine a free energy scale by analysis with a modified Lifson-Roig helix coil theory that includes a parameter for the N3 energy (n3). The most stabilizing residues at N3 in rank order are Ala, Glu, Met/Ile, Leu, Lys, Ser, Gln, Thr, Tyr, Phe, Asp, His, and Trp. Free energies for the most destabilizing residues (Cys, Gly, Asn, Arg, and Pro) could not be fitted. The results correlate with N1, N2, and helix interior energies and not at all with N-cap preferences. This completes our work on studying the structural and energetic preferences of the amino acids for the N-terminal positions of the alpha-helix. These results can be used to rationally modify protein stability, help design helices, and improve prediction of helix location and stability.     

Specificity and reversibility of the transpeptidation reaction catalyzed by the Streptomyces R61 D-Ala-D-Ala peptidase

The specificity of the Streptomyces R61 penicillin-sensitive D-Ala-D-Ala peptidase has been re-examined with the help of synthetic substrates. The products of the transpeptidation reactions obtained with Gly-L-Xaa dipeptides as acceptor substrates are themselves poor substrates of the enzyme. This is in apparent contradiction with the classically accepted specificity rules for D-Ala-D-Ala peptidases. The Gly-L-Xaa dipeptide is regenerated by both the hydrolysis and transpeptidation reactions. The latter reaction is observed when another Gly-L-Xaa peptide or D-Alanine are supplied as acceptors. Utilization of substrates in which the terminal -COO(-) group has been esterified or amidated shows that a free carboxylate is not an absolute prerequisite for activity. The results are discussed in the context of the expected reversibility of the transpeptidation reaction.     

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of these disease vectors. We have recently identified a bitter sensing neuron in the labellum of female Aedes aegypti that responds to DEET and other repellents, as well as bitter quinine, through direct electrophysiological investigation. These gustatory receptor neuron responses prompted our sequencing of total mRNA from both male and female labella and tarsi samples to elucidate the putative chemoreception genes expressed in these contact chemoreception tissues. Samples of tarsi were divided into pro-, meso- and metathoracic subtypes for both sexes. We then validated our dataset by conducting qRT-PCR on the same tissue samples and used statistical methods to compare results between the two methods. Studies addressing molecular function may now target specific genes to determine those involved in repellent perception by mosquitoes. These receptor pathways may be used to screen novel repellents towards disruption of host-seeking behavior to curb the spread of harmful viruses.     

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the development of more effective treatments is the relative lack of understanding of the molecular underpinnings of the effects of ethanol on behavior. The nematode, Caenorhabditis elegans (C. elegans), provides a useful model in which to generate and test hypotheses about the molecular effects of ethanol. Here, we describe an assay that has been developed and used to examine the roles of particular genes and environmental factors in behavioral responses to ethanol, in which locomotion is the behavioral output. Ethanol dose-dependently causes an acute depression of crawling on an agar surface. The effects are dynamic; animals exposed to a high concentration demonstrate an initial strong depression of crawling, referred to here as initial sensitivity, and then partially recover locomotion speed despite the continued presence of the drug. This ethanol-induced behavioral plasticity is referred to here as the development of acute functional tolerance. This assay has been used to demonstrate that these two phenotypes are distinct and genetically separable. The straightforward locomotion assay described here is suitable for examining the effects of both genetic and environmental manipulations on these acute behavioral responses to ethanol in C. elegans.     

Life at the extreme: lessons from the genome

Extremophile plants thrive in places where most plant species cannot survive. Recent developments in high-throughput technologies and comparative genomics are shedding light on the evolutionary mechanisms leading to their adaptation.     

Ubiquitination Regulates the Neuroprotective Function of the Deubiquitinase Ataxin-3 in Vivo

Deubiquitinases (DUBs) are proteases that regulate various cellular processes by controlling protein ubiquitination. Cell-based studies indicate that the regulation of the activity of DUBs is important for homeostasis and is achieved by multiple mechanisms, including through their own ubiquitination. However, the physiological significance of the ubiquitination of DUBs to their functions in vivo is unclear. Here, we report that ubiquitination of the DUB ataxin-3 at lysine residue 117, which markedly enhances its protease activity in vitro, is critical for its ability to suppress toxic protein-dependent degeneration in Drosophila melanogaster. Compared with ataxin-3 with only Lys-117 present, ataxin-3 that does not become ubiquitinated performs significantly less efficiently in suppressing or delaying the onset of toxic protein-dependent degeneration in flies. According to further studies, the C terminus of Hsc70-interacting protein (CHIP), an E3 ubiquitin ligase that ubiquitinates ataxin-3 in vitro, is dispensable for its ubiquitination in vivo and is not required for the neuroprotective function of this DUB in Drosophila. Our work also suggests that ataxin-3 suppresses degeneration by regulating toxic protein aggregation rather than stability.     

辽宁医巫闾山种子植物区系研究

医巫闾山约有野生种子植物718种,隶属于94科362属之中。依据它们的现代分布,这些种被归纳成23个分布区类型和20个亚型。研究表明,医巫闾山植物区系属于温带性质,但在其发生发展过程中曾经历过与热带相联系的历史渊源。本区系地理联系广泛,尤以与东亚地区特别是与日本的联系较为密切,其次则是与北温带的联系,并且这些成分构成本区系种类组成的主体。     

Harnessing the placebo effect: the need for translational research

Laboratory research recently has greatly enhanced the understanding of placebo and nocebo effects by identifying specific neuromodulators and brain areas associated with them. However, little progress has been made in translating this knowledge into improved patient care. Here, we discuss the limitations in our knowledge about placebo (and nocebo) effects and the need for translational research with the aim of guiding physicians in maximizing placebo effects and minimizing nocebo effects in their routine clinical practice. We suggest some strategies for how, when and why interventions to promote beneficial placebo responses might be administered in the clinical setting.     

In the right place at the right time: Parnassia resolves the herkogamy dilemma by accurate repositioning of stamens and stigmas

Background and Aims

Spatial (herkogamy) and temporal (dichogamy) separation of pollen presentation and stigma receptivity have been interpreted as reducing interference between male and female functions in hermaphroditic flowers. However, spatial separation leads to a potential conflict: reduced pollination accuracy, where pollen may be placed in a location on the pollinator different from the point of stigma contact.

Methods

To understand better how herkogamous flowers resolve this conflict, a study was made of a subalpine herb, Parnassia epunctulata, the nectariferous flowers of which exhibit sequential anther dehiscence (staggered pollen presentation) and stamen movements; usually one newly dehisced anther is positioned each day over the central gynoecium, while the older stamens bend away from the central position.

Key Results

The open flowers were visited by a variety of pollinators, most of which were flies. Seed set was pollinator-dependent (bagged flowers set almost no seeds) and pollen-limited (manual pollination increased seed set over open pollination). Analyses of adaptive accuracy showed that coordinated stamen movements and style elongation (movement herkogamy) dramatically increased pollination accuracy. Specifically, dehiscing anthers and receptive stigmas were positioned accurately in the vertical and horizontal planes in relation to the opposite sexual structure and pollinator position. By contrast, the spatial correspondence between anthers and stigma was dramatically lower before the anthers dehisced and after stamens bent outwards, as well as before and after the period of stigmatic receptivity.

Conclusions

It is shown for the first time that a combination of movement herkogamy and dichogamy can maintain high pollination accuracy in flowers with generalized pollination. Staggered pollen and stigma presentation with spatial correspondence can both reduce sexual interference and improve pollination accuracy.     

Toward the solution of the protein structure prediction problem

Since Anfinsen demonstrated that the information encoded in a protein’s amino acid sequence determines its structure in 1973, solving the protein structure prediction problem has been the Holy Grail of structural biology. The goal of protein structure prediction approaches is to utilize computational modeling to determine the spatial location of every atom in a protein molecule starting from only its amino acid sequence. Depending on whether homologous structures can be found in the Protein Data Bank (PDB), structure prediction methods have been historically categorized as template-based modeling (TBM) or template-free modeling (FM) approaches. Until recently, TBM has been the most reliable approach to predicting protein structures, and in the absence of reliable templates, the modeling accuracy sharply declines. Nevertheless, the results of the most recent community-wide assessment of protein structure prediction experiment (CASP14) have demonstrated that the protein structure prediction problem can be largely solved through the use of end-to-end deep machine learning techniques, where correct folds could be built for nearly all single-domain proteins without using the PDB templates. Critically, the model quality exhibited little correlation with the quality of available template structures, as well as the number of sequence homologs detected for a given target protein. Thus, the implementation of deep-learning techniques has essentially broken through the 50-year-old modeling border between TBM and FM approaches and has made the success of high-resolution structure prediction significantly less dependent on template availability in the PDB library.     

On the spermatophore of ixodid ticks

Structure and formation of the spermatophore and the extrusion of its contents in ixodid ticks were studied. The structure of the ectospermatophore resembles that of argasid ticks, whereas that of the endospermatophore differs completely; it is composed of several organelles which are absent in argasids.The chemical nature of the components of the spermatophore was studied by histochemical methods. The male forms the spermatophore by ejaculating the components in a definite order. The components of the ectospermatophore are deposited one into another—while those of the endospermatophore one on top of the other and are linked together in a chain. The last component to be ejected is a plug that closes the endospermatophore.During extrusion of the endospermatophore all of the components burst out of the ectospermatophore. The endospermatophore sheath evaginates and forms the capsule into which the sperm is inserted. Each capsule has two tube-like appendices.     

Functional Analysis of the Larval Feeding Circuit in Drosophila

The serotonergic feeding circuit in Drosophila melanogaster larvae can be used to investigate neuronal substrates of critical importance during the development of the circuit. Using the functional output of the circuit, feeding, changes in the neuronal architecture of the stomatogastric system can be visualized. Feeding behavior can be recorded by observing the rate of retraction of the mouth hooks, which receive innervation from the brain. Locomotor behavior is used as a physiological control for feeding, since larvae use their mouth hooks to traverse across an agar substrate. Changes in feeding behavior can be correlated with the axonal architecture of the neurites innervating the gut. Using immunohistochemistry it is possible to visualize and quantitate these changes. Improper handling of the larvae during behavior paradigms can alter data as they are very sensitive to manipulations. Proper imaging of the neurite architecture innervating the gut is critical for precise quantitation of number and size of varicosities as well as the extent of branch nodes. Analysis of most circuits allow only for visualization of neurite architecture or behavioral effects; however, this model allows one to correlate the functional output of the circuit with the impairments in neuronal architecture.     

Seeing beyond the limit: A guide to choosing the right super-resolution microscopy technique

Super-resolution microscopy has become an increasingly popular and robust tool across the life sciences to study minute cellular structures and processes. However, with the increasing number of available super-resolution techniques has come an increased complexity and burden of choice in planning imaging experiments. Choosing the right super-resolution technique to answer a given biological question is vital for understanding and interpreting biological relevance. This is an often-neglected and complex task that should take into account well-defined criteria (e.g., sample type, structure size, imaging requirements). Trade-offs in different imaging capabilities are inevitable; thus, many researchers still find it challenging to select the most suitable technique that will best answer their biological question. This review aims to provide an overview and clarify the concepts underlying the most commonly available super-resolution techniques as well as guide researchers through all aspects that should be considered before opting for a given technique.     

Understanding the development of roots exposed to contaminants and the potential of plant-associated bacteria for optimization of growth

Background and Scope

Plant responses to the toxic effects of soil contaminants, such as excess metals or organic substances, have been studied mainly at physiological, biochemical and molecular levels, but the influence on root system architecture has received little attention. Nevertheless, the precise position, morphology and extent of roots can influence contaminant uptake. Here, data are discussed that aim to increase the molecular and ecological understanding of the influence of contaminants on root system architecture. Furthermore, the potential of plant-associated bacteria to influence root growth by their growth-promoting and stress-relieving capacities is explored.

Methods

Root growth parameters of Arabidopsis thaliana seedlings grown in vertical agar plates are quantified. Mutants are used in a reverse genetics approach to identify molecular components underlying quantitative changes in root architecture after exposure to excess cadmium, copper or zinc. Plant-associated bacteria are isolated from contaminated environments, genotypically and phenotypically characterized, and used to test plant root growth improvement in the presence of contaminants.

Key Results

The molecular determinants of primary root growth inhibition and effects on lateral root density by cadmium were identified. A vertical split-root system revealed local effects of cadmium and copper on root development. However, systemic effects of zinc exposure on root growth reduced both the avoidance of contaminated areas and colonization of non-contaminated areas. The potential for growth promotion and contaminant degradation of plant-associated bacteria was demonstrated by improved root growth of inoculated plants exposed to 2,4-di-nitro-toluene (DNT) or cadmium.

Conclusions

Knowledge concerning the specific influence of different contaminants on root system architecture and the molecular mechanisms by which this is achieved can be combined with the exploitation of plant-associated bacteria to influence root development and increase plant stress tolerance, which should lead to more optimal root systems for application in phytoremediation or safer biomass production.