Application of Amphipols for Structure–Functional Analysis of TRP Channels

Amphipathic polymers (amphipols), such as A8-35 and SApol, are a new tool for stabilizing integral membrane proteins in detergent-free conditions for structural and functional studies. Transient receptor potential (TRP) ion channels function as tetrameric protein complexes in a diverse range of cellular processes including sensory transduction. Mammalian TRP channels share ~20 % sequence similarity and are categorized into six subfamilies: TRPC (canonical), TRPV (vanilloid), TRPA (ankyrin), TRPM (melastatin), TRPP (polycystin), and TRPML (mucolipin). Due to the inherent difficulties in purifying eukaryotic membrane proteins, structural studies of TRP channels have been limited. Recently, A8-35 was essential in resolving the molecular architecture of the nociceptor TRPA1 and led to the determination of a high-resolution structure of the thermosensitive TRPV1 channel by cryo-EM. Newly developed maltose-neopentyl glycol (MNG) detergents have also proven to be useful in stabilizing TRP channels for structural analysis. In this review, we will discuss the impacts of amphipols and MNG detergents on structural studies of TRP channels by cryo-EM. We will compare how A8-35 and MNG detergents interact with the hydrophobic transmembrane domains of TRP channels. In addition, we will discuss what these cryo-EM studies reveal on the importance of screening different types of surfactants toward determining high-resolution structures of TRP channels.     

The Semantic Morphology of Adolf Portmann: A Starting Point for the Biosemiotics of Organic Form?

This paper develops the ideas of the Swiss zoologist Adolf Portmann or, more precisely, his concept of organic self-representation, wherein Portmann considered the outer surface of living organisms as a specific organ that serves in a self-representational role. This idea is taken as a starting point from which to elaborate Portman’s ideas, so as to make them compatible with the theoretical framework of biosemiotics. Today, despite the many theories that help us understand aposematism, camouflage, deception and other phenomena related to the category of mimicry, there still is a need for a general theory of self-representation that would re-synthesize evolutionary, morphogenetic and semiotic aspects of the surface of organisms. Here, Adolf Portmann’s concept of self-representation is considered as an important step towards the biosemiotics of animal form.

Combined Liquid Chromatography–Tandem Mass Spectrometry Analysis of Progesterone Metabolites

Progesterone has a number of important functions throughout the human body. While the roles of progesterone are well known, the possible actions and implications of progesterone metabolites in different tissues remain to be determined. There is a growing body of evidence that these metabolites are not inactive, but can have significant biological effects, as anesthetics, anxiolytics and anticonvulsants. Furthermore, they can facilitate synthesis of myelin components in the peripheral nervous system, have effects on human pregnancy and onset of labour, and have a neuroprotective role. For a better understanding of the functions of progesterone metabolites, improved analytical methods are essential. We have developed a combined liquid chromatography—tandem mass spectrometry (LC-MS/MS) method for detection and quantification of progesterone and 16 progesterone metabolites that has femtomolar sensitivity and good reproducibility in a single chromatographic run. MS/MS analyses were performed in positive mode and under constant electrospray ionization conditions. To increase the sensitivity, all of the transitions were recorded using the Scheduled MRM algorithm. This LC-MS/MS method requires small sample volumes and minimal sample preparation, and there is no need for derivatization. Here, we show the application of this method for evaluation of progesterone metabolism in the HES endometrial cell line. In HES cells, the metabolism of progesterone proceeds mainly to (20S)-20-hydroxy-pregn-4-ene-3-one, (20S)-20-hydroxy-5α-pregnane-3-one and (20S)-5α-pregnane-3α,20-diol. The investigation of possible biological effects of these metabolites on the endometrium is currently undergoing.     

PCR–SSCP: A Method for the Molecular Analysis of Genetic Diseases

Single strand conformation polymorphism (SSCP) is a reproducible, rapid and quite simple method for the detection of deletions/insertions/rearrangements in polymerase chain reaction amplified DNA. All the details for the use of PCR–SSCP are presented in the direction of genetic diseases (β-thalassaemia, cystic fibrosis), optimum gel conditions, sensitivity and the latest modifications of the method, which are applied in most laboratories. This non-radioactive PCR–SSCP method can be reliably used to identify mutations in patients (β-globin, CFTR), provided suitable controls are available. Moreover, it is widely used for mutation identification in carriers (β-thalassaemia, cystic fibrosis), making it particularly useful in population screening.     

Phylogenetic and Exon–Intron Structure Analysis of Fungal Subtilisins: Support for a Mixed Model of Intron Evolution

Phylogenetic and exon–intron structure analyses of intra- and interspecific fungal subtilisins in this study provided support for a mixed model of intron evolution: a synthetic theory of introns-early and introns-late speculations. Intraspecifically, there were three phase zero introns in Pr1A and its introns 1 and 2 located at the highly conserved positions were phylogentically congruent with coding region, which is in favor of the view of introns-early speculation, while intron 3 had two different sizes and was evolutionarily incongruent with coding region, the evidence for introns-late speculation. Noticeably, the subtilisin Pr1J gene from different strains of M. ansiopliae contained different number of introns, the strong evidence in support of introns-late theory. Interspecifically, phylogenetic analysis of 60 retrievable fungal subtilisins provided a clear relationship between amino acid sequence and gene exon–intron structure that the homogeneous sequences usually have a similar exon–infron structure. There were 10 intron positions inserted by highly biased phase zero introns across examined fungal subtilisin genes, half of these positions were highly conserved, while the others were species-specific, appearing to be of recent origins due to intron insertion, in favor of the introns-late theory. High conservations of positions 1 and 2 inserted by the high percentage of phase zero introns as well as the evidence of phylogenetic congruence between the evolutionary histories of intron sequences and coding region suggested that the introns at these two positions were primordial.Reviewing Editor:Dr. Manyuan Long     

A Comparative Structure–Function Analysis and Molecular Mechanism of Action of Endonucleases from Serratia marcescens and Physarum polycephalum

Structural and functional characteristics were compared for wild-type nuclease from Serratia marcescens, which belongs to the family of DNA/RNA nonspecific endonucleases, its mutational forms, and the nuclease I-PpoI from Physarum polycephalum, which is a representative of the Cys-His box-containing subgroup of the superfamily of extremely specific intron-encoded homing DNases. Despite the lack of sequence homology and the overall different topology of the Serratia marcescens and I-PpoI nucleases, their active sites have a remarkable structural similarity. Both of them have a unique magnesium atom in the active site, which is a part of the coordinatively bonded water–magnesium complex involved in their catalytic acts. In the enzyme–substrate complexes, the Mg²⁺ ion is chelated by an Asp residue, coordinates two oxygen atoms of DNA, and stabilizes the transition state of the phosphate anion and 3"-OH group of the leaving nucleotide. A new mechanism of the phosphodiester bond cleavage, which is common for the Serratia marcescens and I-PpoI nucleases and differs from the known functioning mechanism of the restriction and homing endonucleases, was proposed. It presumes a His residue as a general base for the activation of a non-cluster water molecule at the nucleophilic in line displacement of the 3"-leaving group. A strained metalloenzyme–substrate complex is formed during hydrolysis and relaxes to the initial state after the reaction.     

Development of Dispersive Liquid–Liquid Microextraction Based on Solidification of Floating Organic Drop for the Determination of Trace Nickel

A liquid-phase microextraction technique was developed using dispersive liquid-liquid microextraction based on solidification of floating organic drop combined with flame atomic absorption spectrometry, for the extraction and determination of trace amounts of nickel in water samples. Microextraction efficiency factors, such as the type and volume of extraction and dispersive solvents, pH, extraction time, the chelating agent amount, and ionic strength, were investigated and optimized. Under optimum conditions, the calibration graph was linear in the range of 4.23-250?μg?L(-1) with a detection limit of 1.27?μg?L(-1). The relative standard deviation for ten replicate measurements of 10 and 100?μg?L(-1) of nickel were 3.21% and 2.55%, respectively. The proposed method was assessed through the analysis of certified reference water or recovery experiments.     

Structure–activity relationships of the thujaplicins for inhibition of human tyrosinase

Tyrosinase inhibitors have become increasingly critical agents in cosmetic, agricultural, and medicinal products. Although a large number of tyrosinase inhibitors have been reported, almost all the inhibitors were unfortunately evaluated by using commercial available mushroom tyrosinase. Here, we examined the inhibitory effects of three isomers of thujaplicin (α, β, and γ) on human tyrosinase and analyzed their binding modes using homology model and docking studies. As the results, γ-thujaplicin was found to strongly inhibit human tyrosinase with the IC₅₀ of 1.15 μM, extremely superior to a well-known tyrosinase inhibitor kojic acid (IC₅₀ = 571.17 μM). MM-GB/SA binding free energy decomposition analyses suggested that the potent inhibitory activity of γ-thujaplicin may be due to the interactions with His367, Ile368, and Val377 (hot spot amino acid residues) in human tyrosinase. Furthermore, the binding mode of α-thujaplicin indicated that Val377 and Ser380 may cause van der Waals clashes with the isopropyl group of α-thujaplicin. These results provide a novel structural insight into the hot spot of human tyrosinase for the specific binding of γ-thujaplicin and a way to optimize not only thujaplicins but also other lead compounds as specific inhibitors for human tyrosinase in a rational manner.     

Critical Analysis of the Bennett–Riedel Attack on Secure Cryptographic Key Distributions via the Kirchhoff-Law–Johnson-Noise Scheme

Recently, Bennett and Riedel (BR) (http://arxiv.org/abs/1303.7435v1) argued that thermodynamics is not essential in the Kirchhoff-law–Johnson-noise (KLJN) classical physical cryptographic exchange method in an effort to disprove the security of the KLJN scheme. They attempted to demonstrate this by introducing a dissipation-free deterministic key exchange method with two batteries and two switches. In the present paper, we first show that BR''s scheme is unphysical and that some elements of its assumptions violate basic protocols of secure communication. All our analyses are based on a technically unlimited Eve with infinitely accurate and fast measurements limited only by the laws of physics and statistics. For non-ideal situations and at active (invasive) attacks, the uncertainly principle between measurement duration and statistical errors makes it impossible for Eve to extract the key regardless of the accuracy or speed of her measurements. To show that thermodynamics and noise are essential for the security, we crack the BR system with 100% success via passive attacks, in ten different ways, and demonstrate that the same cracking methods do not function for the KLJN scheme that employs Johnson noise to provide security underpinned by the Second Law of Thermodynamics. We also present a critical analysis of some other claims by BR; for example, we prove that their equations for describing zero security do not apply to the KLJN scheme. Finally we give mathematical security proofs for each BR-attack against the KLJN scheme and conclude that the information theoretic (unconditional) security of the KLJN method has not been successfully challenged.     

Spatial Imprint of Food Consumption: A Historical Analysis for Sweden, 1870–2000

Depending on quantity and composition of food as well as on production conditions and techniques, the space needed to sustain an individual’s nourishment varies. The amount of space needed also depends on the use of resources such as energy, water, and fertilizers, as well as potential land degradation and water pollution. Our study focuses on the changing spatial imprint of an average inhabitant of an expanding Swedish city, Linköping, from 1870 to 2000 taking into account both shifts in consumption as well as agricultural productivity and practices. Despite the distinctly larger amount of animal food products, such as meat and fish, consumed in 2000, we calculate the area needed to sustain an individual’s annual food consumption could be less than one fourth of that needed in 1870. However, if the import of various globally produced foods is included in our calculations, the land needed to sustain the consumption of an inhabitant of Linköping in 2000 doubles. We also argue that an examination of this regional imprint can be used to explore and evaluate possibilities for regional development.     

Structure–cytotoxic activity relationship of sesquilignan,morinol A

The cytotoxic activities of sesquilignans, (7S,8S,7′R,8′R)- and (7R,8R,7′S,8′S)-morinol A and (7S,8S,7′S,8′S)- and (7R,8R,7′R,8′R)-morinol B were compared, showing no significant difference between stereoisomers (IC₅₀ = 24–35 μM). As a next stage, the effect of substituents at 7, 7′, and 7″-aromatic ring on the activity was evaluated to find out the higher activity of (7S,8S,7′R,8′R)-7,7′,7″-phenyl derivative 18 (IC₅₀ = 6–7 μM). In the research on the structure–activity relationship of 7″-position of (7S,8S,7′R,8′R)-7,7′,7″-phenyl derivative 18, the most potent compounds were 7,7′,7″-phenyl derivative 18 (IC₅₀ = 6 μM) against HeLa cells. Against HL-60 cells, 7″-(4-nitrophenyl)-7,7′-phenyl derivative 33 and 7″-hexyl-7,7′-phenyl derivative 37 (IC₅₀ = 5 μM) showed highest activity. We discovered the compounds showed four to sevenfold potent activity than that of natural (7S,8S,7′R,8′R)-morinol A. It was also confirmed that the 7′-benzylic hydroxy group have an important role for exhibiting activity, on the other hand, the resonance system of cinnamyl structure is not crucial for the potent activity.     

Liquid chromatography–mass spectrometry-based metabolomics for authenticity assessment of fruit juices

High performance liquid chromatography?Cmass spectrometry (HPLC?CMS) technique, employing a hybrid triple quadrupole/linear ion trap (QqQ/LIT) mass analyzer, was used for comprehensive metabolomic fingerprinting of several fruit juices types, prepared from expensive (orange) or relatively low-priced (apple, grapefruit) fruits. Following the automated data mining and pre-treatment step, the suitability of the multivariate HPLC?CMS metabolomic data for authentication, i.e., classification of fruit juice and adulteration detection, was assessed with the use of advanced chemometric tools (principal component analysis, PCA, and linear discrimination analysis, LDA). The LDA classification model, constructed and validated employing a highly variable samples set, was able to reliably detect 15% addition of apple or grapefruit juice to orange juice. In the final stage of this study, high performance liquid chromatography?Cquadrupole?Cquadrupole-time-of-flight mass spectrometry (HPLC?CQqTOFMS) measurements were performed in order to obtain data for identification of pre-selected marker compounds using elemental formula calculation and online databases search.     

Structure–activity relationships of non-opioid [des-Arg7]-dynorphin A analogues for bradykinin receptors

In our earlier studies, bradykinin receptors (BRs) were identified as a potential target for the neuroexcitatory effects of dynorphin A (Dyn A) in the central nervous system (CNS), and [des-Arg⁷]-Dyn A-(4–11) (6) was discovered as a lead ligand to modulate Dyn A-(2–13) induced neuroexcitatory effects in the CNS as an antagonist. In an effort to gain insights into key structural features of the Dyn A for the BRs, we pursued further structure–activity relationships (SAR) study on the [des-Arg⁷]-Dyn A analogs and confirmed that all of the [des-Arg⁷]-Dyn A analogues showed good binding affinities at the BRs.     

Identification of reactive toxicants: Structure–activity relationships for amides

A diverse series of amides were evaluated for aquatic toxicity (IGC₅₀) assessed in the Tetrahymena pyriformis population growth impairment assay and for reactivity (EC₅₀) with the model soft nucleophile thiol in the form of the cysteine residue of the tripeptide glutathione. All alkylamides along with some halo-substituted amides are well predicted by the simple hydrophobicity (log K _ow)–electrophilicity (E _lumo) response-surface model [log(IGC⁻¹ ₅₀) = 0.45(log K _ow) − 0.342(E _lumo) − 1.11]. However, 2-halo amides with the halogen at the end of the molecule and α,β-unsaturated primary amides are among those derivatives identified as being more toxic than predicted by the model. Amides, which exhibit excess toxicity, were capable of forming covalent bonds through an S_N2 displacement or a Michael addition. Moreover, only those amides exhibiting excess toxicity were reactive with thiol, suggesting that the reactivity with model nucleophiles such as the thiol group may provide a means of accurately defining reactive toxicants.     

Structure–activity relationships of dibenzoylhydrazines for the inhibition of P-glycoprotein-mediated quinidine transport

We previously demonstrated that dibenzoylhydrazines (DBHs) are not only P-glycoprotein (P-gp) substrates, but also inhibitors. In the present study, we evaluated the inhibition of P-gp-mediated quinidine transport by two series of DBHs and performed a classical QSAR analysis and docking simulation in order to investigate the mechanisms underlying P-gp substrate/inhibitor recognition. The results of the QSAR analysis identified the hydrophobic factor as the most important for inhibitory activities, while electronic and steric effects also influenced the activities. The different substituent effects observed in each series suggested the different binding modes of each series of DBHs, which was supported by the results of the docking simulation.     

Determining a Cut-Off Point for Scores of the Breastfeeding Self-Efficacy Scale–Short Form: Secondary Data Analysis of an Intervention Study in Japan

Background

Breastfeeding self-efficacy can be measured with the Breastfeeding Self-Efficacy Scale-Short Form (BSES-SF). Mothers with low BSES-SF scores stop exclusive breastfeeding prematurely, but specific interventions can prevent that undesirable outcome. Because those interventions can be expensive, often one must decide which mothers will receive them. For that purpose, a cut-off BSES-SF score would be useful, but none is available. Therefore, we aimed to assess the overall accuracy of BSES-SF scores as predictors of not practicing post-discharge exclusive breastfeeding, and to choose an appropriate cut-off score for making that prediction.

Methods

This is a secondary data analysis of an intervention study. Data from 378 women in two non-Baby-Friendly Hospitals were analyzed. Participants were women in their third trimester who were 16 years of age or older, were able to read and write Japanese, were expected to have a singleton birth, and had completed the BSES-SF before discharge. BSES-SF scores were measured before discharge. Breastfeeding status was assessed 4 weeks and 12 weeks postpartum. Receiver Operating Characteristic (ROC) curves were used to assess the predictive ability of the BSES-SF and to inform the choice of a cut-off point.

Results

For both of the ROC curves (4 and 12 weeks postpartum) the area under the curve was 0.74. To obtain a high sensitivity, a cut-off score of 50 was chosen. With that cut-off score the sensitivity was 79% and the specificity was 52% 4 weeks postpartum, and they were 77% and 52%, respectively, 12 weeks postpartum.

Conclusion

In conclusion, the BSES-SF has moderate overall accuracy to distinguish women who will not practice exclusive breastfeeding after discharge from those who will. At non-Baby-Friendly hospitals in Japan, interventions to support exclusive breastfeeding might be considered for new mothers who have BSES-SF scores that are less than or equal to 50.     

Structure–Function Analysis of the DNA Translocating Portal of the Bacteriophage T4 Packaging Machine

Tailed bacteriophages and herpesviruses consist of a structurally well conserved dodecameric portal at a special 5-fold vertex of the capsid. The portal plays critical roles in head assembly, genome packaging, neck/tail attachment, and genome ejection. Although the structures of portals from phages φ29, SPP1, and P22 have been determined, their mechanistic roles have not been well understood. Structural analysis of phage T4 portal (gp20) has been hampered because of its unusual interaction with the Escherichia coli inner membrane. Here, we predict atomic models for the T4 portal monomer and dodecamer, and we fit the dodecamer into the cryo-electron microscopy density of the phage portal vertex. The core structure, like that from other phages, is cone shaped with the wider end containing the “wing” and “crown” domains inside the phage head. A long “stem” encloses a central channel, and a narrow “stalk” protrudes outside the capsid. A biochemical approach was developed to analyze portal function by incorporating plasmid-expressed portal protein into phage heads and determining the effect of mutations on head assembly, DNA translocation, and virion production. We found that the protruding loops of the stalk domain are involved in assembling the DNA packaging motor. A loop that connects the stalk to the channel might be required for communication between the motor and the portal. The “tunnel” loops that project into the channel are essential for sealing the packaged head. These studies established that the portal is required throughout the DNA packaging process, with different domains participating at different stages of genome packaging.