pH-dependent structural change of reduced spinach plastocyanin studied by perturbed angular correlation of γ-rays and dynamic light scattering

In this study the pH-dependent structural changes of reduced spinach plastocyanin were investigated using perturbed angular correlation (PAC) of γ-rays and dynamic light scattering (DLS). PAC data of Ag-substituted plastocyanin indicated that the coordinating ligands are two histidine residues (His37, His87) and a cysteine residue (Cys84) in a planar configuration, whereas the methionine (Met92) found perpendicular to this plane is not a coordinating ligand at neutral pH. Two slightly different conformations with differences in the Cys–metal ion–His angles could be observed with PAC spectroscopy. At pH 5.3 a third coordination geometry appears which can be explained as the absence of the His87 residue and the coordination of Met92 as a ligand. With DLS the aggregation of reduced plastocyanin could be observed below pH 5.3, indicating that not only the metal binding site but also the aggregation properties of the protein change upon pH reduction. Both the structural changes at the metal binding site and the aggregation are shown to be reversible. These results support the hypothesis that the pH of the thylakoid lumen has to remain moderate during steady-state photosynthesis and indicate that low pH induced aggregation of plastocyanin might serve as a regulatory switch for photosynthesis.     

Hydrodynamic properties of nitrogenase — the MoFe protein from Azotobacter vinelandii studied by dynamic light scattering and hydrodynamic modelling

Experimental results for the nitrogenase MoFe protein from Azotobacter vinelandii obtained by dynamic light scattering (DLS) are presented. The translational diffusion coefficient was determined to D=(4.0±0.2)×10⁻⁷ cm²/s. Complementary, we have performed hydrodynamic model calculations based on the X-ray crystallographic data of the MoFe protein. The calculated transport coefficient suggests that the size and shape of the protein in solution is consistent with that in the crystal structure.     

Detection of Pb²⁺ at attomole levels by using dynamic light scattering and unmodified gold nanoparticles

Lead ion (Pb²⁺) accumulation in nature can affect the environment and human health severely. Thus, rapid and sensitive detection is of great importance. One-step detection of Pb²⁺ at attomole levels was realized by using dynamic light scattering (DLS) technique coupled with unmodified gold nanoparticles (AuNPs). Pb²⁺-dependent DNAzyme was double-stranded and could not adsorb on the surface of AuNPs, while the substrate strand could be cleaved into ssDNA fragments on addition of Pb²⁺. The ssDNA fragments could adsorb on the surface of AuNPs and prevent them from aggregating in the presence of NaCl. Therefore, the disperse state of AuNPs changed on addition of Pb²⁺ in the presence of DNAzyme and NaCl, which was estimated with an average hydrodynamic diameter by using DLS. Under optimum conditions, the average diameter of the solution decreased linearly with the concentration of Pb²⁺ over the range from 10 to 300 pM, with a detection limit of 6.2 pM. Moreover, satisfactory results were obtained when the proposed method was applied in the detection of Pb²⁺ in water samples.     

Structural basis of protein–protein interaction studied by NMR

This paper describes efforts of the structural genomics project in the nuclear magnetic resonance (NMR) laboratory at the University of Science and Technology of China. This structural genomics project is biological-functional driven. Targets are mainly selected from two systems: proteins related with regulation of gene expression in humans and other eukaryotes, and proteins existing in the cell junction in humans. The majority of proteins selected from these two systems are related with human health and diseases, and some are potential drug targets. Twenty-five protein structures from Homo sapiens and other eukaryotes have been determined during last 5 years in this laboratory. Nuclear magnetic resonance (NMR) spectroscopy is highly suited to investigate molecular interactions at a close physiological condition and is particularly suited for the study of low-affinity, transient complexes. It can provide information on protein surface interaction, their complex structure, and their dynamic properties during protein recognition. Several examples are given in this paper.     

Study of kinetics of thermal aggregation of mitochondrial aspartate aminotransferase by dynamic light scattering: protective effect of α-crystallin

Thermal aggregation of aspartate aminotransferase from pig heart mitochondria (mAAT) has been studied at various temperatures and various protein concentrations by dynamic light scattering. The character of the dependence of protein aggregate size on time indicates that aggregation of mAAT proceeds in the regime of diffusion-limited cluster–cluster aggregation. Suppression of mAAT aggregation by α-crystallin is due to transition of the aggregation process into the regime of reaction-limited cluster–cluster aggregation. Realization of this regime of aggregation means that the sticking probability for the colliding particles is less than unity.     

Affinity enhancement by multivalent lectin–carbohydrate interaction

The binding of simple carbohydrate ligands by proteins often requires affinity enhancement to attain biologically relevant strength. This is especially true for endocytotic receptors and the molecules that engage in the first-line of defense. For such purposes, nature often utilizes a mode of affinity enhancement that arises from multiple interactions between the binding proteins and the carbohydrate ligands, which we term glycoside cluster effect. In this review article we give a number of examples and describe important factors in the multi-valent interactions that govern the degree of affinity enhancement.     

Host–parasitoid interaction as affected by interkingdom competition

Although still underrepresented in ecological research, competitive interactions between distantly related organisms (so-called “interkingdom competition”) are expected to be widespread in various ecosystems, with yet unknown consequences for, e.g. trophic interactions. In the model host–parasitoid system Drosophila melanogaster–Asobara tabida, toxic filamentous fungi have been shown to be serious competitors that critically affect the density-dependent survival of host Drosophila larvae. This study investigates the extent to which the competing mould Aspergillus niger affects key properties of the well-studied Drosophila–parasitoid system and how the host–parasitoid interaction influences the microbial competitor. In contrast to slightly positive density-dependent host mortality under mould-free conditions, competing A. niger mediated a strong Allee effect for parasitised larvae, i.e. mortality decreased with increasing larval density. It was found that the common toxic fungal metabolite kojic acid is not responsible for higher death rates in parasitised larvae. Single parasitised Drosophila larvae were less harmful to fungal reproduction than unparasitised larvae, but this effect vanished with an increase in larval density. As predicted from the negative effect of fungi on host survival and thus on parasitoid fitness at low larval densities, A. tabida females spent less time foraging in fungus-infested patches. Interestingly, even though high host larval densities increased host survival, parasitoids still reduced their search efforts in fungus-infested patches, indicating a benefit for host larvae from feeding in the presence of noxious mould. Thus, this experimental study provides evidence of the potentially important role of interkingdom competition in determining trophic interactions in saprophagous animal communities and the dynamics of both host–parasitoid and microbial populations.     

Control of glioma cell death and differentiation by PKM2–Oct4 interaction

Glioma stem cells are highly resistant to cell death and as such are supposed to contribute to tumor recurrence by eluding anticancer treatments. Here, we show that spheroids that contain rat neural stem cells (NSCs) or rat glioma stem cells (cancer stem cells, CSCs) express isoforms 1 and 2 of pyruvate kinase (PKM1 and PKM2); however, the expression of PKM2 is considerably higher in glioma spheroids. Silencing of PKM2 enhances both apoptosis and differentiation of rat and human glioma spheroids. We establish that PKM2 was implicated in glioma spheroid differentiation through its interaction with Oct4, a major regulator of self-renewal and differentiation in stem cells. The small molecule Dichloroacetate (DCA), a pyruvate dehydrogenase kinase inhibitor, increases the amount of PKM2/Oct4 complexes and thus inhibited Oct4-dependent gene expression. Taken together, our results highlight a new molecular pathway through which PKM2 can manage gliomagenesis via the control of glioma stemness by Oct4.     

Analysis and prediction of drug–drug interaction by minimum redundancy maximum relevance and incremental feature selection

Drug–drug interaction (DDI) defines a situation in which one drug affects the activity of another when both are administered together. DDI is a common cause of adverse drug reactions and sometimes also leads to improved therapeutic effects. Therefore, it is of great interest to discover novel DDIs according to their molecular properties and mechanisms in a robust and rigorous way. This paper attempts to predict effective DDIs using the following properties: (1) chemical interaction between drugs; (2) protein interactions between the targets of drugs; and (3) target enrichment of KEGG pathways. The data consisted of 7323 pairs of DDIs collected from the DrugBank and 36,615 pairs of drugs constructed by randomly combining two drugs. Each drug pair was represented by 465 features derived from the aforementioned three categories of properties. The random forest algorithm was adopted to train the prediction model. Some feature selection techniques, including minimum redundancy maximum relevance and incremental feature selection, were used to extract key features as the optimal input for the prediction model. The extracted key features may help to gain insights into the mechanisms of DDIs and provide some guidelines for the relevant clinical medication developments, and the prediction model can give new clues for identification of novel DDIs.     

Obsolescence in LCA–methodological challenges and solution approaches

Purpose

Obsolescence, as premature end of use, increases the overall number of products produced and consumed, and thereby can increase the environmental impact. Measures to decrease the effects of obsolescence by altering the product or service design have the potential to increase use time (defined as the realized active service life) of devices, but can themselves have (environmental) drawbacks, for example, because the amount of material required for production increases. As such, paying special attention to methodological choices when assessing such measures and strategies using life cycle assessment (LCA) needs is crucial.

Methods

Open questions and key aspects of obsolescence, including the analysis of its effects and preventative measures, are discussed against the backdrop of the principles and framework for LCA given in ISO 14040/44, which includes guidance on how to define a useful functional unit and reference flow in the context of real-life use time.

Results and discussion

The open and foundational requirements of ISO 14040/14044 already form an excellent basis for analysis of the phenomenon obsolescence and its environmental impact in product comparisons. However, any analysis presumes clear definition of the goal and scope phase with special attention paid to aspects relevant to obsolescence: the target product and user group needs to be placed into context with the analysed “anti-obsolescence” measures. The reference flow needs to reflect a realized use time (and not solely a technical lifetime when not relevant for the product under study). System boundaries and types of data need to be chosen also in context of the anti-obsolescence measure to include, for example, the production of spare parts to reflect repairable design and/or manufacturer-specific yields to reflect high-quality manufacturing.

Conclusions

Understanding the relevant obsolescence conditions for the product system under study and how these may differ across the market segment or user types is crucial for a fair and useful comparison and the evaluation of anti-obsolescence measures.

     

Fractionation of functional polystyrenes,poly(ethylene oxide)s and poly(styrene)–b–poly(ethylene oxide) by liquid chromatography at the exclusion–adsorption transition point

The paper reports the fractionation of functional polystyrenes (PSs) and poly(ethylene oxide)s (PEOs) as well as their block copolymers, by liquid chromatography at the exclusion adsorption transition point (EATP–LC), also called “critical conditions” mode. In this specific elution mode (EATP–LC), the fractionation is only governed by the nature and the number of functions attached to the polymer backbone, independent of the molar mass distribution of the whole sample. Functional polystyrenes (α- and/or α,ω-alcohol-, acetal-, aldehyde- and acidic-PS) could be readily separated from non-functional polystyrenes under various chromatographic conditions. The technique also allowed the fractionation of poly(ethylene oxide)s and PS–PEO block copolymers. In the latter cases, moderately polar columns (grafted silica) and water-based polar eluents were required to obtain a satisfactory fractionation.     

Assessing the dispersive and electrostatic components of the selenium–aromatic interaction energy by DFT

Selenium has been increasingly recognized as an important element in biological systems, which participates in numerous biochemical processes in organisms, notably in enzyme reactions. Selenium can substitute sulfur of cysteine and methionine to form their selenium analogues, selenocysteine (Sec) and selenomethionine (SeM). The nature of amino acid pockets in proteins is dependent on their composition and thus different non-covalent forces determine the interactions between selenium of Sec or SeM and other functional groups, resulting in specific biophysical behavior. The discrimination of selenium toward sulfur has been reported. In order to elucidate the difference between the nature of S-π and Se-π interactions, we performed extensive DFT calculations of dispersive and electrostatic contributions of Se-π interactions in substituted benzenes/hydrogen selenide (H₂Se) complexes. The results are compared with our earlier reported S-π calculations, as well as with available experimental data. Our results show a larger contribution of dispersive interactions in Se-π systems than in S-π ones, which mainly originate from the attraction between Se and substituent groups. We found that selenium exhibits a strong interaction with aromatic systems and may thus play a significant role in stabilizing protein folds and protein–inhibitor complexes. Our findings can also provide molecular insights for understanding enzymatic specificity discrimination between single selenium versus a sulfur atom, notwithstanding their very similar chemical properties.     

Anionic polymer,poly(methyl vinyl ether–maleic anhydride)-coated beads-based capture of human influenza A and B virus

An anionic magnetic beads-based method was developed for the capture of human influenza A and B viruses from nasal aspirates, allantoic fluid and culture medium. A polymer, poly(methyl vinyl ether–maleic anhydride) [poly(MVE-MA)], was used to endow magnetic beads with a negative charge and bioadhesive properties. After incubation with samples containing human influenza virus, the beads were separated from supernatants by applying a magnetic field. The absorption of the virus by the beads was confirmed by hemagglutinin assay, immunochromatography, Western blotting, egg infection, and cell infection. Successful capture was proved using 5 H1N1 influenza A viruses, 10 H3N2 influenza A viruses, and 6 influenza B viruses. Furthermore, the infectivity in chicken embryonated eggs and Madin–Darby canine kidney (MDCK) cells of the captured human influenza virus was similar to that of the total viral quantity of starting materials. Therefore, this method of capture using magnetic beads coated with poly(MVE-MA) can be broadly used for the recovery of infectious human influenza viruses.     

The interaction of amyloid Aβ(1-40) with lipid bilayers and ganglioside as studied by P solid-state NMR

Amyloid β-peptide (Aβ) is a major component of plaques in Alzheimer's disease, and formation of senile plaques has been suggested to originate from regions of neuronal membrane rich in gangliosides. We analyzed the mode of interaction of Aβ with lipid bilayers by multinuclear NMR using ³¹P nuclei. We found that Aβ (1-40) strongly perturbed the bilayer structure of dimyristoylphosphatidylcholine (DMPC), to form a non-lamellar phase (most likely micellar). The ganglioside GM1 potentiated the effect of Aβ (1-40), as viewed from ³¹P NMR. The difference of the isotropic peak intensity between DMPC/Aβ and DMPC/GM1/Aβ suggests a specific interaction between Aβ and GM1. We show that in the DMPC/GM1/Aβ system there are three lipid phases, namely a lamellar phase, a hexagonal phase and non-oriented lipids. The latter two phases are induced by the presence of the Aβ peptide, and facilitated by GM1.     

Single-molecule study of the CUG repeat–MBNL1 interaction and its inhibition by small molecules

Effective drug discovery and optimization can be accelerated by techniques capable of deconvoluting the complexities often present in targeted biological systems. We report a single-molecule approach to study the binding of an alternative splicing regulator, muscleblind-like 1 protein (MBNL1), to (CUG)_{n = 4,6} and the effect of small molecules on this interaction. Expanded CUG repeats (CUG^exp) are the causative agent of myotonic dystrophy type 1 by sequestering MBNL1. MBNL1 is able to bind to the (CUG)_n–inhibitor complex, indicating that the inhibition is not a straightforward competitive process. A simple ligand, highly selective for CUG^exp, was used to design a new dimeric ligand that binds to (CUG)_n almost 50-fold more tightly and is more effective in destabilizing MBNL1–(CUG)₄. The single-molecule method and the analysis framework might be extended to the study of other biomolecular interactions.