Physical Chemistry of Cellular Liquid-Phase Separation

Compartmentalization of biochemical processes is essential for cell function. Although membrane-bound organelles are well studied in this context, recent work has shown that phase separation is a key contributor to cellular compartmentalization through the formation of liquid-like membraneless organelles (MLOs). In this Minireview, the key mechanistic concepts that underlie MLO dynamics and function are first briefly discussed, including the relevant noncovalent interaction chemistry and polymer physical chemistry. Next, a few examples of MLOs and relevant proteins are given, along with their functions, which highlight the relevance of the above concepts. The developing area of active matter and non-equilibrium systems, which can give rise to unexpected effects in fluctuating cellular conditions, are also discussed. Finally, our thoughts for emerging and future directions in the field are discussed, including in vitro and in vivo studies of MLO physical chemistry and function.     

Engineered Artificial Membraneless Organelles in Saccharomyces cerevisiae To Enhance Chemical Production

Microbial cell factories provide a green and sustainable opportunity to produce value-added products from renewable feedstock. However, the leakage of toxic or volatile intermediates decreases the efficiency of microbial cell factories. In this study, membraneless organelles (MLOs) were reconstructed in Saccharomyces cerevisiae by the disordered protein sequence A-IDPs. A regulation system was designed to spatiotemporally regulate the size and rigidity of MLOs. Manipulating the MLO size of strain ZP03-FM, the amounts of assimilated methanol and malate were increased by 162 % and 61 %, respectively. Furthermore, manipulating the MLO rigidity in strain ZP04-RB made acetyl-coA synthesis from oxidative glycolysis change to non-oxidative glycolysis; consequently, CO₂ release decreased by 35 % and the n-butanol yield increased by 20 %. This artificial MLO provides a strategy for the co-localization of enzymes to channel C₁ starting materials into value-added chemicals.     

Metabolic study of grapevine leaves infected by downy mildew using negative ion electrospray – Fourier transform ion cyclotron resonance mass spectrometry

Grapevine is of worldwide economic importance due to wine production. However, this culture is often affected by pathogens causing severe harvest losses. Understanding host–pathogen relationships may be a key to solve this problem. In this paper, we evaluate the direct flow injection by electrospray – Fourier transform ion cyclotron resonance mass spectrometry (MS) of leaf extracts as a rapid method for the study of grapevine response to downy mildew (Plasmopara viticola) attack. The comparison of MS profiles obtained from control and infected leaves of different levels of resistant grapevines highlights several classes of metabolites (mainly saccharides, acyl lipids, hydroxycinnamic acids derivatives and flavonoids) which are identified using high resolution MS and tandem MS (MS/MS). Statistical analyses of 19 markers show a clear segregation between inoculated and healthy samples. This study points out relative high levels of disaccharides, acyl lipids and glycerophosphoinositol in inoculated samples. Sulfoquinovosyl diacylglycerols also emerge as possible metabolites involved in plant defense.     

Potential Mitigation of Smoke Taint in Wines by Post-Harvest Ozone Treatment of Grapes

When bushfires occur near grape growing regions, vineyards can be exposed to smoke, and depending on the timing and duration of grapevine smoke exposure, fruit can become tainted. Smoke-derived volatile compounds, including volatile phenols, can impart unpleasant smoky, ashy characters to wines made from smoke-affected grapes, leading to substantial revenue losses where wines are perceivably tainted. This study investigated the potential for post-harvest ozone treatment of smoke-affected grapes to mitigate the intensity of smoke taint in wine. Merlot grapevines were exposed to smoke at ~7 days post-veraison and at harvest grapes were treated with 1 or 3 ppm of gaseous ozone (for 24 or 12 h, respectively), prior to winemaking. The concentrations of smoke taint marker compounds (i.e., free and glycosylated volatile phenols) were measured in grapes and wines to determine to what extent ozonation could mitigate the effects of grapevine exposure to smoke. The 24 h 1 ppm ozone treatment not only gave significantly lower volatile phenol and volatile phenol glycoside concentrations but also diminished the sensory perception of smoke taint in wine. Post-harvest smoke and ozone treatment of grapes suggests that ozone works more effectively when smoke-derived volatile phenols are in their free (aglycone) form, rather than glycosylated forms. Nevertheless, the collective results demonstrate the efficacy of post-harvest ozone treatment as a strategy for mitigation of smoke taint in wine.     

Mass spectrometry in the study of anthocyanins and their derivatives: differentiation of Vitis vinifera and hybrid grapes by liquid chromatography/electrospray ionization mass spectrometry and tandem mass spectrometry

A mass spectrometric-based procedure for anthocyanin profiling was set up to distinguish authentic Vitis vinifera from hybrid red grapevine cultivars. 3-O-Monoglucoside and the related acetyl-, p-coumaryl- and caffeoyl-monoglucoside anthocyanins occurred only in Vitis vinifera, whereas 3,5-O-diglucoside and the substituted acetyl-, p-coumaryl-, feruloyl- and caffeoyl-diglucoside anthocyanins were the additional pigments in hybrid grapevines. The procedure was applied expressly to identify red grape cultivars based on the anthocyanin chemo-type determination. In particular, a red grape cultivar, having 3,5-O-diglucoside anthocyanins and a novel class of anthocyanin monoglucosides, such as cyanidin-3-O-, cyanidin-3-O-(6-O-acetyl)- and cyanidin-3-O-(6-O-p-coumaryl)pentoside, was classified as hybrid. A second vine cultivar, characterized exclusively by 3-O-monoglucoside anthocyanins, was included among the Vitis vinifera species. Anthocyanin profiling by mass spectrometry could represent the core of a chemotaxonomic procedure for distinguishing American and European grapevines based on the identification of post-synthetic anthocyanidin modification.     

Metabolisation of eutypine by plant tissues: an HPLC determination

Eutypine, 4-hydroxy-3-(3-methyl-3-butene-1-ynyl) benzaldehyde, is a toxin produced by Eutypa lata, the causal agent of eutypa dieback of grapevine. The tolerance of some grapevine cultivars to the disease has been ascribed to the potential reduction of eutypine into its corresponding non-toxic alcohol, eutypinol. In the present study, eutypine biotransformation in different tissues of grapevine was investigated by HPLC and LC-MS. Grape callus tissues were able to biotransform eutypine into eutypinol within the first 3 h of culture. The grape plantlets cultured in vitro can also transform eutypine into eutypinol. Grape plantlet leaves do not have any effect on the uptake of eutypine, which goes through the tissues following a concentration gradient. Results revealed that the toxicity of eutypine in grape tissues is an active process showing that eutypinol is rapidly metabolised into other compounds. The use of micro-cuttings and in vitro plants showed that eutypine strongly accumulates in the bottom part of the diseased plant stems.     

Coacervates as models of membraneless organelles

Coacervates are condensed liquid-like droplets, usually formed with oppositely charged polymeric molecules. They have been studied extensively in colloid and interface science for their remarkable material properties. The liquid–liquid phase separation underlying coacervate formation also plays an important role in the formation of various membraneless organelles (MLOs) that are found in many living cells. Therefore, there is an increasing interest to use well-characterized coacervates as in vitro models that mimic specific aspects of MLOs. Here, we review five aspects – physical and chemical properties, hierarchical organization, uptake selectivity, formation dynamics, and maturation – that are of particular interest and discuss how useful coacervates are to better understand these aspects of MLOs.     

The main phytotoxic metabolite produced by a strain of Fusarium oxysporum inducing grapevine plant declining in Italy

A strain of Fusarium oxysporum was isolated from grapevine showing heavy decline disease in a vineyard of Veneto region in Italy. The fungus showed to produce phytotoxic metabolites when grown in liquid culture. The main metabolite was identified as fusaric acid produced for the first time as a phytotoxin by a strain of F. oxysporom isolated from diseased grapevine plants. Its quantification in the fungus cultures filtrates was accomplished by HPLC. When tested on tobacco by leaf-puncture assay fusaric acid at 0.5 mg/mL induced the formation of extensive necrosis.     

Emission of volatile sesquiterpenes and monoterpenes in grapevine genotypes following Plasmopara viticola inoculation in vitro

The grapevine (Vitis vinifera) is one of the most widely cultivated fruit crops globally, and one of its most important diseases in terms of economic losses is downy mildew, caused by Plasmopara viticola. Several wild Vitis species have been found to be resistant to this pathogen and have been used in breeding programs to introduce resistance traits to susceptible cultivars. Plant defense is based on different mechanisms, and volatile organic compounds (VOCs) play a major role in the response to insects and pathogens. Although grapevine resistance mechanisms and the production of secondary metabolites have been widely characterized in resistant genotypes, the emission of VOCs has not yet been investigated following P. viticola inoculation. A Proton Transfer Reaction‐Time of Flight‐Mass Spectrometer (PTR‐ToF‐MS) was used to analyze the VOCs emitted by in vitro‐grown plants of grapevine genotypes with different levels of resistance. Downy mildew inoculation significantly increased the emission of monoterpenes and sesquiterpenes by the resistant SO4 and Kober 5BB genotypes, but not by the susceptible V. vinifera Pinot noir. Volatile terpenes were implicated in plant defense responses against pathogens, suggesting that they could play a major role in the resistance against downy mildew by direct toxicity or by inducing grapevine resistance. The grapevine genotypes differed in terms of the VOC emission pattern of both inoculated and uninoculated plants, indicating that PTR‐ToF‐MS could be used to screen hybrids with different levels of downy mildew resistance. Copyright © 2015 John Wiley & Sons, Ltd.     

Protolytic properties of silica modified with maleinized linseed oil

The protolytic equilibria on the surface of silicas coated with a layer of maleinized linseed oil (MLO), a new type of carboxylic cation exchangers, were studied using potentiometric titration. The ionexchange capacity of the sorbents was determined, a parameter that first increases and then decreases with increasing concentration of MLO on the silica surface. For the dibasic carboxy groups of MLO, pK _a=3.99±0.04 and 4.73±0.04, values close to pK _a for succinic acid, a homogeneous analogue of the system under study.     

Synthetic Membraneless Droplets for Synaptic-Like Clustering of Lipid Vesicles

In eukaryotic cells, the membraneless organelles (MLOs) formed via liquid-liquid phase separation (LLPS) are found to interact intimately with membranous organelles (MOs). One major mode is the clustering of MOs by MLOs, such as the formation of clusters of synaptic vesicles at nerve terminals mediated by the synapsin-rich MLOs. Aqueous droplets, including complex coacervates and aqueous two-phase systems, have been plausible MLO-mimics to emulate or elucidate biological processes. However, neither of them can cluster lipid vesicles (LVs) like MLOs. In this work, we develop a synthetic droplet assembled from a combination of two different interactions underlying the formation of these two droplets, namely, associative and segregative interactions, which we call segregative-associative (SA) droplets. The SA droplets cluster and disperse LVs recapitulating the key functional features of synapsin condensates, which can be attributed to the weak electrostatic interaction environment provided by SA droplets. This work suggests LLPS with combined segregative and associative interactions as a possible route for synaptic clustering of lipid vesicles and highlights SA droplets as plausible MLO-mimics and models for studying and mimicking related cellular dynamics.     

Modeling Alleviative Effects of Ca,Mg, and K on Cu-Induced Oxidative Stress in Grapevine Roots Grown Hydroponically

The aim of this study was to determine the pattern of alleviation effects of calcium (Ca), magnesium (Mg), and potassium (K) on copper (Cu)-induced oxidative toxicity in grapevine roots. Root growth, Cu and cation accumulation, reactive oxygen species (ROS) production, and antioxidant activities were examined in grapevine roots grown in nutrient solutions. The experimental setting was divided into three sets; each set contained a check (Hoagland solution only) and four treatments of simultaneous exposure to 15 μM Cu with four cation levels (i.e., Ca set: 0.5, 2.5, 5, and 10 mM Ca; Mg set: 0.2, 2, 4, and 8 mM Mg; K set: 0.6, 2.4, 4.8, and 9.6 mM K). A damage assessment model (DAM)-based approach was then developed to construct the dose-effect relationship between cation levels and the alleviation effects on Cu-induced oxidative stress. Model parameterization was performed by fitting the model to the experimental data using a nonlinear regression estimation. All data were analyzed by a one-way analysis of variance (ANOVA), followed by multiple comparisons using the least significant difference (LSD) test. The results showed that significant inhibitory effects on the elongation of roots occurred in grapevine roots treated with 15 μM Cu. The addition of Ca and Mg significantly mitigated phytotoxicity in root growth, whereas no significant effect of K treatment on root growth was found. With respect to oxidative stress, ROS and malondialdehyde (MDA) contents, as well as antioxidant (superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX)) activities, were stimulated in the roots after exposure to 15 μM Cu for three days. Moreover, H₂O₂ levels decreased significantly as Ca, Mg, and K concentrations increased, indicating that the coexistence of these cations effectively alleviated Cu-induced oxidative stress; however, alleviative effects were not observed in the assessment of the MDA content and antioxidant enzyme activities. Based on the DAM, an exponential decay equation was developed and successfully applied to characterize the alleviative effects of Ca, Mg, and K on the H₂O₂ content induced by Cu in the roots. In addition, compared with Mg and K, Ca was the most effective cation in the alleviation of Cu-induced ROS. Based on the results, it could be concluded that Cu inhibited root growth and Ca and Mg absorption in grapevines, and stimulated the production of ROS, lipid peroxidation, and antioxidant enzymes. Furthermore, the alleviation effects of cations on Cu-induced ROS were well described by the DAM-based approach developed in the present study.     

Improvement of Impact Strength of Polylactide Blends with a Thermoplastic Elastomer Compatibilized with Biobased Maleinized Linseed Oil for Applications in Rigid Packaging

This research work reports the potential of maleinized linseed oil (MLO) as biobased compatibilizer in polylactide (PLA) and a thermoplastic elastomer, namely, polystyrene-b-(ethylene-ran-butylene)-b-styrene (SEBS) blends (PLA/SEBS), with improved impact strength for the packaging industry. The effects of MLO are compared with a conventional polystyrene-b-poly(ethylene-ran-butylene)-b-polystyrene-graft-maleic anhydride terpolymer (SEBS-g-MA) since it is widely used in these blends. Uncompatibilized and compatibilized PLA/SEBS blends can be manufactured by extrusion and then shaped into standard samples for further characterization by mechanical, thermal, morphological, dynamical-mechanical, wetting and colour standard tests. The obtained results indicate that the uncompatibilized PLA/SEBS blend containing 20 wt.% SEBS gives improved toughness (4.8 kJ/m²) compared to neat PLA (1.3 kJ/m²). Nevertheless, the same blend compatibilized with MLO leads to an increase in impact strength up to 6.1 kJ/m², thus giving evidence of the potential of MLO to compete with other petroleum-derived compatibilizers to obtain tough PLA formulations. MLO also provides increased ductile properties, since neat PLA is a brittle polymer with an elongation at break of 7.4%, while its blend with 20 wt.% SEBS and MLO as compatibilizer offers an elongation at break of 50.2%, much higher than that provided by typical SEBS-g-MA compatibilizer (10.1%). MLO provides a slight decrease (about 3 °C lower) in the glass transition temperature (T_g) of the PLA-rich phase, thus showing some plasticization effects. Although MLO addition leads to some yellowing due to its intrinsic yellow colour, this can contribute to serving as a UV light barrier with interesting applications in the packaging industry. Therefore, MLO represents a cost-effective and sustainable solution to the use of conventional petroleum-derived compatibilizers.     

Complexes of Group 12 Metal Dihalides with 2‐Acetylpyridine‐N‐oxide 4N‐methylthiosemicarbazone (H4MLO). The Crystal Structures and Organization of [Zn(H4MLO)X2] (X = Br,I)

Reaction of group 12 metal dihalides with 2‐acetylpyridine‐N‐oxide ⁴N‐methylthiosemicarbazone (H4MLO) in ethanol afforded compounds [M(H4MLO)X₂] (M = Zn^II, Cd^II, Hg^II; X = Cl, Br, I), the structures of which were characterized by elemental analysis and by IR and ¹H and ¹³C NMR spectroscopy. In addition, the complexes of ZnBr₂ and ZnI₂ were analysed structurally by X‐ray diffractometry. In [Zn(H4MLO)Br₂] the ligand is O,N,S‐tridentate and the metal is pentacoordinated, while in [Zn(H4MLO)I₂] the thiosemicarbazone is S,O‐bis‐monodentate and the Zn^II cation has a distorted tetrahedral coordination polyhedron. In assays of antifungal activity against Aspergillus niger and Paecilomyces variotii, only the mercury compounds showed any activity, and only [Hg(H4MLO)Cl₂] and [Hg(H4MLO)I₂] were competitive with nystatin against A. niger.     

Micropropagation and production of Camptothecin from in vitro plants of Ophiorrhiza rugosa var. decumbens

Camptothecin and its derivatives are used as anticancer alkaloids. At present, they are obtained by the extraction from the Chinese tree Camptotheca acuminata and the indigenous tree Nothapodytes foetida. Identification of alternate species of plants like Ophiorrhiza species and development of tissue culture methods may be a suitable alternative for large-scale micropropagation as well as for the production of camptothecin. Plantlets were successfully regenerated from shoot cultures of Ophiorrhiza rugosa initiated from axillary meristems on medium containing benzyladenine (BA) (4.0 mg L(-1)) + alpha-naphthalene acetic acid (NAA) (0.05 mg L(-1)). Rooting was initiated in half concentration of Murashige and Skoog's (MS) basal medium devoid of growth hormones. Tissue culture derived plants of O. rugosa were similar to the normal plants in their morphological characteristics and chemical constitution. Ten O. rugosa plants that were obtained through micropropagation showed when analysed a higher alkaloid content compared to the normal plant. Chemical analysis of the different organs of the tissue culture plant of O. rugosa established in soil indicated 0.002% dry weight of camptothecin in the roots, 0.011% dry weight in the stems, 0.090% dry weight in the leaves and 0.015% in the floral parts.     

Metabolites from Eutypa species that are pathogens on grapes

Structural and synthetic studies of the metabolites isolated from Eutypa lata are reviewed. This fungus is the causative agent of Eutypa dieback disease, also known as eutyposis or dying-arm disease, a perennial canker that affects grapevines and many other woody fruit plants. The review, which encompasses all the literature in this field up to the present and in which 76 references are cited, also includes a detailed study of the biological activity of the metabolites, especially the role of toxins in the development of the plant disease. Some aspects of the synthesis and biosynthesis of these metabolites and related compounds are discussed.     

Phytotoxic metabolites by nine species of Botryosphaeriaceae involved in grapevine dieback in Australia and identification of those produced by Diplodia mutila,Diplodia seriata,Neofusicoccum australe and Neofusicoccum luteum

Botryosphaeria dieback is one of the main trunk diseases of grapevine caused by several species of Botryosphaeriaceae. Twenty-four fungal isolates representing the eight most widespread and most virulent Botryosphaeriaceae were tested for their ability to produce phytotoxic metabolites. The chromatographic profiles of their culture filtrates organic extracts showed the ability of all isolates to produce several and different metabolites. When tested on grapevine leaves and tomato cuttings the organic extracts phytotoxicity varied among isolates and species. To our knowledge, this is the first study on phytotoxic compounds produced by Botryosphaeriaceae species found in Australian vineyards. The phytotoxic metabolites produced by Diplodia seriata, Diplodia mutila, Neofusicoccum australe and, for the first time, by Neofusicoccum luteum were isolated and chemically identified essentially by spectroscopic methods.

     

Microbial liquefaction of lignite pretreated with dilute acid at elevated temperature and pressure

Two microbial cultures—ML-13 (aCandida sp.) and LSC (a fungal isolate from the University of Arkansas)—have been employed in the direct liquefaction of Louisiana lignite. Lignite samples were pretreated with nitric acid and microbial culture broths at elevated temperatures and pressures. Subsequent treatment with active cultures and culture derivatives resulted in significant solubilization of the lignite. Up to 50% liquefaction of pretreated coal (20% HNO₃ at ambient temperature and pressure) was observed in 4 d with ML-13 cultures, whereas almost 80% liquefaction occurred in a similar time period when exposing pretreated lignite to an autoclaved, cell-free culture broth.     

Interaction of cylindrical polymer brushes in dilute and semi-dilute solution

We present a systematic study of flexible cylindrical brush-shaped macromolecules in a good solvent by small-angle neutron scattering (SANS), static light scattering (SLS), and by dynamic light scattering (DLS) in dilute and semi-dilute solution. The SLS and SANS data extrapolated to infinite dilution lead to the shape of the polymer that can be modeled in terms of a worm-like chain with a contour length of 380 nm and a persistence length of 17.5 nm. SANS data taken at higher polymer concentration were evaluated by using the polymer reference interaction site model (PRISM). We find that the persistence length reduce from 17.5 nm at infinite dilution to 5.3 nm at the highest concentration (volume fraction 0.038). This is comparable with the decrease of the persistence length in semi-dilute concentration predicted theoretically for polyelectrolytes. This finding reveals a softening of stiffness of the polymer brushes caused by their mutual interaction.