Perspectives on antimicrobial properties of Paulownia tomentosa Steud. fruit products in the control of Staphylococcus aureus infections.

ETHNOPHARMACOLOGICAL RELEVANCE: Paulownia tomentosa Steud. (P. tomentosa) is a medium-sized tree traditionally used in Chinese folk medicine for the treatment of infectious diseases. It is a rich source of prenylated phenolic compounds that have been extensively studied for their promising biological activities. AIM OF THE STUDY: Due to the increasing development of antibiotic resistance, our study investigated plant-derived natural products from the fruits of P. tomentosa that could control Staphylococcus aureus infections with novel targets/modes of action and reduce antimicrobial resistance. MATERIALS AND METHODS: The ethanolic extract was fractionated and detected by liquid chromatography. The antistaphylococcal effects of the plant formulations were studied in detail in vitro by various biological methods, including microdilution methods for minimum inhibitory concentration (MIC), the checkerboard titration technique for synergy assay, fluorescence measurements for membrane disruption experiments, autoinducer-2-mediated bioassay for quorum sensing inhibition, and counting of colony-forming units for relative adhesion. Morphology was examined by transmission electron microscopy. RESULTS: Total ethanolic extract and chloroform fraction showed MICs of 128 and 32 µg/mL, respectively. Diplacol, diplacone, and 3'-O-methyl-5'-hydroxydiplacone inhibited S. aureus growth in the range of 8-16 µg/mL. Synergistic potential was shown in combination with mupirocin and fusidic acid. The ethanolic extract and the chloroform fraction destroyed the cell membranes by 91.61% and 79.46%, respectively, while the pure compounds were less active. The ethanolic extract and the pure compounds reduced the number of adhered cells to 47.33-10.26% compared to the untreated control. All tested plant formulations, except diplacone, inhibited quorum sensing of S. aureus. Transmission electron microscopy showed deformation of S. aureus cells. CONCLUSIONS: The products from the fruit of P. tomentosa showed antimicrobial properties against S. aureus alone and in combination with antibiotics. By affecting intracellular targets, geranylated flavonoids proposed novel approaches in the control of staphylococcal infections.

Diversity and Pathobiology of an Ilarvirus Unexpectedly Detected in Diverse Plants and Global Sequencing Data.

High-throughput sequencing (HTS) and sequence mining tools revolutionized virus detection and discovery in recent years, and implementing them with classical plant virology techniques results in a powerful approach to characterize viruses. An example of a virus discovered through HTS is Solanum nigrum ilarvirus 1 (SnIV1) (Bromoviridae), which was recently reported in various solanaceous plants from France, Slovenia, Greece, and South Africa. It was likewise detected in grapevines (Vitaceae) and several Fabaceae and Rosaceae plant species. Such a diverse set of source organisms is atypical for ilarviruses, thus warranting further investigation. In this study, modern and classical virological tools were combined to accelerate the characterization of SnIV1. Through HTS-based virome surveys, mining of sequence read archive datasets, and a literature search, SnIV1 was further identified from diverse plant and non-plant sources globally. SnIV1 isolates showed relatively low variability compared with other phylogenetically related ilarviruses. Phylogenetic analyses showed a distinct basal clade of isolates from Europe, whereas the rest formed clades of mixed geographic origin. Furthermore, systemic infection of SnIV1 in Solanum villosum and its mechanical and graft transmissibility to solanaceous species were demonstrated. Near-identical SnIV1 genomes from the inoculum (S. villosum) and inoculated Nicotiana benthamiana were sequenced, thus partially fulfilling Koch's postulates. SnIV1 was shown to be seed-transmitted and potentially pollen-borne, has spherical virions, and possibly induces histopathological changes in infected N. benthamiana leaf tissues. Overall, this study provides information to better understand the diversity, global presence, and pathobiology of SnIV1; however, its possible emergence as a destructive pathogen remains uncertain. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

The Role of luxS in Campylobacter jejuni Beyond Intercellular Signaling.

The full role of the luxS gene in the biological processes, such as essential amino acid synthesis, nitrogen and pyruvate metabolism, and flagellar assembly, of Campylobacter jejuni has not been clearly described to date. Therefore, in this study, we used a comprehensive approach at the cellular and molecular levels, including transcriptomics and proteomics, to investigate the key role of the luxS gene and compared C. jejuni 11168ΔluxS (luxS mutant) and C. jejuni NCTC 11168 (wild type) strains. Transcriptomic analysis of the luxS mutant grown under optimal conditions revealed upregulation of luxS mutant metabolic pathways when normalized to wild type, including oxidative phosphorylation, carbon metabolism, citrate cycle, biosynthesis of secondary metabolites, and biosynthesis of various essential amino acids. Interestingly, induction of these metabolic pathways was also confirmed by proteomic analysis, indicating their important role in energy production and the growth of C. jejuni. In addition, genes important for the stress response of C. jejuni, including nutrient starvation and oxidative stress, were upregulated. This was also evident in the better survival of the luxS mutant under starvation conditions than the wild type. At the molecular level, we confirmed that metabolic pathways were upregulated under optimal conditions in the luxS mutant, including those important for the biosynthesis of several essential amino acids. This also modulated the utilization of various carbon and nitrogen sources, as determined by Biolog phenotype microarray analysis. In summary, transcriptomic and proteomic analysis revealed key biological differences in tricarboxylic acid (TCA) cycle, pyruvate, nitrogen, and thiamine metabolism as well as lipopolysaccharide biosynthesis in the luxS mutant. IMPORTANCE Campylobacter jejuni is the world's leading foodborne bacterial pathogen of gastrointestinal disease in humans. C. jejuni is a fastidious but widespread organism and the most frequently reported zoonotic pathogen in the European Union since 2005. This led us to believe that C. jejuni, which is highly sensitive to stress factors (starvation and oxygen concentration) and has a low growth rate, benefits significantly from the luxS gene. The role of this gene in the life cycle of C. jejuni is well known, and the expression of luxS regulates many phenotypes, including motility, biofilm formation, host colonization, virulence, autoagglutination, cellular adherence and invasion, oxidative stress, and chemotaxis. Surprisingly, this study confirmed for the first time that the deletion of the luxS gene strongly affects the central metabolic pathway of C. jejuni, which improves its survival, showing its role beyond the intercellular signaling system.

Correction: Jahic Mujkic et al. Synergy of the Inhibitory Action of Polyphenols Plus Vitamin C on Amyloid Fibril Formation: Case Study of Human Stefin B. Antioxidants 2021, 10, 1471.

In the original publication [...].

Extracellular vesicle-bound DNA in urine is indicative of kidney allograft injury.

Extracellular vesicle-bound DNA (evDNA) is an understudied extracellular vesicle (EV) cargo, particularly in cancer-unrelated research. Although evDNA has been detected in urine, little is known about its characteristics, localization, and biomarker potential for kidney pathologies. To address this, we enriched EVs from urine of well-characterized kidney transplant recipients undergoing allograft biopsy, characterized their evDNA and its association to allograft injury. The SEC-based method enriched pure EVs from urine of kidney transplant recipients, regardless of the allograft injury. Urinary evDNA represented up to 29.2 ± 8% (mean ± SD) of cell-free DNA (cfDNA) and correlated with cfDNA in several characteristics but was less fragmented (P < 0.001). Importantly, using DNase treatment and immunogold labelling TEM, we demonstrated that evDNA was bound to the surface of urinary EVs. Normalised evDNA yield (P = 0.042) and evDNA copy number (P = 0.027) significantly differed between patients with normal histology, rejection injury and non-rejection injury, the later groups having significantly larger uEVs (mean diameter, P = 0.045) and more DNA bound per uEV. ddDNA is detectable in uEV samples of kidney allograft recipients, but its quantity is highly variable. In a proof-of-principle study, several evDNA characteristics correlated with clinical and histological parameters (P = 0.040), supporting that the potential of evDNA as a biomarker for kidney allograft injury should be further investigated.

First Insights into the Antiviral Activity of Chitosan-Based Bioactive Polymers towards the Bacteriophage Phi6: Physicochemical Characterization, Inactivation Potential, and Inhibitory Mechanisms.

The outbreak of the worrisome coronavirus disease in 2019 has caused great concern among the global public, especially regarding the need for personal protective equipment with applied antiviral agents to reduce the spread and transmission of the virus. Thus, in our research, chitosan-based bioactive polymers as potential antiviral agents were first evaluated as colloidal macromolecular solutions by elemental analysis and charge. Three different types of low and high molecular weight chitosan (LMW Ch, HMW Ch) and a LMW Ch derivative, i.e., quaternary chitosan (quart-LMW Ch), were used. To explore their antiviral activity for subsequent use in the form of coatings, the macromolecular Chs dispersions were incubated with the model virus phi6 (surrogate for SARS-CoV-2), and the success of virus inactivation was determined. Inactivation of phi6 with some chitosan-based compounds was very successful (>6 log), and the mechanisms behind this were explored. The changes in viral morphology after incubation were observed and the changes in infrared bands position were determined. In addition, dynamic and electrophoretic light scattering studies were performed to better understand the interaction between Chs and phi6. The results allowed us to better understand the antiviral mode of action of Chs agents as a function of their physicochemical properties.

Optimization of isolation protocol and characterization of urinary extracellular vesicles as biomarkers of kidney allograft injury.

AIMS: Long-term kidney allograft survival requires a personalized approach to allograft injury recognition in a timely and reliable manner. Kidney biopsy is invasive and unsuitable for continuous function assessment. Alternatively, in urine, we find extracellular vesicles (uEVs), stable carriers of kidney pathology signals. Analysis of uEVs and their cargo could allow for more frequent and non-invasive assessment of allograft function. We aimed to optimize the uEVs isolation method applicable for kidney allograft injury biomarker studies. MATERIALS AND METHODS: To this end, we optimized several steps of size-exclusion chromatography (SEC)-based method for uEVs isolation from second morning urine of kidney allograft recipients. uEVs were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), western analysis, and quantitative PCR. RESULTS: According to TEM and NTA, SEC isolated high concentrations (8.64 × 108 EVs/mL of urine) of EVs that showed typical morphology and mean size (171 nm), but addition of EDTA and filtration step were needed to remove impurities. Additionally, typical EV proteins Hsc70, CD63, flotillin, tubulin, GAPDH, and miR hsa-let-7i were detected in isolated uEVs, further confirming their identity. CONCLUSION: Optimized method based on SEC was effective and adequate in isolating pure EVs from urine of kidney allograft recipients and could be used in future biomarker studies.

Synergy of the Inhibitory Action of Polyphenols Plus Vitamin C on Amyloid Fibril Formation: Case Study of Human Stefin B.

In order to study how polyphenols and vitamin C (vitC) together affect protein aggregation to amyloid fibrils, we performed similar in vitro studies as before using stefin B as a model and a potentially amyloid-forming protein (it aggregates upon overexpression, under stressful conditions and some progressive myoclonus epilepsy of tape 1-EPM1-missense mutations). In addition to the chosen polyphenol, this time, we added a proven antioxidant concentration of 0.5 mM vitC into the fibrillation mixture and varied concentrations of resveratrol, quercetin, and curcumin. Synergy with vitC was observed with curcumin and quercetin.

Inactivation of Pepper Mild Mottle Virus in Water by Cold Atmospheric Plasma.

Water scarcity is one of the greatest threats for human survival and quality of life, and this is increasingly contributing to the risk of human, animal and plant infections due to waterborne viruses. Viruses are transmitted through polluted water, where they can survive and cause infections even at low concentrations. Plant viruses from the genus Tobamovirus are highly mechanically transmissible, and cause considerable damage to important crops, such as tomato. The release of infective tobamoviruses into environmental waters has been reported, with the consequent risk for arid regions, where these waters are used for irrigation. Virus inactivation in water is thus very important and cold atmospheric plasma (CAP) is emerging in this field as an efficient, safe, and sustainable alternative to classic waterborne virus inactivation methods. In the present study we evaluated CAP-mediated inactivation of pepper mild mottle virus (PMMoV) in water samples. PMMoV is a very resilient water-transmissible tobamovirus that can survive transit through the human digestive tract. The efficiency of PMMoV inactivation was characterized for infectivity and virion integrity, and at the genome level, using test plant infectivity assays, transmission electron microscopy, and molecular methods, respectively. Additionally, the safety of CAP treatment was determined by testing the cytotoxic and genotoxic properties of CAP-treated water on the HepG2 cell line. 5-min treatment with CAP was sufficient to inactivate PMMoV without introducing any cytotoxic or genotoxic effects in the in-vitro cell model system. These data on inactivation of such stable waterborne virus, PMMoV, will encourage further examination of CAP as an alternative for treatment of potable and irrigation waters, and even for other water sources, with emphasis on inactivation of various viruses including enteric viruses.

Enrichment of plasma extracellular vesicles for reliable quantification of their size and concentration for biomarker discovery.

Human plasma is a complex fluid, increasingly used for extracellular vesicle (EV) biomarker studies. Our aim was to find a simple EV-enrichment method for reliable quantification of EVs in plasma to be used as biomarker of disease. Plasma of ten healthy subjects was processed using sedimentation rate- (sucrose cushion ultracentrifugation-sUC) and size- (size exclusion chromatography-SEC) based methods. According to nanoparticle tracking analysis (NTA), asymmetrical flow field-flow fractionation coupled to detectors (AF4-UV-MALS), miRNA quantification, transmission electron microscopy and enzyme-linked immunosorbent assay, enrichment of EVs from plasma with sUC method lead to high purity of EVs in the samples. High nanoparticle concentrations after SEC resulted from substantial contamination with lipoproteins and other aggregates of EV-like sizes that importantly affect downstream EV quantification. Additionally, sUC EV-enrichment method linked to quantification with NTA or AF4-UV-MALS is repeatable, as the relative standard deviation of EV size measured in independently processed samples from the same plasma source was 5.4% and 2.1% when analyzed by NTA or AF4-UV-MALS, respectively. In conclusion, the sUC EV-enrichment method is compatible with reliable measurement of concentration and size of EVs from plasma and should in the future be tested on larger cohorts in relation to different diseases. This is one of the first studies using AF4-UV-MALS to quantify EVs in blood plasma, which opens new possible clinical utility for the technique.

The effect of three polyphenols and some other antioxidant substances on amyloid fibril formation by Human cystatin C.

Human cystatin C (CysC) is an amyloid forming protein involved in the hereditary cerebral amyloid angiopathy (HCCAA) that affects arteries in the brain and the peripheral nervous system. In this study we measured the influence of several substances on human CysC aggregation and amyloid fibril formation, induced at pH 4 in vitro. The effect of three polyphenols: resveratrol, quercetin and curcumin and of two antioxidants: vitamin C (VitC) and N-acetyl-L-cysteine (NAC) was explored as well as the effect of sulphoraphane (SF) and α-lipoic acid (AL). The formation of amyloid fibrils was followed by Thioflavin T (ThT) fluorescence and by transmission electron microscopy (TEM). Effects on the length of the lag phase were revealed by following the increase of ThT fluorescence intensity with time. The amount and morphology of fibrils in comparison to prefibrillar aggregates and globular oligomers were evaluated by TEM at the plateau stage of the reaction. Thermal stabilization of the CysC monomer by the small compounds was measured by differential scanning fluorimetry (DSF). NAC, VitC and SF exhibited the largest inhibitory effect on amyloid fibril growth. The effects of polyphenols were not significant, apart from resveratrol, which partly inhibited the amyloid fibril growth.

Precision transcriptomics of viral foci reveals the spatial regulation of immune-signaling genes and identifies RBOHD as an important player in the incompatible interaction between potato virus Y and potato.

Whereas the activation of resistance (R) proteins has been intensively studied, the downstream signaling mechanisms leading to the restriction of the pathogen remain mostly unknown. We studied the immunity network response conditioned by the potato Ny-1 gene against potato virus Y. We analyzed the processes in the cell death zone and surrounding tissue on the biochemical and gene expression levels in order to reveal the spatiotemporal regulation of the immune response. We show that the transcriptional response in the cell death zone and surrounding tissue is dependent on salicylic acid (SA). For some genes the spatiotemporal regulation is completely lost in the SA-deficient line, whereas other genes show a different response, indicating multiple connections between hormonal signaling modules. The induction of NADPH oxidase RBOHD expression occurs specifically on the lesion border during the resistance response. In plants with silenced RBOHD, the functionality of the resistance response is perturbed and the spread of the virus is not arrested at the site of infection. RBOHD is required for the spatial accumulation of SA, and conversely RBOHD is under the transcriptional regulation of SA. Using spatially resolved RNA-seq, we also identified spatial regulation of an UDP-glucosyltransferase, another component in feedback activation of SA biosynthesis, thus deciphering a novel aspect of resistance signaling.

L-Amino Acid Oxidases From Mushrooms Show Antibacterial Activity Against the Phytopathogen Ralstonia solanacearum.

Ralstonia solanaceraum is the quarantine plant pathogenic bacterium that causes bacterial wilt in over 200 host plants, which include economically important crops such as potato, tomato, tobacco, banana, and ginger. Alternative biological methods of disease control that can be used in integrated pest management are extensively studied. In search of new proteins with antibacterial activity against R. solanacearum, we identified L-amino acid oxidases (LAOs) from fruiting bodies of Amanita phalloides (ApLAO) and Infundibulicybe geotropa (CgLAO). We describe an optimized isolation procedure for their biochemical characterization, and show that they are dimeric proteins with estimated monomer molecular masses of 72 and 66 kDa, respectively, with isoelectric point of pH 6.5. They have broad substrate specificities for hydrophobic and charged amino acids, with highest Km for L-Leu, and broad pH optima at pH 5 and pH 6, respectively. An enzyme with similar properties is also characterized from the mycelia of I. geotropa (CgmycLAO). Fractionated aqueous extracts of 15 species of mushrooms show that LAO activity against L-Leu correlates with antibacterial activity. We confirm that the LAO activities mediate the antibacterial actions of ApLAO, CgLAO, and CgmycLAO. Their antibacterial activities are greater against Gram-negative versus Gram-positive bacteria, with inhibition of growth rate, prolongation of lag-phase, and decreased endpoint biomass. In Gram-positive bacteria, they mainly prolong the lag phase. These in vitro antibacterial activities of CgLAO and CgmycLAO are confirmed in vivo in tomato plants, while ApLAO has no effect on disease progression in planta. Transmission electron microscopy shows morphological changes of R. solanacearum upon LAO treatments. Finally, broad specificity of the antibacterial activities of these purified LAOs were seen for in vitro screening against 14 phytopathogenic bacteria. Therefore, these fungal LAOs show great potential as new biological phytoprotective agents and show the fruiting bodies of higher fungi to be a valuable source of antimicrobials with unique features.

Viromics and infectivity analysis reveal the release of infective plant viruses from wastewater into the environment.

Viruses represent one of the most important threats to agriculture. Several viral families include highly stable pathogens, which remain infective and can be transported long distances in water. The diversity of plant viruses in wastewater remains understudied; however, their potential impact is increasing with the increased irrigation usage of reclaimed wastewater. To determine the abundance, diversity and biological relevance of plant viruses in wastewater influents and effluents we applied an optimized virus concentration method followed by high-throughput sequencing and infectivity assays. We detected representatives of 47 plant virus species, including emerging crop threats. We also demonstrated infectivity for pathogenic and economically relevant plant viruses from the genus Tobamovirus (family Virgaviridae), which remain infective even after conventional wastewater treatment. These results demonstrate the potential of metagenomics to capture the diversity of plant viruses circulating in the environment and expose the potential risk of the uncontrolled use of reclaimed water for irrigation.

Accurate Quantification and Characterization of Adeno-Associated Viral Vectors.

One of the main challenges in the gene therapy viral vector development is to establish an optimized process for its large scale production. This requires optimization for upstream and downstream processes as well as methods that enable the step-by step analytical characterization of the virus, the results of which inform the iterative refinement of production for yield, purity and potency. The biggest problem here is a plethora of viral vector formulations, many of which interfere with analytical techniques. We took adeno-associated virus (AAV) as an example and showed benefits of combined use of molecular methods and transmission electron microscopy (TEM) for viral vectors' characterization and quantification. Results of the analyses showed that droplet digital PCR (ddPCR) performs better than quantitative real-time PCR (qPCR), in terms of robustness and assay variance, and this was especially relevant for partially purified (in-process) samples. Moreover, we demonstrate the importance of sample preparation prior to PCR analysis. We evaluated viral structure, presence of aggregates and impurities with TEM analysis and found that these impacted the differences in viral titers observed by qPCR and ddPCR and could be altered by sample preparation. These results serve as a guide for the establishment of the analytical methods required to provide measures of identity and purity for AAV viral vectors.

Prolines Affect the Nucleation Phase of Amyloid Fibrillation Reaction; Mutational Analysis of Human Stefin B.

Proline residues play a prominent role in protein folding and aggregation. We investigated the influence of single prolines and their combination on oligomerization and the amyloid fibrillation reaction of human stefin B (stB). The proline mutants influenced the distribution of oligomers between monomers, dimers, and tetramers as shown by the size-exclusion chromatography. Only P74S showed higher oligomers, reminiscent of the molten globule reported previously for the P74S of stB-Y31 variant. The proline mutants also inhibited to various degree the amyloid fibrillation reaction. At 30 and 37 °C, inhibition was complete for the P74S single mutant, two double mutants (P6L P74S and P74S P79S), and for the triple mutant P6L P11S P74S. At 30 °C the single mutant P6L completely inhibited the reaction, while P11S and P79S formed amyloid fibrils with a prolonged lag phase. P36D did not show a lag phase, reminiscent of a downhill polymerization model. At 37 °C in addition to P36D, P11S, and P79S, P6L and P11S P74S also started to fibrillate; however, the yield of the fibrils was much lower than that of the wild-type protein as judged by transmission electron microscopy. Thus, Pro 74 cis/trans isomerization proves to be the key event, acting as a switch toward an amyloid transition. Using our previous model of nucleation and growth, we simulated the kinetics of all the mutants that exhibited sigmoidal fibrillation curves. To our surprise, the nucleation phase was most affected by Pro cis/trans isomerism, rather than the fibril elongation phase.

Comparative ultrastructure of cells and cuticle in the anterior chamber and papillate region of Porcellioscaber (Crustacea, Isopoda) hindgut.

Isopod hindgut consists of two anatomical and functional parts, the anterior chamber, and the papillate region. This study provides a detailed ultrastructural comparison of epithelial cells in the anterior chamber and the papillate region with focus on cuticle ultrastructure, apical and basal plasma membrane labyrinths, and cell junctions. Na+/K+-ATPase activity in the hindgut epithelial cells was demonstrated by cytochemical localisation. The main difference in cuticle ultrastructure is in the thickness of epicuticle which is almost as thick as the procuticle in the papillate region and only about one sixth of the thickness of procuticle in the anterior chamber. The apical plasma membrane in both hindgut regions forms an apical plasma membrane labyrinth of cytoplasmic strands and extracellular spaces. In the papillate region the membranous infoldings are deeper and the extracellular spaces are wider. The basal plasma membrane is extensively infolded and associated with numerous mitochondria in the papillate region, while it forms relatively scarce basal infoldings in the anterior chamber. The junctional complex in both hindgut regions consists of adherens and septate junctions. Septate junctions are more extensive in the papillate region. Na+/K+-ATPase was located mostly in the apical plasma membranes in both hindgut regions. The ultrastructural features of hindgut cuticle are discussed in comparison to exoskeletal cuticle and to cuticles of other arthropod transporting epithelia from the perspective of their mechanical properties and permeability. The morphology of apical and basal plasma membranes and localisation of Na+/K+-ATPase are compared with other arthropod-transporting epithelia according to different functions of the anterior chamber and the papillate region.

Kinetically Stable Triglyceride-Based Nanodroplets and Their Interactions with Lipid-Specific Proteins.

Understanding of the interactions between proteins and natural and artificially prepared lipid membrane surfaces and embedded nonpolar cores is important in studies of physiological processes and their pathologies and is applicable to nanotechnologies. In particular, rapidly growing interest in cellular droplets defines the need for simplified biomimetic lipid model systems to overcome in vivo complexity and variability. We present a protocol for the preparation of kinetically stable nanoemulsions with nanodroplets composed of sphingomyelin (SM) and cholesterol (Chol), as amphiphilic surfactants, and trioleoylglycerol (TOG), at various molar ratios. To prepare stable SM/Chol-coated monodisperse lipid nanodroplets, we modified a reverse phase evaporation method and combined it with ultrasonication. Lipid composition, ζ-potential, gyration and hydrodynamic radius, shape, and temporal stability of the lipid nanodroplets were characterized and compared to extruded SM/Chol large unilamellar vesicles. Lipid nanodroplets and large unilamellar vesicles with theoretical SM/Chol/TOG molar ratios of 1/1/4.7 and 4/1/11.7 were further investigated for the orientational order of their interfacial water molecules using a second harmonic scattering technique, and for interactions with the SM-binding and Chol-binding pore-forming toxins equinatoxin II and perfringolysin O, respectively. The surface characteristics (ζ-potential, orientational order of interfacial water molecules) and binding of these proteins to the nanodroplet SM/Chol monolayers were similar to those for the SM/Chol bilayers of the large unilamellar vesicles and SM/Chol Langmuir monolayers, in terms of their surface structures. We propose that such SM/Chol/TOG nanoparticles with the required lipid compositions can serve as experimental models for monolayer membrane to provide a system that imitates the natural lipid droplets.

Newly Isolated Bacteriophages from the Podoviridae, Siphoviridae, and Myoviridae Families Have Variable Effects on Putative Novel Dickeya spp.

Soft rot pathogenic bacteria from the genus Dickeya cause severe economic losses in orchid nurseries worldwide, and there is no effective control currently available. In the last decade, the genus Dickeya has undergone multiple changes as multiple new taxa have been described, and just recently a new putative Dickeya species was reported. This study reports the isolation of three bacteriophages active against putative novel Dickeya spp. isolates from commercially produced infected orchids that show variable host-range profiles. Bacteriophages were isolated through enrichment from Dickeya-infected orchid tissue. Convective interaction media monolith chromatography was used to isolate bacteriophages from wastewaters, demonstrating its suitability for the isolation of infective bacteriophages from natural sources. Based on bacteriophage morphology, all isolated bacteriophages were classified as being in the order Caudovirales, belonging to three different families, Podoviridae, Myoviridae, and Siphoviridae. The presence of three different groups of bacteriophages was confirmed by analyzing the bacteriophage specificity of bacterial hosts, restriction fragment length polymorphism and plaque morphology. Bacteriophage BF25/12, the first reported Podoviridae bacteriophage effective against Dickeya spp., was selected for further characterization. Its genome sequence determined by next-generation sequencing showed limited similarity to other characterized Podoviridae bacteriophages. Interactions among the bacteriophages and Dickeya spp. were examined using transmission electron microscopy, which revealed degradation of electron-dense granules in response to bacteriophage infection in some Dickeya strains. The temperature stability of the chosen Podoviridae bacteriophage monitored over 1 year showed a substantial decrease in the survival of bacteriophages stored at -20°C over longer periods. It showed susceptibility to low pH and UV radiation but was stable in neutral and alkaline pH. Furthermore, the stability of the tested bacteriophage was also connected to the incubation medium and bacteriophage concentration at certain pH values. Finally, the emergence of bacteriophage-resistant bacterial colonies is highly connected to the concentration of bacteriophages in the bacterial environment. This is the first report on bacteriophages against Dickeya from the Podoviridae family to expand on potential bacteriophages to include in bacteriophage cocktails as biocontrol agents. Some of these bacteriophage isolates also showed activity against Dickeya solani, an aggressive strain that causes the soft rot of potatoes, which indicates their broad potential as biocontrol agents.

1-Aminocyclopropane-1-Carboxylate Oxidase Induction in Tomato Flower Pedicel Phloem and Abscission Related Processes Are Differentially Sensitive to Ethylene.

Ethylene has impact on several physiological plant processes, including abscission, during which plants shed both their vegetative and reproductive organs. Cell separation and programmed cell death are involved in abscission, and these have also been correlated with ethylene action. However, the detailed spatiotemporal pattern of the molecular events during abscission remains unknown. We examined the expression of two tomato ACO genes, LeACO1, and LeACO4 that encode the last enzyme in ethylene biosynthesis, 1-aminocyclopropane-1-carboxylate oxidase (ACO), together with the expression of other abscission-associated genes involved in cell separation and programmed cell death, during a period of 0-12 h after abscission induction in the tomato flower pedicel abscission zone and nearby tissues. In addition, we determined their localization in specific cell layers of the flower pedicel abscission zone and nearby tissues obtained by laser microdissection before and 8 h after abscission induction. The expression of both ACO genes was localized to the vascular tissues in the pedicel. While LeACO4 was more uniformly expressed in all examined cell layers, the main expression site of LeACO1 was in cell layers just outside the abscission zone in its proximal and distal part. We showed that after abscission induction, ACO1 protein was synthesized in phloem companion cells, in which it was localized mainly in the cytoplasm. Samples were additionally treated with 1-methylcyclopropene (1-MCP), a competitive inhibitor of ethylene actions, and analyzed 8 h after abscission induction. Cell-layer-specific changes in gene expression were observed together with the specific localization and ethylene sensitivity of the hallmarks of cell separation and programmed cell death. While treatment with 1-MCP prevented separation of cells through inhibition of the expression of polygalacturonases, which are the key enzymes involved in degradation of the middle lamella, this had less impact on the occurrence of different kinds of membrane vesicles and abscission-related programmed cell death. In the flower pedicel abscission zone, the physical progressions of cell separation and programmed cell death are perpendicular to each other and start in the vascular tissues.