Silver-Containing Humic Substance-Based Nanocomposites—Agents for Healing of Potatoes from the Ring Rot

The influence of silver-containing humic substance (HS)-based nanocomposites (NCs) obtained from mud, shale, and coals of three kinds of deposits in Mongolia on the viability and ability to biofilm formation of a phytopathogenic gram-positive bacterium Clavibacter michiganensis subsp. sepedonicus (Cms), as well as peroxidase activity and potato plant growth in vitro, has been investigated. The maximum reduction of the viability of bacterial cells was found in the case of incubation with HS-mu/Ag NC and HS-coal/Ag NC. It was found that HSs, including HS-mu and HS-sl, and NCs synthesized on the base of these HSs, HS-mud/Ag NC and HS-sl/Ag NC reduce more than twofold the activity of peroxidase in potato tissues. It was also found that HS-co and HS-coal/Ag NC stimulate the potato peroxidase activity, as well as biofilm formation of Cms bacterium. No negative effect of the studied NCs on the growth of potato has been revealed. Moreover, NC HS-mud/Ag based on mud had a stimulating effect on leaf formation in plants.     

Effect of coculturing canine notochordal,nucleus pulposus and mesenchymal stromal cells for intervertebral disc regeneration

IntroductionEarly degenerative changes in the nucleus pulposus (NP) are observed after the disappearance of notochordal cells (NCs). Thus, it has been suggested that NCs play an important role in maintaining the NP and may have a regenerative potential on other cells of the NP. As the number of resident NP cells (NPCs) decreases in a degenerating disc, mesenchymal stromal (stem) cells (MSCs) may be used for cell supplementation. In this study, using cells of one species, the regenerative potential of canine NCs was assessed in long-term three-dimensional coculture with canine NPCs or MSCs.MethodsCanine NCs and canine NPCs or MSCs were cocultured in alginate beads for 28 days under hypoxic and high-osmolarity conditions. Cell viability, cell morphology and DNA content, extracellular matrix production and expression of genes related to NC markers (Brachyury, KRT18) and NP matrix production (ACAN, COL2A1, COL1A1) were assessed after 1, 15 and 28 days of culture.ResultsNCs did not completely maintain their phenotype (morphology, matrix production, gene expression) during 28 days of culture. In cocultures of NPCs and NCs, both extracellular matrix content and anabolic gene expression remained unchanged compared with monoculture groups, whereas cocultures of MSCs and NCs showed increased glycosaminoglycan/DNA. However, the deposition of these proteoglycans was observed near the NCs and not the MSCs. Brachyury expression in the MSC and NC coculture group increased in time. The latter two findings indicate a trophic effect of MSCs on NCs rather than vice versa.ConclusionsNo regenerative potential of canine NCs on canine NPCs or MSCs was observed in this study. However, significant changes in NC phenotype in long-term culture may have resulted in a suboptimal regenerative potential of these NCs. In this respect, NC-conditioned medium may be better than coculture for future studies of the regenerative potential of NCs.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0569-6) contains supplementary material, which is available to authorized users.     

Effect of polyethyleneglycol-phospholipids on aggregate structure in preparations of small unilamellar liposomes.

Phospholipids with covalently attached poly(ethylene glycol) (PEG lipids) are commonly used for the preparation of long circulating liposomes. Although it is well known that lipid/PEG-lipid mixed micelles may form above a certain critical concentration of PEG-lipid, little is known about the effects of PEG-lipids on liposome structure and leakage at submicellar concentrations. In this study we have used cryogenic transmission electron microscopy to investigate the effect of PEG(2000)-PE on aggregate structure in preparations of liposomes with different membrane compositions. The results reveal a number of important aggregate structures not documented before. The micrographs show that enclosure of PEG-PE induces the formation of open bilayer discs at concentrations well below those where mixed micelles begin to form. The maximum concentration of PEG-lipid that may be incorporated without alteration of the liposome structure depends on the phospholipid chain length, whereas phospholipid saturation or the presence of cholesterol has little or no effect. The presence of cholesterol does, however, affect the shape of the mixed micelles formed at high concentrations of PEG-lipid. Threadlike micelles form in the absence of cholesterol but adapt a globular shape when cholesterol is present.     

Photostability of quantum dot micelles under ultraviolet irradiation

Phospholipid quantum dot micelles are useful for bio‐applications because of their amphiphilicity and exceptional biocompatibilities. We investigated the uptake of phospholipid [polyethylene glycol (PEG), biotin, and folic acid terminated] modified CdSe/ZnS quantum dot micelles by cancer cells and its photostability under ultrviolet light in the C spectrum (UV‐C) (254 nm) or UV‐A (365 nm) light irradiation. The stability of micelles to the exposure of UV‐C and UV‐A light was assessed. Biotin‐modified quantum dot micelles give photoluminescence enhancement under UV‐C light irradiation. Folate modified micelle under UV‐C and UV‐A results show considerable photoluminescence enhancement. Photoluminescence lifetime measurements showed 7.04, 8.11 and 11.42 ns for PEG, folate, and biotin terminated phospholipid micelles, respectively. Folate and biotin‐modified quantum dot micelles showed excellent uptake by HeLa cells under fluorescence confocal microscopy. Phospholipid CdSe/ZnS quantum dot micelles can be potentially used for diagnosis and treatment of cancer in the future.     

Characterization of the nonlinear optical properties of nanocrystals by Hyper Rayleigh Scattering

Background

Harmonic Nanoparticles are a new family of exogenous markers for multiphoton imaging exerting optical contrast by second harmonic (SH) generation. In this tutorial, we present the application of Hyper-Rayleigh Scattering (HRS) for a quantitative assessment of the nonlinear optical properties of these particles and discuss the underlying theory and some crucial experimental aspects.

Methods

The second harmonic properties of BaTiO₃, KNbO₃, KiTiOPO₄ (KTP), LiNbO₃ and ZnO nanocrystals (NCs) are investigated by HRS measurements after careful preparation and characterization of colloidal suspensions.

Results

A detailed analysis of the experimental results is presented with emphasis on the theoretical background and on the influence of some experimental parameters including the accurate determination of the nanocrystal size and concentration. The SH generation efficiency and averaged nonlinear optical coefficients are then derived and compared for six different types of NCs.

Conclusions

After preparation of colloidal NC suspensions and careful examination of their size, concentration and possible aggregation state, HRS appears as a valuable tool to quantitatively assess the SH efficiency of noncentrosymmetric NCs. All the investigated nanomaterials show high SH conversion efficiencies, demonstrating a good potential for bio-labelling applications.

     

Elimination of nurse cell nuclei that shuttle into oocytes during oogenesis

Drosophila oocytes develop together with 15 sister germline nurse cells (NCs), which pass products to the oocyte through intercellular bridges. The NCs are completely eliminated during stages 12–14, but we discovered that at stage 10B, two specific NCs fuse with the oocyte and extrude their nuclei through a channel that opens in the anterior face of the oocyte. These nuclei extinguish in the ooplasm, leaving 2 enucleated and 13 nucleated NCs. At stage 11, the cell boundaries of the oocyte are mostly restored. Oocytes in egg chambers that fail to eliminate NC nuclei at stage 10B develop with abnormal morphology. These findings show that stage 10B NCs are distinguished by position and identity, and that NC elimination proceeds in two stages: first at stage 10B and later at stages 12–14.     

Development of PEGylated Cu nanoclusters: A nontoxic,multifunctional colloidal system for bioimaging and peroxide sensing

This study introduces the development of blue-emitting colloidal Cu NCs through a novel and easy PEGylation method using different functional groups, including -SH and -COOH. The surface functionalization controls the size, cellular toxicity, and emission properties of Cu NCs. The combination of PEG, thiol, and carboxylic groups protects the particle surface from aggregation and oxidation. Among the samples, CAGP (Surface modified Cu NCs with -SH-COOH-PEG combination) emerges as an amazing candidate with the lowest toxicity and enhanced blue emission properties. The bright blue fluorescence emission from Hela cells after treatment with CAGP demonstrated this property. It also has excellent peroxide sensing potential, with a detection limit of 1.4 μM. Because of their excellent bioimaging and peroxide sensing properties, these Cu NCs could be a promising candidate for cellular oxidative stress sensing applications with high clinical relevance.     

Synthesis and characterization of bovine serum albumin-coated nanocapsules loaded with indocyanine green as potential multifunctional nanoconstructs

We have synthesized and characterized bovine serum albumin (BSA)-coated polymeric nanocapsules (NCs) loaded with indocyanine green (ICG), an FDA-approved near infrared chromophore. Poly(allylamine) hydrochloride was electrostatically crosslinked with phosphate anions to form nanoconstructs encapsulating ICG. BSA was conjugated onto the polymeric NCs via glutaraldehyde. BSA-coated ICG-containing nanocapsules (BSA-ICG NCs) were characterized by FTIR and optical spectroscopy, SEM, and zeta-potential measurements. Using normal human endocervical epithelial cells, we demonstrate the effectiveness of BSA-ICG NCs for intracellular optical imaging in vitro. These nanoconstructs may potentially serve as a multifunctional platform for combined optical imaging, phototherapy, and drug delivery.     

Detection of hydrogen peroxide with chemiluminescent micelles

The overproduction of hydrogen peroxide is implicated in the progress of numerous life-threatening diseases and there is a great need for the development of contrast agents that can detect hydrogen peroxide in vivo. In this communication, we present a new contrast agent for hydrogen peroxide, termed peroxalate micelles, which detect hydrogen peroxide through chemiluminescence, and have the physical/chemical properties needed for in vivo imaging applications. The peroxalate micelles are composed of amphiphilic peroxalate based copolymers and the fluorescent dye rubrene, they have a ‘stealth’ polyethylene glycol (PEG) corona to evade macrophage phagocytosis, and a diameter of 33 nm to enhance extravasation into permeable tissues. The peroxalate micelles can detect nanomolar concentrations of hydrogen peroxide (>50 nM) and thus have the sensitivity needed to detect physiological concentrations of hydrogen peroxide. We anticipate numerous applications of the peroxalate micelles for in vivo imaging of hydrogen peroxide, given their high sensitivity, small size, and biocompatible PEG corona.     

Micelles as delivery vehicles for oligofluorene for bioimaging

With the successful development of organic/polymeric light emitting diodes, many organic and polymeric fluorophores with high quantum efficiencies and optical stability were synthesized. However, most of these materials which have excellent optical properties are insoluble in water, limiting their applications in biological fields. Herein, we used micelles formed from an amino-group-containing poly(ε-caprolactone)-block-poly(ethylene glycol) (PCL-b-PEG-NH₂) to incorporate a hydrophobic blue emitter oligofluorene (OF) to enable its application in biological conditions. Although OF is completely insoluble in water, it was successfully transferred into aqueous solutions with a good retention of its photophysical properties. OF exhibited a high quantum efficiency of 0.84 in a typical organic solvent of tetrahydrofuran (THF). In addition, OF also showed a good quantum efficiency of 0.46 after being encapsulated into micelles. Two cells lines, human glioblastoma (U87MG) and esophagus premalignant (CP-A), were used to study the cellular internalization of the OF incorporated micelles. Results showed that the hydrophobic OF was located in the cytoplasm, which was confirmed by co-staining the cells with nucleic acid specific SYTO 9, lysosome specific LysoTracker Red®, and mitochondria specific MitoTracker Red. MTT assay indicated non-toxicity of the OF-incorporated micelles. This study will broaden the application of hydrophobic functional organic compounds, oligomers, and polymers with good optical properties to enable their applications in biological research fields.     

Assembly and budding of Ebolavirus

Ebolavirus is responsible for highly lethal hemorrhagic fever. Like all viruses, it must reproduce its various components and assemble them in cells in order to reproduce infectious virions and perpetuate itself. To generate infectious Ebolavirus, a viral genome-protein complex called the nucleocapsid (NC) must be produced and transported to the cell surface, incorporated into virions, and then released from cells. To further our understanding of the Ebolavirus life cycle, we expressed the various viral proteins in mammalian cells and examined them ultrastructurally and biochemically. Expression of nucleoprotein alone led to the formation of helical tubes, which likely serve as a core for the NC. The matrix protein VP40 was found to be critical for transport of NCs to the cell surface and for the incorporation of NCs into virions, where interaction between nucleoprotein and the matrix protein VP40 is likely essential for these processes. Examination of virus-infected cells revealed that virions containing NCs mainly emerge horizontally from the cell surface, whereas empty virions mainly bud vertically, suggesting that horizontal budding is the major mode of Ebolavirus budding. These data form a foundation for the identification and development of potential antiviral agents to combat the devastating disease caused by this virus.     

Synthesis and characterization of a new fluorescent phospholipid

A novel fluorescent phospholipid, whose structure was tentatively assigned as 1-(2′-thio-1′-hydroxyethyl)-2-(ethylphosphatidyl)isoindole), was synthesized by reacting O-phthalaldehyde and β-mercaptoethanol with phosphatidylethanolamine. The fluorescent lipid product was purified by silicic acid chromatography. The purity was demonstrated by thin-layer chromatography. This fluorescent phospholipid could not form stable lipid vesicles. However, a mixture of phosphatidylcholine and this fluorescent phospholipid did form stable vesicles after sonication, as demonstrated by Sepharose 4B column chromatography and electron microscopy. The absorption and fluorescence properties of this lipid, both as aqueous micelles or incorporated into vesicles, have been determined. The potential usage of this new fluorescent phospholipid in membrane studies is discussed.     

LIPID COMPOSITION AND METABOLISM OF CULTURED HAMSTER BRAIN ASTROCYTES

Abstract— The lipid composition and metabolism of confluent cultures of cells derived from newborn hamster brain and having morphology characteristic of immature astrocytes or spongioblasts was investigated and compared to that of newborn hamster brain dispersions and cloned glioma cells (C6). The cells displayed stable morphology for at least 30 subcultures; thereafter spontaneous transformation occurred. No appreciable changes were observed in either composition or metabolic characteristics of any major neutral lipid or phospholipid class in successive subcultures or following transformation. The overall lipid composition of the hamster astrocyte cultures closely resembled that of newborn hamster brain, but the phospholipid composition showed substantial differences. The cells contained as a percent of lipid P relatively more ethanolamine plasmalogen, choline plasmalogen and sphingomyelin and somewhat less phosphatidylcholine and phosphatidylethanolamine. The phospholipids of the hamster astrocyte and C6 cells were similar. Of the lipid precursors examined, [U-¹⁴C]glucose was incorporated best into all preparations. C6 glioma cells incorporated both [U-¹⁴C]glucose and [1-¹⁴C]acetate most actively. From 69–88% of ³²P incorporated into hamster astrocyte phospholipids was present in choline phosphoglycerides, whereas the corresonding figure for hamster brain dispersions was 53%. The ratio of specific activities of phosphatidylcholine to phosphatidylinositol was substantially higher in the cultured cells than in the brain preparations. The small pool of choline plasmalogen in the hamster astrocytes usually achieved the highest specific activity of any phospholipid. When [U-¹⁴C]glucose and [1-¹⁴C]acetate were precursors, the bulk of label in the astrocytes appeared in choline phosphoglycerides and triacyglycerol. Our results indicate that the hamster astrocyte cell line as grown expresses distinctive features of lipid composition and metabolism which are nearly constant through many generations.     

Fluorine-18-labeled phospholipid quantum dot micelles for in vivo multimodal imaging from whole body to cellular scales

We have designed new nanoprobes applicable for both positron emission tomography (PET) and optical fluorescence in vivo imaging. Fluorine-18, which is commonly used for clinical imaging, has been coupled to phospholipid quantum dot (QD) micelles. This probe was injected in mice and we demonstrated that its dynamic quantitative whole body biodistribution and pharmacokinetics could be monitored using PET as well as the kinetics of their cellular uptake using in vivo fibered confocal fluorescence imaging. Phospholipid micelle encapsulation of QDs provides a highly versatile surface chemistry to conjugate multiple chemicals and biomolecules with controlled QD:molecule valency. Here, we show that, in contrast with several previous studies using other QD polymer coatings, these phospholipid QD micelles exhibit long circulation half-time in the bloodstream (on the order of 2 h) and slow uptake by reticulo-endothelial system.     

The Interaction of the Polyene Antibiotic Lucensomycin with Cholesterol in Erythrocyte Membranes and in Model Systems: I. A Fluorometric and Spectrophotometric Study

The increase of fluorescence emission of lucensomycin occurring upon interaction with cholesterol, either as colloidal suspension or included in phospholipid micelles or in erythrocyte membranes, was described. Colloidal cholesterol differed from that contained in membranes by the kinetic behavior of its interaction with lucensomycin, the reaction being very slow in the former case, very fast in the latter one. Variations of optical density also occurred, though neither the kinetics nor the titration curves were superimposable on those obtained fluorometrically. The stoichiometry was, however, the same.     

Formation and characterization of mixed micelles of the nonionic surfactant Triton X-100 with egg, dipalmitoyl, and dimyristoyl phosphatidylcholines

Mixed micelles of the nonionic surfactant Triton X-100 and egg phosphatidylcholine were isolated by column chromatography on 6% agarose and by centrifugation at 35,000g. It was found that egg phosphatidylcholine bilayers are able to incorporate Triton X-100 at molar ratios of Triton to phospholipid below about 1:1, whereas above a molar ratio of about 2:1 Triton/phospholipid all of the phospholipid is converted into mixed micelles. Mixed micelles at a molar ratio of about 10:1 Triton/phospholipid were found to be in the same size range as pure micelles of Triton X-100. The formation of mixed micelles with dipalmitoyl phosphatidylcholine at room temperature, when the phospholipid is below its thermotropic phase transition, is shown to require relatively high concentrations of Triton X-100. The point at which dimyristoyl phosphatidylcholine bilayers are converted to mixed micelles was found to be less clear cut than with egg phosphatidylcholine, but above a molar ratio of about 2:1 Triton/phospholipid, all of this phospholipid is also in mixed micelles. The relevance of these results to the solubilization of membrane-bound proteins with Triton X-100 and the action of phospholipase A₂, which hydrolyzes phosphatidylcholine when it is in mixed micelles with Triton X-100, is discussed.     

Maturation-Associated Destabilization of Hepatitis B Virus Nucleocapsid

The mature nucleocapsid (NC) of hepatitis B virus containing the relaxed circular (RC) DNA genome can be secreted extracellularly as virions after envelopment with the viral surface proteins or, alternatively, can be disassembled to release RC DNA (i.e., uncoating) into the host cell nucleus to form the covalently closed circular (CCC) DNA, which sustains viral replication and persistence. In contrast, immature NCs containing the viral single-stranded DNA or the pregenomic RNA are incompetent for either envelopment or uncoating. Little is currently known about how mature NCs, and not the immature ones, are specifically selected for these processes. Here, we have carried out a biochemical analysis of the different NC populations upon their separation through sucrose gradient centrifugation. We have found that the maturation of NCs is associated with their destabilization, manifested as increased protease and nuclease sensitivity, altered sedimentation during sucrose gradient centrifugation, and retarded mobility during native agarose gel electrophoresis. Also, three distinct populations of intracellular mature NCs could be differentiated based on these characteristics. Furthermore, mature NCs generated in vitro under cell-free conditions acquired similar properties. These results have thus revealed significant structural changes associated with NC maturation that likely play a role in the selective uncoating of the mature NC for CCC DNA formation and/or its preferential envelopment for virion secretion.     

Preparation of pH‐stimuli‐responsive PEG–TGA/TGH‐capped CdTe QDs and their application in cell labeling

A pH‐sensitive and double functional nanoprobe was designed and synthesized in a water‐soluble system using thioglycolic acid (TGA) and mercapto‐acetohydrazide (TGH) as the stabilizers. TGA is biocompatible because the carboxyl group is easily linked to biological macromolecules. At the same time, the hydrazide on TGH reacts with the aldehyde on poly(ethylene glycol) (PEG) and forms a hydrazone bond. The hydrazone bond ruptured at specific pH values and exhibited pH‐stimuli‐responsive characteristics. As an optical imaging probe, the PEG–TGA/TGH‐capped CdTe quantum dots (QDs) had high quality, with a fluorescence efficiency of 25–30%, and remained stable for at least five months. This pH‐responsive factor can be used for the effective release of CdTe QDs under the acidic interstitial extracellular environment of tumor cells. This allows the prepared pH‐stimuli‐responsive nanoprobes to show fluorescence signals for use in cancer cell imaging. Copyright © 2014 John Wiley & Sons, Ltd.     

Structural organization of the phospholipid component of Streptomyces hygroscopicus cell membranes as determined from neutron diffraction data

The nanoparameters of an actinobacterial membrane have been estimated by neutron diffraction on multilamellar lipid membranes. The lamellar repeat distance in a partly hydrated membrane prepared with the phospholipid fraction from Streptomyces hygroscopicus is 85.8 ± 0.5°C and decreases to 83.5 ± 0.5 0°C. Some lipids are not incorporated into the bilayer and form a liquid phase of micelles 54.2 ± 0.2