DNA-oligonucleotide encapsulating liposomes as a secondary signal amplification means

A novel liposome-based signal amplification system was developed by encapsulating DNA oligonucleotides within antibody-tagged liposomes and subsequently detecting the oligonucleotide with dye-encapsulating liposomes for double signal enhancement. In this sandwich immunoassay, the model analyte, protective antigen protein from B. anthracis, was captured by one set of antibodies immobilized in microtiter plate wells and detected using a second antibody conjugated to oligonucleotide-encapsulating liposomes. Bound liposomes were lysed releasing the encapsulated fluorescein-tagged DNA 25-mer probe, which was then permitted to hybridize with its complementary sequence immobilized in a second plate. Finally, the amount of oligonucleotide was detected through the addition of anti-fluorescein antibody tagged dye-encapsulating liposomes. These secondary liposomes allowed for a approximately 400x lower LOD than detection of the fluorescein-labeled probe alone. Several aspects were investigated, including the encapsulation of various oligonucleotide concentrations within liposomes; oligonucleotide hybridization times and buffers; degree of anti-fluorescein antibody coverage on the liposomes; and immobilized anti-protective antigen antibody concentration. We found that the encapsulation efficiency increased with the starting oligonucleotide concentration. As many as 4000 DNA 25-mers were successfully entrapped in the liposome, and minimal leakage was observed over the course of 8 months. When used in the sandwich immunoassay, a limit of detection of 4.1 ng/mL protective antigen was observed with an upper limit of 5000 ng/mL. Due to the endless combination of DNA oligonucleotide sequences, this assay lends itself perfectly for multiplexing on the order of tens to hundreds of analytes.     

Application of ganglioside-sensitized liposomes in a flow injection immunoanalytical system for the determination of cholera toxin

Cholera, an acute infectious disease associated with water and seafood contamination, is caused by the bacterium Vibrio cholerae, which lives and colonizes in the small intestine and secretes cholera toxin (CT), a causative agent for diarrhea in humans. Based on earlier lateral flow assays, a flow injection liposome immunoanalysis (FILIA) system with excellent sensitivity was developed in this study for the determination of CT at zeptomole levels. Ganglioside (GM1), found to have specific affinity toward CT, was inserted into the phospholipid bilayer during the liposome synthesis. These GM1-sensitized, sulforhodamine B (SRB) dye-entrapping liposomes were used as probes in the FILIA system. Anti-CT antibodies were immobilized in its microcapillary. CT was detected by the formation of a sandwich complex between the immobilized antibody and GM1 liposomes. During the assay, the sample was introduced first into the column, and then liposomes were injected to bind to all CT captured by the antibody in the microcapillary. Subsequently, the SRB dye molecules were released from the bound liposomes via the addition of the detergent octyl glucopyranoside. The released dye molecules were transported to a flow-through fluorescence detector for quantification. The FILIA system was optimized with respect to flow rate, antibody concentration, liposome concentration, and injected sample volume. The calibration curve for CT had a linear range of 10-16 to 10-14 g mL-1. The detection limit of this immunosensor was 6.6 x 10(-17) g mL-1 in 200-microL samples (equivalent to 13 ag or 1.1 zmol).     

Evaluation of internal standards in a competitive nucleic acid sequence-based amplification assay

An end-point quantitative nucleic acid sequence-based amplification (NASBA) reaction with two exogenous internal standards for the detection of the model analyte E. coli clpB mRNA was developed and statistically analyzed. Electrochemiluminescence was chosen as a highly sensitive detection means allowing careful evaluation of the internal standards used. The two internal standards examined had been designed previously using a novel and rapid NASBA-based method. Initially, each standard was used separately in a NASBA reaction; subsequently, two internal standards were added into one reaction at different concentrations. The accuracy and precision of the data obtained were analyzed using linear and multiple regression analysis. In the case of single-standard reactions, the accuracy was >95% and the precision >98.5%. In the case of double-standard reactions, the accuracy increased to >97%. With a single internal standard, 3 orders of magnitude of target sequence could be quantified; using three different concentrations of one internal standard, the dynamic range increased to 5 orders of magnitude. In both cases, a detection limit as low as 0.14 pg of target sequence was obtained. In the case of double-internal standard reactions, a dynamic range with 5 orders of magnitude and a detection limit of 1.76 pg was determined. The high-performance quality of the internal standards was assumed to be in part due to the unique synthesis process using two NASBA reactions rather than traditional cloning techniques.     

Bulk Ceramic Composites Derived from a Preceramic Polysilazane with Alumina and Zirconia Fillers

Bulk composites for the production of green‐machinable, complex‐shaped ceramic parts with tailorable properties for applications under mechanical stress are fabricated by a novel combination of a poly(vinyl)silazane‐derived ceramic matrix with Al₂O₃ or ZrO₂ fillers. Green‐machinable composites were produced by coating of the filler powders with the preceramic polymer, followed by subsequent warm‐pressing. The final ceramic materials are obtained by a thermal conversion process. A systematic variation of preceramic polymer content, pressure during warm‐compaction, or pyrolytic conversion temperature is used to identify the main factors responsible for differences in densification behavior, microstructure, composition, and mechanical properties. The procedure of composite powder preparation as well as the optimization of the subsequent warm‐pressing step are found to be decisive in the successful production of crack‐free bulk ceramic composites.     

Two Defensive Lines in Juvenile Leaf Beetles; Esters of 3-nitropropionic Acid in the Hemolymph and Aposematic Warning

Juveniles of the leaf beetles in subtribe Chrysomelina have efficient defense strategies against predators. When disturbed, they transiently expose volatile deterrents in large droplets from nine pairs of defensive glands on their back. Here, we report on an additional line of defense consisting of the non-volatile isoxazolin-5-one glucoside and its 3-nitropropanoyl ester in the larval hemolymph. Because isoxazolin-5-one derivatives were not detectable in related leaf beetle taxa, they serve as a diagnostic marker for the Chrysomelina subtribe. Conjugation of isotopically labelled 3-nitropropionic acid to isoxazolin-5-one glucoside in vivo demonstrates its function as a carrier for the 3-nitropropanoyl esters. The previous identification of characteristic glucosides as precursors of the volatile deterrents underlines the general importance of glucosides for sequestration from food plants, and the subsequent transport in the hemolymph to the defense system. The combination of repellent volatiles with non-volatile toxic compounds in the hemolymph has the potential to create synergistic effects since the odorant stimulus may help predators learn to avoid some foods. The combination of the two defense lines has the advantage, that the hemolymph toxins provide reliable and durable protection, while the repellents may vary after a host plant change.     

A Method for Viability Testing of Pectobacterium carotovorum in Postharvest Processing by Means of Flow Cytometry

Rapid detection methods such as flow cytometric analysis enable the detection of phytopathogenic and human pathogenic bacteria and hence, the monitoring and optimisation of inactivation processes. The aim of this study was to develop a method for viability testing of the soft-rot-causing Gram-negative bacteria Pectobacterium carotovorum spp. carotovorum by flow cytometry based on a combination of carboxyfluorescein diacetate (cFDA) and propidium iodide. Due to the cell membrane composition of Gram-negative bacteria, the uptake ability of cFDA indicating esterase activity is limited. In this study, an adequate dye concentration (0.83 mM cFDA) and incubation time (45 min) at 37 °C for bacteria suspensions with a theoretical optical density (OD₆₂₀) above 5 were defined, which enables a reliable determination of esterase activity of Gram-negative bacteria. This developed staining procedure was successfully applied to monitor inactivation treatments. It was shown that the test bacteria (Escherichia coli and P. carotovorum) lose their culturability due to the applied thermal treatment, but physiological activities were still detectable using flow cytometry. The remaining physiological activities may still result in product spoilage and may also cause human diseases. For example, E. coli cells still showed esterase activity after 10 min of thermal treatment at 70 °C. The degree of bacterial damage due to the inactivation processes is highly dependent on treatment parameters as well as on treated bacteria.     

Basic Properties of Magnetic Shape-Memory Materials from First-Principles Calculations

The mutual influence of phase transformations, magnetism, and electronic properties of magnetic-shape memory Heusler materials is a basic issue of electronic structure calculations based on density functional theory. In this article, we show that these calculations can be pursued to finite temperatures, which allows to derive on a first-principles basis the temperature versus composition phase diagram of the pseudo-binary Ni-Mn-(Ga, In, Sn, Sb) system. The free energy calculations show that the phonon contribution stabilizes the body-centered-cubic (bcc)-like austenite structure at elevated temperatures, whereas magnetism favors the low-temperature martensite phase with body-centered-tetragonal (bct) or rather face-centered-tetragonal (fct) structure. The calculations also allow to make predictions of magnetostructural and magnetic field induced properties of other (new) magnetic Heusler alloys not based on NiMn such as Co-Ni-(Ga-Zn) and Fe-Co-Ni-(Ga-Zn) intermetallic compounds.     

The time course of name retrieval during multiple-object naming: Evidence from extrafoveal-on-foveal effects.

The goal of the study was to examine whether speakers naming pairs of objects would retrieve the names of the objects in parallel or in sequence. To this end, we recorded the speakers’ eye movements and determined whether the difficulty of retrieving the name of the 2nd object affected the duration of the gazes to the 1st object. Two experiments, which differed in the spatial arrangement of the objects, showed that the speakers looked longer at the 1st object when the name of the 2nd object was easy than when it was more difficult to retrieve. Thus, the easy 2nd-object names interfered more with the processing of the 1st object than the more difficult 2nd-object names. In the 3rd experiment, the processing of the 1st object was rendered more difficult by presenting it upside down. No effect of 2nd-object difficulty on the gaze duration for the 1st object was found. These results suggest that speakers can retrieve the names of a foveated and an extrafoveal object in parallel, provided that the processing of the foveated object is not too demanding. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Enhanced Antiviral Function of Magnesium Chloride-Modified Heparin on a Broad Spectrum of Viruses

Previous studies reported on the broad-spectrum antiviral function of heparin. Here we investigated the antiviral function of magnesium-modified heparin and found that modified heparin displayed a significantly enhanced antiviral function against human adenovirus (HAdV) in immortalized and primary cells. Nuclear magnetic resonance analyses revealed a conformational change of heparin when complexed with magnesium. To broadly explore this discovery, we tested the antiviral function of modified heparin against herpes simplex virus type 1 (HSV-1) and found that the replication of HSV-1 was even further decreased compared to aciclovir. Moreover, we investigated the antiviral effect against the new severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) and measured a 55-fold decreased viral load in the supernatant of infected cells associated with a 38-fold decrease in virus growth. The advantage of our modified heparin is an increased antiviral effect compared to regular heparin.     

“Dialdehyde Cellulose” Nanofibers by Electrospinning as Polyvinyl Alcohol Blends: Manufacture and Product Characterization

A nanofiber was obtained by electrospinning of “dialdehyde cellulose” (periodate-oxidized cellulose, DAC) and polyvinyl alcohol (PVA), using only water as the solvent. Celluloses of four different origins were fully oxidized with sodium periodate to water-soluble DAC. Aqueous solution of DAC showed inadequate spinnability regardless of the polymer concentration and the electrospinning conditions used. Addition of PVA improved the solution's viscoelasticity and, consequently, the solution's spinnability. We examined the effects of DAC/PVA composition and electrospinning parameters on fiber morphology. Highly homogeneous nanofibers were prepared from 1:1 up to 2:1 (weight) DAC/PVA blends while samples of lower viscosity or higher relative DAC contents resulted in continuous, beaded fiber networks. Characterization of the electrospun fabrics revealed a highly crosslinked DAC structure reinforced with PVA, strongly interacting through hemiacetal bonds and hydrogen bonding. Fluorescence labeling confirmed the presence of reactive aldehyde functionalities in the electrospun web. The versatile properties of DAC as reactive material can now be imparted on electrospun fiber and nanofiber material – which was not possible so far –further widening the application scope of this interesting cellulose derivative.