Yuri M. Shlyapnikov

Bacterial production of latarcin 2a, a potent antimicrobial peptide from spider venom

Natural venoms are promising sources of candidate therapeutics including antibiotics. A recently described potent antimicrobial peptide latarcin 2a (Ltc 2a) from Lachesana tarabaevi spider venom shows a broad-spectrum antibacterial activity. This peptide consists of 26 amino acid residues and therefore its production using chemical synthesis, although trivial, is costly. We describe an easy approach to Ltc 2a production in Escherichia coli using the conventional fusion partner thioredoxin. Latarcin 2a synthetic gene was cloned into the expression vector pET-32b, which was then used to transform E. coli BL21(DE3) strain. His-tagged fusion purification was achieved using metal-chelate affinity chromatography. Since no methionine residues are present in the latarcin 2a sequence, cyanogen bromide could be effectively utilized to separate the target product from the carrier protein. Reverse-phase HPLC was used as the final step of purification; the final yield was approximately 3 mg/L of bacterial culture. To increase the yields, we attempted incorporation of Ltc 2a tandem repeats into the fusion protein; however, production rates greatly decreased due to enhanced fusion toxicity. Moreover, we probed constructs to produce an Ltc 2a dimer and the Ltc 2a propeptide to study their functional properties. Recombinant peptides were produced at appreciable yields and biological tests to determine their activities were performed. Latarcin 2a is the first linear peptide from spider venom and one of the first membrane-active peptides from venomous animals to be biosynthetically produced.

Are Reactive Oxygen Species Generated in Electrospray at Low Currents

It was demonstrated that electrospraying (ES) of solvents from a glass capillary proceeds without emission of light provided that the current is kept below a certain critical level (<100 nA at positive potential and <25 nA at negative potential for 96% ethanol; < 40 nA at positive potential for water). Though the onset of corona, as detected by the appearance of light, was always accompanied by a break in the current-voltage slope, such breaks also happened before the onset of corona, so they cannot be used as an adequate indicator of corona ignition. Of four ROS studied (hydrogen peroxide, ozone, hydroxyl radicals, and superoxide anions), only H2O2 and ozone were found to be generated at a current of 150-200 nA in detectable quantities: with a yield of 0.5-1 H2O2 molecules per electron at positive potential and 1.5-3 at negative potential. Despite the low yield of the ROS, jack bean urease was shown to be inactivated when the enzyme solution with a concentration below 20 μg/mL was electrosprayed at a current of 200 nA. Addition of 0.1 mM EDTA totally protected the activity of the electrosprayed urease.

Carboxymethyl cellulose film as a substrate for microarray fabrication.

Magnetic beads (MB) are widely used for quick and highly sensitive signal detection in microarray-based assays. However, this technique imposes stringent requirements for smoothness and adhesive properties of the surface, which most common substrates do not satisfy. We report here a new type of substrate for microarrays with a low adhesion to MB-thermally cross-linked carboxymethyl cellulose (CMC) film. This substrate can be readily fabricated on a conventional glass slide. A highly cross-linked CMC film (∼1 cross-link per monomer unit) possesses a surface smooth on a nanometer scale and a low adhesion to protein-coated MB, which partly originates from electrostatic repulsion of MB from negatively charged CMC surface. The efficiency of the CMC substrate is demonstrated hereby in fabrication of microarrays for the detection of three bacterial toxins: cholera toxin, staphylococcal enterotoxin A, and toxic shock syndrome toxin. The assay employing a primary antibodies arrayed on a CMC surface and detection of the bound bacterial toxins with a biotinylated secondary antibodies and streptavidin-coated MB resulted in a limits of detection as low as 0.1 ng/mL. The CMC-based microarrays demonstrated very high storage stability; their activity did not change after one year storage at room temperature.

Non-invasive lung disease diagnostics from exhaled microdroplets of lung fluid: Perspectives and technical challenges

Abstract The combination of ultra-sensitive assay techniques and recent improvements in the instrumentation used to collect microdroplets of lung fluid (MLF) from exhaled breath has enabled the development of non-invasive lung disease diagnostics that are based on MLF analysis. In one example of this approach, electrospun nylon filters were used to collect MLFs from patients with pulmonary tuberculosis. The filters were washed to obtain liquid probes, which were then tested for human immunoglobulin A (h-IgA) and fractions of h-IgA specific to ESAT-6 and Psts-1, two antigens secreted by Mycobacterium tuberculosis. Probes collected for 10 min contained 100–1500 fg of h-IgA and, in patients with pulmonary tuberculosis, a portion of these h-IgA molecules showed specificity to the secreted antigens. Separate MLFs and their dry residues were successfully collected using an electrostatic collector and impactor developed especially for this purpose. Visualization of MLF dry residues by atomic force microscopy made it possible to estimate the lipid content in each MLF and revealed mucin molecules in some MLFs. This exciting new approach will likely make it possible to detect biomarkers in individual MLFs. MLFs emerging from an infection site (‘hot’ microdroplets) are expected to be enriched with infection biomarkers. This paper discusses possible experimental approaches to detecting biomarkers in single MLFs, as well as certain technological problems that need to be resolved in order to develop new non-invasive diagnostics based on analysing biomarkers in separate MLFs.

Reversible and Irreversible Mechanical Damaging of Large Double-Stranded DNA upon Electrospraying

Electrohydrodynamic spraying (or electrospaying, ES) of DNA solutions is an attractive technique for applications in mass spectrometry, in microarray fabrication, and in generation of DNA nanoaerosols. Here we report how ES affects DNA structure and evaluate possible ways to reduce DNA damage upon ES. It is shown that under any ES conditions, linear λ-phage DNA is subjected to intensive rupture producing a mixture of fragments. In addition to such fragmentation, notable reversible changes in the DNA structure were revealed by a slight increase in DNA electrophoretic mobility. The degree of fragmentation was shown to decrease with decreased DNA length and with increased flow rate through the ES capillary. Fragments shorter than 5 kbp did not show any notable damage upon ES. Both experimental data and theoretical estimations of the forces acting on DNA during ES indicate that DNA is damaged by mechanical forces, and the damage takes place in the vicinity of the Taylor cone tip, presumably due to the high shear stress or/and viscous drag forces operating there. Condensation of λ-DNA with hexamminecobalt(III) ions completely protected it from any damage upon ES.

Ballistic Penetration of Highly Charged Nanoaerosol Particles through a Lipid Monolayer

To be used as a drug, inhaled nanoaerosol particles (NAPs) must first penetrate the lipid layer on top of the lung fluid before they will be able to reach the lung epithelium. We investigated how the penetration of NAPs through a model lipid monolayer (LM) depends upon their charging level and size. It was shown that deposition of NAPs 20-200 nm in diameter and charged to the Rayleigh limit gradually increased the surface tension of a dipalmitoylphosphatidylcholine monolayer (DPPC), indicating a loss of lipid molecules from the monolayer. This phenomenon was reproduced with a variety of NAPs produced from glucose, proteins, and polymers. Transfer of the lipid material into the subphase was documented by direct visualization of lipid nanoparticles in the subphase with atomic force microscopy after deposition of glucose NAPs on a DPPC monolayer, followed by collection of the lipid nanoparticles on a mica surface. Partial restoration of tension upon storage indicates that some of the lipid may return to the monolayer. Experiments with the deposition of highly charged calibrated polystyrene nanoparticles showed that the amount of lipid removed from the surface was roughly proportional to the overall surface area of the deposited NAPs. When the number of charges on the NAPs was reduced from their Rayleigh level of 103-104 units to 1-10 units, no notable changes in monolayer surface tension were observed even with prolonged deposition of such NAPs. It was therefore concluded that only highly charged NAPs of a certain size acquire sufficient speed from their attraction by mirror charges to enable ballistic penetration through a lipid monolayer.

Exposure to bleomycin nanoaerosol does not induce fibrosis in mice

Abstract Bleomycin (BLM), a cytostatic drug widely used in the treatment of cancer, often induces lung fibrosis as a side effect. This phenomenon has been exploited in biomedical studies to create a fibrosis model. Inhalation of BLM nanoaerosol particles (NAPs) was expected to induce severe fibrosis in mice. Instead, inhalation of BLM NAPs for 13 days with a total inhaled dose of ~1 mg/kg did not cause pneumofibrosis in CD-1 mice; only marked pneumonitis with infiltration of macrophages in all parts of the lungs was observed in all eight mice exposed. These changes were notably reversible within 2 weeks of cessation of exposure. Despite the limited number of animals it was confidently concluded that slow administration of BLM in the form of NAPs did not cause fibrosis as the bolus instillation of similar doses does.