Viral Quantitative Capillary Electrophoresis for Counting and Quality Control of RNA Viruses

The world of health care has witnessed an explosive boost to its capacity within the past few decades due to the introduction of viral therapeutics to its medicinal arsenal. As a result, a need for new methods of viral quantification has arisen to accommodate this rapid advancement in virology and associated requirements for efficiency, speed, and quality control. In this work, we apply viral quantitative capillary electrophoresis (viral qCE) to determine (i) the number of intact virus particles (ivp) in viral samples, (ii) the amount of DNA contamination, and (iii) the degree of viral degradation after sonication, vortexing, and freeze–thaw cycles. This quantification method is demonstrated on an RNA-based vesicular stomatitis virus (VSV) with oncolytic properties. A virus sample contains intact VSV particles as well as residual DNA from host cells, which is regulated by WHO guidelines, and may include some carried-over RNA. We use capillary zone electrophoresis with laser-induced fluorescent detection to separate intact virus particles from DNA and RNA impurities. YOYO-1 dye is used to stain all DNA and RNA in the sample. After soft lysis of VSV with proteinase K digestion of viral capsid and ribonucleoproteins, viral RNA is released. Therefore, the initial concentration of intact virus is calculated based on the gain of a nucleic acid peak and an RNA calibration curve. After additional NaOH treatment of the virus sample, RNA is hydrolyzed leaving residual DNA only, which is also calculated by a DNA calibration curve made by the same CE instrument. Viral qCE works in a wide dynamic range of virus concentrations from 10⁸ to 10¹³ ivp/mL. It can be completed in a few hours and requires minimum optimization of CE separation

The anticancer activity of diethyldithiocarbmamate (DDC) is dependent on copper.

<p><b>(A)</b> Disulfiram is metabolized to diethyldithiocarbamate (DDC) and DDC complexes with Copper (Cu) (II). <b>(B)</b> Cytotoxicity curves for DSF (●) and DSF + CuSO₄ (■) were obtained with the IN CELL Analyzer using U87 glioblastoma cells where cell viability was assessed based on loss of plasma membrane integrity 72 hours following treatment; i.e. total cell count and dead cell count were determined using Hoechst 33342 and ethidium homodimer staining, respectively. <b>(C)</b> Cytotoxicity curves for DDC (●) and DDC + CuSO₄ (■); where cytotoxicity was measured as described above. <b>(D)</b> DDC and Cu(DDC)₂ IC₅₀ for U251, MDA-231-BR, and A549 cancer cell lines as well as HBEcP a normal cell line; averages (±SEM) are reported from three separate experiments each done in triplicate. <b>(E)</b> Photograph of DDC, CuSO₄ and Cu(DDC)₂ solutions in water.</p

Preliminary tolerability and plasma elimination profiles for liposomal formulations of Cu(DDC)₂, Cu(CQ)₂, CuQu and CuCX-5461 after intravenous injection into CD-1 mice.

<p>Mice were injected with a single dose of 15 mg/kg Cu(DDC)₂ (-●-), 30mg/kg Cu(CQ)₂ (-■-), 70mg/kg CuQu (-▲-) and 50 mg/kg Cu-CX-5461 (-▼-). <b>(A)</b> Changes in body weight following administration of the indicated liposomal formulation where body weights were measured over 14 days after injection (n = 3). <b>(B)</b> Preliminary plasma elimination profiles of the indicated liposomal formulations where the copper-complexed compound was measured at 1, 4, 8 and 24 hrs after administration (n = 4); concentrations were measured using HPLC or AAS as described in the Methods.</p

Diethyldithiocarbamate (DDC) loading into DSPC/Chol (55:45) liposomes prepared with encapsulated 300 mM CuSO₄.

<p><b>(A)</b> Photograph of solutions consisting of DDC (5mg/mL) and added to CuSO₄-containing DSPC/Chol (55:45) liposomes (20 mM liposomal lipid) over a 1 hour at 25°C. <b>(B)</b> Formation of Cu(DDC)₂ inside DSPC/Chol liposomes (20 mM) as a function of time over 1 hour at 4(●), 25(■) and 40(▲)°C following addition of DDC at a final DDC concentration of (5 mM); Cu(DDC)₂ was measured using a UV-Vis spectrophotometer and lipid was measured using scintillation counting. <b>(C)</b> Cu(DDC)₂ formation inside DSPC/Chol (55:45) liposomes over time where the external pH was 7.4 (▲) and 3.5 (▼). <b>(D)</b> Measured Cu(DDC)₂ as a function of increasing DDC added, represented as the theoretical Cu(DDC)₂ to total liposomal lipid ratio; where the lipid concentration was fixed at 20 mM and final DDC concentration was varied. <b>(E)</b> Cryo-electron microscopy photomicrograph of CuSO₄- containing DSPC/Chol (55:45) liposomes and the same liposomes after formation of encapsulated Cu(DDC)₂. <b>(F)</b> Size of the CuSO₄- containing liposomes and liposomes with encapsulated Cu(DDC)₂ as determined by quasi-electric light scattering and cryo-electron microscopy; data points are given as mean ± SD.</p

Characterization of copper-complex loading method.

<p><b>(A)</b> Measured (AAS) copper to liposomal lipid ratio (black bars) compared to measured Cu(DDC)₂ (UV-Vis spectrophotometer) to liposomal lipid ratio (grey bars) after DDC was added to CuSO₄-containing DSPC/Chol liposomes prepared with different amounts of DSPE-PEG₂₀₀₀ (ranging from 0 to 5 mole%). <b>(B)</b> Formation of Cu(DDC)₂ inside CuSO₄-containing DSPC/Chol liposomes as a function of the CuSO₄ concentration used to prepare the liposomes (ranging from 0 to 300 mM); where the measured copper (AAS) to liposomal lipid ratio (black bar) is compared to the measured Cu(DDC)₂ (UV-Vis spectrophotometer) to liposomal lipid ratio (grey bar). <b>(C)</b> Linear regression analysis comparing measured (AAS) copper concentration (assuming encapsulated copper was free in solution) to measured Cu(DDC)₂ (UV-Vis spectrophotometer) concentration (assuming encapsulated Cu(DDC)₂ was free in solution); R² = 0.9754; each data point represents a mean ± SEM determined from at least three separate experiments done in duplicate.</p

Development of Bacteriostatic DNA Aptamers for Salmonella

<i>Salmonella</i> is one of the most dangerous and common food-borne pathogens. The overuse of antibiotics for disease prevention has led to the development of multidrug resistant <i>Salmonella</i>. Now, more than ever, there is a need for new antimicrobial drugs to combat these resistant bacteria. Aptamers have grown in popularity since their discovery, and their properties make them attractive candidates for therapeutic use. In this work, we describe the selection of highly specific DNA aptamers to <i>S. enteritidis</i> and <i>S. typhimurium</i>. To evolve species-specific aptamers, twelve rounds of selection to live <i>S. enteritidis</i> and <i>S. typhimurium</i> were performed, alternating with a negative selection against a mixture of related pathogens. Studies have shown that synthetic pools combined from individual aptamers have the capacity to inhibit growth of <i>S. enteritidis</i> and <i>S. typhimurium</i> in bacterial cultures; this was the result of a decrease in their membrane potential