Viral Infectivity Quantification and Neutralization Assays Using Laser Force Cytology.

Despite the widespread need to assess cell-based viral infectivity during vaccine development and production, as well as viral clearance monitoring and adventitious agent testing for viral safety, traditional methods, including the end-point dilution assay (TCID50) and viral plaque assay, are slow, labor-intensive, and can vary depending upon the skill and experience of the user. LumaCyte's Radiance instrument uses Laser Force Cytology (LFC), a combination of advanced optics and microfluidics, to rapidly analyze the viral infectivity of cell populations in a quantitative fashion. LFC applies optical and fluidic forces to single cells in order to measure their intrinsic biophysical and biochemical properties without the use of stains, antibodies or fluorescent labels.

Rapid quantification of vesicular stomatitis virus in Vero cells using Laser Force Cytology.

The ability to rapidly and accurately determine viral infectivity can help improve the speed of vaccine product development and manufacturing. Current methods to determine infectious viral titers, such as the end-point dilution (50% tissue culture infective dose, TCID50) and plaque assays are slow, labor intensive, and often subjective. In order to accelerate virus quantification, Laser Force Cytology (LFC) was used to monitor vesicular stomatitis virus (VSV) infection in Vero (African green monkey kidney) cells.

CHO-Omics Review: The Impact of Current and Emerging Technologies on Chinese Hamster Ovary Based Bioproduction.

CHO cells are the most prevalent platform for modern bio-therapeutic production. Currently, there are several CHO cell lines used in bioproduction with distinct characteristics and unique genotypes and phenotypes. These differences limit advances in productivity and quality that can be achieved by the most common approaches to bioprocess optimization and cell line engineering.

Construction and Analysis of Two Genome-Scale Deletion Libraries for Bacillus subtilis.

A systems-level understanding of Gram-positive bacteria is important from both an environmental and health perspective and is most easily obtained when high-quality, validated genomic resources are available. To this end, we constructed two ordered, barcoded, erythromycin-resistance- and kanamycin-resistance-marked single-gene deletion libraries of the Gram-positive model organism, Bacillus subtilis. The libraries comprise 3,968 and 3,970 genes, respectively, and overlap in all but four genes.

The Bacillus subtilis GntR family repressor YtrA responds to cell wall antibiotics.

The transglycosylation step of cell wall synthesis is a prime antibiotic target because it is essential and specific to bacteria. Two antibiotics, ramoplanin and moenomycin, target this step by binding to the substrate lipid II and the transglycosylase enzyme, respectively. Here, we compare the ramoplanin and moenomycin stimulons in the Gram-positive model organism Bacillus subtilis.

Reduction in membrane phosphatidylglycerol content leads to daptomycin resistance in Bacillus subtilis.

Daptomycin (DAP) is a cyclic lipopeptide that disrupts the functional integrity of the cell membranes of Gram-positive bacteria in a Ca(2+)-dependent manner. Here we present genetic, genomic, and phenotypic analyses of an evolved DAP-resistant isolate, Dap(R)1, from the model bacterium Bacillus subtilis 168. Dap(R)1 was obtained by serial passages with increasing DAP concentrations, is 30-fold more resistant than the parent strain, and displays cross-resistance to vancomycin, moenomycin, and bacitracin.

Genetic analysis of factors affecting susceptibility of Bacillus subtilis to daptomycin.

Daptomycin is the first of a new class of cyclic lipopeptide antibiotics used against multidrug-resistant, gram-positive pathogens. The proposed mechanism of action involves disruption of the functional integrity of the bacterial membrane in a Ca(2+)-dependent manner. We have used transcriptional profiling to demonstrate that treatment of Bacillus subtilis with daptomycin strongly induces the lia operon including the autoregulatory LiaRS two-component system (homologous to Staphylococcus aureus VraSR).

In vitro mutagenesis of Bacillus subtilis by using a modified Tn7 transposon with an outward-facing inducible promoter.

A Tn7 donor plasmid, pTn7SX, was constructed for use with the model gram-positive bacterium Bacillus subtilis. This new mini-Tn7, mTn7SX, contains a spectinomycin resistance cassette and an outward-facing, xylose-inducible promoter, thereby allowing for the regulated expression of genes downstream of the transposon. We demonstrate that mTn7SX inserts are obtained at a high frequency and occur randomly throughout the B.

Regulatory overlap and functional redundancy among Bacillus subtilis extracytoplasmic function sigma factors.

Bacillus subtilis encodes seven extracytoplasmic function (ECF) sigma factors that regulate partially overlapping regulons related to cell envelope homeostasis and antibiotic resistance. Here, we investigated their physiological role by constructing a mutant set of single, double, triple, and quadruple ECF sigma factor deletions in the undomesticated B. subtilis strain NCIB3610.