Gary S. Kath

Enhanced in vivo transgene expression and immunogenicity from plasmid vectors following electrostimulation in rodents and primates

Safe and efficient methods for in vivo delivery of transgenes of interest must be developed so that the promise of these therapies can be practically used in the clinic. In this work, we describe the use of electrostimulation to enhance the in vivo efficiency of plasmid DNA delivery. The method was optimized to work over a range of moderate frequencies, utilizing low field strengths and simple symmetrical waveforms. After studying several parameters of delivery in mice, we demonstrate how this methodology can be employed to significantly improve both gene expression (over 16-fold) and the immunogenicity of HIV-1 vaccines (over 28-fold) compared to naked DNA in non-human primates. Compared to an efficient viral Ad5 vector system, the gene expression levels of DNA+electrostimulation were surprisingly within a factor of four of the viral delivery system.

Automated rodent in situ muscle contraction assay and myofiber organization analysis in sarcopenia animal models

Age-related sarcopenia results in frailty and decreased mobility, which are associated with increased falls and long-term disability in the elderly. Given the global increase in lifespan, sarcopenia is a growing, unmet medical need. This report aims to systematically characterize muscle aging in preclinical models, which may facilitate the development of sarcopenia therapies. Naïve rats and mice were subjected to noninvasive micro X-ray computed tomography (micro-CT) imaging, terminal in situ muscle function characterizations, and ATPase-based myofiber analysis. We developed a Definiens (Parsippany, NJ)-based algorithm to automate micro-CT image analysis, which facilitates longitudinal in vivo muscle mass analysis. We report development and characterization of translational in situ skeletal muscle performance assay systems in rat and mouse. The systems incorporate a custom-designed animal assay stage, resulting in enhanced force measurement precision, and LabVIEW (National Instruments, Austin, TX)-based algorithms to support automated data acquisition and data analysis. We used ATPase-staining techniques for myofibers to characterize fiber subtypes and distribution. Major parameters contributing to muscle performance were identified using data mining and integration, enabled by Labmatrix (BioFortis, Columbia, MD). These technologies enabled the systemic and accurate monitoring of muscle aging from a large number of animals. The data indicated that longitudinal muscle cross-sectional area measurement effectively monitors change of muscle mass and function during aging. Furthermore, the data showed that muscle performance during aging is also modulated by myofiber remodeling factors, such as changes in myofiber distribution patterns and changes in fiber shape, which affect myofiber interaction. This in vivo muscle assay platform has been applied to support identification and validation of novel targets for the treatment of sarcopenia.

Synthesis of di- and trisubstituted guanidines on multivalent soluble supports

Abstract A library of 48 di- and trisubstituted guanidines was synthesized on a soluble, tetravalent support. Support-bound intermediates and cleaved products were isolated in parallel by size exclusion chromatography using a semiautomated system. Products were generally obtained in good yield and purity.

Non-invasive muscle contraction assay to study rodent models of sarcopenia

BackgroundAge-related sarcopenia is a disease state of loss of muscle mass and strength that affects physical function and mobility leading to falls, fractures, and disability. The need for therapies to treat age-related sarcopenia has attracted intensive preclinical research. To facilitate the discovery of these therapies, we have developed a non-invasive rat muscle functional assay system to efficiently measure muscle force and evaluate the efficacy of drug candidates.MethodsThe lower leg muscles of anesthetized rats are artificially stimulated with surface electrodes on the knee holders and the heel support, causing the lower leg muscles to push isometric pedals that are attached to force transducers. We developed a stimulation protocol to perform a fatigability test that reveals functional muscle parameters like maximal force, the rate of fatigue, fatigue-resistant force, as well as a fatigable muscle force index. The system is evaluated in a rat aging model and a rat glucocorticoid-induced muscle loss modelResultsThe aged rats were generally weaker than adult rats and showed a greater reduction in their fatigable force when compared to their fatigue-resistant force. Glucocorticoid treated rats mostly lost fatigable force and fatigued at a higher rate, indicating reduced force from glycolytic fibers with reduced energy reserves.ConclusionsThe involuntary contraction assay is a reliable system to assess muscle function in rodents and can be applied in preclinical research, including age-related sarcopenia and other myopathy.

Development of an integrated semi-automated system for in vitro pharmacodynamic modelling

OBJECTIVES The aim of this study was to develop an integrated system for in vitro pharmacodynamic modelling of antimicrobials with greater flexibility, easier control and better accuracy than existing in vitro models. METHODS Custom-made bottle caps, fittings, valve controllers and a modified bench-top shaking incubator were used. A temperature-controlled automated sample collector was built. Computer software was developed to manage experiments and to control the entire system including solenoid pinch valves, peristaltic pumps and the sample collector. The system was validated by pharmacokinetic simulations of linezolid 600 mg infusion. The antibacterial effect of linezolid against multiple Staphylococcus aureus strains was also studied in this system. RESULTS An integrated semi-automated bench-top system was built and validated. The temperature-controlled automated sample collector allowed unattended collection and temporary storage of samples. The system software reduced the labour necessary for many tasks and also improved the timing accuracy for performing simultaneous actions in multiple parallel experiments. The system was able to simulate human pharmacokinetics of linezolid 600 mg intravenous infusion accurately. A pharmacodynamic study of linezolid against multiple S. aureus strains with a range of MICs showed that the required 24 h free drug AUC/MIC ratio was approximately 30 in order to keep the organism counts at the same level as their initial inoculum and was about > or = 68 in order to achieve > 2 log(10) cfu/mL reduction in the in vitro model. CONCLUSIONS The integrated semi-automated bench-top system provided the ability to overcome many of the drawbacks of existing in vitro models. It can be used for various simple or complicated pharmacokinetic/pharmacodynamic studies efficiently and conveniently.

Prescreening bacterial colonies for bioactive molecules with Janus plates, a SBS standard double-faced microbial culturing system

Despite the availability of many culture-based antibiotic screening methods, the lack of sensitive automated methods to identify functional molecules directly from microbial cells still limits the search for new biologically active compounds. The effectiveness of antibiotic detection is influenced by the solubility of the assayed compounds, indicator strain sensitivity, culture media and assay configuration. We describe a qualitative high throughput screening system for detecting cell-perturbing molecules from bacterial colonies employing two opposed agar layers sequentially formed in prototype Society for Biomolecular Screening (SBS) plates, named Janus plates. Direct assay of microbial colonies against target organisms in opposed agar layers overcomes some of the limitations of agar overlay methods. The system enables the rapid detection of extracellular cell-perturbing molecules, e.g., antibiotics, excreted directly from environmental isolates. The source bacterial colonies remain separate from the target organism. The growth layer is prepared and grown independently, so environmental strains can be grown for longer intervals, at temperatures and in media that favor their growth and metabolite expression, while the assay layer with pathogens, usually requiring nutrient-rich medium and elevated temperatures, are added later. Colonies to be tested can be precisely arrayed on the first agar surface, thus avoiding dispersion and disturbance of potential antibiotic-producing colonies by overlaying agar with the target strain. The rectangular SBS configuration facilitates factorial replication of dense microbial colony arrays for testing with multiple assays and assay conditions employing robotic colony pickers and pin tools. Opposed agar layers only slightly reduced the effectiveness for detecting growth inhibition from pure antibiotics compared to single-layer agar diffusion assays. The Janus plate enabled an automation-assisted workflow where a lone operator can effectively identify and accumulate bioactive soil bacterial strains within a few weeks. We also envisage the method’s utility for functional prescreening colonies of clones from genomic and metagenomic libraries or improved strains originating from mutagenized cells.

Automated Agar Plate Streaker: A Linear Plater on Society for Biomolecular Sciences Standard Plates

Several protocols for bacterial isolation and techniques for aerobic plate counting rely on the use of a spiral plater to deposit concentration gradients of microbial suspensions onto a circular agar plate to isolate colony growth. The advantage of applying a gradient of concentrations across the agar surface is that the original microbiological sample can be applied at a single concentration rather than as multiple serial dilutions. The spiral plater gradually dilutes the sample across a compact area and therefore saves time preparing dilutions and multiple agar plates. Commercial spiral platers are not automated and require manual sample loading. Dispensing of the sample volume and rate of gradients are often very limited in range. Furthermore, the spiral sample application cannot be used with rectangular microplates. Another limitation of commercial spiral platers is that they are useful only for dilute, filtered suspensions and cannot plate suspensions of coarse organic particles therefore precluding the use of many kinds of microorganism-containing substrata. An automated agar plate spreader capable of processing 99 rectangular microplates in unattended mode is described. This novel instrument is capable of dispensing discrete volumes of sample in a linear pattern. It can be programmed to dispense a sample suspense at a uniform application rate or across a decreasing concentration gradient.

Automatic sample loader for LKB model 9000 mass spectrometer

An automatic sample loading system is designed for use with the LKB model 9000 mass spectrometer. The apparatus permits solid samples previously placed in glass vials to be automatically entered one by one into the jet separator of the mass spectrometer for analysis. An 8085 based microcomputer controls the movement of the sample into the vacuum, positions the sample in a heating zone for vaporization, initiates a number of mass scan measurements, dumps the sample when complete, and prompts the data system for the next sample. The apparatus can analyze up to 50 samples with no operator intervention.

FIZICS: Fluorescent Imaging Zone Identification System, A Novel Macro Imaging System

Constantly improving biological assay development continues to drive technological requirements. Recently, a specification was defined for capturingwhite light and fluorescent images of agar plates ranging in size from theNUNCOmnitray (96-well footprint, 128 · 85 mm) to the NUNC Bio Assay Dish (245 · 245 mm). An evaluation of commercially available products failed to identify any systemcapable of fluorescentmacroimaging with discretewavelength selection. To address the lack of a commercially available system, a customimaging systemwas designed and constructed. This systemprovides the same capabilities ofmany commercially available systemswith the added ability to fluorescently image up to a 245 · 245mmarea using wavelengths in the visible light spectrum.

High‐performance liquid chromatography fraction marker‐timer controller

A fraction marker‐timer controller has been designed that partially automates peak collection and provides event marking during high‐performance liquid chromatography of partially purified samples having complex chromatograms. The device is activated via a pushbutton switch when a peak of interest elutes. The apparatus places an event mark on the detector’s chart recording, provides a time delay to account for detector‐to‐fraction‐collector dead volume, and then advances a fraction collector to capture the peak in a test tube. If more than one peak elutes in the dead volume window, up to three independent time delays may be triggered.