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A novel antifungal strategy targeting the inhibition of calcineurin is described. To develop a calcineurin based inhibitor of pathogenic fungi, analogs of FK506 were synthesized that were able to permeate mammalian but not fungal cells. Antagonists in combination with FK506 were not immunosuppressive and retained antifungal activity in A. fumigatus. To reduce the dosage burden of the antagonist, murine oral PK was improved an order of magnitude relative to previous FK506 antagonists.

The utility of acoustic droplet ejection (ADE), originally used to transfer dimethyl sulfoxide (DMSO) solutions, is expanded beyond the transfer of DMSO to a wide variety of aqueous solutions common to biochemical experiments and assays. Aqueous-based liquids are transferred with high precision (coefficient of variation

Acoustic energy can precisely and accurately eject a droplet of liquid from a reservoir, enabling delivery of picoliter and nanoliter volumes (Ellson, R.; Mutz, M.; Browning, B.; Lee, L.; Miller, M.; Papen, R. Transfer of low nanoliter volumes between microplates using focused acoustics—automation considerations. Journal of the Association for Laboratory Automation 2003, 8(5), 29–34). Acoustic droplet ejection has been shown to be extremely precise (coefficients of variation < 2%) over a wide range of dispensed volumes (Ellson, R.; Mutz, M.; Browning, B.; Lee, L.; Miller, M.; Papen, R. Transfer of low nanoliter volumes between microplates using focused acoustics—automation considerations. Journal of the Association for Laboratory Automation 2003, 8(5), 29–34). However, measuring the performance of low-volume fluid transfers can be difficult because the data are often masked by variability in bulk dispensers and fluorescence readers used as part of the overall measurement process (Petersen, J.; Nguyen, J. Comparison of absorbance and fluorescence methods for determining liquid dispensing precision. Journal of the Association for Laboratory Automation 2005, 10(2), 82–87; Rhode, H.; Schulze, M.; Renard, S.; Zimmerman, P.; Moore, T.; Cumme, G.; Horn, A. An improved method for checking HTS/uHTS liquid handling systems. Journal of Biomolecular Screening 2004, 9, 726–733). The fluorophore used must also be stable so that thermal bleaching and photobleaching do not contribute additional variability to the measurements. This study assesses the suitability of fluorescein to measure the precision of fluid transfers of 2.5-nL DMSO droplets. The short-term and long-term stabilities of fluorescein are first qualified using a reference standard. Next, we determine the noise contribution of the filler and reader. Lastly, data are presented for the precision of 5- and 50-nL fluid transfers using this fluorescein measurement process. (JALA 2006;11:233–9)

Acoustic auditing is a non-destructive, non-invasive technique to monitor the composition and volume of fluids in open or sealed microplates and storage tubes. When acoustic energy encounters an interface between two materials, some of the energy passes through the interface, while the remainder is reflected. Acoustic energy applied to the bottom of a multi-well plate or a storage tube is reflected by the fluid contents of the microplate or tube. The amplitude of these reflections or echoes correlates directly with properties of the fluid, including the speed of sound and the concentration of water in the fluid. Once the speed of sound in the solution is known from the analysis of these echoes, it is easy to determine the depth of liquid and, thereby, the volume by monitoring how long it takes for sound energy to reflect off the fluid meniscus. This technique is rapid (100,000 samples per day), precise (1% coefficient of variation for hydration measurements,4% coefficient of variation for volume measurements), and robust. It does not require uncapping tubes or unsealing or unlidding microplates. The sound energy is extremely gentle and has no deleterious impact upon the fluid or compounds dissolved in it.

Acoustic droplet ejection (ADE) gently and precisely aliquots nanoliter and picoliter liquid volumes without any physical contact with the solution being transferred. The technology is very automation-friendly, as it is compatible with conventional microplates. Focused energy from an acoustic transducer induces droplet ejection into an inverted standard microplate. The commercial system transfers low-nanoliter volumes of dimethyl sulfoxide–dissolved compound libraries and thereby enables cell-based assays to be performed in 1536-well plates.

Cryptococcosis is one of the most important invasive fungal infections and is a significant contributor to the mortality associated with HIV/AIDS. As part of our program to repurpose molecules related to the selective estrogen receptor modulator (SERM) tamoxifen as anti-cryptococcal agents, we have explored the structure-activity relationships of a set of structurally diverse SERMs and tamoxifen derivatives. Our data provide the first insights into the structural requirements for the antifungal activity of this scaffold. Three key molecular characteristics affecting anti-cryptococcal activity emerged from our studies: 1) the presence of an alkylamino group tethered to one of the aromatic rings of the triphenylethylene core; 2) an appropriately sized aliphatic substituent at the 2 position of the ethylene moiety; and 3) electronegative substituents on the aromatic rings modestly improved activity. Using a cell-based assay of calmodulin antagonism, we found that the anti-cryptococcal activity of the scaffold correlates with calmodulin inhibition. Finally, we developed a homology model of C. neoformans calmodulin and used it to rationalize the structural basis for the activity of these molecules. Taken together, these data and models provide a basis for the further optimization of this promising anti-cryptococcal scaffold.

The invention relates to methods and devices that use focused radiation to handle and/or monitor pathogenic fluids. In particular, a method is provided for dispensing one or more droplets of a fluid containing a pathogen. The method involves providing the pathogen-containing fluid in a reservoir and applying focused radiation to the pathogen-containing fluid in the reservoir in a manner effective to eject a droplet of the fluid therefrom. Often, a pathogen-impermeable enclosure is used.

The invention provides a device for controlling the composition and/or volume of a fluid within a reservoir. The device includes a reservoir adapted to contain at least one fluid, a means for monitoring a characteristic of the fluid contained in the reservoir, and a means for introducing additional fluid into the reservoir according to the fluid characteristic monitored by the monitoring means. The device also includes an acoustic generator for generating acoustic radiation. A dispensing means may be provided as well. Other devices and methods that use acoustic radiation to control the composition and/or volume of a fluid within a reservoir are included.

The present invention provides a method and device for the acoustic ejection of fluid droplets from each of a plurality of fluid-containing reservoirs. The droplets are ejected toward sites on a substrate surface for deposition thereon. The device is comprised of: a plurality of reservoirs each adapted to contain a fluid; an ejector comprising a means for generating acoustic radiation and a means for focusing the generated acoustic radiation so as to eject fluid droplets from the reservoir fluids; and a means for positioning the ejector in acoustically coupled relationship to each of the reservoirs. The invention is useful in a number of contexts, particularly in the preparation of biomolecular arrays.