Frank Tao

Gangliosides are important signaling molecules in the cell membrane and are processed by several enzymes. Deficiencies in these enzymes can cause human lysosomal storage diseases. Building an understanding of the pathways of glycosphingolipid catabolism requires methods for the analysis of these enzymatic activities A GM3-derived FRET probe was synthesized chemoenzymatically for the detection and quantitation of a range of ganglioside-degrading enzymes, both in cell lysates and in living cells. This is the first substrate that enables the ratiometric fluorogenic assay of sphingolipid ceramide N-deacylase and endoglycoceramidase and can detect and localize neuraminidase activity in living cells. It is therefore a valuable tool for building a better understanding of membrane-confined enzymology. It also enables the robust and reliable assay of ganglioside-degrading enzymes in a microtiter plate, thus opening the door to screening for novel or engineered biocatalysts or for new inhibitors.

Clear-cut: A small-molecule FRET probe was designed and synthesized for monitoring at least three key enzymatic activities involved in ganglioside degradation. The substrate, which contains BODIPY (Fluor) and Coumarin (Coum) fluorophores at opposite termini, enables sensitive fluorogenic assay in both cell lysates and living cells and should be useful for dissecting the mechanisms of these enzymes, as well as for protein engineering and inhibitor development.

Current methods for the detection of Mycobacterium tuberculosis (Mtb) are either time consuming or require expensive instruments and are thus are not suitable for point-of-care diagnosis. The design, synthesis, and evaluation of fluorogenic probes with high specificity for BlaC, a biomarker expressed by Mtb, are described. The fluorogenic probe CDG-3 is based on cephalosporin with substitutions at the 2 and 7 positions and it demonstrates over 120 000-fold selectivity for BlaC over TEM-1 Bla, the most common β-lactamase. CDG-3 can detect 10 colony-forming units of the attenuated Mycobacterium bovis strain BCG in human sputum in the presence of high levels of contaminating β-lactamases expressed by other clinically prevalent bacterial strains. In a trial with 50 clinical samples, CDG-3 detected tuberculosis with 90 % sensitivity and 73 % specificity relative to Mtb culture within one hour, thus demonstrating its potential as a low-cost point-of-care test for use in resource-limited areas.

TB or not TB? A fluorogenic probe (CDG-3) was developed for BlaC, a biomarker expressed by Mycobacterium tuberculosis (Mtb). CDG-3 is based on cephalosporin with substitutions at both the 2 and 7 positions and it demonstrates over 120 000-fold selectivity for BlaC over the common β-lactamase TEM-1 Bla. This rapid, low cost, sensitive, and selective method shows great potential for point-of-care tuberculosis (TB) testing in resource-limited settings.