No abstract

Meningitis and encephalitis are leading global causes of central nervous system (CNS) disability and mortality. Current diagnostic workflows remain inefficient, requiring costly pathogen-specific assays and sometimes invasive surgical procedures.

The molecular chaperone, Hsp104, is a hexameric protein belonging to the triple-A ATPase family (1). Along with its roles in protein folding and conferring thermotolerance, this chaperone is essential for propagation of prions in budding yeast. Prions are infectious proteins that occur in two conformations, a properly folded non-infectious conformation and a misfolded infectious amyloid conformation (2). Yeast has more than a dozen prions, which pass from mother to daughter cells as prion seeds. The steady-state number of prion seeds is maintained by Hsp104, which severs the seeds; this severing reaction is dependent on the ATPase activity of Hsp104 and involves the presentation of the prion to Hsp104 by the molecular chaperones, Sis1 and Ssa1/Ssa2, followed by threading of the prions through the Hsp104 channel (3). Yeast prions are cured by inhibiting the ATPase activity of Hsp104; this inhibition can be caused by either overexpressing a dominant-negative Hsp104 mutant or by inactivating Hsp104 with guanidine. Curing is then caused by absence of prion seed severing, in combination with dilution of the seeds by cell division (4 -6).Surprisingly A red/white colony assay is the standard method of detecting these two conformations. In this assay, yeast having nonsense mutations in the adenine synthesis pathway are plated on limiting adenine medium. Another method of detecting these two conformations is by fluorescence imaging using GFP-labeled Sup35. Using these methods, we previously showed that the curing of [PSI ϩ ] by overexpression of Saccharomyces cerevisiae Hsp104 (Sc-Hsp104) requires a new activity of Hsp104 called trimming (13). Like severing, trimming reduces the size of the prion seeds. However, unlike severing, trimming, which may occur through removal of Sup35 molecules from the ends of the prion fibers, is not accompanied by an increase in seed number. The dissolution of the prion seeds caused by overexpression of Sc-Hsp104 was shown to require the trimming activity of Hsp104. Mutants of Hsp104 that cannot trim also cannot cure [PSI ϩ ] prion by overexpression (14). For example, overexpression of the T160M point mutant of Sc-Hsp104 that lacks trimming activity but has severing activity comparable with wildtype Sc-Hsp104 does not cure [PSI ϩ ]. Consistent with our in vivo observations, in vitro evidence for dissolution of the prion seeds by Hsp104 has been obtained by Shorter and Lindquist (15). Other chaperones and cochaperones such as Hsp70, Sti1,

Rapid bacterial identification remains a critical challenge in infectious disease diagnostics. We developed a novel molecular approach to detect and identify a wide diversity of bacterial pathogens in a single, simple assay, exploiting the conservation, abundance, and rich phylogenetic content of ribosomal RNA in a rapid fluorescent hybridization assay that requires no amplification or enzymology. Of 117 isolates from 64 species across 4 phyla, this assay identified bacteria with >89% accuracy at the species level and 100% accuracy at the family level, enabling all critical clinical distinctions. In pilot studies on primary clinical specimens, including sputum, blood cultures, and pus, bacteria from 5 different phyla were identified.