The target of rapamycin (TOR) proteins regulate various cellular processes including autophagy1, which may play a protective role in certain neurodegenerative and infectious diseases2. Here we show that a primary small-molecule screen in yeast yields novel small-molecules modulators of mammalian autophagy. We first identified novel small-molecule enhancers (SMER) and inhibitors (SMIR) of the cytostatic effects of rapamycin in Saccharomyces cerevisiae. Three SMERs induced autophagy independently of rapamycin in mammalian cells, enhancing the clearance of autophagy substrates like mutant huntingtin and A53T α-synuclein, associated with Huntington's disease (HD) and familial Parkinson's disease, respectively3-5. These SMERs, which appear to act either independently, or downstream, of TOR, attenuated mutant huntingtin-fragment toxicity in HD cell and Drosophila models, suggesting therapeutic potential. We also screened structural analogs of these SMERs and identified additional candidate drugs enhancing autophagy. Thus, we have demonstrated proof-of-principle for a novel approach for discovery of small-molecule modulators of mammalian autophagy.The autophagy/lysosome and ubiquitin/proteasome pathways are the two major routes for protein clearance in eukaryotic cells. Proteasomes predominantly degrade short-lived nuclear and cytosolic proteins, which need to be unfolded to pass through the narrow pore of the proteasome barrel, precluding clearance of large membrane proteins and protein complexes (including oligomers and aggregates). Mammalian lysosomes, on the other hand, can degrade substrates like protein complexes and organelles. The bulk degradation of cytoplasmic proteins or organelles is largely mediated by macroautophagy, generally referred to as autophagy1. It involves the formation of double-membrane structures called 7 Joint corresponding authors. Correspondence to D.C. Rubinsztein -E-mail: dcr1000@cam.ac.uk , Telephone: (0)1223 762608, Fax: (0)1223 331206; S.L. Schreiber -E-mail: stuart_schreiber@harvard.edu. 6 These authors contributed equally to this work. Competing financial interestsThe authors declare no competing financial interests. Europe PMC Funders Group Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts autophagosomes/autophagic vacuoles (AVs), which fuse with lysosomes to form autolysosomes (also called autophagolysosomes) where their contents are then degraded by acidic lysosomal hydrolases. Autophagosomes are generated by elongation of small membrane structures, whose precise origins have yet to be elucidated1. Autophagy can be induced under physiological stress conditions such as starvation. Several protein kinases regulate autophagy, the best characterised being the mammalian target of rapamycin (mTOR), which negatively regulates the pathway in organisms from yeast to man1. However, the targets of mTOR-dependent and -independent signalling in the autophagy apparatus are not well understood in mammalian systems. Recently, we described an mTOR-independent path...

This new edition of the universally acclaimed and widely-used textbook on fungal biology has been completely re-written, drawing directly on the authors' research and teaching experience. The text takes account of the rapid and exciting progress that has been made in the taxonomy, cell and molecular biology, biochemistry, pathology and ecology of the fungi. Features of taxonomic relevance are integrated with natural functions, including their relevance to human affairs. Special emphasis is placed on the biology and control of human and plant pathogens, providing a vital link between fundamental and applied mycology. The book is richly illustrated throughout with specially prepared drawings and photographs, based on living material. Illustrated life-cycles are provided, and technical terms are clearly explained. Extensive reference is made to recent literature and developments, and the emphasis throughout is on whole-organism biology from an integrated, multidisciplinary perspective.

To facilitate genus and species level identification of a broad range of bacteria without the requirement of presumptive identification, we have developed a unified set of primers and polymerase chain reaction conditions to amplify spacer regions between the 16S and 23S genes in the prokaryotic rRNA genetic loci. Spacer regions within these loci show a significant level of length and sequence polymorphism across both genus and species lines. A generic pair of priming sequences was selected for the amplification of these polymorphisms from highly conserved sequences in the 16S and 23S genes occurring adjacent to these polymorphic regions. This single set of primers and reaction conditions was used for the amplification of 16S-23S spacer regions for over 300 strains of bacteria belonging to eight genera and 28 species or serotypes, including Listeria, Staphylococcus, and Salmonella species and additional species related to these pathogenic organisms. When the spacer amplification products were resolved by electrophoresis, the resulting patterns could be used to distinguish all of the species of bacteria within the test group. Unique elements in the amplification product patterns generally clustered at the species level, although some genus-specific characteristics were also observed. On the basis of the results obtained with our test group of 300 bacterial strains, amplification of the 16S-23S ribosomal spacer region is a suitable process for generating a data base for use in a polymerase chain reaction-based identification method, which can be comprehensively applied to the bacterial kingdom.

In an effort to explore the origin and/or reservoirs of the genetic determinant(s) of methicillin resistance in Staphylococcus aureus, we examined over 200 strains representing 13 different species within the genus Staphylococcus for the presence of the mecA gene, using a DNA probe internal to this gene prepared from a methicillin-resistant strain of S. aureus. Occasional mecA- positive isolates were detected among several staphylococcal species. On the other hand, each one of the 134 isolates of Staphylococcus sciuri, a species considered taxonomically the most primitive among staphylococci and found primarily on rodents and primitive mammals, gave positive reaction with the DNA probe when tested under conditions of high stringency. About two thirds (99) of these isolates, all of which belonged to S. sciuri subspecies "sciuri," as well as 9 of the 11 species carnaticum isolates, showed only marginal, if any, resistance to methicillin (minimal inhibitory concentration of 0.75-6.0 micrograms/ml), while most of the remaining isolates that belonged to the subspecies "rodentius" (13 isolates in all) expressed antibiotic resistance with a heterogeneous phenotype similar to those seen in many methicillin-resistance strains of S. aureus In SmaI digests of chromosomal DNA isolated from such "methicillin-resistant S. aureus-like" strains, the mecA probe hybridized with DNA fragments in the range of 145-180 kb, while in subspecies "sciuri" and carnaticum isolates the mecA hybridizing fragment was located in the SmaI fragment with the highest molecular size (> or = 400 kb). A DNA probe comprising an internal sequence to the regulatory gene mecI from Staphylococcus epidermidis identified the presence of sequences with low degree of homology in isolates of the three S. sciuri subspecies. The mecA-reacting sequences in these bacteria differed from mecA of S. aureus in several respects (e.g., by the absence of a ClaI restriction site from mecA of subspecies "sciuri" and carnaticum, and in some isolates of subspecies "rodentius." The uniform presence of mecA in each one of a large number of S. sciuri strains belonging to distinct ribotypes and macrorestriction patterns and recovered over a 20-year period from a wide variety of animal sources and geographic sites suggests that mecA may be a native genetic element with an as yet unidentified physiologic function in this staphylococcal species.