Inhibition of cholesterol recycling impairs cellular PrPSc propagation

The infectious agent in prion diseases consists of an aberrantly folded isoform of the cellular prion protein (PrPc), termed PrPSc, which accumulates in brains of affected individuals. Studies on prion-infected cultured cells indicate that cellular cholesterol homeostasis influences PrPSc propagation. Here, we demonstrate that the cellular PrPSc content decreases upon accumulation of cholesterol in late endosomes, as induced by NPC-1 knock-down or treatment with U18666A. PrPc trafficking, lipid raft association, and membrane turnover are not significantly altered by such treatments. Cellular PrPSc formation is not impaired, suggesting that PrPSc degradation is increased by intracellular cholesterol accumulation. Interestingly, PrPSc propagation in U18666A-treated cells was partially restored by overexpression of rab 9, which causes redistribution of cholesterol and possibly of PrPSc to the trans-Golgi network. Surprisingly, rab 9 overexpression itself reduced cellular PrPSc content, indicating that PrPSc production is highly sensitive to alterations in dynamics of vesicle trafficking

Dissociation of Infectivity from Seeding Ability in Prions with Alternate Docking Mechanism

Previous studies identified two mammalian prion protein (PrP) polybasic domains that bind the disease-associated conformer PrPSc, suggesting that these domains of cellular prion protein (PrPC) serve as docking sites for PrPSc during prion propagation. To examine the role of polybasic domains in the context of full-length PrPC, we used prion proteins lacking one or both polybasic domains expressed from Chinese hamster ovary (CHO) cells as substrates in serial protein misfolding cyclic amplification (sPMCA) reactions. After ∼5 rounds of sPMCA, PrPSc molecules lacking the central polybasic domain (ΔC) were formed. Surprisingly, in contrast to wild-type prions, ΔC-PrPSc prions could bind to and induce quantitative conversion of all the polybasic domain mutant substrates into PrPSc molecules. Remarkably, ΔC-PrPSc and other polybasic domain PrPSc molecules displayed diminished or absent biological infectivity relative to wild-type PrPSc, despite their ability to seed sPMCA reactions of normal mouse brain homogenate. Thus, ΔC-PrPSc prions interact with PrPC molecules through a novel interaction mechanism, yielding an expanded substrate range and highly efficient PrPSc propagation. Furthermore, polybasic domain deficient PrPSc molecules provide the first example of dissociation between normal brain homogenate sPMCA seeding ability from biological prion infectivity. These results suggest that the propagation of PrPSc molecules may not depend on a single stereotypic mechanism, but that normal PrPC/PrPSc interaction through polybasic domains may be required to generate prion infectivity

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Mortalidade e fatores associados em uma UTI de cirurgia torácica Mortality and associated factors in a thoracic surgery ICU

OBJETIVO: Determinar a mortalidade e identificar fatores de riscos associados em pacientes em uma UTI de cirurgia torácica. MÉTODOS: Foram avaliados retrospectivamente 141 pacientes admitidos na UTI de cirurgia torácica do Hospital Estadual de Denizli, localizado na cidade de Denizli, Turquia, entre janeiro de 2006 e agosto de 2008. Foram coletados dados sobre gênero, idade, causa de admissão, intervenções invasivas e operações, status de ventilação mecânica invasiva, infecções e tempo de permanência na UTI. RESULTADOS: Dos 141 pacientes, 103 (73,0%) eram do sexo masculino e 38 (23,0%) do sexo feminino. A média de idade foi de 52,1 anos (variação: 12-92 anos), e a taxa de mortalidade foi de 16,3%. A causa de admissão mais frequente foi trauma. A mortalidade correlacionou-se com idade avançada (p < 0,05), uso de ventilação mecânica invasiva (OR = 42,375; p < 0,05), longa permanência na UTI (p < 0,05) e causas de admissão específicas - trauma, injúria por arma de fogo, injúria por arma branca e malignidade (p < 0,05 para todos). CONCLUSÕES: Os pacientes em uma UTI de cirurgia torácica têm alta morbidade e mortalidade. Um conhecimento maior dos fatores de risco de mortalidade pode melhorar a eficiência do tratamento, resultando em diminuição da morbidade e mortalidade, o que gerará economia de tempo e reduzirá os custos financeiros<br>OBJECTIVE: To assess mortality and identify mortality risk factors in patients admitted to a thoracic surgery ICU. METHODS: We retrospectively evaluated 141 patients admitted to the thoracic surgery ICU of the Denizli State Hospital, located in the city of Denizli, Turkey, between January of 2006 and August of 2008. We collected data regarding gender, age, reason for admission, invasive interventions and operations, invasive mechanical ventilation, infections, and length of ICU stay. RESULTS: Of the 141 patients, 103 (73.0%) were male, and 38 (23.0%) were female. The mean age was 52.1 years (range, 12-92 years), and the mortality rate was 16.3%. The most common reason for admission was trauma. Mortality was found to correlate with advanced age (p < 0.05), requiring invasive mechanical ventilation (OR = 42.375; p < 0.05), prolonged ICU stay (p < 0.05), and specific reasons for admission-trauma, gunshot wound, stab wound, and malignancy (p < 0.05 for all). CONCLUSIONS: Among patients in a thoracic surgery ICU, the rates of morbidity and mortality are high. Increased awareness of mortality risk factors can improve the effectiveness of treatment, which should reduce the rates of morbidity and mortality, thereby providing time savings and minimizing cost