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ImportanceNew guidelines recommend that molecular testing replace sputum-smear microscopy to guide discontinuation of respiratory isolation in patients undergoing evaluation for active tuberculosis (TB) in health care settings.ObjectiveTo evaluate the implementation and impact of a molecular testing strategy to guide discontinuation of isolation.Design, setting, and participantsProspective cohort study with a pragmatic, before-and-after-implementation design of 621 consecutive patients hospitalized at Zuckerberg San Francisco General Hospital and Trauma Center who were undergoing sputum examination for evaluation for active pulmonary TB from January 2014 to January 2016.InterventionsImplementation of a sputum molecular testing algorithm using GeneXpert MTB/RIF (Xpert; Cepheid) to guide discontinuation of isolation.Main outcomes and measuresWe measured the proportion of patients with molecular testing ordered and completed; the accuracy of the molecular testing algorithm in reference to mycobacterial culture; the duration of each component of the testing and isolation processes; length of stay; mean days in isolation and in hospital; and mean cost. We extracted data from hospital records and compared measures before and after implementation.ResultsClinicians ordered sputum testing for TB for 621 patients at ZSFG during the 2-year study period. Of 301 patients in the preimplementation period with at least 1 sputum microscopy and culture ordered, clinicians completed the rapid TB testing evaluation process for 233 (77%).Among 320 patients evaluated in the postimplementation period, clinicians ordered molecular testing for 234 (73%) patients and received results for 295 of 302 (98%) tests ordered. Median age was 54 years (interquartile range, 44-63 years), and 161 (26%) were women. The molecular testing algorithm accurately diagnosed all 7 patients with culture-confirmed TB and excluded TB in all 251 patients with Mycobacterium tuberculosis (MTB) culture-negative results. Compared with the preimplementation period, there were significant decreases in median times to final rapid test result (39.1 vs 22.4 hours, P

Yeast macroautophagy begins with the de novo formation of a double-membrane phagophore at the preautophagosomal structure/phagophore assembly site (PAS), followed by its expansion into the autophagosome responsible for cargo engulfment. The kinase Atg1 is recruited to the PAS by Atg13 through interactions between the EAT domain of the former and the tMIM motif of the latter. Mass-spectrometry data have shown that, in the absence of Atg13, the EAT domain structure is strikingly dynamic, but the function of this Atg13-free dynamic state has been unclear. We used structure-based mutational analysis and quantitative and superresolution microscopy to show that Atg1 is present on autophagic puncta at, on average, twice the stoichiometry of Atg13. Moreover, Atg1 colocalizes with the expanding autophagosome in a manner dependent on Atg8 but not Atg13. We used isothermal titration calorimetry and crystal structure information to design an EAT domain mutant allele ATG1DD that selectively perturbs the function of the Atg13-free state. Atg1DD shows reduced PAS formation and does not support phagophore expansion, showing that the EAT domain has an essential function that is separate from its Atg13-dependent role in autophagy initiation.

The class III PI 3-kinase, VPS34 forms distinct complexes essential for cargo sorting and membrane trafficking in endocytosis as well as for autophagosome nucleation and maturation. We used integrative structural biology approach to provide insights into the conformational dynamics of the complex and mechanisms that regulate VPS34 activity at the membrane.

Autophagy is a physiological process for the recycling and degradation of cellular materials. Forming the autophagosome from the phagophore, a cup-shaped double-membrane vesicle, is a critical step in autophagy. The origin of the cup shape of the phagophore is poorly understood. In yeast, fusion of a small number of Atg9-containing vesicles is considered a key step in autophagosome biogenesis, aided by Atg1 complexes (ULK1 in mammals) localized at the preautophagosomal structure (PAS). In particular, the S-shaped Atg17-Atg31-Atg29 subcomplex of Atg1 is critical for phagophore nucleation at the PAS. To study this process, we simulated membrane remodeling processes in the presence and absence of membrane associated Atg17. We show that at least three vesicles need to fuse to induce the phagophore shape, consistent with experimental observations. However, fusion alone is not sufficient. Interactions with 34-nm long, S-shaped Atg17 complexes are required to overcome a substantial kinetic barrier in the transition to the cup-shaped phagophore. Our finding rationalizes the recruitment of Atg17 complexes to the yeast PAS, and their unusual shape. In control simulations without Atg17, with weakly binding Atg17, or with straight instead of S-shaped Atg17, the membrane shape transition did not occur. We confirm the critical role of Atg17-membrane interactions experimentally by showing that mutations of putative membrane interaction sites result in reduction or loss of autophagic activity in yeast. Fusion of a small number of vesicles followed by Atg17-guided membrane shape-remodeling thus emerges as a viable route to phagophore formation.

The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) is required for the initiation of essentially all macroautophagic processes. PI3KC3-C1 consists of the lipid kinase catalytic subunit VPS34, the VPS15 scaffold, and the regulatory BECN1 and ATG14 subunits. The VPS34 catalytic domain and BECN1:ATG14 subcomplex do not touch, and it is unclear how allosteric signals are transmitted to VPS34. We used EM and crosslinking mass spectrometry to dissect five conformational substates of the complex, including one in which the VPS34 catalytic domain is dislodged from the complex but remains tethered by an intrinsically disordered linker. A "leashed" construct prevented dislodging without interfering with the other conformations, blocked enzyme activity in vitro, and blocked autophagy induction in yeast cells. This pinpoints the dislodging and tethering of the VPS34 catalytic domain, and its regulation by VPS15, as a master allosteric switch in autophagy induction.

The ULK1 complex initiates autophagosome formation, linking cellular nutrient status to downstream events in autophagy. Recent work suggests that the ULK1 complex might also be activated in selective autophagy independent of nutrient or energy status. In this review we will discuss our current understanding of how the ULK1 complex is regulated by different signals, as well as how this complex then regulates other components of the autophagy machinery. Recently obtained structural data both on ULK1 and the orthologous yeast Atg1 complex are beginning to shed light on the higher-order organization of ULK1 complex. Ultimately, these insights might make it possible to understand how cargo organization and structure recruits and regulates ULK1 in selective autophagy initiation.

The primary cilium is a microtubule-based organelle that functions in sensory and signalling pathways. Defects in ciliogenesis can lead to a group of genetic syndromes known as ciliopathies (1-3). However, the regulatory mechanisms of primary ciliogenesis in normal and cancer cells are incompletely understood. Here we demonstrate that autophagic degradation of a ciliopathy protein, OFD1 (oral-facial-digital syndrome 1), at centriolar satellites promotes primary cilium biogenesis. Autophagy is a catabolic pathway in which cytosol, damaged organelles and protein aggregates are engulfed in autophagosomes and delivered to lysosomes for destruction (4). We show that the population of OFD1 at the centriolar satellites is rapidly degraded by autophagy upon serum starvation. In autophagy-deficient Atg5or Atg3 null mouse embryonic fibroblasts, OFD1 accumulates at centriolar satellites, leading to fewer and shorter primary cilia and a defective recruitment of BBS4 (Bardet-Biedl syndrome 4) to cilia. These defects are fully rescued by OFD1 partial knockdown that reduces the population of OFD1 at centriolar satellites. More strikingly, OFD1 depletion at centriolar satellites promotes cilia formation in both cycling cells and transformed breast cancer MCF7 cells that normally do not form cilia. This work reveals that removal of OFD1 by autophagy at centriolar satellites represents a general mechanism to promote ciliogenesis in mammalian cells. These findings define a newly recognized role of autophagy in organelle biogenesis., During autophagy, the membrane anchored LC3 (microtubule-associated protein 1 light chain 3, also known as MAP1LC3B) interacts with cargo and cargo-adaptor proteins, recruiting cargoes to the autophagosome for subsequent degradation [...]

In this study the mechanism of synergistic toxicity between malononitrile and p -nitrobenzaldehyde was revealed by examining their toxicodynamic and toxicokinetic interactions. The investigation of the toxicodynamic interactions focused on the interactions among malononitrile, p -nitrobenzaldehyde, and luciferase, and found that malononitrile does not induce luciferase to bind p -nitrobenzaldehyde. This result suggested that the synergistic toxicity does not arise from toxicodynamic interactions. Further research on toxicokinetic interactions compared the amounts of p -nitrobenzaldehyde being transported from the water phase to the biotic phase in two circumstances--in the presence of malononitrile and in its absence. The results of this research indicated that in the presence of malononitrile, more p -nitrobenzaldehyde is transported, and it is the increased amount of p -nitrobenzaldehyde that leads to the synergistic toxicity of malononitrile and p -nitrobenzaldehyde. [ABSTRACT FROM AUTHOR]

This article identifies the types of common and toxic mold that are found in homes, schools, and child care centers; explains the negative health effects of mold for infants and young children; discusses ways of preventing and controlling mold in homes, schools, and child care centers; and recommends appropriate responses to the international health issue of maintaining indoor air quality. [ABSTRACT FROM AUTHOR]