23 research outputs found

    Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

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    In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

    Factors Associated with Revision Surgery after Internal Fixation of Hip Fractures

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    Background: Femoral neck fractures are associated with high rates of revision surgery after management with internal fixation. Using data from the Fixation using Alternative Implants for the Treatment of Hip fractures (FAITH) trial evaluating methods of internal fixation in patients with femoral neck fractures, we investigated associations between baseline and surgical factors and the need for revision surgery to promote healing, relieve pain, treat infection or improve function over 24 months postsurgery. Additionally, we investigated factors associated with (1) hardware removal and (2) implant exchange from cancellous screws (CS) or sliding hip screw (SHS) to total hip arthroplasty, hemiarthroplasty, or another internal fixation device. Methods: We identified 15 potential factors a priori that may be associated with revision surgery, 7 with hardware removal, and 14 with implant exchange. We used multivariable Cox proportional hazards analyses in our investigation. Results: Factors associated with increased risk of revision surgery included: female sex, [hazard ratio (HR) 1.79, 95% confidence interval (CI) 1.25-2.50; P = 0.001], higher body mass index (fo

    Differential Anti-Glycan Antibody Responses in <em>Schistosoma mansoni</em>-Infected Children and Adults Studied by Shotgun Glycan Microarray

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    <div><h3>Background</h3><p>Schistosomiasis (bilharzia) is a chronic and potentially deadly parasitic disease that affects millions of people in (sub)tropical areas. An important partial immunity to <em>Schistosoma</em> infections does develop in disease endemic areas, but this takes many years of exposure and maturation of the immune system. Therefore, children are far more susceptible to re-infection after treatment than older children and adults. This age-dependent immunity or susceptibility to re-infection has been shown to be associated with specific antibody and T cell responses. Many antibodies generated during <em>Schistosoma</em> infection are directed against the numerous glycans expressed by <em>Schistosoma</em>. The nature of glycan epitopes recognized by antibodies in natural schistosomiasis infection serum is largely unknown.</p> <h3>Methodology/Principal Findings</h3><p>The binding of serum antibodies to glycans can be analyzed efficiently and quantitatively using glycan microarray approaches. Very small amounts of a large number of glycans are presented on a solid surface allowing binding properties of various glycan binding proteins to be tested. We have generated a so-called shotgun glycan microarray containing natural N-glycan and lipid-glycan fractions derived from 4 different life stages of <em>S. mansoni</em> and applied this array to the analysis of IgG and IgM antibodies in sera from children and adults living in an endemic area. This resulted in the identification of differential glycan recognition profiles characteristic for the two different age groups, possibly reflecting differences in age or differences in length of exposure or infection.</p> <h3>Conclusions/Significance</h3><p>Using the shotgun glycan microarray approach to study antibody response profiles against schistosome-derived glycan elements, we have defined groups of infected individuals as well as glycan element clusters to which antibody responses are directed in <em>S. mansoni</em> infections. These findings are significant for further exploration of <em>Schistosoma</em> glycan antigens in relation to immunity.</p> </div

    Unsupervised clustering analyses.

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    <p>Hierarchical clustering analysis (A) and PCA (B), percentage of individuals clustering together in the HCA (C) and percentage of glycan fractions in clusters C2 and C4 (D). For HCA, individuals are reported in horizontal dimension and glycan fractions in the vertical dimension. In the PCA, high and low response clusters are represented by red and blue dots, respectively. Percentages of individuals per response cluster in the unsupervised HCA are shown in C. Red and blue bars represent percentage of individuals clustering in high and low response clusters, respectively. White bars represent individuals with low IgG+high IgM and high IgG+low IgM response (mixed). In (D) the number of glycan fractions present in glycan clusters C2 and C4 is depicted as a percentage of the total number of glycan fractions from each source. White and black bars represent results for IgG and IgM, respectively.</p

    Glycan subclusters analyses.

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    <p>HCA for IgG glycan cluster C2 (A) and IgM glycan cluster C4 (D), egg counts (B and E), and antibody responses against synthetic glycan structures (C and F) in different clusters of individuals. Results are shown for IgG (A–C) and IgM (D–F). Black bars represent individuals in the low response clusters originating from the unsupervised HCA. Black dotted and checkered bars represent individuals from these low response clusters that show a low (C1<sup>low</sup>C2<sup>low</sup>/C3<sup>low</sup>C4<sup>low</sup>) or high response (C1<sup>low</sup>C2<sup>high</sup>/C3<sup>low</sup>C4<sup>high</sup>) for glycan clusters C2 and C4, respectively. Asterisks represent statistical differences between these two groups.</p

    Shotgun glycan microarray.

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    <p>Binding of serum antibodies from <i>S. mansoni</i> infected individuals <12 years (A and B) and >20 years (C and D) to glycan fractions from different life stages of <i>S. mansoni</i>. Average background subtracted median fluorescence intensities are shown for IgG (A and C) and IgM (B and D).</p

    Age groups.

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    <p>Comparisons of anti-glycan responses between age group <12 years and >20 years. Supervised HCA and PCA of differentially expressed glycan fractions are shown for IgG (A+C) and IgM (B+C), percentage of individuals per age group clustering together in the supervised HCA (D) and percentage of differentially recognized glycan fractions (E). For HCA, individuals are reported in horizontal dimension and differentially recognized glycan fractions in the vertical dimension. In (C) and (D), individuals clustering in high, intermediate (int.), and low response clusters are represented by red, white, and blue dots and (dashed) bars, respectively. In (E), the number of differentially recognized glycan fractions is depicted as a percentage of the total number of glycan fractions from each source. White and black bars represent results for IgG and IgM respectively.</p
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