Population distribution analyses reveal a hierarchy of molecular players underlying parallel endocytic pathways.

Single-cell-resolved measurements reveal heterogeneous distributions of clathrin-dependent (CD) and -independent (CLIC/GEEC: CG) endocytic activity in Drosophila cell populations. dsRNA-mediated knockdown of core versus peripheral endocytic machinery induces strong changes in the mean, or subtle changes in the shapes of these distributions, respectively. By quantifying these subtle shape changes for 27 single-cell features which report on endocytic activity and cell morphology, we organize 1072 Drosophila genes into a tree-like hierarchy. We find that tree nodes contain gene sets enriched in functional classes and protein complexes, providing a portrait of core and peripheral control of CD and CG endocytosis. For 470 genes we obtain additional features from separate assays and classify them into early- or late-acting genes of the endocytic pathways. Detailed analyses of specific genes at intermediate levels of the tree suggest that Vacuolar ATPase and lysosomal genes involved in vacuolar biogenesis play an evolutionarily conserved role in CG endocytosis

Transposon libraries identify novel Mycobacterium bovis BCG genes involved in the dynamic interactions required for BCG to persist during in vivo passage in cattle

Background BCG is the most widely used vaccine of all time and remains the only licensed vaccine for use against tuberculosis in humans. BCG also protects other species such as cattle against tuberculosis, but due to its incompatibility with current tuberculin testing regimens remains unlicensed. BCG’s efficacy relates to its ability to persist in the host for weeks, months or even years after vaccination. It is unclear to what degree this ability to resist the host’s immune system is maintained by a dynamic interaction between the vaccine strain and its host as is the case for pathogenic mycobacteria. Results To investigate this question, we constructed transposon mutant libraries in both BCG Pasteur and BCG Danish strains and inoculated them into bovine lymph nodes. Cattle are well suited to such an assay, as they are naturally susceptible to tuberculosis and are one of the few animal species for which a BCG vaccination program has been proposed. After three weeks, the BCG were recovered and the input and output libraries compared to identify mutants with in vivo fitness defects. Less than 10% of the mutated genes were identified as affecting in vivo fitness, they included genes encoding known mycobacterial virulence functions such as mycobactin synthesis, sugar transport, reductive sulphate assimilation, PDIM synthesis and cholesterol metabolism. Many other attenuating genes had not previously been recognised as having a virulence phenotype. To test these genes, we generated and characterised three knockout mutants that were predicted by transposon mutagenesis to be attenuating in vivo: pyruvate carboxylase, a hypothetical protein (BCG_1063), and a putative cyclopropane-fatty-acyl-phospholipid synthase. The knockout strains survived as well as wild type during in vitro culture and in bovine macrophages, yet demonstrated marked attenuation during passage in bovine lymph nodes confirming that they were indeed involved in persistence of BCG in the host. Conclusion These data show that BCG is far from passive during its interaction with the host, rather it continues to employ its remaining virulence factors, to interact with the host’s innate immune system to allow it to persist, a property that is important for its protective efficacy.</p

Population distribution analyses reveal a hierarchy of molecular players underlying parallel endocytic pathways.

Single-cell-resolved measurements reveal heterogeneous distributions of clathrin-dependent (CD) and -independent (CLIC/GEEC: CG) endocytic activity in Drosophila cell populations. dsRNA-mediated knockdown of core versus peripheral endocytic machinery induces strong changes in the mean, or subtle changes in the shapes of these distributions, respectively. By quantifying these subtle shape changes for 27 single-cell features which report on endocytic activity and cell morphology, we organize 1072 Drosophila genes into a tree-like hierarchy. We find that tree nodes contain gene sets enriched in functional classes and protein complexes, providing a portrait of core and peripheral control of CD and CG endocytosis. For 470 genes we obtain additional features from separate assays and classify them into early- or late-acting genes of the endocytic pathways. Detailed analyses of specific genes at intermediate levels of the tree suggest that Vacuolar ATPase and lysosomal genes involved in vacuolar biogenesis play an evolutionarily conserved role in CG endocytosis

Parody as positive dissent in Hindi theatre

Parody (etymologically a voice alongside another voice) involves imitation, but what is crucial is the co-presence of these two voices, the parodying and the parodied. It is the dialogue between two enunciative spheres, two utterances, hence its preeminent position in the Bakhtinian concept of dialogism. The two points of view, set against each other dialogically, represent two utterances, speakers, styles, languages, and axiological systems, even if they issue from a single speaker. As a reflexive device and critical manipulation of canonized forms, parody has often been considered as the epitome of postmodernism in European and North American literature and artistic expression. The paper aims to show that, in Hindi theatre, parody is politically significant. The article focuses on Bhartendu Hariścandra (1850—1885) and Habīb Tanvīr (1923—2009). It argues that the use of the quotes of Nazīr Akbarābādīin Tanvīr’s most famous play Āgrā Bāzār, a poet who himself parodies the traditional poetical canons, enhances a literary reflexivity that is one of the deepest creative devices of Indian culture

Quantitative profiling of two endocytic routes at single cell resolution.

<p>(A) Experimental workflow outline for cell seeding, transfection and multiplex endocytic assays to obtain multifeature data across 7131 gene depletions. The entire procedure was performed on a cell array (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100554#pone.0100554.s001" target="_blank">Figure S1A</a>; details in SOM) and the positions of negative and positive (dsRNA against <i>sec23</i>, <i>arf1</i>, <i>shi</i>) controls are highlighted in their respective colours. (B) Table grouping the 27 quantitative features into categories. The top half of the table contains direct measurements of intensity, while the lower half contains geometric parameters of the cell, endosomes and nucleus. Various measurements are made from each fluorescent channel, including those utilizing different pixel radii for local background subtraction while detecting endosomes. (C) Representative brightfield (bf) and fluorescent micrographs of a field of view of individual cells (zoomed in insets) labeled with four different fluorescent probes: Hoechst; FITC-Dextran (Fdex); Alexa568-Tf (Tf); Alexa647-αOkt9 (Okt9); (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100554#pone.0100554.s018" target="_blank">Methods S1</a> for details). The psuedocolour merge image is a composite of the Fdex (green), TfR (red) and Okt9 (blue) channels. Scale bar = 15 µm; inset = 3×. (D) Grayscale heatmap representing the fraction of four control genes (<i>arf1</i> (<i>arf79f</i>); <i>shi</i>; <i>sec23</i>; <i>chc</i>) picked up as hits (above a Z-score threshold of 3) across all 27 features in the entire dataset. Higher values on the grayscale bar denote higher pick-up rates. The features with higher pick-up rates correspond to the known endocytic roles of these genes.</p

Primary hits validated in a secondary classification assay.

<p>(A–B) Schema (A) and positional patterning (B) on cell arrays of secondary endocytic classification assays carried out for all CG features (upper schema) or a subset of CD features (lower schema).All the test genes were surrounded with local positive controls, and negative controls (see legend in (B)). With this patterning, each gene was tested in triplicate, with three local positive controls and six local negative controls. (C) Heatmap representing raw mean fluorescence intensities (in the pulse channel) across a test cell array used to validate the CG secondary endocytic assay described in (A). Only the means of control wells are shown in the top panel and the inter-control variation in means is representative of a typical experiment. For comparison, the lower panel depicts the mean fluorescence intensities of test genes. (D) The green bars show the fraction of genes predicted as hits for each feature in the primary screen that were also picked up as hits for that feature in the secondary. The gray bars show the fraction of genes not predicted as a hit for each feature in the primary screen that were nevertheless picked up as hits for that feature in the secondary. With a single exception (Tnum) we find that the green bars exceed the gray (p-value 5×10⁻⁶ for 22 fair coin flips) demonstrating the selectivity and reproducibility of our primary assay. (E) Psuedocoloured fluorescence micrographs of representative control and <i>drab5</i>- and <i>dvps4</i>- dsRNA treated populations of cells that were subjected to the CG pulse-chase assay from (<b>A</b>). Both Drab5 and Dvps4 depleted cells were affected in the chase (with Fdex, green) portion of the assay, while the pulse portion (with Rdex, red) was unaffected (see quantitation in bar graphs on the right, normalized to control). Scale bar = 10 µm.</p

Endocytic phenotypes in mutant primary hemocytes from <i>Drosophila</i>.

<p>(A–D) dsRNA treated S2R+ cells phenocopy corresponding allelic mutants in primary hemocyte cultures in a secondary assay. Scatter plots (A, B) show normalized fold change in fluorescence intensity of dextran that was pulsed (A) or chased (B) in S2R+ cells treated with different dsRNAs (y axis) or in hemocytes (x axis) from the corresponding mutant flies. In all cases, representative values were normalized to those from negative controls (CS hemocytes or zeo dsRNA treated S2R+ cells) and are plotted as mean± SEM. (n>30 for hemocyte assays, n>200 for S2R+ assays in all cases). For the chase assay in (B), we utilized <i>dor⁴</i> and <i>car¹</i> mutant hemocytes as positive controls (shown in light blue; Sriram et al., 2003). (C) Representative micrographs of hemocyte cultures from flies carrying hypomorphic alleles of <i>vps35</i>, <i>epac</i>, <i>α-cop</i> and <i>CG1418</i> assayed as in (B). (D) Summary of the experiment in (A–B) displaying statistically significant (Student's T-test, p<0.05) changes in uptake/retention of mutant hemocytes or gene-depleted S2R+ cells as colour coded maps. Scale bar in (C) = 5 µm.</p

Role of lysosomal genes.

<p>(A) Network map depicting known and predicted interactions (green lines: genetic; blue lines: physical; brown lines: predicted based on conserved data) between the ‘Granule group’ set of eye colour mutants (pink) and selected hits (gray). In this network, genes encoding Carnation (<i>car</i>; the fly homolog of VPS33), Deep orange (<i>dor</i>), Carmine (<i>cm</i>) and Rab7 were identified with roles in CG endocytosis in this study (denoted by black asterisks), while White (<i>w</i>) depletion affected at least one Tf pathway feature (white asterisk). (B) Localization of Carnation on early fluid endosomes. <i>Drosophila</i> S2R+ cells were pulsed with TMR-Dextran for two minutes and fixed and labeled with antibodies to Carnation (αCar). Micrographs show a representative cell imaged in two channels and a pseudo colour merge image (labeled TMRdex and αCar), in red, green and merge respectively). Carnation (green) is seen enriched on peripheral, small, early fluid endosomes (red). Three examples of such endosomes (white arrows in merge panel) are shown in the magnified inset. (C) Fluorescent micrographs depict the levels of fluid uptake in representative S2R+ cells treated with dsRNA against <i>car</i> (first lower panel) or <i>syx1A</i> (last lower panel) or in hemocytes from <i>car¹</i> mutant flies (middle lower panel), with their respective controls (upper panels). Bar graph represents mean and SD of normalized fluorescent integrated intensity per cell from 2–3 experiments, with 100–150 cells per treatment (S2R+ cells) or 40 cells per genotype (hemocytes). (D) Representative fluorescent micrographs depict fluid uptake measured in hemocytes as in (C), in flies that were: homozygous for a mutant allele of <i>car</i> (<i>car¹</i>); a hetero-allelic combination of <i>car¹</i>/+;<i>syx1</i>/+;or wild type (CS). Also tested were flies heterozygous for <i>syx1</i>/+ and <i>car¹</i>/+. Bar graph represents mean and SD of normalized fluorescent integrated intensity per hemocyte from 2–3 experiments with 40 cells per genotype. (E) Representative micrographs show human AGS cells treated with control siRNA or siRNA to hSYX1A and hVPS33A/B and pulsed with FITC-Dextran for 5 min. Right panel - Bar graphs show population averaged mean fluorescence intensity uptake per cell (representative experiment with n>50 cells per replicate, 2 replicates). Scale bar in (B–E) main panel = 5 µm, inset = 1 µm. Double asterisks denote significance <i>p</i> values lower than 0.01 with the Student's T-Test.</p