Organic contaminants in Ganga basin: from the Green Revolution to the emerging concerns of modern India

The Ganga basin includes some of the most densely populated areas in the world, in a region characterised by extremely high demographic and economic growth rates. Although anthropogenic pressure in this area is increasing, the pollution status of the Ganga is still poorly studied and understood. In the light of this, we have carried out a systematic literature review of the sources, levels and spatiotemporal distribution of organic pollutants in surface water and sediment of the Ganga basin, including for the first time emerging contaminants (ECs). We have identified 61 publications over the past thirty years, with data on a total of 271 organic compounds, including pesticides, industrial chemicals and by-products, artificial sweeteners, pharmaceuticals and personal care products (PPCPs). The most studied organic contaminants are pesticides, whereas knowledge of industrial compounds and PPCPs, among which some of the major ECs, is highly fragmentary. Most studies focus on the main channel of the Ganga, the Yamuna, the Gomti and the deltaic region, while most of the Ganga’s major tributaries, and the entire southern part of the catchment, have not been investigated. Hotspots of contamination coincide with major urban agglomerations, including Delhi, Kolkata, Kanpur, Varanasi and Patna. Pesticides levels have decreased at most of the sites over recent decades, while potentially harmful concentrations of polychlorinated biphenyls (PCBs), organotin compounds (OTCs) and some PPCPs have been detected in the last ten years. Considering the limited geographical coverage of sampling and number of analysed compounds, this review highlights the need for a more careful selection of locations, compounds and environmental matrices, prioritizing PPCPs and catchment-scale, source-to-sink studie

Old World Arenaviruses Enter the Host Cell via the Multivesicular Body and Depend on the Endosomal Sorting Complex Required for Transport

The highly pathogenic Old World arenavirus Lassa virus (LASV) and the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) use α-dystroglycan as a cellular receptor and enter the host cell by an unusual endocytotic pathway independent of clathrin, caveolin, dynamin, and actin. Upon internalization, the viruses are delivered to acidified endosomes in a Rab5-independent manner bypassing classical routes of incoming vesicular trafficking. Here we sought to identify cellular factors involved in the unusual and largely unknown entry pathway of LASV and LCMV. Cell entry of LASV and LCMV required microtubular transport to late endosomes, consistent with the low fusion pH of the viral envelope glycoproteins. Productive infection with recombinant LCMV expressing LASV envelope glycoprotein (rLCMV-LASVGP) and LCMV depended on phosphatidyl inositol 3-kinase (PI3K) as well as lysobisphosphatidic acid (LBPA), an unusual phospholipid that is involved in the formation of intraluminal vesicles (ILV) of the multivesicular body (MVB) of the late endosome. We provide evidence for a role of the endosomal sorting complex required for transport (ESCRT) in LASV and LCMV cell entry, in particular the ESCRT components Hrs, Tsg101, Vps22, and Vps24, as well as the ESCRT-associated ATPase Vps4 involved in fission of ILV. Productive infection with rLCMV-LASVGP and LCMV also critically depended on the ESCRT-associated protein Alix, which is implicated in membrane dynamics of the MVB/late endosomes. Our study identifies crucial cellular factors implicated in Old World arenavirus cell entry and indicates that LASV and LCMV invade the host cell passing via the MVB/late endosome. Our data further suggest that the virus-receptor complexes undergo sorting into ILV of the MVB mediated by the ESCRT, possibly using a pathway that may be linked to the cellular trafficking and degradation of the cellular receptor

GLUT1 and GLUT3 in non-small cell lung cancer

While altered metabolism is a well-established tumor cell trait, the specifics of metabolic rewiring during cancer development and progression are yet to be fully understood. The Warburg effect, or aerobic glycolysis, has been described in a wide array of malignancies and has long been considered as an inefficient mean to fill energetic requirements. Emerging evidence however supports a role that extends beyond that, with glycolytic intermediates diverting to anabolic pathways to allow growth and proliferation. Importantly, the first and most critical rate-limiting step in glycolysis is glucose uptake. Fittingly, high affinity, high capacity glucose transporters such as GLUT1 and to a lesser extent GLUT3, are upregulated in cancer. Given the centrality of glycolysis in tumor biology and reported clinical observations relating to glucose transporter expression patterns, the present work focuses on characterizing the role of GLUT1 and GLUT3 in the context of non-small cell lung cancer (NSCLC), the most prevalent histological subtype of lung cancer. Linking glucose metabolism and a program involved in early steps of metastasis, part of the findings uncovers an aberrant expression of GLUT3 as integral to the epithelial-mesenchymal transition (EMT) in NSCLC. Indeed, a unique association between GLUT3 and a mesenchymal status is observed in a panel of human NSCLC cell lines. Furthermore, GLUT3 expression is increased during EMT by a mechanism that involves direct binding of EMT mediating transcription factor ZEB1 to the GLUT3 (SLC2A3) gene. Data also supports a functional role of GLUT3 in proliferation, as inhibiting GLUT3 expression reduces glucose import and proliferation of mesenchymal lung tumor cells, whereas ectopic expression in epithelial cells sustains proliferation in low glucose. In an analysis of a large microarray data collection of human NSCLC samples, GLUT3 expression is found to correlate with EMT markers, and is prognostic of poor overall survival. Taken collectively, the data reveal an important role for GLUT3 in lung cancer, when tumor cells loose their epithelial characteristics to become more invasive. Owing to the restricted expression of this transporter in healthy individuals, its presence in lung tumor cells may represent a noteworthy prospect for targeted therapy. The contrastingly ubiquitous nature of GLUT1 in normal and transformed tissue warrants in vivo investigation to determine expression patterns and contribution tumor development. To that end, generation of autochthonous lung cancer mouse models allows assessment of the effect of tumor exclusive GLUT1 loss. Ensuing preliminary results allude to a possible role of GLUT1 in specific histological lesions. The significance of this specificity is currently under investigation

Working model for the cell entry of the Old World arenaviruses LASV and LCMV.

<p>For details please see text.</p

A role of Vps4 in rLCMV-LASVGP and LCMV entry.

<p>(A) HEK293 cells were transiently transfected with FLAG-tagged wild-type Vps4A and the DN mutant Vps4AQE and expression of the recombinant proteins detected in Western-blot. (B) Expression of DN Vps4A reduced viral infection. Cells were transiently transfected with FLAG-tagged wild-type and DN Vps4A. After 36 hours, cells were infected with rLCMV-LASVGP and LCMV (MOI = 1). After 16 hours, cells were fixed and stained for the Vps4A variants (anti-FLAG) and LCMV NP. Cells were analyzed by FACS separating transfected “expressing” from untransfected “non-expressing” cells with gating based on the intensity of the anti-FLAG signal. The percentage of cells infected within each population was quantified. (C) Quantitation of (B). All data presented are means (n = 3 ± SD).</p

Cell entry of LASV and LCMV into human macrophages depends on the MVB and ESCRT proteins.

<p>(A) Differentiation of THP-1 cells into macrophages. THP-1 cells were treated with 50 ng/ml PMA for 48 hours and changes in cell morphology assessed by differential interference contrast microscopy (bar = 20 µM). (B) THP1 cells were seeded in 96 well plates (2×10⁴ cells/well) and differentiated into macrophage-like cells as in (A). Cells were then transfected with siRNAs for Hrs, Tsg101, Alix, or control siRNA and efficiency of depletion assessed after 48 hours by Western-blot as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002232#ppat-1002232-g004" target="_blank">Fig. 4A</a>. For normalization, α-tubulin (α-Tu) was used. (C) Cells were transfected with siRNAs for Vps22 and Vps24 or control siRNA and efficiency of depletion assessed after 48 hours by quantification of mRNA levels by RT-qPCR as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002232#ppat-1002232-g004" target="_blank">Fig. 4B</a>. (D) Cell entry of LASV and LCMV into human macrophage-like cells depends on PI3K and microtubules. THP1-derived macrophage-like cells generated as in (A) were treated with the indicated concentrations of wortmannin and nocodazole. Cells were then infected with rVSVΔG-LASVGP (LASV), rVSVΔG-LCMVGP (LCMV), and rVSVΔG-VSVG (VSV) either via fusion at the plasma membrane (2000 PFU/well) or via the normal route of infection (200 PFU/well) as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002232#ppat-1002232-g002" target="_blank">Fig. 2</a>. Infection was detected by scoring EGFP positive cells (mean ± SD; n = 3). (E) Infection of VSV pseudotypes of LASV, LCMV, and VSV is perturbed by anti-LBPA treatment. THP-1-derived macrophage-like cells were pre-incubated with no antibody (1), 50 µg/ml mAb anti-LBPA (2) or isotype antibody control (3) for 14 hours. Cells were then infected with rVSVΔG-LASVGP (LASV), rVSVΔG-LCMVGP (LCMV), rVSVΔG-VSVG (VSV), and AdV5-EGFP at 300 PFU/well. In specimens subjected to pretreatment only (4), the cells were incubated for 1 h at 4°C with viruses in presence of the antibody, unbound virus washed out, and cell incubated at 37°C in normal medium. Cells were fixed after 16 hours and EGFP positive cells counted (mean ± SD; n = 3). (F) Cell entry of LASV and LCMV into human macrophages depends on Tsg101, Vps22, Vps24, and Alix, but not Hrs. THP-1-derived macrophage-like cells were subjected to RNAi silencing of Hrs, Tsg101, Vps22, Vps24, and Alix as in (B) and (C). After 48 hours cells were infected with rVSVΔG-LASVGP (VSV-LASV), rVSVΔG-LCMVGP (VSV-LCMV), rVSVΔG-VSVG (VSV-VSV), rLCMV-LASVGP, LCMV, and AdV5-EGFP (AdV) at 300 PFU/well. Infection was assessed after 16 hours by IFA (mean ± SD; n = 3).</p

Incoming LCMV transiently co-localized with Tsg101 prior to reaching late endosomes.

<p>(A) Co-localization of LCMV WE54 with Tsg101 and Rab7. A549 cells were cooled on ice for 30 min and LCMV WE54 added at an MOI of ∼100. After incubation for 1 hour on ice, unbound virus was washed off, cells shifted to 37°C and fixed at the indicated time points. Cells were then immunostained to detect endogenous Tsg101 or Rab7 and incoming viral particles. Representative images are shown. Left: LCMV (green) and Tsg101 (red) at 20 min after temperature shift; right: LCMV (green) and Rab7 (red) at 60 min after temperature shift. Scale bar = 5 µm. (B) Quantification of co-localization. Ten randomly selected cells per time point were analyzed by confocal microscopy and the percentage of co-localizing viruses determined as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002232#s4" target="_blank">Materials and Methods</a>. Data presented are means ± SD (n = 10).</p

Alix is required for cell entry of LASV and LCMV.

<p>(A) A549 cells were transfected with siRNAs specific for Alix or control siRNA and efficiency of depletion assessed after 72 hours by Western-blot. For normalization, α-tubulin (α-Tu) was used. (B, C) Depletion of Alix perturbed cell entry of LASV and LCMV. A549 cells were treated with specific siRNAs to Alix and control siRNA as in (A), followed by infection with (B) rVSVΔG-LASVGP (LASV), rVSVΔG-LCMVGP (LCMV), rVSVΔG-VSVG (VSV), and AdV5-EGFP (AdV) at 500 PFU/well or (C) rLCMV-LASVGP, LCMV, rVSVΔG-VSVG (VSV), and AdV5-EGFP (AdV) (500 PFU/well). Cells were fixed after 16 hours and infection detected by IFA as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002232#ppat-1002232-g004" target="_blank">Fig. 4</a> (mean ± SD; n = 3). (D) Representative specimens of rLCMV-LASVGP infected cells in cultures treated with the indicated siRNAs in (C). LCMV NP is in green and cell nuclei in blue (bar = 20 µM). (E) Depletion of Alix does not interfere with transferrin uptake. Cells treated as in (A) were subjected to transferrin uptake assay as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002232#ppat-1002232-g003" target="_blank">Fig. 3D</a>. The cellular distribution of transferrin (red) and cell nuclei stained with DAPI in blue are shown (bar = 10 µM). (F) Depletion of Alix does not affect cell surface expression of α-DG. Cells were subjected to RNAi as in (A) and cell surface staining for α-DG performed after 72 hours as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002232#ppat-1002232-g004" target="_blank">Fig. 4H</a>. In histograms, y-axis represents cell numbers and x-axis Alexa 594 fluorescence intensity. Shaded area: primary and secondary antibody, empty area: secondary antibody only. The broken line indicates the superimposition of the shaded peak (primary+secondary antibody) from cells treated with control siRNA.</p