The Caenorhabditis elegans homolog of human copper chaperone Atox1, CUC-1, aids in distal tip cell migration

Cell migration is a fundamental biological process involved in for example embryonic development, immune system and wound healing. Cell migration is also a key step in cancer metastasis and the human copper chaperone Atox1 was recently found to facilitate this process in breast cancer cells. To explore the role of the copper chaperone in other cell migration processes, we here investigated the putative involvement of an Atox1 homolog in Caenorhabditis elegans, CUC-1, in distal tip cell migration, which is a key process during the development of the C. elegans gonad. Using knock-out worms, in which the cuc-1 gene was removed by CRISPR-Cas9 technology, we probed life span, brood size, as well as distal tip cell migration in the absence or presence of supplemented copper. Upon scoring of gonads, we found that cuc-1 knock-out, but not wild-type, worms exhibited distal tip cell migration defects in approximately 10–15% of animals and, had a significantly reduced brood size. Importantly, the distal tip cell migration defect was rescued by a wild-type cuc-1 transgene provided to cuc-1 knock-out worms. The results obtained here for C. elegans CUC-1 imply that Atox1 homologs, in addition to their well-known cytoplasmic copper transport, may contribute to developmental cell migration processes

The Global Burden of Snakebite: A Literature Analysis and Modelling Based on Regional Estimates of Envenoming and Deaths

Janaka de Silva and colleagues estimate that globally at least 421,000 envenomings and 20,000 deaths occur each year due to snakebite

Membrane Fluidity Regulation: From C. elegans to mammalian cells

Biological membranes are primarily an assembly of lipids and proteins. Collectively, these constituents within a phospholipid bilayer determine the physical membrane properties such as fluidity, viscosity, thickness, packing and permeability. Maintenance of membrane properties within acceptable margins, i.e. membrane homeostasis, is fundamentally important for cellular processes. For example, it is a well-established phenomenon that poikilothermic organisms, that cannot control their body temperature, constantly adapt their membrane lipid composition in order to maintain optimal membrane fluidity for membrane functions in spite of variation in ambient temperatures. Regulatory mechanisms must also exist in mammals to maintain membrane lipid heterogeneity across the secretory pathway and to compensate for dietary lipid variation. However, the molecular mechanisms of such an adaptive response in mammals remain poorly understood. Here, using systematic genetics, lipidomics and membrane property assays, we have established that the PAQR-2/IGLR-2 complex in C. elegans and AdipoR2 (a PAQR-2 homolog) in mammalian cells specifically respond to the toxic membrane-rigidifying effects of dietary saturated fatty acids (SFAs) and promote fatty acid desaturation to restore membrane composition and fluidity. In an attempt to understand other mechanisms essential to prevent SFA-mediated cellular toxicity, we also performed an unbiased forward genetic screen in C. elegans. Strikingly, this screen for SFA-tolerance genes led only to the isolation of novel paqr-2 and iglr-2 alleles; this strongly indicates that paqr-2 and iglr-2 are important genes specifically essential to respond to toxic effects of dietary saturated fats. In particular, we noted that in worms and cells that lack PAQR-2/AdipoR2 function, exogenous SFAs becomes rapidly incorporated into membrane phospholipids, leading to membrane rigidification. This was accompanied by an abnormal transcriptional response, impaired mitochondrial respiration and increased ER-UPR as measured in HEK293 cells. Interestingly, we noticed that the toxic effects of exogenous SFAs can be completely mitigated by supplying the cultured cells with small amounts of membrane fluidizing unsaturated fatty acids (UFAs). Consistently, we also found that facilitating the accumulation of UFAs either with mutations in fld-1 in worms or silencing the fld-1 mammalian homologs TLCD1/2, which normally function to limit the incorporation of polyunsaturated fatty acids in membrane phospholipids, is protective and able to attenuate SFA-mediated cellular toxicity. Altogether, these results suggest that maintenance of an optimal SFA/UFA ratio is crucial for normal cellular function and that the PAQR-2/AdipoR2 proteins essentially act as “guardians of membrane homeostasis”

Supplemental Material for Bodhicharla et al., 2018

<p><b>Fig. S1. Mosaic analysis of <i>iglr-2;Ex[sur-5::gfp(NLS) pIGLR-2]</i>worms selected for growth to adulthood on 20 mM glucose.</b>Cell lineages and tissues carrying the extrachromosomal arrays were identified by the expression of GFP. Only worms not expressing GFP in the intestine were selected for the analysis because expression in the organ obscures expression elsewhere (Yochem <i>et al.</i>1998).<b></b></p><p><br></p><p></p><p><b>Fig. S2. Tissue-specific <i>paqr-2 </i>and <i>fat-6 </i>rescue and <i>paqr-2 </i>vitellogenin defect at L4 stage. (A)</b>Length of 1 day old adult worms with the indicated genotypes cultivated on normal plates (NGM) or 20 mM glucose (n≥20). <b>(B) </b>Brood size of worms with the indicated genotypes cultivated on normal plates (n=10). <b>(C) </b>Photographs of worms 72 hours after placing them as L1s on normal culture plates (NGM) or culture plates containing 20 mM glucose. <b>(D) </b>Length of 1 day old adult worms with the indicated genotypes cultivated on normal plates (NGM) or 20 mM glucose (n≥20). <b>(E) </b>Tail tip phenotype of 1 day old adult worms with the indicated genotypes and cultivated on normal plates (n=100). <b>(F) </b>Visualization of the membranes within the developing gonad of wild type N2 and <i>paqr-2 </i>L4 larvae (the bright puncti in the N2 GFP image are auto-fluorescent gut granules); all L4s had normal gonads (n=50).</p><p><br></p><p><b>Fig. S3. Laurdan dye measurement of fluidity and tracking of ³H-labelled PA in transwell experiments. (A-B) </b>Representative confocal images of HEK293 treated with PA 400 μM or vehicle for 24 h and stained with Laurdan dye. <b>(C-D)</b>Pseudo-color images of A and B showing GP index. Red colors indicate high membrane order, whereas blue colors indicate low order. <b>(E)</b>Histogram and <b>(F)</b>average of the GP values from HEK293 treated with PA 400 μM or vehicle for 24 h. The histogram shows how many pixels have each GP value in the region of interest (white circle, n=11 images).<b>(G) </b>³H activity in the acceptor HEK293 cells exposed to different media for 24 h (<b>H-I</b>) Activity recorded in the transfering media after 24 h. (<b>J</b>) Activity registered in the donor cells at the end of the experiment. (<b>K-L</b>) CPM measured in the loading media after 4 h cell cultivation. (n=3 wells per condition for <b>G-L</b>). <i></i></p

Characterization of bla CTX-M sequences of Indian origin and thirteen uropathogenic Escherichia coli isolates resistant to multiple antibiotics

Abstract Objectives ESBL-producing isolates of the Enterobacteriaceae occur throughout the world. The objectives of this study were to characterize uropathogenic Escherichia coli isolated at a tertiary care hospital in southern India, and shed light on bla CTX-M sequences of Indian origin. Results A cohort of 13 urinary isolates of E. coli (obtained from patients at the Sri Sathya Sai Institute of Higher Medical Sciences, Prasanthigram, Andhra Pradesh, India) were characterized and found to be resistant to multiple antibiotics, including extended-spectrum cephalosporins. All 13 isolates contained bla CTX-M-15, and many of them transferred this genotype to at least one laboratory strain of E. coli after conjugation. Analyses of bla CTX-M-15 sequences (n = 141) of Indian origin showed that > 85% of them were obtained from bacteria not associated with the urinary tract, and that E. coli isolates account for majority of all bla CTX-M-15-carrying bacteria reported from India. Other types of bla CTX-M appear to be rare in India, since only six such sequences were reported as of July 2015. The results indicate that ‘selection pressure’ exerted by extended-spectrum cephalosporins may have stabilized the bla CTX-M-15 genotype among E. coli in India. The rarity of other bla CTX-M suggests that they lack the survival advantage that bla CTX-M-15 may have

The adiponectin receptor AdipoR2 and its <i>Caenorhabditis elegans</i> homolog PAQR-2 prevent membrane rigidification by exogenous saturated fatty acids

<div><p>Dietary fatty acids can be incorporated directly into phospholipids. This poses a specific challenge to cellular membranes since their composition, hence properties, could greatly vary with different diets. That vast variations in diets are tolerated therefore implies the existence of regulatory mechanisms that monitor and regulate membrane compositions. Here we show that the adiponectin receptor AdipoR2, and its <i>C</i>. <i>elegans</i> homolog PAQR-2, are essential to counter the membrane rigidifying effects of exogenously provided saturated fatty acids. In particular, we use dietary supplements or mutated <i>E</i>. <i>coli</i> as food, together with direct measurements of membrane fluidity and composition, to show that diets containing a high ratio of saturated to monounsaturated fatty acids cause membrane rigidity and lethality in the <i>paqr-2</i> mutant. We also show that mammalian cells in which AdipoR2 has been knocked-down by siRNA are unable to prevent the membrane-rigidifying effects of palmitic acid. We conclude that the PAQR-2 and AdipoR2 proteins share an evolutionarily conserved function that maintains membrane fluidity in the presence of exogenous saturated fatty acids.</p></div

Leveraging a gain-of-function allele of Caenorhabditis elegans paqr-1 to elucidate membrane homeostasis by PAQR proteins.

The C. elegans proteins PAQR-2 (a homolog of the human seven-transmembrane domain AdipoR1 and AdipoR2 proteins) and IGLR-2 (a homolog of the mammalian LRIG proteins characterized by a single transmembrane domain and the presence of immunoglobulin domains and leucine-rich repeats in their extracellular portion) form a complex that protects against plasma membrane rigidification by promoting the expression of fatty acid desaturases and the incorporation of polyunsaturated fatty acids into phospholipids, hence increasing membrane fluidity. In the present study, we leveraged a novel gain-of-function allele of PAQR-1, a PAQR-2 paralog, to carry out structure-function studies. We found that the transmembrane domains of PAQR-2 are responsible for its functional requirement for IGLR-2, that PAQR-1 does not require IGLR-2 but acts via the same pathway as PAQR-2, and that the divergent N-terminal cytoplasmic domains of the PAQR-1 and PAQR-2 proteins serve a regulatory function and may regulate access to the catalytic site of these proteins. We also show that overexpression of human AdipoR1 or AdipoR2 alone is sufficient to confer increased palmitic acid resistance in HEK293 cells, and thus act in a manner analogous to the PAQR-1 gain-of-function allele

Correction: Caenorhabditis elegans PAQR-2 and IGLR-2 Protect against Glucose Toxicity by Modulating Membrane Lipid Composition.

[This corrects the article DOI: 10.1371/journal.pgen.1005982.]

Palmitic acid causes membrane rigidity in the paqr-2 mutant.

<p><b>(A)</b> Outline of the experimental design. <b>(B)</b> PA content and PA/OA ratio among the PEs of <i>E</i>. <i>coli</i> grown with or without PA; vehicle is ethanol. <b>(C)</b> <i>E</i>. <i>coli</i> grown pre-loaded with PA inhibits the growth of the <i>paqr-2</i> mutant but not of wild-type N2 worms. <b>(D)</b> PA content among the PEs and PCs of worms fed control or PA-loaded <i>E</i>. <i>coli</i>. Note that the <i>paqr-2</i> mutant accumulates more PA than wild-type N2 worms <b>(E-F)</b> FRAP of N2 and <i>paqr-2</i> mutants on normal plates or plates seeded with PA-loaded <i>E</i>. <i>coli</i>, respectively. Note the loss of membrane fluidity in <i>paqr-2</i> worms fed PA-loaded <i>E</i>. <i>coli</i>. <b>(G)</b> The growth of <i>paqr-2</i> is greatly ameliorated by pre-loading <i>E</i>. <i>coli</i> with both PA and OA rather than PA alone, with photographed worms shown in <b>(H)</b>. <b>(I)</b> FRAP showing that the rigidifying effects of PA pre-loaded dietary <i>E</i>. <i>coli</i> on the <i>paqr-2</i> mutant are abrogated by pre-loading the <i>E coli</i> with both PA and OA. The dashed line in <b>(C)</b> and <b>(G)</b> represents the approximate length of the L1s at the start of the experiments.</p