Acinetobacter baumannii ATCC 19606, where mutants lacking enzymes occurring early in lipid A biosynthesis , and correspondingly lacking LPS, can grow. In contrast, we show here that LpxH, an enzyme that occurs downstream of LpxD in the lipid A biosynthetic pathway, is essential for growth in this strain. Multiple attempts to disrupt lpxH on the genome were unsuccessful, and when LpxH expression was controlled by an isopropyl β-d-1-thiogalactopyranoside (IPTG) inducible promoter, cell growth under typical laboratory conditions required IPTG induction. Mass spectrometry analysis of cells shifted from LpxH-induced to uninduced (and whose growth was correspondingly slowing as LpxH was depleted) showed a large cellular accumulation of UDP-2,3-diacyl-GlcN (substrate of LpxH), a C14:0(3-OH) acyl variant of the LpxD substrate (UDP-3-O-[(R)-3-OH-C14]-GlcN), and disaccharide 1-monophosphate (DSMP). Furthermore, the viable cell counts of the LpxH depleted cultures dropped modestly, and electron microscopy revealed clear defects at the cell (inner) membrane, suggesting lipid A intermediate accumulation was toxic. Consistent with this, blocking the synthesis of these intermediates by inhibition of the upstream LpxC enzyme using CHIR-090 abrogated the requirement for IPTG induction of LpxH. Taken together, these data indicate that LpxH is essential for growth in A. baumannii ATCC19606, because, unlike earlier pathway steps like LpxA or LpxC, blockage of LpxH causes accumulation of detergent-like pathway intermediates that prevents cell growth.The lipid A moiety of lipopolysaccharide (LPS) is the main constituent of the outer leaflet of the Gram-negative bacterial outer membrane (OM) and is essential in many Gram-negative pathogens. An exception is  O-[(R)-3-OH-C12]-GlcNAc, with the length of the incorporated β-hydroxyacyl chain varying among species [O-[(R)-3-OH-C12]-GlcNAc, catalyzed by LpxC, to produce UDP-3-O-[(R)-3-OH-C12]-GlcN -GlcN . LpxD th12]-GlcN . Again, 12]-GlcN . The mem12]-GlcN . DSMP is12]-GlcN  yielding-GlcN -GlcN (LpxD substrate) may somewhat impair growth due to its suspected detergent-like properties -GlcNAc through lipid IVA showed a profound accumulation of UDP-2,3-diacyl-GlcN (LpxH substrate) comprised of acyl chain lengths of two C12:0(3-OH) fatty acids or one C14:0(3-OH) and one C12:0(3-OH) fatty acid at the 5 hour time point (0.5 OD600) along the depletion growth curve compared to induced conditions (+IPTG) or the wild type parent cells -3-OH-C12]-GlcNAc (LpxA product) or the predicted LpxC product containing the C12:0(3-OH) fatty acid (UDP-3-O-[(R)-3-OH-C12]-GlcN) [O-[(R)-3-OH-C14]-GlcN) was observed . Alternative enzymes that hydrolyze UDP-2,3-diacyl-GlcN have been found in other Gram-negatives including Cdh (E. coli), LpxI (Caulobacter crescentus) and LpxG (Chlamydia trachomatis) [A. baumannii ATCC 19606. Since we harvested our cells at a point of strong growth inhibition, but not complete cessation  , 40, howobserved . This maobserved . This wahomatis) –47 but Bessation , the celessation , and thepathways .A. baumannii mutant lacking LPS , perhaps surprisingly did not exhibit any gross morphological defects when examined by electron microscopy [A. baumannii ATCC 19606 requires LpxH to grow and as described above, cells depleted of LpxH accumulate the detergent-like molecule UDP-diacyl-GlcN (and other pathway intermediates). If the accumulation of these intermediates is toxic and occurs at the cell membrane we surmised that, unlike the case for cells deficient in LpxA, cells deficient in LpxH would exhibit severe morphological defects. Consistent with this, electron microscopy of LpxH depleted cells revealed profound morphological changes, particularly at the cell envelope/inner membrane  should offset the dependence on LpxH for growth. Strongly supporting this notion, growth of LpxH depleted cells (-IPTG) was rescued by exposure to the well-characterized LpxC inhibitor, CHIR-090 [A. baumannii ATCC19606  depletion of LpxH (using a strain with the native lpxH deleted and complemented by a temperature sensitive plasmid carrying lpxH) caused an accumulation of the lipid A precursor UDP-diacyl-GlcN and a loss of viability under non-replicative conditions at 44°C [N. meningitidis, lpxH could be inactivated on the genome, and genomic sequencing of a LPS-deficient meningococcal isolate revealed a glycine to aspartic acid substitution (G95D) in LpxH believed to cause the loss of LPS [A. baumannii ATCC 19606, this phenomenon remains to be fully explored for other Gram-negatives.As described above, LpxH was essential for growth of CHIR-090 –53 in a TCC19606 . A similTCC19606 . These degatives , 38. In  at 44°C . In thats of LPS . This coA. baumannii ATCC19606 under standard laboratory conditions. A. baumannii ATCC 19606, which can tolerate loss of LPS, provided an elegant platform to explore how toxic accumulation of LPS pathway intermediates might mediate this dependence on LpxH, in turn suggesting that other LPS biosynthetic steps downstream of LpxH could also be essential for growth. Inhibiting targets such as LpxA or LpxC, which are not essential in some A. baumannii strains, would still be expected to provide coverage in the clinic since the cells inhibited in these enzymes would lack the LPS-containing outer membrane required for virulence and drug resistance [per se, and the contribution of toxic accumulation may extend to additional Gram-negatives making inhibition of these pathway steps particularly detrimental.In conclusion, we have established that LpxH is the earliest occurring (and so far only) lipid A biosynthetic enzyme that is essential for growth of sistance . NonetheS1 FiglacI-Ptac promoter fragment was inserted in front of lpxH by double homologous recombination to enable IPTG controlled expression of lpxH.The (PDF)Click here for additional data file.S2 FigKm::lacI, bla, alaS.pBR322 ori, pWH1266 P(PDF)Click here for additional data file.S3 FigalaS gene was replaced with a gentamicin resistance cassette (accC1) using double homologous recombination.The (PDF)Click here for additional data file.S4 Fig600 was placed in a 15-mL conical and centrifuged at 6000 x g  for 10 minutes to pellet cells. RNA isolation was performed using the RNeasy Mini Kit according to manufacturer’s instructions  and samples were diluted to a concentration of 20 ng/μL. RT-qPCR reaction were performed according to the qScriptTM XLT One-Step RT-qPCR ToughMix protocol  utilizing primer/probe set 5'-TGC TTG GAT TGG TGA TGA CTA T-3', 5'-CTC GGT TAC CCA CTT GGA AAT A-3', and 5'-/56-FAM/CGC CTT GGC/ZEN/TCG ACG AAA TTG TC/3IABkFQ/-3' (Integrated DNA Technologies). Briefly, in a 96-well PCR plate , 20 μL of the RT mix containing the appropriate primers and probe mixture was combined with 5 μL of the RNA sample with each reaction performed in duplicate. The plate was sealed , centrifuged, and RT-qPCR reaction were monitored using the Bio-Rad CFX96 Real Time system and data analysis with Bio-Rad CFX Manager 3.1. Raw data was reformatted in Excel.The LpxH depletion experiment was repeated as described in the manuscript. Five milliliter of culture at 0.5 OD(PDF)Click here for additional data file.S5 FigA  and 1x C12:0(3-OH) / 1x C14:0(3-OH) variants were selected for analysis. For DSMP and lipid IVA, the 3x C12:0(3-OH) / 1x C14:0(3-OH) fatty acids were selected from previously reported species [Precursor ion scans were run to determine the abundant acyl-chain variants for LpxC product , LpxD prA . Data ar reports . Based u species  and the  species .(PDF)Click here for additional data file.S6 FigChromatograms are provided for LpxA acyl chain variants from both experimental samples and authentic standards. The specific MRM transition being monitored as described in (PDF)Click here for additional data file.S7 FigChromatograms are provided for LpxA acyl chain variants from both experimental samples and authentic standards. The specific Extracted Ion Chromatogram (EIC)being monitored as described in (PDF)Click here for additional data file.S8 FigChromatograms are provided for LpxC acyl chain variants from both experimental samples and authentic standards. The specific MRM transition being monitored as described in (PDF)Click here for additional data file.S9 FigChromatograms are provided for LpxC acyl chain variants from both experimental samples and authentic standards. The specific Extracted Ion Chromatogram (EIC) being monitored as described in (PDF)Click here for additional data file.S10 Fig12 / C14, C14/ C12, or a mixture of these.Chromatograms are provided for LpxD acyl chain variants from both experimental samples and authentic standards. The specific MRM transition being monitored as described in (PDF)Click here for additional data file.S11 Fig12 / C14, C14/ C12, or a mixture of these.Chromatograms are provided for LpxD acyl chain variants from both experimental samples and authentic standards. The specific Extracted Ion Chromatogram (EIC) being monitored as described in (PDF)Click here for additional data file.S12 Fig12 / C14, C14/ C12, or a mixture of these.Chromatograms are provided for Lipid X acyl chain variants from both experimental samples and authentic standards. The specific MRM transition being monitored as described in (PDF)Click here for additional data file.S13 Fig12 / C14, C14/ C12, or a mixture of these.Chromatograms are provided for Lipid X acyl chain variants from both experimental samples and authentic standards. The specific Extracted Ion Chromatogram (EIC) being monitored as described in (PDF)Click here for additional data file.S14 Fig12 / C14 / C12 / C14 or C12 / C12 / C14 / C14, or a mixture of these.Chromatograms are provided for DSMP acyl chain variants from both experimental samples and authentic standards. The specific MRM transition being monitored as described in (PDF)Click here for additional data file.S15 Fig12 / C14 / C12 / C14 or C12 / C12 / C14 / C14, or a mixture of these.Chromatograms are provided for DSMP acyl chain variants from both experimental samples and authentic standards. The specific Extracted Ion Chromatogram (EIC) being monitored as described in (PDF)Click here for additional data file.S16 Fig12 / C14 / C12 / C14 or C12 / C12 / C14 / C14, or a mixture of these.Chromatograms are provided for Lipid IVa acyl chain variants from both experimental samples and authentic standards. The specific MRM transition being monitored as described in (PDF)Click here for additional data file.S17 Fig12 / C14 / C12 / C14 or C12 / C12 / C14 / C14, or a mixture of these.Chromatograms are provided for Lipid IVa acyl chain variants from both experimental samples and authentic standards. The specific Extracted Ion Chromatogram (EIC) being monitored as described in (PDF)Click here for additional data file.S18 Figm/z in the 1- charge state displayed. Acyl chain positions are for illustrative purposes only, based upon the final Lipid A structure. For example, it is not known if the species is C12 / C14 / C12 / C14 or C12 / C12 / C14 / C14, or a mixture from our analysis. See Proposed species are provided, consistent with reported MS/MS fragmentation for LPS intermediates and observed product ions. Species are drawn as neutral molecules with exact mass and (PDF)Click here for additional data file.S19 FigA. baumannii ATCC 19606 parent and NB48062-JWK0133 under inducing and non-inducing conditions. (B) LCMS-MRM quantification of lipid X with 1 acyl group 12:0(3-OH) and 1 acyl group 14:0(3-OH) compared with 2 acyl groups 14:0(3-OH) is shown for A. baumannii ATCC 19606 parent and NB48062-JWK0133 under inducing and non-inducing conditions. (C) Lipid IVA with three acyl groups with 12:0(3-OH) and one acyl group of 14:0(3-OH), compared with two acyl groups 12:0(3-OH) and two acyl groups 14:0(3-OH) for A. baumannii ATCC 19606 parent and NB48062-JWK0133 under inducing and non-inducing conditions. Experiments were performed in triplicate and bars show the mean value and SD) between NB46082-JWK0133 in the presence or absence of IPTG. Data shown was normalized to an internal standard (IS) as previously described [(A) LCMS-MRM quantification of LpxA product with 1 acyl group 12:0(3-OH) and 1 acyl group 14:0(3-OH) is shown for escribed .(PDF)Click here for additional data file.S20 FigE. coli LPS standard; Lane 2, A. baumannii ATCC 19606 parent; Lane 3, A. baumannii ATCC 19606 + 8 μg/ml CHIR-090. Experiments were performed as previously described [Lane 1, escribed .(PDF)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 TableLipid A pathway intermediates are described with acyl chain variants. Pathway enzymes producing each intermediate are also listed. Charge of the targeted transition, precursor ions, product ions, and collision energies are noted.(PDF)Click here for additional data file.S3 TableA in the QTOF system may also relate to interaction with the plastic surfaces in our Agilent system. Our AB Sciex system uses all glass and metal surfaces post-column.Lipid A pathway intermediates are described with acyl chain variants. Retention times by QQQ MRM analysis are noted, with standard peaks within 0.2 minutes of experimental peaks. Differences may be due to matrix effects for the poorly behaved detergent like molecules. QTOF theoretical and experimental monoisotopic m/z values are reported with mass errors in parts per million (ppm). Mass errors are <5 ppm for all species detected in the QTOF. Retention times by QTOF LC-MS are noted, with standard peaks within 0.2 minutes of experimental peaks. Differences may be due to matrix effects for the poorly behaved detergent like molecules. Peak times differ between QTOF and QQQ systems due to substantially larger dead-volume in the QTOF system. The relatively poor behavior observed for lipid IV(PDF)Click here for additional data file.S4 Tablem/z or QTOF theoretical m/z are provided depending on where the specific spectra was acquired—this is also noted in the reported precision. Product ions of interest are indicated and if they can be assigned to a putative structure as in m/z is provided. Peaks are also noted if a) shared between acyl chain variants (e.g. UDP fragments of LpxD product) or b) shifted by 28 Da between acyl chain variants (e.g. acyl-GlcN fragments form LpxD product) with the second class in bold. Peaks matching a specific MRM transition are italic underlined as reported in Lipid A pathway intermediates are described. QQQ precursor (PDF)Click here for additional data file.


Clostridium perfringens iota toxin is a binary toxin composed of the enzymatically active component Ia and receptor binding component Ib. Ia is an ADP-ribosyltransferase, which modifies Arg177 of actin. The previously determined crystal structure of the actin-Ia complex suggested involvement of Asp179 of actin in the ADP-ribosylation reaction. To gain more insights into the structural requirements of actin to serve as a substrate for toxin-catalyzed ADP-ribosylation, we engineered Saccharomyces cerevisiae strains, in which wild type actin was replaced by actin variants with substitutions in residues located on the Ia-actin interface. Expression of the actin mutant Arg177Lys resulted in complete resistance towards Ia. Actin mutation of Asp179 did not change Ia-induced ADP-ribosylation and growth inhibition of S. cerevisiae. By contrast, substitution of Glu270 of actin inhibited the toxic action of Ia and the ADP-ribosylation of actin. In vitro transcribed/translated human β-actin confirmed the crucial role of Glu270 in ADP-ribosylation of actin by Ia. Clostridium botulinum, C. difficile, C. perfringens, C. spiroforme, and Bacillus cereus, produces actin-ADP-ribosylating “binary toxins”. These toxins consist of a binding component, which is involved in toxin up-take, and an enzymatically active component, which harbors ADP-ribosyltransferase activity. Both components are secreted as separated proteins  yeast variant was transformed with YCplac33 vector containing ACT1 gene with the minus-827 promoter- and plus-274 terminator-containing regions. The resulting strain was subsequently sporulated and dissected . After tetrad analysis a haploid [Δact1 + YCplac33-ACT1] isolate was selected and used for subsequent experiments. YCplac33-ACT1 was replaced by pRS313-based plasmids encoding different ACT1 mutants via 5-fluoroorotic acid  plasmid shuffling method [e method . To gened strain , before ACT1 was cut out from the YCplac33-ACT1 plasmid and ligated into pBluescript vector. The resulting plasmid was used as a matrix in QuikChange reactions  aimed at substitutions of amino acid residues of interest. All mutated genes were initially subcloned back into XhoI/KpnI endonuclease restriction sites of YCplac33-ACT1 and then transferred en bloc with the upstream promoter and downstream terminator regions into pRS313 using EcoRI/SalI sites.For site-directed mutagenesis XhoI/KpnI fragment of 595 measurements were spotted onto agar plates containing the required supplements or YPD. Plates were incubated for 3–4 days at 30°C.The influence of actin site-substitutions and Ia gene expression upon growth behavior of the resulting yeast variants was analyzed on minimal agar plates by drop-test. To that end, 5-fold serial dilutions of suspensions prepared from overnight agar cultures normalized by ODS. cerevisiae  was constructed using YEplac555 backbone by introducing GAL1/10 promoter and 3’ untranslated region from pESC-His using SacI/PstI restriction endonucleases with subsequent introduction of NcoI site immediately following BamHI site of the plasmid. A gene coding for the A-subunit of C. perfringens iota toxin was PCR amplified from chromosomal DNA of C. perfringens strain E342  and cloned into YEpGal555 using NcoI/SalI endonuclease sites. For protein production Ia-coding sequence was cut out from the latter plasmid with BamHI/SalI restriction endonucleases and ligated into pET28a.The yeast vector for cloning of the Ia-coding gene in E. coli BL-21 Codon Plus strain transformed with Ia-coding gene within pET28a plasmid was grown on a shaker at 37°C until OD595 = 0.8. Expression of ia gene was induced by 1 mM IPTG overnight at 28°C. Cells were collected by centrifugation and suspended in a 6xHis-binding buffer (20 mM phosphate buffer (pH 7.4), 0.5 M NaCl, and 5 mM imidazole) supplemented with 1 mM PMSF and protease inhibitor cocktail . Bacteria were lysed by sonication. Bacterial extract, clarified by centrifugation, was loaded onto a column with Ni-IDA resin . The column was washed sequentially with 6xHis-binding buffer and with 6xHis-binding buffer plus 30 mM imidazole. Ia-containing preparation was eluted with 500 mM imidazole and dialyzed overnight against 20 mM Tris-HCl (pH 7.4) with 50 mM NaCl. After dialysis, the sample was applied onto Mono Q 5/50 GL  column. Flow-through fraction, containing Ia, was collected, concentrated and subjected to gel chromatography on Superdex 75 10/300 GL  equilibrated with TBS (20 mM Tris-HCl (pH 7.4) and 150 mM NaCl).For protein production et al [595 = 5–7, collected by centrifugation, resuspended in a small amount of lysis buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2 mM DTT, 1 mM PMSF and protease inhibitor cocktail) and frozen drop-wise in liquid nitrogen. Frozen yeast cells were lysed using Mixer Mill MM400 device . Obtained yeast powder was resuspended in a lysis buffer, cleared by centrifugation and applied onto a column, containing DNase I (Sigma) immobilized on Affi-Gel 10 (Bio-Rad) according to the manufacturer’s protocol. After application of yeast extract, the column was washed with G-buffer (20 mM Tris-HCl (pH 7.4), 0.5 mM ATP, 0.2 mM DTT and 0.2 mM CaCl2) and wash buffer (G-buffer with 10% formamide). Actin was eluted with 50% formamide in G-buffer. The protein-containing fractions were immediately diluted 5-fold with G-buffer and dialyzed against it overnight. Afterwards, samples were concentrated using Vivaspin 20 , dispensed into aliquots, frozen in liquid nitrogen and stored at -80°C until use.Purification of yeast actin was performed according to the DNase I affinity method originally described by Kron et al  with modet al . To thisMass-analysis of purified wild type actin and actin R177K variant was performed on 4800 MALDI TOF/TOF Analyzer  at TOPLAB GmbH, .in vitro was performed either with crude yeast lysate (2.5 mg/ml) or with purified actin (100 μg/ml) by recombinant Ia or CDT-A (0.25 μg/ml of Ia or CDT-A for crude yeast extract and 1 μg/ml of Ia for the purified protein) in a total volume of 10 μl. Reaction mixtures contained 1 μM non-radiolabeled NAD, 0.1 μCi 32P-NAD , 0.5 mM DTT, 20 mM Hepes-KOH (pH 7.4), 100 mM KCl and 2 mM MgCl2 (ADP-ribosylation with yeast extract) or 1 μM non-radiolabeled NAD, 0.1 μCi 32P-NAD, 20 mM Tris-HCl (pH 7.4), 0.5 mM ATP, 0.2 mM DTT and 0.2 mM CaCl2 (ADP-ribosylation with purified actin). Incubation was at 30°C for the indicated times. Reactions were stopped by the addition of Laemmli sample buffer and heating at 95°C for 5 min. Then, the samples were subjected to SDS-PAGE and autoradiography using Storm 820 phosphorimager . Autoradiograms were quantified by ImageQuant 5.2 .Actin modification in vitro transcription/translation reaction  using the plasmids, coding for the proteins with the amino acid substitutions, and 35S-methionine . Afterwards, 1 μl of the in vitro transcription/translation mix, containing co-translationally 35S-labeled actin, was ADP-ribosylated with 0.2 mM NAD in 20 mM Tris-HCl (pH 7.4), 0.5 mM ATP, 0.2 mM DTT and 0.2 mM CaCl2 and various concentrations of Ia and subjected to polyacrylamide gel electrophoresis under non-denaturing conditions [Point mutations in human β-actin gene, resulting in actin variants with Arg177Lys, Asp179Ala, Glu270Asp and Glu270Gln substitutions, were produced by QuikChange reaction using the matrix plasmid β-actin-pET28a  and the nditions . Finally2, 0.5 mM DTT, 1 mM PMSF and protease inhibitor cocktail. Yeast extracts and purified proteins were analyzed by polyacrylamide gel electrophoresis in sodium dodecylsulfate buffer and Western blotting. Protein concentrations were estimated using Coomassie Brilliant Blue G-250 dye. For actin immunodetection monoclonal antibodies  were used at 1/2000 dilution.Crude yeast extracts for Western blotting and enzymatic assays were prepared by glass-beads disruption in 20 mM Hepes-KOH (pH 7.4) with 100 mM KCl, 2 mM MgClS1 Fig(TIFF)Click here for additional data file.S2 Fig(TIFF)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S1 Legends(DOCX)Click here for additional data file.S1 Results(DOCX)Click here for additional data file.


Drosophila fGSCs and that their complex formation is required for accurate abscission timing in GSCs in vivo.Abscission is the final step of cytokinesis that involves the cleavage of the intercellular bridge connecting the two daughter cells. Recent studies have given novel insight into the spatiotemporal regulation and molecular mechanisms controlling abscission in cultured yeast and human cells. The mechanisms of abscission in living metazoan tissues are however not well understood. Here we show that ALIX and the ESCRT-III component Shrub are required for completion of abscission during Drosophila female germline stem cell (fGSC) division. Loss of ALIX or Shrub function in fGSCs leads to delayed abscission and the consequent formation of stem cysts in which chains of daughter cells remain interconnected to the fGSC via midbody rings and fusome. We demonstrate that ALIX and Shrub interact and that they co-localize at midbody rings and midbodies during cytokinetic abscission in fGSCs. Mechanistically, we show that the direct interaction between ALIX and Shrub is required to ensure cytokinesis completion with normal kinetics in fGSCs. We conclude that ALIX and ESCRT-III coordinately control abscission in  Drosophila melanogaster as a model organism. We show that the scaffold protein ALIX and the ESCRT-III component Shrub are required for completion of abscission in Drosophila female germline stem cells (fGSCs). ESCRT-III has been implicated in topologically similar membrane scission events as abscission, namely intraluminal vesicle formation at endosomes and virus budding. Here we demonstrate that ALIX and Shrub co-localize and interact to promote abscission with correct timing in Drosophila fGSCs. We thus show that ALIX and ESCRT-III coordinately control abscission in Drosophila fGSCs cells and report an evolutionarily conserved function of the ALIX/ESCRT-III pathway during cytokinesis in a multi-cellular organism.Cytokinesis, the final step of cell division, concludes with a process termed abscission, during which the two daughter cells physically separate. In spite of their importance, the molecular machineries controlling abscission are poorly characterized especially in the context of living metazoan tissues. Here we provide molecular insight into the mechanism of abscission using the fruit fly  We complex .Nanos-GAL4 and ALIX was detected with our anti-ALIX antibody. Interestingly, GFP-Shrub localized at MRs and MBs during cytokinesis in fGSCs  and ALItions by ). Furthetions by . Consistshrub using the Nanos-GAL4 driver. Control germaria displayed normal fGSC and egg chamber phenotypes    and ~50%ranched) . Importaem cysts , ~10% ofem cysts  and ~50%em cysts . Consistgermaria  suggestishrub- and alix-RNAi using Nanos-GAL4. We detected about 55% of the fGSCs in stem cysts   gave, in addition to stem cysts, rise to the appearance ~10% of germaria with polyploid fGSCs, ~10% of agametic germaria as well as fewer stem cells per germarium than normal suggesting that reduction of the ALIX and Shrub levels in all cell types in the germarium both caused abscission defects and affected germ cell viability  . The inciability . The facDrosophila GFP-ALIX (GFP-ALIX-F198D and GFP-ALIX-I211D) of residues which have previously been shown to mediate the interaction with CHMP4 in human cells -CmR-BW (http://bacpac.chori.org/home.htm) were injected into strains y1 1118; PBac{y+-attP-9A}VK00018w  and y1 1118; PBac{y+-attP-3B}VK00037w  and integrated into predetermined attP docking sites in the genome using PhiC31 integrase-mediated germline transformation. The methodology is decribed in “Versatile P[acman] BAC libraries for transgenesis studies in Drosophila melanogaster” [Drosophila embryos was performed by BestGene (http://www.thebestgene.com/). Males with integrated constructs were obtained from BestGene, balanced and crossed to generate 1/TM6B, TbCH322–119C06/CyO; alix , 3/TM6B, TbCH322–119C06/CyO; alix , 1/TM6B, TbCH321–50C24/CyO; alix  and 3/TM6B, TbCH321–50C24/CyO; alix  stocks.Genomic rescue constructs . The dechorionated embryos were extensively rinsed with PBS + 0.02% Triton X-100, and blotted dry on paper towels. The embryos were transferred from the nylon mesh and to a small flask with 5 mL heptane. An equal amount of 4% formaldehyde in PBS was added, and the two-phase mixture was incubated with vigorous shaking for 17 minutes. The embryos were now between the two phases. The formaldehyde phase was removed and replaced with methanol, and the embryos were gently shaken for 1 minute with gentle heating for removal of the vitelline membrane. The heptane phase was removed along with the embryos still remaining in the interphase. The embryos that sank to the bottom of the flask were washed three times in methanol and stored at -20°C. Immunofluorescent staining of embryos was performed as follows. The embryos were rehydrated by first putting them in 3:4 methanol and 1:4 4% formaldehyde in PBS for 2 minutes, and then 1:4 methanol and 3:4 formaldehyde for 5 minutes. Post fixation was done for 10 minutes in 4% formaldehyde, before the embryos were rinsed six times using PBS with 1% BSA and 0.05% Triton X-100. The embryos were incubated with α-spectrin antibodies  over night at 4°C. After incubation the embryos were rinsed three times and washed for one hour with PBS with 1% BSA and 0.05% Triton X-100, and then incubated with secondary antibody for two hours. The antibodies were diluted in PBS with 1% BSA and 0.05% Triton X-100. The embryos were again rinsed three times and washed for one hour with PBS with 1% BSA and 0.05% Triton X-100, before being labeled with Hoechst 33342 (2 µL/mL) for 10 minutes, and then rinsed 3 times in PBS to remove detergent. The embryos were mounted using Vectashield (Vector laboratories). For quantifications of mono- and binucleate cells, images of homozygous wild type, 1alix and 3alix mutant stage 16 embryos were captured at the confocal microscope and more than 1000 cells analyzed for each genotype.Embryo collection, permeabilization and fixation were based on the protocol described by Rothwell and Sullivan . The DroS1 Fig(A-C) ALIX co-localizes with Centrosomin (Cnn) at centrosomes in meta-, anaphase and early telophase . No signal is detected at centrosomes using the pre-immune (pre-im) serum . (D) ALIX co-localizes with Cnn at centrosomes and in addition appears at the intercellular bridge in mid telophase where it overlaps with the mitotic spindle (D’). No signal is detected at centrosomes nor at the intercellular bridge using the pre-immune serum (D). (E) In late telophase/cytokinesis, ALIX localizes to the dark region in the α-tubulin staining at the centre of the intercellular bridge, indicating its localization at the midbody ring (E’). ALIX also shows a vesicular pattern within the cell at this stage (E’). No signal is detected at the midbody ring using the pre-immune serum (E). A weak vesicular pattern detected, but is much weaker than in (E’). In all panels, S2 cells were fixed and stained with antibodies against ALIX (red), Cnn (green) and α-tubulin (white), and with Hoechst (blue). Images in all panels were captured with the same intensity. Scale bars represent 5 µm.(TIF)Click here for additional data file.S2 Fig(A) Western blot showing ALIX expression levels in Drosophila embryos, L3 larvae, pupae, adult males and females as well as testes and ovaries. α-tubulin was used as a loading control. (B) Western blot showing loss of ALIX protein in 1alix mutant males and females. Heterozygote 1/TM6B, Tbalix males and females show reduced protein levels compared to wild type. α-tubulin was used as a loading control. (C) Left: Image of wild type stage 16 embryonic epithelium. Middle and right: Images of homozygous 1alix and 3alix mutant stage 16 embryonic epithelia. Embryos were fixed and stained with antibodies against α-spectrin (red) and with Hoechst (green). More than 1000 cells from five embryos of each genotype were analyzed for the presence of mono- and bi-nucelate cells and no evident bi-nucleation could be detected for any of the genotypes. Scale bars represent 10 µm. (D) Left: Image of wild type follicle cell epithelium of stage 6 egg chamber. Middle and right: Images of 1alix and 3alix mutant follicle cell epithelia of stage 6 egg chambers. Bi-nucleate cells are indicated with asterisks. Ovaries were fixed and stained to visualize F-actin (red) and nuclei . Scale bars represent 5 µm. See also (E) Left: Stage 10 EC with GFP-positive mono-nucleate control follicle cell clones. Middle and right: 3alix and 1alix mutant GFP-positive clones with bi-nucleate cells (asterisks). Ovaries were fixed and stained to visualize F-actin (red) and nuclei . Scale bars represent 20 µm. See also (F) Left: Stage 14 EC with GFP-positive mononucleate control follicle cell clones. Middle and right: 3alix and 1alix mutant GFP-positive clones with bi-nucleate cells (asterisks). The bi-nucleation in the alix mutant clones may arise via alternative mechanisms. It is possible that loss of ALIX function leads to loss of the connection of the stable intercellular bridge between follicle cells with the plasma membrane as the egg chamber develops from stage 10 to stage 14 and thus that ALIX is required to maintain separate cells at late stages of oogenesis in somatic Drosophila follicle cells. Alternatively, the bi-nucleation could be caused by abscission failure, but an abscission event in follicle cells at late stages of oogenesis has not, to our knowledge, been described as the stable intercellular bridges formed via incomplete cytokinesis in the follicle epithelium are thought to persist throughout Drosophila oogenesis [ogenesis , 79, 80.(TIF)Click here for additional data file.S3 Fig(A) Graph showing the average egg lay rates for wild type and 1alix mutant females crossed to either wild type or 1alix mutant males from three independent experiments. Data are presented as mean ± STD. (B) Graph showing average hatch rates for the eggs laid in the crosses from the three independent experiments in (B). Data are presented as mean ± STD. (C) Western blot showing expression of ALIX protein in wild type ovaries and loss of ALIX protein in ovaries of 1alix homozygote mutant females, of females in which the 1alix allele is combined with two different deficiences (1/Df(3R)BSC499, alix1/Df499alix and 1/Df(3R)BSC739, alix1/Df739alix) or with the 3alix allele , of 3alix homozygote mutant females, or females in which the 3alix allele is combined with the two different deficiences . α-tubulin served as a control for protein loading. (D) Images showing four ring canals (arrows) to the oocyte in a wild type egg chamber and five ring canals (arrows) to the oocyte in egg chambers of the genotypes in (C). Ovaries were fixed and stained to visualize F-actin (white). Scale bars represent 5 µm. (E) Graph showing the percentages of egg chambers with 16, 32 or more germ cells the indicated genotypes in (C-D). Wild type, three independent experiments, n = 362 egg chambers; 1alix, two independent experiments, n = 154 egg chambers; 1/Df(3R)BSC499alix, two independent experiments, n = 242 egg chambers; 1/Df(3R)BSC739alix, two independent experiments, n = 38; 1/alix3alix, one experiment, n = 42 egg chambers, 3alix, two independent experiments, n = 150 egg chambers, 3/Df(3R)BSC499alix, two independent experiments, n = 139; 3/Df(3R)BSC739alix, one experiment, n = 88 egg chambers. Data are presented as mean ± STD.(TIF)Click here for additional data file.S4 Fig(A) Western blot showing ALIX expression in wild type ovaries, lack of ALIX protein in 1alix homozygote mutant ovaries and ALIX expression in ovaries of two lines with one copy of either of the two genomic rescue constructs  in the 1alix mutant background . Levels of α-tubulin show equal protein loading. Rescue constructs and lines are described in the Materials and Methods. (B) Images showing four ring canals (arrows) to the oocyte in a wild type egg chamber, five ring canals to the oocyte (arrows) in an 1alix mutant egg chamber and four ring canals (arrows) to the oocyte upon reexpression of ALIX in the 1alix mutant background from either of the two rescue constructs . Ovaries were fixed and stained to visualize F-actin (white). Images are planar projections of several sections of a z-stack. Scale bars represent 5 µm. (C) Graph showing the average percentage of egg chambers with 16, 32 or more germ cells of the genotypes in (A) and (B). Wild type, three independent experiments, n = 326 egg chambers; 1alix, three independent experiments, n = 236 egg chambers; 1alix-s/CyO; alix, three independent experiments, n = 301 egg chambers; 1alix-l/CyO; alix, three independent experiments, n = 226 egg chambers. Data are presented as mean ± STD. (D) Western blot showing ALIX expression in wild type ovaries, lack of ALIX protein in 3alix homozygote mutant ovaries and ALIX expression in ovaries from two lines with one copy of either of the two genomic rescue constructs described in (A) in the 3alix mutant background . Levels of α-tubulin show equal protein loading. (E) Images showing four ring canals to the oocyte (arrows) in a wild type egg chamber, five ring canals (arrows) to the oocyte in an 3alix mutant egg chamber and four ring canals (arrows) to the oocyte upon reexpression of ALIX in the 3alix mutant background from either of the two rescue constructs . Ovaries were fixed and stained to visualize F-actin (white). Scale bars represent 5 µm. (F) Graph showing the average percentage of egg chambers with 16, 32 or more germ cells from the genotypes in (D) and (E). Wild type, three independent experiments, n = 340 egg chambers; 3alix, three independent experiments, n = 275 egg chambers; 3alix-s/CyO; alix, three independent experiments, n = 310 egg chambers; 3alix-l/CyO; alix, two independent experiments, n = 235 egg chambers. Data are presented as mean ± STD. (G) Western blot showing ALIX expression in control ovaries (MTD-GAL4/+) and reduced levels of ALIX protein in ovaries in which RNAi-mediated gene silencing was performed in germ cells using MTD-GAL4 and the TRiP-alix-RNAi line . (H) Images showing four ring canals (arrows) to the oocyte of a control egg chamber and five ring canals (arrows) to the oocyte in an alix-RNAi egg chamber. Ovaries were fixed and stained to visualize F-actin (white). Images are planar projections of several sections from z-stacks. Scale bars represent 10 µm. (I) Graph showing the average percentage of egg chambers with 16, 32 or other phenotypes from control and alix-RNAi females. Control, four experiments, n = 423 egg chambers; alix-RNAi, four experiments, n = 602 egg chambers. Data are presented as mean ± STD.(TIF)Click here for additional data file.S5 Fig(A)Control (Nos-GAL4/+) egg chamber with four ring canals (arrows) to the oocyte (left image) and egg chamber with alix-RNAi  expression in fGSCs (right image) with five ring canals (arrows) to the oocyte are shown. Nos, Nanos; TRiP-alix = alix-RNAi line from TRiP. (B)Control  egg chamber with four ring canals (arrows) to the oocyte (left image) and egg chamber with alix-RNAi and Dicer  expression (right image) with five ring canals to the oocyte are shown. Dcr2, Dicer 2. (C) Egg chambers from females with the genotypes Bam-GAL4/+ (left image) and Bam-GAL4/TRiP-alix (right image) with four ring canals (arrows) to the oocyte are shown. In (A-C) ovaries were fixed and stained with fluorescently labeled phalloidin (green) and with Hoechst (not shown). Scale bars represent 20 μm. (D) Graph showing the average percentage of egg chambers with 16 or 32 or more germ cells for the genotypes in (A-C). Nos-Gal4/+, three independent experiments, n = 181 egg chambers; Nos-Gal4/+; TRiP-alix/+, three independent experiments, n = 159 egg chambers; Dcr2/+; Nos-GAL4/+, three independent experiments, n = 149 egg chambers; Dcr2/+; Nos-GAL4/+; TRiP-alix /+, three independent experiments, n = 146; Bam-GAL4/+, three independent experiments, n = 179 egg chambers; Bam-GAL4/TRiP-alix, three independent experiments, n = 192 egg chambers. Data are presented as mean ± STD. (E) Germarium from control female (Nos-GAL4/+) with Nanos-positive fGSCs with spectrosomes (left image) and germarium with alix-RNAi expression in germ cells, including fGSCs, using Nos-GAL4  (right image) are shown. In the right image an fGSC interconnected to three other Nanos-positive cells via fusome is seen in the anterior tip of the germarium. (F) Germarium from control female  (left image) with Nanos-positive fGSCs with spectrosomes and germarium in which alix-RNAi and Dicer were expressed  (right image) are shown. Two fGSCs interconnected to multiple Nanos-positive cells via fusomes are seen in the anterior tip of the germarium in the right image. (G) Germarium from control female (Bam-GAL4/+) (left image) with Nanos-positive fGSCs with spectrosomes and germarium in which RNAi-mediated depletion of alix was performed using Bam-GAL4  (right image) are shown. fGSCs with spectrosomes are seen in the anterior tip of the germaria. In (E-G) ovaries were fixed and stained with antibodies against Nanos (green) and hts-F (red). Scale bars represent 10 µm. (H) Graph showing the average percentages of germaria with normal or abnormal (linear or branched elongated) fusome morphologies in germaria of females of the genotypes in (E-G). Nos-Gal4/+, three independent experiments, n = 155 germaria; Nos-Gal4/+; TRiP-alix/+, three independent experiments, n = 168 germaria; Dcr2/+; Nos-GAL4/+, three independent experiments, n = 158 germaria; Dcr2/+; Nos-GAL4/+; TRiP-alix/+, three independent experiments, n = 139 germaria; Bam-GAL4/+, three independent experiments, n = 174 germaria; Bam-GAL4/TRiP-alix, three independent experiments, n = 172 germaria. Data are presented as mean ± STD.(TIF)Click here for additional data file.S6 Fig(A) Examples of wild type, 1alix and 3alix germaria stained for p-MAD (red) are shown. The wild type p-Mad-positive fGSC (red) is connected to a daughter cell CB via fusome (white). The 1alix and 3alix p-Mad-positive fGSCs (red) are connected chains of daughter cell via elongated fusomes. CC, cap cell. Scale bars represent 5μm. (B-D) Images showing Bam protein staining of wild type, 1alix and 3alix germaria. An fGSC-CB pair (B) or stem cysts (C-D) are outlined. Ovaries were stained with antibodies against Bam (green) and Cindr (red), and with Hoechst (blue). Scale bars represent 10 µm.(TIF)Click here for additional data file.S7 Fig(A-B) Shown are examples wild type germaria with normal fGSC morphologies: (i) a single fGSCs with a spectrosome , (ii) fGSC-CB pairs undergoing cytokinesis with midbody rings  and fused fusomes (red) and (iii) an fGSC-CB pair in late cytokinesis with a midbody  and fusome with exclamation point morphology (red). (C-D)1alix mutant germaria show abnormal fGSC morphologies. Shown are fGSCs connected to more than one daughter cell in (iv) linear (C) or (v) branched (D) chains via midbody rings (green) and fusome (red). Cells interconnected are marked with asterisks. Ovaries in (A-D) were fixed and stained with antibodies against Cindr (green) and hts-F (red), with phalloidin to visualize F-actin (white) and with Hoechst (blue). Scale bars in (A-D) represent 5 µm. (E) Graph showing the average percentage of fGSCs with the indicated phenotypes from wild type and 1alix mutant flies. Wild type, three independent experiments, n = 110, 30 germaria; 1alix, three independent experiments, n = 70, 29 germaria. The larger proportion of polyploid fGSCs in the 1alix compared to the 3alix mutant germaria might be explained by the fact that the 1alix mutant flies were older than the 3alix mutant females (7 days compared to 2–4 days old), allowing for more time for cleavage furrow regression. Data are presented as mean ± STD. See also (F)Wild-type germarium with normal fGSC morphologies. Shown are fGSCs with spectrosomes . (G-H) In 1alix (G) and 3alix (H) mutant germaria, bi- and multinucleate fGSCs can be detected. Nuclei of cells with more than one nucleus are marked with asterisks. Bi- and multi-nucleate phenotypes were mostly detected in flies older than four days and in some cases the midbody ring was still visible in the cell (H), indicating that it lost the connection to the plasma membrane. Ovaries in (F-H) were fixed and stained with antibodies against Cindr (green) and hts-F (blue), with rhodamine-phalloidin to visualize F-actin (red) and with Hoechst (white). Scale bars represent 10 µm. (I) Germarium from control female (Nos-GAL4/+) with fGSC-CB pair in abscission with an MB  dividing the fusome (red) (left image) and germarium with alix-RNAi expression  (right image) are shown. In the right image an fGSC is interconnected to several daughter cells via MRs  and fusome (red). (J) Germarium from control female  with fGSC-CB pair in abscission with an MB  dividing the fusome (red) (left image) and germarium with alix-RNAi and Dicer expression  (right image) are shown. In the right image an fGSC is interconnected to several daughter cells via MRs  and fusome (red). (K) Germarium from control female (Bam-GAL4/+) (left image) and germarium in which RNAi-mediated depletion of alix was performed using Bam-GAL4  (right image) are shown. fGSCs in both images display normal abscission . In (I-K) ovaries were fixed and stained with antibodies against Cindr (green) and hts-F (red). Scale bars represent 10 µm (full germaria) and 5 µm (enlarged images). (L-N) Shown in (L) is an fGSC-CB pair in abscission . In chains of cells interconnected to fGSCs (marked with asterisks and outlined) in 3alix (M) and 1alix (N) mutant germaria, MBs are detected (arrows), indicating abscission events of 2-cell cysts with MRs/RCs (arrowheads). Ovaries were fixed and stained with antibodies against Cindr  and hts-F , and with Hoechst (blue). F-actin is stained in red in N. Scale bars represent 5 µm.(TIF)Click here for additional data file.S8 Fig(A) Western blot showing the lack of ALIX protein 1alix and 3alix mutant testes. Equal protein loading is validated by the levels of α-tubulin. (B)Wild type testis tip with mGSC-gonialblast (GB) pair (outlined) in cytokinesis interconnected by an MR  and fusome . (C-D) Testis tips in 1alix and 3alix mutants with mGSCs connected to chains of daughter cells (outlined) via MRs (green) and fusome (red). Testes in (B-D) were fixed and stained with antibodies against Cindr (green), α-spectrin (red) and FasIII (red), and with Hoechst (blue). Hubs are indicated with asterisks. Scale bars represent 5 µm. (E) Graph showing the average percentage of mGSCs with the indicated phenotypes. Wild type, three independent experiments, n = 166 mGSCs, 17 testes; 1alix, three independent experiments, n = 201 mGSCs, 17 testes; 3alix, three independent experiments, n = 119 mGSCs, 17 testes.(TIF)Click here for additional data file.S9 Fig(A-D) Ovaries of 1–2 day-old UASp-GFP-ALIX/+; Nanos-GAL4/+ (A-C) or Nanos-GAL4, UASp-GFP-Shrub (D) flies were dissected, fixed and stained with anti-α-tubulin (red), GFP Booster (green), anti-hts-F (white) and Hoechst (blue). Arrows indicate localization of GFP-ALIX to fusome plugs (A), MRs in G1/S , S phase  and G2 . Intercellular bridge MTs are present early when the fusome has plug morphology. At this point only weak GFP-Shrub signal is detected and in G2 phase GFP-Shrub is detected at an MR (D). Scale bars represent 5 µm.(TIF)Click here for additional data file.S10 Fig(A-D) Control EC with 4 RCs (arrows) to the oocyte (A) and alix-RNAi (B), shrub-RNAi (C) as well as shrub- and alix-RNAi (D) ECs with 5 RCs (arrows) to the oocyte. Ovaries were fixed and stained with phalloidin to visualize F-actin (white) and with Hoechst (blue). Scale bars represent 10 µm (left images) and 5 µm (right images). (E) Graph showing the average percentage of egg chambers (ECs) with 16, 32, more than 32 GCs, tumor phenotype and other phenotypes from control, alix-RNAi, shrub-RNAi, and shrub & alix-RNAi flies. Control, four independent experiments, n = 493 ECs; alix-RNAi, four independent experiments, n = 509 ECs; shrub-RNAi, four independent experiments, n = 231 ECs; shrub & alix-RNAi, 3 independent experiments, n = 215 ECs. (F) Graph showing the average percentages of germaria with the indicated phenotypes from wild type, 1, shrubG5/+alix and G5/+; alix1shrub germaria. Wild type, four independent experiments, n = 29 germaria; 1alix, three independent experiments, n = 23 germaria; G5/+shrub, three independent experiments, n = 27 germaria; G5/+; alix1shrub, three independent experiments, n = 33 germaria. (G) Graph showing the average number of stem cells per germarium from the wild type, 1, shrubG5/+alix and G5/+; alix1shrub germaria in (F). Wild type, n = 80 fGSCs; 1alix, n = 50 fGSCs; G5/+shrub, n = 70 fGSCs; G5/+; alix1shrub, n = 54 fGSCs. Data are presented as mean ± STD.(TIF)Click here for additional data file.S11 Fig(A) Images showing the localization of wild type GFP-ALIX, GFP-ALIX-F198D or GFP-ALIX-I211D at MRs/MBs in fGSC-CB pairs or at MRs in stem cysts in germaria of the indicated genotypes. Ovaries were fixed and stained with antibodies against Cindr (red) and hts-F (white) and with Hoechst (blue). CC, cap cell. Scale bars represent 5 µm. (B) Graph showing the frequencies of stem cyst lengths for the indicated genotypes from the experiment in (C) Graph showing the average percentages of egg chambers with the indicated phenotypes in ovaries of females with the indicated genotypes from three independent experiments. Nanos-GAL4/+, n = 391 ECs; Nanos-GAL4/UASp-GFP-ALIX, 381 ECs; Nanos-GAL4/UASp-GFP-ALIX-F198D, 357 ECs; Nanos-GAL4/UASp-GFP-ALIX-I211D, 485 ECs; 1alix, 508 ECs; 1Nanos-GAL4/UASp-GFP-ALIX; alix, 392 ECs; Nanos-GAL4/UASp-GFP-ALIX-F198D; 1alix, 500 ECs; 1Nanos-GAL4/UASp-GFP-ALIX-I211D; alix, 378 ECs. Data are presented as mean ± STD.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


Odors evoke gamma 60 - 100 Hz) and beta 20 - 30 Hz) oscillations in the local field potential (LFP) of the rat olfactory bulb (OB). These oscillations arise from activity in the dendrodendritic microcircuit between excitatory mitral cells (MCs) and inhibitory granule cells (GCs) [00 Hz and0 - 30 Hz


Similarly, pharmacologic inhibition of aerobic glycolysis with moderate doses of 2-deoxyglucose (2-DG) slowed B-ALL cell proliferation, but extensive apoptosis only occurred at high doses. Nevertheless, 2-DG induced the pro-apoptotic protein Bim and sensitized B-ALL cells to the tyrosine kinase inhibitor Dasatinib in vivo. Together, these data show that despite expression of multiple glucose transporters, B-ALL cells are reliant on Glut1 to maintain aerobic glycolysis and anabolic metabolism. Further, partial inhibition of glucose metabolism is sufficient to sensitize cancer cells to specifically targeted therapies, suggesting inhibition of aerobic glycolysis as a plausible adjuvant approach for B-ALL therapies.The metabolic profiles of cancer cells have long been acknowledged to be altered and to provide new therapeutic opportunities. In particular, a wide range of both solid and liquid tumors use aerobic glycolysis to supply energy and support cell growth. This metabolic program leads to high rates of glucose consumption through glycolysis with secretion of lactate even in the presence of oxygen. Identifying the limiting events in aerobic glycolysis and the response of cancer cells to metabolic inhibition is now essential to exploit this potential metabolic dependency. Here, we examine the role of glucose uptake and the glucose transporter Glut1 in the metabolism and metabolic stress response of BCR-Abl+ B-cell acute lymphoblastic leukemia cells (B-ALL). B-ALL cells were highly glycolytic and primary human B-ALL samples were dependent on glycolysis. We show B-ALL cells express multiple glucose transporters and conditional genetic deletion of Glut1 led to a partial loss of glucose uptake. This reduced glucose transport capacity, however, was sufficient to metabolically reprogram B-ALL cells to decrease anabolic and increase catabolic flux. Cell proliferation decreased and a limited degree of apoptosis was also observed. Importantly, Glut1-deficient B-ALL cells failed to accumulate  Indeed, aerobic glycolysis readily supports both generation of ATP and biosynthesis of lipids, nucleic acids, and amino acids.1 Given the high rates of glucose consumption and aerobic glycolysis in most cancers, targeting glucose metabolism has become of significant interest as an approach to eliminate cancer cells. It is now important to establish mechanisms of aerobic glycolysis and the response of cancer cells to metabolic inhibition.Many cancer cells have elevated rates of glycolysis and lactate production even in the presence of oxygen. This program, termed aerobic glycolysis, occurs in a wide range of both solid and liquid tumors and is driven by oncogenic signals and microenvironmental pressures.t chromosomal translocation that generates the oncogenic kinase BCR-Abl occurs in ~25% of adult B-cell acute lymphoblastic leukemia cells (B-ALL) and is associated with poor prognosis.2 The metabolic program of B-ALL cells is undefined, although diffuse large B-cell lymphoma (DLBCL) can either be highly glycolytic or use oxidative phosphorylation and mitochondrial metabolism.3 It has been suggested that BCR-Abl signaling is associated with elevated glucose metabolism, as BCR-Abl can promote glucose uptake and trafficking of glucose transporter Glut1 to the cell surface. Conversely, inhibition of BCR-Abl in leukemic cells suppresses glucose uptake and glycolysis.7 This regulation of glucose metabolism may be critical for survival of BCR-Abl B-ALL, as enforced expression of Glut1 protected B-ALL cells from imatinib-induced apoptosis.8 These data show that BCR-Abl promotes glucose uptake and aerobic glycolysis, and BCR-Abl-transformed cells may rely on this pathway.The 9 Mechanistic understanding of cancer cell metabolic requirements or response to inhibition using pharmacologic approaches, however, has been limited. It has been shown using the glycolytic inhibitor 2-deoxyglucose (2-DG) or glucose deprivation culture conditions that inhibition of glucose metabolism impacts cancer cell growth and viability through several different mechanisms, including cell cycle arrest or cell death by activating AMPK pathway and inactivating mTOR signaling.10 Reduced glucose metabolism has also been found to impact the stability and synthesis of Bcl-2 family proteins. Glucose deprivation induces expression of pro-apoptotic molecules, including Bim15 and can induce apoptosis in cells transformed with oncogenic K-Ras through the unfolded protein response pathway.16Targeting glucose metabolism can have efficacy against a variety of cancers.17 and B-ALL cells expressed multiple family members. Conditional deletion of Glut1, however, demonstrated that B-ALL cells are reliant on this specific glucose transporter to sustain anabolic metabolism, proliferation, and resistance to cell death. Consistent with our data showing a key role for glucose uptake, we found that pharmacologic inhibition of glycolysis sensitized B-ALL cells to caspase activation and apoptosis to reduce leukemia burden in vivo. Glut1 and glucose uptake have a key role, therefore, to maintain BCR-Abl B-ALL cell growth and resistance to cell death.Here we examine the mechanism and role of glucose uptake in B-ALL metabolism and leukemia progression by genetically targeting glucose transport. The Glut family of hexose transporters consists of 14 members18 However, usage of glucose in B-ALL and the response of B-ALL cells to inhibition of aerobic glycolysis are poorly understood. To examine glucose metabolism activity in B-ALL, real-time extracellular flux was measured in six human B-ALL cell lines, including BCR-Abl+ and BCR-Abl− cell lines, and peripheral B cells from three healthy donors in the absence of nutrients, after addition of glutamine, then upon addition of glucose. Extracellular acidification rate (ECAR), reflective of lactate production, was similar in each cell type in the absence of nutrients . Treatment of Glut1fl/fl/Ubi-Cre-ERT2 B-ALL with 4-OHT led to efficient deletion of Glut1 with minimal compensation from other Glut transporters, and 4-OHT had no effect on control B-ALL cells ; Sup-B15 from ATCC ) were cultured in complete RPMI 1640  or complete IMDM  with 10 or 20% fetal bovine serum . De-identified primary human B-ALL samples were cultured in Hybridoma SFM media (Gibco) supplemented with cytokines (refer to −/− (Jackson Labs), Ubi-Cre-ERT2 transgenic (Jackson Labs), and Glut1fl/fl mice crossed to Ubi-Cre-ERT2 mice were cultured in IL-7 (10 ng/ml) (eBioscience) in IMDM supplemented with 20% fetal bovine serum and infected with MSCV-BCR-Abl-IRES-GFP retrovirus 7 with polybrene (4 μg/ml) . Infected cells were cultured in methylcellulose medium containing IL-7 (Stem Cell Technologies) and individual colonies were isolated on day 7 and transferred into complete IMDM media with 20% fetal bovine serum, but no IL-7. After 7 days, viable GFP+ colonies were selected for expansion. The Institutional Animal Care and Use Committee of Duke University approved all animal protocols.Bone marrow cells from C57B6/J , Bim12 Primary antibodies used were rabbit anti-Bim , rabbit anti-Glut1 , rabbit anti-phospho-BCR , rabbit anti-BCR , rabbit anti-phospho-CRKL , rabbit anti-CRKL , rabbit anti-Bcl-xL , rabbit anti-Mcl-1 , rabbit anti-Bax , rabbit anti-Bak , rabbit anti-Bid , rabbit anti-PDI , rabbit anti-IRE1α , rabbit anti-Bip , mouse anti-CHOP , mouse anti-actin (Sigma-Aldrich) and were detected with anti-rabbit horseradish peroxidase-labeled antibody  and fluorescent-labeled anti-mouse antibody . Blots were visualized using Supersignal West Pico Chemiluminescent Substrate  or the Odyssey infrared imaging system (LiCor).Cell lysates for immunoblots were prepared as described.13 Apoptotic cell populations were assessed by annexin V/PI staining. Cells were incubated with Alexa Fluor 488 annexin V (Invitrogen) for 15 min in dark and then 2 μg/ml PI was added. Cell cycle profile and DNA content were measured in cells fixed in ethanol and stained with PI. BrDU incorporation was measured by culture of cells with 10 uM BrDU (Sigma-Aldrich) for 1.5 h followed by ethanol fixation, denaturation with 2 M HCl for 20 min and staining with anti-BrdU. Flow cytometry data were collected on MACSQuant  or FACScan  flow cytometers and analyzed using FlowJo software .Antibodies for cytometry include anti-human CD34, CD19, CD10 and CD20 (eBioscience), annexin V  and anti-BrDU (Invitrogen). Cell viability was measured by flow cytometry for propidium iodide  exclusion as described.μg of RNA was reverse transcribed  to perform SYBR Green (Bio-Rad) semi-quantitative real-time-PCR for Glut1. Relative expression levels were calculated using the ΔCt/ΔCt method, with expression normalized to 18S or actin RNA. Primers for mouse glucose transporters and mouse actin: Glut1, 5'-AGCCCTGCTACAGTGTAT-3', 5'-AGGTCTCGGGTCACATC-3'; Glut3, 5'-TAAACCAGCTGGGCATCGTTGTTG-3', 5'-AATGATGGTTAAGCCAAGGAGCCC-3' Glut6, 5'-TTGGTGCTGTGAGGCT-3', 5'-TGGCACAAACTGGACGTA-3'; Glut8, 5'-ACATCTCGGAAATCGCCT-3', 5'-ACACAGCCCAGCACG-3'; Glut9, 5'-TGCTTCCTCGTCTTCGCCACAATA-3', 5'-CTCTTGGCAAATGCCTGGCTGATT-3'; actin, 5'-CCTTCCTTCTTGGGTATGGA-3', 5'-TGGTACCACCAGACAGCACT-3'. Primers for human glucose transporters and 18S: Glut1, 5'-CACTCCTGTTACTTACCTAA-3', 5'-CACTTACTTCTGTCTCACT-3'; Glut3, 5'-GACCCAGAGATGCTGTAATGGT-3', 5'-GGGGTGACCTTCTGTGTCCC-3'; Glut4, 5'-CTTCCAACAGATAGGCTCCG-3', 5'-CCCCAATGTTGTACCCAAAC-3'; Glut5, 5'-GCAACAGGATCAGAGCATGA-3', 5'-CCATACTGGAAGGATGACCC-3'; Glut6, 5'-GTCCATCTTCGACAGCACCG-3', 5'-GCAAACATGATGGCCGCTGA-3'; Glut7, 5'-CACCGTCTCCATGTTTCCTC-3', 5'-TGTTGTTGATCAGCAGGGTC-3'; Glut8, 5'-TCCTGGTTCGGGGCTGTC-3', 5'-GAGCACAGCAAGAGGCTCAG-3'; Glut9, 5'-GAGTATCGTGGGCATTCTGG-3', 5'-AGTTGGAGAGCCAGTTGACG-3'; 18S, 5'-GTAACCCGTTGAACCCCATT-3', 5'-CCATCCAATCGGTAGTAGCG-3'.Total mRNA was extracted  and 1 22 In non-targeted metabolomics analyses, Glut1fl/fl CreER and WT CreER cells were treated with vehicle or 0.4 μM 4-OHT for 96 h followed by an additional 48 h in complete IMDM media containing 1 mM sodium pyruvate. For 13C-glucose flux studies, cells were cultured as described above for 5 days culture, washed with PBS (Mediatech), and cultured in glucose-free RPMI without sodium pyruvate (Gibco) supplemented with 10% fetal bovine serum, glutamine (2 mM) and uniformly labeled 13C-glucose  for 24 h. Metaboanalyst was used to range-scale data and provide PCA and KEGG pathway analysis of metabolites significantly changed .43Metabolomic analyses were performed as described before using la iquid chromatography Q Exactive Mass Spectrometer (LC-QE-MS) (Thermo Scientific).44 XF24 extracellular flux assays  were performed as described.19 Cells were initially incubated in XF basic media in the absence of glutamine or glucose. Glutamine, glucose, oligomycin and 2-DG were sequentially injected in the test to the following final concentrations: glutamine 2 mM, glucose 25 mM, oligomycin 1 μM, 2-DG 20 mM. Measurements of ECAR and OCR were normalized to cell number.Glucose uptake, lipid oxidation, and pentose phosphate pathway were measured as described.Caspase 3/7 activity was measured using caspase-Glo 3/7 assay kit (Promega). Equal number of live cells from different treatment conditions was used for the assay. Cells were incubated with substrates for 1 h at room temperature and a luminescent signal was measured on a luminescence plate reader.5) were injected via the tail vein into NOD/SCID/Gamma-immunodeficient mice . Two days after transfer, mice were treated with vehicle  or tamoxifen  by i.p. injection for four consecutive days. GFP+B-ALL cells were observed in spleen and bone marrow by flow cytometry. In some cases, animals were treated with 2-DG  or Dasatinib  through oral gavage, as indicated.B-ALL cells .Statistical analysis on data involving two groups was performed with unpaired two-tailed Student's


Although circulating Th-17 (cTh-17) cells are related to inflammatory diseases, its role in HIV immunopathogenesis is not well known. We investigated the association of cTh-17 cells with immune activation (IA), immune exhaustion (IE) and regulatory T-cells (T-regs) in HAART treated virologically controlled (<40 HIV RNA copies/ml) HIV-1 infected patients with poor CD4 T-cell recovery (discordant).Discordant patients (n=20) with mean absolute CD4 numbers (absCD4: 273±29) were compared with virologically controlled patients on HAART  with optimal CD4 T-cell recovery (absCD4:852±51) and 10 healthy controls (HCs). cTh17 cells were measured in PBMCs, as frequencies of CD4+IL-17+ cells, after PMA+Ionomicin stimulation by flow cytometry, and correlated with markers of IA (HLA-DR+CD38+) and IE  of CD4 and CD8 T-cells and frequencies of T-regs (CD4+CD25brightFoxP3+). Plasma levels of lipopolysaccharide (LPS) and soluble-CD14 (sCD14) were measured to determine gut microbial translocation (MT). Results obtained for discordant patients were compared with concordant patients and HCs.Frequencies of cTh-17 cells were significantly higher in discordant patients than concordant patients (P < 0.0001) and HCs (P = 0.014). cTh-17 cells frequency correlated inversely with CD4 T-cell percentages and absolute count and directly with CD4-IA and T-reg frequencies. Discordant patients showed higher CD4-IA (HLA-DR+CD38+) than concordant patients which directly correlated with MT. CD4 T-cells of discordant patients showed higher expression of multiple IE markers (PD1+Tim3+) that correlated with IA. Although naive CD4+ T-cells were lower, effector memory cells were higher in discordant patients.Persistent CD4 T-cell IA and inflammation in discordant patients may favor the differentiation of activated CD4 T-cells towards cTh-17 phenotype. cTh-17 cell frequency could be an indicator of ongoing IA and inflammation in discordant patients. Analysis of the nature and function of cellular subsets associated with IA and immune regulation would assist in understanding HIV immunopathogenesis and developing strategies to target disease progression.


Gamma-herpesviruses colonise lymphocytes. Murid Herpesvirus-4 (MuHV-4) infects B cells via epithelial to myeloid to lymphoid transfer. This indirect route entails exposure to host defences, and type I interferons (IFN-I) limit infection while viral evasion promotes it. To understand how IFN-I and its evasion both control infection outcomes, we used Mx1-cre mice to tag floxed viral genomes in IFN-I responding cells. Epithelial-derived MuHV-4 showed low IFN-I exposure, and neither disrupting viral evasion nor blocking IFN-I signalling markedly affected acute viral replication in the lungs. Maximising IFN-I induction with poly(I:C) increased virus tagging in lung macrophages, but the tagged virus spread poorly. Lymphoid-derived MuHV-4 showed contrastingly high IFN-I exposure. This occurred mainly in B cells. IFN-I induction increased tagging without reducing viral loads; disrupting viral evasion caused marked attenuation; and blocking IFN-I signalling opened up new lytic spread between macrophages. Thus, the impact of IFN-I on viral replication was strongly cell type-dependent: epithelial infection induced little response; IFN-I largely suppressed macrophage infection; and viral evasion allowed passage through B cells despite IFN-I responses. As a result, IFN-I and its evasion promoted a switch in infection from acutely lytic in myeloid cells to chronically latent in B cells. Murine cytomegalovirus also showed a capacity to pass through IFN-I-responding cells, arguing that this is a core feature of herpesvirus host colonization. Gamma-herpesviruses establish chronic infections and cause cancers. They achieve this by immune evasion. Immune responses nonetheless suppress infection to a degree. By understanding how immune responses and viral evasion come together we can potentially control infection and prevent disease. MuHV-4 provides an accessible model with which to define outcomes. It enters new hosts via epithelial cells, passes to macrophages, then persists in B cells. Type I interferons—a key anti-viral defence—controlled epithelial and B cell infections but poorly restricted the intervening macrophage infection. Therefore for maximal effect interferons must act before B cells are infected. The γ-herpesviruses persist in lymphocytes and cause lymphoid and epithelial cancers. MuHV-4, like Epstein-Barr virus (EBV) and the Kaposi's Sarcoma-associated Herpesvirus (KSHV), persists in B cells . After eImmune cell colonization makes host defences an important feature of the γ-herpesvirus infection landscape. Type 1 interferons (IFN-I) are a core vertebrate anti-viral defence . Myriad + T cells [Most viruses also evade IFN-I . MuHV-4  T cells ; and M2  T cells –21 and p T cells . Thus, tin vivo infection is more complicated, with MuHV-4 spreading between cell types and between anatomic sites [Qualitative questions also remain. ORF11, ORF36, ORF37, ORF54 and M2 all limit IFN-I signalling to infected cells, so ongoing viral gene expression should preclude a cellular IFN-I response; but in cells already exposed to IFN-I, viral signalling blocks should be ineffective and infection should be suppressed. In a uniform cell population such mutual inhibition would manifest as transient viral replication; but ic sites . Splenicic sites , suggestic sites . Most st-/- fibroblasts transfected with IFNAR encoding DNA) may have responded poorly to murine IFNα. Myeloid cells play a central role in MuHV-4 host colonization [MuHV-4 replicates in a wide range of primary and transformed cells, suggesting that it either induces little IFN-I or evades its effects. Weak inhibition of fibroblast infection by MuHV-4-expressed IFNα  suggestsnization , and RAWnization . Therefo-/- mice given MuHV-4 i.n. have normal virus titers in the lungs for at least 4d [+ MuHV-4  and type 1 alveolar epithelial cells (AEC1s) . Again wleast 4d , implyinleast 4d , and con+ MuHV-4  and plaq+ MuHV-4  showed I+ MuHV-4 . This im+ or eGFP+ we could determine whether they had passed through a cre+ cell; and as Mx1 induction is essentially specific to IFN-I [To track how IFN-I responses and viral replication overlap in single cells, we infected Mx1-cre mice, in which cre is transcribed from an IFN-I-inducible Mx1 promoter , with Muto IFN-I –39, thisAs lymphoid infection follows epithelial infection, greater fluorochrome switching in spleens than in lungs could also have reflected time-dependent IFN-I induction. To analyse spleen infection without a preceding lung infection, we gave MHV-RG to Mx1-cre mice i.p., when it reaches splenic MZ macrophages directly—presumably via the thoracic duct and blood . At d3 apDCs are important IFN-I producers in some settings . Depletiex vivo (in vivo (+eGFP-). Even when IFN-I was induced with poly(I:C), essentially all the cells infected at d4 were unswitched. +PDP+), but of fluorescent AMs (CD68+ or CD169+) 50% were eGFP+. By d6, all fluorescent cells were again mCherry+. Therefore MuHV-4 initiated lytic gene expression in IFN-I responding AMs, but the subsequent loss of eGFP+ AMs, lack of eGFP spread to AEC1s (+ AEC1s after poly(I:C) treatment suggested that these cells made little Mx1 response, although mCherry+ cells might still produce eGFP+ virions, as switching could occur after viral fluorochrome expression.The M3 promoter used to drive MuHV-4 fluorochrome expression is active in the lytic cycle . It is tex vivo , but als, and at d3-4 >90% of fluorescent B cells were eGFP+. >80% of these were in the MZ and stained for IgM  . Now at  for IgM . As in l for IgM  indicate+ cells, even after IFN-I induction with poly(I:C)  injection. We reasoned that it could also reveal cellular exposure to IFN-I, via activation of a floxed cellular fluorochrome. To this end we crossed Mx1-cre with ROSA26-YFP mice, in which cre-dependent removal of a floxed translational stop activates YFP expression, and infected them with wild-type MuHV-4 . Infecteoly(I:C) . The YFPnfection . Thus, v+ macrophages  and B cells (B220+)  injection (Infected spleens contained contrastingly large numbers of YFP (B220+) . However (B220+) . The orinjection . Therefo- MuHV-4 delivered i.n. shows an IFNAR-dependent infection defect in lungs and spleens [- MuHV-4 with wild-type also by i.p. inoculation, which reaches the spleen directly [B cell colonization despite viral exposure to IFN-I implied an important role for IFN-I evasion. The MuHV-4 ORF36 inhibits IRF3 signalling, and ORF36 spleens . Because- MuHV-4 showed a relatively minor defect in direct lung infection when given i.n.  to red pulp  macrophages   and F4/80+ (RP macrophage) populations  , CD206+ ulations , so IFNAulations . Thus in+) in IFNAR-blocked mice than in controls, so more lytic infection in MZ macrophages eventually fed through to more latent infection in WP B cells. However IFN-I blockade did not increase viral antigen+/ viral eGFP+ cell ratios in the WP at d7—in fact they were reduced—so the proportion of lytic infection in WP B cells did not increase. We conclude that splenic macrophage infection was strongly restricted by IFN-I, but that acute viral reactivation in WP B cells was largely IFN-I-independent.We tracked latent infection in spleens by viral eGFP expression from a constitutive promoter . Again b5 p.f.u.). Lung virus titers after 7d showed restoration of the M50 replication defect by IFN-I blockade  replicates normally in mice for 3d, but is then progressively attenuated . This viblockade . Thus, a-tdTomato+ cells around lymphoid follicles, consistent with MCMV infecting IFN-I-responding MZ macrophages [-tdTomato+ cells with the morphology of hepatocytes. Infected cells were more switched than recovered virions, consistent with IFN-I exerting some restriction on virion production [MuHV-4 passage through IFN-I responding cells was surprising: we expected that either viral evasion would prevent an IFN-I response in infected cells, or IFN-I would prevent viral replication. To test whether it was a unique to MuHV-4, we infected Mx1-cre mice with murine cytomegalovirus that cre switches from eGFP to tdTomato expression (MCMV-GR). We induced IFN-I or not with poly(I:C), gave MCMV-GR i.p., and recovered infectious virus from livers and spleens by plaque assay at d3 . As withrophages . Liver soduction , but proMuHV-4 provides an experimental window onto the γ-herpesviruses, whose colonization of lymphoid tissue directly confronts host immune defences. IFN-I attenuated macrophage infection, whereas B cell infection was protected by viral evasion. This cell type-dependent outcome explained how IFN-I and its evasion both control infection, with each dominating in a different setting . TogetheIn the lungs, AMs provide MuHV-4 with a gateway to AEC1s, which then support lytic replication . AEC1s m+ DC) are not an acute infection target in spleens [An important role for the ORF36 IFN-I evasion gene in splenic infection was surprising, as its inhibition of IRF3 should limit IFN-I induction, and the abundant exposure of wild-type splenic virus to IFN-I implied that MuHV-4 evades its effector functions rather than relying solely on limiting induction. A possible explanation is that ORF36, as a lytic gene, operates more in macrophages than in B cells, as macrophages support lytic infection  and are pace cell differences in IFN-I response, macrophage infection may be more susceptible than B cell infection to inhibition by IFN-I because it is more lytic [The recovery of fluorochrome-switched virions implied that viral IFN-I evasion also operates down-stream of IFN-I induction or signalling, that is downstream of ORF36. Switching was evident early after i.p. virus inoculation, when splenic infection is mostly lytic . Establire lytic , 46.in vivo viral tropism. A corollary is that IFN-I-based therapies are likely to have only a small window of efficacy—mainly reducing acute gamma-herpesvirus replication in myeloid cells. Downstream of this, virions entering IFN-I-exposed B cells would still establish a viable infection. IFN-I deficiency increases ex vivo MuHV-4 reactivation rates [in vitro cultures that contain both virus-infected and immune cells. Reactivation rates are low, particularly from B cells [in vitro antibody production and cytokines. The failure of IFNAR blockade to increase the viral antigen / eGFP staining ratio of d7 splenic WP B cells argued that here IFN-I is not a major regulator of in vivo lytic reactivation. The capacity of MCMV also to re-emerge acutely after Mx1-dependent tagging suggested that many herpesviruses can enter IFN-I responding cells and rapidly re-emerge. How far IFN-I can restrict MCMV was not explored, but clearly there are limits on what it alone is likely to achieve.Together the IFN-I response and its evasion promoted B cell over macrophage infection, making IFN-I an important determinant of on rates . However B cells , and may4 p.f.u.) under isofluorane anesthesia or i.p. (105 p.f.u.). Luciferase+ MuHV-4 infection (MHV-LUC) [BALB/c, C57BL/6J and Mx1-cre mice  were maiMHV-LUC)  was imagAll animal experiments were approved by the University of Queensland and Walter and Eliza Hall Animal Ethics Committees in accordance with Australian National Health and Medical Research Council (NHMRC) guidelines. Project 301/13.XcmI site (nucleotide 53027 of Genbank sequence NC001826) of the ORF36 coding sequence (52848–54161) an oligonucleotide with multiple stop codons and an EcoRI restriction site. Correct mutagenesis was identified by EcoRI digestion of BAC DNA, and confirmed by sequencing of viral DNA across the insertion site. Infectious virus was recovered by BAC DNA transfection into BHK-21 cells, and the loxP-flanked BAC cassette removed by virus passage through NIH-3T3-cre cells. Luciferase+ [+ MuHV-4 [g, 120min) and cell debris was removed by low speed centrifugation .Bovine Hamster Kidney (BHK-21) fibroblasts (American Type Culture Collection CCL-10), RAW-264 monocytes (American Type Culture Collection TIB-71), NIH-3T3 cells (American Type Culture Collection CRL-1658), NIH-3T3-cre cells  and fibriferase+ , floxed + MuHV-4  are desc+ MuHV-4  was growTo titer infectious virus, culture-grown stocks or freeze-thawed organ homogenates were plated on BHK-21 (MuHV-4) or embryonic fibroblast (MCMV) monolayers . To titeMurine IFNβ was assayed by ELISA (PBL Verikine). Mx1 mRNA was quantitated in lung tissue  by quantitative PCR  with Mx1-specific primers , and normalized by parallel amplification of Nidogen-1 .Organs were fixed in 1% formaldehyde / 10 mM sodium periodate / 75 mM L-lysine , equilibrated in 30% sucrose , then frozen in OCT. Sections (6μm) were air-dried , washed 3x in PBS, blocked with 0.3% Triton X-100 / 5% normal donkey serum , then incubated  with combinations of antibodies to eGFP , CD68  (AbCam), B220 (rat mAb RA3-6B2), F4/80 (rat mAb CI:A3–1) (Santa Cruz Biotechnology), mCherry , CD206 (rat mAb MR5D3), CD169 (rat mAb 3D6.112) (Serotec), podoplanin , and MuHV-4 . Sections were washed 3× in PBS, incubated  with combinations of Alexa568-donkey anti-rat IgG pAb, Alexa488 or Alexa647-donkey anti rabbit IgG pAb, Alexa647-donkey anti-mouse IgM pAb, Alexa488-donkey anti-chicken IgG pAb (Abcam), and Alexa488-donkey anti-goat pAb (Life Technologies), then washed 3× in PBS, stained with DAPI and mounted in Prolong Gold (Life Technologies). TdTomato fluoresence was visualized directly. Images were captured with a Zeiss LCM510 confocal microscope or a Nikon epifluorescence microscope and analyzed with Zen imaging software or ImageJ.S1 Fig4 p.f.u. i.n. in 30μl under anesthesia) with wild-type MuHV-4 (MHV-WT), colour switching MuHV-4 (MHV-RG), or the same virus pre-switched in vitro by passage through cre+ NIH-3T3 cells (MHV-G). Lungs were titered for infectious virus after 6 days by plaque assay. Spleens were titered for total recoverable virus after 13 days by infectious center assay. Crosses show means, other symbols show individual mice. No significant difference was seen between MHV-WT, MHV-RG and MHV-G.C57BL/6 mice were infected in lungs (10(PDF)Click here for additional data file.S2 Fig+ heterozygote progeny (Mx1-cre) and non-transgenic littermates (C57BL/6) were infected i.n. when 6–8 weeks old with wild-type MuHV-4 (104 p.f.u. i.n. in 30μl under isoflurane anesthesia). Infectious virus in lungs was titered after 6 days by plaque assay, and lytic plus latent virus in spleens was titered after 13 days by infectious center assay. Crosses show means; other symbols show individual mice. No significant difference was observed between Mx1-cre+ and C57BL/6 mice.Mx1-cre mice were bred as heterozygote x non-transgenic. Mx1-cre(PDF)Click here for additional data file.S3 Fig5 p.f.u.). 3 days later footpads, popliteal lymph nodes (PLN) and spleens were titered for virus by infectious center assay. Bars show mean ± SEM for 3–6 mice. Virus titers were significantly reduced in footpads by Student’s unpaired 2 tailed t test, but not in PLN or spleens .Mice were gives anti-pDC mAb  or not i.p., then MuHV-4 into footpads (10(PDF)Click here for additional data file.S4 Fig5 p.f.u.). M50 MuHV-4 has the proximal 416bp of the Murine cytomegalovirus IE1 promoter inserted in its ORF50 exon1 5’ untranslated region. ORF50 encodes the MuHV-4 lytic switch protein. M50 MuHV-4 shows increased ORF50 transcription and an incapacity to remain latent  Forced lytic replication impairs host colonization by a latency-deficient mutant of murine gammaherpesvirus-68. J Gen Virol 85: 137–146). At 7 days after infection, lungs were titered for infectious virus by plaque assay. Crosses show means, other symbols show individual mice. Without aIFN mAb M50 titers were significantly less than wild-type (p<0.001 by Student’s unpaired 2-tailed test); with IFNAR blockade M50 and WT titers were not significantly different.Mice were given anti-IFNAR blocking mAb  or not (nil) then wild-type (WT) or M50 MuHV-4 i.n. (10(PDF)Click here for additional data file.S5 FigType 1 alveolar epithelial cells made no detectable Mx1 response to MuHV-4 infection or to p(I:C), and IFN-I induction had little effect on viral replication in the lungs, where these cells are abundantly infected. Thus, their interaction was dominated by poor responsiveness to IFN-I. Macrophages contrastingly showed viral fluorochrome switching but propagated switched virions poorly, and IFN-I blockade increased massively the extent of their infection. Thus, in macrophages IFN-I was protective. B cells were different again. They showed abundant viral fluorochrome switching and switched virion production. IFN-I blockade had little effect on infection, but viral evasion gene disruption caused marked attenuation, indicating that B cell infection is normally dominated by IFN-I evasion. This implies that virions can enter IFN-I-responding B cells and establish a latent infection that is stably maintained and can reactivate, presumably when IFN-I signalling has diminished.(PDF)Click here for additional data file.


Lung ultrasonography (LUS) has been used for noninvasive detection of pulmonary edema. LUS visual scores (V-LUS) based on B-lines are poorly correlated with either pulmonary capillary wedge pressure (PCWP) or extravascular lung water (EVLW). A new quantitative LUS analysis (Q-LUS) has been recently proposed ,2. The a2O (n = 47) or 10 cmH2O (n = 30) and PCWP (n = 77) or EVLW (n = 38) monitored were studied.Thirty-nine patients mechanically ventilated with PEEP of 5 cmHr2 = 0.64) but weakly with V-LUS B-line score (r2 = 0.19). EVLW was significantly and strongly correlated with QLUS Grey Unit mean value (r2 = 0.65) more than with V-LUS B-line score (r2 = 0.42). Q-LUS showed a better diagnostic accuracy than V-LUS for the detection of PCWP >15 mmHg or EVLW >10 ml/kg. With a PEEP of 5 cmH2O, the correlations with PCWP or EVLW were stronger with Q-LUS than V-LUS. With a PEEP of 10 cmH2O, the correlations with PCWP or EVLW were still significant for Q-LUS but insignificant for V-LUS. Intraobserver repeatability and interobserver reproducibility were much better for Q-LUS than V-LUS.PCWP was significantly and strongly correlated with Q-LUS Grey Unit value (Both V-LUS and Q-LUS are acceptable indicators of pulmonary edema in patients mechanically ventilated with low PEEP but at high PEEP only Q-LUS provides data that are significantly correlated with. Computer-aided Q-LUS has the advantages of being not only independent of operator perception but also of PEEP.


FMR1) on the X-chromosome [FMR1 premutation (PM) is about 1:855 in males and 1:291 in females [FMR1 mRNA (2 to 8 fold in leucocytes) and normal to slightly reduced FMR1 protein (FMRP) levels [FMR1 gene a polyGlycine-containing protein, FMRpolyG, is expressed. This protein accumulates in ubiquitin-positive inclusions in Drosophila, cell culture, mouse disease models and brain from FXTAS patients. To investigate the link between FMRpolyG expression and the co-morbid medical problems associated with the PM we have developed two novel mouse monoclonal antibodies against polyGlycine; 8FM and 9FM . Interestingly, a very recent report suggests that RAN translation products in C9FTD/ALS, toxic dipeptide repeat proteins (poly-(glycine-arginine) and poly-(proline-arginine)), are toxic in Drosophila [Fragile X-associated Tremor/Ataxia syndrome (FXTAS), a late-onset monogenetic neurodegenerative disorder, is caused by a CGG-repeat expansion 55-200) in the 5′ UTR of the fragile-X mental retardation 1 gene  levels ,7. So fa


C. elegans. Confirming the specificity of this screen, we identified the Rho GTPase cdc-42, which mediates invadopodia formation in many cancer cell lines. Using live-cell imaging, we show that CDC-42 localizes to the AC-BM interface and is activated by an unidentified vulval signal(s) that induces invasion. CDC-42 is required for the invasive membrane localization of WSP-1 (N-WASP), a CDC-42 effector that promotes polymerization of F-actin. Loss of CDC-42 or WSP-1 resulted in fewer invadopodia and delayed BM breaching. We also characterized a novel invadopodia regulator, gdi-1 (Rab GDP dissociation inhibitor), which mediates membrane trafficking. We show that GDI-1 functions in the AC to promote invadopodia formation. In the absence of GDI-1, the specialized invadopodial membrane was no longer trafficked normally to the invasive membrane, and instead was distributed to plasma membrane throughout the cell. Surprisingly, the pro-invasive signal(s) from the vulval cells also controls GDI-1 activity and invadopodial membrane trafficking. These studies represent the first in vivo screen for genes regulating invadopodia and demonstrate that invadopodia formation requires the integration of distinct cellular processes that are coordinated by an extracellular cue.Invadopodia are specialized membrane protrusions composed of F-actin, actin regulators, signaling proteins, and a dynamically trafficked invadopodial membrane that drive cell invasion through basement membrane (BM) barriers in development and cancer. Due to the challenges of studying invasion in vivo, mechanisms controlling invadopodia formation in their native environments remain poorly understood. We performed a sensitized genome-wide RNAi screen and identified 13 potential regulators of invadopodia during anchor cell (AC) invasion into the vulval epithelium in  Caenorhabditis elegans, we have conducted the first large-scale screen to isolate genes that control invadopodia in live animals. Our screen isolated 13 genes and we confirmed two are key invadopodia regulators: the Rho GTPase CDC-42 that promotes F-actin polymerization at invadopodia to generate the force to breach basement membranes, and the Rab GDI-1 that promotes membrane addition at invadopodia that may allow invadopodia to extend through basement membranes. This work provides new insights into invadopodia construction and identifies potential novel targets for anti-metastasis therapies.During animal development specialized cells acquire the ability move and invade into other tissues to form complex organs and structures. Understanding this cellular behavior is important medically, as cancer cells can hijack the developmental program of invasion to metastasize throughout the body. One of the most formidable barriers invasive cells face is basement membrane–-a thin, dense, sheet-like assembly of proteins and carbohydrates that surrounds most tissues. Cells deploy small, protrusive, membrane associated structures called invadopodia (invasive feet) to breach basement membranes. How invadopodia are formed and controlled during invasion has been challenging to understand, as it is difficult to examine these dynamic structures in live animals. Using the nematode worm  Basement membrane (BM) is a dense, highly cross-linked extracellular matrix that surrounds most tissues and acts as a barrier to migrating cells . During C. elegans is a genetically and visually tractable model to dissect invadopodia formation and activity during BM invasion in vivo .,50.gdi-1controls . To furtargeting . Expresscdc-42, we found that loss of gdi-1 decreased the rate of invadopodia formation by nearly 50%  mutants significantly enhanced the cdc-42(gk388) invasion defect  to examine invasion and invadopodia dynamics. This treatment slightly enhanced the invasion defect associated with loss of either gdi-1 or cdc-42 alone  that converge to promote invadopodia formation, loss of the vulval precursor cells should more severely perturb invadopodia formation relative to loss of see Figs  and 4E. or gdi-1 . These d2 (mCherry::PLCδPH) and the RAC GTPases MIG-2 and CED-10 revealed that RNAi-mediated knockdown of gdi-1 led to inappropriate localization of the invadopodial membrane in apical and lateral plasma membrane domains  ; qyIs17[cdh-3>mCherry]; qyIs43; qyEx45[cdc-42>GFP::cdc-42]; qyIs46[emb-9>emb-9::mCherry]; qyIs57[cdh-3>mCherry::moeABD]; qyIs61[cdh-3>GFP::unc-34]; qyIs204[wsp-1>GFP]; qyIs211[cdh-3>lmp-1::GFP]; qyIs212[cdh-3>GFP::wsp-1]; qyIs219[cdh-3>GFP::PLCδPH]; qyIs220[cdh-3>mig-2::GFP]; qyIs221[cdh-3>ced-10::GFP]; qyIs409[cdh-3>GFP::cup-5]; qyIs410[cdh-3>GFP::cdc-42]; qyIs412[cdh-3>GFP::GBDwsp-1]; qyIs427[lam-1>lam-1::mCherry]; qyEx507[cdh-3>GFP::gdi-1]; qyEx515[gdi-1>GFP]; qyEx533[cdh3>GFP::Cbrgdi-1]; urIs; LGI, unc-40(e271); ayIs4[egl-17>GFP]; LGII, cdc-42(gk388); mIn1; LGIII, unc-119(ed4); rhIs23[him-4>him-4::GFP]; LGIV, wsp-1(ng324); qyIs10[lam-1>lam-1:GFP]; gdi-1(tm660); nT1; LGV, qyIs50[cdh-3>mCherry::moeABD]; LGX, qyIs7[lam-1>lam-1::GFP]; qyIs24[cdh-3>mCherry:: PLCδPH].Culturing and handling of escribed . Wild tygdi-1(tm660) was obtained from the National Bioresource Project . The gdi-1(tm660) allele is a 541 bp deletion in the first exon that shifts the frame of the open reading such that only the first 37 amino acids of the protein are predicted to be made (out of 549 amino acids in the full protein). The gdi-1(tm660) allele is thus likely a null for gdi-1 activity. The gdi-1(tm660) allele was balanced using the nT1 translocation by creating the strain, gdi-1(tm660) IV/nT1[qIs51] . The qIs51 transgene, which is linked to nT1, expresses GFP in the pharynx. The presence of gdi-1 in the balanced strains was confirmed using the published NBRP primers: ExtRev:TCAAGGAGTGCATCATCTCG, ExtFwd:CCTGATCATTCAACGACAAG, IntFwd:GTGAGTGATGTTGGTGAAGT, IntRev:CTCGGGAATGCTGTCGGTTT. Balanced gdi-1/nT1 mothers never segregated non-fluorescent gdi-1 homozyogous progeny (n = 0/54 examined) in timed egg-lays. Dead embryos were observed on the plates, but dead larvae were not seen. These results suggest that gdi-1 is required for embryonic viability.The deletion allele cdh-3(mk62-62) AC-specific regulatory element ) result in vulvaless worms that lack specification of primary vulval precursor cells and, therefore, do not generate the vulval cue(s) ; qyIs10[lam-1>lam-1:GFP]; rde-1(ne219); qyIs24[cdh-3>mCherry:: PLCδPH] worms for uterine-specific RNAi and rrf-3(pk1426); qyIs138[unc-62>rde-1]; rde-1(ne219) worms for vulval-specific RNAi [Tissue-specific RNAi experiments were preformed as previously described using fic RNAi ,28. Briefic RNAi . A functunc-40(e271) mutant background, 50–100 synchronized L1-arrested worms were plated on RNAi for 70 hours at 20°C. Egg production was assayed by counting the number of laid eggs at this time point. Worms on control plates laid an average of 100 eggs and experimental worms were scored as (-) when no eggs were laid, (+) when there was a moderate reduction in the number of eggs laid, and (++) when the number of eggs laid was decreased by more than 50%. Worms were also scored for the protruding vulval phenotype in three categories: (#) when 10% of the worms had protruding vulvas, (##) when 20% of worms had protruding vulvas, and (###) when more than 50% of worms exhibited this phenotype. Candidate genes for the secondary screen for AC invasion defects were identified using AmiGO v1.8 [In the primary genome-wide RNAi screen in the iGO v1.8 . These gImages were acquired using an EM-CCD camera (Hamamatsu Photonics) and a spinning disk confocal  mounted on a microscope  with a Plan-APOCHROMAT 100x/1.4 oil differential objected controlled by MicroManager software . AcquireQuantitative analyses of AC invadopodia and BM breach were done using ImageJ, Imaris, or both. For time-lapse microscopy, worms were anesthetized in 0.2% tricaine and 0.02% levamosile in M9 and then transferred to 5% noble agar pads, sealed with VALAP, and imaged at 23°C at one minute time intervals . For conQuantification of GFP::CDC-42, GTP-CDC-42, and GFP::WSP-1 foci was performed in a blinded manner using the manual threshold and particle counting with the watershed filter functions in ImageJ on max projections of the two confocal z-planes that comprised the invasive membrane. The size of BM breach was calculated by using the threshold and measurement functions in ImageJ. Colocalization measurements were performed using the Imaris coloclaization module on blinded images. The enrichment of invadopodial membrane components and PAT-3::GFP at the invasive membrane were performed using ImageJ. For markers of the invadopodial membrane compartment, polarity measurements were calculated from sum projections that were thresholded manually. The proportion of thresholded signal in a region of interest (ROI) containing only the invasive membrane relative to the entire AC was then calculated. PAT-3::GFP calculations were performed as previously described ,24. BrieStatistical analysis was preformed using JMP version 9.0 (SAS Institute) or Graphpad PRISM v. 5, using two-tailed Fisher’s Exact test, a two-tailed unpaired Student’s t-test, or nonparametric Wilcoxon rank-sum test. Figure legends specify when each test was used.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S1 Figcdh3>GFP::unc-34) and PIP2  colocalize with F-actin . Bottom panels: Knockdown of cdc-42 by RNAi reduces the number of invadopodia, but GFP::UNC-34 (A) and PIP2 (B) still colocalize with F-actin (arrowheads). The correlation coefficients are not different between wild type animals and animals treated with cdc-42 RNAi  and 0.26 P2, Student’s t-tests). Scale bar, 5 μm. Ventral views showing the composition of invadopodia. Top panels: the invadopodial markers GFP::UNC-34 Click here for additional data file.S2 Figgdi-1>GFP) revealed gdi-1 expression in the AC and vulval precursor cells throughout invasion. Expression of gdi-1 is specifically upregulated in the AC. (B) An AC-specific full-length fusion of GFP to GDI-1 (cdh3>GFP::gdi-1) showed cytosolic distribution with no specific subcellular enrichment.  The gdi-1 RNAi construct reduced levels of an AC expressed GDI-1 reporter .  The gdi-1 RNAi construct from C. elegans did not alter the fluorescence intensity of a GDI-1 reporter made from the related nematode C. briggsae . AU = arbitrary units; scale bar, 5 μm.(A) A transcriptional reporter ((TIF)Click here for additional data file.S3 Fig2 , GFP::MIG-2 , GFP::CUP-5 , GFP::CED-10 , and LMP-1::GFP . RNAi mediated knockdown of gdi-1 (middle panels) resulted in mis-trafficking of the invadopodial membrane components PI2, GFP::MIG-2, and GFP::CED-10, as well as GFP::CUP-5 and LMP-1::GFP  relative to wild type (left panels). RNAi targeting of cdc-42 did not affect the distribution of PIP2, GFP::MIG-2, or GFP::CUP-5 (right panels). Box plots  display the percentage of the total fluorescent signal at or near the basal invasive cell membrane of the AC. For all conditions a minimum of 9 animals were analyzed (n is noted on each graph). In (A-C) comparisons were made using Tukey’s multiple comparisons tests, ** p < 0.01, *** p < 0.001. In (D-E) comparisons were made using a Student’s t-test, * p < 0.05. Scale bar, 5 μm.(A-E) 3D renderings showing the distribution of PIP(TIF)Click here for additional data file.S1 MoviemCherry::moeABD; left) and with spots  that are overlaid on fluorescence. Isosurface renderings built in place of F-actin were used to determine a threshold for assigning the spots that mark AC invadopodia. Frames were acquired for 24 minutes in one-minute intervals using a spinning disc confocal microscope . The frames are played at 10 frames per second. This video corresponds to Figs Ventral view time-lapse showing spot tracking analysis of a wild type animal prior to BM breach. Invadopodia are marked by F-actin ((MOV)Click here for additional data file.S2 Moviecdc-42 RNAi treated animal prior to BM breach. Invadopodia are marked by F-actin  and with spots  that are overlaid on fluorescence. Isosurface renderings built in place of F-actin were used to determine a threshold for assigning the spots that mark AC invadopodia. Loss of cdc-42 reduced the number invadopodia and decreased the rate of invadopodia formation but did not affect invadopodia lifetimes. Frames were acquired for 24 minutes in one-minute intervals using a spinning disc confocal microscope . The frames are played at 10 frames per second. This video corresponds to Ventral view time-lapse showing an AC of a (MOV)Click here for additional data file.S3 Movielin-3 RNAi treated animal prior to BM breach. Invadopodia are marked by F-actin  and with spots  that are overlaid on fluorescence. Isosurface renderings built in place of F-actin were used to determine a threshold for assigning the spots that mark AC invadopodia. Loss of the vulval precursors cells (via lin-3 RNAi) and thus loss of a cue(s) generated by the vulval precursor cells reduced the number invadopodia, decreased the rate of invadopodia formation, and increased invadopodia lifetimes. Frames were acquired for 24 minutes in one-minute intervals using a spinning disc confocal microscope . The frames are played at 10 frames per second. This video corresponds to Ventral view time-lapse showing an AC of a (MOV)Click here for additional data file.S4 Moviegdi-1 RNAi treated animal prior to BM breach. Invadopodia are marked by F-actin  and with spots  that are overlaid on fluorescence. Isosurface renderings built in place of F-actin were used to determine a threshold for assigning the spots that mark AC invadopodia. Reduction of gdi-1 by RNAi reduced the number invadopodia, decreased the rate of invadopodia formation, and increased invadopodia lifetimes. Frames were acquired for 24 minutes in one-minute intervals using a spinning disc confocal microscope . The frames are played at 10 frames per second. This video corresponds to Ventral view time-lapse showing an AC of a (MOV)Click here for additional data file.S5 Moviecdh-3>mCherry::PLCδPH) and the basement membrane . The invadopodial membrane is trafficked dynamically to the invasive membrane throughout the 40-minute time-lapse. The images were acquired in one-minute intervals using a spinning disc confocal microscope . The frames are played at 10 frames per second. This video corresponds to A 3D reconstruction of a lateral view time-lapse of a wild type AC visualizing the invadopodia membrane with a probe for PIP2 Click here for additional data file.S6 Moviegdi-1 RNAi treated AC visualizing the invadopodial membrane component PIP2  and the basement membrane . Loss of gdi-1 resulted in mis-trafficking of the invadopodial membrane to lateral and apical plasma membrane. The time-lapse takes place over a 40-minute period. The images were acquired in one-minute intervals using a spinning disc confocal microscope . The frames are played at 10 frames per second. This video corresponds to A 3D reconstruction of a lateral view time-lapse of a (MOV)Click here for additional data file.S7 Movielin-3 RNAi treated AC visualizing the invadopodial membrane component PIP2  and the basement membrane .). lin-3 RNAi treatment results in loss of the vulval precursor cells, which inhibits generation of the vulval cue. Loss of the vulval precursor cells resulted in mis-trafficking of the invadopodial membrane to the lateral and apical plasma membrane. The time-lapse takes place over a 40-minute period. The images were acquired in one-minute intervals using a spinning disc confocal microscope . The frames are played at 10 frames per second. This video corresponds to A 3D reconstruction of a lateral view time-lapse of a (MOV)Click here for additional data file.S8 Moviecdc-42 RNAi treated AC visualizing the invadopodial membrane component PIP2  and the basement membrane . The invadopodial membrane traffics normally to the invasive membrane throughout the 40-minute time-lapse. The images were acquired in one-minute intervals using a spinning disc confocal microscope . The frames are played at 10 frames per second. This video corresponds to A 3D reconstruction of a lateral view time-lapse of a (MOV)Click here for additional data file.