Ptgs2/COX-2 mRNA and IL-6, IL-1β, and CXCL1 protein expression, as well as neutrophil infiltration after cisplatin. GTS-21 blunted cisplatin-induced renal ERK1/2 activation, as well as renal ATP depletion and apoptosis (p<0.05). GTS-21 suppressed the expression of CTR1, a cisplatin influx transporter and enhanced the expression of cisplatin efflux transporters MRP2, MRP4, and MRP6 (p<0.05). Using breast, colon, and lung cancer cell lines we showed that GTS-21 did not inhibit cisplatin’s tumor cell killing activity. GTS-21 protects against cisplatin-AKI by attenuating renal inflammation, ATP depletion and apoptosis, as well as by decreasing renal cisplatin influx and increasing efflux, without impairing cisplatin-mediated tumor cell killing. Our results support further exploring the cholinergic anti-inflammatory pathway for preventing cisplatin-induced AKI.Acute kidney injury (AKI) is the most common side effect of cisplatin, a widely used chemotherapy drug. Although AKI occurs in up to one third of cancer patients receiving cisplatin, effective renal protective strategies are lacking. Cisplatin targets renal proximal tubular epithelial cells leading to inflammation, reactive oxygen species, tubular cell injury, and eventually cell death. The cholinergic anti-inflammatory pathway is a vagus nerve-mediated reflex that suppresses inflammation via α7 nicotinic acetylcholine receptors (α7nAChRs). Our previous studies demonstrated the renoprotective and anti-inflammatory effects of cholinergic agonists, including GTS-21. Therefore, we examined the effect of GTS-21 on cisplatin-induced AKI. Male C57BL/6 mice received either saline or GTS-21  twice daily for 4 days before cisplatin and treatment continued through euthanasia; 3 days post-cisplatin mice were euthanized and analyzed for markers of renal injury. GTS-21 significantly reduced cisplatin-induced renal dysfunction and injury (p<0.05). GTS-21 significantly attenuated renal  Cisplatin is a highly effective antineoplastic drug used for treating a myriad of solid tumors , with complete remission achieved in up to 90% of men diagnosed with stage III testicular cancer . Despiteex vivo [GTS-21, also known as 3- anabaseine (or DMXB-A), is a selective α7 nicotinic acetylcholine receptor (α7nAChR) agonist. GTS-21 is a relatively safe drug candidate in humans and has been shown to significantly improve cognitive function and attention when compared to placebo . Currentex vivo –15. Actiex vivo , 16, 17 ex vivo , as wellex vivo , atherosex vivo , and sepex vivo . Given t1 porcine renal epithelial, MCF7 human differentiated breast cancer and CT26 mouse colon carcinoma cell lines were purchased from ATCC . The H460 human large cell lung cancer cell line was provided by Dr. H Simpkins .Male C57BL/6 mice (8–12 wks old) were purchased from Taconic Farms . Animals were allowed to acclimate for at least one week prior to experimentation. All animals were housed under standard conditions (room temperature 22°C with a 12h light–dark cycle) with access to chow and water. All animal experiments were performed in accordance with the National Institutes of Health Guidelines and according to protocol #2009–002, which was approved by the Institutional Animal Care and Use Committee (IACUC) of the Feinstein Institute for Medical Research at Northwell Health. The LLC-PKPhospho-ERK1/2 , total ERK1/2 (rabbit anti-mouse), MRP4 (rabbit anti-mouse) and GAPDH (rabbit anti-mouse) antibodies were purchased from Cell Signaling Technology . MRP2 (rabbit anti-mouse) and MRP6 (rabbit anti-mouse) antibodies were purchased from Santa Cruz Biotechnologies . GTS-21 was synthesized in the laboratory of Dr. Yousef Al-Abed . Cisplatin (cis-Dicholorodiammineplatinum(II)) was purchased from Acros Organics  and 2′, 7′-dichlorodihydrofluorescein diacetate (DCFH-DA) was purchased from Molecular Probes/Thermo Fisher Scientific .2 asphyxiation/and exsanguination and blood was collected via cardiac puncture into heparinized needles/syringes; after centrifugation, plasma was collected and frozen at -80°C until analysis. Kidneys (outer medulla and cortex) were collected and either flash frozen in liquid N2 or fixed in 10% formalin.C57BL/6 mice  were acclimatized under normal environmental conditions and allowed free access to standard chow and tap water for at least 1 wk before experimentation. Mice were divided into 3 groups: (1) Control (CTRL); (2) cisplatin-treated (CIS) alone; and (3) GTS-21 plus cisplatin treated group (GTS+CIS). The GTS+CIS mice were injected with GTS-21  for 4 days prior to cisplatin  administration; GTS-21 treatments continued until euthanasia. Mice in the CTRL group received saline injections instead of GTS-21 and cisplatin. The mice were euthanized 72 hrs post- cisplatin  by COPlasma BUN and creatinine levels were determined using the respective Quantichrom kits , according to the manufacturer’s protocols.Ptgs2 (COX-2) primers , the Eurogentec One Step RT qPCR mastermix , 60ng RNA and the Roche 480 Light Cycler . Mouse Gapdh was used as the housekeeping gene for normalizing transcript levels (F: 5’-GAGCCAAACGGGTCATCA-3’ and R: 5’-CATATTTCTCGTGGTTCACACC-3’). Changes in gene expression were calculated as relative fold-changes using the comparative Ct (ΔΔCt) method [qPCR was performed using high quality RNA (OD 260/280 and 260/230 ≥2.0) isolated from frozen kidneys using the RNeasy Universal Plus Mini kit with elimination of genomic DNA . qPCR reactions were performed in triplicate using the mouse Approximately 100 mg kidney  per mouse was homogenized in 200μl lysis buffer  in a Dounce homogenizer; supernatants were collected after centrifugation. Homogenates were assayed for cytokines and chemokines using a multiplex platform  and the MSD Sector Imager 2400 plate reader . The electrochemiluminescent signals generated (raw data) were analyzed using the Discovery Workbench 3.0 software (MSD). The values were adjusted for protein concentration using the BioRad protein assay .For assessment of ATP and MPO levels, kidneys (cortex and outer medulla) were homogenized in lysis buffer (as described above). ATP levels were measured using a colorimetric ATP assay kit , according to manufacturer’s instructions. Tissue MPO levels were measured in the homogenate using an ELISA kit . Renal tissue ATP and MPO values were normalized to protein concentrations using the BioRad protein assay, as described above.Kidneys were homogenized in lysis buffer (as described above). Proteins  were separated by NuPAGE/SDS-PAGE electrophoresis  and then transferred to Immobilon-FL PVDF transfer membranes . After blocking, the membranes were incubated with each primary antibody according to the manufacturer’s instructions. After washing, the blots were incubated with the appropriate near-infrared-fluorescently labeled secondary antibody  for 45min and washed before revealing the bands using the Odyssey infrared imaging system (LI-COR Biosciences). Membranes were stripped using Re-blot Plus (Millipore) and re-probed with additional antibodies. Quantitation of the band densities was determined using Image J Software  and normalized using appropriate controls or housekeeping protein, GAPDH.Formalin-fixed kidneys were embedded in paraffin and sagittal sections (5μm) were prepared. To assess AKI-associated tubular injury, sections were stained with Periodic Acid-Schiff (PAS)  and scored by a pathologist blinded to the experimental groups using a semi-quantitative scale to determine tubular injury . Tubular injury scores  were based on the percentage of tubules affected , as previously described . Leder sKidney platinum (Pt) levels were measured by inductively coupled plasma mass spectrometry (ICP-MS) at the University of North Carolina at Chapel Hill using an Agilent 7500cx , as previously described . Pt conc1 renal epithelial cells were grown in M199 containing 10% fetal bovine serum (FBS), penicillin-streptomycin (PS), and glutamine (Q) to 90% confluency. After 24hrs, the media was replaced with minimum essential media (MEM) containing 5%FBS, Ca (0.2 g/L), Mg , non-essential amino acids and PSQ. On the next day, cells were treated with GTS-21 (1μM) or N-Acetyl Cysteine . After 16 hrs, cells were harvested, resuspended (1x106cells/ml) in either MEM (plus vehicle) or MEM containing cisplatin (50µg/mL) ± GTS-21 (1μM) or NAC (2mM) and incubated at 37°C/5% CO2 for 3.5 hrs. After the incubation period, cells were washed with HBSS and labeled with 2′,7′-Dichlorofluorescin diacetate  for 20 min. Labeled cells were washed and analyzed for oxidative stress by the conversion of the non-fluorescent DCFH-DA into a fluorescent compound, 2′,7′-dichlorofluorescein (DCF) by ROS intermediates [DCFH-DA assay: LLC-PKmediates , 26, whi1 renal epithelial cells were grown as described above and cells were treated with vehicle or GTS-21 (1μM) for 1hr followed by the addition of cisplatin (0 or 30 μM). After 20hrs, cells were harvested and the amount of reduced glutathione (GSH) in the cell lysates was determined using a glutathione fluorometric assay kit  and corrected for cellular protein using BCA Protein Assay Kit (Pierce/Thermo Fisher Scientific).Glutathione assay: LLC-PK4), non-essential amino acids and PSQ. After 24 hrs, cells were pretreated with saline or GTS-21 . After 1 hr, vehicle or cisplatin (0–230μM) was added and cytotoxicity was measured using the MTT assay (OD570/690) 24 hrs later. The IC50 values for the tumor cell killing by cisplatin were calculated using non-linear regression to fit the data to the log (inhibitor-cisplatin dose) vs. response (variable slope-percent viable) curve using GraphPad Prism . The same procedures were followed for MCF7 human differentiated breast cancer and CT26 mouse colon carcinoma cell lines, except the MCF7 cell line was originally grown in DMEM containing 10% FBS, PSQ.H460 human large cell lung cancer cells were grown in RPMI 1640 media containing 10% FBS, PSQ in 96-well plates and allowed to reach ~60% confluence. The media was replaced with (MEM) containing 5%FBS, Ca (0.2 g/L), Mg , as indicated. One-way ANOVAs were used for multiple comparisons followed by Bonferroni post-hoc testing using GraphPad Prism . P values <0.05 were considered significant.C57BL/6 mice treated with cisplatin alone exhibited elevated BUN  and plasPtgs2 (COX-2) mRNA expression  and the chemokine CXCL1 . In our Energy depletion in kidney cells promotes injury and necrosis , 29. We 1 renal epithelial cell line. Cisplatin administration significantly reduced glutathione (GSH) levels in LLC-PK1 renal epithelial cells in vitro  and MRP4 (ABCC4) protein levels, while MRP6 (ABCC6) protein expression remained unchanged when compared to control mice . GTS-21 50).Next, we used the H460 human lung cancer, MCF7 human breast cancer and CT26 mouse colon carcinoma cell lines to investigate whether GTS-21 treatment impaired the anti-tumor efficacy of cisplatin. As previously reported, all tumor cell lines were sensitive to cisplatin-mediated killing, with different sensitivities ranging from 10 to 43μM to kill one half of the cells . Pre-tre2 causes approximately one-third of cancer patients to develop nephrotoxicity that requires dose reduction or drug discontinuation [Although cisplatin is a highly effective chemotherapeutic agent, a dose of 50–100 mg/minuation , as wellinuation , 33. In inuation , 6. Thus2in humans) [Rodents and humans develop renal injury at fairly comparable cisplatin doses   and exhi humans) , 16 and  humans) . More re humans) , 35. Fur humans) –15. Chol humans) , 10. How humans) . Our pre humans)  and beca humans) . AlthougPtgs2 (COX-2) mRNA expression  were not protective in a similar mouse model of cisplatin-AKI [Ptgs2 mRNA expression in the kidneys   and otheHere we demonstrate for the first time that treatment with GTS-21, a selective α7nAChR agonist, attenuates cisplatin-induced acute tubular injury and renal dysfunction in mice. Several studies have demonstrated that α7nAChRs mediate the effects of cholinergic agonists (e.g. GTS-21) , 72, inc50 values between approximately 10 and 40μM; the H460 cell line was the most sensitive  show that GTS-21 does not compromise cisplatin’s anti-tumor effects. These findings warrant additional studies to identify the precise mechanisms of GTS-21’s renoprotective effects and to explore the use of prophylactic GTS-21 treatment for suppressing AKI following cisplating administration.Currently, there are no therapies available to treat or prevent cisplatin-induced AKI. Our results show that cisplatin-mediated AKI can be significantly reduced by GTS-21 administration in mice. Additional


Adipose tissue secretes a plethora of adipokines as evidenced by characterization of subcutaneous and visceral adipose tissue secretomes. However, adipose tissue composition and secretion pattern is depot and disease dependent, influencing the adipose tissue secretome. We investigated the secretome of cancer-associated adipose tissue (CAAT) explants from breast cancer patients and explored its role in breast cancer proliferation. CAAT proteins were identified by LC-MS/MS and human protein antibody arrays and stimulated proliferation of three breast cancer cell lines. Kinomics and transcriptomics of MCF-7 breast cancer cells treated with the secretome of CAAT revealed activation of Akt-, ERK- and JNK-pathways and differential expression of activator protein 1 (AP-1) and cAMP responsive element-binding protein (CREB) target genes. The cyclin-dependent kinase (CDK)4/6-inhibitor palbociclib significantly abrogated CAAT-enhanced breast cancer cell proliferation. Our work characterizes the specific breast CAAT protein secretome and reveals its pro-proliferative potency in breast cancer. In breast cancer, adipose tissue is the main component of the tumor environment and the heterogeneous cell types of the adipose tissue stimulate breast cancer cells towards further progression. Adipocytes can drive breast cancer progression by lipid transfer, androgen-to-estrogen production and secretion of several adipokines –3. HumanAdipose tissue can be found throughout the body but its composition and secretion pattern differs and is influenced by its localisation in the body and disease states such as obesity and cancer –11. ThisWe performed the first proteomic analysis of the human breast CAAT secretome and explored its influence on biological processes, pathways and transcription factors involved in breast cancer growth.CAAT from 6 patients from which 2 CMCAAT were used for LC-MS/MS analysis and 4 CMCAAT for antibody array as low levels of highly bioactive proteins are often not detected by gel-electrophoresis-based techniques [CAAT from an additional 18 patients. In 16 patients, the concentration of 5 identified proteins was measured by ELISA, i.e. leptin (9.1 ± 5.1 ng/ml), adiponectin (267.2 ± 101.5 ng/ml), IL-6 (25.1 ± 7.5 ng/ml), CSF-1 (2.8 ± 1.0 ng/ml) and CCL22 (0.1 ± 0.1 ng/ml) . In contrast, a 5-fold higher IL-6 (p = 0.005) and a 4-fold higher CCL22 expression (p = 0.005) in SVF compared to TAA were observed. CSF-1 did not significantly differ in both fractions (p = 0.959)  Figure . Westerns Figure . As adip) Figure . Compari) Figure . A detaip < 0,0001)   Figure . We next) Figure . Positivd Figure . A phospt Figure .CAAT treated MCF-7 cells when compared to control [CAAT revealed several AP-1 transcriptional targets of which the upregulated gene ETS2  and the downregulated genes RGS2 , EMP1 , DUSP2  and GADD45B  are linked with a pro-proliferative signature in breast cancer [CAAT treated MCF-7 cells and found an upregulation of LDHA  and downregulation of GEM , ATF3 , DUSP1  and PER1 , known to affect breast cancer proliferation and growth [CAAT revealed 30 proteins  involved in CREB-mediated transcription. RT-qPCR validated the differential expression of 4 of the above mentioned AP-1 and CREB transcriptional targets  , Akt- (1.75-fold), JNK- (1.94-fold) and CREB- (2.02-fold) pathways in CM control . Western control . Micro-at cancer –22  Figure .CAAT enhanced proliferation of MCF-7, T47D and MDA MB 231 breast cancer cells. Palbociclib significantly reduced the stimulatory effect of CAAT in all cell lines   like oleate and palmitate on Akt-activation and subsequent cell proliferation in MDA MB 231 cells demonstrating that not only proteins but also FFAs can exert a pro-proliferative effect [CAAT treated MDA MB 231 cells. As our research focused on breast cancer-associated adipose tissue proteins, further research will be necessary to elucidate the role of FFAs in breast cancer.c-Jun and c-Fos are both proto-oncogenes and their homo- or heterodimers form the transcription factor AP-1, linked with cell proliferation, differentiation and survival , 42, 43.e effect . This coIn vitro experimentation showed that palbociclib is most effective in estrogen receptor positive breast cancer cell lines [CAAT). A possible explanation of these findings may be that CDK4/6 inhibition induces a compensatory mitogenic response as has been described in ER+/HER2- models [Palbociclib has recently been FDA approved for use in combination with letrozole for the treatment of postmenopausal women with estrogen receptor-positive, human epidermal growth factor (HER)2-negative advanced breast cancer as initial endocrine-based therapy for their metastatic disease. Palbociclib inhibits CDK4 and 6 to bind cyclin D1, keeping the tumor suppressor retinoblastoma protein (pRb) in its activated form and halting cell cycle progression , 50. In ll lines . This co- models . In thesin vivo results correlate with our results of palbociclib-induced inhibition of CMCAAT-stimulated proliferation of MDA MB 231 cells given the fact that CMCAAT-treated MDA MB 231 cells resemble more closely the in vivo situation (MDA MB 231 cells injected into the mammary fat pad) than MDA MB 231 cells grown in absence of adipose tissue-derived factors (in vitro situation with control medium).In contrast with the results in MDA MB 231 cells in control conditions, palbociclib was able to significantly inhibit CAAT-enhanced proliferation of all three breast cancer cell lines. This might signify that CAAT shifts the cell cycle control in MDA MB 231 cells from a non-pRb-regulated to a pRb-regulated way, sensitizing the breast cancer cells to CDK4/6 inhibitory treatment. This is supported by the fact that MDA MB 231 is a pRb-positive cell line, which is an absolute necessity for sensitivity to palbociclib treatment , 54. MorThis the first report identifying patient-derived breast cancer-associated adipose tissue proteins representing potential therapeutic targets of paracrine signalling implicated in breast cancer growth.MCF-7, T47D and MDA MB 231 cell lines were obtained from American Type Culture Collection . Cells were maintained in Dulbecco's minimal essential medium (DMEM) supplemented with 10% fetal calf serum, 100 U/mL penicillin, 100 μg/mL streptomycin  and 2.5 μg/mL fungizone . Every month cell cultures were tested for Mycoplasma contamination using MycoAlert Plus kit .Primary antibodies used for Western blot analysis and immunohistochemistry: mouse monoclonal anti-Ki67 (clone MIB-1) , mouse monoclonal anti-cyclin A and anti-cyclin E , mouse monoclonal anti-p27 and anti-p21 , mouse monoclonal anti-tubulin , rabbit monoclonal anti-phospho-p44/42 MAPK (Erk1/2), anti-p44/42 MAPK (Erk1/2), anti-phospho-Akt, anti-Akt, anti-phospho-c-Jun, anti-c-Jun, anti-phospho-c-Fos, anti-c-Fos, anti-phospho-CREB, anti-CREB . Secondary antibodies coupled to horseradish peroxidase were obtained from Amersham Pharmacia Biotech . Palbociclib (PD-0332991) was purchased at Selleck Chemicals .CAAT preparation and separation of adipose tissue in TAA and SVF were previously described [2. After 24 h, CMCAAT is harvested, centrifuged, filtered, aliquoted and stored at –80°C until experimentation. For separation of CAAT into TAA and SVF, CAAT is washed, thoroughly minced and digested with collagenase. After centrifugation, the upper layer containing TAA is separated from SVF and RNA of both fractions was extracted using RNeasy Plus Universal Kit . CAAT throughout the article.CAAT was obtained from breast cancer patients undergoing mastectomy at Ghent University Hospital in accordance with local ethics committee and written informed consent was obtained from all subjects . CMCAAT escribed . In shorCAAT samples were run on NuPAGE 4%-20% Bis-Tris gradient gels (Invitrogen) in SDS-containing denaturation buffer, stained with 0.5% Coomassie Brilliant Blue (Bio-Rad) in 40% methanol and 10% acetic acid for 20 minutes, and destained. Gel bands were processed and analyzed by LC-MS/MS as previously described [CMescribed , 55.CAAT. Human phospho-kinase antibody array (R&D systems) was used to detect relative phosphorylation levels of 44 kinases. Concentrations of human leptin, adiponectin, IL-6, C-C motif chemokine 22 (CCL22) and colony stimulating factor 1 (CSF-1) in CMCAAT were measured with a Duoset or Quantikine ELISA Kit (R&D systems). Scanning densitometry was carried out with the Quantity One Program . Functional analysis of detected proteins was performed using FunRich version 2.1 [www.cmbi.ru.nl/cdd/biovenn/).Samples for western blot were prepared, runned and immunostained as described in . Human Lsion 2.1 . Venn diTotal RNA was isolated using the Nucleospin RNA II kit  including DNAse I treatment. Quality control was performed using Agilent 2100 Bioanalyser (Agilent Technologies). Total RNA (0.5 μg) was processed and analysed on Human GE Agilent 4 × 44K microarrays. Four biological samples were studied. Data can be found on GEO (GEO accession number GSE58574).RNA was isolated using RNeasy Plus Universal Kit (Qiagen) including DNAse I treatment. cDNA synthesis and SYBR Green I RT-qPCR were carried out as described in . Prime PCAAT with control medium (ratio 1:1) was added. On day 3, 6 and 9, cells were trypsinized and counted with Countess Automated Cell Counter .For proliferation tests, 20.000 cells were seeded in culture flasks with normal growth medium for 24 h. Every three days, cells were washed with serum-free medium and control medium or CMCAAT with control medium (ratio 1:1) and/or palbociclib were added and refreshed after 2 days. On day 5, 40 μl/well of 1 mg/ml MTT-solution was added and incubated for 2 h protected from light. Supernatant was removed and the formazan crystals were dissolved in 100 μl/well DMSO. Optical density was measured with a Spectramax Paradigm Multi-Mode Microplate Reader .For MTT-assays, 2000 cells were seeded in a 96-well plate with normal growth medium for 24 h. Cells were washed with serum-free medium and control medium, CMU test (mean ± standard deviation) or Student's t-test  where appropriate. Statistical tests were two-sided, p-values less than 0.05 were considered statistically significant.Statistical analyses were performed using IBM SPSS Statistics 21.0 software . Continuous data were analysed with Mann-Whitney


Tuberculosis (TB) is a global health burden, and a major cause of mortality and morbidity in West Africa. Here, we select keyconserved pathogen epitopes of proven immunogenicity to form a potential TB epitope ensemble vaccine. We compared two vaccineformulations: one comprising class I epitopes from the 13 most prevalent class I epitope-bearing antigens and class II epitopesderiving from the 20 most prevalent class II epitope-bearing antigens and another consisting of epitopes derived solely from 5 antigensidentified as the most immunogenic by VaxiJen. In the prevalence analysis, 279 class I and 561 class II epitopes were collected and asubset selected using our published methodology, yielding 32 conserved epitopes. Combining 9 conserved epitopes gave a putativevaccine with predicted population coverage (PPC) over 95%. This consists of ISSGVFLLK, AVAGAAILV, WYYQSGLSI, YQSGLSIVM,MPVGGQSSF, QSSFYSDWY, WDINTPAFEWYYQSGLSIVM, DAPLITNPGGLLEQAVAVEE and NQAVTPAARALPLTSLTSAA. 5immunogenic antigens VaxiJen-identified yielded 187 epitopes, which we again analyzed using published protocol. This identified 11conserved epitopes. From this set the highest PPC value (<85%) was obtained by combining: GQQYQAMSAQAAAFH,DDIKATYDKGILTVSVAVSE and AVAGAAILV. We conclude that prioritizing epitope selection using predicted immunogenicityalone is likely to be unduly restrictive and is currently not an optimal or advisable strategy in the design of epitope ensemble vaccines. Mycobacterium Tuberculosis (TB) is a global health burden withan estimated 2015 death toll of 1.8 million, with 10.5 million newTB cases .The immune response against TB predominantly involves T cellmediated immunity, specifically CD4+ and CD8+ T cells, whichbind to complexes of class I or class II MHC bound to specificepitopes. Once activated, both CD4+ and CD8+ cells secretecytokines inducing an immune response. CD8+ T cells alsomediate cytotoxicity and the lysis of infected cells.The Bacillus Calmette-Guérin (BCG) anti-TB vaccine has beenused for disease prophylaxis and control of TB progression forover 80 years. BCG is an attenuated form of mycobacterium bovisand is currently the only anti-TB vaccine in use worldwide.However, BCG has a well-attested variable efficacy (0-80%) inadult populations presenting with pulmonary TB , limitinSeveral live vaccines are currently in development against TBincluding recombinant BCG VPM1002 and MTBVAC . A keyiHere, we seek to identify pathogen epitopes able of form anepitope ensemble vaccine against TB. The complete genomesequence of Mycobacterium Tuberculosis H37Rv strain,comprising over 4 million base pairs and approximately 4,000genes  andinflWe applied an epitope ensemble vaccine design protocol alreadydescribed ,9 to TB.TB Antigens were processed using VzxiJen (URL:http://www.ddgpharmfac.net/vaxijen/Vaxijen.html), whichpredicts immunogenicity scores, allowing classification ofantigens as either an antigen capable of inducing protectiveimmunity or as a non-antigen ,11. BactTwo sets of epitope-bearing antigens were collected. The firstdataset comprised proteins with greater than 10 epitopes. Therewere 13 Class I epitope-bearing antigens (Ag85B (59 class Iepitopes), Putative ATP-dependent 6-phosphofructokinaseisozyme 2 (28 epitopes), Alanine and proline-rich secretedprotein Apa (26), Probable membrane protein Rv1733c (26),ESAT-6-like protein EsxB (23), ESAT-6-like protein EsxH (19),Low molecular weight T-cell antigen (17), 6 kDa early secretoryantigenic target (16), Lipoprotein LpqH (14), PPE68 (14), Ag85A(13), Alpha-crystallin (13), MPT64 (11)) and 20 class II-bearingantigens (Ag85B (55 class II epitopes), 6 kDa early secretoryantigenic target (55 epitopes), ESAT-6-like protein EsxB (45), 60kDa chaperonin 2 (42), Uncharacterized PPE family proteinPPE19 (36), Ag85A (34), Putative ATP-dependent 6-phosphofructokinase isozyme 2 (32), Alanine and proline-richsecreted protein Apa (30), Phosphate-binding protein PstS 1 (29), Uncharacterized PPE family immunogenic protein PPE68 (27),MPT64 (24), Alpha-crystallin (22), Lipoprotein LpqH (21),Probable membrane protein Rv1733c (21), MPT70 ( 19), 10 kDachaperonin (18), MPT83 (15), Putative phthioceroldimycocerosate transporter LppX (12), ESAT-6-like protein EsxH(12), MPT63 (12).PVS identified conserved epitopes. From the 13 class I epitopebearingantigens, only 9 had conserved epitopes. Overall, 22conserved epitopes were found. The greatest number of epitopeswas collected from diacylglycerol acyltransferase Ag85B (11),followed by lipoprotein LpqH (3), diacylglycerol acyltransferaseAg85A (2), alanine and proline-rich secreted protein Apa (2),PPE68 (1), ATP-PFK 2 (1) and alpha-crystallin (1). From the 20class I epitope-bearing antigens, only 5 had a total of 11conserved epitopes: ATP-PFK 2 (1), alanine and proline-richsecreted protein Apa (1), lipoprotein LpqH (1), diacylglycerolacyltransferase Ag85B (3), PPE19 (5).The second dataset comprised antigens with 3 or more epitopesfor analysis using VAXIJEN. For class I, 22 antigens wereidentified. Antigens with the top 5 immunogenicity scores wereidentified: Lipoprotein LpqH ,Isoniazid-induced protein , ESAT-6-like protein EsxB, Alpha-crystallin , PPE68 . Forclass II, 61 antigens were identified. Antigens with the top 5immunogenicity scores were identified: PE_PGRS14 , Lipoprotein LpqH , ESAT-6-like proteinEsxB , Alpha-crystallin ; PstS 1 .PVS identified conserved epitopes: for class I only 4 antigens hadconserved epitopes, returning a total of 6 epitopes, and for class IIonly 3 of the 5 antigens had conserved epitopes, returning a totalof 5 epitopes.For both VaxiJen and non-VaxiJen analysis, combinations ofepitopes were assessed for population coverage. For the high prevalenceset, non-VaxiJen analysis, the highest PPC valuefound was 98.75  and 95.60 (West Africa). For the VaxiJenanalysis, the highest PPC value found was 83.79  and68.62 (West Africa). See Compared to the unrestricted analysis, results from VaxiJenranking were disappointing. Due to the small number ofconserved epitopes identified, limited combinations could beundertaken. The conserved epitopes from VAXIJEN have similarHLA binding profiles. For class I, A*02:06, A*02:01 and A*02:03were the most common restrictions seen. The limited variation inbinding profiles contributed to the low PPC values seen. Thehighest combined PPC values for both subsets contained the classI conserved epitope AVAGAAILV, derived from lipoproteinLpqH, consistent with the epitope being highly immunogenicand promiscuous.In this vaccine design exercise, we sought to limit selection toepitopes from major highly immunogenic antigens. Suchconstrained selection should concentrate the immune responseon antigens more likely repeatedly to evoke strong responses invivo. As an alternative to using all experimentally verifiedepitopes available, we used VaxiJen - the first and, ostensibly, theonly on-line server for whole protein immunogenicity prediction- to rank antigens in terms of potential efficacy. However,prioritizing antigens using VaxiJen did not prove effective. Thisidentifies a shortcoming of this method, due in no small part tothe dearth of adequate training data. This is something we willseek to rectify in due course. Prevalence analysis, however,allowed for the total pool of available epitopes to be tractable yet large enough to generate an optimal hypothetical vaccinecandidate. Epitope combinations derived through epitopeprevalenceanalysis had sufficiently high population coverage togenerate effective potential universal epitope ensemble vaccines.However, due to the low immunogenicity traditionallyassociated with peptide vaccines, such candidates are likely torequire the addition of adjuvants. Overall, we have shown howcomputational approaches can identify a potential ensemblevaccine against a major bacterial pathogen by selecting epitopesof known, validated immunogenicity with broad populationcoverage. These will now need experimental validation todetermine the efficacy and safety of the vaccine in live subjects.


Providencia rettgeri is an opportunistic bacterial pathogen of clinical significance due to its association with urinary tract infections and multidrug resistance. Here, we report the first complete genome sequence of P. rettgeri. The genome of strain RB151 consists of a 4.8-Mbp chromosome and a 108-kbp blaNDM-1-positive plasmid. Providencia rettgeri is an opportunistic human pathogen mainly associated with urinary tract infections  and repA (plasmid). The final assembly was verified using Circlator version 1.4.0 (https://github.com/sanger-pathogens/ariba).Colombia . Total gsion 2.3 , revealision 2.3 , and lowon 1.4.0 . The genP. rettgeri RB151 has an average G+C content of 41.7% and consists of a 4,780,676-bp chromosome and a 108,417-bp NDM-1-encoding plasmid (pRB151-NDM). The automated genome annotation predicted 4,497 coding sequences (CDSs), 22 rRNAs, 77 tRNAs, and one transfer-messenger RNA (tmRNA). The antimicrobial resistome of the RB151 chromosome evaluated using ARIBA included resistance genes to aminoglycosides , β-lactams (blaTEM-1B and blaOXA-2), fluoroquinolones [aac(6′)Ib-cr], sulfonamides (sul1 and sul2), and trimethoprim (dfrA31). The plasmid pRB151-NDM only contained the blaNDM-1 gene, which confers resistance to β-lactams.The complete genome of P. rettgeri, this genome sequence will be a useful reference genome and could be utilized to contribute further insights into this species.As the first complete genome of Providencia rettgeri RB151 has been deposited in DDBJ/EMBL/GenBank under the GenBank accession numbers CP017671 (chromosome) and CP017672 (plasmid pRB151-NDM). The version described in this paper is the first version.The complete genome of


S1 FigPast1110-1 mutant and GMRGal4>UAS-GFP-Past1B larval eye discs. Shown are Z-projections of confocal sections.Boss (grey) staining of wild type, (TIF)Click here for additional data file.S2 FigPast1110-1 homozygous mutant early-mid pupal eyes (42-48h after puparium formation). (B) Quantification of fluorescent intensity in the pupal eyes of wild type and Past1110-1 homozygous mutant. Results represent the mean ± SD of 26 eyes of wild type and Past1110-1 homozygous mutant from five independent experiments, statistically analyzed using the student t-test.(A) Notch intracellular domain (green) staining of wild type and (TIF)Click here for additional data file.


Inhibitors of apoptosis (IAP) proteins contribute to cell death resistance in malignancies and emerged as promising targets in cancer therapy. Currently, small molecules mimicking the IAP-antagonizing activity of endogenous second mitochondria-derived activator of caspases (SMAC) are evaluated in phase 1/2 clinical trials. In cancer cells, SMAC mimetic (SM)-mediated IAP depletion induces tumor necrosis factor (TNF) secretion and simultaneously sensitizes for TNF-induced cell death. However, tumor cells lacking SM-induced autocrine TNF release survive and thus limit therapeutic efficacy. Here, we show that hyperosmotic stress boosts SM cytotoxicity in human and murine cells through hypertonicity-induced upregulation of TNF with subsequent induction of apoptosis and/or necroptosis. Hypertonicity allowed robust TNF-dependent killing in SM-treated human acute lymphoblastic leukemia cells, which under isotonic conditions resisted SM treatment due to poor SM-induced TNF secretion. Mechanistically, hypertonicity-triggered TNF release bypassed the dependency on SM-induced TNF production to execute SM cytotoxicity, effectively reducing the role of SM to TNF-sensitizing, but not necessarily TNF-inducing agents. Perspectively, these findings could extend the clinical application of SM. SM free caspases from XIAP-mediated inhibition and trigger degradation of cIAP1/2, which is death promoting in two interdependent ways: it initiates autocrine secretion of tumor necrosis factor (TNF) and concomitantly undermines the function of cIAP1/2 to counteract TNF-induced TNF-receptor 1 (TNFR1)-mediated cytotoxicity.7 Usually, TNFR1 activation first triggers formation of a receptor-associated signaling complex (termed ‘complex I’). cIAP1/2-mediated ubiquitination of complex I provides a scaffold for recruitment of kinases activating the survival-promoting canonical nuclear factor kappa B (NFκB)-pathway. SM abolish complex I ubiquitination and facilitate its transition to a secondary cytosolic complex (termed ’complex II’), which can induce apoptotic and necroptotic cell death via caspase-8 and RIPK1/RIPK3, respectively.8 How SM trigger TNF production is not fully understood, but SM-mediated loss of cIAP1/2 impairs constitutive ubiquitination and subsequent proteasomal degradation of NFκB-interacting kinase (NIK). Increasing NIK levels activate the non-canonical NFκB pathway and transcription of downstream target genes such as TNF.10Imbalances in pro- and anti-apoptotic proteins disturb cellular death pathways and allow tumor cells to escape from chemotherapy-induced apoptosis. Newer therapeutic approaches aim to reinstate the cell’s death machinery by targeting the pro-survival BCL-2 family members or the cellular inhibitor of apoptosis proteins (IAP) cIAP1, cIAP2 and X-linked IAP (XIAP). Mechanistically, XIAP blocks apoptosis by direct caspase inhibition whereas cIAP1/2 shut down cell death promoting activities of receptor-interacting protein kinase-1 (RIPK1). Overexpression of IAP contributes to the onset of cancer and drug resistance,7 Cancer cells that do not release TNF upon SM treatment are consequently resistant even though SM-mediated IAP depletion sensitizes for TNF-induced cell death. Combining SM with exogenously added TNF could overcome this limitation, but is clinically challenging due to the severe cardio-vascular side effects of the cytokine. However, aside from SM-triggered activation of the non-canonical NFκB pathway, a plethora of stimuli can induce cellular TNF production.11 Here, we show that hyperosmotic stress boosts SM cytotoxicity in human and murine cells through hypertonicity-induced upregulation of TNF with subsequent initiation of apoptotic and/or necroptotic cell death. Human acute lymphoblastic leukemia (ALL) cells resisted SM treatment under isotonic conditions due to poor SM-induced TNF secretion. Under hypertonic conditions, however, SM treatment displayed a robust, TNF-dependent cytotoxicity. Mechanistically, hypertonicity-triggered TNF secretion bypassed the dependency on SM-triggered TNF production for SM cytotoxicity and reduced the role of SM to TNF-sensitizers, but not necessarily simultaneous TNF-inducers. Perspectively, our findings could extend the clinical application of SM.Efficient killing by SM alone requires both autocrine TNF secretion and TNFR1-mediated cytotoxicity.12 Synergism in terms of cell death induction was observed for both, BV6 and LCL161 with NaCl  expectedly abrogated cytotoxicity of BV6/NaCl  generates an osmotic pressure gradient between the intra- and extracellular space (hyperosmotic stress or hypertonicity). Hypertonicity generated by supplementing the cell culture medium with 75 mM NaCl or 150 mM mannitol (both increasing osmolarity by 150 mOsm/l) drastically increased cell death in L929 cells upon challenge with the SM BV6, LCL161 and birinapant compared with isotonic conditions . Althougith NaCl . This isBV6/NaCl . As the ng RIPK3 . Collect21 In human ALL cells (REH) and rhabdomyosarcoma cells (KYM-1), hypertonicity (established by addition of 50 mM NaCl or 100 mM mannitol to the cell culture medium) enhanced BV6-, LCL161- and birinapant-mediated cytotoxicity compared with isotonic controls  detection of annexin-V and 7-AAD in flow cytometry  incytometry  and (c) ytometry . In sum,κB pathway or itself triggered TNF production. Birinapant-induced non-canonical NFκB activation (as seen by decline of p100) was not enhanced under hypertonic conditions, potentially pointing to an additional, hypertonicity-activated TNF-producing pathway  NaC pathway  and (c)  pathway .24 Notably, NFAT5 also potently initiates TNF transcription under hyperosmotic stress.25 We observed hypertonicity-induced increase in TNF mRNA levels  along with its hypertonicity-induced activation through the mitogen-activated protein kinase (MAPK) p38.A levels . The notA levels  and 3a–cA levels  promptedA levels  and phosA levels  were dettoxicity  and  to treat early stages of bladder cancer triggered local inflammation and TNF release from recruited neutrophils. In combination with SM, neutrophil-derived TNF potently induced apoptosis.26 mediated through binding of the transcription factor NFAT5 to the TNF promoter.25 Hypertonicity-induced TNF release from cancer cells  could therefore substitute for lacking SM-induced TNF production, while still relying on the TNF-sensitizing activity of SM. Our study strengthened this concept by showing that hyperosmotic stress (a) initiated TNF release in human and murine cells (κB-mediated upregulation of pro-inflammatory cytokines (including TNF).31 However, artificially establishing (transient) hypertonic conditions in a narrowly defined area such as the tumor environment could attenuate systemic toxicity by spatially restricting TNF production and lowering SM concentrations necessary for cancer cell elimination  enhne cells , 4 and 7ne cells  and 7. Amination  and 3d–f32 Hyperosmotic stress activates p38,33 raising the possibility of reduced SM cytotoxicity (due to lower TNF production) under hypertonic conditions. Our results, however, indicate the opposite. Although p38 activation was clearly detectable under hypertonic conditions , TF-1 (#ACC334) and L929 (#ACC-2) cells were obtained from the German Collection of Microorganisms and Cell Cultures , KYM-1 cells were kindly provided by Harald Wajant (University of Wuerzburg). All cell lines were grown RPMI 1640 medium  supplemented with 10% (v/v) fetal calf serum . TF-1 cells were additionally supplemented with 5 ng/ml human granulocyte–macrophage colony-stimulating factor . Antibodies used in the study: TNF #3707, p38 #9212, phospho-p38 #9211, ERK #4695, phospho-ERK #4370 ; tubulin #MS-581: Dunnlab ; NFAT5 #PA1-023: Thermo Fisher Scientific . Chemicals: MTT: Biomol ; zVAD-fmk -fluoromethylketone): Bachem ; BIRB796, PH797804, SB203580, BV6, LCL161 and birinapant: Selleck Chemicals . Necrostatin-1 (Nec-1): Stress-Marq ; Etanercept was obtained from Pfizer .4 cells/well), REH cells (7 × 104 cells/well) and TF-1 cells (7 × 104 cells/well) were seeded in 96-well plates and challenged with the indicated concentrations of SM in triplicates . For co-culture assays, 1:1 mixtures of TF-1 and REH cells (5 × 104 cells of each cell line/well) were seeded in 96-well plates and stimulated as above. Cell viability was determined 18 h after stimulation using MTT staining (2 h at 37 °C). Staining intensity was measured at 595 nm and the mean was calculated from the technical replicates of each experiment. The mean value for untreated controls was set to 100%. For any other condition, the MTT staining intensity is given relative to the corresponding untreated group (% of control). Data points shown are mean values  of independent experiments (n≥3).L929  SDS, 0.1 M dithiothreitol, 40% (v/v) glycerol, 0.2 M Tris, pH 8.0) supplemented with phosphatase inhibitor cocktails-I and -II (Sigma). Samples were sonicated and boiled for 5 min at 96 °C before proteins were separated by SDS-PAGE and transferred to PVDF membranes. To block non-specific binding sites, membranes were incubated in TBS containing 0.1% (v/v) Tween 20 and 5% (w/v) dry milk before primary antibodies of the specificity of interest were added. Antigen–antibody complexes were visualized using horseradish peroxidase-conjugated secondary antibodies  and ECL technology .12 In this model, CI-values <1 are considered to be synergistic and CI >1 indicate antagonistic effects. Strength of synergism can be further graded: <0.1 very strong synergism, 0.1–0.3 strong synergism, 0.3–0.7 synergism, 0.7–0.85 moderate synergism, 0.85–0.9 slight synergism, 0.9–1.1 no synergism but nearly additive effects.CI-values were calculated with the freely available software CompuSyn version 1.0 using the median effect/combination index isobologram method.4/well) or REH cells (1 × 106/well) were seeded in 96-well tissue culture plates and cultured overnight. The next day, cells were challenged with SM in the presence and absence of NaCl. The supernatant was collected and TNF was quantified using an enzyme-linked immunosorbent assay . All groups were analyzed as triplicates .For measuring TNF secretion, L929 cells  according to the manufacturer’s instructions. Emitted fluorescence was quantified using a Victor3 Multilabel Reader .35 In brief, total RNA was isolated using the Qiagen RNeasy mini kit  according to the manufacturer’s instructions. Two micrograms of total RNA were transcribed into complementary DNA using the high-capacity cDNA reverse transcription kit . Tnf mRNA levels were quantified using the TaqMan human Tnf (Hs00174128_m1) gene expression assay (Applied Biosystems) and an ABI Prism 7900 sequence detector (Applied Biosystems). qRT-PCR reactions were performed in duplicates for each sample of an experiment and normalized to the housekeeping gene Hprt1 (Hs02800695_m1). mRNA levels were calculated using the SDS 2.1 software (Applied Biosystems).qPCR was essentially performed as described previously.μM LCL161 and birinapant for 16 h in the presence and absence of 50 mM NaCl. Afterwards, cells were stained with 7-AAD and Annexin-V (4 °C for 15 min in the dark) and analyzed immediately using a FACSCanto flow cytometer (BD Biosciences) following standard procedures.Cell death was assessed by Annexin-V and 7-AAD staining. In brief, REH cells were challenged with 2.5


C. elegans, young larvae form mature motor circuits through a dramatic switch in GABAergic neuron connectivity, by concomitant elimination of existing synapses and formation of new synapses that are maintained throughout adulthood. We have previously shown that an increase in microtubule dynamics during motor circuit rewiring facilitates new synapse formation. Here, we further investigate cellular control of circuit rewiring through the analysis of mutants obtained in a forward genetic screen. Using live imaging, we characterize novel mutations that alter cargo binding in the dynein motor complex and enhance anterograde synaptic vesicle movement during remodeling, providing in vivo evidence for the tug-of-war between kinesin and dynein in fast axonal transport. We also find that a casein kinase homolog, TTBK-3, inhibits stabilization of nascent synapses in their new locations, a previously unexplored facet of structural plasticity of synapses. Our study delineates temporally distinct signaling pathways that are required for effective neural circuit refinement.Neural circuits are dynamic, with activity-dependent changes in synapse density and connectivity peaking during different phases of animal development. In  C. elegans. Our work characterizes the interaction between molecular motors kinesin and dynein, which carry cargo and move towards opposite ends of microtubules during synapse formation. We also address the role of a protein kinase gene TTBK-3 in maintaining synapse structure once synaptic components have reached the sites of new synapses. Our findings shed mechanistic insight into the coordination of molecular motors and the cytoskeleton in neural circuit function.In this study, we identify pathways that regulate the formation and maintenance of synapses, the functional connections between neurons, in the nervous system of the nematode  Cothe head  [11]. Weping see . Below, tba-1(gf) alters a conserved glycine residue to arginine (G414R) in the C-terminal H11-12 loop of α-tubulin (tba-1(0) null mutants appeared wild-type, presumably due to functional redundancy among the nine α-tubulin homologs in C.elegans . Ad. Adju127 animals . We alsoju972 and ju977) to be novel alleles of the anterograde motor, Kinesin-1/UNC-116 . Ki. Kiju972ving SVs  33–35].–35.ju972ransport . These oirection .tba-1(gf) dlk-1(0), ju978, to the kinase ttbk-3 (F32B6.10) (ttbk-3(tm4006), a deletion allele that removes the N-terminus and part of the kinase domain, also suppressed the synapse remodeling defects of tba-1(gf) dlk-1(0)  was causative for suppression of tba-1(gf) dlk-1(0) by overexpressing wild type TTBK-3 in tba-1(gf) dlk-1(0); ttbk-3(ju978) animals, and observed a block in synapse remodeling in the transgenic animals  dlk-1(0) animals.We mapped another suppressor mutation of 32B6.10) . ttbk-3(dlk-1(0) . Single  defects . We veri animals . These rC. elegans, sharing 32% identity in the kinase domain to human tau-tubulin kinases (TTBK1 and TTBK2) [ju978 generated a STOP codon in the C-terminal end of TTBK-3, which could result in a truncated protein with an intact kinase domain that lacked a coiled-coil domain further downstream (ttbk-3(ju978) suppressed tba-1(gf) dlk-1(0) to a similar extent as ttbk-3(tm4006), we asked whether the catalytic activity of ttbk-3 was required for synapse remodeling. Overexpressing kinase-dead versions of TTBK-3 (K115A or D209A) [tba-1(gf) dlk-1(0); ttbk-3(ju978) (TTBK-3 belongs to a large expansion of the Casein-kinase (CK1) superfamily in d TTBK2) . Tau-tubd TTBK2) , 38. ju9wnstream . Since tr D209A)  failed t3(ju978) . We also3(ju978) . These rttbk-3 was weak and extremely variable [ttbk-3 promoter. Expression of GFP tagged TTBK-3 in D motor neurons (Punc-25-TTBK-3-GFP) showed both diffuse and punctate GFP accumulation in the cell body and neurites during the L3 and L4 developmental stages, with a more diffuse distribution of GFP observed in adult animals (flp-13), which rescued the suppression of defective remodeling by ttbk-3(ju978) to a similar degree as full-length ttbk-3. As a control, expression of TTBK-3 from a muscle specific promoter (myo-3) failed to do so  did not have any effect on synapse formation or remodeling in wild type animals, and also failed to suppress the synapse remodeling defects of tba-1(gf) dlk-1(0) animals (ttbk-7 (R90.1), the closest homolog of mammalian TTBK1/2 (65% sequence identity), which also failed to suppress tba-1(gf) dlk-1(0)  . These otba-1(gf) dlk-1(0); ttbk-3(tm4006) animals when compared to tba-1(gf) dlk-1(0) animals . Following heat-shock in young adult animals TTBK-3-GFP formed punctate aggregates in neurons, the pharynx, the intestinal lumen and posterior intestinal cells  dlk-1(0); ttbk-3(ju978) animals expressing TTBK-3-GFP significantly reduced the suppression of both behavioral and synapse remodeling defects by ttbk-3(ju978), when compared to non-transgenic animals undergoing the same heat shock treatment  dlk-1(0) animals from the L2-L4 stage, which were then eliminated as the animal achieved adulthood [ttbk-3(tm4006) animals, both in the wild type and tba-1(gf) dlk-1(0) backgrounds (tba-1(gf) dlk-1(0); ttbk-3(ju978) animals, we concluded that TTBK-3 antagonizes the stability of nascent synapses that are formed during synapse remodeling.We next imaged MT dynamics and SV transport and found that neither changed in  animals . Taken tal cells . To assadulthood . Heat-shdulthood . On the reatment . We obsedulthood . Indeed,kgrounds . This suC. elegans model of structural synaptic plasticity, where synapses are eliminated and re-assembled along different neurites of the same neuron during a developmentally defined time scale [Modifications to synapse architecture occur throughout the lifetime of an animal, either dependent or independent of large scale neurite rewiring –9. In thme scale .tba-1(gf) dlk-1(0) animals failed to complete synapse remodeling because of enhanced MT stability in DD neurons [tba-1(gf) dlk-1(0) animals following EMS mutagenesis. Since we performed a non-clonal screen for homozygous viable adult animals, we were not able to isolate suppressors that might cause either lethality or sterility. Of the viable suppressors that we characterized, more than half were missense or nonsense alleles of tba-1 that either reverted tba-1(gf) function back to wild type or resulted in null mutation of tba-1. Multiple mutations in human α-tubulin have been implicated in neuronal disorders like lissencephaly and ALS, and their effects on MTs have only been studied using in vitro overexpression models [tba-1 alleles that we identified in this screen alter invariant residues adjacent to disease-linked mutations in the C-terminal H12, the GTP binding domain and the loop between H8 and S7 (annotated in in vivo.We had previously shown that  neurons . To idenn models –44. All tba-1(gf) dlk-1(0). On their own, none of these alleles had any effect on synapse formation or animal behavior. This might result from multiple factors. First, as was the case with motor proteins and their adaptors , the screen identified novel mutations that subtly altered protein function in such a way that embryonic development was completed and the animals were viable. Next, loss of function alleles in ttbk-3(ju978) behaved superficially wild type, likely because of the presence of other redundant kinases, which might also explain the incomplete suppression on tba-1(gf) dlk-1(0). Finally, any suppressors with strong behavioral deficits were likely missed since the design of our screen aimed to identify animals with wild type behavior first, and then assayed for restoration of DD synapse remodeling.The non-tubulin suppressor mutations displayed incomplete suppression of the synapse remodeling defects of tba-1(gf) dlk-1(0). We previously identified kinesin-1 mutations that increased motor motility, and here, we characterized dynein-dynactin mutations that could alter cargo binding, with both strategies increasing anterograde SV transport along the DD neuron commissure during remodeling. However, while there was an overall increase in bidirectional SV transport in unc-116(ju972) animals, dhc-1(ju1279) selectively increased anterograde SV transport. This likely reflects the distinct motor domains targeted by ju972 and ju1279 on kinesin and dynein, respectively, with MT-motor interactions affecting bidirectional transport and variations in motor-cargo binding biasing transport in the direction with more engaged motors. Our results thus highlight both the interdependence and the competition between anterograde and retrograde motors during bidirectional cargo transport in vivo. The combination of unc-116(ju972) and dhc-1(ju1279) had an additive effect on SV transport, with an increase in both the total and anterogradely moving vesicle pools. Interestingly, dhc-1(ju1279); unc-116(ju972) did not enhance the number of dorsal neurite synapses in tba-1(gf) dlk-1(0) compared to unc-116(ju972) alone (tba-1(gf) alone [A bottleneck to successful DD synapse remodeling is the motor dependent transport of SVs from the ventral to the dorsal neurite. Consistently, a majority of the suppressors altered either anterograde or retrograde motor function to compensate for a reduction in SV transport in 2) alone , indicatf) alone  could notba-1(gf) dlk-1(0) animals, the lack of sufficient SVs at sites of new synapse formation on the dorsal neurite resulted in their destabilization and subsequent elimination at the L4 stage [C. elegans member of the casein kinase superfamily, promotes synapse destabilization along the dorsal neurite, and loss of ttbk-3 was sufficient to maintain newly formed synapses in tba-1(gf) dlk-1(0). Synapse destabilization by TTBK-3 is likely phosphorylation dependent, and future studies will be required to identify potential substrates. The coiled-coil domain was also required for TTBK-3 function, leading us to speculate a possible regulatory role for the domain on kinase function. We previously showed that the initiation of dorsal neurite synapse formation was mediated by dlk-1 at the L2 stage; however ttbk-3 does not appear to play a role at this stage [ttbk-3 is also not involved in ventral neurite synapse elimination during DD neuron remodeling, which is complete by L3 stage [Studies from various experimental models and cultured neurons indicate that nascent synapse stability is dependent on the transport of sufficient numbers of SVs to the site of new synapse formation –47. In tL4 stage . Here, wis stage . ttbk-3 L3 stage –13. Thes°C on NGM plates unless noted otherwise [Strains were maintained at 20therwise . Informadnc-4 and ttbk-3 constructs was amplified from purified genomic or cDNA by PCR using Phusion HF DNA polymerase (Finnzyme) . DNA for innzyme) , and subinnzyme)  and Pgcy°C overnight, and day 1 adults were imaged using a Zeiss LSM 710 confocal microscope. Animals were anaesthetized in 0.6 mM levamisole on 2% agar pads for image acquisition. Z-stacks were generated from slices of 0.6 μm thickness. Images were processed using Zen lite software. Synaptic puncta number was quantified manually using a Zeiss Axioplan 2 microscope equipped with Chroma HQ filters.L4 animals were cultured at 20°C) were collected for analysis, and anesthetized using 30 mM muscimol on 10% agarose pads. 4-D imaging was performed using a Yokogawa CSU-W1 spinning disc confocal head placed on a Leica DMi8 confocal microscope equipped with a piezo Z stage for fast Z- acquisition controlled using MetaMorph (Molecular Devices). The entire DD commissure was visualized in 5–6 slices and images were collected for a total of 20 frames. The resulting movies were analyzed using Metamorph to generate kymographs for analysis of number and direction of movement of synaptic vesicles.L2 stage animals  to generate kymographs for analysis.tba-1(gf) dlk-1(0) animals were mutagenized using Ethyl Methane Sulphonate (EMS) following standard procedures [tba-1(gf) by Sanger sequencing the genic region of tba-1 for any additional mutations. Two of the extragenic suppressors, ju972 and ju977, were determined to be extragenic and mapped to the gene unc-116 following whole genome sequence analysis by MAQGene [https://usegalaxy.org/) workflow, and the causative mutations were identified by linkage analysis of the suppression to the SNPs identified in the whole sequence analysis.ocedures . F2 anim MAQGene . The resPhsp-16.2TTBK-3-GFP; Pmyo-2 mCherry in the wild type and tba-1(gf) dlk-1(0); ttbk-3(0) backgrounds were selected by positive pharyngeal mCherry expression. L1, L2, L3, L4 and young adult animals were heat shocked at 34°C for 2 hours in an incubator. Heat shocked animals were maintained at 20°C after heat shock until they reached day 1 adulthood, when they were imaged using a Zeiss LSM 710 confocal microscope.Transgenic animals expressing Statistical analysis was performed using GraphPad Prism 5. Normal distribution of samples was tested using D'Agostino & Pearson omnibus normality test. Significance was determined using One way ANOVA followed by Tukey’s multiple comparison tests and two way ANOVA followed by Bonferroni posttests for multiple samples.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.S1 MovieSynaptic vesicles move bidirectionally along the DD commissure towards the ventral and dorsal neurites.(WMV)Click here for additional data file.S1 Figtba-1, with tba-1(ju89gf) and all the intragenic suppressors listed. Sequence alignment of parts of C.elegans, H. sapiens and M. musculus homologs of TBA-1. Sequence conservation of ju89, ju962, ju965, ju973 and ju987 is shown, as well as the location of various helices (H) and beta sheets (B). Also annotated are mutations linked to ALS (in green) and lissencephaly (in blue) disease phenotypes in patient samples. (B) Gene structure of tbb-2, with ju1535 and reference allele gk129 marked. Sequence alignment of parts of C.elegans, H. sapiens and M. musculus homologs of TBB-2, highlighting the conserved Proline that is altered in ju1535, which lies between H9 and H10 helices of tbb-2. (C) Structure prediction of C. elegans TBA-1 (based on PDB#4i4tc) modeled on SWISS-MODEL and rendered using PyMOL, with ju89 (G414R) and the various missense mutations identified during the suppressor screen are also marked. (D) Structure prediction of C. elegans TBA-1 and TBB-2 (based on PDB# 1JFF) modeled on SWISS-MODEL and rendered using PyMOL, with the position of the internal and external MT surface, and the positions of ju89 and ju1535 highlighted.(A) Gene structure of (PDF)Click here for additional data file.S2 FigC.elegans, H. sapiens and M. musculus, surrounding the conserved Proline residue that is mutated in ju1279. (B) Bright field images of various dhc-1 alleles  cultured at 25°C.  Representative kymographs of EBP-2 movement in the adult VNC of the respective genotypes. Red lines indicate EBP-2 comets moving in the anterograde direction.  Quantification of number of EBP-2 comets (D) and their direction of movement (E) for various genotypes. Data are mean ± SEM; n = number of animals (shown on (E)). Statistics: One-way ANOVA followed by Tukey’s posttest; ***p<0.001, n.s.-not significant. (G) Bright field images of various dnc-4 alleles  cultured at 25°C.(A) Alignment of sequences of dynein heavy chains from (PDF)Click here for additional data file.S3 Figflp-13-SNB-1-GFP (juIs137). Scale bar: 10 μm. (B) Quantification of synaptic puncta in the DNC of adult animals. Data are mean ± SEM; n>10 animals per genotype. Statistics: One-Way ANOVA followed by Tukey’s posttest; ***p<0.001, ns- not significant. (C) Representative images of synaptic puncta along the DNC imaged using Punc-25-SNB-1-GFP (juIs1). Scale bar: 10 μm. (D) Representative images of TTBK-3-GFP expression in the GABAergic D motor neurons (driven by the unc-25 promoter) in L3, L4 and adult animals. (E) Quantification of number of EBP-2 comets for various genotypes. Data are mean ± SEM; Statistics: One-way ANOVA followed by Tukey’s posttest; ***p<0.001, n.s.-not significant. (F) Representative images of young adult wild type animals carrying the Phsp-16.2-TTBK-3-GFP transgene, 4 hours after a 2hr heat shock period. Punctate structures are seen in the pharynx, some head neurons, the intestinal lumen, CAN neuron and a posterior intestinal cell. Scale bar: 10 μm.(A) Representative images of DD synapses along the DNC in adult animals using P(PDF)Click here for additional data file.S4 Figflp-13-SNB-1-GFP (juIs137)) in the DNC of L4 animals, anaesthetized using 30mM muscimol. Data are mean ± SEM; n = 10 animals per genotype. Statistics: Mann-Whitney test, p-values are displayed on graph. (C) Quantification of synaptic puncta in the DNC of adult animals. Data are mean ± SEM; n>10 animals per genotype. Statistics: One-Way ANOVA followed by Tukey’s posttest; ***p<0.001, ns- not significant. Quantification of SNB-1::GFP intensity (P(PDF)Click here for additional data file.


Notably, STAT5 interacted with minichromosome maintenance (MCM) complex, suggesting that STAT5 directly facilitates viral DNA replication by recruiting the helicase complex of the cellular DNA replication machinery to viral DNA replication centers. The FDA-approved drug pimozide dephosphorylates STAT5, and it inhibited B19V replication in ex vivo expanded human erythroid progenitors. Our results demonstrated that pimozide could be a promising antiviral drug for treatment of B19V-related diseases.Productive infection of human parvovirus B19 (B19V) exhibits high tropism for burst forming unit erythroid (BFU-E) and colony forming unit erythroid (CFU-E) progenitor cells in human bone marrow and fetal liver. This exclusive restriction of the virus replication to human erythroid progenitor cells is partly due to the intracellular factors that are essential for viral DNA replication, including erythropoietin signaling. Efficient B19V replication also requires hypoxic conditions, which upregulate the signal transducer and activator of transcription 5 (STAT5) pathway, and phosphorylated STAT5 is essential for virus replication. In this study, our results revealed direct involvement of STAT5 in B19V DNA replication. Consensus STAT5-binding elements were identified adjacent to the NS1-binding element within the minimal origins of viral DNA replication in the B19V genome. Phosphorylated STAT5 specifically interacted with viral DNA replication origins both  ex vivo expanded human EPCs; therefore, pimozide has the potential to be used as an antiviral drug for treatment of B19V-caused hematological disorders.Human parvovirus B19 (B19V) infection can cause severe hematological disorders, a direct consequence of the death of infected human erythroid progenitor cells (EPCs) of the bone marrow and fetal liver. B19V replicates autonomously in human EPCs, and the erythropoietin (EPO) and EPO-receptor (EPO-R) signaling is required for productive B19V replication. The Janus kinase 2 (JAK2)-signal transducer and activator of transcription 5 (STAT5) signaling plays a key role in B19V replication. Here, we identify that phosphorylated STAT5 directly interacts with B19V replication origins and with minichromosome maintenance (MCM) complex in human EPCs, and that it functions as a scaffold protein to bring MCM to the viral replication origins and thus plays a key role in B19V DNA replication. Importantly, pimozide, a STAT5 phosphorylation-specific inhibitor and an FDA-approved drug, abolishes B19V replication in  Erythroparvovirus of the Parvoviridae family  ATP. Each 20 μl binding reaction contained 3 μg/ml of poly dI-dC (MilliporeSigma).Electrophoretic mobility shift assay (EMSA) was performed as previously reported . Complem2O, and used for PCR or quantitative PCR (qPCR) analysis.Chromatin immunoprecipitation (ChIP) assay was performed essentially as described previously ,75 with Immunoprecipitated viral DNA from ChIP assay was subjected to PCR analysis using either F1 and R1 or F1 and R2 primers spanning the viral origin region: F1 (nt 5036–5053), 5’-CCT GCC CCC TCC TAT ACC-3’, R1 (nt 5308–5285), 5’-CAG GAA ATG ACG TAA TTG TCC GCC-3’, and R2 (nt 5393–5376), 5’-ACG TCA ACC CCA AGC GCT-3’. q-PCR analysis was done as described previously , using t+ EPCs were cultured in Wong expansion medium and were treated with pimozide at various concentrations on Day 7. After 48 hours, ≥3 × 104 cells from each well were cultured in semi-solid methyl cellulose-based medium for 10–12 days, at which time colony counts were assessed by someone who was blinded to the experimental conditions.The colony formation assay was performed with methyl cellulose-based medium  according to the manufacturer’s instructions, with modifications. Briefly, CD36Co-immunoprecipitation (Co-IP) assay was performed as previously described ,76. BrieThe following primary antibodies were purchased: mouse anti-STAT5 (cat# sc-74442), rabbit anti-STAT5 (cat# sc-835), anti-STAT5A (cat# sc-271542) and anti-STAT5B (cat# sc-1656), anti-BrdU (IIB5) (cat# sc-32323) were from Santa Cruz ; anti-MCM2 (cat# 12079), and anti-pSTAT5(Y694) (cat# 4322) were from Cell Signaling ; anti-STAT5a/b pan-specific antibody (cat # AF2168) and normal IgG rabbit (cat# AB-105-C) were from R&D Systems Inc ; anti-MCM5 antibody (cat# 2380–1) was from Epitomics ; anti-B19V capsid (cat# Mab8293) was from Millipore ; anti-BrdU (clone B44) was from BD ; and anti-β-actin (cat# A5441) was from Sigma; anti-MCM3 (cat# A300-124A), anti-MCM5 , and MCM7(cat#A300-128A) were from Bethyl Laboratories ; anti-ATM(pS1981) (cat#ab81292) were from Abcam ; and anti-ATR(pT1989) (cat#GTX128145) from GeneTex . Rat anti-NS1 polyclonal antibody was prepared in our lab as previously reported .Horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit secondary antibodies were purchased from Sigma, and fluorescein isothiocyanate (FITC)-, Texas Red-, and Dylight405-conjugated anti-mouse, anti-rat, and anti-rabbit secondary antibodies were all purchased from Jackson ImmunoResearch .Statistical analysis was performed using GraphPad Prism Version 7.0. Statistical significance was determined by using 1-way ANOVA analysis, followed by Tukey-Kramer post-test for comparison of three or more groups and unpaired (Student) t-test for comparison of two groups. Error bars show mean and standard deviation (Mean ± SD) unless otherwise specified.S1 Fig+ EPCs were treated with either DMSO or pimozide and then incubated with BrdU to perform a BrdU incorporation assay. (C) Results of a representative cell-cycle analysis. (D) Relative fold changes of the cell population in S-phase are shown with means and standard deviations, which were obtained from three independent experiments. P values are calculated using one-way ANOVA followed by Tukey-Kramer post-test, n.s. (P> 0.05) denotes no statistical significance.(A&B) Inhibition of B19V DNA replication. UT7/Epo-S1 cells were pre-incubated with DMSO or pimozide , 6 h prior to M20 transfection. Transfected cells were cultured under hypoxic conditions. (A) At 48 h post-transfection, cells were collected for Hirt DNA extraction. The DNA samples were subjected to Southern blotting with a B19V M20 probe (upper panel). Mitochondrial (mito) DNA was probed as a loading control (lower panel). (B) At 48 h post-transfection, cells were collected for Western blotting with anti-pSTAT5(Y694). The blot was reprobed for β-actin. (C&D) Evaluation of the effect of pimozide on cell proliferation. CD36(TIF)Click here for additional data file.S2 Fig(A) Southern blot analysis. UT7/Epo-S1 cells were incubated with either DMSO or STAT5-SH2 inhibitor  at 6 h prior to transfection, and then the cells were transfected with M20 DNA and cultured under hypoxic conditions. At 48 h post-transfection, cells were collected for Hirt DNA extraction. The DNA samples were subjected to Southern blotting with an M20 DNA probe. Mitochondrial (Mito) DNA was probed as a loading control (lower panel). (B&C) Evaluation of the effect of STAT5 inhibitor on cell proliferation. UT7/Epo-S1 cells were treated with either DMSO or STAT5-SH2i (at 250 μM or 500 μM), and then incubated with BrdU for BrdU incorporation assays. (B) Results of a representative cell-cycle analysis. (C) Relative fold changes of the cell population in S-phase are shown, with means and standard deviations shown. P values are calculated using one-way ANOVA followed by Tukey-Kramer post-test, compared with DMSO group. *** denotes P<0.001 and n.s. (P>0.05) for no statistical significance.(TIF)Click here for additional data file.S3 Fig(A&B). Differential expression of STAT5A and STAT5B. (A) Cell lysates of UT7/Epo-S1 and EPCs were subjected to Western blotting with STAT5A/B pan-specific, STAT5A-specific, or STAT5B-specifc antibodies. Asterisks indicate dimerized or degraded or non-specific protein bands. (B) Purified STAT5 of UT7/Epo-S1 cells was subjected to Western blotting with a STAT5A/B pan-specific antibody. (C) Both STAT5A and STAT5B interact with the MCM2 complex. UT7/Epo-S1 cells were collected and lysed with RIPA buffer. The lysates were incubated with an anti-MCM2 or control IgG antibody for co-immunoprecipitation (Co-IP). Immunoprecipitated proteins were blotted for the presence of STAT5A, STAT5B, and MCM2 with anti-STAT5A, anti-STAT5B, and anti-MCM2 antibodies, respectively. The precipitated IgG heavy chain is also shown.(TIF)Click here for additional data file.S4 FigOri) at the left ITR (L-ITR). The starting nucleotide numbers of both forward and reverse (F and R) and the location of the probe are indicated. (C) UT7/Epo-S1 cells were treated with either DMSO, STAT5-SH2i inhibitor (at 500 μM) or pimozide (at 15μM), as indicated in the figure, at 6h prior to transfection. Then, the cells were transfected with M20 and cultured under hypoxic conditions for 48 h. Cells were collected for ChIP analysis using an anti-STAT5 and Ori-qPCR. Error bars represent standard deviation taken from at least three experiments. P values were calculated using one-way ANOVA followed by Tukey-Kramer post-test, compared with the M20 group. **** denotes P<0.0001. (D) Mock- or M20- transfected UT7/Epo-S1 cells, cultured under hypoxic conditions for 48 h, were collected for ChIP assay using an anti-STAT5 antibody, followed by PCR using primers: forward , 5’- GGA CTG TAG CAG ATG AAG AGC T-3’, and reverse , 5’-GTG GCC CCC TCA CTC CAC AT-3’, primes as indicated in the diagram. Rabbit IgG was used as a negative control of pull-down, and M20 DNA was used as PCR positive control.(A) UT7/Epo-S1 cells were transfected with M20 and allowed to replicate for 48 h under hypoxic conditions. Cells were collected for ChIP analysis. Anti-MCM2, anti-MCM3, anti-MCM5, anti-MCM7, and control IgG antibodies were used to pull down DNA-protein complex. Recovered DNA was analyzed by qPCR targeting the viral origin (Ori-qPCR). Error bars represent standard deviations taken from at least three experiments. P values were calculated using a Student’s t test, compared to the IgG control. ** P<0.01; * P<0.05. (B) A diagram of the Ori-qPCR amplicon targeting the viral replication origin ((TIF)Click here for additional data file.S5 Fig+ EPCs were treated with pimozide (at 15 μM). At 3 h post-treatment, cells were incubated with hydroxyurea at 10 mM for 24 h under hypoxic conditions. Then, the same numbers of the cells were collected for Western blot analysis of proteins, as indicated, using anti-ATM(pST1981), anti-ATR(pT1989), pSTAT5, and β-actin, respectively. (B) Mock and B19V-infected CD36+ EPCs, cultured under hypoxic conditions, were used for Western blot analysis of pSTAT5. The membrane was reprobed for β-actin.(A) CD36(TIF)Click here for additional data file.S6 Fig(A) A diagram of lentivirus Lenti-ATF/P6-GFP. The virus was made as we reported previously . (B) UT7(TIF)Click here for additional data file.S7 Fig+ EPCs were pre-incubated with DMSO or pimozide (at 15 μM), STAT5-SH2i (at 250 μM), or pimozide plus STAT5-SH2i , 6 h prior to B19V infection under hypoxic conditions. (A) At 48 h post-infection, cells were subjected to flow cytometry analysis using anti-B19V capsid antibody. Error bars represent standard deviation taken from at least three experiments. P values are calculated using one- way ANOVA followed by Tukey-Kramer post-test, compared with DMSO group. **** denotes P<0.0001. (B) Similarly, at 48 h post-treatment, uninfected cells were labeled with BrdU for cell cycle analysis. Numbers shown are percentages of cells at G1, S, and G2 phase, respectively.CD36(TIF)Click here for additional data file.


Drosophila tumour model, we show that Egfr cooperates with oncogenic Ras via Arf6, which functions as a novel regulator of Hh signalling. Oncogenic Ras induces the expression of Egfr ligands. Egfr then signals through Arf6, which regulates Hh transport to promote Hh signalling. Blocking any step of this signalling cascade inhibits Hh signalling and correspondingly suppresses the growth of both, fly and human cancer cells harbouring oncogenic Ras mutations. These findings highlight a non-canonical Egfr signalling mechanism, centered on Arf6 as a novel regulator of Hh signalling. This explains both, the puzzling requirement of Egfr in oncogenic Ras-mediated overgrowth and the cooperation between Egfr and Hedgehog.Multiple signalling events interact in cancer cells. Oncogenic Ras cooperates with Egfr, which cannot be explained by the canonical signalling paradigm. In turn, Egfr cooperates with Hedgehog signalling. How oncogenic Ras elicits and integrates Egfr and Hedgehog signals to drive overgrowth remains unclear. Using a  Drosophila tumour model the authors demonstrate that depletion of Arf6, a Ras-related GTP-binding protein activated by EGFR, supresses oncogenic Ras driven overgrowth via modulation of Hedgehog signalling.EGFR signalling is required for oncogenic Ras driven tumorigenesis. In this study, using a  Ras gene are highly prevalent in human cancers and give rise to some of the most aggressive tumours13457Activating mutations of the On the other hand, Egfr has been shown to cooperate with Hedgehog (Hh) signalling, another oncogenic pathway, to drive basal cell carcinoma and melanomas91011Using a fly tumour model of oncogenic Ras we have identified Egfr as a positive regulator of oncogenic Ras-mediated overgrowth. Our characterization of Egfr's role in oncogenic Ras-mediated overgrowth led the finding that oncogenic Ras signalling stimulates the expression of the Egfr ligand spitz (spi) to recruit Egfr signalling and achieve tumour overgrowth. Egfr promotes tumour overgrowth independent of the canonical the Sos/Ras signalling, instead it acts via the ADP-Ribosylation Factor 6 (Arf6). Arf6 belongs to a family of highly conserved small Ras-related GTP-binding proteins and is largely known for its role in regulating endocytosis, vesicle transport and secretion1415161718192021Ras (RasV12) gives rise to hyperplastic tumours in Drosophila tissues2325RasV12 tumour overgrowthEgfr mutation EgfrCo  assays. We did not detect ectopic cell death inside RasV12, Egfr_ double mutant clones compared with RasV12 clones  and spitz (Spi)3030RasV12 clones  in RasV12 cells and scored clone sizes compared with RasV12 clones. Similar to Egfr_, the spi_ mutation produced small clones and suppressed the overgrowth phenotype of RasV12  family of Egfr ligands in cancer cells42 clones , an anti2 clones . This arates Spi . Next, wf RasV12 . In addifunction . This alergrowth . Moreovesla ref. . We examergrowth . CollectRasV12 tumour overgrowth. While the expression of Sos-RNAi caused a small eye phenotype in adult animals, consistent with Egfr/Ras signalling inhibition   ref. , produce0A) ref.  but failhenotype . Taken tDrk and Arf6) by RNAi and asked whether this suppress oncogenic Ras-mediated tumour overgrowth, and identified Arf6 as a mediator of Ras tumour overgrowth. Arf6 belongs to a family of highly conserved small Ras-related GTP-binding proteins and mediates Egfr signalling in mammals and flies153738RasV12 tumour overgrowth . In addition, Egfr directly recruits Arf6 GEFs to stimulate Arf6  in complementing biochemical and immunostaining experiments. Using antibody against active Ci, we found that Ci protein levels were elevated in lysates derived from dissected RasV12 discs compared with controls (RasV12 mosaic tissues against Ci. In wild-type discs ci is suppressed posteriorly while Hh activates ci anteriorly (RasV12 clones and this upregulation was more pronounced in larger clones (see discussion)  showed similar effects  of the developing wing disc where it induces Hh expression but represses cici and patched (ptc) to control wing size and patterning43Arf6-RNAi clones in developing eye and wing discs, examined Hh signalling in the Hh responsive anterior cells, and found that Arf6-RNAi suppressed Hh signalling in both tissue types  and asked whether this upregulates Hh. Posteriorly produced Hh normally activates ptc in a 8–10 cells diameter domain along the A/P boundary. Consistent with enhanced Hh signalling, ap-Gal4>UAS-ARF6 discs showed elevated Ptc protein levels and an expansion of the Ptc domain specifically in the apterus-dorsal portion of the disc  , a processes regulated by Hh signallingAL4>GFP) . Taken tRasV12 clones induced in tissues heterozygotes for a hypomorphic allele of Hh, Hhfse  in cells expressing either RasV12 alone or co-expressing Arf6-DN or Arf6-RNAi, and stained tissues against Arl8. We found that Hh-GFP preferentially localizes to clusters of Arl8-positive vesicles in RasV12, Arf6-DN tissues   . Moreovendosomes . Furtherndosomes . Consistng cells . TogetheDrosophila tumour model we found that oncogenic Ras transcriptionally stimulates the TGF-α Egfr ligand spitz to recruit Egfr signalling and increase tumour growth. Egfr's role is likely tissue and/or context-dependent as inhibition of Egfr shows variable effects in different cancer types45RasV12 appeared to cause a non-autonomous growth effect  y w; FRT82B; (2) y w; FRT82B, UAS-RasV12/TM6B; (3) EgfrTop−CA ; (4) UAS-DER(DN) ; (5) UAS-spi-RNAi (VDRC # 3922); (6) cn1 Egfrtslabw1/TTSTL, CyO: TM6B, Tb1(BDSC# 6501);(7) FRT40, rasc40A ;UAS-ARF6-RNAi ; (8) UAS-loner-RNAi (VDRC #106168 and BDSC#39060); (9) UAS-ci-RNAi (BDSC# 28984); (10) spi, FRT40A/CyO (BDSC#114338); (11) yw,ey-Flp1;act>y+>GAL4,UAS-GFP.S65T;FRT82B,tub-GAL80; (12) Hhfse (BDSC# 35562); (13)yw,ey-Flp1;act>y+>GAL4,UAS-myrRFP;FRT82B,tub-GAL80; (14) yw,ey-Flp1; FRT42D,tub-GAL80; act>y+>GAL4,UAS-GFP; (15) UAS-ARF6-DN ; (16)yw,ey-Flp1;tub-GAL80,FRT40A;act>y+>GAL4,UAS-GFP.S65T; (17) Star-RNAi (BDSC# 38914); (18) Sos-RNAi (BDSC#31174); (19) Arf6-GFP line is a previously generated and functionally validated GFP knock-in line54Arf6 gene is under endogenous control and a GFP is inserted in frame at the carboxyl terminus of Arf6 protein; (20) UAS-steppke-RNAi (BDSC#32374); (21) UAS-Hh-GFP and UAS-Hh-N-GFP, a gift from A. O'Reilly ; (22) yw, hsp70-Flp; act-y+-GAL4, UAS-GFP. See e BDSC# 362; (13)yClones. Clones were generated using standard MARCM techniquesey-FLP MARCM system, to the exception of hs-Flp. For these particular experiments, 2 days larvae were heat-shocked for 1 h at 37 °C and raised for two days at 25 °C before processing. Images presented in Apoptag Red kit from Chemicon.Eye-antenna discs were dissected, fixed and stained as described previously25RasV12 single or EgfrTop−CA, RasV12 or DerDN(EGFR-DN), RasV12 double mutant clones were homogenized in lysis buffer (50 mM HEPES (pH 7.5), 150 mM KCl, 5 mM MgCl2, 0.01% Triton-X) supplemented with protease inhibitor tablets; Roche). Global protein concentrations was normalized across all genotypes and the lysate was pre-cleared with protein agarose-A beads for 1 h at 4 °C, incubated either with 2 μl of anti-Arf6  for 4 h at 4 °C. Lysates were then incubated with protein agarose A beads for 1 h at room temperature. For pull downs, beads were precipitated and washed three times in modified lysis buffer containing 0.5% TritonX-100. Samples were run on SDS–polyacrylamide gel electrophoresis, transferred on nitrocellulose membrane, and blotted with anti-Hh  and anti-αTubulin (Sigma), loading control. For cancer cell lines, cells were lysed in lysis-loading buffer (0.5 M Tris (pH 6.8), 20% SDS, Glycerol, 10% β-mercaptoethanol and Bromophenol Blue), homogenized further by hydrodynamic shearing  or anti-Arf6  or anti β-actin  antibodies. Full images of the blots are presented in For immunoprecipitation experiments, imaginal discs from 50 third-instar animals carrying Rp49 as endogenous control. The following primers were used: Rp49, 5′-GGCCCAAGATCGTGAAGAAG-3′ and 5′-ATTTGTGCGACAGCTTAGCATATC-3′; Spitz, 5′-CGCCCAAGAATGAAAGAGAG-3′ and 5′-AGGTATGCTGCTGGTGGAAC-3′Total RNA from Eye-antenna imaginal discs containing wild-type or mutant clones was isolated using a Trizol RNA extraction method (Invitrogen). The SuperScriptIII First-Strand Synthesis System (Invitrogen) kit was used to synthesize complementary DNA. Real-time PCR was carried out on an Applied Biosystems machine using the SYBR green fast kit (Applied Biosystems) following the manufacturer's instructions. Relative gene expression was obtained from triplicate runs normalized to Clones of mutant cells in the eye-antennal discs were generated using standard MARCM system5 cells were seeded onto 6-well-plates and treated with 2 nM of either ARF6 SiRNA  or universal control scrambled  and supplemented with 5 μl of DhamaFect 2 (GE- Dharmacon).In all, 2 × 105 and grown overnight before GANT61  treatment at the final concentration of 10 μm ml−1. Cell numbers were scored 96 h after GANT61 treatment.Cells were seeded onto 6-well-plates at a density of 2 × 10All data supporting the findings of this study are available within the article and its How to cite this article: Chabu, C. et al. EGFR/ARF6 regulation of Hh signalling stimulates oncogenic Ras tumour overgrowth. Nat. Commun.8, 14688 doi: 10.1038/ncomms14688 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures and Supplementary Note.


Letrozole decreased IGF-1R and IRS-1/2 tumor levels. These data suggest that ER drives PI3K/AKT activation in response to mTORC1 inhibition, providing molecular rationale for therapeutic combinations of anti-estrogens and mTORC1 inhibitors in endocrine-sensitive disease.The mTORC1 inhibitor RAD001 (everolimus) is approved for treatment of recurrent/metastatic estrogen receptor (ER)-positive breast cancer in combination with the aromatase inhibitor (AI) exemestane. The benefits of A) continued anti-estrogen therapy for anti-estrogen-resistant disease in the context of mTORC1 inhibition, and B) adjuvant everolimus in combination with anti-estrogen therapy for early-stage disease are being tested clinically, but molecular rationale remains unclear. We hypothesized that mTORC1 inhibition activates the IGF-1R/InsR/IRS-1/2 axis in an ER-dependent manner to drive PI3K/AKT and promote cancer cell survival, implicating ER in survival signaling induced by mTORC1 inhibition. Anti-estrogen treatment synergized with RAD001 to inhibit ER+ breast cancer cell growth. Inhibition of ER, IGF-1R/InsR, or IRS-1/2 suppressed AKT activation induced by mTORC1 inhibition. RAD001 primed IGF-1R/InsR for activation, which was enhanced by ER signaling. Post-menopausal patients with early-stage ER+ breast cancer were treated presurgically +/− the AI letrozole. Viable tumor fragments from surgical specimens were treated with RAD001 and/or OSI-906  Approximately 70% of primary breast tumors express estrogen receptor α (ER) and/or progesterone receptor (PR). Patients with such hormone receptor-positive breast cancers are typically treated with adjuvant anti-estrogen therapies such as tamoxifen or aromatase inhibitors (AIs) for 5-10 yrs after surgical removal of the primary tumor , 3.e.g., letrozole, anastrozole), treatment with the steroidal AI exemestane plus the mTORC1 inhibitor everolimus  significantly improved median progression-free survival  compared to exemestane/placebo (11 vs. 4.1 months) in the BOLERO-2 study . The first 10 patients received no presurgical anti-cancer therapy (Arm A), and an interim analysis was performed. The next 7 patients were presurgically treated with the AI letrozole for 10-21 d (Arm B). Primary tumors were surgically resected as standard of care, and tumor fragments for research use were then dissected from the surgical specimen within 1.5 h of removal from the patient. Tumor fragments were transported in serum-free DMEM on ice. Using a 1-mm biopsy punch device (Miltex), ~40 viable tumor cores were dissected from each tumor specimen within 30 min. Tumor cores (~10 per treatment group) were cultured S473, P-AKTT308, AKT, P-p70S6KT389, P-S6S240/244, P-IGF-1RβY1135/6/P-InsRβY1150/1, IGF-1Rβ, InsRβ, IRS-1, IRS-2, actin, vinculin , p85 (Abcam), or ER (Santa Cruz). HRP-labeled secondary antibodies  and ECL substrate (Pierce) were used for signal detection. Densitometry analysis of immunoblot film was performed using ImageJ software, and relative signal values were analyzed by ANOVA followed by Bonferroni multiple comparison-adjusted post-hoc test between groups.Immunoblotting of lysates from cells and frozen tumor fragments was performed as described . ImmunopFive-micron sections of formalin-fixed, paraffin-embedded (FFPE) tumor tissues acquired from patients at baseline (diagnostic biopsy) and at the time of surgery  were used for IHC staining with antibodies against Ki67 , ER , PR , or IGF-1R . IHC evaluation was performed by a breast pathologist blinded to treatment arm; details are provided in Supplementary Information. In Arm A, two patients had bilateral tumors, and two patients had two ipsilateral tumors; all were analyzed by IHC.