Giardia lamblia and enteroaggregativeEscherichia coli (EAEC), we provide new insights intointersecting pathogen-specific mechanisms that enhance malnutrition. We show forthe first time that during protein malnutrition, the intestinal microbiotapermits persistent Giardia colonization and simultaneouslycontributes to growth impairment. Despite signals of intestinal injury, such asIL1α, Giardia-infected mice lack pro-inflammatory intestinalresponses, similar to endemic pediatric Giardia infections.Rather, Giardia perturbs microbial host co-metabolites ofproteolysis during growth impairment, whereas host nicotinamide utilizationadaptations that correspond with growth recovery increase. EAEC promotesintestinal inflammation and markers of myeloid cell activation. Duringco-infection, intestinal inflammatory signaling and cellular recruitmentresponses to EAEC are preserved together with aGiardia-mediated diminishment in myeloid cell activation.Conversely, EAEC extinguishes markers of host energy expenditure regulatoryresponses to Giardia, as host metabolic adaptations appearexhausted. Integrating immunologic and metabolic profiles during co-pathogeninfection and malnutrition, we develop a working mechanistic model of howcumulative diet-induced and pathogen-triggered microbial perturbations result inan increasingly wasted host.Diverse enteropathogen exposures associate with childhood malnutrition. Toelucidate mechanistic pathways whereby enteric microbes interact duringmalnutrition, we used protein deficiency in mice to develop a new model ofco-enteropathogen enteropathy. Focusing on common enteropathogens inmalnourished children,  Giardia lamblia and enteroaggregative Escherichiacoli in mice fed a protein deficient diet, we identify host growthand intestinal immune responses that are differentially mediated bypathogen-microbe interactions, including parasite-mediated changes in intestinalmicrobial host co-metabolism, and altered immune responses during co-infection.Our data model how early life cumulative enteropathogen exposures progressivelydisrupt intestinal immunity and host metabolism during crucial developmentalperiods. Furthermore, studies in this co-infection model reveal new insightsinto environmental and microbial determinants of pathogenicity for presentlycommon, but poorly understood enteropathogens like Giardialamblia, that may not conform to existing paradigms of microbialpathogenesis based on single pathogen-designed models.Malnourished children are exposed to multiple sequential, and oftentimes,persistent enteropathogens. Intestinal microbial disruption and inflammation areknown to contribute to the pathogenesis of malnutrition, but how co-pathogensinteract with each other, with the resident microbiota, or with the host toalter these pathways is unknown. Using a new model of enteric co-infection with Escherichia coli and theoftentimes persistent protozoan Giardia lamblia among the mostcommonly detected  and the EAECaap gene (forward 5’-CTTGGGTATCAGCCTGAATG-3’ and reverse5’-AACCCATTCGGTTAGAGCAC-3’ primers) for EAEC detection are described elsewherepropionate (TSP). Samples were mixed by vortex and spun  for 10 minutes before transfer to a 1.7 mm NMR tube.Spectroscopic analysis was carried out on a 700 MHz Bruker NMR spectrometerequipped with a cryo-probe. Standard one-dimensional 1H NMR spectraof the urine samples were acquired with water peak suppression using a standardpulse sequence. For each sample, 8 dummy scans were followed by 128 scans andcollected in 64K data points. A recycle delay of 2 s, a mixing time of 10 μs andan acquisition time of 3.8 s was used. The spectral width was set at 20 ppm.Chemical shifts in the spectra were referenced to the TSP singlet at δ 0.0.Spectra were manually phased and corrected for baseline distortions.1H NMR spectra (δ 0.2–10.0) were digitized into consecutiveintegrated spectral regions  of equal width (0.00055 ppm). The regionsbetween δ 4.50–5.00 were removed in order to minimize the effect of baselineeffects caused by imperfect water suppression. Each spectrum was then normalizedto unit area. Multivariate modeling was performed in Matlab using in-housescripts. This included principal components analysis (PCA) using pareto scalingand orthogonal projection to latent structures-discriminant analysis (OPLS-DA)constructed using unit variance scaling. OPLS-DA models were constructed toassist model interpretation. Here, 1H NMR spectroscopic profiles wereused as the descriptor matrix and class membership was usedas the response variable. The predictive performance (Q2Y) of themodel was calculated using a 7-fold cross validation approach and model validitywas established by permutation testing (1000 permutations) [R) with the class membership and summarized in aheat map.Urine samples were on individual mice at each timepoint indicated. Samplecollections occurred regularly at 2 pm on each collection data. Urines wereplaced immediately on ice, and stored at -80°C before shipping on dry ice to JMPand JS. Urines were then analyzed individually by tations) . MetabolFlow cytometry of lamina propria cells was performed according to our previouslypublished protocols .For isoFor mucosal cytokine and chemokine responses, 0.5–1.0 cm of ileum wereimmediately placed in liquid nitrogen at the time of euthanasia and stored at-80°C until use. Protein was collected from ileum lysates, which were made usinga lysis buffer containing 50 mM HEPES, 1% Triton X-100, and Halt proteaseinhibitor on ice and homogenized in Zirconia beads (Biospec) using aMini-Beadbeater (Biospec) for 60 seconds. Clarified supernatants were stored at-80°C. Multiplex protein quantification was performed using Luminex 100 ISSystem at the University of Virginia Biomolecular Core facility.Markers of inflammation were measured in the cecal contents or stool usingmethods described previously , 16.Brit tests were used for parametrically distributed data andMann-Whitney tests for non-parametrically distributed data where applicable.Differences were considered significant at P<0.05. Data arerepresented as means ± standard errors of the mean.Data analyses were performed with GraphPad Prism 6.0 and 7.0 software. Allstatistical analyses were done from raw data with the use of One-way and Two-wayanalysis of variance with Dunn’s or Bonferroni post hoc analysis frommulti-group comparisons. For comparisons between only two groups, StudentS1 FigGiardia burden in mice fed CD 35 daysafter 106G. lamblia H3 cyst challenge. Micereceived Abx beginning 8 days prior to challenge and through 21 dayspost-challenge. B) Giardia stool shedding (28 through 42days post-infection) by 18S small ribosomal subunit qPCR in weaned mice fedcontrol diet (CD) (left) or protein deficient diet (PD) (right) receivingcontinuous antibiotics  in drinkingwater beginning 8 days prior to 106G. lamblia H3 cyst challenge. C)Comparison of G. lamblia H3 colonizationin duodenum 7 days following challenge with either axenized trophozoites(107/100 mcl) or gerbil-passaged purified cysts(106/100 mcl) in mice fed PD with or without antibiotics asdetermined by light microscopy (left) and in a subset comparingquantification by light microscopy and qPCR (right). * = P<0.05 asindicated; *** = P<0.001 for % infectivity trophozoites  vscysts . D) Ratio of Firmicutes:Bacteroidetes (F:B) in smallintestine of mice fed either CD or PD after 20 days on respective diets.. E) Impact of continuous antibiotics onfecal (left) and duodenal (right) 16S V3-V4 community taxa in mice fed PDdiet with and without Giardia infection through 15 dayspost-challenge. Only high abundance  taxa are showndue to high-proportion low-abundances of unassigned taxa inantibiotic-treated animals (see A) Small intestinal mals see . *P<0.05(PDF)Click here for additional data file.S2 Fig(A) Flow cytometry of ileum lamina propria leukocytes (LPL) day 17post-challenge in PD-fed mice. (B) Representative chemokine and cytokineproteins (by Luminex) secreted in ileum of mice fed PD. n = 6-12/group,*P<0.05 as indicated.(PDF)Click here for additional data file.S3 FigGiardia (d7 (row a) and d13 post-challenge (rowb)), EAEC ), or both (rows d-f) on 16S V3-V4 fecalbacterial community during protein deficiency. Sub-phyla level OTU relativeabundances as indicated. *P<0.05 for genus-level abundances highlightedin green  or red  in the table. OTUs included in statisticaltests were filtered by two criteria: presence in at least two samples in thedataset, and total relative abundance across all samples > 0.5%. B-D) PCAscore plots (left) and OPLS-DA correlation coefficient plots (right)indicating the differences between: B) Mice at day 7 post infection withEAEC  versus age and diet-matcheduninfected controls; D) Mice infected with Giardia (d13)versus age and diet-matched co-infected mice. E-G) Giardiafacilitates catch-up growth in mice fed a protein deficient diet. E) Growthin mice fed protein-deficient diet (PD) through 42 days afterG. lamblia H3 cyst (106)infection. F) Growth as % initial weight after transitioning mice from PD tocontrol diet (day 0 on x-axis) and through 25 days post-refeeding(*P<0.05). C) Persistence of Giardia in feces and smallintestine after re-feeding (D42 = day of re-feeding). (n = 2-4/group).A) Effect of (PDF)Click here for additional data file.S1 Table(PPTX)Click here for additional data file.


Scientific Reports 10.1038/s41598-017-15342-4, published online 13 November 2017Correction to: The Acknowledgements section in this Article is incomplete.“We thank Yasamin Dabiri for her support to prepare Stat3 plasmids. This work was supported by Deutsche Forschungsgemeinschaft (CH 1690/2-1) and the BMBF grant programs Drug-iPS (FKZ 0315398B) and SysToxChip (FKZ 031A303E).”should read:“We thank Yasamin Dabiri for her support to prepare Stat3 plasmids. This work was supported by Deutsche Forschungsgemeinschaft (CH 1690/2-1) and the BMBF grant programs Drug-iPS (FKZ 0315398B) and SysToxChip (FKZ 031A303E).We acknowledge the financial support of the Deutsche Forschungsgemeinschaft and Ruprecht-Karls-Universität Heidelberg within the funding programme Open Access Publishing.”


Xanthomonas oryzaepv. oryzae(Xoo). We previously reported that XA21 is cleaved in transgenic plants overexpressing XA21 with a GFP tag (Ubi-XA21-GFP) and that the released C-terminal domain is localized to the nucleus. XA21 carries a predicted nuclear localization sequence (NLS) that directs the C-terminal domain to the nucleus in transient assays, whereas alanine substitutions in the NLS disrupt the nuclear localization.The rice receptor kinase XA21 confers robust resistance to the bacterial pathogen in planta, we generated transgenic plants overexpressing an XA21 variant carrying the NLS with the same alanine substitutions (Ubi-XA21nls-GFP).To determine if the predicted NLS is required for XA21-mediated immunity Ubi-XA21nls-GFP plants displayed slightly longer lesion lengths, higher Xoobacterial populations after inoculation and lower levels of reactive oxygen species production compared with the Ubi-XA21-GFP control plants. However, the Ubi-XA21nls-GFP plants express lower levels of protein than that observed in Ubi-XA21-GFP.These results demonstrate that the predicted NLS is not required for XA21-mediated immunity. Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of blight disease in rice (required for activation of XA21-mediated immunity X) derived from Xoo. Tyrosine sulfation is required for RaxX’s activity. In rice plants expressing XA21, sulfated RaxX activates XA21-mediated immune responses, including production of reactive oxygen species (ROS), ethylene, and induction of defense gene expression (Pattern recognition receptors (PRRs) recognize conserved microbial signatures, activating immune responses . The ric in rice . XA21 repression .Ubi-Myc-XA21 transgenic plants and a 70-kDa carboxy-terminal cleavage product was observed in Ubi-XA21-CFP and Ubi-XA21-GFP plants . The Ubi-XA21nls-GFP transgenic lines displayed partial resistance to Xoo infection, as reflected in slightly longer lesion lengths and higher Xoo bacterial populations compared with Ubi-Xa21-GFP control plants. A tyrosine-sulfated 21-amino acid derivative of RaxX (RaxX21-sY) triggered ROS production in Ubi-XA21nls-GFP plants. The expressed protein levels of XA21nls-GFP in Ubi-XA21nls-GFP lines were lower than those observed in Ubi-XA21-GFP plants. These results suggest that lower levels of expressed protein account for the slightly longer lesions observed in Ubi-XA21nls-GFP transgenic plants and indicate that the predicted NLS is not critical for XA21-mediated immunity.To assess the biological relevance of the predicted XA21 nuclear localization sequence Oryza sativa ssp. japonica cultivar Kitaake and a transgenic line expressing XA21 , were used in this study. The Kitaake genetic background lacks XA21 and is fully susceptible to the Xoo strain used in this study. Rice seeds were surfaced-sterilized with 15% bleach, rinsed with water, and soaked in water for one week. Well-germinated seedlings were transplanted into a soil mixture  in an environmentally-controlled greenhouse.ing XA21 , Ubi-XA2XA21nls-GFP (KRTKK678-682AATAA) construct was generated using site-directed mutagenesis. The DNA sequence containing alanine substitutions were amplified from an XA21-GFP/pENTR vector  Philippines race 6 PXO99A (referred to as PXO99) was used in this study. Xoo was cultured on peptone sucrose agar plates supplemented with 20 mg L−1 cephalexin for two days, then washed off and re-suspended in sterilized water. The concentration of bacterial suspension was adjusted to an OD600 of 0.5 (approximately 1 × 108 cfu/mL) for inoculation.Xoo inoculum. The lesion lengths were measured 14 days post-inoculation. In planta bacterial growth was assessed as described previously  to acclimate the chamber conditions for one week. Plants were inoculated using the scissor clipping method . For eaceviously . BrieflyProtein extraction from rice leaves and western blot assays were performed as previously described . Briefly2 pieces and floated on water overnight. Leaf pieces were treated with water, 1 µM nonsulfated 21-amino acid synthetic RaxX peptides (RaxX21-Y) or tyrosine sulfated RaxX21 peptides (RaxX21-sY). For each treatment, four biological replicates were included and in each replicate two leaf pieces were used. Chemiluminescence was recorded every 30 s for 3 h in a high-sensitivity TriStar plate reader .ROS assays were performed as previously described . Fully-eUbi-XA21nls-GFP lines were obtained, and three lines that expressed detectable full-length XA21nls-GFP protein were selected for further analysis. We observed reduced gene and protein expression levels of XA21-GFP in these transgenic lines  as compared with those in the Ubi-XA21-GFP control plants  were assessed for resistance to Xoo. Genotyping revealed that all three Ubi-XA21nls-GFP lines segregated for the transgene in the T1 generation . Five-week-old Kitaake, Ubi-XA21-GFP and Ubi-XA21nls-GFP plants were inoculated with PXO99. Fourteen days after inoculation, Kitaake plants developed long water-soaked lesions typical of the disease (15.6 ± 2.5 cm), whereas Ubi-XA21-GFP developed very short lesions (3.4 ± 1.2 cm)   and 2C.  in #7–3 . The les Kitaake . A seconransgene , which iransgene .Xoo growth in planta, the same plants from the first inoculation experiment were used for a bacterial growth assay. Inoculated leaves were collected 0, 4, 8 and 12 days post-inoculation (dpi). As shown in 8 cfu/leaf at 4 dpi, whereas the populations in Ubi-XA21-GFP and Ubi-XA21nls-GFP plants reached over 107 cfu/leaf. At 8 dpi, the Xoo population grew in Ubi-XA21nls-GFP plants but remained unchanged in the Ubi-XA21-GFP control plants. At 12 dpi, the bacterial populations in three Ubi-XA21-GFP transgenic lines were significantly higher than the Ubi-XA21-GFP plants but lower than that in the Kitaake plants , that the released GFP tagged C-terminal domain is localized to the nucleus and that a predicted NLS directs this domain to the nucleus in transient assays (Ubi-XA21nls-GFP) to Xoo.We previously reported that XA21 is cleaved in transgenic plants overexpressing XA21 with a GFP tag , and T1 progeny derived from three independent Ubi-XA21nls-GFP T0 lines . The inoculation experiments were carried out separately on 8/19/14 (upper panel) and 9/23/14 (lower panel). The genotyping results of Ubi-XA21nls-GFP plants were shown in the gel picture under the columns (segregants represented by closed columns and null segregants by open ones). Bars represent mean ± SD from about six leaves in each plant. The statistical analysis was performed using Tukey’s honestly significant difference test to compare the lesion lengths between groups. In the upper panel, Kitaake, null segregants from XA21nls line #1 and #2 are in group “a”, XA21 in “b”, #1 segregants in “c”, and #2 segregants in “bc”; in the lower panel, Kitaake is in group “a”, XA21in “b”, XA21nls#7 segregants in “b”, and null segregants in “c” (α < 0.05). The lesion lengths on the T1 progeny from Ubi-XA21nls-GFP line #7 were comparable with those on Ubi-XA21-GFP, due to the Ubi-XA21nls-GFP transgenic plants slightly stressed as reflected in browning at leaf tips.Lesion lengths and genotyping results of Kitaake, Click here for additional data file.10.7717/peerj.2507/supp-2Figure S2Ubi-XA21-GFP (XA21) and T2 progeny derived from three Ubi-XA21nls-GFP T1 lines  were measured 14 days post-inoculation. The genotyping results were shown in the gel pictures under the columns (segregants represented by closed columns and null segregants by open ones). Bars represent mean ± SD from about six leaves. The statistical analysis was performed using Tukey’s honestly significant difference test to compare the lesion lengths between groups. Kitaake is in group “a”, XA21 in “b”, Ubi-XA21nls-GFP line #1-4 segregants and null segregants in “d” and “ac”, line #2–1 segregants and null segregants in “d” and “c”, and line #7-10 segregants and null segregants in “e” and “ac”, respectively (α < 0.05).Leison lengths of Kitaake, Click here for additional data file.10.7717/peerj.2507/supp-3Table S1Click here for additional data file.


In vivo, oral administration of XL388 inhibited in nude mice 786-0 RCC tumor growth, and its anti-tumor activity was sensitized with co-administration of the MEK-ERK inhibitor MEK162. Together, these results suggest that concurrent inhibition of mTORC1/2 by XL388 may represent a fine strategy to inhibit RCC cells.XL388 is a mammalian target of rapamycin (mTOR) kinase inhibitor. We demonstrated that XL388 inhibited survival and proliferation of renal cell carcinoma (RCC) cell lines (786-0 and A549) and primary human RCC cells. XL388 activated caspase-dependent apoptosis in the RCC cells. XL388 blocked mTOR complex 1 (mTORC1) and mTORC2 activation, and depleted hypoxia-inducible factor 1α (HIF1α) and HIF-2α expression in RCC cells. Yet, XL388 was ineffective in RCC cells with mTOR shRNA knockdown or kinase-dead mutation. Notably, XL388 was more efficient than mTORC1 inhibitors  in killing RCC cells. Further studies showed that activation of MEK-ERK might be a key resistance factor of XL388. Pharmacological or shRNA-mediated inhibition of MEK-ERK pathway sensitized XL388-induced cytotoxicity in RCC cells.  Growing evidences have proposed a critical function of mammalian target of rapamycin (mTOR) in renal cell carcinoma (RCC) carcinogenesis and progression –4. It isi.e. AKT and ERK-MAPK)  were applied. Both of them dramatically downregulated mTOR in 786-0 cells Figure . IntriguThe results above showed that XL388 blocked mTORC1 and mTORC2 activation simantanuously. We thus compared its cytotoxicity in RCC cells with known mTORC1 blockers or rapalogs . As showExisting evidences have suggested that several mTOR inhibitors could provoke feedback activation of oncogenic MEK-ERK signaling, which serves as a major resistance factor . Others The above pharmacological evidences suggest that MEK-ERK inhibition could potentiate XL388's cytotoxicity in RCC cells. Next, shRNA strategy was applied to knockdown MEK1/2 and ERK1/2 in RCC cells. As shown in Figure in vivo, using a 786-0 cell xenograft tumor model [3 [in vivo  and RCC's poor prognosis . EpidemiIn vivo, XL388-mediated anti-tumor activity was further sensitized with co-administration of MEK162. It would be interesting to test the underlying signaling mechanisms of MEK-ERK inhibition-mediated XL388 sensitization. Further studies will also be needed to explore the same phenomenon in other cancer cells.Another key finding of this study is that MEK-ERK could be a key resistance factor of XL388 in RCC cells. MEK-ERK inhibitors (MEK162 and AZD-6244) or shRNA knockdown of MEK1/2-ERK1/2 dramatically sensitized XL388-induced killing of RCC cells. XL388 was provided by MedChem Express . Rapamycin, everolimus (RAD001), temsirolimus, MEK162 and AZD-6244 were obtained from Selleck . The caspase-9 inhibitor z-LEHD-CHO, the caspase-3 inhibitor z-DEVD-CHO and the pan caspase inhibitor z-VAD-CHO were obtained from Invitrogen . Antibodies of p-AKT , p-AKT , AKT (9272), p-p44/42 MAPK , ERK1/2 (#9102), p-MEK1/2 (#9121), MEK1/2 (#9122), p70-S6 Kinase (S6K1 #9202), p-S6K1 , mTOR (#2983), p-mTOR (#2971), HIF-1α (#3716), HIF-2α (#7096), and (β-) tubulin (#2128) were all obtained from Cell Signaling Tech .The established human RCC cell lines (786-0 and A498) and HK-2 human proximal tubule epithelial cell line were provided by Dr. Zheng's Group at Nantong University , 4, 43. Fresh RCC specimens were obtained from two different RCC patients  with nephroureterectomy. The two patients were enrolled at Huashan Hospital Affiliated to Fudan University , both received no treatment prior to surgery. RCC tissues were thoroughly washed and then minced into small pieces, which were then digested via collagenase I for 30 min. Afterwards, primary cancer cells were pelleted and washed, and then cultured in the described DMEM medium [Following treatment, cell viability was evaluated via the Cell Counting Kit-8  assay with the manufacturer’s instructions. Absorbance was measured at 490 nm through a Microplate Reader.786-0 cells with indicated treatment were plated onto 6-well plate at 2000 cells per well. Following incubation of 7 days, the remaining large proliferative colonies were fixed, stained, and counted manually.To assay of cell proliferation, BrdU ELISA assay kit  was used . The ELIAfter indicated treatment, cells were subjected tothe single-stranded DNA (ssDNA) apoptosis ELISA  assay. The detailed protocol can be seen in other studies , 46.After treatment, 20 μg protein lysates (per treatment) were added to caspase assay buffer  along wiCell apoptosis was examined by the TUNEL staining assay . PercentCells or tumor tissues were lysed by the lysis buffer described . The quaMEK1/2 shRNA, MEK1/2 shRNA and two mTOR lentiviral shRNAs were purchased from Santa Cruz Biotech , which contain a puromycin resistance gene . RCC celThe kinase-dead mTOR  construct and the empty vector (pSuper-puro) were from Dr. Liu's group . The con6 786-0 cells were injected into the left flank. Within three weeks, the xenograft tumors were established. Mice were divided into four groups. Mice body weight and bi-dimensional tumor measurements were recorded every 7 days for a total of 35 days. Tumor volume was estimated using the standard formula: (length × width2)/2. All animal protocols were approved by the authors' institution IACUC.As reported , female Data were always expressed as mean ± standard deviation (SD). Statistical analyses were performed via one-way analysis of variance . IC-50 was calculated by SPSS 16.0 using a sigmoidal dose-response curve model. Multiple comparisons were performed using Tukey’s honestly significant difference procedure.


Modern MRI myocardial perfusion protocols use rapid venous bolus injections, typically 3-5 ml/s of 5-15 ml of agent over a few seconds. The resulting arterial input functions are rapidly varying with high agent concentrations Fig.  and 1B aNumerical simulations of whole body vascular systems were used to design optimized venous injection protocols for the generation of step-input-like arterial-input functions targeting the idealized step-input function show in Fig. In-vivo experiments were performed on a Siemens Aera 1.5T . ECG-gated saturation-recovery (TS=100 ms) bSFFP images were acquired for 120 heartbeats . Matrix size 224 × 136, rate 2 GRAPPA, 8 mm slice, 1.03 ms TE, 2.5 ms TR, 70° flip. All contrast injections were single dose (0.1 mmol/kg) of Magnevist (Bayer). In-vivo data was acquired in 3 healthy controls and 3 CAD patients, all ~90 days post MI . Blood/tissue signal intensities were converted to contrast agent concentrations using a Bloch equation look-up-table approach and myocardial perfusion was estimated with an exponential deconvolution approach.Optimized venous injection protocols comprised decaying injection rates over ~1 min. with contrast agent dilution to ~60 ml . Sample blood and tissue time-intensity curves  in a healthy subject are shown in Fig. 2* and T1 saturation effects and long injection duration avoids recirculation effects. The conventional short tissue "dynamic" window (~10 seconds, Fig. A generalizable injection protocol can generate a pseudo arterial step-input function for a range of subject sizes and heart function, offering several advantages over conventional bolus injections: slower tissue dynamics enable multi-slice imaging with single-slice per heart-beat acquisitions, lower concentrations mitigate T


An immunofluorescence microscopy analysis revealed that RAD52 acetylation at DSBs sites is counteracted by SIRT2- and SIRT3-mediated deacetylation, and that non-acetylated RAD52 initially accumulates at DSB sites, but dissociates prematurely from them. In the absence of RAD52 acetylation, RAD51, which plays a central role in HR, also dissociates prematurely from DSB sites, and hence HR is impaired. Furthermore, inhibition of ataxia telangiectasia mutated (ATM) protein by siRNA or inhibitor treatment demonstrated that the acetylation of RAD52 at DSB sites is dependent on the ATM protein kinase activity, through the formation of RAD52, p300/CBP, SIRT2, and SIRT3 foci at DSB sites. Our findings clarify the importance of RAD52 acetylation in HR and its underlying mechanism.The p300 and CBP histone acetyltransferases are recruited to DNA double-strand break (DSB) sites where they induce histone acetylation, thereby influencing the chromatin structure and DNA repair process. Whether p300/CBP at DSB sites also acetylate non-histone proteins, and how their acetylation affects DSB repair, remain unknown. Here we show that p300/CBP acetylate RAD52, a human homologous recombination (HR) DNA repair protein, at DSB sites. Using  DNA double strand breaks (DSBs) are the most dangerous type of DNA damage in cells. Homologous recombination (HR) is a DSB repair system in which a central player, RAD51, functions with several proteins, including RAD52. DSBs activate the DNA damage response signaling network, in which the ataxia telangiectasia mutated (ATM) protein plays a chief role, by phosphorylating numerous target proteins. As compared to phosphorylated proteins, relatively few acetylated proteins have been functionally characterized in DNA repair. In addition, beyond the roles in phosphorylation signaling, much less is known about whether ATM functions are linked with other protein modifications, such as acetylation. Here, we found that RAD52 at DSB sites is acetylated by p300/CBP acetyltransferases and then deacetylated by SIRT2/SIRT3 deacetylases. RAD52 acetylation is required for sustained RAD51 colocalization at DSB sites, and is therefore essential in HR. ATM is required for the recruitment of RAD52, p300/CBP and SIRT2/SIRT3 to DSB sites, and therefore is essential for RAD52 acetylation. Thus, the RAD52-acetylation state is critical for HR, and its regulation is linked to ATM signaling. Our work demonstrates the importance of the regulation of RAD52 acetylation in HR and its underlying mechanism. Ionizing radiation (IR) induces deleterious DNA lesions, such as DNA double-strand breaks (DSB). In response to DSBs, DNA damage response (DDR) signaling is induced. Ataxia telangiectasia mutated (ATM) protein kinase is one of the central players for phosphorylation-mediated DDR signaling, which is activated at DSB sites and phosphorylates numerous proteins, including the histone variant H2AX, and cell cycle checkpoint and DNA repair proteins . HomologRAD52 reduces HR and may be involved in certain types of DSB repair processes Ac-CoA was added. The reactions were analyzed by Coomassie Brilliant Blue staining (left) or autoradiography (right). (B) RAD52 (3 μg), DNA polymerase β (3 μg), or BSA (3 μg) was incubated with FLAG-p300 (2 μg) where indicated.  RAD52 (1.5 μg), RAD51 (1.5 μg), or DNA polymerase κ (1.5 μg) was incubated with 1 μg of CBP-FLAG (C) or FLAG-p300 (D). (E) Silver staining of the RAD52, RAD51, DNA polymerase κ, FLAG-p300 and CBP-FLAG proteins used in (PDF)Click here for additional data file.S2 FigMus musculus (NCBI accession number AAA85794), Rattus norvegicus (NCBI accession number NP_001100087), Cricetulus griseus (NCBI accession number NP_001233693), Homo sapiens (NCBI accession number AAA85793), Pan troglodytes (NCBI accession number JAA24777), Rhesus monkey (NCBI accession number AFH33435), Gallus gallus (NCBI accession number NP_001161231), and Xenopus laevis (NCBI accession number NP_001089585), was performed using the Clustal 2.1 multiple sequence alignment program.Alignment of RAD52 proteins from (PDF)Click here for additional data file.S3 FigSchematic representation of RAD52 wild-type and acetylation-site mutants used in this study. Mutations were introduced in functional domains, such as the highly conserved region , the RPA binding region (K262R), and the RAD51 binding region (K323R), and also introduced outside the domains (190/192R). The 13xR and 11xR mutants contain multiple mutations including the NLS sequence, whereas the acetylation sites in the NLS sequence are normal in the 10xR and 8xR mutants. The NLS sequence is conjugated at the N-terminal in NLS-RAD52 (Wt) and NLS-RAD52 (13xR). The 10xQ mutant contains multiple glutamine (Q) substitutions at the same mutated sites as in the 10xR mutant.(PDF)Click here for additional data file.S4 FigssDNA binding activity of the RAD52 11xR mutant. (A) Electrophoretic mobility shift assay (EMSA) was performed using a 50-mer oligonucleotide (10 μM in nucleotides) with a Cy5 dye attached to the 5' end (oligo 1), and the indicated concentrations of RAD52 or the RAD52 11xR mutant. (B) Percentages of ssDNA bound by RAD52  and the RAD52 11xR mutant  as a function of the protein concentration.(PDF)Click here for additional data file.S5 Figin vivo.Human RAD52 is acetylated by p300/CBP  (A) Acetylation of the FLAG-RAD52-HA protein purified from T-Rex-293 (HEK293) cells was detected as described in the Supporting Materials and Methods, using the indicated antibodies. pRc/RSV-CBP-HA was transfected into cells, and FLAG-RAD52-HA was purified from cell extracts 24 h after transfection.  MRC5V1 cells were transfected with the expression plasmid for HA-p300 (B) or CBP-HA (C), and were unirradiated or irradiated with γ-rays (8 Gy) at 24 h after the transfection. At the indicated time after irradiation, the cells were subjected to immunofluorescent staining with an anti-HA (green) antibody, an anti-γH2AX (red) antibody, and 4',6-diamidino-2-phenylindole .  T-Rex-293 cells expressing Myc-RAD52 were transfected with the expression plasmid for HA-p300 (D) or CBP-HA (E), and were irradiated with γ-rays (8 Gy) at 24 h after the transfection. At the indicated time after irradiation, the cells were subjected to immunofluorescent staining with anti-HA (green) and anti-Myc (red) antibodies.  DSP-mediated cross-linking experiments were performed in the presence or absence of doxorubicin, as described in the Supporting Materials and Methods. RAD52-HA or GST-HA was expressed, as indicated. Whole cell extracts  or immunoprecipitates  with anti-p300 (F) or anti-CBP (G) antibodies.  T-Rex-293 cells expressing FLAG-RAD52-HA were transfected with either a negative control siRNA or mixture of p300 and CBP-specific siRNAs. Cell extracts were subjected to immunoblotting analyses with the indicated antibodies.  The relative band intensities normalized to those of the HA or GAPDH bands are shown below the immunoblots.(PDF)Click here for additional data file.S6 FigImmunostaining of empty expression vector containing cells with acetylated RAD52 antibody. T-Rex-293 cells containing the empty pT-Rex-DEST30 vector were irradiated with γ-rays (8 Gy). At 1 hour after irradiation, the cells were subjected to immunofluorescent staining with an anti-γH2AX antibody (red), an anti-acetylated RAD52 at K323 antibody (blue or white), and DAPI (blue).(PDF)Click here for additional data file.S7 FigConfirmation of siRNA-mediated knockdown. T-Rex-293 , HEK293  or HeLa pDR-GFP  cells were transfected with the negative control (shown by “C”) or the indicated siRNA, as described in the Supporting Materials and Methods. The mixture of siRAD52 , the mixture of siRAD51 (#1 and #2), and the mixture of siBRCA2  were used in panels D, F, and G, respectively. Whole cell extracts  or total RNA from the cells (D and G) were prepared 72 h after siRNA transfection, and were subjected to immunoblotting  and RT-PCR (D and G) analyses, respectively.(PDF)Click here for additional data file.S8 FigATM inhibition does not affect cellular protein levels of RAD52, p300, CBP, SIRT2 and SIRT3. (A-E) Cell extracts prepared under the same experimental conditions as in Figs (PDF)Click here for additional data file.S9 FigCRISPR-Cas9-mediated genome editing. (A) The donor plasmid DNA used for the CRISPR-Cas9-mediated knock-in of FLAG and the HA-tagged RAD52 coding sequence (CDS) into the genomic region of the targeted RAD52 gene. Multisite Gateway technology was used for the construction of the donor plasmid DNA, as described in the Supporting Materials and Methods.  PCR analysis of knock-in HeLa pDR-GFP and HEK293 cells.  The DNA sequences of the forward (P1) and reverse (P2 and P3) primers used in the PCR analysis are described in the Supporting Materials and Methods. The primers P1, P2 and P3 anneal the genomic DNA region upstream from the start codon of the RAD52 gene, the SV40 polyA region from pT-Rex-DEST30, and the intron region of RAD52, respectively.  Agarose gel electrophoresis of the PCR products. The genomic DNAs purified from HeLa pDR-GFP (D) and HEK293 (E) knock-in cells were analyzed by PCR with the indicated primers. As controls, genomic DNAs from untargeted cells were used in lane C.  Whole cell extracts of the HeLa pDR-GFP (F) and HEK293 (G) knock-in cells were subjected to immunoblotting analyses with the indicated antibodies.(PDF)Click here for additional data file.S10 FigEffect of acetylation-deficient mutations on ionizing radiation-induced foci formation of RAD52 expressed by the native promoter. (A) RAD52 (Wt or 10xR) knock-in HeLa pDR-GFP cells, as shown in p<0.001 by t-test).(PDF)Click here for additional data file.S11 FigComparison of knockdown effects on HR repair between RAD52 and BRCA2. HeLa pDR-GFP cells were transfected with the control or indicated siRNA, and were subjected to the DR-GFP assay as described in the Supporting Materials and Methods. In the column labeled siBRCA2 (mix), the mixture of siBRCA2  was used. For RAD52 depletion, siRAD52 (CDS #1) was used. Error bars indicate the standard error of the mean from three samples. Asterisks indicate statistically significant differences of each protein-depleted sample, as compared with the control (+I-SceI) . The samples connected by lines were also compared.(PDF)Click here for additional data file.S12 FigInteractions of human RAD52 with human RAD52, RAD51 and RPA subunits by a yeast two-hybrid analysis. Yeast MAV203 cells were transformed with expression vectors encoding the indicated NLS-GAL4-activation domain (AD)- and GAL4-DNA binding domain (DBD)-fused proteins. As controls, the empty vector for the expression of the GAL4-DBD fusion protein was used (1–5). The negative control (C1) and positive controls (C2 and C3) were also included. Each transformant was examined for growth on an SC-Leu-Trp agar plate and an SC-Leu-Trp-His agar plate containing 25 mM 3-Amino-1,2,4-Triazole (3AT).(PDF)Click here for additional data file.S13 Fig(A) EMSA was performed using a 50-mer oligonucleotide (10 μM in nucleotides) with a Cy5 dye attached to the 5' end (oligo 1), and the indicated concentrations of RAD52 or acetylated RAD52. (B) Quantification of (A). Percentage of ssDNA bound by RAD52  and acetylated RAD52  as a function of protein concentration. Error bars indicate standard deviation (n = 3).(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.


Escherichia coli (EHEC) O157, the leading cause of life-threatening hemolytic uremic syndrome, is poorly understood. Using proteomics, electron and confocal laser scanning microscopy, immunoblotting, and bioassays, we investigated OMVs secreted by EHEC O157 clinical isolates for virulence factors cargoes, interactions with pathogenetically relevant human cells, and mechanisms of cell injury. We demonstrate that O157 OMVs carry a cocktail of key virulence factors of EHEC O157 including Shiga toxin 2a (Stx2a), cytolethal distending toxin V (CdtV), EHEC hemolysin, and flagellin. The toxins are internalized by cells via dynamin-dependent endocytosis of OMVs and differentially separate from vesicles during intracellular trafficking. Stx2a and CdtV-B, the DNase-like CdtV subunit, separate from OMVs in early endosomes. Stx2a is trafficked, in association with its receptor globotriaosylceramide within detergent-resistant membranes, to the Golgi complex and the endoplasmic reticulum from where the catalytic Stx2a A1 fragment is translocated to the cytosol. CdtV-B is, after its retrograde transport to the endoplasmic reticulum, translocated to the nucleus to reach DNA. CdtV-A and CdtV-C subunits remain OMV-associated and are sorted with OMVs to lysosomes. EHEC hemolysin separates from OMVs in lysosomes and targets mitochondria. The OMV-delivered CdtV-B causes cellular DNA damage, which activates DNA damage responses leading to G2 cell cycle arrest. The arrested cells ultimately die of apoptosis induced by Stx2a and CdtV via caspase-9 activation. By demonstrating that naturally secreted EHEC O157 OMVs carry and deliver into cells a cocktail of biologically active virulence factors, thereby causing cell death, and by performing first comprehensive analysis of intracellular trafficking of OMVs and OMV-delivered virulence factors, we provide new insights into the pathogenesis of EHEC O157 infections. Our data have implications for considering O157 OMVs as vaccine candidates.Outer membrane vesicles (OMVs) are important tools in bacterial virulence but their role in the pathogenesis of infections caused by enterohemorrhagic  Escherichia coli (EHEC) O157, the leading EHEC group causing diarrhea and the life-threatening hemolytic uremic syndrome in humans, produce several virulence factors which play distinct roles in the pathogenesis of these diseases. However, the mechanisms of their secretion and host cell injury are poorly understood. We show here that EHEC O157 strains isolated from patients shed nanostructures termed outer membrane vesicles (OMVs) which contain major EHEC O157 virulence factors including Shiga toxin 2a (Stx2a), cytolethal distending toxin V (CdtV), EHEC hemolysin, and flagellin. The OMVs are taken up by human intestinal epithelial and renal and brain microvascular endothelial cells, which are the major targets during EHEC O157 infections, and deliver the virulence factors intracellularly. Inside cells the virulence factors separate from OMVs and are transported via different pathways to their target compartments including the cytosol (Stx2a), nucleus (CdtV-B subunit), and mitochondria (EHEC hemolysin). Cells exposed to EHEC O157 OMVs develop G2 cell cycle arrest induced by CdtV-B-mediated DNA damage. This is followed by apoptotic cell death triggered by Stx2a and CdtV via caspase-9 activation. OMVs thus serve as novel tools of EHEC O157-mediated host injury and are quite likely involved in the pathogenesis of human diseases.Enterohemorrhagic  Escherichia coli (EHEC) O157, the leading EHEC serogroup causing human diseases including life-threatening hemolytic uremic syndrome (HUS) (pQE60/cdtV-B493/89) and purified (Protein concentration in OMVs was determined with Roti-Nanoquant (Carl Roth). Stx2a, EHEC-Hly, and H7 concentrations were determined as described , 27 usinpurified . CdtV-B E. coli O157 LPS antibody (culture sections), or with anti-Stx2a, anti-CdtV-A, anti-CdtV-B, anti-EHEC-Hly, and anti-H7 antibody (OMV sections) and Protein A Gold (15 nm) . Staining with Protein A Gold alone, without first antibodies, served as a control of specificity of the immunogold staining. For negative staining, OMVs in OptiPrep fractions of 5791/99 and 493/89 OMVs were fixed and stained with 0.5% uranyl acetate. Samples were analyzed at 80 kV on a FEI-Tecnai 12 electron microscope  and photographed (Ditabis imaging plates).Electron microscopy of ultrathin cryosections of LB agar cultures of strains 5791/99 and 493/89, or of ultrathin cryosections of OptiPrep-purified 5791/99 OMVs was performed as described . BrieflyThe size of OMVs in OptiPrep fractions was determined with DLS  using a Caco-2 and DLD-1 cells  were cultured in Quantum 286 epithelial medium (PAA), HBMEC  in EndotTo determine the kinetics of OMV cellular uptake, cells grown in 96-well plates with black frames (Greiner Diagnostics) were incubated for 15 min to 24 h at 37°C with rhodamine isothiocyanate B-R18-labeled  OMVs (R1E. coli O157 LPS antibody (O157 OMVs) or anti-E. coli LPS antibody (non-O157 OMVs) and Alexa Fluor 488-conjugated goat anti-rabbit IgG. Actin was counterstained with phalloidin-tetramethyl rhodamine (TRITC) (Sigma-Aldrich) and nuclei with DRAQ5 . Preparations were mounted in fluorescence mounting medium (Dako) and analyzed with a confocal laser-scanning microscope . To determine the effect of dynasore on OMV uptake by CLSM, cells were pretreated with 80 μM dynasore for 1 h, and without removing the inhibitor, exposed to OMVs for 4 h and processed as above. Alexa Fluor 488-conjugated transferrin (20 μg/ml) and Alexa Fluor 488-conjugated cholera toxin B subunit (10 μg/ml) (Molecular Probes) incubated with dynasore-untreated and dynasore-treated cells for 4 h served as positive controls.For CLSM, cells grown in 8-well chamber slides (ibidi) were incubated with OMVs (4 μg/ml of protein) for 30 min at 4°C (binding) followed by 30 min, 90 min, 4 h, or 24 h at 37°C . Cells exposed to OMV buffer  served as controls. After washing, fixation , quenching (0.2 M glycine pH 7.2), permeabilization (0.25% Triton X-100) and blocking  , OMVs were stained with anti-stx2a (40 μg/ml of protein containing ~3.4 μg/ml of CdtV), TA153 (35 μg/ml of protein containing ~3.4 μg/ml of CdtV), TA154 (35 μg/ml of protein) or without OMVs (negative control). The cells were extensively washed and lysed with SDS-PAGE loading buffer. After heating , cell lysates were centrifuged  and supernatants  were analyzed by immunoblot with antibodies against OmpA, Stx2a, CdtV-A, -B, and -C, EHEC-Hly, H7 flagellin, and actin (loading control).Cell monolayers in 12-well microtiter plates were incubated for 30 min or 4 h with OMVs 5791/99 , 493/89 (40 μg/ml of protein containing ~3.4 μg/ml of CdtV and ~4.6 μg/ml of Stx2a), 493/89ΔE. coli O157 antibody and Cy3-conjugated goat anti-mouse IgG, and Stx2a, CdtV-A, -B, -C, and EHEC-Hly with the respective rabbit antibodies and Alexa Fluor 488-conjugated goat anti-rabbit IgG. To monitor intracellular trafficking of OMVs and OMV-delivered toxins/toxin subunits, OMVs were stained with rabbit anti-E. coli O157 LPS antibody, and the toxins/toxin subunits with the respective toxin-specific antibodies, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG. Early endosomes, late endosomes/lysosomes, Golgi complex, endoplasmic reticulum, and mitochondria were stained with mouse anti-Rab5, anti-CD63, anti-K58 Golgi protein, anti-PDI, and anti-mitochondria (MTC02) antibodies, respectively, and Cy3-conjugated goat anti-mouse IgG. Nuclei were stained with DRAQ5. Preparations were analyzed with a confocal laser-scanning microscope (LSM 510 META microscope equipped with a Plan-Apochromat 63x/1.4 oil immersion objective). Single channels and the merged images  are shown. To quantify colocalizations between signals of interest, digital colocalization images were imported into BioImageXD6 [To analyze intracellular trafficking of O157 OMVs and OMV-delivered virulence factors, HBMEC grown in 8-well ibidi slides were preincubated with 5791/99 OMVs  for 30 min at 4°C (OMV cell binding), and without washing, postincubated for 15 min, 30 min, 90 min, 4 h, and 20 h at 37°C . Cells were then washed, fixed and quenched as above, and permeabilized/blocked with PBS containing 5% goat serum, 1% bovine serum albumin and 1 mg/ml saponin (Sigma-Aldrich). To monitor colocalizations of OMV-delivered toxins/toxin subunits with OMVs, OMVs were stained with mouse anti-ImageXD6 , signalscdtV-B) (containing ~140 ng/ml of CdtV-B), BL21(cdtV-A), or BL21(cdtV-C); OMVs from BL21(cdtV-ABC) (containing ~142 ng/ml of CdtV-B and ~370 ng/ml of CdtV), BL21(cdtV-ACΔB) or from the vector control BL21(pET23) served as controls. To monitor colocalizations of CdtV subunits with OMVs, OMVs were stained with mouse anti-E. coli LPS antibody and Cy3-conjugated goat anti-mouse IgG, and CdtV-A, -B, and -C with the respective rabbit antibodies and Alexa Fluor 488-conjugated goat anti-rabbit IgG. To monitor intracellular trafficking of OMVs and OMV-delivered CdtV subunits, OMVs were stained with rabbit anti-E. coli LPS antibody, and CdtV-A, -B, and -C with their respective antibodies, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG. The Golgi complex, endoplasmic reticulum, and late endosomes/lysosomes were stained with primary antibodies described above and Cy3-conjugated goat anti-mouse IgG. Colocalizations were quantified as above.To analyze intracellular trafficking of OMV-delivered recombinant CdtV subunits, HBMEC were preincubated for 30 min at 4°C, and, without washing, postincubated  with OMVs (4 μg/ml of protein) from strains BL21(stx2a OMVs (4 μg/ml of protein) for 30 min at 4°C followed by postincubation at 37°C for 20 h (HBMEC) or 4 h (DLD-1). Free purified Stx2a [2) [PPMP treatment was performed by growing HBMEC for 6 days in Endothelial medium containing 5 μM PPMP (Sigma-Aldrich) as described previously for HeLa cells . PPMP-tred Stx2a . Cells wtx2a [2)  before f7) were fixed with 3.7% paraformaldehyde, permeabilized with 0.2% Triton X-100, and stained with FITC-conjugated anti-CD77/Gb3 antibody (1 h on ice). After washing, the fluorescence of at least 104 cells was acquired with FACSCalibur (Becton Dickinson) using green channel. The data were analyzed with CellQuest Pro (Becton Dickinson) and expressed as geometric means of fluorescence from 10,000 events. Unstained cells served as controls.Gb3 in HBMEC, PPMP-treated HBMEC and DLD-1 cells was visualized by CLSM of permeabilized cells using rat anti-CD77/Gb3 IgM antibody and Alexa Fluor 488-conjugated goat anti-rat IgM. Quantification of Gb3 was performed by FACS analysis. Cells  were incubated  in 20 mM TRIS-HCl buffer with pH ranging from 8.0 to 5.0. The samples were then ultracentrifuged , and the pellets (OMVs) and supernatants (OMV-released proteins) were analyzed for Stx2a and CdtV-B by immunoblot. Signals were quantified densitometrically and the percentage of each protein present in the pellet and supernatant at each particular pH was calculated from the total signal. To determine the effect of bafilomycin A1, HBMEC were pretreated with 100 nM bafilomycin A1 (Sigma-Aldrich) for 1 h at 37°C, and without removing the inhibitor, exposed (30 min at 4°C followed by 4 h at 37°C) to OMVs 5791/99. The presence of Stx2a and CdtV-B in the endoplasmic reticulum was analyzed by CLSM as described above.6 cells) were incubated with 5791/99 OMVs  for 30 min at 4°C, and postincubated for 30 min to 72 h at 37°C. Untreated cells and cells treated for 72 h with OMV buffer instead of OMVs served as controls. Lysosomal and mitochondrial fractions were isolated using the Lysosome Enrichment Kit for Tissue and Cultured Cells and the Mitochondria Isolation Kit for Cultured Cells (both Thermo Scientific), respectively, as described previously [Confluent HBMEC monolayers ~8 x 106 cells wcdtV-A), BL21(cdtV-B) (containing ~140 ng/ml of CdtV-B), BL21(cdtV-C), BL21(cdtV-ABC) (containing ~142 ng/ml of CdtV-B and ~370 ng/ml of CdtV) (positive control), BL21(cdtV-ACΔB), or from the vector control BL21(pET23) (negative control) for 30 min at 4°C followed by 90 min to 20 h at 37°C. Endoplasmic reticulum, nuclear and lysosomal fractions isolated as above were analyzed for the respective CdtV subunits by immunoblot.To determine the presence of recombinant CdtV-A, -B and -C in subcellular fractions, HBMEC were exposed to OMVs (4 μg/ml of OMV protein) from strains BL21(stx2a OMVs (4 μg/ml of protein), EDL933 OMVs (4 μg/ml of protein containing ~430 ng/ml of Stx2a and ~ 210 ng/ml of Stx1a) or free Stx2a (~460 ng/ml) as described for CLSM. Endoplasmic reticulum and lysosomal fractions were isolated as above and analyzed for Stx2a by immunoblot.To analyze the presence of Stx2a in subcellular fractions of PPMP-treated HBMEC or DLD-1 cells, cells were incubated with 5791/99 OMVs , Stx2a-negative 493/89Δstx2a (4 μg/ml of protein containing ~340 ng/ml of CdtV), TA153 (3.5 μg/ml of protein containing ~340 ng/ml of CdtV) or TA154 (3.5 μg/ml of protein) or remained untreated. Cells were washed and lysed with SDS-PAGE loading buffer. Cell lysates were heated , centrifuged , and the supernatants  were analyzed by immunoblot with anti-γ-H2AX, anti-phospho-ATM, anti-phospho-Chk2, anti-phospho-cdc2 or anti-actin (loading control) antibodies. To detect the DNA damage signaling induced by recombinant CdtV-B, HBMEC were incubated for 20 h with OMVs (4 μg/ml of protein) from strains BL21(cdtV-B) (containing ~140 ng/ml of CdtV-B), BL21(cdtV-ABC) (containing ~142 ng/ml of CdtV-B and ~370 ng/ml of CdtV) (positive control), BL21(cdtV-ACΔB), or the vector control BL21(pET23) (negative controls); cell lysates prepared as above were analyzed for γ-H2AX and p-cdc2 by immunoblot.To detect the DNA damage signaling, Caco-2 cells, HBMEC and HRGEC were incubated for 20 h with OMVs 5791/99 , 493/89 (4 μg/ml of protein containing ~340 ng/ml of CdtV and ~460 ng/ml of Stx2a), 493/89ΔcdtV-B), BL21(cdtV-ACΔB), BL21(cdtV-ABC), and the vector control BL21(pET23) was determined after 48 h of exposure. After staining of nuclei with propidium iodide (Sigma-Aldrich), G2 arrested cells (4n DNA content) were quantified by flow cytometry  . Data from 104 nuclei were analyzed by CellQuest software (Becton Dickinson). To determine dose-dependence of the G2 arrest, cells were exposed to O157 or TA153 OMVs containing CdtV in doses ranging from 340 ng/ml to 5.3125 ng/ml for 24 h (HRGEC) or 48 h  and analyzed for DNA content as above.The cell cycle was analyzed as described previously  using cecdtV-B), BL21(cdtV-ACΔB), BL21(cdtV-ABC), and BL21(pET23) were incubated for 72 h with HBMEC. Morphology was examined in native cells (HRGEC) or cells fixed with 70% ethanol and stained with 10% Giemsa using Axio Imager A1 microscope (Carl Zeiss).In the distension assay, freshly seeded Caco-2 cells, HBMEC or HRGEC were exposed to O157, TA153 or TA154 OMVs (concentrations as above) for 72 h or remained untreated. OMVs from the recombinant strains BL21, purified Stx2a [4 nuclei were analyzed by CellQuest software.To measure apoptosis, cells were exposed for 24 h to 96 h to 5791/99, 493/89, 493/89Δed Stx2a  and the Caspase-9 and caspase-8 activities were assayed in cells exposed to the above OMVs, OMV buffer or purified Stx2a for 48 h or left untreated using Caspase Colorimetric Substrate Kit I (Biozol Diagnostica) . The colPLUS (Roche) was performed as described [405 absorbance values of sample-treated cells with those of untreated cells. To determine apoptotic potential of EDL933 OMVs in DLD-1 cells, the Cell Death Detection ELISA was performed as above after 96 h incubation of the cells with EDL933 OMVs (4 μg/ml of protein containing ~430 ng/ml of Stx2a and ~ 210 ng/ml of Stx1a), free Stx2a (460 ng/ml), staurosporine (1 μM) (positive control) and OMV buffer (negative control).The Cell Death Detection ELISAescribed  using cet-test or paired Student´s t-test. p values < 0.05 were considered significant.Data were analyzed with one-way ANOVA , two-tailed unpaired Student´s GenBank NC_002695.1 (1266965..1267924) Shiga toxin 2 subunit A gene (ECs1205)GenBank NC_002695.1 (1267936..1268205) Shiga toxin 2 subunit B gene (ECs1206)Escherichia coli O157:H7UniProtKB/Swiss-Prot Q7DI68 Shiga toxin 2 subunit A Escherichia coli O157:H7UniProtKB/Swiss-Prot A7UQX3 Shiga toxin 2 subunit B Escherichia coli cdtA gene, cdtB gene and cdtC geneGenBank AJ508930.1 UniProtKB/Swiss-Prot Q8GJ13 Cytolethal distending toxin-V A subunitUniProtKB/Swiss-Prot O32586 Cytolethal distending toxin-V B subunitUniProtKB/Swiss-Prot Q8GJ12 Cytolethal distending toxin-V C subunithlyA geneGenBank X79839.1 EHEC-Escherichia coli (EHEC-Hly)UniProtKB/Swiss-Prot Q47262 Hemolysin fliC gene Escherichia coli O157:H7 (ECs2662)GenBank NC_002695.1 (2624379..2626136) Escherichia coli O157:H7UniProtKB/SwissProt Q7AD06 Flagellin ompA gene (ECs1041)GenBank NC_002695.1 (1148484..1149524) Escherichia coli O157:H7UniProtKB/SwissProt P0A911 Outer membrane protein A espP geneGenBank X97542.1 (2571..6473) Escherichia coli O157:H7UniProtKB/SwissProt Q7BSW5 Serine protease EspP S1 Fig600. Data are means ± standard deviations from three independent experiments. (D) Distribution of OMV-associated proteins identified with nano-LC-MS/MS according to their subcellular localization determined by PsortB prediction tool. Strains were grown in LB broth, OMVs were isolated at indicated times, subjected to immunoblot with anti-OmpA antibody, and quantified by densitometry of OmpA signals . OMV-free supernatants served as controls. Bacterial growth was monitored by measuring OD(TIF)Click here for additional data file.S2 Figstx2a were incubated in HEPES buffer alone (control), or in HEPES buffer with the indicated chemicals. After ultracentrifugation, pellets  and supernatants  were analyzed by immunoblot with antibodies against the virulence factors or their subunits.OptiPrep-purified OMVs from strains 5791/99, 493/89, and 493/89Δ(TIF)Click here for additional data file.S3 FigOptiPrep gradient fractions of 5791/99 OMVs (F1 –F8) and 493/89 OMVs (F1 –F9) were negatively stained with 0.5% uranyl acetate and analyzed with a FEI-Tecnai 12 electron microscope. Scale bars are 200 nm.(TIF)Click here for additional data file.S4 FigThe x-axis displays the OMV size distribution in each fraction and the y-axis the scattered light intensity of the OMVs. In (C) and (D) the average diameter (Z-average) of OMVs and the index of the particle size distribution  in each fraction is shown . The Z-averages and PDIs were calculated by the method of cumulants using the Zetasizer software.(TIF)Click here for additional data file.S5 FigE. coli O157 LPS (A) or anti-E. coli LPS (C) antibody and Alexa Fluor 488-conjugated goat anti-rabbit IgG, actin (red) with phalloidin-TRITC and nuclei (blue) with DRAQ5. Confocal Z-stack projections are included at upper/right sides. Crosshairs show the position of the xy and yz planes. Scale bars are 10 μm. (D) CLSM of control cells incubated with OMV buffer instead of OMVs for 24 h and stained and processed as described in A and C. (B) Distribution of CdtV-A, CdtV-B, and CdtV-C proteins in OMVs and OMV-free supernatants of strains TA153 (containing the cdtV-ABC operon from strain 493/89 in SuperCos I) and TA154 (vector control) determined by immunoblot with antibodies against OmpA (an OMV marker) and the respective CdtV subunits. CLSM visualization of binding (panels 0 min) and uptake of O157 (A) and TA153 and TA154 OMVs (C) by Caco-2 cells and HBMEC after 90 min, 4 h and 24 h of incubation. OMVs (green) were detected with anti-(TIF)Click here for additional data file.S6 Figp < 0.01, and *** p < 0.001 compared to inhibitor-untreated cells (one-way ANOVA). (C-F) Effect of dynasore on the uptake of OMVs  and control endocytosis markers  by Caco-2 cells  and HBMEC  visualized by CLSM after 4 h of incubation of cells with the indicated samples. Panels marked Dynasore show dynasore-pretreated cells. Green, OMVs  or Alexa Fluor 488-conjugated transferrin (Tf-AF488) or Alexa Fluor 488-conjugated cholera toxin B subunit (CT-B-AF488) ; red, actin; blue, nuclei. Confocal Z-stack projections are included at upper/right sides. Crosshairs show the position of the xy and yz planes. Scale bars are 10 μm. For evaluation of the effect of dynasore on OMV uptake, compare the OMV amounts in dynasore-treated cells  with those of the respective OMVs in dynasore untreated cells  Activities of inhibitors of endocytosis used in this study against markers of different endocytosis pathways including clathrin-mediated endocytosis , lipid rafts/caveolae-mediated endocytosis , and macropinocytosis . Caco-2 cells (A) and HBMEC (B) either untreated (no inhibitor) or pretreated with the indicated inhibitors were incubated with Tf-TMR, CT-B-AF647 or Dextran-TMR for 4 h and fluorescence was measured with FLUOstar OPTIMA fluorometer. The uptake of each marker in the presence of inhibitors was expressed as the percentage of its uptake by inhibitor-untreated cells (100%). Data are means ± standard deviations from three independent experiments. ** (TIF)Click here for additional data file.S7 FigImmunoblot detection of OMVs (anti-OmpA antibody) and OMV-associated virulence factors in lysates of Caco-2 cells, HBMEC, and HRGEC which were incubated with O157 OMVs  or control CdtV-containing (TA153) or CdtV-lacking (TA154) OMVs (C) for 30 min and 4 h. Untreated cells (no OMV) were negative controls. Actin served as a loading control. .(TIF)Click here for additional data file.S8 Figp < 0.05; two-tailed unpaired Student’s t-test) compared to the previous time interval or between the spanned time intervals.Graphical presentations of CLSM colocalizations between (A) 5791/99 OMVs and OMV-delivered virulence factors, (B) 5791/99 OMVs and subcellular compartments, and (C-G) the indicated OMV-delivered virulence proteins and subcellular compartments during time. The subcellular compartments investigated and their markers were: early endosomes (Rab5), late endosomes/lysosomes (CD63), Golgi complex (K58), endoplasmic reticulum (PDI), mitochondria (MTC02), and nucleus (DNA). The percentages of colocalizations between signals of interest were determined with the BioImageXD6 tool. Data are shown as means ± standards deviations from measurements of at least five (for CdtV-A/CdtV-C of at least three) different samples. *significantly increased or decreased ((TIF)Click here for additional data file.S9 FigScale bars are 10 μm.(TIF)Click here for additional data file.S10 FigScale bars are 10 μm.(TIF)Click here for additional data file.S11 Fig(A) Lysosomal (lyso), mitochondrial (mito), endoplasmic reticulum (ER), nuclear (nucl), and cytosolic (cyto) fractions were isolated from HBMEC which had been incubated for the indicated times with 5791/99 OMVs or for 72 h with OMV buffer  or left untreated (no OMV). The fractions were analyzed by immunoblot with antibodies against the indicated compartment-specific marker proteins. 5791/99 OMVs without cells (OMV ctrl) served as a negative control. (B) Whole cell lysates (WCL) (positive control) and subcellular fractions prepared from HBMEC exposed to 5791/99 OMVs for 20 h were analyzed by immunoblot with antibodies against homologous and heterologous compartment-specific marker proteins.(TIF)Click here for additional data file.S12 FigScale bars are 10 μm. Ee, early endosomes; Le/Lyso, late endosomes/lysosomes; ER, endoplasmic reticulum; Mito, mitochondria.(TIF)Click here for additional data file.S13 FigScale bars are 10 μm. Ee, early endosomes; Le/Lyso, late endosomes/lysosomes; ER, endoplasmic reticulum; Mito, mitochondria.(TIF)Click here for additional data file.S14 Fig(A) Immunoblot detection of Stx2a B subunit in isolated subcellular fractions of HBMEC which were incubated for the times indicated with 5791/99 OMVs or for 72 h without OMVs or with OMV buffer (20 mM TRIS-HCl) (negative controls); OMVs without cells (OMV ctrl) served as a positive control. (B) Densitometric quantification of Stx2aB signals in endoplasmic reticulum and lysosomal fractions shown in A. Abbreviations used: ER, endoplasmic reticulum; Cyto, cytosol; Lyso, lysosomes; Mito, mitochondria; Nucl, nucleus; DU, densitometric unit.(TIF)Click here for additional data file.S15 FigScale bars are 10 μm. Ee, early endosomes; ER, endoplasmic reticulum; Le/Lyso, late endosomes/lysosomes; Mito, mitochondria.(TIF)Click here for additional data file.S16 FigScale bars are 10 μm. Ee, early endosomes; ER, endoplasmic reticulum; Le/Lyso, late endosomes/lysosomes; Mito, mitochondria.(TIF)Click here for additional data file.S17 Fig(A) 5791/99 OMVs were incubated in TRIS-HCl with the indicated pH range for 1 h and then ultracentrifuged. The pellets (P) containing OMV-associated proteins and supernatants (S) containing proteins that separated from OMVs were analyzed by immunoblot with antibodies against Stx2a or CdtV-B. The Stx2a and CdtV-B signals were quantified densitometrically and the percentage of each protein present in the P and S fraction at each particular pH was calculated from the total signal. (B) HBMEC were not (no BafA1) or were pretreated (+ BafA1) for 1 h with 100 nM bafilomycin A1 (BafA1), incubated for 4 h with 5791/99 OMVs, and analyzed for the presence of Stx2a or CdtV-B in the endoplasmic reticulum by CLSM. The right panels show the indicated single fluorescence channels, and the left panels the merged images . Scale bars are 10 μm. Note the differences between the intensities of Stx2a and CdtV-B signals, respectively, in BafA1-untreated and BafA1-treated cells.(TIF)Click here for additional data file.S18 Figp < 0.001 (paired Student´s t-test) for Gb3 content in PPMP-treated compared to PPMP-untreated HBMEC.  Visualization of Gb3 in PPMP-untreated and PPMP-treated HBMEC (C) and in DLD-1 cells (D) by CLSM. Green, Gb3; blue, nuclei.  Negative controls to stx2a for 20 h (F) or left untreated (no OMV) (G). (I) Negative controls to stx2a for 90 min or 4 h or left untreated (no OMV) before they were processed for CLSM directly (no Triton) or after 1 min extraction with Triton X-100-containing buffer (+ Triton). In C, D, F, G and I, the indicated single fluorescence channels are shown in the right panels and the merged images in the left panels. Scale bars are 10 μm.  Graphical presentations of CLSM data shown in p < 0.001 (paired Student´s t-test) for colocalization rates in PPMP-treated compared to PPMP-untreated HBMEC. Graphical presentations of FACS analyses of Gb3 content in PPMP-untreated (PPMP-) and PPMP-treated (PPMP+) HBMEC (A) and in DLD-1 cells (B) stained with anti-CD77/Gb3-FITC antibody or unstained (control). Geometric mean fluorescence ± standard deviations from three independent experiments are shown. ***(TIF)Click here for additional data file.S19 Fig(A) HBMEC , and (B)(TIF)Click here for additional data file.S20 Fig(A) Images shown in (TIF)Click here for additional data file.S21 FigScale bars are 10 μm. Ee, early endosomes; ER, endoplasmic reticulum; Le/Lyso, late endosomes/lysosomes; Mito, mitochondria.(TIF)Click here for additional data file.S22 FigScale bars are 10 μm. Ee, early endosomes; ER, endoplasmic reticulum; Le/Lyso, late endosomes/lysosomes; Mito, mitochondria.(TIF)Click here for additional data file.S23 FigScale bars are 10 μm. Ee, early endosomes; Le/Lyso, late endosomes/lysosomes; ER, endoplasmic reticulum; Mito, mitochondria.(TIF)Click here for additional data file.S24 FigScale bars are 10 μm. Ee, early endosomes; Le/Lyso, late endosomes/lysosomes; ER, endoplasmic reticulum; Mito, mitochondria.(TIF)Click here for additional data file.S25 FigScale bars are 10 μm. Ee, early endosomes; Le/Lyso, late endosomes/lysosomes; ER, endoplasmic reticulum; Mito, mitochondria.(TIF)Click here for additional data file.S26 FigScale bars are 10 μm. Ee, early endosomes; Le/Lyso, late endosomes/lysosomes; ER, endoplasmic reticulum; Mito, mitochondria.(TIF)Click here for additional data file.S27 FigcdtV-ABC) OMVs carrying CdtV holotoxin were used as a control.(A) Immunoblot analyses of OMVs from the indicated strains with anti-CdtV-A, anti-CdtV-B, and anti-CdtV-C antibodies. OMVs from BL21(pET23) (vector control) served as a negative control. OmpA is an OMV marker. (B) Intravesicular localization of the recombinant CdtV subunit proteins demonstrated by the proteinase K (PK) assay. PK-untreated (PK-) or PK-treated (PK+) OMVs from the indicated strains, either intact (EDTA-) or lysed with 0.1 M EDTA (EDTA+), were separated by SDS-PAGE and analyzed by immunoblot with the indicated antibodies. BL21((TIF)Click here for additional data file.S28 FigcdtV-A) and BL21(cdtV-C), respectively) or together (OMVs BL21(cdtV-ACΔB)) with the endoplasmic reticulum (ER) and late endosomes/lysosomes (Le-Lyso), and association of CdtV-A and CdtV-C with OMVs after 20 h determined by CLSM. The indicated single fluorescence channels are shown in the right panels and the merged images in the left panels . The percentages of colocalizations of the respective signals (white numbers) were calculated with the BioImageXD6 tool . Scale bars are 10 μm. (D) Controls to CLSM data shown in cdtV-ABC) carrying CdtV holotoxin (positive control), or with CdtV-B lacking OMVs from cdtV-B deletion mutant BL21(cdtV-ACΔB) (negative control). The CLSM data were analyzed and are presented as given in B and C. Green, CdtV-B; red, OMV or compartment-specific marker proteins, as indicated; blue, nuclei; yellow, colocalized green and red signals .  Immunoblot detection of CdtV-A (E) and CdtV-C (F) in isolated endoplasmic reticulum (ER) and lysosomes (Lyso) of HBMEC incubated with the indicated OMVs for 20 h. BL21(cdtV-ABC) OMVs were a positive control and BL21(pET23) OMVs and untreated cells (no OMV) negative controls; lanes OMV ctrl contain 5791/99 OMVs without cells.HBMEC were preincubated  with OMVs from the indicated recombinant strains and postincubated at 37°C for 90 min to 20 h. (A) OMV uptake was determined by CLSM after 4 h. Green, OMVs; red, actin; blue, nuclei. Confocal Z-stack projections are included at upper/right sides. Crosshairs show the position of the xy and yz planes. Scale bars are 10 μm.  Colocalization of OMVs and OMV-delivered CdtV-A and CdtV-C expressed either singly (OMVs BL21((TIF)Click here for additional data file.S29 Fig(A) Images shown in (TIF)Click here for additional data file.S30 Fig(A) Images shown in (TIF)Click here for additional data file.S31 Fig(A) CLSM of HBMEC preincubated with OMVs 5791/99 for 30 min at 4°C, and postincubated at 37°C for the times indicated. The indicated single fluorescence channels are shown in the right panels and the merged images in the left panels ; red, compartment-specific marker proteins; blue, nuclei; yellow, colocalized green and red signals). The percentages of EHEC-Hly colocalizations with compartment-specific marker proteins (white numbers) and with nucleus (blue numbers in panels ER) were calculated with the BioImageXD6 tool. Scale bars are 10 μm. (B) Graphical summary of EHEC-Hly colocalizations with subcellular compartments based on CLSM data shown in A, and with OMVs (based on data shown in (TIF)Click here for additional data file.S32 FigFlow cytometry histograms of (A) HBMEC and (C) Caco-2 cells treated for 24 h to 96 h with O157 OMVs or OMVs from CdtV-positive control strain TA153. (B) HBMEC and (D) Caco-2 cells treated for 24 h or 96 h with CdtV-negative OMVs from strain TA154 (vector control) or left untreated (negative controls). Positions of the G1 (2n DNA) and G2 (4n DNA) peaks are indicated in the first histograms in A and C. The proportions (%) of cells in G1 and G2 cell cycle phase, respectively, are shown in all histograms. Arrows depict sub-G1 populations (apoptotic cells).(TIF)Click here for additional data file.S33 Figstx2a) or TA153 OMVs containing the indicated amounts of CdtV. Proportions of cells in G2 arrest (4n DNA content) were determined by flow cytometry. Data are means ± standard deviations from three independent experiments. *p < 0.05, **p < 0.01 or ***p < 0.001 (one-way ANOVA) for G2 arrest caused by the indicated CdtV doses compared to untreated cells (cell control).(A) Caco-2 cells, (B) HBMEC, and (C) HRGEC were incubated for 24 h (HRGEC) or 48 h  with two-fold dilutions of O157 Click here for additional data file.S34 Fig405 absorbance values of sample-treated cells with those of untreated cells. Staurosporine (1 μM) was a positive control and OMV buffer and free Stx2a which is not internalized by DLD-1 cells .(A) Presence of Stx2a and Stx1a in EDL933 OMVs as detected by immunoblot. (B) EDL933 OMVs are internalized by Gb3-negative DLD-1 cells after 4 h of incubation as determined by CLSM. Green, OMVs; red, actin; blue, nuclei. Confocal Z-stack projections are included at upper/right sides. Crosshairs show the position of the xy and yz planes. (C) Colocalizations of EDL933 OMVs and OMV-delivered Stx2a with late endosomes/lysosomes (Le-Lyso) and the endoplasmic reticulum (ER) after 4 h of incubation of the OMVs with DLD-1 cells. The indicated single fluorescence channels are shown in the right panels and the merged images in the left panels . The percentages of colocalization of the respective signals (white numbers) were calculated with the BioImageXD6 tool . Scale bars in B and C are 10 μm.  Immunoblot detection of Stx2a (D) and Stx1a (E) in isolated endoplasmic reticulum (ER) and lysosomal (Lyso) fractions of DLD-1 cells which were incubated for 4 h with EDL933 OMVs or left untreated (no OMV) (negative control). EDL933 OMVs without cells were a positive control (OMV ctrl). (F) Apoptosis caused in DLD-1 cells by the indicated samples after 96 h of incubation as determined by Cell Death Detection ELISA. Enrichment factors were calculated by dividing OD-1 cells  negative(TIF)Click here for additional data file.S35 FigSamples were separated by SDS-PAGE and subjected to immunoblot with rabbit anti-Stx2a antibody . Sizes of Stx2a A and B subunits are indicated on the right side.(TIF)Click here for additional data file.S36 FigE. coli O157 LPS antibody and Alexa Fluor 488-conjugated goat anti-rabbit IgG or with mouse anti-E. coli O157 antibody and Cy3-conjugated goat anti-mouse IgG to detect OMVs. To detect virulence proteins, cells were stained with anti-Stx2a, anti-CdtV-A, -B, -C, or anti-EHEC-Hly rabbit antibodies and Alexa Fluor 488-conjugated goat anti-rabbit IgG. (B) HBMEC were incubated with 5791/99 OMVs for 20 h and stained with Cy3-conjugated goat anti-mouse IgG and with Alexa Fluor 488-conjugated goat anti-rabbit IgG, or with Alexa Fluor 488-conjugated goat anti-rat IgM in the absence of first antibodies. (C) DLD-1 cells were incubated for 4 h with 5791/99 OMVs and stained with Alexa Fluor 488-conjugated goat anti-rat IgM or with Cy3-conjugated goat anti-mouse IgG and Alexa Fluor 488-conjugated goat anti-rabbit IgG in the absence of first antibodies. Nuclei in all panels were stained with DRAQ5. Scale bars are 10 μm.(A) HBMEC were incubated for 20 h with OMV buffer (20 mM TRIS-HCl) instead of OMVs. Cells were stained with rabbit anti-(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S1 Text(PDF)Click here for additional data file.


Therefore, p19Arf-/-Rag1-/- (AR) mice were crossed with Aid-deficient mice (ARA). Surprisingly, loss of Aid expression in pro-B cells accelerated pro-B ALL incidence from 30% (AR) to 98% (ARA). This effect was Aid dose dependent, since Aid+/- animals of the same background displayed a significantly lower incidence (83%). Furthermore, B-cell-specific Aid up-regulation was observed in Aid-competent pro-B ALLs. Additional whole exome/sanger sequencing of murine pro-B ALLs revealed an accumulation of recurrent somatic Jak3  and Dnm2 (p.G397R) mutations, which highlights the importance of active IL7R signaling in the pro-B ALL blast cells. These findings were further supported by an enhanced proliferative potential of ARA pro-B cells compared to Aid-competent cells from the same genetic background. In summary, we show that both Aid and Rag1 act as a negative regulators in pro-B cells, preventing pro-B ALL.Activation-induced cytidine deaminase (AID) is essential for somatic hypermutation and class switch recombination in mature B-cells, while AID was also shown to play a role in developing pre-BCR/BCR-positive B-cells of the bone marrow. To further elucidate a potential function of Aid in the bone marrow prior to V(D)J-recombination, we utilized an  RAG1 is the key player in this context [Over the last decade, various studies gave novel insights that improved our understanding about the molecular basis of childhood acute lymphoblastic leukemia (ALL) –5. Genom context –11. Unex context , which s context , the act context . By deam context , 16.Rag1-/- background (p19Arf-/-Rag1-/-Aid-/- ARA). Utilizing this model, we were able to assess the influence of Aid at the pro-B cell stage [in vivo evidence, that the combined absence of Aid and Rag1 in tumor prone murine pro-B cells accelerates pro-B ALL incidence, which suggests a functional role of Aid in Rag1 deficient BM pro-B-cells even before the expression of a pre-BCR.While AID expression was shown to have implications in B-cell lymphomas , 18, whill stage , in the ll stage  and in tIn order to explore the physiological relevance of Aid at the pro-B cell stage, and independent of Rag1 off-target activity, we designed a mouse model which allows the investigation of an arrested tumor-prone pro-B cell population in combination with Aid deficiency. Aid knockout mice (Aid-/-) were crossed with p19Arf-/-Rag1-/- (AR) mice to obtain p19Arf-/-Rag1-/-Aid-/- (ARA) and p19Arf-/-Rag1-/-Aid+/- (ARa) mice. P19Arf-/-Rag1-/- mice are known to develop pro-B ALL at a rate of 26% , which w+ B-cells, which co-express a functional IgM heavy chain product [p19Arf-/-Rag1-/- (AR) mice, which were classified as Hardy Fractions B, resembling a pro-B stage [+ BM cells from healthy AR mice (data not shown), significant Aid expression was observed in AR pro-B ALLs  while differences in IL-7 sensitivity could not be observed between the groups after 24 hours of IL-7 withdrawal . Nevertheless, the proliferation rate of healthy ARA B220+ B-cells was significantly higher  compared to AR B220+ B-cells  in the DNA synthesis phase (S-phase)  Figure . Since tet al., showing that AID gene dosage regulates central B-cell tolerance in humans [in vivo and in vitro [In the BM, AID induction through inflammatory stimuli drives the clonal evolution of B-ALL , while in humans . AID-medin vitro , 37–39, in vitro , our datIn normal pro-B and pre-BI cells AID and RAG1 are repressed by IL-7R-signaling (active JAK-STAT/PI3K-AKT) –42, sincDnm2 and Jak3. Loss of function mutations in Dnm2 have recently been linked to increased IL-7R density on the surface of murine Lmo2 transgenic T-ALLs [Although it was shown that the IL-7R safeguards pre-B cells against premature AID activation , pro-B cc T-ALLs . By up-rc T-ALLs . Additioc T-ALLs , render + sorted cells from healthy ARA mice showed increased proliferative potential, when compared to Aid competent AR mice, suggests that loss of Aid already affects certain intrinsic cell properties of IL-7 dependent pro-B cells. So far, increased proliferation of Aid-deficient cells has been shown to be significant in immature B-cells, which displayed an advantage through increased self-renewal capacity, seen by comparing Aicda+/+ against Aicda-/- stem cell populations [in vitro results by elucidating the differences between healthy AR and ARA pro-B cells on a general gene expression level, or even on an epigenetic basis.The observation that B220ulations . TherefoWhile it was previously shown that AID expression is regulated by PI3K , these fIn summary, we propose Aid as a negative regulator in Rag1 deficient pro-B cells, whereby Aid clears aberrant pro-B cells that are leukemia prone. Therefore, we extend the role of Aid in clearing autoreactive B-cells to establish B-cell tolerance, to the negative regulation of pro-B cells that are susceptible to malignant transformation in the specific context of Rag1 deficiency and pro-B ALL development.p19Arf-/-/Rag1-/- mice were generously provided by Marina Cavazzana [p19Arf-/-/Rag1-/-/Aid-/- and p19Arf-/-/Rag1-/-/Aid+/-. Recipient C57BL/6 wildtype mice were obtained from Janvier Laboratories. Animals were housed in a specific, pathogen-free animal facility at the ZETT/Heinrich-Heine University Duesseldorf and experiments were performed in compliance with the German State Agency for Nature, Environment and Consumer Protection (LANUV).The avazzana , Imagineavazzana  kindly p+ (Miltenyi Biotec) from mouse BM were cultured on Mitomycin-C-treated ST2 feeder cells in IMDM medium containing IL-7 (5 ng/ml) (R&D Systems).Iscove's modified Dulbecco's medium supplemented with 50 μM β-mercaptoethanol, 1 mM L-glutamine, 2% heat-inactivated fetal calf serum, 1mM penicillin-streptomycin (Life Technologies) and 0.03% (w/v) primatone RL (Sigma) was used for pro-B cell culture experiments. Pro-B cells isolated by MACS-sorting for B2205 healthy pro-B cells of p19Arf-/-Rag1-/-, p19Arf-/-Rag1-/-Aid+/- and p19Arf-/-Rag1-/-Aid-/- mice were seeded on day 1 in a 6-well plate. Cell numbers were determined every 24 hours using a Vi-Cell XR (Beckman Coulter). Cells from day 3 were further analyzed for proliferation with the FITC BrdU Flow Kit (BD Biosciences). The cells were pulse labeled with BrdU for 1 hour and analyzed on a FACSCaliburTM Flow Cytometer according to the manufacturer's protocol .3×10p19Arf-/-Rag1-/-, p19Arf-/-Rag1-/-Aid+/- and p19Arf-/-Rag1-/-Aid-/- mice, switch culture stimulated spleen cells from WT C57BL/6 mice (LPS (20 μg/ml) + IL4 (25 ng/ml) treatment for 4 days) or BaF3 cells (with and without expression of murine Jak3 V670A and after 4h of IL-3 depletion) were lysed in RIPA buffer  containing protease/phosphatase inhibitors (Roche Diagnostics). Immunoblotting was carried out using the following antibodies: anti-AID clone L7E7 1:1000 , anti-phospho-Stat5 D47E7 (Tyr694) 1:1000 , anti-Stat5 1:1000  and anti-β-Actin clone AC-74 1:10000 (Sigma-Aldrich). Detection was carried out using anti-mouse horseradish peroxidase conjugates (Santa Cruz Biotechnology) with an ECL system (Thermo Scientific) (n = 3).Leukemic bone barrow cells from


Activation of NF-E2-related factor 2 (Nrf2) signaling could protect cells from ultra violet (UV) radiation. We aim to provoke Nrf2 activation via downregulating its inhibitor Keap1 by microRNA-141 (“miR-141”). In both human retinal pigment epithelium cells (RPEs) and retinal ganglion cells (RGCs), forced-expression of miR-141 downregulated Keap1, causing Nrf2 stabilization, accumulation and nuclear translocation, which led to transcription of multiple antioxidant-responsive element (ARE) genes . Further, UV-induced reactive oxygen species (ROS) production and cell death were significantly attenuated in miR-141-expressing RPEs and RGCs. On the other hand, depletion of miR-141 via expressing its inhibitor antagomiR-141 led to Keap1 upregulation and Nrf2 degradation, which aggravated UV-induced death of RPEs and RGCs. Significantly, Nrf2 shRNA knockdown almost abolished miR-141-mediated cytoprotection against UV in RPEs. These results demonstrate that miR-141 targets Keap1 to activate Nrf2 signaling, which protects RPEs and RGCs from UV radiation. Existing evidences have shown that excessive ultra violet (UV) radiation may induce direct damages to resident retinal pigment epithelium (RPE) cells (RPEs) and retinal ganglion cells (RGCs) , 2, whici.e. heme oxygenase-1 (HO1), NAD(P)H quinone oxidoreductase 1 (NQO1) and γ-glutamyl cysteine ligase catalytic subunit (GCLC) [NF-E2-related factor 2 (Nrf2) transcriptionally controls the expression of several key anti-oxidative enzymes –13. Exprt (GCLC) , would lt (GCLC) –13. Nrf2t (GCLC) –13. In tt (GCLC) –13. Actit (GCLC) –13. Nrf2t (GCLC) –13.Growth evidences have shown that microRNAs are important in almost all cellular biological processes including oxidative and anti-oxidant responses –16. micrIn the current study, a miR-141 expression vector was constructed . The miR-141 vector was transfected to ARPE-19 cells (“RPEs”) , 17. ThrHO1, NQO1 and GCLC [Keap1 binds to Nrf2, causing it ubiquitination and proteasome degradation . On the and GCLC , were siOur previous studies have demonstrated that Nrf2 activation could protect RPEs from UV , 17, 20.Nrf2 is a well-established anti-oxidant signaling , 21. AboNext, antagomiR-141, an anti-sense RNA molecule complementary to miR-141 , was traThe results above have shown that miR-141 activates Nrf2 signaling and protects cells from UV. Next, we wanted to explore the link between the two. shRNA strategy was utilized to stably knockdown Nrf2 in miR-141-expressing RPEs. Two different Nrf2 shRNAs  with non-overlapping sequences were applied, each of them efficiently downregulated Nrf2 expression (protein and mRNA) in miR-141-expressing RPEs (“L1/2”) Figure . Meanwhiet al., demonstrated that Salvianolic acid A-induced RPE cell protection against oxidative stress requires Nrf2-HO1 signaling [et al., showed that Nrf2 activation by 3H-1,2-dithiole-3-thione (D3T) offers dramatic cytoprotection in UV-radiated RPEs [Nrf2-inducing agents were shown to result in decreased oxidative stress and significant cytoprotection . For exaignaling . Similarted RPEs .HO1, NOQ1 and GCLC). Importantly, activation of Nrf2 by miR-141 expression significantly ameliorated UV-induced oxidative stress and subsequent death/apoptosis of RPEs and RGCs. shRNA-mediated knockdown of Nrf2 in these cells, on the other hand, almost abolished miR-141-cytoproteciton against UV. We therefore conclude that miR-141 protects RPEs and RGCs from UV radiation via activating Nrf2-Keap1 signaling.In this study, we showed that miR-141 expression activated Nrf2 signaling in both RPEs and RGCs. First, miR-141 targeted and degradated Nrf2 inhibitor Keap1 in the above eye cells. Second, Nrf2 was stabilized in the miR-141-expressing cells, which was evidenced by upregulation of Nrf2 protein, but not mRNA. Third, stabilized Nrf2 was shown to translocate to cell nuclei, leading to transcription of several ARE-genes . Other antibodies utilized in this study were purchased from Cell Signaling Tech : HO-1 , NQO1 , Nrf2 , KEAP1 , Erk1/2  and Lamin B1 . Cell culture reagents were obtained Gibco .Culture of human retinal pigment epithelial cells (ARPE-19 cell line) was described previously , 25, 26.Fresh donor human eyes were obtained from ocular trauma patients administrated at the authors institutions. The human retina was separated very carefully from the globe and dissected to give a flat retinal preparation, which was then maintained in the DMEM/HamF12 medium plus 50 μg/mL gentamicin in the 35mm culture dish . Retinal cells were then isolated by enzymatic dissociation , and werThe pre-has-miR-141 was obtained from Applied Biosystem , which was sub-cloned into pSuper-puro-GFP vector  to construct miR-141 expression vector. The miR-141 construct or the empty vector was transfected to the RPEs and RGCs through Lipofectamine 2000 reagents . The transfection took 48 hours. Afterwards, puromycin (1.0 μg/mL) was added to the complete medium to select stable cells, which lasted 3-4 passages, until at least 95% of cells were GFP positive. Control cells were transfected with non-sense scramble microRNA-control (“miR-C”) (Applied Biosystem). Mature has-miR-141-3p expression in the stable cells was tested by RT-qPCR assay.RGCs or RPEs were transfected with 200 pmol of miR-141 inhibitor   or negatΔΔCt method was utilized to quantify mRNA expression, and GAPDH was tested as an internal control. Primers were described in our previous studies [After applied treatment, cellular RNA was extracted via the Trizol reagents (Invitrogen), which was then utilized to perform the reverse transcription assay , 20. The studies , 20 and  studies . Express studies . All theUV radiation (UVB and UVA2) to cultured cells was performed as reported –8, 17.Following treatment, the survival of cells was tested via the routine MTT assay; Cell death was examined by trypan blue staining assay. The detailed protocols were described in our previous publications , 20, 30.After treatment, cell apoptosis was examined by previously described assays, including Histone DNA apoptosis enzyme-linked immunosorbent assay (ELISA) assay and the caspase-9 activity assay. Detailed protocols were described in our previous studies , 31–33.As previously reported , 34, theWestern blot assay was performed as described , 20, 31.The two non-overlapping lentiviral Nrf2 shRNAs, Nrf2 shRNA-A (sc-37030-V) and Nrf2 shRNA-B (sc-44332-V), along with lentiviral scramble control shRNA, were purchased from Santa Cruz Biotech (Santa Cruz); The lentiviral shRNA (10 μL/mL) was added directly to cultured cells for 36 hours. Cells were then subjected to puromycin (1.0 μg/mL) selection for another 6 days. Nrf2 expression in the stable cells was tested by Western blot and RT-qPCR assays.p < 0.05.Quantitative results were normalized to the control values of each assay, and were presented as mean ± standard deviation (SD). Data were analyzed by one-way ANOVA. Significance was chosen as


DNMT1 promoter and drives DNMT1 transcription. We also show that targeting MUC1-C substantially reduces gene promoter-specific DNA methylation, and derepresses expression of tumor suppressor genes, including CDH1, PTEN and BRCA1. In support of these results, we demonstrate that combining GO-203 with the DNMT1 inhibitor decitabine is highly effective in reducing DNMT1 levels and decreasing AML cell survival. These findings indicate that (i) MUC1-C is an attractive target for the epigentic reprogramming of AML cells, and (ii) targeting MUC1-C in combination with decitabine is a potentially effective clinical approach for the treatment of AML.Aberrant DNA methylation is a hallmark of acute myeloid leukemia (AML); however, the regulation of DNA methyltransferase 1 (DNMT1), which is responsible for maintenance of DNA methylation patterns, has largely remained elusive. MUC1-C is a transmembrane oncoprotein that is aberrantly expressed in AML stem-like cells. The present studies demonstrate that targeting MUC1-C with silencing or a pharmacologic inhibitor GO-203 suppresses DNMT1 expression. In addition, MUC1 expression positively correlates with that of DNMT1 in primary AML cells, particularly the CD34+/CD38− population. The mechanistic basis for this relationship is supported by the demonstration that MUC1-C activates the NF-κB p65 pathway, promotes occupancy of the MUC1-C/NF-κB complex on the  NPM1 and CEBPA, among others , 3028, 3DNMT1 promoter in AML cells, we transfected THP-1/CshRNA and THP-1/MUC1shRNA cells with a pDNMT1-Luc reporter that contains a consensus NF-κB binding site (GGGGTATCCC) at positions −833 to −824 upstream of the transcription start site  and MOLM-14 (ATCC) cells were cultured in RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, 100 ug/ml streptomycin and 2 mM L-glutamine. Authenticity of the cells was confirmed by short tandem repeat (STR) analysis . Cells wCells were lysed using NP-40 lysis buffer containing protease cocktail inhibitor (Thermo Scientific), DTT and PMSF. Soluble proteins were analyzed by immunoblotting with anti-MUC1-C antibody (Thermo Scientific), anti-DNMT1 (Abcam), anti-NF-κB p65 (Santa Cruz Biotechnology), anti-phospho-NF-κB p65, anti-IKKβ, anti-phospho-IKKβ, anti-E-cadherin, anti-PTEN , anti-BRCA1 (Santa Cruz Biotechnology) and anti-β-actin (Sigma). Detection of immune complexes was achieved using horseradish peroxidase-conjugated secondary antibodies and enhanced chemuluminescence  , 46.t-test.Quantitative qRT-PCR analysis was performed as described . BrieflyRenilla-Luc in the presence of Lipofectamine 3000 Reagent (Invitrogen). At 24 h after transfection, the cells were lysed with passive lysis buffer and analyzed using the Dual Luciferase assay kit (Promega). Firefly luciferase values were normalized to that for Renilla-Luc. The normalized values for pDNMT1-Luc were then compared to that obtained with pGL3.Cells cultured in 96-well plates were transfected with the pGL3-Basic Luc vector, wild-type pDNMT1-Luc or mutant pDNMT1-Luc (GGGGTATCCC→CCCCTATCCC) and SV-40 DNMT1 promoter and control regions are listed in the Soluble chromatin was precipitated with anti-MUC1-C (Thermo Scientific) or a control non-immune IgG (Santa Cruz Biotechnology). For re-ChIP analysis, anti-NF-κB p65 (Santa Cruz Biotechnology) complexes from the primary ChIP were eluted and re-immunoprecipitated with anti-MUC1-C. For real-time ChIP qPCR, the SYBR green system was used with the ABI Prism 7300 sequence detector (Applied Biosystems). Data are reported as relative fold enrichment . PrimersThe EpiTect Methyl II PCR System (Qiagen) was used to assess the methylation of TSG promoters according to the manufacturer's protocol.CDH1 promoter region using primers listed in For MeDIP analysis, genomic DNA was extracted using the DNAeasy kit (Qiagen), digested with MseI (300-1000 kbp fragments) and immunoprecipitated using an anti-5-methylcytidine (5-mC) antibody (Active Motif). The resulting enriched methylated DNA in the immunoprecipitated fraction was subjected to PCR analysis of Primary AML cells from patients were isolated as described . BrieflyCells were seeded in a 96-well plate and treated with GO-203  and/or dIntracellular staining was performed as described , 47. BriCDH1 (cg16739895) gene in AML (n = 62) and normal bone marrow progenitor cells  were compared using the Student's t-test.Datasets of AML patient samples were downloaded from gene expression omnibus (GEO) under the accession numbers GSE17855 and GSE30375. Data was RMA normalized (Bioconductor) and Log-2 transformed . Multipl


White blood cells did not exceed 12.0x109/L, while the fraction of blast cells increased to 10%. Elevated levels of teardrop cells were observed and the nucleated red blood cell count gradually increased from non-detectable to 2.4x1012/L. Recent peripheral blood smears showed bi- and tri-nucleated red blood cells, and even more notably, erythroid precursors with internuclear chromatin and cytoplasmic bridging An 84-year-old male diagnosed with primary myelofibrosis based on WHO grade 2-3 fibrosis