MCAK) core promoter from ‒266 to +54, relative to the transcription start site, has been determined. The MCAK mRNA and protein levels were 2.1- and 3.0-fold higher, respectively, in HCT116 (p53‒/‒) than in HCT116 (p53+/+) cells. Enforced down-regulation of p53 levels either in HCT116 (p53+/+) cells by p53 RNAi treatment or in MCF-7 cells using shRNA for p53 (shp53) resulted in a remarkable increase in the MCAK protein level. Site-directed mutagenesis and ChIP analyses showed that p53-mediated repression of the MCAK core promoter activity was not directly exerted by p53-binding to putative p53-response elements (p53-RE1 at −173/−166 and p53-RE2 at −245/−238), but indirectly by attenuating Sp1 binding to GC-motifs (GC1 at −93/−84 and GC2 at −119/−110). Treatment of HEK-293 cells bearing the MCAK core promoter-reporter (pGL2-320-Luc) with mithramycin A, which down-regulates Sp1 gene expression, reduced the promoter activity as well as endogenous MCAK levels. Exposure of HCT116 (p53+/+) cells to nutlin-3a, a validated activator of p53, caused a simultaneous reduction in Sp1 and MCAK protein levels, but not in HCT116 (p53−/−) cells. In contrast to wild-type (wt)-p53, tumor-derived p53 mutants  failed to repress the Sp1-dependent activation of the MCAK promoter and to down-regulate endogenous levels of Sp1 and MCAK proteins. Collectively, these findings demonstrate that p53 can repress MCAK promoter activity indirectly via down-regulation of Sp1 expression level, and suggest that MCAK elevation in human tumor cells might be due to p53 mutation.The repressive role of p53 on the human mitotic centromere-associated kinesin ( MCAK has been the best-characterized member of the kinesin-13 family in terms of subcellular localization and microtubule-depolymerizing activity regulation. During cell cycle progression, MCAK is dominantly located at the centrosomes, centromere and kinetochores, spindle poles, and midbody , hSgo2, Chromosomal aneuploidy, an abnormality in the number of chromosomes, is considered one of the most common features of cancer. Defects in segregation fidelity during mitosis can cause chromosome instability (CIN), which is defined as persistent mis-segregation of whole chromosomes during cell division . SeveralMCAK promoter region from −1097 to +54 (pGL2-1151-Luc) relative to the transcription start site in order to elucidate the transcriptional regulation of the human MCAK gene [MCAK core promoter region (−266/+54) in HEK-293 cells showed the GC-motifs (GC1 at −93/−84 and GC2 at −119/−110) are crucial for promoter activation. Additionally, the analysis with the serial deletion constructs of the MCAK promoter suggested that the promoter region (–476/–267) possessing a putative p53-response element  might be associated with promoter repression. The putative p53-RE appeared to resemble the consensus p53 binding sequence, consisting of two repeats of a 10-bp motif, 5′-PuPuPuC(A/T)(T/A)GPyPyPy-3′, separated by a 0- to13-bp spacer [et al. also demonstrated that ectopic overexpression of p53 caused a significant repression of MCAK gene expression in human breast cancer cells [In a previous study, we cloned the human CAK gene . Mutatiop spacer . Previouer cells . HoweverMCAK core promoter region (−266/+54), which was previously shown to contain two Sp1-binding motifs (GC1 at −93/−84 and GC2 at −119/−110) for positive regulation of the promoter activity [MCAK core promoter activity is repressed through p53 binding to putative p53-RE1 and p53-RE2. Additionally, we investigated whether p53-dependent down-regulation of Sp1 level contributes to p53-mediated repression of the MCAK promoter activity, because Sp1 gene expression was reported to be negatively regulated by p53 [MCAK promoter activity is not exerted by p53 binding to its response elements (p53-REs), but by p53-dependent down-regulation of Sp1 which is crucial for transcriptional activation of the MCAK core promoter. The results also suggest that tumor-associated elevation of MCAK levels might result from p53 mutation.Recently, we found two additional putative p53-response elements (p53-RE1 at −173/−166 and p53-RE2 at −245/−238) within the human d by p53 . The resWAF1/Cip1, anti-Sp1, and anti-β-actin antibodies were purchased from Santa Cruz Biotechnology . Anti-p-p53 (Ser-15) and anti-poly (ADP-ribose) polymerase (PARP) antibodies were obtained from Cell Signaling Technology , and anti-p-Sp1(Thr-453) antibody was obtained from Abcam . Human colorectal adenocarcinoma cell lines HCT116 (p53+/+) and p53-null HCT116 (p53−/−) were kindly provided by Dr. B. Vogelstein . Human embryonic kidney cell line HEK-293, human cervical cancer cell line HeLa, and human colon adenocarcinoma cell line SW480 were purchased from ATCC . Human breast adenocarcinoma cell line stably transfected with shRNA control vector (MCF-control) and MCF7 cells stably transfected with shp53 expression vector (MCF7-shp53) were kindly provided by Chris R. Harris . Cells were grown in Dulbecco's modified Eagle's medium  supplemented with 10% fetal calf serum and 100 μg/mL of gentamycin.All restriction and modifying enzymes, reporter gene vectors  were purchased from Promega . An ECL Western Blotting Kit was purchased from GE Healthcare Life Sciences  and a ChIP Assay Kit was purchased from EMD Millipore . Mithramycin A and normal mouse IgG were`purchased from Sigma-Aldrich . A rabbit polyclonal anti-human MCAK antibody was raised against the N-terminal region (187 amino acids) of human MCAK . Anti-CDMCAK promoter-reporter plasmids containing DNA fragments (−476/+54) or (−266/+54) of the 5'-regulatory region was previously described [V143A, pCMV-Neo-mutant p53R248W, and pCMV-Neo-mutant p53R273H) were provided by Dr. B. Vogelstein (Johns Hopkins University). The mdm2-luciferase construct as a p53-response positive reporter control was provided by Dr. M. Oren . The pG5-5x(GC)-Luc construct containing five tandem Sp1-binding sites was provided by Dr. Man-Wook Hur [Generation of escribed . The expWook Hur . The expWook Hur .HEK-293 or HCT116 cells were transfected by using Lipofectamine 2000 reagent (Invitrogen). The pSV-β-galactosidase control vector was used to normalize the transfection efficiency. After transfection for 36−48 h, luciferase activity was measured using the Luciferase Reporter Assay System (Promega) and β-galactosidase activity was determined using the Galacto-Light Plus Kit .5'-ACGGAATTCCTGAGATATGCAGAC-3'; MCAK-reverse, 5'-AATGGAT-CCTCAATGCTTGGCTTGCTG-3'; p53-forward, 5'-ACCACCATCCACTACAACTAC-3'; p53-reverse, 5'-AAGGATCCTCAGTCTGAGTCAGGCC-3'; p21-forward, 5'-GTTCCTTGTGGAGCCG-GAGC-3'; p21-reverse, 5'-GGTACAAGACAGTGACAGGTC-3'; glyceraldehyde 3-phosphate dehydrogenase (GAPDH)-forward, 5'-CCACTGGCGTCTTCACCAC-3'; GAPDH-reverse 5'-CCTG-CTTCACCACCTTCTTG-3'. The PCR products were electrophoresed using 1.5% agarose gels, and visualized after ethidium bromide staining under UV light.Total RNA (10 μg) was transcribed using Superscript II (Invitrogen) and random hexamer primers, according to the manufacturer’s instructions. PCR amplification was performed using AccuPrime Tag DNA polymerase high fidelity (Invitrogen) and specific primers. The following primers were used for PCR: MCAK-forward, -/-) cells transiently transfected with wild-type p53-expression vector (pCMV-Neo/p53) or empty-vector (pCMV-Neo), and was immunoprecipitated with anti-Sp1 antibody. Normal mouse IgG was also included as a negative control. PCR-amplification of the MCAK 320-bp promoter region (−266/+54) was performed using the SacI-forward primer (GAGCTCGGTGACCGAGGAC-AAT-3′5′-AG) and the BglII-reverse primer-2 (AGATCTCGGAGAGTCAGCAAGGAAGAG-3′5′-AG).Formaldehyde cross-linking and ChIP assay were conducted using a ChIP Assay Kit as described previously . Briefly+/+) cells, Stealth RNAi for p53  was transfected into the cells at a concentration of 200 nM by using Lipofectamine 2000. Cell lysates were prepared 48 h after transfection. Synthetic Stealth RNAi duplex for p53 and scrambled Stealth RNAi duplex (Cat. #12395–100) for a negative control were designed and supplied by Invitrogen.To reduce the level of p53 protein in HCT116 . One-way ANOVA followed by Dunnett’s multiple comparison test was also used for statistical analysis using the SPSS Statistics version 23 .Unless otherwise indicated, each result in this paper is representative of at least three separate experiments. Statistical analyses were performed using the Students t-test to evaluate the significance of differences between two groups and one-way ANOVA for between three or more than three groups. In all graphs, * indicates +/+) and HCT116 (p53−/−) cells. The level of p21WAF1/Cip1, the expression of which is known to be activated by p53 [−/−) cells, respectively, in comparison to those in HCT116 (p53+/+) cells (WAF1/Cip1 protein became barely detectable in the HCT116 (p53−/−) cells. These results indicate that the expression of the MCAK gene could be repressed at the transcriptional level by the function of p53.To examine the involvement of p53 as a transcriptional repressor in the MCAK gene expression, the MCAK-specific mRNA and protein levels were compared between human colorectal adenocarcinoma HCT116  cells . Additio+/+). Although treatment with the control scramble RNAi did not influence p53 protein level in the cells, treatment with two different kinds of p53-specific RNAi could reduce p53 protein levels by 0.16- and 0.13-fold  involved in promoter repression, the proximal promoter sequences (−476/+54) were analyzed using two transcription factor binding prediction programs (http://www.cbil.upenn.edu/cgi-bin/tess/tess and http://motif.genome.jp). As shown in MCAK proximal promoter sequences. To investigate the functional role of the three putative p53-REs in the repression of MCAK promoter activity, the promoter activities of pGL2-530-Luc (the reporter construct containing the promoter region from −476 to +54), pGL2-530-Luc with p53-RE3 mutation, and pGL2-320-Luc (the reporter construct containing the promoter region from -266 to +54) were compared in HCT116 (p53+/+) and HCT116 (p53−/−) cells. As shown in CACCGCGCCCGGCCAAGTCTC → CACCGCGCCCGGCaggagCTC) nor the deletion of p53-RE3 produced a remarkable enhancement in the promoter activity in HCT116 (p53+/+) cells. The luciferase activities of these reporter constructs  were enhanced ~16.6-, 14.3-, and 9.0-fold in HCT-116 (p53−/−) cells, respectively, as compared with the activities in HCT116 (p53+/+) cells. However, the enhanced activities of the individual reporter constructs were abrogated when these reporter constructs were cotransfected with wt-p53 expression vector (pCMV-Neo-wt-p53) into HCT116 (p53−/−) cells (MCAK core promoter activity.To examine whether the human −) cells . These rMCAK core promoter, mutant constructs of pGL2-320-Luc, possessing site-directed mutations in p53-RE1 at −182/−155 and p53-RE2 at −256/−228 were transfected into HCT116 (p53+/+) and HCT116 (p53−/−) cells, and then individual promoter activities were analyzed. The nucleotide sequence, putative transcription factor binding sites, and transcription start site (TSS) are shown in the MCAK 320-bp promoter region (−266/+54) (CAGACGCAT-GGGCGGTGCCAAGCGTAGG → CAGACGCATGGtttGTGCCAAGCGTAGG) and in p53-RE2 at −256/−228 (ATACCAGAAATGGGAGCCCC-AAACCCCG → ATACCAGAAATGttgaCCCC-AAACCCCG) failed to influence the promoter activity in both HCT116 (p53+/+) and HCT116 (p53−/−) cells (+/+) cells were reduced to 19−21% of those detected in HCT116 (p53-/-) cells. These results demonstrate that there was no contribution from either p53-RE1 or p53-RE2 to p53-mediated repression of the MCAK core promoter activity. On the other hand, mutation in the distal GC-motif (GC2) at −119/−110 (GCAGGGCGCC→ GCAGttaGCC) completely abrogated the promoter activity irrespective of the presence of p53, whereas mutation in the proximal GC-motif (GC1) at −93/−84 (GAGGGCGGGG→ GAGttaGGGG) resulted in a decrease in the promoter activity to ~85% and ~77% of the pGL2-320-Luc activity in HCT116 (p53−/−) and HCT116 (p53+/+) cells, respectively. These results indicate that although both GC-motifs (GC1 and GC2) were required for positive regulation of the MCAK promoter in HCT116 cells, the GC2-motif was more critical than GC1 for the MCAK promoter activity.To investigate the involvement of p53-RE1 and p53-RE2 in the p53-mediated repression of the 266/+54) . Mutatio−) cells . At the MCAK core promoter, the effect of p53 on Sp1-binding to the MCAK core promoter was investigated by ChIP assay in HCT116 (p53−/−) cells following transfection with pCMV-Neo-wt-p53. As shown in −/−) resulted in a remarkable decrease in the level of Sp1-binding to GC-motifs located within the MCAK core promoter. Western blot analysis also showed that the expression levels of endogenous Sp1 and MCAK proteins were reduced 0.28- and 0.38-fold by the ectopic expression of wt-p53 -Luc, pGL2-mdm2-Luc or pGL2-320-Luc into HCT116 (p53−/−) cells. After incubation for 48 h, the effects of Sp1 and p53 expressions on individual promoter-reporter constructs were measured. As shown in −/−) cells after transfection with individual expression plasmids. Additionally, the level of Sp1 protein markedly decreased when ectopically expressed wt-p53 was accumulated in HCT116 (p53−/−) cells. These data indicate that the expressed wt-p53 as well as Sp1 following transfection into HCT116 (p53−/−) cells was functional, and support the notion that p53-mediated repression of the MCAK core promoter activity was indirectly exerted by p53-dependent down-regulation of Sp1 level.To confirm the roles of Sp1 and p53 in regulation of the WAF1/Cip1, PUMA, and BAK genes [WAF1/Cip1 level, consistent with a previous report demonstrating mithramycin-mediated inhibition of Sp1 binding to the p21WAF1/Cip1 promoter [To verify the contribution of Sp1 to MCAK gene expression, we investigated the effect of mithramycin A on the Sp1-dependent activation of pGL2-320-Luc, because mithramycin A was previously reported to not only down-regulate Sp1 at the transcriptional level but also inhibit sp1 binding to the Sp1 target genes including the p21AK genes . When HEpromoter .MCAK core promoter activity in HCT-116 (p53+/+) and HCT-116 (p53−/−), using nutlin-3a that blocks the p53-mdm2 interaction to stabilize and activate p53 and thus induce the expression of the p21WAF1/Cip1 gene [+/+) cells treated with nutlin-3a (1−10 μM) exhibited a concomitant accumulation of p53 and p21WAF1/Cip1 proteins. In accordance with the nutlin-3a-induced enhancement of p53 level in HCT116 (p53+/+) cells, the Sp1 and MCAK levels were significantly reduced. However, these nutlin-3a-induced alterations including the decrease in Sp1 and MCAK levels were not detected in HCT116 (p53−/−) cells. Since the human Sp1 gene was reported to be positively regulated by Sp1 protein through its binding to multiple GC-motifs located within the proximal promoter region [Sequentially, we further compared the contribution of down-regulation of Sp1 level to p53-mediated repression of the ip1 gene , 35. As r region , the p53MCAK promoter is associated with down-regulation of the cellular Sp1 level by p53; however, a concurrent inhibition of Sp1 activity by p53 via protein-protein interaction cannot be excluded.These previous and current results indicate that p53-mediated repression of the Sp1-dependent activation of the MCAK promoter activity as efficiently as wt-p53.Although current results shows that the human MCAK gene is a target for p53-mediated repression, the mechanism for tumor-associated elevation of MCAK levels still remains obscure. As one of the most frequent genetic alterations detected in human cancer cells (>50%), several missense mutation patterns have been found in p53 , 38. TheMCAK core promoter activity as efficiently as wt-p53, pGL2-320-Luc was cotransfected with individual expression vectors for wt p53, p53V143A, p53R248W, and p53R273H into HCT116 (p53−/−) cells and then the luciferase activity of pGL2-320-Luc was measured. As shown in −/−) cells following cotransfection with pCMV-Neo-wt p53. However, cotransfection of pGL2-320-Luc with each p53 mutant expression vector for p53V143A, p53R248W, or p53R273H, resulted in only ~50%, ~40%, and ~48% reduction of the luciferase activity, respectively. These results demonstrate that mutant p53V143A, mutant p53R248W, or mutant p53R273H repressed the MCAK promoter activity to a much lower extent as compared to wt-p53. Under these conditions, western blot analysis also showed that p53 mutants failed to repress the endogenous MCAK level as efficiently as wt-p53 in HCT116 (p53−/−) cells (−/−) cells were similar in their quantities, the endogenous MCAK level in HCT116 (p53−/−) cells was reduced to ~22% in the presence of wt-p53, but the levels of 78%, 88%, and 74% were maintained in the presence of mutants p53V143A, p53R248W, and p53R273H, respectively. At the same time, the endogenous Sp1 level in HCT116 (p53−/−) cells appeared to decrease to ~19% in the presence of wt-p53, whereas it decreased to 52%−57% in the presence of the p53 mutants. Additionally, only wt-p53, in contrast to the p53 mutants, was able to enhance the expression level of p21WAF1/Cip1 in HCT116 (p53−/−) cells, confirming that the DNA-binding domain of p53 mutants was not functional. These results demonstrate that wt-p53, but none of the p53 mutants, could efficiently repress the MCAK core promoter activity via down-regulating the expression level of Sp1 that is crucial for the MCAK promoter activity. Consequently, these results suggest that the elevation of MCAK levels in tumors is due to the functional impairment of p53 resulting from tumor-associated mutations, which have frequently been observed in many types of tumors [To examine whether tumor-derived p53 mutants are able to down-regulate the −) cells . While tf tumors , 38.V143A, p53R248W, and p53R273H), alterations of which reside in the DNA-binding domain [V143A, p53R248W, and p53R273H mutants in the nuclear fraction were ~34.5%, ~30.4%, and ~28.7%, respectively, indicating that nuclear localization of p53 could be impaired by mutation of the DNA-binding domain of p53. Under these conditions, the p53 mutations appeared to restore the wt-p53-dependent decrease in the cellular Sp1 level; however, the p53 mutations did not influence the nuclear localization of Sp1 or its phosphorylation at Thr-453. Consequently, these results indicate that p53 mutations  could inhibit the nuclear translocation of p53, and thus reduce both p53-interactions with Sp1 and p53-mediated down-regulation of the Sp1 level in the nucleus.Previously, it has been shown that p53 mutations mainly reside in the DNA-binding domain , 40. Mutg domain , 38, couOn the other hand, ectopic expression of wt-p53 in HeLa and SW480 cells, both of which are known to carry compromised p53 , 42, dowMCAK core promoter  is repressed by wt-p53, whereas tumor-derived p53 mutants, such as p53V143A, p53R248W, and p53R273H, cannot repress the MCAK promoter as efficiently as wt-p53. The tumor suppressor protein p53-mediated repression of the MCAK promoter was not directly mediated by p53-binding to p53-REs in the promoter, but indirectly by interfering with the role of transcriptional activator Sp1 which could bind to the GC-motifs within the core positive regulatory region of the MCAK promoter. The p53-mediated repression of the Sp1-dependent activation of the MCAK promoter is caused by down-regulation of the Sp1 level by p53 at the transcriptional level. Tumor-associated elevation of MCAK levels appears to be mainly due to malfunction of tumor-derived p53 mutants, which failed to repress the MCAK promoter as efficiently as did wt-p53.In conclusion, this is the first report to demonstrate that the human


Bacillus anthracis, disrupts endocytic recycling mediated by the small GTPase Rab11. As a result, cargo proteins such as cadherins fail to reach inter-cellular junctions. In the present study, we provide further mechanistic dissection of Rab11 inhibition by EF using a combination of Drosophila and mammalian systems. EF blocks Rab11 trafficking after the GTP-loading step, preventing a constitutively active form of Rab11 from delivering cargo vesicles to the plasma membrane. Both of the primary cAMP effector pathways -PKA and Epac/Rap1- contribute to inhibition of Rab11-mediated trafficking, but act at distinct steps of the delivery process. PKA acts early, preventing Rab11 from associating with its effectors Rip11 and Sec15. In contrast, Epac functions subsequently via the small GTPase Rap1 to block fusion of recycling endosomes with the plasma membrane, and appears to be the primary effector of EF toxicity in this process. Similarly, experiments conducted in mammalian systems reveal that Epac, but not PKA, mediates the activity of EF both in cell culture and in vivo. The small GTPase Arf6, which initiates endocytic retrieval of cell adhesion components, also contributes to junctional homeostasis by counteracting Rab11-dependent delivery of cargo proteins at sites of cell-cell contact. These studies have potentially significant practical implications, since chemical inhibition of either Arf6 or Epac blocks the effect of EF in cell culture and in vivo, opening new potential therapeutic avenues for treating symptoms caused by cAMP-inducing toxins or related barrier-disrupting pathologies.Various bacterial toxins circumvent host defenses through overproduction of cAMP. In a previous study, we showed that edema factor (EF), an adenylate cyclase from  Bacillus anthracis. Indeed, two toxins secreted by this pathogen—edema toxin (ET) and lethal toxin (LT)—can cause death in face of effective antibiotic treatment. ET, a potent adenylate cyclase, severely impacts host cells and tissues through an overproduction of the ubiquitous second messenger cAMP. Previously, we identified Rab11 as a key host factor inhibited by ET. Blockade of Rab11-dependent endocytic recycling resulted in the disruption of intercellular junctions, likely contributing to life threatening vascular effusion observed in anthrax patients. Here we present a multi-system analysis of the mechanism by which EF inhibits Rab11 and exocyst-dependent trafficking. Epistasis experiments in Drosophila reveal that over-activation of the cAMP effectors PKA and Epac/Rap1 interferes with Rab11-mediated trafficking at two distinct steps. We further describe conserved roles of Epac and the small GTPase Arf6 in ET-mediated disruption of vesicular trafficking and show how chemical inhibition of either pathway greatly alleviates ET-induced edema. Thus, our study defines Epac and Arf6 as promising drug targets for the treatment of infectious diseases and other pathologies involving cAMP overload or related barrier disruption.Recent anthrax outbreaks in Zambia and northern Russia and biodefense preparedness highlight the need for new therapies to counteract fatal late-stage pathologies in patients infected with  Bacillus anthracis (B. a.), CyaA from Bordetella pertussis, and ExoY from Pseudomonas aeruginosa. Other toxins modify host proteins to induce cAMP production by endogenous cellular machineries. For example, cholera toxin (Ctx) from Vibrio cholerae, and the related heat-labile toxin from enterotoxigenic Escherichia coli, both ADP-ribosylate the α subunit of trimeric G proteins to stimulate cAMP synthesis by host AC, while pertussis toxin (Ptx) from Bordetella pertussis ADP-ribosylates and inactivates Gi subunits that normally inhibit endogenous ACs .B.a., the etiological agent of anthrax, produces two A-subunit toxins, edema factor (EF) and lethal factor (LF), which are secreted together with a shared B-subunit, protective antigen (PA), and then assemble to form edema toxin (ET) and lethal toxin (LT), respectively . We. We33]. Drosophila wings, both PKA* and Rap1* cause phenotypes similar to that of EF , had little if any effect on the EF phenotype  and Sec (dRip11 ), as wel (dRip11 . We firs (dRip11  in the sDrosophila) results in an EF-resistant association between Rab11* and dRip11DN, it is possible that PKA phosphorylation may contribute to this dissociation.In contrast to full-length dRip11, a truncated dominant-negative form of dRip11 (dRip11DN) retaining only the C-terminal Rab11-binding domain, did notDrosophila salivary glands, EGFP-Rip11 accumulation at cell boundaries was reduced by ET-treatment  cells, in which the role of Rab11 in cadherin trafficking has been well established . Co-exprreatment . Interacreatment , the forreatment . When threatment . CumulatJunctional homeostasis is also established by a balance of Rab11-mediated delivery of junctional cargo and retrieval of proteins via endocytic processes. Arf6, a small GTPase of the Arf subfamily (ADP-ribosylation factors) is involved in early steps of endocytosis from the plasma membrane, exocytosis, and endosomal recycling, and is predominantly localized to the plasma membrane and endosomes . Arf6 acDrosophila. We wondered whether the same might be true in mammalian systems and thus examined the relative contributions of each of these pathways in EF-induced toxemia in various experimental models relevant to B.a. infection in mammals. In HBMECs, ET treatment reduced total cadherin levels and weakened AJs as indicated by staining with an anti-pan-cadherin (p-Cad) antibody , ultDrosophila homolog of human DENND4A) is the only known Rab11-dedicated GEF  [57 [57Droso of Rap1  and play of Rap1  and 59]Drosophil of Rap1 . Thus, RDrosophila salivary glands. This reduction in Rab11 levels is unlikely to derive from transcriptional inhibition, as infection of HBMECs with B. a Sterne did not result in any change in levels of Rab11 transcripts . Similarly, in Drosophila wings, where EF also triggers great reduction in Rab11 protein levels, mRNA transcript levels again were not greatly affected . In HBMECs, where Rab11 levels are reduced by ET treatment, we observed that total levels of cadherins were also severely reduced in ET-treated cells. Although the precise mechanism responsible for the loss of these proteins following ET treatment remains to be explored, it is worth noting that degradation of VE-cadherins has been observed following silencing of Rab11 in human endothelial cells [We previously showed that EF caused a drastic reduction in total Rab11 levels in wing epithelial cells. Here, wal cells , in whicNumerous studies have demonstrated the positive role of physiological induction of cAMP in junction establishment and stabilization, through stimulation of both PKA and Epac ,62. It mThe small GTPase Arf6 initiates retrieval of membrane proteins from cell junctions in a wide variety of cells types . Arf6, aB.a, blocked exocyst-mediated vesicular docking downstream of Rab11 via inhibition of MAPK signaling. It will be interesting to explore how the various effects of EF and LF are integrated to achieve an efficient inhibition of junctional delivery, and if any compound identified in this study can also block some of the downstream effects of LF. Altogether, our study suggests that a broad range of barrier disruptive diseases ranging from cAMP related toxemia to inflammatory autoimmune diseases that involve positive feedback loops between immune activation and barrier disruption, could potentially be treated with compounds that inhibit Arf6 or Epac/Rap1, or by yet undiscovered compounds that may boost Rab11 activity.An emerging lesson from the current and prior studies is that blocking multiple steps of branching pathways that converge on critical nodes in endocytic recycling may allow pathogens to weaken host protective mechanisms that rely on junctional integrity . For exaAll experiments were performed in strict accordance with guidelines from the National Institute of Health and the Animal Welfare Act, approved by the Animal Care and Use Committee of University of California, San Diego and the National Institute of Allergy and Infectious Diseases, National Institutes of Health (approved protocols s00227m and LPD-8E). Anesthesia and euthanasia were performed using Isoflurane and CO2, respectively. All efforts were made to minimize suffering of animals employed in this study.rd chr. # 9791), UAS-Rab11DNYFP (#23261), UAS-CragRNAi (2nd chr. #53261), UAS-CragHA (3rd chr. #58463), UAS-Arf6RNAi (3rd chr. #51417), UAS-PkaDN/CyO (#5282), and Pka-C1B10 (#32018) lines were obtained from Bloomington Drosophila Stock Center (BDSC). UAS-EpacRNAiv50272 and UAS-EpacRNAiv50273 were obtained from Vienna Drosophila Resource Center (VDRC). UAS-Rap1*/TM6 and UAS-PKA*/CyO were generated by I. Hariharan (UCB), and D. Kalderon (Columbia University), respectively. UAS-Rip11GFP and UAS-Rip11DNGFP were kindly provided by Don Ready (Purdue University). UAS-Arf6* was generated in the Olson laboratory (UT Southwestern).UAS-EF construct and line were described previously,23. UAS-Imaginal discs were dissected, fixed and stained using standard procedures. Salivary glands were dissected similarly, fixed for 30 minutes, and left attached to carcasses until ready to mount in SlowFade (LifeTechnologies #S36936), using double sided tape as a spacer to prevent tissue squashing. Antibodies: rabbit anti-GFP antibody , rat-anti GFP antibody , mouse anti-Rab11 , mouse anti Rab11-GTP , D-Ecad . The rabbit anti-Rip11 (1/1000) was a gift from D. Ready (Purdue University) and A. Satoh , and guinea pig anti-Sec15 (1/1000) was kindly provided by Hugo Bellen (Baylor College of Medicine). Images were collected by confocal microscopy on a Leica TCS SP5. All images were acquired using a 40X or 63X objective, and all higher magnifications were obtained using a 4X digital zoom. Co-localization quantifications in 2 atmosphere. Cells were gently dislodged with 0.05% trypsin (Mediatech) and were electroporated with cDNA expressing DsRed-Rab11A (Addgene) and Rip11-EGFP  using Neon Transfection system (Life Technologies) according to manufacturer’s protocol. Briefly, cells were rinsed once with PBS and resuspended at a density of 107 cells/ml. cDNA expressing DsRed-Rab11A and Rip11-EGFP were added to the suspension, and cells were electroporated with a 10 μl Neon tip at 1650 V, 20 ms width and 1 pulse. Cells were transferred to 600 μl pre-warmed medium of which 300 μl cell suspension was plated on each well of 8 chamber tissue culture treated glass slide . Cells were treated with 10 μg/ml EF +20 μg/ml PA for 4 h before fixation with 4% para-formaldehyde in PBS for 30 min at 37°C and processed for imaging. Fluorescence images were collected using a Delta Vision RT microscope. Colocalization between Rab11 and Rip11 was determined by measuring the Pearson's correlation coefficient (PCC) using the Velocity 6.3 imaging and analysis software (PerkinElmer). Costes automatic thresholding method [MDCK cells (ATCC CCL-34) were maintained in DMEM  containing 10% FBS, 1% Penicillin/streptomycin, 2 mM L-glutamine and were incubated in 37°C, 5% COg method  was appl2 atmosphere. Cells were gently dislodged with 0.05% trypsin (Mediatech Inc.) and cultured on glass poly-D-lysine coated chamber slides . At about 80% confluence, EF and PA  were added to cells. Drug co-treatments included: Slit2 , ESI-09 , and H89 . After 24 h (HBMEC cultures were maintained in DMEM (Corning) containing 10% FBS, 1% Penicillin/streptomycin, 2 mM L-glutamine, and were incubated in 37°C, 5% COter 24 h , cells wBALB/cJ mice  were intraperitoneally injected with drugs. SecinH3 (TOCRIS #2849), AG1024 , ESI-09 (TOCRIS #4773) or H89 (TOCRIS #2910), or with vehicle  2–3 h prior to injection of ET  or PBS , and in the case of SecinH3, also 2 h post toxin injection. ESI-09, AG1024 and H89 were administered at 10 mg/kg and SecinH3 was administered as 250 μl of 2.5 mM solution. Edema was assessed at 8–10 h, and 18–24 h by dorsal/plantar measurements using digital calipers.Untreated or ET intoxicated (24h) HBMEC cells were lysed in RIPA buffer  supplemented with mammalian protease inhibitor cocktail (Sigma Aldrich). The lysates were clarified by centrifugation at 1000 g for 10 min at 4°C and LDS sample buffer (NuPAGE) was added. Samples were boiled at 95°C, run on a 4–12% SDS polyacrylamide gel (Life Technologies) and transferred onto PVDF membrane (Bio-RAD). After incubation with primary antibodies against Rab11 , Cadherins , and actin , blots were probed with respective HRP conjugated secondary antibodies and developed using SuperSignal West Pico chemiluminescent substrate (Thermo Scientific).S1 Fig(A-C) Expression of Rab11wt in 1096GAL4>Rab11wtYFP wing discs. (A) Rab11wtYFP detected with a rabbit anti-GFP antibody appears as a peppered stain near the apical surface. (B) D-Ecad/GFP double stain. (C) corresponding D-Ecad stain marking AJs. (D-F) Expression of Rab11* in 1096GAL4>Rab11*YFP wing discs. (D) Rab11*YFP detected with a rabbit anti-GFP antibody. In addition to a peppered apical stain, Rab11* shows a distinctive net-like pattern at cell borders. (E) D-Ecad/GFP double stain, revealing that Rab11*YFP tends to accumulate at the AJs. (F) Corresponding D-Ecad single stain. (TIF)Click here for additional data file.S2 FigRNAi construct. (C) A 1096GAL4>UAS-CragHA salivary gland stained with an anti-HA antibody. (D) CragHA expression is suppressed by co-expression of a CragRNAi construct. (E) A 1096GAL4>UAS-Arf6Myc gland stained with an anti-Myc antibody. (F) Arf6Myc expression is blocked by co-expression of an Arf6RNAi construct.(A) A 1096GAL4>UAS-Sec15GFP salivary gland, stained with an anti-GFP antibody. (B) Sec15GFP expression in strongly inhibited by co-expression of a Sec15(TIF)Click here for additional data file.S3 FigRNAi glands. (C) Rab11*YFP fails to accumulate at the AJs in 1096GAL4>Rab11*YFP +Sec15RNAi salivary glands.(A-C) Rab11*YFP detected with a rabbit anti-GFP antibody in salivary glands. (A) Rab11*YFP selectively accumulates at the AJs in 1096GAL4>Rab11*YFP salivary glands. (B) Rab11* distribution is unchanged in 1096GAL4>Rab11*YFP+Crag(TIF)Click here for additional data file.S4 FigImages from experiment described in (TIF)Click here for additional data file.S5 Fig(A-D) Salivary glands stained with an anti-D-Ecad antibody. (A) A wild-type salivary gland showing D-Ecad accumulation at AJs. (B) A SglGAL4>Rab11DN salivary gland, in which Rab11 inhibition in this tissue leads to D-Ecad accumulation in broad zones around intercellular gaps. (C-D) Higher magnifications. (C) A wild-type salivary gland showing D-Ecad forming AJs (arrows). (D) A SglGAL4>Rab11DN salivary gland, revealing gaps between cells, and broad accumulation of D-Ecad around them (arrows). D-Ecad fails to form AJs.(TIF)Click here for additional data file.S6 FigRNAi. (D) 1096GAL4>EF+EpacRNAi. Inhibition of Epac expression potentlyreduces the EF phenotype. (E) PKA-C1B10/+ . (F) 1096GAL4>EF; PKA-C1B10/+. Reduction of PKA-C1 levels, either in a heterozygote loss-of-function PKA-C1 alleles (B10/+) or in flies expressing a dominant negative form of PKA-C (C1-DN), does not obviously alter the EF phenotype. (G) The surface areas of wings of the indicated genotypes were measured in Photoshop. Results were plotted as a histogram, with relevant p-values indicated. EF expression reduces wing size significantly compared to widl-type (wt) (****p<0.0001). EpacRNAi ameliorates the EF phenotype (****p<0.0001).(A-F) wings of the following genotypes: (A) Wild-type (+/+). (B) 1096GAL4>EF. (C) 1096GAL4>Epac(TIF)Click here for additional data file.S7 Fig(A-C) 1096GAL4>Rip11DN-GFP salivary glands, stained with (A) a rabbit anti GFP antibody, (B) a mouse anti Rab11 antibody, and (C) both antibodies, showing that Rab11 and Rip11DN-GFP co-localize in punctate vesicles. (D-F) 1096GAL4>Rip11DN+EF salivary glands stained with a rabbit anti-GFP antibody (D), a mouse anti-Rab11 antibody (E), and both antibodies (F), showing that Rab11 and Rip11DN still co-localize in EF-expressing glands. However, EF alters the distribution of both proteins, transforming small punctate staining into a ring-shaped halo surrounding secretory vesicles.(TIF)Click here for additional data file.S8 Fig-9).Co-localization between Rip11-GFP and DsRed-Rab11A in co-transfected MDCK cells measured by the Pearson's correlation coefficient (PCC) is reduced by ET treatment Click here for additional data file.S9 Fig(A-C) HBMECs, untreated. (D-F) HBMECs treated with ET for 6hours. Co-localization of Rab11* with Sec15 (B and E) and Rab11* with Rip11 (C and F) can be visualized following transfection of cells with Sec15-GFP. High-level expression of Sec15-GFP, and staining with an anti-Rab11* antibody (A) reveals a high degree of Sec15/Rab11* co-localization (B). In ET-treated cells, this co-localization is severely reduced (E). A double label Rab11*/Rip11 stain, reveals Rab11*/Rip11 co-localization (C), which is also abrogated by ET (F).(TIF)Click here for additional data file.S10 FigApical levels of D-Ecad in wing discs was measured using ImageJ. Arf6RNAi restores normal levels of apical D-Ecad in 1096GAL4>EF+Arf6RNAi discs (p<0.0001). Arf6RNAi does not notably affect apical levels of D-ECad. Surface areas of wings of the same genotypes were also measured, and Arf6RNAi showed a modest yet significant restorative effect in EF-expressing wings .(TIF)Click here for additional data file.S11 Fig(A-B) Western blot analysis of HBMECs cells. (A) Rab11A (~28 kD) and pan-Cadherin (~97 kD) levels are severely decreased in ET-treated cells (24hrs), while control actin (~42 kD) levels are only slightly reduced. (B) dcAMP, a cell-permeant stable analog of cAMP that is insensitive to phosphodiesterase, also causes a severe loss of Rab11, suggesting that the reduction of Rab11 levels induced by ET is mediated by cAMP. (TIF)Click here for additional data file.


The potential effect of icariside II on dexamethasone-induced osteoblast cell damages was evaluated here. In MC3T3-E1 osteoblastic cells and the primary murine osteoblasts, co-treatment with icariside II dramatically attenuated dexamethasone- induced cell death and apoptosis. Icariside II activated Akt signaling, which is required for its actions in osteoblasts. Akt inhibitors  almost abolished icariside II-induced osteoblast cytoprotection against dexamethasone. Further studies showed that icariside II activated Nrf2 signaling, downstream of Akt, to inhibit dexamethasone-induced reactive oxygen species (ROS) production in MC3T3-E1 cells and primary osteoblasts. On the other hand, Nrf2 shRNA knockdown inhibited icariside II-induced anti-dexamethasone cytoprotection in MC3T3-E1 cells. Finally, we showed that icariside II induced heparin-binding EGF (HB-EGF) production and EGFR trans-activation in MC3T3-E1 cells. EGFR inhibition, via anti-HB-EGF antibody, EGFR inhibitor AG1478 or EGFR shRNA knockdown, almost blocked icariside II-induced Akt-Nrf2 activation in MC3T3-E1 cells. Collectively, we conclude that icariside II activates EGFR-Akt-Nrf2 signaling and protects osteoblasts from dexamethasone. Icariside II might have translational value for the treatment of dexamethasone-associated osteoporosis/osteonecrosis. In vitro, exogenously-added Dex could induce direct damages to the cultured osteoblastic cells and osteoblasts  H: quinone oxidoreductase 1 (NQO1) [HO-1 and NQO1). Remarkably, icariside II (5 μM) significantly inhibited Dex-induced ROS production in MC3T3-E1 cells  production and oxidative stress in osteoblasts, which cause subsequent cell damages , 22. On 1 (NQO1) , 22 in M1 (NQO1) , 22, whis Figure . Very sis Figure  and inhis Figure .HO-1 and NQO1 mRNA expression in icariside II-treated MC3T3-E1 cells was almost blocked with Nrf2 silence -induced pro-survival Figure  and antiHO-1 and NQO1 mRNA expression in icariside II-treated cells  could protect osteoblasts from Dex , 18. Thus Figure  and 5F, To further support the requirement of EGFR in icariside II-induced actions, shRNA method was again applied to stably knockdown EGFR in MC3T3-E1 cells. The applied EGFR shRNA efficiently downregulated EGFR, and inhibited activation of EGFR-Akt Figure  and Nrf2et al., showed that Compound 13 (C13) activates AMP-activated protein kinase (AMPK)-dependent signaling to inhibit Dex-induced oxidative stress and protect osteoblasts [et al., showed that microRNA- 135b (“miR-135b”) expression similarly provokes AMPK signaling to inhibit Dex-induced oxidative damages in osteoblasts [Dex could induce significant ROS production and oxidative stress in osteoblasts/osteoblastic cells, which could be a major contributor of subsequent cell death , 7–9, 22eoblasts . Fan et HO-1 and NOQ-1). Therefore, Akt is very likely the upstream signaling of Nrf2 activation by icariside II.Nrf2 phosphorylation at Ser-40 is required for Nrf2 stabilization, nuclear translocation and activation by a number of stimuli , 33. RecFan et al., showed that activation of EGFR-Akt-mTOR signaling mediates epiregulin-induced osteoblast cytoprotection against Dex [Besides being directly activated by the above ligands, EGFR may also be activated under a number of stimuli, the process known as “trans-activation” –40. In tinst Dex . Similarinst Dex . Furtherinst Dex , 12.Icariside II, Dex and LY294002 were purchased from Sigma Chemicals . Perifosine and MK-2206 were obtained from Selleck . All the antibodies of this study were purchased from Cell Signaling Technology .The culture and differentiation of the murine osteoblastic MC3T3-E1 cells were described in our previous studies –7.In brief, the calvariae of neonatal mice was isolated, washed, and digested –7. DigesThe Cell Counting Kit-8  assay kit was utilized to test the survival of osteoblasts with indicated treatment –7.The cell apoptosis histone-DNA ELISA plus kit  was utilized to quantify cell apoptosis in osteoblasts with indicated treatment, the detailed protocol was described previously –7.As described in our previous studies –7, cell Cellular protein lysates (30 μg per lane) were separated by the SDS-PAGE gels, and were transferred onto polyvinylidene difluoride (PVDF) membranes, which were blocked and incubated with indicated primary and secondary antibodies. Antibody-antigen binding was visualized though enhanced chemiluminescence  reagents. The quantification of each Band was performed by the ImageJ software –7.The two non-overlapping lentiviral shRNAs against murine Nrf2 were synthesized by Shanghai Genepharm Co. . The EGFR shRNA lentiviral particles were purchased from Santa Cruz Biotech . The lentiviral shRNA  was added to cultured MC3T3-E1 cells (plated with 50% confluence) for 24 hours. Stable cells were selected by puromycin  for 4–5 passages. Knockdown of targeted protein (EGFR or Nrf2) in the stable cells was confirmed by Western blot assay. The control MC3T3-E1 cells were infected with lentiviral scramble control shRNA (Santa Cruz).After treatment of cells, the HB-EGF content in the conditional medium was tested by a commercial HB-EGF ELISA kit ROS content in the cells was tested by a DCFH-DA fluorescent dye . Briefly, after treatment, cells were incubated with 10 μM of DCFH-DA at room temperature for 30 min, which were and analyzed for fluorescence via a Fluorescence/Multi-Detection Microplate Reader . ROS intensity in the treatment group was expressed as fold change of that in the control group.Nrf2, HO-1 and NQO1, as well as NADPH were provided by Dr. Jiang's group [ΔΔCt was then calculated to yield fold expression relative to GAPDH.Following treatment of cells, total cellular RNA was extracted through TRIzol reagents . 500 ng of total RNA per sample was reverse-transcribed (RT) by the commercial RT-PCR kit  according to the attached procedure. Real-time quantitative PCR (“RT-qPCR”) via SYBR green was performed in an ABI7300 machine . The primers of Nrf2-associated genes, 's group , 34, 35.p values < 0.05 (one-way ANOVA) were considered statistically significant.Experiments were repeated at least three times and consistent results were always obtained. Collectively, we conclude that icariside II activates EGFR-Akt-Nrf2 signaling and protects osteoblasts from Dex. Icariside II might have translational value for treatment of Dex-associated osteoporosis and osteonecrosis.


In Supporting Information files S1 and S2 Figs, an extra un-lettered panel in the upper left corner was included in error. Please see the corrected S1 FigROSA26-CRE-luc mice infected with adenovirus encoding Cre recombinase (Ad-Cre) or GFP (Ad-GFP). Both viruses encode GFP. Scale bar = 100 μm. (B) Total bioluminescence (photons/sec) in brain ROI in images shown in Fig 1B and four additional mice . (C) Bioluminescence images on male heterozygous ROSA26-CRE-luc animals, ad libitum fed (ZT8) or fasted for 16 h (ZT8 through day2 ZT0). (D) Total bioluminescence in regions of interest (ROI) drawn over livers in C . (E) Diagram of fasting plus glucagon experiment in panels F and G. (F) Bioluminescence images on a separate set of male heterozygous ROSA26-CRE-luc animals fasted for 16 h  followed by glucagon injection (100 μg/kg) and imaging 4 hours later right . (G) Total bioluminescence in liver ROI of animals shown in F plus 4 additional littermates .(A) tdTomato fluorescence in primary hepatocytes from (EPS)Click here for additional data file.S2 FigROSA26-CRE-luc mice in running experiment shown in Fig 2A . (B) Luciferase activity in liver lysates shown in Fig 2C . (C) Correlation between bioluminescence shown in Fig 2A and 2B and luciferase activity shown in B. Pearson’s R coefficient = 0.8865. (D), (F) Western blot of phospho-CREB(S133)/ATF-1 and total CREB from quadriceps and gastrocnemius muscle of female ROSA26-CRE-luc knock-in mice shown in Fig 2A, static housed  and after 12 h voluntary wheel running exercise (ZT12-day2 ZT0). Proteins for probing with phospho-CREB and total CREB antibodies were run on two separate gels. (E), (G) Quantification of pCREB western blots shown in D and F. (H) Luciferase activity in primary myocytes from ROSA26-CRE-luc mice treated with FSK/ IBMX for 4 h . **p<0.01, ***p<0.001 by t-tests.(A) Bioluminescence in brain of (EPS)Click here for additional data file.


Nature Communications8:251 doi:10.1038/s41467-017-00228-w; Article published online 15 August 2017This Article contains typographical errors in Equation (2), where two division operators were omitted. The correct version of Equation (2) appears below


In vivo vector-based production of bNAbs represents an alternative strategy. We investigated adenovirus serotype 5 (Ad5) and adeno-associated virus serotype 1 (AAV1) vectors to deliver the HIV-1-specific bNAb PGT121 in wild-type and immunocompromised C57BL/6 mice as well as in HIV-1-infected bone marrow-liver-thymus (BLT) humanized mice. Ad5.PGT121 and AAV1.PGT121 produced functional antibody in vivo. Ad5.PGT121 produced PGT121 rapidly within 6 h, whereas AAV1.PGT121 produced detectable PGT121 in serum by 72 h. Serum PGT121 levels were rapidly reduced by the generation of anti-PGT121 antibodies in immunocompetent mice but were durably maintained in immunocompromised mice. In HIV-1-infected BLT humanized mice, Ad5.PGT121 resulted in a greater reduction of viral loads than did AAV1.PGT121. Ad5.PGT121 also led to more-sustained virologic control than purified PGT121 IgG. Ad5.PGT121 afforded more rapid, robust, and durable antiviral efficacy than AAV1.PGT121 and purified PGT121 IgG in HIV-1-infected humanized mice. Further evaluation of vector delivery of HIV-1 bNAbs is warranted, although approaches to prevent the generation of antiantibody responses may also be required.Broadly neutralizing antibodies (bNAbs) are being explored for HIV-1 prevention and cure strategies. However, administration of purified bNAbs poses challenges in resource-poor settings, where the HIV-1 disease burden is greatest. IMPORTANCE Broadly neutralizing antibodies (bNAbs) are being explored for HIV-1 prevention and cure strategies, but delivery of purified antibodies may prove challenging. We investigated adenovirus serotype 5 (Ad5) and adeno-associated virus serotype 1 (AAV1) vectors to deliver the HIV-1-specific bNAb PGT121. Ad5.PGT121 afforded more rapid, robust, and durable antiviral efficacy than AAV1.PGT121 and purified PGT121 IgG in HIV-1-infected humanized mice. The therapeutic efficacy of potent neutralizing human immunodeficiency virus type 1 (HIV-1)-specific broadly neutralizing antibodies (bNAbs) has been shown in both humanized mice  and is u3–in vivo should be explored.Despite the therapeutic potential of potent HIV-1 bNAbs, there are challenges to the administration of purified antibodies in the developing world. These include (i) the necessity of repeated intravenous or subcutaneous dosing , (ii) hiin vivo. Ads encoding MAbs have previously shown therapeutic efficacy against infectious diseases, including Yersinia pestis  and adeno-associated viruses (AAVs) that encode the HIV-1-specific bNAb PGT121 to produce the therapeutic monoclonal antibody (MAb) a pestis , and Wesa pestis , as wella pestis , but Ads in mice , nonhuma in mice , and hum in mice . In thisin vivo PGT121 expression from Ad5 and AAV vectors. BALB/c mice were injected intramuscularly (i.m.) once with either 1010 viral particles (vp) of Ad5 expressing luciferase (Ad5.Luc) or 1010 genome copies (GC) of AAV2/8 expressing luciferase (AAV2/8.Luc), and luciferase expression was measured by IVIS imaging following viral injection. Ad5.Luc induced high levels of expression within 6 h, but expression levels then declined markedly by day 7 (3 relative luminescence units [RLU]) on day 1, followed by increased expression by day 4 and a plateau of >105 RLU from day 14 onwards , reaching a concentration in serum of ∼1 μg/ml at 12 h and >10 μg/ml by 24 h (10 vp) in Rag knockout (KO) mice yielded sustained levels of serum PGT121 for over 400 days  titers similar to those of purified PGT121 IgG against two PGT121-sensitive pseudoviruses (6811.v7.c18 and P1981_C5_3) and one PGT121-resistant pseudovirus (R2184.c04) (To assess whether the Ad5- and AAV1-produced PGT121 bound the HIV-1 envelope (Env) gp140 protein similarly to purified PGT121, we performed surface plasmon resonance (SPR). Ad5- and AAV1-expressed PGT121 bound C97ZA012 and 92UG037 gp140s with affinity comparable to that of purified PGT121 IgG . To meas184.c04) . These d11 vp) or AAV1.PGT121 (1011 vp) or received by intravenous (i.v.) infusion 20 μg purified PGT121 IgG, and serum PGT121 concentrations were measured by enzyme-linked immunosorbent assay (ELISA)   and E. ILISA  and (ELISA)  and G, AAll immunocompetent mice that received Ad5.PGT121 induced potent anti-PGT121 antibodies by day 7. AAV1.PGT121 elicited slightly delayed anti-PGT121 antibodies by days 7 to 10. Purified PGT121 did not elicit detectable levels of anti-PGT121 antibodies for up to 14 days . Thus, tIn contrast, in Rag KO mice, Ad5.PGT121 produced detectable levels of PGT121 by day 1, and serum PGT121 levels were then durably sustained at the remarkably high levels of approximately 6 mg/ml for >28 days. AAV1.PGT121 exhibited a delay in detectable PGT121 levels until day 5 and was sustained at levels of approximately 44 μg/ml. Intravenous injection of purified PGT121 IgG resulted in detectable serum PGT121 with declining levels over 28 days, similar to what was observed with immunocompetent mice . No antiin vivo, 12 BLT (bone marrow/liver/thymus) humanized mice were infected with HIV-1 strain JR-CSF. HIV-1 infection was confirmed in all mice after infection, with stable setpoint viral loads prior to PGT121 administration (data not shown). Ad5.PGT121 (1011 vp i.m.), AAV1.PGT121 (1011 vp i.m.), and PGT121 IgG (20 μg i.v.) were administered on day 17 (n = 4/group). Serum PGT121 concentrations following the treatments in BLT mice showed kinetics similar to those of Rag KO mice  . Se. Sein viopies/ml . In cont< 0.001) .Serum PGT121 levels following Ad5.PGT121, AAV1.PGT121, and PGT121 IgG administration correlated with the reductions in viral loads, suggesting that serum PGT121 levels of >100 ng/ml are required to suppress HIV-1 replication in this model . SolubleN-linked glycosylation site (PNGS) in the V3 loop  monoclonal antibody, against lethal toxin challenge  protein, including the CD4-binding site , 27, the32–We observed viral rebound in mice that received intravenous PGT121 IgG, which correlated with the decline of serum PGT121 levels to <100 ng/ml. Rebound did not appear to be due to the emergence of resistance, consistent with previous studies with PGT121 in chronically SHIV-infected rhesus monkeys . These dFor this study, we used Ad5 for proof of concept, but we have previously demonstrated that baseline Ad5 seroprevalence is extremely high in sub-Saharan Africa . We therIn conclusion, our data demonstrate that Ad5.PGT121 reduced viral loads in HIV-1-infected humanized mice more effectively than did AAV1.PGT121 and purified PGT121 IgG. The potential role of bNAbs in HIV-1 cure strategies is under investigation . AntiretReplication-deficient Ad5 vectors expressing PGT121 were constructed essentially as previously described . Briefly2 and harvested 72 h after transfection. Virus was released from the cells by freeze-thawing and treated with benzonase to remove cellular nucleic material. AAV1 purification was performed by iodixanol gradient centrifugation  promoter, PGT121 heavy and light chains separated by an F2A linker, and simian virus 40 (SV40) poly(A) sequence, was cloned into an AAV2-ITR plasmid. AAV1-RepCap and AAV helper plasmid pHELP  were cotransfected with AAV2-ITR-PGT121 in 293T cells plated 1 day prior to transfection. Cells were incubated at 37°C and 10% COfugation  followedAAV2/8 expressing luciferase (AAV2/8.Luc) was purchased from Vector Biolabs, PA.Female 6- to 10-week-old C57BL/6, BALB/c, and RagKO (B6.129S7-Rag1tm1Mom/J) mice  were maintained in accordance with the Institutional Animal Care and Use Committee guidelines of Beth Israel Deaconess Medical Center.Ad and AAV vectors were administered at specified viral particle dosages in PBS by the intramuscular route in the quadriceps in a 50-μl volume. Purified PGT121 IgG was diluted in 100 μl PBS and administered by the intravenous route. After vector administration, mice were bled to determine PGT121 (or anti-PGT121) antibody concentration in the serum.450–550) with SoftMax Pro GxP v5 software.A quantitative immunoglobulin ELISA for PGT121 detection was performed essentially as previously described . BrieflyTo quantify the concentration of anti-PGT121 antibodies, the quantitative murine immunoglobulin ELISA previously described  was usedSurface plasmon resonance experiments were conducted on a Biacore 3000  instrument at 25°C utilizing HBS-EP  as the running buffer. Immobilization of protein A (Thermo Scientific) to CM5 chips was performed following the standard amine-coupling procedure as recommended by the manufacturer . RagKO mouse serum  was diluted in HBS-EP, resulting in about 600 response units (RU) of total captured IgG. Binding experiments were conducted with a flow rate of 50 μl/min with a 2-min association phase and a 5-min dissociation phase. Regeneration was conducted with one injection (3 s) of 10 mM glycine hydrochloric acid (pH 2.1)  at 100 μl/min followed by a 3-min equilibration phase in HBS-EP. Identical injections over blank surfaces were subtracted from the binding data for analysis. Binding kinetics were determined using BIAevaluation software  and the Langmuir 1:1 binding model. All samples were run in duplicate and yielded similar kinetic results. Single curves of the duplicates are shown in all figures.In vitro neutralization assays in luciferase reporter cells  were performed as previously described  was determined .Ad5-specific neutralizing antibodies were measured using a luciferase-based neutralization assay as previously described . BrieflyEquivalent amounts of serum PGT121 IgG from Ad- and AAV-injected RagKO mice, as determined by ELISA, were brought to a total equivalent volume in naive RagKO mouse serum. Purified PGT121 IgG was added to an equivalent volume in naive RagKO mouse serum as a control. Samples were added to reducing sample buffer (Pierce), heated for 5 min at 95°C, and run on a 4 to 15% SDS-PAGE gel (Bio-Rad). Protein was transferred to a polyvinylidene difluoride (PVDF) membrane using the iBlot dry blotting system (Invitrogen), and membrane blocking was performed overnight at 4°C in PBS-T . The membrane was incubated for 1 h with PBS-T containing a 1:5,000 dilution of anti-human Fc HRP (Jackson ImmunoResearch), washed, and developed using the Amersham ECL plus Western blotting detection system  and CL-Xposure film (Thermo Fisher Scientific).−/− mice were subjected to sublethal whole-body irradiation before human fetal thymus and liver fragments and CD34+ cells were injected. Maximal peripheral blood human T cell numbers were reached ∼14 weeks after reconstitution, with mice deemed suitable for experiments if >40% of peripheral blood lymphocytes were hCD45+. Mice were infected intraperitoneally (i.p.) with 105 50% tissue culture infectious doses (TCID50) of HIV-1 JR-CSF, to achieve 100% infection. Bleeds were obtained by submandibular puncture, and plasma was isolated and stored at −80°C. All mice were kept in accordance with Institutional Animal Care and Use Committee guidelines.BLT (bone marrow/liver/thymus) humanized mice were generated as previously described . BrieflyM38429.1) was utilized as a standard. RNA standards were generated using the AmpliCap-Max T 7 High Yield Message Maker kit  and purified with the RNA Clean and Concentrator kit . RNA quality and concentration were assessed by the BIDMC Molecular Core Facility. Log dilutions of the RNA standard were reverse transcribed and included with each RT-PCR assay. Viral loads were calculated as virus particles (vp) per milliliter. Assay sensitivity was >100 copies/ml.HIV-1 viral loads were assessed as previously described, adapted to target Gag of HIV-1 . BrieflyThe single genome amplification assay was adapted from Keele et al. . Brieflyd-luciferin Ultra . Imaging was performed for a 60-s exposure time, 1.2 f/stop, and medium binning.Luciferase transgene expression through luminescence was quantified using an IVIS Series 100 imaging device and Living Image software v2.50.1 (Xenogen). At selected time points, mice were anesthetized by 1.5% isoflurane inhalation and injected i.p. with 150 μl of XenoLight RediJect t test (for two groups) was used. P values of <0.05 were considered significant .Statistical analyses were performed using Prism 6.0 (GraphPad Software). Nonparametric data were analyzed by the Kruskal-Wallis test with Dunn's multiple-comparison posttest (more than two groups) or the two-tailed Mann-Whitney U test (for two groups). For parametric data, a one-way analysis of variance (ANOVA) with Bonferroni's multiple comparison posttest (more than two groups) or an unpaired env sequences discussed in this paper have been deposited in GenBank (accession numbers MG706260 to MG706443).All SGA HIV-1


Drosophila lacks full-length Trks, raising the question of how these processes occur in the fly. Paradoxically, truncated Trk isoforms lacking the TyrK predominate in the adult human brain, but whether they have neuronal functions independently of full-length Trks is unknown. Drosophila has TyrK-less Trk-family receptors, encoded by the kekkon (kek) genes, suggesting that evolutionarily conserved functions for this receptor class may exist. Here, we asked whether Keks function together with Drosophila neurotrophins (DNTs) at the larval glutamatergic neuromuscular junction (NMJ). We tested the eleven LRR and Ig-containing (LIG) proteins encoded in the Drosophila genome for expression in the central nervous system (CNS) and potential interaction with DNTs. Kek-6 is expressed in the CNS, interacts genetically with DNTs and can bind DNT2 in signaling assays and co-immunoprecipitations. Ligand binding is promiscuous, as Kek-6 can also bind DNT1, and Kek-2 and Kek-5 can also bind DNT2. In vivo, Kek-6 is found presynaptically in motoneurons, and DNT2 is produced by the muscle to function as a retrograde factor at the NMJ. Kek-6 and DNT2 regulate NMJ growth and synaptic structure. Evidence indicates that Kek-6 does not antagonise the alternative DNT2 receptor Toll-6. Instead, Kek-6 and Toll-6 interact physically, and together regulate structural synaptic plasticity and homeostasis. Using pull-down assays, we identified and validated CaMKII and VAP33A as intracellular partners of Kek-6, and show that they regulate NMJ growth and active zone formation downstream of DNT2 and Kek-6. The synaptic functions of Kek-6 could be evolutionarily conserved. This raises the intriguing possibility that a novel mechanism of structural synaptic plasticity involving truncated Trk-family receptors independently of TyrK signaling may also operate in the human brain.Neurotrophism, structural plasticity, learning and long-term memory in mammals critically depend on neurotrophins binding Trk receptors to activate tyrosine kinase (TyrK) signaling, but  kekkon (kek) genes, suggesting that deep evolutionary functional conservation for this receptor class could be unveiled. Here, we show that Kek-6 is a receptor for Drosophila neurotrophin 2 (DNT2) that regulates structural synaptic plasticity via CaMKII and VAP33A. The latter are well-known factors regulating synaptic structure and plasticity and vesicle release. Furthemore, Kek-6 cooperates with the alternative DNT2 receptor Toll-6, and their concerted functions are required to regulate structural homeostasis at the NMJ. Our findings suggest that in mammals truncated Trk-family receptors could also have synaptic functions in neurons independently of Tyrosine kinase signaling. This might reveal a novel mechanism of brain plasticity, with important implications for understanding also the human brain, in health and disease.A long-standing paradox had been to explain how brain structural plasticity, learning and long-term memory might occur in Drosophila in the absence of canonical Trk receptors for neurotrophin (NT) ligands. NTs link structure and function in the brain enabling adjustments in cell number, dendritic, axonal and synaptic patterns, in response to neuronal activity. These events are essential for brain development, learning and long-term memory, and are thought to depend on the tyrosine-kinase function of the NT Trk receptors. However, paradoxically, the most abundant Trk isoforms in the adult human brain lack the tyrosine kinase, and their neuronal function is unknown. Remarkably, Drosophila has kinase-less receptors of the Trk family encoded by the  Brain plasticity, neurotrophism in development, structural and synaptic plasticity during learning and long-term memory in humans critically depend on the receptor TrkB binding its neurotrophin (NT) ligand BDNF ,2. DurinDrosophila. However, neurotrophism, structural and synaptic plasticity, learning and long-term memory all occur in fruit-flies, implying that either in the course of evolution insects and humans found different molecular solutions to elicit equivalent functions, or that undiscovered mechanisms contribute to brain plasticity in both humans and fruit-flies. Finding out what happened to the Trks in Drosophila is important, as it could uncover novel fundamental mechanisms of structure-function relationships in any brain.No canonical, bona fide, full-length, TyrK Trk receptors have been found in Drosophila. Trks  bear the unique combination of Cysteine Rich Repeats (CRR), Leucine Rich Repeats (LRR) and Immunoglobulin (Ig) domains extracellularly, and an intracellular TyrK domain kek6e000907 and PBac[WH]kek6f05733; for DNT2: PBac[RB]spz5e03444 and PBac[WH]Shabf05893.. Mutants were selected using genetics and PCR, using primers as recommended in VAP33AG0231). Toll-6 mutants used have been described in [GAL4 lines:kek5GAL4 line P[GawB]NP5933 (from BSC); w;;elavGAL4 drives expression in all neurons (insertion on the third chromosome); Toll-6-GAL4 was generated by RMCE from MIMICToll-6MIO2127 and kek-6-GAL4 from MIMICkek-6MI13953; w;;D42GAL4 and w;Toll-7GAL4 drive expression in motoneurons and have been previously described; P{ (Bloomington). Double mutant lines were generated by conventional genetics. MIMIC-GFP lines:y w; MIMICkek6MI13953 and has a GFP insertion in the coding region (BSC). All stocks were balanced over SM6aTM6B or TM6B to identify balancer chromosomes, and all were generated from a yw or w mutant background. In all figures, controls are: (1) F1 from yw x Oregon; (2) y w; GAL4/+cribed in, Df(3L)Bon lines : for Kekended in . kek634 ribed in . GAL4 lidescribed; w; MhcRkek1, 2, 3, 4, 5 and 6 were obtained either from cDNA clones , by PCR from cDNA libraries , or by reverse transcription PCR from larvae (kek4), using primers designed for Gateway cloning into the pDONR plasmid: kek1 forward: GGGGACAAGTTTGTAC AAA AAA GCA GGC TCA TCC AGG AAA ATG CAT ATC A and reverse: GGGGACCAC TTT GTA CAA GAA AGC TGG GTA GTC AGT TCT TGG TTT GGT TT; kek2: forward: GGGGACAAGTTTGTAC AAA AAA GCA GGC TCA ATG AGT GGT CTG CCA ATC T and reverse: GGGGACCAC TTT GTA CAA GAA AGC TGG GTA AAT GTC GCT GGT TTC CTG GC; kek3: forward: GGGGACAAGTTTGTAC AAA AAA GCA GGC TCA TAT GCG ATG GCA GCG GGA A and reverse: GGGGACCAC TTT GTA CAA GAA AGC TGG GTA GCT CTT GAA AAT ATC CTG TC; kek4: forward: GGGGACAAGTTTGTAC AAA AAA GCA GGC TCA CTA GAC CTT CCG TTC CTT, and reverse: GGGGACCAC TTT GTA CAA GAA AGC TGG GTA TAT TGA GAT ATC AAC ACC AG; kek5: forward: GGGGACAAGTTTGTAC AAA AAA GCA GGC TAG CTA GAC GCA GAC TTA GAG and reverse: GGGGACCAC TTT GTA CAA GAA AGC TGG GTA GAC CTC GGT GCC ATC CTC GC; kek6: forward: GGGGACAAGTTTGTAC AAA AAA GCA GGC TCA ATG CAT CGC AGC ATG GAT C and reverse: GGGGACCAC TTT GTA CAA GAA AGC TGG GTA GAG CGA CAC GAA CTC GCC AG. CaMKII and VAP33A full-length cDNAs were cloned using the Gateway System first into pDONR, and then into pAct5-attR-HA.Full length cDNAs for pUASt-attR-mRFP destination vector for conventional transgenesis, injected by BestGene (www.thebestgene.com). For expression in S2 cells, they were subcloned into tagged pAct destination vectors pAct5c-attR-mCFP, pAct5c-attR-FLAG and pAct5c-attR-HA. Constructs used for expression of S2 cells of DNT1 and 2, Toll-6 and 7 were as previously described[kek and Toll-6 sequences, and attB sites were introduced in the primers at the 5′ and 3′ ends of the chimaeric insert for Gateway cloning into pAct5c-attR-3xHA destination vector. Unfortunately, we were not able to generate kek1-Toll-6 and kek2-Toll-6 chimaeric receptor constructs. To clone chimaeric kek3,4,5,6-Toll-6 receptor constructs, reverse primers at the juxtamembrane of keks were as follows: kek3-Toll-6: CGAT-GAATTC-AGGTACAGAGTTCCAGAGAC; kek4-Toll-6: CGCG-GAATTC-TTGCAAATAAGTGTGCTGGC; kek5-Toll-6: CTAT-GGATCC-GCTCATCATGGTGGTGTCCT; kek6-Toll-6: GTAT-GAATTC-ACGCCGGCCTTGTTGGCATG. Toll-6 primers from chimaeras were: Forward primers from juxtamembraneCATG-GAATTC-AACTTCTGCTACAAGTCACC  or CATG-GGATCC-AACTTCTGCTACAAGTCACC (compatible with kek5 chimaera) and reverse from C terminus: GGGGACCAC TTT GTA CAA GAA AGC TGG GTC CGC CCA CAG GTT CTT CTG CT.For expression in flies under UAS/GAL4 control, cDNAs were subcloned to a Gateway described. ChimaerUn-tagged full-length DNT2 was cloned into the pUASt-attB vector by conventional cloning, DNT2-full length (DNT2-FL) was PCR-amplified from cDNA libraries, and cloned into pUASt-attB for ΦC31 transgenesis . UAS-DNTGAPDH: GAPDH Fw TCACCACCATTGACAAGGC; GAPDH Rev:; CGGTAAGATCCACAACGGAG; (2) DNT2: DNT2 0.5kDa Rwd TGGAAACCCGCTCTTTGTCAG; DNT2 0.9kDa Rev:.TGATGAACTGCGATGTCGTCT. The genotypes of larvae tested were y w control, and transheterozygous mutants DNT237/Df(3L)6092.Reverse Transcription PCR was carried out following standard procedures after mRNA straction from wandering larvae, using the following primers: (1) Control: drosomycin-luciferase were maintained at 27°C in air in Insect-Xpress medium (Lonza) supplemented with penicillin/streptomycin/L-glutamine mix (Lonza) and 10% foetal bovine serum (Lonza). 1ml suspended cells were passaged every two days into 4ml fresh medium. 3x106 cells in 2ml media were seeded per well of a 6-well plate 24 hours prior to transfection. Per well of experiment, 250μl serum-free media, 3μl TransIT-2020 (Mirus), 2μg of HA tagged chimaeric receptors kek-toll6-HA plus 1μg of pAct-renilla-luciferase were incubated at room temperature for 30 minutes, supplemented with 350μl serum-free media and added to aspirated cells. After 4 hours, transfection mixture was removed and 2ml supplemented medium added. All experiments were conducted 48 hours after transfection; for imaging membrane targeting of Kek-Toll6-HA protein S2 cells were starved for 6h prior to fixation.For Dif signaling assays using chimaeric kek-Toll-6 receptors, S2 cells stably transfected with For signaling assays, S2 cells were stimulated with 50nM/well of purified baculovirus DNT2 protein (generated as previously described) and DifpAct5C-Pro-TEV6HisV5-DNT2-CK and pAct5C-Kek6-3xHA; (2) Co-IP Kek6-DNT2 or Kek6-DNT1: pAct5C-Kek6-3xHA, pAct5C-Pro-TEV6HisV5-DNT1-CK-CTD, pAct5C-Pro-TEV6HisV5-DNT2-CK; (3) Co-IP Kek5-DNT2: pAct5C-Kek5-3xFlag and pAct5C-Pro-TEV6HisV5-DNT2-CK. (4) Co-IP Kek2-DNT2: pAct5C-Kek2-3xFlag and pAct5C-DNT2-FL-HA. (5) Co-IP Kek3-DNT2: pAct5C-Kek3-3xFlag and pAct5C-DNT2-FL-HA. (5) Co-IP kek6-Toll-6: pAct5C-Kek6-HA and pAct5C-Toll-6CY-3xFlag, pAct5C-Toll-7CY-3xFlag.(6) Co-IP Kek6-CaMKII: pAct5-CaMKII-HA and pAct-kek-6-FLAG. (7) Co-IP kek6-VAP33A: pAct5-Vap33A-HA and pAct-kek-6-FLAG. 48h after transfection cells were harvested and washed in PBS. Final cell pellets were lysed in 600 μL NP-40 buffer  supplemented with protease inhibitor cocktail (Pierce). For V5 and HA immuno-precipitations,500 μL of lysates from single or co-transfected cells were incubated with 1 μg of mouse anti-V5 or 1 μg of mouse anti-HA antibodies overnight at 4°C, then the lysate plus antibody mixtures were supplemented with 25 μL of protein-A/G megnetic beads (Pierce) and incubated for 1 h at room temperature. For Flag immunopreciptations, lysates were incubated with anti-Flag antibody conjugated magnetic beads (Sigma-Aldrich) overnight at 4°C or for 2h at room temperature. Beads were washed thoroughly in NP-40 buffer and/or PBS, or in TBST buffer. Proteins were eluted in 40 μL of 2x Laemmli-buffer and analysed by Western blot following standard procedures.Coimmunoprecipitations were carried out as previously described, after tpAct5C-Kek6-3xFlag expression construct. For controls mock-transfected cells (i.e. no construct) were used. After transfection cells were processed as described above for co-immunoprecipitation. 600 μL of cell lysates were incubated with 20 μL of anti-Flag conjugated magnetic beads overnight at 4°C. Beads were washed thoroughly in PBS, then proteins were eluted in 40 μL 2x Laemmli-buffer for 10 min at room temperature. Eluted proteins were loaded onto 10% polyacryalamide gels. Alternatively, proteins were not eluted after overnight incubation with anti-Flag beads, but they were re-incubated with whole fly (OregonR) head lysates. Here, 60 heads were lysed in 600 μL of NP-40 buffer. After overnight incubation at 4°C, proteins were eluted and analysed as for S2 cell lysate proteins. Thus, eluted proteins were loaded onto 10% polyacryalamide gels, and the gels were stained with Coomassie Blue and cut into several pieces. Gel pieces were subjected to in-gel digestion with trypsin using a standard protocol. Tryptic peptides were analysed by LC-MS/MS using Ultimate 3000 HPLC coupled to a LTQ Orbitrap Velos ETD mass-spectrometer. Peptide separation, mass spectrometric analysis and database search were carried out as specified at the University of Birmingham Proteomics Facility. Candidate binding proteins were identified on these criteria: (1) Proteins were accepted only if they were identified with at least two high confident peptides. (2) Mock-transfected controls and Kek6-3xFlag samples were compared, and only proteins identified from Kek6-3xFlag lysates or heads, but absent form controls, were considered as possible interacting partners for Kek6.S2 cells were transfected with 4 μg Western blot was carried out following standard procedures using antibodies listed here: mouse anti-V5 , mouse anti-HA , chicken anti-HA , rabbit anti-Flag , HRP-conjugated anti-mouse IgG , HRP-conjugated anti-chicken IgG , HRP-conjugated anti-rabbit IgG , mouse dCaMKII (Cosmo CAC-TNL-001-CAM 1:1000), rabbit α-p-CaMKIIα (Thr 286) (Santa Cruz sc-12886-R 1:1000), mouse Tubulin DM1A (Abcam ab7291 1:10000).MhcGAL4>UAS-DNT2-FL-GFP, L3 larvae were placed in agar plates (2%) and left at 29°C for 90 minutes to potentiate the NMJ before dissections [NMJ preparations were carried out according to . For GFPsections . A hundrsections , and fixT286 1:150 (Santa Cruz), raised against mammalian pCaMKIIT286 detects phosphorylation at Thr287 in Drosophila, the constitutively active form; mouse plain anti-Synapsin at 1:25 (DSHB 3C11). Secondary antibodies were: anti-guinea pig-Alexa 488 at 1:250 (Molecular Probes); biotynilated anti-mouse at 1:300 (Jackson Labs) followed by the ABC Elite Kit (Vector Labs); biotynilated anti-guinea pig at 1:300 (Jackson Labs) followed by Streptavidin-Alexa-488 at 1:400 (Molecular Probes); anti-rabbit-Alexa 488 at 1:250 (Molecular Probes); anti-mouse-Alexa 488 1:250 (Molecular Probes); anti-rabbit-Alexa 647 at 1:250 (Molecular Probes); anti-mouse-Alexa 647 1:250 (Molecular Probes).Antibody stainings in embryos and S2 cells were carried out following standard procedures, using the following primary antibodies at the indicated dilutions: mouse anti-FasII at 1:5 ; rabbit anti-GFP 1:1000 (Molecular Probes); rabbit anti-GFP 1:4,000 (abcam ab290); mouse anti-Dlg 1:20 ; rabbit anti-HRP 1:250 (Jackson Immunoresearch); mouse anti-Brp 1:100 ; rabbit anti-p-CaMKIIlambik: a 551 nucleotides fragment was cloned into pDONR with primers: forward GGGGACAAGTTTGTAC AAA AAA GCA GGC TAG AAA CTA CGC ATG AGC CTG and reverse GGGGACCAC TTT GTA CAA GAA AGC TGG GTA CCG CTC AAA TGT CCA CTG T; then linearised with HpaI and transcribed with T7 RNA polymerase. CG15744: a 569 nucleotides fragment was cloned into pDONR with primers: forward GGGGACAAGTTTGTAC AAA AAA GCA GGC TGG ATT GGA TAG CCT TGG TGA and reverse GGGGACCAC TTT GTA CAA GAA AGC TGG GTT TCG CTT CCA TCT CCA TCT C ; CG16974: a 548 nucleotides fragment was cloned into pDONR with primers: forward GGGGACAAGTTTGTAC AAA AAA GCA GGC TTA TAT GAA TCC CGA AGG CGC and reverse GGGGACCAC TTT GTA CAA GAA AGC TGG GTT TGG GGG GAG TAG ATG GTA A ; kek1 ; kek2 ; kek3 ; kek4 ; kek6 ; CG15744 ; CG16974 ; lambik . Colorimetric reaction was using Alkaline Phosphatase conjugated anti-DIG conjugated.In situ hybridizations were carried out following standard procedures, using antisense mRNA probes from 5’ linearised plasmids and transcribed as follows: Wide-field Nomarski optics images were taken with a Zeiss Axioplan microscope, 63x lens and JVC 3CCD camera and Image Grabber graffics card (Neotech) and a Zeiss AxioCam HRc camera and Zen software. Laser scanning confocal microscopy was carried out using inverted Leica SP2 AOBS, upright Leica SP8 or inverted Zeiss LSM710 laser scanning confocal microscopes, with 40x lens, 1024 x 1024 resolution, 0.25μm step for anti-Brp, and 0.5 μm step for the rest. Images were compiled using ImageJ, Adobe Photoshop and Illustrator.T286 were carried out in 3D throughout the stacks of images using the DeadEasy Synapse ImageJ plug-in that we previously validated [For NMJ data, all images provided in the figures are projections from Z stacks; all the quantitative anlyses were carried out in the raw stacks, not in projections. Muscle surface area was measured from bright field images using ImageJ. Anti-HRP was used to measure total terminal axonal length using ImageJ, and branching points. Boutons were visualized with anti-Dlg, and total boutons as well as separately Is and Ib boutons, were counted manually with the aid of the ImageJ Cell Counter plug-in. Automatic quantification of anti-Brp and anti-pCaMKIIalidated , and antDeadEasy Synapse has already been described and validated [T286 and anti-Synapsin stainings. Data were normalized to muscle surface area or axonal length.alidated . DeadEasalidated  was usedL3 wandering larvae were placed one at a time on an agar plate (2%) and left it crawl for 40 seconds. Larvae were filmed crawling across the agar plate and then discarded. Plates were cleared before placing another larva. At least 50 larvae were filmed per genotype, and the test was always done in the morning. Films of 400 frames per larva were analysed using FlyTracker software developed in our lab to obtain the trajectory and speed, as previously described.2, and a Bonferroni correction was applied for multiple comparisons. Continuous data were first tested for normality by determining the kurtosis and skewness, and a Levene’s Test was applied to test for homogeneity of variance. Data were considered not normally distributed if absolute kurtosis and skewness values for each genotype were greater than 1.96 x standard error of kurtosis/skewness. Variance of the populations of different samples were considered unequal if Levene’s test for homogeneity of variance gave a p value of <0.05. If samples were normally distributed and variances were equal, Student-t tests were applied for 2-sample type graphs, and One-Way ANOVA was used to compare means from >2 samples. When data were normally distributed, but variances were unequal, Welch ANOVA was used instead. Multiple comparison corrections to normal data were applied using a post-hoc Dunnett test of comparisons to a control, Games-Howell or Bonferroni post-hoc comparing all samples. Non-parametric continuous data were compared using a Mann-Whitney U-test when 2 sample types were being analysed, and a Kruskal-Wallis test for >2 samples, and multiple comparison corrections were applied using a post-hoc Dunn test. See Data were analysed in SPSS Statistics 21 (IBM) and GraphPad Prism 6. Confidence interval was 95% (p<0.05). Categorical data were tested using χS1 FigMIMIC-GFP larvae, stained with anti-GFP and (A) anti-Eve and (B) the pan-glial nuclear marker anti-Repo. (A) Eve colocalises with GFP in many cells (arrows indicate examples). Interestingly, most Eve+ neurons are also Toll-6+ . (B) Re(TIF)Click here for additional data file.S2 Fig(A)kek634 and kek635 null mutant alleles are deletions lacking the entire coding region for kek-6. (B) Following mutagenesis, these alleles were identified by two-sided PCR. (C) The deletion alleles were confirmed by demonstrating the lack of genomic regions internal to the deletion site. (D) The DNT237 null mutant allele bears a deletion with a breakpoint at the site of PBac{RB}spz5e03444. It lacks the regulatory region, all 5’UTR, the start site, and the first two exons. (E) Reverse Transcription PCR (RT-PCR) using primers to test if DNT2 mRNA is transcribed downstream of the breakpoint, which should result in a 464bp band. No transcripts were was detected (arrows). See also DNT237 is a null allele.(TIF)Click here for additional data file.S3 Figkek-6 mutants and upon over-expression of kek-6 in all neurons (with elavGAL4). Arrows indicate stereotypic projections in wild-type, and mistargeting in other genotypes. Chi-square p<0.0001 and ***p<0.001 Boferroni corrections, see The motoneuron marker FasII reveals motoraxon targeting phenotypes at muscle 6,7,12,13 in stage 17 embryos, in (TIF)Click here for additional data file.S4 Figkek-6 in motoneurons (MN) with D42GAL4 did not affect bouton number . (C-E) Over-expression of kek-6 induced pre-synaptic ghost boutons lacking a post-synaptic component , (D) higher magnification; (E) quantification. Both bouton number and area increased, albeit not significantly. Mann-Whitney U-tests. See Genotypes: Controls: y w/+; D42GAL4/+. kek-6–/–: kek634/Df(3R)6361; MN>kek6: D42GAL4>UAS-kek6-RFP (with one or two copies of UAS-kek-6). Over-expression of (TIF)Click here for additional data file.S5 FigT287 for the constitutively active form.  Quantification.  Inhibiting CaMKII function with Ala in motoneurons (D42GAL4>UASAla) decreased NMJ terminal axonal length , and active zones. Student t-tests, p<0.005, **p<0.01.  Pre-synaptic over-expression of constitutively active CaMKII (D42GAL4>UASCaMKIIT287D) increased pCaMKII levels , axonal length , Ib bouton number , and active zones . See (A) Confocal images of muscle 6/7 NMJs, in A3-4, labeled with anti-HRP for the pre-synaptic terminal, anti-Brp for active zones, anti-Dlg for post-synaptic boutons and anti-pCaMKII(TIF)Click here for additional data file.S1 TableFor all genotypes, sample sizes, tests and p values, see this table.(XLSX)Click here for additional data file.S2 TableList of S2 cell lysate proteins binding Kek6-Flag, but not mock-transfection controls, and detected with more than one peptide.(XLSX)Click here for additional data file.S3 TableList of adult fly head proteins binding Kek6-Flag, but not mock controls, and detected with more than one peptide.(XLSX)Click here for additional data file.


Salmonella enterica serotype 4,[5],12:i:- sequence type 34 is present in Europe and Asia. Using genomic surveillance, we determined that this sequence type is also endemic to Australia. Our findings highlight the public health benefits of genome sequencing–guided surveillance for monitoring the spread of multidrug-resistant mobile genes and isolates.Multidrug- and colistin-resistant  Salmonella enterica serotype 4,[5],12:i:- has increased sharply among humans, livestock, and poultry  sequence type (ST) 34. Before this study, the ST34 clone, already emerged in Europe and Asia, was yet to be detected in Australia as a drug-resistant pathogen of humans. We therefore investigated the circulation of drug-resistant Salmonella 4,[5],12:i:- ST34 in New South Wales (NSW), Australia.Since the 1990s, the global incidence of infection with Salmonella isolates referred to the NSW Enteric Reference Laboratory  have undergone whole-genome sequencing in addition to serotyping and multilocus variable-number tandem-repeat analysis (MLVA) performed as described  of these underwent whole-genome sequencing. Five duplicate isolates were excluded. In our retrospective study, we included 54 isolates from humans and 2 isolates from pork meat obtained from independent butchers during a routine survey conducted by the NSW Food Authority in 2016.Since October 2016, all https://card.mcmaster.ca) by using ABRicate version 0.5 (https://github.com/tseemann/abricate). Markers of colistin resistance were examined by using CLC Genomics Workbench . We identified Salmonella 4,[5],12:i:- genomes recovered in Europe and Asia by using Enterobase (https://enterobase.warwick.ac.uk/). We confirmed phenotypic resistance on a randomly selected subset of isolates by using the BD Phoenix system  or Etest .We extracted genomic DNA by using the chemagic Prepito-D  and prepared libraries by using Nextera XT kits and sequenced them on a NextSeq-500  with at least 30-fold coverage. We assessed genomic similarity and STs by using the Nullarbor pipeline (We obtained 54 isolates from 53 case-patients who had a median age of 25 years (range <1 to 90 years). We detected 20 MLVA profiles; however, 2 profiles predominated: 3-13-10-NA-0211 (45%) and 3-13-11-NA-0211 (14%). All but 2 case-patients resided in areas of distinct postal codes distributed throughout NSW; we found no apparent temporal or geographic clustering. Recent overseas travel was reported by 5 case-patients: 2 to Cambodia and 1 each to Thailand, Vietnam, and Indonesia. Salmonella 4,[5],12:i:- isolates were classified as ST34. The diversity between isolates was higher than that suggested by MLVA; we detected up to 112 single-nucleotide polymorphism (SNP) differences between isolates. The isolates from Australia clustered with each other and with isolates from the United Kingdom  genes (We detected AMR genes in 95% of ST34 isolates from NSW. The number of AMR genes (up to 13) was equivalent to that reported for ST34 isolates from the United States and United Kingdom , panel BB) genes , panel BstrA-strB, aph(3′)-Ia, blaTEM-1b, tet(A)-tet(B), sul2, and dfrA5 (which confers resistance against trimethoprim). Six isolates collected from case-patients who resided in the Sydney region over a 3-week period in 2017 shared R-type ASSuTmGK: genes aac -IV, aph -Ia, aph(3′)-Ic, blaTEM-1B, sul1, and dfrA5  (R-type ASSuTTmK was found for 12 (23%) isolates from humans: genes thoprim) , panel BqnrS1 (from 3 case-patients) and aac(6′)lb-cr (from 1 case-patient) were detected (aac(6′)lb-cr (aacA4-cr) gene was plasmid borne (IncHI2 plasmid) and was typically a class 1 integron–associated gene cassette (mcr-1 or mcr-2 genes nor mutations in the pmrAB, phoPQ, and mgrB genes were present (mcr-3, carried on a plasmid (Fluoroquinolone resistance–conferring genes detected , panel Bmcr-3 and indications that multidrug-resistant Salmonella 4,[5],12:i:- ST34 has established endemicity in Australia. Our findings highlight the public health benefits of genome sequencing–guided surveillance for monitoring the spread of multidrug-resistant mobile genes and isolates.Using genomic surveillance, we identified the presence of novel colistin resistance gene Salmonella sequence type 34 genomes used in study of multidrug-resistant Salmonella sequence type 34, New South Wales, Australia, 2016–2017.Publicly available


Caenorhabditis elegans, initiation of axon regeneration is positively regulated by SVH-2 Met-like growth factor receptor tyrosine kinase (RTK) signaling through the JNK MAPK pathway. Here we show that SVH-4/DDR-2, an RTK containing a discoidin domain that is activated by collagen, and EMB-9 collagen type IV regulate the regeneration of neurons following axon injury. The scaffold protein SHC-1 interacts with both DDR-2 and SVH-2. Furthermore, we demonstrate that overexpression of svh-2 and shc-1 suppresses the delay in axon regeneration observed in ddr-2 mutants, suggesting that DDR-2 functions upstream of SVH-2 and SHC-1. These results suggest that DDR-2 modulates the SVH-2–JNK pathway via SHC-1. We thus identify two different RTK signaling networks that play coordinated roles in the regulation of axonal regeneration.The ability of specific neurons to regenerate their axons after injury is governed by cell-intrinsic regeneration pathways. However, the signaling pathways that orchestrate axon regeneration are not well understood. In  C. elegans JNK MAP kinase pathway is required for the regrowth of neurons after injury. Previously, we identified several svh genes involved in JNK-mediated signaling. Among them, the svh-1 and svh-2 genes encode a growth factor and its receptor tyrosine kinase (RTK), respectively. This SVH-1–SVH-2 signaling cascade positively regulates axon regeneration through the JNK pathway. In the present study, we investigate the role of the svh-4/ddr-2 gene, which encodes an RTK containing a discoidin domain that is activated by collagen. Indeed, DDR-2 functions downstream of EMB-9 collagen type IV. Here, we show that the ddr-2 and emb-9 mutations delay initiation of regeneration after axon injury. Furthermore, we demonstrate that DDR-2 modulates the SVH-1–SVH-2–JNK pathway through the scaffold protein SHC-1. Thus, two different RTK signaling networks play coordinated roles in the regulation of axonal regeneration.An axon’s ability to regenerate after injury is governed by cell-intrinsic regeneration pathways. The  The ability of a neuron to regenerate in response to injury is modulated by a balance of extrinsic factors that promote or inhibit axon outgrowth, and by intrinsic processes that regulate axon growth potential. Most invertebrate neurons are able to regenerate, as are neurons in the mammalian peripheral nervous system. By contrast, neurons in the mammalian central nervous system have limited regenerative capability . AlthougCaenorhabditis elegans has been successfully used as a model system to study the mechanisms of axon regeneration . The. TheCaenin MLK-1 .svh-4/ddr-2 gene in the regulation of axon regeneration. The ddr-2 gene encodes an RTK that contains a discoidin domain activated by collagen. We demonstrate a link between the extracellular matrix and cell intrinsic pathways in the regulation of axonal regeneration.The ability of neurons to regenerate their axons following injury is modulated by interactions between the intrinsic axon growth machinery and the local extracellular environment. In the present study, we investigate the role of the svh-4 gene was identified as the ddr-2 gene, which encodes a protein homolog of the discoidin domain receptors (DDRs)  . DDRs arnd DDR-2  10]. Th. Thsvh-4ddr-2 loss-of-function mutants. The ok574 deletion allele disrupts the kinase domain and the ddr-2(tm797) knock-out causes a premature translation stop in the extracellular domain (C. elegans using single-neuron laser axotomy (ddr-2(ok574) and ddr-2(tm797) mutants was reduced (ddr-1(ok874) deletion mutation (ddr-2(ok574) mutants  after ablation. We observed that the frequency of axon regeneration in ddr-2(ok574) mutants was increased at 72 hr after axon injury, whereas regeneration in svh-2(tm737) mutants was not  mutation   .C. elegans DDR-2 corresponds to Arg-105 of the mammalian DDR2 (ddr-2 mutant [ddr-2(R100A)], in which Arg-100 is replaced with Ala. We found that the DDR-2(R100A) mutated form failed to rescue the defect in axon regeneration . The Arg-105 residue in the discoidin domain of mammalian DDR2 is essential for collagen binding . Sequencian DDR2  and therneration . Thus, tC. elegans, three collagens are associated with the basement membrane: the type IV collagens EMB-9 and LET-2 , kmEx1201 [Pddr-2a::ddr-2a (50 ng) + Pmyo-2::dsredmonomer (25 ng)], kmEx1202 [Punc-25::ddr-2a (25 ng) + Pmyo-2::dsredmonomer (25 ng)], kmEx1204 [Punc-25::ddr-2a(R100A) (25 ng) + Pmyo-2::dsredmonomer (25 ng)], kmEx1205 [Punc-25::ddr-2a(K554E) (25 ng) + Pmyo-2::dsredmonomer (25 ng)], kmEx1206 [Punc-25::svh-2 (50 ng) + Pmyo-2::dsredmonomer (25 ng)], kmEx1207 [Pshc-1::shc-1(R234K) (50 ng) + Pmyo-2::dsredmonomer (25 ng)], kmEx1208 [Pshc-1::shc-1(R136K) (50 ng) + Pmyo-2::dsredmonomer (25 ng)], kmEx1209 [Punc-25::ddr-2a::gfp (25 ng) + Punc-47::mcherry (50 ng) + Pmyo-2::dsredmonomer (25 ng)], kmEx1210 [Pddr-2a::nls::venus (25 ng) + Punc-25::nes::cfp (50 ng) + Pmyo-2::dsredmonomer (25 ng)], kmEx1213 [Punc-25::ddr-1 (25 ng) + Pmyo-2::dsredmonomer (25 ng)], kmEx1214 [Punc-25::ddr-2a::gfp (25 ng) + Punc-47::mcherry::caax (50 ng) + Pmyo-2::dsredmonomer (25 ng)], kmEx1215 [Punc-25::ddr-2a(R100A)::gfp (25 ng) + Punc-47::mcherry::caax (50 ng) + Pmyo-2::dsredmonomer (25 ng)], and kmEx1216 [Punc-25::ddr-2a(K554E)::gfp (25 ng) + Punc-47::mcherry::caax (50 ng) + Pmyo-2::dsredmonomer (25 ng)]. The kmEx505 and kmEx507 extrachromosomal arrays have been previously described [Transgenic animals were obtained by the standard n method . Pmyo-2:Axotomy was performed as previously described . Young aStandard fluorescent images of transgenic animals were observed under a X100 objective of a Nikon ECRIPSE E800 fluorescent microscope and photographed with a Hamamatsu 3CCD camera. Confocal fluorescent images were taken on an Olympus FV-500 confocal laser-scanning microscope with a X100 objective.unc-47 promoter (Punc-47::mcherry::caax) within these areas were determined. Then each net fluorescent intensity was calculated by subtracting the background fluorescence outside of the neurons. The relative fluorescent intensity (RFI) for each region was obtained by dividing the value of GFP fluorescence with that of mCherry fluorescence in the same region. Then the RFI at the proximal end of the injured axon was divided by the RFI at the axon region below the proximal end in the same axon.Localization of DDR-2::GFP fluorescence was quantified using the ImageJ program (NIH). First, regions to be analyzed were outlined with closed polygons and the fluorescent intensities of GFP and mCherry::CAAX expressed in D neurons by the http://www.graphpad.com/quickcalcs/). The Welch’s t -test was performed using a t-test calculator .Statistical analyses were carried out as described previously . BrieflyTransfection of transgenes into COS-7 cells, preparation of the cell lysates, immunoprecipitation and immunoblotting using anti-FLAG, ant-HA, anti-T7 and anti-phospho-tyrosine antibodies have been described previously [S1 Fig(A) Discoidin (DS) and DS-like domains. The DS domain is boxed. Identical and similar residues are highlighted with black and gray shading, respectively. Conserved Arg residue targeted for mutagenesis in this study is indicated by asterisk. (B) Protein kinase domain. Conserved Lys residue targeted for mutagenesis in this study is indicated by asterisk.(PDF)Click here for additional data file.S2 FigYellow arrowheads indicate the positions of nuclei. NLS::VENUS and NES::CFP are localized to the nucleus and cytoplasm, respectively, in D neurons. Scale bar = 10 μm.(PDF)Click here for additional data file.S3 FigWhole-cell extracts and immunoprecipitated complexes obtained with anti-FLAG antibody (IP: FLAG) were analyzed by immunoblotting. An arrow indicates the position of FLAG-DDR-2C.(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.