Some low molecular weight heparins (LMWHs) prolong survival of cancer patients and inhibit experimental metastasis. The underlying mechanisms are still not clear but it has been suggested that LMWHs (at least in part) limit metastasis by preventing cancer cell-induced destruction of the endothelial glycocalyx.To prove or refute this hypothesis, we determined the net effects of the endothelial glycocalyx in cancer cell extravasation and we assessed the anti-metastatic effect of a clinically used LMWH in the presence and absence of an intact endothelial glycocalyx. We show that both exogenous enzymatic degradation as well as endogenous genetic modification of the endothelial glycocalyx decreased pulmonary tumor formation in a murine experimental metastasis model. Moreover, LMWH administration significantly reduced the number of pulmonary tumor foci and thus experimental metastasis both in the presence or absence of an intact endothelial glycocalyx.In summary, this paper shows that the net effect of the endothelial glycocalyx enhances experimental metastasis and that a LMWH does not limit experimental metastasis by a process involving the endothelial glycocalyx. In experimental animal models and clinical studies it has been well established that some low molecular weight heparins (LMWH) inhibit experimental metastasis and prolong survival in vivo remains to be elucidated however.The endothelial glycocalyx is a negatively charged, organized network of membranous glycoproteins, proteoglycans and glycosaminoglycans that affects several biological processes with potential importance for cancer cell extravasation. First, the endothelial glycocalyx is essential for vascular barrier function. Its disruption by pro-inflammatory cytokines, including tumor necrosis factor (TNF-α) and glycocalyx-degrading enzymes such as heparanase and hyaluronidase, leads to increased vascular permeability Interestingly, cancer cells produce enzymes that are known to degrade the endothelial glycocalyx, such as heparanase and hyaluronidase In the current manuscript, we aimed to assess whether the effect of a LMWH on experimental metastasis depends on restoration of the endothelial glycocalyx. To this end, we first determined the net effect of the endothelial glycocalyx in experimental metastasis. Next, we assessed the effect of a LMWH in the presence or absence of an intact endothelial glycocalyx to determine the contribution of the glycocalyx to the effect of this LMWH on the reduction of experimental metastasis.To assess the net effect of the endothelial glycocalyx on experimental metastasis, wild type mice were treated with hyaluronidase in order to remove hyaluronan and, in part, heparan sulphates from the endothelial glycocalyx. As it has previously been shown that one hour after hyaluronidase treatment vascular leakage is evident To confirm the pro-metastatic effect of the glycocalyx and to exclude “side effects” like acute immunological responses As already indicated, the glycocalyx is considered as an integrated and balanced carbohydrate layer in which both hyaluronan and heparan sulfate chains are key structural components. Importantly, our data show that targeting either hyaluronan  or the heparan sulphate chains (genetic ablation of syndecan-1) of the glycocalyx leads to reduced experimental metastasis. As these two different interventions have a similar effect on experimental metastasis, our data imply that barrier protective-properties of the glycocalyx are less essential for metastasis than its functions in cancer cell adhesion or growth factor storage As mentioned before, some LMWHs protect against cancer progression in experimental animal models and clinical trails, including the B16F10 melanoma model of experimental metastasis. As suggested previously, these LMWHs may inhibit metastasis through competitive binding of heparanase or hyaluronidase thereby protecting the vascular endothelium and its barrier function from disruption caused by these enzymes. To assess whether the inhibitory effect of the administration of a LMWH on cancer progression are dependent on its protective effects on the glycocalyx, we compared the effect of enoxaparin administration on experimental metastasis in syndecan-1 deficient and wild type mice. As shown in Some aspects of the experimental set-up require further comments. First, a lower amount of cancer cells was injected in the second experiment  in orderIn conclusion, our data show that targeted interference of either hyaluronan or heparan sulfate limits experimental metastasis suggesting that the net effect of the glycocalyx is pro-metastatic. Moreover, the effect of enoxaparin on cancer progression and cancer cell metastasis is glycocalyx independent.Bovine testicular hyaluronidase  dissolved in 0.9% NaCl was administered intravenously in a dose of 100 units per mouse 1h prior to cancer cell inoculation Murine B16F10 melanoma cells were obtained from the American Type Culture Collection . Cells were cultured in Dulbecco Modified Eagle Medium  supplemented with 10% fetal calf serum (Sigma-Aldrich), 1% penicillin-streptomycin solution and 1% L-glutamine at 37°C as described before Eight to ten week-old C57Bl/6 male mice  were maintained at the animal care facility of the Academic Medical Centre, Amsterdam, The Netherlands according to institutional guidelines. Syndecan-1 −/− male mice on a C57Bl/6 background were housed and bred in the Radboud University Nijmegen Medical Centre and handled as described 5 cancer cells were administered per mouse, whereas in the second experiment 2.0×105 cells were used in order to lower the amount of secondary tumor foci for purpose of countability. After 14 days, mice were sacrificed and lungs were prepared as described before Cancer cells (suspended in 200 µl PBS) were injected into the lateral tail vein as described before Statistical analysis was carried out in GraphPad Prism version 4.03. Data are expressed as means +/− SEM or medians with interquartile range. For normally distributed data, significance was assessed with the Student t-test. For not normally distributed data, non-parametric testing was performed using the Mann-Whitney test. Statistical significance was assumed when the p-value was <0.05.


Jatropha curcas L.) are rich in fatty acids/lipids, storage proteins, polysaccharides, and a number of other secondary metabolites that could either bind and/or co-precipitate with RNA, making it unsuitable for downstream applications. Existing RNA isolation methods and commercial kits often fail to deliver high-quality total RNA from immature jatropha seeds for poly(A)+ RNA purification and cDNA synthesis.RNA quality and quantity is sometimes unsuitable for cDNA library construction, from plant seeds rich in oil, polysaccharides and other secondary metabolites. Seeds of jatropha This is a simple CTAB + silica column method to extract high quality RNA from oil rich immature jatropha seeds that is suitable for several downstream applications. This method takes less time for RNA extraction and is equally effective for other tissues where the quality and quantity of RNA is highly interfered by the presence of fatty acids, polysaccharides and polyphenols. The RNA extracted with this method was good for several downstream applications such as cDNA library construction, RT-PCR, gene isolation and Northern blot analysis. This method was equally good for seeds of mustard (Brassica spp.) and starchy rice (Oryza sativa).Cetyltrimethylammonium bromide (CTAB) based method was developed for RNA extraction from tissues containing high levels of polysaccharides and phenols . The proJatropha curcas) seeds, at 4-5 weeks after fertilization were selected for total RNA extraction. Rice (Oryza sativa) seeds 21 days after fertilization and mustard (Brassica spp.) seeds 20 days after fertilization were also used. Before grinding, the kernels of immature jatropha seeds were separated from the seed shell, whereas for rice seed husks were removed using sterilized scissors or forceps and stored at -80°C until use. Mustard seeds were removed from pods and used as such.Immature jatropha (g for 20 minutes at 4°C. The aqueous supernatant (1 ml/tube) above the white phase was carefully transferred into 2.0 mL RNase-free microcentrifuge tubes and an equal volume of Chloroform: Isoamylalcohol was added, mixed with a vortex and centrifuged in a desktop centrifuge at 10,000 g for 10 minutes at 4°C. Without touching the white layer, the supernatant (1.0 ml) was distributed to Rnase free1.5 mL microcentrifuge tubes and 0.5 mL of 96-100% ethanol was added. The supernatant-ethanol mixture was immediately loaded onto RNA binding columns (0.75 mL/column) skipping filtration step (Qiagen RNA Mini extraction kit or any other similar kit) and spun at 10,000 g for 30 seconds at room temperature. Leftover samples were loaded on the same columns to process the entire sample. The kit protocol was followed in subsequent steps to wash and desalt the samples bound with the silica membrane of the column. Finally, the RNA from each column was eluted using 50 μL of RNase free water and stored at -80°C.About 0.5 g of each seed sample was ground in liquid nitrogen using oven baked RNase-free mortar and pestle and the seed powder was then transferred to a pre-chilled 50-mL polypropylene  tube. Five mL of pre-heated (65°C) total RNA extraction buffer {2% (w/v) CTAB (Sigma), 2% (w/v) polyvinylpyrrolidone (PVP-40) (Sigma), 100 mM Tris HCl (pH 8.0), 25 mM EDTA, 2 M NaCl, 0.1% spermidine (Sigma) and 2% β-mercaptoethanol} was added to the powdered seeds in each tube and samples were incubated for 30 min at 65°C in a water bath. The samples were placed on a vortex every 5 minutes to help tissue disruption and RNA extraction in the buffer. After incubation, an equal volume of Chloroform: Isoamylalcohol (24:1) was added to each sample in a fume hood and samples were mixed with a vortex for 30 seconds. Thereafter the samples were centrifuged at 10,000 260/230 and A260/280 nm. The integrity of total RNA was determined by running samples on 1.2% denaturing agarose gel . The intensity of 28S and 18S bands was quantified with Molecular Imaging software version 5.1 . Aliquots of RNA were stored at -80°C.The quality of RNA was checked using a spectrophotometer  at two wavelength ratios of A+ RNA was purified from total RNA of immature jatropha seeds using the Oligotex® Midi mRNA kit , dissolved in RNase-free water, quantified with spectrophotometer , and stored at -80°C. The cDNA library was constructed using CloneMiner™ cDNA Library Construction Kit (Invitrogen). The first strand of cDNA was synthesized using 5 μg poly(A)+ RNA and converted into double strand cDNA (ds cDNA) containing attB sequences on each end followed by ligating attB1 adapter to the 5' end of cDNA. The cDNA was size fractionated by column chromatography to remove excess of primers, adapters, and small cDNAs. A non-radio labeled method was used to determine the cDNA yield. About 75-100 ng of cDNA obtained from different pooled fractions was used in site-specific recombination and attB-flanked cDNA was cloned into an attP-containing donor vector (pDONR™ 222). The BP reactions were transformed into ElectroMAX DH10B T1 phage resistant cells using an electroporator (Life technologies) and the transformed cells were plated on kanamycin (50 μg/mL) added LB agar media. Twenty positive clones were picked for verification of cDNA inserts. The mini-prepared plasmids were digested with BsrG1 enzyme (New England Biolabs) and electrophoresed on 1% agarose gel to determine average insert size of cDNA.Poly(A)Primers from the Arabidopsis 3-ketoacyl-acyl carrier protein reductase, (AT1G24360) (KAR) involved in Fatty acid biosynthesis were selected from the GenBank database  for RT-P+ nylon membranes (Amersham Pharmacia) and stained for visualization of the RNA bands [For Northern blotting, 15 μg of RNA was isolated from immature jatropha seeds and leaves and fractionated on 1.2% agarose-formaldehyde denaturing gel (Qiagen RNeasy Mini handbook). The RNA was blotted onto Hybond-NNA bands . The KAR® Plant Mini Kit) and skipped the LiCl precipitation step to reduce the extraction time from two days to ~3 h.We tried a few protocols of RNA extraction based on CTAB ,7,8,17, 260/230 ratio was lower than 2. 00. The average 28S:18S ratio was 1.49+0.21, RNA showed smear on the gel and the bands were not sharp, indicating that the total RNA was still bound with contaminants or some degradation had occurred. Similar trends were observed with other published CTAB based methods and that involving phenol-guanidinium thiocyanate resulting in low RNA yield and quality (data not shown). The commercial kits for RNA extraction were also not successful as the silica columns were usually blocked with viscous extracts and the yield was extremely low and the quality was not good , II (CTAB + silica column), and III (RNA extraction kit) were electrophoresed on denatured 1.2% agarose gel to determine the quality and integrity of RNA bands  + RNA was precipitated with isopropanol and NaAc to a concentration of 1 μg/μL for first-strand cDNA synthesis. Size fractionated double-strand cDNA was visualized as a smear on the agarose gel (1.2%) with a size ranging from 0.5 kb to 5 kb  and the positive clones were selected on kanamycin added LB plates. This cDNA library consisted of 1 × 107 clones, which should be enough to represent most of the genes expressed in immature jatropha seeds.The suitability of isolated RNA in downstream enzymatic procedures was also determined by constructing a cDNA library. The total RNA extracted with the modified method II produced a high quality poly(A)) Figure , Panel BBsrG1 enzyme (New England Biolabs) and separated on 1% agarose gel. The insert size of different cDNA clones ranged from 300 bp to 2.3 Kb with an average size of 1.3 Kb (Figure To evaluate the cDNA library, plasmids were isolated from 20 randomly picked cDNA clones, digested with Silica columns and silica particles have been used previously in combination with CTAB  based meThe information on RNA extraction protocols is limited for jatropha. An acid phenol-silica particles based method was used to extract RNA from jatropha leaf and dry seeds . The metThe modified protocol is simple and highly effective for extracting good quality RNA from oil rich immature jatropha seeds as well as mustard and rice seeds. It should be equally useful for other tree plants for molecular characterization where the quality and quantity of RNA is highly dependent on the presence of lipids/fatty acids, polysaccharides and polyphenols.The authors declare that they have no competing interests.JSS conducted the experiments. JK drafted the manuscript. JSS and KG constructed cDNA library. ZY supervised and drafted the manuscript. All authors have read and approved the final manuscript.


Caenorhabditis elegans counterpart of the Receptor for Activated C Kinase (RACK-1) interacts physically with the actin-binding protein UNC-115/abLIM and that RACK-1 is required for axon pathfinding. Genetic interactions indicate that RACK-1 acts cell-autonomously in the UNC-115/abLIM pathway in axon pathfinding and lamellipodia and filopodia formation, downstream of the CED-10/Rac GTPase and in parallel to MIG-2/RhoG. Furthermore, we show that RACK-1 is involved in migration of the gonadal distal tip cells and that the signaling pathways involved in this process might be distinct from those involved in axon pathfinding. In sum, these studies pinpoint RACK-1 as a component of a novel signaling pathway involving Rac GTPases and UNC-115/abLIM and suggest that RACK-1 might be involved in the regulation of the actin cytoskeleton and lamellipodia and filopodia formation in migrating cells and growth cones.Migrating cells and growth cones extend lamellipodial and filopodial protrusions that are required for outgrowth and guidance. The mechanisms of cytoskeletal regulation that underlie cell and growth cone migration are of much interest to developmental biologists. Previous studies have shown that the Arp2/3 complex and UNC-115/abLIM act redundantly to mediate growth cone lamellipodia and filopodia formation and axon pathfinding. While much is known about the regulation of Arp2/3, less is known about regulators of UNC-115/abLIM. Here we show that the  Caenorhabditis elegans called rack-1. This gene is conserved in vertebrates and is involved in cellular signaling. We show that it interacts in a novel manner with other cell signaling genes (the Rac GTPase genes) and a gene involved in lamellipodia and filopodia formation, called unc-115/abLIM. We think that rack-1 is involved in a novel cellular signaling event involving Rac GTPases that regulates lamellipodia and filopodia protrusion in the growth cone during nervous system development.In the developing nervous system, the growth cone guides axons of neurons to their correct targets in the organism. The growth cone is a highly dynamic specialization at the tip of the axon that senses cues and responds by crawling toward its target, leaving the axon behind. Key to growth cone motility are dynamic cellular protrusions called lamellipodia and filopodia. These protrusions are required for growth cone movement and steering. The genes that are involved in lamellipodia and filopodia formation in the growth cone are still being discovered, and studies to understand how these genes act together in cell signaling events that control growth cone movement are in their infancy. Here we report discovery of a new gene necessary for growth cone movement in  The actin cytoskeleton is necessary for the formation of cellular protrusions, lamellipodia and filopodia, that underlie morphogenetic events such as cell migration and axon pathfinding Caenorhabditis elegansC. elegans, the Arp2/3 complex is required for both lamellipodial and filopodial formation C. elegans growth cones C. elegans axon pathfinding, WVE-1/WAVE acts downstream of CED-10/Rac and WSP-1/WASP acts downstream of the MIG-2/RhoG GTPase to regulate Arp2/3 In cultured cells, the actin-nucleating Arp2/3 complex controls the formation of lamellipodial networks C. elegans showed that UNC-115/abLIM acts downstream of the CED-10/Rac GTPase in neuronal lamellipodia and filopodia formation While much is known about the Arp2/3 signaling pathway, less is known about the control of UNC-115/abLIM in lamellipodia and filopodia formation. The conserved UNC-115/abLIM proteins have multiple LIM domains at the N terminus and an actin-binding villin headpiece domain at the C terminus C. elegans Receptor for Activated C Kinase molecule (Rack1), called RACK-1 in C. elegansC. elegans, RACK-1 has been shown to be involved in embryonic cytokinesis C. elegans RACK-1 regulates membrane trafficking and recycling endosome distribution via interaction with dynactin, and thus might regulate the microtubule motor dynein. As a consequence, rack-1 loss of function leads to defects in cytokinesis and chromosome separation in the early embryo.A two-hybrid screen with the central region of UNC-115 identified the rack-1 mutants, indicating that RACK-1 acts cell autonomously in axon pathfinding. Furthermore, we show that RACK-1 is involved in migration of the gonadal distal tip cells, and that the signaling pathways involved in this process might be distinct from those involved in axon pathfinding. In sum, these studies pinpoint RACK-1 as a component of a signaling pathway involving Rac GTPases and UNC-115/abLIM, and suggest that RACK-1 might be involved in the regulation of the actin cytoskeleton and lamellipodia and filopodia formation in migrating cells and growth cones.Here we show that RACK-1 interacts with the actin-binding protein UNC-115/abLIM, and that RACK-1 is required for axon pathfinding. Genetic interactions indicate that RACK-1 acts in the UNC-115/abLIM pathway in axon pathfinding, downstream of the CED-10/Rac GTPase and in parallel to MIG-2/RhoG and the UNC-34/Enabled. Neuron-specific expression of RACK-1 is sufficient to rescue the axon pathfinding defects of UNC-115, abLIM, dematin)  in the C-terminus, and a middle region with unknown function that contains a highly conserved region across species, the UAD domain (dematin)  [17]. ThHIS5 expression in a liquid-based microtiter screening procedure  were found. All seven cDNAs were found to activate when retested, and all seven cDNAs displayed bait-dependence and did not activate in the absence of the UNC-115 bait (data not shown).In order to identify molecules that interact physically with the non-LIM-domain region UNC-115, the central region of UNC-115 (residues 243 to 553 of the F09B9.2b molecule as described on Wormbase) was used as bait in a yeast two-hybrid screen . This twdure see . From a The seven cDNAs represented five independent isolates (i.e. represented five different 5′ ends), with two of the isolates having two representatives each . Three oC. elegans , called RACK-1 in  elegans  [27]. RAs RACK-1 . Two of s RACK-1 .C. elegans extracts. We generated a transgene expressing MYC-tagged RACK-1 under its endogenous promoter and made animals transgenic for this construct. This transgene produced functional RACK-1::MYC, as it rescued the sterility, gonadal distal tip cell migration defects, and axon pathfinding defects caused by the rack-1(tm2262) deletion (see below). We immunoprecipitated MYC-tagged RACK-1 (RACK-1::MYC) using an anti-MYC antibody from animals harboring a rack-1::myc integrated gene (see C. elegans extracts and that it was immunoprecipitated by this treatment (C. elegans not expressing RACK-1::MYC were immunoprecipitated with the MYC antibody (data not shown). We repeated this co-immunoprecipitation two additional times, and the results of one representative experiment are shown in To confirm that RACK-1 and UNC-115 interact in a complex, we determined if RACK-1 and UNC-115 co-immunoprecipitated from gene see . Using areatment . Westernreatment . In the reatment . FurtherC. elegans RACK-1 is a 325 amino-acid protein that has two regions similar to the PKC binding sites of vertebrate RACK and a conserved tyrosine that is phosphorylated by Src in vertebrate RACK. C. elegans PKC and Src isoforms are expressed in the nervous system, and both PKC and Src have been implicated in growth cone pathfinding and cell migration C. elegansC. elegans.unc-25::gfp transgene (juIs76) to image the VD/DD neurons and their axons unc-115(ky275) disrupts axon pathfinding in these neurons, yielding in an uncoordinated movement phenotype rack-1 function using RNAi by injection (see rack-1(RNAi) disrupted the proper pathfinding of the VD and DD commissural axons (unc-115(ky275)The VD and DD motor neurons are GABAergic neurons that control the coordination and movement of the nematode tion see . In 22% al axons . The defrack-1 locus, called tm2262, was isolated and kindly provided by the National Bioresource Project for the Experimental Animal “Nematode C. elegans” (S. Mitani). The tm2262 deletion was an in-frame deletion that removed part of the first WD repeat, all of the second, and most of the third, including the predicted PKC interaction site in WD3  might be a hypomorph. However, RNAi did not worsen the low brood size or axon defects of rack-1(tm2262) , indicating that it might be a strong loss of function allele.A deletion of the e in WD3 . Since track-1, the deletion allele rack-1(tm2262) caused pathfinding defects in the VD and DD motor neurons (rack-1(tm2262) displayed VD/DD commissures aberrantly extending up the left side of the animal ; juIs76 showed VD/DD commissures on the left side (p<0.001) ; juIs76 animals (rack-1(tm2262); juIs76 animals displayed VD/DD axon guidance and outgrowth defects such as axonal wandering, branching or termination  . In juIs animals . In addimination , whereasrack-1(tm2262) were due to rack-1 perturbation and not a background mutation, we rescued the VD/DD axon defects with a rack-1::myc transgene. rack-1::myc rescued both left-right defects and commissural guidance defects (60% to 32% (p<0.001) and 42% to 10% (p<0.001)) (To ensure that the axon guidance defects observed in <0.001)) . Togetherack-1(tm2262) animals were slow growing and had very low brood size. In a progeny count, ten wild type and ten rack-1(tm2262) animals were individually plated and then transferred to a new plate every day until egg laying ceased. The number of viable adult progeny resulting from each animal were counted and averaged. The average progeny count for a wild-type N2 animal was of 278.4 (s.d. = 32.72), while for rack-1(tm2262) the count dropped to 23.3 (s.d. = 9.9) (p<0.0001). A transgene containing the rack-1 gene under its native promoter fused to the gfp coding region (rack-1::gfp) (see rack-1(tm2262) animals to 73.78 (s.d. = 17.18) (p<0.0001), suggesting that rack-1::gfp was functional and could rescue the brood size defect in rack-1 animals. rack-1::myc could also rescue the low brood size of rack(tm2262) (data not shown). Thus, the reduction in brood size was due to rack-1 and not due to genetic background in the tm2262 strain.gfp) see  increaserack-1(tm2262) seems to be predominantly due to decreased production of fertilized embryos. rack-1 might affect oogenesis or spermatogenesis, but the nature of this sterility has not been explored. Previous studies indicate that rack-1 also affects embryogenesis by regulating membrane trafficking and recycling endosome distribution via interaction with dynactin to control cytokinesis and chromosome separation in the early embryo The reduced brood size of rack-1 gene is expressed, we constructed a reporter transgene consisting of the promoter region of rack-1 fused to the gfp coding region . rack-1::gfp rescued the sterility and gonadal distal tip cell migration defects of rack-1(tm2262) mutants. RACK-1::GFP was present in the cytoplasm of cells and showed little if any nuclear accumulation  coding region lacking the upstream promoter region was fused to gfp downstream of the unc-25 promoter. The Ex[unc-25 promoter::rack-1::gfp] transgene was expressed specifically in the GABAergic neurons including the VD/DD neurons and nowhere else (rack-1(tm2262) as did the genomic rack-1(+) transgene (data not shown), indicating that expression was specific to the VD/DD neurons. Ex[unc-25 promoter::rack-1(+)] rescued the lateral asymmetry defects and axon wandering defects of rack-1(tm2262) animals (Ex[unc-25 promoter::rack-1(+)] transgene. These data indicate that rack-1 acts cell autonomously in neurons in axon pathfinding.ere else . This tr animals  , ced-10(n1993), and unc-115(ky275), alone had low-penetrance defects in PDE axon pathfinding on their own  also had very few defects in PDE axon pathfinding ; unc-115(ky275) double mutants  displayed levels of PDE axon pathfinding defects  and rack-1(tm2262) alone, suggesting that UNC-115/abLIM and RACK-1 might act in the same pathway. In contrast, rack-1(tm2262M+); mig-2(mu28) double mutants showed significantly increased levels of defects compared to either single alone  can synergize with other mutants to cause axon defects, and that RACK-1 and MIG-2/RhoG might act in parallel pathways in axon pathfinding.Previous results show that CED-10/Rac and MIG-2/RhoG act redundantly in PDE axon pathfinding, and UNC-115/abLIM works downstream of CED-10/Rac, in parallel to MIG-2/RhoG in PDE pathfinding ects 6%;  that werone 28%; . This rerack-1(tm2262M+) ced-10(n1993M+) double mutants displayed no significant increase in PDE defects compared to either single alone  ced-10(n1993M+); unc-115(ky275) triple mutant to be no more severe than any double mutant combination alone. As previously reported, ced-10(n1993); unc-115(ky275) double mutants were significantly more severe than either single alone  ced-10(n1993M+) mutants did not show this interaction. Possibly, RACK-1 is not the only molecule regulating UNC-115 and has a weaker effect. In any case, the rack-1(tm2262M+) ced-10(n1993M+); unc-115(ky275) triple mutant was not significantly more severe than ced-10(n1993); unc-115(ky275) alone ; unc-34(e951M+) double mutants displayed significantly increased pathfinding defects compared to unc-34(e951) alone  loss of function might have on overactive Rac GTPases. Constitutively-activated Rac GTPases transgenes harbor a guanine-12-valine mutation in the GTPase binding pocket, which favors the active GTP-bound state of the GTPases. Previous studies showed that CED-10(G12V) and MIG-2(G16V)  both caused axon pathfinding defects and drove the formation of ectopic neurites, lamellipodia, and filopodia when expressed in PDE neurons ; ced-10(G12V) animals displayed 45% ectopic lamellipodia and filopodia, a significant reduction (p = 0.004) from CED-10(G12V) alone (rack-1(tm2262) partially suppressed activated CED-10(G12V) and that functional RACK-1 might be required for the formation of ectopic lamellipodia and filopodia induced by activated CED-10. In contrast, rack-1(tm2262) did not suppress ectopic lamellipodia and filopodia associated with MIG-2(G16V) and in fact slightly enhanced these defects  and slightly enhanced MIG-2(G16V) We determined if RACK-1 was required for the effects of CED-10(G12V) and MIG-2(G16V). CED-10(G12V) alone caused 66% of PDE neurons to have ectopic lamellipodia and filopodia in young adults . rack-1(V) alone . These dC. elegans neurons and in cultured mammalian fibroblasts Previous studies showed that UNC-115 tagged with an N-terminal myristylation sequence (MYR) caused activation of the molecule rack-1(tm2262) loss of function background. MYR::UNC-115 was expressed from the unc-115 promoter (the lqIs62 transgene), which drives expression in most neurons including PDE and the VD/DDs myr::unc-115 transgene scored in lqIs62). We found that lqIs62[MYR::UNC-115] caused 8% ectopic lamellipodia and filopodia in PDE neurons (rack-1(tm2262) mutation  in rack-1(tm2262); myr::unc-115 animals displayed lateral asymmetry defects similar to each alone (rack-1(tm2262); myr::unc-115 displayed significantly increased defects compared to the additive effects of each alone (rack-1(tm2262) might enhance myr::unc-115 in VD/DD pathfinding.To study the interactions of ch alone . Thus, rmyr::unc-115 by rack-1(tm2262) in the PDE neurons and the VD/DD neurons. The results are consistent with RACK-1 acting upstream of or together with UNC-115 in axon pathfinding. Some context-specific interactions were observed, such as rack-1(tm2262) enhancing VD/DD commissural axon pathfinding, indicating that RACK-1 and UNC-115 might have distinct interactions in different contexts or developmental events.In summary, we detected no strong suppression of myr::rack-1::gfp was expressed in the PDE neurons by the osm-6 promoter. MYR::RACK-1::GFP displayed a membrane-associated distribution ; myr::rack-1 and unc-115(ky274); myr::rack-1, respectively) (unc-115(mn481) allele myr::rack-1, indicating that possibly only a small amount of UNC-115 activity is required for MYR::RACK-1 to drive ectopic lamellipodia. These studies support the model that RACK-1 acts upstream of UNC-115 in lamellipodia formation.The above results indicate that RACK-1 is not required for the effects of MYR::UNC-115, suggesting that RACK-1 might act upstream of UNC-115. To test this idea, we constructed a myristylated version of RACK-1, similar to MYR::UNC-115. We reasoned that constitutive membrane localization might activate RACK-1 as it does UNC-115. ctively) . The hyprack-1 and unc-115, we could detect no change in distribution of UNC-115::GFP in loss of function rack-1(tm2262) or in the activated myr::unc-115 transgenics (data not shown). In each case, UNC-115::GFP was present uniformly throughout the cytoplasm, similar to wild-type animals, and showed no membrane localization.Despite the strong genetic interactions of myr::unc-115 caused lateral displacement of GABAergic motor neuron cell bodies such that they often were found outside of the ventral nerve cord  suppressed the displaced VD cell body defect of myr::unc-115 ; myr::unc-115 (p = 0.011). This result suggests that RACK-1 might act downstream of or participate with MYR::UNC-115 in impeding P nucleus migration, again suggesting context-dependent interactions of UNC-115 and RACK-1.Activated rve cord . The VD :unc-115 : 30% of C. elegans gonad is derived from two somatic cells (Z1 and Z4) surrounding the two germ cells (Z2 and Z3) C. elegans (rack-1(tm2262) animals were misrouted (rack-1::gfp transgene rescued gonad misrouting defects in rack-1(tm2262)  (rack-1 perturbation. It should be noted that rack-1(tm2262) homozygotes from a heterozygous mother (rack-1(tm2262M+)) had less severe DTC migration defects compared to the rack-1(tm2262) animals without maternal contribution . This indicates that DTC migration defects were partially rescued by wild-type maternal rack-1(+) activity. unc-115 mutants displayed no defects in DTC migration, and the gonad defects of rack-1 were not affected by unc-115 (data not shown). rack-1::myc also rescued the DTC migration defects of rack-1(tm2262) (data not shown). Thus, DTC migration is controlled by RACK-1 and is independent of UNC-115/abLIM.The  elegans . If DTC isrouted . Misroutp<0.005) , indicatCED-10/Rac and MIG-2/RhoG have previously been shown to control gonad distal tip cell migration ced-10(n1993) and mig-2(mu28) mutations caused defects in DTC migration (27% for mig-2(mu28), 12% for ced-10(n1993)) ; mig-2(mu28) showed no significant difference (26%) compared to mig-2(mu28) (27%), and rack-1(tm2262M+) ced-10(n1993) showed no significant difference compared to rack-1(tm2262M+) (21% in each case).As reported previously, both (n1993))  [48]. Weced-10; mig-2 double mutants did not display enhanced DTC migration defects compared to either single alone CED-10/Rac and MIG-2/RhoG interact differently in the DTCs than they do in other tissues such as axons, in which they act in parallel redundant pathways. In summary, we have presented data indicating that RACK-1 is required cell autonomously for axon pathfinding, and that RACK-1 is required for migration of the distal tip cells of the gonad. We show that RACK-1 interacts physically with UNC-115/abLIM, and that RACK-1 and UNC-115/abLIM might act in the same pathway in axon pathfinding. Consistent with this idea, RACK-1 was required for ectopic lamellipodia and filopodia induced by the activated CED-10/Rac GTPase, similar to UNC-115/abLIM. RACK-1-like molecules have been implicated in a wide variety of cellular events and have been shown to interact with a large number of distinct protein complexes, consistent with the idea of RACK molecules as scaffolds and integrators C. elegans, RACK-1 depletion by RNAi resulted in embryos with cytokinesis defects, including shorter astral microtubules, defects in chromosome separation, and defects in membrane organization and recycling endosome distribution rack-1(tm2262) was very sick and slow growing, and gave very few progeny. rack-1(tm2262) produced very few embryos, suggesting that the animals had defects in sperm and/or oocyte production. Indeed, rack-1 RNAi resulted in defects in germline membrane organization rack-1(tm2262) mutant.Previous studies showed that in rack-1(tm2262) mutants displayed a variety of axon pathfinding defects, including left-right choice and guidance defects of the VD and DD commissural motor axons and guidance defects of the PDE axons. VD/DD axons are dorsally directed, and PDE axons are ventrally directed, indicating that rack-1 is not specific for any particular guidance direction. VD/DD axon pathfinding defects were rescued when rack-1(+) was expressed under a promoter that specifically drives expression in the GABAergic neurons including VD/DD and nowhere else, demonstrating that RACK-1 is required cell-autonomously for axon pathfinding. As RACK-1 is likely involved in many different developmental events, this result shows that the effects of RACK-1 on axon pathfinding are due to defects in the neuron itself and not a substrate or guidepost tissue such as the hypodermis or other neurons. Indeed, RACK-1 was expressed in neurons, and functional RACK-1::GFP fusion protein accumulated in the growth cones of neurons, consistent with a role of RACK-1 in growth cone cytoskeletal regulation. RACK-1::GFP also accumulated in the cell bodies and axons of neurons.rack-1(tm2262) mutants display defects in the structure of the gonad arms consistent with a defect in distal tip cell migration. rack-1 is expressed in the migrating distal tip cells. The Rac GTPases CED-10/Rac and MIG-2/RhoG also each affect distal tip cell migration, but do not show the phenotypic synergy in DTC migration as is observed in axon pathfinding We have shown that mig-2; rack-1 and ced-10 rack-1 double mutants were not significantly different than the stronger single mutants alone. This result suggests that RACK-1 might act in a pathway independent of MIG-2 and CED-10, or that all three act in a common pathway. This again points to context dependent differences in the function of RACK-1 and suggests that RACK-1 might interact with different effectors in different ways in different cells and cellular events. Indeed, the effect of RACK-1 on DTC migration is likely to be independent of UNC-115/abLIM, as unc-115 mutants have no effect on DTC migration alone or in any double mutant combination analyzed so far, including ced-10 and mig-2.DTC migration defects in rack-1(tm2262) synergized with mig-2/RhoG and unc-34/Enabled in PDE axon pathfinding, similar to unc-115/abLIM and ced-10/Rac. rack-1(tm2262) did not synergize with unc-115/abLIM or ced-10/Rac, consistent with the idea that they act in the same pathway in parallel to mig-2/RhoG and unc-34/Enabled.A model of RACK-1 interaction with CED-10/Rac and UNC-115/abLIM is shown in unc-115 loss of function suppresses this effect rack-1(tm2262) also partially suppressed ectopic lamellipodia and filopodia caused by CED-10(G12V), indicating that RACK-1 is required downstream of CED-10/Rac in lamellipodia and filopodia formation (Activated CED-10(G12V) drives the formation of ectopic lamellipodia and filopodia in PDE neurons, and ormation . This rerack-1(tm2262) and myr::unc-115. One interpretation of these data is that RACK-1 does not act downstream of UNC-115/abLIM and instead might act together with or upstream of UNC-115/abLIM. Indeed, unc-115 mutations suppressed the ectopic lamellipodia caused by MYR::RACK-1, indicating that UNC-115 acts downstream of RACK-1. These results are consistent with a model in which RACK-1 acts downstream of CED-10/Rac and upstream of UNC-115/abLIM in axon pathfinding  suppressed the lateral displacement of VD cell bodies caused by myr::unc-115. Laterally misplaced VD cell bodies are indicative of a defect in the ventral migration of the nuclei of the P cells. UNC-115 is not normally involved in P nucleus migration, but ced-10/Rac and mig-2/RhoG act redundantly in the process myr::unc-15 ectopically interferes with P nucleus migration, and RACK-1 is required for this effect. In this case RACK-1 might normally positively regulate MYR::UNC-115. In any case, these data indicate that RACK-1 and UNC-115 might have distinct interactions in different cellular contexts.Second, In summary, these studies suggest that RACK-1 acts in a common pathway with CED-10/Rac and UNC-115/abLIM in axon pathfinding . These sC. elegans culture and techniques were performed using standard protocols rack-1(tm2262) allele was provided to us by the National Bioresource Project for the Experimental Animal “Nematode C. elegans” (S. Mitani), and was outcrossed to wild-type N2 animals three times before analysis. Polymerase chain reaction (PCR) was used to verify the homozygosity of rack-1(tm2262) in strains. The following mutations and genetic constructs were used: LGII: juIs76[unc-25::gfp]; LGIV: ced-10(n1993), rack-1(tm2262), nT1 IV:V, lqIs3[osm-6::gfp]; LGV: unc-34(e951); LGX: unc-115(ky275), mig-2(mu28), lqIs2[osm-6::gfp]; LG?: lqIs62[myr::unc-115(+)]. C. elegans transformation was performed by standard techniques using DNA microinjection into the syncytial germline of hermaphrodites All micrographs were obtained on a Leica DMRE microscope with a Qimaging Rolera MGi EMCCD camera or a Qimaging Retiga CCD camera. Openlab and IPlab software were used to obtain images.All coding regions amplified by PCR were sequenced to ensure the absence of mutations in the sequence. PCR, recombinant DNA and other molecular biology techniques were performed according to standard techniques unc-25 promoter::gfp integrated transgene (juIs76 II) were used osm-6 promoter::gfp integrated transgene (lqIs2 X or lqIs3 IV) were used Axon pathfinding defects were scored with fluorescence microscopy of hermaphrodite animals in the fourth larval stage (L4) or young adults expressing a green fluorescent protein transgene for specific cells. To visualize and score the axons of VDs and DDs, animals harboring an VD/DD commissural axons normally extend up the right side of the animal . The number of animals with aberrant left-side extension of commissural axons was scored.A VD/DD commissural axon that failed to reach the dorsal nerve cord or that wandered laterally before reaching the dorsal nerve cord was considered mutant. The percent of animals with pathfinding defects was noted, and the percentage of defective axons was noted.The VD neurons are descendants o the P cells. If the P nuclei fail to migrate ventrally, the resulting VD cell bodies can be laterally displaced out of the ventral nerve cord. The percentage of animals with laterally displaced VD cell bodies was scored.The cell bodies of the PDE neurons (PDEL and PDER) are situated in the posterior lateral post-deirid ganglion. PDEs extend an axon ventrally to the ventral nerve cord, which then bifurcates and extends anteriorly and posteriorly in the ventral nerve cord. If the axon failed to reach the ventral nerve cord or wandered beyond a 45° angle from a straight line ventrally from the cell body, it was considered mutant. Significance was determined using Fisher Exact Analysis.ced-10(G12V) and mig-2(G16V) transgenes under the control of the osm-6 promoter were used as described previously myr::unc-115 transgene under the control of the unc-115 promoter was used as described previously Gonadal distal tip cell migration defects were scored by Differential Interference Microscopy in young adult hermaphrodite animals. Any deviation from the normal U-shape of gonad arms was scored as defective, including failure to migrate fully, failure to make a dorsal turn, failure to make an anterior or posterior turn, or extra dorsal-ventral or anterior-posterior turns. Significances of differences  were determined using Fisher Exact Analysis.rack-1(+) transgene was generated by PCR from genomic DNA (based on the Wormbase gene model K07D7.1) and included the entire upstream rack-1 region (∼2.5 kb), the coding region, and the downstream region past the poly-A addition site  activity (for lacZ) and growth on 25 mM 3-aminotriazole (3-AT) (for HIS5). This analysis identified 244 potential interacting cDNAs that had β-gal activity and grew on 25 mM 3-AT. From these 244, 142 isolates activated both lacZ and HIS5 similarly when re-tested. Of these, 124 were bait-specific and did not activate when the bait plasmid was removed. These cDNAs were sequenced, and seven of these were found to represent the K07D7.1 gene in Wormbase (rack-1)The two-hybrid screen was conducted at the Molecular Interaction Facility at the University of Wisconsin-Madison (thanks to E. Maher). In a liquid multi-well format, approximately 36 million C. elegans protein extract, animals carrying an integrated rack-1::myc transgene were raised at room temperature in a liquid culture containing 2.5 mg cholesterol, 0.05 mg/mL streptomycin, Escherichia coli strain HB101 and M9 buffer (up to 500 mL). After about a week, these animals were harvested and snap-frozen in liquid nitrogen. We then added lysis buffer  in a 1∶1 ratio, and then 1 mM of phenylmethanesulphonylfluoride. We lysed the animals with glass beads in a beater for two cycles of 1 minute each. The supernatant was then collected and stored at −80°C for further experiments. We based our immunoprecipitation assays in Clonetech Laboratories' protocol No. PT3407-1 (Clonetech). We performed the standard immunoprecipitation assays as described in In order to obtain large amounts of


Small molecule inhibitors of RNA virus replication are potent antiviral drugs and useful to dissect selected steps in the replication process. To identify antiviral compounds against Tomato bushy stunt virus (TBSV), a model positive stranded RNA virus, we tested acridine derivatives, such as chlorpromazine (CPZ) and quinacrine (QC), which are active against prion-based diseases.Here, we report that CPZ and QC compounds inhibited TBSV RNA accumulation in plants and in protoplasts. In vitro assays revealed that the inhibitory effects of these compounds were manifested at different steps of TBSV replication. QC was shown to have an effect on multiple steps, including: (i) inhibition of the selective binding of the p33 replication protein to the viral RNA template, which is required for recruitment of viral RNA for replication; (ii) reduction of minus-strand synthesis by the tombusvirus replicase; and (iii) inhibition of translation of the uncapped TBSV genomic RNA. In contrast, CPZ was shown to inhibit the in vitro assembly of the TBSV replicase, likely due to binding of CPZ to intracellular membranes, which are important for RNA virus replication.Since we found that CPZ was also an effective inhibitor of other plant viruses, including Tobacco mosaic virus and Turnip crinkle virus, it seems likely that CPZ has a broad range of antiviral activity. Thus, these inhibitors constitute effective tools to study similarities in replication strategies of various RNA viruses. Viruses, including plant viruses, cause many diseases that lead to significant economic losses. Therefore, it is important to develop antiviral strategies to efficiently combat viral infections. One intriguing approach is the use of small molecule inhibitors of viral infections as demonstrated by the use of ribavirin against hepatitis C virus (HCV), respiratory syncytial virus and treatment of Lassa fever as well as amantadine and rimantadine M2 channel inhibitors and zanamivir and oseltamivir neuraminidase inhibitors against influenza virus Tomato bushy stunt virus (TBSV), to test small molecule inhibitors. TBSV, a small (+)RNA virus of plants, has emerged as a highly suitable model virus for studying viral RNA replication and recombination pol replication proteins pol is the RNA-dependent RNA polymerase (RdRp) TDH2 and TDH3 in yeast), pyruvate decarboxylase (Pdc1p), Cdc34p ubiquitin conjugating enzyme and eukaryotic translation elongation factor 1A (eEF1A) In spite of the above progress with small molecule inhibitors of human and animal viruses, identification of small molecule inhibitors to combat plant virus infections is much less advanced. Therefore, we have chosen a simple model virus, One of the major advantages of tombusviruses is the development of yeast as a model host for virus - host interactions, allowing the utilization of powerful genomics and proteomics tools developed for yeast Based on the above observation, we have tested in this paper if inhibitors of prion propagation active against both yeast and mammalian prions Nicotiana benthamiana protoplasts, whereas CPZ and QC were added to the culture media either prior to electroporation or after electroporation (N. benthamiana cells (not shown). We found that both CPZ and QC inhibited TBSV accumulation by ∼70 and 50%, respectively, when applied prior to electroporation (We have tested two acridine derivatives chlorpromazine (CPZ) and quinacrine (QC), which are known anti-prion molecules active against both yeast poration . We haveporation , lane 6.pol replication proteins and a yeast cell-free extract To define the mode of action for these acridine derivatives leading to inhibition of TBSV RNA accumulation, we took advantage of efficient in vitro replication assays developed for tombusviruses. First, we used an in vitro replicase assembly assay that includes purified recombinant TBSV p33 and p92pol proteins in combination with the soluble fraction of the yeast extract. Under these conditions, the TBSV replicase can be successfully assembled in vitro and accordingly, replication of TBSV (+)repRNA was detected in the in vitro replication assay in the presence of the control DMSO -SL, is absolutely essential for TBSV replication in plants, plant protoplasts, in yeast cells and in the authentic in vitro TBSV replication system pol that would lead to reduced amount of replicase complex and low level of replication. To test this possibility, we used the in vitro wheat germ translation assay that can be programmed with added RNA templates. When capped and polyadenylated mRNA for p33 (similar to that expressed in yeast from plasmid to support TBSV replication) N. benthamiana protoplasts RNA to produce p33 and p92toplasts , was inhBased on the above in vitro experiments, CPZ and QC might inhibit TBSV replication directly, encouraging us to further test the antiviral activities of these compounds in plant leaves as well. Our preliminary experiments have indicated that both compounds were needed at higher concentrations for treatment of plant leaves (not shown) than for the protoplasts experiments . InfiltrN. benthamiana leaves via infiltration of 1600 µM QC 30 min prior to inoculation with TBSV virion preparation also resulted in inhibition of TBSV RNA accumulation in the infiltrated leaves  and the unrelated Tobacco mosaic virus  in N. benthamiana protoplasts. These experiments were conducted in a similar way to those shown for TBSV in N. benthamiana protoplasts -SL] required for recruitment of the TBSV RNA into replication The inhibitory effect of QC on TBSV replication is unlikely due to a putative general cytotoxic effect, since TMV RNA replication in protoplasts was not inhibited by QC under similar conditions used in the TBSV studies . TMV RNAThe in vitro experiments have revealed that CPZ inhibits TBSV replication using a different mechanism from that of QC. We found that CPZ reduced drastically the in vitro activity of the TBSV replicase at the early time points, but CPZ had only weak inhibitory effect on binding of p33 to (+)RNA. In addition, CPZ did not inhibit the translation of TBSV gRNA. Also, CPZ did not inhibit (−) or (+)strand synthesis in an assay with the solubilized/purified tombusvirus replicase . In vitrRibosome-borne Protein Folding Activity or RPFA) Therefore, the respective effects of QC and CPZ against plant viruses parallel their putative mode of action on prion propagation. Indeed, antiprion activity of both QC and CPZ may be related to their ability to down regulate the protein folding activity of the ribosome  and quinacrine (QC) were purchased from Sigma. Both compounds were dissolved in DMSO and stored at −20°C as a stock at concentration of 100 mM. Further dilutions were made with water just before use.N. benthamiana callus culture was treated with 1 g cellulysin and 0.2 g macerase  as described For protoplast isolation, 5′-TCTGGTTTGGTTTGGACCTC-3′) and #2889 (5′-GTAATACGACTCACTATAGGGATTCGAACCC-3′). The normalization of samples was done based on quantified ribosomal RNA levels in total RNA samples visualized by ethidium-bromide staining of gels.Total RNA was extracted 40 hrs after electroporation, followed by agarose gel analysis and Northern blotting using radiolabeled TBSV probe as described Cell-free yeast extract was prepared and replication assay for TBSV DI-72 RNA was performed as described N. benthamiana plants infected with Cucumber necrosis virus, a close relative of TBSV, which can replicate TBSV RNA templates as efficiently as the TBSV replicase For the preparation of the tombusvirus replicase, we used 5′- GTAATACGACTCACTATAGAGGTTTGTGAGAAGGTTGG- 3′) and #1301 (5′-CTGGCTCGTGTGTAAGTACG-3′) and DI-72 cDNA clone as a template E. coliThe sequence for RII(+)-SL RNA (containing the cis-acting p33RE that is involved in selective binding to p33) was amplified with PCR using primers #1300 ( 5′-GTAATACGACTCACTATAGGG AAGCTATACCAAGCATACAATC-3′) and #2145 (5′-TTTTTTTTT-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAAAACCTAAGAGTCAC-3′). In vitro translation of RNA transcripts was carried out in a wheat germ extract (Promega) according to manufacturer's recommendation. Briefly, 0.3 pmol of RNA transcripts, 3 µl wheat germ extract and 0.3 µl amino acid mix without methionine (provided by the manufacturer) were mixed in 50 mM potassium acetate with a final volume of 6 µl. In addition, 1 µl of CPZ , QC (10 or 20 µM), and DMSO (0.05%) was also added. Reactions were conducted for 1 hour in the presence of [35S] methionine (1000 Ci/mmol) at room temperature. In vitro translation was stopped by denaturation at 100°C for 3 minutes. Products were analyzed using 10% SDS-PAGE and quantified by Typhoon 9400 and ImageQuant .Uncapped, nonpolyadenylated TBSV genomic RNA transcripts were obtained with the T7 transcription using linearized DNA template N. benthamiana plants (four leaf-stage) were infiltrated with 2 ml of a 600 µM solution of CPZ or of a 1600 µM solution of QC. Control plants were infiltrated with the same percentage of DMSO that was present in the CPZ and QC solutions. The infiltrated leaves were inoculated with a purified TBSV virion preparation 30 min after infiltration. Total RNA was extracted 4 days post inoculation (dpi) and 1% agarose gel electrophoresis was done to analyze viral and ribosomal RNAs. Northern blot analysis was performed with a TBSV specific probe as described above for the protoplast experiments.Two leaves of


It has been observed that following viroid infection, there is an accumulation of viroid-derived siRNAs in infected plants. Some experimental results suggest that these small RNAs may be produced by the plant defense system to protect it from infection, indicating that viroids can elicit the RNA-silencing pathways. The objective of this study is to identify in the peach latent mosaic viroid (PLMVd), a model RNA genome, the regions that are most susceptible to RNA interference machinery.The RNA isolated from an infected tree have been used to sequence in parallel viroid species and small non-coding RNA species. Specifically, PLMVd RNAs were amplified, cloned and sequenced according to a conventional approach, while small non-coding RNAs were determined by high-throughput sequencing. The first led to the typing of 18 novel PLMVd variants. The second provided a library of small RNAs including 880 000 sequences corresponding to PLMVd-derived siRNAs, which makes up 11.2% of the sequences of the infected library. These siRNAs contain mainly 21-22 nucleotide RNAs and are equivalently distributed between the plus and the minus polarities of the viroid. They cover the complete viroid genome, although the amount varies depending on the regions. These regions do not necessarily correlate with the double-stranded requirement to be a substrate for Dicer-like enzymes. We noted that some sequences encompass the hammerhead self-cleavage site, indicating that the circular conformers could be processed by the RNA-silencing machinery. Finally, a bias in the relative abundance of the nature of the 5' nucleotides was observed .The approach used provided us a quantitative representation of the PLMVd-derived siRNAs retrieved from infected peach trees. These siRNAs account for a relatively large proportion of the small non-coding RNAs. Surprisingly, the siRNAs from some regions of the PLMVd genome appear over-represented, although these regions are not necessarily forming sufficiently long double-stranded structures to satisfy Dicer-like criteria for substrate specificity. Importantly, this large library of siRNAs gave several hints as to the components of the involved silencing machinery. Pospiviroidae and the Avsunviroidae. The members of the first family, for which the typical representative is the potato spindle tuber viroid (PSTVd), share a conserved central region, a nuclear localization and an asymmetric rolling circle replication that appears to involve host enzymes. Members of the Avsunviroidae, including the peach latent mosaic viroid (PLMVd), which is the viroid model in the present study, have been shown to accumulate in the chloroplasts [Viroids are small , single-stranded circular RNA pathogens that self-replicate in the plants causing important economical losses in agricultural industries . Their gPlants accumulate a multitude of small non-coding RNAs (i.e. 18-25 nucleotides in length). The actions of most of these small RNAs result in gene repression through what is called post-transcriptional gene silencing (PTGS) to protect the plants from biotic stresses, like viroid or virus infection. Besides their role in response to pathogens, these small RNAs are also implicated in development and genome integrity . Based oIn the case of PLMVd, some aspects of the RNA-silencing mechanism, including how it might trigger Dicer-like enzyme activity, were studied using a cell-free wheat germ assay . In addiHigh-throughput sequencing (HTS) is a method that provides a relatively unbiased and direct digital readout of cDNA sequences generated from an RNA sample ,16. The Total RNA was isolated from the leaves of one healthy and one PLMVd-infected tree. PLMVd infection was confirmed in the leaves of the infected tree by RT-PCR amplification, and could not be detected in the sample taken from the leaves of the healthy tree Figure . SubsequArabidopsis thaliana rRNA since the Prunus persica genome sequence remains incomplete  present in the RNA sample above were isolated after fractionation on a 15% denaturing polyacrylamide gel. After the ligation of 5' and 3' adapters, these RNAs were amplified by RT-PCR. After purification on a 10% polyacrylamide gel, the resulting DNA samples were high-throughput sequenced using the Illumina Solexa technology. Reads that did not contain the 3' linker, those shorter than 16 nt, and those with ambiguous nucleotides were discarded so as to reduce, as much as possible, any further bias or wrong identification. This trimming steps yielded libraries of 12 476 493 and 7 862 905 total reads for the healthy and the PLMVd-infected samples, respectively  [Clearly, the accumulated siRNAs differ significantly for the two PLMVd polarities. The secondary structures adopted by the PLMVd strands of both polarities ,25 diffe6 (RDR6) . Based o6 (RDR6) . AnotherArabidopsis argonaute (AGO) complexes is influenced by the 5' terminal nucleotide [Prunus genus. More generally, it is tempting to suggest that the active AGO proteins in the infected plant cell differ from those retrieved in the healthy ones. However, this does not exclude that other AGO proteins may be involved in the binding of those small RNAs since plants may encode up to ten different AGO proteins with redundant functions [Recent reports have shown that the sorting of small RNAs into cleotide -30. In ocleotide ,30. Takiunctions .Lastly, in the previous analysis, the use of a minimal set of siRNAs , led to For the first time, all of the RNA isolated from an infected tree have been used in parallel to sequence viroid species and small non coding RNA species. On the one hand, this work led to the determination of 18 novel PLMVd variants. On the other hand, the use of high throughput sequencing provided the largest library of siRNA reported yet for a viroid infection. It was shown that the entire PLMVd genome is retrieved in the siRNA population. Moreover, PLMVd strands of both polarities appeared susceptible for targeting, although specific regions for each one are over-represented in terms of siRNAs. Those regions, which are not the same for each polarity, do not necessarily correlate with double-stranded regions that could be substrates for Dicer-like enzymes. The nature of the first 5' nucleotide in the 21-22 nt population revealed a bias toward a C or a U in viroid-derived siRNAs. In fact this bias is also observed in the non-PLMVd-siRNAs. Unlike the infected library, the majority of the healthy sequences has A or G at their 5'. Importantly, the large libraries of siRNAs created in this study led to several observations concerning the silencing mechanism associated with PLMVd infection.in vivo. These small RNAs map the PLMVd genome adopting a hotspot profile which is different from one polarity to the other (plus vs minus), although their libraries of PLMVd-associated siRNAs were smaller. However, the Di Serio et al. study has the advantage of reporting data isolated from two peach plants. The present study offers two important differences: Firstly, the fact that the sequencing of the small ncRNAs was also performed on a healthy plant ensures that there are no species showing homologies with PLMVd. Secondly, the PLMVd sequences used to identify viroid-associated siRNAs were obtained from the same sample as the small RNAs instead of using sequences reported several years ago [Pospiviroidae family) [At the same time this manuscript was being submitted, another study describing deep sequencing of small RNAs associated to PLMVd infection was released . The autears ago . It has ears ago . There i family) . ClearlyTotal RNA was extracted from the leaves of peach (Siberian C cultivar) and nectarine trees grown on the West Coast of Canada  using Trizol (Invitrogen) according to the manufacturer's instructions. PLMVd can infect nectarine as well as peach trees . The sam4 and 1 mM CaCl2. Following two phenol-chloroform extractions and an ethanol precipitation, the RNA was reverse transcribed for 1 h at 45°C in the presence of the reverse primer 5'-AGTGCTCCGAATAGGGCACCCCAAGGTGG-3' using SuperScript III (Invitrogen) in 20 μL reactions as recommended by the manufacturer. Upon completion of the reverse transcription reactions, 10 μg of RNase A were added and the mixtures incubated for 15 min at room temperature. Ten microliters of the reverse transcriptase reactions were then used in PCR amplification reactions performed using purified Pfu DNA polymerase. The reactions were carried out in a final volume of 100 μL containing 2 μM of each DNA primer, 2 mM MgSO4, 200 μM dNTP, 10 mM Tris-HCl (pH 8.85), 25 mM KCl and 5 mM (NH4)2SO4. The reactions were incubated at 94°C for 1 min prior to the addition of the enzyme, and were then subjected to 25 amplification cycles . The reverse primer was the same as that used previously in the reverse transcription reactions, while the forward primer was 5'-GCAGTTCCCGATAGAAAGGCTAAGCACC-3'. The amplifications were analyzed by loading 10 μL aliquots on a 1% agarose gel. The gel band corresponding to a fragment of 338 nt in length was cut out and the DNA extracted using Spin-X columns (Corning Science Products). The resulting DNA was then incubated in the presence of Taq DNA polymerase (New England Biolabs) for 20 min at 72°C and ligated into pGEM-T vector (Promega). After transformation into E.coli, 23 clones were sequenced at the Platefome de séquençage et de génotypage des genomes (CRCHUL) . All sequences were deposited to NCBI, accession numbers GQ499305 to GQ499322.For PLMVd cloning, 5 μg of total RNA were incubated in the presence of 3 U of RQ1 DNAse (Promega) at 37°C for 1 h in a total volume of 20 μL containing 40 mM Tris-HCl pH 8.0, 10 mM MgSOFor each library  10 μg of total RNA was size fractionated on a 15% denaturing polyacrylamide gel and the 18-30 nt fraction excised from the gel. Following elution of the RNA, 5' and 3' adapters were added to the RNA species using the Digital Gene Expression for Small RNA Sample Prep Kit (Illumina). After reverse transcription, the samples were PCR amplified, purified on 10% polyacrylamide gels and then eluted according to the manufacturer's protocol. High-throughput Illumina Solexa sequencing was performed at The Center for Applied Genomics .Unfiltered sequence reads from both sets were trimmed of the 3' adapter. Reads that did not contain the 3' linker, and those shorter than 16 nt, were discarded. The resulting reads were aligned on the PLMVd sequences, as well as on other control RNA sequences, using the Exonerate software . No mismJ-PP conceived the study. PS-P isolated and cloned small RNAs that had been sent to Solexa sequencing. CH cloned the new PLMVd variants and contributed to bioinformatic analysis. FB conceived and performed the bioinformatic analysis. J-PP and FB wrote the manuscript. All authors read and approved the final manuscript.New PLMVd sequences and their NCBI accession number.Click here for file


The objective of this study was to investigate the effects of interleukin-6 (IL-6) overexpression in androgen-dependent prostate cancer LNCaP cells on their phenotype under an androgen-deprived condition.We established IL-6-overexpressing LNCaP (LNCaP/IL-6) by introducing the expression vector containing IL-6 cDNA. Changes in the phenotype in LNCaP/IL-6 were compared with that in LNCaP transfected with control vector alone (LNCaP/Co).In vitro, the growth of LNCaP/IL-6 was significantly inferior to that of LNCaP/Co under an androgen-deprived condition. Similarly, LNCaP/IL-6 tumour in nude mice rapidly regressed after castration; however, LNCaP/Co tumour growth was transiently inhibited after castration and then continuously accelerated. After androgen withdrawal, expression levels of phosphorylated p44/42 mitogen-activated protein kinase (MAPK) and Akt in LNCaP/IL-6 were markedly upregulated compared with those in LNCaP/Co; however, additional treatment with specific inhibitor of the MAPK or Akt signalling pathway significantly inhibited the growth of LNCaP/IL-6 compared with that of LNCaP/Co. Furthermore, gene microarray analyses showed that androgen deprivation resulted in differential expression of genes involved in growth, apoptotsis and tumorigenesis between LNCaP/Co and LNCaP/IL-6.Excessive secretion of IL-6 by LNCaP cells in an autocrine manner may have a suppressive function in their growth and acquisition of androgen-independent phenotype under an androgen-deprived condition. The prognosis of patients with organ-confined prostate cancer has been remarkably improved with technical advances in surgical as well as radiological treatments; however, androgen withdrawal therapy remains the only effective form of systemic therapy for men with advanced prostate cancer. Initially, >80% of such patients respond favourably to this therapy; however, progression to the lethal and untreatable stage of androgen independence ultimately occurs within a few years in the majority of these patients . RecentlDespite intensive efforts in the field of prostate cancer research, the precise molecular mechanism of progression to androgen independence has not been fully elucidated; however, several hypotheses explaining the complex process during AI progression have been reported . One potThe biological activities of IL-6 are mediated by the IL-6 receptor; that is, the binding of IL-6 to its receptor results in the activation of intracellular signalling, including mitogen-activated protein kinase (MAPK), phosphoinositol 3′-kinase (PI3K)/Akt and Janus-activated kinase (JAK)/signal transducers and activation of transcription (STAT) pathways . In receIn this study, therefore, we evaluated the effects of IL-6 overexpression in human androgen-dependent (AD) prostate cancer LNCaP cells on changes in their phenotype before and after androgen withdrawal to assess the functional role of IL-6 secreted in an autocrine manner as a ligand-independent activator of AR.LNCaP and PC3, derived from human prostate cancer, were purchased from the American Type Culture Collection . Cells were maintained in RPMI  supplemented with 10% heated inactivated fetal bovine serum. To investigate the androgen deprivation effect, steroid hormone-depleted charcoal-stripped medium (CSM) containing 10% CS serum was prepared as described earlier  was added to LNCaP cells after preincubation for 30 min with 5 μg of lipofectamine reagent and 3 ml of serum-free OPTI-MEM (Life Technologies). Drug selection in 300 μg ml−1 Geneticin (Life Technologies) was begun 3 days after transfection. Three weeks after the drug selection, colonies were harvested with cloning cylinders and expanded to cell lines.The cDNA fragment encoding human IL-6 was directly cloned into the expression vector pcDNA3.1  according to the manufacturer's instructions. The pcDNA3.1/IL-6 construction was transfected into LNCaP cells by the liposome-mediated gene transfer method (6 LNCaP sublines were measured using a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) kit for human IL-6 according to the manufacturer's instructions . Similarly, a sandwich ELISA kit for human prostate-specific antigen (PSA)  was used to determine the concentrations of PSA in overnight culture supernatants of 5 × 106 LNCaP sublines and those in sera from nude mice bearing the subcutaneous tumours of LNCaP sublines.The concentrations of IL-6 in overnight culture supernatants of 5 × 10in vitro proliferation of LNCaP sublines, 5 × 103 cells of each cell line were seeded in each well of 12-well plates, and the number of cells in each cell line was counted daily by triplicate. In addition, the effect of androgen deprivation on the proliferation of LNCaP sublines with and without additional treatment with IL-6 , UO126  or LY294002 (SA Bioscience) was also examined; that is, following the culture of LNCaP sublines in the standard medium for 3 days, the medium was replaced with CSM with and without supplementary IL-6, UO126 or LY294002, and the number of cells was counted.To compare the μg) from lysates of the LNCaP sublines cultured in either standard medium or CSM were electrophoresed on an SDS–polyacrylamide gel and transferred to a nitrocellulose filter. The filters were blocked in PBS containing 5% nonfat milk powder at 4°C overnight and then incubated for 1 h with antibodies against IL-6 receptor (R&D Systems), AR, β-actin  and total and phosphorylated STAT3, p44/42 MAPK and Akt . The filters were then incubated for 30 min with horseradish peroxidase-conjugated secondary antibodies , and specific proteins were detected using an enhanced chemiluminescence western blotting analysis system (Amersham Pharmacia Biotech).Western analysis was performed as described earlier , and assessed using the 3DNA Array 350 Expression Array Detection kit according to the manufacturer's instructions . After overnight hybridization and washing, arrays were scanned on a Scan Array Express Microarray Scanner , and signal quality and quantity were determined using the ImaGene 8.0 software . Comparative analysis of the gene expression profiles in each cell line was conducted to identify genes that are differentially regulated by overexpression of IL-6. Genes were considered to be significantly different if the mean expression level in the IL-6-overexpressing cells was at least 2.0-fold greater or 2.0-fold less than that seen in the control cells.Gene microarrays of 34 580 (70-mer) human oligos representing 24 650 genes and 37 123 gene transcripts printed on aminosilane-coated microarray slides were supplied by the microarray facility of the Prostate Centre at Vancouver General Hospital , and microarray analysis was performed as described earlier  into right flank of mice. The subcutaneous tumour growth was measured once per week using calipers as described earlier  were purchased from Clea Japan  and housed in a controlled environment at 22°C on a 12-h light, 12-h dark cycle. Animals were maintained in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Each experimental group consisted of 20 mice. The tumour cells of each cell line were trypsinized, washed twice with PBS, and 5 × 10t-test. All statistical calculations were performed using Statview 5.0 software , and P-values <0.05 were considered significant.Differences between the two groups were compared using the unpaired LNCaP was transfected with pcDNA3.1/IL-6 or pcDNA3.1 alone as a control. After drug selection, several Geneticin-resistant stable transfectants were randomly isolated. The culture supernatants of LNCaP sublines and PC3 were analysed by sandwich ELISA to measure the IL-6 concentrations. All of the IL-6-transfected LNCaP (LNCaP/IL-6#1 to LNCaP/IL-6#4) secreted abundant immunoreactive IL-6 protein in amounts equal to those of PC3 originally expressing the IL-6 gene . Furtherin vitro experiments, almost identical findings were obtained from IL-6-transfected cell lines (LNCaP/IL-6#1 to LNCaP/IL-6#4) or their control cell lines (LNCaP/P and LNCaP/Co); therefore, we subsequently presented the data for LNCaP/IL-6#1 and LNCaP/Co only.In the following in vitro growth of LNCaP under conditions with and without androgen, the growth of LNCaP sublines cultured in the standard medium and that in CSM were assessed. As shown in To examine the effects of IL-6 overexpression on in vivo effect of IL-6 overexpression on tumour growth, 5 × 106 cells of each cell line were injected subcutaneously into 20 nude mice; then 10 of the 20 mice were castrated when the tumour size reached 80 mm2 or greater. In intact mice without castration, the growth of LNCaP/IL-6#1 tumour was significantly faster than that of LNCaP/Co tumour , exogenous IL-6 rapidly activated MAPK and STAT3 pathways in LNCaP/Co, but not in LNCaP/IL-6#1; that is, phosphorylated p44/42 MAPK and STAT3, which were not induced in LNCaP/IL-6#1, became detectable in LNCaP/Co 5 and 15 min after treatment with IL-6, respectively .To determine whether androgen withdrawal modulates the activation patterns of signal transduction pathways through AR, changes in expression of both phosphorylated and total MAPK, Akt and STAT3 in LNCaP sublines were evaluated. There were no significant differences in the expression levels of both phosphorylated and total STAT3 between LNCaP/Co and LNCaP/IL-6#1 under an androgen-deprived condition (data not shown). As shown in Consistent with the evaluation of changes in the downstream signalling pathways of IL-6, we investigated the effect of additional treatment with UO126 or LY294002, a selective inhibitor of mitogen-activated protein kinase kinase (MEK) or PI3K on the growth of LNCaP sublines cultured in the androgen-deprived condition. Treatment with UO126 or LY294002 inhibited the growth of LNCaP/Co and LNCaP/IL-6#1; however, growth inhibitory effects of both UO126 and LY294002 on LNCaP/IL-6#1 were significantly greater than those on LNCaP/Co .Changes in the expression of AR in LNCaP sublines were then analysed. As shown in To further address the molecular mechanism involved in the enhanced sensitivity of androgen withdrawal in LNCaP/IL-6#1, the influence of IL-6 overexpression on global gene expression was assessed by gene microarray analysis. We analysed the gene expression profiles in LNCaP/Co and LNCaP/IL-6#1 cultured in both the standard medium and CSM. Excluding genes differentially expressed between LNCaP/Co and LNCaP/IL-6#1 cultured in the standard medium, a total of 223 genes were found to be differentially expressed in LNCaP/IL-6#1 at least 2.0-fold greater or 2.0-fold less than those of LNCaP/Co under an androgen-deprived condition. On the basis of the biological relevance and magnitude of changes, 40 of these 223 genes were selected, and presented in Since reports showing elevated levels of circulating IL-6 in men with advanced prostate cancer (in vitro and in vivo were initially compared. Before androgen ablation, despite the lack of a significant difference in the in vitro growth between LNCaP sublines, LNCaP/IL-6#1 tumour grew significantly faster than LNCaP/Co tumour. Under an androgen-deprived condition, however, the in vitro growth of LNCaP/IL-6#1 was significantly suppressed compared with that of LNCaP/Co. This enhanced sensitivity of LNCaP/IL-6#1 to androgen withdrawal was more remarkable in vivo; that is, after castration, LNCaP/IL-6#1 tumour rapidly regressed and completely disappeared. According to previously reported studies, the effects of IL-6 on the growth of LNCaP cells seem to be puzzling with some groups showing growth stimulation, whereas others showing growth inhibition (in vitro, this study is the first to demonstrate the following evidence that overexpression of IL-6 in LNCaP cells enhanced their sensitivity to androgen withdrawal both in vitro and in vivo, resulting in marked growth inhibition immediately after androgen ablation. Furthermore, it would be of interest to investigate the effects of an antiandrogen, such as bicalutamide, on the growth of LNCaP sublines.The growth patterns of LNCaP sublines both in vitro and that subcutaneously implanted, respectively, under both conditions with and without androgen. Interestingly, the amount of baseline PSA production by LNCaP/IL-6#1 was significantly smaller than that by LNCaP/Co both in vitro and in vivo, when androgen was supplemented; however, several earlier studies have shown that treatment of LNCaP cells with IL-6 enhances their AR-mediated PSA gene expression through an increase in PSA reporter activity (PSA is a reliable tumour marker showing a proportional change in relation to the degree of prostate cancer progression (Considering the unique proliferative potential and PSA production of LNCaP/IL-6#1 cells, it would be of interest to investigate whether LNCaP/IL-6#1 exhibits a different response to exogenous IL-6 compared with that of LNCaP/Co. As reported earlier (Another point of interest is the mechanism whereby overexpression of IL-6 enhances sensitivity of LNCaP cells to androgen withdrawal. To address this point, it was investigated whether the downstream signalling pathways of IL-6 are differentially regulated by androgen between LNCaP sublines. Although the growth of LNCaP/IL-6#1 was significantly inhibited compared with that of LNCaP/Co under an androgen-deprived condition, Akt and MAPK pathways in LNCaP/IL-6#1, which are generally regarded as a positive regulator of prostate cancer cell growth through the transactivation of AR (γ receptor, negatively regulating cancer progression (We further investigated the molecular mechanism underlying the enhancement of androgen withdrawal-induced apoptotic cell death of LNCaP/IL-6#1 using gene microarray analysis. Gene expression profiling showed that 223 genes were differentially expressed between LNCaP/Co and LNCaP/IL-6#1 under androgen-deprived condition, and 40 of these 223 were listed considering the biological relevance and magnitude of changes. These outcomes were not confirmed by an alternative method; however, expression profiles in some genes in LNCaP/Co before and after androgen withdrawal were consistent with those in parental LNCaP reported in earlier studies (In conclusion, this is the first study to show enhanced sensitivity to androgen withdrawal due to overexpression of IL-6 in AD human prostate cancer LNCaP cells. Furthermore, the present findings suggest that the difference in response to exogenous IL-6 between LNCaP/Co and LNCaP/IL-6#1 may, at least in part, be involved in the differential sensitivity to androgen withdrawal between these two sublines, and that despite the significant activation of Akt and MAPK pathways in LNCaP/IL-6#1 compared with that in LNCaP/Co under an androgen-deprived condition, this might be due to the response to proapototic stimuli representing an adaptive cell survival mechanism. Although gene microarray analysis identified several potentially important candidate genes responsible for the different sensitivities to androgen ablation between LNCaP/Co and LNCaP/IL-6#1 by globally evaluating their gene expression profiles, it would be further required to accumulate the intensive investigation to elucidate the molecular mechanism underlying changes in the phenotype of LNCaP cells by overexpression of IL-6.