In essence, the scFv in gH substituted for gD-mediated activation and rendered a functional gD non-essential for entry via HER2. The activation of the gH moiety in the chimera was carried out by the scFv in cis, not in trans as it occurs with wt-gD. With respect to the design of oncolytic-HSVs, previous retargeting strategies were based exclusively on insertion in gD of ligands to cancer-specific receptors. The current findings show that (i) gH accepts a heterologous ligand. The viruses retargeted via gH (ii) do not require the gD-dependent activation, and (iii) replicate and kill cells at high efficiency. Thus, gH represents an additional tool for the design of fully-virulent oncolytic-HSVs retargeted to cancer receptors and detargeted from gD receptors.Herpes simplex virus (HSV) enters cells by means of four essential glycoproteins - gD, gH/gL, gB, activated in a cascade fashion by gD binding to one of its receptors, nectin1 and HVEM. We report that the engineering in gH of a heterologous ligand – a single-chain antibody (scFv) to the cancer-specific HER2 receptor – expands the HSV tropism to cells which express HER2 as the sole receptor. The significance of this finding is twofold. It impacts on our understanding of HSV entry mechanism and the design of retargeted oncolytic-HSVs. Specifically, entry of the recombinant viruses carrying the scFv-HER2–gH chimera into HER2 in cis. Cumulatively, the receptor-binding and activating functions of gD were no longer essential and were replaced by the heterologous ligand in gH. Relevance to translational medicine rests in the fact that gH can serve as a tool to retarget HSV tropism to cancer-specific receptors. This expands the toolkit for the design of fully-virulent oncolytic-HSVs.To enter cells, all herpesviruses use the core fusion glycoproteins gH/gL and gB, in addition to species-specific glycoproteins responsible for specific tropism, etc. In HSV, the additional glycoprotein is the essential gD. We engineered in gH a heterologous ligand to the HER2 cancer receptor. The recombinant viruses entered cells through HER2, independently of gD activation by its receptors, or despite deletion of key residues that are part of the receptors’ binding sites in gD. The ligand activated gH  Herpesviridae family; gB exhibits features typical of viral fusion glycoproteins [-/- non-infectious virions and to promote fusion in a cell-cell fusion assay; the ability of soluble gD receptor to mediate virus entry into receptor-negative cells [Entry of herpes simplex virus (HSV) into the cell is a multistep process that involves four virion glycoproteins , all of which are required. gD is species-specific, and a major determinant of HSV tropism. gH/gL and gB constitute the conserved fusion apparatus across the proteins –6. Many proteins . Specifiproteins –10—resulproteins –15. Sincproteins ,17, mostproteins . Evidencproteins ,20; the ve cells –23.134.5 gene and of ICP47 gene, and encoding the GM-CSF cytokine to boost the host immune response against the tumor [134.5 genes restricts o-HSVs replication to cells defective in the PKR-dependent innate response. To overcome these limitations, non-attenuated o-HSVs retargeted to cancer-specific receptors and detargeted from the natural receptors were designed. They preserve the killing ability of wt-viruses [There is intense interest in HSV as an oncolytic agent (o-HSV) –27. In the tumor . In a phhe tumor . A drawb-viruses ,31. So f-viruses ,32–38. T-viruses ). In the+ glioblastoma [The cancer-specific receptor selected in our laboratory is HER2 , a member of the EGFR  family, overexpressed in breast, ovary, gastric carcinomas, glioblastomas, etc . Two fulblastoma –35,42. IHere, we engineered a heterologous ligand in gH. The aims were twofold, i.e. to better elucidate the respective roles of gD and gH/gL in HSV entry, and define whether gD is an absolute requirement for HSV entry, and to explore novel avenues in the design of retargeted o-HSVs. We report that the engineering in gH of a scFv to HER2 confers to the recombinant viruses the ability to use HER2 as the sole receptor, in the absence of gD receptors, or upon deletion of residues that form the nectin1/HVEM binding sites in gD.+ cell line derived from human ovary carcinoma, and resistant to trastuzumab [r of 130 K (R-VG803 carries the insertion of the scFv to HER2 (herein named scFv-HER2) at the N-terminus of gH, the mCherry red fluorescent marker in the UL37–UL38 intergenic region, and the LoxP-bracketed BAC sequences between UL3 and UL4  directed to HER2—in gH conferred to HSV an expanded tropism for cells which express HER2 as the sole receptor. Virus entry mediated by the chimeric scFv-HER2–gH could occur in the absence of gD receptors, despite deletion of the receptor-binding sites in gD, or presence of gD-neutralizing MAbs. Basically, the key functions of gD are no longer essential, and can be replaced by a ligand in gH. This finding impacts on current view of how HSV enters cells, and on the strategies for retargeting the HSV tropism to receptors of choice.When wt-HSV enters target cells, gD serves two major functions. It serves as major receptor binding glycoprotein, and determinant of the viral tropism, i.e. it dictates which cells HSV will or will not infect. Secondly, the encounter of HSV with a cell carrying a gD receptor is signaled to gH/gL and gB, to trigger fusion. In essence, the receptor-bound gD initiates the cascade of activation of the entry glycoproteins –3,47,48 trans-signaling, as opposed to a signaling that occurs intramolecularly, herein referred to as cis-signaling. A novelty of our results is that the activation of gH can occur in cis, i.e. the scFv activates the gH moiety in the chimera. In the past, Klupp and Mettenleiter generated a non-viable PrV recombinant, carrying a deletion in gL [-/- gH+ virus, or of a gH-/- gD+ virus. It was not tested for complementation of a double deletion gD-/- gH-/- virus. There are two key differences between the previous report [-/- gD+ virus had the possibility to activate in trans the gH moiety in the gD-gH chimera. Conversely, the gD moiety in the chimera had the possibility to activate in trans the wt-gH present in the gD-/- gH+ virions. In either case, the activation may have taken place in-trans, as concluded by the Authors. Formal evidence for cis-activation of the gD-gH chimera was not provided [In wt virus, the activation exerted by the receptor-bound gD on gH/gL necessarily occurs through intermolecular signaling. We refer to it as on in gL . Upon seon in gL . Subsequon in gL . In comps report  and our provided . Secondlprovided –16,52, aprovided . Whetherprovided ), or recprovided . Hence, As regards the field of oncolytic HSVs, our data show that gH accepted the insertion of a hetelogous ligand and became a tool for the retargeting of HSV tropism to a heterologous receptor. The ligand may be at least 270 AA in size, i.e. about 1/3 of gH ectodomain. The gH-mediated retargeting could be combined with detargeting, through a suitable deletion in gD. This ensued in the fully retargeted R-VG809, whose replication and killing capacity for SK-OV-3 cells did not substantially differ, or were even better than those of the gD-retargeted R-LM113 and R-LM249. In essence, changes in tropism through modifications in gH or in gD yield o-HSVs with substantially similar growth and lytic properties. Remarkably, both the gH- and the gD-retargeted o-HSVs grew almost as efficiently as the wt R-LM5. They represent an improvement over the first generation retargeted o-HSVs, which were marred by a relatively low replication capacity ,54. We h+ and trastuzumab-resistant [via gH and via gD opens the way to the design of double-retargeted o-HSVs, which may be better suited to counteract cancer cell heterogeneity than singly-retargeted o-HSVs.Current findings expand the toolkit for generation of non attenuated retargeted o-HSVs. Two prospective applications are worth noting. The anti-HER2 huMAbs and small molecule inhibitors of HER2 signaling now in clinical trials have non-overlapping mechanisms of action, and patients clearly benefit from combinations ,57. Howeesistant . The obsesistant ,35, can The receptor negative J cells, their counterparts expressing HER2, nectin1, HVEM and CHO-HER2 were described ,43. HFF14)2SO4, 100 mM KH2PO4, 1.8 μg FeSO4·7H2O, adjusted to pH 7) supplemented with 1 mg/L D-biotin, 0.2% galactose, 45 mg/L L-leucine, 1 mM MgSO4·7H2O and 12 μg/ml chloramphenicol. To exclude galK false positive colonies, the recombinant clones were plated on McConkey agar base plates, supplemented with 1% galactose and 12 μg/ml chloramphenicol, and checked by colony PCR with primer galK_129_f ACAATCTCTGTTTGCCAACGCATTTGG and galK_417_r CATTGCCGCTGATCACCATGTCCACGC. Next, the trastuzumab scFv cassette, bracketed by Ser-Gly linkers and by upstream and downstream homology arms to gH, was amplified using pSG-ScFvHER2-SG (a gift from Alfredo Nicosia) as template. pSG-ScFvHER2-SG was obtained by inserting the synthetic antiHER2 scFv cassette, designed on the basis of published information [L and VH had the sequence SDMPMADPNRFRGKNLVFHS. The recombinant bacterial clones carried the scFv-HER2 cassette in place of the galK cassette. They were selected on M63 plates, supplemented with 1 mg/L D-biotin, 0.2% deoxy-2-galactose, 0.2% glycerol, 45 mg/L L-leucine, 1 mM MgSO4·7H2O and 12 μg/ml chloramphenicol. Bacterial colonies were checked for the presence of inserted sequence by colony PCR.First, we engineered R-VG801, by insertion of the sequence encoding the trastuzumab scFv between AA 23 and 24 of gH. Subsequently we engineered R-VG803 by insertion of mCherry sequences into the UL37-UL38 intergenic region of R-VG801. To generate R-VG801, the starting viral genome was pYEBac102, which carries LOX-P-bracketed p-BeloBAC sequences inserted between UL3 and UL4 of HSV-1 genome . All engormation ; SequencThe mCherry red fluorescent protein, under the CMV promoter, was inserted in the UL37-UL38 intergenic region of R-VG801 (coordinates 84156–84157), to generate R-VG803, following the two step procedure outlined above. Briefly, we first inserted the galK cassette, amplified by means of oligonucleotides UL37/38_galK_f CCGCAGGCGTTGCGAGTACCCCGCGTCTTCGCGGGGTGTTATACGGCCACCCTGTTGACAATTAATCATCGGCA and UL37/38_galK_r TCCGGACAATCCCCCGGGCCTGGGTCCGCGAACGGGATGCCGGGACTTAATCAGCACTGTCCTGCTCCTT. Subsequently, the galK sequence was replaced with the promoter-mCherry cassette, amplified by means of oligonucleotides UL37/38_CMV_mcherry_f CCGCAGGCGTTGCGAGTACCCCGCGTCTTCGCGGGGTGTTATACGGCCACCGATGTACGGGCCAGATATACG and UL37/38_pA_mcherry_1958_r TCCGGACAATCCCCCGGGCCTGGGTCCGCGAACGGGATGCCGGGACTTAACCATAGAGCCCACCGCATCC. The starting material for R-VG809 was the R-VG803 BAC genome. To generate the AA 6–38 deletion in gD, a galK cassette flanked by homology arms to gD was amplified by means of primers gD5_galK_f TTGTCGTCATAGTGGGCCTCCATGGGGTCCGCGGCAAATATGCCTTGGCGCCTGTTGACAATTAATCATCGGCA and gD39_galK_r ATCGGGAGGCTGGGGGGCTGGAACGGGTCCGGTAGGCCCGCCTGGATGTGTCAGCACTGTCCTGCTCCTT. Next, we replaced the galK sequence with a synthetic double stranded oligonucleotide gD_aa5_39_f_r TTGTCGTCATAGTGGGCCTCCATGGGGTCCGCGGCAAATATGCCTTGGCGCACATCCAGGCGGGCCTACCGGACCCGTTCCAGCCCCCCAGCCTCCCGAT. In all cases, the recombinant viruses were generated by transfection of SK-OV-3 cells with the appropriate recombinant BAC DNA (500 ng) by means of Lipofectamine 2000 (Life Technologies). Virus growth was monitored by red fluorescence. The structure of the viral recombinants was verified by sequencing the gH and mCherry ORFs, and gD ORF for R-VG809. Virus stocks were generated and titrated in SK-OV-3 cells, or in J-HER2 cells, as specified.Lysates of Vero cells infected with R-VG803, R-VG809 or R-LM5 (3 PFU/cell) were subjected to PAGE, transferred to PVDF membranes. Immunoblot reactivity to polyclonal antibody (PAb) to gH was assayed as detailed .The indicated cells were infected with R-VG803, or R-VG809 at 2 and 20 PFU/cell, respectively. Red fluorescence was monitored by fluorescence microscopy.Replicate monolayers of J-HER2 or SK-OV-3 cells in 12 well plates were preincubated with trastuzumab or non-immune mouse IgG  for 1 h, and then infected with R-VG803, R-VG809, R-LM113, R-LM249 or R-LM5 , in the same medium. Alternatively, virions were preincubated with MAbs HD1 , or MAb 52 S (ascites fluid 1:25) for 1 h at 37°C, and then allowed to absorb to cells for 90 min, in the absence or presence of trastuzumab, as indicated. Viral inocula were then removed, and cells were overlaid with medium containing the indicated antibodies. Virus replication was monitored at 24 h after infection by BD Accuri C6 flow cytometer. Results are expressed ad the mean of three independent experiments ± SD.To determine R-VG803 and R-VG809 growth in J-HER2, SK-OV-3, J-nectin, and Vero cells, the cells were infected with R-VG803, R-LM113, R-LM249, R-LM5 at the indicated MOI. Unabsorbed virus was inactivated by rinsing cells with pH 3 solution . Cells were harvested at 3 (0 time), 24 and 48 h after infection and progeny virus (intracellular plus extracellular) was titrated in J-HER2 or SK-OV-3 cells, as indicated.3 cell/well, and infected with R-VG803, R-VG809, R-LM113, R-LM249 and R-LM5 (2 PFU/cell) or mock-infected. AlamarBlue  was added to the culture media at indicated times after infection and incubated for 4 h at 37°C. Plates were read at 560 and 600 nm with GloMax Discover System (Promega). For each time point, cell viability was expressed as the percentage of AlamarBlue reduction in infected versus uninfected cells, excluding for each set of samples the contribution of medium alone. Each point represents the average of at least triplicate samples ± SD.SK-OV-3 and J-nectin cells were seeded in 96 well plates at 8x10HSV-1 strain F, Genbank GU73477. gH coordinates 43783–46299HSV-1 strain F, gD: Genbank L09242scFv to HER2 Genbank CQ877234, CS276173mCherry Genbank HM771696S1 FigRegions of interest are highlighted as follows. Signal sequence, yellow. scFv to HER2, blue. Upstream and downstream Ser-Gly linkers, green. scFv intermediate linker, red. Two residues implicated in MAb 52S epitope, S536 and A537 in wt-gH, fuchsia.(PDF)Click here for additional data file.


Cofilin, an actin-binding protein which disassembles actin filaments, plays an important role in invasion and metastasis. Here, we discover that JG6, an oligomannurarate sulfate, binds to cofilin, suppresses the migration of human breast cancer cells and cancer metastasis in breast cancer xenograft model. Mechanistically, JG6 occupies actin-binding sites of cofilin, thereby disrupting cofilin modulated actin turnover. Our results highlight the significance of cofilin in cancer and suggest JG6, a cofilin inhibitor, to treat metastatic cancer. Cancer metastasis constitutes the major cause of death in cancer patients. The cellular basis of cancer invasion and metastasis is the up-regulated cell motility of cancer cells, which is the hallmark of invasion and an essential step in metastasis. TargetiActin framework is widely accepted as the basic engine for cell motility. The molecular machinery controlling the assembly/disassembly of actin filaments consists of several actin-binding proteins that regulate the dynamic behavior of actin cytoskeleton. Of them, the ubiquitously distributed cofilin protein is the most important regulator for accelerating actin-filament turnover and generating free barbed ends by facilitating pointed end depolymerization and filament severing. In migrAlthough there is as yet no direct evidence for a role of deregulated cofilin activity in the etiology of human cancers, the increased activity of the cofilin pathway and its output has been demonstrated in cancer cells. There aJG6 Fig. , a novelOur pilot experiment using JG6 affinity chromatography in combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis has suggested that JG6, a novel marine-derived oligosaccharide possessing interesting bioactivities, binds to actin-regulating protein cofilin . Before We then took the advantage of a specific antibody raised against JG6 to verify its binding to cofilin in cancer cells. Immunoprecipitaion was performed using JG6 antibody in cellular extracts derived from breast adenocarcinoma MTLn3 cells, which were pretreated with 100 μg/ml JG6 for 24 hours. The presence of cofilin in the immunoprecipitates was examined using immunoblotting. Cofilin was clearly detected in the complexes pulled down by JG6 antibody. Moreover, it appeared that JG6 bound equally to un-phosphorylated and phosphorylated forms of cofilin Fig. , suggestCofilin is known to importantly regulate the dynamics of the actin cytoskeleton via its actin-severing and depolymerization activity, 18. We The reduction in the rate of actin depolymerization suggests a blockage in reorganizing F-actin into monomeric G-actin molecules. We hence assessed the effect of JG6 on the ratio of G-actin to F-actin-22. MDA-Defect in actin depolymerization may also result in the accumulation of actin filament in the form of stress fibers in cancer cells. We thusOur results thus far have suggested that JG6 binds to cofilin and inhibits its actin-severing and depolymerization activity. It has been known that F-actin depolymerizing/severing activities of cofilin are regulated at the binding sites. We thus presumed that JG6 might compete with actin in accessing to binding sites of cofilin. To test this possibility, we measured the effect of JG6 on the interaction between cofilin and actin. MTLn3 cells were incubated with JG6 100 μg/ml for 24 hours. The detection of actin-cofilin interaction was facilitated by serum stimulation, which was known to accelerate the dynamics of the actin cytoskeleton and in turn increase its interaction with cofilin. Cell lyIn addition to cofilin, the assembly and disassembly of actin filaments are regulated by a variety of actin-binding proteins. We also investigated the impact of JG6 on these proteins. As shown in Fig. It suggests to us that JG6 can disrupt the interaction between cofilin and actin, and thereby inhibit actin turnover by restricting actin depolymerization.To gain a better understanding of how JG6 binds to cofilin, molecular docking was used to predict the potential binding sites and possible binding mode of JG6 to cofilin. A JG6 trimmer was selected as the docking probe. Our results indicated that JG6 binds to the actin-binding pocket of cofilin with high affinity. A close-up view of the binding interface was developed for an improved resolution Fig. . It was Based on the simulation prediction, the impacts of these suggested amino acids on JG6 binding was examined. Surface plasmon resonance (SPR) assay was utilized to measure the binding affinity of JG6 to E42A, K44A, D79A, E42A/D79A and K44A/D79A cofilin mutants, which were expressed and purified in E. coli cells. It was revealed that only K44A/D79A among the 5 mutants significantly disrupted JG6 binding, resulting in nearly a 3600-fold decrease in binding affinity to JG6 Fig. . MeanwhiFor further confirmation, we examined whether introduction of K44A/D79A mutants would eliminate the impact of JG6 on actin polymerization. MTLn3 cells were transfected with cofilin siRNA to eliminate endogenous cofilin. Silent mutations were introduced to wildtype cofilin and K44A/D79A mutant to generate resistance to siRNA disruption. By measuring F-actin/G-actin ratio in cells transfected with wildtype cofilin and K44A/D79A mutant, we found that K44A/D79A mutant also completely eliminated JG6 modulated F-actin/G-actin ratio change Fig. .All the data above support a mechanism that JG6 binds to cofilin, blocks the actin binding pocket, disrupts the interaction between cofilin and actin, and thereby hinders cofilin-dependent actin organization.P < 0.001). Meanwhile, JG6 treatment exhibited negligible cytotoxic effects on 6 different breast cancer cell lines . Though the structural components responsible for interactions between cofilin and sulfated oligosaccharides have not been well characterized, sugar backbone, distribution of sulfation as well as chain length seems important. With studies continuing to explore the structure-based mechanisms of action of JG6, it is hoped that these results will add impetus to the search for cofilin-targeted carbohydrate-based anti-cancer agents. Our study provides important insights into oligosaccharides as a new and hitherto unrecognized therapeutic class. The uniTaken together, this study demonstrates for the first time that an oligosaccharide JG6 binds cofilin at the actin binding pocket and thereby inhibits actin turnover by restricting actin depolymerization. This mechanism allows JG6 anti-metastasis efficacy and highlights JG6 as a lead molecule in cancer therapy. In addition, our results identify cofilin as a key regulator in motility-driven cancer metastasis, which reinforces the view that cofilin may be a tractable and attractive target for cancer therapy.3H in formamide. Briefly, oligomannurarate was added to sulfating reagents containing formamide and ClSO3H, and reacted for 3 hours. The pH of the products was adjusted to 7.0 with 4 mol/L NaOH and desalted with Sephadex G-10. The product peak was pooled and freezedried. The molecular weights of JG6 and its precursor were analyzed by high-performance gel permeation chromatography with a G3000PW×l column (300 mm×7.8 mm) . Cofilin, p-cofilin, Erizin, p-Erizin, p-VASP antibodies were from Cell Signaling Technology (Actin Reorganization Antibody Sampler Kit #9967); PLCγ1 (#2822) and p-PLCγ1 (#2821) antibodies were from Cell Signaling Technology. N-WASP (sc-20770), Arp2 (sc-15389), Arp3 (sc-15390), β-Actin(sc-130301), GAPDH (sc-166574) antibodies were from Santa Cruz Biotechnology: JG6 mAb was generated by hybridoma fusions of BALB/C mouse spleen cells and NS-1 myeloma cells. This mAb displays high affinity for JG6 with a KD value of 2.3×10−9 M, as determined using SPR. Analysis of the rate of cross-reactivity in an ELISA assay showed that the mAb did not react with the carrier proteins, BSA or OVA.JG6 was obtained by semisynthesis following sulfate modification by reacting its precursor with ClSOVector pcDNA3.1-myc-His(A), pBAD-His(A) were obtained from Invitrogen. Cofilin mutants were generated using the Muta-direct Site-directed Mutagenesis Kit . Primers for generating cofilin siRNA-resistant mutations were: sense-GATGGTGTCATCAAAGTCTTTAACGACATGAAGGTGCG; antisense-CGCACCTTCATGTCGTTAAAGACTTTGATGACACCATC. Primers for generating cofilin mutants were: E42A sense- GCT CTTCTGCCTGAGTGCGGACAAGAAGAACATCA; E42A antisense- TGATGTTCTTCTTGTCCGCACTCAGGCAGAAGAGC; K44A sense- CTCTTCTGCCTGAGTGAGGACGCGAAGAACATCA; K44A antisense- TGATGTTCTTCGCGTCCTCACTCAGGCAGAAGAG; D79A sense- TGCTGCCAGATAAGGCCTGCCGCTATGCCC; D79A antisense- GGGCATAGCGGCAGGCCTTATCTGGCAGCA.Shanghai GenePharm  synthesized short interfering RNA (siRNA) sequences specifically targeting cofilin: sense-AAGGUGUUCAAUGACAUGAAATT, antisense-UUUCAUGUCAUUGAACACCUUTT.Human breast carcinoma cells MCF-7, MDA-MB-231, MDA-MB-468, MDA-MB-435 and BT-474 were obtained from ATCC and maintained in appropriate medium as suggested by ATCC. MTLn3 and GBA-MTLn3 cells , rat mammary adenocarcinoma cell lines described by Chan et al.[2 for 24 hours and then 100 μg/mL JG6-FITC was added. After incubated with JG6-FITC for the indicated time, cells were harvested and washed three times with PBS, then analyzed by FCM with a 488-nm laser excitation and a 513-nm emission filter. Data were analyzed with CellQuest software. For live cell fluorescence analysis, cells were seed into Nunc™ glass bottom dishes for 24 hours. Before and after the addition of JG6-FITC, cells were analyzed and photographed under a fluorescence microscope (OLYMPUS IX71).JG6-fluorescein-5-isothiocyanate (FITC) was used to detect the binding and entry of JG6 to cells. MDA-MB-435 and MTLn3 cells were plated into 6-well plates incubated at 37°C in a humidified incubator with 5% CO4 cells/well) in the presence or absence of JG6 with serum-free culture media. The lower compartment contained 600 μL of complete culture media supplemented with 10% FBS. After 12-hours incubation at 37°C, cells were fixed and stained with 0.1% crystal violet. The inhibition rate was calculated as:[Migration of MDA-MB-435 and MTLn3 cells were evaluated using a Transwell assay as previously described. BrieflyDOCK 4.0 was employed for conformational screening, based on the X-ray crystal structure of the cofilin protein reported in the Brookhaven Protein Database. Residues within 5Å of the active center, actin binding domain of cofilin was showed as the binding pocket for docking. During docking simulation, different conformational isomers of tri-mannurarate were used to present JG6.3PO4, pH 8.0, 10 μM PLP, 0.1 M NaCl, 1 mM PMSF, 2 mM EDTA, and protease inhibitor. The cells were lysed by sonication. The precipitate was removed by centrifugation. The supernatant was applied to Ni-NTA agarose column. After the column was washed with washing buffer  three times, the His-cofilin proteins were eluted with elution buffers that had increasing imidazole concentrations at 500 mM.E. coli BL21 transformants harboring the expression vectors pBAD-His-cofilin or cofilin mutation were cultured in RM media  plus 0.2% glucose with 150 μg/mL ampicillin at 37 °C in a buffered flask with constant shaking at 300 rpm, until the cell cultures reached an OD600 of 0.8. L-(+)-arabinose was added to culture to a final concentration 0.2% (w/v), and the cell cultures were then allowed to grow for an additional 4 h at 30 °C. Cells were pelleted by centrifugation at 3,000 × g for 15 min, washed once in half of the original volume with 10 mM Tris-HCl buffer, pH 8.0, containing 0.1 M NaCl. Cell pellets were resuspended in 1:20 of the original volume in extraction buffer containing 50 mM KSPR biosensor measurements (Bio-Rad ProteOn XPR36) were used to evaluate the interactions between JG6 and cofilin or cofilin mutants. In this assay, 10 mM Tris-HCl buffer (pH 8.0) containing cofilin or cofilin mutants at concentrations of 0.312, 0.625, 1.25, 2.5, or 5 μM were passed over the JG6 sensor chip surface for 2 minutes at a flow rate of 5 μl/min. Changes in mass were measured, and the sensorgrams were recorded in real time and analyzed after subtracting the control.2O containing 2 μm cytochalasin D at a volume equivalent to the G-actin-containing supernatant volume. The resuspended F-actin pellet was kept on ice for 60 min with mixing by pipette every 15 min to dissociate F-actin. After dissociation, dissociated F-actin was centrifuged at 14,000 g for 10 min at 4 °C. The F-actin and G-actin preparations were then assayed for protein.The concentration of F-actin and G-actin in cells was obtained using an F-actin/G-actin assay kit . Briefly, cells were rinsed with phosphate-buffered saline at 25 °C and scraped and homogenized in a lysis and F-actin stabilization buffer (LAS1). F-actin was then separated from G-actin by centrifugation at 100,000 × g for 60 min at 37 °C. The F-actin-containing pellet was resuspended in ddH2+-bound actin  was polymerized for 12 h at 20 °C. Depolymerization was initiated by a 12-fold dilution in 10 mM Hepes/KOH/16 mM Tris/HCl/2 mM MgCl2/100 mM KCl/0.5 mM EGTA/0.1 mM dithiothreitol (pH 7.2) containing 2 μM WT or mutant cofilin. The decline in fluorescence was monitored immediately. JG6 was added when necessary, depending on certain application.MgHuman breast cancer cells were seeded into 96-well plates, attached overnight and subsequently exposed to different concentrations of JG6 for 48 h. Cytotoxicity to breast cancer cells were assessed by MTT assay as previously described. The cyt2, 0.1 mM EDTA, 1 mM sodium orthovanadate, 1% Nonidet P-40, and supplemented with a protease inhibitor cocktail  and then centrifuged at 12,000 g at 4°C for 15 minutes. For co-IP, the protein lysates (1 mg) were incubated with the JG6 antibodies with rocking for 4 hours at 4°C, followed by the incubation of protein A/G plus Agarose (Santa Cruz) for 6 hours to overnight at 4°C with rocking. The bound proteins were then eluted using 0.2% sodium dodecyl sulfate.For total protein extraction, the indicated cell lines were lysed in buffer consisting of 25 mM HEPES (pH 7.5), 150 mM NaCl, 10 mM MgClBoth total protein lysates and eluted proteins from the co-IP experiment were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to Hybond C Extra nitrocellulose membranes . Membranes were incubated in blocking solution containing 5% nonfat dry milk for 1 hour at room temperature. The membranes were incubated at 4°C overnight with the appropriate primary antibodies immunoblotting for GAPDH or β-actin served as a protein loading control. All experiments were performed at least three independent times.7) through a 27-gauge needle. The well-developed tumors were cut into 1-mm3 fragments and transplanted s.c. into the right flank of nude mice using a trocar. When the tumor reached a volume of 100 to 200 mm3, mice were divided into three experimental groups, specifically (a) untreated (n = 12); (b) JG6 10 mg/kg (n = 6); and (c) JG6 20 mg/kg (n = 6). JG6 was s.c. administrated through the tail vein once a day for 6 weeks thereafter. Tumors and body weight of mice were measured individually twice per week. Mice were sacrificed 5 days later after the final therapy. The lungs were removed, some were fixed with Bouin's solution, and metastatic nodules were counted.Female athymic nude mice, ages 4 to 5 weeks, were anesthetized with chloral hydrate, Hypodermic injection an inoculum of human breast cancer MDA-MB-435 cells  were performed using Statview. P < 0.05 was considered significant and P < 0.01 as highly significant. Five parallel samples were prepared in each group and all experiments were replicated at least three times.


The Qiangic languages in western Sichuan (WSC) are believed to be the oldest branch of the Sino-Tibetan linguistic family, and therefore, all Sino-Tibetan populations might have originated in WSC. However, very few genetic investigations have been done on Qiangic populations and no genetic evidences for the origin of Sino-Tibetan populations have been provided. By using the informative Y chromosome and mitochondrial DNA (mtDNA) markers, we analyzed the genetic structure of Qiangic populations. Our results revealed a predominantly Northern Asian-specific component in Qiangic populations, especially in maternal lineages. The Qiangic populations are an admixture of the northward migrations of East Asian initial settlers with Y chromosome haplogroup D (D1-M15 and the later originated D3a-P47) in the late Paleolithic age, and the southward Di-Qiang people with dominant haplogroup O3a2c1*-M134 and O3a2c1a-M117 in the Neolithic Age. The Sino-Tibetan languages are a family of some 460 languages, including two subfamilies, namely Chinese and Tibeto-Burman. They are spoken by over a billion people all over East Asia and Southeast Asia, and second only to the Indo-European languages in terms of the population size of native speakers Despite intense linguistic and archaeological researches, little has been known about how the Sino-Tibetan people dispersed from western China? During the past two decades, the characterization of genetic diversity has shed light on the history of Sino-Tibetan populations, especially the diversity defined by the maternal mtDNA and the paternal Y chromosome. In the maternal side, mtDNA evidence suggested a northern Asian origin of Tibetans, due to the high frequencies of northern Asian specific haplogroup A, D, G, and M8 Given that a correlation is emerging that suggests language change in an already-populated region may require a minimum proportion of immigrant males, while mtDNA types represent more ancient settlement Y chromosome evidence indicates that Qiang people might be the origin source for the Sino-Tibetan populations We collected blood samples of 407 healthy and unrelated individuals from four Qiaingic populations in western Sichuan province . Our stuThe samples were typed through seven panels of 100 SNPs as listed in the latest Y chromosome phylogenetic tree Haplogroup O panel: M175, M119, P203, M110, M268, P31, M95, M176, M122, M324, M121, P201, M7, M134, M117, 002611, P164, L127 (rs17269396), and KL1 (rs17276338).Corset Panel: M130, P256, M1, M231, M168, M174, M45, M89, M272, M258, M242, M207, M9, M96, P125, M304, M201 and M306.Haplogroup C panel: P54, M105, M48, M208, M407, P33, M93, P39, P92, P53.1, M217, M38, M210, M356, P55, and M347.Haplogroup D panel: P47, N1, P99, M15, M125, M55, M64.2, M116.1, M151, N2, and 022457.Haplogroup N panel: M214, LLY22g, M128, M46/Tat, P63, P119, P105, P43, and M178.Haplogroup R panel: M306, M173, M124, M420, SRY10831.2, M17, M64.1, M198, M343, V88, M458, M73, M434, P312, M269, and U106/M405.Haplogroup Q panel: P36.2, M3, M120, MEH2, M378, N14/M265, M25, M143, M346, L53, and M323.Those binary markers were hierarchically genotyped by SNaPshot (ABI SNaPshot Multiplex Kit) and fluorescent allele-specific PCR. PCR products were electrophoresed on a 3730xl Genetic Analyzer .Seventeen Y chromosomal STRs  were amplified using the AmpFlSTR Yfiler PCR Amplification kit . Amplified products were separated and detected using the ABI 3730xl Genetic Analyzer  according to the manufacturer’s recommended protocol. The data were analyzed using GeneMapper ID v3.2 . For use in the analyses, DYS389II was calculated by subtracting the DYS389I allele size.http://www.phylotree.org/). The mtDNA sequences have been deposited in Genbank with accession numbers KJ783504-KJ783899.The hypervariable segment I (HVS-I) of the control region was amplified by primers L15974 and H16488 4 samples of the program’s output representing 106 MCMC cycles were taken after discarding the first 3×103 samples as burn-in. The Time to the Most Recent Common Ancestor (TMRCA) is calculated using the product of the estimated population size N and the height of the tree T  Principal component analysis (PCA) was performed using SPSS 18.0 software . Networks of Y chromosomal STR data and the mtDNA HVS-I motifs were constructed by reduced median-joining method According to the nomenclature of Y Chromosome Consortium (YCC) The paternal genetic relationships among Qiangic, Tibeto-Burman, and other East Asian populations were discerned with the aid of additional published Y chromosome datasets. We used a PCA based on the distribution of Y chromosome haplogroup frequencies of 51 populations to show the overall clustering pattern . ResultsTo discern the detail relationship between the D3a-P47, O3a2c1a-M117, D1-M15, and O3a2c1*-M134 haplogroups in Tibeto-Burman and other related populations, a median-joining network was constructed based on Y-STR haplotypes of those haplogroups . A clearWe then estimated the coalescence and expansion time of Y chromosome lineages in Qiangic populations . The ageMtDNA HVS-I sequences of 396 individuals from the four studied Qiangic populations have been successfully typed. A total of 214 different haplotypes were defined by 134 polymorphic sites in the HVS-I dataset. The haplotype diversity of those Qiangic groups ranged from 0.978 to 0.994, with the lowest haplotype diversity observed in Horpa-Daofu (0.978) and the highest in Horpa-Danba (0.994). The mean number of pairwise differences (MNPD) and nucleotide diversity (ND) show a similar pattern with the haplotype diversity, as the highest diversity was observed in Horpa-Danba and the lowest in Horpa-Daofu. However, Tibetan-Yajiang has a higher diversity in haplotype but lower diversity in MNPD and ND than Tibetan-Xinlong. Measures of population growth  all gave the negative values for each population, but Tajima’s D, Fu & Li’s D* and F* were not statistically significant in Horpa-Daofu . The not397 samples were successfully assigned to mtDNA haplogroups using a combination of HVS-I sequence motifs and single nucleotide polymorphisms (SNPs) distributed around the coding region of the mtDNA genome. A total of 79 haplogroups or paragroups (unclassified lineages within a clade marked with an asterisk [*]) were identified , all witWe performed a PCA using the mtDNA haplogroup frequencies of Qiangic groups in this study and other 68 populations to see the detailed genetic patterns of those populations . The firMacrohaplogroup M and its subhaplogroups represent the majority of the Qiangic maternal lineages, with frequencies ranging from 65.22% in Horpa-Danba to 57.98% in Tibetan-Xinlong. Haplogroup D4 and G are the most frequent sub-clades of macrohaplogroup M in Qiangic populations, each comprising 13.60%. Haplogroup D4, which is prevalent throughout Central Asia Haplogroup G is found at high frequencies in northeastern Siberia but it is also common among populations of Japanese Archipelago and Korean Peninsula. This haplogroup also comprises an average of 20% of the maternal gene pool of the Tharus from Nepal Haplogroup M8 has two sublineages, haplogroup C and Z. Haplogroup C is a common lineage, which is widespread in East Asia and Siberia and is one of the founder lineages among Native Americans M9a’b is widely distributed in mainland East Asia Haplogroup M13a has been found at its greatest frequency and diversity in Tibet, but it has also been detected at very low frequencies in Siberian Buryat, Yakut, Altaian Kazakh, and Ewenki Qiangic populations also exhibit some basal Eurasian mtDNA lineages. Haplogroup M62, for example, was first reported in Northeast India Haplogroup R and its subhaplogroups (B and F) represent the majority of the lineages branching from the basal N trunk, accounting for 26.09%, 22.50%, 28.57%, and 23.44% of the maternal diversity in Horpa-Danba, Horpa-Daofu, Tibetan-Xinlong, and Tibetan-Yajiang, respectively. Subhaplogroup B4* is the most frequent lineage of haplogroup B in Qiangic, comprising 4.53% of all the samples. In the network of B4*, the root clade composed almost exclusively of non-Tibetan-Burman samples, however, the Tibetan-Burman samples only formed some small clusters or shared the terminal types, suggesting that B4* had already differentiated before its arrival in Tibet. Subhaplogroup F1* is the most frequent lineage of haplogroup F in Qiangic, accounting for 5.54% of all the samples, and even comprising as high as 12.5% of Horpa-Daofu. Age estimate generated for F1* in Qiangic was around 5 kya . The excHaplogroup N* is almost exclusively represented by haplogroup A in our samples. Haplogroup A is widely distributed in northern and eastern Asia, occurring at frequencies of 5%–10% in different populations The Sino-Tibetan linguistic family comprises some 460 languages distributed in East Asia, Southeast Asia, and parts of South Asia, including the Chinese and Tibeto-Burman subfamilies From the paternal Y chromosome perspective, haplogroup D1-M15 originated from D*-M174 during its migration into mainland East Asia Around 20–40 kya, a population with dominant haplogroup O3-M122 Y chromosomes  finally reached the upper and middle Yellow River basin and formed the Di-Qiang populations. During the Neolithic period, the Di-Qiang people experienced relatively huge population expansion. A subgroup of the Di-Qiang people with dominant haplogroup O3a2c1*-M134 and O3a2c1a-M117, now called the Proto-Tibeto-Burman people left their Yellow River homeland, probably also moved along the Tibeto-Burman corridor, embarking on large-scale westward migrations to nowadays Qinghai province and then southward to the Himalayas, or southward migration directly via the WSC corridor to Yunnan and Guangxi, where they mixed with D-M174 linages and developed into Tibeto-Burman populations. However, haplogroup O3a2c1*-M134 might have already reached Tibet predated the above southward migration together with O3a2c1a-M117, judging from the high diversity in the network of O3a2c1*-M134 . In addiThe role of haplogroup O3-M122 lineages played in the origin of Tibeto-Burman populations has suggested extensive genetic input from northern Asians. This suggestion has been supported by previous studies employing autosomal STR Table S1Y chromosome and mtDNA haplogroup frequencies of Qiangic populations.(XLS)Click here for additional data file.Table S2Y chromosome SNP and STR data, mtDNA haplogroups and HVS-I motif of Qiangicpopulations.(XLS)Click here for additional data file.Table S3Y chromosome and mtDNA haplogroup frequencies used in PCA plot and haplogroupcontributions to each PC.(XLS)Click here for additional data file.Table S4Shared Y-STR haplotypes between Qiangic populations and other East Asian populations.(XLS)Click here for additional data file.Doc S1Geographic distribution of Y chromosome haplogroup D1 and D3a, Y-STR neighbor-joining tree based on genetic distance.(DOC)Click here for additional data file.Doc S2Three representative complete mtDNA haplotypes compared to rCRS.(DOC)Click here for additional data file.


In all organisms, RecA-family recombinases catalyze homologous joint formation in homologous genetic recombination, which is essential for genome stability and diversification. In homologous joint formation, ATP-bound RecA/Rad51-recombinases first bind single-stranded DNA at its primary site and then interact with double-stranded DNA at another site. The underlying reason and the regulatory mechanism for this conserved binding order remain unknown. A comparison of the loop L1 structures in a DNA-free RecA crystal that we originally determined and in the reported DNA-bound active RecA crystals suggested that the aspartate at position 161 in loop L1 in DNA-free RecA prevented double-stranded, but not single-stranded, DNA-binding to the primary site. This was confirmed by the effects of the Ala-replacement of Asp-161 (D161A), analyzed directly by gel-mobility shift assays and indirectly by DNA-dependent ATPase activity and SOS repressor cleavage. When RecA/Rad51-recombinases interact with double-stranded DNA before single-stranded DNA, homologous joint-formation is suppressed, likely by forming a dead-end product. We found that the D161A-replacement reduced this suppression, probably by allowing double-stranded DNA to bind preferentially and reversibly to the primary site. Thus, Asp-161 in the flexible loop L1 of wild-type RecA determines the preference for single-stranded DNA-binding to the primary site and regulates the DNA-binding order in RecA-catalyzed recombinase reactions. Escherichia coli (E. coli) RecA has been most thoroughly studied for decades ((Homologous genetic (or DNA) recombination plays essential roles in DNA double-strand break repair to maintain genome stability and in genetic diversification, and defects in homologous recombination in mitotic cells cause carcinogenesis and various diseases see –4 for re for re4 decades . The prsistance , as wellsistance –19. ATP-sistance ). The ba4-Mg2+-bound RecA–DNA complexes , respectively. The bases or base pairs of the bound DNA form triplets, which are sandwiched between adjacent L2 β-hairpins  in loop L1, in selecting single-stranded DNA for the primary DNA-binding step by RecA at the start of homologous joint formation.2Cl, 0.3-g KH2PO4 and 0.6-g Na2HPO4·7H2O per 100 ml of distilled water.LB (liquid) contained 1% Bacto-tryptone, 0.5% Bacto-yeast extract and 1% NaCl at pH 7.5; LB plates contained 1.5% agar in LB (liquid) medium. M9 buffer contained 0.1-g NHThe sequence of the 90-nucleotide single-stranded DNA used in the homologous joint formation by RecA is 5′-AAATCAATCT AAAGTATATA TGAGTAAACT TGGTCTGACA GTTACCAATG CTTAATCAGT GAGGCACCTA TCTCAGCGAT CTGTCTATTT-3′, which has least secondary structures and is the complementary sequence from positions 1932 to 2022 of the pBluescript SK(-) double-stranded DNA . This 90Amounts of DNA are expressed in nucleotides, unless otherwise stated.E. coli cells, since we experienced some experimental difficulties with the wild-type RecA purified from old 60% glycerol stock of partially purified RecA in a freezer .These are described in the Supplementary data. It is advised to use freshly purified RecA preparation from freshly grown E. coli MV1184 , rpsL, thi (Φ80 lacZΔM15), Δ(srl-recA) 306::Tn10 r) cells, with the pKK223-3 plasmid bearing recA under the control of the tac promoter, or with the empty pKK223-3 plasmid, were cultured at 37°C in 1 ml of LB medium containing 50-μg/ml ampicillin and 10-μg/ml tetracycline for ∼18 h (overnight).600nm 2.3–2.6; GE Ultrospec 2100 pro spectrophotometer), the cell culture was diluted with M9 buffer to 1.0 at OD600nm, and 5.0-μl portions of the diluted cell culture were spotted on LB agar plates containing 50-μg/ml ampicillin, 10-μg/ml tetracycline and the indicated amounts of methyl methanesulfonate (MMS). The spotted plates were incubated at 37°C for the indicated times.For spot tests, 20-μl portions of the overnight cultures were inoculated into 1 ml of fresh LB medium containing the same amounts of antibiotics, and shaken at 37°C. When the cells reached early stationary phase . The plates were incubated at 37°C for 1 or 2 days, as indicated. The numbers of colonies formed on the plates were counted.2, 1.3-mM ATP, 1.8-mM dithiothreitol and 88-μg/ml bovine serum albumin , as described previously (The standard reaction buffer consisted of 31-mM Tris-HCl (pH 7.5), 13-mM MgCleviously ,26, unle33P-labeled at the 5′ terminus) was incubated with the indicated amounts of RecA in a 10-μl standard reaction buffer at 37°C for 5 min and fixed by 0.01% fresh glutaraldehyde at 37°C for 3 min. The samples were mixed with 1/10 volume of loading buffer B ), loaded onto a 1.2% agarose gel, and electrophoresed in 0.5x TBE buffer  at 50 V/12.5 cm for 75 min at room temperature. After electrophoresis, the gel was dried and the 33P-signals were analyzed with a BAS-2000 or BAS-2500 image analyzer . In some experiments, the 90-nucleotide single-stranded DNA was labeled at 5′ terminus with FAM, and analyzed by a LAS-1000 Luminescent Image Analyzer .For single-stranded DNA binding, the 90-nucleotide single-stranded DNA  double-stranded DNA  was incubated with the indicated amounts of RecA, in a 10-μl standard reaction buffer at 37°C for 15 min. The samples were then mixed with 1 μl of the loading buffer B and loaded onto a 1.0% agarose gel, and electrophoresed at 40 V/12.5 cm for 120 min at room temperature. The gel was stained with 1-μg/ml ethidium bromide and was photographed with UV illumination at 312 nm. Smart Ladder  markers were used as DNA size markers.14C or α-32P]ATP (26 nmol at 1.3 mM) was incubated with the indicated amounts of RecA, MgCl2 and indicated DNA, at 37°C for 30 min. The sample was subjected to thin layer chromatography as described (Adenosine triphosphatase (ATPase) activities were assayed as described previously . In the escribed , but the2, containing an ATP-regenerating system. The ATP-regenerating system used in this study consisted of 4.8-mM phosphocreatine and 7.8 units/ml of creatine phosphokinase . LexA cleavage was initiated by the addition of LexA repressor protein (4.7 μM) to the reaction mixture , followed by an incubation at 37°C. At the indicated times, 5-μl aliquots were withdrawn. The samples were mixed with 5-μl 2×SDS sample buffer (125 mM Tris-HCl (pH 6.8), 10% 2-mercaptoethanol, 4% sodium dodecyl sulphate (SDS), 10% sucrose, 0.01% bromophonol blue (BPB)) at 0°C, and then heated at 95°C for 5 min to terminate the reaction. The products and unreacted substrates were then separated by electrophoresis through a 15% polyacrylamide gel containing 0.4% SDS. The gel was stained by 0.25% Coomassie Brilliant Blue and photographed. Precision Plus Protein™ All Blue Standards , purchased from Bio-Rad Laboratories, Inc., were used as protein size markersM13 single-stranded DNA or pBluescript SK(-) double-stranded DNA (18 μM) and RecA (1.0 μM) were first incubated at 37°C for 20–30 min in the standard reaction buffer, except with 5.0-mM MgCl33P]DNA (0.05 μM) and RecA (2.0 μM) were first incubated at 37°C for 10 min, in the standard reaction buffer containing the ATP-regenerating system. The ATP-regenerating system used in this study consisted of 4-mM phosphocreatine and 5 units/ml of creatine phosphokinase . Homologous joint formation was initiated by the addition of homologous form I (negatively supercoiled closed circular double-stranded) DNA (pBluescript SK(-); 18 μM) to the reaction mixture , followed by an incubation at 37°C. At the indicated times, 4.5-μl aliquots were withdrawn. The samples were mixed with 1.7-μl 66.7-mM EDTA and 5% SDS solution on ice to terminate the reaction and were treated with 2.4-mg/ml  Proteinase K at 0°C or 37°C for 15–45 min to remove the proteins. The samples were mixed with 1/10 volume of 10x loading buffer H , and the products and unreacted substrates were then separated by 1.0% agarose gel electrophoresis in 0.5x TBE buffer . After the gel was dried, the 33P-signals were analyzed quantitatively with a BAS-2000 or BAS-2500 image analyzer . The Proteinase K treatment at 0°C yielded somewhat increased amounts of the RecA-catalyzed homologous joint-formation products, as compared to the treatment at 37°C, probably because of the heat instability of some of the homologous joints, as observed previously  in the P61 space group and determined the structure at 2.8-Å resolution . The RecA helical filaments in the crystals had six protomers per turn, with a 72.1-Å helical pitch , suggesting that this crystallographic filament reflects an inactive state . The core and the C-terminal domains in each protomer of the inactive DNA-free RecA and those of the activated RecA–DNA complexes . This 90-nucleotide single-stranded DNA was prepared for a homologous joint-formation assay, with minimal effects of the secondary structure. After the incubation of the 90-nucleotide single-stranded [33P] DNA under the standard buffer conditions for homologous joint formation, the samples were fixed by glutaraldehyde and subjected to agarose gel electrophoresis. The intensity of the free DNA signal (arrow i) was decreased, with a concomitant increase in the signal of RecA-bound DNA, along with the increase in the amount of either recA-D161A or recA-D161N, or of the wild-type RecA (RecA-wt) in the presence or absence of ATP . Before we tested whether these replacements of Asp-161 (D161A) stimulated double-stranded DNA binding by an electrophoresis mobility shift assay, we analyzed the effects of the D161A and D161N substitutions on single-stranded DNA binding, using the 90-nucleotide single-stranded [Since we intended to understand how RecA selected single-stranded DNA at the start of homologous joint formation, we analyzed DNA binding by use of 90-nucleotide single-stranded DNA as described above and double-stranded DNA (form I DNA), both of which were used for homologous joint formation. Form I DNA was incubated under the same conditions and analyzed by agarose gel electrophoresis without fixation. As expected from the assumption that Asp-161 inhibits the binding of the second DNA strand, the D161A substitution clearly enhanced double-stranded DNA (form I DNA) binding, in either the presence or absence of ATP  for homologous joint formation. The stimulation of double-stranded DNA binding and a slight effect on single-stranded DNA binding of RecA by D161A and D161N substitutions were not significantly altered by changing MgCl2 concentrations .We repeated the DNA binding assays with the 90-nucleotide single-stranded DNA and the form I DNA in the presence of 1.0-mM MgClAlthough the D161A substitution extensively stimulated double-stranded DNA binding, recA-D161A still showed higher affinity toward single-stranded DNA than double-stranded DNA, especially in the presence of ATP, as determined by comparing the minimum amounts of the protein required to detect the mobility shift (ca. 0.1 μM versus 2 μM) and those to generate the entire shift . We compared the ATP hydrolysis by the mutant recAs and RecA-wt, in the presence of single-stranded DNA or double-stranded DNA, to test their primary DNA-binding abilities which induces the conformational change for ATP hydrolysis.2 at the concentration required for homologous joint formation , double-stranded DNA is a poor cofactor for ATP hydrolysis by RecA-wt , as expected, the D161A substitution extensively enhanced the double-stranded DNA-dependent ATPase activity, especially in the presence of MgClan 5 mM; ), and wat Figure . Under t RecA-wt ,28. RecAnd 1 mM . Form I DNA has some characteristics of single-stranded DNA, because its topological stress is favorable for the local melting of a double helix, as shown by the sensitivity to the single-stranded DNA-specific S1 nuclease . In the  Figures  and 4A aThese results suggested that recA-D161A can bind single-stranded DNA with secondary structure, which prevents the binding of RecA-wt. Supporting this suggestion, in the presence of a sufficient amount of DNA relative to RecA, the mutant and wild-type RecAs showed similar levels of ATP hydrolysis Figure  and C. A2 .in vitro by RecA-family recombinases depend on the DNA substrates: when the substrates are closed circular double-stranded DNA with negative supercoils and single-stranded DNA-fragments or tails, the RecA- or Dmc1-promoted branch migration results in the dissociation of the homologous joint , stimulates the joint formation, as compared to the reaction started by the simultaneous addition of single-stranded and double-stranded DNAs . We also varied the preincubation times of RecA with single-stranded DNA (standard order-of-addition for homologous joint-formation assay of RecA and Rad51) or with double-stranded DNA before the addition of their partner DNA. The amounts of homologous joints formed at 30 s of incubation were plotted, as representatives of the initial velocity of homologous joint formation, against the amounts of protein Figure  and D. TThe stimulating and suppressing effects of the preincubation of RecA with single-stranded DNA and double-stranded DNA, respectively, on the initial velocity of homologous joint formation were time dependent. The effects of the preincubation with double-stranded DNA appeared more quickly (finished within 1 min) than the slow activation by preincubation with single-stranded DNA  and a multi-copy plasmid bearing either the wild-type or mutant recA. The E. coli cells with recA-D161A did not show filamentous growth , indicating that recA-D161A did not cause SOS constitutive. As shown in Supplementary Figure S8A, the cells with recA-D161A appeared more resistant to methyl MMS than the cells with +recA. A quantitative analysis more clearly showed that the cells with recA-D161A are more resistant to MMS than the cells with +recA . Notably, under these conditions, the SOS genes are inducible by activated RecA.Finally, we tested whether the alanine replacement of Asp-161 (D161A) inhibited or stimulated the repair of DNA lesions in 2+ concentration requirements  structure  (E. coli RecA) also sterically clashes with the complementary strand located in the activated RecA-double-stranded DNA complex, in both the absence and presence of nucleotide cofactors  –38, Asp-s Figure  and E. TThe primary DNA binding induces the hydrolysis of ATP and LexA-repressor cleavage. The enhancement of double-stranded DNA binding by the D161A replacement Figure  correlatE. coli mutant bearing recA-D161N was previously classified as +recA . To understand these observations, we need to consider the recombination mediators required by RecA, and the SOS-mediated DNA repair functions in E. coli cells.The selectivity for single-stranded DNA in the primary binding step of RecA appears to enable RecA targeting to double-strand break sites to be repaired, since the resection from a break end yields a single-stranded tail, which is unique to the break sites. Then, one may suppose that the lower selectivity of RecA by the D161A replacement would decrease the DNA repair efficiency. The as recA+ . In contE. coli cells, and the recombination mediator recruits RecA to the SSB-bound single-stranded region. Thus, the preference of RecA for single-stranded DNA is not essential to initiate recombination at the single-stranded region.In cells, RecA requires a recombination mediator (RecOR or RecBCD) to bind to single-stranded DNA regions, where SSB binds first. The single-stranded regions are recognized by SSB in E. coli cells used in this study, the original +recA gene was removed, and the mutant and wild-type recAs to be tested were solely expressed under the tac promoter on a multiple copy-number plasmid (pKK223-3), with a pBR322-derived replication origin , indicating the absence of spontaneous SOS induction. This observation suggests that the enhanced double-stranded DNA-binding capacity of recA-D161A by itself is not sufficient for SOS induction in vivo.In the n origin . Althougn origin . Thus, tThe most prominent effect of the D161 replacement on homologous joint formation is the reduced suppression in the preincubation reaction of recA-D161A with double-stranded DNA before the addition of single-stranded DNA, as shown by the order-of-addition experiments Figure , which iE. coli RecA and yeast Rad51, respectively). This study revealed that this inhibitory role of the contact of RecA with double-stranded DNA, before the binding to single-stranded DNA, is actively regulated by Asp-161 on loop L1, and may have a biological function, which is under investigation.The incubation of RecA/Rad51 recombinases with double-stranded DNA, before the addition of single-stranded DNA, is known to prevent homologous joint formation by these recombinases (see Figure The structural coordinates of the DNA-free inactive RecA crystal were deposited in the Protein Data Bank, under the accession code 4TWZ.Supplementary Data are available at NAR Online.


In the Materials and Methods section, there is an error in the equation of the section titled “The uptake rate model for amino acid uptake studies.” Please review the complete, correct equation here: There is an error in the legend for There are errors in the Supporting Information file and the legend. The line numbers were inadvertently left in File S1Supporting files. Figure S1, Southern blot analysis of WT, stopEO1472F (A) and InsOE1475F (B). A, Lane 1: size marker (in bp), lane2: SacI digested DNA from WT, lane 3: SacI digested DNA from StopOE1472F. B, Lane 1: size marker (in bp), lane2: NotI digested DNA from WT, lane 3: NotI digested DNA from InsOE1472F. Figure S2, AroAA regulated CHY and HY ORFs that are closely adjacent, and possibly organized in operons. Arrows show the relative positions and orientations of ORFs, but are not drawn to scale. The fold regulations are indicated below. The ORFs shown in panels A-D are described in Table 1. The ABC transporter ORFs shown in panel E is described in Table S2 in File S1. Table S1, Formation of 3-dehydroshikimate (DHS) from 3-dehydroquinate (DHQ) under different conditions. Table S2, Masses detected by LC-MS after derivatization with NBHA. Table S3, Expression of AroAA-related genes and transport-related genes in H. salinarum R1 cells grown in synthetic medium without AroAA relative to synthetic medium with AroAA. Table S4, Strains used in this study. Table S5, The composition of the chemically defined medium, pH  =  7.0. Table S6, Plasmids used in this study. Table S7, List of oligonucleotides used in this study.(DOCX)Click here for additional data file.


Drosophila Neto (Neuropillin and Tolloid-like protein) is an essential auxiliary subunit of ionotropic glutamate receptor (iGluR) complexes required for the iGluRs clustering at the neuromuscular junction (NMJ). Here we show that optimal levels of Neto are crucial for stabilization of iGluRs at synaptic sites and proper NMJ development. Genetic manipulations of Neto levels shifted iGluRs distribution to extrajunctional locations. Perturbations in Neto levels also produced small NMJs with reduced synaptic transmission, but only Neto-depleted NMJs showed diminished postsynaptic components. Drosophila Neto contains an inhibitory prodomain that is processed by Furin1-mediated limited proteolysis. neto null mutants rescued with a Neto variant that cannot be processed have severely impaired NMJs and reduced iGluRs synaptic clusters. Unprocessed Neto retains the ability to engage iGluRs in vivo and to form complexes with normal synaptic transmission. However, Neto prodomain must be removed to enable iGluRs synaptic stabilization and proper postsynaptic differentiation.Stabilization of neurotransmitter receptors at postsynaptic specializations is a key step in the assembly of functional synapses.  Drosophila NMJ is a glutamatergic synapse similar in composition and physiology with mammalian central excitatory synapses. Like mammals, Drosophila utilizes auxiliary subunit(s) to modulate the formation and function of glutamatergic synapses. We have previously reported that Neto is an auxiliary protein essential for functional glutamate receptors and for organization of postsynaptic specializations. Here we report that synapse assembly and NMJ development are exquisitely sensitive to postsynaptic Neto levels. Furthermore, we show that Neto activity is controlled by Furin-type proteases, which regulate the processing and maturation of many developmentally important proteins, from growth factors and neuropeptides to extracellular matrix components. Such concerted control may serve to coordinate synapse assembly with synapse growth and developmental progression.Synapse development is initiated by genetic programs, but is coordinated by neuronal activity, by communication between the pre- and postsynaptic compartments, and by cellular signals that integrate the status of the whole organisms and its developmental progression. The molecular mechanisms underlining these processes are poorly understood. In particular, how neurotransmitter receptors are recruited and stabilized at central synapses remain the subject of intense research. The  Synapse development is a highly orchestrated process that enables proper establishment of neural circuits and development of the nervous system. Crucial to synapse assembly is the recruitment and stabilization of neurotransmitter receptor complexes at synaptic sites . ReceptoDrosophila NMJ provides an ideal genetic system to examine the mechanisms that couple synapse assembly with synapse growth and development. The fly NMJ is a glutamatergic synapse similar in composition and physiology to vertebrate AMPA/kainate central synapses .,16.G14>nneto and importin-β11 mutants  attP40). The control is y1w1118 unless otherwise specified.Fly lines were generated by standard germline transformation of pUAST-based plasmids containing various neto109 , netoRNetoRNAi , G14-Galneto transgenes were introduced into neto36 null mutant background using tissue-specific promoters. Since neto is on the X-chromosome we used only FM7-GFP balanced stocks to eliminate any meiotic non-disjunction event. The F1 progenies were genotyped during late embryogenesis and reared at the indicated temperatures. After 24 hours, crawling larvae were removed, counted, and kept at the same temperatures for further analyses or adult viability testing.For rescue analyses, 51-Q130 and loops out the Neto prodomain. PM-Neto has two point mutations: R123I and R126I. Double-tagged Neto constructs were generated by QuikChange loop-in of various Neto fragments in a previously described AcPA-SP-Myc-V5/His plasmid [Neto variants were generated using QuikChange site-directed mutagenesis kit (Stratagene) as described previously . CA-Neto plasmid . This acFor RNA interference, PCR primers for Furins that carry the T7 promoter sequence at the 5’ end were designed as previously described . The pridFur1-F 5’-TAATACGACTCACTATAGGGACGCAAAGATCCTCTGTGGCA;dFur1-R 5’- TAATACGACTCACTATAGGGACATTGCTCCCGGAACTGC;dFur2-F 5’- TAATACGACTCACTATAGGGACGCTAGAGGCCAATCCGGAA;dFur2-R 5’- TAATACGACTCACTATAGGGACCCTTCTCGCCCCAAAAGTG;Amon-F 5’- TAATACGACTCACTATAGGGACCCACATGGAGCTGGCTGT;Amon-R 5’- TAATACGACTCACTATAGGGACCCTGACTTTGCCGCCATT.PCR products were amplified from genomic DNA or S2 cells cDNA. In vitro transcribed dsRNA was produced using the MEGAscript kit (Ambion). RNAi treatment was carried out by transfections of 5 mg/ml of dsRNA into S2 cells.S2 cells were transfected with indicated constructs and harvested after five days incubation. Total RNA was extracted using TRIZOL reagent (Invitrogen) according to manufacturer's instructions. AccuScript High Fidelity First-Strand cDNA Synthesis Kit (Agilent) was used to generate cDNAs from the extracted total RNAs according to manufacturer’s instructions. PCR reaction for each target gene was executed using the cDNAs as templates with specific primer pairs (above) and β-Actin as a reaction standard .2+-free HL-3 solution [Wandering third instar larvae were dissected as described previously in ice-cooled Casolution ,66. Disssolution , rabbit solution ; rabbit solution ; rabbit solution ; FITC-, Drosophila S2 cells were used for producing recombinant proteins as previously described [6 cells/ml. For the transfection, dimethyldioctadecyl-ammonium bromide (DDAB) solution (250 μg/ml) was mixed with M3 media at 1:2 ratio and incubated 5 min at RT, then the DNA was added to the DDAB-M3 mixture (1μg of plasmid DNA to 100 μl suspension). The mixture was incubated for 20 min and transfected into S2 cells (100 μl mixture to 2 X 106 cells/ml culture). After five days, the secreted proteins were collected for analysis, and membrane proteins were extracted by homogenizing cells in lysis buffer  for 30 min on ice. The lysates were collected by centrifugation at 13,000rpm for 30 min at 4°C, separated by SDS-PAGE on 4%–12% NuPAGE gels (Invitrogen) and transferred onto PVDF membranes (Millipore). Primary antibodies were used at the following dilutions: rat anti-Neto, 1:1000; chicken anti-GFP (Abcam), 1:2000; anti-GluRIIC, 1:1000; anti-tubulin (Sigma), 1:1000. Immune complexes were visualized using secondary antibodies coupled with IR-Dye 700 or IR-Dye 800 followed by scanning with the Odyssey infrared imaging system (Li-Cor Biosciences).Transiently transfected escribed . The S2 To analyze muscle proteins, wandering third instar larvae were dissected, and the body walls were mechanically homogenized in lysis buffer for 30 min on ice. The lysates were analyzed by Western blotting. For co-immunoprecipitation, the lysates were incubated with rabbit anti-GFP antibody (Invitrogen) for 1 hr at 4°C. Protein A/G UltraLink Resin  was added and incubated overnight at 4°C. The beads were washed with lysis buffer. Proteins were eluted with 1x SDS sample buffer and analyzed by Western blotting.Secreted and processed Neto fragment (CUB1-V5/His) was purified using His-Trap affinity column equipped with AKTA FPLC system (Pharmacia) and separated by SDS-PAGE. A specific gel band was isolated and analyzed at Microchemistry and Proteomics Analysis Facility, Harvard University.2+ and processed as previously described [p-formaldehyde, 1% glutaraldehyde, 0.1 M sodium cacodylate, and 2 mM MgCl2, pH 7.2) for 20 min at room temperature followed by incubation overnight at 4°C, then washed extensively . The samples were processed and analyzed at the Microscopy and Imaging Core Facility, NICHD.Wandering third instar larvae were dissected in Jan's saline containing 0.1 mM Caescribed . Dissect2+ using a custom microscope stage system [p < 0.05.The standard larval body wall muscle preparation first developed by Jan and Jan 1976) was used for electrophysiological recordings ,72. Wande system . The nere system . To calce system . In addie system ,76. Corr was usede system ,77. DataS1 Figneto null mutants was measured as percentage of embryos hatching into larval stages (B) and adults (C). (D-E) The length and number of branch points of NMJs at various Neto levels, as illustrated in (A) Confocal images of third instar NMJ controls as indicated  immunostained against Neto (green), and HRP (red). Bars: 10 μm. (B-C) The ability of various transgenes to rescue (TIF)Click here for additional data file.S2 Figp<0.001, ***; p<0.05. Bars: 10 μm, 1 μm in details.(A-D) Confocal images of third instar NMJs  and bouton details labeled for HRP (in red) and PAK (A), Dlg (B), α-spectrin (C), or CSP (D) (in green). (E-F) Quantification of the number of synaptic clusters (E), and relative intensity (F) are indicated. Error bars indicate SEM. *; (TIF)Click here for additional data file.S3 Figneto null NMJs rescued with tagged neto transgenes  exhibited normal Dlg synaptic signals (B) but reduced NMJ size (C). (D-G) Muscle expression of Neto-GFP rescued the neurotransmission in neto mutants to control levels . The numbers of NMJs examined are indicated in each bar. Error bars indicate SEM. *; p<0.001. Bars: 10 μm, 1 μm in details.(A) Confocal images of third instar NMJ  immunostained against Dlg (green), and HRP (red). Compared with the control, and similar to NMJ with excess Neto, the (TIF)Click here for additional data file.S4 FigG14>fur1RNAi and 24B>fur1RNAi) or neurons (elav>fur1RNAi) induced mild reduction in the number of synaptic clusters but drastic decrease in the intensity of all synaptic signals examined (quantified in C-D). Knockdown of Fur1 also produced significant reduction of NMJ size (E). The numbers of NMJs examined were indicated in each bar. Error bars indicate SEM. *; p<0.001, **; p<0.01, ***; p<0.05. Bars: 10 μm.(A-B) Confocal images of third instar NMJ  immunostained against Brp (green) and GluRIIC (red) (A); or GluRIIA (green) and pMad (red) (B) and Neto (blue). Knockdown of Fur1 in either muscles ((TIF)Click here for additional data file.S5 FigG14>PM-neto-GFP-D2) third instar larvae.Large field from an electron micrograph of synaptic boutons from PM-Neto rescued ((TIF)Click here for additional data file.


HSF-1, and its downstream effectors played as common genetic modulators of sensitivity to addictive substances. Using Caenorhabditis elegans, an exemplary model organism with substance dose-dependent responses similar to mammals, we demonstrate that HSF-1 altered sensitivity to both alcohol and nicotine. Using a combination of a targeted RNAi screen of downstream factors and transgenic approaches we identified that these effects were contingent upon the constitutive neuronal expression of HSP-16.48, a small heat shock protein (HSP) homolog of human α-crystallin. Furthermore we demonstrated that the function of HSP-16.48 in drug sensitivity surprisingly was independent of chaperone activity during the heat shock stress response. Instead we identified a distinct domain within the N-terminal region of the HSP-16.48 protein that specified its function in comparison to related small HSPs. Our findings establish and characterize a novel genetic determinant underlying sensitivity to diverse addictive substances.Addiction to drugs is strongly determined by multiple genetic factors. Alcohol and nicotine produce distinct pharmacological effects within the nervous system through discrete molecular targets; yet, data from family and twin analyses support the existence of common genetic factors for addiction in general. The mechanisms underlying addiction, however, are poorly described and common genetic factors for alcohol and nicotine remain unidentified. We investigated the role that the heat shock transcription factor,  A receptors  on nicotine and alcohol dependence behaviors have identified potential contributing factors  family of cellular chaperones. Indeed acute exposure to stressful concentrations of ethanol in  of HSPs . Here weC. elegans strains were grown under standard conditions on nematode growth medium (NGM) agar plates at 20° with Escherichia coliOP50 as a food source as previously described (N2 (wild type), hsf-1 (sy441), hsp-3 (ok1083), hsp-16.1/hsp-16.48 (ok577), and hsp-16.11/hsp-16.49 (tm1221). To analyze potential effects of pat-10 overexpression, we used the AGD1101 strain (DM8005 strain containing a GFP-tagged myo-3 protein (NM306 strain containing a GFP-tagged snb-1 protein (Table S2. The transgenic strains used in this study were: hsf-1 (sy441);Ex[Phsf-1::hsf-1], hsf-1 (sy441);Ex[Prab-3::hsf-1], hsf-1 (sy441);Ex[Pmyo-3::hsf-1], hsf-1 (sy441);Ex[Pvha-6::hsf-1]; hsf-1 (sy441);Ex[Prab-3::hsp-16.48]; N2;hsf-1Ex[P::hsf-1]; N2;rab-3Ex[P::hsp-16.48], N2;rab-3Ex[P::hsp-16.48ΔN] (HSP-16.48AA54-143), N2;rab-3Ex[P::hsp-16.48ΔC] (HSP-16.48AA1-128), N2;rab-3Ex[P::hsp-16.48ΔNC] (HSP-16.48AA54-128), N2;rab-3Ex[P::hsp-16.48Δ38-44], N2;rab-3Ex[P::hsp-16.1 chimera] (a fusion construct of the N terminus of hsp-16.48 (HSP-16.48AA1-70) with the crystallin domain and C-terminus of hsp-16.1 (HSP-16.1AA67-145)), N2;rab-3Ex[P::hsp-16.48 E118N]; N2;hsf-1Ex[P::hsp-16.48], N2;hsf-1Ex[P::hsp-16.1], and N2;hsf-1Ex[P::hsp-1]. For study of HSP-16.48 expression, we generated and examined green fluorescent protein (GFP) expression in unc-18 (e81);[Phsp-16.48::unc-18GFP+P::unc-18]. protein . Transgerrf-3 (pk1426) strain, as it has been widely characterized and has enhanced efficiency in neurons  and 25 µg/ml carbenicillin. NGM plates were dried at least 4 days before seeding. To each RNAi plate, five L3–L4 worms were added and cultured at 20°. Phenotypic analysis was performed on first generation progeny  fed with the indicated individual RNAi bacterial clones at 20°. For each feeding clone, the locomotion rate of animals was quantified as thrashing in Dent’s solution (ethanol experiments) or body bends on unseeded NGM agar plates (nicotine experiments) as defined below in behavioral assays. Negative controls for the RNAi screen were feeding with the empty feeding vector and RNAi of an HSP unrelated to the heat shock stress response, the endoplasmic reticulum chaperone hsp-3. Positive controls for the RNAi ethanol screen were feeding with clones targeting rab-3 and slo-1 both of which would be predicted from mutant analysis to decrease ethanol sensitivity , which is described previously (myo-3P (pPD133.61) , tissue and cell type-specific promoters were amplified from Bristol N2 genomic DNA and cloned into pPD117.01 (kind gift of A. Fire) in place of mec-7P. The following primers were used for promoter cloning:With the exception of hsf-1P forward: ACTGGCGCGCCGGAATGCTATTTCGCAATCACGhsf-1P reverse: AGTCGGATCCTTTACGAACTAGCACGCGGTATCvha-6P forward: GTAAGGCGCGCCAAGTTGAACCATGTATGGGAACCGvha-6P reverse: AAAGGGATCCGGGTTTTGGTAGGTTTTAGTCGhsp-16.48P forward: GTAAGGCGCGCCGATTGTAGTTTGAAGATTTCACAATTAGAGTGhsp-16.48P reverse: GCATGGATCCTCTTGAAGTTTAGAGAATGAACAGTAAGC.N2 genomic DNA and cloned into pDONR201 and recombined into these DEST vectors to produce tissue-specific expression vectors. The following primers were used for gene cloning:These constructs were converted to Gateway DEST vectors with the use of a conversion cassette (Life Technologies). Genes of interest were amplified from hsf-1 attB forward: GGGGACAAGTTTGTACAAAAAAGCAGGCTTCAAAATGCAGCCAACAGGGAATCAAhsf-1 attB reverse: GGGGACCACTTTGTACAAGAAAGCTGGGTCTTAAACCAAATTAGGATCCGATGGhsp-16.48 attB forward: GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGCTCATGCTCCGTTCTCChsp-16.48 attB reverse: GGGGACCACTTTGTACAAGAAAGCTGGGTCAGATTAATGTTTTGCAACAAAATTAATGGGhsp-16.1 attB forward: GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTCACTTTACCACTATTTCCGTChsp-16.1 attB reverse: GGGGACCACTTTGTACAAGAAAGCTGGGTCTTATTCAGAAGTTTTTTGTTCAACGGGhsp-1 attB forward: GGGGACAAGTTTGTACAAAAAAGCAGGCTTCAAAATGAGTAAGCATAACGCTGTTGhsp-1 attB reverse: GGGGACCACTTTGTACAAGAAAGCTGGGTCAGATTAGTCGACCTCCTCGATCG.g at 4° for 15 min. Following two separations of the aqueous upper phase with chloroform, RNA was precipitated by dropwise addition of equal volume 70% ethanol made in nuclease-free water. RNA was then treated with RNAse-free DNAse I (Qiagen) and then purified using the RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions. RNA concentration and purity were quantified using a NanoDrop 1000 spectrophotometer . First-strand cDNA was synthesized using the ProtoScript First Strand cDNA Synthesis kit (New England Biolabs) using equal amounts of RNA and the random primer mix according to the manufacturer’s instructions. The cDNA was diluted (1:50) with diethylpyrocarbonate (DEPC)-treated water and stored at −80° until required. For gene expression analysis, master mixes for diluted cDNA samples mixed with DEPC water and selected qRT-PCR primers mixed with iTaq Universal SYBR Green Supermix (BioRad) were made and added to 96-well plates sequentially to make a final reaction volume of 10 μl per well. Reactions were performed using an IQ5 real-time PCR detection system (Bio-Rad). The thermocycler was programmed to first heat to 95° for 3 min, followed by 40 cycles of a 10-sec denaturation step at 95°, a 30-sec annealing step at 58°, and a 30-sec elongation step at 72°. qPCR reactions were carried out with three technical repeats of three individual biological replicates and transcript expression was analyzed using Bio-Rad CFX Manager 3.0 software using the comparative Ct method. Internal controls were the geometric mean of cdc-43, pmp-3, and Y45F10D.4, which are optimal internal controls for worm qCPR , and frozen at −80°. Following one freeze–thaw cycle, worm samples were disrupted and homogenized on a vortex shaker at 4° for 40 min. A further 200 μl of TRIzol was added and vortexed before addition of 140 μl of chloroform. Samples were then centrifuged at 12,000 × cdc-42 forward: GGTGGCGAGCCATACACATTAGGcdc-42 reverse: CTCTCCAACATCCGTTGACACTGGpmp-3 forward: TGGTGTCGCGATTACTGTAGpmp-3 reverse: GATTTGTTGTCGCAGAGTGGY45F10D.4 forward: CCTGCACAAGTTTGCGTTGCCY45F10D.4 reverse: CGAATCTGCAATTTCATGACATCTCCAChsp-16.48 forward: TCATGCTCCGTTCTCCATTTTCTGATTChsp-16.48 reverse: CTTCTTTGGAGCCTCAATTTGAAGTTTTCChsp-16.1 forward: TTTTGTTCAACGGGCGCTTGChsp-16.1 reverse: GAGGCTCTCCATCTGAATCTTCTGAGhsp-70 forward: GAAGGAGAACGTGCTATGACTCGhsp-70 reverse: CAGTGATCCATGTTCTTCGAGAGCpat-10 forward: GGCCACTCAGATCGGTCAAATCATGpat-10 reverse: GTTGTTGATCGGTGAGGTCATCGG.Fifty worms were picked into 12.5 μl of nuclease-free water, mixed with an equal volume of 2× Tris-glycine SDS sample buffer (Life Technologies), and frozen at −80°. Following one freeze–thaw cycle, worm samples were boiled, separated on Novex 4–12% Tris-glycine gels (Life Technologies), and transferred to nitrocellulose. Antibodies used for immunoblotting were: 1:1000 anti-β-actin (Sigma) and 1:1000 anti-GFP (Roche).Ethanol concentrations were measured following a previously published protocol . Ethanol was previously mixed with Dent’s at the indicated concentrations. For acute ethanol exposure, locomotion (as thrashes per minute) was quantified following a 10-min exposure and normalized as a percentage of the mean thrashing rate of untreated worms measured each day (at least 10 control worms per strain). For nicotine-sensitivity experiments, single adult hermaphrodites were removed from NGM plates and placed on a fresh unseeded NGM plate supplemented with the appropriate nicotine concentration. Locomotion (body bends per minute) was quantified following a 14-min exposure and normalized as a percentage of the mean body bend rate of untreated worms measured each day (at least 10 control worms per strain). For acute ethanol tolerance experiments, we also quantified locomotion as body bends per minute on unseeded NGM plates supplemented with the appropriate ethanol concentration at 10 and 30 min. Locomotion rate here was again normalized to the mean body bend rate of each untreated strain at the indicated times. For heat shock (HS) preconditioning, worms were transferred to seeded, uncrowded NGM plates; the plates were parafilmed and then placed in an incubator at 30° for 1 hr. The plates were then removed to 20° following preconditioning to recover for at least 1 hr before behavioral analysis. Thrashing and body bend rates were quantified within 4 hr post-HS preconditioning. For thermotolerance assays, worms (30 per strain per experiment) were transferred to seeded NGM plates; the plates were parafilmed and then placed in an incubator at 33° for ∼16–18 hr before scoring worms for lethality. All data are expressed as mean ± SE. Significance was tested by Student’s t-test, Mann-Whitney U-test, or analysis of variance (ANOVA) with Tukey post hoc comparisons as indicated.All experiments were performed on young adult hermaphrodite animals from sparsely populated plates grown at 20°. Behavioral experiments were performed in a temperature-controlled room at 20°. Locomotion rate was measured either as thrashing in solution (ethanol experiments) or as body bends on agar plates (ethanol and nicotine experiments) as previously described  that inhibits its transcriptional activity  and RNAi knockdown of hsf-1 demonstrate two well-established phenotypes of an increase in temperature sensitivity and a decrease in lifespan  and hsf-1sy441 worms (59.8 ± 8.1 mM) at levels comparable with previously published concentrations  background had no significant additional effect to ethanol sensitivity. Finally, like mammals, nematodes display acute tolerance to alcohol .In comparison to wild-type worms, comotion  that coupromoter . OverexpHSF-1 transcription factor including members of the HSP-70 and HSP-16 family of proteins . We next investigated whether a preconditioning HS would have the opposite effect on alcohol sensitivity as the hsf-1sy441 mutation. As predicted, HS preconditioned Bristol N2 worms showed a reduction in the depressive effects of alcohol over the tested range of concentrations, whereas the preconditioning HS actually enhanced the ethanol sensitivity for the hsf-1sy441 mutants (Exposure to heat shock (HS) classically upregulates the expression of many HSPs downstream of the  mutants .HSF-1 in acute sensitivity to nicotine. Wild-type C. elegans has an acute dose-dependent response to nicotine at concentrations similar to humans  background again had no significant additional effect to nicotine sensitivity. From these results we conclude that HSF-1 is a novel common modulator of substance sensitivity.Following these results, we tested for a role for pressant . As seennicotine . OverexpHSF-1 is a transcription factor expressed ubiquitously in all cell types. Therefore we wanted to determine the localization of HSF-1 required for rescue of both the acute alcohol and nicotine phenotypes by tissue-specific transgenic expression. Both transgenic rescue of hsf-1 under its endogenous promoter  or using a panneuronal promoter  rescued the alcohol phenotype equally in hsf-1sy441 mutants, whereas transgenic expression using a body-wall muscle promoter  was insufficient  was insufficient to rescue the ethanol phenotype. Similar results for the nicotine phenotype of hsf-1sy441 mutants was achieved by transgenic expression of hsf-1 under a panneuronal promoter , but also a body-wall muscle promoter  in comparison to rescue with the endogenous promoter  . From these results we conclude that neural expression of HSF-1 is sufficient for modulation of substance sensitivity.The alteration in locomotor speeds following ethanol or nicotine could reflect changes in the coordinated patterns of motor activity generated by the worms’ CNS or an effect on the strength of the end-point signal at the body wall neuromuscular junction. We and others have demonstrated that the effects of ethanol are independent of signaling strength and basal locomotion rates (fficient . To test:hsf-1]) . A requi elegans . SimilarHSF-1 functions as a transcription factor that regulates the expression of a host of downstream targets, in both the stressed and unstressed conditions. Control of HSP expression is an integral component of either condition. HSPs are ubiquitous proteins that function to block protein aggregration, assist in protein folding or refolding, facilitate protein degradation, and stabilize protein–protein interactions  (trp-2 and acr-15) . The RNAi screen was performed by feeding . RNAi of the additional positive controls, rab-3 and slo-1, reduced ethanol sensitivity as has been shown for loss-of-function mutants of these genes  or nicotine , specifically, they did not affect sensitivity to the other drug. We found that RNAi of only one gene target, hsp-16.48, was able to phenocopy hsf-1 RNAi for both alcohol and nicotine sensitivity. We therefore concentrated our efforts on HSP-16.48 acting downstream of HSF-1 in both phenotypes and repeated the RNAi experiments, statistically verifying that, in comparison to either the empty vector control or RNAi or hsp-3, RNAi of both hsf-1 and hsp-16.48 significantly reduced alcohol and nicotine sensitivity to an equivalent level , a ubiquitously expressed protein associated with cataract and a number of neurodegenerative diseases and cancer  and comparing with the hsf-1sy441 mutant (hsp-3 (ok1083) gene had no effect on either sensitivity to alcohol or nicotine. In comparison to Bristol N2 wild types, however, we found that the hsp-16.48ok577 mutant only phenocopied hsf-1sy441 for alcohol sensitivity . We also tested the mutant allele of hsp-16.49 and hsp-16.11 (tm1221), but found that this too was phenotypically similar to Bristol N2s. Given this genetic duplication, a lack of complete phenocopy is perhaps unsurprising. Although RNAi will knockdown both hsp-16.48 and hsp-16.49, the mutant allele only deletes one of two copies.1 mutant . Consistsitivity . We predhsp-16.48 was indeed acting downstream of hsf-1 in both alcohol and nicotine sensitivity, we tested whether transgenic overexpression of hsp-16.48 alone could alter sensitivity to both alcohol and nicotine. We found that neuronal expression of hsp-16.48 in Bristol N2 wild-type worms indeed had the opposite effect to the hsf-1sy441 phenotype , transgenic overexpression of closely homologous hsp-16.1 or an unrelated HSP-70 chaperone, hsp-1, did not phenocopy hsp-16.48 overexpression . Taken together, these results indicate that, although HSP-16.48 overexpression alone is not sufficient to compensate completely for a lack of functional HSF-1 in the assayed phenotypes, the HSF-1-dependent effects on drug sensitivity require HSP-16.48 and that HSP-16.48 expression alone is sufficient to alter both alcohol and nicotine sensitivity.To support further the hypothesis that henotype . We alsoHSF-1 transcription factor is activated, stimulating a large increase in expression of many genes. The classically defined genes that are overexpressed in response to temperature stress are the HSPs . Finally the extent of GFP expression in response to temperature stress, ethanol, or nicotine were quantified by Western blot, emphasizing the inability of ethanol or nicotine at these concentrations to activate the classical HS stress response . Second, we visualized basic aspects of muscle integrity in response to ethanol or nicotine exposure. Upon HS, muscle filaments become disorganized and this is thought to be a contributing factor to thermotolerance . Using a GFP-tagged synaptobrevin/VAMP marker for synapses, we could also find no evidence for overt effects of ethanol or nicotine on neuronal morphology . Finally, overexpression of pat-10 has been previously demonstrated to increase thermotolerance and lifespan of nematodes . Therefore, our preliminary evidence suggests that the pharmacological effects of these drugs at these experimental conditions are independent of pat-10.In addition to increasing HSP expression hundreds-fold, HS can also affect the expression of other genes. One such gene encodes the calcium binding protein hsp-16.1 and hsp-16.48 are expressed in most cells of C. elegans and their expression is upregulated in response to stress; yet, they appear to have completely separate functions. Given the similarity of protein structure, we were interested in identifying the section of the protein that determined this drug sensitivity phenotype of HSP-16.48. Structurally, all small HSPs comprise a central crystallin domain with N- and C-terminal domains of variable length . The distinction in phenotype between hsp-16.1 and hsp-16.48 indicates that the inclusion of a crystallin domain does not necessarily predetermine protein function. We first constructed and analyzed the phenotypic effects of a number of truncation mutations  of HSP-16.48  was truncated (hsp-16.48 ΔC+) only eliminated the nicotine sensitivity. Closer examination of the N-terminal sequences of HSP-16.1 and HSP-16.48 identified a short stretch of seven amino acids that distinguished the HSP-16.48 sequence . Neuronal expression of a mutant protein with only these seven amino acids removed (hsp-16.48 Δ38-44+) was phenotypically indistinguishable to the full N-terminal deletion, showing a complete abolition of the effects of HSP-16.48 overexpression on alcohol and nicotine sensitivity  into HSP-16.48 by switching only their N-terminal regions (hsp-16.1 chimera+). Consistent with the C-terminal deletion results, expression of the HSP-16.1 chimera only affected the ethanol sensitivity effects  altered ethanol and nicotine sensitivity to equivalent levels as overexpression of HSP-16.48  and transgenics (N2 + hsp-16.48ΔC) have altered ethanol responses, but no difference in nicotine sensitivity. These data clearly demonstrate that the pharmacological effects of nicotine and ethanol can be specific, depending on the genetic background. Finally, we show that the small heat shock protein hsp-16.48 acts downstream of hsf-1 in ethanol and nicotine; yet, is independent of impairment due to temperature stress. The closely related hsp-16.1 conversely does not act downstream of hsf-1 in ethanol and nicotine, but does act downstream of hsf-1 in temperature-dependent neurodegeneration , sequence analysis indicates that this mutant should be extremely similar to HSP-16.1 which does act as a chaperone during temperature-dependent neurodegeneration  of the protein’s N terminus. Potential binding partners could be those proteins previously identified in genetic experiments of addiction using model organisms such as mice, Drosophila, and C. elegans (HSP-16.48 are exacted through cytoskeletal interactions. As there have been no descriptions for specific binding partners for HSP-16.48, these hypotheses would require substantial further investigation.Small HSPs are thought to function solely as molecular chaperones, stabilizing proteins and preventing irreversible aggregation (hsp-16.48 can dramatically alter ethanol and nicotine sensitivity in the wild type; yet, its overexpression in the hsf-1sy441 genetic background is statistically beneficial but more limited. This is perhaps not surprising as small HSPs are well known to act in cooperation with other ATP-dependent chaperones, such as the HSP70s, that are downstream of hsf-1 (hsp-70 could phenocopy hsf-1 in ethanol sensitivity, but not nicotine sensitivity. In contrast, RNAi of an alternative HSP70, F11F1.1, phenocopied hsf-1 in nicotine sensitivity only. It is possible that HSP-16.48 is a common factor in drug sensitivity, but associates with divergent HSP70 isoforms dependent upon environmental circumstances. Nevertheless the RNAi and overexpression data demonstrate that hsp-16.48 is both necessary and sufficient for drug sensitivity phenotypes and indicate that, in a wild-type background at least, hsp-16.48 expression level is the limiting factor.Overexpression of hsf-1sy441 phenotype in drug sensitivity, which was surprisingly the opposite to the effect of HS in wild-type worms. This effect may be a consequence of the HS protocol itself damaging the temperature-sensitive hsf-1sy441 strain, as HS did negatively affect endogenous rates of locomotion. Alternatively, this finding may imply an HSF-1-independent effect of HS acting detrimentally in the organism that becomes apparent only in the hsf-1sy441loss-of-function  strain. If the second hypothesis was true, then the HSF-1-dependent effects of HS in the wild-type worm would be greater than that estimated here.The preconditioning HS protocol was found to enhance the effect of the hsf-1 overexpression could increase thermotolerance and lifespan of C. elegans through an upregulation in pat-10 and a subsequent protection of the cytoskeleton (hsf-1-dependent drug sensitivity phenotypes appear to be of a different origin for the following reasons. First, overexpression of hsf-1 does not alter basal sensitivity to either ethanol or nicotine. Second, neither drug upregulates the expression of pat-10, whereas temperature stress does. Third, overexpression of pat-10 does not alter ethanol sensitivity, whereas it does protect against temperature stress. Finally, pat-10 acts to protect the cytoskeleton in the face of temperature stress and our evidence, at least qualitatively, does not point to an acute effect of ethanol or nicotine on the cytoskeleton.A recent report indicated that There is considerable evidence for the conservation of the genetic basis of addiction, from invertebrate models to mammals, including humans. For example the large conductance calcium-activated potassium channel, the voltage-insensitive cation leak channel, and the exocytotic proteins Rab-3 and Munc18 have effects on both ethanol sensitivity in nematodes and have altered complex alcohol phenotypes in mice (HSF-1-dependent transcriptional program that is independent of stress (HSF-1 transcriptional activity and downstream HSP effectors within the nervous system specifically can also regulate addiction independent of stress. HSF-1 knockouts in mice show abnormal affective behavior, including increased susceptibility to depression-like behavior and aggression (HSF-1 and HSPs are more intrinsic than as simple stress response proteins. Our research expands upon these functions, potentially implicating HSF-1 and small HSPs as a central intracellular hub linking genetic predisposition to multiple, complex neurological disorders, including susceptibility to addiction. These findings could then facilitate new avenues for pharmacological intervention to addiction in general.Genetic risk factors for diverse complex disorders are increasingly found to be linked, such as addiction with cancer (