Ribonuclease L (RNase L) is a metal-ion–independent endoribonuclease associated with antiviral and antibacterial defense, cancer and lifespan. Despite the biological significance of RNase L, the RNAs cleaved by this enzyme are poorly defined. In this study, we used deep sequencing methods to reveal the frequency and location of RNase L cleavage sites within host and viral RNAs. To make cDNA libraries, we exploited the 2′, 3′-cyclic phosphate at the end of RNA fragments produced by RNase L and other metal-ion–independent endoribonucleases. We optimized and validated 2′, 3′-cyclic phosphate cDNA synthesis and Illumina sequencing methods using viral RNAs cleaved with purified RNase L, viral RNAs cleaved with purified RNase A and RNA from uninfected and poliovirus-infected HeLa cells. Using these methods, we identified (i) discrete regions of hepatitis C virus and poliovirus RNA genomes that were profoundly susceptible to RNase L and other single-strand specific endoribonucleases, (ii) RNase L-dependent and RNase L-independent cleavage sites within ribosomal RNAs (rRNAs) and (iii) 2′, 3′-cyclic phosphates at the ends of 5S rRNA and U6 snRNA. Monitoring the frequency and location of metal-ion–independent endoribonuclease cleavage sites within host and viral RNAs reveals, in part, how these enzymes contribute to health and disease. Endoribonucleases include metal-ion–dependent enzymes and metal-ion–independent enzymes . Metal-ix5’A(2’p5’A)n; x = 1-3; n ≥ 2] (Ribonuclease L (RNase L) is a well-studied endoribonuclease associated with lifespan , cancer ; n ≥ 2] ,35, the ; n ≥ 2] . Charact; n ≥ 2]  and RNas; n ≥ 2]  cleavageHepatitis C virus (HCV), which is restricted by the 2-5A/RNase L pathway in tissue culture cells , is ableArabidopsis thaliana tRNA ligase, an enzyme that specifically recognizes RNA fragments with 2′, 3′-cyclic phosphate termini  (PV (Type 1 Mahoney) and HCV (genotype 1a) RNAs were used in this study. A plasmid encoding PV cDNA  were purified as previously described , RNase A (0.5 nM), or RNase L (25 nM), and 2-5A (25 nM). These reaction conditions were optimized empirically to give limited and increasing amounts of viral RNA cleavage over time. RNase A, even at relatively low concentrations, cleaved the viral RNAs readily . Higher concentrations of RNase L (25 nM) were needed for optimal enzymatic activity, due in part to the required dimerization of RNase L following 2-5A binding  or a dominant-negative R667A mutant form of RNase L (M25 HeLa cells) were grown as previously described  in Dulbe6 cells per 35 mm well). Following 1 h of virus adsorption at room temperature, the inoculum was removed and replaced with 2 ml of DMEM containing fetal bovine serum, pen-strep and G418. Mock infections were performed using phosphate buffered saline without virus. Mock-infected and PV-infected cells were incubated at 37°C. At the indicated times , RNA was isolated from the cells using guanidine thiocyanate disruption , P:C:I extraction and ethanol precipitation. Host and viral RNAs in each sample were separated by agarose gel electrophoresis and visualized using ethidium bromide and UV light. PV was isolated from a parallel set of cells and quantified by plaque assays to monitor the magnitude and kinetics of virus replication (Cells were infected with 10 plaque forming units (PFU) per cell of PV diluted in phosphate buffered saline  were purified as previously described , A. thaliana tRNA ligase (0.75 µM), Riboguard (Epicenter) at 1U per µl and RNA from in vitro reactions  or RNA from cells . Saturating RNA linker concentrations (20 µM), in excess to RNA fragments with 2′, 3′-cyclic phosphates, maximized ligation efficiencies (data not shown). After incubation at 37°C for 1 h, RNAs were purified using P:C:I extraction and ethanol precipitation. GlycoBlue (Ambion) was included in all ethanol precipitation steps associated with the preparation of cDNA libraries. The RNAs were fragmented for 10 min at 70°C using 1X Fragmentation Reagents (Ambion), followed by an ethanol precipitation. Because a 2′-phosphate persists at the junction between RNA fragments and RNA linkers, fragmented RNAs were dephosphorylated at 30°C for 1 h in reactions (20 µl of volume) containing Tpt1 buffer , NAD+ (10 mM) and Tpt1 (4.5 µM). These RNAs were P:C:I extracted and ethanol precipitated.escribed . An RNA Following the ligation and Tpt1 reactions, RNAs were resuspended in denaturing sample buffer , heat denatured (10 min at 65°C) and fractioned on a 6% urea-polyacrylamide gel. RNAs in the gel were visualized using SYBR-Gold (Invitrogen) and blue light transillumination. RNAs 100–500 nt in length were excised from the gel, crushed into a slurry with 0.3 M sodium acetate and incubated at 42°C for 2 h. RNA was purified from polyacrylamide by running samples through DTR cartridges (EdgeBio) followed by ethanol precipitation.Superscript III (Invitrogen) and a DNA primer (5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT C-3′) were incubated with the gel-purified RNAs to make cDNA (per the manufacturer’s instructions). Exonuclease I (0.5 U) and recombinant shrimp alkaline phosphatase (0.25 U) (USB/Affymetrix) were added to each RT reaction, followed by additional incubation at 37°C for 30 min, to remove excess DNA primer and dNTPs. RT reactions were then heated to 95°C to inactivate the enzymes. RNAs were hydrolyzed using 1 N NaOH treatment at 100°C for 10 min and neutralized with 1 N HCl. cDNA was P:C:I extracted and ethanol precipitated.2 and 1 mM DTT). Linked cDNAs were P:C:I extracted and ethanol precipitated. Linked cDNAs were resuspended in formamide sample buffer and fractioned on a 6% urea-polyacrylamide gel. cDNAs >150 bases long were gel purified and resuspended in 30 µl of water.A DNA linker was attached to the 3′-end of the cDNA in reactions (10 µl of volume) containing 1 µM miRNA cloning linker 1 (5′-rApp-CTG TAG GCA CCA TCA AT-ddC-3′ [Integrated DNA Technologies]), 10 units of T4 RNA ligase I (New England Biolabs) and T4 RNA ligase buffer  was used to amplify cDNA libraries before Illumina sequencing. PCR reactions (50 µl) contained 1 U Phusion High Fidelity DNA polymerase (New England Biolabs), HF or GC Phusion buffer, 0.4 mM dNTPs, 5–8 µl of cDNA template, 0.8 µM forward indexed primer (5′-CAA GCA GAA GAC GGC ATA CGA GAT XXX XXX GTG ACT GGA GTT CAT TGA TGG TGC CTA CAG-3′ where XXX XXX = a distinct 6-base index sequence used to distinguish one cDNA library from another when mixing multiple libraries into one Illumina sequencing run), 0.8 µM reverse primer (5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC T-3′) and 1.0–4.6% DMSO. Thermal cycling conditions were an initial 98°C for 30 s, 28–30 cycles of , a final extension at 72°C for 10 min and a 4°C hold.PCR products 250–1000 bp in length were gel purified from a 6% nondenaturing polyacrylamide gel and resuspended in 10 mM Tris–HCl [pH 8.5] with 0.1% Tween-20. The amounts of DNA in each library were determined using the Qubit fluorometer (Invitrogen). Five to seven DNA libraries were combined for multiplexed sequencing for a combined total of 10 nM of DNA for each Illumina sequencing run. A multiplexing index read primer (5′-CTG TAG GCA CCA TCA ATG AAC TCC AGT CAC-3′) was used to identify the library after sequencing on the MiSeq or GAIIx .Illumina sequence data were trimmed of residual linker sequences and then aligned to the following sequences using Bowtie  with a 5′-end of each cDNA read was plotted against the nucleotide position of each genome using R  and are accessible through GEO Series accession number GSE52489 .RNase L, RNase A and other metal-ion–independent endoribonucleases target single-stranded regions of RNA, leaving 2′, 3′-cyclic phosphates at the end of RNA fragments . We explInitially, we used viral RNAs and purified endoribonucleases to optimize and validate the 2′, 3′-cyclic phosphate cDNA synthesis and Illumina sequencing methods. HCV and PV RNAs were incubated for 0–20 min in reactions containing RNase L and 2-5A or RNase A, followed by agarose gel electrophoresis to characterize the RNA fragments . RNase LSupplemantary Tables S1 and S2 and Supplementary Figures S2–S5). RNase L and RNase A cleavage sites in the viral RNAs were extremely reproducible across RNA samples from independent time points .The viral RNAs from each time point shown in Supplementary Figures S4 and S5). A peak of cleavage was evident in the E2 gene of HCV RNA, with especially large amounts of RNase L cleavage at UA1755, UA4422 and UU4465 and especially large amounts of RNase A cleavage at UC1671, UC1682 and CU1754 . As highlighted in Supplementary Figure S5, UA961 was the most frequent RNase L cleavage site within region 1 (1108 UMI reads after 5 min of cleavage), UA1803 was the most frequent RNase L cleavage site within region 2 (4223 UMI reads after 5 min of cleavage) and UU3102 was the most frequent RNase L cleavage site within region 3 (5347 UMI reads after 5 min of cleavage). Intriguingly, these regions of PV RNA encode portions of the capsid proteins recognized by neutralizing antibodies .RNase L cleaved HCV and PV RNAs predominantly at UpN dinucleotides (UA and UU > UG), with prominent amounts of cleavage at distinct locations in the viral RNAs . Pyrimidd CU1754 A. Three Supplementary Figures S2 and S4 for HCV RNA/Supplementary Figures S3 and S5 for PV RNA). This is most clearly presented in Supplementary Figure S4, focusing on four prominent RNase L cleavage sites in HCV RNA. The number of UMI-corrected cDNA reads at UA1755, UA4422, UU4465 and UU4466 increased coordinate with the time of incubation with RNase L . Note that UU4465 and UU4466 are adjacent cleavage sites, where cleavage at UU4465 would preclude detectable cDNA reads at UU4466. These data suggest that viral RNAs were partially digested, even after 20 min of incubation with RNase L, and that prominent cleavage sites detected at early times did not disappear after more prolonged periods of incubation.Consistent with increasing amounts of viral RNA cleavage over time, the number of UMI-corrected cDNA reads at prominent cleavage sites in HCV and PV RNAs increased coordinate with the time of incubation with RNase L . cDNA reads corresponding to PV RNA increased from undetectable levels at early times after infection to 5% of the cDNA at 6 hpa in W12 HeLa cells and 26.1% of the cDNA at 6 hpa in M25 HeLa cells (Supplementary Table S3). Abundant amounts of cDNA in each sample aligned to rRNAs (28S/18S/5.8S/5S rRNAs) and U6 snRNA (Supplemantary Table S3). cDNA reads corresponding to host mRNAs and other nonribosomal RNAs were present in each sample, ranging from 2.6% of all cDNA from PV-infected W12 HeLa cells at 2 hpa to 16.3% of cDNA in mock-infected W12 HeLa cells at 8 hpa.cDNA libraries were prepared and sequenced using the RNAs from HeLa cells  and in vivo . Surprisingly, there were no other obvious RNase L-dependent cleavage sites in PV RNA isolated from W12 HeLa cells. The increased frequency of UA and UU dinucleotides in cDNAs from W12 HeLa cells at 6 and 8 hpa was consistent with the activation of RNase L at these times . 28S rRNA was cleaved in an RNase L-independent fashion at specific sites, most frequently at AG409, GU432, CA1699, UG2055, GC2083, GA2093, GU2097, AA2396, UG2427, AU4512 and UA4729 . There were no obvious RNase L-dependent cleavage sites in 28S rRNA under the conditions of this experiment. Interestingly, expansion segments of 28S rRNA were largely resistant to cleavage . The most common cleavage sites identified in 18S rRNA include CG130, AG225, CC502, 541UU, 743UU, AC1403, CC1419, GU1432, UC1433, GU1462, UU1463, AU1477, UA1506, AG1507 and UU1721 (Supplementary Table S5). UU541 and UU743 were clearly RNase L-dependent cleavage sites based on their increased frequency in PV-infected W12 HeLa cells at 6 and 8 hpa and the low frequency of cleavage at these sites in RNA isolated from uninfected and PV-infected M25 HeLa cells (Supplementary Table S5).18S rRNA was cleaved reproducibly at specific sites, including two RNase L-dependent cleavage sites , which had relatively few 2′, 3′-cyclic phosphates relative to other rRNAs (Supplementary Table S3). The most common cleavage sites identified in 5.8S rRNA include cleavage at A80, A108, C109, U110, U111, C113, A137, U142 and U156 (Supplementary Table S6). Notably, 2′, 3′-cyclic phosphates were frequently detected at the 3′-end of mature 5.8S rRNA .Reproducible distribution of RNase L-independent cleavage sites was exhibited by 5.8S rRNA , was exhibited by 5S rRNA, which consistently had large amounts of 2′, 3′-cyclic phosphate-dependent cDNA relative to other RNAs from HeLa cells (Supplementary Table S3).A striking distribution of RNase L-independent cleavage sites, with the majority of signal at the 3′-end of mature 5S rRNA , as well as the tertiary structure of 80S ribosomes (The location of these RNase L-dependent and RNase L-independent endoribonuclease cleavage sites were mapped onto the secondary structures of rRNAs (ibosomes  27). RN. RNSupplibosomes A and B. ibosomes C. RNase ibosomes D. Cleavaibosomes D.FigureSupplementary Table S3). The U6 snRNA cDNA reads in our libraries align almost exclusively to the 3′-end of mature U6 snRNA . U6 snRNA, an RNA polymerase III (Pol III) transcript, is posttranscriptionally modified by a nuclease encoded by gene C16orf57, which leaves 2′, 3′-cyclic phosphates at the end of mature U6 snRNAs . Overall, these methods reliably and reproducibly reveal the location and relative frequency of 2′, 3′-cyclic phosphates in host and viral RNAs. These cDNA synthesis and Illumina sequencing methods did not detect RNA fragments corresponding to RNase P or RNase MRP activity, consistent with the specificity of the method for RNA fragments with 2′, 3′-cyclic phosphates.RNase L, like other metal-ion–independent endonucleases, is expected to produce RNA fragments with 2′, 3′-cyclic phosphate termini . Our datSupplementary Figure S15), a cellular enzyme known to leave cyclic phosphates at the end of mature U6 snRNA in cells . Thus, a metal binding pocket at this site in 28S rRNA, under physiologic conditions within cells, may lead to rRNA cleavage. Carefully controlled experimental variables are needed to attribute specific cleavage sites to particular endonucleases or to metal binding pockets.Several lines of evidence suggest that cyclic phosphates within host and viral RNAs were due to metal-ion–independent endonucleolytic cleavage of RNA within cells rather than cleavage during RNA processing or within linker reactions. Cyclic phosphates were not frequent in viral RNA transcripts isolated from control reactions lacking endonucleases, confirming the absence of endonuclease activity within RNA linker reactions or other steps of RNA processing. The integrity of host and viral RNAs purified from cells was confirmed by agarose gel electrophoresis B, host ain cells . Nonethein cells . Thus, ireported . This isin vitro . We entertained two hypotheses as we considered these data. First, PV RNA may assume a secondary and tertiary structure that renders some portions of the genome particularly susceptible to single-strand–specific endoribonucleases while rendering other regions resistant . Second, antigenic variation in the capsid proteins, as a consequence of antibody selection, could render portions of the capsid genes more sensitive to cleavage by single-strand–specific endoribonucleases, due to the counter-selective forces of antigenic variation and resistance to endoribonucleases . The predicted secondary structures of HCV and PV RNAs do explain much of the data . In addition, atomic force microscopy of HCV and PV RNA genomes reveals globular structures, where some regions of the viral RNA genomes might be more exposed to endoribonucleases than others . A complete understanding of the discrete distribution of endoribonuclease cleavage sites in PV RNA awaits further investigation.Endoribonuclease cleavage sites across PV RNA genomes have not been previously described. The striking distribution of RNase L and RNase A cleavage sites in PV RNA B, with pin vitro B and in . Unlike many studies, which actively exclude rRNAs from deep sequencing experiments to enrich for other RNA populations, we intentionally retained rRNAs in our study to identify RNase L-dependent and RNase L-independent cleavage sites in rRNAs. Although we did not detect RNase L-dependent cleavage sites in 28S rRNA under the conditions of our experiment, others have reported RNase L-dependent cleavage of 28S rRNA . Each of the sites mapped to single-stranded regions of rRNA , and most were located at the surface of 40S and 60S ribosomal subunits  and to the reduced frequency of Illumina cDNA reads in GC-rich regions of RNA  are distant from the mRNA decoding channel , is analogous to an oxidative stress-induced rRNA cleavage site identified in yeast , and almost all of the 5S rRNA signal was associated with the 3′-end of mature 5S rRNA . The 3′-ends of 5.8S and 5S rRNAs are on the surface of 60S ribosomal subunits .U6 snRNA is a Pol III transcript modified posttranscriptionally by an exonuclease that leaves 2′, 3′-cyclic phosphates at the end of mature U6 snRNAs . The abuWe used 2′, 3′-cyclic phosphate cDNA synthesis and Illumina sequencing to identify and quantify metal-ion–independent endoribonuclease cleavage sites in host and viral RNAs. With these methods, we identified discrete regions of HCV and PV RNA genomes that were profoundly susceptible to RNase L and other single-strand specific endoribonucleases, RNase L-dependent and RNase L-independent cleavage sites within rRNAs, and 2′, 3′-cyclic phosphates at the ends of 5S rRNA and U6 snRNA. We expect these methods will prove useful in the characterization of other metal-ion–independent endoribonucleases, like the NendoU of arteriviruses and coronaviruses whose RNA substrates remain unknown , and theSupplementary Data are available at NAR Online.Mucosal and Vaccine Research Colorado (MAVRC), Golfers Against Cancer, the University of Colorado Cancer Center, the March of Dimes [Basil O’Connor Starter Scholar Award5-FY10-478 to J.R.H.]; a Damon Runyon-Rachleff Innovation Award [DRR-17-12 to J.R.H.]; the American Cancer Society [RSG-13-216-01-DMC to J.R.H.]; and the National Institutes of Health (NIH) [CA044059 to R.H.S. and AI042189 to D.J.B.]. Funding for open access charge: NIH.Conflict of interest statement. None declared.


Kv7.2/Kv7.3 dysfunction resulting from KCNQ2 mutations predominantly causes self-limited or benign epilepsy in neonates, but also causes early onset epileptic encephalopathy. Retigabine (RTG), a Kv7.2/ Kv7.3-channel opener, seems to be a rational antiepileptic drug for epilepsies caused by KCNQ2 mutations. We therefore evaluated the effects of RTG on seizures in two strains of knock-in mice harboring different Kcnq2 mutations, in comparison to the effects of phenobarbital (PB), which is the first-line antiepileptic drug for seizures in neonates. The subjects were heterozygous knock-in mice (Kcnq2Y284C/+ and Kcnq2A306T/+) bearing the Y284C or A306T Kcnq2 mutation, respectively, and their wild-type (WT) littermates, at 63–100 days of age. Seizures induced by intraperitoneal injection of kainic acid  were recorded using a video-electroencephalography (EEG) monitoring system. Effects of RTG on KA-induced seizures of both strains of knock-in mice were assessed using seizure scores from a modified Racine’s scale and compared with those of PB. The number and total duration of spike bursts on EEG and behaviors monitored by video recording were also used to evaluate the effects of RTG and PB. Both Kcnq2Y284C/+ and Kcnq2A306T/+ mice showed significantly more KA-induced seizures than WT mice. RTG significantly attenuated KA-induced seizure activities in both Kcnq2Y284C/+ and Kcnq2A306T/+ mice, and more markedly than PB. This is the first reported evidence of RTG ameliorating KA-induced seizures in knock-in mice bearing mutations of Kcnq2, with more marked effects than those observed with PB. RTG or other Kv7.2-channel openers may be considered as first-line antiepileptic treatments for epilepsies resulting from KCNQ2 mutations.The hetero-tetrameric voltage-gated potassium channel K Kv7.2/Kv7.3 is predominantly expressed in the hippocampus, neocortex, and the granular layer of the cerebellum [v7.2/Kv7.3 thus plays an important role in limiting network excitability in the neonatal brain, where GABAergic action is depolarizing and excitatory [Krebellum –4 and gecitatory .KCNQ2 and KCNQ3 are known to cause predominantly benign familial or non-familial neonatal epilepsy (BFNE or BNE) [KCNQ2 mutations also cause early onset epileptic encephalopathies (EOEEs) or early infantile epileptic encephalopathies (EIEE), such as Ohtahara syndrome [v7.2/Kv7.3 is thus urgently needed.Mutations in  or BNE) ,7, both syndrome –12, whicsyndrome ,12. The v7.2/Kv7.3 opener, increases open channel probability and leads to hyperpolarization of the membrane potential. Hence, RTG may stabilize the resting membrane potential and suppress repetitive firing caused by KCNQ2 mutations. Several in vitro electrophysiological studies on reconstituted Kv7.2/Kv7.3 have suggested that seizures caused by KCNQ2 mutations might respond to RTG [Kcnq2 mutations, or compared these effects with the effects of phenobarbital (PB). At present, PB is the first-line and most commonly used anti-epileptic drug (AED) for neonatal seizures, including BFNE or EOEEs. Therefore, we here used knock-in mice bearing mutations in Kcnq2, the mouse orthologue of KCNQ2, to compare the effects of RTG on drug-induced seizures in the animals with those of PB.Retigabine (RTG), a Kd to RTG . HoweverKcnq2Y284C/+ and Kcnq2A306T/+, which harbor heterozygous Y284C or A306T Kcnq2 mutations, respectively, were produced using the “kick-in” system as described elsewhere [Kcnq2Y284C/+ and Kcnq2A306T/+ mice used in this study were congenic strains produced by more than 10 repeated backcross to C57BL/6J strain mice. Heterozygous mutations of Y284C and A306T have previously been identified in KCNQ2 in individuals with BFNE [Kcnq2Y284C/+ and Kcnq2A306T/+ mice to strengthen the results to evaluate potential differences, if any, between the two mutants.Two strains of heterozygous knock-in mice, lsewhere . Kcnq2Y2ith BFNE . Both Y2lox technology [Kcnq2Y284C/+ and Kcnq2A306T/+ mice have been found to exhibit occasional spontaneous seizures. In contrast to these sparse spontaneous seizures, seizures can be more readily induced by proconvulsants, such as pentylenetetrazol, in both Kcnq2Y284C/+ and Kcnq2A306T/+ mice compared to their wild-type (WT) littermates [The “kick-in” system that we previously developed is a modified knock-in procedure making use of Cre/mutant chnology . The systermates .Kcnq2Y284C/+ mice, n = 40; Kcnq2A306T/+ mice, n = 33). To conduct all experiments blindly, genotyping to distinguish knock-in mice from their WT littermates was performed after each experiment, according to methods described elsewhere [Mice were housed at 23 ± 2°C with 12 h light−12 h dark cycle (light on 7:00 to 19:00) and were given free access to commercial chow and tap water. Mice used for all experiments were 63–100 days of age  injection. The cause of death is unknown, but may have involved asphyxia or asystole. After the experiments, the mice were euthanized using sodium pentobarbital to minimize suffering.The experimental protocols were approved by the Committee for Animal Care and Use of Fukuoka University  and all experiments were conducted in compliance with the animal experimentation guidelines “Basic policies on animal experimentation" issued by the Ministry of Education, Sports, Science, and Technology (MEXT) and the Ministry of Health, Labor, and Welfare (MHLW) in Japan. All surgery was performed under sodium pentobarbital anesthesia, and efforts were made to minimize stress to the animals. Because all drug-induced seizures were monitored using a video-electroencephalography (EEG) monitoring system, we were able to observe in real time to determine whether mice were overstressed during seizures. During the course of the study, one v7.2/Kv7.3 channel transmembrane domain was remodeled from a PDB file provided by Prof. Bernard Attali  [V2.1 paddle (PDB entry 2R9R). Tetrameric reconstruction and energy minimization were calculated using Molecular Operating Environment (MOE), 2013.08 .The stereoscopic protein structure model of the human K Israel) , which iEEGs of mice were recorded with video monitoring as previously described . In brieKcnq2Y284C/+ and Kcnq2A306T/+ mice and their WT littermates were compared.A spike burst is a cluster of high-amplitude and high frequency spikes, each of which lasts a few seconds to several minutes on EEG during KA challenge tests  Fig 1C)Fig 1C). To induce seizures, mice received 12 mg/kg body weight KA  intraperitoneally 30 min after they had acclimated, as previously reported . KA stocThe seizures and behaviors were recorded continuously for more than 120 min with EEGs and video monitoring after drug injection. Seizures were scored based on behavior and EEG findings using a modified Racine’s scale  Table 1Table 1 . Spikes Kcnq2Y284C/+ and Kcnq2A306T/+ mice. Regardless of the dose, the injection volume was adjusted to 10 ml/kg bodyweight. We evaluated seizure scores and EEG findings as described above.RTG dihydrochloride  and PB sodium salt  were dissolved in physiological saline. All drugs were administered intraperitoneally 30 min before KA injection. Two doses of each AED (5 mg/kg and 15 mg/kg) were given for Kcnq2 mutations with the seizure score, which is an ordinal measure, was examined using an exact test for ordered differences [Kcnq2Y284C/+, and Kcnq2A306T/+ mice or among mice administered vehicle, PB, or RTG were examined using the Jonckheere−Terpstra test [Statistical data analyses were performed using the SAS  Software Package  at Fukuoka University. The association of heterozygous ferences . Differetra test  and the tra test . The numtra test . Additiotra test . Negativtra test ,23. The tra test ,23. The Kcnq2Y284C/+ and Kcnq2A306T/+ mice showed significantly more KA-induced seizures, with a higher seizure score, than WT mice , as assessed by the logistic regression analysis. These results indicate that Kcnq2Y284C/+ and Kcnq2A306T/+ mice have significantly higher sensitivity to KA-induced seizures than WT mice, and Kcnq2Y284C/+ mice have higher sensitivity to KA-induced seizures than Kcnq2A306T/+ mice.Both  WT mice . In WT m WT mice . The difKcnq2Y284C/+ mice exhibited frequent and prolonged spike bursts during KA challenges . Furthermore, Kcnq2Y284C/+ mice had significantly more and longer spike bursts than Kcnq2A306T/+ mice , after adjusting for dose by the logistic regression analysis.Administration of PB or RTG (5 mg/kg and 15 mg/kg) prior to KA challenges reduced the incidence of KA-induced seizures in the C/+ mice . The trep = 0.0041, 15 mg/kg PB vs. vehicle), although the effect of 5 mg/kg of PB was not statistically significant , as assessed by the Jonckheere−Terpstra test. There was no significant difference between the effects of 5 mg/kg and 15 mg/kg PB in Kcnq2Y284C/+ mice , as assessed by the Jonckheere−Terpstra test. There was no significant difference in the preventative effects between the two doses of RTG , as assessed by using Negative binomial model. There was no significant difference between these two doses of PB in terms of the number of spike bursts. RTG, in contrast, reduced the number of spike bursts significantly at both 5 mg/kg and 15 mg/kg doses . The effects of RTG on the number of spike bursts were significantly better than those of PB in Kcnq2Y284C/+ after adjusting for dose (p = 0.0026).Both PB and RTG reduced the number of spike bursts . PB at ap = 0.0851, 5 mg/kg PB vs. vehicle, p = 0.0011, 5 mg/kg RTG vs. vehicle; and p = 0.0003, 15 mg/kg RTG vs. vehicle, p = 0.5815, 15 mg/kg vs. 5 mg/kg RTG). The effects of RTG on shortening the total duration of spike bursts were significantly better than those of PB after adjusting for dose (p < 0.0001).In accordance with the effects on the number of spike bursts, PB and RTG were effective in shortening the total duration of spike bursts, and the effects of RTG were more marked than that of PB . PB, at Kcnq2A306T/+ mice , as assessed by the Jonckheere−Terpstra test. Furthermore, RTG had better preventive effects on KA-induced seizures than PB in Kcnq2A306T/+ mice, although the difference did not reach statistical significance (p = 0.1857), after adjusting for dose by the logistic regression analysis.The effects of PB and RTG were also evaluated and compared in heterozygous T/+ mice . Adminis6T/+mice . The treKcnq2A306T/+ mice , but the effect of RTG was not significantly dose-dependent , as assessed by using Negative binomial model. The effects of RTG in terms of the number of spike bursts were significantly better than those of PB in Kcnq2A306T/+ mice after adjusting for dose (p = 0.0603).RTG demonstrated the ability to significantly reduce the number of spike bursts during KA challenges at both 5 mg/kg and 15 mg/kg doses .In accordance with the effects on the number of spike bursts, PB and RTG were effective in shortening the total duration of spike bursts, and the effect of RTG was more marked than that of PB . The effKcnq2Y284C/+ and Kcnq2A306T/+ heterozygous mutant mice showed significantly higher sensitivity to KA-induced seizures than WT mice. Accordingly, both mutant mice exhibited more frequent and prolonged spike bursts during KA challenges. RTG, a pan Kv7.2-Kv7.5-channel opener [KCNQ2 mutations.In the present study, both l opener –28, suppKcnq2Y284C/+ and Kcnq2A306T/+ mice, using pentylenetetrazole (PTZ) [Szt1 mice, a spontaneous mutant mouse strain that harbors a microdeletion affecting the C-terminus of mouse Kcnq2 and also its adjacent two genes, Chnra4 and Arfgap-1 [Kcnq2 knock-out mice have also demonstrated hypersensitivity to PTZ [Kcnq2 and the G311V mutation in Kcnq3, which is an ortholog of a KCNQ3 mutation found in human BFNE [Kcnq2Y284C/+ and Kcnq2A306T/+ mice, these heterozygous mutant mice did not show spontaneous seizures, although their homozygotes did. The Kcnq2A306T/+ and Kcnq3G311V/+ mice had significantly higher sensitivity to electroconvulsive seizure and kindling acquisition [Kcnq2Y284C/+ mice were more prone to KA-induced seizures than Kcnq2A306T/+ mice in terms of the frequency and duration of seizure bursts upon EEG, suggesting that the BFNE mutations vary in sensitivity.We previously showed high sensitivity to proconvulsive treatment in le (PTZ) . SimilarArfgap-1 –33. Hetey to PTZ . Furtherman BFNE ,36. Simiuisition ,36. InteKcnq2-deficient mice and the resulting impairment of M-currents hamper the stabilization of the resting membrane potential and subthreshold levels of membrane excitability [in vitro [Thus, the hypersensitivity to proconvulsants observed in tability . Therefotability , supportin vitro –44.Kcnq2Y284C/+ and Kcnq2A306T/+ mice, RTG, an opener of Kv7 channels, was more effective in preventing KA-induced seizures than PB, not only in terms of the incidence of seizures, but also in terms of suppressing the spike bursts on EEG.In both Kcnq2 BFNE mutations. The effects of another potassium channel opener, flupirtine, which is an analogue of RTG, were previously evaluated and compared with PB, albeit in experimental seizure models involving WT rodents. Flupirtine has a better effect on experimental seizures than PB [To our knowledge, this is the first application and comparison of RTG with other AEDs in genetically engineered animal models bearing  than PB .Szt1 mice, but was found less effective in reducing electroconvulsions in Szt1 mice than in their WT controls [v7.2.In addition, RTG has been used in controls . Howevercontrols . Therefoin vitro electrophysiological findings that RTG ameliorates the dysfunction of Kv7.2 caused by KCNQ2 mutations, obtained either from reconstituted channels in Xenopus laevis oocytes [KCNQ2 microdeletion [v7.2 in Xenopus laevis oocytes revealed that the effect of RTG on lowering action potential in WT Kv7.2 was significantly greater than that on Kv7.2 harboring EOEE mutations [KCNQ2 mutations cause haploinsufficiency, while EOEE mutations cause a dominant-negative effect. Hence, given that Kv7.2/Kv7.3 is a hetero-tetramer, RTG may exert its function in both Kcnq2Y284C/+ and Kcnq2A306T/+ mice by ameliorating the pore region, which consists of two KCNQ3 subunits and one normal KCNQ2 subunit derived from WT alleles Click here for additional data file.


Drosophila melanogaster (Dm) mutant for PTEN-induced putative kinase 1 (B9PINK1) gene is a powerful tool to investigate physiopathology of Parkinson's disease (PD). Using B9PINK1 mutant Dm we sought to explore the effects of Mucuna pruriens methanolic extract (Mpe), a L-Dopa-containing herbal remedy of PD. The effects of Mpe on B9PINK1 mutants, supplied with standard diet to larvae and adults, were assayed on 3–6 (I), 10–15 (II) and 20–25 (III) days old flies. Mpe 0.1% significantly extended lifespan of B9PINK1 and fully rescued olfactory response to 1-hexanol and improved climbing behavior of B9PINK1 of all ages; in contrast, L-Dopa  ameliorated climbing of only B9PINK1 flies of age step II. Transmission electron microscopy analysis of antennal lobes and thoracic ganglia of B9PINK1 revealed that Mpe restored to wild type (WT) levels both T-bars and damaged mitochondria. Western blot analysis of whole brain showed that Mpe, but not L-Dopa on its own, restored bruchpilot (BRP) and tyrosine hydroxylase (TH) expression to age-matched WT control levels. These results highlight multiple sites of action of Mpe, suggesting that its effects cannot only depend upon its L-Dopa content and support the clinical observation of Mpe as an effective medication with intrinsic ability of delaying the onset of chronic L-Dopa-induced long-term motor complications. Overall, this study strengthens the relevance of using B9 DmPINK1 as a translational model to study the properties of Mucuna pruriens for PD treatment.The fruit fly  B9PINK1) Parkinsonism, both in humans B9Drosophila melanogaster (Dm) PINK1 mutant model recapitulates several of the essential features of PD B9PINK1 model provides major information regarding pathogenic molecular basis of early onset PD and mitochondrial dysfunction PINK1 mutation enhances mitochondrial stress-induced neurodegeneration in mice Parkinson’s disease (PD) is, after the Alzheimer’s disease, the second most prevalent neurodegenerative disease first affecting medulla oblongata, olfactory bulb and substantia nigra L-Dopa is the most effective symptomatic medication of PD and is still considered the gold standard in its treatment, although other drugs such as dopamine (DA) agonists, DA uptake and mono amino oxidase-B inhibitors are commonly used in the clinical management of PD patients Mucuna pruriens (Mp) Mp seeds in PD. In addition to L-Dopa, Mp seeds contain genistein and polyunsaturated fatty acids which support its antiparkinsonian and neuroprotective actions Mp constituent with antioxidant and iron sequestrant activity, has been reported to suppress methyl-phenyl-tetrahydropyridine (MPTP) induced hydroxyl radical generation Mp, the present study was aimed at verifying if Mpe’s ability to ameliorate symptoms in this PD model might be attributable to L-Dopa only or to the Mp extract as a whole in which L-Dopa is present along with other ingredients. On these bases we evaluated the antiparkinsonian profile of the standardized methanolic extract of the seeds of Mp (Mpe) on lifespan, climbing activity and olfactory function in B9PINK1 as compared to either wild type (WT) and untreated B9 Dm.PINK1 In addition, in order to gain mechanistic insights on the neuroprotective and neuro-rescue properties of Mpe, we also evaluated the expression of bruchpilot protein and tyrosine hydroxylase, as well as the morphology of presynaptic active zones and mitochondria in flies’antennal lobes, i.e. the olfactory bulbs-equivalent structure, and thoracic ganglia, of both WT as well as untreated and Mpe-treated B9PINK1 mutants.Several reports on antiparkinsonian activity of B9PINK1 (w[*] Pink1[B9]) mutant Drosophila melanogaster (Dm) males . After emergence from pupae, male WT and B9PINK1 mutant flies were separated. WT and mutant flies were reared on a standard cornmeal-yeast-agar medium in controlled environmental conditions . In detail, four groups of mutant flies were reared on a standard medium supplemented with Mucuna pruriens methanolic extract (Mpe) . B9PINK1 mutants were supplied with Mpe at different concentrations (0 (i.e. untreated B9PINK1 mutants), 0.1, 1 and 10% w/w in their standard diet) both as larvae and adults (L+/A+). In addition, another group was reared on a standard medium supplemented with 0.01% (0.5 mM) L-Dopa , a percentage similar to that at which L-Dopa was supplemented with 0.1% MpeMpe were assayed at different age steps . A series of experiments on life span, using various concentrations of Mpe  provided the basis for selecting the optimal concentration at which conduct the behavioral, morphological, and protein expression assessments. In particular, based on lifespan results, the olfaction behavior assessments, transmission electron microscopy (TEM) and western blot analyses were restricted to group II flies after 0.1% Mpe administration as L+/A+. Standard genetic procedures were used during the study.For these experiments we used adult wild type (WT) Oregon-R (Oregon-R-C) and PTEN-induced putative kinase 1 Mpe’s concentration, Dm were grown on standard diet supplemented with different concentrations of Mpe at 25°C. Cohorts of 40 flies (4 flies/tube) from each experimental group  were monitored every 2 days for their survival. Mortality was analyzed using Kaplan-Meier survival curves and the statistical comparisons were made with a Gehan-Breslow-Wilcoxon test. Experiments were done in duplicate with the exception of those on WT, untreated mutants, 0.1% Mpe- and 0.01% L-Dopa-treated B9PINK1 that were done in triplicate.With the aim of selecting the optimal B9PINK1 and treated B9PINK1).Each experiment was conducted with the appropriate control group  and tapped to the bottom. Climbing time was recorded upon crossing a line drawn at 6 cm from the bottom. The number of flies that could climb unto, or above, this line within 10 seconds was recorded and expressed as percentage of total flies. Data were expressed as average + SEM from at least three separate experiments. The statistical evaluation was made by two-way ANOVA (p<0.05) followed by HSD post-hoc test.The climbing assay (negative geotaxis assay) was used to assess locomotor ability Dm and untreated, Mpe- and L-Dopa-treated B9PINK1 from group II (n = 12/each strain) were singly positioned under the view of an Olympus BX51WI light microscope . Electrodes were silver wires inserted in glass capillaries filled with a saline solution (NaCl 150 mM).The recording glass electrode was positioned on the tip of the left antenna while the reference was pierced through the compound eye. The EAG signal was amplified with an AC/DC probe and then acquired with an IDAC-4 interface board . The antennae were constantly blown by a flow of charcoal purified and humidified air (speed 0.5 m/s) via a glass tube. Odor stimuli were administered by injecting a puff of purified air (0.5 s at 10 mL/s airflow) through the pipette using the stimulus delivery controller .In vivo electroantennogram recordings (EAG) were performed following a previously described protocol Dm. Mean values of EAG amplitude were calculated and then analyzed by comparing the results obtained in untreated B9PINK1, Mpe- and L-Dopa-treated flies with matched WT. The significance of differences was tested by one-way ANOVA (followed by HSD post hoc test) with a threshold level of statistical significance set at p<0.05. EAG results are expressed as average values ± S.E.M and represented by histograms.Odor stimuli were prepared in 3 step-dose concentration  diluted in hexane. Odor stimulus, 1-hexanol, was chosen according to Fishilevich and Vosshall B9PINK1 and, as L+/A+, 0.1% Mpe- and 0.01% L-Dopa-treated B9PINK1 mutants were given the opportunity to choose between vials containing water with or without odor. Two 4 mL glass vials were placed symmetrically in a large petri dish (arena) and then fitted with truncated pipette tips. The vials were filled with 300 µL of water with 0.25% Triton X  with or without the odorant (0.1% (v/v) 1-hexanol; Sigma-Aldrich, Milan, Italy). As mentioned above, in order to allow detection of possible Mpe’s effects independently from the circuit, octopaminergic -for appetitive- and dopaminergic -for aversive stimuli Free-walking bioassay was performed following the experimental procedures used by Dekker et al. B9PINK1 and, as L+/A+, 0.1% Mpe-treated B9PINK1 mutants were anesthetized using carbon dioxide and carefully decapitated. The brains and the thoracic ganglia, once rapidly removed, were fixed in a mixture of 2% glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate buffer, washed several times in the same buffer, post-fixed in 1% osmium tetroxide in distilled water for 2 hours, and stained overnight at 4°C in an aqueous 0.5% uranyl acetate solution. After several washes in distilled water, the samples were dehydrated in a graded ethanol series, and embedded in SPURR resin. To identify the antennal lobes (ALs), semi-thin coronal sections of the whole brains were cut with a Leica EM UC6 ultramicrotome, stained with toluidine blue and observed with a Leica DM2700 P light microscope. Sections of about 70 nm corresponding to portions of the ALs and thoracic ganglia were cut with a diamond knife on a Leica EM UC6 ultramicrotome. Transmission electron microscopy (TEM) images were collected with a FEI Tecnai G2 F20  and a Jeol JEM 1011  electron microscopes, working respectively at an acceleration voltage of 80 and 100 kV, and recorded with a 1 and 2 Mp charge-coupled device (CCD) camera . T-bars density (expressed as number of T-bars/m2) in both ALs and thoracic ganglia presynaptic boutons was assessed on a total of ten animals . 459 and 683 T-bars were randomly sampled respectively in the ALs and the thoracic ganglia on a total 496 non-overlapping micrographs at a final magnification of 6000, corresponding to a total sampled area of more than 6000 µm2. T-bars were unambiguously identified at presynaptic active zones by the presence of T-shaped electron-dense projections typically tethered by a large number of presynaptic vesicles.Group II WT, untreated B9PINK1 and 0.1% Mpe-treated B9PINK1 mutants. More than 3000 mitochondria were randomly sampled on 191 non-overlapping micrographs at a final magnification of 4000, corresponding to a total sampled area of more than 5000 µm2. Damaged mitochondria were recognized for the presence of swollen external membrane, clearly fragmented cristae and inhomogeneous electron transparent mitochondrial matrix. The mean differences were tested using a two tailed t-test and a p<0.01 level was considered statistically significant.The number of damaged mitochondria within ALs  was evaluated in WT, untreated B9PINK1 and, as L+/A+, 0.1% Mpe- and 0.01% L-Dopa-treated B9PINK1 mutants flies were collected and immediately stored at −80°C. Head lysate preparations of adult males were performed by homogenization in RIPA buffer  containing fresh protease inhibitor cocktails . Two centrifugations were performed at 4°C at 10,000 g for 15 minutes, before protein quantification by DC Protein assay . 20 µg of proteins were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis using the mini-PROTEIN 3-electrophoresis module assembly  and then transferred to immobilon-polyvinylidenedifluoride membranes (Amersham Biosciences). The membranes were incubated with primary antibodies overnight at 4°C. Immune complexes were detected with horseradish peroxidase–conjugated secondary antibodies and chemiluminescence reagents  and visualized by Image Quant LAS 4000. Densitometric analysis was performed by Image Studio Lite software for quantitative assessment.Group II WT, untreated Primary antibodies used in this study were against nc82 ; Tyrosine Hydroxylase ; actin ; Horseradish-peroxidase–conjugated secondary antibodies were purchased from Life Technologies. Statistical significance of the results was evaluated by one-way ANOVA (p<0.05) followed by a HSD post-hoc test.B9PINK1 mutants displayed a significantly shorter lifespan with respect to WT flies. To assess the ability of Mpe to affect lifespan of B9PINK1 mutants, they were supplied Mpe at different concentrations (0 (untreated), 0.1, 1 and 10% w/w in their standard diet) both as adults only (L−/A+) (+/A+)  on life span of B9PINK1 are also reported in Mpe-treated B9PINK1, as shown by Kaplan-Meier survival curves, revealed a statistically significant effect of Mpe on lifespan of B9PINK1 mutants only when L+/A+ flies were fed 0.1% Mpe  , and as ) (+/A+) . The effard diet .B9PINK1 at different age steps  with a worsening trend with aging, while WT flies fulfilled the evaluation criterion without differences among age groups. As shown in To investigate the locomotor ability the negative geotaxis assay, as described previously Mpe 0.1% treatment significantly ameliorated the climbing activity in mutants and also reduced the worsening trend with aging although the score obtained by treated mutants still remained higher than that measured in WT. Interestingly, the climbing time of L-Dopa-treated mutants from groups I and III did not significantly differ with respect to age-matched untreated B9PINK1, the performance of only group II flies being significantly ameliorated.The +/A+ 1% Mpe-treated mutants reached similar rescue of climbing activity as observed in 0.1% Mpe-treated ones only when tested at early ages (groups I and II). On the other hand, 10% Mpe administration failed to significantly ameliorate motor behavior in groups I and III with respect to untreated B9PINK1 mutants, while a significant effect was detected in treated flies from group II. We also considered the percentages of flies that were able to complete the test and the results are depicted in histograms shown in B9PINK1 from group I, 46% from group II and 36% from group III accomplished it, showing a clear age-dependent worsening. Administration of 0.1% Mpe, as L+/A+, greatly rescued B9PINK1 mutants (groups I–III) from motor impairment and restored to WT values the percentages (86–94%) of flies able to accomplish the task according to the evaluation criterion (10 sec). Furthermore, at variance with the above results, the effects of L-Dopa worsened over time. In particular, 0.01% L-Dopa administration determined a decrease of the number of flies able to complete the task showing a negative trend with aging. In fact, percentages of flies were 91%, 82% and 62%, in groups I, II and III, respectively.As shown in As expected, the olfactory stimulations of flies’ antennae elicited responses with the typical EAG wave form, i.e. a rapid depolarization followed by a slower recovery phase, ending with the hyperpolarized wave before complete reversal to the baseline.B9PINK1, 0.1% Mpe- and 0.01% L-Dopa-treated mutants from age group II. In details, the average EAG signal amplitudes evoked by stimuli were significantly lower in B9PINK1 specimens in respect to WT thus substantially confirming data previously reported The results, summarized in The stimulation with 1-hexanol at 1% did not elicit a significant increase in the EAG amplitude as compared with the stimulation at 0.1% in all strains of flies with the exception of mutants flies treated with L-Dopa 0.01%. This result indicates that at the highest odor concentrations (0.1 and 1%) a saturation of response was reached by all groups but by the L-Dopa treated mutants. Besides, we observed that the responses to stimuli in WTs elicited a greater hyperpolarized phase in the EAGs  and S2.Mpe- and L-Dopa-treated B9PINK1 was not detected. The lowest odor concentration tested elicited a significantly higher response in WT as compared to the all strains of mutants (p<0.05). This difference shrank when the 0.1% concentration of odor was administered. A reduced response, although not statistically significant (p>0.05), was still detected in untreated mutants with respect to WTs (p<0.05), while treated flies showed on average an increased response with respect to untreated flies. The response measured in treated flies was therefore halfway between the highest of WTs and the lowest of untreated B9PINK1. Samples of EAGs responses are shown in Even if a positive trend in treated mutants exists in the signal amplitude in response to 1-hexanol, a statistical difference between untreated, B9PINK1, 0.1% Mpe- and 0.01% L-Dopa-treated, as L+/A+, B9PINK1 mutants. As expected, the analysis of the result, shown in B9PINK1 flies p<0.004). B9PINK1 flies treated with 0.1% Mpe and 0.01% L-Dopa were able to reach the stimuli as WT controls (p>0.05). In fact, percentages of trapped flies were 45.2±5.8% and 44.2±3.6% for 0.1% Mpe- and 0.01% L-Dopa-treated mutants, respectively. Similar results were obtained concerning the numbers of trapped flies in the blank bait (H2O) .The olfactory behavior assay was restricted to flies of group II, by testing the responses to 1-hexanol (0.1% v/v) of WT, untreated B9PINK1 and 0.1% Mpe-treated, as L+/A+, B9PINK1 mutants and results are shown in B9PINK1 mutants with respect to WT controls . More importantly, a significant increase of T-bars density was detected in the ALs and thoracic ganglia of B9PINK1 treated with 0.1% Mpe, as L+/A+, with respect to untreated B9PINK1 . Moreover the number of damaged, swollen and with clearly fragmented cristae, mitochondria was significantly lower in presynaptic boutons of ALs of B9PINK1 mutants treated with 0.1% Mpe, compared with untreated mutants .Transmission electron microscopy (TEM) analysis was restricted to flies of group II of untreated WT, untreated B9PINK1 0.1% Mpe- and 0.01% L-Dopa-treated, as L+/A+, B9PINK1 mutants. As shown in B9PINK1 mutants was significantly lower (p<0.05) than in WT. Diet supply of 0.1% Mpe to B9PINK1 mutants significantly recovered BRP and TH expression to WT controls levels (p<0.05) and these values did not differ statistically from those of WT. Notably, BRP and TH expression in B9PINK1 mutants fed 0.01% L-Dopa resulted similar to BRP and TH expression in untreated B9PINK1 mutants. ANOVA also revealed that both BRP and TH expression resulted statistically different as compared to their expression of both WT and B9PINK1 mutants fed 0.1% Mpe.Mucuna pruriens seeds, known for possible neuroprotective effects in neurotoxin-induced models of PD B9PINK1 mutant DmPINK1 gene model a number of features of early onset PD such as cell energy maintenance B9PINK1-related molecular, morphological and functional bases of PD. The present results show that addition of 0.1% Mpe to the feeding medium of B9PINK1 mutants significantly a) improved climbing ability and olfaction, b) rescued compromised T-bars density and damaged mitochondria in the ALs and thoracic ganglia, c) restored to WT control values the expression of BRP and TH proteins. Moreover, these results suggest that Mpe is an effective medication with intrinsic ability of delaying the onset of chronic L-Dopa-induced long-term motor complications  no matter the concentration tested  even if not significant, a worsening trend was observed : Lifespan, expressed as % survival rates of untreated and Mpe-treated B9PINK1 at the 4 dose-step tested: 0, 0.1, 1 and 10% (w/w) only when adults (L−/A+). (B): lifespan of untreated and Mpe-treated B9PINK1 at the 4 dose-step tested: 0, 0.1, 1 and 10% (w/w) only when adults (L+/A−).(TIF)Click here for additional data file.Figure S2EAGs samples. Dose-response relationships for olfactory stimulations in WT and B9PINK1 adult flies and their differences in signal amplitude and shape.(TIF)Click here for additional data file.


Aim. To investigate the correlation between PPP1R12A gene copy number and clinical outcomes of oxaliplatin-based regimen in stage III colorectal cancer (CRC). Methods. A total of 139 paraffin-embedded tissue samples of stage III CRC patients who received oxaliplatin-based treatment after radical surgery were recruited. Genomic DNA was extracted and purified from paraffin-embedded sections. Quantitative PCR methods were used to detect the relative copy number (RCN) of PPP1R12A. Results. Statistical analysis demonstrated that low PPP1R12A RCN was associated with poor RFS  and OS . Additionally, when patients were stratified according to subgroups of stage III and tumor location, poor RFS and OS were also observed in the low PPP1R12A RCN group with significance . Conclusion. Our findings suggest the copy number of PPP1R12A can independently predict recurrence and overall survival of stage III colorectal cancer patients receiving oxaliplatin-based chemotherapy. Colorectal cancer (CRC) is the third most commonly diagnosed cancer in males and the second in females worldwide , 2 and tδ  to form the PP1cδ-MYPT1 complex. Through modulating the catalytic activity and specificity of PP1cδ, PPP1R12A has been reported to participate in diverse cellular functions, such as smooth muscle contraction [δ [β-catenin signaling [PPP1R12A (protein phosphatase 1 regulatory subunit 12A) belongs to the myosin phosphatase targeting protein (MYPT) family and is also known as myosin phosphatase target subunit 1 (MYPT1). PPP1R12A molecule possesses a PP1c-binding motif, via which PPP1R12A recognizes and binds PP1ctraction –9, cell traction , 11, celtraction , and embtraction . In addiction [δ . Merlin ignaling  and PI3Kignaling , both ofignaling , 20. ThuIn this study, we employed the quantitative PCR to detect the relative copy number (RCN) of PPP1R12A in stage III CRC patients receiving oxaliplatin-based regimen and compute the association of PPP1R12A gene copy number with clinical outcomes.This study included a total of 139 paraffin-embedded tissue samples of the patients who received curative resection from 2006 to 2011 in the Ruijin Hospital affiliated to Shanghai Jiao Tong University and the Tenth People's Hospital affiliated to Shanghai Tongji University. All the patients were histologically and clinically diagnosed as the stage III of CRC according to the criteria proposed by the Standard American Joint Committee on Cancer (AJCC) and only patients receiving at least 6 cycles of oxaliplatin-based chemotherapy after operation were included in this study. None of the patients received chemotherapy or radiotherapy before radical surgery. Written informed consent was obtained from all participants. This study was approved by the Medical Ethical Committee of Shanghai Jiaotong University and Tongji University.μm thick formalin-fixed paraffin-embedded sections (tumor cells account for at least 70%) of the 139 tumors using a DNeasy kit  according to the manufacturer's instructions.Genomic DNA was extracted and purified from 5 μL with housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) used as an internal control. The primers for GAPDH were forward, 5′-ACG AAT TTG GCT ACA GCA ACA GG-3′, and reverse, 5′-CCA GCA GTG AGG GTC TCT CTC TT-3′. The primers for PPP1R12A were forward, 5′-AGG TGA AGT TCG ACG ATG GC-3′, and reverse, 5′-TCC GTC CAC ATT GGC GTA AT-3′. Each thermal cycle consisted of 15 s of denaturation at 95°C, 20 s of primer annealing at 60°C, and 20 s of extension at 72°C. All the samples (both PPP1R12A and GAPDH) were run in triplicate.To detect PPP1R12A gene DNA copy number, real-time quantitative polymerase chain reaction (qPCR) was performed using IQ5 Multicolour Real-Time PCR Detection system . qPCR was carried out using the SYBR Green kit  in a final volume of 20 −ΔΔCt method [Each assay was tested in triplicate. Data points that generated triplicate Ct values with over one cycle variance were excluded from analysis. Since the samples were analyzed in triplicate, values for each triplicate sample were averaged to the final data use. The mean Ct value obtained from each sample was normalized to the averaged copy number and then subjected to analysis with the 2t method , 22.χ2) to compare the distributions of categorical variables. Recurrence-free survival (RFS) was calculated from the surgery date to the date of disease recurrence or death. Overall survival (OS) was defined as the time from the date of surgery to that of death or the end of follow-up. We dichotomized patients into high RCN and low RCN groups at median, tertile and quartile cut-off points, and it showed that the most significant associations of PPP1R12A RCN with RFS and OS were achieved at first tertile point . So we chose the first tertile of PPP1R12A copy number as the cut-off point in our study [P < 0.05 was considered statistically significant.We performed the Pearson Chi-square  and rectum . The patients with stage IIIA, IIIB, and IIIC disease accounted for 9.4% (n = 13), 71.2% (n = 99), and 19.4% (n = 27), respectively. After a median follow-up of 49 months , 56 patients (40.3%) exhibited tumor recurrence and 44 patients (31.7%) died.The demographic and clinical characteristics in the 139 patients with CRCs were summarized in n = 93) and low RCN  groups with a tertile cut-off of PPP1R12A relative copy number (RCN = 0.37). During the follow-up, 56 (40.3%) of the 139 patients suffered recurrence (P = 0.007), high TNM stage , and low RCN of PPP1R12A  were significantly associated with poor RFS. After adjusting for age, location, gender, TNM, lymph node metastasis, and histology grade, PPP1R12A remained an independent factor associated with RFS. In the Kaplan-Meier analysis, the curves showed that patients with low PPP1R12A copy number were significantly associated with poor RFS (P = 0.003) (P < 0.001) (P = 0.037)   . To inve< 0.001) . Also, l= 0.037) . In the  Figures .P = 0.011), high lymph node metastasis , high TNM stage , and low PPP1R12A RCN  were shown to be significantly associated with poor OS. Furthermore, the multivariate analysis showed that poor differentiation , TNM stage , and low PPP1R12A  remained independent predictive factors of poor OS. The Kaplan-Meier analysis was also used to assess the correlation between PPP1R12A copy number and overall survival. The log-rank test showed that patients with low PPP1R12A copy number were associated with a statistically significant poor OS (P < 0.001)  and IIIC  patients  .Among the 139 stage III CRC patients, 44 (31.7%) patients died during the follow-up. The median overall periods were 47 (±11.1) months in the low RCN group while the OS were not reached in the high gene copy number group . Both th< 0.001) . Further Figures . In the = 4.229) . SimilarColorectal cancer remains one of the most prevalent cancer and leading causes of cancer deaths worldwide. Adjuvant chemotherapy is thus required to reduce relapse rate and enhance survival. Oxaliplatin-based regimen is widely used as first-line therapy for advanced colorectal cancer; however, the recurrence is still high. So identification of patients who will likely or unlikely benefit from chemotherapy would allow personalized treatment of CRC patients.β-catenin signaling [β-catenin pathway has been revealed to enhance chemoresistance in many cancer cell types including hepatocellular carcinoma and neuroblastoma and this kind of chemoresistance is thought to be associated with overexpression of multidrug resistance 1 gene (MDR1) [β-catenin signaling and leads to inhibition of drug resistance and enhances the apoptosis induced by oxaliplatin.Here for the first time we conducted the study to examine the possibility of PPP1R12A as a possible biomarker for the prediction of clinical outcomes in stage III colorectal cancer patients treated with oxaliplatin-based chemotherapy. The results showed that patients with low copy number of PPP1R12A had a poor OS and RFS with statistical significance, indicating PPP1R12A was able to predict recurrence and overall survival. PPP1R12A is essential in the activation of Merlin . Serine ignaling , 18. Recignaling . In addie (MDR1) , 26. ProConventional TNM staging system is not able to predict the risk of recurrence among certain subgroup patients such as IIIB and IIIC. In this study, we showed that PPP1R12A RCN was significantly correlated with OS and RFS even in IIIB and IIIC. This indicates PPP1R12A can not only predict OS and RFS of stage III CRC patients, but also predict the OS and RFS of IIIB and IIIC patients. This compensates the weakness of TNM staging and allows clinicians to make the more accurate therapeutic decision.In conclusion, our study demonstrates that the copy number of PPP1R12A could independently predict recurrence and overall survival of stage III colorectal cancer patients receiving oxaliplatin-based adjuvant chemotherapy.Supplementary Material: Median, tertile and quartile points of the PPP1R12A RCN distribution were chosen to dichotomize patients into high RCN and low RCN groups. The associations of PPP1R12A RCN with RFS and OS at these cut-offs were analyzed and the results were shown in supplemental table 1 and 2.


PIEZO1 is a mechanosensitive eukaryotic cation-selective channel that rapidly inactivates in a voltage-dependent manner. We previously showed that a fluorescent protein could be encoded within the hPIEZO1 sequence without loss of function. In this work, we split the channel into two at this site and asked if coexpression would produce a functional channel or whether gating and permeation might be contained in either segment. The split protein was expressed in two segments by a bicistronic plasmid where the first segment spanned residues 1 to 1591, and the second segment spanned 1592 to 2521. When the “split protein” is coexpressed, the parts associate to form a normal channel. We measured the whole-cell, cell-attached and outside-out patch currents in transfected HEK293 cells. Indentation produced whole-cell currents monotonic with the stimulus. Single channel recordings showed voltage-dependent inactivation. The Boltzmann activation curve for outside-out patches had a slope of 8.6/mmHg vs 8.1 for wild type, and a small leftward shift in the midpoint (32 mmHg vs 41 mmHg). The association of the two channel domains was confirmed by FRET measurements of mCherry on the N-terminus and EGFP on the C-terminus. Neither of the individual protein segments produced current when expressed alone. The Piezo proteins are eukaryotic mechanosensitive cation channels that rapidly inactivate in a voltage dependent manner , 2 and aFam38a and implicated in integrin activation and cell adhesion [The physiological function of Piezo channels is currently under study. This protein was originally designated adhesion . Geneticadhesion , 11 invoadhesion , 13 thatadhesion . Piezo1 adhesion , 16. Pieadhesion  and in sadhesion . The reladhesion –21 and padhesion . Interesadhesion .In this work we show that the PIEZO1 protein can be split in two, such that when coexpressed, form a functional wild type channel. These whole-cell currents were robust and monotonic with the stimulus. In cell-attached mode, we observed voltage-dependent activation, slowing with depolarization. For outside-out patches we calculated the Boltzmann curve of activation and showed that the slope sensitivity was similar to wild type, but had a small leftward shift (to lower pressures) in the gating curve. The proximity of the two protein segments was confirmed by energy transfer (FRET) between two fluorophores.2, 1 CaCl2, 10 HEPES, pH 7.3 (adjusted with KOH) and 160 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 HEPES, pH 7.3 (adjusted with NaOH) for whole cell mode. Mechanical stimulation for patches was applied by suction for the cell-attached configuration and positive pressure for the outside-out configuration using an ALA high speed pressure clamp  controlled by QuBIO software . Whole-cell mechanical stimulation utilized a fire-polished glass pipette (diameter of 2–4 μm) positioned at an angle of 30° with respect to the cover glass. The probe was coarsely positioned ~20 μm from the cell using an MP-285 manipulator (Sutter Instruments Co.), and from that position the probe had vertical movements using a trapezoidal waveform with a piezoelectric stage . The indentation depth (with 40 nm resolution) was controlled using a home built LabVIEW program. The probe velocity was 0.15 μm/ms during the transitions, and the stimulus was held constant for 300 ms. Currents were typically recorded at a holding potential of -60 mV at room temperature. The experiments were performed using an Axopatch 200B amplifier (Molecular Devices), sampled at 10 kHz and filtered at 1 kHz. Data acquisition and stimulation were all controlled by QUBIO and analyzed by QuB software.HEK-293 cells (purchased from ATTC) transfected with 500 ng cDNA using TransIT-293 reagent (Mirus), were tested between 24-48h later. All recordings were done as previously described , 24, 25.A Spe1 site was first inserted into pIRES2-EGFP (after the GFP) by site-directed mutagenesis with the following primers:GCATGGACGAGCTGTACAAGACTAGTTAAAGCGGCCGCGACTCTAGqcIEGFPSpeF CTAGAGTCGCGGCCGCTTTAACTAGTCTTGTACAGCTCGTCCATGCqcIEGFPSpeR To incorporate the protein portion from the N terminal to the end of mCherry, we used human  PIEZO1-1ATCAAGTGTATCATATGCCAAGTACInFIENdeFwd–GGAGAGGGGCGGATCCTACTTGTACAGCTCGTCCATGCInF1591mChBamR–PCR was performed using Prime Star GXL DNA polymerase according to manufacturer’s specification. This 5.9kb product was inserted into Spe1 modified pIRES2-EGFP treated with the endonucleases Nde1 and BamH1, by InFusion HD cloning (Clontech/Takara).The C-terminus of HekP1 was amplified from amino acid 1591 to the end using Prime Star GXL DNA polymerase and the following primers:GCTGTACAAGACTAGTAGTGGGCTGGGCGCGGAGGAGInFHP1ctSpeF–GGCCGCTTTAACTAGTCTACTCCTTCTCACGAGTCCACTInFHP1ctSpeR–The 2.8kb product was inserted into the vector containing the N-terminal fragment at the Spe1 site by InFusion HD cloning.Nt1591mCh IE GFP vector with the endonucleases BamH1 and Spe1. This DNA was then used as template for PCR to amplify a segment from Nde1 site in the CMV promoter to Age1 site after mCherry (contains DNA that codes for PIEZO1 protein 1–1591 linked to mCherry). A plasmid DNA with GFP inserted into position 1591 was digested with the endonucleases Nde1 and Age1. The PCR produce (1–1591 linked to mCherry) was combined with the plasmid (Nde1/Age1) and ligated by InFusion HD.To prevent priming from a secondary site, the DNA encoding GFP was removed from The IRES HP1 double mutant C-terminus  was ampl(ex480)/680 nm(ex560) images. The ratio of the 520 nm (ex480) to 680 nm(ex560) was used to confirm that the ratio of expression levels of the two fusion proteins in different cells remained constant. Image acquisition and microscope components were controlled using MicroManager software [Cells were visualized on a Zeiss Axio Observer Z1 inverted microscope with a 63x 1.4 NA oil immersion objective, DIC optics, motorized Z-axis and DefiniteFocus Z-axis positioning . Cells expressing the GFP and mCherry fusion proteins were excited with the BDX (450-490nm) and BGX (505-545nm) LEDs (respectively) from an X-Cite XLED1 illuminator, Excelitas Technologies . The filter sets in the Zeiss microscope contained: for GFP—a 480/20x excitation filter and a 495lp dichroic, and for mCherry—a 560/55x excitation filter, a 595lp dichroic and a 645/75m emission filter. Images were collected with an Andor iXon DV897U cooled, EM multiplying, back illuminated, CCD camera . A Dual-View splitter was mounted in front of the camera for simultaneous imaging of GFP and mCherry fluorophores. The Dual-View contained a 565lpxr splitter with 525/50 (GFP) and 680/35 nm (mCherry) emission filters. Images were acquired at 525 nm and 680 nm for both 480 nm and 560 nm excitation wavelengths for a total of 4 images of each cell. FRET ratio was the slope of the 680 nmsoftware , and imaWe have previously shown that a fluorescent protein could be encoded in the hPIEZO1 protein without disrupting the properties of the channel . In thisWe transfected HEK293 cells with 250–500 ng of plasmid (hPIEZO1 NtCt), and after 24–48 hrs measured whole-cell currents at -60 mV  using a + conductance in the absence of divalent cations was 49 pS at -100mV, similar to the wild type channel [Results shown in  channel .1/2 ‾ Boltzmann) to be 34 ± 0.9 mmHg (R2 = 0.99), slightly leftward shifted compared to wt at 40 ± 2.1 mmHg (R2 = 0.99) [no significant change of the slope (sensitivity) which was ~ 8.5 ± 1.8 vs 8.6 ± 0.9 mmHg-1 for wt  . There w1 for wt  indicati1 for wt . NeitherWe have previously shown that two mutations made to the C-terminus remove inactivation. When weTo confirm intermolecular interactions between the coexpressed Nt and Ct halves, we determined the proximity of the two proteins by calculating the FRET ratio using the 680 nm emission of mCherry at 480 and 560 nm excitation. As a control for minimal FRET ratio , we coexpressed TREK1-mCherry and MscL-EGFP, two proteins that have different distributions and are assumed not to interact . To asseNtCt) supporting nm-scale interactions between the two proteins. The FRET ratio data was supported by the Ct and Nt expression patterns which showed very different distributions when expressed separately ((Ct) and(Nt)). The Ct half primarily resides in cytoplasmic and nuclear regions, while the Nt half localizes preferentially to membranes. Coexpression of the two halves caused the Ct protein to distribute in similar pattern with the Nt protein, suggesting the Nt protein escorts the Ct protein to more membrane localized regions when the two interact.The ratio of the expected minimal energy transfer in this system was 1.15 ± 0.17 based on the TREK-1/MscL pair , while tWe previously showed that encoding a fluorescent protein at position 1591 of hPIEZO1 did not alter the biophysical properties of the channel . The abiThe structure of mouse Piezo1 elucidated by cryo EM to 4.8 Å resolution showed that the pore is defined by a trimer of the C-terminal segment . These rMycobacterium tuberculosis was also divided into two parts [The split-protein technique has been applied to the potassium channel, TOK1, where the pore was split in two  but the wo parts , generatWe were unable to generate channel activity from either PIEZO1 segment when expressed alone. While the Nt segment expressed alone was targeted to membrane domains, the Ct half did not, but became membrane localized with coexpression see  Figs. ThS1 FigOptical sections (0.25 μm thick) showing DIC and fluorescent images of a representative HEK cell expressing TREK1-mCherry and MscL-EGFP bicistronic vector. A section near the glass coverslip and in the middle of the cell are shown. EGFP images at 525 nm (green) and mCherry image at 680 nm (orange). TREK1 primarily labels plasma membrane and ER regions. MscL primarily labels internal membrane structrues like nuclear membrane, vesicles, ER and reticulate stuctures farther from the nucleus. An “*” designates nucleus position, and “#” designates likely endoplasmic reticulum (ER) region.(TIFF)Click here for additional data file.S2 FigMiddle section images taken from two groups of cells. (A) shows two cells expressing higher concentration of Ct-EGFP with cytoplasmic and nuclear distribution. (B) Shows two cells with lower expression having primarily diffuse cytoplasmic distribution. An “*” designates nucleus position, and “#” designates likely endoplasmic reticulum (ER) region.(TIFF)Click here for additional data file.S3 FigIn addition, most Nt-mCherry expressing cells are highly vesiculated, though the reason for this was not investigated further. An “*” designates nucleus position, and “#” designates likely endoplasmic reticulum (ER) region.Two representative cells are shown (A and B) with image sections near the glass coverslip and in the middle of the cell. Many punctae form on the surface and dense staining occurs in the ER and at the ends of cell protrusions. (TIFF)Click here for additional data file.S4 FigWhile not complete colocalization, most of the Ct protein is localized to Nt regions, and no Ct protein no longer goes to the nucleus. Single representative cell showing image sections near glass coverslip and near the middle of the cell. Cells expressing both halves do not show vesiculation. An “*” designates nucleus position, and “#” designates likely endoplasmic reticulum (ER) region.(TIFF)Click here for additional data file.


Sheifer et al indicated that undetected infarction account for at least 20% of all infarctions and carry a prognosis as poor as detected ones . Large m3 microemboli in the LAD , 2) large infarction by occluding LAD for 90 min  or 3) combined large and diffuse infarction by occluding LAD for 90 min and infusion of 32 mm3 microemboli . Three days following coronary intervention, contrast enhanced 3D (TE/TR/TI = 2.3/4.8/230-240 ms) and 2D (TE/TR/TI = 1.8/6.8/230-240 ms) was performed for measuring myocardial infarction in beating hearts. The animals were then euthanized inside the scanner and reimaged using the same imaging sequences. At postmortem, histochemical triphenyltetrazolium chloride (TTC) and microscopic histopathology (hematoxylin-eosin) stains were used for validation.Pigs were subjected to: 1) diffuse infarction by infusing 32 mmThere was no significant difference in infarction size between delayed contrast enhanced 3D and 2D MRI in both beating and non-beating hearts Table . FurtherMyocardial infarction measured on 3D MRI is highly correlated and in a good agreement with infarction measured microscopically. This imaging sequence has the potential to measure diffuse and large acute myocardial infarction and has minimal motion artefacts.N/A


Various types of human cancers exhibit amplification or deletion of KDM4A-D members, which selectively demethylate H3K9 and H3K36, thus implicating their activity in promoting carcinogenesis. On this basis, it was hypothesized that dysregulated expression of KDM4A-D family promotes chromosomal instabilities by largely unknown mechanisms. Here, we show that unlike KDM4A-B, KDM4C is associated with chromatin during mitosis. This association is accompanied by a decrease in the mitotic levels of H3K9me3. We also show that the C-terminal region, containing the Tudor domains of KDM4C, is essential for its association with mitotic chromatin. More specifically, we show that R919 residue on the proximal Tudor domain of KDM4C is critical for its association with chromatin during mitosis. Interestingly, we demonstrate that depletion or overexpression of KDM4C, but not KDM4B, leads to over 3-fold increase in the frequency of abnormal mitotic cells showing either misaligned chromosomes at metaphase, anaphase–telophase lagging chromosomes or anaphase–telophase bridges. Furthermore, overexpression of KDM4C demethylase-dead mutant has no detectable effect on mitotic chromosome segregation. Altogether, our findings implicate KDM4C demethylase activity in regulating the fidelity of mitotic chromosome segregation by a yet unknown mechanism. Nucleus of the eukaryotic cells is composed of DNA and proteins that are organized in higher-order structure termed chromatin . One maiChromatin structure is regulated by the dynamic interplay between histone-interacting proteins and post-translational modifications (PTMs) of histones ,9,10. ThLysine methylation is one of the most common modifications of histone tails, which acts as a platform for chromatin modifier proteins and leads to either gene activation or repression. Tri-methylation of histone H3 lysine 9 (H3K9me3) is enriched in condensed pericentric heterochromatin, while di-methylation of histone H3 lysine 9 (H3K9me2) and mono-methylation of histone H3 lysine 9 (H3K9me1) are associated with transcriptionally silent regions within euchromatin . FurtherTwo families of lysine demethylases (KDM) have been identified, confirming that histone methylation is a reversible and dynamically regulated process . One fam2+ and α-KG-dependent manner . These enzymes specifically catalyze the demethylation of H3K9me2/me3, H3K36me2/me3 and H1.4K26me2/me3 in a Fet manner . Besidest manner . In addit manner ,41. On tt manner ,41. In at manner . Thus, bt manner .in vivo  by impairing the fidelity of mitotic chromosome segregation. These observations suggest that CIN, found in cancer cells driven by KDM4C dysregulation, could result from mitotic chromosome missegregation.The constructions of all plasmids used in this study are described in Table U2OS-TetON stable cell lines that conditionally express the fusions EGFP-KDM4B and EGFP-KDM4C were established as previously described . Cell li2 at 37°C. U2OS cells were grown in DMEM. U2OS-Tet-ON cells were grown in Dulbecco's modified Eagle's medium (DMEM) in the presence of 200 μg/ml Geneticin (G418). U2OS-Tet-ON-EGFP-KDM4A-C cells were grown in DMEM containing 200 μg/ml G418 and either 0.6 μg/ml Puromycine (for KDM4A-B) or 200 μg/ml Hygromycin B (for KDM4C).All cells lines were supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 μg/ml penicillin, 100 μg/ml streptomycin and grown in a humidified incubator containing 5% COCell transfections with plasmid DNA or siRNA were performed using Poly Jet (Bio-Consult) and Lipofectamine 2000 (Invitrogen), respectively, following the manufacturer's instructions. siRNAs used in this study include Stealth KDM4B-C siRNA (Invitrogen) and Stealth RNAi negative control. All constructs and siRNA sequences are available upon request.Protein lysates were prepared using two different methodologies. First, cells were lysed using NP40 lysis buffer ) for 30 min on ice, centrifuged at 14 000  rpm for 25  min at 4°C, and supernatant was recovered. Second, protein lysates were prepared using hot-lysis buffer , boiled for 15 min, sonicated with two 15 s pulses of 35% amplitude, and centrifuged at 14 000 rpm for 15 min at room temperature; supernatant was then treated with benzonaze for 30 min, centrifuged and recovered. Protein concentration was determined using bicinchoninic acid (BCA) protein determination reagent (Sigma). Immunoblots were performed using appropriate antibodies . We sought therefore to determine the localization of KDM4A-C members, which share common domain architecture consisting of JmjN, JmjC, two PHD and two Tudor domains Figure . Toward To determine the localization of EGFP-KDM4A-C fusions during the different stages of mitosis, U2OS-TetON-EGFP-KDM4A-C cells were treated with Dox for 36 h, fixed and stained with DAPI to visualize mitotic cells. Results show that KDM4C protein is localized to mitotic chromatin from prometaphase to telophase. In striking contrast to KDM4C, EGFP-KDM4A-B fusions are excluded from mitotic chromatin Figure . InteresNext, to assess whether the mitotic localization of the endogenous KDM4A-C proteins is similar to their overexpressed EGFP-fused forms, we first tested the suitability of commercial KDM4A-C antibodies to detect the native forms of KDM4A-C proteins by immunofluorescence analysis. U2OS cells were transfected with expression constructs expressing EGFP-KDM4A-C fusions (green) and immunostained with KDM4A-C antibodies (red). Results show that the intensity of the red signal in cells expressing the EGFP-KDM4A-C fusions is much higher than the untransfected cells . This result confirms that these antibodies detect KDM4A-C proteins in cells and can be used for immunofluorescence-based studies. MCF7 cells were then immunostained using KDM4A-C antibodies to detect their mitotic localization. Results show that, similar to the localization of EGFP-KDM4A-C fusions, the endogenous KDM4A-B are excluded from mitotic chromatin, while KDM4C protein is associated with chromatin during the different mitotic stages Figure . AltogetTo map KDM4C region that mediates its localization to mitotic chromatin, we performed domain-swapping analysis between KDM4A and KDM4C proteins. First, we swapped the regions containing the two PHD and the two Tudor domains between KDM4A and KDM4C proteins. As a result, two chimeras were produced Table : chimeraTo identify the domain that mediates the association of KDM4C with mitotic chromatin, we repeated the domain-swapping analysis and exchanged the regions containing only the two Tudor domains between KDM4A and KDM4C proteins. This analysis produced chimera3 and chimera4 Table . ChimeraTo determine whether both Tudor domains are required for KDM4C mitotic localization, we swapped the C-terminus region containing the distal Tudor domain between KDM4C and KDM4A Table . ChimeraRDTF/DNLY mutant was determined. Results show that EGFP-KDM4CRDTF/DNLY mutant is excluded from mitotic chromatin . Results show that KDM4C depletion using either one of the three siRNA sequences leads to over 3-fold increase in percentage of metaphase cells with misaligned chromosomes compared to cell transfected with control siRNA (n = 107 metaphase cells)  compared to control cells (n = 101)  Figure . Next, w) Figure  and G. AImportantly, KDM4B-C members are overexpressed in several types of human cancer and its depletion impairs cancer cell proliferation ,71,74,75To gain further insights into KDM4C mitotic function, we sought to address whether its demethylase activity is implicated in regulating chromosome segregation. Toward this, we generated KDM4C ‘demethylase-dead’ mutant. As we have previously reported , Ser198MNext, we sought to address whether the defective chromosome segregation in cells overexpressing KDM4C is due to its localization on mitotic chromatin. To this end, we overexpressed EGFP-KDM4C-R919D mutant, which is not associated with mitotic chromatin Figure , and detThis study shows a unique localization of KDM4C on mitotic chromatin that differs from the closely related members, KDM4A and KDM4B, which are excluded from chromatin during mitosis Figure . This diOur data suggest that the two Tudor domains are involved in the regulation of KDM4C localization during mitosis. Interestingly, the Tudor domain was shown to mediate the subcellular localization of proteins. For example, the Tudor domain of TDRD3 is both required and sufficient for its localization to stress granules, which are cytoplasmic structures involved in RNA metabolism . SimilarIn addition, we reveal a novel role of KDM4C in regulating mitotic chromosome segregation. Our results show that the levels of KDM4C protein are critical for the proper chromosome segregation. KDM4C, but not KDM4B, knockdown or overexpression increases the frequency of abnormal mitotic cells showing misaligned chromosomes during metaphase, anaphase bridge and chromosome lagging. Further, overexpression of KDM4C-S198M demethylase-dead mutant has no detectable effect on the fidelity of chromosome segregation. These results imply that CIN can result from loss or gain of KDM4C demethylase activity. In accord with this, recent analysis of the Cancer Genome Atlas revealed that KDM4C is lost in some cancer types and overexpressed in others . It shouDetermining the cellular localization of protein is often a crucial step toward understanding its biological functions . Our datHow does KDM4C misregulation disrupt the fidelity of mitotic chromosome segregation? There are three main possibilities which are not necessarily mutually exclusive. First, KDM4C serves as a scaffold protein for recruiting other proteins that are required for proper chromosome segregation. In accord with this, cells overexpressing KDM4C-R919D mutant  show no increase in the percentage of metaphases with misaligned chromosome Figure . Second,Supplementary Data are available at NAR Online.


Hepatitis C virus (HCV) is a public health problem with almost 185 million people estimated to be infected worldwide and is one of the leading causes of hepatocellular carcinoma. Currently, there is no vaccine for HCV infection and the current treatment does not clear the infection in all patients. Because of the high diversity of HCV, protective vaccines will have to overcome significant viral antigenic diversities. The objective of this study was to predict T-cell epitopes from HCV genotype 5a sequences.HCV near full-length protein sequences were analyzed to predict T-cell epitopes that bind human leukocyte antigen (HLA) class I and HLA class II in HCV genotype 5a using Propred I and Propred, respectively. The Antigenicity score of all the predicted epitopes were analysed using VaxiJen v2.0. All antigenic predicted epitopes were analysed for conservation using the IEDB database in comparison with 406, 221, 98, 33, 45, 45 randomly selected sequences from each of the HCV genotypes 1a, 1b, 2, 3, 4 and 6 respectively, downloaded from the GenBank. For epitope prediction binding to common HLA alleles found in South Africa, the IEDB epitope analysis tool was used.A total of 24 and 77 antigenic epitopes that bind HLA class I and HLA class II respectively were predicted. The highest number of HLA class I binding epitopes were predicted within the NS3 (63%), followed by NS5B (21%). For the HLA class II, the highest number of epitopes were predicted in the NS3 (30%) followed by the NS4B (23%) proteins. For conservation analysis, 8 and 31 predicted epitopes were conserved in different genotypes for HLA class I and HLA class II alleles respectively. Several epitopes bind with high affinity for both HLA class I alleles and HLA class II common in South Africa.The predicted conserved T-cell epitopes analysed in this study will contribute towards the future design of HCV vaccine candidates which will avoid variation in genotypes, which in turn will be capable of inducing broad HCV specific immune responses. Hepatitis C virus (HCV) is estimated to infect 185 million people worldwide . The WorProphylactic vaccines against viral infections are generally aimed at inducing a humoral (B-cell) immune response, while therapeutic vaccines preferably activate both humoral and cellular (T-cell) immune responses . A succeThe development of an effective HCV vaccine requires an understanding of the host’s adaptive immune response to natural infection. As with other viral infections, viral antigens are presented to CD4+ and CD8+ T-cells via human leukocyte antigen (HLA) class II and class I molecules, respectively . Differein-silico prediction of peptide binding affinities to HLA proteins [HCV infection evades the host’s immune system by generating immune escape variants through alteration of the virus HLA-restricted epitopes to avoid being recognized by T-cells and neutralizing antibodies . Thus efproteins .Genotype 5 accounts for over 50% of HCV infections in South Africa , and is 1325–1333 covered 30 of the 47 HLA class I alleles that were analysed assuring high binding affinity to different alleles. For conservation analysis with other genotypes, 8 of 24 epitopes were 100% conserved for specific genotypes. Epitopes NS31332–1340 and NS5B2557–2565 were highly conserved in all genotypes analysed, and epitope E2684–692 was conserved in all genotypes except for genotype 2, while 5 other epitopes were conserved in either 2 or 3 genotypes analysed. In addition epitope NS4B1832–1840 was conserved in genotype 1a, 1b, 2 and 4 epitope variants at anchor residues position 2 and 9 while E2677–685, NS31325–1333 and NS31357–1365 were conserved in at least 3 genotypes epitope variants each , while other epitopes were conserved in at least 1 to 4 of the genotypes analysed. Epitope NS4B1886–1894 was conserved at anchor residues position 1, 4, 6 and 9 in all 6 genotypes epitope variants while E2692–700, NS2964–972, NS31418–1426 and NS41561–1569 were conserved in at least 5 genotypes epitope variants each , followed by NS5B (21%), and for the HLA class II, the highest number of epitopes were predicted in the NS3 (30%) followed by the NS4B (23%) proteins . Four of the epitopes binding to HLA-B*35:01 followed by HLA-A*02:01 with 3 epitopes binding to it, however, none of the epitopes bind to HLA-C*04:01, HLA-C*06:01 and HLA-C*07:02 alleles. The NS31359–1367 shows a level of promiscuity to HLA-A and HLA-B alleles. NS31359–1367 HPNIEEVAL bind with intermediate affinity to HLA-B*07:02, while its genotype 2, 3 and 4 variant HSNIEEVAL bind with poor affinity and genotype 6 variant HPNITETAL bind with high affinity. For HLA-B*35:01, the NS31359–1367 HPNIEEVAL and genotype 6 variant HPNITETAL bind with high affinity while genotype 2, 3 and 4 variant HSNIEEVAL bind with poor affinity. Three  of the thirteen epitopes were predicted by both Propred 1 and IEDB analysis tool , HLA-B , and HLA-C  were analysed. The limitation of Propred 1 is that it does not cover most of the main HLA class I alleles: HLA-A , HLA-B (HLA-B*08:01) and HLA-C  that are observed in South Africa. As a results the IEDB epitope analysis tool was used to predict epitopes of all the 11 most common HLA-A, HLA-B, HLA-C covering HLA class I alleles found in South Africa with the ANN prediction server. Thirteen antigenic epitopes with high binding affinity score of <50 IC50nM were predicted. The HLA-DRB1*11:01 has the highest number of binding epitopes (10) followed by HLA-DRB1*04:01 and HLA-DRB1*15:01 (7 each). Epitopes NS4B1919–1927 LIAFASRGN was predicted to be the most promiscuous epitope binding to HLA-DRB1*04:01, HLA-DRB1*11:01 and HLA-DRB1*15:01 with high affinity and HLA-DRB1*03:01 with intermediate affinity. This epitope is conserved in all genotypes. Epitopes E2507-515, NS4B1774–1782, NS4B1919–1927, and NS4B1920–1928 were highly conserved in all genotypes  were analysed. Nineteen antigenic epitopes with high binding affinity score of <50 IC#) in Tables Seven of the predicted epitopes were previously confirmed experimentally by other studies as true positives in comparison with the epitopes analysed in the IEDB resource database. Majority of the epitopes predicted in this study have not been previously tested experimentally. The ‘true epitopes’ are highlighted by .http://www.imtech.res.in/raghava/propred1/) at a 4% default threshold by keeping the proteosome and immunoproteosome filters on at 5% threshold. ProPred 1 predicts antigenic epitopes for 47 HLA class I alleles [http://www.imtech.res.in/raghava/propred/) at a 3% default threshold. ProPred predicts antigenic epitopes for 51 HLA class II alleles [Genotype 5a full-length sequences available in the GenBank and 6 of the near full length sequences generated from a previous study conducted by our group  were ali alleles . For HLA alleles .http://www.ddg-pharmfac.net/vaxijen/). Epitopes having antigenic score >0.5 were selected as antigenic. Vaxijen server performed well with 87% accuracy at a threshold of 0.5 antigenic score for viruses. VaxiJen v2.0 allows antigen classification based on the physicochemical properties of proteins without recourse to sequence alignment.The Antigenicity score of all the predicted epitopes were analysed using VaxiJen v2.0 online antigen prediction (http://tools.immuneepitope.org/tools/conservancy/iedb_input) at a threshold of 100% conservation in comparison with 406, 221, 98, 33, 45, 45 randomly selected sequences from each of the HCV genotypes 1a, 1b, 2, 3, 4 and 6 respectively. The epitopes were considered conserved in another genotype if it shows 100% identity across the epitope in at least 70% of sequences in that genotype in the randomly selected sequences used in this study, downloaded from the public database. In addition, epitope variants that were conserved in at least 70% of the sequences were analysed for conservancy for anchor residues at positions 2 and 9 for HLA class I and positions 1, 4, 6 and 9 for HLA class II.All predicted epitopes were analyzed for conservation using the IEDB database (http://www.immuneepitope.org/) to confirm if they had been tested previously by other studies. The immuneepitope database contains experimentally confirmed data about antibody, T-cell epitopes, HLA binding, HLA restriction and HLA class.All the predicted epitopes were submitted to IEDB database (http://tools.immuneepitope.org/tools/conservancy/iedb_input) was used for HLA class I using the artificial neural network (ANN) algorithm [http://www.imtech.res.in/raghava/propred/) at a 3% default threshold was used. ProPred predicts antigenic epitopes for 51 HLA class II alleles [For epitope prediction binding to common HLA alleles found in South Africa, the IEDB epitope analysis tool (lgorithm  on the I alleles . The mos alleles .