Caenorhabditis elegans, the Rac proteins CED-10, RAC-2 and MIG-2 act in parallel to control axon outgrowth and guidance. Here, we have identified a single glycine residue in the CED-10/Rac1 Switch 1 region that confers a non-redundant function in axon outgrowth but not guidance. Mutation of this glycine to glutamic acid (G30E) reduces GTP binding and inhibits axon outgrowth but does not affect other canonical CED-10 functions. This demonstrates previously unappreciated domain-specific functions within the CED-10 protein. Further, we reveal that when CED-10 function is diminished, the adaptor protein NAB-1 (Neurabin) and its interacting partner SYD-1 (Rho-GAP-like protein) can act as inhibitors of axon outgrowth. Together, we reveal that specific domains and residues within Rac GTPases can confer context-dependent functions during animal development.Rac GTPases act as master switches to coordinate multiple interweaved signaling pathways. A major function for Rac GTPases is to control neurite development by influencing downstream effector molecules and pathways. In  Caenorhabditis elegans to model neurite development, we found that a single amino acid in a conserved domain of the Rac GTPase CED-10 is crucial for controlling neurite outgrowth in a partially non-redundant manner. Further, we revealed that lesions in discrete domains in the CED-10 protein lead to distinct developmental defects. Therefore, our in vivo study proposes that regulation of distinct signalling pathways through Rac GTPase protein domains can drive different developmental outcomes.Brain development requires that neurite outgrowth and guidance are precisely regulated. Previous studies have shown that molecular switch proteins called Rac GTPases perform redundant functions in controlling neurite development. Using a pair of bilateral neurons in the nematode  Caenorhabditis elegans ventral nerve cord (VNC) contains two fascicles housing multiple neurons that project axons along the length of the animal , 36rp100 alleles . As prev animals  [36]. In animals . To ask ogenesis . We founhatching . We nextgulfment . Double D-6/GULP , or the D-6/GULP , had no ced-10(rp100) mutant animals do not adversely affect apoptotic corpse engulfment. In a reciprocal experiment, we asked whether signalling components through which CED-10 controls apoptotic cell recognition, engulfment and removal are required for PVQ axon outgrowth [ced-5(tm1949) mutant animals exhibit ~35% penetrant defects in PVQ guidance (ced-10(rp100) mutation on PVQ axon outgrowth is not likely due to defective interpretations of signals from the core apoptotic corpse engulfment machinery.We have shown that the molecular pathways dysregulated in utgrowth . We examguidance , which srp100, n3246 and n1993 mutant animals  mutation on neurodevelopment compared to the n3246 and n1993 mutations, we crossed all three alleles into fluorescent reporter strains to permit visualization of neuronal development at single-neuron resolution  choice of the VD motor neurons, and in the PLM and PVM neurons  and candidate genes that encode known Rac GTPase interactors or are known to regulate cytoskeletal remodelling at the growth cone  mutation on axon outgrowth could be due to a change in affinity with interacting molecules and/or altered activity or expression of its effector molecules. To determine the pathways through which defective CED-10(G30E) protein may cause PVQ outgrowth defects, we performed single and double mutant analysis with wth cone .ced-10(rp100) PVQ defects is not affected by the rin-1(gk431) mutation (mig-10(ct41) null mutant animals exhibit minimal PVQ axon outgrowth defects (2%), albeit with highly penetrant guidance defects (75%), and does not affect the PVQ outgrowth penetrance of ced-10(rp100) mutant animals (C. elegans abLIM homolog UNC-115 is an actin modulatory protein that functions to control growth cone filopodia formation [unc-115(ky275) null mutant animals exhibit a low penetrant axon guidance defect (unc-115(ky275); ced-10(rp100) animals is additive when compared to either single mutant suggesting that these factors regulate PVQ guidance in parallel  nor max-2(ok1904) mutants caused PVQ outgrowth defects. However, max-2(ok1904) animals do exhibit PVQ axon guidance defects, consistent with the finding that MAX-2 can regulate axon guidance independently of Rac proteins [ced-10(rp100) PVQ axon outgrowth defects. In contrast, we found that two independent alleles of pak-1 suppress the PVQ outgrowth defects of ced-10(rp100) mutant animals, without affecting the penetrance of PVQ guidance defects (ced-10(rp100)-induced defects are suppressed. Alternatively, our observations could mean that CED-10(G30E) protein specifically alters PAK-1-regulated outgrowth.Next, we analysed the function of the p21-activated kinases (PAKs), which are known downstream Rac GTPase effectors that are important for controlling cytoskeletal dynamics. Dimerized PAKs interact with GTP-bound Rac proteins, which relieves PAK self-inhibition and permit kinase domain activation . We anal-2 genes . We founproteins .Furtherm defects . One intced-10(rp100) PVQ axon outgrowth defects, we performed a forward genetic suppressor screen (ced-10(rp100) animals exhibit PVQ outgrowth defects when cultivated at 25°C (ced-10(rp100); oyIs14 strain with ethyl methanesulfonate and screened for reduced PVQ outgrowth defects in the progeny of 1000 F2 animals cultivated at 25°C. Of the four independent alleles that significantly suppress ced-10(rp100) PVQ outgrowth defects, we focus on rp117, a recessive allele that reduces ced-10(rp100) PVQ outgrowth defects at 20°C from ~35% to ~10%  PVQ outgrowth defects (nab-1 does not suppress PVQ axon guidance defects (nab-1 is dispensable for correct PVQ development in a wild type background (ced-10(rp100) mutant background, we expressed nab-1 cDNA using a PVQ specific promoter (nab-1 cDNA reversed the suppression of PVQ axon outgrowth defects observed in the ced-10(rp100); nab-1(ok943) double mutant  axon outgrowth defects. To test this hypothesis, we generated compound mutants between ced-10(rp100) and two, independent, syd-1 alleles (ju82 and tm6234). We found that both syd-1 alleles suppress ced-10(rp100) PVQ outgrowth defects (nab-1 and syd-1 did not further suppress ced-10(rp100) PVQ outgrowth defects, suggesting that these genes act in the same pathway to regulate neuronal outgrowth  with putative protein and RNA binding function –56. Prevus actin . SYD-1 ius actin . We hypo defects . Furtherutgrowth .ced-10(rp100); mig-2(mu28) double mutant, which exhibits highly penetrant defects in PVQ axon outgrowth (ced-10(rp100); mig-2(mu28) animals. However, we found that PVQ axon outgrowth defects of ced-10(rp100); mig-2(mu28) animals are suppressed when nab-1 or syd-1 are absent (mig-2(mu28) is a null allele, these data suggest that NAB-1 and SYD-1 can regulate the activity of alternative GTPases, or may directly regulate CED-10 to control PVQ axon outgrowth.Our data suggest that NAB-1 and SYD-1 are required to suppress Rac GTPase activity during PVQ axon outgrowth. We have shown that CED-10 and MIG-2 act redundantly in PVQ axon outgrowth , and a putgrowth . If NAB-e absent . As mig-C. elegans CED-10/Rac1 that is crucial for regulating neuronal development  mutant animals exhibit similar PVQ axon outgrowth defects. In addition, PVQ axon outgrowth defects caused by ced-10(rp100) are fully recessive and are rescued by expressing wild type ced-10 cDNA. We also demonstrate that animals harboring a known Rac1 hyperactive mutation (proline to serine substitution at position 29) in CED-10 did not exhibit PVQ axon outgrowth defects. Most importantly, using in vitro GTP activation assays we found that Rac1(G30E) is GTP activated ~50% less than wild type Rac1. The reduced affinity of CED-10(G30E) for GTP may result in diminished and/or atypical interactions between CED-10(G30E) and unknown effector molecule(s), thereby causing the PVQ axon outgrowth phenotype.Our multiple lines of evidence show that the G30E substitution reduces CED-10 function. We have shown that We found that the CED-10(G30E) mutant protein causes profound effects on PVQ outgrowth. However, unlike other CED-10 mutant proteins, CED-10(G30E) does not cause defects in apoptotic corpse engulfment or fecundity. This suggests that defects in cell behaviour caused by the CED-10(G30E) protein specifically affects nervous system development. It could also suggest that the activity of CED-10(G30E) is not sufficiently reduced to impinge upon apoptotic corpse engulfment or generation of progeny. Focusing our analysis on the nervous system, we found that the neuronal guidance defects caused by CED-10(G30E) are similar to another mutation affecting the Switch 2 region, CED-10(G60R). However, we only found PVQ axon outgrowth defects in CED-10(G30E) animals. To our knowledge, this is the first report where different regions of Rac GTPases have been shown to differentially regulate axon outgrowth and guidance. Our data therefore suggest that PVQ outgrowth is especially sensitive to CED-10(G30E)-defective signaling and is an ideal model to delineate previously unknown functions for the Switch 1 region of Rac GTPases in regulating cell behaviour. Further analysis of the actin cytoskeletal architecture in the PVQ neurons during embryogenesis—the time of PVQ outgrowth, is unfortunately not presently feasible, as the tools to observe PVQ-specific reporter proteins during embryogenesis are unavailable.C. elegans Neurabin homolog, suppresses ced-10(rp100) PVQ axon outgrowth defects. In C. elegans, NAB-1 regulates synaptogenesis and synaptic function by binding to F-actin and recruiting the synaptic active zone proteins SYD-1 (RhoGAP) and SYD-2 (liprin-α) , 54. In mammals, Neurabin I was originally shown to promote neurite formation in primary rat hippocampal neurons [We performed a genetic suppressor screen to identify the regulators controlling CED-10-directed PVQ axon outgrowth. This approach revealed that loss of NAB-1, the  neurons . Further neurons . Neurabi neurons . Subsequ neurons . Support neurons , 64. The neurons . These d neurons .C. elegans. Rac GTPase activation status is central for regulating downstream effector pathways, and we found that the scaffolding protein NAB-1 and RhoGAP SYD-1 likely enhance CED-10 activity and therefore modify CED-10 output. Intriguingly, we found that although reduced NAB-1 and SYD-1 function promoted axon outgrowth, it did not restore PVQ axon guidance. This supports the hypothesis that there are intracellular mechanisms in growth cones that are specifically regulated by effector molecule binding to Rac GTPases, which differentially promote/inhibit axon outgrowth and guidance. We propose that identifying the precise effector molecules and pathways controlled by CED-10 under different levels of activation will uncover novel insights into how correct axon outgrowth and guidance is achieved.Taken together, our work reveals that a conserved amino acid (glycine 30) in the CED-10/Rac1 protein is important for the activation status of this Rac GTPase and for correct axon outgrowth of the PVQ neurons in C. elegans strains were maintained at 20°C on NGM plates seeded with Escherichia coli OP50 bacteria, unless otherwise stated [All e stated . StrainsoyIs14, hdIs26, rpEx1640; Mechanosensory neurons:  zdIs5; HSNs: rpEx6; PDEs: IqIs2; PVPs: hdIs26; AVGs: otIs182; D-type motor neurons: oxIs12; AQR: rpIs8. All neuronal scoring was repeated in triplicates on independent days, n = 75, except for oxIs12 scoring, n = 35.Neurons were scored, blinded to phenotype, in L4/young adult stages, unless otherwise stated. Scoring criteria for each neuron is detailed in nDf67(mir-51 mir-53 deletion); oyIs14 [nDf67 deletion, from which we singled animals and genotyped for loss of the nDf67 deletion. These animals still exhibited the PVQ outgrowth phenotype. We named the mutant allele rp100 and used the one-step whole-genome sequencing and SNP mapping strategy to map the genetic lesion [rp100; oyIs14 hermaphrodites. Ten F1 progeny carrying the oyIs14 transgene were picked to individual plates and allowed to self-fertilize. F2 progeny carrying the oyIs14 transgene were picked to 250 individual plates and allowed to self-fertilize. Approximately 20 F3 progeny from each of the 250 F2 plates were scored for PVQ axon outgrowth defects. 47 plates were homozygous for the phenotype causing mutation, based on the presence of PVQ outgrowth defects. Progeny of these animals were pooled and DNA was isolated. Pooled genomic DNA was sequenced using Ilumina sequencing and the resultant sequencing data was analyzed using the Galaxy platform. Our mapping identified a single lesion (GGA-GAA) at base-pair position 89 within exon 1 of the ced-10 gene, which was independently confirmed by Sanger sequencing.We initially observed PVQ axon outgrowth and guidance defects in the following strain—; oyIs14 . After tc lesion . Males oced-10(rp100); oyIs14 animals were induced with ethyl methanesulfonate (EMS) following the modified version of a previously described protocol [ced-10(rp100), screening for suppressor mutations was conducted at 25°C. The germlines of ced-10(rp100); oyls14 animals (L4 stage) were mutagenised using 50mM EMS (Sigma) in M9 buffer  for 4 hours (room temperature), after which they were transferred to standard OP50 NGM plates for 72 hours (20°C). F1s were allowed to self-fertilise on individual plates (20°C). Four F2s (L4 stage) were randomly picked from each F1 and separated into individual plates for incubation at 25°C. F3 populations with PVQ outgrowth defects lower than 20% (n = 50) were selected and retested in subsequent generations for heritability of suppression.Random mutations in protocol . Since iced-10(rp100); oyls14 and 200 recombinant F2s from each cross were separated into individual plates for incubation at 25°C. Recombinant F2s homozygous for the suppressor mutation were identified by scoring for suppression of PVQ outgrowth defects of F3 offspring. 40 F2 plates exhibited the suppressor phenotype and worms were washed off with M9 buffer and pooled for DNA extraction with Gentra Puregene kit (Qiagen). Paired-end whole genome sequencing (WGS) with Illumina NextSeq500 was performed and the causative genetic lesion identified using Mutation Identification in Model Organism Genomes (MiMoDd) (version 0.1.7.3).Suppressor gene identification was carried out using whole genome sequencing and bulk-segregant mapping . CandidaPlasmid inserts were confirmed using Sanger sequencing prior to use.sra-6prom::ced-10cDNAbRJP273 ced-10 cDNA isoform b was amplified from an oligodT amplified cDNA library using the oligos 5’-ttggctagcgtcgacggtacatgcaagcgatcaaatgtg-3’ and 5’-agatatcaataccatggtacttagagcaccgtacactt-3’ and ligated into a sra-6prom plasmid with a pPD49.26 backbone using KpnI.rgef-1prom::ced-10cDNAbRJP272 ced-10 cDNAb from RJP273 was ligated downstream of a rgef-1prom in a pPD49.26 backbone using NheI-SpeI.sra-6prom::GFPRJP296 sra-6prom plasmid with a pPD49.26 backbone using NheI-SpeI.Sequence encoding GFP was ligated into a sra-6prom::mCherryRJP297 sra-6prom plasmid with a pPD49.26 backbone using NheI-SpeI.Sequence encoding mCherry protein was ligated into a npr-11prom::nab-1RJP370 nab-1 cDNA was amplified from an oligodT amplified cDNA library using the oligos 5’- gctagcatgacaacggcttccgagc -3’ and 5’- ggtacctcacatgggaattgtgtgtgc-3’ and ligated into a npr-11prom plasmid with a pPD49.26 backbone using NheI-KpnI.3. Examination and imaging of neurons was performed using an automated fluorescence microscope  and ZEN software (version 3.1).Animals were mounted on 5% agarose pads and immobilized using 50mM NaNced-10(rp100); oyIs14. The WRM0639dH09 fosmid spanning the entire ced-10 locus was injected at 1ng/μl. For pan-neuronal rescue, RJP272 (rgef-1prom::ced-10cDNAb) was injected at 10ng/μl. For cell-autonomous rescue, RJP273 (sra-6prom::ced-10cDNAb) was injected at 50ng/μl and RJP370 npr-11prom::nab-1 at 10ng/μl. RJP370 (npr-11prom::nab-1) was injected into nab-1(ok943); ced-10(rp100); oyIs14 animals at 20ng/μl. myo-2prom::mCherry was injected at 5ng/μl as a co-injection marker in all transgenic animals.Transgenic animals were generated as previously described . Rescue rgef-1prom::ced-10cDNAb) at 10ng/μl, RJP297 (sra-6prom::mCherry) at 10ng/μl and myo-2prom::mCherry at 5ng/μl into the ced-10(rp100); oyIs14 strain. A transgenic line was selected that exhibited rescue of the PVQ axon outgrowth and guidance defects. Transgenic animals from this line were then scored for phenotypic rescue of the PVQ defects in the presence and absence of the rescuing extrachromosomal array in the PVQ neurons by detection of mCherry fluorescence.For mosaic analysis, transgenic animals were generated by injecting RJP272 .1-177 was obtained from Christina Lucato [pGEX-4T-1-TEV-Rac1a Lucato . Point mEscherichia coli BL21(DE3) CodonPlus cells and GST-Rac1 protein expression was induced by IPTG at 18°C overnight. Cells were pelleted and resuspended in lysis buffer . Cells were lysed by sonication and cell debris was cleared by centrifugation. Soluble lysate was filtered at 0.8 μm. GST-Affinity purification was carried out with glutathione-Sepharose 4B resin  at 4°C for 90 min. The GST tag was cleaved with overnight incubation with TEV protease. Rac1 was eluted and further purified with size-exclusion chromatography on a HiLoad Superdex 75 16/60 column , equilibrated in SEC buffer .pGEX-4T-1-TEV-Rac1 wild type and mutant plasmids were transformed into 2 . EDTA and excess GDP is removed from GDP-loaded Rac1 with PD-10 desalting columns and equilibrated with 20 mM Tris, pH 8.0, 150 mM NaCl, 10 mM MgCl2, 2mM DTT. 30 μM Rac1-GDP was incubated with 500 μM GTPγS with or without 15 mM EDTA for 1 hour at room temperature and the reaction was terminated by adding 20 mM MgCl2 . 5 μg Rac1-nucleotide complexes were allowed to bind with 5 μg PAK-PDB beads (Cytoskeleton) in 500 μl of pulldown buffer  Igepal CA-630) for 1 hour at 4°C. Beads were washed three times with cold pulldown buffer. Active Rac1 bound to PAK beads were eluted in Lithium Dodecyl Sulfate loading buffer and subjected to SDS-PAGE. Eluted Rac1 protein was detected by western blotting and anti-Rac1 mouse monoclonal antibody (Cytoskeleton). Western quantification was carried out with ImageJ with intensities normalised to loading controls. Method adapted from [Purified wild type and mutant Rac1 proteins (500 μM) in SEC buffer was preloaded with 10 mM GDP and 5 mM EDTA, for 1 hour at room temperature. The reaction was terminated by adding 10 mM MgClted from .Statistical analysis was carried out using Graphpad Prism 7 using one-way ANOVA with Tukey’s Multiple Comparison Test or t test, where applicable. Values are expressed as mean ±S.D. Values <0.05 were considered statistical significant.S1 Figced-10(rp100) rescue and mosaic analysis.Homo sapiens Rac1 (HsRac1) and C. elegans CED-10 (CeCED-10). The ced-10 point mutants used in this study are marked with an asterisk: rp100 (G30E - Switch 1 region), n3246 (G60R - Switch 2 region) and n1993 (V190G - membrane targeting region).(A) Alignment of the amino acid sequences of ced-10(rp100) animals with ced-10 cDNA (ced-10(+)). Data are expressed as mean ±SD and statistical significance was assessed using one-way ANOVA, with Tukey’s multiple comparison test. ****<0.0001, comparing wild type and rp100 animals; #<0.0001 and +<0.006 and comparing the three transgenic rescue lines to ced-10(rp100). n.s. not statistically significant from rp100 animals. n = 90 per strain, each dot represents independent scoring replicates.(B) Quantification of pan-neuronal and PVQ-specific transgenic rescue of total PVQ defects of Prgef-1::ced-10(+), Psra-6::mCherry and Pmyo-2::mCherry plasmids was expressed in ced-10(rp100); oyIs14 animals. PVQ outgrowth and guidance was scored in animals in which the array was absent , present in the PVQs  or missing from the PVQs . Number of animals scored is shown in parentheses.(C) An unstable transgenic array containing (PDF)Click here for additional data file.S2 Figced-10 allelesgenetic analysis of .ced-10 mutant homozygotes, heterozygotes and transheterozygotes. Data are expressed as mean ±SD and statistical significance was assessed using one-way ANOVA, with Tukey’s multiple comparison test. ****<0.0001, n.s. not significantly different to wild type. n = 90 per strain, each dot represents independent scoring replicates.(A) Quantification of total PVQ defects (outgrowth and guidance) in wild type, ced-10 mutant homozygotes, heterozygotes and transheterozygotes. Data are expressed as mean ±SD and statistical significance was assessed using one-way ANOVA, with Tukey’s multiple comparison test. ****<0.0001, n.s. not significantly different to wild type. n = 90 per strain, each dot represents independent scoring replicates.(B) Quantification of PVQ outgrowth defects in wild type, ced-10(tm597) L1 escapers. Data are expressed as mean ±SD and statistical significance was assessed using t test. n = 9 per strain, each dot represents independent scoring replicates.(C) Quantification of PVQ outgrowth defects in wild type L1 larvae and (PDF)Click here for additional data file.S3 Figapoptotic pathway function.(A) Images of the heads of freshly hatched L1 animals. Persistent apoptotic cell corpses are marked with white arrows.ced-10 mutant animals. The number of persistent cell corpses was counted at the bean stage of embryogenesis. Data are expressed as mean ±SD and statistical significance was assessed using one-way ANOVA, with Tukey’s multiple comparison test. ****<0.0001, n.s., not statistically significant from wild type. n = 21, from three independent scorings.(B) Quantification of apoptotic corpse engulfment in ced-10(rp100) mutant animals. Data are expressed as mean ±SD and statistical significance was assessed using one-way ANOVA, with Tukey’s multiple comparison test. ****<0.0001, *<0.01, n.s., not statistically significant from wild type. n = 15, from three independent scorings.(C) Quantification of apoptotic corpse engulfment in wild type and ced-10(rp100) L1 larvae combined with mutations in CED-6/GULP and CED-12/ELMO. Data are expressed as mean ±SD and statistical significance was assessed using one-way ANOVA, with Tukey’s multiple comparison test. n.s. not statistically significant comparing ced-6 and ced-12 single mutants combined with ced-10(rp100). n = 17, from three independent scorings.(D) Quantification of persistent cell corpses present in the head of freshly hatched (E) Quantification of PVQ outgrowth defects in wild type and mutants of apoptotic pathway genes. Data are expressed as mean ±SD and statistical significance was assessed using one-way ANOVA, with Tukey’s multiple comparison test. ****<0.0001, n.s. not significantly different to wild type. n = 75 per strain, each dot represents independent scoring replicates.(F) Quantification of total PVQ defects (outgrowth and guidance) in wild type and mutations in apoptotic pathway genes. Data are expressed as mean ±SD and statistical significance was assessed using one-way ANOVA, with Tukey’s multiple comparison test. ****<0.0001, n.s. not significantly different to wild type. n = 75 per strain, each dot represents independent scoring replicates.(PDF)Click here for additional data file.S4 Figgenetic analysis of NAB-1 function in PVQ development. Quantification of total PVQ defects (outgrowth and guidance) in wild type, ced-10(rp100) and compound mutants between ced-10(rp100) and nab-1 alleles . Data are expressed as mean ±SD and statistical significance was assessed using one-way ANOVA, with Tukey’s multiple comparison test. ****<0.0001, comparing wild type and rp100 animals, n.s. not statistically significant when compared to either wild type (for single mutants) or ced-10(rp100) for compound mutants. n = 90 per strain, each dot represents independent scoring replicates.(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.


From analysis of TCGA data, we found that PNI is significantly associated with lymphovascular invasion, pathological nodal invasion, and pathological tumor staging . In adjusted models, we observed that the NGF receptor p75NTR (NGFR) and the TNF-α receptor 1 (TNFRSF1A) were associated with PNI (both p<0.05) and significantly correlated to each other . High levels of TNF-α were present in HSC-3 cell lines and the mouse xenograft cancers. In mice with cancer pain, combined treatment with anti-NGF and anti-TNF-α together provided more effective pain control compared with either anti-NGF or anti-TNF-α treatment alone (p<0.05). We found significantly increased levels of MIP3a, IL-1b, IL-2, IL-4, IL-28b, IL-23, IL17a, IL-31, and IL-33 in cancer mice compared with normal mice . The combination therapy significantly reduced cytokines MIP3a, IL-1b, IL-4, IL-28b, IL-31, and IL-33 . DISCUSSION/SIGNIFICANCE OF IMPACT: We show that targeting both NGF and TNF-α provides more effective pain relief in an oral cancer model. These results suggest that therapeutic strategies aimed at both pathways could yield improved pain management for oral cancer patients.OBJECTIVES/SPECIFIC AIMS: We hypothesize that both NGF and TNF-α contribute to oral cancer pain by upregulating pro-nociceptive inflammatory cytokines. METHODS/STUDY POPULATION: In total, 48 oral cancer patients were evaluated and their pain scores were measured using a validated oral cancer pain questionnaire. Presence of perineural invasion (PNI) was identified from patients’ pathology reports. We utilized The NIH Cancer Genome Atlas (TCGA) Head and Neck Cancer cohort to investigate the association between pain and genes related to NGF, TNF-α, and their receptors  in oral cancer samples by employing PNI as a surrogate for pain. Demographic characteristics, clinical characteristics, and genes were analyzed using logistic regression models. A xenograft cancer pain model was created by inoculating human oral cancer cells (HSC-3) into the mouse hind paw. Mice (n=6 per group) were treated with anti-NGF alone, anti-TNF-α alone, a combination of anti-NGF and anti-TNF-α, or PBS (vehicle control). Nociceptive behaviors were measured using an electronic paw withdrawal assay. Paw volume was measured using a plethysmometer. Cytokines in the paw tissues were measured using a multiplex assay kit with 28 cytokines. RESULTS/ANTICIPATED RESULTS: Oral cancer patients with PNI report significantly more pain compared with patients without PNI in our patient cohort (


C9ORF72 expansion is the most common genetic alteration in both familial and sporadic ALS [C9ORF72 transcripts and may be toxic to cells [C9ORF72-associated amyotrophic lateral sclerosis (C9ALS), being identified in small neurons of hippocampus and cerebellum, as well as in neocortical neurons [to cells , 14 in ato cells . DPRs in neurons  and rare neurons . DPRs ma neurons  and are  neurons .C9ORF72 model demonstrated DPRs in skeletal muscle [C9ORF72 model showed distinct perinuclear poly-GP inclusions in muscle [Several studies have also suggested that DPRs are not limited to neurons. One study of human tissue, applying antibodies against GGGGCC RAN-translated peptides, showed DPRs predominantly in neurons, but also in Sertoli cells . Subsequl muscle , as welln muscle . This suTo address this question, we examined DPR inclusion pathology in 68 C9ALS skeletal muscle samples obtained from autopsies at Houston Methodist Hospital (HMH) and Mayo Clinic Jacksonville (MCJ). We assessed samples for the most common DPRs in human disease , poly-GR, as well as p62, N-terminal TDP-43, and phospho-TDP-43 (pTDP-43) pathology, using previously described methods  . Ultrastructural evaluation of a DPR-positive sample showed homogeneous intranuclear material and haphazardly arranged tubule-like structures in close proximity to subsarcolemmal nuclei. These ultrastructural findings were not seen in non-C9ALS postmortem muscle samples; however, DPRs were too sparse to definitively identify without immunoelectron microscopy, which will be a focus of future studies. Phospho-TDP-43 inclusions, also shown by N-terminal TDP-43, were present in 22 samples (32.4%) from 17 patients (46.0%) and co-localized with p62 . TDP-43-positive foci were uniformly negative for phospho-tau and beta-amyloid and their ultrastructural correlates have previously been shown [DPRs were identified in 28 of 68 samples 41.2%) from 18 of 37 patients (48.6%). Figure .2% from Z = 4.4, P = 1.12E − 05) . DPR pathology did not significantly associate with age at death, disease duration, or pTDP-43 pathology. Among samples with any inclusion pathology, 40% of patients had DPRs and pTDP-43, 32% had DPRs only, and 28% had pTDP-43 only. DPR-positive muscle samples were from patients with lower extremity (44.4%), upper extremity (22.2%) and bulbar (22.2%) onset sites. pTDP-43-positive and negative muscle samples did not significantly differ with respect to disease duration (P = 0.37), though a trend was seen for pTDP-43 pathology in younger C9ALS patients (P = 0.06). DPR-positive and pTDP-43-positive samples did not significantly differ in frequency by axial muscle group.DPRs were significantly associated with more severe atrophy in samples . RAN-translated peptides were not seen in three patient muscle samples in a prior study . A separxpansion . Future Supplementary file1 (PDF 960 kb)Below is the link to the electronic supplementary material.


Non-small cell lung cancer (NSCLC) is among the leading causes of human mortality due to a lack of effective treatments. Conventional chemotherapies affect healthy cells and cause multidrug resistance, while tumors may eventually develop resistance to less-toxic targeted therapies. Thus, the need to develop novel therapies for NSCLC is urgent. Here, we show that Nile tilapia-derived Tilapia piscidin (TP) 4 is cytotoxic to a panel of NSCLC cells with different genetic profiles. We observed that TP4 triggers NSCLC cell death through the necrosis and combining TP4 with potent Epidermal growth factor receptor (EGFR)- tyrosine kinase inhibitors (TKI)s, Erlotinib, and Gefitinib, improved drug responses in EGFR-mutated NSCLC cells, but not in EGFR-wild-type NSCLC cells. This work provides novel insights into potential NSCLC treatments, which may utilize antimicrobial peptide TP4 as monotherapy or in combination with EGFR-TKIs. Lung cancer is the leading cause of cancer mortality worldwide . Most luOreochromis niloticus)-derived cationic AMP, tilapia piscidin (TP4) [Antimicrobial peptides (AMPs) are evolutionarily conserved peptides that function to combat microbial infections  and havein (TP4) , binds tin (TP4) . NSCLC cin (TP4) ,31.In this study, we aimed to investigate the therapeutic potential of TP4 in NSCLC. We show that TP4 is highly cytotoxic to multiple NSCLC cell lines with different EGFR status. Moreover, NSCLC cells with wild-type EGFR were equally susceptible to TP4 alone and in combination with EGFR-TKIs, but combined treatment of TP4 with EGFR-TKIs showed enhanced cytotoxicity over TKIs or TP4 alone in EGFR-mutated NSCLC cells. Furthermore, TP4 was found to induce necrotic death in NSCLC cells. Combining TP4 with EGFR-TKIs enhanced necrosis in NSCLC cells with EGFR mutations. These findings support the notion that TP4 is a promising candidate drug for treatment of various NSCLC types.50) of over 15.48 and 26.50 μM, respectively  were used to test the efficacy of combined treatments. In A549 cells, a combination of 10 μM Erlotinib (Erlo) or Gefitinib (Gef) with a range of TP4 concentrations (3.35–6.71 μM) showed enhanced cytotoxicity A. In NCIWe next examined the cell death pathway triggered by TP4 in NSCLC cells. Treatment of TP4 for six and 24 h induced lactate dehydrogenase (LDH) release from NSCLC cells A,B, suggIn this work, we show that the antimicrobial peptide, TP4, shows excellent cytotoxicity toward NSCLC cells with different EGFR status, and combining TP4 with potent EGFR-TKIs enhanced cytotoxicity in EGFR-mutated cells. The ratio of surviving EGFR-mutated H1975 cells and HCC827 cells was decreased from 17.6−25.6% (10 μM TKIs) to 1.7%−13.6% (10 μM TKIs + 3.35−6.71 μM of TP4) and 47.1−50.7% (1 μM TKIs) to 3%−25.5% (1 μM TKIs + 3.35−6.71 μM of TP4) E,F, suggFOSB knockdown attenuated TP4-mediated cytotoxicity [FOSB (or FOSΔB) requires Ca2+ signaling and mitochondrial dysfunction to trigger necrotic death [2+-dependent FOSB signaling is involved in TP4-induced cytotoxicity. In addition, TP4 was shown to induce apoptosis in an osteosarcoma cell-line, MG63, through activation of extrinsic Fas/FasL- and intrinsic mitochondria-mediated pathways [2+ homeostasis are also involved in TP4-mediated cytotoxicity in NSCLC cells remains to be further addressed.The mechanism of TP4-mediated cytotoxicity may differ depending on the cancer cell type, transcriptome, or regulation of specific genes. For example, TP4 was found to induce FBJ murine osteosarcoma viral oncogene homolog B (FOSB) activation in all tested TNBC cells. Interestingly, in MDA-MB-231 and MCF7 cells, full-length FOSB is induced by TP4, while the alternatively spliced form (truncated) of FOSB (FOSΔB) is not; conversely, TP4 mainly induces FOSΔB in MB453 cells . Moreovetoxicity . TP4 indic death , suggestpathways . Pretreapathways . These f50 of 2.5 μM. Meanwhile, magainin analogs (magainin A and G) were shown to exhibit antitumor activity in SCLC cell lines but were less-toxic to normal human fibroblast cell lines [50 of MAG A and G in the SCLC cell lines were 8.64 and 8.82 µM, respectively, while the average IC50 of MAG A and G against normal human fibroblast cell lines were 21.1 and 29.2 µM, respectively. In addition, two sequence-modified magainin analogues (MSI-136 and MSI-238), which were designed to enhance the amphiphilic structure, showed more toxicity to A549 cells compared to magainin 2 [50) of all L-form MSI-136 and all D-form MSI-238 to the A549 cells are 6 μg/mL (2.44 μM) and 10 μg/mL (4.06 μM), respectively. The all L-form of TP4 has an IC50 value about 1.922 μM -7-methoxy-6-(3-morpholin-4-ylpropoxy)quinazolin-4-amine (Gefitinib) and N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine hydrochloride (Erlotinib) were purchased from Sigma-Aldrich . Primary antibodies were purchased from the Cell Signaling   and EMD Millipore  . The Structural model and the helical projection of TP4 were generated, as previously described [TP4 (FIHHIIGGLFSAGKAIHRLIRRRRR) were synthesized and purified by GL Biochem Ltd.  as previously described . Necrosiescribed .3 cells were seeded into the wells of a 96-well plate and cultured overnight. During the drug treatment assay, inhibitors were added 30 minutes prior to TP4, and cell viability was determined at indicated time-points. LDH release was quantified with a Cytotoxicity Detection KitPLUS (LDH) , as previously described [Cell-lines (A549 (BCRC 60074), NCI-H661 (BCRC 60125), NCI-H209 (BCRC 60123), NCI-H1975 (ATCC CRL-5908), BEAS-2B (ATCC CRL-9609), and MRC-5 (BCRC 60023)) were purchased from the Bioresource Collection and Research Center (BCRC)  and the American Type Culture Collection (ATCC)  and cultured as suggested by BCRC and ATCC. For the cell viability assay, 4–5 × 10escribed .For Western blots, sample preparations were performed, as previously described , and werp < 0.05.Cells were plated at least in quadruplicate for the multi-well based assay. Data were collected from repeated experiments (n ≥ 3) and were analyzed by Prism5 software ). Statistical significance was determined by two-tailed Student’s t-test or one-way analysis of variance (ANOVA) with Bonferroni post hoc test. Differences were considered statistically significant at


Repetitive double (D)-TBI (two months after the S-TBI at a dose of 5Gy) further weakened HSPC function via mitochondrial ROS accumulation and senescence-associated β-galactosidase  activity. Oral administration of CA (20 mg/kg) five times before and once immediately after TBI ameliorated ROS generation and TBI-induced HSC senescence and its radioprotective effect was long lasting in S-TBI mice but not in D-TBI mice. Further, supplementation of CA also induced apoptotic cell death of colon cancer cells. Collectively, these findings indicate that CA has a dual effect, ameliorating HSC senescence-accompanied long-term BM injury in S-TBI mice and stimulating apoptotic cell death of colon cancer cells.Total body irradiation (TBI) serves as an effectively curative therapy for cancer patients and adversely causes long-term residual bone marrow (BM) injury with premature senescence of hematopoietic stem cells (HSCs), which is mediated by increased production of reactive oxygen species (ROS). In the present study, we investigated how the exposure time of TBI in a mouse model affects HSCs and whether the treatment of caffeic acid (CA), a known dietary phenolic antioxidant, has a radioprotective effect. Single (S)-TBI at a sublethal dose (5 Gy) caused relatively higher induction of mitochondrial ROS and senescence-related factors in HSCs than those in hematopoietic progenitor cells (HPCs) and Lineage Although moderate or high doses of TBI are administered to cancer patients for treatment, this treatment causes acute myelosuppression and long-term residual BM injury with senescence of HSCs ,2. BM daAll experimental procedures were approved by the Animal Welfare Committee of Chonbuk National University. C57BL/6 female mice (6 weeks old) were purchased from Damul Science  and equilibrated for seven days before the experiment. During the experimental period, mice were housed at 22 ± 1°C and 55 ± 5% humidity on an auto-cycling 12 h light/dark cycle with free access to food and water. Mice were exposed to 5 Gy TBI with Ɣ-rays by regulating dosage time that was based on the radioactive half-life of Ɣ-rays on a rotating platform . Two months after the first TBI, mice were again exposed to the same dose. CA was obtained from Sigma-Aldrich Co. LLC  and dissolved in 99% ethanol and diluted with distilled water before use. A final volume of 200 μl CA (20 mg/kg body weight) was administered five times (once every three days) before and once immediately after TBI to mice via oral gavage . The dose of CA for animal studies was determined on the basis of previous studies . To meas°C in a humidified atmosphere of 5% CO2.Mouse colon cancer (CT-26), human liver cancer (HepG2) and human breast cancer (MCF-7) cells lines were purchased from ATCC and cultured in RPMI 1640 and DMEM medium supplemented with 10% FBS and antibiotics (penicillin G and streptomycin) at 37-Sca-1-c-Kit+), Lin-Sca-1+c-Kit+ (LSK) cells, and hematopoietic stem cells  from BM were characterized with the following lineage markers: PE-Cy7-conjugated anti-CD3, anti-CD4, anti-CD8, anti-CD45R, anti-CD11b, anti-Gr-1, and anti-TER-119 (BD Biosciences); FITC or PE-conjugated anti-Sca-1 (BD Biosciences); APC-conjugated anti-c-Kit (BD Biosciences); PerCP/Cy5.5-conjugated anti-CD150 (eBioscience); or APC-Cy7-conjugated anti-CD48 (BD Biosciences). SA-β-gal activity and mitochondrial superoxide anion levels were assessed using C12FDG (Molecular Probes) and MitoSoxTM Red (Invitrogen), respectively. Expression of p16INK4a was determined with Alexa Flour 488-conjugated antibody (Santa Cruz Biotechnology) after fixation and permeabilization (BD Bioscience).Numbers of hematopoietic cells derived from BM were analyzed by multicolor flow cytometry . These cell populations were further analyzed with FlowJo software for phenotypical identification. Hematopoietic progenitor cells  were cultured in 35-mm dishes with MethoCult® methylcellulose-based medium (MethoCult® GF M3434) and the number of colonies formed was counted using standard criteria on day 12. For pre-B assays, BM cells (2×105 cells per dish) were divided into 35-mm dishes with MethoCult® methylcellulose-based medium (MethoCult® M3630), followed by the colony counting on day 7.For colony-forming unit (CFU) assay, BM cells  were mixed with an equal number of cells from competitor mice (CD45.1) and then transplanted into recipient mice exposed to lethal irradiation (10 Gy). B6 × SJL F1 hybrid mice (CD45.1/CD45.2 double-positive) were used as recipients to distinguish donor-derived cells from host-derived cells. CD150+CD48-LSK cells (1×103) from BM of control or S-TBI+CA mice  were co-transplanted with cells from competitor mice  into conditioned recipient mice (CD45.1/2). Peripheral blood (PB) cells were collected from recipient mice 5 months after transplantation. The ratio of CD45.1/CD45.2 was estimated by flow cytometry using FITC- and PE-conjugated antibodies (BD Biosciences).For donor-derived repopulating potential assays, 5×102EDTA and measured in the Department of Laboratory Medicine of Chonbuk National University Hospital. To analyze levels of white blood cells (WBCs), RBCs, and platelets in samples, an automated complete blood cell counter  was used.PB samples collected from cardiac puncture of mice were saved in Vacutainer®  plastic tubes coated with KA stock solution of 2’, 7’-dichlorodihydrofluorescein-diacetate (DCFH-DA)  was prepared in DMSO and stored at -20°C in the dark. When CT-26 cells reached 90% confluence on 60-mm culture dishes, the cells were treated with CA for 24 h and incubated with 10 µM DCFH-DA for 30 min. The fluorescence of DCF was recorded at 515 nm (FL-1H) using a FACS Calibur® system  and 10,000 events were counted per sample.Protein lysates were prepared from CT-26 cells treated with CA. Protein extracts (20 µg per sample) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 15% gels and electroblotted onto polyvinylidene difluoride membranes. Blots were washed with a solution containing 10 mM Tris-HCl (pH 7.6), 150 mM NaCl, and 0.05% Tween-20, blocked with 5% skim milk for 1 h, and then incubated with primary antibodies such as cleaved caspase-3  and β-Actin . After washing, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG. Immunoreactive bands were visualized by enhanced chemiluminescence  followed by exposure to X-ray film .CT-26 cells were sub-cultured for 24 h on cover slips in twenty-four-well culture plates. For the observation of cell morphological change, the cells were treated with CA for 24 h, washed with ice-cold PBS, and then fixed with 2% formaldehyde and 0.2% glutaraldehyde for 30 min. After washing with PBS, cell nuclei were stained with 4,6-Diamidino-2-phenylindole dihydrochloride  and nuclear morphology was visualized by fluorescence microscopy .t-test was used to determine significant differences; * < 0.05, ** < 0.01, *** < 0.001.All results were expressed as the mean ± standard deviation (SD). One-way analysis of variance was used for multiple comparisons using SPSS version 16.0 software . Student’s -Sca-1-c-kit+ cells), Lin-Sca-1+c-Kit+ cells (LSKs) and HSCs (CD150+CD48-LSK cells) [We compared mitochondrial ROS levels in hematopoietic stem progenitor cells (HSPCs) such as HPCs  from control, S-TBI, or D-TBI mice (CD45.2) and cells from competitor mice (CD45.1) into lethally irradiated recipient mice  of CD150+CD48-LSK cells from control, S-TBI, CA-treated S-TBI, D-TBI or CA-treated D-TBI mice (CD45.2) and competitor mice (CD45.1) into conditioned recipient mice (CD45.1/2). The low repopulating ability of S-TBI mice-derived HSCs in the recipient mice was undoubtedly improved by transplanting CA-treated S-TBI mice-derived HSCs up to the control mice-derived HSCs’ basal level  . Not onlal level . CA-treaal level . Taken a+CD48-LSK cells (5×102) from control or CA-treated S-TBI mice (CD45.2) and competitor mice (CD45.1) . Furthermore, CA-treated S-TBI mice maintained similar numbers of WBCs, RBCs and platelets in the PB compared to corresponding control mice . These findings suggest that the radioprotective effect of supplementary CA consistently lasts, granting normal HSC function and hematopoiesis, and thereby prolonging the survival of S-TBI mice.We subsequently investigated whether the oral administration with CA extends survival rate of mice exposed to sublethal TBI. Compared with control mice that showed 90% survival rate until 19 months, all mice in S-TBI and D-TBI groups died at 16 and 12 months, respectively, after TBI exposure . Oral adSince a delicate balance of intracellular ROS levels is required for regulating cancer cell function , we furtAlthough TBI is an everlasting curative therapy, it causes long-term residual BM injury accompanied with ROS-associated HSC senescence . In the in vitro studies showed that micromolar concentrations of CA did not induce apoptosis in cancer cells and required a higher concentration. Previous reports of anti-cancer effects of CA in other cancer cell lines used comparatively high concentrations of CA, consistent with our observations [ It was found that the toxicity of CA was extremely low and the dose not lethal in animals . Similarvations  -25. Of nvations  . Althougvations   and bonevations  , 29.Taken together, these findings indicate that CA could be clinically used as a supplemental drug to autonomously ameliorate HSC senescence-accompanied long-term BM injury and to stimulate apoptotic death of cancer cells in patients who need radiotherapy with chemotherapy. Further understanding of cell autonomous and non-autonomous mechanisms by which repetitive irradiation induces BM injury, as well as on the efficacy of dietary phenolic antioxidants in clinical applications for radiotherapy-required cancer patients will be needed.


Anaerosphaera aminiphila WN036T) indicated that GS7-6-2 represents a putative novel species within the genus Anaerosphaera (family Peptoniphilaceae).Strain GS7-6-2 was isolated from a mesophilically operated biogas fermenter. The 16S rRNA gene sequence (93.27% identity to  Anaerosphaera aminiphila WN036T) indicated that GS7-6-2 represents a putative novel species within the genus Anaerosphaera (family Peptoniphilaceae). Here, we report the draft genome sequence of GS7-6-2 as established by Illumina paired-end sequencing.Strain GS7-6-2 was isolated from a mesophilically operated biogas fermenter. The 16S rRNA gene sequence (93.27% identity to  Peptoniphilaceae  were recently defined as a group of organisms belonging to the genera Anaerococcus, Anaerosphaera, Helcococcus, Finegoldia, Gallicola, Murdochiella, and Parvimonas (Anaerosphaera aminiphila WN036T (SAMN10397725), isolated from a mesophilically operated biogas fermenter fed with maize silage, as established by Illumina paired-end sequencing.Organisms within the family rvimonas . The spervimonas . Most ofrvimonas . RemarkaAnaerosphaera sp. strain GS7-6-2 genome, 1 µg chromosomal DNA of a clonal pure culture, cultivated anaerobically in GS2 medium  indicated that GS7-6-2 represents a putative novel species within the genus Anaerosphaera (family Peptoniphilaceae) (For sequencing of the 2 medium  at 30°C,2 medium . The libAnaerosphaera sp. GS7-6-2 provided here will broaden the knowledge of the genus Anaerosphaera and the putative importance of its members in biogas reactor-related environments.The genome sequence of RLIH00000000. The version described in this paper is version RLIH01000000. Raw sequencing reads are available in the Sequence Read Archive under accession no. SRR8641363. The whole project is summarized in the NCBI under accession no. PRJNA504539.This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession no.


C. elegans generated by transposon- or CRISPR/Cas9- mediated genome editing of the endogenous sod-1 gene. Introduction of ALS patient amino acid changes A4V, H71Y, L84V, G85R or G93A into the C. elegans sod-1 gene yielded single-copy/knock-in ALS SOD1 models. These differ from previously reported overexpression models in multiple assays. In single-copy/knock-in models, we observed differential impact of sod-1 ALS alleles on glutamatergic and cholinergic neurodegeneration. A4V, H71Y, G85R, and G93A animals showed increased SOD1 protein accumulation and oxidative stress induced degeneration, consistent with a toxic gain of function in cholinergic motor neurons. By contrast, H71Y, L84V, and G85R lead to glutamatergic neuron degeneration due to sod-1 loss of function after oxidative stress. However, dopaminergic and serotonergic neuronal populations were spared in single-copy ALS models, suggesting a neuronal-subtype specificity previously not reported in invertebrate ALS SOD1 models. Combined, these results suggest that knock-in models may reproduce the neurotransmitter-type specificity of ALS and that both SOD1 loss and gain of toxic function differentially contribute to ALS pathogenesis in different neuronal populations.Mutations in Cu/Zn superoxide dismutase 1 (SOD1) lead to Amyotrophic Lateral Sclerosis (ALS), a neurodegenerative disease that disproportionately affects glutamatergic and cholinergic motor neurons. Previous work with SOD1 overexpression models supports a role for SOD1 toxic gain of function in ALS pathogenesis. However, the impact of SOD1 loss of function in ALS cannot be directly examined in overexpression models. In addition, overexpression may obscure the contribution of SOD1 loss of function in the degeneration of different neuronal populations. Here, we report the first single-copy, ALS knock-in models in  e.g. A4V) primarily affect cholinergic spinal neurons, while other alleles affect both cholinergic and glutamatergic neurons. New genome editing techniques allowed us to create the first C. elegans knock-in/single-copy models for SOD1 ALS by directly editing the C. elegans sod-1 gene to recreate SOD1 amino acid changes that cause ALS in patients. These new models are complementary to previously described overexpression models, which revealed mutant SOD1 toxic gain of function properties. By contrast, in the new C. elegans knock-in models, we find that both loss and gain of sod-1 function contribute to neurodegeneration. C. elegans cholinergic motor neuron loss is primarily driven by toxic gain of function, but glutamatergic neuron loss is primarily driven by loss of function. Only cholinergic and glutamatergic neurons degenerate in C. elegans knock-in models; dopaminergic, serotoninergic and GABAergic neurons do not. This pattern of neuronal loss is reminiscent of the pattern of neuronal loss seen in SOD1 ALS patients. Strikingly, in the C. elegans A4V model, only cholinergic neurons are lost. Our results suggest that an underlying premise of the ALS field–that identical pathological mechanisms lead to degeneration of cholinergic and glutamatergic neurons–should be reconsidered. Mechanisms that predominantly drive glutamatergic and cholinergic neuron degeneration in ALS may not be identical.In all SOD1 ALS patients, cholinergic spinal motor neurons degenerate, but degeneration of cortical glutamatergic neurons is less common. Despite decades of work, it remains unclear why some disease alleles ( Amyotrophic lateral sclerosis (ALS) is an adult-onset fatal neurodegenerative disorder marked by the progressive loss of glutamatergic and cholinergic motor neurons. It is the most common motor neuron disorder affecting adults. The first ALS-linked mutations were discovered in a gene encoding the antioxidant enzyme Cu/Zn superoxide dismutase 1 (SOD1) [In patients, misfolded SOD1 is a major constituent of proteinaceous inclusions in the affected neurons –4 and paPublished studies to date have primarily relied on human mutant SOD1 protein overexpression models to examine neuronal dysfunction –15. OverHowever, SOD1 overexpression models may not permit the study of disease mechanisms in entirety for several reasons. First, overexpression of wild type SOD1 protein has deleterious effects in model organisms ,18, makiC. elegans demonstrate neuronal and muscular dysfunction [C. elegans. We edited the C. elegans sod-1 gene to create A4V, H71Y, L84V, G85R and G93A missense mutations. In the resulting single-copy ALS model animals, we observed oxidative stress induced glutamatergic and cholinergic neuron degeneration. While H71Y and G85R affected both cholinergic and glutamatergic neurons, A4V and G93A models affected cholinergic neurons only. However, other neuron classes were relatively spared. Overall, we found that cholinergic and glutamatergic neurons are differentially sensitive to SOD-1 loss and gain of toxic function. Our results suggest that both loss and gain of toxic SOD-1 function may be involved in disease pathogenesis.Previously reported ALS SOD1 overexpression models in function ,22–24. TC. elegans ALS SOD1 models, we compared human SOD1 and C. elegans SOD-1 protein sequences. Alignment of the two proteins revealed 71% similarity and 56% identity between species  rescue construct. Therefore, an additional “empty” negative control carrying the unc-119(+) construct alone was generated; these lack any of the sod-1 sequences inserted in the remainder of the MosSCI models. All alleles generated by MosSCI were named with an “M” superscript , to distinguish them from the endogenous C. elegans sod-1 gene on chromosome II. Subsequently, each transgene was crossed into the sod-1(tm776) loss of function background, referred to hereafter as sod-1(-). The resulting strains were homozygous for control or single-copy ALS sod-1 transgenes. All the comparisons between ALS sod-1 models and controls reported here were made in sod-1(-) background, unless indicated otherwise.Using Mos1-mediated single copy insertion (MosSCI ), we recequences . To contosome IV . To faciC. elegans sod-1 gene to recreate ALS SOD1 mutations for L84V, G85R and G93A in the endogenous sod-1 gene on chromosome II . To demonstrate reproducibility across strains generated by MosSCI and CRISPR/Cas9, we created the G85R allele twice, once using each technique , we directly edited the endogenous osome II . CRISPR/utations . Models echnique .sod-1 models to previously published neuronal overexpression models provided by the Horwich lab [sod-1 model animals carrying patient amino acid changes had overt locomotion defects . While single-copy ALS sod-1 models increased the formation of hSOD1WT-YFP protein inclusions, loss of sod-1 did not alter formation of hSOD1WT-YFP inclusions in these neurons (P = 0.61 for emptyM vs sod-1WTM and P = 0.12 for sod-1(-) vs sod-1(+); sod-1(+) refers to the standard N2 wild type unedited allele). We conclude that loss of sod-1 function does not alter propensity to form neuronal SOD1 inclusions in C. elegans motor neurons, but that most C. elegans single-copy ALS sod-1 models show increased hSOD1WT-YFP inclusion formation.ALS SOD1 mutations lead to formation of SOD1-rich proteinaceous inclusions in motor neurons –4. Consi muscles ,23. We fsod-1 function, coupled with an increase in oxidative damage, could exacerbate defects in ALS SOD1 models. To determine the impact of oxidative stress in single-copy ALS sod-1 models, we exposed animals to paraquat, an oxidative stress inducing herbicide [sod-1 function decreased survival under paraquat-induced oxidative stress (P < 0.001 for emptyM vs sod-1WTM and sod-1(-) vs sod-1(+), log-rank test), replicating a previously published finding [sod-1A4VM . By contrast, without external stress, survival was unaffected or slightly decreased in these genotypes . A significantly larger fraction of sod-1H71YM and sod-1G85RM animals had more neuronal inclusions per animal compared to wild type controls . Moreover, sod-1A4VM, sod-1H71YM and sod-1G85RM animals had larger neuronal inclusions compared to wild type controls , compared to appropriate wild type controls . Conversely, degeneration was not observed in control animals lacking sod-1 or in sod-1L84VC animals after paraquat treatment  allele on chromosome II into sod-1A4VM, sod-1H71YM or sod-1G85RM animals did not rescue paraquat-induced cholinergic motor neuron degeneration  into the hSOD1G85R-YFPOE animals failed to rescue stress induced degeneration  dysfunction is an early defect in ALS patients . Heterolfunction , based oP < 0.05, log-rank test and [C. elegans . HoweveC. elegans . However, sod-1G85RC and sod-1H71YM animals were hypersensitive to aldicarb compared to wild type controls . Thus, even though single-copy models are not uniform in their response to aldicarb, none of these strains were resistant to aldicarb-induced paralysis. Overall, we observed dramatically different consequences for C. elegans NMJ function in overexpression versus single-copy ALS model animals.Next, we examined the impact of single-copy ALS models on NMJ function. The response of wild type control animals was indistinguishable from non-transgenic  elegans . And, insod-1 loss of function on C. elegans NMJ function has not been reported previously. We found that loss of endogenous sod-1 function lead to aldicarb hypersensitivity, compared to standard N2 strain sod-1(+) controls (P < 0.001 for sod-1(-) vs sod-1(+)). To confirm this change was due to decreased sod-1 activity, and to establish conservation of SOD1 function across species, we undertook phenotypic rescue studies. Introduction of C. elegans sod-1WTM to sod-1(-) fully restored normal response to aldicarb (sod-1WTM vs emptyM), and introduction of neuronal human SOD1WT-YFP partially restored aldicarb response (sod-1(-); hSOD1WT-YFPOE vs sod-1(-)). These results suggest that NMJ functional defects in ALS sod-1 model animals may be, in part, driven by loss of sod-1 function.The impact of sod-1 loss of function contributes to NMJ defects, we also examined the consequences of altering sod-1 dosage. First, we confirmed that restoring sod-1 function would restore normal NMJ function in sod-1H71YM animals. We crossed the unedited endogenous sod-1(+) allele into sod-1H71YM allele background. These animals had normal response to aldicarb (sod-1(+); sod-1H71YM vs sod-1(+); sod-1WTM, P = 0.05).To confirm that sod-1 animals is driven primarily by sod-1 loss of function, we examined aldicarb response in animals heterozygous for ALS sod-1 transgenes. Homozygous ALS sod-1 animals were crossed to homozygous sod-1WTM or emptyM males carrying a GFP-expressing transgene. Then, the heterozygous GFP-positive cross-progeny were examined for aldicarb resistance after paraquat treatment. As control, we confirmed that emptyM/emptyM cross-progeny were hypersensitive to aldicarb compared to sod-1WTM/sod-1WTM cross-progeny . Next, we determined if the sod-1H71YM aldicarb response defect is recessive. We found that heterozygous sod-1WTM/H71YM animals had normal aldicarb response . Taken together, these findings suggest that loss of sod-1 function contributes to the NMJ functional defects in both sod-1(-) and sod-1H71YM animals.Introduction of one functional copy of sod-1 models on NMJ proteins, we determined the localization and intensity of fluorescently-labelled pre-synaptic proteins in live animals using previously described transgenic strains and protocols in adult sod-1H71YM and sod-1G85RM animals. Size, distribution and intensity of presynaptic synaptobrevin/SNB-1 and intersectin-1/ITSN-1 fluorescent punctae were normal in sod-1H71YM and sod-1G85RM animals  animals compared to appropriate wild type controls ; Activity Index, Travel Speed, and Wave Initiation Rate metrics were all increased, consistent with increased activity. Combined, NMJ defects and locomotion assays results suggest that loss of sod-1 function may increase NMJ signaling, without dramatic perturbation in synapse structure and number. Cholinergic motor neuron defects might be expected to change locomotion in sod-1G85RM model animals; we found that oxidative stress also increased their locomotion activity, compared to controls. At first glance, this is counterintuitive as cholinergic motor neuron degeneration should impair locomotion. However, modest loss of cholinergic motor neurons does not dramatically impair C. elegans locomotion based on laser ablation studies [C. elegans can respond to noxious environments with a coordinated escape response by decreasing spontaneous reversals and increasing forward locomotion speed [sod-1G85RM, sod-1H71YMsod-1G85RC, or sod-1 loss of function animals overnight on culture dishes containing paraquat resulted in high rates of escape; after 24 hours, roughly 40% of sod-1H71YM, 47% of emptyM, and 38% of sod-1G85RM animals left the 2.5mM paraquat culture dishes, compared to only 2% of sod-1WTM control animals. Similarly, 23% of sod-1G85RC animals left after overnight exposure to paraquat, compared to only 1% of sod-1WTC animals . Combined, our results suggest that the increased locomotion activity observed in sod-1G85RC animals is likely an escape response and that single-copy ALS sod-1 models differ significantly from previously described human SOD1 overexpression models in their impact on NMJ function.Considering that oxidative stress exacerbated defects in single-copy ALS comotion  using co studies ,33. Addion speed . Consisti.e. dat-1p::GFP, tph-1::GFP, unc-47p::GFP, and osm-11p::GFP. As the single-copy sod-1G85R models show robust cholinergic motor neuron loss after paraquat stress, this genetic model was used to assess loss of other GFP-labelled neurons after paraquat-induced oxidative stress. For dopaminergic neurons, all four CEPs and both ADE neurons were scored, as well as ADE neuron sensory processes. For serotonergic neurons, both NSM and both ADF neurons were scored. For GABAergic neurons, nineteen GABAergic motor neurons in the ventral cord were scored. For glutamatergic neurons, both ASH neurons were scored.Neuron loss in ALS patients is generally limited to specific subsets of cholinergic and glutamatergic neurons . We examC. elegans found significant dopaminergic neuron degeneration in younger animals after a more extended exposure period to paraquat [sod-1G85RC, sod-1(-), or control animals after paraquat-induced oxidative stress  animals had less than 10% affected animals  animals after paraquat treatment, while virtually all neurons were intact in sod-1WTC animals . Degenerative sensory process loss or cell death completely stops dye uptake by individual neurons, as shown in C. elegans models of Huntington’s Disease polyglutamine toxicity [C. elegans ALS sod-1 model animals and in animals lacking sod-1 function . ASH, PHA and PHB neurons failed to take up DiD in sod-1(-) animals after paraquat-induced oxidative stress  animals. DiD uptake defects were dramatically reduced in the resulting sod-1(-); hSOD1WT-YFPOE animals  animals did not ameliorate the dye uptake defects  had no degeneration, confirming that loss of sod-1 function is not dominant. When we examined animals hemizygous for sod-1H71YM or sod-1G85RM (sod-1H71YM/emptyM and sod-1G85RM/emptyM), they had ASH, PHA and PHB dye-uptake defects after paraquat treatment, suggesting that these alleles cause sod-1 loss of function  allele was present, based on dye-filling after paraquat treatment. By contrast, a modest level of degeneration was observed in sod-1(+); hSOD1G85R-YFPOE animals  were relatively spared, suggesting that single-copy ALS SOD1 knock-in models in C. elegans may recapitulate the selective sensitivity of cholinergic and glutamatergic neurons in ALS caused by SOD1 mutations. Furthermore, we found that sod-1 loss of function is a major contributor to glutamatergic neuron degeneration after oxidative stress. However, sod-1 gain of function likely drives cholinergic motor neuron degeneration. Combined, these results suggest that C. elegans knock-in models, at a minimum, complement overexpression models and provide unique insights into why different SOD1 mutations lead to degeneration in different types of neurons.Herein, we report the first single-copy/knock-in models for ALS SOD1 in C. elegans single-copy/knock-in models for A4V, H71Y, G85R and G93A increased accumulation of the YFP-tagged human wild type SOD1 protein in small cytosolic inclusions within C. elegans motor neurons, even under optimal growth conditions. These hSOD1WT-YFP inclusions were possibly seeded by misfolded mutant C. elegans SOD-1 protein in single-copy/knock-in models. Are these inclusions caused by loss or gain of sod-1 function? Because inclusions were not increased in C. elegans with decreased sod-1 function, this is likely a gain of function defect. It seems likely that introduction of the ALS patient amino acid changes into the C. elegans SOD-1 protein conferred a neomorphic/novel gain of function that increased inclusion propensity. In contrast with these four alleles, we found that hSOD1WT-YFP inclusions were not increased in the motor neurons of sod-1L84VC animals. Previous studies have shown that driving human L84V SOD1 expression in cells or transgenic mice results in SOD1 aggregation [sod-1L84VC to increase inclusions in C. elegans neurons may arise from differences between human and C. elegans SOD1 proteins. Consequently, the usefulness of the C. elegans sod-1L84VC model remains unclear.Cytoplasmic SOD1 aggregates are found in ALS SOD1 patients and in most ALS SOD1 overexpression models ,23,40–42regation . The faiC. elegans cholinergic motor neurons do not die in young adult animals, in either single-copy/knock-in models described herein or in human SOD1 ALS models overexpression models described previously [sod-1 models were hypersensitive to oxidative stress, except sod-1L84VC. Oxidative stress lead to motor neuron loss in A4V, H71Y, G85R and G93A single-copy/knock-in animals, and in hSOD1G85R-YFPOE overexpression model animals [sod-1 function and introduction of wild type sod-1 did not rescue cholinergic motor neuron degeneration in A4V, H71Y or G85R single-copy/knock-in models. Oxidative stress exposure also increased the number and/or size of neuronal SOD1 inclusions in these ALS SOD1 models. These results are consistent with previous work showing misregulation of cellular stress response pathways in ALS SOD1 models [C. elegans cholinergic neurons.ALS is characterized by degeneration of lower cholinergic motor neurons. However, a significant fraction of patients present with cortical glutamatergic neuron degeneration . Given teviously . We foun animals , which h1 models  and incr1 models ,46. CombC. elegans single-copy/knock-in G85R and H71Y SOD1 ALS model animals, glutamatergic neurons were also hypersensitive to oxidative stress; paraquat treatment led to degeneration of neurons in these animals, but not in control animals. Similarly, loss of sod-1 function resulted in glutamatergic neuron degeneration after oxidative stress. The defects in H71Y and G85R single-copy/knock-in animals are recessive and introduction of hSOD1WT-YFP ameliorated defects in sod-1(-) animals. Combined, these results suggest that oxidative stress hypersensitivity in glutamatergic neurons is a C. elegans sod-1 loss of function defect. This is consistent with studies in mice; SOD1 loss of function results in degenerative changes [C. elegans glutamatergic and cholinergic neurons stands in contrast to the lack of neurodegeneration observed in dopaminergic and serotonergic neurons under the same oxidative stress conditions. Additionally, even after oxidative stress, C. elegans GABAergic motor neurons are relatively spared in single-copy/knock-in models. This level of specificity is unusual and suggests that the mechanisms underlying ALS specificity for specific neuronal classes/neurotransmitter subtypes may be recapitulated in C. elegans knock-in models.Defects in glutamatergic corticospinal tracts can be detected in many ALS patients ,29 and g changes ,47. The C. elegans; hSOD1WT-YFPOE animals paralyze more slowly than normal animals in the presence of aldicarb  decreased sod-1 function may be more deleterious for glutamatergic neurons and 2) gain of function may be the major contributor to cholinergic neuron degeneration. To our knowledge, this hypothesis has not been previously explored and it may shed light on the connections between ALS and Frontotemporal Dementia (FTD). There is considerable genetic and pathological overlap between these diseases [Results presented herein suggest that an underlying premise of the ALS field–that identical pathological mechanisms lead to degeneration of cholinergic and glutamatergic neurons–should perhaps be reconsidered. Mechanisms contributing to glutamatergic and cholinergic neurons may not be identical. The differential susceptibility of cholinergic and glutamatergic neurons in diseases , but it sod-1 gene, including the sod-1 promoter, introns and UTRs, was amplified from wild type N2 genomic DNA with primers sodF and sod-1RC using the Roche Expand Long Range Kit (SN:04829034001). The resulting sod-1 fragment was subcloned into the pPD#49.26 vector with NheI+XmaI and mutagenized using the QuickChange II Site-Directed Mutagenesis Kit with primer pairs specific for A4V, H71Y and G85R. These primers are listed under sod-1 fragment was excised from the corresponding pPD#49.26 plasmid with NheI+XmaI, and ligated into the pCFJ178 Mos vector with AvrII+XmaI to generate final constructs pHA#725 sod-1p::sod-1A4VM, unc-119(+); pHA#702 sod-1p::sod-1H71YM, unc-119(+) and pHA724 sod-1p::sod-1G85RM, unc-119(+). The wild type sod-1 fragment was cloned directly into the pCFJ178 Mos vector following PCR amplification and NheI+XmaI digestion to generate pHA#720 sod-1p::sod-1WTM, unc-119(+). Another plasmid carrying unc-119(+) alone, pHA#723 unc-119(+), was generated as “empty” negative control.A 4391 bp fragment spanning the endogenous wild-type sod-1 allele, ALS mutant sod-1 alleles and the empty control carrying unc-119(+) rescue alone was integrated into chromosome IV using the Mos1/transposase-mediated homologous recombination system . The final constructs pHA#720 sod-1p::sod-1WTM, unc-119(+); pHA#725 sod-1p::sod-1A4VM, unc-119(+); pHA#702 sod-1p::sod-1H71YM, unc-119(+); pHA#724 sod-1p::sod-1G85RM, unc-119(+) and pHA#723 unc-119(+) were injected at 50 ng/ml with the standard MosSCI cocktail into EG6700 (unc-119(ed3)III; cxTi10882 IV) animals along with co-injection markers. Single-copy insertion events were detected by rescue of unc-119(ed3) in non-fluorescent animals, and confirmed by PCR genotyping and sequencing. All strains were backcrossed at least four times. The endogenous unc-119(ed3) allele was PCR amplified and sequenced to check the replacement of unc-119(ed3) with wild type unc-119(+) in all strains. Transgenic animals were subsequently crossed into the sod-1(tm776) null background to generate single-copy insertion models homozygous for wild-type sod-1, mutant sod-1 and for sod-1 deletion.The wild type sod-1 guide RNA was amplified from the pU6::klp-12::sgRNA (Addgene plasmid # 46170) [klp-12 targeting sequence with sod-1 specific primer pair sod-1_guide2_f and sod-1_guide2_r. The resulting DNA was circularized to generate the final construct pHA0816 pU6::sod-1::guide2.A U6 promoter-driven # 46170)  vector tsod-1 alleles L84VC, G85RC and G93AC were introduced into the endogenous sod-1 locus on chromosome II using CRISPR/Cas9-mediated oligo-templated homologous recombination and pha-1(ts) co-conversion, using previously published methods [pU6::sod-1::guide2 was injected at 50 ng/ul into temperature-sensitive GE24 (pha-1(e2123) III) animals with 50 ng/ul of Peft-3::Cas9, 50ng/ul of pJW1285 pha-1 sgRNA, 10 uM of 200mer sense pha-1(+) rescue oligo [sod-1WTC control was also generated to control for the silent mutations. Putative recombinants among pha-1(+) rescued animals were screened for insertion events with PCR amplification using primers sod-1cIIgenoF and sod-1cIIgenoRC1, followed by MscI digestion. Isolated alleles were verified by sequencing and backcrossed four times. The endogenous pha-1 locus was PCR amplified and sequenced to verify the replacement of repaired pha-1 allele with wild type pha-1(+) in all strains. methods . The finue oligo  and 10uMunc-17p::GFP or cho-1p::mCherry), GABAergic (unc-47p::GFP), glutamatergic (osm-10p::GFP), serotonergic (tph-1p::GFP) and dopaminergic (dat-1p::GFP) specific neuronal markers were individually crossed into ALS models to assess neuron loss. A full list of strains used in this study can be found in unc-17p::GFP or cho-1p::mCherry) neurons, animals missing at least two neurons were scored as defective. For scoring unc-47p::GFP, nineteen GABAergic ventral nerve cord motor neurons were scored. For scoring osm-10p::GFP, only ASH amphid sensory neurons neurons were scored due to variable/faint GFP expression in ASI and PHB neurons. For paraquat trials, animals were exposed to 2.5 mM paraquat overnight on plates.Cholinergic  D307) in a microfuge tube as in . After 1oC were scored for survival on alternating days starting from the first day of adulthood. FUDR was omitted from the growth medium as it alters C. elegans lifespan for some genotypes [Animal reared under normal culture conditions at 25 enotypes . To avoiDay 1 adult animals expressing human SOD1WT-YFP were mounted on 2% (vol/vol) agar pads and immobilized with 30 mg/mL 2-3-butanedione monoxime (BDM) (Sigma) in M9 buffer. Animals were quantified for inclusions within the motor neurons along the ventral nerve cord under 63x or 100x objectives (Zeiss AxioImager ApoTome and AxioVision software v4.8). For paraquat trials, animals were exposed to 2.5 mM paraquat for 3 hours on plates, as overnight exposure to paraquat problematically increased background fluorescence.oC were scored for paralysis on 1 mM aldicarb (Sigma-Aldrich 33386) over the course of 7 hours. NGM plates containing 1 mM aldicarb were freshly poured the day before the assay. Aldicarb plates were seeded with 30 ul of OP50, and left to dry open-lid under the hood for 30 minutes. Day 1 adult animals were then transferred onto aldicarb plates, and scored for paralysis every hour. Aldicarb-induced paralysis was scored as inability to move/pump to sequential prodding with a metal wire twice in the tail and then twice in the head. Paralyzed animals were removed from the plate and not re-counted.Animals reared at 25 n > 20 animals in total/genotype) was analyzed. Puncta total intensity, width, and linear density were quantified using the Punctaanalyser program in Matlab  [For synaptic puncta imaging, day 1 adult animals were mounted on 2% (vol/vol) agar pads and immobilized using 30 mg/mL 2-3-butanedione monoxime (BDM) (Sigma) in M9 buffer. Images were captured in z-stacks from dorsal cord posterior to vulva . Data from three independent trials (_001622) .Nose touch assays were performed as described elsewhere . AnimalsDay 1 adult animals treated overnight with 2.5 mM paraquat were transferred into 60 ul of M9 buffer pipetted into a 1 mm diameter ring on a microscope slide. Videos were recorded at 18 frames per second for 30 seconds, and analyzed with computer vision software CeleST .P < 0.05 was used to establish statistical significance. Error bars in figures represent error of the mean (S.E.M.).Data collection and analysis were performed by experimenters blinded to genotype and, when possible, treatment. Quantitative data was analyzed using Graph Pad Prism 6 software . Statistical significance for the survival and aldicarb resistance assays was determined with log-rank test. Kruskal-Wallis test was used to determine statistical significance for the swimming locomotion assays. For the remainder of the assays, two-tailed t-test or chi-square test was used to determine significance. A value of S1 Fig(A and B) To assess lifespan in C. elegans knock-in models, we scored survival at 25 oC on alternating days from pre-adulthood (L4 stage) until death. Single-copy sod-1G85RM (Panel A) and sod-1G85RC (Panel B) lifespan did not differ from those of appropriate wild type controls . Single-copy/knock-in animals sod-1A4VM, sod-1H71YM, sod-1L84VC and sod-1G93AC, as well as animals lacking endogenous sod-1 or emptyM controls, showed a modest decrease in lifespan, compared to their respective wild type controls. The largest difference in median lifespan was observed in sod-1H17YM animals; median lifespan decreased from 14 to 9 days. sod-1L84VC indicated with #. sod-1(+) is standard N2 strain. Survival was scored in the absence of FUDR under standard culture conditions, moving animals to new plates every other day to avoid progeny contamination. Animals that left the plate or animals with internal egg hatching were censored; these animals were included in lifespan determinations until the day before censoring. N was 30 for all genotypes in both panels in each of 3 independent trials. P < 0.05; ** P < 0.01; *** P < 0.001.(C and D) The impact of neuronal overexpression of human SOD1 on C. elegans lifespan has not been examined previously. We found that neuronal overexpression of human SOD1G85R (hSOD1G85R-YFPOE) did not decrease survival, relative to hSDOD1WT-YFPOE controls . And, lifespan of animals overexpressing the human wild type SOD1 protein (hSDOD1WT-YFPOE) in neurons was not different than sod-1(+) wild type controls . However, neuronal overexpression of human wild type SOD1 did not rescue lifespan in sod-1(-) animals, which lack the endogenous sod-1 gene (P = 0.44 for sod-1(-); hSOD1WT-YFPOE vs sod-1(-), Panel D). However, neuronal overexpression of human SOD1G85R in sod-1(-) background modestly restored lifespan compared to sod-1(-) animals; this difference is seen predominantly at midlife. Genotypes, analysis and methods as in Panels A-B, with 30 animals for all genotypes in both panels in each of three independent trials. (TIF)Click here for additional data file.S2 Fig(A and B) To determine the impact of oxidative stress in ALS SOD1/sod-1 models, we scored survival at 25°C every day from pre-adulthood (L4 stage) in animals exposed to 2.5 mM paraquat. sod-1H71YM and sod-1G85RM animals had decreased survival, compared to sod-1WTM animals under oxidative stress (Panel A). Loss of sod-1 in emptyM controls similarly decreased survival, relative to sod-1WTM wild type control animals. sod-1A4VM animals had increased survival under oxidative stress, compared to sod-1WTM controls. sod-1L84VC, sod-1G85RC and sod-1G93AC animals had decreased survival compared to sod-1WTC controls (Panel B). sod-1L84VC indicated with #. sod-1(+) is the standard N2 strain. Survival was scored in the absence of FUDR, moving animals to new plates every other day to avoid progeny contamination. Animals that left the plate or animals with internal egg hatching were censored; these animals were included in survival determinations until the day before censoring. N was 30 for all genotypes in both panels in each one of the three independent trials, except for one trial that was started with 20 sod-1G85RC animals in Panel B. Supplemental P < 0.05; ** P < 0.01; *** P < 0.001. sod-1(-): sod-1(tm776).(C and D) Neuronal overexpression of human SOD1G85R-YFP further decreased survival under paraquat-induced oxidative stress compared to hSOD1WT-YFPOE animals (Panel C). Loss of sod-1 decreased survival under oxidative stress compared to sod-1(+) wild type controls (Panel D). Additionally, oxidative stress sensitivity in sod-1(-) animals was partially rescued by neuronal hSOD1WT-YFPOE (Panel D). Genotypes, analysis and methods as in Panels A-B. N was 30 for all genotypes in both panels in each one of the three independent trials. (TIF)Click here for additional data file.S3 FigC. elegans NMJ can be detected as changes in the accumulation of fluorescently-tagged pre-synaptic proteins. We found that GFP-tagged SNB-1 (Panel A) and ITSN-1 (Panel B) accumulation and levels were unperturbed in single-copy sod-1H71YM and sod-1G85RM animals compared to sod-1WTM controls. Previous work reported that SNB-1::GFP is altered in human SOD1G85R overexpression animals [Perturbations in presynaptic signaling or loss of synapses at the  animals . N > 22 (TIF)Click here for additional data file.S4 Fig(A) Serotonergic neurons NSM, ADFL and ADFR (scored using tph-1p::GFP) were intact in sod-1G85RC and sod-1(-) animals after paraquat treatment. sod-1(+) designates the unedited wild type gene at the endogenous locus; this is the same allele present in standard N2 strain. For panels A-C: three independent trials. Error bars indicate ±SEM. N > 30 per genotype. Two-tailed t-test: * P < 0.05; ** P < 0.01; *** P < 0.001.(B) Dopaminergic neurons CEPVL, CEPVR, CEPDL, CEPDR, ADEL, ADER, and ADEL/R sensory processes (scored using dat-1p::GFP) were intact in sod-1G85RC and sod-1(-) animals after paraquat treatment. sod-1(+) as in Panel A.(C) ASH neurons (scored using osm-10p::GFP) were lost in sod-1G85RC and sod-1(-) animals after paraquat treatment, compared to sod-1WTC and sod-1(+) controls, respectively. PHA and PHB neurons were not scored due to inconsistent PHB GFP expression in wild type controls.(D) GABAergic motor neurons (scored using unc-47p::GFP) were lost only in the sod-1G85RM animals compared to sod-1WTM controls. Three independent trials. N > 20 per genotype. Chi-square test.(E) To examine the consequences of altering gene dosage and assess recessive/dominance of single-copy/knock-in ALS alleles, homozygous ALS sod-1 model animals and controls were crossed to homozygous sod-1WTM or emptyM males carrying a GFP-expressing transgene and cross-progeny tested for DiD dye-uptake in ASH neurons after paraquat treatment. emptyM/emptyM cross-progeny had defects after paraquat treatment, compared to sod-1WTM/sod-1WTM cross-progeny, while sod-1WTM/emptyM cross-progeny had intact glutamatergic neurons. sod-1H71YM/emptyM and sod-1G85RM/emptyM animals were defective compared to sod-1WTM/emptyM animals. Conversely, sod-1WTM/sod-1H71YM or sod-1WTM/sod-1G85RM animals had intact glutamatergic neurons after paraquat treatment. Three independent trials. N > 25 per genotype. Error bars indicate ±SEM. Two-tailed t-test: * P < 0.05; ** P < 0.01; *** P < 0.001.(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 TableoC on alternating days from pre-adulthood (L4 stage) until death. In oC every day from pre-adulthood (L4 stage) in animals exposed to 2.5 mM paraquat. sod-1(+) is the standard N2 strain. Survival was scored in the absence of FUDR. Animals were transferred to new plates every other day to avoid progeny contamination. Animals that left the plate or animals with internal egg hatching were censored; these animals were included in lifespan determinations until the day before censoring. N was 30 for all genotypes in all panels in each of 3 independent trials, except for sod-1G85RC in “+” paraquat R1 trial which had 20 animals Click here for additional data file.S5 TableDirectly comparable data sets are flanked by double lines. Appropriate controls are indicated within each experimental set. Genotypes with increases and decreases are indicated with a shade of red and blue, respectively. n.s., not significant; n.a., not available.(PDF)Click here for additional data file.


Mesua beccariana, Mesua ferrea and Mesua congestiflora were studied. Eleven xanthone derivatives identified as mesuarianone (1), mesuasinone (2), mesuaferrin A (3), mesuaferrin B (4), mesuaferrin C (5), 6-deoxyjacareubin (6), caloxanthone C (7), macluraxanthone (8), 1,5-dihydroxyxanthone (9), tovopyrifolin C (10) and α-mangostin (11) were isolated from the three Mesua species. The human cancer cell lines tested were Raji, SNU-1, K562, LS-174T, SK-MEL-28, IMR-32, HeLa, Hep G2 and NCI-H23. Mesuaferrin A (3), macluraxanthone (8) and α-mangostin (11) showed strong cytotoxicities as they possess significant inhibitory effects against all the cell lines. The structure-activity relationship (SAR) study revealed that the diprenyl, dipyrano and prenylated pyrano substituent groups of the xanthone derivatives contributed towards the cytotoxicities.The cytotoxic structure-activity relationships among a series of xanthone derivatives from  Mesua, from the Clusiaceae family was selected for preliminary pharmacognosy analysis as some previous chemical investigations on this genus have revealed their activities to be due to the phloroglucinols, xanthones and neoflavonoids present in these plants [6) has been reported for its growth inhibitory effect against HL-60 (leukemia) cells [7) and macluraxanthone (8) having good activities on Plasmodium falciparum [8) has also been reported to exhibit potent cholinesterase inhibitory [9) has been shown to display moderate cytotoxicity towards KB cells [α-Mangostin (11) however, showed anti-proliferative and pro-apoptotic effects on chronic B leukemia cells [1–5 and 10 have not been reported. We report here the biological activities for compounds 1–11  cells , antioxia) cells  as well a) cells . There axanthone  having ghibitory , strong hibitory . On the KB cells . α-Mangoia cells  and was ia cells . The bionds 1–11  as well M. beccariana, M. ferrea and M. congestiflora are listed in 50 values of tested cancer cells treated with the isolated xanthone derivatives are summarized in Cytotoxicities of all the isolated xanthone derivatives were evaluated against a panel of cancer cell lines which included Raji (human B lymphocyte), SNU-1 (human gastric carcinoma), K562 (human erythroleukemia cells), LS-174T , HeLa , SK-MEL-28 , NCI-H23 (human lung adenocarcinoma), IMR-32 (human neuroblastoma) and Hep G2 (human hepatocellular liver carcinoma). All the xanthone derivatives isolated from 3), caloxanthone C (7) and macluraxanthone (8), or diprenylated like α-mangostin (11) or geranylated as in mesuasinone (2) and have a fused pyrano ring showed prominent inhibitory effects towards Raji cells. Interestingly, the hydroxylation of the xanthone nucleus at C-6 of mesuaferrin A (3) and macluraxanthone (8) led to a significant increase in the cytotoxic effect, as shown in 1) was probably due to the indirectly attached prenyl group at the pyrano moiety. Xanthone derivatives such as 6-deoxyjacareubin (6) and mesuaferrin B (4) with no prenyl chain and mesuaferrin C (5) with a furano ring have their cytotoxic effects further reduced. On the other hand, the simple xanthone, 1,5-dihydroxyxanthone (9) as well as tovopyrifolin C (10) which is methoxylated at C-2 of the xanthone nucleus did not inhibit the growth of the Raji cells or any of the other cancer cell lines tested.Xanthones with a prenyl group such as mesuaferrin A  and the dipyrano group in mesuaferrin B (4) induce inhibitory activity towards K562 cells. Moreover, the presence of the prenylated pyrano with an oxygen atom at R3, as in macluraxanthone (8) (Differences in the activity of compounds bserved. . Apparenhone (8)  reduces hone (8) A.α-mangostin (11), dipyrano rings as in mesuaferrin B (4), and for prenylated tri-hydroxylated xanthones with a pyrano ring like mesuaferrin A (3) and macluraxanthone (8). The prenylated dihydroxylated monopyranoxanthone, caloxanthone C (7) showed a significant cytotoxicity towards Hep G2. However, the cytotoxicity towards SNU-1 cells was reduced due to the absence of one OH group. The methoxylated diprenylated xanthone derivative, α-mangostin (11) revealed significant anti-proliferative effects against both SNU-1 and NCI-H23 while the inhibitory effect towards Hep G2 was reduced due to the presence of its diprenyl groups. On the other hand, derivatives having a fused furano ring, mesuaferrin C (5), indirectly prenylated dipyrano ring, mesuarianone (1), monopyrano ring, 6-deoxyjacareubin (6), geranyl group with pyrano ring, mesuasinone (2), as well as dihydroxylated xanthones, 1,5-dihydroxyxanthone (9) have their cytotoxicities towards SNU-1 cells reduced. Meanwhile, the monopyrano derivative, 6-deoxyjacareubin (6) and furano derivative, mesuaferrin C (5) have reduced inhibitory effects against NCI-H23 cells. The latter furano derivative also possesses weak cytotoxicity towards Hep G2 cells. Both NCI-H23 and Hep G2 cells were less vulnerable to the rest of the xanthone derivatives , macluraxanthone (8), α-mangostin (11) and mesuaferrin B (4) demonstrated significant cytotoxic activity towards LS-174T cells. This could be due to the prenylated pyrano moiety together with dihydroxylation at C-5 and C-6 or the presence of diprenyl or dipyrano moieties. However, caloxanthone C (7) and mesuasinone (2) which are prenylated or geranylated dihydroxy pyranoxanthone derivatives showed a decrease in inhibitory activity against the LS-174T cells. 6-Deoxyjacareubin (6), with a fused monopyrano ring and mesuaferrin C (5) with a fused furano ring, had their inhibitory activity towards LS-174T cell line diminished. The non-prenylated 1,5-dihydroxyxanthone (9) along with mesuarianone (1) with a prenylated methyl group at the pyrano ring also had no inhibitory activity towards LS-174T cell line (Mesuaferrin A (ell line C.6) and a furano ring in mesuaferrin C (5) when compared to the LS-174T cells. The replacement of OH by H in caloxanthone C (7) as compared in macluraxanthone (8) astonishingly reduces the activity of the xanthone. On the other hand, the indirectly-attached prenylated dipyrano ring in mesuarianone (1), the geranylated pyrano ring in mesuasinone (2) and the dihydroxyl group in 1,5-dihydroxyxanthone (9) were inactive towards SK-MEL-28 cells  and caloxanthone C (7). The inhibitory potency of the dipyrano derivative, mesuaferrin B (4) towards IMR-32 cells remarkably decline compared to SK-MEL-28 while the absence of the OH group at C-6 of caloxanthone C (7) was associated with a higher cytotoxicity compared to that on SK-MEL-28 cells (The difference in the structure-activity relationship between SK-MEL-28 and IMR-32 cell lines was noticed for mesuaferrin B (28 cells C.1), dipyrano-mesuaferrin B (4), monopyrano-6-deoxyjacareubin (6), dihydroxylated 1,5-dihydroxyxanthone (9) and diprenylated α-mangostin (11) which did not show significant inhibitory growth of the cells. Meanwhile, the presence of a furano moiety in mesuaferrin C (5) further diminished remarkably its inhibitory activity towards HeLa cells . Cell stocks were placed in a −86 °C ultra- low temperature freezer  and preserved in a liquid nitrogen tank .All the apparatus used for cell culture were sterilized and decontaminated using a Hirayama HICLAVE HVE-50 . Cell culture handling was carried out in an ESCO Class II BSC Biosafety Cabinet . The healthy cells were spun down, adherent together and separated from unhealthy and dead cells by using Thermo Scientific Sorvall ST 16R centrifuge . All cultures were incubated in 5% COM. beccariana, root bark of M. ferrea and roots of M. congestiflora were collected from the Sri Aman district in Sarawak, Malaysia. All the plant materials were identified by Associate Professor Dr. Rusea Go, Department of Biology, Faculty of Science, Universiti Putra Malaysia and deposited in the herbarium of the Department of Biology, Faculty of Science, Universiti Putra Malaysia.The stem bark of M. beccariana stem bark (3 Kg) was extracted successively with n-hexane, dichloromethane, ethyl acetate and methanol . The extracts were dried under reduced pressure using a rotary evaporator to obtain the hexane (15.6 g), dichloromethane (21.2 g), ethyl acetate (15.8 g) and methanol (80.5 g) extracts, respectively. Column chromatographic purification  of the hexane extract gave two xanthones, mesuarianone (1) and mesuasinone (2) [6) [M. ferrea afforded hexane (49.6 g), dichloromethane (19.5 g), ethyl acetate (16.7 g) and methanol (62.2 g) extracts. Column chromatography purification of the hexane extract yielded four compounds which were identified as mesuaferrin A (3) [5) [7) [8) [4) [9) [10) [M. congestiflora roots were macerated consecutively with hexane, ethyl acetate and methanol at room temperature. The extracts were desiccated under reduced pressure using a rotary evaporator to yield hexane (5.5 g), ethyl acetate (61.0 g) and methanol (120.5 g) extracts. Column chromatographic separation of the hexane extract afforded a xanthone α-mangostin (11) [1–11 are in agreement with literature data [The air-dried and powdered none (2) . Meanwhie hexane .6 g, dic acetate .7 g and  (3) [5) , caloxane hexane .6 g, dic [7) [8) . Meanwhi acetate .7 g and omethane .5 g, eth[9) [10) . Furtheromethane .2 g, ethe hexane .6 g, dic acetate .7 g and ure data ,19,20,2150 values ranging from 6.89–72.45 µM except for LS-174T and IMR-32 cell lines.The cytotoxic assays were performed by the MTT assay as described by Mosmann . In thisPreliminary insights towards the structure-activity relationships among a series of xanthone derivatives are proposed. The xanthone derivatives bearing substituent groups such as diprenyl, dipyrano and prenyl-pyrano displayed cytotoxicity towards almost all the tested cancer cell lines.


S. typhimurium; 0.5 mg/kg i.p.) or saline. Twenty-four hours later, hearts were isolated, perfused for 10 min, and then used for biochemical analyses. LPS pretreatment enhanced cardiac formation of the peroxynitrite marker 3-nitrotyrosine. LPS pretreatment also increased cardiac levels of the peroxynitrite precursor nitric oxide (NO) and superoxide. The activities of Ca2+-independent NO synthase and xanthine oxidoreductase increased in LPS-pretreated hearts. LPS pretreatment resulted in significantly enhanced phosphorylation of STAT3 and non-significantly increased phosphorylation of Akt without affecting the activation of Erk. In separate experiments, isolated working hearts were subjected to 30 min global ischemia and 20 min reperfusion. LPS pretreatment significantly improved ischemia-reperfusion-induced deterioration of cardiac function. We conclude that LPS pretreatment enhances cardiac peroxynitrite formation and activates STAT3 24 h later, which may contribute to LPS-induced late preconditioning.Administration of low-dose endotoxin  24 h before a lethal ischemia induces pharmacological late preconditioning. The exact mechanism of this phenomenon is not clear. Here we aimed to investigate whether low-dose LPS exerts late effects on peroxynitrite formation and activation of Akt, Erk, and STAT3 in the heart. Male Wistar rats were injected with LPS ( Cardiovascular diseases including myocardial infarction are the leading cause of death in western societies. Cardiac injury associated with myocardial infarction and subsequent reperfusion due to therapeutic restoration of blood supply  includes cell death, life-threatening arrhythmias, and myocardial contractile dysfunction . Precond2•−) may also be involved in endotoxin-induced late cardioprotection arginine, EGTA, NG-monomethyl-l-arginine, l-citrulline, pterin and methylene blue, protease inhibitor cocktail (#8340) were purchased from Sigma Aldrich . Sucrose, Na2EDTA, and FeSO4·7H2O were from Reanal . N-methyl-d-glucamine-dithiocarbamate (MGD) was synthetized by Fülöp F . BCA Protein Assay Kit was from Pierce . Saline was from TEVA . Lactate dehydrogenase (LDH)-P kit was purchased from Diagnosticum . Peroxynitrite marker 3-nitrotyrosine enzyme-linked immunosorbent assay (ELISA) was from Cayman Chemical . Superoxide dismutase (SOD) assay was from Randox Laboratories . Western blotting reagents were from Bio-Rad . Radioimmunoprecipitation assay (RIPA) buffer and primary antibodies were purchased from Cell Signaling Technology : anti-phospho(Tyr705)-STAT3 (#9145), anti-phospho(Ser473)-Akt (#9271), anti-phospho(Thr202/204)-Erk1/2 (#9101), anti-total STAT3 (#4904), anti-total Akt (#9272), anti-total ERK (#9102), anti-glyceraldehyde 3-phosphate dehydrogenase . HRP-conjugated secondary antibody was from Dako Corporation . Price Western blotting Detection Reagent was from Amersham .Bovine serum albumin (BSA), lipopolysaccharid from Salmonella enterica serotype typhimurium. Twenty four hours after LPS treatment, rats were anesthetized with diethyl ether and were given 500 U·kg−1 heparin intravenously. Hearts were then isolated and perfused according to Langendorff for 5 min at 37 °C with Krebs-Henseleit buffer containing NaCl 118 mM, NaHCO3 25 mM, KCl 4.3 mM, CaCl2 2.4 mM, KH2PO4, 1.2 mM, MgSO4 1.2 mM, glucose 11 mM, gassed with 95% O2 and 5% CO2 arginine to l-[14C]citrulline was measured in supernatants with or without EGTA (1 mM) or EGTA plus NG-monomethyl-l-arginine (1 mM) to estimate Ca2+-dependent and -independent NOS activities, respectively. NOS activities were expressed in pmol/min/mg protein.To determine enzymatic NO production in the hearts, Caescribed ,51. The Since XOR is a major source of superoxide in the rat heart , the actSOD activity was measured by a spectrophotometric assay based on the inhibition of superoxide-induced formazan dye formation .w/v BSA  were incubated with primary antibodies generated against the following antigens: phospho(Ser473)-Akt (1:500), total Akt (1:2000); phospho(Thr202/Tyr204)-Erk1/Erk2 (1:2000), total Erk1/Erk2 1:1000; phospho(Tyr705)-STAT3 (1:1000), total STAT3 (1:2000) ; or GAPDH . Then the membranes were incubated with a HRP-conjugated secondary antibody . Enhanced chemiluminescence kit was used to develop the membranes.To assess the activation of Akt, Erk and STAT3, the phosphorylation rate of these proteins was determined by Western blot as described earlier ,55 with t-test or two-way analysis of variance (ANOVA) as appropriate. If a difference was established in ANOVA, Fisher’s Least Significant Difference (LSD) post hoc test was applied. Differences were considered significant at p < 0.05.Data were expressed as mean ± S.E.M. and analyzed with unpaired