K2P gene twk-40 suppressed the reduced body curvatures of C. elegans NALCN(lf) mutants. twk-40(lf) caused a deep body curvature and extended backward locomotion, and these phenotypes appeared to be associated with neuron-specific expression of twk-40 and distinct twk-40 transcript isoforms. To survey the functions of other less studied K2P channels, we examined loss-of-function mutants of 13 additional twk genes expressed in the motor circuit and detected defective body curvature and/or locomotion in mutants of twk-2, twk-17, twk-30, twk-48, unc-58, and the previously reported twk-7. We generated presumptive gain-of-function (gf) mutations in twk-40, twk-2, twk-7, and unc-58 and found that they caused paralysis. Further analyses detected variable genetic interactions between twk-40 and other twk genes, an interdependence between twk-40 and twk-2, and opposite behavioral effects between NALCN and twk-2, twk-7, or unc-58. Finally, we found that the hydrophobicity/hydrophilicity property of TWK-40 residue 159 could affect the channel activity. Together, our study identified twk-40 as a novel modulator of the motor behavior, uncovered potential behavioral effects of five other K2P genes and suggests that NALCN and some K2Ps can oppositely affect C. elegans behavior.Two-pore domain potassium channels (K2P) are a large family of “background” channels that allow outward “leak” of potassium ions. The NALCN/UNC80/UNC79 complex is a non-selective channel that allows inward flow of sodium and other cations. It is unclear how K2Ps and NALCN differentially modulate animal behavior. Here, we found that loss of function (lf) in the  Caenorhabditis elegans as a model to study the behavioral effects of K2P TWK-40 and NALCN, we found opposite effects of TWK-40 and NALCN on the body curvature. We identified five other K2P genes, including twk-2, twk-17, twk-30, twk-48 and unc-58, as potential modulators of the motor behavior and confirmed the behavioral effect of the previously studied twk-7. These twk genes interacted genetically with twk-40 in a variable manner, and twk-2 exhibited an interdependent relationship with twk-40. In addition, twk-2 and unc-58 appeared to affect the body curvature in opposition to NALCN, and twk-7 appeared to affect the locomotion in opposition to NALCN. Finally, we found that the chemical property of TWK-40 amino acid 159 can affect the channel activity. Together, we suggest that TWK-40 is a novel regulator of C. elegans motor behavior, five other K2Ps can potentially modulate the behavior, and some K2Ps and NALCN may oppositely affect the behavior.Two-pore domain potassium channels (K2P) allow outward “leak” of potassium ions. The NALCN channel complex allows inward flow of sodium and other cations. K2Ps and NALCN are implicated in severe human diseases. Using the nematode  Ion channels are pore-forming membrane proteins that allow specific ions to pass through lipid membranes via the channel pore along the concentration gradient . The maj+ channels (K2P) are a large group of “leak” channels containing the characteristic four transmembrane domains and two pore-forming domains (4T2P)  (wwwwww.cengen.org) [twk genes with obvious expression in neurons of the motor circuit. The motor circuit primarily includes backward premotor interneurons AVA/AVD/AVE, forward premotor interneurons AVB/PVC, excitatory backward motor neurons DA/VA, excitatory forward motor neurons DB/VB, and inhibitory motor neurons DD/VD [twk genes, including twk-40   for twk ns DD/VD ,45. The twk genes might affect the locomotion, we generated two independent presumptive loss-of-function mutations in each gene using the CRISPR/Cas9 method. We also generated a mutation in twk-3, which was included as a negative control because it was not obviously expressed in neurons of the motor circuit  mutants exhibited a moderately deep curvature (urvature  and a wetwk-7(lf) mutants exhibited hyperactive forward locomotion  mutants ( mutants  exhibitetwk-30(lf) mutants (twk-40(lf) mutants. mutants  exhibitetwk-48(lf) mutants exhibited an irregular curvature (urvature . These murvature .unc-58(lf) mutants exhibited a deep curvature (twk-40(lf) mutants. These mutants also exhibited reduced forward and backward locomotion  mutants, including the twk-3(lf) mutant that was treated as a negative control (twk mutations did not obviously enhance or suppress the curvature or backward locomotion of twk-40(lf) mutants  and twk-2(lf), twk-7(lf), twk-17(lf), twk-30(lf), twk-48(lf) or unc-58(lf) (To understand how the c-58(lf) . Their ptwk-2(lf) or twk-7(lf) suppressed the deep curvature of twk-40(lf) mutants (twk-30(lf) had no effect, and twk-17(lf) (twk-48(lf) or unc-58(lf) enhanced. mutants  Movies, k-17(lf) , twk-48(twk-2(lf) or twk-30(lf) had no effect on the slightly reduced forward locomotion of twk-40(lf) mutants, twk-7(lf) improved it, and twk-17(lf) or unc-58(lf) enhanced it.twk-40(lf) mutants was not obviously affected by other twk(lf) mutations (twk-48 and unc-58), suggesting a dominant effect of twk-40 on this behavior.However, the extended backward locomotion of twk-48(lf); twk-40(lf) double mutants tended to coil or make delta turns upon head or tail touch (twk-40(lf); unc-58(lf) double mutants by head touch but not by tail touch  mutants raised a question as to how gain-of-function mutations in these genes might affect the behavior. To examine this, we used the CRISPR/Cas9 method to generate mutations that changed a residue located in the 2nd transmembrane domain inner helix of the channels (named TM2.6) to asparagine (N). A recent study found that similar substitutions can generate hyperactive K2P channels in vertebrates and C. elegans [The variable phenotypes of  elegans . We weremac505gf) (twk-40a(mac505gf)_cDNA transgenes driven by Ptwk-40F and Ptwk-40L, and partially by Ptwk-40R and Pnmr-1 (twk-40(mac505gf) with unc-80(lf) (nca-1(gf) mutants (twk-40(mac505gf)  mutation (ac505gf)  caused ad Pnmr-1 . Crossinc-80(lf) . Interesac505gf) .twk-2(gf) and twk-7(gf) mutants were flaccid, like twk-40(mac505gf) mutants. The body shape of unc-58(gf) mutants was circled , TWK-7(L309N) and UNC-58a(F332N) all caused strong paralysis ( circled  or in so circled  Tables. twk-40(gf) mutants might be affected by other twk genes, we crossed twk-40(mac505gf) with loss-of-function mutations in twk-2, twk-7, twk-17, and unc-58. Interestingly, twk-2(lf) partially suppressed the paralysis of twk-40(mac505gf) mutants to slow locomotion (Tables twk-7(lf), twk-17(lf) or unc-58(lf) failed to do so  also suppressed the paralysis of twk-2(gf) mutants to slow locomotion (Tables twk-40(lf) did not obviously affect the paralysis of twk-7(gf) or unc-58(gf) mutants . In these animals, we observed co-expression of the reporters in AVA and AVE neurons  mutations with unc-80(lf) and twk(gf) mutations with nca-1(gf).To investigate how these newly analyzed twk(lf) mutations failed to improve the slow locomotion of unc-80(lf) mutants except twk-7(lf), which did it weakly (twk-2(lf) or unc-58(lf), like twk-40(lf), suppressed the reduced curvature of unc-80(lf) mutants to deeper ones, while twk-7(lf) or twk-17(lf) failed to do so , twk-2(gf), twk-7(gf) and unc-58(gf) all suppressed the coiler phenotype of nca-1(gf) mutants to paralysis phenotypes comparable with those of the individual twk(gf) mutants (Like  mutants .twk-40(mac505gf) mutation, we obtained an in-frame deletion of I158 and L159 in TWK-40a (mac504). twk-40(mac504) mutants were sluggish and moderately flaccid (twk-40a(mac504) cDNA transgene driven by Ptwk-40F caused a locomotion defect phenocopying that of twk-40(mac504) mutants  and unc-80(lf) together caused a strong paralysis (twk-40(mac504gf) suppressed the coiler phenotype of nca-1(gf) mutants to a non-coiler phenotype with very deep curvature (twk-40(mac505gf), the reduced curvature and defective locomotion of twk-40(mac504gf) mutants were also suppressed by twk-2(lf)  .It is intriguing that the deletion of I158 and L159 would confer a hyperactivity in TWK-40a. We examined whether deletion of either residue alone would affect the channel activity using transgene phenocopy experiments.twk-40a cDNA (I158del) transgene driven by Ptwk-40F failed to affect the forward locomotion of wildtype animals, so did the transgene with an L159 deletion (We found that a deletion .twk-40a(I158-L159del) cDNA transgene, we mutated T161 to asparagine (N) or lysine (K). Both mutations caused paralysis in the transgenic animals  mutants also exhibited reduced locomotion (twk-40(mac504gf) mutants showed weakly improved locomotion, both forward and backward, in K+-deficient plates  C. elegans mutants, providing an early piece of evidence that NALCN and K2Ps might act as opposite regulators of animal behaviors. Our results are consistent with their findings. The opposite effect was observed between twk-40(lf) and NALCN(lf) and was corroborated by the suppression of the coiler phenotype of nca-1(gf) mutants by twk-40(gf). Opposite effects on specific behaviors were observed between NALCN and twk-2, twk-7, or unc-58 as well. Besides K2Ps, other channels may also affect the behaviors regulated by NALCN, e.g., increasing Ca2+ channel activity pharmacologically improved the locomotion of NALCN(lf) mutants [Recently, Kasap et al.  found th mutants . HoweverPtwk-40L and Ptwk-40R promoters, which appeared to be active in different neurons, exhibited similar rescuing activities for twk-40(lf) suppression of unc-80(lf) curvature. They also showed rescuing activities for the deep curvature of twk-40(lf) single mutants, though Ptwk-40L appeared to be stronger. However, Ptwk-40L and Ptwk-40R exhibited quite different rescuing activities for the extended backward locomotion of twk-40(lf) mutants: Ptwk-40R was effective while Ptwk-40L was not. We recently made a similar observation about the differential effects of promoters on behaviors in our study of C. elegans avoidance response to the odorant methyl salicylate [PSunc-79) of the full-length unc-79 promoter (PLunc-79) exhibited a rescuing activity like PLunc-79 for the avoidance defect of unc-79(lf) mutants. However, PSunc-79 failed to rescue the locomotion defect of unc-79(lf) mutants, which could be strongly rescued by PLunc-79.It is intriguing that the licylate . In thisPnmr-1 promoter showed rescuing activities for both the curvature and backward locomotion phenotypes of twk-40(lf) mutants. However, Pnmr-1 appeared to be active in a set of neurons that only partially overlapped with those for Ptwk-40L, e.g., in AVA/AVE/AVG, or with Ptwk-40R, e.g., in PVC.Another intriguing finding was that the NALCN might affect an array of behaviors by regulating the activity of a neural network. A component of the network may affect a specific behavior redundantly with other components, or only affect component-specific behaviors, or both. TWK channels, through restricted expression and regulated activities, might be involved in compartmentalizing the expression of some behaviors oppositely regulated by NALCN.To elucidate the underlying mechanism of these phenomena is beyond the scope of our current study. Nevertheless, it is tempting to speculate a scenario that might accommodate these findings. In this scenario, NALCN and twk genes. We found that several tail neurons in which twk-40 promoters were active, including PVC, DVA, DVB and DVC, did not obviously affect the curvature or backward locomotion. However, our analyses were insufficient to reveal whether one or more of the head neurons in which TWK-40 and NALCN were co-expressed, e.g., AVA, AVB and AVE, can affect these behaviors or were the sites where TWK-40 and NALCN exert opposite behavioral effects. Future experiments that can directly measure the activities of these neurons, e.g., calcium imaging or electrophysiology experiments, will provide insights into how TWK-40 and NALCN affect neuronal activities. Also, optogenetic approaches will be important for associating the behaviors affected by these channels with the neurons expressing them.We primarily used genetic and behavioral experiments to analyze the effects of Ptwk-40L (upstream of the twk-40b isoform) exhibited a stronger rescuing effect on the curvature phenotype of twk-40(lf) mutants while Ptwk-40R (upstream of the twk-40c and twk-40a isoforms) exhibited a stronger rescuing effect on the backward locomotion phenotype implies that twk-40 isoforms might have functional differences. We examined this possibility using presumptive isoform-specific twk-40 mutants.That twk-40(mac564) (twk-40a-specific) or twk-40(mac557) (twk-40c-specific) had no obvious effect. However, twk-40(mac554) (twk-40b-specific) caused a moderately deep curvature, and with the disruption of more isoforms, e.g., in twk-40(mac558) (disruption of twk-40b and twk-40c) and twk-40(mac560) , the mutants exhibited gradually increased deep curvature. Based on these analyses, we speculate that the curvature is more significantly affected by twk-40b and less by twk-40a or twk-40c.For the curvature phenotype, twk-40(mac554) (twk-40b-specific), twk-40(mac557) (twk-40c-specific) or twk-40(mac558) (disruption of twk-40b and twk-40c) had no obvious effect. However, twk-40(mac564) (twk-40a-specific) caused a moderately extended backward locomotion, and twk-40(mac560)  caused a more severe phenotype. Therefore, the backward locomotion appeared to be more significantly affected by twk-40a and less by twk-40b or twk-40c. Together, the isoform-specific mutant phenotypes are consistent with the transgene rescue results, suggesting differential behavioral effects of twk-40 isoforms.For the backward locomotion phenotype, twk-40 expression. In addition, the expression patterns of twk-40 isoforms should not be extracted simply from the reporters, as the promoters may regulate expression of adjacent isoforms as well as distant ones. It is also possible that the presumptive twk-40 isoform-specific mutations may affect the expression of other isoforms, as they were within the full-length promoter region and/or might affect all isoforms. Therefore, new reagents and methods that provide higher-resolution and more accurate pictures about twk-40 expression, e.g., TWK-40 isoform-specific antibodies, isoform-specific knockin of reporters or single-copy transgene experiments, are warranted for validating our findings.Caution needs to be exercised in interpreting the results of the transgenes and isoform-specific mutants. For example, transgenes may cause non-specific expression in neurons, the promoters may not be complete, and the fluorescent reporters probably did not provide a full picture about C. elegans genome contains 47 K2P channel encoding genes [www.wormbase.org). The functions of most TWK channels are largely unclear, with the exception of TWK-18 [unc-58 was unclear.f TWK-18 , SUP-9  by generating loss-of-function mutations in them. Focusing on the body curvature and touch-triggered forward or backward locomotion, we found that five such twk genes, including twk-2, twk-17, twk-30, twk-48, and unc-58, might be modulators of motor behavior. Our study also confirmed the hyperactive forward locomotion of twk-7(lf) mutants as previously described [Prompted by the findings on  circuit   mutants are often displayed by wildtype animals under different circumstances [C. elegans motor behavior. However, our analyses were mostly descriptive and qualitative. Extensive future studies are needed for understanding the molecular mechanisms underlying the effects of these twk genes.The behavioral patterns exhibited by these mstances . Therefotwk-2 or twk-40 can partially suppress the paralysis phenotype caused by gain-of-function mutations in the other. However, twk-40 did not interact with other twk genes in such a manner. During the study, we also performed a screen for mutations that can suppress the paralysis phenotype of twk-40(mac505gf) mutants (twk-2 predicted to change TWK-2 G181 to glutamate (E), and G181 is a conserved residue within selectivity filter 1 of K2P channels [Ptwk-40F and Ptwk-2 promoters were both active in AVA, AVE, and other neurons, we postulate that twk-40 and twk-2 might function in same neurons for their full activities.We found that loss-of-function mutations in  mutants . From thchannels . Realiziunc-58(gf) mutants confirmed that of unc-58(gf) mutants generated by Ben Soussia et al. [Recently, Ben Soussia et al. found that changing TM2.6 to asparagine can generate hyperactivity in multiple K2Ps . We genea et al. .mac504gf) that deleted I158 (TM2.5) and L159 (TM2.6) of TWK-40a. mac504gf caused a moderately hyperactive channel. Amino acid substitutions in wildtype TWK-40a and TWK-40(mac504gf) channels and transgene phenocopy analyses suggest that the residue at TM2.6 can significantly affect channel activity. Specifically, hydrophilic residues appeared to be related to hyperactivity while hydrophobic residues appeared to be related to inactivity or highly regulated activity. Our results are consistent with recent findings that such a hydrophobic barrier on the mammalian K2P TWIK1 can oppose efficient K+ passage through the channel pore [We also isolated an in-frame mutation  mutants. For example, among the twk genes with expression in interneurons , extended backward locomotion was observed if the expression was in AVA neurons , while reduced forward locomotion was observed if the expression was in AVB neurons (twk-40 and twk-17). Alternatively, deep curvature was observed for twk genes expressed in AVA/AVE neurons (twk-40 and twk-2). However, twk expression in interneurons might not be required for the mutants to show these phenotypes: loss-of-function mutations in twk-30, twk-48, and unc-58, genes not obviously expressed in these interneurons (based on CeNGEN), resulted in similar phenotypes  L4 animals were mutagenized with EMS (ethyl methanesulfonate) [0 animals were picked to 20 NGM plates, with 20 animals per plate. We estimated that each P0 animal would lay at least 100 eggs within 24 hrs of becoming young adult. After these first-day F1 eggs  became young adults, they were bleached to generate synchronized F2 progeny. Once growing into young adults, F2 animals were examined under a dissecting microscope to identify individuals with apparently improved locomotion or body curvature. From the screen, we obtained 5 independent isolates.Synchronized lfonate) . 400 P0 mac420 and mac422 were assigned to Chr. X because F1 male progeny from the cross between unc-80(lf) males and unc-80(lf); mac420 or unc-80(lf); mac422 hermaphrodites were suppressed for the defective locomotion, while F1 hermaphrodite progeny were not. F1 males from the cross between unc-80(lf) males and unc-80(lf); mac424/mac425/mac426 hermaphrodites were not suppressed for their respective phenotype, suggesting that mac424, mac425 and mac426 were autosomal. Complementation test suggested that mac420 and mac422 might affect different genes. We did not further map mac420 and mac422 using SNPs.All five suppressors were recessive. mac425 and mac426 similarly suppressed the reduced body curvature of unc-80(lf) mutants and did not complement each other, suggesting that they might affect a same gene. We mapped mac425 using published single nucleotide polymorphisms (SNP) [ms (SNP) .unc-80(lf); mac425 hermaphrodites to generate F1 cross progeny , and their F2 progeny with reduced curvature, which were probably of unc-80(lf); mac425/+ or unc-80(lf); +/+ genotype, were picked to individual plates. Individual F3 progeny with suppressed curvature from different F2 plates were established as mapping lines.To generate mapping lines, we crossed Hawaiian (CB4856) males with mac425, homozygous CB4856 genotypes were not detected at W06F12 (Chr. III: 21 cM) or Y17D7B (Chr. V: 18 cM. Note: unc-80 is at Chr. V: 26 cM) but were detected at other selected SNPs in at least one line. At the same time, genomic sequencing of unoutcrossed mac425 and mac426 mutants found that twk-40 was the only gene on Chr. III affected in both strains. Hence we pursued twk-40 as the candidate gene for mac425 and mac426.In 10 mapping lines for mac424 is ongoing.The mapping of twk-40(mac505gf) P0 animals at the L4 larval stage with EMS. From synchronized F2 adult progeny of ~40,000 F1 progeny, we isolated four recessive mutants with improved locomotion. Three isolates exhibited locomotion and body curvature like twk-40(lf) mutants and failed to complement twk-40(lf). We postulated that these isolates probably carried intragenic loss-of-function mutations in twk-40 and did not further analyze them.We mutagenized twk-2((lf) could suppress the locomotion of twk-40(mac505gf) mutants (twk-2(lf); twk-40(mac505gf) hermaphrodites and found that the male progeny were still suppressed for the locomotion phenotype. Sanger sequencing identified a mutation in twk-2 (named mac537). mac537 caused a G181E change in TWK-2a. TWK-2a G181 is a conserved residue in selectivity filter 1 of K2P channels [In earlier study, we found that  mutants . We croschannels .unc-80(lf); mac420), CSM742 (unc-80(lf); mac422), CSM757 (unc-80(lf); mac424), CSM758 (unc-80(lf); mac425) and CSM759 (unc-80(lf); mac426) before outcrossing, was grown on a single 9 cm NGM plate and washed from the plates before food was completely consumed. Animals were washed twice with M9, resuspended in M9 and starved for several hours. Genomic DNAs were extracted by proteinase K digestion, followed by RNase A treatment and two rounds of phenol–chloroform extraction. Three genomic DNA libraries (380-bp inserts) were constructed by Berry Genomics Co., Ltd (Beijing) using Illumina’s paired-end protocol and paired-end sequencing (100-bp reads) was performed on the Illumina HiSeq 2000. Over 4G clean bases were mapped to the N2 genome (Wormbase release 220) after removal of duplicated reads. SNP calling was performed using Genome Analysis Toolkit (GATK) with the N2 genome as reference. 4449 (mac420), 4623 (mac422), 4633 (mac424), 4712 (mac425), and 4675 (mac426) sequence variants were detected in these mutants. Sequence variants shared among strains were excluded as they were likely derived from common ancestors. To enhance the stringency for mutation identification, we set the depth of reference base (WT) to be < 6 in these mutants. Exon or splice site variants carrying effective mutations with a variant quality greater than 30 were selected. Based on these criteria, we obtained 32 (mac420), 45 (mac422), 46 (mac424), 62 (mac425), and 74 (mac426) variants overall. The potential target genes for each mutant were further narrowed down to the mapped chromosomes.Each of the original isolates, CSM740  plasmid as co-injection marker and 20 ng/μl of the transgene of interest.For For transcriptional reporters, a transgene solution containing 20~50 ng/μl of the reporter plasmid was injected to wildtype animals.Promoter:: twk-40_cDNA and 20 ng/μl pPD95_86 (Pmyo-3::GFP) plasmid or 2.5 ng/μl pCFJ90 (Pmyo-2::mCherry) plasmid [For neuron-specific rescue experiments, the transgenic mixtures contained 10~30ng/μl  plasmid  as co-intwk-40. The transgenic mixtures contained 25 ng/μl Promoter::Cas9::NLS::3’UTR, 25 ng/μl PU6::twk-40_sgRNA #1 and #2, and 20 ng/μl pPD95_86 (Pmyo-3::GFP) plasmid as co-injection marker.We used a previously described method  with modPromoter::twk-40a(mutations)_cDNA and 20 ng/μl pPD95_86 (Pmyo-3::GFP) plasmid as co-injection marker.For phenocopy experiments, transgenic mixtures contained 10~20 ng/μl twk-40 insertion/deletion mutation, the injected DNA mixture contained 50 ng/μl pPD162 (Peft-3::Cas9-SV40_NLS), 25 ng/μl PU6::sgRNAs (two specific sgRNAs for a twk gene), and 20 ng/μl pPD95_86 (Pmyo-3::GFP) plasmid as co-injection marker. To generate twk-40 knockin mutations, we included a synthesized oligo (500 nM) containing the designed mutation as repair template in the injection mixture.We followed the method  with mod0 animals for knockout or knockin mutations. For twk-40(mac469) and twk-40(mac472), we injected unc-80(lf) P0 animals. ~40 F1 animals with strong GFP signals in body-wall muscles (co-injection marker) were picked to individual plates, and F2 progeny were sequenced until the expected mutations were detected.We normally injected 20 to 40 wildtype Ptwk genes. For each of twk-2, twk-3, twk-7, twk-13, twk-17, twk-43, twk-46 and unc-58, knockout or knockin mutations were generated using an injection solution containing the aforementioned injection mix together with a repair template. We obtained two or more deletion/insertion mutations for each of the eight genes, one knockin mutation for twk-2, twk-7 or twk-43, three knockin mutations for unc-58, and no knockin mutation for twk-3, twk-13, twk-17 and twk-46. For twk-26, twk-29, twk-30, twk-35, twk-48 and twk-49, we generated deletion/insertion mutations using an injection solution without repair template . Multiple animals from each of two transgenic lines were observed for consistency of reporter expression and a subset of animals with strong fluorescence were examined further for neuronal identification. Pictures shown were representative images. We determined the identities of 2HPO4/ KH2PO4 with equal molar amount of Na2HPO4/ NaH2PO4. Synchronized L1 animals were allowed to grow on the K+-deficient plates until reaching the L4 larval stage. The animals were then transferred to fresh K+-deficient NGM plates with thin bacterium lawn. Touch-triggered body bends were measured in 18 to 24 hours.NGM plates were prepared by replacing Kwww.cengen.org to search for the graphed expression of each twk gene in C. elegans neurons. The top 20 neuronal classes in which a twk gene was expressed were listed in We followed the instruction at Maximum likelihood phylogenetic tree was constructed with the Molecular Evolutionary Genetics Analysis software MEGA X .P values were determined by two-tailed unpaired Student’s t-test for pairwise comparison or Bonferroni multiple comparison with one-way ANOVA for multiple comparison using GraphPad Prism 7.0 software.Strains used in this study and plasmid constructions are shown in S1 Figunc-80(lf); sup mutants. mac420, mac422 and mac424 could significantly improve the defective locomotion of unc-80(lf) mutants. (B) Curvature indices of nca-1(lf) single, nca-2(lf) single and nca-2(lf); nca-1(lf) double mutants. 40 animals were quantified for each genotype. Statistics: Bonferroni multiple comparison with one-way ANOVA. ***, p < 0.001.(A) Touch-triggered forward locomotion of (TIF)Click here for additional data file.S2 FigDrosophila CG43155 were omitted. C-terminal regions of TWK-40a (aa 338–393), Drosophila CG43155 (aa 364–411), M. musculus KCNK3 (aa 285–409), H. sapiens KCNK9 (aa 287–401) and H. sapiens KCNK16 (aa 260–262) were not included in the alignment.The four transmembrane domains are enclosed in blue boxes, and the two pore domains are in red boxes. The amino acid at TM2.6 is indicated with a red arrowhead. The first 50 amino acids of the (TIF)Click here for additional data file.S3 FigPtwk-40F, Ptwk-40L and Ptwk-40R promoters. (B) GFP driven by the Ptwk-40L promoter labeled some head neurons, ventral nerve cord and tail neurons. Ventral cord motor neurons were not obviously labeled. (C) mCherry driven by the Ptwk-40R promoter labeled multiple head neurons, ventral nerve cord, ventral motor neurons and tail neurons. a: anterior; v: ventral.(A) The genomic positions of (TIF)Click here for additional data file.S4 FigPtwk-40R::mCherry (left panel) and Ptwk-40L::GFP (middle panel). The merged picture was shown on the right. (B) Confocal pictures of the same animal as in (A) at a different focal plane. (C) A tail neuron(s) at the position of DVA/DVB/DVC neurons was co-labeled by mCherry and GFP. (D) Ventral nerve cord in the middle region of an animal showing the mCherry-labeled motor neurons and GFP-labeled cord. For all pictures, a: anterior; v: ventral.(A) Confocal pictures of head neurons labeled by (TIF)Click here for additional data file.S5 FigPtwk-40L::mCherry transgene (left panel) and the Punc-80::GFP transgene (middle panel). (B) Confocal picture of the same animal as in (A) on a different focal plane. (C) A tail neuron(s) at the position of DVA/DVB/DVC was co-labeled by mCherry (left panel) and GFP (middle panel). The merged pictures were shown on the right. For all pictures, a: anterior; v: ventral.(A) Confocal pictures of adult head neurons expressing the (TIF)Click here for additional data file.S6 FigPtwk-40R::mCherry transgene (left panel) and the Punc-80::GFP transgene (middle panel). The merged picture was shown on the right. (B) Confocal picture of the same animal as in (A) at a different focal plane. (C) A tail neuron(s) at the position of DVA/DVB/DVC appeared to be co-labeled by mCherry (left panel) and GFP (middle panel). (D) Ventral cord in the middle region of an animal showing several motor neurons co-labeled by mCherry and GFP. For all pictures, a: anterior; v: ventral.(A) Confocal pictures of adult head neurons expressing the (TIF)Click here for additional data file.S7 FigPtwk-40R::GFP transgene (left panel) and the Pnmr-1::mCherry transgene (middle panel). The merged picture was shown on the right. (B) Confocal pictures of adult tail neurons expressing the Ptwk-40R::GFP transgene (left panel) and the Pnmr-1::mCherry transgene (middle panel). (C) Confocal pictures of adult head neurons expressing the Ptwk-40L::GFP transgene (left panel) and the Pnmr-1::mCherry transgene (middle panel). For all pictures, a: anterior; v: ventral.(A) Confocal pictures of adult head neurons expressing the (TIF)Click here for additional data file.S8 FigPtwk-40F::mCherry transgene (left panel) and the Ptrp-4::GFP transgene (middle panel). (B) Confocal pictures of adult tail neurons expressing the Ptwk-40F::mCherry transgene (left panel) and the Plim-6int4::GFP transgene (middle panel). (C) Confocal pictures of adult tail neurons expressing the Ptwk-40L::mCherry transgene (left panel) and the Plim-6int4::GFP transgene (middle panel). (D) Confocal pictures of adult tail neurons expressing the Ptwk-40R::mCherry transgene (left panel) and the Plim-6int4::GFP transgene (middle panel). The merged pictures were shown on the right. For all pictures, a: anterior; v: ventral.(A) Confocal pictures of adult tail neurons expressing the (TIF)Click here for additional data file.S9 Figtwk-40 isoform-specific mutations. (B) Genomic positions of twk-40 isoform-specific mutations.(A) List of presumptive (TIF)Click here for additional data file.S10 Fignd transmembrane domains of indicated TWK channels. TM2.6 was substituted with asparagine (N). (B) Representative pictures of twk(gf) mutants.(A) Alignment of the 2(TIF)Click here for additional data file.S11 FigPtwk-40F::GFP transgene (left panel) and a Ptwk-2::mCherry transgene (middle panel). (B) An unidentified tail neuron(s) at the position of DVA/DVB/DVC was co-labeled by GFP (left panel) and mCherry (middle panel). The merged pictures were shown on the right. (C) Ventral cord in the middle region of a transgenic animal showing two motor neurons co-labeled by GFP and mCherry. For all pictures, a: anterior; v: ventral.(A) Confocal pictures of adult head neurons expressing the (TIF)Click here for additional data file.S12 Figt-test. ***, p < 0.001.40 animals were quantified for each assay condition. Statistics: two-tailed unpaired Student’s (TIF)Click here for additional data file.S1 Tableunc-80(lf); mac425 and unc-80(lf); mac426 mutants are shown at the bottom.Chromosomal locations of the suppressors are shown at the top. Genetic variations identified from genomic sequencing of unoutcrossed (XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Tabletwk-40 was not annotated to be obviously expressed in PVC neurons at CeNGEN. twk-3 was chosen as a presumptive negative control due to its lack of obvious expression in the motor circuit. The relative expression levels of twk genes (based on CeNGEN) in each neuronal type are qualitatively indicated with the numbers of asterisks: *, weak; **, moderate; ***, strong.Genes in red were expressed in the motor circuit and exhibited detectable effects on the motor behavior. Genes in blue were expressed in the motor circuit and did not obviously affect the motor behavior. Genes in bold black were previously studied but not analyzed in this study. Behaviors of loss-of-function mutants were considered here. (XLSX)Click here for additional data file.S4 Tabletwk genes. For each gene, top 20 neuron classes with high to low transcript signals (www.cengen.org) were listed. Mutations in bold were used as reference alleles. Key neurons in the motor circuit are shown in red.We used the CRISPR/Cas9 method to generate mutations in these (XLSX)Click here for additional data file.S5 Tableunc-119 promoter were used. 20 animals from each of two transgenic lines were quantified and the data were used together for comparison. Obvious phenotypes of twk-17(lf) and twk-48(lf) mutants other than the body curvature or locomotion were not quantified due to strong rescuing effects by the transgenes. Statistics: two-tailed unpaired Student’s t-test. ***, p < 0.001. NA: not applicable.Wildtype transgenes under control of the (XLSX)Click here for additional data file.S6 Tablep < 0.001.40 animals were measured in each dataset. Statistics: Bonferroni multiple comparison with one-way ANOVA, ***, (XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S8 Table(XLSX)Click here for additional data file.S9 Tabletwk genes, two sgRNAs were used simultaneously. The use of twk-40 sgRNAs was as follows: twk-40a sgRNA # a1 and # a2 together with a repair template Click here for additional data file.S10 Tabletwk-40a #1 repair template was used for generating mac564 and twk-40a #2 for mac505. Nucleotides in red are for introducing the missense mutation and nucleotides in blue are silent mismatch mutations of the target sequences.The (XLSX)Click here for additional data file.S1 Movie(MP4)Click here for additional data file.S2 Movie(MP4)Click here for additional data file.S3 Movie(MP4)Click here for additional data file.S4 Movie(MP4)Click here for additional data file.S5 Movie(MP4)Click here for additional data file.S6 Movie(MP4)Click here for additional data file.S7 Movie(MP4)Click here for additional data file.S8 Movie(MP4)Click here for additional data file.S9 Movie(MP4)Click here for additional data file.S10 Movie(MP4)Click here for additional data file.S11 Movie(MP4)Click here for additional data file.S12 Movie(MP4)Click here for additional data file.S13 Movie(MP4)Click here for additional data file.S14 Movie(MP4)Click here for additional data file.S15 Movie(MP4)Click here for additional data file.S16 Movie(MP4)Click here for additional data file.S17 Movie(MP4)Click here for additional data file.S18 Movie(MP4)Click here for additional data file.S19 Movie(MP4)Click here for additional data file.S20 Movie(MP4)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


Parthanatos is programmed cell death mediated by poly(ADP-ribose) polymerase 1 (PARP1) after DNA damage. PARP1 acts by catalyzing the transfer of poly(ADP-ribose) (PAR) polymers to various nuclear proteins. PAR is subsequently cleaved, generating protein-free PAR polymers, which are translocated to the cytoplasm where they associate with cytoplasmic and mitochondrial proteins, altering their functions and leading to cell death. Proteomic studies revealed that several proteins involved in endocytosis bind PAR after PARP1 activation, suggesting endocytosis may be affected by the parthanatos process. Endocytosis is a mechanism for cellular uptake of membrane-impermeant nutrients. Rab5, a small G-protein, is associated with the plasma membrane and early endosomes. Once activated by binding GTP, Rab5 recruits its effectors to early endosomes and regulates their fusion. Here, we report that after DNA damage, PARP1-generated PAR binds to Rab5, suppressing its activity. As a result, Rab5 is dissociated from endosomal vesicles, inhibiting the uptake of membrane-impermeant nutrients. This PARP1-dependent inhibition of nutrient uptake leads to cell starvation and death. It thus appears that this mechanism may represent a novel parthanatos pathway. Parthanatos is a poly(ADP-ribose) polymerase1 (PARP1)-dependent programmed cell death, which occurs after DNA damage ,3. PARP1Endocytosis, the process by which cells take up membrane-impermeant substances from the extracellular space through vesicular transport, plays important roles in intracellular signaling, nutrient uptake, immune responses and cell motility, as well as in pathological processes such as tumorigenesis ,21. FollRecent studies have shown that proteins interact with PAR polymers through a PAR-binding motif (PBM), PAR-binding domain (PBD) or WWE domain ,26. In aTo investigate whether poly(ADP-ribosyl)ation catalyzed by PARP1 regulates endocytic events, the DNA alkylating agent N-methyl-N’-nitro-N-nitrosoguanidine (MNNG)  was added to NIH3T3 and HeLa cells to induce PARP1 activation before addition of fluorescently labelled ovalbumin (OVA-FITC and OVA-Alexa647). MNNG dose-dependently inhibited the percentage of NIH3T3 cells that took up OVA-FITC and the amount of OVA-FITC taken up cells, which was reversed by pretreatment with the PARP inhibitors PJ34, ABT-888 or XAV-989 . To asseThe inhibition of OVA uptake developed at least 20 min after exposure to MNNG in NIH3T3 cells A,B. SimuBecause MNNG-induced PARP1 activation triggers parthanatos, we assessed the effect of PARP1-mediated inhibition of OVA uptake on cell viability. NIH3T3 cells exposed to MNNG for 24 h exhibited diminished viability, and this effect was suppressed by pretreatment with the PARP1 inhibitor PJ34 H. In conEndocytosis contributes to cell survival by supplying cells with nutrients and macromolecules that cannot permeate the cell membranes and by controlling receptor signaling to adapt cellular stress ,21. WhenTo identify the molecular mechanism underlying PARP1-mediated inhibition of OVA uptake, we observed the subcellular localization of endocytosis-related proteins . ExposurThe changes in the subcellular localization of Rab5 and EEA1 appeared 20 min after exposure to MNNG, which was consistent with the time required for PAR synthesis in the nucleus and its translocation to the cytoplasm C. MoreovBecause only proteins localized in EEs were dissociated upon PARP1 activation, we investigated whether Rab5, a master regulator of early endosomal fusion, is inactivated through PAR binding. To detect PAR-Rab5 binding, pull-down assays using GST-Af1521 macrodomain were performed to isolate PAR-bound proteins  28]. As. As28]. GTP-bound Rab5 recruits Vps34 to EEs where it catalyzes the phosphorylation of PI to phosphatidylinositol-3-phosphate (PI3P) . PI3P inInhibition of Rab5 insertion into the membrane of vesicles by GDI  appears 1-X2-X3-[AIQVY]4-[KR]5-[KR]6-[AILV]7-[FILPV]8) has been identified through proteomic analysis [5-[KR]6) are important for binding to the negatively charged phosphate group of PAR. Rab5 appears to have two putative PAR-binding motifs located at around amino acid residues 140 and 180, respectively , rabbit monoclonal anti-mCherry antibody (E5D8F), rabbit monoclonal anti-Rab5 antibody (C8B1), and rabbit polyclonal anti-PARP1 antibody were purchased from Cell Signaling Technology . Mouse monoclonal anti-PAR antibody (10H) was from Enzo Life Sciences . Mouse monoclonal anti-GST antibody, OVA-FITC, OVA-Alexa647, OVA, Tf-Alexa647, dextran-rhodamine B 7000 MW, Lipofectamine 3000, and Alexa Fluor-conjugated secondary antibodies were from Thermo Fisher Scientific . HRP-conjugated secondary antibodies were from Promega . Rabbit polyclonal anti-Rabenosyn5 antibody, rabbit polyclonal anti-GDI1 antibody, and rabbit polyclonal anti-GDI2 antibody were from Proteintech . Rabbit polyclonal anti-EEA1 antibody (H-300), anti-Clathrin heavy chain antibody (A-8), mouse monoclonal anti-Rabex5 antibody (C-4), mouse monoclonal anti-LAMP1 antibody (H4A3), mouse monoclonal anti-LAMP2 antibody (H4B4), mouse monoclonal anti-Cathepsin S antibody (E-3), PJ34, and ADP-HPD were from Santa Cruz Biotechnology . SB203580 was from AdipoGen Life Sciences . U0126 was from Cayman Chemical Company . Rapamycin and dorsomorphin were from Fujifilm . Mouse monoclonal anti-Flag antibody (OTI4C5) was from Origene .http://n2t.net/addgene:27679, accessed on 31 May 2019; RRID:Addgene_27679) [http://n2t.net/addgene:24398, accessed on 12 November 2018; RRID:Addgene_24398) [http://n2t.net/addgene:35138, accessed on 26 April 2018; RRID:Addgene_35138) [http://n2t.net/addgene:42635, accessed on 9 November 2017; RRID:Addgene_42635) [http://n2t.net/addgene:79801, accessed on 9 November 2017; RRID:Addgene_79801) [pET-GST-R5BD-hRABEP1 was purchased from Vector Builder. Rab5a-pmCherryC1 was a gift from Christien Merrifield  . pcDNA4-e_24398) . mCherrye_42635) . pTag-BFe_79801) . RFP-Rab2. shRNA plasmids targeting human or mouse PARP1 or scrambled shRNA (Origene) were introduced into NIH3T3 and HeLa cells using Lipofectamine 3000 transfection reagent according to manufacturer’s instructions. Cells stably expressing shRNA plasmids were selected in medium containing 1 μg/mL puromycin.NIH3T3 and HeLa cells were incubated in DMEM containing 10% FBS, 100 units of penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere with 5% COd-lysine-coated dishes. Half the volume of the medium was replaced every 5 days. The protocols used in this study were approved by the Ethical Committee of Doshisha Women’s College of Liberal Arts .Primary hippocampal neurons were isolated using neuron dissociation solution  and cultured in neurobasal medium with B-27 supplement on poly-5 cells) were seeded onto 24-well plates and incubated for 3 h in serum-free DMEM with non-essential amino acids cell culture supplement . PARP inhibitors were added for 10 min before 20-min exposure to MNNG. After washing with PBS, the cells were incubated with OVA-FITC (50 μg/mL), OVA-Alexa 647 (50 μg/mL), Tf-Alexa 647 (15 μg/mL), or dextran-rhodamine B 7000 MW (1.5 μg/mL) for the indicated times. Cells were trypsinized and suspended in PBS. Fluorescence was measured using a Cytoflex flow cytometer. The data were analyzed and processed using Cytexpert .For flow cytometric measurement of the uptake of OVA-FITC, OVA-Alexa647, Tf-Alexa647 or dextran-rhodamine, NIH3T3 or HeLa cells (1 × 104 cells) seeded onto 96-well plates were incubated for 30 min with a PARP inhibitor and then exposed to MNNG. Cell numbers were then counted using cell counting reagent SF  according to the manufacturer’s instructions by measuring the absorbance at 450 nm .NIH3T3 and HeLa cells (1 × 105 cells) and primary hippocampal neurons were seeded onto 8-well chamber plates, fixed with 4% paraformaldehyde , permeabilized, and blocked with Blocking One  containing 0.5% Triton X-100 . After incubation  with primary antibodies, the cells were treated  with Alexa-488-conjugated goat anti-rabbit IgG or Alexa-564-conjugated goat anti-mouse IgG (1:500), washed three times with PBS, and incubated  with 300 nM DAPI (Thermo Fisher Scientific) to stain the nuclei. The cells were then imaged using a confocal microscope  equipped with an oil-immersion objective . Fluorescence data were processed and analyzed using FijiJ.NIH3T3 and HeLa cells (1 × 105 cells) seeded onto glass-bottomed dished were transfected using Lipofectamine 3000 according to the manufacturer’s instructions. After incubation for 1 day, fluorescence was observed with a confocal microscope (Zeiss LSM 700 Meta) equipped with an oil-immersion objective . Hoechst 33258  was added to enhance DNA damage. Micro-irradiation of whole nuclei was carried out with a 405 nm diode laser set to 100% transmission [NIH3T3 cells (1 × 10smission . The mic5 cells) seeded onto 6-well plates were incubated  in DMEM with 10% FBS. Cell lysates were prepared with 2% SDS in 20 mM Tris-HCl (pH 7.4) containing complete protease inhibitor cocktail . After adjustment of the protein concentration using a BCA kit (Thermo Fisher Scientific), cell lysates were subjected to Bis-Tris SDS-PAGE (Thermo Fisher Scientific) and then transferred to nitrocellulose membranes (Thermo Fisher Scientific). The membranes were blocked with Blocking One for 30 min at room temperature and then incubated with primary antibodies. After incubation with HRP-conjugated anti-mouse or rabbit IgG secondary antibodies, an ECL system  was used for detection.Cells  for 16 h after addition of 1 mM IPTG at room temperature [GST-fused-Af1521 macrodomain and Rab5-binding domain (R5BD) were expressed in perature . GST-Af15 cells) seeded onto 6-well plates were incubated  in DMEM with 10% FBS. Cell lysates were prepared with lysis buffer containing 50 mM Tris-HCl (pH 7.4), 200 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10% glycerol, 1 mM DTT, 10 μM PJ34, 1 μM ADP-HPD, and protease inhibitor cocktail. The resultant lysate was mixed with GST-Af1521 macrodomain and R5BD immobilized on Glutathione Sepharose 4B beads  on a rotating wheel. After washing three times with the lysis buffer, complexes were collected in LDS sample buffer.HeLa cells (3 × 103:12N HCl (80:40:1) and then separated by addition of CHCl3:0.1 N HCl (1:1). The organic layer was collected and concentrated using a vacuum evaporator. 9-Aminoacridine  was used as a matrix. Mass spectrometry was performed using matrix-assisted laser desorption and ionization/time of flight mass spectrometry (MALDI-TOF-MS)  [Cells were precipitated with 0.5 M trichloroacetic acid (TCA). After washing with 5% TCA with 1 mM EDTA, membrane fractions were lysed with MeOH:CHClMA, USA) . The mast-test between two samples or one-way and two-way ANOVA with post hoc Tukey’s test for three or more groups. Data are means ± S.E.M of values from the indicated number of experiments. p values < 0.05 were considered significant. All representative experiments were repeated three times.Statistical analysis was performed using Sigmaplot 13 . Significance was determined using Student’s


The first-line colon cancer chemotherapies oxaliplatin and 5-fluorouracil were assessed in a syngeneic colon cancer model, either as monotherapies or in combination with PD1, comparing radiopharmaceutical uptake to memory-associated immune cells in the tumour. [18F]AlF-NOTA-KCNA3P reliably separated tumours with immunological memory responses from non-responding tumours and could be used to measure Kv1.3-expressing TEM cells responsible for durable immunological memory response to combination therapy in vivo.Often, patients fail to respond to immune checkpoint inhibitor (ICI) treatment despite favourable biomarker status. Numerous chemotherapeutic agents have been shown to promote tumour immunogenicity when used in conjunction with ICIs; however, little is known about whether such combination therapies lead to a lasting immune response. Given the potential toxicity of ICI–chemotherapy combinations, identification of biomarkers that accurately predict how individuals respond to specific treatment combinations and whether these responses will be long lasting is of paramount importance. In this study, we explored [ We used flow cytometry to determine which tumour-infiltrating immune cell populations were associated with response to these treatment combinations in a murine syngeneic colorectal cancer model.Immune checkpoint inhibitors (ICIs) have transformed the field of immunotherapy; however, many patients do not respond to ICI monotherapy, despite favourable biomarker status  ,3. The e T-cells ,11,12,13 T-cells ,14,15. T T-cells ,16. Howe T-cells ,18,19; iM cells) . TEM celmulation . Furthersurvival . ActivatO+CCR7+) ,24,25; h18F]AlF-NOTA-KCNA3P was isolated with a non-decay-corrected radiochemical yield of 12 ± 6 % within 50 min (n = 6) from aqueous [18F]fluoride. The radiochemical purity was greater than 99% and molar activity was 59 ± 16 GBq/µmol at the end of synthesis (n = 6).The precursor NOTA-KCNA3P peptide was custom synthesized by Chinese Peptide Company (CPC) and radiolabelling was performed as previously described . [18F]Al5 cells prepared in a 1:1 v:v ratio in Matrigel, Sigma-Aldrich, Singapore) into the right shoulder. Callipers were used to assess tumour volumes for the duration of the experiment (modified ellipsoid formula 1/2Length × Width2 AlF-NOTA-KCNA3P tumour retention (>0.8%ID/g) and TNR animals with low [18F]AlF-NOTA-KCNA3P tumour retention (<0.5%ID/g) were re-challenged, implanting CT26 tumour cells  into the contralateral left shoulder. Contralateral tumour re-growth was measured using callipers for a further 15 days.After the animals had been separated into treatment responders (TR) and treatment non-responders (TNRs) based on their response to therapy, TR animals with high [18F]AlF-NOTA-KCNA3P (~10 MBq) via the lateral tail vein, static PET acquisitions were acquired and analysis of reconstructed calibrated images was performed using Amide software . Volumes of interest, delineated by CT imaging, were used to determine uptake in tissues. Data are expressed as percentages of the injected dose per gram (%ID/g).Tumoured animals underwent imaging using a Siemens Inveon PET-CT 13 days after tumour implantation as previously described . BrieflyThe flow cytometry procedure has been reported previously . BrieflyTime-gated, size-gated, live, singlet CD45-positive cells from the fcs files were exported from FlowJo and used for dimension reduction analysis as reported previously  for fur.www.graphpad.com, accessed from 1 January 2022). p < 0.05 was considered statistically significant. Data are expressed as means ± S.D. unless otherwise indicated.Kruskal Wallis one-way ANOVA was used to assess the non-parametric datasets, with a Dunn’s post-test for multiple comparisons. All statistics were evaluated using GraphPad Prism version 8.3.4 . Treatment arms including oxaliplatin, 5-FU and αPD1 were administered on their own or in pairwise ICI–chemotherapy combinations . Combined ICI–chemotherapy treatment arms had both greater response rates and response magnitudes than αPD1 or chemotherapy monotherapy arms . The control-treated arm and TNRs showed little tumour retention for [18F]AlF-NOTA-KCNA3P. The ⍺PD1- and 5-FU-responsive tumours both showed significant increases compared to TNRs (** p < 0.01 for both). The OXA responders (*** p < 0.001) and the ⍺PD1 + OXA and ⍺PD1 + 5-FU responders (*** p < 0.001 for both) showed significantly higher tumour uptake of [18F]AlF-NOTA-KCNA3P when compared to TNRs  in the contralateral shoulder and tumour growth was measured for 15 days after implantation. TR animals with high tumour uptake of [18F]AlF-NOTA-KCNA3P on day 13 (>0.8%ID/g) showed negligible tumour regrowth after re-challenge up to 15 days post-re-implantation in the contralateral shoulder, whereas TNR animals with low tumour uptake of [18F]AlF-NOTA-KCNA3P showed significant tumour growth after re-implantation  compared to tumours that did not respond to therapy TNR; . Earlier T-cells ,11,12,13 T-cells ,14,15. HEM cells . In contEM cells .18F]AlF-NOTA-KCNA3P has previously been shown to reliably stratify tumours responding to immune checkpoint inhibitors from non-responding tumours, measuring tumour-associated Kv1.3-expressing TEM cells responsible for durable immunological memory response to combination therapy in vivo; however, little is known about whether adjuvant therapies that modulate the immune environment will synergise with the effects of PD1 blockade to promote a lasting immunological memory response at the tumour. Immunological memory occurs when naïve T-cells are repeatedly exposed to antigens and differentiate into memory T-cells, antigen-specific T-cells that remain long-term and rapidly proliferate in response to antigen re-exposure. Effector memory T-cells (TEM), in particular, are associated with durable tumour response to ICIs AlF-NOTA-KCNA3P  [18F]AlF-NOTA-KCNA3P could be a useful addition to [18F]FDG imaging in the clinic, aiding in therapy management and helping distinguish tumour response from pseudoprogression or immune-related adverse events.5-FU treatment led to increases in tumour-infiltrating NK cells as previously reported , but no observed B. FurtheEM cells D. Unlikehaustion . Despite events) ,36, and  events) . Overall+ TEM tumour infiltration induced by PD1 blockade. Imaging with [18F]AlF-NOTA-KCNA3P accurately measured Kv1.3-expressing TEM cells associated with durable response to combination therapy in vivo. With further development, [18F]AlF-NOTA-KCNA3P may serve as a clinical biomarker to support investigations into new therapy combination strategies to enhance responsiveness to ICIs.Although the chemotherapeutic agents assessed in this study affected the tumour microenvironment in different ways, both led to effective immunological memory responses in tumours and complemented the CD8


CMV infection can cause substantial morbidity and mortality in kidney transplant (KT) recipients due to an impairment in cell-mediated immunity (CMI) from immunosuppressive drugs. We investigated the role of CMI monitoring prior to and after transplant to predict CMV infection after KT.A prospective study was performed between December 2020 and December 2021. All adult KT recipients underwent CMI measurement by investigating IFN-γ-producing T cells using enzyme-linked immunosorbent (ELISpot) assay before and one month post-transplant. The incidence of CMV infection within six months after transplant was reported, and predictors of CMV infection were analyzed using the Cox proportional hazard model.5 PBMCs, p < 0.001). Forty (42.9%) KT recipients who developed CMV infection appeared to have significantly less IFN-γ-producing T cells compared to those did not develop CMV infection (47.1%) . In univariate analysis, predictors for CMV infection included higher panel-reactive antibody , receiving ATG for induction therapy  and lack of CMI one month after transplant defined as IFN-γ-producing T-cells of < 250 SFUs/2.5x105 PBMCs . In multivariate analysis, lack of CMI one month after transplant is the only predictor which remained independently associated with CMV infection  (Table 1).We included 93 KT recipients with a mean (SD) age of 44 (11) years; 59.1% were male, and 98.9% were CMV seropositivity. Twenty-two (23.7%) participants received anti-thymocyte globulin (ATG) for induction therapy. A median (IQR) of IFN-γ-producing T cells measured one month after transplant was significantly lower compared to before transplant (148 [54-389] vs. 763  SFUs per 2.5 x 10KT recipients with low IFN-γ-producing T cell responses are more likely to develop CMV infection post-transplant. Quantification of CMI using ELISpot assay could potentially predict those at risk of CMV infection after KT.All Authors: No reported disclosures.


A/GliA activity by gradually alleviating Sufu-mediated inhibition.The Hh gradient is translated into a phosphorylation gradient of Ci/Gli at multiple Fu/CK1 phosphorylation sites, which progressively increases Ci Graded Hedgehog (Hh) signaling is mediated by graded Cubitus interruptus (Ci)/Gli transcriptional activity, but how the Hh gradient is converted into the Ci/Gli activity gradient remains poorly understood. Here, we show that graded Hh induces a progressive increase in Ci phosphorylation at multiple Fused (Fu)/CK1 sites including a cluster located in the C-terminal Sufu-binding domain. We demonstrated that Fu directly phosphorylated Ci on S1382, priming CK1 phosphorylation on adjacent sites, and that Fu/CK1-mediated phosphorylation of the C-terminal sites interfered with Sufu binding and facilitated Ci activation. Phosphorylation at the N-terminal, middle, and C-terminal Fu/CK1 sites occurred independently of one another and each increased progressively in response to increasing levels of Hh or increasing amounts of Hh exposure time. Increasing the number of phospho-mimetic mutations of Fu/CK1 sites resulted in progressively increased Ci activation by alleviating Sufu-mediated inhibition. We found that the C-terminal Fu/CK1 phosphorylation cluster is conserved in Gli2 and contributes to its dose-dependent activation. Our study suggests that the Hh signaling gradient is translated into a Ci/Gli phosphorylation gradient that activates Ci/Gli by gradually releasing Sufu-mediated inhibition. Drosophila to human (Drosophila homolog Cubitus interruptus (Ci) exist in two distinct forms. In the absence of Hh, full-length Gli/Ci (GliF/CiF) is proteolytically processed into a truncated repressor form (GliR/CiR), which lacks the C-terminal coactivator–binding domain and actively represses the expression of a subset of Hh target genes (A/CiA) that enters the nucleus to activate Hh target genes (The Hedgehog (Hh) family of secreted proteins plays critical roles in embryonic development, adult tissue homeostasis and regeneration, and cancer progression . Initialto human . Gli andet genes . In the et genes .R/CiR and GliA/CiA with increasing levels of Hh progressively decrease the levels of GliR/CiR activity but increase the levels of GliA/CiA activity. Much is known about how GliR/CiR is regulated. The absence of Hh allows GliF/CiF to be phosphorylated by multiple kinases including PKA, glycogen synthase kinase 3 (GSK3), and casein kinase 1 (CK1)  and a Ser/Thr kinase Fused (Fu), which forms a complex with Ci and its kinases including PKA, GSK3, and CK1 to facilitate Ci phosphorylation and processing  , which tGliR/CiR . In Drosocessing . Hh signocessing . In mammocessing .R/CiR, how Hh signaling converts GliF/CiF into GliA/CiA is less understood  or kinase-dead (CC-FuKR) Fu purified from insect cells as the kinase source (EE but not CC-FuKR phosphorylated the GST-CiC fusion protein containing the wild-type (WT) sequence. Substitution of S1382 (S1382A) or both S1382 and S1385 to Ala (2SA) completely abolished the phosphorylation of corresponding GST-CiC fusion proteins  of Ci from aa1371 to aa1397, which is conserved in both Gli2 and Gli3 . Inspect5E/D   provide an optimal spacing between the phospho-acceptor site and D/E/(p)S/T  abolished CiC phosphorylation detected by the pSer antibody in the in vitro kinase assay  and S1230 (middle site), which primed Ci phosphorylation by CK1 on adjacent sites . We founE/(p)S/T . To detecubation . As expese assay . These rWT but not GST-CiCS1382A or GST-CiC2SA after incubation with purified CC-FuEE (EE or CC-FuKR and a non-processed form of Ci (Myc-Ci-PKA), which has three PKA sites  mutated to Ala . The in vitro kinase assay described above showed that pS1382 specially recognized GST-CiC CC-FuEE . Next, Cd to Ala , were co CC-FuKR . Mutatin2 signal , suggest-PKA-WT, Myc-Ci-PKA-S218AS1230A, or Myc-Ci-PKA-S1382A with CC-FuEE in S2R+ cells and detected the phosphorylation of individual sites with the corresponding phospho-specific antibodies. As showed in Because Fu can also phosphorylate Ci on S218 and S1230 , we aske+ cells transfected with Myc-Ci-PKA were treated with Hh-conditioned medium. We found that Myc-Ci-PKA was phosphorylated on S1382 upon Hh treatment . GST pull-down assay showed that GST-CiCSD bound less HA-Sufu derived from S2R+ cell extracts than GST-CiCWT  reporter gene, and this repression was alleviated by coexpression of CC-FuEE (-PKA only slightly reduced the expression ptc-luc compared with wild-type (WT) Ci-PKA. Combined mutations of two or more Fu phosphorylation sites resulted in a more dramatic reduction in ptc-luc expression with SA123 exhibiting the strongest defect , SA2 (S1230A), SA3 (S1382A), SA12 , SA13 , SA23 , and SA123  . These mF to CiA . When trr levels . To dete CC-FuEE . As showt defect . Althougt defect . Hence, ptc expression  to mimic different levels of phosphorylation : SD1 hasr levels . In the activity . When inactivity . In anotactivity .A by antagonizing Sufu  consistently exhibited higher pathway activity than those with less or no phospho-mimetic mutations (SD1 and WT) before reaching a saturated amount of Sufu which inhibited all the Ci constructs  compartment of wing imaginal discs express and secrete Hh, whereas anterior (A) compartment cells express Ci, and consequently, only A-compartment cells near the anterior-posterior (A/P) compartment boundary receive and transduce the Hh signal to activate the Hh pathway target genes such as ptc  that drives the expression of UAS transgenes at low levels throughout wing discs  but not a kinase dead form (HA-Ulk3KR) phosphorylated a GST fusion protein carrying the Gli2 C-terminal region  attenuated its activation by Shh in NIH3T3 cells . To detePtch1 mRNA expression  , we founpression , suggestWT, Gli2SD1, Gli2SD2, and Gli2SD12 were expressed at increasing levels by transfecting increasing amounts of DNA constructs and their relative activities (measured by Gli-luc activity) were compared at a given expression level. As shown in To determine whether increasing levels of Gli2 phosphorylation by Ulk3/CK1 could progressively increase Gli2 activity, we generated phospho-mimetic mutations in N- (SD1) or C-terminal sites (SD2) either alone or in combination (SD12) . These GSD1 and Gli2SD12 were more resistant to Sufu-mediated inhibition than Gli2WT  alone or RA plus ShhN for 24 h, followed by RT-qPCR to determine the expression of Shh responsive genes  equally well  were induced to differentiate into spinal cord neural progenitor cells that can respond to different levels of Shh and express different Shh target genes that specify progenitor subtypes . We deplnfection . mESCs tve genes . Foxa2 aet genes . On the lly well , consistlly well . These rR/GliR and CiA/GliA. It has been well-established that PKA/GSK3/CK1–mediated multisite phosphorylation regulates the formation of CiR/GliR. Here, we provided evidence that Fu/Ulk3/CK1–mediated multisite phosphorylation plays a conserved role in the regulation of the CiA/GliA activity gradient. We showed that Hh induces Ci/Gli phosphorylation at conserved Fu/Ulk3/CK1 sites in a dose- and time-dependent manner. Furthermore, we found that increasing the number of phospho-mimetic mutations of the physiologically relevant Fu/Ulk3/CK1 sites progressively increased CiA/GliA activity by gradually alleviating Sufu-mediated inhibition  from a closed and inactive formation to an open and active formation, leading to dimerization/oligomerization of Smo C-tails  to Ci (How does Fu/Ulk3/CK1–mediated phosphorylation of Ci/Gli contribute to Ci/Gli activation? Our previous studies identified multiple Sufu-binding sites in Ci and Gli proteins including Gli2 and Gli3 . BindingP) to Ci  (Han et P) to Ci , suggestP) to Ci . Indeed,P) to Ci . Further-PKA_SD123 was still activated by CC-FuEE and Hh  and Ulk3KR (HA-Ulk3KR) were subcloned into the pcDNA3.1(+) vector using EcoRI and XbaI sites.Myc-Cieviously . Myc-Ci-T vector . All GSTDrosophila culture and crosses were carried out according to standard procedure. Transgenic flies expressing Ci constructs  were generated using the phiC31 integration system (C765 (BL#36523) was used to drive the expression of UAS transgenes.n system . Gal4 drDrosophila S2R+ cells were cultured in Drosophila serum-free medium (Gibco) containing 10% FBS (Gibco) at 24°C. Clone-8 cells were cultured in Shields and Sang M3 Insect Medium (Sigma-Aldrich) with 2.5% FBS (Gibco), 2.5% fly extract , insulin  at 24°C. The Calcium Phosphate Transfection Kit  was used to perform the transfection experiments according to the manufacturer’s instruction. The NIH3T3 cells were cultured in DMEM (Sigma-Aldrich) containing 10% bovine calf serum (Gibco) and 1% penicillin/streptomycin (Sigma-Aldrich). The transfection was carried out by using the GenJet Plus in vitro DNA transfection kit (SignaGen). HEK293T cells were cultured in DMEM (Sigma-Aldrich) supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin (Sigma-Aldrich). The PolyJet in vitro DNA transfection kit (SignaGen) was used to do the transfection according to the manufacturer’s instruction. For lentivirus production, the N-terminal 6× Myc-tagged mouse WT and Gli2SD12 (Myc-Gli2SD12) and Gli2SA12 (Myc-Gli2SA12) were subcloned into the lentiviral vector FUGW. The expression vectors and package vectors (PSPAX2 and PMDG2) were co-transfected into HEK293T cells using PolyJet (SignaGen). After 48 h, the recombinant viruses were harvested for cell line infection. The recombinant viruses were infected into the NIH3T3 cells using the standard method.+ cells stably expressing the HhN  which is responsible for the hedgehog signaling. The cells were cultured in the medium without hygromycin but with 0.7 mM CuSO4 for 1 d. Then the medium was harvested and sterilized by filtration. Hh-conditioned medium was used at a 6:4 dilution ratio with fresh medium .In vitro kinase assay was performed by incubating 25 μl of reaction mixtures containing 150 mM Tris–HCl (pH 7.5), 0.2 mM Mg2+/ATP, 1 μg of purified GST-Ci/Gli2 fusion proteins, together with appropriate amount of kinase for 30 min at 30°C. The reaction was terminated by adding 2× SDS loading buffer. The resultant samples were load on SDS–PAGE and subjected to the pIMAGO phospho-protein detection kit with the fluor-680 (Sigma-Aldrich) detection kit and LI-COR Odyssey platform for Western blot analyses of phosphorylated proteins (Millipore Sigma). Flag-CC-Fueviously . HA-Ulk3+ cells were cultured in 12-well plates, and on the second day, each well was transfected with 0.5 μg ptc-luc reporter construct, 25 ng RL-PolIII Renilla construct, and 0.5 μg Ci construct. Samples were collected after 48-h incubation. For Hh treatment, 1 d after the transfection, two-thirds of the medium was replaced with HhN-conditioned medium, and cells were incubated for 24 h before harvest. Each sample was measured in triplicate by using a FLUOstar OPTIMA plate reader (BMG Labtech). The Gli-luc reporter assay was carried out by transfecting the NIH3T3 cells in 12-well plate with 0.4 mg 8XGli-Luc reporter, 0.1 mg PRL-SV40 Renilla vector, and 0.5 mg Gli2 construct. The luciferase activity of each sample was measured in triplicate by using a FLUOstar OPTIMA plate reader (BMG Labtech). dsRNAs were generated using the MEGAscript High Yield Transcription Kit (Ambion). The dsRNA targeting the coding sequence of GFP was used as a negative control. Fu dsRNA targets the coding sequence between amino acids 324–434. For RNAi experiments, cells were treated with corresponding dsRNA in serum-free culture medium for 8 h at 24°C before switching to regular growth medium. The dsRNA-treated cells were cultured for 2 d before analysis. shRNA plasmids against eGFP  and mGli2 (TRCN0000219066) were purchased from Sigma-Aldrich.The Dual-Luciferase Reporter Assay System (Promega) was applied to detect the dual–luciferase activity according to the manufacturer’s instructions. Briefly, S2R6 cells using the RNeasy Plus Mini Kit (QIAGEN), and cDNA was synthesized with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems), and qPCR was performed using Fast SYBR Green Master Mix (Applied Biosystems) and a Bio-Rad CFX96 real-time PCR system. qRT-PCR was performed in triplicate for each of three independent biological replicates. Quantification of mRNA levels was calculated using the comparative CT method. Primers: Ptc, 5′-ATGGACCGCGACAGCCTCCCA-3′ and 5′-CGACGCAGAAGGTGCTCAGCA-3′; Gli1, 5′-GTGCACGTTTGAAGGCTGTC-3′ and 5′-GAGTGGGTCCGATTCTGGTG-3′; Ptch1, 5′-GAAGCCACAGAAAACCCTGTC-3′ and 5′-GCCGCAAGCCTTCTCTACG-3′; Foxa2, 5′-GGAGTGTACTCCAGGCCTATTA-3′ and 5′-CTCCACTCAGCCTCTCATTTC-3′; Nkx2.2, 5′-CAGCCTCATCCGTCTCAC-3′ and 5′-TCACCTCCATACCTTTCTCC-3′; GAPDH, 5′-GTGGTGAAGCAGGCATCTGA-3′ and 5′-GCCATGTAGGCCATGAGGTC-3′.Total RNA was extracted from 1 × 10A stable cell line derived from HM1 mESCs harboring 8XGBS-Venus reporter at hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus was first transduced by shRNA lentivirus targeting 3′ UTR of Gli2. The Knock down efficiency was validated by qRT-PCR (>90%). Myc-tagged WT and mutant mGli2 were introduced by lentivirus and further G418 selection. The resultant mESCs were cultured with feeder MEFs onto dishes pre-coated with 0.1% gelatin in medium . For differentiation, feeder cells were eliminated by trypsinization followed by 2× 10 min incubation for settlement. mESC were then cultured in CellBIND plates at the density of 60,000 cells per six well in N2B27 medium . For the first 2 d (day 0 and 1) after the seeding, cells were cultured in N2B27 medium supplemented with 10 ng/ml bFGF. On day 2, the differentiation was induced by adding RA  for 12 h. After that, the cells were treated with Shh N  for another 12 h. Then samples were collected for qRT-PCR.


To the Editor: We commend our colleagues in Brazil for completing a multicenter, open-label, randomized controlled trial (RCT) of coronavirus disease (COVID-19) convalescent plasma (CCP) against wild-type severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (https://www.medrxiv.org/content/10.1101/2021.12.10.2126748v1) and 1 that provided inpatient treatment within 3 days of symptom onset   than controls (35%), given that the prevailing view is that this therapy functions as an antiviral and should not be effective in late disease. A similar finding is apparent in most other RCTs of hospitalized patients (


Drosophila, expression of pro-apoptotic genes, including reaper (rpr), head involution defective (hid), grim, and sickle (skl), is sufficient to induce cell death. Here, we demonstrate that the chromatin remodeler Dmp18, the homolog of mammalian Znhit1, plays a crucial role in regulating apoptosis in eye and wing development. We showed that loss of Dmp18 disrupted eye and wing development, up-regulated transcription of pro-apoptotic genes, and induced apoptosis. Inhibition of apoptosis suppressed the eye defects caused by Dmp18 deletion. Furthermore, loss of Dmp18 disrupted H2Av incorporation into chromatin, promoted H3K4me3, but reduced H3K27me3 modifications on the TSS regions of pro-apoptotic genes. These results indicate that Dmp18 negatively regulates apoptosis by mediating H2Av incorporation and histone H3 modifications at pro-apoptotic gene loci for transcriptional regulation. Our study uncovers the role of Dmp18 in regulating apoptosis in Drosophila eye and wing development and provides insights into chromatin remodeling regulating apoptosis at the epigenetic levels.Programmed Cell Death (PCD) or apoptosis is a highly conserved biological process and plays essential roles both in the development and stress context. In  Drosophila. Znhit1 encodes a Zinc finger HIT-type containing protein and works as a component of the chromatin remodeling complex in yeast and mammals. In the current study, we identified that Dmp18, the homolog of Znhit1, regulates apoptosis by mediating the histone variant H2Av incorporation and H3K4me3 as well as H3K27me3 modifications around the transcription start site (TSS) regions of pro-apoptotic genes. Dmp18 deletion up-regulated the transcription of pro-apoptotic genes including rpr and hid, triggered massive cell death, and resulted in eye and wing defects. Importantly, loss of Dmp18 disrupted H2Av incorporation into chromatin, increased H3K4me3, but decreased H3K27me3 modifications on the TSS regions of pro-apoptotic genes for transcriptional activation. Together, this study reveals the mechanism by which Dmp18 regulates apoptosis in the eye and wing discs.Apoptosis is an important biological process in the development and stress context by removing unwanted or damaged cells. Dysregulation of apoptosis causes many diseases including cancers. Transcriptional induction of pro-apoptotic genes is sufficient to induce apoptosis in  Drosophila, expression of the pro-apoptotic genes, including reaper (rpr), head involution defective (hid), grim, and sickle (skl), is sufficient to induce cell death [Maintaining the balance between cell death, proliferation, and differentiation is needed for organogenesis and histogenesis in multicellular organisms. Programmed Cell Death (PCD) or apoptosis plays essential roles both in the development and stress context by removing unwanted or damaged cells to keep the balance , 2. Dysrll death –10, whicll death –14. ThusH2A.Z in mice leads to embryonic death [Mammals have multiple H2A variants, including H2A.Z and H2A.X. H2A.Z is considered the most universal variant and is highly conserved in eukaryotes. Deletion of ic death , suggestic death –18. In aic death –28. Receic death –32 and tic death –35. In aic death , 37.Drosophila only has a single H2A variant, H2Av, which belongs to the H2A.Z family and is necessary for animal survival [Drosophila H2Av may serve the functions of mammalian both H2A.Z and H2A.X in transcriptional regulation and DNA damage response [survival , 39. ItsZnhit1 in mice disrupts organ development and homeostasis maintenance [Drosophila as a model system and generated the Drosophila Znhit1 (referred to as Dmp18) mutant fly, Dmp18d1, to investigate its role in eye and wing development. Our study showed that Dmp18 regulates apoptosis by controlling the transcription of pro-apoptotic genes in the eye and wing discs. We further demonstrated that Dmp18 mediates the incorporation of histone variant H2Av into chromatin and the modifications of both H3K4me3 and H3K27me3 on the TSS regions of pro-apoptotic genes for transcriptional regulation. Thus, our study reveals the role of Dmp18 in regulating apoptosis at the epigenetic levels in the eye and wing discs.The dynamic exchange of histone H2A with histone variant H2A.Z on chromatin is catalyzed by the ATP-dependent chromatin remodeling complex SWR1/SRCAP , 41. Znhntenance –51. HoweCG31917 caused rough and small eyes  by P-element mediated imprecise excision, which contained a 224 bp deletion  activity in the Dmp l8 mutant. As shown in Dmp18 mutant clones both in the eye and wing discs. We also observed massive Cas3* staining in the eye-antennae disc and the wing pouch region in homozygous Dmp18d1  in the Dmp18 mutant clones and examined the Cas3* activity. The result showed that over-expression of Dmp18-V5 completely inhibited the Cas3* activity in the Dmp18 mutant clones H99 (referred to as H99) removes three pro-apoptotic genes, including rpr, hid, and grim (RHG), and suppresses apoptosis both in the development and stress response context [Dmp18 deletion-induced apoptosis. The results showed that the Cas3* activity in the Dmp18 mutant clones was significantly inhibited , was up-regulated in homozygous Dmp18d1  and the effector caspase Death related ICE-like caspase (Drice) were also up-regulated  was unchanged in Dmp18d1  nor knocking down p53 by RNAi could inhibit Cas3* activity in the Dmp18 mutant clones  [H2Av increased the Cas3* activity and TUNEL signals both in the eye and wing discs   induced ng discs . The RNAted Figs . The GO ted Figs , which ited Figs . The tra H2Av810 . Interesdeletion . The hidng discs . Moreovenockdown . Inhibitegulated  Figs. ThDmp18 deletion and H2Av deletion generate similar phenotype, we hypothesized that Dmp18 regulates apoptosis by controlling H2Av deposition. To test our hypothesis, we examined the H2Av deposition on the polytene chromosome in the homozygous Dmp18d1. As expected, loss of Dmp18 disrupted H2Av deposition on chromatin  and performed the rescue experiment with an over-expression of H2Av-myc in the Dmp18 mutant clones to examine the Cas3* activity. The result showed that over-expression of H2Av-myc alone did not activate Cas3*  assay and sequencing. The result showed that the H2Av-binding peaks around the TSS of rpr were lower than that of hid in wild-type . The results from the anti-H2Av CUT&Tag assay showed that the H2Av-binding peaks on the rpr locus were lower than those of hid in wild-type, indicating that rpr may belong to the low H2Av-binding gene relative to hid. Our data revealed that loss of Dmp18 has limited effects on H2Av incorporation in low H2Av-binding genes such as rpr. Although the H2Av binding at rpr locus is not changed, the active histone mark H3K4me3 is increased and the silencing histone mark H3K27me3 is slightly reduced at the TSS region of rpr, which may induce the transcriptional up-regulation of rpr. These results suggest that Dmp18 may control rpr transcription via being involved in regulating histone modifications. Further studies are needed to define the involved mechanisms.Znhit1 regulates gene transcription by controlling the incorporation of H2A.Z , 50, 51.hromatin . The CUT H2Av810 , but itso change . These dDrosophila, the single DOMINO chromatin remodeling complex combines the functions of histone acetylation activity and histone variant exchange [H2Av or domino causes a global down-regulation of gene expression [H2Av deletion enhanced the phenotypes of Polycomb (Pc) mutation and suppressed of the phenotypes of Trithorax mutant [Distalless (Dll) transcription in Drosophila embryo [Dmp18 or H2Av deletion leads to high expression of pro-apoptotic genes by modifying the binding of epigenetic marks H3K4me3 and H3K27me3 at gene loci  was generated from yw;P{EP}CG31917G5894Tfb5G5894 (BL26915) by P-element-mediated imprecise excision which deleted 224 bp. The primer used to detect the deletion as followed: Forward: 5’-CTATCGATGACGCTCACAGC-3’; Reverse: 5’-CGACAAGGACGCTTAGAACC-3’. The fly lines: hid-lacZ, puc-lacZ, mirror-Gal4, ptc-Gal4, en-Gal4, tubulin-Gal4, H2Av810 (BL9264), UAS-H2Av IR (BL28966 and BL44056), UAS-luciferase IR, Df(3L)H99, UAS-BSKDN (BL6409 and BL9311), yw, hs-FLP; FRT40A-M(2L)-Ubi-GFP/BCG and yw, ey-Gal4, UAS-FLP; neoFRT40A, GMR-hid/cyo;UAS-CD8-GFP/TM2 were obtained from Bloomington Drosophila Stock Center . The H2Av810 was described previously [Df(3L)H99 is a deficiency line that deleted the pro-apoptotic genes including rpr, hid, and grim [yw,hs-FLP,tub-Gal4,UAS-nlsGFP/FM7;tub-Gal80,neoFRT40A/cyo  and UAS-p53 IR (THU2533 and THU5318) were obtained from Tsing Hua Fly Center . The UAS-Dmp18-V5 and UAS-H2Av-myc transgenic flies were generated in this study.All eviously . The Df, mouse anti-V5 , chicken anti-beta Galactosidase , rabbit anti-Hid (d-300) , rabbit anti-H2Av , goat anti-Myc , rat anti-Elav . The primary antibodies were detected by fluorescent-conjugated secondary antibodies from Invitrogen . The nuclear was detected by Hoechst 33258 . The TUNEL assay kit was from Roche  and performed as described previously . All picp<0.05 was considered statistically significant.All animals were crossed at the same time and raised under identical circumstances. The eye and wing imaginal discs were obtained from the third instar larvae by randomly with the correct genotype, and the pictures were taken by confocal laser scanning microscope at the same magnification. The adult eyes were obtained from females by microscope  at the same magnification. For SEM, adult flies were anesthetized, mounted on the stage, and observed under the scanning electron microscope  in the vacuum mode. The pictures were taken at the same magnification. The relative size of eye discs, wing discs, and adult eyes was measured by Photoshop software . For size measure, we used the wand tool in Photoshop to select the region that needs to be measured and recorded the pixel value in the histogram (cache level is set to 1). The pixel value represented the relative size. All data were analyzed by Graphpad Prism 8 , using the two-tailed unpaired t-test or the Mann Whitney test (means ± SEM). RPL32. The data represented an average of at least three independent assays and were analyzed by Graphpad Prism 8 , using the two-tailed unpaired t-test or Mann Whitney test for two groups and one-way ANOVA with Tukey’s multiple comparisons test for multiple groups (means ± SEM). p<0.05 was considered statistically significant. The primers used for qPCR were listed in the Total RNA was extracted from 1 third instar larva (for single larva qPCR) or 3–5 third instar larvae with TRIzol . The complementary DNA (cDNA) was prepared with the M-MLV Reverse transcriptase . The qPCR reactions were performed using SYBR select Master Mix  on Quant Studio 5 . Quantification was normalized to Drosophila BDGP6 version 92 reference genome by HISAT2, the uniquely mapped reads were used to estimate the expression values at the gene level by FPKMs. Statistical significant tests of differentially expressed genes were performed by DEseq with R. Genes with absolute log2 fold change > 1 were regarded as differentially expressed genes and p < 0.05 was used. Hierarchical clustering of log2-transformed FPKMs was generated by Cluster 3.0 and visualized by Java TreeView. The raw NGS data were deposited to the NCBI SRA database under Bioproject ID: PRJNA761186 (the accession numbers are from SRR15734269 to SRR15734277).RNA from 10 third instar larvae were isolated by TRIzol and then converted into cDNA libraries. High-throughput sequencing was performed using Illumina Novaseq 6000 platform for 3 biological replicates. Over 40 million reads were obtained for each sample. The RNA-seq data were mapped to w1118, Dmp18d1, and H2Av810 third instar larvae. The CUT&Tag was performed using the Hyperactive In-Situ ChIP Library Prep Kit for Illumina . The antibodies for CUT&Tag assay: rabbit anti-H2Av , rabbit anti-H3K4me3 , rabbit anti-H3K27ac , and mouse anti-H3K27me3 . The sequencing was performed using the Illumina Novaseq 6000 platform. The raw NGS data were deposited to the NCBI SRA database under Bioproject ID: PRJNA761186 (the accession numbers are from SRR15734939 to SRR15734950).The eye and wing discs for the CUT&Tag assay were isolated from S1 FigDmp18 by RNAi induced adult eye defects.(A) Control. (B-C) Knockdown of (TIF)Click here for additional data file.S2 FigDmp18, Tfb5, and Dmp18d1. The red arrows labeled the primer location for detecting Tfb5 transcription. (B) Total RNA was extracted from control and homozygous Dmp18d1 larvae. The mRNA level of Tfb5 was measured by RT-qPCR and normalized to RPL32. The mRNA level of Tfb5 was not affected.(A) The Schematic diagram of the genomic region of (TIF)Click here for additional data file.S3 FigDmp18 deletion. The Elav was expressed in the Dmp18 mutant clones in the eye disc. GFP-free region marked the Dmp18 mutant clones. (B-B”) The control eye disc staining with photoreceptor marker Elav. (C-C”) Homozygous Dmp18d1 eye disc staining with photoreceptor marker Elav. The photoreceptor cells showed normal Elav staining. (D-D”) Some homozygous Dmp18d1 eye disc showed an abnormal arrangement of photoreceptor cells. Genotypes: A-A”: yw, hs-FLP; FRT40A-M(2L)-Ubi-GFP/FRT40A-Dmp18d1; B-B”: FRT40A/ FRT40A; C-D”: FRT40A-Dmp18d1/FRT40A-Dmp18d1.(A-A”) The differentiation of photoreceptor cells was not affected by (TIF)Click here for additional data file.S4 FigDmp18 mutant clones generated by the Minute clone technique. GFP-free region marked the Dmp18 mutant clones. (C-F’) TUNEL signals were increased in homozygous Dmp18d1 discs (E-E’ and F-F’) compared to the control discs (C-C’ and D-D’). Genotype: A-B”’: yw, hs-FLP; FRT40A-M(2L)-Ubi-GFP/FRT40A-Dmp18d1; C-D’: FRT40A/FRT40A; E-F’: FRT40A-Dmp18d1/FRT40A-Dmp18d1.(A-B”‘) TUNEL signals were increased in the (TIF)Click here for additional data file.S5 FigDmp18 activated Cas3* in the eye and wing discs. (C-D”’) Over-expression of Dmp18-V5 in the Dmp18 mutant clones suppressed Cas3* activity in the eye (C-C”’) and wing (D-D”’) discs. GFP marked the mutant cells or mutant cells with over-expressed Dmp18-V5. Genotypes: A-B”: yw, hs-FLP, tub-Gal4, UAS-nls-GFP/+; tub-Gal80, neoFRT40A/FRT40A-Dmp18d1; C-D”’: yw, hs-FLP, tub-Gal4, UAS-nls-GFP/+; tub-Gal80, neoFRT40A/FRT40A-Dmp18d1; UAS-Dmp18-V5/+.(A-B”) Loss of (TIF)Click here for additional data file.S6 FigDmp18d1. 1266 genes were up-regulated, and 987 genes were down-regulated in Dmp18d1. The x-axis showed the log2 fold change and the y-axis showed corresponding -log10 p values. Red and blue dots marked differentially expressed genes , and the gray dots marked no differentially expressed genes. (B) The Gene Ontology (GO) analysis of the Dmp18-regulated genes. The Dmp18-regulated genes were involved in multiple biological processes such as tissue development, metabolism, and stress response. (C) The KEGG pathway enrichment analysis of the Dmp18-regulated genes. The GO and KEGG enrichment analyses were performed by Metascape online (https://metascape.org) corresponding to the mini overlap was 3 and p <0.05.(A) The volcano plot showed differentially expressed genes in homozygous (TIF)Click here for additional data file.S7 FigDmp18 activated Cas3* in the eye and wing discs. (B) Inhibition of JNK signaling by expressing dominant negative JNK (BSKDN) did not inhibit Cas3* activity in the Dmp18 mutant clones in the eye and wing discs. (C) The Hid expression was up-regulated in the Dmp18 mutant clones. (D-E) Expression of BSKDN did not reduce the expression of Hid (D) and hid-lacZ (E). (F) Expression of BSKDN suppressed the up-regulated puc-lacZ expression induced by Dmp18 deletion in the eye and wing discs. GFP marked the mutant cells or the mutant cells with expressed UAS-BSKDN. Genotypes: A and C: yw, hs-FLP, tub-Gal4, UAS-nls-GFP/+; tub-Gal80, neoFRT40A/FRT40A-Dmp18d1; B and D: yw, hs-FLP, tub-Gal4, UAS-nls-GFP/UAS-BSKDN; tub-Gal80, neoFRT40A/FRT40A-Dmp18d1; E: yw, hs-FLP, tub-Gal4, UAS-nls-GFP/UAS-BSKDN; tub-Gal80, neoFRT40A/FRT40A-Dmp18d1; hid-lacZ/+; F: yw, hs-FLP, tub-Gal4, UAS-nls-GFP/UAS-BSKDN; tub-Gal80, neoFRT40A/FRT40A-Dmp18d1; puc-lacZ/+.(A) Loss of (TIF)Click here for additional data file.S8 FigDmp18 activated Cas3* in the eye and wing discs. (B) Knockdown of p53 did not suppress Cas3* activity in the Dmp18 mutant clones in the eye and wing discs. (C) Knockdown of p53 did not reduce the up-regulated Hid expression in the wing disc. (D) The RT-qPCR results showed the transcription of rpr. The rpr still showed high expression when p53 was knocked down by RNAi in the homozygous Dmp18d1. (E) The RT-qPCR results showed the knockdown efficiency of p53 in the wild-type and homozygous Dmp18d1. The transcription of p53 was reduced in the wild-type and Dmp18d1 when p53 was knocked down by RNAi. The GFP marked mutant cells or mutant cells with knocking down p53. Genotypes: A: yw, hs-FLP, tub-Gal4, UAS-nls-GFP/+; tub-Gal80, neoFRT40A/FRT40A-Dmp18d1; B: yw, hs-FLP, tub-Gal4, UAS-nls-GFP/+; tub-Gal80, neoFRT40A/FRT40A-Dmp18d1; UAS-p53 IR5318/+; C: yw, hs-FLP, tub-Gal4, UAS-nls-GFP/+; tub-Gal80, neoFRT40A/FRT40A-Dmp18d1 and yw, hs-FLP, tub-Gal4, UAS-nls-GFP/+; tub-Gal80, neoFRT40A/FRT40A-Dmp18d1; UAS-p53 IR5318/+.(A) Loss of (TIF)Click here for additional data file.S9 FigH2Av810. 1365 genes were up-regulated, and 881 genes were down-regulated in H2Av810. The x-axis showed the log2 fold change and the y-axis showed corresponding -log10 p values. The red and blue dots marked differentially expressed genes , and the gray dots marked no differentially expressed genes. (B) The GO analysis of the H2Av-regulated genes. The H2Av-regulated genes were involved in multiple biologic processes including tissue development, metabolism, and stress response. (C) The KEGG pathway enrichment analysis of the H2Av-regulated genes. The GO and KEGG enrichment analyses were performed by Metascape online (https://metascape.org) corresponding to the mini overlap was 3 and p <0.05.(A)The volcano plot showed differentially expressed genes in homozygous (TIF)Click here for additional data file.S10 Fighttps://metascape.org) corresponding to the mini overlap was 3 and p <0.05.Go and KEGG analyses showed that the Dmp18 and H2Av co-regulated genes were involved in multiple biological processes including tissue development, metabolism, and stress response. The GO and KEGG enrichment analyses were performed by Metascape online ((TIF)Click here for additional data file.S11 FigH2Av was knocked down by RNAi. (C-C”) H99 suppressed the TUNEL signals induced by H2Av knockdown. Genotypes: A-A”: ptc-gal4/+; UAS-luciferase IR/+; B-B”: ptc-gal4/UAS-H2Av IR44056; C-C”: ptc-gal4/UAS-H2Av IR44056; H99/+.(A-A”) Control. (B-B”) TUNEL signals were increased when (TIF)Click here for additional data file.S12 FigDN driven by en-gal4 did not affect H2Av expression and TUNEL signals. (B-B”’) Expression of BSKDN did not suppress the TUNEL signals induced by H2Av knockdown. (C-C’) The TUNEL signals were increased when H2Av was knocked down by RNAi. (D-D’) Knockdown of H2Av up-regulated hid-lacZ expression. (E-E”’) Expression of BSKDN driven by ptc-gal4 failed to suppress TUNEL signals (E’) and reduce the hid-lacZ expression (E”) in H2Av knockdown cells. (F-F”) Expression of BSKDN driven by ptc-gal4 suppressed the puc-lacZ expression induced by H2Av knockdown. Genotypes: A-A”’: en-gal4, UAS-GFP/+; UAS-BSKDN/+; B-B”’: en-gal4, UAS-GFP/UAS-H2Av IR44056; UAS-BKSDN/+; C-C’: ptc-gal4/UAS-H2Av IR44056. D-D’: ptc-gal4/UAS-H2Av IR44056; hid-lacZ/+; E-E”’: ptc-gal4/UAS-H2Av IR44056; UAS-BSKDN/hid-lacZ; F-F”: ptc-gal4/UAS-H2Av IR44056; UAS-BSKDN/puc-lacZ.(A-A”’) Expression of BSK(TIF)Click here for additional data file.S13 FigH2Av knockdown cells. (B-C’) knockdown of p53 did not reduce TUNEL signals induced by H2Av knockdown. (D-D’) Knockdown of H2Av increased hid-lacZ expression. (E-E’) Knockdown of p53 did not suppress hid-lacZ expression induced by H2Av knockdown. (F) Statistical analysis of relative hid-lacZ fluorescence intensity in the D and E (the number of D = 17 and number of E = 15). To calculate the hid-lacZ fluorescence intensity, we subtracted the average fluorescence intensity near the ptc region (marked by a rectangle) from the average fluorescence intensity in the ptc region in the wing pouch. The data represented the relative hid-lacZ fluorescence intensity and were analyzed by Graphpad Prism 8 , using the Mann Whitney test (means ± SEM). Genotypes: A-A’: ptc-gal4/UAS-H2Av IR44056; B-B’: ptc-gal4/UAS-H2Av IR44056; UAS-p53 IR5318/+; C-C’: ptc-gal4/UAS-H2Av IR44056; UAS-p53 IR2533/+; D-D’: ptc-gal4/UAS-H2Av IR44056; hid-lacZ/+; E-E”: ptc-gal4/UAS-H2Av IR44056; UAS-p53 IR5318/hid-lacZ.(A-A’) TUNEL signals were increased in (TIF)Click here for additional data file.S14 Figen-gal4 did not activate Cas3* in the posterior region of wing disc.(A-A”) Control. (B-B”’) Over-expression of H2Av-myc driven by (TIF)Click here for additional data file.S15 FigH2Av mutant clones in the eye and wing discs. The GFP marked the H2Av mutant clones. (C-C’) The commercial H2Av antibody detected the binding of H2Av at the polytene chromosome in homozygous H2Av810. (D-D’) The commercial H2Av antibody detected the H2Av staining in some salivary gland cells in homozygous H2Av810. Genotypes: A-B’: yw, hs-FLP, UAS-GFP/+; tub-Gal4, FRT82B, tub-Gal80/FRT82B-H2Av810; C-D’: H2Av810/H2Av810.(A-B’) The H2Av expression level was reduced in the (TIF)Click here for additional data file.S16 FigDmp18d1 larvae showed increased hid transcription. (B-C) Compared with control 2 and control 3, two of the four homozygous Dmp18d1 larvae showed increased hid transcription. (D) Compared with control 4, none of the four homozygous Dmp18d1 larvae showed increased hid transcription. Except for the mutant 3, which did not show an obvious change in rpr transcription compared with control 4 (the fold change is 1.6), all other Dmp18 mutant larvae showed increased rpr transcription compared with controls. (The fold change >2 was considered up-regulated).(A) Compared with control 1, none of the four homozygous (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.