Drosophila by using histone lysine-to-alanine mutants.Histone modifications are critical in regulating neuronal processes. However, the impacts of individual histone modifications on learning and memory are elusive. Here, we investigated the contributions of histone H3 lysine modifications to learning and memory in Drosophila overexpressing mutated H3K23A with a CBP inhibitor did not aggravate the learning defect.Behavioural analysis indicated that compared to the H3WT group, mutants overexpressing H3K23A displayed impaired courtship learning. Chromatin immunoprecipitation analysis of H3K23A mutants showed that H3K23 acetylation (H3K23ac) levels were decreased on learning-related genes. Knockdown of CREB-binding protein (CBP) decreased H3K23ac levels, attenuated the expression of learning-related genes, led to a courtship learning defect and altered development of the mushroom bodies. A decline in courtship learning ability was observed in both larvae and adult treatments with ICG-001. Furthermore, treatment of dCBP, contributes to Drosophila learning, likely by controlling the expression of specific genes. This is a novel epigenetic regulatory mechanism underlying neuronal behaviours.H3K23ac, catalysed by the acetyltransferases The online version of this article (10.1186/s12861-018-0179-z) contains supplementary material, which is available to authorized users. Learning and memory can be broadly defined as lasting alterations to behavioural outputs that are produced in response to a transient environmental input . UnderstH2A, H2B, H3 and H4, which make up the octamer around which the DNA duplex is wound. Histone acetylation, which usually affects the interaction between DNA and histones and further regulates gene transcription [MOZ, Ybf2/Sas3, Sas2 and Tip60) and p300/CBP [Histone modifications are chemical groups, such as acetyl and methyl groups, that are enzymatically added to and removed from particular amino acids in the histones cription –8, is cacription , 10. HATp300/CBP . With hip300/CBP .Aplysia, when a sensory neuron is stimulated by 5-hydroxytryptamine (5-HT), CBP is recruited to the C/EBP (CAAT box enhancer binding protein) promoter, followed by the acetylation of specific lysine residues on different histones, which further induce gene expression [CBP function exhibit reduced histone acetylation levels and impairments in long-term memory ability [HDAC2 exhibit impaired memory formation, whereas HDAC2 knockout mice exhibit enhanced memory formation [Drosophila, HDAC4 overexpression in adult mushroom bodies impairs long-term courtship memory [PP1) [EZH2, the major methyltransferase of histone H3K27, regulates adult hippocampal neurogenesis. EZH2 knockout mice have impaired spatial learning and memory, contextual fear memory, and pattern separation [According to recent genetic and pharmacological evidence, changes in the activity of histone-modifying enzymes impact cognition . For inspression . Moreove ability –19. HDAC ability , 21. Micormation . In Drosp memory . Based op memory . Thus, Hp memory . In addiry [PP1) . EZH2, tparation .Furthermore, correlational evidence indicates that histone post-translational modifications, predominantly histone acetylation, are modulated by experiences, such as learning and memory . Early sDrosophila strains, characterized the effects of each mutant on learning and memory, and identified an association between H3K23 acetylation and courtship learning in Drosophila. Furthermore, we observed that a loss of dCBP, a histone H3 lysine 23 acetyltransferase, also impaired neuronal gene activation and resulted in defects in courtship learning. However, inhibition of dCBP in the H3K23A overexpression line did not have additive effects on learning. Therefore, dCBP-related H3K23 acetylation is required for successful learning.Histone-modifying enzymes, such as HATs and HDACs, have multiple functions and substrates, and genetic evidence alone may be insufficient to definitively demonstrate their involvement in a specific transcriptional process . As therDrosophila strains with UAS-histone H3 lysine-to-alanine mutations were constructed. These mutant strains overexpressed histone H3 with specific lysine residues changed to alanine residues. First, to validate whether the overexpressed histone H3 mutants, which were fused with GFP, were incorporated into the chromatin, we examined the expression of GFP on polytene chromosomes from third-instar larvae of the histone mutant lines via immunofluorescence assays. We found that GFP was indeed expressed in the polytene chromosomes . Unsuccessful courtship reduces the subsequent courtship behaviour of male flies.[Drosophila  , ligand-gated channels , phospholipase C (norpA), and phosphodiesterase (dnc) [eag and shakB, significantly decreased in the H3K23A overexpression line.  –40. The Cngl and gfA can regulate the activity of calcium signalling pathways [2+ responses induced by KCl stimulation of larval brains. Fura-red was used as a red Ca2+ indicator dye combined with GFP-tagged brain. Ca2+ signals can be presented as values (ΔR/R0) of the relative change rate in fluorescence intensity (ΔR) normalized to the baseline fluorescence rate (R0). The H3K23A mutant was driven by Elav-GAL4. H3K23A mutant larval brains showed a lower calcium response after KCl stimulation than the control group  assay with larval brains using antibodies against H3K23ac or H3K23me1 followed by real-time PCR. The PCR primers were designed to amplify the sequences of the coding regions of the affected neuronal genes. The intergenic regions were used as controls. CompareDrosophila. GCN5 can acetylate the H3K23 site in yeast [Drosophila[dCBP catalyse H3K23 acetylation in flies [dCBP is one of the enzymes related to H3K23ac, that regulates courtship learning. Therefore, we constructed transgenic RNAi flies with an Elav-GAL4 driver to disrupt the expression of dCBP. The RNAi efficiency was validated by RT-qPCR, and the mRNA level of dCBP was greatly reduced in larval brains with the pan-neuronal driver Elav-GAL4 . Indeed, the levels of H3K23ac were significantly decreased in the dCBP RNAi line . As expected, when tested in the behavioural assay, the dCBP RNAi line exhibited learning defects  and dCBP RNAi larval brains using an antibody against H3K23ac followed by real time-PCR. Compared with those in the control group, H3K23ac enrichment levels were significantly decreased in most of these neuronal genes, except for eag  in memory index in H3K23A overexpression flies, additional studies are needed to elucidate the mechanisms determining whether H3K23ac is combined with other factors to regulate learning and memory.Given the multiple functions and substrates of HATs and HDACs, genetic evidence alone may be insufficient to definitively demonstrate the involvement of these enzymes in a specific transcriptional process . Here, w targets , 53, 54.CBP, a histone acetyltransferase, produced similar phenotypes and attenuated the expression of learning-related genes. In particularly, these two factors did not exert addictive effect on learning regulation in Drosophila. This indicating that H3K23ac, which is catalysed by the acetyltransferases CBP, regulates the Drosophila learning, likely by controlling specific genes.Histone modifications are highly conserved in eukaryotes and have been shown to regulate gene transcription in neuronal processes. Our results presented here suggest an important role for H3K23ac in regulating courtship learning. Overexpression the H3K23A mutant impaired courtship learning. Knocking down RNAi(TH00781.N), UAS-dCBPRNAi (THU1718), OK107-GAL4, Elav-GAL4 and SG-GAL4; Histone mutants: UAS-H3.3WT-GFP, UAS-H3K23A-GFP, UAS-H3K4A-GFP, UAS-H3K18A-GFP, UAS-H3K37A-GFP, and UAS-H3K122A-GFP. UAS-GFPRNAi and UAS-H3WT-GFP lines were used as controls.All flies were raised on a standard cornmeal-agar medium at 25°C under 12:12 light/dark conditions unless otherwise stated. The following strains were from the TsingHua Fly Center: UAS-GFPThe methods were based on previous work by us and others , 55. FliFor the learning assay, males were placed into cells  of a wheel chamber via a mouth aspirator. The males adapted to the chamber for 3-5 min. Each trained male was paired with a mated female for 1 hour, and video was recorded during the initial 10 min and last 10 min of 1 hour. After training, males were transferred to a new chamber. For controls (sham training), males were isolated in the chamber for 1 hour.For the memory training, a male was paired with a mated female in a small food tube (1.5 ml centrifuge tubes with food) for 5 hours. The naïve males were isolated in the tube. After training, males were transferred to a new tube, and their memory was immediately tested. Memory in males was tested by pairing the male with a freeze-kill virgin female, and the test was recorded for 10 min.initial, CIfinal, CItest and CIsham. The CI is the percent of time males exhibited courtship behaviour  during the 10 min recording. If copulation occurred during training, or the male scored CIinitial<0.1, the data were discarded. The learning index (LI) was also calculated. The LI is the ratio of the courtship level during the final 10 min of training  to the initial 10 min. The memory index was calculated by dividing CItest by the mean of the sham control courtship levels, CIsham. An LI or MI score ≥1 indicates no learning or memory.For each 10 min recording, we calculated a courtship index (CI) for each male: CILI=CIfinal/ CIinitialMI= CItest/ mean CImsha2O) for 10 min. The samples were washed in PBS three times. Then, the samples were incubated with the following antibodies at 4°C overnight: H3K23ac ; Fas2 . After the primary antibody incubation, the salivary glands were incubated with secondary antibody  for 1 hour and stained with DAPI (1:1000) for 10 min. Finally, the samples were observed with a fluorescence microscope.We performed immunofluorescence as previously described. The salg for 3 min at 4°C to collect the supernatants. Protein concentration was measured with a BCA protein assay kit (cwbiotech), and equal amounts of protein were loaded to perform western blotting. The following antibodies were used: H3K23ac , H3 , H3K23me1 .In total, 120 pairs of salivary glands or brains from third-instar larva were lysed in 200 μl of 4% SDS. The lysates were incubated in 50 μl of 5× SDS loading buffer and boiled for 10 min followed by centrifugation at 14,000 × A total of 50 larval heads for one sample were homogenized with TRIzol reagent (Takara) according to the manufacturers’ instructions. 2 μg of total RNA was utilized for cDNA synthesis with an OligodT primer (Takara) and M-MLV reverse transcriptase (Promega). The generated cDNA was utilized for RT-qPCR. All PCR reactions were performed in an ABI 7500 system. The following primers were used:para F- AGGACTTCTGGGAGGATCTGTpara R- AGCTTCTTCCGCTTCACGTAshakB F- GCAAAATATGGAGTTTTTACGCGGshakB R- CATCTTCGGGAATGTCTCTTGTATGHk F- CAATCGTAATGCCGCTCTGTCHk R- ACTGGCTCATGTTTCAGCGASh F-GCACAAAAATCGAGGAAGACGASh R-GGACATGCGAGGAACCTGAcngl F- ATATACGATGGAAGCGCACGAcngl R-GAGATACTGGTGCCCGTGGSK F-TCGCCTGGGAAAGAGGAAAGSK R-CGAGTAGAACGATGCCTTTGTGdnc F-TACATCGTCCACCCGCTATGdnc R-GGGTTACTTGAAAGCGTATCCTGnorpA F-CGTCATGCCAGGGACATCAAnorpA R-GCCGACGATCCTTCTCGTTgfA F-GTGGACCTACGATGGGTTTCAgfA R-TACCGGGCACACCTAACAAGeag F- ACACGAAGGGAGTTTTAGGTCTCeag R- GGAGTATGTGTGGTGGCGTdCBP F- TCGCAAACCCAAATCAAAAAGGAdCBP R-CAGTTATCGCACACGAAGCCrp49 F-AAGCTGTCGCACAAATGGCrp49 R-CTTCTTGAATCCGGTGGGCAact5c F-AGTCGGTTTATTCCAGTCATTCCTTact5c R- AGAGCAGCAACTTCTTCGTCAαTub84B F- CTGTGAATTTTCCTTGTCGCGTαTub84B R- CCAGCAGGCGTTTCCAATCβTub56D F-GCGCCAAGTTCTGGGAGATβTub56D R-CACCGGACGCCTCATTGTAgapdh1 F- CCCAATGTCTCCGTTGTGGAgapdh1 R-CATCGGTGTAGCCCAGGATTrp49 was considered a reference gene.Elav-GAL4 males, and the GFP RNAi strain was crossed with the same GAL4 as a control. For one chromatin immunoprecipitation, approximately 400 brain-disc complexes were dissected from third-instar larvae and stored at -80°C. Tissue fixation was conducted in PBS with 1% formaldehyde for 20 min at room temperature, and the process was stopped by the addition of 125 mM glycine for 5 min. After two washes with PBS, the tissues were lysed in RIPA buffer  and were sonicated for 10 min . This sonication method yields genomic DNA fragments of 150-500 bp. The soluble chromatin was collected by centrifugation at 4°C for 10 min at 12000 rpm. 10 μl aliquots of supernatants were used to estimate chromatin concentrations after reverse cross-linking (input DNA). A total of 15 μg of genomic DNA was incubated with 10 μl of antibody at 4°C overnight. The antibody-chromatin complexes were captured with the addition of 30 μl of Protein A/G-Agarose beads suspension (Santa Cruz Biotechnology). The beads were washed three times with RIPA buffer and once with elution buffer . After reversal crossing-linking, input DNA and immunoprecipitation DNA were purified via phenol-chloroform extraction. RT-qPCR was performed to measure the enrichment of H3k23ac on target genes.The ChIP assay was performed as previously described . dCBP RNThe following primers were used:SK F-TGATGGACACGCAGTTGACSK R-TTGGTGGGTTCTTACACGGpara F-CTGCCACAAAGACCCATACpara R-AAATCGAGCCAACACCACeag F-GTTACCACCATCATCCAGCeag R- CTCGGTATCCAGACCCTTGshakB F-TGAGACGGCAGACAGAGCshakB R-TTCTTCCTGGGTCCTTCAGCSh F-TACTTGTTGCTGGCGGGTCSh R-AATGGGTTCAAAGTGTTGCGcngl F-GTGGTCACTCCCTATCCGcngl R-CATGTCCGTCTCACTAAATTCCnorpA F-TCAACAGCTCGCATAATACCnorpA R-CTCGTCCTCACCCTTTCCgfA F-AGAAGCCATCACGCAGTCgfA R-AAGTCCCAGGCCATAGACCHk F- ACGGGCGGTCATCTTGATTTHk R- ACCCGCTTTTGCCCAGATTdnc F- GACAGTATGCACGCTCCCdnc R-CCAGCTCATTCTCCCTGGintergenic region F-GCTGATGCTTCCTGAAATCCintergenic region R-GTTTGGTGTGCTCGTCCTTT0) was calculated by the fluorescence intensity of fura-red dividing by that of GFP preceding the stimulus. △R/R0 was calculated by relative change in fluorescence intensity normalized to the baseline fluorescence. This is a negative deflection when the intracellular Ca2+ concentration increases using fura-red as the Ca2+ indicator dye. So, we multiplied the ΔR/R0 values with -1 to obtain positive signals to display a rise in Ca2+.Calcium imaging was performed following previously described protocols , 57. LarAdditional file 1:Histone mutants are incorporated into the chromatin. Salivary gland immunostaining was performed in histone H3 mutant flies with anti-GFP. The overexpression of Histone mutants was driven by SG-GAL4. Scale bars: 50 μm. (DOCX 440 kb)Additional file 2:Training for 5 hours made the learning index of H3K23A overexpression group at the similar level to the H3WT group. A courtship learning experiment was conducted to value the learning levels after training 5 hours. Each male fly was allowed to train for 5 hours as the method described. The learning index of courtship was the time spent during the final 10 min vs. the initial 10 min. Unpaired t-test was used for statistics. Error bars represent the standard error of the mean; the number of samples was indicated in the bar. n.s., not significant. p=0.1379. (DOCX 50 kb)Additional file 3:p<0.05, **p<0.01. (DOCX 240 kb)The total initial courtship time do not affect courtship learning indexes. (a-e) The data of total courtship time were from the courtship learning and memory experiments. Flies overexpressing H3K23A, H3K4A, H3K18A and H3K122A were compared to H3WT overexpression group, respectively. Except for the group of overexpressing the H3K37A, there were no significant change in the total courtship time among other groups of flies. (f-k) The learning index data were divided into two subgroups by the median. Then the learning indexes are compared to another subgroups. These analyses were used to study whether lower CI has an impact on the learning index. Unpaired Two-tailed Student’s t-test was used. Error bars represent the standard error of the mean; the number of samples was indicated in the bar. n.s., not significant. *Additional file 4:Overexpression of the H3K23A mutants affected the gene expression and Calcium signaling. (a) Four housekeeping genes were examined by RT-qPCR. The samples were from larval brains. The expression levels were normalized to the levels of rp49. No significant difference was observed in this experiment. (b) Calcium signalling was impaired in H3K23A mutants during KCl stimulus. The baseline rate (R0) was calculated by the fluorescence intensity of fura-red dividing by that of GFP preceding the stimulus. △R/R0 was calculated by relative change in fluorescence intensity normalized to the baseline fluorescence. n= 6 for each group. (DOCX 187 kb)Additional file 5:dCBP RNAi flies. (a) The data of total courtship time were from the flies with Ok107-GAL4 derived RNAi in courtship learning experiments. (b-c) The learning index data of either GFP RNAi groups or dCBP RNAi groups were divided into two subgroups by the median. Then the learning indexes are compared to another subgroups. Unpaired Two-tailed Student’s t-test was used. Error bars represent the standard error of the mean; the number of samples was indicated in the bar. n.s., not significant. ***p<0.001. (DOCX 149 kb)The total initial courtship time in the GFP RNAi flies and the Additional file 6:The total initial courtship time of the groups with or without treatment of ICG-001. The data of total courtship time were from the courtship learning experiments. Unpaired t-test was used. Error bars represent the standard error of the mean; the number of samples was indicated in the bar. n.s., not significant. *p<0.05, ##p<0.01 (elav-GAL4 group compared to H3WT group). (DOCX 71 kb)Additional file 7:dCBP RNAi flies. (a) Immunostaining of H3K23ac in the adult brains were conducted for observing the morphology of brains. (b-d) The mushroom bodies were stained by anti-fas2 antibodies. (b) It seems to be no significant change in mushroom bodies in the group of overexpressing the H3K23A mutant. (c) The defects of mushroom bodies were observed in the flies knockdown dCBP. (d) The same experiment was used for detecting whether ICG-001 impaired the development of mushroom bodies. n=3. (DOCX 1213 kb)The structure of mushroom body in H3K23A overexpression and Additional file 8:Treatment with ICG-001 in adult flies affected the courtship learning. (a) A courtship learning experiment was included to test whether treatment with ICG-001 in adult flies led to impair the learning ability. The flies were treated with 10μM of ICG-001 for 4 to 5 days after eclosion. The learning index of courtship was the time spent during the final 10 min vs. the initial 10 min. Unpaired t-test was used for statistics. Error bars represent the standard error of the mean; the number of samples was indicated in the bar. *, p<0.05. (b) The mushroom bodies were stained by anti-fas2 antibodies. Scale bars: 50 μm. n=5. (DOCX 443 kb)Additional file 9:dCBP RNAi fly courtship learning. Videos were recorded during the initial and final 10 min of the 60 min training session. Video 1: H3WT flies exhibited courtship suppression after training. Video 2: H3K23A flies exhibited a reduction of courtship suppression and a high courtship index after training. Video 3: GFP RNAi flies exhibited normal courtship suppression after training. Video 4: dCBP RNAi flies exhibited less courtship suppression after training. (ZIP 25330 kb)Representative movies of H3WT, H3K23A, GFP RNAi and


Although the high-throughput sequencing technique is useful for evaluating gastric microbiome, it is difficult to use clinically. We aimed to develop a predictive model for gastric microbiome based on serologic testing.Helicobacter pylori [HP] antibody or pepsinogen [PG] levels).This study was designed to analyze sequencing data obtained from the Hanyang University Gastric Microbiome Cohort, which was established initially to investigate gastric microbial composition according to the intragastric environment. We evaluated the relationship between the relative abundance of potential gastric cancer-associated bacteria (nitrosating/nitrate-reducing bacteria or type IV secretion system [T4SS] protein gene-contributing bacteria) and serologic markers (IgG anti-2) was 53.7% and 70.0% in the model for nitrosating/nitrate-reducing bacteria and T4SS protein gene-contributing bacteria, respectively.We included 57 and 26 participants without and with HP infection, respectively. The relative abundance of nitrosating/nitrate-reducing bacteria was 4.9% and 3.6% in the HP-negative and HP-positive groups, respectively, while that of T4SS protein gene-contributing bacteria was 20.5% and 6.5% in the HP-negative and HP-positive groups, respectively. The relative abundance of both nitrosating/nitrate-reducing bacteria and T4SS protein gene-contributing bacteria increased exponentially as PG levels decreased. Advanced age (only for nitrosating/nitrate-reducing bacteria), a negative result of IgG anti-HP antibody, low PG levels, and high Charlson comorbidity index were associated with a high relative abundance of nitrosating/nitrate-reducing bacteria and T4SS protein gene-contributing bacteria. The adjusted coefficient of determination (RNot only the negative results of IgG anti-HP antibody but also low PG levels were associated with a high abundance of nitrosating/nitrate-reducing bacteria and T4SS protein gene-contributing bacteria. Helicobacter pylori (HP) infection is the most potent known risk factor for gastric cancer, and over 50% of the global population over 40 years old has HP colonization in the stomach . However, the PG I/II ratio in the HP-negative group was higher than that in the HP-positive group . The Charlson comorbidity index did not differ between the groups (P = 0.413).S1 Fig. The proportion of Firmicutes and non-HP Proteobacteria was 29.8% and 29.5%, respectively, in the HP-negative group, and 4.2% and 6.6%, respectively, in the HP-positive group. In the HP-positive group, the proportion of Helicobacter was 81.0%. In the linear discriminant analysis, various bacterial taxa were abundant in the HP-negative group, whereas a few bacterial taxa, including HP, were significant in the HP-positive group .The relative abundance of bacteria at the phylum level in the HP-negative and HP-positive groups is shown in S3 Fig. The relative abundance of nitrosating/nitrate-reducing bacteria was 4.9% and 3.6% in the HP-negative and HP-positive groups, respectively, while Neisseria was the predominant bacterial taxon . The relative abundance of T4SS protein gene-contributing bacteria was 20.5% and 6.5% in the HP-negative and HP-positive groups, respectively.The relative abundance of nitrosating/nitrate-reducing bacteria and T4SS protein gene-contributing bacteria is shown in S4 Fig. Two outliers (one each for PG I and PG II) were identified using the Bonferroni outlier test, and they were excluded in the analysis of predictive model development due to avoid overfitting of the model.PG I and II levels according to the HP infection are shown in Fig 1 shows the relationship between the relative abundance of bacteria (nitrosating/nitrate-reducing bacteria or T4SS protein gene-contributing bacteria) and PG levels, which seemed to exhibit an exponential inverse correlation. Specifically, the bacterial abundance increased exponentially as PG levels decreased. The relationship between relative abundances and PG levels according to the results of HP infection status are shown in Fig 2. Participants with negative IgG anti-HP antibody results had a higher abundance of both nitrosating/nitrate-reducing and T4SS protein gene-contributing bacteria than those with positive results did. Bacterial abundance increased as PG levels decreased in each subgroup according to the results of the IgG anti-HP antibody analysis.Table 2 illustrates predictive models for the logarithm of the abundance of nitrosating/nitrate-reducing bacteria. In the multivariable analysis, advanced age, negative IgG anti-HP antibody results, low PG I, low PG II (model 1) or high PG I/II ratio (model 2), and high Charlson comorbidity index were identified as factors associated with the relative abundance of nitrosating/nitrate-reducing bacteria. The adjusted coefficient of determination (R2) values of models 1 and 2 were 53.7% and 49.1%, respectively.Table 3. The factors associated with T4SS genes-contributing bacteria were similar to those with nitrosating/nitrate-reducing bacteria. In the multivariable analysis, a negative result in the IgG anti-HP antibody test, low PG I, low PG II (model 1) or high PG I/II ratio (model 2), and high Charlson comorbidity index were identified as factors associated with the relative abundance of T4SS protein gene-contributing bacteria. The adjusted R2 of models 1 and 2 were 70.0% and 66.7%, respectively.The predictive model for T4SS gene-contributing bacteria is shown in 2 = 5.0%, P < 0.001; PG I: R2 = 9.3%, P < 0.001; PG II: R2 = 10.0%, P < 0.001; IgG anti-HP antibody: R2 = 16.8%, P < 0.001; Charlson comorbidity index: R2 = 1.6%, P = 0.046).The permutational analysis of variance showed that age, PG I, PG II, IgG anti-HP antibody, and Charlson comorbidity index had significant effects on the overall bacterial community (age: R). There appeared to be an inverse relationship between PG levels and the relative abundance of Cyanobacteria and T3SS protein gene-contributing bacteria, as shown in S5 Fig; however, the relative abundance of Cyanobacteria did not significantly correlate with PG I and II . Additionally, no significant association was observed between the relative abundance of T3SS protein gene-contributing bacteria and PG I levels (P = 0.469).To evaluate whether IgG anti-HP antibody and PG levels were associated with bacterial subgroups other than nitrosating/nitrate-reducing bacteria and T4SS protein gene-contributing bacteria, the relative abundance of Cyanobacteria and T3SS protein gene-contributing bacteria was presented according to the IgG anti-HP antibody and PG levels . Besides, the interpretation of sequence data obtained from the next-generation sequencing technique is relatively difficult. Therefore, it is not widely used clinically. On the other hand, conventional methods such as rapid urease test or histologic examination can test only the presence of HP among various intragastric bacteria. In our study, we suggested that potential gastric carcinogenesis-associated bacteria may be predicted using serologic testing of IgG anti-HP antibody and PG levels. Although the RIn contrast, patients with positive anti-HP antibody results and high PG levels may not need to be overly concerned about gastric cancer because they may have fewer gastric carcinogenesis-related bacteria other than HP. However, over time, chronic gastritis may progress to atrophic gastritis or intestinal metaplasia, and PG levels are expected to decrease gradually. Therefore, periodic serological testing may be needed to assess changes in the gastric environment of these patients.The relatively high abundance of nitrosating/nitrate-reducing bacteria and T4SS protein gene-contributing bacteria in patients with negative results of anti-HP antibody and high PG levels should be cautiously interpreted. These patients may not have current or past HP infection, and the risk of gastric cancer is extremely low , despiteFig 3) [Prediction of bacterial abundance in the stomach based on serologic testing is similar to the ABCD method, which is a risk stratification method for gastric cancer prediction based on the IgG anti-HP antibody and PG levels  . In the In our study, the relative abundance of nitrosating/nitrate-reducing bacteria and T4SS protein gene-contributing bacteria increased as PG levels decreased. Additionally, Cyanobacteria and T3SS protein gene-contributing bacteria tended to be more abundant in patients with low PG levels. Generally, low PG levels are associated with decreased gastric acid secretion and high intragastric pH. Therefore, various bacterial taxa may have the opportunity to increase in patients with low PG levels. However, the relative abundance of Cyanobacteria and T3SS protein gene-contributing bacteria did not significantly correlate with PG levels. In other words, PG levels may be more helpful to predict potential gastric carcinogenesis-related bacteria, such as nitrosating/nitrate-reducing bacteria and T4SS protein gene-contributing bacteria.2 values of the models were not high enough for the accurate prediction of gastric microbial abundance. Many other factors, including diet, lifestyle, and genetic factors as well as interactions among intragastric microorganisms, have the potential to affect the gastric microbiome composition. To evaluate the exact abundance of the gastric microbiome, gastric microbiome analysis based on next-generation sequencing should be considered. However, we do not think that determining the exact abundance of gastric microbiomes such as nitrosating/nitrate-reducing bacteria is imperative in clinical practice. Just knowing whether their abundance is high or low could help stratify the risk of gastric cancer. Second, our study was performed in a single institute. Although gastric microbiome analysis tools and serologic testing methods could be unified, a generalization of our results to other institutions or countries may be limited. Different prevalence of HP or differences in dietary habits among countries may influence the prediction model.Although our study suggests that bacterial abundance in the stomach may be predicted using serologic testing of IgG anti-HP antibody and PG, it has some limitations. First, the RDespite these limitations, our data enhance the understanding of the relationship between the results of serologic testing of IgG anti-HP antibody and PG and abundance of nitrosating/nitrate-reducing bacteria and T4SS protein gene-contributing bacteria. Not only HP but also low PG levels may be important to predict the abundance of nitrosating/nitrate-reducing bacteria and T4SS protein gene-contributing bacteria.S1 FigHelicobacter Proteobacteria (Brown) and Helicobacter (OliveDrab) are demonstrated separately.For phylum Proteobacteria, non-Helicobacter pyloriHP, (TIF)Click here for additional data file.S2 FigP < 0.05 by the Kruskal-Wallis test).For all bacterial taxa evaluated, a significant difference was observed between the groups Click here for additional data file.S3 FigHelicobacter pylori.HP, (TIF)Click here for additional data file.S4 FigHelicobacter pylori; Hollow gray circle indicates outlier.HP, (TIF)Click here for additional data file.S5 FigCyanobacteria and (A) PG I or (B) PG II. T3SS protein gene-contributing bacteria and (C) PG I or (D) PG II.PG, pepsinogen; T3SS, type III secretion system.(TIF)Click here for additional data file.S6 FigCyanobacteria and (A) PG I or (B) PG II. T3SS protein gene-contributing bacteria and (C) PG I or (D) PG II.P = 0.469), although it was associated with PG II (P = 0.003).No statistically significant relationship was observed between the relative abundance of Cyanobacteria and PG I or II. The proportion of Cyanobacteria was 0% in three patients with negative IgG anti-HP antibody results, despite relatively low PG I and II levels. Additionally, the relative abundance of T3SS protein gene-contributing bacteria was not significantly correlated with PG I Click here for additional data file.


Solanum spp.) possess gametophytic self-incompatibility that is primarily controlled by a single multiallelic locus called the S-locus which is composed of tightly linked genes, S-RNase (S-locus RNase) and multiple SLFs (S-locus F-box proteins), which are expressed in the style and pollen, respectively. Using S-RNase genes known to function in the Solanaceae gametophytic SI mechanism, we identified S-RNase alleles with flower-specific expression in two diploid self-incompatible potato lines using genome resequencing data. Consistent with the location of the S-locus in potato, we genetically mapped the S-RNase gene using a segregating population to a region of low recombination within the pericentromere of chromosome 1. To generate self-compatible diploid potato lines, a dual single-guide RNA (sgRNA) strategy was used to target conserved exonic regions of the S-RNase gene and generate targeted knockouts (KOs) using a Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (Cas9) approach. Self-compatibility was achieved in nine S-RNase KO T0 lines which contained bi-allelic and homozygous deletions/insertions in both genotypes, transmitting self compatibility to T1 progeny. This study demonstrates an efficient approach to achieve stable, consistent self-compatibility through S-RNase KO for use in diploid potato breeding approaches.Potato breeding can be redirected to a diploid inbred/F1 hybrid variety breeding strategy if self-compatibility can be introduced into diploid germplasm. However, the majority of diploid potato clones ( Solanum tuberosum L.) is the third most important food crop after rice and wheat (n = 4x = 48) relies on the discovery and introgression of genes from wild species for traits such as disease resistance, the polyploid nature of cultivated tetraploid potato hampers the fixation of desirable alleles in new cultivars. For example, the introgression of critical dominant alleles such as the potato virus Y (PVY) disease-resistance gene in a triplex or quadruplex allelic configuration can take up to 15 years (n = 2x = 24) would allow the application of efficient breeding methods  and SLFs (S-locus F-box) genes known also as S-haplotype-specific F-box brothers (SFBB), expressed in the style and pollen, respectively  inactivates the gametophytic SI system  potato lines, the inbred line M6 was generated from the wild tuber-bearing species, hacoense . In M6, I system  leading Genome editing by Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated protein 9 (Cas9) system has been widely used to generate gene knockouts (KOs) of candidate genes related to agronomic traits in important crops . Cas9 inS-RNase ribonuclease activity in S. peruvianum and S. pennellii, leading to self-compatibility  DRH-195 and DRH-310 F1 lines were generated from a cross between S. tuberosum Gp. Phureja DM 1-3 516 R44 (DM) and S. tuberosum Gp Tuberosum RH 89-039-16 (RH) at Virginia Tech and used in this study. Plants were maintained in vitro, propagated on Murashige and Skoog (MS) medium    and cultS-RNase gene was selected using the top blast hits. Expression abundances across a range of developmental stages, tissues, and organs were determined using available gene expression atlases for DM and RH  of the National Center for Biotechnology Information ] searches were performed using reported S-RNase protein sequences  from theM and RH . To idenormation  and aligormation . Duplicaols v1.2  with theols v1.2 .S-RNase gene in DRH-195 and DRH-310 using the detected S-RNase variants  with the following thermocycler conditions: one cycle of initial denaturation for 4 min at 94°C, followed by 34 cycles for 15 s at 94°C, 45 s at 56°C and 1 min at 72°C and a final extension of 5 min at 72°C. S-RNase amplicons were gel-purified using the QIAquick PCR Purification Kit  and cloned into the Zero Blunt TOPO PCR Cloning vector . Ten colonies for each line were sequenced by the Sanger method and aligned using Clustal Omega  and second (sgRNA 2) EN tools . A gene 01 pMP90  by electAgrobacterium-mediated transformation was performed using leaf segments from 4-week-old tissue culture plants of DRH 195 and DRH 310 as described by D) for 4 days and inoculated with Agrobacterium. After 3 days, explants were rinsed with sterile distilled water containing 250 mg/l cefotaxime and 200 mg/l carbenicillin and placed onto step II media . Explants were transferred to fresh step II media every week. After approximately 30 days, transformation events (T0 lines) were selected from step II media and transferred to root induction media containing MS medium supplemented with antibiotics for selection as described above.0 plants was isolated as described above. PCR was carried out using the GoTaq DNA polymerase  with the following thermocycler conditions: one cycle of initial denaturation for 4 min at 94°C, followed by 34 cycles for 15 s at 94°C, 45 s at 56°C and 1 min at 72°C and a final extension of 5 min at 72°C. Amplicons were visualized on 1% (w/v) agarose gels. Allelic mutations of positive transformation events were identified by insertion/deletion presence. Selected transformation events were amplified with the Q5 High-Fidelity DNA Polymerase . Then, purified PCR products were cloned into the Zero Blunt TOPO PCR Cloning vector , and transformed into DH5α competent cells . Colonies carrying the alleles from each event were Sanger sequenced.DNA from Tin vitro plants were planted in one gallon plastic pots with a peat and perlite growth medium mixture  and placed into a greenhouse with a light intensity of 250 μmoles m-2 s-1, 16/8-h light/dark photoperiod and a temperature of 25°C. Plants were fertilized with Peters Professional® 20: 20: 20 fertilizer  at a rate of 500 mg/l twice a week. Around 50 flowers per plants were hand self-pollinated to test for self compatibility. Pollen staining with acetocarmine-glycerol  were self-pollinated at anthesis. Pollinated pistils were excised 24-hour post pollination (hpp) and preserved in -80°C until use. Total RNA was isolated using the RNeasy Plant Mini Kit  and DNase treated using the TURBO DNA-free kit  following manufacturer’s instructions. RNA was quantified using a NanoDrop spectrophotometer  and a reverse-transcription polymerase chain reaction (RT-PCR) was carried-out with 1 μg of total RNA using the Super-Script One-Step RT-PCR kit . Primers designed to amplify the S-RNase ORF and the elongation-factor one alpha (EF1α) housekeeping internal control were used for the RT-PCR reaction (Twenty-five flowers from wild-type (WT) and DRH-195/310-derived Treaction .S-RNase gene, the DM genome sequence was selected as the reference genome and utilized in sequence similarity searches using seven Solanaceae S-RNase genes  predicted protein composed of five conserved and two hypervariable regions, characteristic of S-RNases  is highly expressed in mature flowers [245.5 Fragments Per Kilobase of exon model per Million mapped reads (FPKM)] compared with no expression in leaves or tubers in DM. High S-RNase expression levels were also detected in carpels (4342.7 FKPM) consistent with its role in preventing self compatibility  and not tubers or leaves. Together with the functional annotation, these results suggest we have identified the S-RNase gene involved in SI.We found that the DM tibility . More litibility  and cons1 diploid self-incompatible lines derived from a cross between DM and RH were used to identify S-RNase allelic variants. Using whole genome resequencing data for these two lines, the DM and RH S-RNase alleles were identified in DRH-195 and DRH-310 and validated using Sanger sequencing. As previously observed for DM, the S. tub_Sp5 predicted 216 amino acid sequence was detected in both lines, beside one of the two RH S-RNase alleles in each line, hereafter referred as S. tub_St5 and S. tub_St6  allele exhibited highest percentage of similarity relative to the other species (Petunia (P. int_S2 and P. hyb_Sx) and S. tuberosum (S. tub_St2) S-RNases clustered in two separate subgroups. Two S. tuberosum Gp Phureja alleles, S. tub_Sp3 and S. tub_Sp4, were located in separate clades , presumably representing the same allele. Moreover, S. tub_Sp5, S. tub_St5, and S. tub_St6 alleles had similar amino acid identity (53–54%) to the reported S. tub_Sp3 allele when compared with S. tub_Sp4 , corresponding to 6.1 and 18.9 Mb of chromosome 1 in the physical map  and inversions (486 bp) were also observed in a bi-allelic configuration. Similarly, besides the described mutation types, chimeric mutations were detected in T0 lines, which has been reported in other species subjected to CRISPR-mediated mutagenesis, potentially due to late embryogenesis editing  was used to efficiently generate  DRH-310 . Multipl DRH-310 . Based uT0 lines . Specifiequenced . A disti editing .0 KOs lines underwent spontaneous chromosome doubling, chloroplasts were counted in stomatal guard cells. One out of 10 S-RNase KO lines (DRH-195.104) revealed chromosome doubling which has also been observed in a related S-RNase KO approach , in addition to the segregation of the mutated S-RNase alleles (1 line (DRH-195.158.6). These results demonstrate the advantage of using CRISPR/Cas9 to generate Cas9-free edited plants and the potential to transmit stable gene mutations through different generations.Viable Thenotype . Cas9 in alleles . Likewis1 hybrid cultivar development in diploid potatoes because efforts involving crossing with wild SC relatives result in many undesirable traits segregating in the progeny. To redirect potato breeding toward an efficient inbred/F1 hybrid generation strategy, we exploited the S-RNase-based SI system in diploid potatoes and generated KO lines using CRISPR-based genome editing to achieve self-compatibility.Self-incompatibility has been a limiting factor for inbred/FS. tub_Sp5, S. tub_St5, and S. tub_St6 alleles . Conversely, a clear intra-specific separation within the S. tuberosum S-RNase alleles  proteins, can directly or indirectly interact with S-RNase contributing to the SI response  associated with the RH allele, fixed in selfed populations, is likely the primary factor for self-fertility in the DRH F1 progeny. Overall, this study demonstrates that S-RNase is the primary component for self-pollen rejection in DRH-195 and DRH-310. However, external evidence suggests that besides RH self-fertility mechanisms, S-RNase-independent stylar factors and environmental conditions could play a role in spontaneous self-compatibility observed in the WT lines in this study.This hypothesis is further supported by S-RNase gene using the CRISPR-Cas9 system. We first computationally identified three new S-RNase alleles in self incompatible diploid lines  and mapped this gene to chromosome 1 within the peri-centromeric region consistent with the localization of the S-locus to a low recombination region on chromosome 1. S-RNase KO lines were obtained using a dual sgRNA strategy in which premature stop codons were generated. After self-pollination, fruits were set in selected KO lines in T0 and T1 lines. Cas9-free KO lines were also identified in T1 lines. Our results demonstrated the inheritance and stablity of the S-RNase KO phenotype, which can contribute to utilization of SC as a first step for the generation of commercial diploid cultivars.In this study, we generated self-compatible potato diploid lines by targeting the Publicly available datasets were analyzed in this study. This data can be found in FE-R and DD conceived the research idea. FE-R, NM-C, SN, CB, DZ, and DD designed the research experiments, analyzed the data, and wrote the manuscript. FE-R performed laboratory experiments. FE-R and NM-C performed self-pollination assays.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Schizosaccharomyces pombe (Sp) has two mating types, Plus (P) and Minus (M). The mating pheromones P-factor and M-factor, secreted by P and M cells, are recognized by the receptors mating type auxiliary minus 2 (Mam2) and mating type auxiliary plus 3 (Map3), respectively. Our recent study demonstrated that a few mutations in both M-factor and Map3 can trigger reproductive isolation in S. pombe. Here, we explored the mechanism underlying reproductive isolation through genetic changes of pheromones/receptors in nature. We investigated the diversity of genes encoding the pheromones and their receptor in 150 wild S. pombe strains. Whereas the amino acid sequences of M-factor and Map3 were completely conserved, those of P-factor and Mam2 were very diverse. In addition, the P-factor gene contained varying numbers of tandem repeats of P-factor (4–8 repeats). By exploring the recognition specificity of pheromones between S. pombe and its close relative Schizosaccharomyces octosporus (So), we found that So-M-factor did not have an effect on S. pombe P cells, but So-P-factor had a partial effect on S. pombe M cells. Thus, recognition of M-factor seems to be stringent, whereas that of P-factor is relatively relaxed. We speculate that asymmetric diversification of the two pheromones might be facilitated by the distinctly different specificities of the two receptors. Our findings suggest that M-factor communication plays an important role in defining the species, whereas P-factor communication is able to undergo a certain degree of flexible adaptation–perhaps as a first step toward prezygotic isolation in S. pombe.In fungi, mating between partners depends on the molecular recognition of two peptidyl mating pheromones by their respective receptors. The fission yeast  An asymmetric pheromone/receptor system in the fission yeast Schizosaccharomyces pombe might allow flexible adaptation of pheromones to mutational changes while maintaining stringent recognition for mating partners, perhaps as a first step toward prezygotic mating isolation. S. pombe each secrete a pheromone (P-factor and M-factor), which binds to a corresponding receptor (mating type auxiliary minus 2 [Mam2] and mating type auxiliary plus 3 [Map3]) on cells of the opposite sex. The interaction between a pheromone and its receptor is essential for successful mating. Here, we explored conservation of the mating pheromone communication system among 150 wild S. pombe strains of different geographical origins and the closely related species S. octosporus. We found that 1) the M-factor/Map3 interaction was completely conserved, whereas the P-factor/Mam2 interaction was very diverse in the strains investigated, and 2) most of the P-factor variants were functional across species. Thus, we have revealed an asymmetric pheromone/receptor system in fungal mating: namely, whereas M-factor communication operates extremely stringently, P-factor communication has the flexibility to create variations, perhaps facilitating prezygotic isolation in S. pombe.The emergence of a new species might occur when two groups can no longer mate. Although such reproductive isolation is considered a key evolutionary process, the mechanisms by which it actually occurs have been confined to conjecture. The two sexes (Plus [P] and Minus [M]) of  S. pombe demonstrated that several mutations in a pheromone and its corresponding receptor created a new prezygotic barrier that can give rise to a new species [Reproductive isolation, which restricts gene flow between sympatric populations, is one of the key mechanisms of speciation . Mating  species . This exS. pombe has two mating types, Plus (P) and Minus (M) [+ (map2+) gene encodes a precursor polypeptide containing four tandem repeats of mature P-factor, which is a mixture of three different peptides in the laboratory strain. P-factor is secreted by the standard secretory pathway [mfm1+, mfm2+, and mfm3+ [S. pombe M cells also produce a pheromone-degrading enzyme encoded by the sexually activated 2+ (sxa2+) gene [mfm genes encoding M-factor might be differentially controlled to facilitate fine tuning. These differences in the two mating pheromones, including chemical structure, secretion pathway, and degradation, are widely common in ascomycetes [inus (M) ,13. Undeinus (M) . P cellsinus (M) ,16. The  pathway . Mature nd mfm3+ ,19. Eachnd mfm3+ . S. pomb2+) gene –23. Sxa22+) gene , and the2+) gene . Yeast c2+) gene . Degrada2+) gene . In contS. pombe, L968, has four copies of the P-factor–encoding sequence and three genes encoding M-factor [S. pombe strains [S. pombe and the related species S. octosporus. Whereas So-M-factor was not functional in S. pombe, all So-P-factors tested were partially functional in S. pombe, enabling these cells to mate successfully using So-P-factors. Thus, recognition of M-factor is highly stringent, whereas that of P-factor is relatively relaxed. Our findings suggest that M-factor communication plays an important role in partner discrimination, whereas P-factor communication allows flexible adaptation to create variations. We speculate that such an asymmetric pheromone/receptor system may have evolved to create a prezygotic barrier in S. pombe.The standard laboratory strain of M-factor . Hence,  strains ,29, findS. pombe mating pheromones and their corresponding receptors in nature, we analyzed 150 wild strains , the receptor gene for M-factor (map3), the P-factor gene (map2), and the receptor gene for P-factor (mam2) of all 150 strains and compared the nucleotide sequences with that of the L968 strain registered in the database . We constructed a phylogenetic tree based on the sequences of these six genes in the 151 S. pombe strains  produced an identical mature M-factor peptide, YTPKVPYMCFar-OCH3 (102 genes have been previously reported [map2 genes were very diverse (map2 gene in L968 carries four P-factor–encoding tandem repeats (P1-P2-P3-P2), extensive variations in the number of repeats were observed in the 150 strains, ranging from four to eight . Many m diverse  Tables. to eight . In addimap2 gene in about half of the 150 strains ; remarkably, however, the zygote frequency of the remaining strains producing the other P-factor peptides  was extremely low or zero (map2+ gene carrying a sequence of repeats (P1-P2-P3-P2) was introduced, the frequency of zygotes was fairly high (76.6% ± 5.1%), despite the production of only two copies of the P2 peptide (map2 gene carrying a permuted sequence of repeats (P2-P3-P2-P1) into the FY23418 strain. Similar to the strain with the native sequence, this strain also mated at high frequency (74.2% ± 1.7%) (To compare the activity of the different P-factor peptides (P1–P6), we introduced a modified FY23418; , in whiceted see . The res or zero . Further peptide . To dete ± 1.7%) ; therefomap2 gene carrying the four tandem P-factor–encoding repeats P5-P2-P5-P2 and introduced it into the FY23418 strain. Surprisingly, however, this strain was almost as sterile as the strain with the 2-repeat ORF  and examined the zygote frequency of these strains in the same way. Remarkably, the strains producing eight copies of peptide showed a much higher frequency of zygotes , we constructed strains carrying two  zygotes  relative zygotes . We consmap2 gene was found to contain at least four tandem repeats encoding multiple P-factor peptides in all 150 wild strains. To assess the effect of producing multiple P-factors, we performed a quantitative competitive mating assay. In this experiment, P cells producing a multiple P-factor (P1-P2-P3-P2) and P cells producing a single P-factor (P2-P2-P2-P2) were inoculated with wild-type M cells onto malt extract agar (MEA) plates at a cell number ratio of 1:1:2. The three strains were differentially marked by different drug-resistant markers  became shmooing cells in response to P-factor after 1 day . Notablysxa2+ ORF was cloned into the pTS111 plasmid downstream of the no message in thiamine 1+ (nmt1+) promoter, which is strongly expressed in absence of thiamine [nmt1+ promoter, and the culture supernatant was assayed to confirm carboxypeptidase activity  were sufficiently degraded after 60 minutes in the presence of Sxa2 . Unexpec sterile . Hence, S. pombe strains analyzed. To determine whether this asymmetry in pheromone diversity is common to other species, we analyzed the nucleotide sequences of both M-factor and P-factor genes in S. octosporus, the species most closely related to S. pombe.As described above, the amino acid sequences of M-factor were completely conserved, whereas those of P-factor were diversified in 150 wild S. octosporus have been determined by the Broad Institute [So-mfm1–So-mfm6”) and one P-factor–encoding gene . The six redundant genes (So-mfm1–So-mfm6) encode M-factor peptides with the same amino acid sequence . The resulting strain, which produced only So-M-factor instead of Sp-M-factor, was found to be completely sterile  in the map2+ gene with the So-P-factor–encoding sequence (7 repeats) (map2So), resulted in a strain with approximately half the mating frequency of the strain with the map2+ gene (35.8% ± 5.1%)  in the So-map2+ gene with the Sp-P-factor–encoding sequence (4 repeats) (So-map2Sp) in S. octosporus cells. The resulting strain, which produced only Sp-P-factor instead of So-P-factor, retained mating ability  . Thus, SS. pombe (Sp-Map3), we carried out a shmooing assay , we treated TS402 with each of the four synthetic So-P-factor peptides. Remarkably, cells formed visible shmoos in all cases (S. pombe cells treated with P2’ peptide (10 nM) elongated a shmoo in response to P-factor after one day, and the proportion of shmooing cells increased to about 80% at a peptide concentration of 100 nM  can degrade So-P-factors. Each of the synthetic So-P-factor peptides was mixed at 200 μM with culture supernatant containing active Sp-Sxa2, and the amount of leucine released from each P-factor was measured at fixed time points, as described above for Sp-P-factors. All four So-P-factor peptides (P1’–P4’) were considerably degraded in the presence of Sp-Sxa2  obtained from the 150 wild strains were integrated into the FY23418 strain, almost all of the resulting strains showed high mating frequency (map2 gene (6 repeats) obtained from the 22CBS10468 strain  to S. pombe. Nielsen’s group developed an alternative technique based on the observation that pheromone stimulation is necessary to undergo meiosis and sporulation [S. pombe. Nevertheless, we believe that the P1, P3, P4, and P6 peptides are certainly expressed because the strains carrying two map2 genes showed a much higher frequency of zygotes  peptides  is that rulation . In our  zygotes  relative zygotes .Candida albicans, because Ste2 and Bar1 recognize the overlapping regions of α-pheromone [We further noticed that there is a positive relationship between the recognition of P-factor by Mam2 and its efficient degradation by Sxa2 . This isheromone . In thisS. pombe: namely, recognition by M-factor is extremely stringent, whereas that by P-factor is relatively relaxed  were obin, L968 , and J. in, L968   medium, EMM2−N medium, and PMG medium were used for mating and sporulation [Cells were grown in yeast extract (YE) medium supplemented with adenine (75 mg/l), uracil 50 mg/l), and leucine (50 mg/l). For solid medium, 1.5% agar was added to yeast extract agar (YEA). Where appropriate, antibiotics  were added to YEA plates at a final concentration of 100 μg/ml. EMM2 was also used for growth [ mg/l, anrulation . Cells wmfm1, mfm2, mfm3, map2, map3, mam2, and sxa2 were amplified using the following primer sets: oTS83/84, oTS507/508, oTS509/510, oTS81/82, oTS91/92, oTS89/90, and oTS624/625, respectively , oTS505 (mfm2), oTS506 (mfm3), oTS85/86 (map2), oTS157/158 (map3), oTS151/152 (mam2), and oTS647/648/658 (sxa2). The sequences obtained were compared with the corresponding sequence of L968. Differences from the L968 sequence are listed in Genomic DNA was extracted from overnight cultures grown in YE medium. Each of the DNA fragments containing map2+ gene (approximately 2.6 kb) containing its promoter and terminator regions was amplified from L968 genomic DNA using the primer set oTS81/82. The DNA fragment was fused by using a Gibson Assembly (New England Biolabs) to a linearized vector derived from the integration vector pBS-ade6 [map2+ gene. The resultant plasmid was referred to as pTS10.The pBS-ade6 , which wmap2 genes constructed) were amplified from pTS10 using the appropriate primers , ligated by T4 ligase (TaKaRa), and transformed into Escherichia coli (DH5α). The introduced mutations were confirmed by sequencing the recovered plasmids. All plasmids carrying 4-repeat Map2 ORFs were then constructed, as described above.To generate pTS54, pTS55, pTS145, pTS146, and pTS233, inverse PCR was carried out using pTS10 and the primer sets oTS202/203, oTS204/205, oTS401/402, oTS403/404, and oTS204/404, respectively. The amplified PCR products were subjected to map2 genes with 4 repeats, the map2 gene (approximately 2.6 kb) containing the promoter and terminator regions was amplified from the appropriate plasmid  using the primer set oTS887/888. Each DNA fragment was fused to a linealized vector derived from the corresponding plasmid, which was prepared by inverse PCR with the primer set oTS885/886. Thus, pTS336–pTS341 were constructed.To construct plasmids carrying two tandem map2 gene (approximately 2.6 kb) containing the promoter and terminator regions was amplified from the genomic DNA of nine wild strains  using the primer set oTS81/82. Each DNA fragment was fused to a linearized vector derived from the integration vector pBS-ade6, which was prepared by inverse PCR with the primer set oTS79/80. All of the obtained plasmids were integrated into the FY23418 strain, as described above.In addition, the 8 cells/ml. A 30-μl aliquot of the resulting suspension was spotted onto sporulation media  and then incubated for 2 days (S. pombe) or 3 days (S. octosporus) at 30 °C unless stated otherwise. The percentage of zygotes was calculated, as described previously [Cells grown on YEA plates overnight were resuspended in sterilized water to a cell density of 1 × 10eviously ,52. In aHeterothallic haploid strains each carrying a chromosomal drug-resistance marker  were cultured on YEA plates overnight, and the same cell numbers of the M-strain and two competing P-strains were mixed in sterilized water. A 30-μl aliquot of the resulting suspension was spotted onto MEA plate and incubated for exactly 24 hours at 30 °C. The mixed cells were allowed to mate, and the resulting hybrid diploids were sporulated to form spores. The cell suspension was diluted and spread on YEA plates containing different combinations of drugs. The number of colonies was counted after 3 days of incubation at 30 °C. The ratio of recombinant frequency was calculated, as described previously . Three s7 cells/ml. The cells were treated with the synthetic pheromone and incubated for exactly 24 hours with gentle shaking.P-factor and M-factor peptides were chemically synthesized (Eurofins) for the shmooing assay. The purity of the preparations was over 95% (HPLC). P-factor was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 500 μM, and M-factor was dissolved in methanol (MeOH) at a concentration of 1 mM. The stock solutions were diluted with culture medium to the appropriate dilution ratio. For the assay, heterothallic haploid cells were grown in YE medium overnight, washed with sterilized water three times, and then resuspended in EMM2−N medium at a cell density of 4 × 10sxa2+ gene (TS402) was used. The cells were incubated with synthetic P-factors at different concentrations  for 24 hours and observed by a DIC microscope. Images were recorded, and the L and W of a cell were measured to determine the L/W ratio. Cells with an L/W ratio of 2.0 or more were defined as shmooing cells; other cells were defined as arrested. In all cases, at least 100 cells each were measured. In the reciprocal experiment, an M-factor-sensitive strain lacking the rgs1+ gene [sxa2Δrgs1Δ double deletion strain (TS578) was used to accurately measure the activity of the P5 peptide.To assess whether Mam2 could recognize different P-factors, a P-factor sensitive strain lacking the s1+ gene  (TS405) sxa2+ under the control of the thiamine-repressible nmt1+ promoter in S. pombe [LEU2 gene was replaced with a KanMX6 cassette. Next, the sxa2+ gene (approximately 1.5 kb) was amplified from L968 genomic DNA using the primer set oTS729/730 and fused to a linearized vector derived from the integration plasmid pTS111, which was prepared by inverse PCR with the primer set oTS305/306. The resultant plasmid pTS284 was transformed into TS402. Thus, a heterothallic strain (TS407) ectopically overexpressing sxa2+ under the control of the nmt1+ promoter was obtained.The pREP vector was used to express S. pombe . First, S. pombe  using thnmt1+ promoter) at a cell density of 1 × 107 cells/ml and incubated with aeration for 2 days. The cell culture supernatant was passed through a 0.22-μm filter (Merck Millipore) to completely remove cell debris and then concentrated 20-fold by ultrafiltration through a Vivaspin 20-10K  because ultrafiltration has been shown to concentrate the carboxypeptidase without loss of Sxa2 activity [sxa2−) was transformed into TS402, and the same preparation of culture medium was carried out.The TS407 strain was precultured overnight in YE medium containing G418 and then washed with sterilized water three times. Cultures were inoculated into EMM2 medium containing G418 without thiamine . The strain produced only So-M-factor and no Sp-M-factor. In contrast, the So-map2+ gene lacking both its signal sequence and two N-linked glycosylation sites was amplified from yFS286 genomic DNA using the primer set oTS57/58. The DNA fragment was fused to a linearized vector derived from pTS13, which was prepared by inverse PCR with the primer set oTS55/56 to replace the map2+ gene. The resultant plasmid pTS15 was integrated into the FY23418 strain, as described above. This strain produced only So-P-factors and no Sp-P-factors.Plasmid pTS189 was integrated at the S. octosporus strain producing only Sp-P-factors. To delete the native So-map2+ gene, the 3′-downstream sequence (1 kb) was first amplified from yFS286 genomic DNA using the primer set oTS47/48. The DNA fragment was fused to a linearized vector derived from pFA6a-kanMX6, which was prepared by inverse PCR with the primer set oTS37/38 to construct the plasmid pTS5. Next, the 5′-downstream sequence (1 kb) was amplified from yFS286 genomic DNA using the primer set oTS46/78. The DNA fragment was fused to a linearized vector derived from pTS5, which was prepared by inverse PCR with the primer set oTS35/36, as described above. Lastly, pTS11 was constructed. The DNA fragment from this plasmid, namely, a 3.5-kb fragment amplified from pTS11 using the primer set oTS48/78, was purified, and 1 μg of the DNA was transformed into yFS286 by our previously described method [So-map2+ gene was successfully disrupted by a homologous recombination, and the resultant strain TS12 was sterile.Next, we constructed an d method . The So-So-map2+ gene (approximately 3.3 kb) containing the promoter and terminator regions was amplified from yFS286 genomic DNA using the primer set oTS78/626. The DNA fragment was fused to a linearized vector derived from the integration vector pFA6a-natMX6, which was prepared by inverse PCR with the primer set oTS35/36 to construct the plasmid pTS247. The Sp-map2+ gene lacking its signal sequence and two N-linked glycosylation sites was amplified from L968 genomic DNA using the primer set oTS635/636. The DNA fragment was fused to a linearized vector derived from pTS247, which was prepared by inverse PCR with the primer set oTS633/634 to replace the So-map2+ gene. The resultant plasmid pTS250 was integrated into the TS12 strain after restriction with BamHI near the center of the terminator region. This strain produced only Sp-P-factors and no So-P-factors.The mam2+ gene (approximateley 3.1 kb) containing its promoter and terminator regions was amplified from L968 genomic DNA using the primer set oTS90/99. The DNA fragment was fused to a linearized vector derived from the integration vector pFA6a-hphMX6, which was prepared by inverse PCR with the primer set oTS35/36 to construct the plasmid pTS14. The mam2 gene (approximately 1.0 kb) was amplified from the genomic DNAs of four wild strains  using the primer set oTS586/587. Each of the DNA fragments was fused to a linearized vector derived from pTS14, which was prepared by inverse PCR with the primer set oTS149/150 to replace the mam2+ gene. All obtained plasmids were integrated into the FY12677 strain after restriction with AfeI near the center of the terminator region.The sxa2+ gene (approximately 3.5 kb) containing its promoter and terminator regions was amplified from L968 genomic DNA using the primer set oTS624/625. The DNA fragment was fused to a linearized vector derived from the integration vector pFA6a-natMX6, which was prepared by inverse PCR with the primer set oTS35/36 to construct the plasmid pTS246. The sxa2 gene (approximately 1.5 kb) was amplified from the genomic DNAs of three wild strains  using the primer set oTS664/665. Each of the DNA fragments was fused to a linearized vector derived from pTS246, which was prepared by inverse PCR with the primer set oTS627/628 to replace the sxa2+ gene. All obtained plasmids were integrated into the TS356 strain after restriction with BamHI near the center of the terminator region.The S1 Figmfm1, mfm2, mfm3, map2, map3, mam2, and sxa2) analyzed in this study are shown. mam, mating type auxiliary minus; map, mating type auxiliary plus; mfm, mating factor minus; sxa, sexually activated.(Related to (TIFF)Click here for additional data file.S2 Figmap2 ORF containing different numbers of P-factor–encoding repeats: WT , D9 , D13 , D11 , D1 , D5 , D8 , D3 , D10 , and D7 . (B) Zygote frequency of strains producing various P-factors (4 repeats). (C) Zygote frequency of strains expressing various mam2 genes: WT (L968), F2 (04CBS2775), F1 (02CBS2628), F5 (26CBS5557), and F3 (06CBS10460). (D) Zygote frequency of strains expressing various sxa2 genes or lacking the sxa2+ gene: WT (L968), G1 (01CBS10391), G4 (04CBS2775), and G7 (101FY29038). At least 300 cells were examined for each sample. Data are the mean ± SD of triplicate samples. The numerical data are included in t test . mam, mating type auxiliary minus; map, mating type auxiliary plus; n.s., not significant; ORF, open reading frame; P, Plus; sxa, sexually activated; WT, wild type.(A) Zygote frequency of strains with a (TIFF)Click here for additional data file.S3 Figsxa2+ gene (TS402) treated with synthetic P-factor at different concentrations  were incubated in EMM2−N medium with gentle shaking for 24 hours. The ability of each P-factor peptide to induce shmooing was assessed by the L/W ratio of a cell. Cells with an L/W ratio of 2.0 or more were defined as shmooing cells (shown in black); those with a ratio of 2.0 less were defined as arrested cells (shown in white). Box-and-whisker plots reperesent the distribution of the L/W ratio; for each peptide, at least 100 cells each were measured. The numerical data are included in t test . EMM2−N, Edinburgh Minimal Medium 2 lacking nitrogen; L, length; M, Minus; P, Plus; sxa, sexually activated; W, width.M cells lacking the (TIFF)Click here for additional data file.S4 Figsxa2+ and rgs1+ (TS578) were treated with 1,000 nM synthetic P5 peptide and incubated in EMM2−N medium with gentle shaking for 24 hours. Box-and-whisker plots represent the distribution of the L/W ratio; at least 100 cells each were measured. The numerical data are included in t test. EMM2−N, Edinburgh Minimal Medium 2 lacking nitrogen; L, length; M, Minus; n.s., not significant; rgs, regulator of G-protein signaling; sxa, sexually activated; W, width.To accurately measure of the activity of the P5 peptide, M cells doubly deleted for (TIFF)Click here for additional data file.S5 FigS. pombe cells. P cells lacking the rgs1+ (TS405) were treated with synthetic M-factor at different concentrations  and incubated in EMM2−N medium with gentle shaking for 24 hours. The ability of each M-factor peptide to induce shmooing was assessed by the L/W ratio of a cell, as described in S. pombe cells. M cells lacking the sxa2+ (TS402) were treated with synthetic So-P-factor at different concentrations  and incubated in EMM2−N medium with gentle shaking for 24 hours. The numerical data are included in S. octosporus; Sp, S. pombe; W, width.(A) Comparison of the amino sequences of Sp-M-factor and So-M-factor. Identical amino acids are shown in gray, indicating that three of the nine amino acid residues  of M-factor differ between the two species. (B) Shmooing assay of synthetic So-M-factor peptide and (TIFF)Click here for additional data file.S6 Figsxa2+ gene, the cells form asci (arrows) containing four spores at a low frequency on MEA plates after 2 days. In the absence of Sxa2, the cells elongate more excessively, resulting in completely sterility. MEA, malt extract agar; sxa, sexually activated.(Related to (TIFF)Click here for additional data file.S7 FigS. pombe.Diagram showing the assembly of multiple DNA fragments containing a P-factor–encoding region with overlaps at the ends. The oligo primers used to amplify each DNA fragment, containing part of the specific sequence of spacers, are shown in (TIFF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S1 Data(ZIP)Click here for additional data file.S2 Data(XLSX)Click here for additional data file.


Locusta migratoria. Here, we report that a conserved transcription factor, cAMP response element-binding protein B (CREB-B), is a key mediator involved in the signaling pathway from NPF2 to NOS in the migratory locust, triggering locomotor activity shift between solitarious and gregarious phases. We find that CREB-B directly activates brain NOS expression by interacting with NOS promoter region. The phosphorylation at serine 110 site of CREB-B dynamically changes in response to population density variation and is negatively controlled by NPF2. The involvement of CREB-B in NPF2-regulated locomotor plasticity is further validated by RNAi experiment and behavioral assay. Furthermore, we reveal that protein kinase A mediates the regulatory effects of NPF2 on CREB-B phosphorylation and NOS transcription. These findings highlight a precise signal cascade underlying environment-induced behavioral plasticity.Gene expression changes in neural systems are essential for environment-induced behavioral plasticity in animals; however, neuronal signaling pathways mediating the effect of external stimuli on transcriptional changes are largely unknown. Recently, we have demonstrated that the neuropeptide F (NPF)/nitric oxide (NO) signaling pathway plays a regulatory role in phase-related locomotor plasticity in the migratory locust,  Locusta migratoria, is a worldwide agricultural pest that displays a remarkable density-dependent phase polyphenism, where being kept in a crowd triggers individuals to transit from the sedentary solitarious phase into the high-active gregarious phase. So, the migratory locust has been regarded as an excellent study model for environment-induced behavioral plasticity. Our previous finding shows that NPF2-regulated NOS transcription plays important roles in phase-related locomotor plasticity in the locust. Here, we further demonstrate that phosphorylated CREB-B directly activates NOS transcription in the pars intercerebralis, thus mediates phase-related locomotor plasticity. Further studies show that the levels of CREB-B phosphorylation is positively correlated with the crowding treatment and suppressed by NPF2. Among several candidate kinases, protein kinase A is demonstrated to transmit the inhibitory effects of NPF2 on CREB-B phosphorylation and NOS transcription. Our study provides deep insight into the precise regulatory mechanisms underlying environment-induced behavioral plasticity.The migratory locust,  Animals can adjust to a changing environment by developing alternative behavioral phenotypes that improve their fitness; this phenomenon is known as “behavioral plasticity” –3. EnvirTranscription factors (TFs) play central roles in the regulation of behavioral plasticity through integrating neural signals and downstream transcriptional events , 12, 13.Locusta migratoria, displays phase-related behavioral plasticity in response to population density variation  vector that can constitutively expression was used as the positive control (PC). The pRL-TK vector that contains a Renilla luciferase (Rr-luc) encoding sequence was used as an internal reference to normalize the cell numbers and the transfection efficiency.Click here for additional data file.S2 FigThree CREB encoding genes were found in the locust genome and were named as LomCREB-A, LomCREB-B, and LomCREB3 (marked with triangles) according to the phylogenetic analysis. The LomCREB-B protein is close to human CREB1, whereas LomCREB-A and LomCREB3 are evolutionarily divided to the cluster containing human CREB3.(TIF)Click here for additional data file.S3 FigLomCREB-A, LomCREB-B, and LomCREB3 share low identity (13.55%).(TIF)Click here for additional data file.S4 FigDNA fragment encoding CREB-A (479 aa), CREB3 (569 aa), or CREB-B (280 aa) followed by a Flag-tag was inserted to pcDNA3.1 expressing vector. Red arrows indicate target proteins. Total protein of cells transiently transfected with pcDNA3.1-CREB-A, pcDNA3.1-CREB3, or pcDNA3.1-CREB-B was used for Western blot analysis. Cells transfected with pcDNA3.1 were used as control. Mouse monoclonal antibody against Flag  was used to validate the expression of these proteins.(TIF)Click here for additional data file.S5 FigP < 0.05).dsRNA of each gene was microinjected to the brains of 3th instar locusts, and a second injection was performed at day 1 of the fourth instar locusts. RNAi effects were detected at 48 h after the final injection. Data are presented as mean ± s.e.m. Click here for additional data file.S6 FigNOS promoter. (B) EMSA of the nuclear proteins extracted from the brain tissues to the CREB R2 of the NOS promoter. (C) EMSA of the nuclear proteins extracted from the brain tissues to the CREB R3 of the NOS promoter. Nuclear proteins extracted (1 μg) from the locust brain were used to bind with the biotin-labeled probe . The corresponding cold CREB probes were unlabeled.(A) EMSA of the nuclear proteins extracted from the brain tissues to CREB R1 of the (TIF)Click here for additional data file.S7 FigLomCREB-B gene knockdown, and Histone 3 (H3) was used as the inner reference.(A) Sequence alignment of the KID domain of LomCREB-B (87–130 aa) and HosCREB1 (108–153 aa). (B), (C), and (D) Validation of LomCREB-B phosphorylation using antibodies against specific phosphorylation sites of HosCREB1. The LomCREB-B was phosphorylated at Ser110 detected by anti-p(S133)-CREB1. (D) Validation of LomCREB-B phosphorylation by transcription knockdown experiment. The western band recognized by anti-p(S133)-CREB1 was significantly decreased by (TIF)Click here for additional data file.S8 FigCREB-B mainly localized in the nuclei. Polyclonal antibody against p(S133)-CREB1 (1:100) were used in the immunohistochemistry assay. Green indicates CREB-B staining, whereas blue indicates nuclei staining. Bar represents 100 μm.(TIF)Click here for additional data file.S9 Fig(TIF)Click here for additional data file.S10 FigTime course of p-CREB-B levels during the (A) the isolation of G-phase locusts and (B) the crowding of S-phase locusts (TIF)Click here for additional data file.S11 FigNPF2 in S-phase locusts(A) Effects on p-CREB-B level after injection NPF2 peptide in G-phase locusts. (B) Effects on p-CREB-B level after transcript knockdown of  .(TIF)Click here for additional data file.S12 Fig(A) Western blot detected by antibody against p-CREB-B. (B) Statistical data for band intensity of (A) .(TIF)Click here for additional data file.S13 Fig(TIF)Click here for additional data file.S14 FigAKT, CAMK4, CAMK2, PKC, and S6K, respectively . (B) CREB-B phosphorylation level after gene knockdown of AKT, CAMK4, CAMK2, PKC, and S6K, respectively . (C) NOS transcription level after the gene knockdown of AKT, CAMK4, CAMK2, PKC, and S6K .(A) RNAi efficiency of the gene knockdown of (TIF)Click here for additional data file.S15 FigThe insect NOS proteins are evolutionally divergent from all three NOS isoforms from vertebrates, including Xenopus, Mouse, Gorilla, and Human.(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 TableRed font indicates T7 promoter sequence.(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.


Anillin (ANLN) is an actin-binding protein essential for assembly of cleavage furrow during cytokinesis. Although reportedly overexpressed in various human cancers, its role in hepatocellular carcinoma (HCC) is unclear. To address this issue, we confirmed that in 436 liver samples obtained from surgically removed HCC tissues, higher ANLN expression was detected in tumor tissues than in adjacent non-tumor tissues of HCC as measured by immunohistochemistry, quantitative real-time PCR and western blotting. Correlation and Kaplan-Meier analysis revealed that patients with higher ANLN expression were associated with worse clinical outcomes and a shorter survival time, respectively. Moreover, ANLN inhibition resulted in growth restraint, reduced colony formation, and a lower sphere number in suspension culture. Mechanistically, ANLN deficiency induced an increasing number of multinucleated cells along with the activation of apoptosis signaling and DNA damage checkpoints. Furthermore, HBV infection increased ANLN expression by inhibiting the expression of microRNA (miR)-15a and miR-16-1, both of which were identified as ANLN upstream repressors by targeting its 3’ untranslated region. Thus, we conclude that ANLN promotes tumor growth by ways of decreased apoptosis and DNA damage. Expression level of ANLN significantly influences the survival probability of HCC patients and may represent a promising prognostic biomarker. Hepatocellular carcinoma (HCC) is the most common primary liver malignancy, accounting for approximately 700,000 deaths per year . CurrentAnillin (ANLN) is a phylogenetically conserved protein that interacts with cytoskeletal components and their regulators . GeneralConsistent with its prominent role in cytokinesis, dysregulated ANLN expression has long been observed in the development and progression of various human cancers . A high In silico pathway prediction analysis revealed that ANLN could be a Wnt/β-catenin responsive gene in gastric tumor. In breast cancer associated fibroblast, Hippo signaling regulates the expression of several cytoskeletal regulators including ANLN, to help remodel the extracellular matrix [In contrast to the large number of articles describing ANLN’s downstream effects, limited research has been designed to investigate the mechanism leading to the dysregulation of ANLN in cancer development. Two reports indicated that inhibition of miR-217 or miR-497 expression may be responsible for ANLN overexpression in pancreatic ductal adenocarcinoma and nasopharyngeal carcinoma ,17, respr matrix . TherefoThe aim of the present study was to investigate the role of ANLN expression in human HCC. The functional impact and potential prognostic predictive value of ANLN expression in HCC were explored. Moreover, microRNAs (miRs) targeting ANLN mRNA expression were also studied. The present study contributed to an enhanced understanding of ANLN expression for HCC cell growth and provided novel insights into its regulatory mechanism in human cancer.vs normal tissue  and low (n = 272) cohorts according to the IHC results . Correla2 = 0.4094, P < 0.05, in vitro as determined by MTT assay  infection is the key risk factor for development of HCC in China . The relIn the present study, we demonstrated that ANLN was upregulated in HCC tissues and cell lines. High ANLN level was associated with a poor prognosis in HCC patients. Through shRNA-mediated knockdown cell models, we observed that ANLN depletion resulted in inhibition of cell proliferation and growth, as well as altered cell shape with multinucleation. We also provided evidence that microRNAs including miR-15a and miR-16-1, functioned upstream and regulated ANLN expression in HCC cells. Thus, our study confirmed that ANLN expression contributed to the development and progression of human HCC and may serve as a promising prognostic biomarker.et al concluding that knockdown of ANLN in liver cells inhibits development of liver cancer in MYC-induced, DEN-induced liver tumorigenesis as well as tumor engraftment mouse models [In fact, elevated ANLN expression was observed in many other human cancers and ANLN expression was proposed to be a prognostic and therapeutic indicator for breast cancer, colorectal cancer, lung cancer and upper urinary tract urothelial carcinoma ,11,12,15e models . In agreDrosophila embryo extracts [ANLN protein was first identified as an F-actin binding protein from extracts . Later, extracts ,28. The extracts . Consistextracts ,31. Indeextracts . TherefoImportantly, our results indicated for the first time a transcriptional mechanism of ANLN in HBV infected HCC. HBV infection accounts for the most important risk factor of HCC in China, and virus replication is commonly seen in almost all cases of Chinese HCC patients . We obseThe results of the present study were convincing in that upregulation of ANLN expression was observed and predicted a poor prognosis in in human HCC. Although there was a trend showing that ANLN was associated with an aggressive cancer phenotype, an overexpression study is needed to fully demonstrate this point of view because our research was mostly based on the loss-of-function experiments. Additionally, our finding that miR-15a and miR-16-1 could regulate the transcription level of ANLN was preliminary and further analysis is needed.In conclusion, we identified ANLN as a highly expressed gene in human HCC, and proved that ANLN could serve as a prognostic biomarker for overall survival and disease free survival of HCC patients. We demonstrated that ANLN played an important role in HCC cell growth by regulating proliferation, colony formation and maintenance of proper morphology. In addition, we revealed that microRNAs, including miR-15a and miR-16-1, are as possible upstream regulators for ANLN expression in HCC.We obtained a total of 436 paraffin-embedded HCC specimens for prognostic survival analysis from Sun Yat-sen University Cancer Center , among which only 201 cases had both observable tumor tissues and adjacent non-tumor tissues under microscopy. To compare ANLN expression between HCC tissues and adjacent non-tumor tissues and to analyze the relationship between ANLN and Ki67, another 81 fresh HCC specimens were collected from the Third Affiliated Hospital of Sun Yat-sen University . A surgical tumor resection was performed on each patient at department of hepatobiliary surgery. Then tissues were cut into proper size and stored in liquid nitrogen directly for RNA and protein extraction or fixed in 4% paraformaldehyde for immunohistochemistry (IHC). The study was approved by the Institute Research Ethics Committee at the Sun Yat-sen University Cancer Center and the Third Affiliated Hospital of Sun Yat-sen University. Written informed consent was obtained from each patient. Relative experiments with these samples were performed in accordance with the relevant regulations.Commercially available antibodies were as follows: ANLN , GFP , α-Tubulin , β-Actin , GAPDH , Ki67 , cleaved-caspase-3 , cleaved-caspase-9 , cleaved-poly ADP-ribose polymerase , phosphorylated ATM (Ser1981) , phosphorylated ATR (Ser428) , phosphorylated Chk1 (Ser345) , phosphorylated Chk2 (Thr68)  and phosphorylated Histone H2A.X (Ser139) . Noble agar (214220) was purchased from BD Biosciences. All other chemical reagents were obtained from Sigma-Aldrich, unless otherwise indicated.2. All cell lines were obtained from the College of Life Science, Sun Yat-sen University. Cells were digested and passaged as previously described [293T cells, eleven HCC cell lines  and two immortalized hepatic cell lines (MIHA and LO2) were employed in this study and were cultured in Dulbecco's Modified Eagle's Medium  containing 10% fetal bovine serum  at 37°C and 5% COescribed .Total RNA was isolated from tissue specimens and HCC cell lines using Trizol reagent  according to the manufacturer’s protocol. Total RNA (1 µg) was reverse transcribed into cDNA by the GoScript™ Reverse Transcription System . Quantitative PCR was performed with Platinum SYBR Green qPCR SuperMix-UDG  with a LightCycler 480 PCR platform . Specific primers were as follows: ANLN forward, 5’-GTGATTCTGTTGCTGTCCCG-3’ and ANLN reverse, 5’-GCAGCCTTTTCCTCTGATGG-3’; GAPDH forward, 5’-GGAGCGAGATCCCTCCAAAAT-3’ and GAPDH reverse, 5’-GGCTGTTGTCATACTTCTCATGG-3’. MiR-15a: RT-qPCR stem-loop primer, 5’-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACCACAAA-3’, qPCR forward primer:5’-GGGTAGCAGCACATAATGG-3’; miR-16-1: RT-qPCR stem-loop primer, 5’-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACCGCCAA-3’, qPCR forward primer: 5’-GGGTAGCAGCACGTAAATA-3’; miR-195: RT-qPCR stem-loop primer, 5’-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACGCCAAT-3’, qPCR forward primer: 5’-GGGTAGCAGCACAGAAAT-3’; miR-424: RT-qPCR stem-loop primer, 5’-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACTTCAAA-3’ qPCR forward primer: 5’-GGGCAGCAGCAATTCATGT-3’; universal reverse primer, 5’-CAGTGCGTGTCGTGGAGT-3’.-1 puromycin  and successful establishment was confirmed by western blotting. The shRNA sequences targeting ANLN mRNA were as follows: shANLN#1, 5’-GCCAATATTCACTACGTATTACTCGAGTAATACGTAGTGAATATTGGCTTTTT-3’. The non-targeting shRNA sequence was served as negative control: shNC, 5’-CTAGCCCGGCCAAGGAAGTGCAATTGCATACTCGAGTATGCAATTGCACTTCCTTGGTTTTTTGTTAAT-3’.The full-length human ANLN cDNA was amplified from cDNA library of QGY-7703 cells and subcloned into pcDNA3.1(+) . Vectors expressing short hairpin RNA (shRNA) against ANLN and scrambled shRNA were kindly gifted from Professor Lunquan Sun . LentiviTo generate the pMIR-Report-ANLN-3’ UTR construct, a region (sense 3599-4720 bp) of ANLN transcript (ENST00000265748.2) containing the predicted targeting microRNAs binding sites was amplified by PCR and cloned into the pMIR-Report luciferase vector (Ambion) between the SpeI and HindIII sites. The PCR primers used were as follows: forward, 5’-GGACTAGTACCGGGAAATTTCCATGCTATCTAG-3’ and reverse, 5’-CCCAAGCTTCCTTTAGACATTTACAGGTATTTATTTGAG-3’. The construct was verified by sequencing.-1 using RNAiMAX reagent (Invitrogen), and oligonucleotide transfection was performed with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. The transfected cells were incubated at 37°C for 24 h in complete medium and harvested at the indicated time points. TargetScan (http://www.targetscan.org/) was used to predict the ANLN targeting microRNAs.The miR-15a, miR-16-1, miR-195, miR-424 mimics and a non-specific miR-control were purchased from Dharmacon. The miR-15a, miR-16-1, miR-195 anti-sense inhibitors and a non-specific miRNA inhibitor control were synthesized by GenePharma. MiRNAs were transfected at a working concentration of 50 nmol·L5 HCC cells were seeded in 12-well plates and cultured for 24 h. For pMIR-Report related assay, the control vector pRL-TK (Promega) encoding Renilla luciferase, pMIR-Report-ANLN-3’ UTR plasmids and 50 nM each of ANLN targeting microRNA mimics or negative control were cotransfected into cells. Cells were harvested 24 h after transfection. Quantification of firefly and Renilla luciferase activities of at least three independent transfections were measured with the Dual Reporter Assay System  using FB12 luminometer . The relative luciferase activities (RLU) were calculated by normalizing the activity of the fluorescent luciferase with that of internal standard Renilla luciferase.Approximately 1×10Cells were lysed in NETN buffer  containing protease and phosphatase inhibitor cocktail . The lysate protein concentration was measured using the BCA protein assay kit ; after normalization to equal amounts, proteins were separated by 8% or 10% SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes and probed with the indicated primary antibodies. The blots were then incubated with species-specific HRP-conjugated secondary antibodies, and the immunoreactive bands were visualized by enhanced chemiluminescence . Quantification of band densitometry was measured with ImageJ software.-1 DAPI  for 5 min at room temperature. Fluorescence signal was imaged using a confocal microscope . Multinucleated cells were defined as cells with more than one nucleus per cell.Cells were plated on chamber slides, and fixed with 4% paraformaldehyde at 37°C for 5 min. To keep the stability of microtubule capture at kinetochores, cells were incubated for 5 min on ice before fixation, to destabilize most of the non-kinetochore microtubules. After fixation, cells were permeabilized with 0.1% Triton X-100 for 5 min. Then cells were blocked with 5% BSA for 20 min and incubated with indicated primary antibodies at 4°C overnight. The fluorescence-visualized secondary antibody was added and incubated for 60 min. Nucleus was staining with 50 ng·mL-1 MTT for 4h at 37°C. Subsequently, 150 μL of 100% dimethylsulfoxide (DMSO) was added to dissolve the precipitates. Viable cells were counted every day by reading the absorbance at 490 nm with a plate reader .Cell proliferation rate was determined using MTT assay  according to the manufacturer’s protocol. Cells were seeded in 5 replicates in a 96-well plate at a density of 2,000 cells per well and cultured with DMEM containing 10% FBS. Cells were incubated with 20 μL of 5 mg·mL6 in 0.1 mL PBS) were injected subcutaneously into the hind flank of NSI mice. Mice were monitored every 12 h for the first 3 days after inoculation of tumor cells, and then daily thereafter. Tumor sizes were measured every 3 days. Mice were sacrificed 8 weeks post-injection via cervical dislocation, and tumors from two groups were extracted and weighed. The perpendicular diameters of the tumors were measured using a caliper, and the tumor volume was calculated using the following formula: tumor volume (V) = π/6 × large diameter × smaller diameter2. The following are general humane endpoints for animals that require euthanasia in this study: 20% decrease in normal body weight; the inability to reach food or water for more than 24 h; a tumor burden greater than 10% body weight or a tumor that exceeds 20 mm in any one dimension. All efforts were made to minimize animal suffering.All the mice were handled according to the Guide for the Care and Use of Laboratory Animals. The procedures were approved by the Institutional Animal Care and Use Committee of Third Affiliated Hospital of Sun Yat-sen University. Female NSI mice  , and dried. Only clearly visible colonies (more than 50 cells) were counted under a light microscope. The test was repeated three times.Cells were seeded into six-well plates at a density of 2000 per well and incubated at 37°C and at an atmosphere of 5% CO2 for 14 days. The number of colonies was monitored manually under a microscope.A 2-mL layer of 0.55% agar (wt/vol) in DMEM with 5% FBS was poured into 6-well plates. Cells were re-suspended in 0.33% agar (wt/vol) in DMEM with 10% FBS at a density of 10,000 cells/mL, and 1 mL of the cell suspension was poured on top of the base layer; the suspension was allowed to solidify, followed by incubation at 37°C in 5% CO-1 EGF  and 20 ng·mL-1 basic FGF . The spheres were observed, imaged and counted under a microscope.Sphere formation assays were performed according to previously reported protocols . BrieflyIHC was performed as previously described . BrieflyThe SPSS software version 20.0 and GraphPad Prism 5 software were used to perform statistical analyses. Correlation of the ANLN staining intensity with clinicopathological characteristics was measured using Pearson’s Chi-Square or Fisher’s exact test. The Cox proportional hazards model and Kaplan-Meier analysis were employed for survival analysis. Spearman’s correlation coefficient was employed for ANLN and Ki67 correlation analysis. The significance of variances between groups was determined by t-test. Each experiment was performed three times in triplicate. Unless otherwise indicated, all error bars indicate standard deviation (SD). All statistical tests were two-sided, and P < 0.05 was considered statistically significant.Supplementary Figure