NSCLC), we previously demonstrated that mesenchymal NSCLC was sensitive to polo‐like kinase (Plk1) inhibitors, but the mechanisms of resistance in epithelial NSCLC remain unknown. Here, we show that cMet was differentially regulated in isogenic pairs of epithelial and mesenchymal cell lines. Plk1 inhibition inhibits cMet phosphorylation only in mesenchymal cells. Constitutively active cMet abrogates Plk1 inhibitor–induced apoptosis. Likewise, cMet silencing or inhibition enhances Plk1 inhibitor–induced apoptosis. Cells with acquired resistance to Plk1 inhibitors are more epithelial than their parental cells and maintain cMet activation after Plk1 inhibition. In four animal NSCLC models, mesenchymal tumors were more sensitive to Plk1 inhibition alone than were epithelial tumors. The combination of cMet and Plk1 inhibition led to regression of tumors that did not regrow when drug treatment was stopped. Plk1 inhibition did not affect HGF levels but did decrease vimentin phosphorylation, which regulates cMet phosphorylation via β1‐integrin. This research defines a heretofore unknown mechanism of ligand‐independent activation of cMet downstream of Plk1 and an effective combination therapy.To address the need for improved systemic therapy for non–small‐cell lung cancer ( We observed differential regulation of cMet phosphorylation after Plk1 inhibition in epithelial and mesenchymal NSCLC. We confirmed cMet's role in Plk1 inhibition–induced apoptosis by inhibiting, silencing, and activating cMet in NSCLC et al, https://portals.broadinstitute.org/ctrp/) database : 33 proteins were associated with sensitivity to BI2536, 36 with sensitivity to GSK461364, 37 with sensitivity to BRD‐K70511574, and 26 with sensitivity to GW‐843682X  and β‐catenin (P < 0.001) was higher and expression of the mesenchymal protein Snail (P < 0.01), as well as ATM and thymidylate synthase, was lower in cell lines resistant to Plk1 inhibitors than in those sensitive to Plk1 inhibitors. We also found that cMet protein expression correlated with drug sensitivity for all Plk1 inhibitors  protein and phosphoprotein expression measured using reverse phase protein array (RPPA) from the MD Anderson Cell Line Project database ] to MET gene copy number in NSCLC cell lines. MET gene copy number was obtained from the MD Anderson Cell Line Project database, CTRPv2, and Kubo et al  with Spearman's rho coefficient values that ranged from −0.428 to 0.430 and associated P‐values that ranged from 0.078 to 0.872. However, this analysis was limited by the fact that there were drug sensitivity data for only two NSCLC cell lines with MET copy number > 5.The correlation of drug sensitivity with cMet protein expression motivated us to compare Plk1 inhibitor sensitivity [area under the curve (AUC) and effective dose 50  with 5 ng/ml TGF‐β for at least 14 days, which led to the expected changes in the expression of vimentin, Snail, Slug, ZEB1, Twist, E‐cadherin, β‐catenin, and claudin 7 Fig . Given tin vitro . The Plket al, P < 0.05, we observed that the mean expression of 33 proteins or phosphoproteins was differentially regulated . The combination therapy led to tumor regression in nine of 10 epithelial and nine of 10 mesenchymal PDX mice  than in the epithelial xenograft models (TC202 and H1975). Co‐treatment with Plk1 and cMet inhibitors significantly increased the percentage of TUNEL‐positive cells in all mouse models. Taken together, these results support cMet as a driver of Plk1 inhibitor resistance in epithelial NSCLC in vivo, and the findings suggest that co‐inhibition of Plk1 and cMet increases apoptosis, leading to tumor regression in NSCLC.To asses for apoptosis, we next performed TUNEL staining in paraffin‐embedded tissues Fig G. SimilaTo test cMet as a driver of resistance, we used constitutively active TPR‐Met, which is a fusion protein that lacks the extracellular, transmembrane, and juxtamembrane domains of the cMet receptor and includes the TPR dimerization motif, which allows constitutive and ligand‐independent activation of the kinase. Expression of the TPR‐Met chimeric protein was biologically active in three mesenchymal cell lines , which in turn increased downstream Akt (S473) and Erk (p42/44 T202/204) phosphorylation Fig A.Compared with the control vector–transfected cells, TPR‐Met–expressing cells showed less apoptosis, as measured by Apo‐BrdU, cleaved PARP, and cleaved caspase 3, after treatment with volasertib Fig A and B. To determine whether we could overcome the effects of cMet activation by using a cMet inhibitor, we combined tepotinib and volasertib. Tepotinib inhibited cMet universally but did not completely abrogate cMet signaling in the TPR‐Met–expressing cell lines. The addition of tepotinib significantly increased apoptosis in control vector–transfected cells, but apoptosis was only moderately increased in TPR‐Met–transfected cells, consistent with a lack of full cMet inhibition Fig A and B. MET amplification and a MET exon 14 skipping mutation  and the one with an activating MET mutation (H596), volasertib did not lead to a decreased in pMet. Both H596 and H920 were resistant to the Plk1 inhibition GSK461364  and a decrease in the mesenchymal genes VIM (0.1‐fold), ZEB1 (0.001‐fold), TWIST (0.3‐fold), and SNAIL  compared with the parental cell line , as measured by Apo‐BrdU and cleaved PARP  in both parental and Calu6‐VAR cells  in conditioned medium and in cellular lysates after Plk1 inhibition in both mesenchymal and epithelial cell lines. Neither the secreted nor cellular HGF levels were affected by Plk1 inhibition in all tested NSCLC cell lines Fig A. LikewiITGB1), and vimentin (VIM) because this is a ligand‐independent pathway that involves proteins associated with EMT. Plk1 maintains cell surface levels of β1‐integrin via phosphorylation of vimentin on S82  and VIM  phosphorylation. Inhibition or silencing of Plk1 by volasertib or siRNA markedly decreased the S82 vimentin phosphorylation in two mesenchymal cell lines. The resulting increase in total vimentin may be a compensatory change. Vimentin levels were very low in epithelial cell lines without consistent effects by Plk1 inhibition , leading to cMet activation in NSCLC.The current study yielded five main findings that follow from our prior discovery that mesenchymal NSCLC is more sensitive to Plk1 inhibitors than epithelial NSCLC  expressing KRASG12D, Narvaez, et al investigated the combination of MET knockdown and cMet inhibition with Poloppin. Both cMet inhibition and knockdown did sensitize five KRAS mutant cell lines and one pancreatic organoid to Poloppin in vitro.Recently published work using a novel drug (Poloppin) that inhibits phosphopeptide binding by the Plk1 polo‐binding domain supports our findings  is underway. MET is also amplified in 5% of newly diagnosed lung adenocarcinomas  in various cancers, including NSCLC. Co‐targeting of these kinases potentially produces synergistic antitumor effects  were used. Human TGF‐β1 was purchased from Cell Signaling Technology . Human fibronectin was purchased from Sigma‐Aldrich . Constitutive active TPR‐Met plasmid and control pBABE plasmid were obtained from Addgene . Antibodies and dilutions used in the study are listed in All drugs were purchased from Selleck Chemicals  and prepared as 10 mmol/l stock solutions in dimethyl sulfoxide. Predesigned sets of four independent siRNA sequences of the target genes et al, et al, Human NSCLC cell lines were obtained, maintained, and genotyped by STR profiling as previously described  and 1 μg of retroviral plasmid pBABE–TPR‐Met or the control vector using Lipofectamine 3000. The HEK293 cell medium was changed 24 h after transfection, and the cells were incubated at 37°C for 48 h to allow for virus production. After 48 h, HEK293 medium containing viral particles was filtered and transferred onto NSCLC cell culture plates and incubated at 37°C for 48 h. After transduction, fresh RPMI 1640 medium with 10% fetal bovine serum was added to the cell culture plates, and the NSCLC cells were allowed to recover for 24 h. NSCLC cells were selected using 3 μg/ml puromycin.et al, For knockdown experiments, NSCLC cells were transfected with predesigned sets of four independent siRNAs against Plk1 and cMet using Lipofectamine RNAiMAX Transfection Reagent , as we previously described  after 72 h of exposure to serial fold drug dilutions, as described previously  according to the manufacturer's protocol. Briefly, control and treated cell lysates were incubated with the indicated antibody–Dynabeads conjugate followed by purification of antigen–antibody–Dynabeads conjugate and separation on SDS–PAGE for immunoblotting.et al, et al, Apoptosis was measured by TUNEL  staining  as described previously  and as we previously described , and complementary DNAs (cDNAs) were synthesized using iScript Reverse Transcription Supermix . Quantitative real‐time PCR assays were done with iTaq Universal SYBR Green Supermix (Bio‐Rad Laboratories) and the CFX96 Real‐time System (Bio‐Rad Laboratories) as described previously  according to the manufacturer's protocol. Briefly, NSCLC cell lines were treated with 50 nM volasertib in serum‐free medium for 24 h, and HGF levels were measured in conditioned medium.et al, 2)/2. When tumor volumes reached 150 mm3, the mice were randomized and treated with 30 mg/kg volasertib intravenously weekly, 25 mg/kg tepotinib daily (5 days on and 2 days off) by oral gavage, both agents, or vehicle control for 5 weeks. Mice were then euthanized and tumors excised.In conducting research using animals, the investigators adhered to the laws of the United States and regulations of the US Department of Agriculture. All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at The University of Texas MD Anderson Cancer Center and conducted in accordance with MD Anderson's Office of Research Administration and IACUC guidelines. Two million NSCLC cells per mouse were injected subcutaneously, or logarithmically growing PDXs  and an autocorrelation structure of order 1 in residuals, given that we had a single grouping variable of mouse identification. The Tukey test was used for pairwise comparisons between treatment groups, and therefore, Harvested tumor tissues were fixed in 10% formalin, embedded in paraffin, and sectioned at 5 μM. Briefly, after deparaffinization and rehydration, tissue sections were subsequently processed for DNA labeling by TdT dNTP followed by Strep‐HRP/DAB detection using the TUNEL Apoptosis Detection Kit . Images were taken at 40× magnification, and image analysis was performed to calculate the percentage of TUNEL‐positive cells using ImageJ software .https://www.r-project.org/), a publically available and widely used statistical computing tool. RPPA protein expression and Plk1 inhibitor  sensitivity data were downloaded from the MD Anderson Cell Line Project  and CTRPv2  databases and curated for NSCLC cell lines on March 5, 2017. RPPA data for 71 cell lines were available for BI2536, 69 cell lines for GSK461364, 68 cell lines for BRD‐K70511574, and 64 cell lines for GW‐843682X. To identify correlation between drug sensitivity and protein expression, we applied Spearman's correlation using R. For RPPA proteins to be compared between cell lines after treatment with a Plk1 inhibitor, it is necessary to treat cell line as a random effect because the cell line variation is dominant in principal component analysis. Further contrasts were conducted to compare changes in epithelial cells after treatment with the Plk1 inhibitor, changes in mesenchymal cells after treatment with the Plk1 inhibitor, and changes between the two, which is the interaction term. The analysis was carried out by the limma package in R. To correct for multiple hypotheses testing, we adjusted the resulting P‐values using the Benjamini–Hochberg method.All statistical analyses were performed using R packages  with three or more technical replicates. Analysis of variance was applied, and various contrasts were set up to test the significance of difference between groups of interest and of the interaction of two factors. Bonferroni or Benjamini–Hochberg correction was applied to adjust for multiple testing. For tumor growth curve analysis, linear modeling using generalized least squares with two variables (treatment and day) and an autocorrelation structure of order 1 in residuals were applied. The Tukey test was used for pairwise comparisons among treatment groups, and P‐values corrected for multiple comparisons were produced. For survival analysis, log‐rank tests were conducted to assess the association of the treatment type with survival. Benjamini–Hochberg correction was applied to P‐values from pairwise comparisons between treatment groups to adjust for multiple comparisons. In addition, Kaplan–Meier curves were generated. A summary of statistical methods for each specific Figure is provided in There are a variety of group comparisons with respect to different study designs, including two‐factor and three‐factor factorial designs. All RS designed and performed most of the experiments and wrote the first draft of the manuscript. SP, PV, and VS assisted with experimental design and conduct of the animal experiments. BF provided and characterized several PDX models. LS, XR, and JW performed the bioinformatics and statistical analyses. FMJ oversaw the study design and wrote the final draft of the paper.Faye M. Johnson has received research funding from PIQUR Therapeutics and Trovagene. Other authors have no conflicts of interest to declare.in vitro and in vivo. However, mechanisms of resistance to Plk1 inhibitors have not been elucidated and this unknown is a major gap in knowledge.Most NSCLC remains incurable despite survival benefits from targeted and immunotherapy. Thus, there is still an urgent need for effective systemic therapy. While Plk1 inhibitors are well tolerated by patients and some striking clinical responses were observed, response rates were generally low. Likewise, Plk1 inhibitors lead to diverse biological effects in cancer cells. Understanding the basis for Plk1 inhibitor–induced apoptosis is essential to maximizing their antitumor efficacy. To address this need, our laboratory previously discovered that mesenchymal NSCLC cell lines are more sensitive to Plk1 inhibitors than epithelial cell lines We used isogenic pairs of epithelial and mesenchymal NSCLC cell lines to measure changes in the expression of 301 proteins after Plk1 inhibition. We observed differential regulation of the cMet/FAK/Src axis, which is intact in both mesenchymal and epithelial cells. However, Plk1 inhibition inhibits cMet phosphorylation only in mesenchymal NSCLC cells, leading to subsequent inhibition of FAK and Src. Constitutively active cMet abrogates Plk1 inhibitor–induced apoptosis. Likewise, cMet silencing or inhibition enhances Plk1 inhibitor–induced apoptosis. Additionally, cells with acquired resistance to Plk1 inhibitors are more epithelial than their parental cells and maintain cMet activation after Plk1 inhibition. In both patient‐derived and cell line xenografts, mesenchymal NSCLC was more sensitive to Plk1 inhibition alone than was epithelial NSCLC. The combination of cMet and Plk1 inhibition led to regression of tumors in three models and marked tumor size reduction in the fourth model. When drug treatment was stopped, tumors treated with the combination did not regrow. Plk1 regulates cMet via the vimentin protein that is only expressed in mesenchymal NSCLC.de novo and acquired resistance to Plk1 inhibitors in patients with NSCLC.NSCLC cell lines have diverse sensitivities to Plk1 inhibition, which is consistent with the results of clinical trials of Plk1 inhibitors in solid tumors. This study reveals a novel mechanism of non‐canonical cMet activation in resistant/epithelial NSCLC after Plk1 inhibition. The addition of cMet inhibitors is a promising therapeutic strategy to overcome AppendixClick here for additional data file.Expanded View Figures PDFClick here for additional data file.Source Data for Expanded View and AppendixClick here for additional data file.Review Process FileClick here for additional data file.Source Data for Figure 2Click here for additional data file.Source Data for Figure 3Click here for additional data file.Source Data for Figure 5Click here for additional data file.Source Data for Figure 6Click here for additional data file.Source Data for Figure 7Click here for additional data file.Source Data for Figure 8Click here for additional data file.


Arabidopsis thaliana, previous forward screens have likely identified almost all the meiotic genes that when mutated lead to a pronounced decrease in fertility. However, the increasing number of genes identified in reverse genetics studies that play crucial roles in meiosis, but do not exhibit strong phenotypes when mutated, suggests that there are still many genes with meiotic function waiting to be discovered. In this study, we produced 897 A. thaliana homozygous mutant lines using Ethyl Methyl Sulfonate (EMS) mutagenesis followed by either single seed descent or haploid doubling. Whole genome sequencing of a subset of lines showed an average of 696 homozygous mutations per line, 195 of which (28%) modify a protein sequence. To test the power of this library, we carried out a forward screen looking for meiotic defects by observing chromosomes at metaphase I of male meiosis. Among the 649 lines analyzed, we identified 43 lines with meiotic defects. Of these, 21 lines had an obvious candidate causal mutation, namely a STOP or splicing site mutation in a gene previously shown to play a role in meiosis . Interestingly, this was the first time that six of these genes were identified in a forward screen in Arabidopsis . These results illustrate the potential of this mutant population for screening for any qualitative or quantitative phenotype. Thus, this new mutant library is a powerful tool for functional genomics in A. thaliana. The HEM (Homozygote EMS Mutants) lines are available at the Versailles Arabidopsis stock center.Genetic screens have been crucial for deciphering many important biological processes, including meiosis. In  This is achieved by screening the phenotype of a population with random mutations throughout the genome, which alter gene function and thus give an identifiable phenotype. On the other hand, the principle of reverse genetics is to test the function of a specific candidate gene by analysing the consequences of its disruption on the process of interest.Genetic screens have improved our knowledge of the genetic basis of biological processes. However, to identify all the genes involved in a biological pathway screening strategies must be sensitive, specific and feasible. In this context, the two classic approaches, which are still widely used, are forward and reverse genetics. On one hand, forward genetics can be used to decipher specific pathways without Arabidopsis  lines that can be used to detect either qualitative or quantitative phenotypes. Thus this new mutant collection is a very useful resource for functional genomics and applied research in A. thaliana.Here, we produced a total of 897 homozygous Figures 1A,B).To produce the HEM lines we generated two collections of almost fully homozygous lines using two different strategies: (i) single seed descent (SSD) and (ii) doubled haploids (DH) . Six parallel rounds of mutagenesis followed by SSD were carried out, giving a total of 698 independent mutant lines (Table 1). With this approach, we expected to obtain a level of homozygosity of 87% in the 4th generation (M4) that was screened (see below). The M5 seeds are available at the Versailles Arabidopsis stock center.For the SSD subset we applied EMS to wild type Col-0 seeds and produced four successive generations by self-fertilization using a single seed in each generation (SSD) . We applied EMS to Col-0 seeds with a homozygous mutation in the GALBRA-1 gene (GL1), which is characterized by the absence of trichomes .For the second population, the strategy was to generate homozygous mutagenized lines by haploidization followed by genome doubling   and 22 DH lines. Among these, 41 lines showed a meiotic defect (see section below) and 6 lines without meiotic defects were randomly chosen among the DH lines.Figure 2A). Of these 897 mutations, 99% were G > A or C > T transitions. When this value was compared for each of the subsets obtained, the SSD lines had more mutations  than the DHs .The HEM populations showed a total mean number of 897 mutations per line, ranging from 500 to 1,500, with a normal distribution : in the SSD lines 70% of the mutations were fixed (676 mutations per line on average), which was lower than the 87% expected for those lines, perhaps due to counter-selection. In the case of the DH lines, we obtained an average of 94% homozygous mutations (720 mutations per line on average). The six randomly chosen lines had a similar number of mutations than the lines having a meiotic phenotype .The percentage of fixed mutations, was different for each of the subsets due to the different approaches used . However, in six lines the percentage of homozygous mutations was 80% (on average) suggesting that the haploidization was not successful or that cross-pollination occurred during their production. Regardless, these lines still show a very high level of homozygosity, equivalent to that in the SSD lines.Figure 2C and Table 2). This category includes: non-synonymous coding mutations that change a single AA ; small indels leading to frame shifts ; new stop codons ; new start codons  and splice site changes . Considering only the mutations resulting in frameshifts, new stop codons or splice site changes, 14 mutations per line should severely disrupt gene functions .In all the HEM lines (SSD and DH), 28% of the fixed mutations (an average of 195 mutations per line) modified a protein sequence  Alexander staining to detect dead pollen grains  and thenAll of the 199 M5 mutant lines from the DH subset were first pre-screened by Alexander staining. The meiotic chromosomal behavior was then examined in lines with more than 10% dead pollen grains. In the case of the SSD lines, we screened 539 M4 mutants  by directly observing meiotic chromosomal behavior at metaphase I. Of these, the lines with a minimum of 20 cells at metaphase I captured were considered as screened, which resulted in a total of 450 mutant lines. For each of the mutants with a meiotic defect at metaphase I, we verified that the same phenotype was observed in the next generation. To optimize the screening procedure, we focused on metaphase I: (i) cells at that stage are relatively easy to find and (ii) most meiotic defects can be detected at metaphase I . A drawback is that some defects cannot be observed at that stage  and would be missed in this screen. However, we occasionally detected meiosis II defects that were included in the study.Table 1): 10% of the lines  had meiotic defects in series 10, whereas no meiotic defects were observed in the other series produced . This variability could reflect differences in the efficiency of the mutagenesis due to slight variations in experimental conditions  that may influence the final outcome of EMS mutagenesis. Therefore, the high number of meiotic mutants observed in the SSD series 10 and the HD lines suggests that these series are especially suitable for carrying out other forward genetic screens.We identified a total of 43 lines with various meiotic defects (18 DH lines and 25 SSD lines), representing 9% (18/199) of the screened DH lines and 6% (25/450) of the SSD lines. However, we observed an important difference among the six different series of mutagenesis used to produce the SSD subset : (i) Different levels of fragmentation , (ii) bivalent shape defects , (iii) the presence of univalent chromosomes at different frequencies suggesting a lack of crossovers , and (iv) bivalent alignment abnormalities .Overall, after screening 80% of the HEM lines the rate of meiotic mutant was high, with 9.4% of the lines showing different types of meiotic phenotypes in the DH and SSD series 10 (43 mutants with a robust meiotic phenotype among 199 DH lines + 261 series 10 SSD lines). The phenotypes described in the 43 HEM lines identified cover a variety of meiotic defects at metaphase I, compared to wild type (Table 3). We considered a candidate mutation to be causal when the mutation was predicted to strongly affect a protein  with a described role in meiosis. Additionally, the observed phenotype had to be consistent with the previously described phenotype. An additional missense mutation in ATM was shown to be causal by genetic mapping (Table 3). In all these lines, the presence of the candidate mutation was confirmed by Sanger sequencing. In addition, Sanger sequencing of the candidate gene SGO1 in two lines that showed premature loss of sister chromatid cohesion identified a stop and a splice site mutation .Of the 41 lines sequenced with meiotic defects, 18 had an obvious candidate mutation (ATM (found in five different mutant lines) plays a role in DSBs repair , and HEI10 are involved in class I crossover formation (FLIP is both an anti- and pro-crossover factor (FANCL promotes crossover formation (PRD2 is required for DSBs formation (REC8 and SGO1 (found in 3 different mutant lines) are both involved in sister chromatid cohesion . Interestingly, most of these mutations have only a mild impact on fertility .The genes described in the 21 lines are involved in a wide range of meiotic mechanisms: s repair , 2003; Mormation , FLIP isr factor ; FANCL pormation ; PRD2 isormation ; REC8 ancohesion ; ASY4 isormation ; and PSSribution ; Table 3In addition, among the 43 lines identified with meiotic phenotypes, in 22 there was no obvious candidate mutation, according to the criteria described above. These lines displayed different meiotic phenotypes and further work is needed to identify their causal mutation.Arabidopsis EMS mutants. These mutants show a high mutation rate per line, 897 mutations per line on average, of which most are fixed. Thus, due to the fixed nature of the mutations, these libraries can be used to repeatedly screen for a specific phenotype and therefore, to analyze either quantitative or qualitative traits.We have described the HEM collections, two libraries of almost fully homozygous Of all the mutations produced, we estimate that the HEM lines contain, >170,000 mutations  with an effect in the protein sequence. Among these >12,000  are mutations that likely knock out the protein’s function .In this study, the HEM lines were used in a forward screen targeting subtle meiotic phenotypes as a new approach to identify novel meiotic mutants. Nine Percent of the mutant lines screened in the DH and SSD series 10 collections showed defects in meiosis (43 lines), which is a direct evidence of the efficiency of mutagenesis in the HEM lines.ATM, MLH3, MLH1, MER3, HEI10, SGO1, ASY4, FLIP, FANCL, PRD2, REC8, and PSS1) involved in different meiotic mechanisms. Interestingly, six of these identified genes  have been found here for the first time in a forward genetic screen. These mutants have only moderate defects in chromosome distribution at meiosis, leading to a subtle reduction in fertility, which is under the threshold of detection by visual examination of fruit length.Within these mutants, there are 12 clear candidate genes  collection, we applied ethyl methanesulfonate (EMS) to wild type ribed in . Seeds wGLABRA1 (GL1). Mutagenized seeds were grown and then crossed as male to the tailswap line (TS) to obtain haploid plants that could be identified due to their lack of trichomes. Diploids were obtained by self-fertilization. M4 seeds were multiplied to obtain the final mutant population of the collection. All plants were cultivated in greenhouses with a 16 h/day and 8 h/night photoperiod, at 20°C and 70% humidity.To generate the DH collection, mutagenesis was performed as in the SSD in Col-0 plants with an existing T-DNA insertion in 1.Alexander staining for pollen viability was performed as described in Genome sequencing was performed with Illumina Hiseq3000 HWIJ00115 with > 8X coverage. The resulting fastq files were analyzed using the Mutdetect pipeline (version 0.0.6-e3ef10e) 1http://www.graphpad.com using TALC-P, VP, MG, and RM contributed to the conception and design of the study. LC-P, VS, AC, AH, AG, VP, DV, and LC performed the experiments. LC and RM analyzed the sequencing data. LC-P wrote the first draft of the manuscript.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.


Giardia lamblia becomes dormant by differentiation into a water-resistant cyst that can infect a new host. Synthesis of three cyst wall proteins (CWPs) is the fundamental feature of this differentiation. Myeloid leukemia factor (MLF) proteins are involved in cell differentiation, and tumorigenesis in mammals, but little is known about its role in protozoan parasites. We developed a CRISPR/Cas9 system to understand the role of MLF in Giardia. Due to the tetraploid genome in two nuclei of Giardia, it could be hard to disrupt a gene completely in Giardia. We only generated knockdown but not knockout mutants. We found that knockdown of the mlf gene resulted in a significant decrease of cwp gene expression and cyst formation, suggesting a positive role of MLF in encystation. We further used mlf as a model gene to improve the system. The addition of an inhibitor for NHEJ, Scr7, or combining all cassettes for gRNA and Cas9 expression into one plasmid resulted in improved gene disruption efficiencies and a significant decrease in cwp gene expression. Our results provide insights into a positive role of MLF in inducing Giardia differentiation and a useful tool for studies in Giardia. Giardia lamblia is a common cause of waterborne diarrhea due to contamination of water with human or animal feces [Giardia can serve as a unique protozoan model for eukaryotic cell differentiation because its life cycle has been completed with the test tube [G. lamblia undergoes differentiation from a pathogenic trophozoite form into a resistant infectious cyst form [al feces ,2. Althoal feces . Chronical feces –6. Giardest tube ,7. Like yst form ,8. The cyst form –11. Syntyst form –16. Histyst form ,18.Giardia for a stable and high expression of a target gene [Giardia for studies of gene function is also needed. A Cre/loxP system has been developed for genetic manipulations [Giardia [Giardia. A CRISPRi system with catalytically inactive Cas9 has been demonstrated for stable transcriptional repression of flagellar and ventral disc genes in Giardia recently [Plasmodium, Toxoplasma, Trypanosoma, Leishmania, and Trichomonas [Stable transfection systems that are involved in maintenance of episomal plasmids under drug selection, including neomycin and puromycin selection have been developed in get gene ,20. A syulations ,22, but [Giardia . Althoug[Giardia ,25, they[Giardia . Developrecently . CRISPR/chomonas –32.Streptococcus pyogenes for defense against viral or plasmid DNA invasion [The type II CRISPR/Cas9 system is from invasion . Cas9 cainvasion ,35. Afteinvasion . NHEJ geinvasion ,37. HR hinvasion , but enainvasion ,37. Scr7invasion ,40, 41. mlf) genes have been identified only in mammals and Drosophila [Giardia genome with a higher expression level during drug selection [mlf gene is upregulated during encystation [The myeloid leukemia factor (obtained from ATCC), were cultured in modified TYI-S33 medium [5 cells/ml.Trophozoites of 3 medium . Encysta3 medium . Briefly6 cells/ml) during vegetative growth as previously described [6 cells/ml. Cells seeded at this density became confluent within 24h. Confluent cultures were maintained for an additional 8h to ensure that the cultures were in stationary phase (at a density of ~2×106 cells/ml). Cyst count was performed on these stationary phase cultures. Cyst count was also performed on 24h encysting cultures.Cyst count was performed on the stationary phase cultures 12-18 and random hexamers and Superscript II RNase H- reverse transcriptase (Invitrogen). Synthesized cDNA was used as a template in subsequent PCR with primers mlfF and mlfR. Genomic and RT-PCR products were cloned into pGEM-T easy vector (Promega) and sequenced . Comparison of genomic and cDNA sequences showed that the mlf gene contained no introns.Synthetic oligonucleotides used are shown in mlf , cwp1 , cwp2 , and ran  genes was performed using primers mlfF (PCR1F) and mlfR (PCR1R), PCR2F and PCR2R, PCR3 and PCR3R, cwp1F and cwp1R, cwp2F and cwp2R, ranF and ranR, respectively. For quantitative real-time PCR, SYBR Green PCR master mixture was used (Kapa Biosystems). PCR was performed using an Applied Biosystems PRISMTM 7900 Sequence Detection System (Applied Biosystems). Specific primers were designed for detection of the mlf, cwp1, cwp2, and ran genes: mlfrealF and mlfrealR; cwp1realF and cwp1realR; cwp2realF and cwp2realR; ranrealF and ranrealR. Two independently generated stably transfected lines were made from each construct and each of these cell lines was assayed three separate times. The results are expressed as a relative expression level over control. Student’s t-tests were used to determine statistical significance of differences between samples.Synthetic oligonucleotides used are shown in G. lamblia cell line during vegetative growth or encystation stages using TRIzol reagent (Invitrogen). For RT-PCR, 5 μg of DNase-treated total RNA was mixed with oligo (dT)12-18 and random hexamers and Superscript II RNase H- reverse transcriptase (Invitrogen). Synthesized cDNA was used as a template in subsequent PCR. Semi-quantitative RT-PCR analysis of mlf , mlf-ha, cwp1 , cwp2 , cwp3 , myb2 , wrky , pax1 , cdk2 , gRNA, ran , and 18S ribosomal RNA  gene expression was performed using primers mlfF and mlfR, mlfHAF and HAR, cwp1F and cwp1R, cwp2F and cwp2R, cwp3F and cwp3R, myb2F and myb2R, wrkyF and wrkyR, pax1F and pax1R, cdk2F and cdk2R, MLFgF and MLFgR, ranF and ranR, 18SrealF and 18SrealR, respectively. For quantitative real-time PCR, SYBR Green PCR master mixture was used (Kapa Biosystems). PCR was performed using an Applied Biosystems PRISMTM 7900 Sequence Detection System (Applied Biosystems). Specific primers were designed for detection of the mlf, cwp1, cwp2, cwp3, myb2, wrky, pax1, cdk2, ran, and 18S ribosomal RNA genes: mlfrealF and mlfrealR; cwp1realF and cwp1realR; cwp2realF and cwp2realR; cwp3realF and cwp3realR; myb2realF and myb2realR; wrkyrealF and wrkyrealR; pax1realF and pax1realR; cdk2realF and cdk2realR; ranrealF and ranrealR; 18SrealF and 18SrealR. Each primer pairs were determined for amplification efficiency ~95% based on the slope of the standard curve. Two independently generated stably transfected lines were made from each construct and each of these cell lines was assayed three separate times. The results are expressed as a relative expression level over control. Student’s t-tests were used to determine statistical significance of differences between samples.Synthetic oligonucleotides used are shown in mlf gene was amplified with oligonucleotides MLF 5HF and MLF 5NR, digested with HindIII/NcoI and cloned into HindIII/NcoI digested 5’Δ5N-Pac, resulting in MLF5. The 700-bp 3’-flanking region of mlf gene was amplified with oligonucleotides MLF 3XF and MLF 3KR, digested with XbaI/KpnI and cloned into XbaI/KpnI digested MLF5, resulting in MLF53. We used gene synthesis services from IDT to obtain the fragment MLF-guide. The NCBI Nucleotide Blast search was used to avoid the potential off-target effects of guide sequence. The MLF-guide was digested with KpnI/EcoRI and cloned into KpnI/EcoRI digested MLF53, resulting in pMLFko. Cas9 gene was amplified using primers Cas9NF and Cas9XR with the template pCas9_GFP (Addgene), digested with NcoI/XhoI and cloned into NcoI/XhoI digested 5’Δ5N-Pac, resulting in pgCas9dC. We used gene synthesis services from IDT to obtain the fragment Cas9CXK. The Cas9CXK was digested with XhoI/KpnI and cloned into XhoI/KpnI digested pgCas9dC, resulting in pgCas9.Synthetic oligonucleotides used are shown in mlf gene and its 324 bp of 5’- flanking region were amplified with oligonucleotides MLFNF and MLFMR, digested with NheI/MluI, and cloned into NheI/MluI digested pPop2NHA [KpnI/EcoRI and cloned into KpnI/EcoRI digested pBluescript SK, resulting in pgU6. To make pgCas9MLFko, Q5 Site Directed Mutagenesis Kit was used to perform a reaction with a pgCas9 template, and primers Cas9insF and Cas9insR to insert multiple restriction sites, resulting in pgCas9ins. The HR template  and gRNA was amplified with the pMLFko plasmid and primers, mlf53UNF and mlf53UMR, digested with NotI/MluI and cloned into NotI/MluI digested pgCas9ins, resulting in pgCas9MLFko. To make pgCas9MLFko gRNA1, Q5 Site Directed Mutagenesis Kit was used to perform a reaction with a pgCas9MLFko template, and primers U6R and MLFgFnew to change the gRNA, resulting in pgCas9MLFko gRNA1. To make pgCas9MLFko gRNA2, Q5 Site Directed Mutagenesis Kit was used to perform a reaction with a pgCas9MLFko template, and primers U6R and control gF to change the gRNA, resulting in pgCas9MLFko gRNA2.To make construct pPMLF, the pPop2NHA . To makepac gene were selected and maintained with 54 μg/ml (100 μM) puromycin [Giardia trophozoites were transfected with plasmid pMLFko and pgCas9, and then selected in 100 μM puromycin. The MLFko stable transfectants were established after selection. Stable transfectants were maintained at 100 μM puromycin and were further analyzed by Western blot, or DNA/RNA extraction. The replacement of the mlf gene with pac gene was confirmed by PCR and sequencing. The control is G. lamblia trophozoites transfected with double amounts of 5’Δ5N-Pac plasmid and selected with puromycin. For removal of puromycin experiments, puromycin was removed from the medium for each stable cell line for a month to obtain MLFko–pu and control–pu cell line. For establishing MLFkoSC stable transfectants, the same plasmids were used, except that the culture medium in the first replenishment contained 6 μM Scr7 and 100 μM puromycin. For establishing Cas9MLFko stable transfectants, Giardia trophozoites were transfected with plasmid pgCas9MLFko, and selected in 25 μM puromycin. The culture medium in the first replenishment contained 1.5 μM Scr7 and 25 μM puromycin. The Cas9MLFko stable transfectants were established after selection. Stable transfectants were maintained at 100 μM puromycin and were further analyzed by Western blot, or DNA/RNA extraction. The control cell line is G. lamblia trophozoites transfected with 5’Δ5N-Pac plasmid and selected with puromycin. Puromycin was then removed from the medium for each stable cell line to obtain Cas9MLFko–pu cell line. Subsequent analysis was performed after removal of the drug for a month. The control cell line is wild type nontransfected WB trophozoites. The single cell population was obtained by dilution of the Cas9MLFko cell line. The cell density was determined first and then diluted to 1 cell/culture tube. The single cell populations were established within 2 weeks.Cells transfected with pPMLF plasmid containing the uromycin . For CRIWestern blots were probed with anti-HA monoclonal antibody , anti-MLF (1/10000 in blocking buffer) (see below), anti-CWP1 (1/10000 in blocking buffer) , anti-MYmlf gene was amplified using oligonucleotides mlfF and mlfR. The product was cloned into the expression vector pET101/D-TOPO (Invitrogen) in frame with the C-terminal His and V5 tag to generate plasmid pMLF. The pMLF plasmid was freshly transformed into Escherichia coli BL21 Star (DE3) (Invitrogen). An overnight pre-culture was used to start a 250-ml culture. E. coli cells were grown to an A600 of 0.5, and then induced with 1mM isopropyl-D-thiogalactopyranoside (IPTG) (Promega) for 4 h. Bacteria expressing pMLF were harvested by centrifugation and sonicated in 10 ml of buffer A  containing 10 mM imidazole and complete protease inhibitor cocktail (Roche). The samples were centrifuged and the supernatant was mixed with 1 ml of a 50% slurry of Ni-NTA superflow (Qiagen). The resin was washed with buffer B  and buffer C  and eluted with buffer E . Fractions containing MLF were pooled, dialyzed in 25 mM HEPES pH 7.9, 40 mM KCl, and 15% glycerol, and stored at -70°C. Protein purity and concentration were estimated by Coomassie Blue and silver staining compared with bovine serum albumin. MLF protein is purified to apparent homogeneity (>95%).The genomic Purified MLF protein was used to generate rabbit polyclonal antibodies through a commercial vendor .6 cells/coverslip) and then fixed and stained [Cells cultured in growth medium or encystation medium for 24 h were harvested, washed in phosphate-buffered saline (PBS), and attached to glass coverslips (2 × 10 stained . Cells wGiardia encystation. The deduced Giardia MLF protein contains 252 amino acids with a predicted molecular mass of ~29.71 kDa and a pI of 6.81. It has one MLF1IP domain (residues 9 to 170) as predicted by Pfam (or residues or 59 to 170 by NCBI Conserved Domain Database) (Drosophila MLF (residues 36 to 199). The Drosophila MLF1IP domain contains a 14-3-3 binding motif, which may involve in interaction with the transcription factor Dref [Giardia MLFIP domain has no 14-3-3 interaction motif, RSXSXPRXSXSX [Giardia MLF is moderately similar to the human MLF1 and MLF2 and Drosophila MLF  . The MLFtor Dref , but theXPRXSXSX . Sequenchila MLF . The fulmlf gene is controlled by its own promoter and contains an HA epitope tag at its C terminus , during vegetative growth and encystation using anti-HA antibody  as compared with strategy 1 (nt 115–134)  but also with very low homology. Scr7 may inhibit Giardia DNA repair since Scr7 can inhibit Giardia growth  as compared with strategy 1 (nt 115–134) . We also performed single cell dilution for the all-in-one plasmid system and found that the obtained two clonal populations have a similar disruption efficiency of 73% and 78% as compared with their original cultures (70%)  , suggest or nonessential (growth) requires successful knockout of mlf gene. A complete knockout of an essential gene may lead to cell death, so it is hard to determine the gene function for that case. We have tried to knockdown 12 genes, including mlf by our CRISPR/Cas9 system, but only have obtained successful growth of 4 mutant cell lines after gene disruption under drug selection. We only generated the knockdown but not knockout mutants for these 4 genes. It is possible that the other 8 genes whose mutants cannot be obtained could be essential genes. Combining the CRISPR/Cas9 system and tetracycline inducible system can allow an adjustable knockdown of a target gene in the future [Because of tetraploid genome and two nuclei, it could be hard to disrupt a gene completely in . This search identified similarity of Giardia MLF to Drosophila MLF human MLF1 and MLF2 in the GenBank database. Sequence of MLF proteins is analyzed by ClustalW 1.83, including Drosophila MLF human MLF1 and MLF2 , and Giardia MLF (orf number 16424 in Giardia genome database). The gray boxes indicate the MLFIP domain of Giardia MLF predicted by pfam (http://pfam.sanger.ac.uk/). (B) Schematic representation of the Giardia MLF protein. The gray box indicates the MLFIP domain, as predicted by pfam. The residue numbers are shown.(A) Alignment of the MLF proteins. Specific sequence similarity search was performed against the GenBank database on NCBI's Web site using the BLASTP algorithm ((PDF)Click here for additional data file.S2 Figmlf-ha, mlf, cwp1, cwp2, cwp3, myb2, ran, and 18S ribosomal RNA genes, respectively. Similar levels of the ran mRNAs and 18S ribosomal RNA were detected. (B) Overexpression of MLF increased the levels of MLF proteins. The 5’Δ5N-Pac and pPMLF stable transfectants were cultured in growth medium and then subjected to SDS-PAGE and Western blot analysis. The blot was probed with anti-MLF antibody. The result is the same as in mlf gene with the pac gene in the MLFko cell line confirmed by PCR2 and sequencing. Genomic DNA was isolated from MLFko and control cell lines cultured in growth medium. PCR was performed using primers specific for pac  Replacement of the mlf gene with the pac gene in the MLFko cell line confirmed by PCR3 and sequencing. Genomic DNA was isolated from MLFko and control cell lines cultured in growth medium. PCR was performed using primers specific for pac (PCR3), which are PCR3F for bold region 1 and PCR3R for bold region 2, to verify the integration of pac gene into the correct region in genomic DNA. The sequence results obtained from the PCR3 product are shown as underlined letters. Capital letters indicate the coding sequence for pac gene, which starts at ATG and stops at TGA. This indicates the replacement of the mlf gene with the pac gene. The region used to clone the mlf 3’ region into the pMLFko plasmid for HR is shown in red, which is also between the sequence of MLF3XF and MLF3KR. The underlined and lower case letters, which are upstream and outside of the red region of MLF3XF and MLF3KR, indicate that HR occurred in the sequence of mlf 3’ region and that the pac gene was integrated in the genomic DNA. Replacement of the mlf gene with the pac gene in the MLFkoSC and Cas9MLFko cell line was also confirmed by PCR3 and sequencing with the same sequencing results. (E) RT-PCR analysis of gene expression in the MLFko cell line during encystation. The control and MLFko cell lines were cultured in encystation medium and then subjected to RT-PCR analysis using primers specific for mlf, cwp1, cwp2, cwp3, myb2, wrky, pax1, cdk2, ran, and 18S ribosomal RNA genes, respectively. Similar levels of the 18S ribosomal RNA were detected. The ran mRNAs slightly decreased.(A) RT-PCR analysis of gene expression in the MLF-overexpressing cell line. The 5’Δ5N-Pac and pPMLF stable transfectants were cultured in growth medium and then subjected to RT-PCR analysis. PCR was performed using primers specific for  PCR2 in , which a(PDF)Click here for additional data file.S3 Figmlf gene decreased the levels of CWP1, MYB2, and other proteins in the MLFko cell line during vegetative growth. The control and MLFko cell lines were cultured in growth medium and then subjected to SDS-PAGE and Western blot analysis, as described in mlf, cwp1, cwp2, cwp3, myb2, wrky, pax1, cdk2, ran, and 18S ribosomal RNA genes, respectively, as described in mlf, cwp1, cwp2, cwp3, myb2, wrky, pax1, cdk2, ran, and 18S ribosomal RNA genes, respectively.(A) Cyst formation decreased by MLF knock down in the MLFko cell line during vegetative growth. The control and MLFko cell lines were cultured in growth medium for 24h (Enc) and then subjected to cyst count as described under “Materials and Methods” and (PDF)Click here for additional data file.S4 Figmlf gene decreased the levels of CWP1, MYB2, and other proteins in the MLFko–pu cell line. The control–pu and MLFko–pu cell lines were cultured in growth medium and then subjected to SDS-PAGE and Western blot analysis, as described in mlf, cwp1, cwp2, cwp3, myb2, wrky, pax1, cdk2, ran, and 18S ribosomal RNA genes, respectively, as described in (A) Quantification of MVs in MLFko–pu and control–pu cell lines using Imaris software. *, P <0.05 (n = 200–300 cells/condition). (B) Knock down of (PDF)Click here for additional data file.S5 Figmlf gene decreased the levels of CWP1, MYB2, and other proteins in the MLFko–pu cell line during encystation. The control–pu and MLFko–pu cell lines were cultured in encystation medium and then subjected to SDS-PAGE and Western blot analysis, as described in mlf, cwp1, cwp2, cwp3, myb2, wrky, pax1, cdk2, ran, and 18S ribosomal RNA genes, respectively, as described in (A) Cyst formation decreased by MLF knock down in the MLFko–pu cell line during encystation. The control–pu and MLFko–pu cell lines were cultured in encystation medium for 24h (Enc) and then subjected to cyst count as described under “Methods” and (PDF)Click here for additional data file.S6 Fig6 cells/ml in growth medium containing 33μM Scr7 for 24 h and then subjected to cell count. An equal volume of Me2SO was added to cultures as a negative control. The sum of total cells is expressed as a relative level over control. Values are shown as means ± S.E. of three independent experiments. *, P <0.05. (B) Partial replacement of the mlf gene with the pac gene in the MLFkoSC cell line confirmed by PCR. The pgCas9 and pMLFko constructs were transfected into trophozoites as described in mlf (PCR1), pac (PCR3), cwp1, cwp2, and ran genes, respectively, as described in mlf gene in the MLFkoSC cell line confirmed by real-time PCR. Real-time PCR was performed using genomic DNA and primers specific for mlf, cwp1, cwp2, and ran genes, respectively, as described in mlf gene decreased the levels of CWP1, MYB2, and other proteins in the MLFkoSC cell line during vegetative growth. The control and MLFkoSC cell lines were cultured in growth medium and then subjected to SDS-PAGE and Western blot analysis as described in mlf, cwp1, cwp2, cwp3, myb2, wrky, pax1, cdk2, ran, and 18S ribosomal RNA genes, respectively, as described in (A) Treatment with Scr7 decreased cell growth. The wild-type non-transfected WB cells were subcultured at an initial density of 1×10(PDF)Click here for additional data file.S7 Figmlf, cwp1, cwp2, cwp3, myb2, wrky, pax1, cdk2, ran, and 18S ribosomal RNA genes, respectively, as described in (A) Cyst formation decreased by MLF knock down in the MLFkoSC cell line during encystation. The control and MLFkoSC cell lines were cultured in encystation medium for 24h (Enc) and then subjected to cyst count as described under “Methods” and (PDF)Click here for additional data file.S8 Figs of the pMLFko plasmid is the same as in mlf (PCR1), pac (PCR2) and ran genes, respectively, as described in mlf, cwp1, cwp2, and ran genes, respectively, as described in mlf (PCR1), pac (PCR2) and ran genes, respectively, as described in mlf, cwp1, cwp2, and ran genes, respectively, as described in (A) Diagram(PDF)Click here for additional data file.S9 Figmlf gene decreased the levels of CWP1, MYB2, and other proteins in the MLFkoSC cell line relative to the MLFko cell line during encystation. The MLFko and MLFkoSC cell lines were cultured in encystation medium and then subjected to SDS-PAGE and Western blot analysis as described in mlf, cwp1, cwp2, cwp3, myb2, wrky, pax1, cdk2, ran, and 18S ribosomal RNA genes, respectively, as described in (A) MLF knock down decreased cyst formation in the MLFkoSC cell line relative to the MLFko cell line during encystation. The MLFko and MLFkoSC cell lines were cultured in encystation medium for 24h (Enc) and then subjected to cyst count as described under “Materials and Methods” and (PDF)Click here for additional data file.S10 Figmlf gene decreased the levels of MLF and CWP1 proteins in the Cas9MLFko cell line during encystation. The control and Cas9MLFko cell lines were cultured in encystation medium and then subjected to SDS-PAGE and Western blot analysis as described in mlf, cwp1, and cwp2 gene expression by MLF knock down in the Cas9MLFko cell line during encystation. The control and Cas9MLFko cell lines were cultured in encystation medium and then subjected to quantitative real-time RT-PCR analysis using primers specific for mlf, cwp1, cwp2, ran, and 18S ribosomal RNA genes, respectively, as described in mlf gene in two single clone populations of the Cas9MLFko cell line confirmed by PCR. Two single clone populations, clones 1 and 2, of the Cas9MLFko cell line were obtained using the single cell dilution method. Genomic DNA was isolated from Cas9MLFko clone 1, clone 2, and control cell lines cultured in growth medium. PCR was performed using primers specific for mlf (PCR1), pac (PCR2), and ran genes, respectively, as described in mlf gene in two single clone populations of the Cas9MLFko cell line confirmed by real-time PCR. Real-time PCR was performed using genomic DNA and primers specific for mlf, cwp1, cwp2, and ran genes, respectively, as described in (A) Cyst formation decreased by MLF knock down in the Cas9MLFko cell line during encystation. The control and Cas9MLFko cell lines were cultured in encystation medium for 24h (Enc) and then subjected to cyst count as described under “Methods” and (PDF)Click here for additional data file.S11 Figmlf gene, has a different sequence compared with pgCas9MLFko in mlf gene with the pac gene in the Cas9MLFko gRNA1 cell line confirmed by PCR. Puromycin was kept in the Cas9MLFko gRNA1 and control cell lines as described in mlf (PCR1), pac (PCR2), and ran genes, respectively, as described in mlf gene in the Cas9MLFko gRNA1 cell line confirmed by real-time PCR. Real-time PCR was performed using genomic DNA and primers specific for mlf, cwp1, cwp2, and ran genes, respectively, as described in mlf gene decreased the levels of MLF and CWP1 proteins in the Cas9MLFko gRNA1 cell line during vegetative growth. The control and Cas9MLFko gRNA1 cell lines were cultured in growth medium and then subjected to SDS-PAGE and Western blot analysis as described in mlf gene with the pac gene in the Cas9MLFko gRNA2 cell line confirmed by PCR. Puromycin was kept in the Cas9MLFko gRNA2 and control cell lines as described in mlf (PCR1), pac (PCR2), and ran genes, respectively, as described in mlf gene in the Cas9MLFko gRNA2 cell line confirmed by real-time PCR. Real-time PCR was performed using genomic DNA and primers specific for mlf, cwp1, cwp2, and ran genes, respectively, as described in (A) Diagrams of the pgCas9MLFko gRNA1 or 2 plasmid are the same as in (PDF)Click here for additional data file.S12 Figmlf gene in the Cas9MLFko–pu cell line confirmed by real-time PCR. Puromycin was removed from the Cas9MLFko cell line to obtain the Cas9MLFko–pu cell line as described in mlf, cwp1, cwp2, and ran genes, respectively, as described in mlf gene decreased the levels of MLF and CWP1 proteins in the Cas9MLFko–pu cell line. The control and Cas9MLFko–pu cell lines were cultured in growth medium and then subjected to SDS-PAGE and Western blot analysis as described in mlf, cwp1, and cwp2 gene expression by MLF knock down in the Cas9MLFko–pu cell line. The control and Cas9MLFko–pu cell lines were cultured in growth medium and then subjected to quantitative real-time RT-PCR analysis using primers specific for mlf, cwp1, cwp2, ran, and 18S ribosomal RNA genes, respectively, as described in (A) Partial disruption of the (PDF)Click here for additional data file.S13 Figmlf gene decreased the levels of MLF and CWP1 proteins in the Cas9MLFko–pu cell line during encystation. The control and Cas9MLFko–pu cell lines were cultured in encystation medium and then subjected to SDS-PAGE and Western blot analysis as described in mlf, cwp1, and cwp2 gene expression by MLF knock down in the Cas9MLFko–pu cell line during encystation. The control and Cas9MLFko–pu cell lines were cultured in encystation medium and then subjected to quantitative real-time RT-PCR analysis using primers specific for mlf, cwp1, cwp2, ran, and 18S ribosomal RNA genes, respectively, as described in (A) Cyst formation decreased by MLF knock down in the Cas9MLFko–pu cell line during encystation. The control and Cas9MLFko–pu cell lines were cultured in encystation medium for 24h (Enc) and then subjected to cyst count as described under “Materials and Methods” and (PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


The existence of extended post-reproductive lifespan is an evolutionary puzzle, and its taxonomic prevalence is debated. One way of measuring post-reproductive life is with post-reproductive representation, the proportion of adult years lived by females after cessation of reproduction. Analyses of post-reproductive representation in mammals have claimed that only humans and some toothed whale species exhibit extended post-reproductive life, but there are suggestions of a post-reproductive stage for false killer whales and Asian elephants. Here, we investigate the presence of post-reproductive lifespan in Asian elephants using an extended demographic dataset collected from semi-captive timber elephants in Myanmar. Furthermore, we investigate the sensitivity of post-reproductive representation values to availability of long-term data over 50 years.We find support for the presence of an extended post-reproductive stage in Asian elephants, and that post-reproductive representation and its underlying demographic rates depend on the length of study period in a long-lived animal.The extended post-reproductive lifespan is unlikely due to physiological reproductive cessation, and may instead be driven by mating preferences or condition-dependent fertility. Our results also show that it is crucial to revisit such population measures in long-lived species as more data is collected, and if the typical lifespan of the species exceeds the initial study period. The evolution of extended post-reproductive lifespan is a long-term puzzle in evolutionary biology, where extended refers to post-reproductive lifespan that is not an artefact of individual variation in somatic and reproductive senescence rates . SpeciesTo distinguish extended post-reproductive lifespan from individual variation in senescence, measures that do not depend on any one individual are preferable to those calculated off specific individuals (e.g. expected lifespan vs maximum recorded lifespan) or individuals meeting specific criteria , 13. IndOrcinus orca), narwhals (Monodon monoceros), beluga whales (Delphinapterus leucas), and short-finned pilot whales  . WhOur second aim was to quantify how values of PrR and its underlying demographic variables may change depending on the length of study. There was large variation in post-reproductive representation through time Fig. , which wOne cannot consider PrR alone though, as its calculation relies heavily on other demographic variables. Whilst sample size is not directly incorporated into the PrR equation, it can have a large effect on the survival and fertility series. For example, one birth will have a greater impact on the fertility rate if the sample size at that age is smaller, which may be important for slow reproducers such as elephants. The same is true for the sampling of old individuals to determine, for example, the maximum lifespan or expected survival to a given age. The number of female elephants used to calculate the demographic series rose steadily, from 998 individuals in 1960 to 3802 individuals in 2018  have changed considerably with the length of the longitudinal data collection , and has remained unchanged in the last 4 years. Note that even though eM has not increased much . Howeveoduction , our resOur results also highlight an important issue in assessing PrR for long-lived species: the need for sufficiently long follow-up in longitudinal datasets. Here, we find a PrR of 0.162, higher than the value calculated in a previous assessment with the same population (0.128) [It is therefore important for future studies to consider when a species is assessed if they are calculating post-reproductive lifespan for long-lived species in which the study period is shorter than the typical adult lifespan. This issue can be alleviated by highly accurate age estimation for individuals of all ages, though there can be difficulties with verifying such estimation methods. For example, size cannot be used for accurate age estimation if growth is determinate and individuals have reached the asymptote of growth , 50. FurDue to the rarity of early reproductive cessation, we still do not know whether its evolution requires a specific driver or drivers. It may be that the prolonged post-reproductive lifespans of Asian elephants are currently driven by behaviour or body condition, rather than reproductive physiology, and may therefore be in an evolutionary transition; an extended post-reproductive life may be a prerequisite of an early end to reproductive capabilities. To properly tackle the puzzle provided by the evolution of early reproductive cessation and post-reproductive lifespan, we first need to know the taxonomic prevalence of these traits, underscoring the importance of long-term studies on known-age individuals.Additional file 1. Dataset containing the lx and mx series of the population.Additional file 2. Dataset containing the changing lx and mx series of the population (1960-2018).


This study aimed to explore the underlying mechanism of linc01014 in oesophagus cancer gefitinib resistance. Gefitinib‐resistant oesophagus squamous cell carcinoma (ESCC gefitinibR) cell lines were constructed by using different gefitinib treatment in FLO‐1, KYAE‐1, TE‐8 and TE‐5 cell lines and confirmed by MTS50 and proliferation assays. Expression of linc01014 was overexpressed/silenced in FLO‐1 cells followed by gefitinib treatment, and then, the apoptosis‐associated markers Bax and Bcl‐2, and PI3KCA in PI3K signalling pathway were determined using Western blotting. MST50 and morphology analyses showed that ESCC gefitinibR cell lines presented obvious gefitinib resistance than their parental ESCC cell lines. ESCC gefitinibR cell lines showed significantly higher proliferation abilities than their parental ESCC cell lines after treating with gefitinib. Overexpression of linc01014 significantly inhibited the apoptosis of FLO‐1 cells induced by gefitinib and silencing linc01014 obviously promoted the apoptosis of FLO‐1 cells induced by gefitinib. Silencing linc01014 could significantly increase the gefitinib chemotherapy sensitivity of oesophagus cancer via PI3K‐AKT‐mTOR signalling pathway. ESCC gefitinib‐resistant cell lines were successfully constructed.Overexpression of linc01014 significantly decreased the apoptosis of FLO‐1 cells induced by gefitinib.Silencing linc01014 obviously promoted the apoptosis of FLO‐1 cells induced by gefitinib.1Oesophageal cancer is one of the most leading cause of mortality cancer worldwide, and oesophageal squamous cell carcinoma (ESCC) is the main type for oesophageal cancer arising from oesophageal epithelial cells.Epidermal growth factor receptor (EGFR), a transmembrane tyrosine kinase, plays critical role in several solid cancers and its chemotherapy resistance regulation.Long non‐coding RNAs (lncRNAs) is a class of transcripts without coding capacity at a length of >200 nt and has reported to play critical roles in the pathogenesis and drug resistance of ESCC.In this study, the role of lncRNA linc01014 involved in gefitinib resistance was explored in ESCC cell lines. According to these investigations, we hope to provide some new insights in solving gefitinib resistance in ESCC and improving the prognosis of patients with ESSC.22.12 in atmosphere. Cells were transfected with linc01014 mimic, si‐linc01014 or negative control (NC) using lipofectamine 2000 (Invitrogen) according to manufacturer's protocol.Human ESCC cell lines KYAE‐1, TE8, TE5 and FLO‐1 were purchased from the Chinese Type Culture Collection, Chinese Academy of Sciences . All cell lines were cultured in PRIM 1640 medium (Gibco) supplemented with 10% fetal bovine serum  and 100 U/mL penicillin/streptomycin (Sigma) at 37°C in a humidified incubator with 5% CO2.2Gefitinib‐resistant cell lines of KYAE‐1, TE8, TE5 and FLO‐1 were constructed via repeatedly subcultured with an increasing concentration of gefitinib (Sigma) starting from 0.1 μM/mL to final concentration of 10.2 μM/mL. Finally, the gefitinib‐resistant cell lines were collected and used for the following investigations.2.33/well and maintained in PRIM 1640 medium contained with 10% FBS overnight. Then, cells were treated with gefitinib in dimethyl sulphoxide  at concentrations of 0, 1, 10, 20 and 30 μmol/L for 48 hours. Concentration of gefitinib that caused 50% inhibition on the MTS activity (MTS50) was determined as previously reported. For proliferation detection, cells were seeded in 96‐well plates at a density of 1 × 103/well and maintained in PRIM 1640 medium contained with 10% FBS overnight. Then, cells were treated with MTS50 determined gefitinib concentration for 0, 48 and 72 hours. Subsequently, cell proliferation was determined. Each experiment was performed in triplicate, and average value was calculated as the results.The cytotoxic effect of gefitinib on ESCC cell lines and the proliferation abilities of these cell lines under different treatments were detected by the MTS ‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulphopheny)‐2H‐tetrazolium]) assay using CellTiter‐96‐AQueous One Solution Cell Proliferation Assay reagent (Promega). Briefly, cells were seeded in 96‐well plates at a density of 5 × 102.4Morphology of cells after treating with or without gefitinib was pictured under light microscope (Olympus CKX41) at 24, 48 and 72 hours at 200× magnification. The morphological appearance of cells was compared at each time‐point.2.5−ΔΔCt method with GAPDH as the internal control. All premier sequences were purchased from RiBio .After treatment, total RNA in cells was isolated using TRIzol reagent (Takara) according to manufacturer's protocol. cDNA was synthesized using the GoScript™ Reverse Transcription System (Promega) according to manufacturer's instruction. Then, the expression of linc01014 was determined using SYBR Green Master Mix (Applied Biosystems) with a total of 2 μg cDNA as a template on a BioRad CFX96 Sequence Detection System (Bio‐Rad company). Relative expression of linc01014 was analysed using 22.6After treatment, cells were lysated with RIPA lysis buffer (Millipore) supplemented with protease inhibitors (Sigma). Protein concentrations were measured using BCA method (Pierce) according to the manufacturer's protocol (Beyotime). Then, proteins were boiled with loading buffer and subjected for 10% SDS electrophoresis followed by transferring to polyvinylidene fluoride membranes (Millipore). Subsequently, membranes were blocked with 5% milk at room temperature for 1 hour. Following this, membranes were incubated with a 1:1000 solution of primary antibodies  at 4°C overnight. Then, membranes were incubated with the secondary antibody at room temperature for 1 hour and visualized using ECL method (Pierce).2.7t test or one‐way ANOVA analysis. P < .05 was considered statistically significant.SPSS 19.0 (SPSS Incorporation) was used to perform statistical analyses in this study. Data in this study were presented with mean ± standard deviation (SD). Comparisons among groups were analysed using Student's 33.1The sensitivity of ESCC and ESCC gefitinibR cells to gefitinib was determined by MTS viability assay and cell morphology. The MTS assay showed that FLO‐1‐gefitinibR cell lines showed remarkably higher relative MTS activity than FLO‐1 cells at concentration of 0.10‐10.0 μmol/L, indicating that FLO‐1‐gefitinibR cell lines presented a higher gefitinib resistance that FLO‐1 cells  was used to treat FLO‐1 cells after altering the expression of linc01014. The result showed that overexpression of linc01014 could significantly increase the expression of Bcl‐2 and PI3CA, but decrease the expression of Bax, indicating that linc01014 could inhibit the apoptosis of FLO‐1 cells induced by gefitinib. However, silencing the expression of linc01014 could significantly increase the expression of Bax, but decrease the expression of Bcl‐2 and PI3KCA, indicating that silencing linc01014 could promote the apoptosis of FLO‐1 cells induced by gefitinib , indicating that linc01014 might play a potential role in regulating the PI3K/AKT/mTOR signalling pathway via PIK3CA. In the current study, we have identified that overexpression of linc01014 could significantly increase the expression of PIK3CA and Bcl‐2, but decrease the expression of Bax. In addition, inhibiting the expression of linc01014 could obviously inhibit the expression of PIK3CA and Bcl‐2, but induce the expression of Bax after treated with gefitinib. These findings suggested that linc01014 could significantly regulate the activity of PI3K/Akt/mTOR signalling pathway via regulating the expression of PIK3CA to altering the sensitivity of ESCC cells to gefitinib.Linc01014 is newly identified PIK3CA corresponding lncRNA (There were still some limitations in this study. First, because of the limited clinical samples, the expression of linc0104 in ESCC and gefitinib‐resistant ESCC tissue samples was not explored, which might impair the significance of our study. Moreover, lncRNAs have many regulatory mechanisms to modulate the pathogenesis of disease, but because of the shortage of research funds, the exact mechanism of linc0104 in regulating gefitinib resistance in ESCC was not further explored. A deepened exploration about linc0104 in gefitinib resistance is still required in the further.In conclusion, linc0104 might play a critical role in the pathogenesis of ESCC. Silencing of linc01014 could significantly promote the sensitivity of ESCC cell lines to gefitinib and increasing the apoptosis of ESCC cells via PI3K/AKT/mTOR signalling pathway. These finding might be of importance in understanding and treatment of ESCC gefitinib resistance in clinic.All of the authors have no conflict of interest in this research.Each author has made an important scientific contribution to the study and has assisted with the drafting or revising of the manuscript.All of the authors have consented to publish this research.


Ovarian cancer (OC) is one of the leading causes of female cancer death. Recent studies have documented its extensive variations as a disease entity, in terms of cell or tissue of origin, pre-cancerous lesions, common mutations, and therapeutic responses, leading to the notion that OC is a generic term referring to a whole range of different cancer subtypes. Despite such heterogeneity, OC treatment is stereotypic; aggressive surgery followed by conventional chemotherapy could result in chemo-resistant diseases. Whereas molecular-targeted therapies will become shortly available for a subset of OC, there still remain many patients without effective drugs, requiring development of groundbreaking therapeutic agents. In preclinical studies for drug discovery, cancer cell lines used to be the gold standard, but now this has declined due to frequent failure in predicting therapeutic responses in patients. In this regard, patient-derived cells and tumors are gaining more attention in precise and physiological modeling of in situ tumors, which could also pave the way to implementation of precision medicine. In this article, we comprehensively overviewed the current status of various platforms for patient-derived OC models. We highly appreciate the potentials of organoid culture in achieving high success rate and retaining tumor heterogeneity. Ovarian cancer (OC) is the most devastating gynecologic cancer. Even after development of many surgical techniques and chemotherapies, its overall five-year survival rate is still as low as 47% . The proTP53 is exclusively mutated in HGSC in a proportion as high as 95% [RAS and BRAF are frequently mutated in LGSC [KRAS mutation is also implicated in mucinous carcinoma [ARID1A, PIK3CA, TERT promoter mutations [PTEN, PIK3CA, ARID1A, and CTNNB1 mutations [With many histological variations, OC has been regarded as a highly heterogeneous disease. Indeed, epithelial OC that comprises nearly 90% of OC is usually classified into many histologically distinct subtypes. Major subtypes include serous carcinoma, endometrioid carcinoma, clear cell carcinoma, and mucinous carcinoma. Recently, epithelial OC are more loosely classified into two general categories by integrating the results from histopathological, molecular biological, and genetic analyses, to underscore biological properties of OC. Type 1 cancers grow slowly and contain low-grade serous carcinoma (LGSC), mucinous carcinoma, endometrioid carcinoma, clear cell carcinoma, and transitional carcinoma. Type 2 cancers progress rapidly and contain high-grade serous carcinoma (HGSC), undifferentiated carcinoma, and carcinosarcioma . Research as 95% , RAS and in LGSC . KRAS muutations ,11,12, autations . HoweverBRACA1 or BRCA2 [Despite the highly heterogeneous nature of OC, standard treatment of ovarian cancer is stereotypically composed of aggressive surgery followed by platinum-taxane chemotherapy. Of the four major subtypes, clear cell carcinoma and mucinous carcinoma tends to be refractory to chemotherapy ,15. Moreor BRCA2 , and molor BRCA2 . Nonetheor BRCA2 . HoweverSuch patient-derived models, especially primary cell culture, have not been intensively developed for OC thus far, unlike for cancers of other vital organs. It is not clear whether this is because of any technical difficulties specific to OC or researchers simply did not attempt to obtain patient-derived material for OC. In this article, we comprehensively overview the current status of various patient-derived platforms  and illuCancer cell lines are special types of cells that acquire infinite proliferation potential on plastic dishes. They can expand as a monolayer sheet, with simple media that typically contains fetal bovine serum (FBS). For their high usability as adherent cells, cancer cell lines have long contributed to many areas of scientific research, including cell biology, biochemistry, physiology, and drug discovery, let alone cancer research . To bettOn the other hand, there are several drawbacks in cell lines. Firstly, they tend to be passaged too many times. As the success rate for establishing cell lines from primary tumor tissues is generally low, only a subset of cell lines has been extensively used, thereby cultured over a significantly long period of time or many passages. With the high potential of FBS-based cultures to introduce mutations and genome instability, cell lines could undergo drastic changes in terms of morphology and biological properties, presumably by selection of some specific clones. Consequently, even cell lines under the same name could harbor significant diversity between laboratories . DiversiTP53 was intact, although this is a hallmark mutation in HGSC. On the other hand, it detected mutations in ARID1A, BRAF, PIK3CA, and PTEN, which are commonly mutated in other subtypes of OC. Besides, OVCAR-3 and CAOV3 were not among the top-ranking HGSC cell lines based on integrative molecular profiling, although they in fact possessed TP53 mutations and substantial copy number change. IGROV1 had a hypermutated phenotype, which is frequently observed in endometrioid carcinoma rather than in HGSC. Strikingly, the 12 cell lines evaluated as best candidates for HGSC models accounted for only 1% of the Pubmed literature on the 47 analyzed cell lines. Morphological characterization of 39 OC cell lines also led to a similar conclusion that questioned SK-OV-3, S2780, and IGROV1 as representative models for HGSC [More than 50 OC cell lines have been so far established and are currently available for research. As predicted, discrepancies in histology or mutation profiles between cell lines and original tumors were documented in a study that investigated in detail the validity of 47 OC cell lines derived from various OC subtypes . Among tfor HGSC . These rfor HGSC , suggestTumorigenicity assay was conducted by inoculating 17 OC cell lines into the subcutis, peritoneal cavity, and ovaries of nude mice, revealing that the tumorigenicity varied among inoculation sites and cell lines . For exaPatient-derived xenografts (PDXs) are usually generated by directly engrafting tumor fragments into immunodeficient mice. Not only surgically resected tumors, but also samples from biopsy, ascites, and pleural effusion can be used. Inoculation sites are most commonly subcutaneous tissues. Depending on the tumor types and purposes of the experiments, orthotopic engraftment or intraperitoneal injection could be an option. Established PDXs can be serially engrafted to multiple mice without a culture step in between. Starting from primary tumors, the tumor take rate of PDX is generally higher than the success rate of establishing cancer cell lines. One possible explanation is that microenvironments reconstituted by interactions between subcutaneous tissues and tumor fragments might be physiological, accelerating tumor development faster than in cell lines, with only epithelial cells in an artificial condition. PDXs have been established from various types of cancer . In mostnull (NSG) mice, which could further involve a significant cost. For that reason, use of PDXs for high-throughput drug screening might be limited. Secondly, PDXs often require long latency to be established [+ cells isolated from blood of the same patient were intravenously inoculated to reconstitute a functional human immune system [On the other hand, there are downsides to PDXs. Firstly, maintaining tumors in immunocompromised mice might be costly and require a more specialized skill in serial passage in mice, compared to simple monolayer cultures of cell lines. Besides, mice with more severely compromised immunity than nude mice might be eventually required to increase the tumor take rate. These mice include non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and NOD/SCID/IL2Rγablished , and couablished . In prine system . PDXs wiThe first xenograft model for OC was described back in 1977, in which tumor tissue was engrafted into subcutaneous tissue of nude mice . SeveralThe responses to platinum chemotherapy in OC PDXs were highly correlated with those in the donor patients ,51,52,53As mentioned earlier, establishment of cell lines from primary cultures of patient-derived samples has only a low efficiency. For example, only 10 breast cancer cell lines were established out of 135 resected primary tumors . Other rIntriguingly, 25 novel cell lines were recently established from primary OC with significantly high efficiency, by using culture media and conditions optimized to each histological subtype . More imSpheroids are sphere-like cell aggregates, which are usually maintained as floating three-dimensional structures. Among various spheroid culture methods so far documented , the sta+ CD133+ stem cells derived from cancerous ascites of OC patients [For OC patients, drug resistance and peritoneal dissemination accompanied by cancerous ascites are two major factors that could profoundly affect prognosis. Given the morphological similarity between tumor cell aggregates within cancerous ascites and tumor spheroids in culture, it was natural that researchers initiated spheroid culture studies of OC with cancerous ascites samples. As was observed in other types of cancer, only a subset of ascites-derived cancer cells exhibited stem-cell like properties, which indeed developed tumors in immune-deficient mice . Resectepatients . ThroughFor spheroid culture, standard preparation of tumor samples begins by obtaining singly dissociated cells. However, this was originally optimized for hematopoietic cells or neuronal cells, by which stem cell biology has considerably evolved. In sharp contrast, destruction of cell-cell adhesion could readily induce anoikis for epithelial cells, which significantly lowers the success rate of spheroid formation for many types of tumors of epithelial origin. To address this issue, only partially digested colorectal cancer (CRC) tissues were selectively subjected to spheroid formation, resulting in substantial improvement of the success rate of primary culture . This ap+ could infinitely proliferate in vitro [An organoid is an emerging concept that is literally a mini-organ exclusively composed of epithelial cells. Without the aid of a stromal niche, even a single normal stem cell can differentiate into all the lineages of the organ, while stem cells self-renew, thereby reconstituting in vivo homeostasis of the whole organ. In 2009, it was first demonstrated that murine intestinal stem cells marked by LGR5in vitro . This stin vitro ,82,83. Iin vitro , developin vitro , and epiin vitro . Organoids can be subjected to genetic engineering by viral introduction of shRNA and cDNA. With highly efficient lentiviral gene transduction into murine organoids , we demoOrganoid culture techniques can be applied to various patient-derived samples, such as resected tissue, biopsy, ascites, and pleural effusions. Obvious advantages of organoid culture over PDX and spheroid culture include that it allows propagation of normal cells and precancerous cells, and a higher success rate for cancer cells. In CRC, the success rate of organoid culture was significantly higher than that of PDX or spheroid culture . Given tEstablishment of tumor-derived organoids from various cancer types has been reported, namely colon , pancreaOn the other hand, there are several shortcomings in organoid culture. Firstly, tumor-derived organoids lack stroma, immune cells, and blood vessels. To address this issue, some co-culture systems involving pancreatic ductal adenocarcinoma-derived organoids and murine pancreatic stellate cells was established to investigate interactions between cancer cells and cancer-associated fibroblasts . RecentlUnlike many other types of cancer, only a few studies have documented organoid cultures of OC . Drug reTo better understand the biological features of OC, thorough elucidation of interactions between tumor cells and the microenvironment is a critical issue. However, standard organoid culture consists of only epithelial cells and lacks such interactions. To address this issue, 3D organotypic models that reproduce a similar situation to that in vivo have been developed . SpecifiUntil very recently, use of patient-derived OC as preclinical models has been quite limited because the success rate of primary cultures was low, and even if cell lines are established, features often differ from those of the original tumors. Recent remarkable advances in 3D culture technique have allowed us to reconstitute many features of the original tumors in an in vitro setting. In the light of rapid implementation of precision medicine, which largely depends on genomic information of each cancer, establishment of patient-derived cell-based assays will be of critical relevance. Among various platforms, the organoid might be the most powerful tool in high-throughput drug screening and establishment of xenografts. Now that OC organoids are becoming available, its nation-wide banking will be readily started as a valuable resource for OC researches. Collectively, OC organoids per se and organoid-derived PDX will likely contribute to the development of novel therapies as well as elucidation of its pathogenesis.