We show that Mitfa represses tfec expression, revealing a likely mechanism contributing to the decision between melanocyte and iridophore fate. Our data are consistent with the long-standing proposal of a tripotent progenitor restricted to pigment cell fates. Moreover, we investigate activation, maintenance and function of tfec in multipotent NCSCs, demonstrating for the first time its role in the gene regulatory network forming and maintaining early neural crest cells. In summary, we build on our previous work to characterise the gene regulatory network governing iridophore development, establishing Tfec as the master regulator driving iridophore specification from multipotent progenitors, while shedding light on possible cellular mechanisms of progressive fate restriction.Understanding how fate specification of distinct cell-types from multipotent progenitors occurs is a fundamental question in embryology. Neural crest stem cells (NCSCs) generate extraordinarily diverse derivatives, including multiple neural, skeletogenic and pigment cell fates. Key transcription factors and extracellular signals specifying NCSC lineages remain to be identified, and we have only a little idea of how and when they function together to control fate. Zebrafish have three neural crest-derived pigment cell types, black melanocytes, light-reflecting iridophores and yellow xanthophores, which offer a powerful model for studying the molecular and cellular mechanisms of fate segregation. Mitfa has been identified as the master regulator of melanocyte fate. Here, we show that an Mitf-related transcription factor, Tfec, functions as master regulator of the iridophore fate. Surprisingly, our phenotypic analysis of  Pigmentation is a conspicuous feature of animal diversity and has broad importance for behaviour and evolution (reviewed in ). Much iThe progressive fate restriction model proposes that neural crest cells (NCCs) become partially fate restricted as development progresses, giving rise to intermediate partially-restricted progenitors, each of which can generate a number, but not all of the NC derivatives –8. Such Characterisation of the phenotypes of mutants affecting multiple NC derivatives has been widely used to infer the identities and potencies of these progenitors. In the pigment cell field, a progressive fate restriction model has been developed based on loss- and gain-of-function studies for single genes, with both a multipotent chromatoblast and a bipotent melano-iridoblast proposed as partially-restricted intermediates , 14–17. colourless/sox10; [nacre/microphthalmia-associated transcription factor a/mitfa, [sox10 function results in lack of all three zebrafish lineages, but also hinders the development of peripheral nervous system components [restricted to the three pigment cell lineages alone) have not been identified.To date, examination of zebrafish mutants provides evidence of complex genetic control of pigment cell development from multipotent NCCs . Of the s/sox10; , 20) or a/mitfa, ). Conseqmponents , 23. Altmponents , mutantsmitfa transcription [dct, silva and tyrosinase). Mitfa is thus dubbed the ‘master regulator’ of melanocyte fate choice [As a first step towards understanding the complex GRN governing NCC fate restriction towards pigment cell lineages, it is important to define the key components involved in fate specification of individual lineages. Of the three zebrafish pigment cells, melanocytes are the currently best studied. In this lineage, Sox10, in conjunction with Wnt signalling, is required to activate and maintain cription , 24–26. e choice , 21, 25.e choice . In conte choice , 28–30. e choice  and, in e choice , 17.purine nucleoside phosphorylase 4a (pnp4a), which encodes an enzyme important in the biosynthesis of guanine, is a robust marker of mature iridophores [shady/ltk mutants fate specification and proliferation of iridophores, but not other pigment cells, are impeded [sox10 mutants, even though ltk expression is strongly detectable by whole-mount in situ hybridisation [Recent studies of key zebrafish iridophore mutants have begun to define the basic genetics of this cell type. In terms of their differentiation, heightened purine synthesis is central to the development of the guanine crystals that form the reflecting platelets. Thus, dophores , 17. Adddophores , while ddophores –35. Furtdophores , 36) recdophores , 38. In  impeded , 36. Nevdisation , 14. Themitfa and tfec are the only ones expressed in the NC. The distinctive expression pattern of tfec in cells along the dorsal and ventral midline in the trunk and tail and two patches over the yolk, is strongly reminiscent of differentiating iridophores [tfec to be one of several transcription factor genes with enriched expression specifically in the iridophore lineage [The zebrafish “MiT” (Mitf/Tfe) family consists of six genes ; of thesdophores . Further lineage . Togethetfec serves as a robust marker of the iridophore lineage, allowing us to define the major stages of iridophore development [in situ hybridisation studies of tfec expression patterns throughout a developmental time-course, and co-expression analysis with both ltk and mitfa, we concluded that tfec is first expressed extensively throughout the premigratory NC progenitors of the trunk and tail at 18 hpf, and then exclusively in the developing iridophore lineage. Focussing on cells in the posterior trunk, we distinguished a subset of tfec-positive cells within the premigratory domain that have downregulated the early NCC marker, foxd3, but do not express definitive pigment cell markers; we refer to these as early chromatoblasts (Cbl early). At approximately 22 hpf, ltk and mitfa are upregulated in the tfec+; foxd3- premigratory cells of the trunk [Cbl late). By 24 hpf, tfec labels migrating progenitors which co-express mitfa and ltk markers, and which we consider fate-specified iridoblasts, but which likely retain at least melanocyte potential too (Ib(sp); [tfec+ cells co-express ltk, but not mitfa, and we now consider these to be either definitive iridoblasts (Ib(df), along the dorsal trunk and on the lateral migration pathway at 30 hpf), or mature iridophores (in iridophore locations from 42 hpf onwards) according to their position and state of visible differentiation. Thus, the established iridophore-associated expression pattern of tfec during zebrafish development reinforces the hypothesis that Tfec might act as the missing iridophore master regulator, but does not eliminate the possibility of additional earlier functions, either in multipotent early NCCs ..tfec serhe trunk , 14, 21,tfec mutants to inform our derivation of a core iridophore GRN. Here, we describe in detail the generation and comprehensive characterization of the effects on NC development of mutations in tfec. Following examination of the development of a wide variety of NC derivatives in tfec mutants, using early and late molecular markers, we conclude that, although neuronal and skeletal derivatives develop normally, specification of all pigment cell fates is delayed in homozygous mutants, suggesting a common early requirement for tfec in the GRN governing specification of all three chromatophore lineages, and providing support for a common chromatoblast precursor. Finally, our previous work identified the GRN governing maintenance of tfec in the iridophore lineage [tfec as a necessary regulator of iridophore specification, yet not alone sufficient to drive NCCs to adopt an iridophore fate. Importantly, we identify its upstream regulators in the multipotent premigratory NC, thus for the first time placing the transcription factor in context in the NCC specification GRN [In our previous study we used a preliminary assessment of  lineage . In the tion GRN . Togethetfec is co-expressed with the established iridophore marker, ltk [tfec expression was prominent in the RNA-seq profiles of iridophores, they also detected low levels of expression in purified melanocytes [in situ hybridisation to detect tfec in individual embryos, following photographic documentation of their individual iridophore patterns, to show definitively its presence in differentiated iridophores  in two different wild-type strains, WIK and NHGRI-1, in addition to frameshifted alleles  promoter did not lead to a significant increase of iridophore numbers ). Here we report that melanogenesis is delayed in 30 hpf homozygous tfec mutant embryos when compared to WT or heterozygous siblings, supporting a functional role for Tfec during its transient expression in melanoblasts. Specifically, we observed a significant reduction in the numbers of differentiating melanocytes along the dorsal trunk, and the medial and lateral migration pathways . We furpathways . Melanocpathways . To testpathways . Furtherpathways , suggestpathways .tfec mutants, expression of the differentiated iridophore marker, pnp4a [tfec mutants. At both 24 hpf and 30 hpf, pnp4a was expressed in a notably reduced number of cells along the dorsal trunk, the migratory pathways and overlying the eye  in these embryos. We assessed expression of tfec itself in tfec mutant embryos, using our tfecba6 allele. At 24 hpf, presumptive homozygous mutants maintained tfec expression along the premigratory NC domain, even in anterior regions, indicating failure of a subset of NC derivatives to become fate specified, since in WT embryos fate specification to non-iridoblast fates is accompanied by loss of tfec expression in the majority of derivatives . At 36 hpf, the number of tfec-positive cells identified as Ib(df) [tfec mutant embryos compared to their siblings  from Cbl(early).We set out to investigate the early specification events that lead to the observed pigmentation phenotypes by studying expression of iridophore markers. In homozygous r, pnp4a , 17, was the eye . Maintenivation   was strosiblings , consistdophores  was not as pnp4a  or ltk [p4a  [tfec mutant. The subsequent recovery of normal melanocyte numbers makes clear that compensatory factors allow melanoblasts to be specified, albeit with a short delay.The delayed melanogenesis phenotype in these mutants might result from delayed specification of melanoblasts, or from slowed differentiation of normally specified melanoblasts. To distinguish between these two possibilities, we performed chromogenic siblings . At 30 hpathways . Consistsiblings . This ea+ Ib(sp)  from lat+ Ib(sp)  that thetfec mutant embryos did not show obvious changes in the number and distribution of mature xanthophores  sensory neurons, as labelled by anti-Hu immunofluorescence, was unaffected by loss of tfec function  in migrating precursors of the posterior lateral line nerve (pLLn) appeared normal in tfec mutants, compared to known WT siblings  [tfec expression in the anterior premigratory NC domain in sox10, sox9b, tfap2a and foxd3 mutants at this stage, consistent with retained premigratory late Cbls  positions), until 36 hpf  . Howeverate Cbls . This pepathways , 20, tfeboundary . Our resl 36 hpf . In eachnts Figs  and S4.sox10 and sox9b have been shown to be functionally redundant in DRG sensory neuron development [tfec expression in early or late Cbls. Examination of embryo batches containing sox10;sox9b double mutants at 18 hpf did not reveal elimination of tfec expression in any of the assessed embryos . We conducted whole-mount in situ hybridisation to assess the effects of loss of mitfa function on tfec expression. Interestingly, presumed homozygous mitfa mutants show ectopic expression of tfec along the dorsal trunk and in NC derivatives along both the medial and lateral migration pathways ) cells, proposed to be able to at least give rise to melanocytes and iridophores, but not in mature melanocytes. Thus, Ib(sp) should also be considered as specified melanoblasts. The first aim of the present study was to determine whether iridophores are indeed the only embryonic differentiated pigment cell expressing tfec; to this end, we conducted analyses showing that tfec expression is maintained at detectable levels only in mature iridophores, but neither in melanocytes nor in xanthophores, in agreement with recently published single cell transcriptomics analyses [Our previous work established e choice , 39. Intanalyses .tfec might have a function in iridophores analogous to that of mitfa in melanocytes; i.e. as the master regulator of the iridophore lineage. We generated mutations in tfec using a CRISPR/Cas9 approach, obtaining several alleles which displayed essentially identical phenotypes. Although tfec mutants have been generated independently [tfec expression; [tfec remain unclear. Nevertheless, data from mosaic adults and a single adult escaper suggest that tfec is required for iridophores throughout the lifetime of the animal  and diee animal .tfec to be considered the iridophore master regulator, the gene must not only be necessary, but also sufficient for iridophore specification. Our loss of function data are strongly suggestive of tfec being required for iridophore development, as mitfa is for melanocytes  may be of an RPE character, rather than neural crest melanocyte character. Transcriptomics analyses and detailed evaluation of the spatio-temporal requirements for melanocyte rescue, as well as Tfec-Mitfa protein-protein and DNA interactions, will be necessary to elucidate the origins of such cells, and the seemingly intricate relationship between Mitfa and Tfec during pigment cell development. Finally, work to define direct and indirect transcriptional targets of Tfec, as well as any other transcriptional co-regulators, similar to the role of Tfap2a for Mitfa [In addition to a complete absence of iridoblasts and mature iridophores our mutants also, surprisingly, presented with delayed differentiation of both NC-derived and RPE melanocytes, as well as of xanthophores. This suggests that Tfec has a subsidiary role in specification of each of the pigment cell-types. The melanocyte specification delay phenotype that we describe may also help explain the otherwise rather surprising observations of lian RPE  and a co closure . Consideor Mitfa , is needtfec expression is a fate-restricted tripotent precursor of the three chromatophore lineages. Decades ago, it was proposed that melanocytes share a common origin with iridophores and xanthophores from a pigment-restricted precursor [ltk expression in sox10 mutants indicated the presence of an ltk+ precursor of pigment cells, consistent with the chromatoblast [tfec loss of function phenotype thus provides further support for the existence of a Cbl, transiently localised within the dorsal trunk of embryos between 18 hpf and 24 hpf  is unaffected. This striking phenotype, with effects restricted to pigment cell specification, is unique amongst the characterised zebrafish pigment mutants and would be consistent with the hypothesis that the cell type defined by recursor , which watoblast . The tfe 24 hpf ; Fig 9)..tfec exptfec is expressed in a more widely multipotent progenitor (Cbl(early), its function is only required for downstream specification of chromatophores. Such a hypothesis is further supported both by ours and others´ data on embryonic tfec expression, which is detected across the entire premigratory domain of multipotent NCCs [tfec is the first zebrafish gene identified that specifically affects all chromatophore lineages without appearing to affect any non-pigment NC derivatives. The key NC transcriptional regulator, sox10, is required for each of the three pigment cell lineages, but loss of sox10 function additionally results in strong reductions or absence of all peripheral glia cells and NC-derived peripheral neurons, suggesting that neuronal and pigment lineages share a common, non-ectomesenchymal progenitor [sox10 manifests itself apparently in different ways; sox10 is required for mitfa expression in late Cbls, and mitfa can promote melanocyte fate in the absence of sox10 if misexpressed [sox10 mutants still strongly express tfec (as well as ltk) within trapped premigratory progenitors, yet fail to produce Ib(sp) [tfec mutants the ability to express the lineage markers of melanocytes and xanthophores is delayed but otherwise unaffected, whereas ltk expression is completely missing, even in premigratory cells, also contrasting the phenotype of sox10 mutants [In a recent single cell lineage tracing study, Singh et al., identified progenitor cells proposed to generate all 3 pigment cell types throughout embryogenesis and into adulthood. However, it was shown that the vast majority of these were also able to give rise to neuronal and glial lineages . These rent NCCs , 45. Basogenitor . Furtherxpressed . In conte Ib(sp) . We show mutants . Thus, Ttfec beyond the iridophore lineage by examining its regulation in early NCCs [tfec expression in the early NC is not affected by loss of tfap2a, foxd3 nor sox10 alone. Notably, loss of sox9b alone caused an apparent delay in induction of tfec expression in posterior NC yet, despite this alteration in the pattern, expression remained strongly present in the progenitor population.Another aim of the work presented here was to extend the GRN surrounding rly NCCs , 39. Initfec expression in double sox10;sox9b mutant embryos is still strongly activated in the NC. It remains to be shown whether loss of function of a single or of both sox10 alleles modifies the degree of tfec expression delay noted in homozygous sox9b mutants.Sox10 and Sox9b have previously been reported to act redundantly during zebrafish development , 22. Ourtfap2a;foxd3 have been shown to eliminate induction of NC [foxd3 and tfap2a are redundantly required for induction of tfec expression in early premigratory NCCs  precursor, the fate of which is influenced by this foxd3/mitfa interaction [mitfa represses tfec expression in melanoblasts, and that tfec mutants have increased melanocytes, indicate that maintenance of tfec activity is also key to the melanocyte versus iridophore cell fate decision. Notably, Mitfa and Tfec have the potential to physically interact as a heterodimer [tfec and ltk mutant iridophore phenotypes, ltk mutants do not show an analogous late increase in melanocytes [Although ectively , 29, 55,ntiation , foxd3 hcription , 56, at cription . In the eraction , 17. Ourerodimer , 58, whianocytes .mitfa [tfec delays xanthoblast migration, raising the question of whether interplay between Mitfa and Tfec might be important for xanthoblast fate choice. Furthermore, it will be of interest to examine potential interactions between not only Tfec and Mitfa, but also between MiT and Pax transcription factors during chromatoblast diversification.Specification and differentiation of the third pigment cell type of zebrafish, the xanthophore, has been shown to depend upon the paired-box transcription factors Pax3 and Pax7a/b , 60. Intmitfa  and we sTo conclude, our study contributes to deepening our understanding of the molecular basis of NC and pigment cell development in zebrafish, providing additional support for the progressive fate restriction of multipotent stem cells. While we show Tfec plays a key role in the GRNs underpinning NC maintenance and chromatophore specification, detailed assessment of the diversity of pigment progenitor states during embryonic stages is needed to test the hypothesis of a tripotent chromatoblast. Furthermore, focused effort on the (redundant) roles of transcription factors in xanthophore development will be decisive in understanding pigment cell fate choice in zebrafish.This study was performed with the approval of the University of Bath ethics committee and in full accordance with the Animals (Scientific Procedures) Act 1986, under Home Office Project Licenses 30/2937 and P87C67227, and in compliance with protocol AM10125 approved by the Institutional Animal Care and Use Committee of Virginia Commonwealth University.sox10t3 [foxd3zdf10 [sox9bfh313 [mitfaw2 [ltkty82 [tfap2ats213 [All embryos were obtained from natural crosses. Staging was performed according to Kimmel et al (1995) . WIK or sox10t3 , foxd3zdxd3zdf10 , sox9bfhx9bfh313 , 65, mit[mitfaw2 , ltkty82[ltkty82 , tfap2atp2ats213 . Adult fhttp://chopchop.cbu.uib.no/; [Target sequences for CRISPR/Cas9 mutagenesis were identified using version 1 of the CHOPCHOP webtool . The teuib.no/; . Primer tfecba6 and assorted tfecvc alleles were generated independently as follows:The Cas9 mRNA per embryo were injected at the flat cell stage. RNAs were diluted in RNase-free water. Approximately 200 injected embryos were raised, from which 30–40 pairs were in-crossed to screen for germline transmission. Adults that transmitted mutant alleles were separated and outcrossed wild-type fish of the WIK line to generate F1 offspring. F1 siblings  were in-crossed to identify mutation carriers. 3 fish were identified, which were outcrossed again to WIK fish and the resulting F2 generation raised. For all experiments the F2 of one of the three identified F1 fish were used. Screening was based on iridophore phenotype and confirmed by a High Resolution Melt Analysis (HRMA) assay for molecular screening (see below). To characterise the mutations, F1 adult genomic DNA extracted by swabbing and genomic DNA extracted from F2 embryos were sent for sequencing.280 pg of CRISPR guide RNA mixed with 700 pg Cas9 mRNA was generated from the plasmid pT3TS-nCas9n [Cas9 mRNA and tfec exon 7 sgRNA were each diluted to 100 ng/μl for microinjection into one-cell embryos of the NHGRI-1 strain [StuI, which cuts in the target sequence. The remaining injected embryos were raised to adulthood and intercrossed or mated to wild-type (NHGRI-1) partners. F1 carriers were identified using the PCR digest assay above, and undigested PCR products  were purified and sequenced.CRISPR guide RNA was synthesized using the MEGAshortscript T7 Transcription Kit  and purified using the miRvana miRNA Isolation Kit  as described by . Capped S-nCas9n , a gift 1 strain . A fract1 strain  to evaluHigh resolution melt analysis. High Resolution Melting (HRM) Software V3.0.1 (Thermo Fisher Scientific) was used to detect and amplify differences between the melting temperature of 150–200 bp q-RT PCR amplicons generated from reference wild-type (WT) samples versus mutagenized embryos or adults. To perform q-RT PCR for HRMA, template genomic DNA was extracted using the KAPA Express Extract Kit  according to manufacturer’s instructions, and was diluted to 8 ng/μl. Amplification reactions were set up as per manufacturer’s instructions using KAPA HRM FAST reagents  and primers designed according to the relevant recommendations  at 25 ng/μl was co-injected with Tol2 transposase mRNA at 25 ng/μl into embryos from an intercross of heterozygous For overexpression experiments, the following additional plasmids were generated using multisite Gateway cloning and the entry vectors pENTR5’_ubi , p5E-soxin situ hybridisation as well as multiplex fluorescent RNAscope can be found in Petratou et al. (2017) [in situ hybridisation, the probes used were sox10 [foxd3 [ltk [pnp4a [aox5 [gch2 [pou3f1/oct6 [dlx2a [mitfa [tfec , tfec .Detailed information on the preparation of generic materials and the protocols for performing chromogenic whole-mount . 2017) . For who17 . For 0 [foxd3 , ltk [114a [aox5 , gch2 [6x5 [gch2 , pou3f1/3f1/oct6 , dlx2a [6 [dlx2a , mitfa [a [mitfa  and tfectfap2a and foxd3 mutant embryos were identified using previously described genotyping protocols [Embryos were observed and imaged, and the Pearson’s chi-squared test was used, as previously described , to procrotocols , 79 follrotocols .tfecvc60 heterozygous adults were sorted at 4 days post-fertilization based on the iridophore phenotype, and then were fixed in separate tubes overnight in 4% PFA. Alcian blue staining was then carried out essentially as described [Larvae from an intercross of escribed . Samplestfec whole-mount in situ hybridisation /Pax7 IHC, the Pax7 antibodies were added simultaneously with the anti-Fab fragments [Immunohistochemistry was carried out as previously described . Primaryragments . Samplestfecvc60 heterozygous adults were treated with tyrphostin/AG1478 (LC Laboratories) at 2 μM or DMSO from 8 hpf to 48 hpf, with or without the addition of MoTP (Enzo Life Sciences) at 50 μM from 24 to 48 hpf, and examined at 4.5 dpf for recovery of melanocytes.Larvae from an intercross of S1 Figtfec crispant (mosaic) adult shows patches of iridophore loss on eye and flank (B). When two tfec CRISPR G0 founder fish were mated, almost all of the offspring lacking iridophores died as larvae, but one escaper survived to adulthood and the absence of iridophores persisted (C). Of the homozygous tfecvc60 embryos injected with a Tol2 transposon containing the tfec promoter and cDNA and Tol2 transposase mRNA, one survived to adulthood and displayed partial rescue of adult iridophore pigmentation (D). Scale bar: A-D: 0.5 cm; D: 0.35 cm.Compared to WT adult (A), G0 (TIF)Click here for additional data file.S2 Figtfec homozygous mutant larvae treated with AG1478 (B) recover melanocytes to levels of DMSO-treated larvae (A) by 4.5 dpf.  Wild-type larvae, treated with MoTP reagent at an early stage to ablate melanocytes, and with AG1478 (D) show strongly reduced melanocyte numbers by 4.5 dpf, compared to MoTP and DMSO-treated siblings (C). Scale bar: 250 μm. (TIF)Click here for additional data file.S3 Figtfecba6/ba6 embryos (red bars) and of wild-type or heterozygous siblings (green bars). *: p-value < 10−3.Positive cells were scored along the dorsal and the ventral stripe of known (TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S1 TableFor melanocyte counts at 4 dpf, to derive the average along the lateral stripe, the cells along both stripes of each embryo were independently scored. Presented p-values derived from unpaired two-tailed t-test between WT (or heterozygous for the melanocyte counts) and the genotype corresponding to each row. DS, dorsal stripe; DT, dorsal trunk; H, head; LS, lateral stripe; MP, migration paths; VS, ventral stripe; VT, ventral trunk.(DOCX)Click here for additional data file.S2 Tablest sub-column of each of the 4 developmental stages) within a batch of WT, heterozygous and homozygous mutant siblings, correlating with homozygosity of the mutant allele in those individuals. Due to the recessive nature of investigated alleles, 25% of embryos in each batch are expected to be homozygous mutants. Therefore, the p-value derived from the chi-squared test indicates whether the number of individuals with an alternative phenotype conform to the expected 25% (null hypothesis), with any deviation being attributable to random chance (p > 0.1), or whether the numbers deviate significantly from the expected . Where observed phenotypes did not significantly correlate with expected Mendelian ratios, i.e. where there is unlikely to be a mutant phenotype, the corresponding counts and p-values are red and bold. For the data on sox10t3/t3; sox9bfh313/fh313 double mutants, the orange cells indicate how many embryos in the sample showed the known sox9bfh313/fh313 phenotype, the green cells indicate number of embryos with the sox10t3/t3 phenotype and blue cells indicate unexpected alternative phenotypes, likely owed to double loss of function.The Pearson’s chi-squared test for goodness of fit is used to calculate the likelihood of a non-WT phenotype, which is consistently present in a number of embryos (1(DOCX)Click here for additional data file.


However, accessibility of public data is limited due to the lack of user-friendly utilities and up-to-date resources covering experiments from third parties. Here, we describe iCSDB, an integrated database of CRISPR screening experiments using human cell lines. We compiled two major sources of CRISPR-Cas9 screening: the DepMap portal and BioGRID ORCS. DepMap portal itself is an integrated database that includes three large-scale projects of CRISPR screening. We additionally aggregated CRISPR screens from BioGRID ORCS that is a collection of screening results from PubMed articles. Currently, iCSDB contains 1375 genome-wide screens across 976 human cell lines, covering 28 tissues and 70 cancer types. Importantly, the batch effects from different CRISPR libraries were removed and the screening scores were converted into a single metric to estimate the knockout efficiency. Clinical and molecular information were also integrated to help users to select cell lines of interest readily. Furthermore, we have implemented various interactive tools and viewers to facilitate users to choose, examine and compare the screen results both at the gene and guide RNA levels. iCSDB is available at  High-throughput screening (HTS) has become an indispensable tool for functional genomics and drug discovery. Genetic loss-of-function (LoF) experiments are frequently used in HTS to identify targets that confer genetic vulnerability in various diseases. RNA interference (RNAi) screening using small-interfering RNA (siRNA) libraries was the method of choice for LoF experiments during the last decade . For exaCRISPR/Cas9-mediated genome editing that generates the double-strand breaks in the target DNA is rapidly replacing the RNAi method for studying LoF consequences of target genes mainly due to high efficiency (knockout rather than knockdown) and high specificity (i.e. low off-target effect by the usage of relatively long sequences in guide RNA design) . Genome-https://depmap.org/portal/) is the largest and latest collection of CRISPR screens integrating three large-scale projects . It als screens . Such coThe overall contents of iCSDB consist of a collection of genome-wide CRISPR screens, molecular characteristics of cell lines and analytical tools Figure . MoleculWe have compiled two major sources of CRISPR-Cas9 screens: DepMap portal (Public 20Q2) and BioGRID ORCS (Ver. 1.0.4). DepMap portal itself is an integrated database of three large-scale projects , including 1129 screens across 921 cell lines Figure  and B. Bet al.  to choose the relevant cell lines and examine the screen result readily.To demonstrate the utility of iCSDB, we compared the screen results for cell lines with KRAS mutations. KRAS is the most commonly mutated oncogene in human cancer including pancreatic, colon, lung and brain tumors. Numerous attempts to develop drugs targeting KRAS mutants directly were unsuccessful and targeting synthetic lethal vulnerabilities have been widely exploited. Several genome-scale LoF screens have been performed with siRNA, shRNA and CRISPR libraries .WEE1, MYC, CHEK1, CDC7, CDC5L and AURKB)  Figure . Gene se) Figure  showed s) Figure   Figure . DNA rep) Figure . Of noteiCSDB is a meta-database to include human CRISPR screen data from the DepMap and BioGRID ORCS resources. As demonstrated in the case study, it provides an opportunity to identify drug targets to overcome resistance in cancer. Combining the screen data with the drug sensitivity data would open a systematic way for finding the mechanism-of-action of drugs  or for igkaa989_Supplemental_FilesClick here for additional data file.


Background: Uterine sarcomas and carcinosarcoma are associated with unfavorable prognosis. The regimens that are used in chemotherapy are associated with high incidence of side effects and usually do not significantly increase patients’ survival rates. In this study we investigated the activity and interactions between gemcitabine and fucoidan, the natural compound known for its anti-tumor properties, in human sarcomas and carcinosarcoma cell models. Methods: SK-UT-1, SK-UT1-B (carcinosarcoma), MES-SA (leiomyosarcoma), and ESS-1  cell lines were used for the experiments. Cells were incubated in the presence of gemcitabine, fucoidan, and mixtures, after the incubation the MTT tests were performed. In order to assess the interactions between tested compounds isobolographic analysis was performed. Additional assessments of apoptosis and cell cycle were done. Results: Additive effect of combined treatment with gemcitabine and fucoidan was observed in ESS-1 and SK-UT-1 cell line. Although the supra-additive (synergistic) effect noticed in SK-UT-1B cell line. It was not possible to determine the interactions of fucoidan and gemcitabine in MES-SA cell line due to insufficient response to treatment. Addition of fucoidan to gemcitabine enhances its proapoptotic activity, what was observed especially in ESS-1 and SK-UT-1B cell lines. The arrest of cell cycle induced by mixture of gemcitabine and fucoidan, superior comparing gemcitabine alone was observed in SK-UT-1B. Conclusions: Obtained data showed that a combination of fucoidan and gemcitabine in uterine endometrial stromal sarcoma and carcinosarcoma cell lines has additive or even synergistic effect in decreasing cell viability. Furthermore, this drug combination induces apoptosis and arrest of cell cycle. The resistance of uterine leiomyosarcoma cell line, justifies searching for other drugs combinations to improve therapy efficacy. Uterine sarcomas are a group of malignancies consist of various types of tumors arising from mesenchymal tissue. The most common type of uterine sarcomas is leiomyosarcoma (about 60% of cases), less common types are endometrial stromal sarcoma (low and high grade), liposarcoma, rhabdomyosarcoma, and many other rare types . The incThe exceptions from the above characteristics are endometrial stromal sarcomas, associated with indolent clinical behavior and favorable prognosis. The majority of these tumors express estrogen receptors and usually respond to hormonal treatment . The retrospective trial comparing gemcitabine plus docetaxel, ifosfamide plus cisplatin, doxorubicin plus ifosfamide, ifosfamide alone, topotecan alone, and observation only in stage I and II leiomyosarcoma revealed no significant differences between groups in recurrence rate . Due to The results obtained in systemic therapy of sarcomas are unsatisfactory. Most authors strongly recommend conducting clinical and pre-clinical trials in this field. The situation described above led to a search for new agents to be active against uterine sarcomas. Fucoidan is sulphated polysaccharide derived from brown seaweed, that recently gained attention due to its biological activities. It is known to affect multiple pathways in cancer cells including PI3K/AKT, MAPK, PTEN, VEGF, and caspases. Its effectiveness was proven in various models including lymphoma, leukemia, prostate cancer, breast cancer, hepatic cancer . RecentlObtained results led us to question the activity of fucoidan in combination with standard chemotherapy among uterine sarcomas. Gemcitabine was selected to be investigated together with fucoidan. Gemcitabine is commonly used anti-tumor drug belonging to the group of antimetabolites. It express antimetabolic effect by interruption of DNA synthesis. It is widely used as an option of standard approach for the systemic therapy in most of uterine sarcomas subtypes ,11,12. FThis study was aimed to test if the combination of gemcitabine and fucoidan will have better therapeutic effect in uterine sarcomas and carcinosarcomas cell lines than the compounds applied alone and to assess the type of interactions between them in concomitant treatment. Undaria pinnatifida) was obtained from Sigma-Aldrich . Fucoidan was diluted in respective complete culture medium at a concentration of 10 mg/mL just before use.Gemcitabine  was purchased from Accord Healthcare (UK), and fucoidan , Eagle’s Minimum Essential Medium (MEM), McCoy’s 5a Medium Modified, fetal bovine serum (FBS), trypsin-EDTA were purchased from PAN-Biotech , penicillin-streptomycin and MTT -2,5-diphenyltetrazolium bromide) were obtained from Sigma-Aldrich . The Cell Proliferation ELISA System assay kit was purchased from Roche . PE Active Caspase-3 Apoptosis Kit and Propidium iodide utilizing the PI/RNase Staining Buffer were obtained from Becton Dickinson Biosciences . Carcinosarcoma cell lines , uterine leiomyosarcoma cell line (MES-SA), and endometrial stromal sarcoma cell line (ESS-1) were obtained from the Laboratorio de Investigación Traslacional . The selection of cell lines was done in order to assess the differences in response to tested compounds among wide range of uterine sarcomas. Each cell line used for experiments is a model of particular tumor what makes obtained data more reproducible and comparable with other research. 2 atmosphere. Cells from the 4th to 9th passage were used for all experiments.The cells were cultured in MEM , McCoy’s 5a Medium Modified (MES-SA), RPMI-1640 (ESS-1), containing 10%  or 20% (ESS-1) FBS and 1% penicillin-streptomycin at 37 °C in a humidified 5% CODetailed characteristic of cell lines is presented in 4 cells/mL) and cells were incubated in the presence of gemcitabine (0.1–200 ng/mL), fucoidan (0.01–5 mg/mL) and mixtures of both compounds for 96 h. The maximum concentration of gemcitabine achieved in human serum was 26.79 ± 10.06 μg/mL, reported by Wang et al. so the concentrations used in our experiments were much lower comparing to ones available in vivo [Cells were platted on 96-well microplates SK-UT-1, SK-UT-1B, MES-SA, and ESS-1 . Labeled cells were analyzed by flow cytometer FACSCalibur , operating with CellQuest software  to quantitatively assess the caspase-3 activity. The methodology of apoptosis assessment were described in detail elsewhere .Cell lines were treated with tested compound and mixture for 48 h (as a control we used cells without exposure to tested compounds) and then fixed in 80% ethanol at −20 °C for 24 h. The experiment was conducted by utilizing PI/RNase Staining Buffer  according to the manufacturer’s instructions. Cell cycle analysis was performed using flow cytometry . Acquisition rate was at least 60 events/sec in low acquisition mode and at least 10,000 events were measured. Methodology of cell cycle analysis was presented in detail previously [50) for gemcitabine and fucoidan in ESS-1, SK-UT-1, and SK-UT-1B cell lines were calculated according to method previously described [50 was not achievable for fucoidan in MES-SA cell line [50 add) for two-drug mixtures were theoretically calculated according to the method described elsewhere [50 mix values for tested drug combinations in a fixed 1:1 ratio. The particular drug concentrations (gemcitabine and fucoidan) in the mixture were calculated by multiplying IC50 mix values accordingly to proportions in additive mixture. Detailed description of isobolographic method was introduced by Tallarida, Grabovsky and Luszczki [p < 0.05 was considered as statistically significant. In order to determine the inhibition rate of cell viability, measured by MTT assay, per dose of gemcitabine and fucoidan, log-probit linear regression analysis was performed according to method described by Litchfield and Wilcoxon ,18. Mediescribed . Due to ell line . Paralleell line . The medlsewhere . The calLuszczki ,21,22. T50 values for gemcitabine in SK-UT-1, SK-UT-1B, ESS-1, and MES-SA cell lines, were 31.173, 25.243, 13.875, and 72.482 ng/mL respectively. Anti-proliferative effects of gemcitabine on tested cell lines is presented on 50 was 0.966, 3.348, and 0.848 mg/mL respectively, it was not possible to determine IC50 for fucoidan in MES-SA cell line due to insufficient response to treatment [50 values are summarized in As we previously reported fucoidan significantly affects SK-UT-1, SK-UT-1B, and ESS-1 cell lines, meanwhile MES-SA cells seem to be resistant for this agent. ICAdditive effect of the combined treatment with gemcitabine and fucoidan was observed in ESS-1 and SK-UT-1 cell lines. Although the supra-additive (synergistic) effect was noticed in SK-UT-1B cell line. The details of results obtained in isobolographic analysis are presented on 50) for gemcitabine (GEM) and fucoidan (FUK) are plotted on the X- and Y-axes, respectively. The solid lines on both axes reflect the S.E.M. for the IC50 values for the studied drugs, when administered alone. The lower and upper isoboles of additivity represent the curves connecting the IC50 values for GEM and FUK administered alone. The dotted line illustrates the fixed-ratio of 1:1 for the combination of GEM with FUK. The points A’ and A” depict the theoretically calculated IC50 add values for both, lower and upper isoboles of additivity. The point M reflects the experimentally-derived IC50 mix value for total dose of the mixture expressed as proportions of GEM and FUK that produced a 50% anti-proliferative effect (50% isobole) in the cancer cell line  was detected in the SK-UT-1 B cell line (comparing to both control and gemcitabine treatment). In the ESS-1 cell line significant differences were noted between cells treated with gemcitabine and control, with no such differences between gemcitabine and mixture. The differences in number of cells in each phase of cell cycle in SK-UT-1 cells exposed to investigated agents were very small in spite of statistical significance observed in particular phases. This paper for our knowledge is the first report of interactions between fucoidan and gemcitabine in any model. The results obtained in our experiments show that both gemcitabine and fucoidan significantly affect cell viability in all tested cell lines (with exception of fucoidan in MES-SA cell line). The problem of MES-SA cell line resistance to fucoidan have been discussed previously . In presIn multidrug regimens, in assumption, lower doses of multiple drugs may cause fewer side effects (or its lower intensity) without decreasing effectiveness or even increasing it . But in The rarity and heterogeneity of uterine sarcomas limit the possibilities of conducting clinical trials, which may be considered as a cause of the poor results of its treatment up to date. Even in preclinical studies number of research aimed to test new drugs combinations in this group of tumors is limited. Coley et al. tested the combination of seliciclib (cyclin-dependent kinases (CDK)-inhibitor) and paclitaxel among selection of uterine sarcomas cell lines. Using isobolography, they observed synergism between both drugs. Interestingly, differences in activity depending on the sequence of each drug administration were reported. Although the study is interesting, seliciclib is not registered for the treatment of uterine sarcomas. Furthermore, paclitaxel in monotherapy is not recommended among this type of malignancies .In the present study we propose a combination of standard chemotherapy with a natural compound that is known to be safe; its intake is not associated with serious adverse events . Both suCell viability was decreased equally or more than the sum of effects of single agents. If these observations are confirmed in animal models, clinical trials of fucoidan as an addition to treatment with gemcitabine may allow a decrease of its dose. Fucoidan is nowadays used as dietary supplement, and seaweeds containing it are widely used in Asian cuisine . There aQuite similar results were obtained by Mathew et al. who assessed IC50 for various types of fucoidan from 0.3 to 1.3 mg/mL (depending on plant that it was derived from). In this study they also performed analysis of fucoidans’ impact on CYP450 and COMT (catechol-O-methyltransferase) pathways, concluding that it is limited, which might be considered as a confirmation of its safety .Our findings are comparable with other studies assessing activity of fucoidan and cytotoxic drugs. Burney et al. assessed activity of 2 types of fucoidan in combination with paclitaxel and tamoxifen on mouse models of human breast or ovarian cancer. They concluded that interaction between tested compounds were additive or supra-additive (depending on combination and model used) .Similar observations were taken by Zhang et al. who tested combinations of fucoidan with cisplatin, tamoxifen, and paclitaxel in breast cancer cell lines. They concluded that fucoidan “enhances the anti-cancer activity” of tested cytostatic drugs. Furthermore, they observed the induction of apoptosis and cell cycle arrest among cells treated with fucoidan what corresponds with our previous findings ,33.Observed additive and supra-additive interactions between various fucoidans and chemotherapeutic drugs require explanation. The mechanism of fucoidans’ activity is not fully investigated. As it was mentioned above there are various cellular pathways that are affected by this compound. Its multipotential activity is, on the one hand, promising, and does not allow the overcoming of single pathway change, but on the other it is very difficult to understand completely. One of possible mechanism of synergistic interaction with chemotherapeutics is the ability to down-regulate expression of Bcl-xL and Mcl-1, known as anti-apoptotic proteins . The addGemcitabine is known to affect the cell cycle in various cell models and in vivo; this feature is the consequence of its mechanism of action by DNA damaging . The proPark et al. reported the effect of cell cycle arrest by fucoidan treatment in bladder cancer cells. They also revealed the its association with down-regulation of cyclin D1, cyclin E, and cyclin-dependent-kinases (Cdks) in a concentration-dependent manner, without any change in Cdk inhibitors  . InteresThe results and survey of literature indicate that testing multicompound regimens gives promising results and further research in the field could reveal a combination that could be beneficial for patients suffering from uterine sarcomas.The study is not without limitations and should be considered carefully. The main limitation of the study is a cancer model that was used, 2D cell cultures, which do not allow investigation of the tumor microenvironment and may not fully reflect the response of tumor cells in vivo. Although promising, obtained results require clinical studies to be considered in practice.Analysis of the mechanisms of the MES-SA cell line relative resistance to fucoidan and gemcitabine that may lead to identification of such mechanisms in tumors and make progress for overcoming them.Due to similarities in clinical course between carcinosarcomas and endometrial carcinomas the results obtained on SK-UT-1 and SK-UT-1B cell lines might be replicated on carcinomas cell lines and extend indications for future practical applications.Studies on 3D cell cultures or animal models have to be performed in order to confirm the activity of proposed combination on tissue models and to assess how the tumor microenvironment affects it.The results obtained in our study indicate 3 potential directions for further investigations:Obtained data showed additive and supra-additive effect of fucoidan combined with gemcitabine in uterine endometrial stromal sarcoma (ESS-1) and carcinosarcoma  cell lines, what confirms it has better or at least equal performance comparing to sum of effects of monotherapies. The addition of fucoidan to gemcitabine enhances proapoptotic effect of gemcitabine in endometrial stromal sarcoma cells (ESS-1) but not in carcinosarcoma  cell lines.Gemcitabine in monotherapy do not induce cell cycle arrest in carcinosarcoma  cell lines. Although the addition of fucoidan to gemcitabine induces it in model of carcinomatous part of carcinosarcoma (SK-UT-1B).Differences in response to applied treatment among tested cell lines (in cell viability apoptosis and cell cycle distribution) can be explained by the multipotential and not-fully-investigated activity of fucoidan as well as differences in cellular mechanisms (such as DNA repairing) in selected models.The relative resistance of uterine leiomyosarcoma cell line (MES-SA) to applied drugs combination justify searching for other therapeutic regimens to improve therapy efficacy.Taking into consideration the disappointingly low effectiveness of systemic therapy among these types of cancer, a combination of gemcitabine and fucoidan seems to be a promising alternative, having the potential to increase effectiveness and safety of the treatment.


Burkholderia gladioli BCC0238 and BCC1622, both isolated from the lungs of cystic fibrosis patients. Bioinfomatics analyses indicated the PKS assembles a novel member of the glutarimide class of antibiotics, hitherto only isolated from Streptomyces species. Screening of a range of growth parameters led to the identification of gladiostatin, the metabolic product of the PKS. NMR spectroscopic analysis revealed that gladiostatin, which has promising activity against several human cancer cell lines and inhibits tumor cell migration, contains an unusual 2‐acyl‐4‐hydroxy‐3‐methylbutenolide in addition to the glutarimide pharmacophore. An AfsA‐like domain at the C‐terminus of the PKS was shown to catalyze condensation of 3‐ketothioesters with dihydroxyacetone phosphate, thus indicating it plays a key role in polyketide chain release and butenolide formation.A gene cluster encoding a cryptic trans‐acyl transferase polyketide synthase (PKS) was identified in the genomes of  Burkholderia gladioli. Gladiostatin contains an unusual 2‐acyl‐3‐hydroxy‐4‐methylbutenolide and exhibits promising activity against human cancer cells. The polyketide synthase responsible for its biosynthesis employs a novel mechanism for chain release, resulting in formation of a phosphorylated butenolide intermediate.Genome mining enabled the discovery of gladiostatin, a novel glutarimide antibiotic from the cystic fibrosis associated bacterium  Burkholderia genus produce a wide array of bioactive specialised metabolites, including the respiratory toxin bongkrekic acid, anti‐proliferative agents such as thailanstatin and spliceostatin, and the antibiotics enacyloxin IIa and gladiolin.[Burkholderia gladioli BCC0238, isolated from the lung of a cystic fibrosis (CF) patient, produces a range of specialised metabolites, dependent on the carbon source. When glycerol is used as the carbon source, this strain produces gladiolin, a novel macrolide with promising activity against Mycobacterium tuberculosis, and the swarming inhibitor icosalide A1, which was originally isolated from a filamentous fungus, but has subsequently been shown to originate from a Burkholderia symbiont.[The constant competition between microbes and their environment has driven the evolution of specialised metabolite production in bacteria, enabling rapid ecological adaptation.ladiolin.5, 6 DB. gladioli BCC0238, analysis of the complete genome sequence of this strain indicated that it contains numerous cryptic BGCs, the metabolic products of which are currently unknown.B. gladioli BCC0238. This unusual metabolite, which contains a rare 2‐acyl‐4‐hydroxy‐3‐methylbutenolide in addition to the 2, 6‐piperidinedione common to all glutarimides, is active against yeast and has promising activity against several human cancer cell lines. Gene disruption experiments confirmed that the BGC directs the biosynthesis of gladiostatin, and chain release from the PKS was reconstituted in vitro, providing insights into the mechanism for formation of the unusual butenolide moiety. These experiments enabled us to propose a biosynthetic pathway for gladiostatin, the first glutarimide to be isolated from Gram‐negative bacteria, illuminating the role played by horizontal gene transfer in trans‐AT PKS evolution.Although several specialised metabolites and their associated biosynthetic gene clusters (BGCs) have already been identified in B. gladioli BCC0238, in silico analysis of the genome sequence revealed a ≈50 kb cryptic BGC encoding a trans‐AT PKS on the second chromosomal replicon . Bioinformatics analyses indicated that several of the proteins encoded by this BGC are very similar to enzymes known to be involved in the biosynthesis of the glutarimide class of polyketide antibiotics in Streptomyces species ,[2),[3)[4; Figure Building on our previous work in s Figure  and b, iigure 1),12, 13smdFGH, chxBCD, ltmHCD and mgsHCD in the 9‐methylstreptimidone, cycloheximide, lactimidomycin and migrastatin BGCs, respectively) are proposed to be involved in the creation of a common malonamyl thioester starter unit for the PKSs that assembles these metabolites  is appended to the C‐terminus of the final PKS module , typified by the methylenomycin furans, and 2‐alkyl‐4‐hydroxy‐3‐methylbutenolides (AHMBs), exemplified by the Streptomyces rochei butenolides and the Streptomyces ansachromogenes butenolides  domain appended to the C‐terminus of the last module, which catalyses release of the fully assembled polyketide chain via hydrolysis or macrocyclisation, but several other chain release mechanisms are known.35, 36e Figure . AccordiB. gladioli BCC0238. However, due to difficulties with creating in‐frame deletions in this strain, we searched the genomes of other B. gladioli strains to see if they contain this BGC. Another CF isolate, B. gladioli BCC1622, which is more amenable to genetic manipulation,B. gladioli BCC1622, grown for 3 days on a minimal agar medium containing glycerol as the sole carbon source, identified a metabolite with the molecular formula C23H37NO5 .We originally identified the BGC encoding the cryptic trans‐AT PKS in 5) was elucidated using 1H, 13C, COSY, HSQC and HMBC NMR experiments  was elucidated using 1H, 13C, COSY, HSQC and HMBC NMR experiments , two fully substituted alkene carbons (C‐20 and C‐2′′), a methyl group (C‐3′′) and a hemiacetal (C‐1′′). HMBC correlations between the C‐3′′ protons and C‐1′′/C‐20 and the C‐1′′ proton and C‐2′′/C‐21 led us to propose that this molecule contains a 2‐subsituted 4‐hydroxy‐3‐methylbutenolide. The NMR data for this moiety are similar to those reported for the AHMBs isolated from S. rochei and S. ansachromogenes  is likely degraded to 5 via conjugate addition of water to the butenolide, followed by ring opening, decarboxylation and retro‐Aldol cleavage .Comparison of the molecular formulae for compound s Figure .[30, 6) against representative members of the ESKAPE panel of bacterial pathogens, Candida albicans and Saccharomyces cerevisiae. While no activity was detected against the bacterial pathogens or C. albicans at concentrations up to 64 μg mL−1, gladiostatin was found to be active against S. cerevisiae, with an MIC of 4 μg mL−1  against a range of cancer cell lines (Table 1), migrastatin (3) and lactimidomycin (4) against various other cell lines (Table S6).[6) was found to be inactive against the A549 lung cancer cell line, indicating it may exhibit some selectivity.Prompted by reports that several glutarimides have anti‐tumour activity,23, 246) can inhibit the migration of A2780 ovarian cancer cells. Strong suppression of cell migration was observed after 24 h exposure to 240 nM gladiostatin , gdsE, which encodes the first polyketide synthase subunit, was disrupted in B. gladioli BCC1622 by insertional mutagenesis. LC‐MS comparison of extracts from the wild type and mutant strains confirmed that gladiostatin (6) production is abolished in the mutant  biosynthesis appear to parallel the assembly of glutarimides in Streptomyces species. GdsB and GdsD are proposed to create a GdsC‐bound malonamyl thioester starter unit, which is elaborated to a 2‐acetyl thioester intermediate by the first PKS module  domain in both modules 3 and 4 of the PKSs, installing methyl branches at C‐8 and C‐10 of the resulting glutarimides. In contrast, the MT domain in module 4 of the gladiostatin PKS appears to methylate only the β‐keto thioester intermediate attached to the module 3 ACP domain  has a methyl branch at C‐8, but not C‐10. Additional studies are needed to understand how PKSs with seemingly identical architectures are able to produce the distinct C‐methylation patterns observed in gladiostatin (6) and the Streptomyces glutarimides.The early stages of gladiostatin  and AT domains fused to a flavin‐dependent ER domain and gdsH encodes an NAD(P)H‐dependent oxidoreductase. We therefore propose that one, or both, of these reductases catalyse enoyl reduction in modules 5, 6, 7 and 8 of the PKS. A recently reported co‐evolutionary categorisation of trans‐AT PKS ACP domains supports this hypothesis, indicating that the module 6, 7 and 8 ACP domains in the gladiostatin PKS clade with ACP domains in modules with a similar architecture from other assembly lines (i.e. KS‐DH‐KR‐ACP and a trans‐acting ER).While it is unclear which domains are responsible for the keto reduction and dehydration reactions in modules 5 and 6, respectively, in silico analysis of the gladiostatin BGC identified two genes encoding putative reductases that could function as trans‐acting ERs in modules 5–8. The Streptomyces glutarimide PKSs are proposed to use TE domains that catalyse hydrolysis or macrolactonisation. In contrast, the gladiostatin PKS appears to employ a PBS domain to catalyse condensation of the β‐ketothioester attached to the last module of the PKS with DHAP. The resulting phosphorylated butenolide is proposed to undergo rearrangement and dephosphorylation to afford a 2‐acyl‐4‐hydroxy‐3‐methylbutenolide . In addition to the PBS (SabA), a phosphatase (SabP) and a ketoreductase (SabD) are known to be required for SAB biosynthesis, but the mechanism for elaboration of the putative phosphorylated butenolide produced by SabA to the 2‐akyl‐4‐hydroxy‐3‐methylbutenolide remains to be elucidated. The gdsA and gdsG genes in the gladiostatin BGC encode putative phosphatases that could play a similar role to SabP in the biosynthesis of the SABs (Table S3).The most striking difference between the gladiostatin PKS and the other glutarimide assembly lines is the mechanism for polyketide chain release. All of the e Figure . This hye Figure . These i6) biosynthesis, we investigated its catalytic activity using simplified mimics of the fully assembled polyketide chain. A synthetic gene encoding the C‐terminal ACP and PBS domains of GdsF was used to overproduce the ACP‐PBS di‐domain in E. coli as an N‐terminal His8‐fusion, which was purified to homogeneity using immobilised metal‐ion affinity chromatography. The identity of the purified protein was confirmed by ESI‐Q‐TOF‐MS analysis .To validate the proposed role of the PBS domain in gladiostatin . The mass of the protein decreased by 86 Da when DHAP was added , consistent with cleavage of the 3‐ketobutyryl group from the ACP domain.The ability of the PBS domain to offload a 3‐ketothioester from the PKS was examined by loading a 3‐ketobutyryl mimic of the fully assembled polyketide chain onto the apo‐ACP‐PBS di‐domain with the N‐acetylcystetamine (NAC) thioester of 3‐ketooctanoate and DHAP, followed by treatment with shrimp alkaline phosphatase yielded a product, absent from the negative control, that gave rise to ions with m/z=213.11 and m/z=235.09 ) in UHPLC‐ESI‐Q‐TOF‐MS analyses  as the product of a cryptic trans‐AT PKS assembly line in B. gladioli BCC0238 and BCC1622 further expands the already rich specialised metabolic repertoire of these and related CF isolates.[Streptomyces species. While some of these structural differences (such as the fully saturated C‐11 to C‐18 chain and the 2‐acyl‐4‐hydroxy‐3‐methylbutenolide) are reflected by alterations in PKS architecture, others (e.g. the lack of a C‐10 methyl group) are not. The discovery of gladiostatin thus offers a golden opportunity to develop a better understanding of the role played by horizontal gene transfer in trans‐AT PKS evolution, which could facilitate biosynthetic engineering approaches to polyketide structural diversification.The discovery of gladiostatin  in the biosynthesis of the methylenomycin furans.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


C. elegans’ stress resistance, and at least 9 extended their lifespan by ∼10–50%. We further evaluated a chalcone that produced relatively large effects on lifespan and were able to implicate the activity of two, stress-response regulators, NRF2/skn-1 and SESN/sesn-1, in its mechanism of action. Our findings suggest that screening for increased stress resistance in human cells can enrich for compounds with promising pro-longevity effects. Further characterization of these compounds may reveal new ways to extend healthy human lifespan.Increased resistance to environmental stress at the cellular level is correlated with the longevity of long-lived mutants and wild-animal species. Moreover, in experimental organisms, screens for increased stress resistance have yielded mutants that are long-lived. To find entry points for small molecules that might extend healthy longevity in humans, we screened ∼100,000 small molecules in a human primary-fibroblast cell line and identified a set that increased oxidative-stress resistance. Some of the hits fell into structurally related chemical groups, suggesting that they may act on common targets. Two small molecules increased  In animals, mutations in nutrient, energy and stress-sensing genes, such as IGF-1-axis and mTOR-signaling genes, extend youthfulness and lifespan and counter age-related disease . These gFOXO3A polymorphisms have been identified in multiple cohorts  selected to maximize the coverage of chemical space. Compounds were provided as 10 mM stocks by the Small Molecule Discovery Center (SMDC), and chemical information, including structures, simplified molecular-input line-entry system (SMILES) IDs, PubChem links and possible 2 incubator at 37° for 24 hr. Small molecules were introduced from stock plates with a 384-well formatted set of 50 nL stainless steel pin-tool (V&P Scientific) on a Biomek FXP (Beckman) automation workstation to 10 μM final concentration (in 0.1% DMSO), a typical single-concentration assay dose shown to produce high structural diversity in many small-molecule screens , measuring luminescence signals on an Analyst HT (Molecule Devices) plate reader.For the screen, we used human lung-derived WI-38 cells at mid population doubling levels . Briefly, WI-38 cells were cultured in OptiMEM (Life Technologies) supplemented with 10% fetal bovine serum (FBS). In a 384 multi-well format, 2,000 cells were added in 50 μl medium per well, using a WellMate (Thermo Fisher) liquid dispenser and cultured in a CO screens . The neg2O2 for 3 hr . Hoechst 33342 (Life Technologies)  and propidium iodide (Life Technologies)  were added for the last 30 min. Fluorescent images of cells were collected on the INCell Analyzer 2000 automated microscope  (10X objective) and further analyzed with the Developer ToolBox software .A high-content imaging assay was developed as a secondary approach to validate our screen hits. Briefly, cells were pre-incubated with small molecules for 24 hr and then subjected to H2O2-resistance in a paralleled experiment – see Supplemental Figure 6) from two different vendors , 0.1% DMSO control or no DMSO mock control (n = 6 each) in the absence of H2O2 for 24 hr and then processed for RNA isolation with the RNeasy kit (Qiagen). Total RNA  was used for each sample to prepare Illumina TruSeq Stranded mRNA library, following the manufacturer’s instructions. Multiplexed samples were analyzed on an Illumina HiSeq 4000 to obtain transcript reads. The resulting reads were processed using our in-house analysis pipeline to obtain transcript abundance. Genes that showed significant change of expression  were identified  and subjected to pathway analysis, using tools including PANTHER  and Enrichr (http://amp.pharm.mssm.edu/Enrichr/). The iLINCS database (http://www.ilincs.org/ilincs/signatures/main/upload) was used for comparison analysis to identify perturbations that produced expression profiles similar to those produced by our small molecules.For RNA-seq analysis, WI-38 cells were treated with Gr-4D . Molecules were analyzed at the highest dose . Liquid culture-based lifespan assays were performed, following the protocol of others (fer-15(rrf-3) (b26)II rol-6(su1006)II; fem-1(hc17)IV], were raised on 10-cm agar plates seeded with OP50 bacteria at 25°. Sterile day-1 adults were transferred onto mini-plates, which were seeded with normal OP50 bacteria  and supplemented with small molecules . Worms were scored every other day. Cumulative survival was analyzed using the STATA software (log-rank test).Given the caveat of lifespan-assay variations for f others . Briefly2-resistance assay, the DPPH assay of ROS scavengers, the AmplexRed assay of hydrogen peroxide, the assay of PARP inhibition, the analysis of small molecules’ effects on cell confluency, DNA-damage markers, mTOR activity and poly(Q) toxicity, plus microarray analysis and qPCR analysis. The H2O2-stress assay and analysis of pathway-activity reporters in C. elegans and methods for C. elegans RNAi are also included in Supplemental Text.Additional methods are described in Supplemental Text: the screen flow with technical details, the CdCl2O2. Table S3 shows that certain small molecules also protect cells from heavy metal cadmium. Table S4 indicates the effects of small molecules on DNA damage-associated markers and ATP contents upon prolonged incubation. Table S5 lists the significant genes identified by RNA-seq (for Gr-4D) and microarrays (for other molecules). Table S6 shows the pathway analysis results. Table S7 shows that certain molecules might inhibit mTOR. Table S8 summarizes the effects of small molecules on C. elegans’ lifespan. Table S9 lists potential targets for our small molecules.Table S1 shows the chemical identities of 209 primary hits, plus 61 top hits and 32 core-set hits analyzed. Table S2 shows the cell death-imaging analysis to confirm protective effects of small molecules against HAKT1 or KEAP1 knockdown. Figure S2 shows Z-prime scores across the screen. Figure S3 indicates the analysis to exclude ROS scavengers. Figure S4 shows no obvious quenching of H2O2 by small molecules in vitro. Figure S5 indicates the long-term effects of small molecules on ATP levels and cell confluency. Figure S6 shows increased cell viability upon H2O2 treatment by small molecules in the RNA-seq and microarray experiment, as well as NRF2 non-dependency for Gr-4D. Figure S7 displays the effects of small molecules on gene expression of WI-38 cells. Figure S8 indicates the analysis to identify PARP inhibitors. Figure S9 shows protective effects of certain small molecules against poly-Q toxicity. Figure S10 shows the effects of Gr-4D on C. elegans expressing reporters of genes related to pathways known to influence longevity, as well as the RNAi experiment to address the sesn-1 dependency of the lifespan extension produced by Gr-4D.Figure S1 shows increased oxidative-stress resistance upon https://doi.org/10.25387/g3.11292143.The RNA-seq data for human WI-38 cells treated with the chalcone Gr-4D can be found at the GEO with the accession number GSE141675. Supplemental material available at figshare: Primary skin fibroblasts from long-lived mouse mutants and long-lived animal species exhibit cellular resistance to the stressors hydrogen peroxide, cadmium and methyl methanesulfonate (MMS)  against the insulin/IGF-1 signal-transducing gene AKT1 and the NRF2 inhibitor gene KEAP1 both increased stress resistance under these conditions , suggesting that we could recover the types of perturbations known to increase lifespan in animals. Altogether, we identified 209 small molecules  that produced signals at least 2.5-fold greater than the DMSO-treated negative control . We retested these candidates at six different concentrations (0.6 μM to 20 μM) and confirmed 127 that consistently produced protective effects against H2O2 at one or more of these doses (data not shown).In the screen, we looked for small molecules that increased cellular resistance to hydrogen peroxide . The scr2O2 stress. Predictably, among the molecules that increased ATP levels but produced little or no protective effect in PI-imaging assays were several inhibitors of poly ADP-ribose polymerase (PARP) . PARP consumes ATP to repair DNA damage; thus, its inhibition increases ATP levels without protecting cells from oxidative stress.To exclude molecules that did not increase stress resistance but instead somehow elevated cellular ATP levels, we also carried out a secondary imaging assay for cell viability. Using propidium iodide (PI), a cell non-permeable dye that stains DNA in late-apoptotic/necrotic cells when membrane integrity is lost, we identified 107 compounds that reduced the percentage of dead cells following H2O2 stress , leaving 61 primary hits , a well-known reactive oxygen species (ROS) scavenger that can protect cells from Hin vitro Amplex Red assay, that these molecules did not quench H2O2 , excluding this as a possible explanation for their protective effects. We then confirmed in multiple independent experiments, by measuring ATP content and performing additional PI-imaging, that 32 molecules  reproducibly protected WI-38 cells during H2O2 treatment . Hence these 32 small molecules became our “core set” of hits for further characterization  contained a second LC-MS peak, likely a modified species. We first verified, by performing an 2O2 . Furthermore, 22 validated hits also protected human primary dermal fibroblasts (isolated from the skin of multiple donors) from H2O2 , demonstrating that their protective capacity was not limited to the WI-38 cell line. We also asked whether any molecules could increase resistance of cells to the DNA-damaging agent MMS, but none of our 32 core-set hits scored positively in this assay.A second cellular stress that correlates well with organismal longevity is resistance to cadmium, which also produces ROS . We found that 25 of our 32 core-set small molecules elicited signs of DNA damage; 20 of these also produced cell toxicity (assayed either by effects on ATP content or cell confluency) , and we also tested their ability to induce proliferation arrest or DNA damage-associated markers in the absence of Hfluency) . HoweverElectrophilic “pan assay interference compounds” (PAINS), due to their reactive nature, can produce non-specific effects in a chemical screen   consistently in cells treated with Gr-4D molecules from two different vendors  showed that, as expected, biological repeats were closely correlated for each condition . SimilarFOXO3, but we did observe a modest induction of a number of NRF2 target genes, including nuclear receptor NR0B1, NAD(P)H dehydrogenase NQO1, aldo-keto reductases AKR1B10 and AKR1C1 . This was noteworthy, because NRF2/SKN-1 is a key regulator of oxidative stress and xenobiotic phase 2 detoxification, and it can extend animal lifespan . These results suggested that Gr-4D could elicit a modest activation of NRF2, consistent with its identity as a chalcone (see Properties of Hit Molecules), compound known to activate NRF2.Among the 550 genes modestly changed upon Gr-4D treatment, we did not observe a strong signature for the longevity-associated gene lifespan . We alsoin vitro and in vivo  and histone deacetylases (HDACs), as well as inhibitors of the proteasome, were among the top chemical perturbations that produced gene expression signatures showing very strong and positive correlations to Gr-4D’s . HSP90 is known to form a complex with aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor that regulates genes such as xenobiotic metabolizing enzymes . DissociWe also analyzed gene expression by microarray analysis of WI-38 cells treated with 3 other non-toxic molecules . For comparison, we included other members of the structural group 4, which produced long-term toxicity. We also included the other group-5 molecule Gr-5B, which, despite its modest induction of TP53BP1, did not appear to exert obvious cell toxicity.2O2 , NQO1 , GCLC  and GSTM1 (glutathione S transferase)  . Consistent with the microarray analysis , we also noted that all the toxic group-4 molecules resulted in significant induction of NRF2 target genes. These data suggested enrichment for NRF2-activating small molecules in our screen, which is expected, as we selected for agents that could promote oxidative stress-resistance.Of the 4 best hits described above , three  induced the expression of two or more NRF2-regulated genes at least modestly , suggesting potential NRF2 activation. In addition to these 3 non-toxic compounds, 11 of the remaining 28 toxic or non-specific compounds also appeared to activate NRF2. Of these, Gr-9A induced NRF2 to be required for the increased resistance. To address this question, we knocked down NRF2 by more than 95% in WI-38 cells via siRNA transfection . Compared with NRF2(+) cells, NRF2-deficent cells showed a high level of propidium iodide staining, and the fraction of PI-positive cells increased further upon treatment with Gr-4D in a dose-dependent manner even in the absence of H2O2, likely reflecting increased xenobiotic sensitivity upon the loss of NRF2 . To compensate for this sensitivity to oxidative stress, we treated the NRF2-deficient cells with a lower dose of H2O2 (500 μM instead of 700 μM), finding that Gr-4D could protect NRF2(+) cells under this condition. As expected, compared with NRF2(+) cells, NRF2-deficient cells were more prone to die upon treatment with H2O2. To our surprise, Gr-4D (at 3 different doses) was still quite protective, significantly reducing the fraction of PI-positive cells . Thus, NRF2 was not required for this chalcone to promote oxidative-stress resistance. Gr-4D must act, at least in part, through a different, NRF2-independent mechanism.NRF2 activation could be one possible mechanism by which our small molecules promote oxidative-stress resistance. In this case, we would expect that FOXO-dependent fashion (SOD2 (superoxide dismutase), GADD45A (cell cycle regulator), CAT , DDB1 (damage-specific DNA binding protein) and TXNIP (thioredoxin interacting protein) .Many perturbations that increase lifespan and stress resistance in animals do so in a  fashion . None ofsesn-1 has been shown to shorten lifespan, while its overexpression promotes longevity of C. elegans .Sestrin genes are regulated by FOXO3 . Many stSESN1 induction. In addition to Gr-4D, 8 of the 28 compounds with liabilities also induced SESN1. We noted that these compounds with liabilities, but not Gr-4D, also scored positively for the DNA damage-markers γH2A.X and TP53BP1, and genotoxic stress previously has been shown to up-regulate sestrins through the induction of p53  is a crucial regulator of cell growth and metabolism and has been implicated in aging and many diseases, including cancer, diabetes and neurological diseases . This coWe examined the effects of our 32 core-set molecules  on the phosphorylation status of ribosomal protein S6 (RPS6), a target of mTORC1, and asked whether they could potentially inhibit mTOR. Rapamycin, as a control, reduced the normalized ratio of phosphorylated RPS6 (p-RPS6) by ∼95%.th structural group and O27) reduced the normalized ratio of p-RPS6 ; note that O27 also modestly induced sestrin, a known inhibitor of mTORC1.None of the non-toxic compounds appeared to inhibit mTOR. However, we found that 4 toxic molecules . Both of these molecules caused toxicity in cultured mammalian cells. We noted that compounds that scored negatively in C. elegans are not necessarily uninteresting. As these molecules were identified in a human cell-based screen, their bioavailability or target engagement could be different in worms.We analyzed our 32 core hits and found that 2 small molecules (Gr-7A & Gr-9A) increased C. elegans’ lifespan increase stress resistance, we analyzed how our small molecules affected lifespan. To ensure robustness, we used several different assay conditions .Because most conditions that extend We analyzed our 32 core-set molecules, and in multiple independent experiments, found that 9 small molecules extended the animals’ lifespan in at least 3 to 4 trials (from ∼10% to ∼50%) . Gr-4D was the only non-toxic compound that consistently produced significant life-extending effects in 4 or more trials  . Among tPhsp-4::gfp, the mitoUPR reporter Phsp-6::gfp, the NRF2/SKN-1 target gene reporter Pgst-4::gfp (for oxidative stress), and the autophagy reporter LGG-1::GFP. We observed that Gr-4D, at a dose that extended lifespan, caused modest induction of the gst-4 transcriptional GFP reporter , without exerting obvious effects on the others. As this glutathione transferase gst-4 gene is up-regulated in response to SKN-1/NRF2 activation upon oxidative stress, these findings suggested that activation of SKN-1/NRF2 by Gr-4D might contribute, at least in part, to the lifespan extension in C. elegans.To investigate the potential mechanism by which the chalcone Gr-4D extended lifespan, we asked whether it might affect gene activities known to influence longevity. To do this, we examined its effects on the expression of pathway-specific reporters, including the ER-stress reporter skn-1 is required for the lifespan extension produced by Gr-4D, we examined this chalcone at multiple doses in RNAi-sensitive mutants fed with control or RNAi bacteria expressing dsRNA of skn-1. When initiated from day 1 of adulthood, RNA interference reduced the level of skn-1 mRNA by ∼80% in 3 days .To test directly whether n 3 days . Whereassesn-1, the only worm ortholog of human sestrins, might be required for the lifespan-extending effects of Gr-4D. Upon modest reduction (∼30%) of sesn-1 mRNA level by RNAi inhibition , Gr-4D also appeared to lose its ability to extend lifespan, even though we observed a trend of lifespan extension in animals treated with high dose of this molecule (P = 0.0518) . Given the caveat of insufficient inhibition of gene expression by RNAi, we further addressed the dependency of sesn-1 by analyzing animals that carried a potential loss-of-function allele of sesn-1. We found that Gr-4D doses that extended wild-type lifespan were not able to extend the lifespan of these mutants .Four of our hits were chalcone-family members, including our most interesting hit Gr-4D, which increased lifespan consistently in worms  with no apparent toxicity . Our 104Chalcones have been reported to produce many beneficial health effects, including anti-cancer, anti-HIV, anti-malarial, anti-inflammatory and anti-allergic activities . They haChalcones, with a backbone of α,β-unsaturated ketone, are known to activate NRF2. In line with this, we observed consistent signatures for NRF2-regulated genes in both worms and human cells; however, genetic epistasis analysis indicated that, while NRF2 may potentially contribute to the stress resistance of human cells, NRF2-independent mechanisms play an important role as well.2O2 in WI-38 cells. The significance of this is not clear, as, for reasons we do not understand, we were also unable to observe an increase in autophagy with DMC in our assay system.A recent study has identified 4,4’-dimethoxychalcone (DMC) as a natural compound that extended lifespan in multiple species, including yeast, worms and flies  to cross-reference our small molecules to other high-throughput screens. We found that at least 9 molecules had been identified in multiple screens and suggested to affect certain human protein targets . Of these, the chalcone Gr-4D, our top non-toxic compound, has been identified in many screens in the PubChem database. For example, it has been reported to kill fibrosarcoma cell lines that produce the oncometabolite 2-hydroxyglutarate (2HG) . Other studies have suggested that Gr-4D can inhibit IL-1B-mediated inflammasome signaling (AID 743279) and activate p53 by inhibiting the MDM2/MDM4 interaction (in multiple assays). Finally, Gr-4D could induce DNA-rereplication in both SW480 colon adenocarcinoma cells and MCF 10A epithelial cells (with a potency of ∼5-6 μM), which in turn can induce the DNA damage response and apoptosis . Given the concern of genome instability that can be caused by DNA re-replication, it would be interesting to analyze whether a lower dose of Gr-4D could be tolerated Among the remaining 8 compounds with liabilities , Gr-3B and Gr-7C were identified in multiple assays that examined different biological targets . This raises a concern about non-specific actions of these molecules in cells. However, except for O6, which contains a reactive phenolic Mannich base, the other molecules do not appear to carry obvious PAINS signatures. By contrast, curcumin, a known PAIN molecule, has been found in at least 2167 assays with 92 reported protein targets . Likewise, the other PAIN molecule resveratrol has been identified in at least 4266 assays with 91 active protein targets .To our surprise, despite the differences in their structures, several different molecules appeared to affect the same protein targets . For example, 4 molecules  were shown previously to affect the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel protein that is involved in multi-drug resistance , consistent with the report from another screen suggesting it as an RELA inhibitor in HUVEC cells . These two studies above also identified Gr-3A, but not Gr-3B (which has a different side group that could introduce steric hindrance), as an NFKBIA-inducing compound . These findings are consistent with our analysis using the in silico docking database ZINC . Curiously, it was also identified in another study as a molecule that inhibited TNF-alpha-induced NF-κB activation in HEK 293T cells (AID 1852), leaving its regulation of NF-κB in question. Among the remaining members of the 32 core-set compounds, Gr-3C was identified previously as a molecule that induced the NF-κB inhibitor NFKBIA in two screens , even though worms do not have apparent NF-κB homologs.NF-κB signaling controls cell survival, differentiation, and proliferation. Key components of the pathway mediate inflammatory responses  and haveAKT1 knockdown, our control that was expected to activate FOXO3, increased stress resistance in our assay. However, we identified only 3 molecules within our 32 core-set hits that may activate FOXO3, suggesting that our stress-resistance screen may not be ideal to look for FOXO3 activators.In animals, the rate of aging can be influenced by many factors, including a network of signaling proteins and transcription factors that also sense nutrients, energy levels and stress. Perturbing many genes in this network can extend healthspan and increase disease-resistance and lifespan . For exadaf-16/foxo. We identified 14 potential NRF2 activators , of which at least 7 molecules  increased resistance to both H2O2 and cadmium (note: scored as “++”). 13 of the 14 molecules activated NRF2 without affecting FOXO3 or mTOR . Importantly, these pathways inhibit a wide variety of cancers , which iFinally, even the small molecules that have cell toxicity could be interesting. In principle, any toxicity caused by these hits, which are administered at relatively high levels, could be off-target and thus dialed out via medicinal chemistry. Alternatively, they could be on-target toxicities. In the latter case, they could potentially increase stress resistance by activating cell-protective mechanisms, as do low levels of the herbicide paraquat in invertebrates, a situation  that can extend lifespan in experimental organisms. Furthermore, toxicity could be cell type-specific. For example, we found that two group-7 molecules (Gr-7A & Gr-7B), which both produced long-term cell toxicity and appeared to inhibit mTOR, reduced the ATP content by ∼40–50% in a human lung adenosquamous carcinoma cell line HTB178 (data not shown). Conversely, when administered overnight at 10μM and assayed in parallel, these same molecules did not produce obvious effects on ATP levels in either the WI-38 primary cell line or in CRL-2081, lung-derived mesothelioma cells. Such selective effects could be due to differences in these tumor cell lines . These preliminary results also suggest an attractive possibility that some of our small molecules, when given at a dose that can be tolerated by normal cells, could selectively kill certain tumor cells. We should note that many cancer drugs, such as doxorubicin, can damage DNA and are intrinsically cell-toxic.in vivo, in C. elegans. By doing these assays, we found several molecules, most notably the chalcone Gr-4D, that could be of potential interest for further translational studies.Our high-throughput screen in human primary cells was designed to enrich for stress resistance-promoting small molecules that could potentially perturb pathways that affect lifespan. We characterized the hit compounds in several different cellular assays, and