The t6A pathway uses two sequentially acting enzymes, YRDC and OSGEP, the latter being a subunit of the multiprotein KEOPS complex. We recently identified mutations in genes encoding four out of the five KEOPS subunits in children with Galloway-Mowat syndrome (GAMOS), a clinically heterogeneous autosomal recessive disease characterized by early-onset steroid-resistant nephrotic syndrome and microcephaly. Here we show that mutations in YRDC cause an extremely severe form of GAMOS whereas mutations in GON7, encoding the fifth KEOPS subunit, lead to a milder form of the disease. The crystal structure of the GON7/LAGE3/OSGEP subcomplex shows that the intrinsically disordered GON7 protein becomes partially structured upon binding to LAGE3. The structure and cellular characterization of GON7 suggest its involvement in the cellular stability and quaternary arrangement of the KEOPS complex.N 6-threonylcarbamoylated adenosine 37 in tRNA (t6A) involves the YRDC enzyme and the KEOPS complex. Here, the authors report mutations in YRDC and the KEOPS component GON7 in Galloway-Mowat syndrome and determine the crystal structure of a GON7-containg subcomplex that suggests a role in KEOPS complex stability.The biosynthesis of N The majority of the complex modifications are concentrated in the anticodon region of the tRNAs and are crucial for accuracy of protein synthesis. The threonylcarbamoylation of the N6 nitrogen of the adenosine at position 37 (t6A) of most ANN-accepting tRNAs represents one of the very few nucleotide modifications that exists in every domain of life3. The t6A biosynthesis pathway consists of two steps: firstly, the YRDC enzyme (Sua5 in yeast) synthesizes an unstable threonylcarbamoyl-AMP intermediate (TC-AMP) and in a second step, the KEOPS protein complex transfers the TC-moiety from TC-AMP onto the tRNA substrate4. Enzymes that synthesize TC-AMP exist in two versions depending on the organism: a short form which only has the YrdC domain (such as human YRDC) and a long form which has an extra Sua5 domain (yeast SUA5 for instance). The eukaryotic KEOPS complex contains five subunits GON7/LAGE3/OSGEP/TP53RK/TPRKB that are arranged linearly in that order8. OSGEP is the catalytic subunit that carries out the TC-transfer reaction and its orthologs are present in virtually all sequenced genomes9. The other subunits are essential for the t6A modification of tRNA, but their precise roles are as yet unknown, especially that of GON7, an intrinsically disordered protein (IDP), which was only recently identified in humans7. Fungal Gon7 was shown to be an IDP that adopts a well-defined structure covering 50% of its sequence upon complex formation with Pcc1 (LAGE3 homolog)8. In humans, GON7 was recently shown to be also structurally disordered in absence of the other KEOPS complex subunits. GON7 was proposed to be a very remote homolog of the yeast Gon7 protein although its structure upon complex formation remains unknown7.Transfer RNAs (tRNA) are subject to multiple post-transcriptional modifications that are important for the stabilization of their ternary structure and the precision of the decoding process10. We recently identified autosomal recessive mutations in genes encoding four of the five subunits of human KEOPS complex, namely LAGE3, OSGEP, TP53RK, and TPRKB in patients with Galloway-Mowat syndrome . GAMOS is a rare neuro-renal disorder characterized by the co-occurrence of steroid-resistant nephrotic syndrome (SRNS) with microcephaly and neurological impairment11. GAMOS is clinically heterogeneous, reflecting a genetic heterogeneity. Indeed, disease-causing mutations have been identified in eight genes to date: four in KEOPS genes and four in other unrelated genes, WDR73, WDR4, NUP133, and NUP107 (refs. 17). SRNS is typically detected in the first months of life and most often rapidly progresses to end-stage renal disease (ESRD) within a few months; however, there are rarer cases with preserved renal function in adulthood18. Kidney lesions range from minimal change disease, to focal segmental glomerulosclerosis (FSGS) that might be of the collapsing type, or diffuse mesangial sclerosis (DMS). Cerebral imaging findings include cerebral and cerebellar atrophy, and gyration and/or myelination defects. These anomalies are associated with neurological deficits such as psychomotor impairment, hypotonia, seizures, and more rarely sensorineural blindness and deafness. Affected children may also present with facial and/or skeletal dysmorphic features. The prognosis of GAMOS is poor, and most affected children die before 6 years of age.tRNA modifications have been demonstrated to play a role in the development of the brain and nervous system, and an increasing number of defects in these modifications are now being linked to various human neurodevelopmental disordersGON7  and YRDC, both genes encoding proteins involved in the biosynthesis of the t6A modification. These data, together with our previous work, show that mutations in genes encoding all the proteins involved in the two chemical steps of t6A lead to GAMOS. Furthermore, we determine the crystal structure of the GON7/LAGE3/OSGEP KEOPS subcomplex showing that GON7 becomes structured upon binding to LAGE3. The structure also explains our observations that GON7 stabilizes the remainder of the KEOPS complex and directs its quaternary organization.Here we present 14 GAMOS-affected individuals from seven families, with mutations in GON7 gene in 11 affected individuals from 5 unrelated families and in the YRDC gene in 3 affected individuals from 2 unrelated families  carried the same homozygous nonsense mutation  which causes a stop codon at position 7 of the protein. These families, all originating from the same region of Algeria, shared a common haplotype at the GON7 locus indicating a founder effect  and a 4-base pair deletion leading to a frameshift . In Family G, we identified a homozygous in-frame deletion of Leucine 265 . For both families, western blot and qPCR analysis on cell extracts from affected children showed the presence of YRDC transcripts and proteins  structural model of human YRDC using the structure of the YRDC domain of the archaeal Sua5  and mapped these mutations onto this in silico model  , and myoclonia. This clinical picture is highly reminiscent of that observed in GAMOS-affected individuals with mutations in LAGE3, OSGEP, TP53RK, and TPRKB13. However, individuals with GON7 mutations presented with milder neurological and renal manifestations, always with post-natal microcephaly and no gyration defects, later onset of proteinuria (median age 18 months vs. 1) and slower progression to ESRD (median age 49 months in 8/11 children vs. 5 months in 3/3 children), and a longer survival compared to the YRDC cases.Early-onset proteinuria was observed in all affected children, with first detection ranging from between birth and 5 years. All but three children reached ESRD between 1.5 months and 6 years of age. All individuals carrying -2) Fig. . Extra-rYRDC mutations, we first used a yeast heterologous expression and complementation assay as previously performed for OSGEP mutations identified in GAMOS individuals13. Indeed, the deletion of SUA5, the YRDC ortholog in S. cerevisiae leads to a very severe growth defect, similarly to the deletion of each of the genes encoding the five KEOPS subunits21. We therefore heterologously expressed the human YRDC cDNAs encoding wild-type (WT) and mutant proteins in the Δsua5 strain to evaluate their ability to rescue the slow growth phenotype. Since the catalytic activity of YRDC does not require protein partners, the WT YRDC protein efficiently complemented the Δsua5 growth defect  and amorphic  alleles, as has been previously shown for OSGEP mutations13. A similar approach could not be applied for GON7 mutations since GON7 failed to complement the growth defect of the Δgon7 yeast strain (see ref. 7 and our data). We therefore measured the t6A content in fibroblasts from two individuals with the p.Tyr7* GON7 mutation, three individuals with YRDC mutations and one individual with the p.Arg325Gln OSGEP mutation. The t6A levels were significantly decreased in both YRDC- and OSGEP-mutated fibroblasts, and to a lesser extent in GON7-mutated fibroblasts  of human KEOPS components tes Fig. . DespiteRNA Fig. . The imping Fig. . By meassis Fig. . Since llls Fig. , even inted Fig. . Altoget6). Based on very weak sequence similarity, it was proposed, that GON7 is a remote homolog of yeast Gon7 (ref. 7). We first investigated the structure of GON7 in solution by collecting a 2D 1H-15N Band-Selective Optimized Flip Angle Short Transient Heteronuclear Multiple-Quantum Correlation (SOFAST-HMQC) NMR spectrum of a 15N-labeled GON7 sample. The poor spectral dispersion in the 1H dimension of the 2D correlation spectrum showed that GON7 lacks well-defined structure, confirming the conclusions of Wan et al.7. Adding non-labeled LAGE3 to the sample, caused the shift and/or disappearance for many crosspeaks, suggesting GON7 interacts with LAGE3 , one can assess qualitative information on the degree of compactness of the scattering object27. The plateau observed for GON7 is characteristic of a fully disordered protein, possibly with very short elements of secondary structure . LAGE3 contains 60 residues at the N-terminus that are absent in the Pcc1 orthologs from yeast and archaea and that are predicted to be disordered. We did not observe any electron density for this N-terminal peptide, confirming this region indeed lacks a stable structure. We cannot exclude however that partial proteolysis removed this peptide during the long crystallization process. LAGE3 is at the center of the complex, binding on one side to OSGEP and on the other to GON7, which does not directly interact with OSGEP  with the scattering curve calculated on the all-atom model built using the program BUNCH from the crystal structure (see Methods)  or good quality 3D models  for all of the KEOPS subunits at our disposal, except for GON7  Fig. . We therGON7 mutations. We first confirmed by mass spectrometry analysis that in a human podocyte cell line stably overexpressing either 2HA-tagged GON7 or V5-tagged LAGE3, the four additional KEOPS subunits significantly co-purified with GON7 or LAGE3, respectively, thus confirming that a five-subunit KEOPS complex is present in these renal glomerular cells  and/or affect the stability of the KEOPS complex (GON7). These results complement our previous findings and establish that mutations in all the genes involved in this pathway lead to GAMOS.In this study, we identified mutations in two genes encoding proteins involved in tGON7 or YRDC mutations present with the clinical features of GAMOS, similarly to the individuals previously reported to have mutations in the genes encoding the four other KEOPS subunits. In addition, we have expanded the GAMOS phenotype spectrum by describing congenital hypothyroidism to be associated with YRDC mutations. Although the two GON7 mutations encode truncated non-functional proteins, we noticed that they unexpectedly result in a less severe clinical outcome compared to that of individuals affected by mutations in other KEOPS subunit genes or in YRDC, for which biallelic null mutations were not found. This suggests that the absence of GON7 has less severe consequences for cell life compared to the other components of the t6A biosynthesis pathway, where four out of six are encoded by genes considered to be essential28. This less severe clinical outcome correlates with our data showing that GON7 loss of function and depletion in fibroblasts and podocytes, respectively, have globally a weaker effect on t6A levels, proliferation, apoptosis, and protein synthesis compared to that of other KEOPS subunits and YRDC mutations or depletion. However, although our data have confirmed that GON7 is a functional homolog of Gon7, the effect of their absence in human and yeast, respectively, is markedly different since in the absence of Gon7, the yeast KEOPS complex has only very low t6A activity and cell growth is dramatically affected3. Altogether, our data in humans suggest that GON7 is not as essential in humans as in yeast for t6A biosynthesis.All individuals bearing 7. The latter stoichiometry has also been observed for the archaeal KEOPS complex, for which no fifth subunit similar to Gon7 has yet been discovered. The Pcc1 subunit constitutes the dimerization unit of archaeal KEOPS29 and this is also very likely the case for the LAGE3 subunit of human KEOPS in absence of GON7 (ref. 7). In line with these results, our structure of the GON7/LAGE3/OSGEP complex shows that GON7 competes with LAGE3 for dimerization, explaining the different stoichiometries of the KEOPS complex observed in the absence or presence of GON7. Indeed, GON7 covers a large hydrophobic surface of LAGE3  quaternary structure as evidenced by the decrease in KEOPS subunits protein levels observed in GON7 patient cells. Taken together, our data demonstrated that GON7 impacts the stability of the KEOPS complex therefore having an effect on its enzymatic activity. This is in line with the in vitro data of Sicheri’s group showing that in presence of GON7, KEOPS t6A activity is potentiated29 and with our in vivo data showing that t6A levels in GON7-mutated patient fibroblasts are slightly decreased compared to YRDC- and OSGEP-mutated fibroblasts.Our biochemical and structural data provide a molecular framework to understand the pathophysiological effects of the GAMOS-associated mutations. The structure of the GON7/LAGE3/OSGEP complex shows that GON7 is bound exclusively to the non-catalytic LAGE3 subunit, distant from the catalytic center of OSGEP. It has been shown in vitro that the intact human KEOPS complex has a 1:1:1:1:1 stoichiometry, in contrast with the complex lacking GON7 which has a 2:2:2:2 stoichiometrygon7 deletion mutant7. Comparison of the GON7/LAGE3 and Gon7/Pcc1 complexes shows that the hydrophobic character of the residues at the interface is very well conserved  methyltransferase that is responsible for the highly conserved m7G modification on a specific subset of tRNAs33. Interestingly, it has been shown that the absence of m7G tRNA modification leads to impaired cell proliferation, neural differentiation, and a decrease in global translation with a less efficient translation of mRNAs involved in cell division and brain development, consistent with the microcephaly and brain anomalies found in individuals with WDR4 mutations34. Similarly, as a consequence of the decrease in t6A levels observed in affected individual’s cells, perturbed protein translation could impact the translation of specific mRNA involved in kidney and brain development and/or podocyte/neuron maintenance. It is likely that the requirement for t6A-modified tRNAs levels is dependent of the cell-type and/or cell cycle as has been previously shown in D. melanogaster where highly proliferative cells of the wing imaginal discs are more affected by the absence of t6A modification than fully differentiated photoreceptors35. Neuronal progenitors that have high mitotic activity probably have higher demands for protein translation, making them more vulnerable to any perturbation in the tight regulation of tRNA modifications. Furthermore, another potential regulatory step to spatiotemporally modulate these tRNA modifications and thus protein translation is the tissue- and developmental stage-specific expression of the tRNA-modifying enzymes37. YRDC and KEOPS subunits could be differentially expressed between specific cell-types in the brain  and the kidney (podocytes) and/or during development/differentiation explaining the tissue involvement and the course of the disease as well as its clinical outcome. Further studies on neuronal/renal progenitors and neurons/podocytes differentiated from induced pluripotent stem cells obtained from individuals with mutations in YRDC and KEOPS subunits will probably provide further insights into the pathogenesis of GAMOS.An increasing number of mutations are being identified in genes encoding tRNA-modifying enzymes that are linked to human neurodevelopmental disorders. Very recently, mutations in 6A biosynthesis pathway in the pathogenesis of GAMOS. Further investigations are needed to fully characterize all the KEOPS mutations at the biochemical, structural, and enzyme activity levels to better understand their impact on KEOPS complex-dependent t6A biosynthesis activity and how they influence the clinical phenotypes. Genes encoding components of the t6A biosynthesis pathway have to be added to the growing list of translation-associated proteins whose loss of function are responsible for rare genetic disorders.Together, our data strongly emphasize the importance and relevance of the tWritten informed consent was obtained from participants or their legal guardians, and the study was approved by the Comité de Protection des Personnes “Ile-De-France II.” Genomic DNA samples were isolated from peripheral blood leukocytes using standard procedures.We performed whole-exome sequencing using Agilent SureSelect All Exon 51 Mb V5 capture-kit on a HiSeq2500 (Illumina) sequencer (paired-end reads: 2 × 100 bases). Sequences were aligned to the human Genome Reference Consortium Human Build 37 (GRCh37) genome assembly with the Lifescope suite from Life Technologies. Variant calling was made using the Genome Analysis Toolkit pipeline. Then, variants were annotated using a pipeline designed by the Paris Descartes University Bioinformatics platform. We assumed the causal variant: (i) segregates with the disease status, (ii) is novel or has a minor allele frequency <1/1000 in gnomAD, (iii) was not found in >10/2352 projects of our in-house database. Missense variant pathogenicity was evaluated using in silico prediction tools . Sanger sequencing was used to validate the variant identified by exome sequencing and to perform segregation analysis in the families. Sequence were analyzed with the Sequencher software  and positions of mutations were numbered from the A of the ATG-translation initiation codon. For Family G, WES, and SNP-array were performed according to standard diagnostic procedures and WES quality criteria at the UMC Utrecht, the Netherlands. The patient–parent quartet WES with sibling-sharing analysis focused on the regions of homozygosity determined by SNP-array (parents are consanguineous in the eighth degree).NheI restriction site, and either two copies of the Human influenza hemagglutinin (HA) tag or one copy of the V5 epitope tag allowing epitope-tagging at the N-terminal of the encoded protein. Human full-length GON7, LAGE3, and YRDC cDNA  were amplified by PCR from IMAGE cDNA clones , and subcloned into the modified pLEX-MCS plasmid using either SpeI and XhoI (for GON7 and LAGE3) or NheI and XhoI (for YRDC). Human YRDC cDNA was also subcloned into the BamHI and SalI sites of pESC-TRP. Site-directed mutagenesis  was used to generate the mutations used in this study. An adapted cloning protocol was used to obtain the C-terminal extension found for the YRDC p.Val241Ilefs*72 mutant. For gene silencing, the shRNA sequences described in Supplementary Table AgeI and EcoRI restriction sites. This vector contains a cassette conferring puromycin resistance. All constructs were verified by Sanger sequencing.The following expression vectors were used in this publication: LentiORF pLEX-MCS (Open Biosystems), pESC-TRP with a c-myc tag (Agilent), and pLKO.1-TRC Cloning vector . The LentiORF pLEX-MCS plasmid was modified by site-directed mutagenesis  to insert one 2 in RPMI 1640 medium supplemented with 10% fetal bovine serum, insulin-transferrin-selenium, glutamine, and penicillin and streptomycin , and human primary fibroblasts, obtained from patient skin biopsies, were cultured in OPTIMEM medium supplemented with 10% fetal bovine serum, sodium pyruvate, glutamine, fungizone, and penicillin and streptomycin  at 37 °C with 7% CO2. Obtention and culture of lymphoblastoid cell lines are detailed in Supplementary Methods. Human podocytes stably overexpressing 2HA-GON7 or V5-LAGE3, or transiently depleted for GON7, LAGE3, OSGEP, or YRDC were obtained by transduction with lentiviral particles and subsequent puromycin selection (2 μg/ml). HEK293T cells (ATCC CRL-3216) were transiently transfected using Lipofectamine® 2000 (ThermoFisher Scientific).The human immortalized podocyte cell line (AB8/13) provided by M. Saleem  was grown at 33 °C with 7% COThe following antibodies were used in the study: mouse anti-α-tubulin , mouse anti-actin , mouse anti-HA , rabbit anti-GON7 , rabbit anti-LAGE3 , rabbit anti-TPRKB , rabbit anti-OSGEP , and mouse anti-GAPDH  from Sigma-Aldrich; mouse anti-V5  from Bio-Rad; rabbit anti-YRDC  from ThermoFisher Scientific; rabbit anti-LAGE3  and mouse anti-OSGEP  from Novus Biologicals; rabbit anti-TP53RK  from Abgent. Secondary antibodies for immunoblotting were sheep: anti-mouse and donkey anti-rabbit HRP-conjugated antibodies , and IRDye 800CW Donkey anti-rabbit (926-32213) and IRDye 680RD Donkey anti-mouse (926-68072) antibodies (LI-COR). Cycloheximide (C7698), Nuclease P1 (N8630), phosphodiesterase I from snake venom (P3243), and alkaline phosphatase (P4252) were purchased from Sigma-Aldrich.38. Media were supplemented with 2% agar for solid media. The S. cerevisiae W303 derived strain, Δsua5::KanMX(YCplac33-SUA5)39, was used as the host for the complementation assay. For each pESC-TRP plasmid derivative to be tested, three independent clones were selected after transformation and grown on GLU-TRP media. Clones were then streaked onto GAL-TRP containing 0.1% 5-fluoroorotic acid (5-FOA) to counter-select the YCplac33-SUA5 plasmid (containing URA3). After two rounds of selection, clones were checked for their acquired Ura- phenotype, their plasmid content was confirmed by sequencing after plasmid rescue before being finally evaluated for fitness by a 10-fold serial dilution spotted onto GAL-TRP minimal supplemented media. Empty pESC-TRP, pESC-TRP-SUA5, and pESC-TRP-SUA5-myc were used as negative and positive controls, respectively.Yeast cells were grown at 28 °C in standard rich medium YEPD  or minimal supplemented media . Cells were transformed using the lithium acetate method® Kit and treated with DNase I. One microgram total RNA was reverse-transcribed using Superscript II, according to the manufacturer’s protocol (Life Technologies). The relative expression levels of the mRNA of interest were determined by real-time PCR using Power SYBR Green ROX Mix (ThermoFisher Scientific) with specific primers listed in Supplementary Table Hgprt). Data were analyzed using the 2−ΔΔCt method.Total mRNA from knocked down podocytes, primary skin fibroblasts, and LCLs was isolated using Qiagen Extraction RNeasy600nm of approximately 3 × 107 cells/ml) with phenol induced cell permeabilization, LiCl-selective precipitation, and subsequent ion exchange-chromatography purification on an AXR-80 column , according to the manufacturer’s instructions. For human primary fibroblasts, the two-step protocol that was applied is detailed in Supplementary Methods. Ten micrograms of yeast tRNAs and approximately 1 µg of human fibroblast tRNAs were then enzymatically hydrolyzed into ribonucleosides with nuclease P1, phosphodiesterase, and alkaline phosphatase, deproteonized by filtration, and finally dried under vacuum according to the protocol of Thuring et al.40. t6A ribonucleoside was analyzed using the quantitative LC/MS-MS protocol of Thüring et al.40. Quantification of t6A was performed by integration of the peaks of interest and expressed relative to the total area of the peaks corresponding to the four canonical unmodified ribonucleosides assessed in the same sample for normalization. tRNA extracted from three independent samples were each measured twice . Detailed information are provided in Supplementary Methods.Yeast tRNAs were extracted and purified from actively growing cells (at ODTM protease inhibitors (Roche), as in Serrano-Perez et al.41. Fifty micrograms of proteins were loaded onto acrylamide gels and blotted onto nitrocellulose membranes (Amersham). The membranes were blocked in 1× Tris-buffered saline, 0.1% Tween 20 (TBST) with 5% bovine serum albumin or in Odyssey (LI-COR Bioscience) blocking buffer. Membranes were then incubated with the indicated primary antibodies, washed, and then incubated with either HRP-conjugated or LI-COR IRDye secondary antibodies. Signals were detected using ECL reagents (Amersham Biosciences) and acquired in a Fusion Fx7 darkroom (Vilber Lourmat) or acquired with Odyssey CLx near-infrared fluorescent imaging system (LI-COR Bioscience). Densitometry quantification was performed either using Bio-1D software or using Image studio lite software (version 5.2). Uncropped and unprocessed blots are provided in the Source Data file.Proteins from KD podocytes, primary fibroblasts, and LCLs were extracted in lysis buffer containing 150 mM NaCl, 50 mM Tris-HCl pH 7, 0.5% Triton-X100 with CompleteTM Epitope Tag Protein Isolation Kit (Miltenyi Biotec). Briefly, fresh lysates (1–1.5 mg of protein) were incubated either with mouse anti-V5 antibodies, followed by a 30-min incubation with magnetic beads coupled to protein A, or directly with magnetic beads coupled to an HA antibody. Immunoprecipitated proteins were isolated using µMACS® Separation Columns in a magnetic µMACS separator and subsequently eluted with 1× Laemmli buffer. Lysates and immunoprecipitated samples were subjected to immunoblot41. To assess rates of protein degradation, HEK293T cells transiently expressing either 2HA-GON7 or V5-LAGE3 alone, or co-expressing both proteins were incubated with cycloheximide at a final concentration of 100 µg/ml for the indicated time periods . Total protein extracts and immunoblotting were performed as described above. Anti-HA and anti-V5 antibodies were used to reveal GON7 and LAGE3, respectively. Relative GON7 and LAGE3 protein amounts were normalized to those of α-tubulin at each time point.For immunoprecipitation, HEK293T cells were transiently transfected with the adequate plasmids  and/or mutants) using calcium phosphate. Forty-eight hours post transfection, cells were lysed in 150 mM NaCl, 25 mM Tris-HCl pH 8, 0.5% Triton with protease inhibitors and HA-tagged GON7 was immunoprecipitated using the µMACSCell proliferation, apoptosis level, and rates of protein synthesis were assessed in KD podocytes using the CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay (MTT) (Promega), the Caspase-3/7 Green detection Reagent , and the Click-iT HPG Alexa Fluor 488 Protein Synthesis Assays , respectively, according to the manufacturer’s instructions. Detailed information are provided in Supplementary Methods.13. Two groups (control IP versus IP HA or IP V5), each containing three biological replicates, were used for statistical analysis. Only proteins that were identified at least three times out of six were retained. A t-test was performed, and the data were represented in a volcano plot .Human podocyte cell lines stably expressing either 2HA-GON7 or V5-LAGE3 were used to perform proteomic studies. 2HA-GON7 and V5-LAGE3 were immunoprecipitated as described in the section above. Eluates were processed according to Braun et al.42.Measurement of the length of the terminal restriction fragments was performed by Southern blotting according to Touzot et al.All structural work was done using the full-length proteins of GON7, LAGE3, and OSGEP.15N-labeled his-tagged GON7 (vector “pET24d-C14_hisTEV_op”) whose sequences are shown in Supplementary Table 15N-GON7 and subcomplex LAGE3/15N-GON7 preparation are described in detail in Supplementary Methods.For NMR experiments, two vectors were ordered from Genscript  for the expression of either unlabeled his-tagged LAGE3 (vector “pET21a-LAGE3_hisTEV_op”) or For SAXS or crystallogenesis experiments, preparation of unlabeled GON7 and GON7/LAGE3/OSGEP subcomplex, and co-expression and purification of the KEOPS subunits are detailed in Supplementary Methods and Supplementary Figs. 43. X-ray diffraction data collection was carried out on beamline Proxima1 at the SOLEIL Synchrotron  at 100 K. Data were processed, integrated, and scaled with the XDS program package44. The crystal belonged to space group P43. The OSGEP and LAGE3 subunits were positioned by molecular replacement with the programs PHASER45 and MOLREP, implemented in the CCP4 suite46 using the structures of MjKae1 (PDB ID: 2VWB) and ScPcc1 (PDB ID: 4WX8) as search models. Residual electron density showed clearly the presence of the GON7 subunit, which was constructed using the program BUCCANEER46. The initial structure was refined using the BUSTER program47 and completed by interactive and manual model building using COOT48. The correctness of the assigned GON7 sequence was verified by omit mFo-DFc, 2mFo-DFc, Prime-and-switch electron density maps49 . To isolate the various species in solution, SAXS data were collected on samples eluting from an online size exclusion high-performance liquid chromatography (SEHPLCBio-SEC3Agilent) column and directly connected to the SAXS measuring cell. sixty-five microliters of GON7/LAGE3/OSGEP and GON7 samples concentrated at 1.5 and 6.7 mg/l, respectively, were injected into the column pre-equilibrated with a buffer composed of 20 mM MES pH 6.5, 200 mM NaCl, and 5 mM 2-mercaptoethanol. Flow rate was 300 µl/min, frame duration was 1.0 s, and the dead time between frames was 0.5 s. The protein concentration was estimated by UV absorption measurement at 280 and 295 nm using a spectrometer located immediately upstream of the SAXS measuring cell. A large number of frames were collected before the void volume and averaged to account for buffer scattering. SAXS data were normalized to the intensity of the incident beam and background (i.e. the elution buffer) subtracted using the program FoxTrot50, the Swing in-house software. The scattered intensities were displayed on an absolute scale using the scattering by water. Identical frames under the main elution peak were selected and averaged for further analysis. Radii of gyration, maximum particle dimensions, and molecular masses were determined using PrimusQT51 . An ultimate adjustment was performed using the program CRYSOL54. The modeling was repeated 10 times and the best model was deposited in SASBDB55 with codes SASDFK8, SASDFM8, and SASDFL8 for GON7, GON7/LAGE3, and GON7/LAGE3/OSGEP, respectively.SAXS experiments were carried out at the SOLEIL synchrotron SWING beamline . The sample to detector (Aviex CCD) distance was set to 1500 mm, allowing reliable data collection over the momentum transfer range 0.008 Ån = 3 independent experiments. For statistical analysis, data sets comparing more than three conditions (to a control group) were analyzed with ANOVA followed by Dunnett’s multiple comparisons test or by using Kruskal–Wallis test followed by a Dunn’s multiple comparisons test. Data sets with only two conditions to compare were analyzed using an unpaired t-test or a Mann–Whitney test. P < 0.05 was considered statistically significant. A standard confidence interval of 95% was applied in all analyses. Displayed in the figures are the mean values of all technical replicates for each of the independent experiments (displayed as single data points). Black lines indicate the mean values of all independent experiments.GraphPad Prism 8.0 software was used for the graphical representation and statistical analysis of cell-based data. Results are presented as mean ± s.e.m. of at least Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Myrica rubra (MEO), which has significant antiproliferative effect in various cancer cell lines. In the present study, we compared the antiproliferative effect of these sesquiterpenes alone and in combination with the cytostatic drug doxorubicin (DOX) in cancer cell lines with different sensitivity to DOX. Two ovarian cancer cell lines (sensitive A2780 and partly resistant SKOV3) and two lymphoblast cancer cell lines (sensitive CCRF/CEM and completely resistant CEM/ADR) were used. The observed effects varied among sesquiterpenes and also differed in individual cell lines, with only VAL being effective in all the cell lines. A strong synergism of DOX with NER was found in the A2780 cells, while DOX acted synergistically with HUM and CAO in the SKOV3 cells. In the CCRF/CEM cells, a synergism of DOX with CAO and NER was observed. In resistant CEM/ADR cells, sesquiterpenes did not increase DOX efficacy, although they significantly increased accumulation of DOX (up to 10-times) and rhodamine-123 (substrate of efflux transporter ABCB1) within cancer cells. In conclusion, the tested sesquiterpenes were able to improve DOX efficacy in the sensitive and partly resistant cancer cells, but not in cells completely resistant to DOX.β-caryophyllene oxide (CAO), α-humulene (HUM), trans-nerolidol (NER) and valencene (VAL) are constituents of the essential oil of  Sesquiterpenes, defined as 15-carbon compounds formed from 3 isoprenoid units, represent an extremely diverse, heterogeneous and large group of natural compounds. Sesquiterpenes are secondary metabolites, formed mainly in plants, fungi, bacteria and marine invertebrates ,3. TogetMyrica rubra essential oil, α-humulene (HUM), caryophyllene oxide (CAO), trans-nerolidol (NER) and valencene (VAL), with doxorubicin (DOX) in the CaCo-2 cancer cell line [In addition, several sesquiterpenes were able to enhance the efficacy of classical anticancer drugs in vitro. For example, a combination of the α-methylene sesquiterpene lacton eremophila-110)-1113)-dien-12, 8β-olide with cisplatin or paclitaxel increased the effect of these cytostatics in the ovarian cell lines SKOV3, OVCAR-3, JC and JCpl . Oxalipl-11(13)-d-dien-12,ell line . Resistaell line ,16.The present study was designed to test and compare the effects of sesquiterpenes HUM, CAO, NER and VAL on cell proliferation and DOX efficacy in ovarian and lymphoblast cancer cells with different sensitivity to DOX. Moreover, the ability of these sesquiterpenes to enhance DOX concentration within cells and to inhibit efflux transporters was also tested. Two ovarian cancer cell lines—A2780 (sensitive to DOX) and SKOV3 (partly resistant to DOX)—and two lymphoblast cancer cell lines—CCRF/CEM (DOX-sensitive) and CEM/ADR (DOX-resistant)—were used for this purpose. The amount of the ABCB1 efflux transporter in these cell lines was quantified and compared.Myrica rubra essential oil and its sesquiterpene components HUM, CAO, NER and VAL to inhibit proliferation of colon cancer cell lines as well as to increase the efficacy of DOX treatment; this is probably via increased production of reactive oxygen species (ROS) and DOX accumulation in these cells. On the other hand, no cytotoxic effect or amelioration of DOX toxicity was observed in rat hepatocytes as a model of non-cancerous cells [The frequent occurrence of resistance of cancer cells to DOX underscores the necessity of determining compounds which are able to suppress this resistance. These compounds might be detected among sesquiterpenes, as these substances are able to enhance drug uptake via increased membrane permeability. In addition, some sesquiterpenes themselves have been shown to have anti-proliferative effects in cancer cells as well as to act synergistically with classic cytostatics . In a prus cells ,17,18. I50 values for individual sesquiterpenes and DOX alone were calculated as presented in Laurus nobilis L. in this cancer cell line [Juniperus, Pinus and Cedrus were able to inhibit cell proliferation in both CCRF/CEM and CEM/ADR lines [The effect of selected sesquiterpenes  on the number of viable cells after 72-h treatment was tested using a neutral red uptake (NRU) or Alamar Blue (AB) assay in ovarian cell lines and lymphoblast cell lines, respectively. The results are demonstrated in ell line . On the DR lines .The use of drugs in combinations with natural compounds can cause significant shifts in efficacy in both positive and negative ways. The present study was aimed at testing and comparing the effect of selected sesquiterpenes on DOX efficacy in cancer cell lines with different sensitivity to DOX. The software CalcuSyn based on the Chou-Talalay method was used to evaluate the effect of DOX + sesquiterpene combinations . ExperimThe cancer cell lines used in our study significantly differed in sensitivity to DOX see . IncreasThe accumulation of DOX within the cells after their treatment with DOX alone and with combinations of DOX + sesquiterpenes is compared in Cell line CEM/ADR (as a DOX-resistant line with high level of ABCB1) was chosen to test the inhibitory effect of sesquiterpenes on Rhodamine 123 efflux. This substrate of the ABCB1 transporter is often used in activity assays in various tissues and cancer cells ,26. In oN-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) buffer, sodium pyruvate, d-glutamine, resazurin, rhodamine 123 (RHO 123) and neutral red were supplied by Sigma-Aldrich . Fetal bovine serum and gentamicin sulfate were purchased from Invitrogen  and bovine serum albumin (BSA) from Fluka . Doxorubicin and pure sesquiterpenes  were supplied by Sigma-Aldrich. Stock solutions of sesquiterpenes were prepared in dimethyl sulfoxide (DMSO) and stored in the dark at 4 °C. All other chemicals used were of HPLC or analytical grade and provided by Sigma-Aldrich.McCoy’s modified medium, RPMI-1640, d-glutamine, and CCRF/CEM and CEM/ADR were cultured in RPMI-1640 with 10% FBS, 1% HEPES, 1% sodium pyruvate. The resistance of CEM/ADR was maintained by incubation in 5 mM DOX for 24 h every week.Cell lines SKOV3 and A2780 were purchased from ATCC . Suspension cell lines CCRF/CEM and CEM/ADR5000 (CEM/ADR) were a gift from Prof. Wink, University of Heidelberg, DE. The cells were multiplied in three passages, frozen in aliquots and stored in liquid nitrogen. The absence of mycoplasma in all the cell lines used in the laboratory was periodically checked by Generi Biotech . For every set of experiments (each lasting 3–9 weeks) new batch of stored cells was used. SKOV3 were cultured in McCoy’s modified medium with 10% FBS, A2780 in RPMI-1640 with 10% FBS and 2 mM The sesquiterpenes and DOX were pre-dissolved in DMSO or in pure ethanol. The cells were exposed to various concentrations of the tested compounds in the culture medium. The final concentration of organic solvent in medium was 0.1%. Cells cultured in medium with pure DMSO or ethanol without the tested compounds were used as control samples. 10% DMSO served as a positive control. The number of viable cells was assayed after 72 h using the neutral red uptake test for SKOV3 and A2780 or Alamar blue for the suspension cell lines.The cells were cultured in 96-well plates. After 72 h exposure, the medium was removed and 200 μL of neutral red-containing medium was added into each well, and the plates were incubated for an additional 3 h at 37 °C. The cells were washed with 100 μL of PBS, then fixed in a solution of 0.5% formaldehyde/1% calcium chloride for 15 min. The neutral red dye was released from the viable cells with a lysing solvent (50% ethanol/1% acetic acid) by shaking for 30 min at room temperature. The absorbance of solubilized dye was measured using a spectrophotometer  at 540 nm. Each sample was assayed in 6 parallels and 3 independent experiments were performed. The viabilities of the treated cells were expressed as a percentage of untreated controls (100%). The effect of sesquiterpenes on DOX efficacy was evaluated using CalcuSyn software.The cells were cultured in 96-well plates, followed by 72 h exposure with 10 µL of Alamar blue solution (0.1 mg resazurin/1 mL PBS). After 1 h incubation at 37 °C, fluorescence was measured  using a fluoresce reader .g, 10 min, 4 °C). The supernatant was filtered through a 0.22 μm filter and the filtrate was used for analysis.A suspension of SKOV3 and A2780 cells  was set to a 12-well plate. After 48 h, 600 μL of solution in medium of the tested substances was added. The cells were treated with DOX (6 μM) alone, or in combination with individual sesquiterpenes (10 or 50 μg/mL) for 3 h. At the end of the experiment, the medium was removed and cells were washed with 500 μL of PBS. 100 μL of lysing buffer  added, at which time the cells were lysed for 15 min, then mixed with 300 μL of ice-cold methanol and centrifuged  equipped with a Zorbax C18 Eclipse Plus  column with a 1290 Infinity inline filter (Agilent). The HPLC method used by Skarka  was adapThe suspension of CEM/ADR cells (106/mL) was incubated with RHO123 . After incubation the cells were washed by PBS and incubated with the tested compounds in serum free medium, with verapamil (20 µM) used as a control. After a 2 h incubation, the cells were washed by PBS and fluoresce measured by a flow cytometer ; excitation was undertaken with a by 488 nm argon laser, 530 nm emission filter. Flow cytometry data were analyzed by Flowing Software 2.The cells were cultivated in 60 mm dishes, washed with PBS and lysed. Whole cell lysates were centrifuged . Supernatant was normalized for protein by BCA assay. For separation, 7.5% acrylamide gel was used. Proteins were transferred to a PVDF membrane by wet blot  and probed with anti-ABCB1 antibody  and anti-ACTB antibody . Secondary antibody with horseradish peroxidase was used and the blots were imaged by chemiluminescence captured on film.p < 0.05. The concentration inducing a 50% decrease of cell viability as compared to control (IC50) was calculated using GraphPad Prism 6.0 nonlinear regression.The data are presented as means ± S.D. of a given number of experiments. Statistical significance  was acceptable at a level of 50 than is normally achievable due to the lower solubility of the compounds. Only VAL had a measurable IC50 in all the tested cell lines, but in combination with DOX did not show a strong synergism in any cell line. Only NER showed a degree of synergism in all cell lines where it was possible to test for it. The effect from the combination of DOX with sesquiterpenes was not different from the effect of sesquiterpenes alone in the CEM/ADR cell line, despite the appearance of the largest shift of intracellular DOX concentrations. Further tests suggested the interaction of sesquiterpenes with efflux transporters as the possible mechanism of synergism with DOX.In summary, drug interactions of sesquiterpenes with DOX were cell line specific. Some of the sesquiterpenes had a higher estimated IC


Chlorinated compounds in a water environment pose serious threats to humanity. A nanoscale zerovalent iron (nZVI) has desirable properties for water dichlorination, but its reactivity is still limited by agglomeration and oxidation. In this study, the mesoporous carbon (MC) derived from biomass waste was prepared for immobilizing nZVI, and the nZVI@MC was further modified by sulfur (S-nZVI@MC) to relieve surface oxidation. The synergistic effect between nZVI and surface modification, the reaction conditions and the removal mechanism were investigated systematically. The characterization results showed nZVI was successfully loaded on the surface of MC, and the aggregation of nZVI was prevented. Moreover, sulfidation modification resulted in the formation of FeS on the surface of nZVI, which effectively alleviated surface oxidation of nZVI and promoted the electron transfer. Batch experiments demonstrated S-nZVI@MC had greatly enhanced reactivity towards 2,4,6-trichlorphenol (TCP) as compared to MC and nZVI, and the removal rate could reach 100%, which was mainly attributed to the significant synergistic effect of MC immobilization and sulfidation modification. Furthermore, the TCP removal process was well described by a Langmuir adsorption model and pseudo-second-order model. The possible mechanism for enhanced removal of TCP is the fast adsorption onto S-nZVI@MC and effective reduction by S-nZVI. Therefore, with excellent reducing activity and antioxidation, S-nZVI@MC has the potential as a pollutant treatment. Nowadays, the scarcity of water resources and increasing water contamination are a worldwide environmental problem that have drawn extensive attention. Chlorophenols are a class of typical organic contaminants that have been utilized widely in various industrial and agricultural fields. Specifically, 2,4,6-trichlorphenol (TCP) has been listed among United States Environmental Protection Agency (US EPA) priority pollutants due to its high toxicity, mutagenicity, and carcinogenicity ,2. VarioIn the past two decades, nanoscale zerovalent iron (nZVI) has been demonstrated to be an effective solution for the degradation/elimination of various pollutants including chlorinated organic compounds, heavy metal ions and antibiotics due to its strong reductive capability, low cost and environmental friendliness ,7,8. HowImmobilizing nZVI particles on porous material supports has been demonstrated as a tactful way to obtain high-efficiency and stable nZVI . On the 2 and other coexisting solutes, which would impede electron transfer from nZVI core to target contaminates [It should be noted that although supported nZVI can prevent the agglomeration and improve the stability of nZVI, it is easily oxidized by water, Oaminates . In viewaminates ,19,20. Oaminates . He et aaminates .4 in the presence of dithionite. The aggregation and surface oxidation of nZVI has been effectively alleviated due to the synergistic effect of loading and sulfidation, which is beneficial to improve its reactivity and life. The S-nZVI@MC was further used for TCP removal from water and the effects of solution pH, TCP initial concentration, S-nZVI@MC dosage were examined. The finding of this study paves the way to developing high-performance and low-cost nZVI for dichlorination and reduction of other pollutants.To take advantage of the recent development in nZVI, a sulfidized nZVI supported on MC (S-nZVI@MC) was prepared and used to remove TCP from aqueous solutions. As seen from −1) was used throughout the experiment.Materials: wheat straw was obtained from Yancheng, Jiangsu, P. R. of China. To remove impurities, the wheat straw is fully washed with deionized water and anhydrous ethanol before use. 2,4,6-Trichlorophenol  was purchased from Aladding Chemistry Co. Ltd., Shanghai, China. Hydrofluoric acid  was obtained from Sinopharm Chemical Reagent Co., Ltd., Shanghai, China. Milli-Q water , and then stirred for 15 min. The above solution was transferred into the Teflon vessel and reacted for 12 h at 180 °C. After cooling to room temperature, the product was washed by deionized water until reaching a pH of 7, and then dried at 80 °C in a conventional oven. Finally, the per-carbonized wheat straw was obtained.Per-carbonization of the wheat straw: in typical synthesis, wheat straw (3.0 g) was added into the H2 atmosphere with the ramp rate of 3 °C/min. After HF etching and water wash, the silica can be removed and MC was finally formed.Synthesis of MC: typically, the per-carbonized wheat straw was soaked in 1.0 wt % colloidal silica aqueous solution, and mass ratio of silica/straw was 3:10. The above solution was evaporated at 85 °C through continuous stirring until becoming a viscous solution. After drying at 80 °C in a conventional oven, a solid powder was formed. To obtain the MC, the precursor was calcinated at 800 °C for 2 h under a N2 inlet; 1.15 g of FeSO4·7H2O was then added to the three-neck flask, and after being stirred for about 20 min in N2 atmosphere, 100 mL containing 7.6 g sodium borohydride and different amounts of Na2S2O6  were added in a dropwise manner to the above FeSO4 solution. After the injection, stirring continued for 30 min to ensure adequate reaction. S-nZVI@MC were harvested by centrifugation and washed with ethanol three times and then dried in a vacuum. The obtained samples were carefully stored in the glove box filled with inert gas.Synthesis of S-nZVI@MC: 0.2 g MC was firstly added into 100 mL DI and ethanol mixture solution (V:V = 7:3). After sonicating for 60–120 min, the above mixture solution was poured into a 250 mL three-necked bottom flask equipped with a mechanical stirrer, and a thermometer with a temperature controller and a NThe detailed morphological information of the samples was further analyzed by transmission electron microscopy  and scanning electron microscopy  tests. Before the TEM testing, the samples were dispersed in ethanol via ultrasonication for 15 min, and then deposited a few drops onto a carbon-coated copper grid. X-ray diffraction  measurements were recorded by the X-ray diffractometerusing Cu Kα radiation . X-ray photoelectron spectroscopy  was tested on a Perkin-Elmer PHI 5000C ESCA  system with a dual X-ray source, using the 45 MgKα (1253.6 eV) anode and a hemispherical energy analyser. Before all tests, the samples were sealed into sample tubes in a glove box, and then shipped to the test instrument.−1) and solution pH (4–11) were further investigated. The solution pH was adjusted by 0.1 M HCl or NaOH solution. The solid-liquid phases were taken out after shaking for a predetermined time, and followed by filtration using 0.22 μm cellulose nitrate membrane filter. The concentration of TCP was measured with an ultraviolet–visible spectrophotometer . The removal efficiency was calculated according to the following equations:0 and Ct (mg L−1) are the initial and final concentrations of TCP in the feed solution, respectively.Batch adsorption experiments were conducted by shaking a mixture of obtained S-nZVI@MC with 100 mL TCP aqueous solution of certain concentration in a series of 150 mL plastic bottles. Such adsorption experiment parameters as contact time (0–180 min), initial concentration of TCP  on the XRD pattern of S-nZVI@MC, which may be due to the low sulfur content or poor crystallinity of sulfur species [2O3 was observed, indicating that only part of the nZVI reacted with TCP. In addition, the reaction between such non-target reactants as H2O and nZVI should be effectively inhibited, which is attributed to the protection effect of FeS [ species . After 3t of FeS .2−, Sn− and SO42−, respectively [2−, Sn− and SO42− is 27.45%, 48.30% and 24.24%. After the reaction with TCP, the peak intensity of S2− decreased obviously, accompany with the increase of Sn−  and Fe (III), respectively [2p1/2 for Fe (III). Besides, the satellite peak positions for Fe2p1/2 and Fe2p3/2 were 718 and 733.4 eV. Before reaction, two broad peaks at 724.8 eV (Fe2p1/2) and 711 eV (Fe2p2/3) corresponding to iron oxides (Fe2+ and Fe3+) are obvious  is strengthened, while the peak corresponding to Fe (II) is weakened  was the removal capacity of TCP at time t (min) and equilibrium, respectively. k1 (min−1) and k2 (g mg−1 min−1) was the equilibrium rate constant of both kinetic models [The adsorption kinetics of MC, nZVI and S-nZVI@MC were further investigated as the following equations:c models ,29.2, the pseudo-second kinetic model can better describe the TCP removal behavior, suggesting that the TCP removal by nZVI and S-nZVI@MC is controlled by chemical processes, and the electronic exchange existed between TCP and S-nZVI@MC. Obviously, the rate constant k2 of S-nZVI@MC was 0.0018 g mg−1 min−1, higher than that of MC and nZVI (0.0012 and 0.0007 g mg−1 min−1), which further reveals a higher affinity between S-nZVI@MC and TCP owing to the hydrophobic effect [k of S-nZVI@MC further indicates that the sulfide treatment can increase the reactivity.The kinetic fitting results were shown in c effect . The hig2- results in ineffective protection of nZVI, and thus the removal efficiency of TCP is lower. The removal efficiency decreased when the S/Fe further increased to 0.25, because excessive iron sulfide, especially FeS2 with a higher band gap (0.95) generated could slow down electron transfer [Fe/S is one of the most critical factors that would influence the reactivity of S-nZVI@MC. The S-nZVI@MC particles sysnthesized at different S/Fe were further used for TCP removal, and the results are presented in transfer . MoreoveAs known, the pH value of aqueous solution has great effect on nZVI. In acidic conditions, nZVI could be rapidly corroded and tends to exhibit better activity. While in alkaline conditions, a large hydroxide passivation layer is formed on the surface of zero-valent iron, thereby inhibiting its activity. It has been demonstrated that sulfidation modification can promote electron transfer and inhibits surface passivation. However, the effect of sulfidation on nZVI reactivity under different pH conditions still needs further discussion. Herein, the removal efficiency of S-nZVI@MC under different pH value ranging from 4 to 11 was investigated. As shown in −1 TCP aqueous solution and the results are shown in −1, respectively. When the dosage is high, there are more surface reactive and adsorptive sites in the reaction system, so the removal rate of TCP is higher. However, an excessive dosage is likely to cause agglomeration of materials and low utilization of reaction sites. Hence, the dosage of the further study is 0.5 g L−1 for TCP removal.The effect of S-nZVI@MC dosage was further investigated under the condition of pH = 4.0, 30 mg L−1. Generally, the TCP removal efficiency is decreased when the initial concentration increased. As the initial concentration increased from 30 to 100 mg L−1, the removal efficiency decreased from 91% to 32%. With higher initial concentration, the surface of S-nZVI@MC is easier to passivate, and thus the diffusion of contaminants and the electron transfer between nZVI and contaminants are inhibited, resulting in a reduced contaminants removal efficiency. Besides, at high concentrations, a large number of target contaminant molecules compete for limited reaction sites on the surface of the S-nZVI@MC, which also leads to reduced TCP removal efficiency [2 (0.993). In contrast, the Freundlich model has a relative lower R2 (0.936). Hence, the adsorption of TCP on the surface of S-nZVI@MC has been dominated by monolayer adsorption [ficiency . The ads−1 TCP solution with the dose of 0.5 g L−1 and reaction time of 3 h. As shown in Reusability is another critical property for the application of nZVI-based material. The reusability of S-nZVI@MC was further tested in 30 mg LIn summary, biomass-derived MC-loaded S-nZVI was successfully prepared for effective TCP removal. MC was selected as the support for nZVI, because its mesoporous structure was beneficial to nZVI dispersion and mass transfer. Moreover, MC prepared from biomass waste was a cost-effective material for practical application. Further surface sulfidation improved the antioxidation of nZVI due to the protection effect of FeS shell. SEM, TEM, XRD and XPS measurements demonstrated that the nZVI particles were successfully loaded on the MC supporter and the FeS was well coated on the surface the nZVI. Furthermore, the aggregation of nZVI and the iron oxide formation was prevented according the structure characterization. S-nZVI@MC was most effective for TCP removal as compared to MC and nZVI, and the removal efficiency reached the highest value at a ratio of S/Fe = 0.125. The effect of ambient conditions such as solution pH, initial concentration and dosage on TCP removal were also studied, and results revealed that the low solution pH value and concentration and higher dosage had positive effect on TCP removal. Furthermore, the removal behavior was fitted with a Langmuir model and pseudo-second kinetic model. The removal mechanism of S-nZVI@MC towards TCP could by analyzed by fast adsorption and effective reduction by S-nZVI. Therefore, the synergistic effect of MC loading and surface sulfide-modification promoted high removal efficiency of S-nZVI@MC towards TCP.


Background: Prunusarmeniaca is a member of the Rosacea family. The most important ingredient of this family is amygdalin that is believed to have anti-tumor and analgesic properties. The aim of this study was to evaluate the anti-proliferative effects of Armeniacae semen extract on the acute leukemia, NALM-6, and KG-1 cell lines, and investigate the effect of the extract on apoptosis of these cell lines and caspase-3 gene expression. Materials and Methods: We prepared aqueous, ethyl acetate, and hydro alcoholic extracts of the Armeniacae semen. The NALM-6 and KG-1 cell lines and mononuclear cells (PBMCs) of healthy controls were treated with different doses of the extracts for 48 hours, and then cell viability was investigated with the MTT test. High-Performance Liquid Chromatography was done for amygdalin identification. The percentage of apoptotic cells was determined using the Annexin V-FITC/PI flow cytometric kit, and caspase-3 gene expression was evaluated.Results: MTT test revealed that the strongest Inhibition Concentration (IC50) in KG-1 and NALM-6 cell lines was related to the ethyl acetate extract. This extract did not have toxic effects on PBMCs. Flow cytometric analysis showed that the ethyl acetate extract at its IC50 concentration led to almost 50% apoptosis in both cell lines after 48 hours. In the molecular examination, after treatment, a significant increase was seen in caspase-3 gene expression in NALM6 and KG1 cells compared to the control .Conclusion: Our data confirmed that the ethyl acetate extract of Prunusarmeniaca could reduce the proliferation of KG-1 and NALM-6 cell lines probably by activating the apoptotic pathway. Based on the type  Acute leukemia refers to rapid and clonal proliferation of lymphoid and myeloid progenitor cells in the bone marrow  7,8. of stem cell involved, it is divided into two major groups: AML (acute myeloid leukemia) and ALL (acute lymphoid leukemia)   10 . Rosaceous plants, which are widely distributed, produce different economically important products, including many edible fruits, as bitter almonds, apricots, peaches, plums, etc   11 . Hee-Young Kwon et al. (2003) showed that Persicae semen extract induces apoptosis in human promyelocytic leukemia (HL-60) cells12. Jasmina Makarevic et al. (2014) reported that amygdalin from apricot kernels affects bladder cancer cell adhesion and invasion in vitro   13 . Because of these features and the lack of coherent studies on various types of leukemia, we decided to use the Armeniacae semen, a member of the Rosacea family, which contains large amount of the amygdalin, to evaluate its anti-proliferative effect on the acute leukemia, NALM-6 (ALL) and KG-1 (AML) cell lines. In addition, we investigated the effect of the Armeniacae semen on apoptosis of these cell lines and caspase-3 gene expression.Amygdalin has an antitumor effect by settling carcinogens in the body, inhibiting the nutritional source of cancer cells, and blocking the growth of the tumor cells. It can also improve the symptoms of patients in the last stages of cancer and increase their survivalCell culture◦C with 5% CO2 and 95% humidity. The medium was changed every 2-3 days.NALM-6 and KG-1 acute leukemia cell lines , which were provided by the Pastor Institute of Iran, were grown and sub cultured in RPMI1640 containing 20mM HEPES-buffer and glutamax 1%  supplemented with 10% heat-inactivated FBS   and 100µg/ml penicillin/streptomycin (Biosera). Mononuclear cells were isolated from the peripheral blood of healthy individuals using Ficoll-Paque. The cultures were incubated at 37Extracts preparationTwo hundred grams of the Armeniacae semen was hatched from the shell and dried in the shade for a week. The seeds were then crushed by a pounder. At first, the dry powder was macerated in a petroleum ether-solvent to remove oils to not disturb the cytotoxic test. After oil extraction, the seed powder was allowed to dry completely and the solvent evaporate. Then, 50 g of the powder was weighed to prepare an aqueous extract. For this purpose, the seed powder was macerated in 90 ° C water for 30-45 minutes. Then, 500 ml of ethyl acetate, was added to the rest of the powder. After 48 hours, the solvent was removed and a new ethyl acetate solvent was added again. The procedure was repeated 3 times. The above processes were also repeated for 60% methanol. The extracts from each step were filtered with filter papers, transferred to a rotary balloon  and concentrated at 100 rpm at 40 ° C.MTT assay4 cells/well. Both cell lines were treated with aqueous, methanol, and ethyl acetate extracts of the Armeniacae semen at concentrations of 0.125, 0.25, 0.5, 1 mg/ml for 48h. PBMCs were treated with the extract that had the best IC50 on the MTT test at concentrations of 0.125, 0.25, 0.5, 1 mg/ml for 48h. After this time, 50µl MTT  was added to each well and incubated for 4h. Then, the supernatant was removed and 100μl DMSO  was added to dissolve MTT. Following incubation for 15 minutes, the OD was read with an ELISA plate reader  at 492 nm. The assay was performed at least three times.To check cell viability, we used the 3--2, 5 di-phenyl tetrazolium bromide (MTT) assay. The cells were seeded in a 96-well plate at a concentration of 5×10SPE methodA solid-phase extraction cartridge (Chromafix-Germany) was used to separate and concentrate the target material of the extract, including a mixture of different materials. At first, the cartridge was washed with 5 ml of methanol and activated with 5ml of water. Then, 5 ml of the extract at a concentration of 20 mg/ml was passed through the cartridge. After washing with 10% methanol aqueous solution, to remove co-adsorbents, the remaining analytes were collected on SPE with 5 ml of pure methanol.Amygdalin identification by reverse phase HPLC18 column (250×4.6mm). The column temperature was 30°C, and the detection wavelength was set at 215 nm. At first, the amygdalin standard was injected to the Agilent 1260 chromatographic system, including a UV detector and ChemStation data system at a concentration of 100μg / ml and a volume of 20 μl, and then the exact location of the peak was determined. This action was repeated 3 times. Then, 20 μl of the specimen (isolated by SPE) was injected and after peak observation, the test was repeated 3 times. By comparing the peak area of the standard and sample, the percentage of amygdalin in the extract was obtained.In order to Amygdalin identification in the extract that had the best IC50 on the MTT test, the gradient method was used to elute other components quickly and reduce the analytical time after amygdalin peak observation. For this purpose, the mobile phase was adjusted to 15: 85 methanol-water for 30 minutes and pure methanol after 30 minutes. The mobile phase was filtered before use with a vacuum filter system containing a 0.45μm filter (Millipore-Germany). Separation was conducted on a CFlow cytometry6 cell/ well on a 6-well plate. Then, the concentration of (the best) IC50 was added to each well. After 48 hours, flow cytometry was performed by the BD FACS Calibur Flow Cytometry Machine (BD Biosciences_ USA), and the results were analyzed using the FlowJo.7.6.1 software. We conducted flow cytometry measurements based on the protocols of eBioscience.In order to determine the amount of primary and secondary apoptosis induced under the influence of the extract in each cell line, the cells were grown at a concentration of 1×10RT-PCRΔΔCT method.RT-PCR was performed to identify caspase-3 gene expressions. For this purpose, the cells were collected after 48 hours of treatment with the extract that had the best IC50 on the MTT test, and their total RNA was extracted using the Trizol  method. Then, 1µg total RNA was converted to cDNA using the PrimeScript 1st strand cDNA Synthesis kit  according to kit manufacturer’s instructions. RQ-PCR assay was performed to investigate the level of caspase-3 gene expression in drug-treated and control groups using primers in Table 1. β-2 microglobulin (B2M) was used  as a housekeeping gene for normalization of RT-qPCR data .It should be noted that all tests were performed in triplicate. The fold change Casp3 mRNA in treated cells in comparison with untreated cells was computed by the 2-Statistical analysisAll Experiments were performed in duplicate and repeated three times. IC50 was calculated using the Excel 2013 software, and flow cytometry analysis was done using the FlowJo.7.6.1 flow cytometry software. The data are expressed as mean ± SD for all experiments. GraphPad Prism 5.0  was used to detect significant differences between the control and treated groups. Statistical significance was defined at *P<0.05, **P<0.01, and ***P<0.001 compared to the corresponding controls.Ethyl acetate extract had the best IC50 on the MTT testWhen cells were treated with the aqueous methanol, and ethyl acetate extracts of the Armeniacae semen at concentrations of 0.125, 0.25, 0.5, 1 mg/ml for 48h, the MTT test showed that cell proliferation was inhibited in a dose-dependent manner with all extracts . The besAmygdalin identification by HPLCThe results of the HPLC test indicated the presence of amygdalin in the ethyl acetate extract of Armeniacae semen. There was 0.67% amygdalin in this extract that was calculated as follows: Area sample/ Area STD×C STD.Analysis of apoptosis by double staining KG-1 and NALM-6 cells with annexin V-FITC and PIKG-1 and NALM-6 cells became apoptotic after they were treated with the ethyl acetate extract of Armeniacae semen. The distribution of apoptotic cells measured by flow cytometry showed that the percentage of early (Q3) and late-apoptotic cells (Q2) was 63±1.48% and 32.7±2.31% in KG-1 and 59.22±1.43% and 30.61±1.94% in NALM-6 cell lines, respectively.Effect of Ethyl acetate extract of Armeniacae semen on casp3 gene expressionp<0.05 in KG1 and p<0.001 Nalm6 compared to the untreated control cells.)Molecular examination 48 hours after treatment of KG-1 and NALM-6 cell lines showed a significant increase in caspase-3 gene expression in test samples compared to the controls.  and KG-1 (acute myeloid leukemia) cell lines. 10,11,14,15 . In 2005, Park HJ et al. reported that the extract from the amygdalin has a dose-dependent cytotoxic effect on human colon cancer (SNU-C4)   11 . They also reported that treatment of human chronic myeloid leukemia (K562 cell line) with amygdalin at different concentrations resulted in decrease in cell viability in a dose-dependent manner    14 . In other studies, cytotoxic effect of amygdalin was shown on prostate cell lines (DU145 and LNCaP) and human cervical cancer HeLa cells 10,15.In the present study, based on the evaluation of cell survival by the MTT assay, the cell viability in both KG-1 and NALM-6 cell lines was dose dependent and the survival rate of the cells reduced, with an increase in the concentration of the mixture. Moreover, the best IC50 was related to the ethyl acetate extract in both cell lines. The effect of the extract on the cell viability seems to be dependent on the cell type. Our results are in line with other studies, showing cytotoxic effect of amygdalin on various cancer cell types 7,10,15,16 . Evaluation of the treatment of UMUC-3, TCCSUP, and RT-112 bladder cancer cell lines with amygdalin at concentrations of 1, 10, 25 mg/ml using the Annexin-V-FITC/PI kit showed  a dose-dependent relationship between amygdalin concentration and Annexin V positive cellsSince most anticancer and cytotoxic compounds induce apoptosis in tumor cells, a quantitative study of apoptosis was performed in both KG-1 and NALM-6 cell lines treated with the ethyl acetate extract of Armeniacae semen. Analysis of flow cytometric results in both cell lines indicated an almost 50% apoptosis at IC50. Moreover, investigation of caspase-3 (executive pathway) gene expression at IC50 showed a significant increase in both cell lines compared to controls. The other studies involving other cancerous cell lines also confirmed apoptosis induction and increased caspase-3 activity by amygdalin    10 . Lee et al. showed that amygdalin induces apoptosis in Hs578T breast cancer cells through the caspase-3 pathway    16 . The key role of Caspase-3 in apoptotic pathways is well established. Therefore, the mRNA expression level of Casp3 was assessed to evaluate the effect of the amygdalin extract on the induction of cell death. Our gene expression study showed that the ethyl acetate extract of Armeniacae semen increases the Casp3 at mRNA level. Chang et al. showed that amygdalin increased caspase-3 activity significantly in both DU145 and LNCaP prostate cancer cell lines17,18. Some other studies suggest that rhodanase, which is capable of eliminating cyanide toxicity, exist in normal tissues, but is ineffective in cancer cells. Combined activity of the two enzymes may be responsible for inducing toxic effects in amygdalin-treated cancer cells, while normal cells remain unaffected11,19.The effect of the ethyl acetate extract of Armeniacae semen on PBMCs isolated from normal blood at different concentrations did not show significant cytotoxic effects compared to the negative control. There are hypotheses about the mechanisms of the differences in the response of normal and cancerous cells to amygdalin. Some studies suggest that the cancer cells are rich in β-glucosidase, which is capable of digesting amygdalin to produce cyanide, leading to toxic effects on cancer cells Taken together, the results of the present in vitro study (which never was done before on these types of leukemia) suggest that the ethyl acetate extract of Armeniacae semen have anticancer effects on AML and ALL. However, future studies should focus on the isolation of the effective ingredients of the ethyl acetate extract and evaluation of their effects on the KG-1 and NALM-6 cell lines in human and animal models as well as the assessment of the effect of the extract on the cell cycle. In addition to measuring the expression of caspase-3 gene, the activity of this enzyme should be assessed through the caspase-3 activity measurement kit.


Spatholobus suberectus Dunn are a traditional Chinese medicine activating blood circulation and removing stasis. They have often been used for the traditional Chinese medicinal treatment of breast cancer in modern China. In this study, four novel isoflavanes (1–3 and 5) and four known analogues (4 and 6–8) were isolated from an ethanolic extract of the rattans of S. suberectus. Their structures were elucidated by extensive spectroscopic analyses and electronic circular dichroism studies. MCF-7 and MDA-MB-231 human breast cancer cell lines were used to evaluate the cytotoxic effects of the isolates. Interestingly, compounds 1 and 2 only inhibited the proliferation of MCF-7 cells, while compound 6 showed a selective cytotoxicity against MDA-MB-231 cells. However, compound 4 had significant cytotoxicity against both MCF-7 and MDA-MB-231 cell lines.The rattans of  Spatholobus suberectus Dunn (Leguminosae) is a representative traditional Chinese medicine historically used to promote blood circulation and remove stasis. As traditional Chinese medicine theory considers cancer as a kind of disease most possibly related to severe blood stasis, S. suberectus is commonly used for the treatment of cancer in China  colorimetric assay using an estrogen-dependent breast cancer cell line (MCF-7) and an estrogen-independent breast cancer cell line (MDA-MB-231). It is interesting that isoflavanes 1, 2, and 6 showed selective cytotoxicity, and isoflavane 4 inhibited the proliferation of both MCF-7 and MDA-MB-231 cell lines.Isoflavanes and isoflavones are two subclasses of isoflavonoids, which are plant secondary metabolites characterized by a B-ring attached at the C-3 position of the C-ring. They are known for their significant estrogen-like activity, and modern research suggests that they also have antiproliferation in many kinds of human cancer cell lines ,15,16,17es (1–8) , includi1 was isolated as a white amorphous powder, and the presence of hydroxy groups (3446 cm−1) and aromatic rings (1614 and 1495 cm−1) was indicated in its infrared (IR) spectrum. High-resolution electrospray ionization mass spectrometry (HRESIMS) data at m/z 317.1389 [M + H]+  and 339.1208 [M + Na]+  revealed the molecular formula of C18H20O5 for 1. The 1H nuclear magnetic resonance (NMR) spectrum of 1 displayed resonances corresponding to a 1,2,3,4-tetrasubstituted phenyl [δH 6.71  and 6.51 ], a 1,2,4-trisubstituted phenyl , two methylenes , a methine [δH 3.56 (m)], three methoxy groups , and an exchangeable hydroxy group (δH 5.63) (13C-NMR and distortionless enhancement by polarization transfer (DEPT) spectra of 1 showed 18 carbon signals , 6 × CH (five aromatic methines), and 7 × C (five oxygenated aromatic carbons). These spectroscopic data indicated that compound 1 was an isoflavane possessing a hydroxy and three methoxy groups  and a methylenedioxy  in 2 replaced the 1,2,4-trisubstituted phenyl and the OMe-4′ in 1, respectively. In the HMBC spectrum of 2, correlations of H-3′ with C-1′, C-2′, C-4′, and C-5′; of H-6′ with C-3, C-2′, C-4′, and C-5′; of OMe-2′ with C-2′; and of 4′-OCH2O-5′ with C-4′ and C-5′ confirmed that the methylenedioxy group in 2 was linked to C-4′ and C-5′. The ECD spectrum of 2 exhibited similar Cotton effects  to 1. Although the specific optical rotation -7-hydroxy-8,2′-dimethoxy-4′,5′-methylenedioxyisoflavane.Compound  2 and 1  suggesteguration . Consequ3 was another isoflavane, as indicated by the HRESIMS, IR, 1H-NMR, and 13C-NMR data. Comparison of the 1H and 13C-NMR data of 3 and 1 suggested that the OMe-2′ in 1 was replaced by a hydroxy group in 3. Thus, the planar structure of 3 was established as 7,2′-dihydroxy-8,4′-dimethoxyisoflavane (8-methoxyvestitol), which was reported in 1979   . These sS)-sativan (4) c 0.08, MeOH); CD (MeOH) 238 (Δε +2.05), 284 (Δε −0.98) nm; IR νmax: 3446, 2939, 2839, 1614, 1495, 1462, 1287, 1201, 1173, 1151, 1088, 1036, 966, 928, 824, 803 cm−1; 1H-NMR data, see 13C-NMR data, see m/z 317.1389 [M + H]+ , 339.1208 [M + Na]+ , 355.0949 [M + K]+ . The original HRESIMS, IR, and 1D- and 2D-NMR spectra of 1 were shown in (3S)-7-Hydroxy-8,2′-dimethoxy-4′,5′-methylenedioxyisoflavane (2): White amorphous powder, [α]c 0.03, MeOH); CD (MeOH) 242 (Δε +1.86), 283 (Δε −0.62) nm; IR νmax: 3370, 2920, 2851, 1579, 1543, 1467, 1261, 1156, 1116, 1041, 799, 721 cm−1; 1H-NMR data, see 13C-NMR data, see m/z 331.1178 [M + H]+ , 353.1000 [M + Na]+ , 369.0738 [M + K]+ . The original HRESIMS, IR, and 1D- and 2D-NMR spectra of 2 were shown in (3S)-7,2′-Dihydroxy-8,4′-dimethoxyisoflavane (3): White amorphous powder, [α]c 0.05, MeOH); CD (MeOH) 234 (Δε +2.43), 284 (Δε −0.50) nm; IR νmax: 3443, 2921, 2851, 1617, 1595, 1463, 1242, 1200, 1165, 1088, 1040, 960, 840, 794, 720 cm−1; 1H-NMR data, see 13C-NMR data, see m/z 325.1052 [M + Na]+ . The original HRESIMS, IR, and 1D- and 2D-NMR spectra of 3 were shown in 7,2′-Dihydroxy-8-methoxy-4′,5′-methylenedioxyisoflavan-3-en-2-one (5): Yellow powder, IR νmax: 3257, 2921, 2851, 1711, 1601, 1505, 1463, 1359, 1317, 1239, 1203, 1087, 1033, 975, 857, 803 cm−1; 1H-NMR data, see 13C-NMR data, see m/z 351.0473 [M + Na]+ . The original HRESIMS, IR, and 1D- and 2D-NMR spectra of 5 were shown in 50 values represented the mean of three independent replicates.The cytotoxic activity of the isolates against the MCF-7 and MDA-MB-231 human breast cancer cells were determined by a colorimetric MTT assay, as described in the literature ,28. PaclSpatholobus suberectus mainly contains flavonoids, including flavones, flavanones, chalcones, isoflavanes, and isoflavones, some of which have a cytotoxic effect against breast cancer cell lines. In this study, four novel isoflavanes (1–3 and 5) and four known analogues (4 and 6–8) were isolated from the rattans of S. suberectus. Compounds 1, 2, and 6 showed selective cytotoxicity against the estrogen-dependent breast cancer cell line (MCF-7) and the estrogen-independent breast cancer cell line (MDA-MB-231) , while compound 4 had cytotoxicity against both cell lines. Preliminary analysis of the structure–cytotoxicity relationship suggested that C-2′ and C-8 substituents may affect the cytotoxicity and selectivity.


The function of human genes can be strongly inferred from their knockout fitness profiles across hundreds of CRISPR screens, illuminating the modular organization of the cell. Genetic interactions mediate the emergence of phenotype from genotype. The systematic survey of genetic interactions in yeast showed that genes operating in the same biological process have highly correlated genetic interaction profiles, and this observation has been exploited to infer gene function in model organisms. Such assays of digenic perturbations in human cells are also highly informative, but are not scalable, even with CRISPR-mediated methods. As an alternative, we developed an indirect method of deriving functional interactions. We show that genes having correlated knockout fitness profiles across diverse, non-isogenic cell lines are analogous to genes having correlated genetic interaction profiles across isogenic query strains and similarly imply shared biological function. We constructed a network of genes with correlated fitness profiles across 276 high-quality CRISPR knockout screens in cancer cell lines into a “coessentiality network,” with up to 500-fold enrichment for co-functional gene pairs, enabling strong inference of gene function and highlighting the modular organization of the cell. Saccharomyces cerevisiae, systematic genetic deletion studies showed that only ∼1,100 of its ∼6,000 genes (∼20%) were required for growth under laboratory conditions  or between members of parallel pathways (“between pathway interactions”) . When asS. cerevisiae genome has less than one-third the number of protein-coding genes as humans, and despite the quantum leap in technology that the CRISPR/Cas system offers to mammalian forward genetics, yeast remains far simpler to perturb reliably in the laboratory. Several groups have applied digenic perturbation technologies, using both shRNA and CRISPR, to find cancer genotype-specific synthetic lethals for drug targeting . We used the log-likelihood score (LLS) to describe the significance of enrichment. Gene pairs are ranked by Pearson correlation, grouped into bins of 1,000 pairs, and each bin is evaluated for the relative abundance of genes annotated to be in the same KEGG pathway (“true positives”) versus genes annotated to be in different pathways . Data from 13 edges . It has 13 edges . We eval13 edges , 2016.BRAF and related genes that are highly specific to BRAF-mutated melanoma cells , indicating their essentiality in BRAF-mutant cells, supporting efforts to incorporate ERK inhibitors into combinatorial therapies to overcome resistance to targeted BRAF treatments , melanogenesis-associated transcription factor, the developmental Sry-related box (Sox) gene SOX10, and mesenchymal marker ZEB2, indicative of the non-epithelial origin of melanocyte cells and providing insight into the genetic requirements for tissue differentiation in this lineage.The data underlying the Cancer Coessentiality Network is derived from well-characterized cancer cell lines from 30+ lineages, representing the major oncogenic mutation profiles common to those cancers. We expected that many clusters in the network could, therefore, be associated with specific tissues and cancer-relevant genotypes. By testing cluster-level essentiality profiles for tissue specificity , we identified only a small number of clusters that correspond to tissue-specific cancers , which ieatments . This exP < 10−7), contains oncogene FLI1 and tissue-specific TF MYB (P < 10−7) and carries lineage-specific TF PAX8, previously shown to be essential in these cells (P < 10−9), contains β-catenin (CTNNB1) and TF partner TCF7L2   , indicatMYCN, the neuroblastoma-specific paralog of the MYC oncogene , MDM4 , and PPM1D  MYCN, the< 10−44) .TP53 shows the characteristic extreme negative essentiality score of a tumor suppressor gene in wild-type backgrounds, surprisingly, it causes a growth defect when knocked out in three cell lines: HCT1143 breast cancer, PC14 lung cancer, and NB4 AML cells , an oxygen sensor that hydroxylates hypoxia response genes HIF1A and HIF2A, marking them for degradation by the VHL complex in normoxic environments .Although ML cells . All thrdetected . Nor is opy loss . In contkgrounds . The essronments . EGLN1 eKRAS amplifications in pancreatic and colorectal cancer (cluster 276), ERBB2 amplifications in breast and other cancers (cluster 52), and CCNE1 overexpression/RB1 mutation (cluster 101), consistent with well-studied patterns of oncogenesis. All network cluster annotations can be found in the master annotation file (Table S7).These examples indicate the breadth and precision of the coessentiality network but represent results from hypothesis-guided queries. In an effort to learn novel associations from the data, we tested each cluster for its correlation with cell lineage as well as correlation with gene expression, mutation, and copy number amplification of all genes both inside and outside the cluster to identify underlying molecular genetic drivers of modular, emergent essentiality. We identified 270 genes in 30 clusters whose essentiality profiles strongly correlated with their own copy number profiles but not their expression profiles . As copyGiven the underlying data, it is not surprising that oncogenic signatures are clearly evident in the coessentiality network. However, the vast majority of the network structure does not appear to be driven by tissue specificity or mutational signatures. The network contains information complementary to prior functional  and physP-value < 10−6, and fitness profiles have been used to implicate additional members of protein complexes  plus additional subunits of all other ETC complexes. The cluster also contains 49 of 51 subunits of the mitochondrial large ribosomal subunit (P < 10−87), 23 of 25 members of the small subunit (P < 10−39), plus 20 mitochondrion-specific tRNA synthases (P < 10−20). This mitochondrial translation machinery is required for the synthesis of proteins in the ETC complexes. These genes’ inclusion in this cluster, where their essentiality profiles are correlated with those of the complexes they support, reflects a fundamental feature of saturating genetic screens: the essentiality of a given enzyme or biological process is matched by the essentiality of the cellular components required for the biogenesis and maintenance of that process.Epistatic interactions frequently underlie covariation in fitness profiles . ClusterGPX4 shows highly variable essentiality across cell lines , the “21st amino acid,” at its active site. Coessential with GPX4 are all the genes required for conversion of serine-conjugated tRNASer to selenocysteine-conjugated tRNASec , as well as selenocysteine-specific elongation factor EEFSEC, which guides Sec-tRNASec to specific UGA codons , are found in a cluster with 10 PEX genes involved in peroxisome biogenesis, maintenance, and membrane transport , RAB3GAP1 and RAB3GAP2, and guanine nucleotide exchange factor (GEF), TBC1D120 ; canonical mTORC1/mTORC2 regulatory and signaling components PDPK1, AKT1, and PIK3CB; plus G-protein subunit GNB2, previously shown to physically interact with mTOR in response to serum stimulation —is reflected in the anti-correlation of fitness profiles connecting the TSC1/2 cluster and the mTOR cluster.A similar network of modules describes the regulation of the mechanistic target of rapamycin (mTOR), in particular, its detection of cellular amino acid levels . Fig 6 smulation . CanonicMTOR response to cellular amino acid levels is modulated by an alternative pathway that functions at the lysosomal membrane (HOPS complex (VPS26/29/35 retromer complex (RRAGA) and RagC (RRAGC) that transmit information on amino acid abundance to mTORC1 (FLCN) (membrane . We iden complex  and the  complex . This sto mTORC1 . The Rago mTORC1  and foll1 (FLCN) , also me1 (FLCN)  and is, 1 (FLCN)  is refleLCMT1, TIPRL, and PTPA), whose strong connectivity to the TSC1/2 complex may suggest a regulatory role for PP2A in MTOR signaling. PP2A has previously been posited to be an activator of TSC1/2 upstream of MTOR . IGF1R is a highly N-glycosylated RTK, and the IGF1R complex is tightly connected to the OST complex in our network. EGFR, also highly glycosylated . Prior work suggests that membrane glycolipid composition can strongly influence EGFR autophosphorylation and signaling . Although the coessentiality network does not capture a large portion of protein–protein interactions  or genetReconstructing biological processes from coessentiality information has some limitations. First of all, interactions between genes that do not affect cell fitness cannot be captured. Second, interactions between gene pairs that are not perturbed in the cell line pool cannot be captured. Last, the presence of genetic alterations like mutation or copy number amplification can generate confounding effects. A critical next step will be to understand the underlying context that drives the emergent essentiality of specific bioprocesses in specific backgrounds. The health implications of this question are profound. In cancer, to understand the causal basis of modular emergent essentiality is to identify matched pairs of biomarkers  and precision targets  for personalized chemotherapeutic treatment. In addition, lineage-specific essential processes could provide explanatory power for germline mutations causing tissue-specific disease presentation, in cancer as well as other diseases.https://hartlab.shinyapps.io/pickles/ (Expanding the coverage of the network will require different screening approaches. Fitness screens in cancer cell lines in rich media will miss cellular dependencies that are present only under stress conditions. In yeast  and nemapickles/  and downhttps://figshare.com/articles/_/5520160). We only kept protein-coding genes for further analysis and updated their names using HGNC (https://github.com/hart-lab/bagel). In comparison with published BAGEL version v0.92  (Table S5). All network figures shown in this article were drawn using Cytoscape , which is the harmonic mean of precision and recall at Bayes factor 5. Then, among 338 cell lines, 276 cell lines were selected for further study, by F-measure (>0.85) and the number of essential genes , to prevent noise from marginal quality of screens .Table S4 Network interactions of coessentiality network.Table S5 Performance benchmark of coessentiality network was conducted by a pathway enrichment test using the KEGG pathway database (downloaded in 2015). Pathways having >200 annotated genes  were discarded to minimize bias. For each network, gene pairs were ranked by PCC and binned into groups of 1,000 pairs. The cumulative LLS  was calcPreprint; https://portals.broadinstitute.org/achilles). For Wang et al screens, we downloaded raw read counts from their article. For https://depmap.org/portal/). We calculated Bayes factor profiles from raw read counts of screens and controls defined in articles through the BAGEL pipeline. To filter low-quality screens out, we applied thresholds of F-measure (BF = 5) 0.80 for To compare performance with other screens, we constructed coessentiality networks for two other CRISPR data sets  and threNetwork clustering was conducted with the Markov Cluster Algorithm (MCL) . We firsGene clusters derived by MCL algorithm.Table S6 Pathway annotations  of major clusters.Table S7 P-values of differential expression, copy number, and mutation between mean essentiality of clusters and gene properties. For RNA-seq expression, we used log2(FPKM + 0.5). For copy number variation, we discretized copy number value into three classes . For mutation data, we made a binary profile of mutation presence, treating silent mutations as wildtype. To measure significance of associated factors, we conducted a t test for expression data, Fisher’s exact test for copy number , and Fisher’s exact test for binary mutation data.To identify molecular genetic factors associated with cluster essentiality, we downloaded RNA -seq , copy number variation, and mutation profiles from the Cancer Cell Line Encyclopedia (CCLE) database  in 2017.P-value was measured by Bonferroni correction of P-value.Tissue-specific essentiality was calculated for 517 clusters and 3,483 essential genes against 19 tissues: upper aerodigestive tract, thyroid, large intestine, autonomic ganglia, soft tissue, central nervous system, haematopoietic and lymphoid tissue, stomach, endometrium, liver, urinary tract, bone, lung, breast, skin, oesophagus, ovary, kidney, and pancreas. For each cluster, we first calculated mean essentiality of member genes per cell line. Then, for each tissue, we conducted Wilcoxon rank sum tests of two groups, a group belonging to the target tissue type, and a group consisting of all other tissue types. Last, adjusted To investigate how many interactions are overlapped with the coessentiality network from Avana screens, we downloaded functional interaction networks  were kept for further steps. To prevent abnormal outlier or zero values, we gave 0.5 pseudo count to all genes, divided each value by the mean of each gene, and took the log form of the resulting value. Expression similarity for all possible combinations of two genes was measured by PCC . Proteinhttps://www.cancerrxgene.org/gdsc1000/GDSC1000_WebResources/Home.html.Drug log(IC50) values used for correlation analyses were taken from the Genomics of Drug Sensitivity in Cancer (GDSC) database . Data tastats (version 3.2.3), based on mean gene BF in a cluster in a cell line against the matching cell line log IC50 value of each drug. Correlations calculated had between 34 and 187 data point pairs  within the overlapping 192 cell lines between GDSC database and Avana project. Pearson correlations that resulted in a negative correlation with a P-value less than 10−4 were added to the annotation text file. Negative correlations imply that IC50 values decrease as mean cluster BF in a given cell line increases . Correlations were calculated for the top 309 co-correlation ranked clusters.Cell line annotation style from TableS4A was altered to match Avana project cell line annotation style. GDSC data contained log (IC50) values from 990 cell lines, which overlapped with 192/276 cell lines used in the Avana project. Log IC50 values contained 265 unique GDSC drug IDs with 250 unique drug names. Pearson correlations were computed using cor.test from the R package https://portals.broadinstitute.org/ccle/data. We used the gene-centric RMA-normalized expression data. Also, we used preprocessed RNA-seq and drug information (log IC50 data) used for other analysis in this article.The CCLE Reverse Phase Protein Array (RPPA) data, RPPA antibody information, and cell line annotations of 1,037 cancer cell lines were retrieved from the CCLE portal at: Essentiality scores for each gene were preprocessed using quantile normalization within each cell line. The quantile-normalized essentiality scores for the selected genes for each of the 276 cell lines were gathered in a matrix. Cell lines with essentiality scores lower than or equal to −10 were set at a Bayes factor of −10.Next, the heat map was plotted sorting the cell lines by the mean Bayes factors for each gene in the cluster by using the matplotlib package in Python. The heat map was annotated by the presence of TP53 mutations (orange) with missing values as black and the Nutlin 3a natural log half maximal inhibitory concentration (IC50) values for each cell line with missing values as light grey.MYC gene was added to Cluster 43 (MYCN) genes, and their quantile-normalized essentiality scores were gathered in a matrix for all high-quality Avana project cell lines. The heat map was plotted sorting the cell lines by the mean Bayes factors for each gene in the cluster. The heat map was annotated with MYC and MYCN expression values as well as a tissue key, specifying the neuroblastoma cell lines in orange.From the 64k network, the From the 64k network, the quantile-normalized essentiality scores of genes from Cluster 17 were gathered in a matrix for all high-quality Avana project screens. The heat map was plotted sorting the cell lines by the mean Bayes factors for each gene in the cluster and annotated by a tissue key specifying the cell lines from the hematopoietic and lymphoid tissues in orange.ERBB2gene, the RPPA values for ERBB2, expression values for ESR1 and CP724714, and Refametinib log IC50 values for each cell line with missing values as light grey.Clusters 52 and 55 from the 64k network were combined and the quantile-normalized essentiality scores for each gene in the clusters were gathered in a matrix after filtering for the cell lines from the breast tissue. The heat map was plotted by sorting with the mean essentiality scores for the genes in the clusters across the breast cell lines. The heat map was annotated with the log2 copy number values for the BRAF, and the log IC50 values for PLX-4720, with missing values in light grey.From the 32k network, quantile-normalized essentiality scores for the genes in cluster 19 were gathered in a matrix for all high-quality Avana cell lines. The cell lines were sorted by the mean Bayes factors and a heat map was plotted. The heat map was annotated with log2 copy number, RPPA values, presence of mutation (in orange) for We filtered for the cell lines in which at least one of the following RTKs: EGFR, ERBB2, ERBB3, FGFR1, IRS2, and IGF1R have a BF of greater than 20. Then for that subset of cell lines, the essentiality scores of the RTKs and their downstream effectors were gathered in a matrix. Next, in Python, the hierarchical clustering package called scipy.cluster.hierarchy was used to cluster the cell lines for each RTK. We used the “average” method representing the UPGMA algorithm and the “Euclidean” distance metric to calculate the distance between the newly formed cluster and each remaining cluster and perform hierarchical clustering. The clustered heat maps were annotated for EGFR and ERBB2 copy number data.We filtered for the cell lines in which at least one of the following genes: KRAS, NRAS, BRAF, and PIK3CA had a BF of greater than 20. Next, we obtained the essentiality scores of the RTKs and their downstream effectors for the filtered cell lines. Later, we performed hierarchical clustering to cluster the cell lines for each RTK as explained above. Next, the clustered heat maps were annotated with mutation status for KRAS, NRAS, BRAF, and PIK3CA in orange.P-values were calculated using the scipy.stats.pearsonr from the scipy package based on the mean cluster essentiality score in a cell line against F-measure value of the matching cell line.For each cluster in the network, the mean Bayes factor of the genes in that cluster was calculated to get a mean essentiality score for the cluster for each cell line. Then, the Pearson correlations and corresponding