Epithelial ovarian cancer (OC) is a heterogenous disease consisting of five major histologically distinct subtypes: high-grade serous (HGSOC), low-grade serous (LGSOC), endometrioid (ENOC), clear cell (CCOC) and mucinous (MOC). Although HGSOC is the most prevalent subtype, representing 70–80% of cases, a 2013 landmark study by Domcke et al. found that the most frequently used OC cell lines are not molecularly representative of this subtype. This raises the question, if not HGSOC, from which subtype do these cell lines derive? Indeed, non-HGSOC subtypes often respond poorly to chemotherapy; therefore, representative models are imperative for developing new targeted therapeutics.Non-negative matrix factorisation (NMF) was applied to transcriptomic data from 44 OC cell lines in the Cancer Cell Line Encyclopedia, assessing the quality of clustering into 2–10 groups. Epithelial OC subtypes were assigned to cell lines optimally clustered into five transcriptionally distinct classes, confirmed by integration with subtype-specific mutations. A transcriptional subtype classifier was then developed by trialling three machine learning algorithms using subtype-specific metagenes defined by NMF. The ability of classifiers to predict subtype was tested using RNA sequencing of a living biobank of patient-derived OC models.Application of NMF optimally clustered the 44 cell lines into five transcriptionally distinct groups. Close inspection of orthogonal datasets revealed this five-cluster delineation corresponds to the five major OC subtypes. This NMF-based classification validates the Domcke et al. analysis, in identifying lines most representative of HGSOC, and additionally identifies models representing the four other subtypes. However, NMF of the cell lines into two clusters did not align with the dualistic model of OC and suggests this classification is an oversimplification. Subtype designation of patient-derived models by a random forest transcriptional classifier aligned with prior diagnosis in 76% of unambiguous cases. In cases where there was disagreement, this often indicated potential alternative diagnosis, supported by a review of histological, molecular and clinical features.This robust classification informs the selection of the most appropriate models for all five histotypes. Following further refinement on larger training cohorts, the transcriptional classification may represent a useful tool to support the classification of new model systems of OC subtypes.The online version contains supplementary material available at 10.1186/s13073-021-00952-5. TP53 mutation, germline and/or somatic mutations in genes involved in homologous recombination (HR) repair and genome-wide copy-number variation (CNV) [TP53 mutations [TP53 is altered in only around 15–20% of ENOC and CCOC [Ovarian cancer (OC) is the most common cause of gynaecological-related cancer death in Europe and North America . Althougon (CNV) –10. Unliutations , 12, TP5and CCOC –15 and land CCOC –18.A greater understanding of the different molecular events that underpin ovarian carcinogenesis is driving the expansion of tailored therapies . CurrentTo support the classification of both established cell lines and novel OC models, we aimed to develop a methodology to classify all subtypes based on molecular features. In particular, now that RNA sequencing (RNAseq) data is widely available, we sought to determine whether a transcriptional ‘fingerprint’ could distinguish subtypes in an unbiased manner. While the utility of RNAseq as a tool for developing biomarkers is in its infancy, techniques are established and are becoming more accessible and less costly. The challenge, however, is in the distilling of robust ‘fingerprints’ from these complex datasets. One approach to reduce complexity is non-negative matrix factorisation (NMF), which has been utilised to reduce the dimensionality of transcriptional profiles from thousands of genes to a subset of important metagenes, concurrently providing meaningful class discovery . Here, wFinally, we sought to translate the NMF clustering into a transcriptional classifier for novel OC models using a living biobank of patient samples. The classifier was first created by training a machine learning algorithm on the subtype metagene profiles defined by NMF of the cell lines. The potential utility of the classifier was then tested using RNAseq generated from our biobank and comparison of a predicted subtype with clinical diagnosis.A literature search was carried out to determine which of the CCLE samples were representative of the major types of epithelial OC. This eliminated three cell lines from the panel of OC cell lines: one malignant Brenner tumour and one granulosa cell tumour and an engineered cell line. The malignant Brenner tumour was removed as, although classified as an epithelial tumour type , it is thttp://www.ebi.ac.uk/ena/; accession PRJNA523380) and remapped raw sequence reads using gene annotations from Gencode v32, to enable comparison with our own RNAseq of patient-derived OCMs .https://www.mcrc.manchester.ac.uk/research/mcrc-biobank/about-the-mcrc-biobank/.Research samples were obtained with informed patient consent from the Manchester Cancer Research Centre (MCRC) Biobank. The MCRC Biobank is licensed by the Human Tissue Authority (licence number: 30004) and is ethically approved as a research tissue bank by the South Manchester Research Ethics Committee (Ref: 07/H1003/161+5). The role of the MCRC Biobank is to distribute samples and does not endorse studies performed or the interpretation of results. For more information, see Ex vivo ovarian cancer models (OCMs) were expanded from 33 clinical specimens from 27 patients, of which 11 were published previously . OC and stromal cells were subsequently cultured in OCMI media supplemented with 5% FBS (Life Science Group) or 5% Hyclone FBS . Cells were passaged at 95% confluence at a ratio of 1:2.OCMs were established as described in Nelson et al. . Briefly2 atmosphere.Kuramochi cells for analysis by RNAseq (JCRB Cell Bank) were cultured in RPMI supplemented with 5% FCS, 100 U/ml penicillin, 100 U/ml streptomycin and 2 mM glutamine and were maintained at 37°C in a humidified 5% CORNA was extracted using RNeasy Plus Mini kit (Qiagen), quantified using a Qubit fluorometer (Life Technologies) and quality/integrity assessed using a 2200 TapeStation (Agilent Technologies). Sequencing libraries were then generated using the TruSeq® Stranded mRNA assay  according to the manufacturer’s protocol. Adapter indices were used to multiplex libraries, which were pooled prior to cluster generation using a cBot instrument . The loaded flow-cell was then paired-end sequenced  on an Illumina HiSeq4000 instrument. The output data was demultiplexed  and BCL-to-Fastq conversion performed using Illumina’s bcl2fastq software. Note: RNAseq was also performed on a selected number of patient-matched stromal cells, as an additional control, and though this data is not used within these analyses, it is included within the deposited data for completeness.The RNAseq data generated by the CCLE , and RNAk), NMF was performed for k of 2 to 10, using 50 random initiation points. Quality measures were computed for each factorisation rank, including the cophenetic coefficients and silhouette width. Inspection of the computed quality metrics revealed two and five clusters fitted the data. Next, 200 iterative runs of NMF were performed from a fixed random initial condition with a k value of two and again for a k value of five. Using annotations given in the primary literature source for each cell line  package was used to apply a variance stabilising transformation to the assembled read count matrix . Transcrk = 5; see the ‘Results and discussion’ section) was used to randomly partition cell lines into four groups, such that each subtype was represented in each. Random partitioning was repeated ten times to achieve a reliable estimate of model performance. Each model was trained to each successive set of three groups, and model performance tested on the omitted group. Quality metrics compared between models were the per-subtype sensitivity, specificity and balanced accuracy. Overall model performance was compared using Cohen’s kappa, which compares observed accuracy with the expected accuracy.The R package caret (v6.0-86) was used for model training and evaluation. The specific modules used were ‘base::knn’, ‘randomForest’ (v4.6-14) and ‘kernlab’ (v0.9-29), respectively. The subtype assignment gleaned from NMF  that enables interactive exploration of multidimensional cancer genomics datasets [TP53 has also been included [The genetic background of the CCLE cell lines is extensively referred to throughout this manuscript. We direct the reader to the mutation datasets generated by the CCLE. The datasets were originally presented in Ghandi et al.  and visudatasets , 46. Datincluded , 47.AKT1, ALK, AR, BRAF, CTNNB1, DDR2, EGFR, ERBB2, FGFR3, GNA11, GNAQ, IDH1, IDH2, KIT, KRAS, MAP2K1, MET, NRAS, PDGFRA, PIK3CA, PTEN, RET, STK11, TP53). For somatic variants in TP53, the target read depth across all coding regions (exon 2 to 9) was a minimum of 350x. Mutations were named according to Human Genome Variation Society guidelines (http://www.hgvs.org/) using reference sequence NM_000546.5. All variant calls were independently reviewed using the BAM files and a genome browser (Integrated Genomic Viewer). At a variant allele frequency ≥ 4%, the call sensitivity was > 90% and specificity > 95% after manual review.Formalin-fixed and paraffin-embedded (FFPE) archival tumour blocks were analysed by immunohistochemistry by collecting 4-μm sections on Superfrost charged slides. After drying overnight at 37°C, samples were processed using a Ventana Benchmark immunohistochemistry platform (Roche) with antibodies against p53 , Cytokeratin7 , PAX8  and WT-1 . Heat-induced epitope retrieval was performed using CC1 (Roche), incubating samples at 95°C for 36, 52, 40 and 64 min for p53, CK7, PAX8 and WT1, respectively. Antibodies were incubated at 37°C for 32, 40, 32 and 40 min for p53, CK7, PAX8, and WT1, respectively. p53 and CK7 were detected using Ultraview universal DAB kit (Roche), while PAX8 and WT1 were detected using Optiview universal DAB kit (Roche), all as per manufacturer’s instructions. Sections were counterstained using Haematoxylin II (Roche) for 12 min and bluing reagent (Roche) for 8 min, and slides imaged using a Leica DM2500 microscope (Leica Microsystems), using a ×20 objective lens under brightfield and processed using Adobe Photoshop. For genotyping, FFPE blocks were assessed for total cellularity and the neoplastic cell content of the sample expressed as a percentage of all nucleated cells on a haematoxylin and eosin (H&E)-stained slide. A neoplastic cell count of ≥10% was required before undertaking DNA extraction. DNA extraction was performed using the cobas® DNA Sample Preparation Kit (Roche). Tumour from 5× 5μM unstained pathology slides was available for DNA extraction. Extracted DNA was quantified using Qubit 2.0 Fluorometer (ThermoScientific). Targeted enrichment was performed using the GeneRead Clinically Relevant Tumour Targeted Panel V2 . The first matrix defines ‘metagenes’ for each cluster, the small set of genes whose co-expression informs cluster assignment, and the second reflects the co-expression levels of those metagenes in each sample. In order to establish the optimum number of clusters for the cell lines, we first performed NMF at k of 2–10  or BRCA1 promoter methylation (~11%) [MYC (31.5%), CCNE1 (21.7%) and PIK3CA (18%), and deletions involving tumour suppressor genes, such as PTEN (6.1%) [HGSOC is the most common histological subtype, characterised by aggressive dissemination. Although most patients respond to primary treatment, nearly all patients with advanced stage disease will relapse, at which point cure is highly unlikely . The pretionship –58. TP53iquitous –61. Halfn (~11%) , 60, 62.N (6.1%) , 46.TP53 (100% of cell lines) and BRCA1/2 (31.25% of cell lines) , ARID1A (~45%), TP53 (~20%) and KRAS (~10%) [CCOC is the second most prevalent subtype of epithelial OC, with the highest frequency reported in Asian countries, whereby it accounts for up to 30% of cases in Japan but only 10% in Europe and North America , 65. AltS (~10%) , 67. UnlS (~10%) .ARID1A (7 of 10), PIK3CA (6 of 10), TP53 (3 of 10) and KRAS , BRAF (~10–40%), ERBB2 (~5%) and NRAS (~10%) [TP53 mutations are rare in LGSOC, ranging from 0–8% prevalence across studies, though some series use the absence of TP53 mutations as an inclusion criterion [LGSOC accounts for only ~3% of epithelial OC and was until recently described as grade 1 serous or well-differentiated serous adenocarcinoma . LGSOC iS (~10%) , 79–82. S (~10%) , 79, 80.riterion –18.KRAS mutations (4 of 8) and additionally show BRAF (2 of 8), NRAS (1 of 8) and ERBB2 (1 of 8) mutations; in fact, 7 of 8 cell lines have a mutation in at least one of these genes. However, none of these cell lines harboured mutations in USP9X, which has recently been found at a high frequency in LGSOC cases [As LGSOC represents a fairly recent descriptor, it is more difficult to infer this classification from literature annotations. Furthermore, 4 of the 8 cell lines in the third cluster were not designated a subtype in their primary literature source. However, we propose that the third cluster of the NMF consensus map may represent LGSOC  has three mutations in MAPK pathway proteins, and ES2 (previously subtype unspecified) has a BRAF mutation, and they are both therefore characteristic of LGSOC [TP53 mutations, an overrepresentation of TP53-mutated cell lines relative to the proportion in respective tumour type has been reported previously [TP53 mutant clone during ex vivo expansion. Indeed, it is difficult to establish cell lines from low-grade, slow-growing indolent tumours [This putative LGSOC cluster contains three cell lines with a top 20 HGSOC-likely score and one ranked ‘possibly-HGSOC’ by Domcke et al.: TYK-nu, 59M and ES2, and JHOM-1, respectively. In agreement with LGSOC designation, TYK-nu has two mutations in of LGSOC , 79. Whiof LGSOC . Althougeviously , which m tumours .Interestingly, proteomic profiling by Coscia et al. also found 59M and TYK-nu to be distinct from other HGSOC cell lines , 30. WhiA cell culture and morphology-based study by Beaufort et al. identified three different morphologies of epithelial OC lines: epithelial, round and spindle, which showed distinct biological and molecular characteristics . InteresKRAS or CDKN2A mutation, progressing to borderline tumours likely to have both events and additional CNV, to overt carcinoma, which display a higher frequency of KRAS and TP53 mutations (both ~60% in MOC), and greater CNV [RNF43, BRAF, PIK3CA and ARID1A (8–12%), as well as amplification of ERBB2 (26%) [MOC are morphologically characterised by epithelium with intestinal differentiation, and thus, it can be challenging to determine whether a disease is primary ovarian or a secondary mucinous adenocarcinoma that originated elsewhere. Historically, many mucinous tumours involving the ovary were in fact metastases from extra-ovarian sites and, after revisions to the diagnostic criteria, the rate of MOC fell from ~10% to only 3% of epithelial OC , 89. Notater CNV , 91, 92.ater CNV . Other mB2 (26%) .ERBB2 amplification, and BRAF and TP53 mutations , PTEN (17–46%), KRAS (33–42%), PIK3CA (27–40%), ARID1A (19–30%), KMT2D (31%), KMT2B (19%) and TP53 (7–19%) [TP53 mutations, HR deficiency and widespread CNV, was also recently identified [ENOC can have a more favourable prognosis than HGSOC, as it tends to present at an earlier stage and at a younger age , 95. The (7–19%) , 15, 96.entified , 97. IndNNB1 25–5%, PTEN , ARID1A (4 of 5), KMT2D (4 of 5), PIK3CA (3 of 5), PTEN (3 of 5), KMT2B (3 of 5) and KRAS  fall into the ENOC cluster  and the remaining two (TOV21G and IGROV1) fall into the CCOC cluster. Indeed, mismatch repair deficiency, which leads to a hypermutated genotype, has been exclusively identified in low-grade, low-stage ENOC and CCOC , in keeping with Lynch syndrome-associated ovarian cancer , 102. ThTP53 mutation and chromosome instability. This model was acknowledged by the WHO classification in 2014, with LGSOC and HGSOC described as the prototypical type I and II tumours, respectively [An alternate, dualistic model of ovarian carcinogenesis (types I and II) has been proposed to consolidate the clinical presentation of OC subtypes with their molecular characteristics , 6. The k = 5. Indeed, all LGSOC-labelled cell lines fell into cluster 1, and all HGSOC-labelled cell lines fell into cluster 2, with the exception of SNU8, which had a poor silhouette score at k = 5; . Given that LGSOC and HGSOC are the prototypic type I and II tumours, the molecular and clinical features that gave rise to the dualistic classification are mirrored within the transcriptional profiles of the corresponding cell lines. However, in addition to LGSOC—ENOC, CCOC and MOC are generally considered type I tumours [To ascertain whether the NMF clustering into two groups reflects the dualistic model, we annotated the cell lines within the two clusters with the subtypes defined by NMF at  tumours . InteresTP53-mutated, we assessed whether TP53 status could explain the differences between the two clusters. There was a trend towards a higher proportion of TP53-mutated cell lines present in cluster 2 , suggesting that TP53 status may be influencing clustering. Furthermore, data suggests no enrichment of chemo-naive or chemotherapy-treated lines in either cluster . However, as treatment history is infrequently given in the original publication that established these cell lines, we cannot rule out a relationship based on available data. Likewise, based on the cell lines annotated with the site of biopsy, those sampled from the ovary or ascites were not enriched in either cluster .As type II tumours have been described as predominantly TP53 mutations, HR deficiency and widespread CNVs [k = 5 suggests they are a distinct and ENOC-derived subset, while demonstrating similarity to HGSOC.In line with our results, it has been questioned whether a dualistic model of OC is reflective of the heterogeneity of so-called type I tumours . Even wiead CNVs , 97. Altead CNVs , our cluk = 2 mirrors concerns that the spectrum of these histotypes is oversimplified by a dualistic model [There are limitations to drawing conclusions on the classification of OC from clustering performed on OC cell lines. Namely, ‘type II-like’ type I cancers may possess an inherent growth advantage and greater ability to adapt to culture conditions than their more indolent counterparts. Meaning, aggressive tumours could constitute a minority of clinical cases but are highly represented among cell lines. It is also possible that the two clusters reflect tissue of origin; however, as the cell-of-origin for some subtypes remains unclear, RNAseq data from tissue from these potential sites would be required to test this. For example, while both CCOC and ENOC are well-known to be endometriosis-associated, cell-of-origin is controversial with proposed sources including endometrium, endometrial cysts, ovarian surface epithelia and fallopian tube-derived cells . Despiteic model . HoweverMutation profiling of the CCLE cell lines within the five NMF clusters supports our histotype designation of each cluster and informs the selection of the most appropriate CCLE cell lines representing all five main subtypes of epithelial OC. However, clonal selection in long-term culture means that cell lines are unlikely to display the true heterogeneity of primary tumours. For example, although single-cell-derived colorectal cancer cell lines display ongoing random instability, a specific karyotype is maintained over time . ConsequTo initially test the potential of a transcriptional classifier, we determined whether the NMF classification of the CCLE cell lines could be used to train a machine learning model to predict the subtype of a ‘hold-out’ set of the lines. Genes with expression levels characteristic for each cluster were first extracted, and the list combined and used to train the models. The largest number of genes was associated with the HGSOC cluster (82 genes), followed by ENOC 40 genes), LGSOC (35 genes), MOC (28 genes) and CCOC . However, although the classifier also predicts OCM.118-7 to be HGSOC, a larger proportion of decision trees predicted LGSOC for the later sample than the earlier sample  and central  histology assessment disagreed for patients 118 and 124, with the final diagnosis confirmed as LGSOC in both. The RF classifier prediction assigns both OCM.124-1 and OCM.124-11 as non-HGSOC therefore supports the revised pathology. While the classifier predicts OCM.124-1 to be LGSOC, the prediction of OCM.124-11 as MOC may reflect lower performance in predicting non-HGSOC subtypes, or possibly molecular alterations that have occurred following treatment .For the two OCMs with pathology given only as ‘suspicious of adenocarcinoma arising from the gynaecological tract’, the RF classifier predicted OCM.87 to be CCOC and OCM.195 to be LGSOC, both with high probability, highlighting the utility of such a classifier when only ascites is available for a cytological diagnosis. Note that we previously concluded from the patient’s case notes that OCM.87 was HGSOC ; howevermutation . This ismutation , 102. Fuin LGSOC , 79–82. Overall, our data demonstrate the potential of a transcriptional classifier as a tool for subtype validation of novel epithelial OC models, identification of atypical clinical presentations, and for classification of new models when clinical annotation is unavailable or if a definitive pathology-based diagnosis is not possible. Inclusion of additional cell lines would improve predictive performance, especially of subtypes that are underrepresented in the CCLE dataset. Alternatively, the classifier may be improved by repeating the NMF clustering on a larger biobank of ex vivo cultures, to optimise the classifier gene sets for these cultures and culture conditions. Additionally, datasets containing patient-derived cell lines could be utilised to further evaluate performance, including expansion of our living biobank and others , 39, 107Classification of disease subtype is important both for clinical decision-making and for selection of appropriate model systems for pre-clinical research into different disease entities. Although it is widely accepted that epithelial OC is a heterogenous disease with five main subtypes, selection of appropriate models representative of each of these subtypes remains a significant challenge for research , 28–30. Attempts to refine OC subtype disease classification include the dualistic model of type I and II tumours , 6. Our Additional file 1. Table S1 (Cell line annotations); Table S2 (Patient demographics); Table S3 (References for subtype mutations).Additional file 2 Fig. S1 , Fig. S2 ; Fig. S3 .


PEPCK) gene knockout (Δpepck) leads to the selection of a deletion event between two tandemly arranged fumarate reductase (FRDg and FRDm2) genes to produce a chimeric FRDg-m2 gene in the Δpepck∗ cell line. FRDg is expressed in peroxisome-related organelles, named glycosomes, expression of FRDm2 has not been detected to date, and FRDg-m2 is nonfunctional and cytosolic. Re-expression of FRDg significantly impaired growth of the Δpepck∗ cells, but FRD enzyme activity was not required for this negative effect. Instead, glycosomal localization as well as the covalent flavinylation motif of FRD is required to confer growth retardation and intracellular accumulation of reactive oxygen species (ROS). The data suggest that FRDg, similar to Escherichia coli FRD, can generate ROS in a flavin-dependent process by transfer of electrons from NADH to molecular oxygen instead of fumarate when the latter is unavailable, as in the Δpepck background. Hence, growth retardation is interpreted as a consequence of increased production of ROS, and rearrangement of the FRD locus liberates Δpepck∗ cells from this obstacle. Interestingly, intracellular production of ROS has been shown to be required to complete the parasitic cycle in the insect vector, suggesting that FRDg may play a role in this process.The genome of trypanosomatids rearranges by using repeated sequences as platforms for amplification or deletion of genomic segments. These stochastic recombination events have a direct impact on gene dosage and foster the selection of adaptive traits in response to environmental pressure. We provide here such an example by showing that the phosphoenolpyruvate carboxykinase ( Leishmania and Trypanosoma species, present several biological singularities in comparison with classical eukaryotic model organisms. For instance, genes are transcribed constitutively as part of long polycistronic units where the precursor mRNA molecules are matured by coupled trans-splicing and polyadenylation  potentially leading to 1848 genetic recombination events, some of them already experimentally validated  have to be regenerated within the organelle by the succinic fermentation pathway composed of PEPCK, malate dehydrogenase , fumarase , and NADH-dependent fumarate reductase  (pepck) and illustrated in Figure 1B (The procyclic form (PCF) of lability . In the lability , 15 and lability . Two pholability . In gluclability . Glycosolability . Becauselability  need to lability . Similarstep 18) . Alternaigure 1B .Figure 1T. brucei genome contains three FRD genes. Two are tandemly arranged in chromosome 5: they encode the glycosomal isoform (FRDg) and a potential FRD isoform for which expression has not been detected so far in trypanosomes . The third gene, located on chromosome 10, codes for the mitochondrial isoform  . FRDg is a 120 kDa protein composed of three domains, a N-terminal ApbE (Alternative pyrimidine biosynthesis protein)-like flavin transferase domain (pfam: PF02424), a central FRD domain (superfamily: SSF56425), and a C-terminal cytochrome b5 reductase (Cytb5R) domain (superfamily: SSF63380)  in the organelles.The step 21)   cell lines, by label-free quantitative mass spectrometry. The effectiveness of this approach was confirmed by the 41.7–54.0-fold reduction observed for the PPDK and/or PEPCK peptide counts in the four mutant cell lines analyzed, compared with the parental cell line . Similarly, the GPDH signal was strongly reduced (20.4-fold) in the Δppdk/Δpepck/RNAiGPDH.i mutant. This analysis also showed that expressions of FRDg and FRDm2 were 6.9-fold decreased and tenfold increased, respectively, in the Δppdk/Δpepck/RNAiGPDH.i cell line, while expression of FRDm1 was not affected . In contrast, expression of the three FRD isoforms remained unaffected in the three other mutant cell lines (PXD020185 data set on the ProteomeXchange Consortium). This FRD expression pattern was confirmed by western blotting using immune sera specific to FRDg (αFRDg) and FRDm2 (αFRDm2), in addition to the αFRD immune serum produced against the conserved FRDg central domain, which is 100% and 71% identical with FRDm2 and FRDm1, respectively (B and C). The αFRD antibodies recognized two proteins in both the parental and Δppdk/Δpepck/RNAiGPDH.i cell lines, including the ∼130 kDa FRDm1 isoform (D and E). As previously reported, the second isoform expressed in the parental cell line (∼120 kDa) was recognized by the αFRDg, while no signal corresponding to FRDm2 was detected using αFRDm2 . Coomassie staining, western blotting , and proteomic analyses  of purified glycosomal fractions confirmed the glycosomal localization of FRDg expressed in the parental cell line. In contrast, none of the FRD isoforms were detectable by western blot in the glycosomal fractions of the mutant cell line, which is consistent with the proteomic analyses.In order to study possible changes in gene expression of mutants missing key enzymes involved in the maintenance of the glycosomal redox and ATP/ADP balances, we have compared the total proteomes of the parental, Δppdk , Δpepck k  but gives a much stronger signal for the FRDg and FRDm2 genes . The restriction pattern obtained with the NcoI-, PvuII-, NdeI-, and XhoI-digested parental genomic DNA  was consistent with the restriction map of the FRDm1  and FRDg/FRDm2  loci deduced from the T. brucei TriTrypDB database (strain 927). Although the FRDm1 locus was identical in the Δppdk/Δpepck/RNAiGPDH genome, the pattern observed for the FRDg/FRDm2 locus differed markedly . For instance, the 6.4 kb PvuII-fragment containing the two FRD genes in the parental genome was converted into a 2.3 kb PvuII-fragment in the mutant genome, suggesting that 4.1 kb had been deleted from the FRDg/FRDm2 locus. Analysis of the three other restriction profiles led to the same conclusion. The size of the deleted DNA fragment (4.1 kb) was consistent with the size of the theoretical DNA fragment (4074 bp) resulting from homologous recombination between the central 1450 bp FRD domains, which are 100% identical in the FRDg and FRDm2 genes.To determine whether the mutually exclusive expression of FRDg and FRDm2 was related to genomic rearrangement inside the e "1" in A but givFRDg and FRDm2 genes in the Δppdk/Δpepck/RNAiGPDH mutant to generate a FRDg-m2 chimeric gene coding for a FRD chimeric protein slightly smaller than FRDg . This DNA rearrangement event was present on both alleles of the locus, since the wild-type FRDg and FRDm2-amplified DNA fragments (C) and the endogenous FRDg protein (D) were not detectable in the mutant cell line.In conclusion, these data showed that a recombination event occurred between the ragments C and the protein D were noFRDg (g5 and g3 primers), FRDm2 (m5 and m3 primers), and FRDg-m2 (g5 and m3 primers) genes (see A). As expected, the FRDg- and FRDm2-specific DNA fragments were amplified from the parental EATRO1125.T7T cell line but not from the Δppdk/Δpepck/RNAiGPDH genomic DNA (B and C), confirming the loss of the wild-type FRDg/FRDm2 locus in the mutant cell population. Also in agreement with the Southern blot data, the FRDg-m2-specific fragment was amplified from the mutant genomic DNA. Interestingly, however, the FRDg-m2-specific fragment was also very weakly PCR-amplified from the parental EATRO1125.T7T cell line, which suggests that the recombination event stochastically occurred in the wild-type cells (B and C). Moreover, a 1.5 kb PCR product was obtained using parental DNA and the g3 and m5 primers; we suggest that the template was a circularized version of the deleted fragment (A–C). No corresponding PCR product was detected in the mutant cell line, suggesting that the circularized deleted DNA fragment was not replicated and thus diluted during cell division to become undetectable. The same PCR analysis conducted on genomic DNA samples showed that the rearrangement event occurred in other strains of T. brucei  (B).To further study this DNA rearrangement event, we used PCR with primer pairs designed for amplification of the central FRD domain of the enes see A. As expomic DNA , B and Cpe cells , B and Cfragment , A–C. Noesiense) B.Figure ppdk/Δpepck/RNAiGPDH cell line being homozygous for the FRDg-m2 recombinant locus to calculate the allele frequency of FRDg-m2 in the EATRO1125.T7T parental cell line. We compared the FRDg-m2 copy number in the two lines by semiquantitative PCR using different amounts of genomic DNA and the g5-m3 primer pair. Primers specific for a control gene  were used for normalization (A). The results showed that the FRDg-m2 gene copy number is 3700-times higher in the Δppdk/Δpepck/RNAiGPDH homogeneous cell line relative to the heterogeneous parental population (B), indicating that at the time of analysis one in 1850 cells in the parental population had a hemizygous recombined allele. This estimation is at best an upper limit since we cannot exclude PCR artifacts due to premature termination of the PCR product in the conserved region of one isoform followed by priming on the other isoform.We took advantage of the Δlization A. The repulation B, indicaFRDg-m2 chimeric gene occurs at low frequency by homologous recombination in the T. brucei genome, but was specifically selected in the Δppdk/Δpepck/RNAiGPDH cell line.Altogether, this analysis suggested that the generation of the pepck mutant obtained and frozen in 2008 , yielded more information about selection of the homologous recombination event in the FRDg/FRDm2 locus. It is noteworthy that this Δppdk/Δpepck/RNAiGPDH cell line was not derived from the Δpepck cell line . Indeed, the Δpepck and the parental cells showed the same PCR profile, while the Δpepck∗ cells maintained for a long term in in vitro culture showed a pattern similar to the Δppdk/Δpepck/RNAiGPDH cell line. Therefore, selection of the recombinant allele occurred independently in a second mutant cell line . However, the selection process was probably less stringent or more recent in the PEPCK null background compared with the Δppdk/Δpepck/RNAiGPDH background, as illustrated by the presence of the wild-type locus in the Δpepck∗ population , even after months of growth. As expected from the PCR analysis, the FRDg-m2 chimeric isoform was expressed in the Δpepck∗ cell line, but not detectable by western blotting in the Δpepck cells . It is noteworthy that proteomics analysis performed on the Δpepck cell line before long-term cultivation showed an intermediate profile of FRD isoform expression between the parental and the Δppdk/Δpepck/RNAiGPDH cell lines (PXD020185 data set on the ProteomeXchange Consortium). These data strongly suggest selection for the FRDg-m2 recombinant locus when PEPCK is missing .Analysis of the ΔA). In contrast, the FRDg-m2 chimeric isoform expressed in the Δpepck∗ cell line was released at lower digitonin concentrations (0.03 mg versus 0.14 mg of digitonin per mg of protein) together with the enolase cytosolic marker (A). The cytosolic location of the FRDg-m2 chimeric isoform expressed in the Δpepck∗ cell line was confirmed by a western blot analysis of glycosomal and cytosolic fractions prepared by differential centrifugation after silicon carbide cell homogenization (B).The glycosomal localization of FRDg was confirmed by a digitonin cell fractionation experiment. Western blot analysis of the supernatant fractions confirmed that as expected, the FRDg isoform was released together with the PPDK and PEPCK glycosomal markers A. In conc marker A. The cynization B.Figure pepck∗ cell lines. As expected, NADH-FRD activity was detected in the glycosomal fraction of cells expressing FRDg (EATRO1125.T7T), but not in the glycosomes of the Δpepck∗ cell line (B). The low level of NADH-FRD activity detected in the cytosolic fraction of the EATRO1125.T7T cell line, compared with the glycosomal fraction (2.3%), was presumably due to the lysis of a few glycosomes during the grinding step. The absence of NADH-FRD activity in the cytosolic fraction of the Δpepck∗ cell line demonstrates that the chimeric FRDg-m2 isoform is inactive (B). These data highlight the role of the Cytb5R domain in the NADH-FRD activity, since only this domain differs between the active FRDg and inactive FRDg-m2 isoforms.The NADH-dependent FRD (NADH-FRD) activity was determined in the glycosomal and cytosolic fractions of the original EATRO1125.T7T and the Δell line B. The loinactive B. These FRDg-m2 recombinant locus in the Δpepck∗ and Δppdk/Δpepck/RNAiGPDH cell lines implied that either expression of the FRDg-m2 isoform in the cytosol or abolition of FRDg expression in the glycosomes provided a selective advantage to both mutant cell lines. To determine which of these two hypotheses is correct, tetracycline-inducible ectopic expression of FRDg and RNAi-mediated downregulation of FRDg-m2 were performed in the Δpepck∗ cell line  (A). These experiments could not be conducted with the Δppdk/Δpepck/RNAiGPDH cell line, because all five available selectable markers had already been used. The glycosomal localization of the recombinant FRDg in the Δpepck∗/OEFRDg.i line was confirmed by western blotting and enzymatic activity assay of glycosomal fractions (B). The doubling time of the Δpepck∗/RNAiFRDg-m2 cell population was identical in the absence (.ni) or the presence (.i) of tetracycline, indicating that expression of the FRDg-m2 chimera was well tolerated by the Δpepck∗ mutant. In contrast, induction of FRDg expression slightly reduced the growth rate of the Δpepck∗/OEFRDg.i cell line (B).Selection of the ctively) A. These ractions B. The doell line B.Figure pepck∗ cell line  was co-cultured with the Δpepck∗ (control), Δpepck∗/RNAiFRDg-m2 or Δpepck∗/OEFRDg cell lines in the presence or the absence of tetracycline, and the proportion of EGFP-positive cells was determined over time by flow cytometry (C). The percentage of EGFP negative cells between induced and noninduced conditions gradually decreased when FRDg was expressed in the Δpepck∗ cell line, while there was no selection against FRDg-m2 expression (D). Calculations revealed that expression of FRDg increased the doubling time of the Δpepck∗ cells from 12.5 h to 14.2 h (E).Selection against FRDg expression was confirmed in a co-culture experiment. EGFP-tagged Δytometry C. The pepression D. Calculo 14.2 h E.pepck∗/OEFRDg.i cell line than the endogenous FRDg in the parental WT cells (B). Interestingly, overexpression of FRDg in the WT background (OEFRDg.i) also induced a significant growth defect (A). FRDg overexpression is therefore detrimental in the absence as well as in the presence of PEPCK. This does not, however, affect our overall conclusions: the native levels of FRDg expression must affect the Δpepck and Δppdk/Δpepck/RNAiGPDH cell lines more than the EATRO1125.T7T parental cell line, since the recombined FRDg/FRDm2 locus has been positively selected in the mutant cell lines.Recombinant FRDg was ∼4.5-times more expressed in the ΔWT cells B. Intereh defect A. FRDg opepck∗ background was dependent on the glycosomal localization of the protein. To address this question, the C-terminal peroxisomal targeting signal (PTS1) composed of the last three amino acids, the SKI tripeptide in FRDg, was removed from the recombinant protein to express a functional FRD in the cytosol of the Δpepck∗ cell line (Δpepck∗/OEFRDg-ΔSKI). The cytosolic localization of the FRDg-ΔSKI was confirmed by western blotting of the glycosomal and cytosolic fractions and NADH-FRD activity was detected in the cytosolic fraction of the Δpepck∗/OEFRDg-ΔSKI.i cell line (B). Co-culture of the Δpepck∗/OEEGFPct cell line with the Δpepck∗/OEFRDg-ΔSKI or OEFRDg-ΔSKI mutants in the presence or the absence of tetracycline, performed as above, revealed only minimal growth retardation upon FRDg-ΔSKI expression in the Δpepck∗ or WT backgrounds (A). Thus the glycosomal expression of FRDg was required to affect growth of the Δpepck∗ cell line.We next tested whether the negative effect of FRDg expression in the Δell line B. Co-culkgrounds A. Thus tB). To determine the FRDg domains responsible for the negative growth effect of the FRDg in the Δpepck background, truncated recombinant FRDg proteins missing the FRD (Δctl), ApbE-like (ΔNterm) or ApbE-like/FRD (ΔNterm/ctl) domains were expressed in the Δpepck∗ cell line . All three recombinant FRDg proteins were successfully expressed in the glycosomes, and as expected none of them showed NADH-FRD activity (A). The Cytb5R domain alone (FRDg-ΔNterm/ctl) or in combination with the FRD domain (FRDg-ΔNterm) did not affect growth, but surprisingly, expression of FRDg-Δctl was even more deleterious than full-length FRDg (B). Clearly, specific NADH-FRD enzymatic activity was not responsible for growth retardation (B). Overexpression of recombinant OEFRDg-Δctl also inhibited growth of the EATRO1125.T7T parental cell line.FRDg is composed of an N-terminal ApbE-like domain, a central fumarate reductase domain (FRD), and a C-terminal cytochrome b5 reductase domain (Cytb5R) B. To detactivity A. The Cygth FRDg B. Clearlardation B. Overexet al. x(s/t)(s/g)AS9]. They suggested that the ApbE-like domain may catalyze the transfer of FMN from FAD to serine 9 of FRDg. Replacement of S9 by an asparagine residue abolished both flavinylation and NADH-fumarate reductase activity of Leptomonas FRDg. We therefore addressed the role of flavinylation for FRD activity in trypanosomes and for the growth phenotype caused by glycosomal expression of FRDg in the Δpepck∗ background. The FRDg-Δ2-9 mutant protein missing the first nine N-terminal residues (the suggested flavinylation motif), expressed in the Δpepck∗ background (Δpepck∗/OEFRDg-Δ2-9_D3 and Δpepck∗/OEFRDg-Δ2-9_T cell lines) did not confer glycosomal FRD activity (A) upon tetracycline-induced expression and caused no growth retardation (B).The absence of an altered growth phenotype upon expression of FRDg-ΔNterm suggested a possible role of the ApbE-like domain. Recently, Serebryakova et al.  showed tactivity A upon teardation B.C and D). For the glycosomally expressed FRD mutant proteins, flavinylation correlates with growth phenotype . The essential role of FRD flavinylation for the altered growth phenotype implicates electron transfer, albeit not to fumarate, in the deleterious effects.The flavinylation of all endogenous and expressed FRD isoforms and mutants was directly assessed by in-gel detection of flavin fluorescence at 526 nm. Using denaturing gels and boiling of protein samples, only covalently linked flavin was detected   from the metabolism of the two main carbon sources used by the parental and mutant cell lines (glucose and proline) using the 1H-NMR profiling approach. The advantage of 1H-NMR spectrometry is the possibility to distinguish protons bound to 12C and 13C carbons, so that end products excreted from two different carbon sources can be distinguished, provided that one is uniformly 13C-enriched. To achieve this, we used [U-13C]-glucose and nonenriched proline   . As expeRDg-m2.i . These dpression .Table 1E2DCFHDA probe, which enters all subcellular compartments before being oxidized to a green fluorescent compound by hydrogen peroxide. The two cell lines showing the largest growth reduction, i.e., Δpepck∗/OEFRDg-Δctl.i and OEFRDg-Δctl.i, produce 1.8 and 3.7 times more ROS, respectively, than the uninduced cell lines, which is consistent with our hypothesis , which facilitate the transmembrane transport of water and small nonionic solutes. In T. brucei, AQP3 plays a role in the osmoregulation and transport of glycerol, while AQP2 is also a carrier involved in importing the trypanocidal melarsoprol and pentamidine drugs by endocytosis  and AQP3 (Tb927.10.14180) genes are 80% identical, with three ∼80 bp direct repeats, and are only separated by 857 bp of noncoding sequence, formation of the AQP2/3 chimeric gene most probably results from drug selection of stochastic homologous recombination within the AQP2/AQP3 locus, as described here for the FRDg/FRDm2 locus.Stochastic recombination leading to amplification of chromosomal regions located between homologous direct or inverted repeated sequences has been observed in ia major . This geia major . Howeverxception . This im. brucei , 29, 30.ocytosis , 31, 32.he drugs , 30. Sinpepck cell line is not active. Indeed, the only difference between the endogenous FRDg and chimeric FRDg-m2 isoforms is the C-terminal domain showing 34%  and 27%  identity with the Cryptococcus neoformans Cytb5R, respectively. Since, the NADH-cytochrome b5 reductases are known to transfer electrons from NADH to cytochrome b5  flanked by the C-terminal Cytb5R domain 222 aa) and the N-terminal ApbE-like flavin transferase domain (287 aa) , 22, 23.2 aa and  aa flankC, . The FRears ago , is surp. brucei  and absepepck and Δppdk/Δpepck/RNAiGPDH cell lines implies that this stochastic recombination event was beneficial for the PCF trypanosomes in the context of the PEPCK null background. We demonstrated that this selection is driven by the deleterious effect of FRDg overexpression in the PEPCK null background, which provided a rational explanation for the selection of the recombined Δpepck∗ cells. It is noteworthy that a ∼5-fold overexpression of FRDg also slightly affected growth of the parental EATRO1125.T7T cells, suggesting that the normal FRDg level is compatible with—and perhaps supports—optimal growth of the wild-type parasite, whereas it impairs growth of the Δpepck mutant. This hypothesis may explain why the recombination event has been selected in the PEPCK null background but not in the wild-type background.Selection of the FRDg-m2 chimera in the ΔBacteroides fragilis exposed to oxygen  . More importantly, overexpression of the recombinant FRDg-Δctl in Δpepck∗ background, as well as in the wild-type cells, induces a significant increase of ROS production, which is most probably responsible for the associate growth retardation. These data are consistent with the proposed competition between fumarate and oxygen for electrons provided by the covalently bound flavin in the ApbE domain. Indeed, in the absence of the central FRD domain, oxygen would become the main electron acceptor, regardless of the amounts of fumarate inside the glycosomes. It is noteworthy that overexpression of a cytosolic version of FRDg does not affect growth of the Δpepck∗ and parental cell lines, unlike glycosomal FRDg, which is probably due to the higher oxidative stress defense capacity of the cytosol versus the glycosomes.Several observations support the view that FRDg, as already observed for FRD from other organisms, can produce ROS, known to be toxic at high concentrations. For instance, FRD is a major contributor to ROS formation in o oxygen . ROS arenditions . However(E. coli . This aufumarate . The nots 20 μM) . Thus, arate see , might sacceptor B. More iet al. recently showed that cytosolic ROS trigger the differentiation of T. brucei procyclics into epimastigotes in the in vitro differentiation model based on overexpression of RNA-binding protein 6 (RBP6)  does not affect growth of the PCF trypanosome in glucose-rich and glucose-depleted conditions, since the parasite has developed alternative means to maintain the glycosomal redox balance and excrete fumarate instead of succinate from glucose metabolism  , 43. Ind6 (RBP6) , hydrogedium see , i.e., ntabolism , 20, 21.T. brucei EATRO1125.T7T (TetR-HYG T7RNAPOL-NEO) was cultivated at 27 °C in the presence of 5% CO2 in SDM79 medium containing 10% (v/v) heat-inactivated fetal calf serum and 3.5 mg ml−1 hemin . After centrifugation, the pellet was resuspended in 0.5 ml of homogenization buffer STE  and ground in a prechilled mortar with 1.5 g of wet-weight silicon carbide per gram of cell pellet. The cells were microscopically checked for at least 90% disruption. The cell lysate was diluted in 7 ml of homogenization buffer, centrifuged at 1000g and then at 5000g for 10 min each, at 4 °C. The supernatant was centrifuged at 33,000g for 10 min at 4 °C to yield the glycosome-enriched pellet, which was resuspended in 2 ml of STE buffer and loaded on a continuous sucrose gradient (1–2 M sucrose in STE). After centrifugation at 39,000 rpm in a vertical rotor, the band corresponding to glycosomes was collected, five times diluted in STE, and centrifuged at 33,000g for 30 min at 4 °C to yield the glycosomal pellet, which was resuspended in 0.5 ml of STE.The PCF of −1 hemin . Subcellribed in , after hΔpepck::BSD/Δpepck::PAC (Δpepck) and double Δppdk::TetR-HYG/Δppdk::T7RNAPOL-NEO Δpepck::BSD/Δpepck::PAC (Δppdk/Δpepck) null mutant cell lines have been generated before  was performed in the Δppdk/Δpepck PCF by expression of stem-loop “sense-antisense” RNA molecules of the targeted sequences corresponding to a 564-bp fragment (position 223–786) of the GPDH gene  of the FRDm2 gene (Tb927.5.940). The resulting pLew100-GPDH-SAS and pLew100-FRDm2-SAS plasmids containing a sense and antisense version of the targeted gene fragment, separated by a 58-bp and 50-bp fragment, respectively, under the control of a PARP promoter linked to a prokaryotic tetracycline operator, were constructed as previously described using the HindIII, XhoI, and BamHI restriction sites  was inserted in the HindIII and BamHI restriction sites of the pLew100 vector to produce pLew100-FRDg plasmid. The FRDg-ΔSKI recombinant gene coding for a FRDg isoform lacking the three C-terminal residues forming the PTS1 (SKI tripeptide) was generated by replacing the 233-bp ApaI/BamHI fragment of the pLew100-FRDg plasmid by the same fragment missing the nine residues coding for the SKI tripeptide. The FRDg-Δctl recombinant gene coding for an FRDg isoform lacking the central FRD domain was generated by removing the 1361-bp PvuII/PspOMI fragment of the pLew100-FRDg plasmid, corresponding to position 997 bp and 2358 bp in the FRDg gene, followed by recircularization of the resulting plasmid. To produce the FRDg-ΔNterm recombinant genes, a 2343-bp PCR fragment corresponding to position 1083 bp to 3426 bp of the FRDg gene was inserted into the HindIII and BamHI restriction sites of the pLew100 vector. To generate the FRDg-ΔNterm/ctl and FRDg-Δ2-9 constructs, the 1504-bp HindIII/XhoI fragment of the pLew100-FRDg plasmid, encoding the first 498 amino acids of FRDg, was replaced by a 498-bp HindIII/XhoI fragment and a 1477-bp HindIII/XhoI fragment, respectively.The single d before , 20, 24.PDH gene , 47, usiPDH gene . Similaron sites , 20. To pepck∗ cell line, the EGFP sequence was inserted in the HindIII and BamHI restriction sites of the pLew100 vector, which was modified by removing the two tetracycline operator sequences. The pLew100-FRDg-m2-SAS, pLew100-FRDg, and pLew100-EGFPct plasmids designed to generate the Δpepck∗/RNAiFRDg-m2, Δpepck∗/OEFRDg, and Δpepck∗/OEEGFPct cell lines were provided by the Genecust company. The plew100 recombinant plasmids were linearized with the restriction enzyme NotI and transfected into the Δppdk/Δpepck (pLew100-GPDH-SAS), Δpepck∗ , or parental  cell lines.To constitutively express EGFP in the Δ−1), neomycin (10 μg ml−1), blasticidin (10 μg ml−1), puromycin (1 μg ml−1), and/or phleomycin (5 μg ml−1). Aliquots were frozen in liquid nitrogen to provide stocks of each line that had not been in long-term culture. Induction of RNAi cell lines was performed by addition of 1 μg ml−1 tetracycline.Selection of all these mutant cell lines was performed in SDM79 medium containing hygromycin (25 μg mlpepck∗/OEEGFPct_F5), which has a doubling time of 14.26 ± 1.06 h. The SDM79 medium was inoculated with the Δpepck∗/OEEGFPct_F5 reference cell line (1.4 × 106 cells ml−1) and a mutant cell line (0.6 × 106 cells ml−1), in the presence or the absence of 1 μg ml−1 tetracycline, and the proportion of EGFP-positive cells was determined every day by flow cytometry using a Guava EasyCyte Flow Cytometer (Merck Millipore). The difference of the percentage of EGFP negative cells between induced and noninduced conditions was plotted as a function of time of growth, in order to estimate the growth difference between the noninduced and tetracycline-induced cell line.The objective of this assay is to determine slight but significant doubling time difference between a conditional mutant and an EGFP-tagged reference cell line, upon co-culture experiments. This assay is based on the co-culture of a tetracycline-inducible conditional mutant cell line and a reference cell line constitutively expressing EGFP (Δ6 cells) or glycosomal extracts of the parental (EATRO1125.T7T) or mutant PCF of T. brucei were separated by SDS-PAGE (8% or 10%) and immunoblotted on TransBlot Turbo Midi-size PVFD Membranes (Bio-Rad)  . ImmunodBio-Rad) , 50 usinBio-Rad) , the rabBio-Rad) , the rabBio-Rad) , the rabBio-Rad) , the rabBio-Rad) , the rabBio-Rad)  and the Bio-Rad)  were useAs described in , gels re2DCFHDA) fluorescent dye. Cells in the exponential growth phase were treated with 10 μM H2DCFHDA for 30 min at 27 °C. A total of 107 cells were pelleted , washed with 1 ml of PBS (pH 7.4), resuspended in 2 ml of PBS, and immediately analyzed by fluorimetry at 520 nm (Ex: 485 nm) with the FLUOstar Omega (BMG Labtech).The cellular ROS levels were determined using the 2′,7′-dichlorofluorescein diacetate (H8 cells ml−1 (corresponding to 3.3 mg of protein per ml) in STE buffer  supplemented with 150 mM NaCl and the Complete Mini EDTA-free protease inhibitor cocktail (Roche Applied Bioscience). Cell aliquots (200 μl) were incubated with increasing quantities of digitonin (Sigma) for 4 min at 25 °C, before centrifugation at 14,000g for 2 min to collect the cellular pellet.Digitonin permeabilization was performed as described before . Brieflyvia oxidation of NADH or NADPH, according to published procedures (Sonicated (5 s at 4 °C) crude extracts of trypanosomes resuspended in cold hypotonic buffer  were tested for enzymatic activities. NADH-dependent FRD, glycerol kinase, and malic enzyme activities were measured at 340 nm ocedures .ppdk/Δpepck/RNAiGPDH cell lines, extracted as previously described . The membrane was hybridized with digoxigenin-labeled DNA probes synthesized with a PCR DIG probe synthesis kit  as recommended by the supplier. The FRD probe was generated by PCR amplification, using the primer pair 5′-GTGTAACGTCGTTGCTCAGTGAGA-3′/5′-GCGAAATTAAATGGGCCCCGCGACG-3′. Probe–target hybrids were visualized by a chemiluminescent assay with the DIG luminescent detection kit , according to the manufacturer's instructions. Blots were exposed to ImageQuant LAS4010  for approximately 20 min.Genomic DNA (10 μg) from the parental (EATRO1125.T7T) and Δescribed , was dig4HCO3), 50% Acetonitrile (ACN) and shrunk in ACN for 10 min. After ACN removal, gel pieces were dried at room temperature. Proteins were first reduced in 10 mM dithiothreitol, 100 mM NH4HCO3 for 30 min at 56 °C, then alkylated in 100 mM iodoacetamide, 100 mM NH4HCO3 for 30 min at room temperature, and shrunken in ACN for 10 min. After ACN removal, gel pieces were rehydrated with 50 mM NH4HCO3 for 10 min at room temperature. Before protein digestion, gel pieces were shrunken in ACN for 10 min and dried at room temperature. Proteins were digested by incubating each gel slice with 10 ng μl−1 of trypsin  in 40 mM NH4HCO3, 10% ACN, rehydrated at 4 °C for 10 min, and finally incubated overnight at 37 °C. The resulting peptides were extracted from the gel by three steps: a first incubation in 40 mM NH4HCO3, 10% ACN for 15 min at room temperature and two incubations in 47.5% ACN, 5% formic acid for 15 min at room temperature. The three collected extractions were pooled with the initial digestion supernatant, dried in a SpeedVac, and resuspended with 0.1% formic acid for a final concentration of 0.05 μg μl−1. Online nanoLC-MS/MS analyses were performed using an Ultimate 3000 system (Dionex) coupled to a nanospray LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific). In total, 0.5 μg of digested protein extracts was loaded on a 300 μm ID × 5 mm PepMap C18 precolumn  at a flow rate of 10 μl min−1. After 5 min desalting, peptides were online separated on a 75 μm ID × 15 cm C18PepMap column  with a 2–40% linear gradient of solvent B (0.1% formic acid in 80% ACN) during 108 min. The separation flow rate was set at 200 nl min−1. The mass spectrometer operated in positive ion mode at a 1.8 kV needle voltage and a 42 V capillary voltage. Data were acquired in a data-dependent mode alternating an FTMS scan survey over the range m/z 300–1700 with the resolution set to a value of 60,000 at m/z 400 and six ion trap MS/MS scans with Collision-Induced Dissociation (CID) as activation mode. MS/MS spectra were acquired using a 3 m/z unit ion isolation window and normalized collision energy of 35. Mono-charged ions and unassigned charge-state ions were rejected from fragmentation. Dynamic exclusion duration was set to 30 s. For protein identification, Sequest HT and Mascot 2.4 algorithms through Proteome Discoverer 1.4 Software (Thermo Fisher Scientific Inc) were used for protein identification in batch mode by searching against a T. brucei protein database . This database was downloaded from http://tritrypdb.org website. Two missed enzyme cleavages were allowed. Mass tolerances in MS and MS/MS were set to 10 ppm and 0.6 Da. Oxidation (M), acetylation (K), and deamidation  were searched as dynamic modifications, and carbamidomethylation (C) was searched as static modification. Peptide validation was performed using Percolator algorithm  were used as the criteria for identification as a differentially expressed protein. Noticeably, only nonconflicting features and unique peptides were considered for calculation at protein level. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE , and incubated in 1 ml  of PBS supplemented with 2 g l−1 NaHCO3 (pH 7.4). Cells were maintained for 6 h at 27 °C in incubation buffer containing 4 mM [U-13C]-glucose and 4 mM nonenriched proline. The integrity of the cells during the incubation was checked by microscopic observation. The supernatant (1 ml) was collected and 50 μl of maleate solution in D2O (10 mM) was added as internal reference. H-NMR spectra were performed at 500.19 MHz on a Bruker Avance III 500 HD spectrometer equipped with a 5 mm cryoprobe Prodigy. Measurements were recorded at 25°. Acquisition conditions were as follows: 90° flip angle, 5000 Hz spectral width, 32 K memory size, and 9.3 s total recycle time. Measurements were performed with 64 scans for a total time close to 10 min 30 s. Resonances of the obtained spectra were integrated and metabolites concentrations were calculated using the ERETIC2 NMR quantification Bruker program.2 × 10http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the data set identifier PXD020185.The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (This article contains The authors declare that they have no conflicts of interest with the contents of this article.


The aim of developing academic resilience and motivational intensity, as two constructs of positive psychology, is to increase learners' capability to compete with each other even in adverse conditions. Different types of academic resilience are conceptualized and germane literature about the relationship between academic resilience and academic achievement is provided. Literature showed that some socio-affective factors , socio-economic factors , and affective factors  can influence learners' academic achievement and policy makers' decision in providing an appropriate context for learning. In the end, the pedagogical implications are expounded to foster the language learning quality and to develop a language educational system. Suggestions for further research are provided to develop the existent literature on the relationship between academic, motivational intensity, and learners' academic achievement. Todays' life is full of challenges and threats. If people do not equip themselves with the necessary abilities, they will face many problems. In the 21st century, a group of psychologists has realized that man must spend his mental energy on the positive aspects of his experience .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Besides the use of maize grain as food and feed, maize stover can be a profitable by-product for cellulosic ethanol production, whereas the whole plant can be used for silage production. However, yield is reduced by pest damages, stem corn borers being one of the most important yield constraints. Overall, cell wall composition is key in determining the quality of maize biomass, as well as pest resistance. This study aims to evaluate the composition of the four cell wall fractions  in diverse maize genotypes and to understand how this composition influences the resistance to pests, ethanol capacity and digestibility.p-coumaric acid and low hemicellulose content; (ii) inbred lines showing cell walls with high cellulose content and high diferulate cross-linking may present higher performance for ethanol production; (iii) and inbreds with enhanced digestibility may have cell walls poor in neutral detergent fibre and diferulates, combined with a lignin polymer composition richer in G subunits.The following results can be highlighted: (i) pests’ resistant materials may show cell walls with low Results evidence that there is no maize cell wall ideotype among the tested for optimal performance for various uses, and maize plants should be specifically bred for each particular application.The online version contains supplementary material available at 10.1186/s12870-021-03040-3. Accessibility, extensibility, and digestibility of tissues would determine important characteristics of maize, such as resistance to stem borers, stem diseases, feedstuff quality and saccharification for ethanol production. These characteristics depend on the cell wall composition and structure . GeneticMaize inbred lines have different mechanisms of defence  that determine the level of corn borer resistance in a particular inbred line . SimilarMaize breeding strategies are normally focused on a particular trait  that produce indirect effects on cell wall components. For example, recurrent selection to improve resistance to corn borer attack increases cell wall bound hydroxycinnamates concentration in the pith tissues of the improved maize cycles . BuendgeIn switchgrass, Sarath et al.  studied sfe) mutant. Lines presenting the mutation showed reduced ether ferulate crosslinking of lignin to arabinoxylans. Cattle fed with this mutant showed greater dry matter intake and milk production.Direct changes towards cell wall structure can also influence the final uses of maize. Successful divergent selection for diferulate ester content in the maize pith resulted in changes in cell wall composition . These cbm 1–4) have been an excellent material to study digestibility and lignification [A final approach to investigate the relationship between cell wall and maize uses consists in mutant or transgenic evaluations. Brown midrib mutants  was determined in all inbred lines, and the aptitude for different uses  was evaluated.The main objective of this research is to know the composition of the four cell wall fractions in different maize genotypes and elucidate how this composition influences pest resistance (R), ethanolic production (E) and forage digestibility (D). Those last three aspects will be further on denoted as R.E.D.The first subset of inbred lines includes maize inbreed lines that have been characterised in relation to resistance to corn borer attack . Among tThe second subset includes five inbred lines from the Agriculture and Agri-Food Canada (AAFC): C103, CO384, CO348, CO442 and CO444. Inbred C103 has high concentration of sucrose in the stalk. Reid et al.  developeThe last group was formed by inbred lines with good performance in hybrid combination. Inbred EC212 has very good general and specific combinatorial aptitude for high animal digestibility . This grA set of 20 inbred lines was evaluated Table . These i−1 after thinning. Local agronomical practices were fulfilled.The inbred panel was evaluated in 2016 and 2017 in Pontevedra, in North Western Spain . A random block design with three repetitions was used in the two trials. The second year, the subset of inbred lines was reduced to nineteen due to the lack of seed for the inbred PB130. Each experimental plot consisted of three rows, each row with 15 double-kernel hills planted manually, spacing between consecutive hills in a row being 0.18 m and 0.8 m between rows, obtaining a final density of ~ 70,000 plants haPor each plot, the days from planting to the date when half of the plants shed pollen or showed silks were recorded as days to anthesis or silking, respectively. Grain and stover yields were recorded at harvest (approx. 70 days after silking); meanwhile forage yield that was recorded 55 days after silking.−1 at 14% humidity. It was determined by the following equation:Grain yield was calculated with the weight of ears collected in a plot and expressed in Mg ha3.The value 8.6 on the equation corresponds to a constant to adjust the yield at 14% humidity. Therefore, yield was calculated per plant and then transformed to Mg ha−1.where humidity, expressed as a percentage, was recorded using a moisture meter Kett (model PM-400) in a sample of 240 cmIn each plot, the weight of 2–10 plants without ears (weight of fresh stover) was recorded, and a stover sample was collected for estimating the percentage of stover dry matter and saccharification efficiency analyses. The fresh stover sample was weighed (sample fresh weight), chopped, pre-dried at 35 °C in a forced air camera, dried at 60 °C in a stove and again weighed after seven days (sample dry weight). Dry stove samples from each plot were grounded in a Wiley mill with a 0.75 mm screen for saccharification assays. Forage yield and forage dry matters were similarly computed but using complete plants collected 55 days after silking.−1 were determined by the following equation:Stover and forage yields in Mg of dry matter haAs for grain yield, yields were calculated per plant and then transformed to Mg / ha.The second internode below the main ear was collected from five plants in each plot. Samples were collected 55 days after silking. Each harvested internode was frozen at − 20 °C until analytical determinations. Then, samples were dried at 60 °C and ground in a Wiley  mill with a 0.75 mm screen before being analysed.Determinations were based in whole dry internode tissues unless mentioned otherwise. Some other biochemical traits  were determined in isolated cell walls from those internodes following the cell wall isolation protocol modified by Melida et al. .Cellulose was quantified by the Updegraff method . The conFor total, and neutral sugars and uronic acids, 5 mg of tissue were weighed and hydrolysed at 212 °C by adding 2 ml of 2 M trifluoroacetic acid (TFA) for 1 h. Total sugar content was determined by the phenol–sulphuric method using glucose as standard . Uronic O-4 linkages.Total lignin content was determined by Klason Lignin protocol . Monomerp-coumaric acid (PCA) were purchased from Sigma-Aldrich Quimica SL, Madrid, Spain Sigma. The identities of FA dimers were confirmed by a comparison with 5 − 5 standard or published retention times and UV spectra [A recently optimized protocol by Santiago and col.  was used spectra . The totFibretec System model 1020 digester .Neutral Detergent Fibre (NDF) and Acid Detergent Fibre (ADF) were determined by Near-Infrared Spectroscopy (NIRs). We included a new variation in the hemicellulose estimation using the hemicellulose content in the fibres, as the difference between ADF and NDF. We will refer to this determination in the text as hemicellulose quantified as percentage (%). The spectral information of the dried and grounded (1 mm) samples was obtained using a Foss NIRSystem 6500 monochromator spectrophotometer , located in an isothermal chamber (24 ± 1° C), provided with a rotation module that performs reflectance measurements in the spectral region between 400 and 2500 nm, at 2 nm intervals. The collection of the spectral data and the chemometric analysis of the data was carried out using the WinISI II v program. 1.5 . In order to detect the presence of extrapolations of the prediction model, the identification of “outliers” samples  was performed using the Mahalanobis (GH). Those samples that presented GH values greater than 3, were selected indicating that the sample did not belong to the calibration group . The sam−1 material−1 h−1) was assessed against a glucose standard curve using the 3-methyl-2-benzothiazolinone hydrozone method.Saccharification assays were performed as described in Gómez et al. . SamplesDigestibility of the Organic Matter (DOM) of the internode was also determined by Near-infrared spectroscopy (NIRs) at LIGAL. The spectral information of the dried and grounded (1 mm) samples was obtained in the same way reported for fibres estimations. The samples recognized as outliers were analysed by the in vitro digestibility procedure described by Tilley and Terry,  modifiedIndividual and combined analyses of variance were made for each trait according to the SAS mixed model procedure (PROC MIXED) of the SAS program (version 9.4) , except Correlation coefficients between R.E.D traits and yield  were calculated using REML estimates according to a published SAS mixed model procedure .After the variance analysis, the inbred lines were classified in high and low groups according to saccharification efficiency. For digestibility of organic matter, lines were classified qualitatively. The inbred lines were also classified by its resistance or susceptibility to corn borer attack based on previous evaluations of these materials . ResistaIn order to understand the relationship between cell wall components and the economically important uses of maize (R.E.D), multiple linear regression models were built for saccharification efficiency and DOM using the cell wall components as probable predictors. The stepwise method following the PROC REG procedure in SAS was used . Only thThe subset of inbred lines evaluated showed variation for all the agronomic traits tested  was, however, close to the significance level , but also for lignin monomeric composition .Correlations between yield traits and R.E.D were performed in order to assess the covariation among traits. Only significant correlations above 0.50 in absolute values are described, but the complete set of correlations is shown in Table To understand the relationship between cell wall components and economically important uses of maize, we performed contrast analysis and multiple linear regression analysis. With the data from the analysis of variance, and the previous data for tunnel length, we classified inbred lines in High, Intermediate and Low for the contrast analysis Tables  and 4. Fp-coumaric acid than resistant inbred lines. Differences between high and low saccharification efficiency groups (E.) were significant for ferulic acid dimers concentration which were larger in the high saccharification efficiency group. Supporting these results, in the multiple linear regression analysis, we identified cellulose and DFA 8–5-b as significantly involved in the variability for saccharification. Regarding to forage feedstocks (D.), highly degradable lines presented lower concentrations of PCA, DFA 5–5, DFA 8–5-l and DFAT, lower proportion of glucuronic acid, and greater proportions of G subunits. In addition, we obtained in the multiple regression model that DOM was negatively affected by the content in NDF, whereas positively affected by FA and G subunits.Overall, susceptible inbred lines presented higher concentration of hemicellulose and The range obtained for days to silking and anthesis indicates that all inbred lines included in the panel were well adapted to the growing conditions at Pontevedra . It is important to highlight two facts from the yield evaluations: (i) the value obtained for each inbred refers to the maximum-potential yield, not to the yield in a plot; and (ii) these values are for inbred lines, not being the standard material to be tested for yield, as it would be hybrid combinations.p-coumaric acid was the most abundant hydroxycinnamic acid, which is consistent with previous studies [Inbred lines differed significantly for every main cell wall fraction, with the exception of hemicellulose. Regarding lignin polymer, and in concordance with previous studies, S subunit represented the largest proportion, and H subunits the lowest proportion . Variati studies , 15.We did not find variation for matrix monosaccharide composition among the 20 genotypes evaluated that could be consequence of lower variability in secondary than in primary cell walls . HoweverMiscanthus, switchgrass or sugarcane, are promising candidates for the industrial production of biofuel as they present high biomass yields (15–25 Mg/ha), broad geographic adaptation, superior carbon sequestration and efficient nutrient utilization [Regarding ethanolic production we found variation for saccharification efficiency. Other researchers have studied the saccharification efficiency of several crops species following the same method and pre-treatment; and variability in saccharification efficiency among different genotypes was also found. Species like lization . Howeverlization ; and canlization . In viewContrarily to expectations, the parental inbreds of hybrids with high soluble sugar content in the stalk  did not show the most promising values for saccharification efficiency, except the inbred line CO442 . ConversRegarding DOM, the rank obtained is in agreement with the previous values published for maize in vitro digestibility . DigestiResistance to corn borer has been associated to shorter galleries produced by the larvae in the stalks ; In viewErinnyis ello feeding on Euphorbia pulcherrima leaves, as opposed to other cell wall components. A larger proportion of hemicellulose could favour the attack of the insect, while other structural components involved in tissue strengthening, such as lignin, deter the larvae advance [Our results relate hemicellulose with pest resistance. Krakowsky et al.  suggeste advance .Regarding DOM, high concentration of fibres  directly affects digestibility by the limitation of the energy intake by the animals . In agreA greater concentration of glucuronic acid was also detrimental for DOM. Hemicellulose could be a potential repository of fermentable sugars , but unlp-coumaric acid. This result appears contradictory with previous results, as high concentration of p-coumaric acid has been related to resistance to corn borer attack [p-coumarate accretion occurs in tandem with lignification and its accumulation can be considered a relevant indicator of lignin deposition. In grasses, lignins are acylated (primarily syringyl units) at the γ-position by p-coumarates [PCA differed in concentration between susceptible and resistant inbred lines; susceptible lines presenting a higher concentration of r attack . On the umarates . Acylatiumarates  with litumarates . S ligniumarates –65. At lumarates , 24.bm3 mutants, characterized by its improved stover digestibility. They also observed that the decrease in the proportion of S subunit was balanced by an increase in proportion of G; which could be, among others, one of the reasons of the increased degradability in bm3 mutants, agreeing with our results.In our study, also in relation with lignin structure, lines included in the high digestibility group showed greater proportion of G subunits. Mechin et al.  observedLignocellulosic polysaccharides, mainly cellulose 40%), serve as principal substrate for fermentation of cell wall sugars in ethanol %, serve .During lignification, the ferulic acid and diferulic esters form cross-links through the etherification of the phenolic hydroxyl group to lignin polymers,  forming Our results show that it is very difficult to find a maize ideotype presenting a cell wall composition that fits the three requirements of R.E.D, as one combination of components would favour one use and negatively affects the other. For example, that is the case of the diferulic acid content, which affects cell wall digestibility and saccharification efficiency, or borer resistance and saccharification, both in opposite ways. Therefore, we could aim to obtain a putative genotype from this subset of lines with the best combination of cell wall compositional traits to be used in a particular usage. Despite this, among this subset of inbred lines, we found inbred lines that present high saccharification efficiency and high digestibility of the organic matter: EP42 and EC212; however, we must note that both are susceptible to corn borer attack.Genotypic variation has been observed for agronomic, cell wall composition and saccharification efficiency in the set of 20 inbred lines analysed. The germplasm represented by these inbred lines is a potential source of variation that can be exploited in the improvement of biofuel production using maize as a resource.p-coumaric acid concentration and low hemicellulose content; (ii) saccharification efficiency must be improved by increasing the presence of cellulose and diferulates; (iii) and, to improve digestibility, cell wall must have poor neutral detergent fiber and diferulate cross-links, combined with a lignin polymer richer in G subunits.It is not possible to define a cell wall ideotype that simultaneously fits the requirements for the three uses proposed. We have to settle for identifying the inbred lines with the better combination of cell wall traits for each final use of maize, to be used in future and specific breeding programs. According to these results, we can propose a maize cell wall ideotype to each specific area to be improved: (i) borers resistance in the internode may involve cell walls with low Additional file 1: Supplementary Table 1. Means for agronomic traits and yields evaluated in 20 inbred lines during two years. Supplementary Table 2. Means of cell wall polysaccharides and matrix monosaccharides in 20 inbred lines evaluated during two years. Supplementary Table 3. Means of Klason lignin, lignin monomeric composition in 20 inbred lines evaluated during two years. Supplementary Table 4. Means of cell wall-bound hydroxycinnamates in 20 inbred lines evaluated during two years. Supplementary Table 5. Values for cell wall fibres in 20 inbred lines evaluated during two years. Supplementary Table 6. Means for traits related to R.E.D evaluated in 20 inbred lines during for two years.


Primary adherent glioblastoma cell lines are an important tool in investigating cellular and molecular tumor biology, as well as treatment options for patients.The phenotypical and immunocytochemical characterization of primary cell lines from glioblastoma specimens during establishment is of great importance, in order to reliably identify these cell lines as primary glioblastoma cell lines.Sixteen primary adherent cell lines out of 34 glioblastoma samples (47%) were established and further characterized. For phenotypical characterization, morphology and growth characteristics of the cells were classified. The cell lines had a high growth rate with a doubling time of 2 to 14 days. Morphologically, the cells displayed spindle‐form or polygonal to amorphous shapes and grow as monolayer or in foci without evidence of contact inhibition. The cells were able to migrate and to form colonies. For further characterization, the protein expression of the astrocyte‐specific protein glial fibrillary acidic protein (GFAP), the glial marker S100B, the neuronal marker TUBB3, and malignancy marker VIM, as well as the progenitor markers NES and SOX2, the proliferation marker MKI67, and the fibroblast marker TE7 were determined. Based on the immunocytochemical validation criterion of a coexpression of GFAP and S100B, 15 out of these 16 cell lines (94%) were defined as primary glioblastoma cell lines (pGCL). All 15 pGCL expressed TUBB3 and VIM. NES and SOX2 were stained positively in 13/15 and 6/15 pGCL. MKI67 was expressed in 11/15 and TE7 in 2/15 pGCL.These results point out that in self‐established primary adherent glioblastoma cell lines, the expression of the specific astrocytic and glial markers GFAP and S100B and of the malignancy and progenitor markers VIM, NES, and SOX2 has to be validated. These data show that primary cell lines of glioblastoma origin with high malignant potential are reliably to establish using standardized validation criteria. Unfortunately, phenotypic as well as immunocytochemical characterization using a panel of marker proteins to confirm the identity of established tumor cells is often missing. Often only single primary glioblastoma cell lines are generated for specific analyses but not described in detail. Four work groupsThis lack of reliable characterization of self‐established primary glioblastoma cell lines demonstrates the urgent need for an accurate immunocytochemical analysis during cell line establishment. There are several well‐established markers used for diagnosis of glioblastoma, but none of them is solely tumor specific. This limiting factor in the investigation of glioblastoma cells in vitro led us to investigate a panel of different markers for immunocytochemical analysis. This marker panel includes the astrocytic marker GFAP, the glial marker S100B , the neuronal marker TUBB3 (tubulin beta 3 class III), the tumor marker VIM (vimentin) and the progenitor markers NES (nestin) and SOX2 (SRY‐Box 2), as well as the proliferation marker MKI67 (Ki‐67) and the fibroblast marker TE7. The immunoprofile of the glial marker GFAP in glioblastomas is similar to astrocytomas.Applying the defined validation criteria, we established 15 adherent primary glioblastoma cell lines (pGCL) under serum conditions. Precisely characterized primary glioblastoma cell lines are an important tool in investigating cellular and molecular biology as well as evaluating treatment options for patients with malignant gliomas. The present technical study aims at simplifying the establishment of individual primary glioblastoma cell lines and defines reliable validation and characterization criteria for the establishment of primary glioblastoma cell lines with regard to expression of marker proteins, morphological characteristics, and growth kinetics.22.1g for 5 minutes at 4°C, resuspended in 1x DMEM supplemented with 10% FBS  and seeded in a cell culture flask, as described previously.Tumor tissue of 34 glioblastoma patients  was collected during resection and transferred cooled to the laboratory. For cell line establishment, tissue from the border of the tumor was used. Under sterile conditions, it was rinsed in 1x PBS. Obvious vessels, clotted blood, and charred tissue were removed. The explant was grossly dissociated using scalpels and further enzymatically dissociated by incubation with Collagenase Type IVa  and Pronase E  in 1xPBS  at 37°C for up to 1 hour. The cells were centrifuged at 3002.2The cell lines A‐1722.3Five thousand cells per well were plated in 24‐well cell culture plates and allowed to attach for 24 hours. The total viable cell number was counted in triplicate every 24 hours for up to 14 consecutive days by Trypan Blue exclusion  and a hemocytometer. Doubling times were then calculated using an online calculator.2.4Five thousand cells per well were plated in triplicate in 24‐well cell culture plates and allowed to attach and proliferate for 48 hours. After fixation  and staining with methylene blue solution , growth type of the cell culture  and the cellular phenotype  was assessed to divide the cell cultures in subgroups according to their morphology.2.5Five thousand cells per well were plated in triplicate in 24‐well cell culture plates and allowed to attach for 24 hours. After fixation, cells were stained overnight for senescence‐associated ß‐galactosidase  as described by Dimri et al.2.6The cells were plated in 24‐well cell culture plates in triplicate and cultivated for 36 hours to achieve confluence. The cells were then synchronized for 12 hours in serum‐free 1x DMEM. In the confluent cell layer, a straight line was scratched using a p10 pipet tip. The cells were incubated for further 24 hours, and images were acquired after 0, 6, 12, and 24 hours from the same field of view. The scratch distance was calculated using ImageJ 1.47v  and staining with methylene blue solution, formation of colonies was assessed .2.8d‐lysine  coated cover slips and stained immunocytochemically. To visualize expression and cellular distribution of different marker proteins, the following primary and secondary antibodies were used: mouse monoclonal anti‐GFAP , mouse monoclonal anti‐S100 (ß‐subunit) , rabbit polyclonal anti‐beta III Tubulin , mouse monoclonal anti‐Vimentin , rabbit polyclonal anti‐Ki‐67 (H‐300) , mouse monoclonal anti‐Nestin (10C2) , mouse monoclonal anti‐Sox2 (L1D6A2) , mouse monoclonal anti‐Human Thymic Fibroblasts (TE7) , goat anti‐Mouse IgG‐FITC, and goat anti‐Rabbit IgG‐FITC . Nuclei were counterstained with bisBenzimide H 33258 . The number of stained cells was examined by fluorescence microscopy and scored as follows: 0, no expression; 1, ≤5% of cells; 2, ≥5% of cells; 3, ≥25% of cells; 4, ≥50% of cells, and 5, ≥75% of cells. GFAP expression was further grouped: 0, no cells; low, ≤5% of cells; medium, >5% to ≤50% of cells; high, >50% of cells. For each cell line, one cover slip per antibody was stained, completely examined and subjectively evaluated. Examples were photographed using an AxioPlan microscope system . All cell lines with cells immunopositive for both GFAP and S100B were defined as “primary glioblastoma cell lines (pGCL).”For analysis of protein expression, cells were grown on Poly‐2.9P ≤ .05. For the analysis of protein expression levels (immunocytochemistry), Spearman's rank correlation was used. Scratch assay results were statistically evaluated with variance analysis (ANOVA). To further test intergroup comparisons for statistical relevance, the Mann–Whitney U test was used.All statistical analyses were carried out with the IBM SSPS Statistics v23 software . Statistical significance was defined as 33.1U‐test, P = .17). The primary cell lines expanded continuously with a doubling time of approximately 2 to 14 days  appearance. These morphological features as first indication of the presence of glioblastoma “multiforme” cells in culture have been described in different studiesFor immunocytochemical characterization, antibodies against glial markers, neuronal, and neural precursor markers, as well as malignancy markers, were selected . To identify the established primary cell lines as glioblastoma cell lines, an immunopositivity for both glial markers GFAP and S100B was defined. GFAP is a protein involved in the structure and function of the cytoskeleton and is commonly used as an astrocytes marker: its expression is increased following brain damage or during degeneration of the central nervous system.The commercially available glioblastoma cell lines A‐172, LN‐229, and U‐251 MG were analyzed as a comparison group for the established pGCL. Only U‐251 MG showed GFAP and S100B expression as expected. Surprisingly, GFAP was not detectable in A‐172 and LN‐229 cell lines. In U‐251, MG, and LN‐229, the expression of S100B was detectable as reported.TUBB3 and VIM have been identified in some human glioma cell lines before.The verified protein expression of the progenitor markers NES and SOX2 in the adherent primary glioblastoma cell lines established here is indicating that the cells retained at least the ability to dedifferentiate, if not potential stem cell properties. Only pGCL positive for NES showed an expression of SOX2. These pGCL also expressed MKI67 as a proliferation marker.To exclude a selection for fibroblasts in the cell cultures, all pGCL were stained for the fibroblast marker TE7Some of the established pGCL could be cultivated over passage 30 (data not shown). This led to a clonal selection and therefore the standardization of the phenotype. These cell lines largely lost GFAP expression. It only remained in individual cells, as indicated by the literature before.5In conclusion, these findings indicate that serum‐based cultivation enables routine expansion of glioblastoma cells and establishment of defined primary glioblastoma cell lines, maintaining heterogeneous cell types over the first passages. The accurate characterization and validation of the cell lines is a mandatory step in the implementation of in vitro glioblastoma research. In principle, the characterization should be performed assessing the expression profiles of the astrocytic markers GFAP and S100B. Using NES as a marker, a proportion of cell lines with potential in terms of malignancy can be determined. Despite the heterogeneous nature of primary cell lines and the broad range of morphological phenotypes displayed by the cells, morphological assessment,and determination of generation time are also important for characterization of self‐established primary cell lines. The migratory behavior and ability to form colonies of the primary cell lines provide additional information for in vitro glioblastoma research.The authors declare no conflicts of interest.This study was approved by the local human research ethics committee of the Friedrich Schiller University Jena (Ethik‐Kommission der Friedrich Schiller Universität Jena: AZ 3253‐10/11) and was performed in accordance with current legislation and the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. All patients gave their written informed consent to study participation.Susanne Grube: Data curation; formal analysis; investigation; methodology; project administration; validation; visualization; writing‐original draft; writing‐review and editing. Diana Freitag: Data curation; formal analysis; investigation; methodology; validation; visualization; writing‐original draft; writing‐review and editing. Rolf Kalff: Conceptualization; resources; writing‐review and editing. Christian Ewald: Conceptualization; data curation; project administration; resources; supervision; writing‐review and editing. Jan Walter: Conceptualization; data curation; project administration; resources; supervision; visualization; writing‐original draft; writing‐review and editing.


The Cancer Genome Atlas using PROGENy. More than half of HPV-negative HNSCC showed a pathway activation in EGFR or PI3K. An amplification in EGFR and a mutation in PI3KCA resulted in a significantly higher activity of the respective pathway (p = 0.017 and p = 0.007). Interestingly, both pathway activations could only be explained by genetic alterations in less than 25% of cases indicating additional molecular events involved in the downstream signaling. Suitable in vitro pathway models could be identified in a published drug screen of 45 HPV-negative HNSCC cell lines. An active EGFR pathway was predictive for the response to the PI3K inhibitor buparlisib (p = 6.36E-03) and an inactive EGFR and PI3K pathway was associated with efficacy of the B-cell lymphoma (BCL) inhibitor navitoclax (p = 9.26E-03). In addition, an inactive PI3K pathway correlated with a response to multiple Histone deacetylase inhibitor (HDAC) inhibitors. These findings require validation in preclinical models and clinical studies.While genetic alterations in Epidermal growth factor receptor (EGFR) and PI3K are common in head and neck squamous cell carcinomas (HNSCC), their impact on oncogenic signaling and cancer drug sensitivities remains elusive. To determine their consequences on the transcriptional network, pathway activities of EGFR, PI3K, and 12 additional oncogenic pathways were inferred in 498 HNSCC samples of  Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide with an incidence that is predicted to increase by 30% by 2030 . Risk faFirst-line palliative treatment includes the immune checkpoint inhibitor pembrolizumab in patients with PDL1-expressing (CPS>1) tumors or tumors with microsatellite instability in the absence of a contraindication to immunotherapy. A combination of pembrolizumab with chemotherapy should be considered for patients with a high tumor burden or a CPS <20 . For PDLvs. placebo and paclitaxel  in HNSCC and are independent of the HPV status 6% in HNS] and in clitaxel . Of notein vitro than genomics . The TCGA-HNSC cohort comprised both HPV-positive (n = 413) and HPV-negative tumors (n = 70). RNA-seq data of the TCGA-HNSC cohort was retrieved recount2 . The RanThe microarray data of the HIPO-HNSC  cohort (n = 79) used in this study is deposited at the Gene Expression Omnibus under the accession number GSE117973. For a detailed description of the clinicopathological characteristics of the cohort, please refer to Schmitt et al. . The rawGene expression microarray data of 45 HPV-negative head and neck carcinoma cell lines from Lepikhova et al.  were avaThe gene-level normalized RNA-seq data  of 26 HNSCC cell lines of the Cancer Cell Line Encyclopedia (CCLE) project were obtained through the Dependency Map (DepMap) portal of the Broad Institute . The DepThe drug sensitivity data of 45 HPV-negative head and neck carcinoma cell lines were obtained from the supplemental material of Lepikhova et al. . The autThe drug sensitivity data of the validation cohort of 26 HNSCC cell lines of the PRISM project (Version 19Q4) were downloaded from the DepMap portal of the Broad Institute .progeny R package . Heatmaps were generated with the lleagues .The activity of EGFR, PI3K, and 12 other key oncogenic pathways was inferred by the PROGENy algorithm from the normalized RNA-seq data of the TCGA-HNSC cohort consisting of 498 tumor samples , while this could not be observed for the few mutated cases  in HPV- tumors within the TCGA-HNSC cohort, it was investigated if suitable lleagues  were anaLepikhova et al. performed a comprehensive drug screen (220 compounds) on the 45 HPV- HNSCC cell lines for which a gene expression microarrray analysis was available. As a measure of efficacy, the authors calculated a drug-sensitivity score (DSS). A higher DSS equals a higher efficacy at lower drug concentrations. The impact of EGFR and PI3K pathway activity on the drug response was modeled as a continuous parameter. Intriguingly, activation of the EGFR signaling did not predict for the EGFR inhibitors afatinib, erlotinib, or gefitinib Table 1.HNSCC cell lines of the DepMap project were used as an independent validation cohort. As the HPV status for the 26 HNSCC cell lines is unknown, a published HPV gene expression signature  was inveWhile the cohort size of the validation cohort was considerably smaller than the cohort by Lepikhova and colleagues, it allowed for an explanatory analysis. As a measure for the drug response associations between pathway activities of EGFR and PI3K and the drug response of the candidate drugs, correlation coefficients were calculated and compared Table S4in vivo activity in preclinical models of head and neck cancer  is accessible Hospital . A validHospital  but matcHospital  was onlyThe original contributions presented in the study are included in the article/Conceptualization, AM, JH, and KZ. Methodology, AM, JH, and KZ. Formal analysis, AM and MT. Resources, SF, DJ. Data curation, AM, MP, and JM. Writing–original draft preparation, AM, MP, JM, JH, MT, and KZ. Writing–review and editing, DJ, JK, and SF. Visualization, AM and MT. All authors contributed to the article and approved the submitted version.AM is supported by the Physician-Scientist Program of the University of Heidelberg, Faculty of Medicine and is a fellow of the DKTK (German Cancer Consortium) School of Oncology and the Cancer Core Europe TRYTRAC program.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.