MYC oncogene is a potent driver of growth and proliferation but also sensitises cells to apoptosis, which limits its oncogenic potential. MYC induces several biosynthetic programmes and primary cells overexpressing MYC are highly sensitive to glutamine withdrawal suggesting that MYC-induced sensitisation to apoptosis may be due to imbalance of metabolic/energetic supply and demand. Here we show that MYC elevates global transcription and translation, even in the absence of glutamine, revealing metabolic demand without corresponding supply. Glutamine withdrawal from MRC-5 fibroblasts depletes key tricarboxylic acid (TCA) cycle metabolites and, in combination with MYC activation, leads to AMP accumulation and nucleotide catabolism indicative of energetic stress. Further analyses reveal that glutamine supports viability through TCA cycle energetics rather than asparagine biosynthesis and that TCA cycle inhibition confers tumour suppression on MYC-driven lymphoma in vivo. In summary, glutamine supports the viability of MYC-overexpressing cells through an energetic rather than a biosynthetic mechanism.The  MYC activation can sensitise cells to apoptosis upon glutamine withdrawal. Here the authors show that MYC activation enhances global transcription and translation that creates a metabolic demand, while glutamine limitation causes a metabolic demand and supply imbalance through loss of TCA energetics and thus, sensitises cells to apoptosis. MYC oncogene is a potent driver of growth and proliferation, and these oncogenic attributes drive its amplification or overexpression in many human cancers1. Deregulation of MYC also drives activation of tumour suppressive pathways, most notably sensitisation to apoptosis2 and activation of the p53 pathway resulting in restriction of cell cycle4. Deregulation of MYC sensitises cells to multiple apoptotic triggers in vitro, including withdrawal of serum2, treatment of cells with death receptor ligands such as TNF5 or TRAIL6 and withdrawal of glutamine7. MYC-induced sensitisation to apoptosis is abrogated in most systems upon expression of antiapoptotic members of the BCL-2 family, such as BCL-2 or BCL-xL8. Similarly, many systems show a requirement for one or both of the proapoptotic BCL-2 family members BAX or BAK1 for the apoptosis induced by MYC11, although the molecular mechanism is not thought to involve direct transcriptional activation of these genes.The 12 and that these target genes mediate both its tumorigenic and tumour suppressive functions. More recently, two studies provided comprehensive analyses of MYC-induced gene targets and promoter occupancy in various contexts14, allowing unparalleled insight into the regulation of gene expression afforded by MYC. A contrasting model holds that MYC activation acts to amplify the existing transcriptional programme of the cell16 and such a model would be hard to reconcile with one in which the apoptotic programme of MYC was based upon specific gene targets. Regardless of which model is correct, no convincing MYC target genes have thus far been identified that carry out its proapoptotic function.Two conceptual models of MYC-induced transcription have been proposed. The longest-standing model is that MYC activates a specific set of target genes, estimated to represent over 10% of the mammalian genome under conditions of MYC overexpression20, which impacts upon the total cellular content of RNA as well as transcriptional and translational load. This potent deregulation of global transcription requires the provision of abundant nucleotide triphosphates (NTPs). This, in turn, creates considerable demand for the energy and metabolic precursors required to feed the pentose phosphate pathway, pathways of purine and pyrimidine nucleotide biosynthesis and synthesis and function of the translational machinery. Indeed, several groups have reported MYC-induced activation of genes in the pentose phosphate and nucleotide biosynthesis pathways in vitro26 and in vivo in D. melanogaster27 and in mouse tumours28.In addition to activation of RNA polymerase II-directed transcription, MYC activates transcription directed by both RNA polymerases I and III29, we identified nucleotide catabolism as an unexpected consequence of MYC activation. Nucleotide catabolism is known to occur in multiple tissues under conditions of hypoxia, particularly in muscle when metabolic demand exceeds supply33 and this led us to hypothesise that MYC may elicit high metabolic demand and that apoptosis may occur when metabolic supply does not meet that demand. Here, we report that MYC drives a striking increase in global transcription and translation and accordingly diverts glycolytic carbon from 3-phosphoglyceraldehyde to ribose 5-phosphate via reversal of the non-oxidative pentose phosphate pathway. This high demand for nucleotides provides a platform for the glutamine-dependence of cells harbouring deregulated MYC34 and we therefore investigated this dependence in the context of metabolic supply and demand. Withdrawal of glutamine in MYC-overexpressing primary human MRC-5 fibroblasts led to a near-complete loss of key TCA cycle metabolites and a striking increase in AMP, ADP and nucleoside/nucleobase levels and activation of AMP kinase, indicating energetic stress due to lack of TCA cycle activity. Withdrawal of serum, which also caused depletion of TCA cycle metabolites, triggered apoptosis in these cells, as did pharmacological inhibition of the TCA cycle enzyme oxoglutarate dehydrogenase (OGDH). Indeed, the latter also elicited tumour suppression in MYC-driven lymphoma in vivo. Perturbations of mitochondrial energetic supply and cellular metabolic demand also revealed an inverse relationship between energetic status and apoptosis. Finally, we showed that asparagine supplementation rescued these cells from apoptosis but so did the knockdown of asparagine synthetase (ASNS), indicating that exogenous asparagine protects from apoptosis through product inhibition of ASNS activity rather than through enabling protein synthesis, as previously suggested34. Taken together, our results demonstrate that MYC-induced sensitisation to apoptosis occurs through an imbalance between metabolic demand and mitochondrial energy supply.Using untargeted stable isotope tracing of glucose and glutamineT2  in the presence of 4-hydroxytamoxifen (4-OHT) are highly sensitive to glutamine withdrawal and undergo apoptosis, as measured by annexin V/propidium iodide staining  for these experiments because they are genetically normal, and all tumour suppressive and homeostatic pathways are therefore intact. MRC-5 fibroblasts expressing MYC-ERing Fig. . We confing Fig. . We alsoing Fig. , as prevT2, MYC activation strongly enhanced global transcription in cells under normal growth conditions . Thus MYC can drive high and continued demand for glutamine and glutamine-derived metabolites for biosynthetic and energetic purposes even when glutamine is withdrawn.Protein translation exerts demand for glutamine as a biosynthetic precursor and energy source to fuel the GTP-dependent reactions of polypeptide chain elongation. Promotion of global translation by MYC has previously been reportedome Fig. . WithdraT2 that were deprived of cystine, although this was slower than the response to glutamine deprivation and only slightly accelerated by MYC activation. Withdrawal of the other amino acids did not lead to apoptosis in either cell type as previously reported7 nor did glutamine withdrawal from cells transduced with the pM6P vector, most likely because of homeostatic attenuation of biosynthetic processes in response to the withdrawal of an essential nutrient. We therefore conclude that glutamine withdrawal does not trigger apoptosis in MYC-expressing cells through impairment of translation. Important cytoprotective metabolic fates of glutamine and cystine are into the biosynthesis of glutathione, which acts to limit ROS-mediated damage and apoptosis. Treatment of cells with 20 μM l-buthionine sulfoximine (BSO), a potent inhibitor of glutathione synthesis, led to a more striking drop in glutathione levels than glutamine withdrawal , nucleobases  and nucleotide-containing lipid precursors . These data indicate that the main metabolic change elicited by MYC activation is a reprogramming of nucleotide metabolism. In particular, they reveal an unexpected MYC-dependent induction of nucleotide catabolism in addition to induction of nucleotide biosynthesis.Having established high MYC-driven metabolic demand, we wished to interrogate metabolite supply in more detail and we chose untargeted stable isotope approaches to examine the usage of two key nutrients, glucose and glutamine, upon MYC activation. Cells transduced with pM6P or pM6P-MYC-ER15N-glycine labelling experiments revealed that withdrawal of glutamine inhibited de novo production of adenine nucleotides and adenosine  upon glutamine withdrawal, and this was significantly higher in cells transduced with pM6P-MYC-ERT2   to visualise metabolic flux through the pentose phosphate pathway. MYC activation led to a striking increase in 6-phosphogluconate levels and label incorporation (m + 6) from 13C6-glucose in the presence of 6-AN, revealing high MYC-driven flux through the pathway  pulse of way Fig. . We obseway Fig. . In contway Fig. . A dramaway Fig. . The isoway Fig. .31 and in the exercise of muscle, particularly under conditions of ischaemia33. These are states in which energetic supply cannot meet demand, and we therefore examined whether MYC might promote nucleotide catabolism through high energy consumption, particularly when energetic supply is limiting . We found that cellular ATP levels were slightly lower upon glutamine withdrawal, and these were further reduced by MYC activation  in a reaction that also releases hydrogen peroxide. The rapid clearance of hydrogen peroxide by peroxidases and catalases renders this reaction irreversible under physiological conditions, and this reaction therefore leads to the loss of purines from the cellular metabolic pool. We observed xanthine accumulation only under conditions of MYC activation and glutamine withdrawal  essentially abrogated MYC expression whilst a second (sh1) gave partial knockdown  and m + 3 (reversed non-oxidative PPP) isotopologues in the parental line transfected with non-silencing shRNA control. Knockdown of MYC led to a reduction in the contribution of both pathways to AMP synthesis, with the sh2 shRNA leading to striking depletion of AMP, ADP and ATP  led to a reduction in expression of MYC  into the adenine nucleotides  withdrawal and also a reduction in levels of fumarate and malate  is that they interconvert adenine nucleotides in a reversible reaction whereby two ADP molecules are converted into one ATP and one AMP molecule. Under conditions of low energy charge (e.g. through extensive hydrolysis of ATP to ADP or lack of ATP production), AKs can act as a source of ATP as well as generating AMP which activates AMP kinase, resulting in cellular energy conservation  sensitises cells to apoptosis through a homeostatic mechanism, including or regardless of the primary transcriptional targets. Here we present data that support a homeostatic metabolic model for MYC-induced sensitisation to apoptosis. MYC induces metabolic demand for nucleotides that is strong enough to divert carbon away from glycolysis into a reversed PPP to support the production of ribose 5-phosphate and nucleotide biosynthesis. Our short-term stable isotope tracing studies with 13C6-glucose not only show enhanced flux through the oxidative arm of the PPP, but also demonstrate flux from glyceraldehyde 3-phosphate to ribose 5-phosphate through reversal of the non-oxidative arm of the PPP. This pathway consists of two bidirectional enzymes, transaldolase and transketolase, that catalyse reactions whose direction is determined by substrate & product concentrations. Thus, reversal of this pathway is a clear indicator of high demand for ribose 5-phosphate driven by MYC. It is also a striking example of how MYC can reprogramme cellular metabolism through a homeostatic mechanism separately from its direct transcriptional activation of metabolic target genes43.MYC requires its transactivation domain to induce apoptosisThe energy crisis that is driven by MYC activation and withdrawal of glutamine does not manifest as a major reduction in ATP levels, but rather as an accumulation of lower-energy nucleotides, nucleosides and bases. Within the nucleotide pools, this remodelling is most likely mediated by the adenylate kinases (with the exception of AK2) that catalyse the reversible conversion of 2 × ADP into ATP and AMP, allowing for the maintenance of ATP concentration whilst accumulating lower-energy species. Here we report that AK2 catalyses the opposite reaction, resulting in ADP production from AMP and ATP. This observation, whilst initially unexpected, is in fact predictable due to the localisation of AK2 to the intermembrane space. The adenine nucleotide translocase (ANT) on the inner mitochondrial membrane provides ATP to the intermembrane space whilst withdrawing ADP from this environment, promoting the production of ADP from ATP and AMP by AK2, particularly under conditions where cytosolic AMP is elevated. Through the generation of ADP, AK2 can therefore fuel mitochondrial ATP production and act to increase cellular energy charge. This non-canonical direction of AK2 catalysis is not only consistent with substrate/product availability but also with the physiological function of the gene family in the conservation of cellular energy charge.33 and hypoxia31 remain obscure. We were unable to identify conditions under which inhibition of nucleotide catabolism was detrimental to cell survival . This may, in large part, be due to redundancy in the catabolic pathways since there are eight currently identified nucleotidases in addition to the nine adenylate kinases that remodel adenine nucleotides in different cellular compartments. A plausible explanation for the catabolism and production of hypoxanthine and xanthine in such cases would be that accumulation of AMP leads to the promotion of nucleotidase activity purely based upon substrate concentration. Such a model would allow for catabolism of AMP and therefore continued activity of the AKs, enabling continued conversion of ADP to ATP in turn. Irrespective of potential benefit to cellular homeostasis, the products of nucleotide catabolism are diagnostic of the shortfall between energetic supply and demand in this context, as they are in others33.The exact reasons for induction of nucleotide catabolism in our experiments, and indeed upon muscle activity34 since there is a remaining requirement for glutamine to support multiple metabolic pathways. Our apoptotic and energetic data, supported by measurements of very high levels of asparagine, indicate that asparagine supplementation rescues from apoptosis through product inhibition of ASNS. Although ASNS activity consumes ATP, it seems unlikely that this could have a major impact on cellular energetics. We propose that a more likely explanation for this is that ASNS removes aspartate from the malate aspartate shuttle and that, in the absence of anaplerosis from glutamine, this has a detrimental impact on mitochondrial energetics.Re-addition of glutamine rescued Myc-overexpressing cells from the apoptotic phenotype and enabled their proliferation, consistent with a homeostatic mechanism based on metabolic supply and demand rather than triggering of an irreversible commitment to apoptosis and cell cycle arrest. In contrast, the rescue from apoptosis afforded by asparagine is not accompanied by proliferation44 and the dependence of MYC expression upon glutamine availability in activated T cells also suggests that glutamine metabolism could be targeted in cancer45. Unfortunately, however, the therapeutic efficacy of glutaminase inhibition in Myc-driven tumours is rarely observed in vivo. Indeed, metabolism of glutamine via amidotransferases and altered utilisation of glucose-derived carbon have recently been identified as compensatory mechanisms in Myc-driven liver tumours46. This illustrates how metabolic redundancy can promote bypass of inhibition of single metabolic nodes and suggests that synthetic lethal approaches targeting multiple pathways are likely to be required. Since the reaction catalysed by OGDH has less redundancy than that catalysed by GLS, we targeted this enzyme in place of GLS inhibition. Genetic approaches cannot be used to recapitulate the metabolic impact of glutamine withdrawal because they lack the requisite rapidity of onset and so we turned to a small molecule inhibitor of OGDH, the lipoate analogue CPI-613. The mechanism of action of CPI-613 is likely to be complex, and it does not inhibit OGDH in all cases40, although we confirmed that it did so in our cells. Indeed, CPI-613 recapitulated the rapid apoptosis elicited by glutamine withdrawal in MYC-overexpressing cells in vitro and achieved tumour suppression in MYC-driven lymphoma in vivo. The development of more specific OGDH inhibitors may therefore be a desirable approach to therapeutic targeting of MYC-overexpressing tumours.Inhibition of glutaminase  was initially proposed as a promising strategy for targeting of MYC-driven tumours47.Our data also indicate that targeting AK2 could be an attractive option for cancer therapy since it should preferentially target cells that exhibit high demand for energy. Indeed, one can envisage a scenario where alternating regimes are applied, based upon inhibition of nucleotide biosynthesis (leading to selection for tumour cells that elevate biosynthetic capacity and therefore elevate metabolic demand) followed by inhibition of the TCA cycle and AK2 (leading to tumour suppression due to lack of energetic support for the excessive metabolic demand). Such ‘metronomic’ delivery of chemotherapy has been effectively demonstrated in a mouse model of melanoma53. BAX has been shown to require the mitochondrial ATPase for its apoptotic function54 and to localise to mitochondria in cardiomyocytes in response to ischaemia and AMPK activation55. The common denominator in most of these studies is the involvement of mitochondrial nucleotide metabolism in the regulation of apoptosis by the BCL-2 family, and our model of MYC-induced sensitisation to apoptosis through energetic demand continues that theme.The robust impact of the BCL-2 family upon MYC-induced apoptosis indicates that common molecular mechanisms are most likely shared between the two. BCL-2 and BCL-xL can protect mitochondria from damage due to reactive oxygen species (ROS), mitochondrial membrane potential (MMP) decline and impaired nucleotide exchange and respiratory controlIn summary, many oncogenes promote growth through activation of various signalling pathways or through transcriptional regulation of fundamental cellular programmes. The deregulation of these growth-promoting programmes inevitably has homeostatic consequences for the cell in terms of biosynthetic and energetic supply and demand. We propose that an increased understanding of these secondary homeostatic responses will open up new possibilities for therapeutic intervention.T2 and selected with blasticidin (5 μg/ml). BCL-xL was expressed with pMSCV-based retrovirus. Cells were cultured in DMEM/10% FCS. Compounds for treatment were obtained from Sigma unless otherwise stated. 4-hydroxytamoxifen (200 nM) was used to activate the MYC-ERT2 and was present in all vector control samples as well. For siRNA experiments, DharmaFect 1 transfection reagent was used to deliver Dharmacon SMARTpool siRNA mix by reverse transfection in six-well plates at 30 nM final concentration. The day after transfection, assays were carried out as described below.MRC-5 human fibroblasts  were transduced with retroviral vector pM6P-blast  or pM6P-blast-MYC-ER2) and 5 μl annexin V-FITC (BD Bioscience). Immediately prior to analysis, propidium iodide was added. Data were acquired using a BD Accuri C6 flow cytometer and analysed using FlowJo v9.6.2, reporting single- and double-positive populations as apoptotic. The gating strategy is provided in Extended Data 1a.Cells were seeded in 6-well plates and on the following day, 4-OHT was added to all wells. After a further 24-h incubation, glutamine was withdrawn (where stated), and cells were incubated overnight. For determination of apoptosis, cells were trypsinized and collected into FACS tubes. Pellets were washed with PBS and resuspended in 100 μl annexin V buffer  was added and apoptosis was measured using an Incucyte live cell imager . Results were obtained as percent apoptotic area based upon the percentage of cell area (determined by phase contrast microscopy) that stained positive for the caspase 3/7 reagent.® RNA Alexa Fluor® 488 HCS Assay (ThermoFisher) was used for the determination of transcription. Cells were plated in 96-well plates, treated as described, incubated with 5-ethynyl uridine (EU) for half an hour before fixation and permeabilization. Alexa Fluor® azide was added for EU detection. Cells were then washed with Click-iT® reaction rinse buffer and counterstained by DAPI. All the images and analysis were done with the ImageXpress Micro XLS Widefield High-Content Analysis System .A Click-iT13C6,15N2-lysine, cell pellets were extracted with 8 M urea. DTT was added to 10 mM and samples were incubated at RT for an hour. Iodoacetamide was then added to 25 mM and incubated at RT for an hour and DTT was then added to final concentration of 25 mM. Samples were incubated overnight at 37*C with lysC (estimated ratio of lysC to the protein of 1:50 to 1:200) and peptides were acidified by addition of TFA to 0.5%. An estimated 10 μg of the resulting peptide solution was loaded onto an activated (20 μl methanol), equilibrated (100 μl 0.1% TFA) C18 StAGE tip and washed with 100 μl 0.1% TFA. The bound peptides were eluted into a Protein LoBind 1.5-ml tube (Eppendorf) with 20 μL 80% acetonitrile, 0.1% TFA and concentrated to less than 4 μl in a vacuum concentrator. The final volume was adjusted to 6 μl with 0.1% TFA. Online LC was performed using a Dionex RSLC Nano HPLC. Following the C18 clean-up, 5 μg peptides were injected onto a C18 packed emitter and eluted over a gradient of 2–80% ACN in 120 minutes, with 0.1% acetic acid throughout. Eluting peptides were ionised at +2 kV before data-dependent analysis on a Thermo Q-Exactive Plus. MS1 was acquired with mz range 300–1650 and resolution 70,000, and top 12 ions were selected for fragmentation with normalised collision energy of 26, and an exclusion window of 30 s. MS2 were collected with resolution 17,500. The AGC targets for MS1 and MS2 were 3e6 and 5e4, respectively, and all spectra were acquired with 1 microscan and without lockmass. Data were analysed using MaxQuant (v 1.5.8.3)56 in conjunction with UniProt FASTA database (UP000005640_9606.fasta (Human) 2017_05 release), with match between runs (MS/MS not required), LFQ with 1 peptide required, and statistical analyses performed in R58.Following labelling of cells for 6 hours with Thr172 , anti-AMPK , anti-β-actin , and for Fig. Cells were lysed in Cell Lysis Buffer (New England Biolabs) supplemented with Complete Protease Inhibitors (Roche) and subjected to SDS-PAGE, and transfer to Immobilon-P membrane (Merck-Millipore). After blocking in the blocking buffer (TBST + 5% non-fat milk), primary and HRP-conjugated secondary antibodies  were applied in the blocking buffer and antigens were revealed with SuperSignal West Pico chemoluminescent reagent (ThermoFisher). Antibody to AK2 (HPA018479) was from Cambridge Biosciences. Antibody to c-MYC (clone Y69—ab32072) was from Abcam. Anti-phospho-AMPKRNA was extracted using an RNEasy Mini kit (Qiagen) and reverse transcribed using qScript cDNA Supermix (Quanta Biosciences) according to the manufacturer’s protocol. qPCR was performed on a StepOne RT-PCR system (ThermoFisher) using Sybr Select Master Mix (Life Technologies) and the following oligonucleotides: AK2 (tggtagtggagctcattgaga and catttctgcctgcctcaca), ASNS (aaaggtggcagatcatattg and atatgacttcatccagagcc).Metabolites were extracted from six-well plates by washing individual wells with ice-cold PBS and adding of cold extraction buffer . Extracts were clarified and stored at −80 °C until required.59.LC-MS was carried out using a 100 mm × 4.6 mm ZIC-pHILIC column (Merck-Millipore) using a Thermo Ultimate 3000 HPLC inline with a Q-Exactive mass spectrometer. A 32 min gradient was developed over the column from 10% buffer A (20 mM ammonium carbonate), 90% buffer B (acetonitrile) to 95% buffer A, 5% buffer B. 10 μl of metabolite extract was applied to the column equilibrated in 5% buffer A, 95% buffer B. Q-Exactive data were acquired in negative mode , and standard ESI source and spectrometer settings were applied . Metabolites were identified based upon m/z values and retention time matching to standards. Quantitation of metabolites and their isotopologs was carried out using AssayR version 0.1.46 Raji cells were injected subcutaneously into the right flank of 8- to 10-week-old male NOD-SCID IL2Rγ-null (NSG) mice. After 2 days, the animals were treated daily with intraperitoneal injections of either vehicle control or 20 mg/kg CPI-613. Tumour weight and engraftment were analysed when animals were euthanized because burden became large (approaching 1.2 cm in any dimension—no mice exceeded these limits) or when animals showed signs of poor health. All mice were maintained in a standard SPF facility . These animal studies have been regulated under the Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012 following ethical review by the University of Cambridge Animal Welfare and Ethical Review Body (AWERB-PPL number P846C00DB). HALO digital pathology software (Indica Labs) was used to analyse the percentage of tumour using a random forest tissue classifier in samples from haematoxylin counterstained formalin-fixed, paraffin-embedded (FFPE) sections. This analysis was carried out by the Histopath Facility at the Cancer Research UK Cambridge Institute.In all, 1 × 1060. P values are from T tests comparing means of lower and upper tertiles of MYC expression.A compendium of 2999 breast tumours from 17 Affymetrix microarray studies was integrated as described previouslyP values ≤ 0.05 were considered to be significant. In the case of X13CMS analysis, P values were calculated using the Student’s T test as part of the X13CMS workflow in R. Experiments were performed a minimum of three times, with the exception of the X13CMS and xenograft analyses that were carried out once.GraphPad Prism software (Version 9.3.1) was used for statistical analyses unless otherwise indicated in the figure legends. Statistical tests are indicated in the figure legends throughout. For all experiments, Further information on research design is available in the Supplementary InformationReporting Summary


Dynamic changes in epigenetic landscape reflect a critical command of lineage-specific gene expression. In an effort to discern the epigenetic regulatory networks of myogenic differentiation, we have used systematic and integrative approaches to explore multi-omics datasets on global myogenic gene expression, histone acetylation and acetyltransferase occupancy in view of distinct chromatin states. In this brief report, we discuss experimental design and provide a comprehensive assessment regarding data quality control, filtering and processing. We also define a gene-level overlap between RNA-seq and ChIP-seq datasets through integrative analyses to offer strategies for future use of the data. Furthermore, our analyses generate a blueprint on chromatin state distribution of residue-specific histone acetylation and concomitant association with histone acetyltransferase p300 in committed skeletal myoblasts and differential histone acetylation signatures at the onset of myoblast differentiation. These datasets can be further utilized to delineate the function of muscle-specific regulatory elements governed by other muscle myogenic regulators or signaling molecules. Epigenetic regulatory mechanisms affect global gene expression patterns underpinning lineage commitment and stem cell differentiation, and thus impact tissue homeostasis including skeletal muscle –4. RecenDifferent regulatory elements are marked by distinct patterns of chromatin modifications, in that H3K9ac, H3K18ac, and H3K27ac are often found near transcription start sites (TSS), while H4K8ac is mostly associated with promoters and gene bodies that are actively transcribed –13. In aPartaking in transcription control of a vast number of genes, p300 is specifically required for the function of muscle-specific transcription factors and essential for myogenic differentiation , 23. UndCell type specific chromatin states can be defined by large-scale analyses of co-occurrence of different histone marks , 27. We Since epigenetic modifiers and transcriptional regulators are essential for myogenic differentiation and their dysfunction is implicated in pathogenesis of skeletal muscles, a better grasp of epigenetic regulatory mechanisms of the myoblast genome is paramount. In this report, we provide technical validation and comprehensive analyses of total of 14 genomic datasets which profile global gene expression, histone acetylation, and p300 occupancy for an unbiased and genome-wide view of chromatin states associated with myoblast differentiation. For an in-depth usage of the datasets, we provide detailed information on the experimental design, data quality control, and integrative analyses. Our objective is to offer strategies for future integrative analyses of the datasets to advance the knowledge of epigenetic regulatory networks on myoblast genome in a specific signaling pathway or pathological context. Moreover, comprehensive analyses of epigenomes and transcriptomes in differentiating myoblasts also provide a platform to identify additional histone acetylation signatures of myoblasts genome.Total RNA was extracted from C2C12 myoblasts using RNeasy kit (Qiagen) according to the manufacturer’s instruction. RNA samples from two biological replicates were processed by the McGill University Genome Quebec Innovation Centre for cDNA library construction and deep sequencing using an Illumina HiSeq 2000 sequencer as single-end 50 base pair reads as previously described . Salmon ChIP-seq library preparation and deep sequencing were performed by the McGill University Genome Quebec Innovation Centre with Illumina HiSeq 2000 as single-end 50 bp reads . The seqThe resulting peak files were used as input into HOMER for gene annotation. ChIP enrichment and p300-associated sites was calculated using ngs.plot, which calculates the coverage vectors for each region based on specified ChIP-seq alignment files. Following normalization and transformation on the coverage, a heatmap as well as an average profile is plotted from the mean of all regions, normalized to the total number of mapped reads in millions. Integrative Genomics Viewer (IGV) was used for visualization of the ChIP-seq read signals. Model-based Analysis of ChIP-seq (MACS) was used to determine the scores of Fraction of Reads in Peaks (FRiP) and to call broad peaks of histone acetylation for chromatin states distribution.We examined myogenic expression and histone acetylation profiles on a genome-wide scale using C2C12 skeletal myoblasts as a system of differentiation. These myoblasts are myogenic precursors of mesenchymal origin and represent a well-established model of myogenic differentiation. They switch from proliferation to differentiation under low mitogen conditions, followed by a fusion process to form multinucleated myotubes and have been a model of choice for many genome-wide studies due to a high correlation with primary myoblasts , 29. TheIn our studies, the C2C12 myoblasts were differentiated for 24 h (Fig. To assess the quality of RNA-seq datasets for differentiating and proliferating myoblasts, base call scores were analyzed using the FastQC program Fig. A. The quNext, the RSeQC gene body coverage tool was used to examine uniformity of read coverage across the gene body for indications of any 5ʹ/3ʹ biases. Figure In RNA-seq, distribution of the reads across known gene features is an additional important measure of experimental validity. Most reads are expected to map to exons compared to introns and intergenic regions . As showIn addition to detecting novel transcripts, a principal benefit of RNA-sequencing has been replicability between samples, which was measured as the correlation of gene expression between samples Fig. F. SpearmThe average base quality scores for all ChIP-seq datasets in the present study fell within the high confidence range Fig. A and disFollowing the confirmation of satisfactory alignment metrics, we also examined the genome coverage and the strength of ChIP-seq simultaneously through cumulative enrichment analysis Fig. D. IntersWe have previously established a model of 14-chromatin states in committed skeletal myoblasts , with muHere, we analyzed ChIP-seq of histone acetylation marks including H4K8ac, H3K9ac, H3K18ac, and H3K27ac after quality verification of the raw sequencing data, since transcriptomes are controlled by epigenetic regulatory networks. PCA plots were first used to assess the clustering of histone acetylation marks into their respective biological groups for chromatin modification and differentiation status (Fig. Next, we examined the chromatin state distribution of residue-specific histone acetylation in early differentiation utilizing our established chromatin state model. As shown in Fig. Tbc1d1, a glucose regulator in skeletal muscle [We also delineated epigenetic networks involved in p300-mediated gene expression, since p300 is a critical HAT in myogenic differentiation , 24, 39.l muscle .Fig. 5HiIntegrative analyses of p300 and histone acetylation ChIP-seq data with corresponding chromatin state and transcriptome offer an invaluable avenue to identify chromatin states distribution of residue-specific histone acetylation and discern the distinct function of HATs in epigenetic landscape and gene regulatory networks within a specific biological milieu. The high throughput sequencing datasets presented here are biologically validated and in high quality, and thus can be used for future studies to determine the roles of muscle regulatory factors, residue-specific histone acetylation and p300 function in early myogenic differentiation, particularly in terms of their impact on chromatin dynamics.


Hepatocellular carcinoma (HCC) is amongst the cancers with highest mortality rates and is the most common malignancy of the liver. Early detection is vital to provide the best treatment possible and liquid biopsies combined with analysis of circulating tumour DNA methylation show great promise as a non-invasive approach for early cancer diagnosis and monitoring with low false negative rates. To identify reliable diagnostic biomarkers of early HCC, we performed a systematic analysis of multiple hepatocellular studies and datasets comprising > 1500 genome-wide DNA methylation arrays, to define a methylation signature predictive of HCC in both tissue and cell-free DNA liquid biopsy samples. Our machine learning pipeline identified differentially methylated regions in HCC, some associated with transcriptional repression of genes related with cancer progression, that benchmarked positively against independent methylation signatures. Combining our signature of 38 DNA methylation regions, we derived a HCC detection score which confirmed the utility of our approach by identifying in an independent dataset 96% of HCC tissue samples with a precision of 98%, and most importantly successfully separated cfDNA of tumour samples from healthy controls. Notably, our risk score could identify cell-free DNA samples from patients with other tumours, including colorectal cancer. Taken together, we propose a comprehensive HCC DNA methylation fingerprint and an associated risk score for detection of HCC from tissue and liquid biopsies. Hepatocellular carcinoma (HCC) is the most common malignancy of the liver accounting for nearly 90% of all cases5. Major risks of HCC include cirrhosis, viral infection with hepatitis B virus (HBV) or hepatitis C virus (HCV), alcoholic liver, non-alcoholic fatty liver disease and inherited traits such as metabolic diseases6. Current HCC diagnostic guidelines report the usage of invasive procedures, such as tissue biopsies, followed by histological and/or contrast-enhanced imaging7. This contributes to HCC being often detected in an advanced stage where it is estimated that 40% of the cases are multinodular or expanded beyond the liver leaving patients with limited therapeutic options8. Screening, surveillance and monitoring programmes are therefore vital to diagnose and detect HCC as early as possible to provide patients with the best treatment possible11. In addition, HCC patients surgically treated often experience relapses and early detection could bring better management of the disease and increase patient’s life quality and span12.Liver cancer is one of the deadliest types of cancer, with a 5-year overall survival rate lower than 20% and death rates increasing around 1.7% each year14. Among others, alpha-fetoprotein (AFP) is often proposed as a diagnostic biomarker present in serum or plasma of high-risk individuals for HCC15, nonetheless official guidelines indicate that AFP has no diagnostic approved role4. High levels of AFP are considered diagnostic of HCC with almost perfect specificity, although sensitivity  rates are frequently low, less than 45%7. Lower thresholds of AFP (20 ng/ml) comprises a balance between specificity and sensitivity with both ranging around 79%7. Of note, in patients with chronic liver disease, the population where screening methods are most needed, the precision of AFP is significantly reduced and insufficient for robust diagnosis17. This is particularly problematic since chronic liver diseases are the major risk factor for HCC, thus novel non-invasive and accurate clinical approaches are needed to improve cancer detection.Body fluids, for example plasma, serum and urine, contain circulating biomarkers that can be measured non-invasively and inexpensively for diagnosis and monitoring of HCC18. LBs contain cell-free DNA (cfDNA) material evocative of cells from the entire body, including varying levels of circulating tumour DNA (ctDNA)20 that is estimated to range between 0.1% and 10% in cancer patients23. Measurement of genetic markers in ctDNA, such as mutations and methylation, can be used as a diagnostic and therapeutic tool32 and provide complementary information to tissue samples, for example circumventing potential tissue heterogeneity which might result in sampling bias34.Liquid Biopsies (LBs) have recently emerged as a promising approach for early detection of tumours by characterising circulating tumour cells or circulating tumour free nucleic acids38. Importantly, DNA methylation changes characteristic of cancer cell formation are often observed in early stages of carcinogenesis42. Hence, ctDNA methylation holds great promise for early cancer detection and monitoring, with systematic studies showing it outperforms other genetic markers like mutations and copy number alterations43. For example, promoter methylation of the gene Septin 9 (SEPT9/ SEPTIN9) is a plasma derived biomarker for colorectal cancer and is being studied for HCC44. Several studies have focused on the identification of DNA methylation biomarkers for HCC48, nonetheless these were limited to either tissue samples only, focused on the identification of small sets of single CpG sites, and/or mostly compared to healthy liver tissue samples. Relying on the accurate measurement of very specific and small sets of methylation biomarkers, mostly derived from tissue samples, may hinder the clinical generalisation of these methylation signatures to LBs and other cohorts. Additionally, it is fundamental to ensure that signatures can distinguish HCC patients from a background of chronic liver diseases, where current non-invasive molecular markers perform worse17.DNA methylation plays an important role in cancer initiation and progression through the repression of tumour suppressor genes by promoter hypermethylation and promoter hypomethylation of many oncogenes58, including both tissue and liquid biopsy samples from HCC, cirrhosis and healthy controls. We developed a machine learning pipeline to harness this resource to identify differentially methylated regions (DMRs) predictive of HCC in both tissue and liquid biopsies, from a background of cirrhotic samples. Our approach benchmarked favourably against 12 independent HCC methylation signatures and supported the development of a novel signature comprising 38 DMRs. Some of the identified regions were associated with transcriptional repression of several members of the Zinc Finger Proteins (ZFNs) family suggesting a potential role with cancer progression and early onset. Lastly, we combined the information of the novel DMR signature into a single score which successfully identified HCC tissue samples in an independent dataset , perfectly classified 13 healthy cfDNA samples, and identified 7 (out of 11) tumour cfDNA samples. Of note, the DMR signature score successfully identified cfDNA from diverse tumours, including colorectal and breast cancer, showing its potential as a diagnostic tool for multiple cancers. Overall, we present a systematic discovery and benchmark of methylation biomarkers for the early detection and monitoring of HCC using tissue and liquid biopsies and propose an improved signature and risk score with the potential to be used for non-invasive clinical diagnostics.Here, we perform a systematic discovery of a HCC methylation signature by compiling 1551 genome-wide DNA methylation arrays from 13 studies60 and ArrayExpress61, using the keywords Hepatocellular Carcinoma, cfDNA and ctDNA. To ensure an exhaustive analysis of methylation markers we focused on studies that provided high-throughput assays and specifically Illumina-based, Infinium 450 K and EPIC assays, as these have been broadly adopted by large-scale studies. Additionally, to minimise potential undesired and technical batch effects while integrating multiple data sources for model training, only studies that provided raw unprocessed files were considered to allow the same processing pipeline to be applied to all samples64. Matching the criteria defined above we assembled 859 samples from 6 different studies58 covering: HCC and cirrhotic samples from tissue and cfDNA, including cirrhotic tissue from multiple aetiologies; healthy controls from both liver tissue and cfDNA; other non-HCC diseased tissue (e.g. liver obesity and Alpha 1 antitrypsin deficiency); and cfDNA from non-HCC patients (e.g. sepsis and other cancer types)  apart. This defined a total of 39,868 CpG clusters with a median size of 700 bp spanning all 22 autosomal chromosomes  Fig. a. We app66. Global changes in methylation affect large swaths of CpG sites and thereby these do not represent optimal candidates for biomarkers due to their lack of specificity . Therefore we considered PC5 as a proxy of tumour purity impact in the DNA methylation measurements  are identified by removing potential cofounder effects, i.e. sex, age, global methylation and tumour purity. Considering that sex and age were not available for all samples, we estimated them from the DNA methylation arraysIn conclusion, the feature selection and model training steps performed in each cross-validated train set avoids information leak between train and test sets, addresses the problem of having many more features than samples and identifies the most predictive DNA methylation biomarkers of HCC.Next, we set to define a DNA methylation signature predictive of HCC and compare it against independently defined sets. We estimated the optimal number of DMRs to consider in the methylation signature by sequentially testing the addition of DMRs into the feature set and tested the increment in precision and recall of the LinearSVC models   and overall show poor agreement between them. This might indicate these signatures are potentially specific to the studies, which could hinder generalisation to other cohorts and the utility for liquid biopsies as these are noisier backgrounds with low available materials, thus affecting the detection of these very specific features. To address this, we assembled > 1500 genome-wide DNA methylation arrays from 13 independent datasets58 making this one of the largest methylation compendium to study HCC to date. We harnessed this rich dataset by implementing a machine learning pipeline that searches, in an unbiased way, for significantly differentially methylated regions (DMRs) in HCC presenting several improvements. Firstly, considering regions spanning multiple CpG sites increases confidence as these can be more robustly measured in liquid biopsies in clinical settings. This procedure reduces the impact of eventual CpG site misdetection in the diagnosis and makes this more amenable for next-generation sequencing readouts, which measure all sites within the specified region. Secondly, training machine learning predictors with a training set equally representing tissue and liquid biopsies ensures the DMRs identified are representative of HCC tumours that can also be measured in ctDNA. Moreover, making this comparison against a cirrhotic background, instead of healthy liver samples, provides a more relevant clinical comparison. Very often patients who develop HCC also suffer from chronic liver disease and cirrhosis, and these are the backgrounds where existing non-invasive alternatives underperform. Lastly, to reduce potential analytical artefacts in the DMR biomarker discovery we processed the training dataset (859 samples from 6 different studies58) from raw data with the same pipeline and applied stringent filters to remove problematic measurements and account for potential confounders, such as sex, age, tumour purity and global methylation, often not considered by other studies. Additionally, we validated our approach using not only hold-out samples and cross-validated procedures, but also an assembled validation dataset (692 samples from 7 independent datasets55), which was never used for training and comprises differently and independently processed datasets, thus testing the robustness of our DMRs to diverse processing pipelines.Hepatocellular carcinoma (HCC) diagnosis is challenging and often misses early detection which is vital to ensure curative options are available to the patient. Non-invasive diagnostic approaches based on serum biomarkers, such as alpha-fetoprotein (AFP), AFP isoforms and micro-RNAs, have shown sub-optimal sensitivity, leaving many patients undiagnosed. Tumour cell-free DNA (cfDNA) from blood liquid biopsies holds great promise to transform clinical oncology diagnosis75 across multiple datasets in both tissue and liquid biopsy samples. We harnessed this to derive a novel methylation fingerprint comprising 38 DMRs and combined it into a single diagnostic metric which detected HCC tissue samples in a validation dataset with 96% recall and 98% precision.Our machine learning approach compared favourably against 12 HCC methylation signatures57, and while not directly related with HCC these samples supported the utility of out approach, by for example showing it could correctly classify all healthy cfDNA samples. Of note, the DMR signature score also successfully identified 7 cfDNA samples (out of 11) from other tumours, including breast, lung and colorectal cancer.A limitation of our analysis is linked with the scarcity of cfDNA methylation samples. While this is ubiquitous across other independent studies, it limits the estimation and extrapolation of evaluation metrics, recall and precision, to other cohorts. To mitigate this, we thoroughly benchmarked our approach by assembling comprehensive and independent training and validation DNA methylation datasets. Specifically, we aimed to integrate as many liquid biopsy samples as possible, e.g. cfDNA analyses from healthy controls, sepsis and different tumours84. Gene expression analysis showed that several DMRs of our signature are significantly associated with transcriptional repression of multiple Zinc Finger Proteins (ZFNs) supporting a potential role of these regions in cancer progression and early onset85. Caution needs to be taken when interpreting this signature as a potential pan-cancer detection as it will be of limited use to identify the tissue of origin of the tumour. This is intended to be used in the context of HCC where, for example, cirrothic patients are clinically followed and this DNA methylation would support the decision for further diagnosis of HCC. Lastly, this approach is currently being considered to monitor HCC patients that have undergone therapies, such as surgical liver resection, radiofrequency ablation and chemoembolization, as a means of clinical follow-up to identify residual disease and guide treatment86.This last point suggests that our ctDNA methylation signature and risk score have the potential for pan-cancer early diagnostics. Indeed other studies have shown that DNA methylation biomarkers can be used for the detection of different cancers, such as promoter methylation of the gene SEPT9 in colorectal cancer and HCCIn this study, we present a machine learning pipeline that harnesses a comprehensive genome-wide DNA methylation resource to build a signature and a diagnostic score for HCC that benchmarks favourably against existing biomarkers. While further work to confirm the clinical utility of this approach is ongoing, it addresses important challenges of the design of reliable non-invasive diagnostic and monitoring approaches for HCC from liquid biopsies, to provide long sought-after alternatives to current suboptimal approaches.58 using Infinium HumanMethylation EPIC and 450 K assays were processed using the R package minfi (v1.32.0)64. Datasets were integrated by considering the overlapping CpG probes between the two Infinium HumanMethylation assays . All datasets were merged into a single matrix containing signal intensities imported from the raw IDAT files and processed using the functional normalisation pipeline63. Lastly, the ratio between the methylation and unmethylated channels was calculated and exported as beta values (β) Eq. (DNA methylation samples from 6 different datasetse minfi v.32.064. https://github.com/markgene/maxprobes) cross-reactive probes of the Illumina methylation arrays were removed90; (3) CpG sites with missing values were discarded; (4) we utilised an updated probe annotation mapped to the hg38 reference build and probes with no available alignments were not considered; and (5) to focus on biomarkers that are sex agnostic CpG sites mapping to sex chromosomes X and Y were removed from downstream analyses. The final filtered DNA methylation matrix covered a total of 390,445 CpG sites without any missing value across all samples.Altogether, we generated a single matrix of DNA methylation beta values spanning 452,567 CpG sites measured across 859 samples, integrating multiple studies processed from the raw signals using the same pipeline. For the downstream analyses several filtering steps were taken: (1) probes containing a single nucleotide polymorphism (SNP) in the CpG site or in the single nucleotide extension at a minor allele frequency (MAF) greater or equal than 0.01 were excluded from downstream analysis; (2) using maxprobes R package 91 we identified CpG clusters with a maximum of 500 base-pairs (bp) distance between any 2 consecutive CpG sites. Then we overlapped the CpG clusters with the filtered CpG sites defined previously and only considered CpG clusters with at least 3 CpG sites with measurements. A final CpG cluster matrix was defined by taking the mean of all filtered CpG sites within each cluster region, generating a DNA methylation matrix spanning 39,868 CpG clusters.To identify DNA methylation regions, CpG clusters, we utilised a similar approach to the one described in Jaffe et al.92.Dimension reduction analysis was performed using Principal Component Analysis (PCA) implemented in scikit-learn Python module (v0.24.0)58 with a Principal Component (PC) 2 lower than 200  to identify differentially methylated regions (DMRs) predictive of HCC.The systematic search of DNA methylation biomarkers of HCC and benchmark against other independent sets of biomarkers64; (2) patient age, this is also largely unavailable and therefore we used66 the R package wateRmelon93 (v1.0.0) to estimate methylation age of the sample using their methylation profile and considered the Hannum65 and Horvath66 approaches; (3) sample global methylation, to mitigate potential biases mediated by the sample overall methylation levels we calculated the sample mean methylation levels and considered it as another independent variable; (iv) tumour purity, this information is only available for the TCGA samples76, CPE purity67, and the varying levels of tumour purity affect the molecular measurements and thereby we included Train & Test PC5 in the model as a proxy to tumour purity estimations ; and lastly (v) we included an intercept term. The full model is fitted and a beta coefficient is estimated for each independent variable. To statistically assess those CpG clusters that are significantly differentially methylated in HCC we also trained a smaller model (null hypothesis) that excludes the HCC status to test the hypothesis that the CpG cluster methylation status provides a significant increase in the classification power of HCC over the covariates. This is estimated using the log-likelihood ratio test for every CpG cluster and the p-values are then adjusted for multiple-hypothesis testing using the Benjamini–Hochberg False Discovery Rate (FDR). We complement this with a ANOVA differential CpG cluster methylation analysis performed with the f_classif function from the scikit-learn (v0.24.0)92 module and statistical assessment using the F-scores associated p-values after adjusting for multiple hypothesis with FDR. Lastly, DMRs are defined as those CpG clusters with a ratio test and ANOVA FDR lower than 1%. This identified a median of 1,355 DMRs across the leave-one-out procedure.Firstly, with the balanced train dataset we defined DMRs using a multivariate linear regression model, LinearRegression class from scikit-learn (v0.24.0), that takes as dependent variables the mean methylation values of the 39,868 CpG clusters contained in the balanced dataset (Samples x CpG clusters) and as independent variable (Samples × 1) the binary classification if a sample is HCC (1) or not (0). Additionally, multiple potential confounding factors, covariates, are included in the model as independent variables: (1) binary variable representing sex , since this information in incomplete, we accurately estimated the sample sex using the methylation profiles and the R package minfi (v1.32.0)Secondly, having identified DMRs in HCC we then estimate the most important DMRs to predict HCC by training linear support vector machines (LinearSVC) using a L1 regularization, with penalty parameter (C) set to 1.5, to reduce the number of DMRs considered in the model. DMRs with non-zero weights in the trained model are then defined as the most predictive DMRs to classify HCC samples. A median of 38 HCC predictive DMRs are identified per model across the 859 folds of the leave-one-out procedure, where 150 unique DMRs are found in at least 5% of all trained models (n = 43).92 which uses a soft voting modality, i.e. taking the argmax of the estimated probabilities to be HCC. For training the ensemble all samples from the Train & Test dataset are used, apart from the one left out for testing. In contrast to the DMR discovery, training of the ensemble is not restricted to the balanced sample set. Lastly, the trained ensemble model is used to make a prediction of the HCC status of the test sample using the soft-voting.For each leave-one-out cross-validation, the top predictive DMRs identified in the Biomarker Discovery section Fig. a .92. For this analysis only DMRs that were found in more than 5% of the leave-one-out optimal models (n = 43 models) were considered (n = 150 DMRs). Considering that 38 is the median optimal number of features in the leave-one-out cross-validated models . Within this subset we systematically tested linear associations between methylation profiles of the top 38 DMRs and 15,341 gene expression profiles using linear regression models implemented in the python module limix (v3.0.4)94. We defined the following linear mixed model Eq.  global methylation; (2) predicted patient sex; (3) predicted patient age using both Hannum and Horvath methods; (4) tumour purity, Train & Test PC5 used as a proxy; and (5) an intercept term. Gene expression measurements were standardized by subtracting the mean and dividing by the standard deviation. For each DMR and gene association a Eq.  is a sample score estimated from our 38 DMR signature. This is calculated using a weighted sum of the methylation of the 38 DMRs recurrently present with non-zero weights in the linear support vector machines (LinearSVCs) trained with the balanced sample sets in the leave-one-out cross-validation. The preponderance (weight) of each DMR is independently estimated using 1000 permutations of the balanced datasets which are used to train a Ridge classifier with an alpha parameter set to 1. This ensures a regularisation of the model’s feature coefficients, while preserving them to non-zero. The mean and standard deviation of each DMR is then calculated across all 1000 iterations. The mean coefficients are used to weight the DMR signature score, where features with larger absolute coefficients have larger preponderance. A score is calculated for each sample using the sample-specific DMR methylation values and the weights calculated before. Recall and precision curves are generated using the risk score and the HCC status of the samples. Optimal threshold and precision and recall rates are estimated based on the best F1 metric possible along the curves. A similar approach is taken for the other 12 independent DNA methylation signatures, where CpG sites are used as features instead.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Information 6.


An iron(III) chloride hexahydrate-catalyzed Friedel–Crafts acylation of benzene derivatives in tunable aryl alkyl ionic liquids (TAAILs) has been developed. Through optimization of the metal salt, reaction conditions and ionic liquids, we were able to design a robust catalyst system that tolerates different electron-rich substrates under ambient atmosphere and allows for a multigram scale. The Friedel–Crafts acylation is one of the oldest metal-catalyzed reactions in organic chemistry  and alloIn the following years, ionic liquids proved to be a remarkable class of compounds due to their high thermal and chemical stability, their negligible vapor pressure and high versatility in terms of chemical structure and usage –24.Successive research for the Friedel–Crafts acylation lead to the development of various reaction protocols using different metal salts –30 and iIn this contribution, we present the use of imidazolium-based tunable aryl alkyl ionic liquids (TAAILs) in a catalytic Friedel–Crafts acylation. This relatively new class of ionic liquids  previous1–6 were synthesized via a three-step procedure starting from commercially available aniline derivatives (see 1–6 are then formed by an anion exchange reaction using lithium bis(trifluoromethylsulfonyl)imide (LiNTf2). All reactions are generally tolerant towards different aryl substitutions, substitution patterns, alkyl chain lengths and can be carried out in a multigram scale ..2O) to a3 nor the hexahydrate were able to catalyze the reaction in TAAILs. The hydrates of several rare-earth metal chlorides  were used as well, but only small amounts of product (less than 5%) were observed, whereas the hydrates of cobalt and iron chloride were able to catalyze the acylation successfully . The reaction time varied between four and 72 hours and the reaction was stopped after full conversion of the benzene derivative (detected via GC–MS). The products were isolated via flash column chromatography to determine the yield. Anthracene was selectively acylated at the 9-position (14) using acetic anhydride. The use of other anhydrides was also tested: propionic anhydride and benzoic anhydride lead to yields between 41% and 92%, with minor formation of the ortho-isomer when using benzoic acid anhydride. Bromobenzene, which is electron poor and less reactive towards acylation, was also used as a substrate in the acylation reaction, but no product was observed. This indicates that the ionic liquid 6, having the same substituent, does not undergo acylation during the catalysis. Finally, we compared our catalytic system with commercially available imidazolium and phosphonium-based ILs , carrying no hydrogen atom at the C2 position of the imidazolium ring, 86% of product were obtained. The phosphonium-based IL [P66614]NTf2 again shows an inferior performance compared to TAAIL 6. Upon changing the anion of the ionic liquid, catalytic activity is suppressed and product formation is not observed at all (The imidazolium-based IL [EMIm]NTf 77% see , entry 1d at all , entry 5In conclusion, we have established a robust system for a catalytic Friedel–Crafts acylation. The reaction was carried out at moderate temperatures of 40 °C to 60 °C under air, was scalable to gram-scale and tolerated different electron-rich benzene derivatives as well as anhydrides. The catalytic performance depends on the choice of ionic liquid and the TAAILs outperform commercially available ILs in our model reaction.File 11H and 13C NMR spectra.Experimental procedures, characterization data, copies of


Malaria vaccines are urgently needed in the fight against this devastating disease that is responsible for almost half a million deaths each year. Here, we discuss recent clinical advances in vaccine development and highlight ongoing challenges for the future. Kurtovic et al. highlight some of the recent advances in the development and clinical evaluation of malaria vaccines. The authors outline key vaccine strategies and clinical trials, and discuss priorities for research into the development of an efficacious malaria vaccine. Malaria is one of the most devastating infectious diseases in humans, responsible for >200 million cases and almost half a million deaths annually. Control programs have significantly reduced disease burden since 2000, but numbers have plateaued since 2015. The disruption caused by the ongoing COVID-19 pandemic will further unwind these efforts and is predicted to increase the number of malaria deaths. New control measures are urgently needed, and lessons learned from emerging diseases such as COVID-19 highlight the enormous potential of effective vaccines. This has been recognized by the World Health Organization (WHO) and key stakeholders who recommend that, within the next decade, a vaccine should be developed that is at least 75% efficacious against malaria over 2 years  and also reduces malaria transmission.This is an exciting time for malaria vaccine development, with a number of promising clinical trials recently completed and others currently underway…Plasmodium falciparum causes the majority of malaria burden and has been the primary focus of vaccine development, and Plasmodiumvivax is the second major cause of malaria. There are several vaccine strategies that target different developmental stages of the malaria-causing parasite 2. Another approach is to combine replication–intact sporozoites with antimalarial drug prophylaxis to prevent malarial illness, known as PfSPZ-CVac. PfSPZ-CVac can induce sterilizing immunity in malaria-naive volunteers, but there is no published data in malaria-endemic populations3. A third approach is to genetically attenuate sporozoites to prevent intrahepatic development. This includes the triple-gene knockout vaccine, GAP3KO, which recently completed an efficacy trial in malaria-naive volunteers (NCT03168854). Although sporozoite-based vaccines are promising, major obstacles include the fact that currently sporozoites cannot be cultured in vitro and are therefore isolated from infected mosquito salivary glands, and they currently require direct venous inoculation of vaccine doses and storage and transportation of vaccines using liquid nitrogen.Vaccines can target sporozoites to prevent or impair infection of the liver, and therefore aim to induce immunity whereby an individual is protected against infection and disease. Live-attenuated sporozoite-based vaccines have the capacity to induce sterilizing immunity (no detectable parasitemia) against CHMI in malaria-naive volunteers. This includes the PfSPZ vaccine that contains radiation-attenuated sporozoites incapable of replicating. It was recently shown that an optimized PfSPZ vaccine dosage induced sterilizing immunity against CHMI in six Tanzanian adults4. Additional trials are underway in African children to evaluate a modified regimen where a fraction of the third dose is administered months later, which appeared to be moderately more efficacious in malaria-naive volunteers (NCT03276962)5. There is also interest in whether RTS,S can synergize with seasonal malaria chemoprevention (NCT03143218).Subunit vaccines aim to target a key antigen, or sub-region, and have the advantage of using existing production technologies and delivery using established infrastructure and systems. The most successful is the RTS,S vaccine based on a truncated form of the major sporozoite surface antigen, circumsporozoite protein (CSP). This is expressed as a virus-like particle and administered with an adjuvant (AS01), which is used to enhance the immune response to vaccination. Promising findings from a phase 3 clinical trial in >15,000 African children led to pilot implementation of a four-dose booster regimen in children aged 5 months in Ghana, Kenya, and Malawi to further evaluate vaccine safety and efficacy, which is ongoing6. Initial results from a pediatric phase 2 clinical trial in Burkina Faso demonstrated 76% efficacy against malaria over 12 months6. Although this was considerably higher than vaccine efficacy achieved in previous RTS,S trials4, R21 was administered prior to the peak malaria season and most malaria occurred in the first 6 months6, making direct comparisons with RTS,S efficacy difficult to interpret. Furthermore, antibody levels had declined markedly by 12 months. Participants are still being monitored to determine vaccine efficacy over 2 years, and children are currently being enrolled in phase 3 clinical trials across multiple African sites (NCT04704830).A recent advance was the development of the R21 malaria vaccine, a virus-like particle that is similar to RTS,S, but has higher density of the CSP antigen on the particle surface and is formulated with Matrix-M adjuvant7. The most recent was RH5.1 comprised of full-length RH5 protein, an indispensable merozoite invasion ligand. In a phase 1/2a clinical trial in malaria-naive volunteers, RH5.1 with AS01 adjuvant was highly immunogenic but only resulted in a 1-day delay until threshold parasitemia levels were reached following CHMI compared with that of the unvaccinated control group8. An upcoming phase 1 clinical trial of RH5.1 will further evaluate safety and immunogenicity in Tanzanian adults and children (NCT04318002).Blood-stage vaccines target the merozoite form or infected red blood cells and aim to control parasitemia and prevent clinical illness. There has been considerably less progress in achieving good efficacy for blood-stage vaccines, with only a handful of candidates evaluated in phase 2 clinical trials, and none having progressed further7. Several vaccines, including RH5.1, have generated substantial GIA activity but shown limited efficacy8. Achieving higher efficacy with merozoite vaccines may require harnessing multiple immunologic mechanisms. Recent studies found that antibodies can activate complement and mediate Fcγ-receptor effector functions against merozoites, which were associated with clinical protection in malaria-endemic population studies9. This creates a new opportunity to evaluate the targets of functional antibody responses and potentially identify new vaccine candidate antigens using these novel approaches9.Merozoites express a multitude of surface proteins and invasion ligands, including many that are polymorphic, which makes it difficult to identify candidate antigens for subunit vaccines. In addition, there are major knowledge gaps on specific mechanisms that mediate protective antimalarial immunity. Antibodies play a critical role, but the current reference growth inhibition assay (GIA) that measures antibody inhibitory activity has neither strongly nor consistently been associated with protection against malaria10. Further studies are needed on this particular vaccine approach, including improvements in vaccine production and formulation to avoid immune responses to the red blood cell membrane.Given the lack of success in developing blood-stage subunit vaccines, there is interest in whole parasite blood-stage vaccines, which present a large antigen repertoire to the immune system and may overcome the challenge of antigen-specific polymorphisms. The first clinical study of chemically attenuated parasite-infected red blood cells promisingly induced cellular immune responses in malaria-naive volunteers, but parasite-specific antibodies were not detected12.Once a merozoite infects a red blood cell and matures, the host cell undergoes extensive changes and expresses parasite-derived proteins on the red blood cell surface. One such protein, VAR2CSA, facilitates the accumulation and sequestration of parasite-infected red blood cells in the placenta. Malaria during pregnancy is, therefore, a major concern and can lead to adverse outcomes such as maternal anemia, preterm delivery, stillbirth, and low birthweight babies. Two VAR2CSA-based vaccines (PAMVAC and PRIMVAC) aimed at inhibiting placental malaria have recently completed phase 1 clinical trials and were immunogenic and had acceptable safety profiles, but are yet to be evaluated for vaccine efficacy13.Vaccines that target the gametocyte and gamete forms aim to prevent parasite transmission to the mosquito and thereby block malaria transmission throughout a population. Although this vaccine approach does not offer any direct clinical protection in humans, blocking malaria transmission is an essential component of effective control and elimination strategies. There has been limited progress for transmission-blocking vaccines, with one candidate based on the gametocyte surface antigen, Pfs230 (Pfs230D1M), showing promise in early clinical trials and a phase 2 clinical trial is ongoing in Mali (NCT03917654)These vaccine approaches targeting different stages of the parasite life cycle have the ability to afford protection against infection, disease, and/or prevent parasite transmission among a population. Although each approach is of value, there may be additional benefits to developing multi-stage subunit vaccines that incorporate antigens expressed at different developmental stages.6. The immunological factors underlying these vaccine limitations remain poorly understood but will be crucial for achieving higher efficacy and wider vaccine implementation in endemic populations. Furthermore, very little progress has been made toward the development of vaccines against P. vivax, which needs to be accelerated.This is an exciting time for malaria vaccine development, with a number of promising clinical trials recently completed and others currently underway, although there are still considerable challenges that stand in the way of achieving vaccine goals within the next decade with several priority areas for research Fig. . Malaria14. There has also been continued interest in heterologous prime-boost with ChAd43 and Modified Vaccinia Ankara viral vectors encoding different malaria antigens alone or in combinations 15.Recent advances in vaccine technologies, including those used in licensed COVID-19 vaccines, have opened new opportunities for malaria vaccine development. This includes mRNA-based vaccine platforms of the CSP malaria antigen, which has already shown encouraging results in animal modelsFurther research on these areas will greatly advance the development of effective malaria vaccines in endemic populations.


Trivalent chromium (Cr) may function to potentiate the action of insulin, but the effects of chromium intakes on metabolic parameters are unclear. Cr is listed as a potentially beneficial element for rodents based on studies that show feeding low quantities affect glucose metabolism. Cr is recommended at 1 mg per kg in rodent diets. This study examined the effects of different levels of dietary Cr on body weight, body composition, energy intake, food efficiency and metabolic parameters of lipid and glucose metabolism in male and female rats when fed from peripuberty to young adult age in the background of a moderately high-fat, high-sucrose diet. Sprague-Dawley CD rats  at 35 days of age were assigned by weight to the low , normal  or high  Cr diets. Diets were fed ad libitum for 12 weeks (83 days). At baseline, body weights and composition were similar (p≥0.05) among diet groups. Compared to the NCr group, the LCr group weighed more (p<0.01) and consumed more energy (food) from Day 56 onwards, but food efficiency was unaffected. Following an oral glucose challenge (Day 77), dietary chromium levels did not affect plasma glucose, but fasting plasma insulin and insulin at 30 and 60 min after dosing were higher in the LCr group compared to the NCr group. At the end of the study, whole-body fat, accrued body fat from baseline and fasting serum triglycerides were higher in the LCr group compared to the NCr group. Effects were similar in both sexes and not observed in the HCr group. These data show that low dietary Cr affects metabolic parameters common in chronic diseases underscoring the need for clinical trials to define the nutritional and/or pharmacological effects of Cr. The National Academy of Medicine considers trivalent chromium (Cr) an essential nutrient with a function in potentiating the action of insulin . A more The American Institute of Nutrition (AIN) Ad Hoc committee lists Cr as a “potentially” beneficial ultra trace element for rodents . More reEarlier studies in male rats conducted in the 1990’s reported that a low Cr diet causes overproduction of insulin in response to a glucose challenge –6. In thAnimal and in vitro experiments have suggested a number of putative mechanisms by which Cr may modulate insulin action and regulate glucose metabolism including increasing the kinase activity of insulin receptor β, enhancing the activity of downstream effectors of insulin signaling phosphoinositide 3-kinase and protein kinase B, downregulation of negative-regulators of insulin signaling, upregulation of AMP-activated protein kinase, regulation of membrane cholesterol efflux and increasing glucose transporter-4 translocation to the cell surface . HoweverOver one-quarter of the adult US population consumes a supplement containing Cr . Cr is aSixty  Sprague-Dawley CD rats  at 35 days of age were assigned (matched by body weight) to 1 of 3 isocaloric high-fat, high-sucrose diets . Diets were fed ad libitum for 12 weeks (83 days). Rats were started on the experimental diets in 5 groups of 12 rats  over 5 successive days to facilitate technical manipulations during the study. The low (LCr), normal (NCr) or high (HCr) Cr diets were formulated using the AIN-93G mineral mix –28.Rats were singly housed in plastic, solid-bottom cages with a wire-grill insert, platform and shelter. Rats were housed without bedding throughout the study. Cages were held in vented racks. Rats were kept on a 12:12-hour light:dark cycle and had free access to food and deionized water throughout the study. Food consumption and body weights were measured weekly. Food consumption was determined by weighing the feeder and determining the weight of food missing from the feeder. Food efficiency was calculated as body weight gain (g) divided by energy intake . Body composition was measured at the start (Day 0) and end (Day 83) of the study using magnetic resonance imaging . During week 6 of the study, all feces were collected for 7 consecutive days. To minimize contamination of the feces, feces were carefully collected each day from an Iso-Pad  placed under the wire-grill insert and cages were fitted with a new Iso-Pad after each collection. Feces from the 7-day collection were pooled and kept frozen at −20°C for the collection period and then stored at −80°C until analysis.After 12 weeks of feeding the diets, rats were fasted overnight (~12 hour) and killed the following morning by exsanguination under general isoflurane anesthesia. Blood was collected from the abdominal aorta by syringe and dispensed into a Trace Element Serum tube . Blood was allowed to clot for 30 min at room temperature and then centrifuged  to isolate serum for analysis of lipid profile. The right leg was excised and immediately frozen on dry ice and then stored at –80˚C until analysis.One male rat from the NCr group was euthanized during Week 8 of the study after biting and ingesting the plastic tubing during a practice oral glucose tolerance test (OGTT). One male rat from the HCr group died unexpectedly (without prior symptoms) inside the EchoMRI chamber while measuring body composition on Day 83 of the study. The experimental protocol was approved by the Health Products and Food Branch Animal Care Committee of Health Canada (Protocol No.: 2020–004).On Day 77 of the study an OGTT was performed on all the rats. Following an overnight fast (~12 hour), rats were weighed to calculate the dextrose dose for each rat (2 g dextrose/kg body weight). A 0.4 g/mL dextrose solution (in deionized water) was administered slowly using a syringe and plastic catheter making sure the rats were swallowing the solution. Blood samples (~250 μl) were drawn from the tail vein before dextrose dosing (0 min) and 30, 60 and 120 min after dosing. To permit rapid blood collection and minimized the stress response, rats were warmed under an infrared heat lamp for 4 min prior to blood collections and briefly restrained during the procedure with a AIMS™ Disposable Plastic Restraint. Blood samples were dispensed into BD Microtainer™ tubes with lithium heparin . Tubes were immediately placed on ice and centrifuged  within 45 min to isolate plasma. Plasma samples were immediately frozen at −80˚C until measurement of glucose and insulin concentrations.3). Femur density was calculated as the wet weight in grams divided by the volume in cubic centimeters.Femurs were isolated from the right leg. Skin and flesh were removed using a scalpel and forceps. Wet and dry femur weights were measured using a Mettler Toledo AT261 delta range analytical balance . Femur length from the greater trochanter to the lateral condyle and mid-diaphysis mediolateral width were measured using a digital caliper . Femur volume was determined using a PYREX™ specific gravity bottle . Femur volume was calculated as the mass of water displaced by the femur in grams divided by the density of water (0.99997 g/cmGround diets (~1.5 g samples) and femurs were placed in preweighted quartz beakers and dried overnight at 100°C in an Isotemp® oven (Thermo Fisher Scientific). Samples were cooled, placed in a desiccator for 1 hour and then weighed to obtain dry weights. Diets and femurs were ashed using a combination of dry ashing with a Thermo Scientific Lindberg/Blue MTM box furnace (Thermo Fisher Scientific) and wet ashing using concentrated trace metal grade nitric acid (Thermo Fisher Scientific). Ashes were solubilized in ~12 mL (weighed accurately) of 1 N (diets) or 3 N (femurs) nitric acid.Frozen feces (one week collection) were ground using disposable grinder cups with the Tube Mill 100 Control, IKA (VWR) and dried as described above. Approximately 1 g of dry feces sample (weighed accurately) was microwave digested (CEM Model: MARS 6) in 10 mL of concentrated nitric acid. Hydrogen peroxide (2 mL) was added to the samples and the samples were microwave digested a second time. The entire sample was transferred into a quartz beaker and the acid evaporated on a hotplate. The sample was dry-ashed in a box furnace and the ashes were solubilized in ~10 mL of 3 N nitric acid.Cr in diet samples was measured using inductively-coupled plasma mass spectrometry with the NexION 300S  equipped with an apex-ST PFA MicroFlow nebulizer and a SC-Fast series autosampler. The instrument was controlled using the Syngistix version 1.1 software and was operated using an RF power of 1600 W and argon flow rates of 1.05, 1.2 and 18 L/min for the nebulizer, auxiliary and plasma, respectively. Analytical accuracy was verified (within 3-sigma limit) using the TM-25.3 and TMDA-61.2 Certified Reference Materials . Percent recoveries for control standards were 93.7−98.9% .Mineral concentrations in solubilized femur and feces ashes were measured using a radial view 700 Series inductively-coupled plasma optical emission spectrometer . Operating conditions have been described previously . StandarPlasma insulin was measured using the Rat Ultrasensitive Insulin ELISA . Level 2 and 3 controls were within the acceptable ranges . Plasma glucose was measured using the Glucose Colorimetric Assay Kit . The CVs of control samples were <5.9%. Serum lipids were measured using the ABX Pentra 400 chemistry analyser  with the ABX Pentra Cholesterol CP, ABX Pentra HDL Direct 100 CP, ABX Pentra LDL Direct CP and the ABX Pentra Triglycerides CP reagents. Quality controls  were within the acceptable ranges .The number of rats per diet group were informed by results from previous rat studies that investigated the effects of dietary Cr on glucose metabolism (4−8). Results are reported as means ± SD. Mixed-design ANOVA was used to determine overall effects and interactions of time, sex and diet. Univariate results are shown for each time point for parameters with a significant (p<0.05) time × sex or time × diet interaction. Two-way ANOVA was used for analysis of parameters measured at a single time point to examine overall effects and interactions of sex and diet. For parameters with a significant diet effect, Dunnett’s test was used to compare the LCr and HCr groups to the NCr group (Control). One-way ANOVA followed by Tukey’s post hoc test was used to determine differences in Cr concentrations among diets. Homogeneity of variances was assessed using Levene’s test. Data that showed unequal variances were log or Box-Cox transformed prior to analysis. AUC for glucose and insulin were calculated using the trapezoidal rule with the AUC function in SigmaPlot 12.5 . Statistical significance was set at p<0.05. Data were analyzed using Statistica 13.1 .Differences in Cr intakes among diet groups were corroborated by measuring Cr concentrations in the feces . The ratFor all parameters, a sex × diet interaction was not detected (p≥0.05) and therefore results for male and female rats were pooled to assess the effect of diet. Male rats weighed more than female rats at all time points . From DaEnergy intake was higher in male rats compared to female rats at all time points . ComparePhysical measurements of femurs differed between male and female rats . Mid-diaSerum lipid profile differed between sexes . Male raAt the 120 min time point, plasma glucose concentration was higher in male rats compared to female rats irrespective of diet . AUC forThis study shows that a low Cr diet with approximately one-third of recommended amounts in the background of a high-fat, high-sucrose diet affects metabolic parameters common in the development and progression of chronic diseases, whereas consuming Cr above normal amounts showed no effects aside from changes in femur mineral concentrations. Rats fed the LCr diet had higher fasting triglycerides and insulin and greater AUC for insulin following an oral glucose challenge. In addition, the LCr diet increased body weights, whole-body fat mass and energy (food) consumption, which contrasts previous observations in rats , 6, 8. TMacronutrient composition of the diet may modify the effect of Cr , 5, 7. TThe HCr diet only affected bone (femur) mineral concentrations; causing higher sodium concentrations and lower iron concentrations compared to the NCr diet. Cr may compete with iron for binding to transferrin, a main iron transport protein . Very hiMost previous studies in rats examined effects of dietary Cr only in males –8. Sex aRats fed the LCr diet consumed more energy and, as expected, weighed more and accumulated more body fat. Food efficiency was not affected; rats fed the LCr and NCr diets gained a similar amount of body weight per kcal of food energy consumed throughout the study. Differences in energy consumption were not statistically different until Day 48−56, approximately at the same time point body weights began to differ. Differences in energy intakes between rats fed the LCr or NCr diets were likely too small to detect at earlier time points given the large variability in energy consumption between rats. As the study advanced, the higher energy intakes led to progressively larger body weights for rats fed the LCr diet which further increased the difference in energy intakes to a degree that was statistically significant by Day 48−56. Together, these results suggest that the effect of the LCr diet on energy consumption was small which may explain why effects on food consumption and body weights were not reported in previous studies with young rats fed a low Cr diet for a similar or longer period . Energy Femur length was not affected by the LCr diet which is further evidence that linear growth and lean body mass was unaffected. Percent lean mass was lower because the rats weighed more. Mid-diaphysis femur width was larger in rats fed the LCr diet. This is likely explained by greater mechanical load caused by the larger body weights of the rats. Femoral structural geometry is known to adapt to mechanical loading . A similThe risk of type 2 diabetes was shown to be lower in US adults taking Cr-containing supplements  and a syCompared to rats fed a standard Cr diet, Di Bona et al.  reportedMetabolic syndrome is a cluster of factors of cardiovascular disease and type 2 diabetes that include abdominal obesity, hypertension, impaired fasting glucose, high triglycerides and low HDL cholesterol. In a 23-year follow-up study, the incidence of metabolic syndrome among young US adults was inversely and longitudinally associated with toenail Cr and the association was largely explained by its relation to blood lipids . Rats feEven though Cr was proposed as an essential trace element in mammals more than six decades ago , 46 its This was a rigorously conducted study in both sexes with accurate measurement of food consumption and body composition using magnetic resonance imaging. Since dietary Cr is poorly absorbed (<2−5%) , 50 withThis study has some limitations. Currently, there is a poor understanding of Cr requirements for rats and our “normal” Cr diet was based on AIN-93G recommendations. The effects were observed in rats fed diets high in fat and sucrose from young age to maturity which may have increased Cr requirements and exaggerated the effects. Given that obesity and dysregulation of lipid and glucose metabolism are manifestations in chronic diseases such as diabetes and metabolic syndrome , 55, theThis study adds to the body of evidence on the effects of dietary Cr intakes on metabolic parameters of health. A low Cr diet (based on rodent standards) negatively affected metabolic parameters of lipid and glucose metabolism and increased energy intake, body weight and whole-body fat mass in male and female rats. These results underscore the need to examine these parameters in clinical trials that will help assess the nutritional and/or pharmacological effects of Cr.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S1 Fig(PDF)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.


Streptococcus viridans. Due to QTc prolongation, her home medication of citalopram was discontinued in favor of escitalopram. Within 24 hours of administration, the patient experienced angioedema with periorbital swelling, lip swelling, and urticaria of the face and arms which was resolved with intravenous (IV) diphenhydramine. In this case report, we present what we believe to be the first recorded case of escitalopram-induced angioedema in a patient without a past medical history of hereditary angioedema (HAE), and how pharmacogenomic testing influenced antidepressant medication selection.Management of psychiatric disorders in high-risk cardiac patients often requires difficult decision making when it comes to acceptable medication side effects. We present the case report of a 28-year-old female with a history of generalized anxiety disorder (GAD), major depressive disorder (MDD), intravenous heroin use disorder, and prior tricuspid valve replacement who presented to the hospital with signs and symptoms of sepsis. She was found to have corrected QT interval (QTc) prolongation and infective endocarditis with blood cultures positive for  CYP450 gene expression.There have been well-documented cases of both paroxetine and fluoxetine-associated angioedema but only a single documented case of escitalopram-induced angioedema in a patient with hereditary angioedema (HAE) -3. This A 28-year-old Caucasian female presented to the emergency department with a one-week history of chest pain, intermittent subjective fevers, chills, night sweats, and shortness of breath upon exertion. The patient was febrile at 38.8 C, had a heart rate of 118 bpm, blood pressure of 118/80 mmHg, and respiratory rate of 18 breaths per minute. Her notable past medical history included intravenous drug use (IVDU), endocarditis, bioprosthetic tricuspid valve replacement eight months prior, generalized anxiety disorder (GAD), and major depressive disorder (MDD). She endorsed using intravenous heroin daily for the previous month.Streptococcus viridans growth.Laboratory testing was notable for a white blood cell (WBC) count of 23.5 cells/L as well as a troponin-I of 22 ng/L. An electrocardiogram (ECG) revealed sinus tachycardia with a prolonged QTc of 513ms  gentamicin 80mg every eight hours and IV ceftriaxone 2g every 24 hours. The patient’s prior diagnosis of MDD and GAD was stabilized with citalopram 40mg daily. However, due to citalopram’s known effect of increasing QTc, this medication was discontinued. Noting the patient’s medical history and the addition of new stressors associated with her current hospitalization, initiation of a different antidepressant was medically necessary. The patient had previously trialed several different selective serotonin reuptake inhibitors (SSRIs), selective serotonin and norepinephrine reuptake inhibitors (SNRIs), and mirtazapine reporting unacceptably severe side effects, among these being worsening mood, drowsiness, and gastrointestinal disturbances. Due to a seizure event on admission day two from suspected benzodiazepine withdrawal, bupropion was not chosen as this medication is known to decrease a patient’s seizure threshold [Escitalopram 5mg daily was chosen as it has been shown to have a smaller effect on QTc prolongation in comparison to citalopram, may reduce the risk of major adverse cardiac events in patients with acute coronary syndrome (ACS), and facilitates abstinence in patients with opioid use disorder . Within Four hours after the resolution of this event, the patient suffered a second, less severe reaction involving urticaria of her left arm and forearm immediately after receiving iopamidol IV contrast during a computerized tomography (CT) scan. Her symptoms quickly resolved after the administration of a second 25mg IV diphenhydramine dose. The patient reported no prior allergic reaction to iopamidol despite having received this IV contrast agent multiple times in the previous two years.Due to the patient’s history of failed antidepressant trials and her reaction to escitalopram, pharmacogenomic testing was pursued. Of interest was the CYP450 family of enzymes; particularly CYP2C19, CYP2D6, and CYP3A4, which are responsible for the majority of antidepressant metabolism . TestingAlthough the mechanism for this patient’s angioedema is unclear, we theorize that reduced enzymatic metabolism and medication clearance could have increased plasma escitalopram concentration beyond therapeutic levels. Medication-induced serotonin release may have then led to increased vascular permeability and angioedema. An alternative theory credits drug molecules interacting non-covalently with T-cell receptors or major histocompatibility molecules (MHC) to activate the immune system and steer inflammatory cells toward the dermis. This theory was described by Tuman et al.  as a potConsidering many antidepressant medications are metabolized hepatically by CYP2C19, CYP2D6, and CYP2C9. Previous drug trial starting doses may have produced higher than intended plasma levels of the medications which could explain why this patient reported such severe side effects .While the Clinical Pharmacogenetics Implementation Consortium (CPIC) issued guidelines in 2015 for SSRI administration in patients with CYP2C19 and CYP2D6 polymorphisms , we feelPresented in this case report is a patient who experienced angioedema and urticaria within 24 hours of her first escitalopram dose. Escitalopram-induced angioedema has been previously described in a patient with HAE, but we believe this to be the first case report in a patient who is not diagnosed with HAE. Similar case reports have also been described in patients taking fluoxetine and paroxetine at therapeutic dosages. Further research is required to understand the mechanism behind these occurrences. This case report also demonstrated the difficulty of choosing an antidepressant in a patient with co-morbid medical conditions and the benefit of using pharmacogenomic testing to advance the therapeutic alliance further.


To investigate changes in the prevalence and nature of hospital admissions towards the end of long-duration workers’ compensation claims (>2 years), and afterwards. To examine differences in hospitalisation when workers’ compensation claims end due to either a 260-week duration limit, or otherwise, and comparisons with hospitalisations of a community comparator.A retrospective cohort study examined 2475 workers, termed the s39 group, whose workers’ compensation ceased due to a 260-week limit in 2017/2018 under s39(1) of the Workers’ Compensation Act New South Wales 2012 legislative amendments . Comparator groups were injured workers with long-duration claims whose compensation ceased independently of s39  and a community group (N=8485). Workers’ compensation records were linked to national social security payments, and hospital admissions. Outcomes describe the prevalence and diagnostic categories of hospital admissions 12 months before, and after, the cessation of workers’ compensation stopped payments.Musculoskeletal health conditions were common diagnoses in overnight hospitalisations for injured workers. Single-day hospital care for mental health disorders were more common for injured workers (17% of same-day admissions) than for members of a community control (3% of same-day admissions) across two years. Exiting the workers’ compensation scheme is associated with significantly fewer annual hospital admissions for the injured control group (OR 0.76), but not for the s39 group (OR 1.01). Injured workers with long-duration compensated work disability were admitted to hospital more often than the community comparator group during the year after workers’ compensation stops . Across all study groups, hospital admission was more common for people receiving disability social security benefits and older age groups.Policy change in the New South Wales workers’ compensation system introduced a 260-week limit on compensation, leaving workers with an elevated need for hospital care after their compensation ended. Welfare policies that disrupt key determinants of health require dedicated inter-agency provisions to support the elevated health needs of those affected.