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Carbapenem-resistant Klebsiella pneumoniae has been classified as an Urgent Threat by the Centers for Disease Control and Prevention (CDC). The combination of two "old" antibiotics, polymyxin and chloramphenicol, displays synergistic killing against New Delhi metallo-β-lactamase (NDM)-producing K. pneumoniae. However, the mechanism(s) underpinning their synergistic killing are not well studied. We employed an in vitro pharmacokinetic/pharmacodynamic model to mimic the pharmacokinetics of the antibiotics in patients and examined bacterial killing against NDM-producing K. pneumoniae using a metabolomic approach. Metabolomic analysis was integrated with an isolate-specific genome-scale metabolic network (GSMN). Our results show that metabolic responses to polymyxin B and/or chloramphenicol against NDM-producing K. pneumoniae involved the inhibition of cell envelope biogenesis, metabolism of arginine and nucleotides, glycolysis, and pentose phosphate pathways. Our metabolomic and GSMN modeling results highlight the novel mechanisms of a synergistic antibiotic combination at the network level and may have a significant potential in developing precision antimicrobial chemotherapy in patients.

The constituents of the gut microbiome are determined by the local habitat, which itself is shaped by immunological pressures, such as mucosal IgA. Using a mouse model of restricted antibody repertoire, we identified a role for antibody-microbe interactions in shaping a community of bacteria with an enhanced capacity to metabolize L-tyrosine. This model led to increased concentrations of p-cresol sulfate (PCS), which protected the host against allergic airway inflammation. PCS selectively reduced CCL20 production by airway epithelial cells due to an uncoupling of epidermal growth factor receptor (EGFR) and Toll-like receptor 4 (TLR4) signaling. Together, these data reveal a gut microbe-derived metabolite pathway that acts distally on the airway epithelium to reduce allergic airway responses, such as those underpinning asthma.

Hyperactivation of oncogenic pathways downstream of RAS and PI3K/AKT in normal cells induces a senescence-like phenotype that acts as a tumor-suppressive mechanism that must be overcome during transformation. We previously demonstrated that AKT-induced senescence (AIS) is associated with profound transcriptional and metabolic changes. Here, we demonstrate that human fibroblasts undergoing AIS display upregulated cystathionine-β-synthase (CBS) expression and enhanced uptake of exogenous cysteine, which lead to increased hydrogen sulfide (H2S) and glutathione (GSH) production, consequently protecting senescent cells from oxidative stress-induced cell death. CBS depletion allows AIS cells to escape senescence and re-enter the cell cycle, indicating the importance of CBS activity in maintaining AIS. Mechanistically, we show this restoration of proliferation is mediated through suppressing mitochondrial respiration and reactive oxygen species (ROS) production by reducing mitochondrial localized CBS while retaining antioxidant capacity of transsulfuration pathway. These findings implicate a potential tumor-suppressive role for CBS in cells with aberrant PI3K/AKT pathway activation. Consistent with this concept, in human gastric cancer cells with activated PI3K/AKT signaling, we demonstrate that CBS expression is suppressed due to promoter hypermethylation. CBS loss cooperates with activated PI3K/AKT signaling in promoting anchorage-independent growth of gastric epithelial cells, while CBS restoration suppresses the growth of gastric tumors in vivo. Taken together, we find that CBS is a novel regulator of AIS and a potential tumor suppressor in PI3K/AKT-driven gastric cancers, providing a new exploitable metabolic vulnerability in these cancers.

BackgroundFibres remain undigested until they reach the colon, where some are fermented by gut microbiota, producing metabolites called short-chain fatty acids (SCFAs). SCFAs lower blood pressure (BP) of experimental models, but their translational potential is unknown. We aimed to determine whether SCFAs lower 24-hour systolic BP (SBP) in untreated participants with essential hypertension.MethodsWe performed a phase II randomized placebo-controlled double-blind cross-over trial using SCFA-supplementation, delivered as acetylated and butyrylated high amylose maize starch (HAMSAB). Twenty treatment-naïve hypertensive participants were recruited from the community and randomised to 40g/day of HAMSAB or placebo. Participants completed each arm for three-weeks, with a three-week washout period between them. The primary endpoint was a 24-hour SBP decrease.ResultsParticipants were on average 55.8±11.2-years old (mean±SD), had a body mass index (BMI) of 25.7±2.5km2/m, 30% were female, baseline 24-hour SBP 136±6mmHg. No adverse effects were reported. After the intervention, the placebo-subtracted reduction in 24-hour SBP was 6.1±9.9mmHg (P= 0.027). This was independent of age, sex, BMI and study arm. There was no statistical significance in the placebo arm. Day and night SBP were reduced by 6.5±12.3mmHg (P=0.01) and 5.7±9.8mmHg (P=0.02), respectively, and 24-h central SBP by 7.2±14.7 mmHg (P=0.005). HAMSAB increased levels of acetate and butyrate by 7.8-fold (P=0.016), shifted the microbial ecosystem, and expanded the prevalence of SCFA-producers.ConclusionsWe observed a clinically relevant reduction in 24-hour SBP in participants with essential hypertension treated with the gut microbial-derived metabolites acetate and butyrate. These metabolites may represent a novel option for lowering BP.

Chemoresistance remains the major barrier to effective ovarian cancer treatment. The molecular features and associated biological functions of this phenotype remain poorly understood. We developed carboplatin-resistant cell line models using OVCAR5 and CaOV3 cell lines with the aim of identifying chemoresistance-specific molecular features. Chemotaxis and CAM invasion assays revealed enhanced migratory and invasive potential in OVCAR5-resistant, compared to parental cell lines. Mass spectrometry analysis was used to analyse the metabolome and proteome of these cell lines, and was able to separate these populations based on their molecular features. It revealed signalling and metabolic perturbations in the chemoresistant cell lines. A comparison with the proteome of patient-derived primary ovarian cancer cells grown in culture showed a shared dysregulation of cytokine and type 1 interferon signalling, potentially revealing a common molecular feature of chemoresistance. A comprehensive analysis of a larger patient cohort, including advanced in vitro and in vivo models, promises to assist with better understanding the molecular mechanisms of chemoresistance and the associated enhancement of migration and invasion.

Current best practice for the treatment of malaria relies on short half-life artemisinins that are failing against emerging Kelch 13 mutant parasite strains. Here, we introduce a liposome-like self-assembly of a dimeric artesunate glycerophosphocholine conjugate (dAPC-S) as an amphiphilic prodrug for the short-lived antimalarial drug, dihydroartemisinin (DHA), with enhanced killing of Kelch 13 mutant artemisinin-resistant parasites. Cryo-electron microscopy (cryoEM) images and the dynamic light scattering (DLS) technique show that dAPC-S typically exhibits a multilamellar liposomal structure with a size distribution similar to that of the liposomes generated using thin-film dispersion (dAPC-L). Liquid chromatography-mass spectrometry (LCMS) was used to monitor the release of DHA. Sustainable release of DHA from dAPC-S and dAPC-L assemblies increased the effective dose and thus efficacy against Kelch 13 mutant artemisinin-resistant parasites in an in vitro assay. To better understand the enhanced killing effect, we investigated processes for deactivation of both the assemblies and DHA, including the roles of serum components and trace levels of iron. Analysis of parasite proteostasis pathways revealed that dAPC assemblies exert their activity via the same mechanism as DHA. We conclude that this easily prepared multilamellar liposome-like dAPC-S with long-acting efficacy shows potential for the treatment of severe and artemisinin-resistant malaria.

Background Resistance to front-line antimalarials (artemisinin combination therapies) is spreading, and development of new drug treatment strategies to rapidly kill Plasmodium spp. malaria parasites is urgently needed. Azithromycin is a clinically used macrolide antibiotic proposed as a partner drug for combination therapy in malaria, which has also been tested as monotherapy. However, its slow-killing ‘delayed-death’ activity against the parasite’s apicoplast organelle and suboptimal activity as monotherapy limit its application as a potential malaria treatment. Here, we explore a panel of azithromycin analogues and demonstrate that chemical modifications can be used to greatly improve the speed and potency of antimalarial action. Results Investigation of 84 azithromycin analogues revealed nanomolar quick-killing potency directed against the very earliest stage of parasite development within red blood cells. Indeed, the best analogue exhibited 1600-fold higher potency than azithromycin with less than 48 hrs treatment in vitro. Analogues were effective against zoonotic Plasmodium knowlesi malaria parasites and against both multi-drug and artemisinin-resistant Plasmodium falciparum lines. Metabolomic profiles of azithromycin analogue-treated parasites suggested activity in the parasite food vacuole and mitochondria were disrupted. Moreover, unlike the food vacuole-targeting drug chloroquine, azithromycin and analogues were active across blood-stage development, including merozoite invasion, suggesting that these macrolides have a multi-factorial mechanism of quick-killing activity. The positioning of functional groups added to azithromycin and its quick-killing analogues altered their activity against bacterial-like ribosomes but had minimal change on ‘quick-killing’ activity. Apicoplast minus parasites remained susceptible to both azithromycin and its analogues, further demonstrating that quick-killing is independent of apicoplast-targeting, delayed-death activity. Conclusion We show that azithromycin and analogues can rapidly kill malaria parasite asexual blood stages via a fast action mechanism. Development of azithromycin and analogues as antimalarials offers the possibility of targeting parasites through both a quick-killing and delayed-death mechanism of action in a single, multifactorial chemotype.

Background The increased incidence of polymyxin-resistant MDR Klebsiella pneumoniae has become a major global health concern. Objectives To characterize the lipid A profiles and metabolome differences between paired polymyxin-susceptible and -resistant MDR K. pneumoniae clinical isolates. Methods Three pairs of K. pneumoniae clinical isolates from the same patients were examined [ATH 7 (polymyxin B MIC 0.25 mg/L) versus ATH 8 (64 mg/L); ATH 15 (0.5 mg/L) versus ATH 16 (32 mg/L); and ATH 17 (0.5 mg/L) versus ATH 18 (64 mg/L)]. Lipid A and metabolomes were analysed using LC-MS and bioinformatic analysis was conducted. Results The predominant species of lipid A in all three paired isolates were hexa-acylated and 4-amino-4-deoxy-l-arabinose-modified lipid A species were detected in the three polymyxin-resistant isolates. Significant metabolic differences were evident between the paired isolates. Compared with their corresponding polymyxin-susceptible isolates, the levels of metabolites in amino sugar metabolism (UDP-N-acetyl-α-d-glucosamine and UDP-N-α-acetyl-d-mannosaminuronate) and central carbon metabolism (e.g. pentose phosphate pathway and tricarboxylic acid cycle) were significantly reduced in all polymyxin-resistant isolates [fold change (FC) > 1.5, P 1.5, P 1.5, P Conclusions To our knowledge, this study is the first to reveal significant metabolic perturbations associated with polymyxin resistance in K. pneumoniae.

Cardiovascular disease is one of the most significant causes of death globally, especially in regions where unhealthy diets are prevalent and dietary fibre intake is low.1,2 Fibre, particularly prebiotic types that feed gut microbes, is essential for maintaining healthy gut microbial ecosystems.3 One assumption has been that cardiovascular health relates directly to lifestyle choices in adult life. Here, we show in mice that some of these benefits operate from the prenatal stage and relate to the diet and gut microbiome of the mother. Intake of fibre during pregnancy shaped the mothers’ gut microbiome, which had a lasting founding effect on the offspring’s microbial composition and function. Maternal fibre intake during pregnancy significantly changed the cardiac cellular and molecular landscape in the offspring, protecting them against the development of cardiac hypertrophy, remodelling, and inflammation. These suggest a role for foetal exposure to maternal-derived gut microbial metabolites, which are known to cross the placenta and drive epigenetic changes. Maternal fibre intake led to foetal epigenetic reprogramming of the atrial natriuretic peptide gene (Nppa), protective against heart failure. These results underscore the importance of dietary intake and the gut microbiome of the mother during pregnancy for cardiovascular disease in the offspring.

Nonalcoholic steatohepatitis (NASH) is a severe form of liver injury that can be caused by a variety of stimuli and has a significant mortality rate. A common technique to induce in vitro steatosis involves culturing primary human hepatocytes (PHH) in fatty acid-enriched media. This study compared the lipidome of PHH cultured in fatty acid-enriched media to hepatocytes from patients with NASH and healthy controls. Hepatocytes from NASH patients displayed increased total cellular abundance of glycerolipids and phospholipids compared to healthy control hepatocytes. PHH cultured in fatty acid-enriched media demonstrated increased glycerolipids. However, these culture conditions did not induce elevated phospholipid levels. Thus, culturing PHH in fatty acid-enriched media has limited capacity to emulate the environment of hepatocytes in NASH patients.

Plasmodium falciparum causes the most lethal form of malaria. Peroxide antimalarials based on artemisinin underpin the frontline treatments for malaria, but artemisinin resistance is rapidly spreading. Synthetic peroxide antimalarials, known as ozonides, are in clinical development and offer a potential alternative. Here, we used chemoproteomics to investigate the protein alkylation targets of artemisinin and ozonide probes, including an analogue of the ozonide clinical candidate, artefenomel. We greatly expanded the list of proteins alkylated by peroxide antimalarials and identified significant enrichment of redox-related proteins for both artemisinins and ozonides. Disrupted redox homeostasis was confirmed by dynamic live imaging of the glutathione redox potential using a genetically encoded redox-sensitive fluorescence-based biosensor. Targeted liquid chromatography-mass spectrometry (LC-MS)-based thiol metabolomics also confirmed changes in cellular thiol levels. This work shows that peroxide antimalarials disproportionately alkylate proteins involved in redox homeostasis and that disrupted redox processes are involved in the mechanism of action of these important antimalarials.

Inducing cell death by the sphingolipid ceramide is a potential anticancer strategy, but the underlying mechanisms remain poorly defined. In this study, triggering an accumulation of ceramide in acute myeloid leukemia (AML) cells by inhibition of sphingosine kinase induced an apoptotic integrated stress response (ISR) through protein kinase R–mediated activation of the master transcription factor ATF4. This effect led to transcription of the BH3-only protein Noxa and degradation of the prosurvival Mcl-1 protein on which AML cells are highly dependent for survival. Targeting this novel ISR pathway, in combination with the Bcl-2 inhibitor venetoclax, synergistically killed primary AML blasts, including those with venetoclax-resistant mutations, as well as immunophenotypic leukemic stem cells, and reduced leukemic engraftment in patient-derived AML xenografts. Collectively, these findings provide mechanistic insight into the anticancer effects of ceramide and preclinical evidence for new approaches to augment Bcl-2 inhibition in the therapy of AML and other cancers with high Mcl-1 dependency.

Plasmodium falciparum, a causative agent of malaria, continues to remain a global health threat since these parasites have developed increasing resistance to all anti-malaria drugs used throughout the world. Accordingly, drugs with novel modes of action are desperately required to combat malaria. P. falciparum parasites infect human red blood cells where they digest the hosts main protein constituent, hemoglobin. Leucine aminopeptidase PfA-M17 is one of several aminopeptidases that have been implicated in the last step of this digestive pathway. Here we utilize both reverse genetics and a compound specifically designed to inhibit the activity of PfA-M17 to show that PfA-M17 is essential for P. falciparum survival as it provides parasites with free amino acids for growth, many of which are highly likely to originate from hemoglobin. We further show that our inhibitor is on-target for PfA-M17 and has the ability to kill parasites at nanomolar concentrations. Thus, in contrast to other hemoglobin-degrading proteases that have overlapping redundant functions, we validate PfA-M17 as a potential novel drug target.

The World Health Organisation (WHO) recommends artemisinin (ART) combinations for treatment of uncomplicated Plasmodium falciparum malaria. Understanding the interaction between co-administered drugs within combination therapies is clinically important to prevent unintended consequences. The WHO guidelines recommend second line treatments that combine artesunate with tetracycline, doxycycline, or clindamycin—antibiotics that target the Plasmodium relict plastid, the apicoplast. In addition, antibiotics can be used simultaneously against other infectious diseases, leading to their inadvertent combination with ARTs. One consequence of apicoplast inhibition is a perturbation to haemoglobin uptake and trafficking—a pathway required for activation of ART derivatives. Here, we show that apicoplast-targeting antibiotics reduce the abundance of the catalyst of ART activation (free haem) in P. falciparum, likely through diminished haemoglobin digestion. We demonstrate antagonism between ART and these antibiotics, suggesting that apicoplast inhibitors reduce ART activation. These data have potential clinical implications due to the reliance on—and widespread use of—both ARTs and these antibiotics in malaria endemic regions.

The scourge of malaria infection continues to strike hardest against pregnant women and children in Africa and South East Asia. For global elimination, testing methods that are ultrasensitive to low-level ring-staged parasitemia are urgently required. In this study, we used a novel approach for diagnosis of malaria infection by combining both electronic ultraviolet-visible (UV/vis) spectroscopy and near infrared (NIR) spectroscopy to detect and quantify low-level (1-0.000001%) ring-staged malaria-infected whole blood under physiological conditions uisng Multiclass classification using logistic regression, which showed that the best results were achieved using the extended wavelength range, providing an accuracy of 100% for most parasitemia classes. Likewise, partial least-squares regression (PLS-R) analysis showed a higher quantification sensitivity (R2 = 0.898) for the extended spectral region compared to UV/vis and NIR (R2 = 0.806 and 0.556, respectively). For quantifying different-stage blood parasites, the extended wavelength range was able to detect and quantify all thePlasmodium falciparum accurately compared to testing each spectral component separately. These results demonstrate the potential of a combined UV/vis-NIR spectroscopy to accurately diagnose malaria-infected patients without the need for elaborate sample preparation associated with the existing mid-IR approaches.

Macrophages can prevent infections from intracellular pathogens by restricting access to essential nutrients, termed nutritional immunity. With the exception of tryptophan depletion, it is unclear if other amino acids are similarly regulated in infected macrophages. Here, we show that the expression of nutrient transporters in Legionella-infected macrophages is modulated by the short chain fatty acid butyrate. Butyrate prevented the upregulation of the cystine/glutamate exchanger, Slc7a11, in macrophages infected with L. pneumophila, which decreased cellular cysteine levels. Butyrate and the Slc7a11 inhibitor erastin impaired intracellular Legionella replication in macrophages in vitro, with these being restored by exogenous supplementation with cysteine. Butyrate caused increased histone acetylation in infected macrophages, and pan- and class II HDAC inhibitors also restricted intracellular Legionella growth in a cysteine-dependent manner. Intranasal administration of butyrate reduced L. pneumophila lung burdens in mice. Our data suggest that butyrate alters the metabolism of macrophages to promote nutritional immunity by decreasing cysteine levels and that this can be harnessed to treat bacterial lung infections.

Novel bis-1,2,4-triazine compounds with potent in vitro activity against Plasmodium falciparum parasites were recently identified. The bis-1,2,4-triazines represent a unique antimalarial pharmacophore and are proposed to act by a novel but as-yet-unknown mechanism of action. This study investigated the activity of the bis-1,2,4-triazine MIPS-0004373 across the mammalian life cycle stages of the parasite and profiled the kinetics of activity against blood and transmission stage parasites in vitro and in vivo. MIPS-0004373 demonstrated rapid and potent activity against P. falciparum, with excellent in vitro activity against all asexual blood stages. Prolonged in vitro drug exposure failed to generate stable resistance de novo, suggesting a low propensity for the emergence of resistance. Excellent activity was observed against sexually committed ring stage parasites, but activity against mature gametocytes was limited to inhibiting male gametogenesis. Assessment of liver stage activity demonstrated good activity in an in vitro P. berghei model but no activity against Plasmodium cynomolgi hypnozoites or liver schizonts. The bis-1,2,4-triazine MIPS-0004373 efficiently cleared an established P. berghei infection in vivo, with efficacy similar to that of artesunate and chloroquine and a recrudescence profile comparable to that of chloroquine. This study demonstrates the suitability of bis-1,2,4-triazines for further development toward a novel treatment for acute malaria.

Inhaled polymyxins are associated with toxicity in human lung epithelial cells that involves multiple apoptotic pathways. However, the mechanism of polymyxin-induced pulmonary toxicity remains unclear. This study aims to investigate polymyxin-induced metabolomic perturbations in human lung epithelial A549 cells. A549 cells were treated with 0.5 or 1.0 mM polymyxin B or colistin for 1, 4, and 24 h. Cellular metabolites were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and significantly perturbed metabolites (log(2) fold change [log(2)FC] ≥ 1; false-discovery rate [FDR] ≤ 0.2) and key pathways were identified relative to untreated control samples. At 1 and 4 h, very few significant changes in metabolites were observed relative to the untreated control cells. At 24 h, taurine (log(2)FC = −1.34 ± 0.64) and hypotaurine (log(2)FC = −1.20 ± 0.27) were significantly decreased by 1.0 mM polymyxin B. The reduced form of glutathione (GSH) was significantly depleted by 1.0 mM polymyxin B at 24 h (log(2)FC = −1.80 ± 0.42). Conversely, oxidized glutathione (GSSG) was significantly increased by 1.0 mM both polymyxin B (log(2)FC = 1.38 ± 0.13 at 4 h and 2.09 ± 0.20 at 24 h) and colistin (log(2)FC = 1.33 ± 0.24 at 24 h). l-Carnitine was significantly decreased by 1.0 mM of both polymyxins at 24 h, as were several key metabolites involved in biosynthesis and degradation of choline and ethanolamine (log(2)FC ≤ −1); several phosphatidylserines were also increased (log(2)FC ≥ 1). Polymyxins perturbed key metabolic pathways that maintain cellular redox balance, mitochondrial β-oxidation, and membrane lipid biogenesis. These mechanistic findings may assist in developing new pharmacokinetic/pharmacodynamic strategies to attenuate the pulmonary toxicities of inhaled polymyxins and in the discovery of new-generation polymyxins.

Background Plasmodium falciparum causes the majority of malaria mortality worldwide, and the disease occurs during the asexual red blood cell (RBC) stage of infection. In the absence of an effective and available vaccine, and with increasing drug resistance, asexual RBC stage parasites are an important research focus. In recent years, mass spectrometry–based proteomics using data-dependent acquisition has been extensively used to understand the biochemical processes within the parasite. However, data-dependent acquisition is problematic for the detection of low-abundance proteins and proteome coverage and has poor run-to-run reproducibility. Results Here, we present a comprehensive P. falciparum–infected RBC (iRBC) spectral library to measure the abundance of 44,449 peptides from 3,113 P. falciparum and 1,617 RBC proteins using a data-independent acquisition mass spectrometric approach. The spectral library includes proteins expressed in the 3 morphologically distinct RBC stages (ring, trophozoite, schizont), the RBC compartment of trophozoite-iRBCs, and the cytosolic fraction from uninfected RBCs. This spectral library contains 87% of all P. falciparum proteins that have previously been reported with protein-level evidence in blood stages, as well as 692 previously unidentified proteins. The P. falciparum spectral library was successfully applied to generate semi-quantitative proteomics datasets that characterize the 3 distinct asexual parasite stages in RBCs, and compared artemisinin-resistant (Cam3.IIR539T) and artemisinin-sensitive (Cam3.IIrev) parasites. Conclusion A reproducible, high-coverage proteomics spectral library and analysis method has been generated for investigating sets of proteins expressed in the iRBC stage of P. falciparum malaria. This will provide a foundation for an improved understanding of parasite biology, pathogenesis, drug mechanisms, and vaccine candidate discovery for malaria.

Lipedema, a poorly understood chronic disease of adipose hyper-deposition, is often mistaken for obesity and causes significant impairment to mobility and quality-of-life. To identify molecular mechanisms underpinning lipedema, we employed comprehensive omics-based comparative analyses of whole tissue, adipocyte precursors (adipose-derived stem cells (ADSCs)), and adipocytes from patients with or without lipedema.We compared whole-tissues, ADSCs, and adipocytes from body mass index-matched lipedema (n = 14) and unaffected (n = 10) patients using comprehensive global lipidomic and metabolomic analyses, transcriptional profiling, and functional assays.Transcriptional profiling revealed4400 significant differences in lipedema tissue, with altered levels of mRNAs involved in critical signaling and cell function-regulating pathways (e.g., lipid metabolism and cell-cycle/proliferation). Functional assays showed accelerated ADSC proliferation and differentiation in lipedema. Profiling lipedema adipocytes revealed900 changes in lipid composition and600 differentially altered metabolites. Transcriptional profiling of lipedema ADSCs and non-lipedema ADSCs revealed significant differential expression of3400 genes including some involved in extracellular matrix and cell-cycle/proliferation signaling pathways. One upregulated gene in lipedema ADSCs, Bub1, encodes a cell-cycle regulator, central to the kinetochore complex, which regulates several histone proteins involved in cell proliferation. Downstream signaling analysis of lipedema ADSCs demonstrated enhanced activation of histone H2A, a key cell proliferation driver and Bub1 target. Critically, hyperproliferation exhibited by lipedema ADSCs was inhibited by the small molecule Bub1 inhibitor 2OH-BNPP1 and by CRISPR/Cas9-mediated Bub1 gene depletion.We found significant differences in gene expression, and lipid and metabolite profiles, in tissue, ADSCs, and adipocytes from lipedema patients compared to non-affected controls. Functional assays demonstrated that dysregulated Bub1 signaling drives increased proliferation of lipedema ADSCs, suggesting a potential mechanism for enhanced adipogenesis in lipedema. Importantly, our characterization of signaling networks driving lipedema identifies potential molecular targets, including Bub1, for novel lipedema therapeutics.

Most eukaryotic mRNAs accommodate alternative sites of poly(A) addition in the 3’ untranslated region in order to regulate mRNA function. Here, we present a systematic analysis of 3’ end formation factors, which revealed 3’UTR lengthening in response to a loss of the core machinery, whereas a loss of the Sen1 helicase resulted in shorter 3’UTRs. We show that the anti-cancer drug cordycepin, 3’ deoxyadenosine, caused nucleotide accumulation and the usage of distal poly(A) sites. Mycophenolic acid, a drug which reduces GTP levels and impairs RNA polymerase II (RNAP II) transcription elongation, promoted the usage of proximal sites and reversed the effects of cordycepin on alternative polyadenylation. Moreover, cordycepin-mediated usage of distal sites was associated with a permissive chromatin template and was suppressed in the presence of an rpb1 mutation, which slows RNAP II elongation rate. We propose that alternative polyadenylation is governed by temporal coordination of RNAP II transcription and 3’ end processing and controlled by the availability of 3’ end factors, nucleotide levels and chromatin landscape.

The mechanism of action of ozonide antimalarials involves activation by intraparasitic iron and the formation of highly reactive carbon-centered radicals that alkylate malaria parasite proteins. Given free intraparasitic heme is generally thought to be the iron source responsible for ozonide activation and its likely close proximity to the activated drug, we investigated heme as a possible molecular target of the ozonides. Using an extraction method optimized for solubilization of free heme, untargeted LC-MS analysis of ozonide-treated parasites identified several regioisomers of ozonide-alkylated heme, which resulted from covalent modification of the heme porphyrin ring by an ozonide-derived carbon-centered radical. In addition to the intact alkylated heme adduct, putative ozonide-alkylated heme degradation products were also detected. This study directly demonstrates ozonide modification of heme within the malaria parasite Plasmodium falciparum, revealing that this process may be important for the biological activity of ozonide antimalarials.

Plasmodium species are evolutionarily distant from model eukaryotes, and as a consequence they exhibit many non-canonical cellular processes. In the post-genomic era, functional "omics" disciplines (transcriptomics, proteomics, and metabolomics) have accelerated our understanding of unique aspects of the biology of malaria parasites. Functional "omics" tools, in combination with genetic manipulations, have offered new opportunities to investigate the function of previously uncharacterized genes. Knowledge of basic parasite biology is fundamental to understanding drug modes of action, mechanisms of drug resistance, and relevance of vaccine candidates. This Perspective highlights recent "omics"-based discoveries in basic biology and gene function of the most virulent human malaria parasite, Plasmodium falciparum.

A continuous, sealed endothelial membrane is essential for the blood-brain barrier (BBB) to protect neurons from toxins present in systemic circulation. Endothelial cells are critical sensors of the capillary environment, where factors like fluid shear stress (FSS) and systemic signaling molecules activate intracellular pathways that either promote or disrupt the BBB. The brain vasculature exhibits complex heterogeneity across the bed, which is challenging to recapitulate in BBB microfluidic models with fixed dimensions and rectangular cross-section microchannels. Here, a Cayley-tree pattern, fabricated using lithography-less, fluid shaping technique in a modified Hele-Shaw cell is used to emulate the brain vasculature in a microfluidic chip. This geometry generates an inherent distribution of heterogeneous FSS, due to smooth variations in branch height and width. hCMEC/D3 endothelial cells cultured in the Cayley-tree designed chip generate a 3D monolayer of brain endothelium with branching hierarchy, enabling the study of the effect of heterogeneous FSS on the brain endothelium. The model is employed to study neuroinflammatory conditions by stimulating the brain endothelium with tumor necrosis factor-α under heterogeneous FSS conditions. The model has immense potential for studies involving drug transport across the BBB, which can be misrepresented in fixed dimension models.

Macrophages have important roles in the immune system including clearing pathogens and wound healing. Metabolic phenotypes in macrophages have been associated with functional phenotypes, where pro-inflammatory macrophages have an increased rate of glycolysis and anti-inflammatory macrophages primarily use oxidative phosphorylation. β-adrenoceptor (βAR) signalling in macrophages has been implicated in disease states such as cancer, atherosclerosis and rheumatoid arthritis. The impact of βAR signalling on macrophage metabolism has not been defined. Using metabolomics and proteomics, we describe the impact of βAR signalling on macrophages treated with isoprenaline. We found that βAR signalling alters proteins involved in cytoskeletal rearrangement and redox homeostasis of the cell. We showed that βAR signalling in macrophages shifts glucose metabolism from glycolysis towards the tricarboxylic acid cycle and pentose phosphate pathways. We also show that βAR signalling perturbs purine metabolism by accumulating adenylate and guanylate pools. Taken together, these results indicate that βAR signalling shifts metabolism to support redox processes and upregulates proteins involved in cytoskeletal changes, which may contribute to βAR effects on macrophage function.

Drug-induced liver injury (DILI) is a significant clinical issue, affecting 1–1.5 million patients annually, and remains a major challenge during drug development—toxicity and safety concerns are the second-highest reason for drug candidate failure. The future prevalence of DILI can be minimized by developing a greater understanding of the biological mechanisms behind DILI. Both qualitative and quantitative analytical techniques are vital to characterizing and investigating DILI. In vitro assays are capable of characterizing specific aspects of a drug’s hepatotoxic nature and multiplexed assays are capable of characterizing and scoring a drug’s association with DILI. However, an even deeper insight into the perturbations to biological pathways involved in the mechanisms of DILI can be gained through the use of omics-based analytical techniques: genomics, transcriptomics, proteomics, and metabolomics. These omics analytical techniques can offer qualitative and quantitative insight into genetic susceptibilities to DILI, the impact of drug treatment on gene expression, and the effect on protein and metabolite abundance. This review will discuss the analytical techniques that can be applied to characterize and investigate the biological mechanisms of DILI and potential predictive biomarkers.

Carbapenem-resistant

The malaria parasite, Plasmodium falciparum, proliferates rapidly in human erythrocytes by actively scavenging multiple carbon sources and essential nutrients from its host cell. However, a global overview of the metabolic capacity of intraerythrocytic stages is missing. Using multiplex 13C‐labelling coupled with untargeted mass spectrometry and unsupervised isotopologue grouping, we have generated a draft metabolome of P. falciparum and its host erythrocyte consisting of 911 and 577 metabolites, respectively, corresponding to 41% of metabolites and over 70% of the metabolic reaction predicted from the parasite genome. An additional 89 metabolites and 92 reactions were identified that were not predicted from genomic reconstructions, with the largest group being associated with metabolite damage‐repair systems. Validation of the draft metabolome revealed four previously uncharacterised enzymes which impact isoprenoid biosynthesis, lipid homeostasis and mitochondrial metabolism and are necessary for parasite development and proliferation. This study defines the metabolic fate of multiple carbon sources in P. falciparum, and highlights the activity of metabolite repair pathways in these rapidly growing parasite stages, opening new avenues for drug discovery., Multiplex stable‐isotope labelling defines the observable metabolome of red blood cell stages of the malaria parasite Plasmodium falciparum and indicates potential roles for metabolic enzymes of unknown function.

Novel 3,3'-disubstituted-5,5'-bi(1,2,4-triazine) compounds with potent in vitro activity against Plasmodium falciparum parasites were recently discovered. To improve the pharmacokinetic properties of the triazine derivatives, a new structure-activity relationship (SAR) investigation was initiated with a focus on enhancing the metabolic stability of lead compounds. These efforts led to the identification of second-generation highly potent antimalarial bis-triazines, exemplified by triazine 23, which exhibited significantly improved in vitro metabolic stability (8 and 42 μL/min/mg protein in human and mouse liver microsomes). The disubstituted triazine dimer 23 was also observed to suppress parasitemia in the Peters 4-day test with a mean ED50 value of 1.85 mg/kg/day and exhibited a fast-killing profile, revealing a new class of orally available antimalarial compounds of considerable interest.

Non-dialysable protein-bound uremic toxins (PBUTs) contribute to the development of cardiovascular disease (CVD) in chronic kidney disease (CKD) and vice versa. PBUTs have been shown to alter sphingolipid imbalance. Dihydroceramide desaturase 1 (Des1) is an important gatekeeper enzyme which controls the non-reversible conversion of sphingolipids, dihydroceramide, into ceramide. The present study assessed the effect of Des1 inhibition on PBUT-induced cardiac and renal effects in vitro, using a selective Des1 inhibitor (CIN038). Des1 inhibition attenuated hypertrophy in neonatal rat cardiac myocytes and collagen synthesis in neonatal rat cardiac fibroblasts and renal mesangial cells induced by the PBUTs, indoxyl sulfate and p-cresol sulfate. This is at least attributable to modulation of NF-κB signalling and reductions in β-MHC, Collagen I and TNF-α gene expression. Lipidomic analyses revealed Des1 inhibition restored C16-dihydroceramide levels reduced by indoxyl sulfate. In conclusion, PBUTs play a critical role in mediating sphingolipid imbalance and inflammatory responses in heart and kidney cells, and these effects were attenuated by Des1 inhibition. Therefore, sphingolipid modifying agents may have therapeutic potential for the treatment of CVD and CKD and warrant further investigation. Refereed/Peer-reviewed

The tumor microenvironment (TME) contains a rich source of nutrients that sustains cell growth and facilitate tumor development. Glucose and glutamine in the TME are essential for the development and activation of effector T cells that exert antitumor function. Immunotherapy unleashes T cell antitumor function, and although many solid tumors respond well, a significant proportion of patients do not benefit. In patients with KRAS-mutant lung adenocarcinoma, KEAP1 and STK11/Lkb1 co-mutations are associated with impaired response to immunotherapy. To investigate the metabolic and immune microenvironment of KRAS-mutant lung adenocarcinoma, we generated murine models that reflect the KEAP1 and STK11/Lkb1 mutational landscape in these patients. Here, we show increased glutamate abundance in the Lkb1-deficient TME associated with CD8 T cell activation in response to anti-PD1. Combination treatment with the glutaminase inhibitor CB-839 inhibited clonal expansion and activation of CD8 T cells. Thus, glutaminase inhibition negatively impacts CD8 T cells activated by anti-PD1 immunotherapy.

The emergence of Plasmodium falciparum resistance to frontline antimalarials, including artemisinin combination therapies, highlights the need for new molecules that act via novel mechanisms of action. Herein, we report the design, synthesis and antimalarial activity of a series of 2-aminobenzimidazoles, featuring a phenol moiety that is crucial to the pharmacophore. Two potent molecules exhibited IC50 values against P. falciparum 3D7 strain of 42 ± 4 (3c) and 43 ± 2 nM (3g), and high potency against strains resistant to chloroquine (Dd2), artemisinin (Cam3.IIC580Y) and PfATP4 inhibitors (SJ557733), while demonstrating no cytotoxicity against human cells (HEK293, IC50 > 50 μM). The most potent molecule, possessing a 4,5-dimethyl substituted phenol (3r) displayed an IC50 value of 6.4 ± 0.5 nM against P. falciparum 3D7, representing a 12-fold increase in activity from the parent molecule. The 2-aminobenzimidazoles containing a N1-substituted phenol represent a new class of molecules that have high potency in vitro against P. falciparum malaria and low cytotoxicity. They possessed attractive pharmaceutical properties, including low molecular weight, high ligand efficiency, high solubility, synthetic tractability and low in vitro clearance in human liver microsomes.

Most eukaryotic mRNAs accommodate alternative sites of poly(A) addition in the 3’ untranslated region in order to regulate mRNA function. Here we present a systematic analysis of 3’ end formation factors, which revealed 3’UTR lengthening in response to a loss of the core machinery, whereas a loss of the Sen1 helicase resulted in shorter 3’UTRs. We show that the anti-cancer drug cordycepin, 3’ deoxyadenosine, caused nucleotide accumulation and the usage of distal poly(A) sites. Mycophenolic acid, a drug which reduces GTP levels and impairs RNA polymerase II (RNAP II) transcription elongation, promoted the usage of proximal sites and reversed the effects of cordycepin on alternative polyadenylation. Moreover, cordycepin mediated usage of distal sites was associated with a permissive chromatin template and was suppressed in the presence of anrpb1mutation, which slows RNAP II elongation rate. We propose that alternative polyadenylation is governed by temporal coordination of RNAP II transcription and 3’ end processing and controlled by the availability of 3’ end factors, nucleotide levels and chromatin landscape.

Soil-transmitted helminths, including hookworms and whipworms, infect billions of people worldwide. Their capacity to penetrate and migrate through their hosts&rsquo, tissues is influenced by the suite of molecules produced by the infective developmental stages. To facilitate a better understanding of the immunobiology and pathogenicity of human hookworms and whipworms, we investigated the metabolomes of the infective stage of Nippostrongylus brasiliensis third-stage larvae (L3) which penetrate the skin and Trichuris muris eggs which are orally ingested, using untargeted liquid chromatography&ndash, mass spectrometry (LC-MS). We identified 55 polar metabolites through Metabolomics Standard Initiative level-1 (MSI-I) identification from N. brasiliensis and T. muris infective stages, out of which seven were unique to excretory/secretory products (ESPs) of N. brasiliensis L3. Amino acids were a principal constituent (33 amino acids). Additionally, we identified 350 putative lipids, out of which 28 (all known lipids) were unique to N. brasiliensis L3 somatic extract and four to T. muris embryonated egg somatic extract. Glycerophospholipids and glycerolipids were the major lipid groups. The catalogue of metabolites identified in this study shed light on the biology, and possible therapeutic and diagnostic targets for the treatment of these critical infectious pathogens. Moreover, with the growing body of literature on the therapeutic utility of helminth ESPs for treating inflammatory diseases, a role for metabolites is likely but has received little attention thus far.

In article number 2000704, Jian Li and co‐workers elucidate the novel mechanism of dependent resistance to the last‐line polymyxins in a high‐priority “superbug” Acinetobacter baumannii. Polymyxindependent resistance cannot be detected by conventional microbiological diagnosis due to the lack of growth on normal agar plates. These polymyxin‐dependent Acinetobacter baumannii isolates utilize the rigid polymyxin molecules as sticking plasters to stabilize their fragile lipopolysaccharide‐deficient and phosphatidylglycerol‐rich outer membrane. [Image: see text]

Sulforaphane, an isothiocyanate found in cruciferous vegetables such as broccoli, shows promise as an adjuvant therapy for preeclampsia. To inform future clinical trials, we set out to determine the bioavailability of sulforaphane in non-pregnant and preeclamptic women. In six healthy female volunteers, we performed a crossover trial to compare the bioavailability of sulforaphane and metabolites afforded by an activated and non-activated broccoli extract preparation. We then undertook a dose escalation study of the activated broccoli extract in 12 women with pregnancy hypertension. In non-pregnant women, an equivalent dose of activated broccoli extract gave higher levels of sulforaphane and metabolites than a non-activated extract (p

There is currently no robust evidence to support prescribing hydroxychloroquine as a treatment or prophylaxis for COVID-19.

Visceral adipose tissue (VAT) encases mesenteric lymphatic vessels and lymph nodes through which lymph is transported from the intestine and mesentery. Whether mesenteric lymphatics contribute to adipose tissue inflammation and metabolism and insulin resistance is unclear. Here we show that obesity is associated with profound and progressive dysfunction of the mesenteric lymphatic system in mice and humans. We find that lymph from mice and humans consuming a high-fat diet (HFD) stimulates lymphatic vessel growth, leading to the formation of highly branched mesenteric lymphatic vessels that 'leak' HFD-lymph into VAT and, thereby, promote insulin resistance. Mesenteric lymphatic dysfunction is regulated by cyclooxygenase (COX)-2 and vascular endothelial growth factor (VEGF)-C-VEGF receptor (R)3 signalling. Lymph-targeted inhibition of COX-2 using a glyceride prodrug approach reverses mesenteric lymphatic dysfunction, visceral obesity and inflammation and restores glycaemic control in mice. Targeting obesity-associated mesenteric lymphatic dysfunction thus represents a potential therapeutic option to treat metabolic disease.

(1) Background: There is increasing understanding of the potential health benefits of cruciferous vegetables. In particular sulforaphane (SFN), found in broccoli, and its metabolites sulforaphane-glutathione (SFN-GSH), sulforaphane-cysteine (SFN-Cys), sulforaphane cysteine-glycine (SFN-CG) and sulforaphane-N-acetyl-cysteine (SFN-NAC) have potent antioxidant effects that may offer therapeutic value. Clinical investigation of sulforaphane as a therapeutic antioxidant requires a sensitive and high throughput process for quantification of sulforaphane and metabolites, (2) Methods: We collected plasma samples from healthy human volunteers before and for eight hours after consumption of a commercial broccoli extract supplement rich in sulforaphane. A rapid and sensitive method for quantification of sulforaphane and its metabolites in human plasma using Liquid Chromatography&ndash, Mass Spectrometry (LC&ndash, MS) has been developed, (3) Results: The LC&ndash, MS analytical method was validated at concentrations ranging between 3.9 nM and 1000 nM for SFN-GSH, SFN-CG, SFN-Cys and SFN-NAC and between 7.8 nM and 1000 nM in human plasma for SFN. The method displayed good accuracy (1.85%&ndash, 14.8% bias) and reproducibility (below 9.53 %RSD) including low concentrations 3.9 nM and 7.8 nM. Four SFN metabolites quantitation was achieved using external standard calibration and in SFN quantitation, SFN-d8 internal standardization was used. The reported method can accurately quantify sulforaphane and its metabolites at low concentrations in plasma, (4) Conclusions: We have established a time- and cost-efficient method of measuring sulforaphane and its metabolites in human plasma suitable for high throughput application to clinical trials.