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The limited availability of therapeutic options for patients with triple-negative breast cancer (TNBC) contributes to the high rate of metastatic recurrence and poor prognosis. Ferroptosis is a type of cell death caused by iron-dependent lipid peroxidation and counteracted by the antioxidant activity of the selenoprotein GPX4. Here, we show that TNBC cells secrete an anti-ferroptotic factor in the extracellular environment when cultured at high cell densities but are primed to ferroptosis when forming colonies at low density. We found that secretion of the anti-ferroptotic factors, identified as monounsaturated fatty acid (MUFA) containing lipids, and the vulnerability to ferroptosis of single cells depends on the low expression of stearyl-CoA desaturase (SCD) that is proportional to cell density. Finally, we show that the inhibition of Sec-tRNAsec biosynthesis, an essential step for selenoprotein production, causes ferroptosis and impairs the lung seeding of circulating TNBC cells that are no longer protected by the MUFA-rich environment of the primary tumour., Competing Interests: Disclosure and competing interests statement ST is the inventor of PlasmaxTM cell culture medium. The remaining authors declare no competing interests., (© 2024. The Author(s).)

Identification of specific and therapeutically actionable vulnerabilities, ideally present across multiple mutational backgrounds, is needed to improve acute myeloid leukemia (AML) patients' outcomes. We identify stearoyl-CoA desaturase (SCD), the key enzyme in fatty acid (FA) desaturation, as prognostic of patients' outcomes and, using the clinical-grade inhibitor SSI-4, show that SCD inhibition (SCDi) is a therapeutic vulnerability across multiple AML models in vitro and in vivo. Multiomic analysis demonstrates that SCDi causes lipotoxicity, which induces AML cell death via pleiotropic effects. Sensitivity to SCDi correlates with AML dependency on FA desaturation regardless of mutational profile and is modulated by FA biosynthesis activity. Finally, we show that lipotoxicity increases chemotherapy-induced DNA damage and standard chemotherapy further sensitizes AML cells to SCDi. Our work supports developing FA desaturase inhibitors in AML while stressing the importance of identifying predictive biomarkers of response and biologically validated combination therapies to realize their full therapeutic potential., (© 2024. The Author(s).)

Osteoporosis and sarcopenia are both common age-related disorders that are associated with increased morbidity and mortality. Bone and muscle are metabolically very active tissues that require large amounts of energy. Bile acids (BAs), a group of liver-derived steroid compounds, are primarily known as emulsifiers that facilitate the resorption of dietary fat and lipids. In addition, they have pleiotropic metabolic functions in lipoprotein and glucose metabolism, inflammation, and intestinal bacterial growth. Through these effects, they are related to metabolic diseases, such as diabetes, hypertriglyceridemia, atherosclerosis, and nonalcoholic steatohepatitis. BAs mediate their metabolic effects through receptor dependent and receptor-independent mechanisms. Emerging evidence suggests that BAs are also involved in bone and muscle metabolism. Under normal circumstances, BAs support bone health by shifting the delicate equilibrium of bone turnover toward bone formation. In contrast, low or excessive amounts of BAs promote bone resorption. In cholestatic liver disease, BAs accumulate in the liver, reach toxic concentrations in the circulation, and thus may contribute to bone loss and muscle wasting. In addition, the measurement of BAs is in rapid evolution with modern mass spectrometry techniques that allow for the detection of a continuously growing number of BAs. This review provides a comprehensive overview of the biochemistry, physiology and measurement of bile acids. Furthermore, it summarizes the existing literature regarding their role in bone and muscle.

Bile acids help facilitate intestinal lipid absorption and have endocrine activity in glucose, lipid and bone metabolism. Obesity and exercise influence bile acid metabolism and have opposite effects in bone. This study investigates if regular exercise helps mitigate the adverse effects of obesity on bone, potentially by reversing alterations in bile acid metabolism. Four-month-old female Sprague Dawley rats either received a high-fat diet (HFD) or a chow-based standard diet (lean controls). During the 10-month study period, half of the animals performed 30 min of running at moderate speed on five consecutive days followed by two days of rest. The other half was kept inactive (inactive controls). At the study's end, bone quality was assessed by microcomputed tomography and biomechanical testing. Bile acids were measured in serum and stool. HFD feeding was related to reduced trabecular (-33%, p = 1.14 × 10 -7 ) and cortical (-21%, p = 2.9 × 10 -8 ) bone mass and lowered femoral stiffness (12-41%, p = 0.005). Furthermore, the HFD decreased total bile acids in serum (-37%, p = 1.0 × 10 -6 ) but increased bile acids in stool (+2-fold, p = 7.3 × 10 -9 ). These quantitative effects were accompanied by changes in the relative abundance of individual bile acids. The concentration of serum bile acids correlated positively with all cortical bone parameters (r = 0.593-0.708), whilst stool levels showed inverse correlations at the cortical (r = -0.651--0.805) and trabecular level (r = -0.656--0.750). Exercise improved some trabecular and cortical bone quality parameters (+11-31%, p = 0.043 to 0.001) in lean controls but failed to revert the bone loss related to the HFD. Similarly, changes in bile acid metabolism were not mitigated by exercise. Prolonged HFD consumption induced quantitative and qualitative alterations in bile acid metabolism, accompanied by bone loss. Tight correlations between bile acids and structural indices of bone quality support further functional analyses on the potential role of bile acids in bone metabolism. Regular moderate exercise improved trabecular and cortical bone quality in lean controls but failed in mitigating the effects related to the HFD in bone and bile acid metabolism.

Intercellular communication between different cell types in solid tumors contributes to tumor growth and metastatic dissemination. The secretome of cancer-associated fibroblasts (CAFs) plays major roles in these processes. Using human mammary CAFs, we showed that CAFs with a myofibroblast phenotype released extracellular vesicles that transferred proteins to endothelial cells (ECs) that affected their interaction with immune cells. Mass spectrometry-based proteomics identified proteins transferred from CAFs to ECs, which included plasma membrane receptors. Using THY1 as an example of a transferred plasma membrane-bound protein, we showed that CAF-derived proteins increased the adhesion of a monocyte cell line to ECs. CAFs produced high amounts of matrix-bound EVs, which were the primary vehicles of protein transfer. Hence, our work paves the way for future studies that investigate how CAF-derived matrix-bound EVs influence tumor pathology by regulating the function of neighboring cancer, stromal, and immune cells.

Background: Non-alcoholic fatty liver disease (NAFLD) is increasing worldwide. A main cause is the obesogenic, so-called Western lifestyle. NAFLD follows a long, unperceived course, and ends potentially fatally. Early diagnosis of aggressive subtypes saves lives. So far, non-invasive means of detection are limited. A better understanding of the pathogenic interplay among insulin resistance, immune inflammation, microbiome, and genetic background is important. Metabolomics may give insight into these interlaced processes., Methods: In this study, we measured bile acids (BA) in the plasma of adult NAFLD and alcohol-associated liver disease (ALD) patients and healthy controls with targeted mass spectrometry. We focused on gender-related bile acid production pathology in NAFLD and ALD., Results: Compared to healthy controls, women with NAFLD had significantly higher concentrations of total BA, total primary BA, total cholic (CA), total chenodeoxycholic (CDCA), total glycine-conjugated, and total non-12-a-OH BA. Concerning subtypes, glycocholic (GCA) and glycochenodeoxycholic (GCDCA), BA were elevated in women with NAFLD. In contrast, men with NAFLD had no significantly altered total BA fractions. However, the subtypes GCA, glycodeoxycholic (GDCA), glycolithocholic (GLCA), lithocholic (LCA), taurolithocholic (TLCA), and tauroursodeoxycholic acid (TUDCA) were elevated, while CA was significantly decreased. In NAFLD, except ursodeoxycholic acid (UDC), all total BA correlated significantly positively in both sexes with the ELF score, while in ALD, only males showed significant correlations exceptive for total UDC BA. In NAFLD, total BA, total primary BA, total secondary BA, total free secondary BA, total CA, total CDCA, total taurine conjugated, total glycine conjugated, total 12-a-OH, and total non-12-a-OH were significantly higher in cases of a high enhanced liver fibrosis (ELF) score above 9.8. In ALD, total UDC was additionally elevated. Between NAFLD with and without NASH, we found no significant differences., Conclusion: Our data show gender-specific bile acid profiles in NAFLD and markedly different BA patterns in ALD. Women with NAFLD had more severe cholestasis. Men may better compensate fat storage-driven bile acid dynamics, indicated by higher levels of taurine-conjugated BA, which associate with beneficial metabolic functions.

Pediatric short bowel syndrome (SBS) is a rare condition characterized by a massive loss of the small intestine, leading to the inability to meet nutritional requirements without the use of parenteral or enteral supplementation. SBS causes profound alterations in the intestinal microbiome and metabolome. The aim of this study was a detailed assessment of the intestinal microbiome and metabolome in a murine model of SBS. We performed a 60% proximal small bowel resection versus a sham operation in C57BL/6 mice. Four weeks postoperatively, the microbial communities of different intestinal segments (jejunum, ileum, colon) and stool were assessed by 16S rRNA gene sequencing. Bile acids in serum and stool and volatile organic compounds (VOCs) in the fecal headspace were assessed using LC-MS and GC-MS techniques. The α-diversity of the different intestinal segments did not significantly differ between the two groups. β-diversity significantly differed between sham and SBS mice. While in the jejunum, Faecalibaculum was significantly increased in SBS animals, a significant reduction in Lactobacillus and Sporosarcina was detected in the ileum of SBS mice. In the colon of SBS mice, a significant decrease in Ruminococcaceae and a significant increase in Proteobacteria such as Faecalibaculum and Escherichia-Shigella were found. Serum levels of deoxycholic, taurocholic and taurochenodeoxycholic acids were significantly higher in the SBS group. Of the 29 VOCs tested, hexane, isoflurane and pentane were significantly higher in the SBS group, and pyrrole was significantly lower. We were able to show that SBS causes shifts in the murine intestinal microbiome and metabolome including serum BAs and fecal VOCs.

Background: The human placenta, a tissue with a lifespan limited to the period of pregnancy, is exposed to varying shear rates by maternal blood perfusion depending on the stage of development. In this study, we aimed to investigate the effects of fluidic shear stress on the human trophoblast transcriptome and metabolism., Results: Based on a trophoblast cell line cultured in a fluidic flow system, changes caused by shear stress were analyzed and compared to static conditions. RNA sequencing and bioinformatics analysis revealed an altered transcriptome and enriched gene ontology terms associated with amino acid and mitochondrial metabolism. A decreased GLUT1 expression and reduced glucose uptake, together with downregulated expression of key glycolytic rate-limiting enzymes, hexokinase 2 and phosphofructokinase 1 was observed. Altered mitochondrial ATP levels and mass spectrometry data, suggested a shift in energy production from glycolysis towards mitochondrial oxidative phosphorylation. This shift in energy production could be supported by increased expression of glutamic-oxaloacetic transaminase variants in response to shear stress as well as under low glucose availability or after silencing of GLUT1. The shift towards amino acid metabolic pathways could be supported by significantly altered amino acid levels, like glutamic acid, cysteine and serine. Downregulation of GLUT1 and glycolytic rate-limiting enzymes, with concomitant upregulation of glutamic-oxaloacetic transaminase 2 was confirmed in first trimester placental explants cultured under fluidic flow. In contrast, high fluid shear stress decreased glutamic-oxaloacetic transaminase 2 expression in term placental explants when compared to low flow rates. Placental tissue from pregnancies with intrauterine growth restriction are exposed to high shear rates and showed also decreased glutamic-oxaloacetic transaminase 2, while GLUT1 was unchanged and glycolytic rate-limiting enzymes showed a trend to be upregulated. The results were generated by using qPCR, immunoblots, quantification of immunofluorescent pictures, padlock probe hybridization, mass spectrometry and FRET-based measurement., Conclusion: Our study suggests that onset of uteroplacental blood flow is accompanied by a shift from a predominant glycolytic- to an alternative amino acid converting metabolism in the villous trophoblast. Rheological changes with excessive fluidic shear stress at the placental surface, may disrupt this alternative amino acid pathway in the syncytiotrophoblast and could contribute to intrauterine growth restriction., (© 2023. Society of Chinese Bioscientists in America (SCBA).)

Rationale: Chronic obstructive pulmonary disease (COPD) is a disease characterized by persistent airway inflammation and disordered macrophage function. The extent to which alterations in macrophage bioenergetics contribute to impaired antioxidant responses and disease pathogenesis has yet to be fully delineated. Objectives: Through the study of COPD alveolar macrophages (AMs) and peripheral monocyte-derived macrophages (MDMs), we sought to establish if intrinsic defects in core metabolic processes drive macrophage dysfunction and redox imbalance. Methods: AMs and MDMs from donors with COPD and healthy donors underwent functional, metabolic, and transcriptional profiling. Measurements and Main Results: We observed that AMs and MDMs from donors with COPD display a critical depletion in glycolytic- and mitochondrial respiration-derived energy reserves and an overreliance on glycolysis as a source for ATP, resulting in reduced energy status. Defects in oxidative metabolism extend to an impaired redox balance associated with defective expression of the NADPH-generating enzyme, ME1 (malic enzyme 1), a known target of the antioxidant transcription factor NRF2 (nuclear factor erythroid 2-related factor 2). Consequently, selective activation of NRF2 resets the COPD transcriptome, resulting in increased generation of TCA cycle intermediaries, improved energetic status, favorable redox balance, and recovery of macrophage function. Conclusions: In COPD, an inherent loss of metabolic plasticity leads to metabolic exhaustion and reduced redox capacity, which can be rescued by activation of the NRF2 pathway. Targeting these defects, via NRF2 augmentation, may therefore present an attractive therapeutic strategy for the treatment of the aberrant airway inflammation described in COPD.

The 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., (© 2022. The Author(s).)

IRE1α is constitutively active in several cancers and can contribute to cancer progression. Activated IRE1α cleaves XBP1 mRNA, a key step in production of the transcription factor XBP1s. In addition, IRE1α cleaves select mRNAs through regulated IRE1α-dependent decay (RIDD). Accumulating evidence implicates IRE1α in the regulation of lipid metabolism. However, the roles of XBP1s and RIDD in this process remain ill-defined. In this study, transcriptome and lipidome profiling of triple negative breast cancer cells subjected to pharmacological inhibition of IRE1α reveals changes in lipid metabolism genes associated with accumulation of triacylglycerols (TAGs). We identify DGAT2 mRNA, encoding the rate-limiting enzyme in TAG biosynthesis, as a RIDD target. Inhibition of IRE1α, leads to DGAT2-dependent accumulation of TAGs in lipid droplets and sensitizes cells to nutritional stress, which is rescued by treatment with the DGAT2 inhibitor PF-06424439. Our results highlight the importance of IRE1α RIDD activity in reprograming cellular lipid metabolism., (© 2022. The Author(s).)

Despite the clinical benefit of androgen-deprivation therapy (ADT), the majority of patients with advanced prostate cancer (PCa) ultimately develop lethal castration-resistant prostate cancer (CRPC). In this study, we identified thioesterase superfamily member 6 (THEM6) as a marker of ADT resistance in PCa. THEM6 deletion reduces in vivo tumour growth and restores castration sensitivity in orthograft models of CRPC. Mechanistically, we show that the ER membrane-associated protein THEM6 regulates intracellular levels of ether lipids and is essential to trigger the induction of the ER stress response (UPR). Consequently, THEM6 loss in CRPC cells significantly alters ER function, reducing de novo sterol biosynthesis and preventing lipid-mediated activation of ATF4. Finally, we demonstrate that high THEM6 expression is associated with poor survival and correlates with high levels of UPR activation in PCa patients. Altogether, our results highlight THEM6 as a novel driver of therapy resistance in PCa as well as a promising target for the treatment of CRPC., (© 2022 The Authors. Published under the terms of the CC BY 4.0 license.)

This article contains supporting information on data collection for the research article entitled "Aircraft noise exposure drives the activation of white blood cells and induces microvascular dysfunction in mice" by Eckrich et al. We found that noise-induced stress triggered microvascular dysfunction via involvement of innate immune-derived reactive oxygen species. In this article, we present the instrumentation of mice with dorsal skinfold chambers for in vivo microscopic imaging of blood flow, interaction of leukocytes with the vascular wall (also by fluorescent labelling of blood cells) and vessel diameter. In addition, we explain the preparation of cerebral arterioles for measurement of vascular reactivity in vitro .•visualization of noise-dependent effects in dorsal skinfold chamber.• in vivo microscopy of noise-dependent activation of white blood cells.•analysis of noise-dependent microvascular dysfunction in dorsal skinfold chamber and cannulated cerebral arterioles., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors. Published by Elsevier B.V.)

Epidemiological studies showed that traffic noise has a dose-dependent association with increased cardiovascular morbidity and mortality. Whether microvascular dysfunction contributes significantly to the cardiovascular health effects by noise exposure remains to be established. The connection of inflammation and immune cell interaction with microvascular damage and functional impairment is also not well characterized. Male C57BL/6J mice or gp91phox -/y mice with genetic deletion of the phagocytic NADPH oxidase catalytic subunit (gp91phox or NOX-2) were used at the age of 8 weeks, randomly instrumented with dorsal skinfold chambers and exposed or not exposed to aircraft noise for 4 days. Proteomic analysis (using mass spectrometry) revealed a pro-inflammatory phenotype induced by noise exposure that was less pronounced in noise-exposed gp91phox -/y mice. Using in vivo fluorescence microscopy, we found a higher number of adhesive leukocytes in noise-exposed wild type mice. Dorsal microvascular diameter (by trend), red blood cell velocity, and segmental blood flow were also decreased by noise exposure indicating microvascular constriction. All adverse effects on functional parameters were normalized or improved at least by trend in noise-exposed gp91phox -/y mice. Noise exposure also induced endothelial dysfunction in cerebral microvessels, which was associated with higher oxidative stress burden and inflammation, as measured using video microscopy. We here establish a link between a pro-inflammatory phenotype of plasma, activation of circulating leukocytes and microvascular dysfunction in mice exposed to aircraft noise. The phagocytic NADPH oxidase was identified as a central player in the underlying pathophysiological mechanisms., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Androgen deprivation therapy (ADT) is the standard of care for treatment of nonresectable prostate cancer. Despite high treatment efficiency, most patients ultimately develop lethal castration-resistant prostate cancer (CRPC). In this study, we performed a comparative proteomic analysis of three in vivo , androgen receptor (AR)-responsive orthograft models of matched hormone-naïve prostate cancer and CRPC. Differential proteomic analysis revealed that distinct molecular mechanisms, including amino acid (AA) and fatty acid metabolism, are involved in the response to ADT in the different models. Despite this heterogeneity, Schlafen family member 5 (SLFN5) was identified as an AR-regulated protein in CRPC. SLFN5 expression was high in CRPC tumors and correlated with poor patient outcome. In vivo, SLFN5 depletion strongly impaired tumor growth in castrated conditions. Mechanistically, SLFN5 interacted with ATF4 and regulated the expression of LAT1, an essential AA transporter. Consequently, SLFN5 depletion in CRPC cells decreased intracellular levels of essential AA and impaired mTORC1 signaling in a LAT1-dependent manner. These results confirm that these orthograft models recapitulate the high degree of heterogeneity observed in patients with CRPC and further highlight SLFN5 as a clinically relevant target for CRPC. SIGNIFICANCE: This study identifies SLFN5 as a novel regulator of the LAT1 amino acid transporter and an essential contributor to mTORC1 activity in castration-resistant prostate cancer., (©2021 American Association for Cancer Research.)

Cytoglobin is important in the progression of oral squamous cell carcinoma but the molecular and cellular basis remain to be elucidated. In the current study, we develop a new cell model to study the function of cytoglobin in oral squamous carcinoma and response to cisplatin. Transcriptomic profiling showed cytoglobin mediated changes in expression of genes related to stress response, redox metabolism, mitochondrial function, cell adhesion, and fatty acid metabolism. Cellular and biochemical studies show that cytoglobin expression results in changes to phenotype associated with cancer progression including: increased cellular proliferation, motility and cell cycle progression. Cytoglobin also protects cells from cisplatin-induced apoptosis and oxidative stress with levels of the antioxidant glutathione increased and total and mitochondrial reactive oxygen species levels reduced. The mechanism of cisplatin resistance involved inhibition of caspase 9 activation and cytoglobin protected mitochondria from oxidative stress-induced fission. To understand the mechanism behind these phenotypic changes we employed lipidomic analysis and demonstrate that levels of the redox sensitive and apoptosis regulating cardiolipin are significantly up-regulated in cells expressing cytoglobin. In conclusion, our data shows that cytoglobin expression results in important phenotypic changes that could be exploited by cancer cells in vivo to facilitate disease progression.

Oncogenic KRAS mutations and inactivation of the APC tumor suppressor co-occur in colorectal cancer (CRC). Despite efforts to target mutant KRAS directly, most therapeutic approaches focus on downstream pathways, albeit with limited efficacy. Moreover, mutant KRAS alters the basal metabolism of cancer cells, increasing glutamine utilization to support proliferation. We show that concomitant mutation of Apc and Kras in the mouse intestinal epithelium profoundly rewires metabolism, increasing glutamine consumption. Furthermore, SLC7A5, a glutamine antiporter, is critical for colorectal tumorigenesis in models of both early- and late-stage metastatic disease. Mechanistically, SLC7A5 maintains intracellular amino acid levels following KRAS activation through transcriptional and metabolic reprogramming. This supports the increased demand for bulk protein synthesis that underpins the enhanced proliferation of KRAS-mutant cells. Moreover, targeting protein synthesis, via inhibition of the mTORC1 regulator, together with Slc7a5 deletion abrogates the growth of established Kras-mutant tumors. Together, these data suggest SLC7A5 as an attractive target for therapy-resistant KRAS-mutant CRC.

BH3-mimetics are a new class of anti-cancer drugs that inhibit anti-apoptotic Bcl-2 proteins. In doing so, BH3-mimetics sensitise to cell death. Venetoclax is a potent, BCL-2 selective BH3-mimetic that is clinically approved for use in chronic lymphocytic leukaemia. Venetoclax has also been shown to inhibit mitochondrial metabolism, this is consistent with a proposed role for BCL-2 in metabolic regulation. We used venetoclax to understand BCL-2 metabolic function. Similar to others, we found that venetoclax inhibited mitochondrial respiration. In addition, we also found that venetoclax impairs TCA cycle activity leading to activation of reductive carboxylation. Importantly, the metabolic effects of venetoclax were independent of cell death because they were also observed in apoptosis-resistant BAX/BAK-deficient cells. However, unlike venetoclax treatment, inhibiting BCL-2 expression had no effect on mitochondrial respiration. Unexpectedly, we found that venetoclax also inhibited mitochondrial respiration and the TCA cycle in BCL-2 deficient cells and in cells lacking all anti-apoptotic BCL-2 family members. Investigating the basis of this off-target effect, we found that venetoclax-induced metabolic reprogramming was dependent upon the integrated stress response and ATF4 transcription factor. These data demonstrate that venetoclax affects cellular metabolism independent of BCL-2 inhibition. This off-target metabolic effect has potential to modulate venetoclax cytotoxicity.

Ageing compromises skeletal muscle mass and function through poorly defined molecular aetiology. Here we have used untargeted metabolomics using UHPLC-MS to profile muscle tissue from young ( n =10, 25±4y), middle aged ( n =18, 50±4y) and older ( n =18, 70±3y) men and women (50:50). Random Forest was used to prioritise metabolite features most informative in stratifying older age, with potential biological context examined using the prize-collecting Steiner forest algorithm embedded in the PIUMet software, to identify metabolic pathways likely perturbed in ageing. This approach was able to filter a large dataset of several thousand metabolites down to subnetworks of age important metabolites. Identified networks included the common age-associated metabolites such as androgens, (poly)amines/amino acids and lipid metabolites, in addition to some potentially novel ageing related markers such as dihydrothymine and imidazolone-5-proprionic acid. The present study reveals that this approach is a potentially useful tool to identify processes underlying human tissue ageing, and could therefore be utilised in future studies to investigate the links between age predictive metabolites and common biomarkers linked to health and disease across age.

There is no effective treatment for autoimmune biliary diseases. Therefore, understanding their immunopathology is crucial. The biliary epithelial cells (BEC), expressing TLR-4, are constantly exposed to gut microbes and bacterial wall LPS, and in settings of inflammation, the immune infiltrate is dense within the peribiliary region of human liver. By dual immunohistochemistry, we affirm human intrahepatic T cell infiltrate includes CCR6 + CD4 + and AhR + CD4 + T cells with potential for plasticity to Th17 phenotype. Mechanistically, we demonstrate that Th1 and Th17 inflammatory cytokines and LPS enhance human primary BEC release of the CCR6 ligand CCL20 and BEC secretion of Th17-polarizing cytokines IL-6 and IL-1β. Cell culture assays with human BEC secretome showed that secretome polarizes CD4 T cells toward a Th17 phenotype and supports the survival of Th17 cells. BEC secretome did not promote Th1 cell generation. Additionally, we give evidence for a mutually beneficial feedback of the type 17 cell infiltrate on BEC, showing that treatment with type 17 cytokines increases BEC proliferation, as monitored by Ki67 and activation of JAK2-STAT3 signaling. This study identifies human BEC as active players in determining the nature of the intrahepatic immune microenvironment. In settings of inflammation and/or infection, biliary epithelium establishes a prominent peribiliary type 17 infiltrate via recruitment and retention and enhances polarization of intrahepatic CD4 cells toward Th17 cells via type 17 cytokines, and, reciprocally, Th17 cells promote BEC proliferation for biliary regeneration. Altogether, we provide new insight into cross-talk between Th17 lymphocytes and human primary biliary epithelium in biliary regenerative pathologies., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Background: Proteomic profiling of extracellular vesicles (EVs) from prostate cancer (PCa) and normal prostate cell lines, led to the identification of new candidate PCa markers. These proteins included the nuclear exportin proteins XPO1 (also known as CRM1), the EV-associated PDCD6IP (also known as ALIX), and the previously published fatty acid synthase FASN. In this study, we investigated differences in expression of XPO1 and PDCD6IP on well-characterized prostate cancer cohorts using mass spectrometry and tissue microarray (TMA) immunohistochemistry to determine their diagnostic and prognostic value., Methods: Protein fractions from 67 tissue samples (n = 33 normal adjacent prostate [NAP] and n = 34 PCa) were analyzed by mass spectrometry (nano-LC-MS-MS). Label-free quantification of EVs was performed to identify differentially expressed proteins between PCa and NAP. Prognostic evaluation of the candidate markers was performed with a TMA, containing 481 radical prostatectomy samples. Samples were stained for the candidate markers and correlated with patient information and clinicopathological outcome., Results: XPO1 was higher expressed in PCa compared to NAP in the MS data analysis (P > 0.0001). PDCD6IP was not significantly higher expressed (P = 0.0501). High cytoplasmic XPO1 staining in the TMA immunohistochemistry, correlated in a multivariable model with high Gleason scores (P = 0.002) and PCa-related death (P = 0.009)., Conclusion: High expression of cytoplasmic XPO1 shows correlation with prostate cancer and has added clinical value in tissue samples. Furthermore, as an extracellular vesicles-associated protein, it might be a novel relevant liquid biomarker., (© 2019 The Authors. The Prostate Published by Wiley Periodicals, Inc.)

Adrenocortical carcinoma (ACC) is an aggressive malignancy with poor response to chemotherapy. In this study, we evaluated a potential new treatment target for ACC, focusing on the mitochondrial reduced form of NAD phosphate (NADPH) generator nicotinamide nucleotide transhydrogenase (NNT). NNT has a central role within mitochondrial antioxidant pathways, protecting cells from oxidative stress. Inactivating human NNT mutations result in congenital adrenal insufficiency. We hypothesized that NNT silencing in ACC cells will induce toxic levels of oxidative stress. To explore this, we transiently knocked down NNT in NCI-H295R ACC cells. As predicted, this manipulation increased intracellular levels of oxidative stress; this resulted in a pronounced suppression of cell proliferation and higher apoptotic rates, as well as sensitization of cells to chemically induced oxidative stress. Steroidogenesis was paradoxically stimulated by NNT loss, as demonstrated by mass spectrometry-based steroid profiling. Next, we generated a stable NNT knockdown model in the same cell line to investigate the longer lasting effects of NNT silencing. After long-term culture, cells adapted metabolically to chronic NNT knockdown, restoring their redox balance and resilience to oxidative stress, although their proliferation remained suppressed. This was associated with higher rates of oxygen consumption. The molecular pathways underpinning these responses were explored in detail by RNA sequencing and nontargeted metabolome analysis, revealing major alterations in nucleotide synthesis, protein folding, and polyamine metabolism. This study provides preclinical evidence of the therapeutic merit of antioxidant targeting in ACC as well as illuminating the long-term adaptive response of cells to oxidative stress.

A wide range of biofluids (urine, serum, plasma, saliva, etc.) as well as cellular and tissue samples can be collected and investigated in clinical metabolomic studies. The choice of sample is dependent on the clinical question being investigated with biofluids typically studied to identify biomarkers, whereas tissues and primary/immortalised cells are typically studied to investigate mechanisms associated with pathophysiological processes. Methods applied to collect samples, quench metabolism and extract samples differ between sample types from simple collect, dilute and analyse methods for urine to complex washing, quenching and biphasic extraction methods for tissues. The range of sample collection and extraction methods are discussed with sample-specific considerations highlighted. Finally, methods for imaging of cells and tissues and for in vivo metabolomic analysis will also be introduced.