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The fight against coronavirus disease 2019 (COVID-19) continues. Since the pandemic's onset, several biomarkers have been proposed to assess the diagnosis and prognosis of this disease. This research aimed to identify potential disease severity biomarkers in serum samples of patients with COVID-19 during the disease course. Data were collected using untargeted and targeted mass spectrometry methods. The results were interpreted by performing univariate and multivariate analyses. Important metabolite classes were identified by qualitative untargeted metabolomics in 15 serum samples from survivors of COVID-19. Quantitative targeted metabolomics on a larger patient cohort including 15 non-survivors confirmed serum 3-sulfate bile acids (i.e. GLCA-3S) were significantly increased in non-survivors compared to survivors during the early disease stage ( p -value < 0.0001). Notably, it was associated with a higher risk of mortality (odds ratio of 26). A principal component analysis showed the ability to discriminate between survivors and non-survivors using the BA concentrations. Furthermore, increased BA-S is highly correlated with known parameters altered in severe clinical conditions.

Persistent organic pollutants (POPs) represent a possible hazard for the ecosystems, with adverse outcomes on wildlife and humans. POPs have always received interest from the scientific community, and they have also been subject to legal restrictions worldwide on their application and commercialization. Among the broad spectrum of POPs, per- and polyfluoroalkyl substances (PFASs) are considered emerging contaminants due to their potential effect on the ecosystem and human health. These contaminants are widely employed in countless applications, from surfactants and building materials to food packaging. On the other hand, their chemical structure gives them the ability to interact with the environment, causing possible toxic effects for humans and environment. Human biomonitoring is a necessary instrument to indagate the impact of PFASs on human health: in recent years several studies have found detectable levels of PFASs in several biological matrices in humans (blood, hair, nails, and urine). Here, we review the most recent scientific literature concerning analytical methods employed in the identification and quantification of PFASs focusing on biological matrices. It has been noted that liquid chromatography coupled with mass spectrometry is the main analytical instrumentation employed, while blood and/or serum samples are the main employed human matrices whereas the use of non-invasive matrices is still at the beginning. Various issues directly related to human metabolism of PFASs and the effective amount of PFAS absorbed from the environment still need to be investigated., Competing Interests: Declaration of competing interest 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., (Copyright © 2023. Published by Elsevier Ltd.)

The release of hazardous chemicals into aquatic environments has long been a known problem, but its full impact has only recently been realized. This study presents a validated liquid chromatography-mass spectrometry (HPLC-MS/MS) method for detecting pharmaceutical and pesticide residues in mussels (Mytilus galloprovincialis). An innovative MS-compatible extraction method was developed and validated, demonstrating successful recovery rates for analytes at three different concentration levels (25-95%). The method detected the target analytes at ng/g concentrations with high accuracy (-7% to 11%) and low relative standard deviation (<10%) for both intra-day and inter-day analyses. After validation, the method was applied to mussel samples collected from a commercial farm near Senigallia, Adriatic Sea, detecting different contaminants in the range of 2-40 ng/g (dry weight). The study provides a valuable tool for investigating the potential threats posed by diverse contaminant classes with high annual tonnage, including analytes with known persistence and/or illegal status., Competing Interests: Declaration of Competing Interest 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., (Copyright © 2023. Published by Elsevier Ltd.)

Bile acids (BAs), endogenous acidic steroids synthetized from cholesterol in the liver, play a key role in the gut-liver axis physiopathology, including in hepatotoxicity, intestinal inflammatory processes, and cholesterol homeostasis. Faecal Oxo-BAs, relatively stable intermediates of oxidation/epimerization reactions of the BA hydroxyls, could be relevant to investigating the crosstalk in the liver-gut axis and the relationship between diseases and alterations in microbiota composition. A paucity of information currently exists on faecal BA profiles in dogs with and without chronic inflammatory enteropathy (CIE). Comprehensive assessment of 31 molecules among faecal BAs and related microbiota metabolites was conducted with high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). Odds ratios (ORs) for associations of BAs with CIE were estimated using logistic regression. Principal component analysis was performed to find differences between the control and pathological dogs. Higher levels of primary BAs and muricholic acids, and lower levels of secondary BAs were found in pathological dogs. Higher concentrations in faecal oxo-metabolites were associated with the absence of CIE (OR < 1). This study shows a marked difference in faecal BA profiles between dogs with and without CIE. Further research will be needed to better understand the role of oxo-BAs and muricholic acids in CIE dogs.

Marine mussels, especially Mytilus galloprovincialis, are well-established sentinel species, being naturally resistant to the exposure to multiple xenobiotics of natural and anthropogenic origin. Even if the response to multiple xenobiotic exposure is well known at the host level, the role of the mussel-associated microbiome in the animal response to environmental pollution is poorly explored, despite its potential in xenobiotic detoxification and its important role in host development, protection, and adaptation. Here, we characterized the microbiome-host integrative response of M. galloprovincialis in a real-world setting, involving exposure to a complex pattern of emerging pollutants, as occurs in the Northwestern Adriatic Sea. A total of 387 mussel individuals from 3 commercial farms, spanning about 200 km along the Northwestern Adriatic coast, and in 3 different seasons, were collected. Multiresidue analysis (for quantitative xenobiotic determination), transcriptomics (for host physiological response), and metagenomics (for host-associated microbial taxonomical and functional features) analyses were performed on the digestive glands. According to our findings, M. galloprovincialis responds to the presence of the complex pattern of multiple emerging pollutants - including the antibiotics sulfamethoxazole, erythromycin, and tetracycline, the herbicides atrazine and metolachlor, and the insecticide N,N-diethyl-m-toluamide - integrating host defense mechanisms, e.g., through upregulation of transcripts involved in animal metabolic activity, and microbiome-mediated detoxification functions, including microbial functionalities involved in multidrug or tetracycline resistance. Overall, our data highlight the importance of the mussel-associated microbiome as a strategic player for the orchestration of resistance to the multixenobiotic exposure at the holobiont level, providing strategic functionalities for the detoxification of multiple xenobiotic substances, as occurring in real world exposure settings. Complementing the host with microbiome-dependent xenobiotic degradative and resistance genes, the M. galloprovincialis digestive gland associated microbiome can have an important role in the detoxification of emerging pollutants in a context of high anthropogenic pressure, supporting the relevance of mussel systems as potential animal-based bioremediation tool., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Idiopathic normal pressure hydrocephalus (iNPH) is the most common form of hydrocephalus in the adult population, and is often treated with cerebrospinal fluid (CSF) drainage using a ventriculoperitoneal (VP) shunt. Symptoms of iNPH include gait impairment, cognitive decline, and urinary incontinence. The pathophysiology behind the symptoms of iNPH is still unknown, and no reliable biomarkers have been established to date. The aim of this study was to investigate the possible use of the oxysterols as biomarkers in this disease. CSF levels of the oxysterols 24S- and 27-hydroxycholesterol, as well as the major metabolite of 27-hydroxycholesterol, 7 alpha hydroxy-3-oxo-4-cholestenoic acid (7HOCA), were measured in iNPH-patients before and after treatment with a VP-shunt. Corresponding measurements were also performed in healthy controls. VP-shunt treatment significantly increased the levels of 7HOCA and 24S-hydroxycholesterol in CSF ( p = 0.014 and p = 0.037, respectively). The results are discussed in relation to the beneficial effects of VP-shunt treatment. Furthermore, the possibility that CSF drainage may reduce an inhibitory effect of transiently increased pressure on the metabolic capacity of neuronal cells in the brain is discussed. This capacity includes the elimination of cholesterol by the 24S-hydroxylase mechanisms.

The Farnesoid X Receptor (FXR) is the master regulator of Bile Acids (BA) homeostasis orchestrating their synthesis, transport and metabolism. Disruption of BA regulation has been linked to gut-liver axis diseases such as colorectal cancer (CRC). In this study, firstly we examined the role of constitutive activation of intestinal FXR in CRC; then we pre-clinically investigated the therapeutic potential of a diet enriched with a synthetic FXR agonist in two models of CRC (chemically-induced and genetic models). We demonstrated that mice with intestinal constitutive FXR activation are protected from AOM/DSS-induced CRC with a significant reduction of tumor number compared to controls. Furthermore, we evaluated the role of chemical FXR agonism in a DSS model of colitis in wild type (WT) and FXR null mice. WT mice administered with the FXR activating diet showed less morphological alterations and decreased inflammatory infiltrates compared to controls. The FXR activating diet also protected WT mice from AOM/DSS-induced CRC by reducing tumors' number and size. Finally, we proved that the FXR activating diet prevented spontaneous CRC in APC Min/+ mice via an FXR-dependent modulation of BA homeostasis. Our results demonstrate that intestinal FXR activation prevented both inflammation- and genetically-driven colorectal tumorigenesis by modulating BA pool size and composition. This could open new avenues for the therapeutic management of intestinal inflammation and tumorigenesis.

Accumulating evidence suggests that high consumption of natural antioxidants promotes health by reducing oxidative stress and, thus, the risk of developing cardiovascular diseases. Similarly, fermentation of natural compounds with lactic acid bacteria (LAB), such as Lactiplantibacillus plantarum , enhances their beneficial properties as regulators of the immune, digestive, and cardiovascular system. We investigated the effects of fermentation with Lactiplantibacillus plantarum on the antioxidant and immunomodulatory effects of Pushgay berries ( Vaccinium floribundum , Ericaceae family) in human umbilical vein endothelial cells (HUVECs) and macrophage cell line RAW264.7. Polyphenol content was assayed by Folin-Ciocalteu and HPLC-MS/MS analysis. The effects of berries solutions on cell viability or proliferation were assessed by WST8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt and Lactate dehydrogenase (LDH) release, Trypan blue exclusion test, and Alamar blue assay. Antioxidant activity was evaluated by a cell-based chemiluminescent probe for the detection of intracellular H 2 O 2 production in HUVECs. Heme oxygenase-1 (HO-1) expression levels were investigated by RT-qPCR. Glutathione reductase (GR), glutathione peroxidase (Gpx), superoxide dismutase (SOD), and catalase (CAT) activities, as markers of intracellular antioxidant defense, were evaluated by spectrophotometric analysis. The immunomodulatory activity was examined in RAW 264.7 by quantification of inducible nitric oxide synthase (iNOS) and Tumor Necrosis Factor-alpha (TNFα) by RT-qPCR. Data showed that fermentation of Pushgay berries ( i ) enhances the content of quercetin aglycone, and ( ii ) increases their intracellular antioxidant activity, as indicated by the reduction in H 2 O 2 -induced cell death and the decrease in H 2 O 2 -induced HO-1 gene expression in HUVECs treated for 24 h with fermented berries solution (10 µg/mL). Moreover, treatment with Pushgay berries for 72 h (10 µg/mL) promotes cells growth in RAW 264.7, and only fermented Pushgay berries increase the expression of iNOS in the same cell line. Taken together, our results show that LAB fermentation of Pushgay berries enhances their antioxidant and immunomodulatory properties.

There is a growing interest in the named "acidic sterolbiome" and in the genetic potential of the gut microbiome (GM) to modify bile acid (BA) structure. Indeed, the qualitative composition of BAs in feces correlates with the bowel microorganisms and their collective genetic material. GM is responsible for the production of BA metabolites, such as secondary and oxo-BAs. The specific BA profiles, as microbiome-host co-metabolic products, could be useful to investigate the GM-host interaction in animals under physiological conditions, as well as in specific diseases. In this context, we developed and validated an ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry method for the simultaneous analysis of up to 21 oxo-BAs and their 9 metabolic precursors. Chromatographic separation was achieved in 7 min with adequate analytical performance in terms of selectivity, sensitivity (LOQ from 0.05 to 0.1 µg/mL), accuracy (bias% < 5%), precision (CV% < 5%) and matrix effect (ME% < 10%). A fast solvent extraction protocol has been fine-tuned, achieving recoveries > 90%. In parallel, the gut microbiota assessment in farming animals was evaluated by 16S rRNA next-generation sequencing, and the correlation with the BA composition was performed by multivariate analysis, allowing to reconstruct species-specific associations between the BA profile and specific GM components., (© 2022. The Author(s).)

Food waste is a global problem due to its environmental and economic impact, so there is great demand for the exploitation of new functional applications. The winemaking process leads to an incomplete extraction of high-value compounds, leaving the pomace still rich in polyphenols. This study was aimed at optimising and validating sustainable routes toward the extraction and further valorisation of these polyphenols, particularly for cosmeceutical applications. New formulations based on red grape pomace polyphenols and natural deep eutectic solvents (NaDESs) were here investigated, namely betaine combined with citric acid (BET-CA), urea (BET-U) and ethylene glycol (BET-EG), in which DESs were used both as extracting and carrying agents for polyphenols. The flavonoid profile determined by HPLC-MS/MS analysis showed similar malvidin content (51-56 μg mL -1 ) in the DES combinations, while BET-CA gave the best permeation performance in Franz cells, so it was further investigated in 3D human keratinocytes (HaCat spheroids) injured with the pro-oxidant agent menadione. BET-CA treatment showed good intracellular antioxidant activity (IC50 0.15 ± 0.02 μg mL -1 in malvidin content) and significantly decreased ( p < 0.001) the release of the pro-inflammatory cytokine IL-8, improving cell viability. Thus, BET-CA formulation is worthy of investigation for potential use as a cosmetic ingredient to reduce oxidative stress and inflammation, which are causes of skin aging.

Small organic molecules, lipids, proteins, and DNA fragments can remain stable over centuries. Powerful and sensitive chemical analysis can therefore be used to characterize ancient remains for classical archaeological studies. This bio-ecological dimension of archaeology can contribute knowledge about several aspects of ancient life, including social organization, daily habits, nutrition, and food storage. Faecal remains (i.e. coprolites) are particularly interesting in this regard, with scientists seeking to identify new faecal markers. Here, we report the analysis of faecal samples from modern-day humans and faecal samples from a discharge pit on the site of the ruins of ancient Pompeii. We propose that bile acids and their gut microbiota oxo-metabolites are the most specific steroid markers for detecting faecal inputs. This is due to their extreme chemical stability and their exclusive occurrence in vertebrate faeces, compared to other ubiquitous sterols and steroids.

Bile acids are known to pass the blood-brain barrier and are present at low concentrations in the brain. In a previous work, it was shown that subdural hematomas are enriched with bile acids and that the levels in such hematomas are higher than in the peripheral circulation. The mechanism behind this enrichment was never elucidated. Bile acids have a high affinity to albumin, and subdural hematomas contain almost as high albumin levels as the peripheral circulation. A subdural hematoma is encapsulated by fibrin which may allow passage of small molecules like bile acids. We hypothesized that bile acids originating from the circulation may be 'trapped' in the albumin in subdural hematomas. In the present work, we measured the conjugated and unconjugated primary bile acids cholic acid and chenodeoxycholic acid in subdural hematomas and in peripheral circulation of 24 patients. In most patients, the levels of both conjugated and free bile acids were higher in the hematomas than in the circulation, but the enrichment of unconjugated bile acids was markedly higher than that of conjugated bile acids. In patients with a known time interval between the primary bleeding and the operation, there was a correlation between this time period and the accumulation of bile acids. This relation was most obvious for unconjugated bile acids. The results are consistent with a continuous flux of bile acids, in particular unconjugated bile acids, across the blood-brain barrier. We discuss the possible physiological importance of bile acid accumulation in subdural hematomas.

Increased epithelial permeability is a key feature of IBD pathogenesis and it has been proposed that agents which promote barrier function may be of therapeutic benefit. We have previously reported the secondary bile acid, ursodeoxycholic acid (UDCA), to be protective in a mouse model of colonic inflammation and that its bacterial metabolism is required for its beneficial effects. The current study aimed to compare the effects of UDCA, LCA, and a non-metabolizable analog of UDCA, 6-methyl-UDCA (6-MUDCA), on colonic barrier function and mucosal inflammation in a mouse model of colonic inflammation. Bile acids were administered daily to C57Bl6 mice by intraperitoneal injection. Colonic inflammation, induced by addition of DSS (2.5%) to the drinking water, was measured as disease activity index (DAI) and histological score. Epithelial permeability and apoptosis were assessed by measuring FITC-dextran uptake and caspase-3 cleavage, respectively. Cecal bile acids were measured by HPLC-MS/MS. UDCA and LCA, but not 6-MUDCA, were protective against DSS-induced increases in epithelial permeability and colonic inflammation. Furthermore, UDCA and LCA inhibited colonic epithelial caspase-3 cleavage both in DSS-treated mice and in an in vitro model of cytokine-induced epithelial injury. HPLC-MS/MS analysis revealed UDCA administration to increase colonic LCA levels, whereas LCA administration did not alter UDCA levels. UDCA, and its primary metabolite, LCA, protect against intestinal inflammation in vivo, at least in part, by inhibition of epithelial apoptosis and promotion of barrier function. These data suggest that clinical trials of UDCA in IBD patients are warranted., (© 2020 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Background: Disruption of bile acid (BA) homeostasis plays a key role in intestinal inflammation. The gut-liver axis is the main site for the regulation of BA synthesis and BA pool size via the combined action of the nuclear Farnesoid X Receptor (FXR) and the enterokine Fibroblast Growth Factor 19 (FGF19). Increasing evidence have linked derangement of BA metabolism with dysbiosis and mucosal inflammation. Thus, here we aimed to investigate the potential action of an FGF19 analogue on intestinal microbiota and inflammation., Methods: A novel engineered non-tumorigenic variant of the FGF19 protein, M52-WO 2016/0168219 was generated. WT and FXR null mice were injected with AAV-FGF19-M52 or the control AAV-GFP and subjected to Sodium Dextran Sulphate-induced colitis., Findings: FGF19-M52 reduced BA synthesis and pool size, modulated its composition and protected mice from intestinal inflammation. These events were coupled with preservation of the intestinal epithelial barrier integrity, inhibition of inflammatory immune response and modulation of microbiota composition. Interestingly, FGF19-M52-driven systemic and local anti-inflammatory activity was completely abolished in Farnesoid X Receptor (FXR) null mice, thus underscoring the need of FXR to guarantee enterocytes' fitness and complement FGF19 anti-inflammatory activity. To provide a translational perspective, we also show that circulating FGF19 levels are reduced in patients with Crohn's disease., Interpretation: Reactivation of the FXR-FGF19 axis in a murine model of intestinal inflammation could bona fide provide positive changes in BA metabolism with consequent reduction of intestinal inflammation and modulation of microbiota. These results point to the therapeutic potential of FGF19 in the treatment of intestinal inflammation with concomitant derangement of BA homeostasis., Funding: A. Moschetta is funded by MIUR-PRIN 2017 <- 2017J3E2W2; Italian Association for Cancer Research (AIRC, IG 23239); Interreg V-A Greece-Italy 2014-2020-SILVER WELLBEING, MIS5003627; HDHL-INTIMIC EuJPI-FATMAL; MIUR PON "R&I" 2014-2020-ARS01&#95;01220. No money has been paid by NGM Biopharmaceuticals or any other agency to write this article., Competing Interests: Declaration of Competing Interest Brian Ko and Jian Luo are employers of NGM Pharmaceuticals that retains the Patent for FGF19-M52 (WO 2013/006486). Brian Ko and Jian Luo had no role in study design, data collection, data analysis, interpretation, writing of the report. Gionatha Fiorino reports personal fees from AbbVie, MSD, Takeda, Janssen, Amgen, Sandoz, Pfizer, Mylan and Ferring, outside the submitted work. Silvio Danese reports personal fees from AbbVie, Allergan, Amgen, AstraZeneca, Biogen, Boehringer Ingelheim, Celgene, Celltrion, Ely Lilly, Enthera, Ferring Pharmaceuticals inc, Gilead, Hospira, Janssen, Johnson and Johnson, MSD, Mundipharma, Mylan, Pfizer, Roche, Sandoz, Sublimity Therapeutics, Takeda, TiGenix, UCB Inc and Vifor outside the submitted work. Antonio Moschetta reports grants from NGM Biopharmaceuticals, during the conduct of the study; grants from Intercept Pharmaceuticals, outside the submitted work. All other authors have declared no conflict of interest., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

The respiratory complexes are organized in supramolecular assemblies called supercomplexes thought to optimize cellular metabolism under physiological and pathological conditions. In this study, we used genetically and biochemically well characterized cells bearing the pathogenic microdeletion m.15,649-15,666 (ΔI300-P305) in MT-CYB gene, to investigate the effects of an assembly-hampered CIII on the re-organization of supercomplexes. First, we found that this mutation also affects the stability of both CI and CIV, and evidences the occurrence of a preferential structural interaction between CI and CIII 2 , yielding a small amount of active CI+CIII 2 supercomplex. Indeed, a residual CI+CIII combined redox activity, and a low but detectable ATP synthesis driven by CI substrates are detectable, suggesting that the assembly of CIII into the CI+CIII 2 supercomplex mitigates the detrimental effects of MT-CYB deletion. Second, measurements of oxygen consumption and ATP synthesis driven by NADH-linked and FADH 2 -linked substrates alone, or in combination, indicate a common ubiquinone pool for the two respiratory pathways. Finally, we report that prolonged incubation with rotenone enhances the amount of CI and CIII 2 , but reduces CIV assembly. Conversely, the antioxidant N-acetylcysteine increases CIII 2 and CIV 2 and partially restores respirasome formation. Accordingly, after NAC treatment, the rate of ATP synthesis increases by two-fold compared with untreated cell, while the succinate level, which is enhanced by the homoplasmic mutation, markedly decreases. Overall, our findings show that fine-tuning the supercomplexes stability improves the energetic efficiency of cells with the MT-CYB microdeletion., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Inflammatory bowel diseases (IBD), ulcerative colitis (UC), and Crohn's disease (CD), represent systematic chronic conditions with a deficient intestinal absorption. We first attempt to investigate the serum bile acids (sBAs) profile in a large cohort of IBD patients to evaluate changes under anti-TNF alpha treatment., Methods: Forty CD and 40 UC patients were enrolled and BAs were quantified by high-pressure liquid chromatography-electrospray-tandem mass spectrometry (HPLC-ES-MS/MS). Up to 15 different sBAs concentrations and clinical biomarkers where added to a Principal Component Analysis (PCA) to discriminate IBD from healthy conditions and treatment., Results: PCA allowed a separation into two clusters within CD (biologic-free patients and patients treated with anti-TNF alpha drugs and healthy subjects) but not UC. The first included CD. CD patients receiving anti-TNF alpha have an increase in total sBAs (4.11 1.23 μM) compared to patients not exposed. Secondary BAs significantly increase after anti-TNF alpha treatment (1.54 0.83 μM). Furthermore, multivariate analysis based on sBA concentration highlighted a different qualitative sBAs profile for UC and CD patients treated with conventional therapy., Conclusion: According to our results, anti-TNF alpha in CD restores the sBA profile by re-establishing the physiological levels. These findings indicate that, secondary BAs might serve as an indirect biomarker of the healing process., Competing Interests: The authors declare no conflict of interest.

Critical regulation of bile acid (BA) pool size and composition occurs via an intensive molecular crosstalk between the liver and gut, orchestrated by the combined actions of the nuclear Farnesoid X receptor (FXR) and the enterokine fibroblast growth factor 19 (FGF19) with the final aim of reducing hepatic BA synthesis in a negative feedback fashion. Disruption of BA homeostasis with increased hepatic BA toxic levels leads to higher incidence of hepatocellular carcinoma (HCC). While native FGF19 has anti-cholestatic and anti-fibrotic activity in the liver, it retains peculiar pro-tumorigenic actions. Thus, novel analogues have been generated to avoid tumorigenic capacity and maintain BA metabolic action. Here, using BA related Abcb4 -/- and Fxr -/- mouse models of spontaneous hepatic fibrosis and HCC, we explored the role of a novel engineered variant of FGF19 protein, called FGF19-M52, which fully retains BA regulatory activity but is devoid of the pro-tumoral activity. Expression of the BA synthesis rate-limiting enzyme Cyp7a1 is reduced in FGF19-M52-treated mice compared to the GFP-treated control group with consequent reduction of BA pool and hepatic concentration. Treatment with the non-tumorigenic FGF19-M52 strongly protects Abcb4 -/- and Fxr -/- mice from spontaneous hepatic fibrosis, cellular proliferation and HCC formation in terms of tumor number and size, with significant reduction of biochemical parameters of liver damage and reduced expression of several genes driving the proliferative and inflammatory hepatic scenario. Our data bona fide suggest the therapeutic potential of targeting the FXR-FGF19 axis to reduce hepatic BA synthesis in the control of BA-associated risk of fibrosis and hepatocarcinoma development.

Berberine (BBR) is a natural alkaloid obtained from Berberis species plants, known for its protective effects against several diseases. Among the primary BBR metabolites, berberrubine (M1) showed the highest plasma concentration but few and conflicting data are available regarding its concentration in biological fluids related to its new potential activity on vascular cells. A combined analytical approach was applied to study biodistribution of M1 in comparison with BBR. The optimization of sample clean-up combined with a fully validated HPLC-ESI-MS/MS tailored for M1 allows sufficient detectability and accuracy to be reached in the different studied organs even when administered at low dose, comparable to that assumed by human. A predictive human vascular endothelial cell-based assay to measure intracellular xanthine oxidase has been developed and applied to study unexplored activities of M1 alongside other common activities. Results showed that oral M1 treatment exhibits higher plasma levels than BBR, reaching maximum concentration 400-fold higher than BBR (204 vs 0.5 ng/mL); moreover, M1 exhibits higher concentrations than BBR also in all the biological compartments analyzed. Noteworthy, the two compounds follow two different excretion routes: M1 through urine, while BBR through feces. In vitro studies demonstrated that M1 inhibited intracellular xanthine oxidase activity, one of the major sources of reactive oxygen species in vasculature, with an IC50 = 9.90 ± 0.01 μg/mL and reduced the expression of the inflammatory marker ICAM-1. These peculiar characteristics allow new perspectives to be opened up for the direct use of M1 instead of BBR in endothelial dysfunction treatment.

Bile acids (BAs) are the end product of cholesterol catabolism. Their synthesis is regulated by the nuclear receptor farnesoid X receptor, also involved in the control of their enterohepatic circulation. Inflammatory bowel diseases (IBD), which include Crohn's disease (CD) and ulcerative colitis (UC), are multifactorial diseases characterized by diarrhea. The pathogenesis of diarrhea in IBD is still debated. The most important factor is the inflammatory process of the intestinal wall, causing alterations of solute and water absorption/secretion, deterioration of epithelial cell integrity, disruption of the intestinal microflora homeostasis, and impairment of specific transport mechanisms within the gut (including that of BAs). In this review, we summarize the current state of the art in this area and we critically evaluate the alterations of BA metabolism in patients with CD and UC., Competing Interests: Conflict of Interest: None

Altered bile acid (BA) signaling is associated with hepatotoxicity. The farnesoid X receptor (FXR) is a nuclear receptor that transcriptionally regulates BA homeostasis. Mice with FXR ablation present hepatocarcinoma (HCC) due to high toxic BA levels. Mice with Abcb4 ablation accumulate toxic BA within the bile ducts and present HCC. We have previously shown that intestinal specific activation of FXR by transgenic VP16-FXR chimera is able to reduce BA pool size and prevent HCC. Here we tested chemical FXR activation by administering for 15 months the dual FXR/ membrane G protein-coupled receptor (TGR5) agonist INT-767 (6α-ethyl-3α,7α,23-trihydroxy-24-nor-5β-cholan-23-sulphate) to Fxr -/- and Abcb4 -/- mice. HCC number and size were significantly reduced by INT-767 administration. In contrast, no changes in HCC tumor number and size were observed in Fxr -/- mice fed with or without INT-767. Notably, INT-767 preserved the hepatic parenchyma, improved hepatic function and down-regulated pro-inflammatory cytokines. Moreover, in Abcb4 -/- mice INT-767 prevented fibrosis by reducing collagen expression and deposition. Thus, long term activation of FXR is able to reduce BA pool, reprogram BA metabolism and prevent HCC. These data provide the impetus to address the bona fide therapeutic potential of FXR activation in disease with BA-associated development of HCC.