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Hyperthermia induced by phenethylamines, such as 3,4-methylenedioxymethamphetamine (MDMA), can lead to life-threatening complications and death. Activation of the sympathetic nervous system and subsequent release of norepinephrine and activation of uncoupling proteins have been demonstrated to be the key mediators of phenethylamine-induced hyperthermia (PIH). Recently, the gut microbiome was shown to also play a contributing role in PIH. Here, the hypothesis that bile acids (BAs) produced by the gut microbiome are essential to PIH was tested. Changes in the serum concentrations of unconjugated primary BAs cholic acid (CA) and chenodeoxycholic acid (CDCA) and secondary BA deoxycholic acid (DCA) were measured following MDMA (20 mg/kg, sc) treatment in antibiotic treated and control rats. MDMA-induced a significant hyperthermic response and reduced the serum concentrations of three BAs 60 min post-treatment. Pretreatment with antibiotics (vancomycin, bacitracin and neomycin) in the drinking water for five days resulted in the depletion of BAs and a hypothermic response to MDMA. Gut bacterial communities in the antibiotic-treated group were distinct from the MDMA or saline treatment groups, with decreased microbiome diversity and alteration in taxa. Metagenomic functions inferred using the bioinformatic tool PICRUSt2 on 16S rRNA gene sequences indicated that bacterial genes associated to BA metabolism are less abundant in the antibiotic-MDMA treated group. Overall, these findings suggest that gut bacterial produced BAs might play an important role in MDMA-induced hyperthermia., (© 2024. The Author(s).)

High-fat diet (HFD) has long been recognized as risk factors for the development and progression of ulcerative colitis (UC), but the exact mechanism remained elusive. Here, HFD increased intestinal deoxycholic acid (DCA) levels, and DCA further exacerbated colonic inflammation. Transcriptome analysis revealed that DCA triggered ferroptosis pathway in colitis mice. Mechanistically, DCA upregulated hypoxia-inducible factor-2α (HIF-2α) and divalent metal transporter-1 (DMT1) expression, causing the ferrous ions accumulation and ferroptosis in intestinal epithelial cells, which was reversed by ferroptosis inhibitor ferrostatin-1. DCA failed to promote colitis and ferroptosis in intestine-specific HIF-2α-null mice. Notably, byak-angelicin inhibited DCA-induced pro-inflammatory and pro-ferroptotic effects through blocking the up-regulation of HIF-2α by DCA. Moreover, fat intake was positively correlated with disease activity in UC patients consuming HFD, with ferroptosis being more pronounced. Collectively, our findings demonstrated that HFD exacerbated colonic inflammation by promoting DCA-mediated ferroptosis, providing new insights into diet-related bile acid dysregulation in UC., 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 © 2024 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

ABCC3 (also known as MRP3) is an ATP binding cassette transporter for bile acids, whose expression is downregulated in colorectal cancer through the Wnt/β-catenin signaling pathway. However, it remained unclear how downregulation of ABCC3 expression contributes to colorectal carcinogenesis. We explored the role of ABCC3 in the progression of colorectal cancer-in particular, focusing on the regulation of bile acid export. Gene expression analysis of colorectal adenoma isolated from familial adenomatous polyposis patients revealed that genes related to bile acid secretion including ABCC3 were downregulated as early as at the stage of adenoma formation. Knockdown or overexpression of ABCC3 increased or decreased intracellular concentration of deoxycholic acid, a secondary bile acid, respectively, in colorectal cancer cells. Forced expression of ABCC3 suppressed deoxycholic acid-induced activation of MAPK signaling. Finally, we found that nonsteroidal anti-inflammatory drugs increased ABCC3 expression in colorectal cancer cells, suggesting that ABCC3 could be one of the targets for therapeutic intervention of familial adenomatous polyposis. Our data thus suggest that downregulation of ABCC3 expression contributes to colorectal carcinogenesis through the regulation of intracellular accumulation of bile acids and activity of MAPK signaling., (© 2024 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Alterations in gut microbiota composition are suggested to contribute to cardiometabolic diseases, in part by producing bioactive molecules. Some of the metabolites are produced by very low abundant bacterial taxa, which largely have been neglected due to limits of detection. However, the concentration of microbially produced metabolites from these taxa can still reach high levels and have substantial impact on host physiology. To explore this concept, we focused on the generation of secondary bile acids by 7α-dehydroxylating bacteria and demonstrated that addition of a very low abundant bacteria to a community can change the metabolic output dramatically. We show that Clostridium scindens converts cholic acid into the secondary bile acid deoxycholic acid (DCA) very efficiently even though the abundance of C. scindens is low, but still detectable by digital droplet PCR. We also show that colonization of germ-free female mice with a community containing C. scindens induces DCA production and affects host metabolism. Finally, we show that DCA correlates with impaired glucose metabolism and a worsened lipid profile in individuals with type 2 diabetes, which implies that this metabolic pathway may contribute to the development of cardiometabolic disease., (© 2024. The Author(s).)

Non-alcoholic fatty liver disease (NAFLD) is regarded as a pandemic that affects about a quarter of the global population. Recently, host-gut microbiota metabolic interactions have emerged as distinct mechanistic pathways implicated in the development of NAFLD. Here, we report that a group of gut microbiota-modified bile acids (BAs), hyodeoxycholic acid (HDCA) species, are negatively correlated with the presence and severity of NAFLD. HDCA treatment has been shown to alleviate NAFLD in multiple mouse models by inhibiting intestinal farnesoid X receptor (FXR) and upregulating hepatic CYP7B1. Additionally, HDCA significantly increased abundances of probiotic species such as Parabacteroides distasonis, which enhances lipid catabolism through fatty acid-hepatic peroxisome proliferator-activated receptor alpha (PPARα) signaling, which in turn upregulates hepatic FXR. These findings suggest that HDCA has therapeutic potential for treating NAFLD, with a unique mechanism of simultaneously activating hepatic CYP7B1 and PPARα., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Tissue engineered bioscaffolds based on decellularized composites have gained increasing interest for treatment of various diaphragmatic impairments, including muscular atrophies and diaphragmatic hernias. Detergent-enzymatic treatment (DET) constitutes a standard strategy for diaphragmatic decellularization. However, there is scarce data on comparing DET protocols with different substances in distinct application models in their ability to maximize cellular removal while minimizing extracellular matrix (ECM) damage., Methods: We decellularized diaphragms of male Sprague Dawley rats with 1 % or 0.1 % sodium dodecyl sulfate (SDS) and 4 % sodium deoxycholate (SDC) by orbital shaking (OS) or retrograde perfusion (RP) through the vena cava. We evaluated decellularized diaphragmatic samples by (1) quantitative analysis including DNA quantification and biomechanical testing, (2) qualitative and semiquantitative analysis by proteomics, as well as (3) qualitative assessment with macroscopic and microscopic evaluation by histological staining, immunohistochemistry and scanning electron microscopy., Results: All protocols produced decellularized matrices with micro- and ultramorphologically intact architecture and adequate biomechanical performance with gradual differences. The proteomic profile of decellularized matrices contained a broad range of primal core and ECM-associated proteins similar to native muscle. While no outstanding preference for one singular protocol was determinable, SDS-treated samples showed slightly beneficial properties in comparison to SDC-processed counterparts. Both application modalities proved suitable for DET., Conclusion: DET with SDS or SDC via orbital shaking or retrograde perfusion constitute suitable methods to produce adequately decellularized matrices with characteristically preserved proteomic composition. Exposing compositional and functional specifics of variously treated grafts may enable establishing an ideal processing strategy to sustain valuable tissue characteristics and optimize consecutive recellularization. This aims to design an optimal bioscaffold for future transplantation in quantitative and qualitative diaphragmatic defects., 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 Elsevier B.V. All rights reserved.)

The ability of bacterial pathogens to establish recurrent and persistent infections is frequently associated with their ability to form biofilms. Clostridioides difficile infections have a high rate of recurrence and relapses and it is hypothesized that biofilms are involved in its pathogenicity and persistence. Biofilm formation by C. difficile is still poorly understood. It has been shown that specific molecules such as deoxycholate (DCA) or metronidazole induce biofilm formation, but the mechanisms involved remain elusive. In this study, we describe the role of the C. difficile lipoprotein CD1687 during DCA-induced biofilm formation. We showed that the expression of CD1687, which is part of an operon within the CD1685-CD1689 gene cluster, is controlled by multiple transcription starting sites and some are induced in response to DCA. Only CD1687 is required for biofilm formation and the overexpression of CD1687 is sufficient to induce biofilm formation. Using RNAseq analysis, we showed that CD1687 affects the expression of transporters and metabolic pathways and we identified several potential binding partners by pull-down assay, including transport-associated extracellular proteins. We then demonstrated that CD1687 is surface exposed in C. difficile, and that this localization is required for DCA-induced biofilm formation. Given this localization and the fact that C. difficile forms eDNA-rich biofilms, we confirmed that CD1687 binds DNA in a non-specific manner. We thus hypothesize that CD1687 is a component of the downstream response to DCA leading to biofilm formation by promoting interaction between the cells and the biofilm matrix by binding eDNA., (© 2023. The Author(s).)

In cancer therapy, a drug delivery system (DDS) has been widely studied to achieve selective drug accumulation at the tumor site. However, DDS still has a major drawback in that it requires multistep processes for intracellular delivery, resulting in low efficiency of drug delivery. To overcome this problem, we recently reported a molecular block (MB) that disrupts cancer cell membranes in the cancer microenvironment using deoxycholic acid (DCA). However, the MB showed considerable cytotoxicity even at neutral pH, possibly due to the structural hydrophobic property of DCA. Herein, we focused on selecting the most suitable bile acid for an MB that possessed high responsiveness to the cancer microenvironment without cytotoxicity at neutral pH. Cell viabilities of the free bile acids such as DCA, chenodeoxycholic acid (CDCA), cholic acid (CA), and ursodeoxycholic acid (UDCA) were evaluated at neutral pH (pH = 7.4) and a cancer acidic environment (pH = 6.3-6.5). The half-maximal inhibition concentration (IC 50 ) value of UDCA at pH = 7.4 showed an approximately 7.5-fold higher IC 50 value than that at pH = 6.3, whereas the other bile acids yielded less than a 4-fold IC 50 value difference between the same pHs. Biocompatible poly(vinyl alcohol) (PVA) was functionalized with UDCA (PVA-UDCA) for the synthesis of higher responsiveness to the cancer microenvironment without cytotoxicity at neutral pH. Importantly, 56% pancreatic cancer cell death was observed at pH = 6.5, whereas only 10% was detected at neutral pH by the PVA-UDCA treatment. However, PVA-DCA indicated almost the same cancer cell death property, independent of pH conditions. These results suggest PVA-UDCA shows great potential for a new class of MB.

It is known that the reduction of blood cholesterol can be accomplished through foods containing a large number of dietary fibers; this process is partially related to the binding of bile salt to fibers. To gain new insights into the interactions between dietary fibers and bile salts, this study investigates the interactions between cationic hydroxyethyl cellulose (catHEC) and sodium deoxycholate (NaDC) or sodium cholate (NaC), which have a similar structure. Turbidity measurements reveal strong interactions between catHEC and NaDC, and under some conditions, macroscopic phase separation occurs. In contrast, the interactions with NaC are weak. At a catHEC concentration of 2 wt %, incipient phase separation is approached at concentrations of NaC and NaDC of 32.5 and 19.3 mM, respectively. The rheological results show strong interactions and a prominent viscosification effect for the catHEC/NaDC system but only moderate interactions for the catHEC/NaC system. Both cryogenic transmission electron microscopy and small-angle X-ray scattering results display fundamental structural differences between the two systems, which may explain the stronger interactions in the presence of NaDC. The surmise is that the extended structures formed in the presence of NaDC can easily form connections and entanglements in the network.

MoS 2 has been increasingly used in place of graphene as a flexible and multifunctional 2D material in many biomedical applications such as cancer detection and drug delivery, which makes it crucial to evaluate downstream compatibility in human immune cells. Molybdenum is a component of stainless-steel stent implants and has previously been implicated in stent hypersensitivity. In view of this, it is important to ascertain the effect of MoS 2 on allergy-relevant cells. Basophils are a less commonly used immune cell type. Unlike mast cells, basophils can be easily derived from primary human blood and can act as a sentinel for allergy. However, merely testing any one type of MoS 2 in basophils could result in different biological results. We thus decided to compare 2D MoS 2 from the two companies BeDimensional© (BD) and Biograph Solutions (BS), manufactured with two different but commonly exploited methods (BD, deoxycholate surfactant in a high-pressure liquid exfoliation, and BS using glycine in ball-milling exfoliation) to elucidate immunological end-points common to both MoS 2 and to demonstrate the need for biological verification for end-users who may require a change of supplier. We report higher histamine production in human basophils with MoS 2 . No effects on either surface basophil activation markers CD63 and CD203c or reactive oxygen species (ROS) production and cell viability were observed. However, different cytokine production patterns were evidenced. IL-6 and IL-1β but not TNF and GM-CSF were increased for both MoS 2 . BS-MoS 2 increased IL-4, while BD-MoS 2 decreased IL-4 and increased IL-13. Molybdate ion itself only increased IL-1β and IL-4. Deoxycholate surfactant decreased viability at 18 h and increased ROS upon basophil activation. Therefore, these results demonstrate the safety of MoS 2 in human basophils in general and highlight the importance of considering manufacturer additives and variability when selecting and investigating 2D materials such as MoS 2 ., 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 B.V.)

Background: Recent studies have demonstrated that dysfunction of the intestinal barrier is a significant contributing factor to the development of severe acute pancreatitis (SAP). A stable intestinal mucosa barrier functions as a major anatomic and functional barrier, owing to the balance between intestinal epithelial cell (IEC) proliferation and apoptosis. There is some evidence that calcium overload may trigger IEC apoptosis and that calcineurin (CaN)/nuclear factor of activated T-cells (NFAT) signaling might play an important role in calcium-mediated apoptosis., Aim: To investigate the potential mechanisms underlying the therapeutic effect of Qingyi decoction (QYD) in SAP., Methods: A rat model of SAP was created via retrograde infusion of sodium deoxycholate. Serum levels of amylase, tumor necrosis factor (TNF-α), interleukin (IL)-6, D-lactic acid, and diamine oxidase (DAO); histological changes; and apoptosis of IECs were examined in rats with or without QYD treatment. The expression of the two subunits of CaN and NFAT in intestinal tissue was measured via quantitative real-time polymerase chain reaction and western blotting. For in vitro studies, Caco-2 cells were treated with lipopolysaccharide (LPS) and QYD serum, and then cell viability and intracellular calcium levels were detected., Results: Retrograde infusion of sodium deoxycholate increased the severity of pancreatic and intestinal pathology and the levels of serum amylase, TNF-α, and IL-6. Both the indicators of intestinal mucosa damage (D-lactic acid and DAO) and the levels of IEC apoptosis were elevated in the SAP group. QYD treatment reduced the serum levels of amylase, TNF-α, IL-6, D-lactic acid, and DAO and attenuated the histological findings. IEC apoptosis associated with SAP was ameliorated under QYD treatment. In addition, the protein expression levels of the two subunits of CaN were remarkably elevated in the SAP group, and the NFATc3 gene was significantly upregulated at both the transcript and protein levels in the SAP group compared with the control group. QYD significantly restrained CaN and NFATc3 gene expression in the intestine, which was upregulated in the SAP group. Furthermore, QYD serum significantly decreased the LPS-induced elevation in intracellular free Ca 2+ levels and inhibited cell death., Conclusion: QYD can exert protective effects against intestinal mucosa damage caused by SAP and the protective effects are mediated, at least partially, by restraining IEC apoptosis via the CaN/NFATc3 pathway., Competing Interests: Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article., (©The Author(s) 2022. Published by Baishideng Publishing Group Inc. All rights reserved.)

Cytochrome P450 3A (CYP3A), the most important class of drug-metabolizing enzymes, participates in the metabolism of half of clinically used drugs. The CYP3A index reactions of dogs, one of the most widely used preclinical nonrodent species, are still poorly understood. This work evaluated the activity and selectivity of 10 CYP3A index reactions, including midazolam (MDZ) 1'- and 4-hydroxylation, alprazolam (APZ) and triazolam (TRZ) α - and 4-hydroxylation, testosterone (T) 6 β -hydroxylation, lithocholate (LCA) 6 α -hydroxylation, deoxycholate (DCA) 1 β - and 5 β -hydroxylation, with quantitative reaction phenotyping and kinetic analysis in human and canine recombinant CYP enzymes (rCYPs). In human studies, all reactions are reconfirmed as mixed index reactions of CYP3A with minor contributions from non-CYP3A isoforms. In canine studies, all reactions are also primarily catalyzed by CYP3A12 with lower contributions from CYP3A26. However, the canine CYP2B11 appreciably contributes to the hydroxylation of benzodiazepines except for APZ 4-hydroxylation. The canine CYP3A isoforms have lower activity than human isoforms toward T 6 β -hydroxylation and LCA 6 α -hydroxylation and both substrates undergo non-CYP3A catalyzed side reactions. DCA 1 β - and 5 β -hydroxylation are validated as the CYP3A index reactions in both humans and dogs with limited non-CYP3A contributions and side reactions. In conclusion, this work provides a comprehensive overview for the selectivity and activity of in vitro CYP3A index reactions in humans and dogs. The validated CYP3A index reactions between humans and dogs may benefit future practices in drug metabolism and drug interaction studies. SIGNIFICANCE STATEMENT: Dogs are one of the most important nonrodent animals with limited studies of cytochrome P450 enzymes than humans. This work provides the most comprehensive quantitative data to date for the selectivity and activity of CYP3A index reactions in humans and dogs. The canine CYP2B11 was found to appreciably contribute to hydroxylation of midazolam, alprazolam and triazolam, the well-known probes for human CYP3A. Deoxycholate 1 β - and 5 β -hydroxylation are validated as the CYP3A index reactions in both humans and dogs., (Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics.)

Purpose: Cholecystectomy (XGB) is widely recognized as a risk factor for colon cancer (CC). Continuous exposure of the colonic epithelium to deoxycholic acid (DCA) post-XGB may exert cytotoxic effects and be involved in the progression of CC. However, the functions of the XGB-induced DCA increase and the underlying mechanism remain unclear., Methods: Colitis-associated CC (CAC) mouse models constructed by AOM-DSS inducement were used to confirm the effect of XGB on the CC progression. Hematoxylin & eosin staining was performed to assess the tumor morphology of CAC mouse models tissues. Various cell biological assays including EdU, live-cell imaging, wound-healing assays, and flow cytometry for cell cycle and apoptosis were used to evaluate the effect of DCA on CC progression. The correlation among XGB, DCA, and CC and their underlying mechanisms were detected with immunohistochemistry, mass spectrometry, transcriptome sequencing, qRT-PCR, and western blotting., Results: Here we proved that XGB increased the plasma DCA level and promoted colon carcinogenesis in a colitis-associated CC mouse model. Additionally, we revealed that DCA promoted the proliferation and migration of CC cells. Further RNA sequencing showed that 120 mRNAs were upregulated, and 118 downregulated in DCA-treated CC cells versus control cells. The upregulated mRNAs were positively correlated with Wnt signaling and cell cycle-associated pathways. Moreover, DCA treatment could reduced the expression of the farnesoid X receptor (FXR) and subsequently increased the levels of β-Catenin and c-Myc in vitro and in vivo. Moreover, the FXR agonist GW4064 decreased the proliferation of CC cells by repressing the expression of β-catenin., Conclusion: We concluded that XGB-induced DCA exposure could promote the progression of CC by inhibiting FXR expression and enhancing the Wnt-β-catenin pathway. Video Abstract., (© 2022. The Author(s).)

Enterococcus faecalis is a natural inhabitant of the human gastrointestinal tract. This bacterial species is subdominant in a healthy physiological state of the gut microbiota (eubiosis) in adults, but can become dominant and cause infections when the intestinal homeostasis is disrupted (dysbiosis). The relatively high concentrations of bile acids deoxycholate (DCA) and taurocholate (TCA) hallmark eubiosis and dysbiosis, respectively. This study aimed to better understand how E. faecalis adapts to DCA and TCA. We showed that DCA impairs E. faecalis growth and possibly imposes a continuous adjustment in the expression of many essential genes, including a majority of ribosomal proteins. This may account for slow growth and low levels of E. faecalis in the gut. In contrast, TCA had no detectable growth effect. The evolving transcriptome upon TCA adaptation showed the early activation of an oligopeptide permease system (opp2) followed by the adjustment of amino acid and nucleotide metabolisms. We provide evidence that TCA favors the exploitation of oligopeptide resources to fuel amino acid needs in limiting oligopeptide conditions. Altogether, our data suggest that the combined effects of decreased DCA and increased TCA concentrations can contribute to the rise of E. faecalis population during dysbiosis., (© 2022. The Author(s).)

A high-fat diet (HFD) leads to long-term exposure to gut microbial metabolite secondary bile acids, such as deoxycholic acid (DCA), in the intestine, which is closely linked to colorectal cancer (CRC). Evidence reveals that vasculogenic mimicry (VM) is a critical event for the malignant transformation of cancer. Therefore, this study investigated the crucial roles of DCA in the regulation of VM and the progression of intestinal carcinogenesis. The effects of an HFD on VM formation and epithelial-mesenchymal transition (EMT) in human CRC tissues were investigated. The fecal DCA level was detected in HFD-treated Apc min/+ mice. Then the effects of DCA on VM formation, EMT, and vascular endothelial growth factor receptor 2 (VEGFR2) signaling were evaluated in vitro and in vivo. Here we demonstrated that compared with a normal diet, an HFD exacerbated VM formation and EMT in CRC patients. An HFD could alter the composition of the gut microbiota and significantly increase the fecal DCA level in Apc min/+ mice. More importantly, DCA promoted tumor cell proliferation, induced EMT, increased VM formation, and activated VEGFR2, which led to intestinal carcinogenesis. In addition, DCA enhanced the proliferation and migration of HCT-116 cells, and induced EMT process and vitro tube formation. Furthermore, the silence of VEGFR2 reduced DCA-induced EMT, VM formation, and migration. Collectively, our results indicated that microbial metabolite DCA promoted VM formation and EMT through VEGFR2 activation, which further exacerbated intestinal carcinogenesis., (© 2021 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

The gut microbiome of vertebrates is capable of numerous biotransformations of bile acids, which are responsible for intestinal lipid digestion and function as key nutrient-signaling molecules. The human liver produces bile acids from cholesterol predominantly in the A/B- cis orientation in which the sterol rings are "kinked", as well as small quantities of A/B- trans oriented "flat" stereoisomers known as "primary allo-bile acids". While the complex multi-step bile acid 7α-dehydroxylation pathway has been well-studied for conversion of "kinked" primary bile acids such as cholic acid (CA) and chenodeoxycholic acid (CDCA) to deoxycholic acid (DCA) and lithocholic acid (LCA), respectively, the enzymatic basis for the formation of "flat" stereoisomers allo-deoxycholic acid (allo-DCA) and allo-lithocholic acid (allo-LCA) by Firmicutes has remained unsolved for three decades. Here, we present a novel mechanism by which Firmicutes generate the "flat" bile acids allo-DCA and allo-LCA. The BaiA1 was shown to catalyze the final reduction from 3-oxo-allo-DCA to allo-DCA and 3-oxo-allo-LCA to allo-LCA. Phylogenetic and metagenomic analyses of human stool samples indicate that BaiP and BaiJ are encoded only in Firmicutes and differ from membrane-associated bile acid 5α-reductases recently reported in Bacteroidetes that indirectly generate allo-LCA from 3-oxo-Δ 4 -LCA. We further map the distribution of baiP and baiJ among Firmicutes in human metagenomes, demonstrating an increased abundance of the two genes in colorectal cancer (CRC) patients relative to healthy individuals.

Streptococcus pneumoniae colonizes the nasopharynx of children and the elderly but also kills millions worldwide yearly. The secondary bile acid metabolite deoxycholic acid (DoC) affects the viability of human pathogens but also plays multiple roles in host physiology. We assessed in vitro the antimicrobial activity of DoC and investigated its potential to eradicate S. pneumoniae colonization using a model of human nasopharyngeal colonization and an in vivo mouse model of colonization. At a physiological concentration, DoC (0.5 mg/ml; 1.27 mM) killed all tested S. pneumoniae strains ( n  = 48) 2 h postinoculation. The model of nasopharyngeal colonization showed that DoC eradicated colonization by S. pneumoniae strains as soon as 10 min postexposure. The mechanism of action did not involve activation of autolysis, since the autolysis-defective double mutants Δ lytA Δ lytC and Δ spxB Δ lctO were as susceptible to DoC as was the wild type (WT). Oral streptococcal species ( n  = 20), however, were not susceptible to DoC (0.5 mg/ml). Unlike trimethoprim, whose spontaneous resistance frequency (srF) for TIGR4 or EF3030 was ≥1 × 10 -9 , no spontaneous resistance was observed with DoC (srF, ≥1 × 10 -12 ). Finally, the efficacy of DoC to eradicate S. pneumoniae colonization was assessed in vivo using a topical route via intranasal (i.n.) administration and as a prophylactic treatment. Mice challenged with S. pneumoniae EF3030 carried a median of 4.05 × 10 5 CFU/ml 4 days postinoculation compared to 6.67 × 10 4 CFU/ml for mice treated with DoC. Mice in the prophylactic group had an ∼99% reduction of the pneumococcal density (median, 2.61 × 10 3 CFU/ml). Thus, DoC, an endogenous human bile salt, has therapeutic potential against S. pneumoniae.

Oral absorption of peptides/proteins is usually compromised by various gastrointestinal tract barriers. To improve delivery efficiency, chitosan-conjugated deoxycholic acid (CS-DCA) coupled with sodium alginate (ALG) was prepared to load insulin into pH-sensitive nanoparticles. The insulin-loaded chitosan-deoxycholic acid/alginate nanoparticles (CDA NPs) were characterized by size (143.3 ± 10.8 nm), zeta potential (19.5 ± 1.6 mV), entrapment efficiency (61.14 ± 1.67%), and insulin drug loading (3.36 ± 0.09%). The CDA NPs exhibited pH-triggered release characteristics in vitro and protected the wrapped insulin from gastric degradation. Stability of the CDA NPs in enzyme-containing simulated gastrointestinal fluids suggested that the NPs could partially protect the wrapped insulin from enzymatic degradation. Additionally, CS-DCA-modified NPs promoted the permeability of Caco-2 cells and enhanced intracellular absorption of FITC-labeled insulin by 9.4 and 1.2-folds, when compared to insulin solution and unmodified NPs, respectively. The positively charged NPs increased intestinal villi adhesion and enhanced insulin absorption in the intestines of diabetic rat models. Furthermore, the hypoglycemic test showed that CDA NPs prolonged insulin release in vivo and exerted a remarkable hypoglycemic effect on diabetic rats with an oral bioavailability of 15%. In conclusion, CDA NPs is a potential oral insulin delivery system.

Unlike in normal cells, ursodeoxycholic acid (UDCA) causes apoptosis rather than protection in cancer cells. Aim of this study was to demonstrate whether UDCA actually inhibits proliferation and induces apoptosis in bile duct cancer cells; the effect of UDCA on the expression of COX-2, PI3K/AKT, ERK, and EGFR; how UDCA affects cancer cell invasiveness and metastasis, since these effects are not established in bile duct cancer cells. SNU-245 cells (human extrahepatic bile duct cancer cells) were cultured. MTT assays were performed to evaluate the effect of UDCA on the cell proliferation. A cell death detection enzyme-linked immunosorbent assay and a caspase-3 activity assay were used to determine apoptosis. Western blot analysis measured expression levels of various proteins. The invasiveness of the cancer cells was evaluated by invasion assay. In cultured bile duct cancer cells, UDCA suppressed cell proliferation in bile duct cancer cells by inducing apoptosis and p53 activation, blocking deoxycholic acid (DCA)-induced activated EGFR-ERK signaling and COX-2, inhibiting DCA-induced activated PI3K-AKT signaling, and suppressing the invasiveness of bile duct cancer cells. In addition, a MEK inhibitor impaired UDCA-induced apoptosis in bile duct cancer cells. UDCA has antineoplastic and apoptotic effects in bile duct cancer cells. Thus, UDCA could be a chemopreventive agent in patients with a high risk of cancer, and/or a therapeutic option that enhances other chemotherapeutics., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Deoxycholic acid (DCA, 3 α , 12 α -dihydroxy-5 β -cholan-24-oic acid) is the major circulating secondary bile acid, which is synthesized by gut flora in the lower gut and selectively oxidized by CYP3A into tertiary metabolites, including 1 β ,3 α ,12 α -trihydroxy-5 β -cholan-24-oic acid (DCA-1 β -ol) and 3 α ,5 β ,12 α -trihydroxy-5 β -cholan-24-oic acid (DCA-5 β -ol) in humans. Since DCA has the similar exogenous nature and disposition mechanisms as xenobiotics, this work aimed to investigate whether the tertiary oxidations of DCA are predictive of in vivo CYP3A activities in beagle dogs. In vitro metabolism of midazolam (MDZ) and DCA in recombinant canine CYP1A1, 1A2, 2B11, 2C21, 2C41, 2D15, 3A12, and 3A26 enzymes clarified that CYP3A12 was primarily responsible for either the oxidation elimination of MDZ or the regioselective oxidation metabolism of DCA into DCA-1 β -ol and DCA-5 β -ol in dog liver microsomes. Six male dogs completed the CYP3A intervention studies including phases of baseline, inhibition (ketoconazole treatments), recovery, and induction (rifampicin treatments). The oral MDZ clearance after a single dose was determined on the last day of the baseline, inhibition, and induction phases, and subjected to correlation analysis with the tertiary oxidation ratios of DCA detected in serum and urine samples. The results confirmed that the predosing serum ratios of DCA oxidation, DCA-5 β -ol/DCA, and DCA-1 β -ol/DCA were significantly and positively correlated both intraindividually and interindividually with oral MDZ clearance. It was therefore concluded that the tertiary oxidation of DCA is predictive of CYP3A activity in beagle dogs. Clinical transitional studies following the preclinical evidence are promising to provide novel biomarkers of the enterohepatic CYP3A activities. SIGNIFICANCE STATEMENT: Drug development, clinical pharmacology, and therapeutics are under insistent demands of endogenous CYP3A biomarkers that avoid unnecessary drug exposure and invasive sampling. This work has provided the first proof-of-concept preclinical evidence that the CYP3A catalyzed tertiary oxidation of deoxycholate, the major circulating secondary bile acid synthesized in the lower gut by bacteria, may be developed as novel in vivo biomarkers of the enterohepatic CYP3A activities., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

Golgi protein 73 (GP73) is upregulated in a variety of liver diseases, yet the detailed mechanism is poorly characterized. We analyzed GP73 in a retrospective cohort including 4211 patients with chronic liver disease (CLD) or hepatocellular carcinoma (HCC). The effect of deoxycholic acid (DCA) and nuclear factor-kappa B (NF-κB) on expression and release of GP73 in Huh-7 and SMMC7721 cells were studied. A mouse study was used to confirm our findings in vivo. A positive correlation was found between serum GP73 and total bile acid (TBA) in cirrhotic patients ( r = 0.540, p < 0.001), higher than that in non-cirrhotic CLD ( r = 0.318, p < 0.001) and HCC ( r = 0.353, p < 0.001) patients. In Huh-7 and SMMC7721 cells, DCA upregulated the expression and release of GP73 in a dose- and time-dependent manner. After overexpressing NF-κB p65, the promoter activity, GP73 messenger RNA (mRNA) level, and supernatant GP73 level were increased. The promotion effect of DCA on GP73 release was attenuated after inhibiting the NF-κB pathway. Mutating the binding sites of NF-κB in the sequence of the GP73 promoter led to a declined promoting effect of DCA on GP73. The upregulation role of DCA in GP73 expression through the NF-κB pathway was confirmed in vivo. In addition, exposure to DCA caused disassembly of Golgi apparatus. In summary, DCA upregulates the expression and release of GP73 via activating the NF-κB pathway and destroying the Golgi structure.

Aims: The progression of myocardial infarction (MI) involves multiple metabolic disorders. Bile acid metabolites have been increasingly recognized as pleiotropic signaling molecules that regulate multiple cardiovascular functions. G protein-coupled bile acid receptor (TGR5) is one of the receptors sensing bile acids to mediate their biological functions. In this study, we aimed to elucidate the effects of bile acids-TGR5 signaling pathways in myocardial infarction (MI)., Methods and Results: Blood samples of AMI patients or control subjects were collected and plasma was used for bile acid metabolism analysis. We discovered that bile acid levels were altered and deoxycholic acid (DCA) was substantially reduced in the plasma of AMI patients. Mice underwent either the LAD ligation model of MI or sham operation. Both MI and sham mice were gavaged with 10 mg/kg/d DCA or vehicle control since 3-day before the operation. Cardiac function was assessed by ultrasound echocardiography, infarct area was evaluated by TTC staining and Masson trichrome staining. Administration of DCA improved cardiac function and reduced ischemic injury at the 7th-day post-MI. The effects of DCA were dependent on binding to its receptor TGR5. Tgr5 - / - mice underwent the same MI model. Cardiac function deteriorated and infarct size was increased at the 7th-day post-MI, which were not savaged by DCA administration. Moreover, DCA inhibited interleukin (IL)-1β expression in the infarcted hearts, and ameliorated IL-1β activation at 1-day post-MI. DCA inhibited NF-κB signaling and further IL-1β expression in cultured neonatal mouse cardiomyocytes under hypoxia as well as cardio-fibroblasts with the treatment of LPS., Conclusions: DCA-TGR5 signaling pathway activation decreases inflammation and ameliorates heart function post-infarction. Strategies that control bile acid metabolism and TGR5 signaling to ameliorate the inflammatory responses may provide beneficial effects in patients with myocardial infarction., (Copyright © 2020. Published by Elsevier Ltd.)

We engineered the cytochrome P450 monooxygenase CYP107D1 (OleP) from Streptomyces antibioticus for the stereo- and regioselective 7β-hydroxylation of lithocholic acid (LCA) to yield ursodeoxycholic acid (UDCA). OleP was previously shown to hydroxylate testosterone at the 7β-position but LCA is exclusively hydroxylated at the 6β-position, forming murideoxycholic acid (MDCA). Structural and 3DM analysis, and molecular docking were used to identify amino acid residues F84, S240, and V291 as specificity-determining residues. Alanine scanning identified S240A as a UDCA-producing variant. A synthetic "small but smart" library based on these positions was screened using a colorimetric assay for UDCA. We identified a nearly perfectly regio- and stereoselective triple mutant (F84Q/S240A/V291G) that produces 10-fold higher levels of UDCA than the S240A variant. This biocatalyst opens up new possibilities for the environmentally friendly synthesis of UDCA from the biological waste product LCA., (© 2020 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

There is an increasing need to explore the mechanism of the progression of non-alcoholic fatty liver disease. Steroid metabolism is closely linked to hepatic steatosis and steroids are excreted as bile acids (BAs). Here, we demonstrated that feeding WKAH/HkmSlc inbred rats a diet supplemented with cholic acid (CA) at 0.5 g/kg for 13 weeks induced simple steatosis without obesity. Liver triglyceride and cholesterol levels were increased accompanied by mild elevation of aminotransferase activities. There were no signs of inflammation, insulin resistance, oxidative stress, or fibrosis. CA supplementation increased levels of CA and taurocholic acid (TCA) in enterohepatic circulation and deoxycholic acid (DCA) levels in cecum with an increased ratio of 12α-hydroxylated BAs to non-12α-hydroxylated BAs. Analyses of hepatic gene expression revealed no apparent feedback control of BA and cholesterol biosynthesis. CA feeding induced dysbiosis in cecal microbiota with enrichment of DCA producers, which underlines the increased cecal DCA levels. The mechanism of steatosis was increased expression of Srebp1 (positive regulator of liver lipogenesis) through activation of the liver X receptor by increased oxysterols in the CA-fed rats, especially 4β-hydroxycholesterol (4βOH) formed by upregulated expression of hepatic Cyp3a2, responsible for 4βOH formation. Multiple regression analyses identified portal TCA and cecal DCA as positive predictors for liver 4βOH levels. The possible mechanisms linking these predictors and upregulated expression of Cyp3a2 are discussed. Overall, our observations highlight the role of 12α-hydroxylated BAs in triggering liver lipogenesis and allow us to explore the mechanisms of hepatic steatosis onset, focusing on cholesterol and BA metabolism., 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 © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

In this study, a system for oral delivery of etoposide (ETP) was designed to avoid the problems associated with low and variable bioavailability of a commercially available ETP emulsion comprised of polyethylene glycol, glycerol, and citric acid anhydrous. ETP was complexed with low-molecular-weight methylcellulose (ETP/LMC) and loaded into a water-in-oil-in-water multiple nanoemulsion to formulate an ETP/LMC-nanoemulsion (ELNE). To further enhance the oral bioavailability, an ionic complex formed by anionic lipid 1,2-didecanoyl-sn-glycero-3-phosphate (sodium salt) and cationic N α -deoxycholyl-l-lysyl-methylester was incorporated into ELNE, yielding ELNE#7. As expected, ELNE#7 showed 4.07- and 2.25-fold increases in artificial membrane and Caco-2/HT29-MTX-E12 permeability ( P app ), respectively, resulting in 224% greater oral bioavailability compared with the commercially available ETP emulsion. In contrast, inhibition of clathrin- and caveola-mediated endocytosis, macropinocytosis, and bile acid transporters by chlorpromazine, genistein, amiloride, and actinomycin D in Caco-2/HT-29-MTX-E12 monolayers reduced the P app by 45.0%, 20.5%, 28.8%, and 31.1%, respectively. These findings suggest that these routes play important roles in enhancing the oral absorption of ELNE#7. In addition, our mechanistic study suggested that P-glycoprotein did not have an inhibitory effect on the permeation of ELNE#7. Notably, ELNE#7 showed significantly enhanced toxicity in LLC and A549 cells compared with ETP-E. These observations support the improved oral absorption of ETP in ELNE#7, suggesting that it is a better alternative than ETP emulsion.

High-fat diet (HFD) leads to systemic low-grade inflammation, which has been involved in the pathogenesis of diverse metabolic and inflammatory diseases. Colon is thought to be the first organ suffering from inflammation under HFD conditions due to the pro-inflammatory macrophages infiltration, however, the mechanisms concerning the induction of pro-inflammatory phenotype of colonic macrophages remains unclear. In this study, we show that HFD increased the percentage of gram-positive bacteria, especially genus Clostridium , and resulted in the significant increment of fecal deoxycholic acid (DCA), a gut microbial metabolite produced by bacteria mainly restricted to genus Clostridium . Notably, reducing gram-positive bacteria with vancomycin diminished fecal DCA and profoundly alleviated pro-inflammatory macrophage infiltration in colon, whereas DCA-supplemented feedings to vancomycin-treated mice provoked obvious pro-inflammatory macrophage infiltration and colonic inflammation. Meanwhile, intra-peritoneal administration of DCA also elicited considerable recruitment of macrophages with pro-inflammatory phenotype. Mechanistically, DCA dose-dependently promoted M1 macrophage polarization and pro-inflammatory cytokines production at least partially through toll-like receptor 2 (TLR2) transactivated by M2 muscarinic acetylcholine receptor (M2-mAchR)/Src pathway. In addition, M2-mAchR mediated increase of TLR2 transcription was mainly achieved via targeting AP-1 transcription factor. Moreover, NF-κB/ERK/JNK signalings downstream of TLR2 are involved in the DCA-induced macrophage polarization. In conclusion, our findings revealed that high level DCA induced by HFD may serve as an initiator to activate macrophages and drive colonic inflammation, thus offer a mechanistic basis that modulation of gut microbiota or intervening specific bile acid receptor signaling could be potential therapeutic approaches for HFD-related inflammatory diseases.

Residual host cell proteins (HCPs) present in biotherapeutics can pose potential safety risks for patients or affect product stability, thus prompting a critical need to monitor HCPs in drug substance or product to ensure product safety and quality. Current approaches for robust HCP identification at or above 10 ppm levels require either concatenated peptide fractionation or enrichment via antibody depletion, which challenges the direct quantitation of HCPs. This paper describes a simple, fast sample preparation method without the need for sample fractionation or enrichment; instead, we utilize trypsin-friendly sodium deoxycholate (SDC) as an advantageous denaturant that can be effectively removed following acidification at the end of sample digestion. This new approach enables the end-to-end one-dimensional liquid chromatography-tandem mass spectrometry (1D LC-MS/MS) workflow (i.e., from sample preparation to HCP identification) to be completed in 7-8 h while demonstrating the ability to consistently identify HCPs across a broad molecular weight range at 10 ppm or above.

Background: Elobixibat, a novel inhibitor of apical sodium-dependent bile acid transporter for treating chronic constipation, increases colonic bile acid concentrations, stimulating bowel function. However, it is not clear which bile acids are altered, or whether altered gut microbiota are associated with functional effects that may alter bowel function., Aims: To investigate the effects of elobixibat on changes in the faecal concentrations of total and individual bile acids and in faecal microbiota., Methods: This was a prospective, single-centre study. After baseline period, patients received 10 mg daily of elobixibat for 2 weeks. We evaluated the effects on bowel function, changes in faecal bile acid concentrations and composition of gut bacteria, before and after elobixibat administration., Results: In the 30 patients analysed, the frequency of pre- and post-treatment bowel movements per fortnight was 7 and 10 (P < 0.001), respectively. The pre-treatment faecal bile acid concentration increased significantly from 10.9 to 15.0 µg/g stool post-treatment (P = 0.030), with a significant increase in faecal deoxycholic acid (pre-treatment 3.94 µg/g stool to post-treatment 5.02 µg/g stool, P = 0.036) and in glycine-conjugated deoxycholic and chenodeoxycholic acids. Shannon index was significantly decreased, but there were no significant changes at the genus and phylum levels., Conclusions: Short term treatment with elobixibat increased the concentrations of total bile acids and deoxycholic acid and decreased the diversity of faecal microbiota. The biological effects of elobixibat are associated with its effects on secretory bile acids, rather than the structural changes of an altered faecal microbiota., (© 2020 John Wiley & Sons Ltd.)

In the Streptococcus pneumoniae, the N-acetylmuramoyl-l-alanine amidase known as LytA protein is a main autolysin and in the presence of sodium deoxycholate, it activates and breaks S. pneumoniae cell wall. In the present study, the interaction between the LytA protein and deoxycholate as ligand was investigated. The Lyt A protein was retrieved from PDB databank and energetically minimized by Molegro Virtual Docker. The binding sites of LytA protein were detected and molecular docking carried out using MolDock algorithm. Finally, the number of hydrogen and electrostatic bonds were obtained for each predicted pose. A total of 5 binding sites predicted on LytA protein. The number of 5 predicted poses for each binding site also detected and molecular docking showed that all the poses have interactions (by H bonds) with deoxycholate. The interaction of the LytA protein with the deoxycholate ligand reveal five binding sites, which are involved in deoxycholate substrate recognition.

Objective: Recent evidence points to the gut microbiome's involvement in postoperative outcomes, including after gastrectomy. Here, we investigated the influence of gastrectomy for gastric cancer on the gut microbiome and metabolome, and how it related to postgastrectomy conditions., Design: We performed shotgun metagenomics sequencing and capillary electrophoresis time-of-flight mass spectrometry-based metabolomics analyses on faecal samples collected from participants with a history of gastrectomy for gastric cancer (n=50) and compared them with control participants (n=56)., Results: The gut microbiota in the gastrectomy group showed higher species diversity and richness (p<0.05), together with greater abundance of aerobes, facultative anaerobes and oral microbes. Moreover, bile acids such as genotoxic deoxycholic acid and branched-chain amino acids were differentially abundant between the two groups (linear discriminant analysis (LDA) effect size (LEfSe): p<0.05, q<0.1, LDA>2.0), as were also Kyoto Encyclopedia of Genes and Genomes modules involved in nutrient transport and organic compounds biosynthesis (LEfSe: p<0.05, q<0.1, LDA>2.0)., Conclusion: Our results reveal alterations of gut microbiota after gastrectomy, suggesting its association with postoperative comorbidities. The multi-omic approach applied in this study could complement the follow-up of patients after gastrectomy., Competing Interests: Competing interests: SF and TY are founders of Metabologenomics. The company is focused on the design and control of the gut environment for human health. The company had no control over the interpretation, writing or publication of this work. The terms of these arrangements are being managed by Keio University and Tokyo Institute of Technology in accordance with its conflict of interest policies., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The gut microbiota synthesize hundreds of molecules, many of which influence host physiology. Among the most abundant metabolites are the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA), which accumulate at concentrations of around 500 μM and are known to block the growth of Clostridium difficile 1 , promote hepatocellular carcinoma 2 and modulate host metabolism via the G-protein-coupled receptor TGR5 (ref. 3 ). More broadly, DCA, LCA and their derivatives are major components of the recirculating pool of bile acids 4 ; the size and composition of this pool are a target of therapies for primary biliary cholangitis and nonalcoholic steatohepatitis. Nonetheless, despite the clear impact of DCA and LCA on host physiology, an incomplete knowledge of their biosynthetic genes and a lack of genetic tools to enable modification of their native microbial producers limit our ability to modulate secondary bile acid levels in the host. Here we complete the pathway to DCA and LCA by assigning and characterizing enzymes for each of the steps in its reductive arm, revealing a strategy in which the A-B rings of the steroid core are transiently converted into an electron acceptor for two reductive steps carried out by Fe-S flavoenzymes. Using anaerobic in vitro reconstitution, we establish that a set of six enzymes is necessary and sufficient for the eight-step conversion of cholic acid to DCA. We then engineer the pathway into Clostridium sporogenes, conferring production of DCA and LCA on a nonproducing commensal and demonstrating that a microbiome-derived pathway can be expressed and controlled heterologously. These data establish a complete pathway to two central components of the bile acid pool.

Bile acids (BAs), well-defined signaling molecules with diverse metabolic functions, play important roles in cellular processes associated with many cancers. As one of the most common BAs, deoxycholic acid (DCA) is originally synthesized in the liver, stored in the gallbladder, and processed in the gut. DCA plays crucial roles in various tumors; however, functions and molecular mechanisms of DCA in gallbladder cancer (GBC) still remain poorly characterized. Here, we analyzed human GBC samples and found that DCA was significantly downregulated in GBC, and reduced levels of DCA was associated with poor clinical outcome in patients with GBC. DCA treatment impeded tumor progression by halting cell proliferation. DCA decreased miR-92b-3p expression in an m 6 A-dependent posttranscriptional modification manner by facilitating dissociation of METTL3 from METTL3-METTL14-WTAP complex, which increased the protein level of the phosphatase and tensin homolog, a newly identified target of miR-92b-3p, and subsequently inactivated the PI3K/AKT signaling pathway. Our findings revealed that DCA might function as a tumor suppressive factor in GBC at least by interfering with miR-92b-3p maturation, and suggested that DCA treatment could provide a new therapeutic strategy for GBC.

The gut contains an enormous diversity of simple as well as complex molecules from highly diverse food sources, together with host-secreted molecules. This presents a large metabolic opportunity for the gut microbiota, but little is known about how gut microbes are able to catabolize this large chemical diversity. Recently, Fe-S flavoenzymes were found to be key in the transformation of bile acids, catalysing the key step in the 7α-dehydroxylation pathway that allows gut bacteria to transform cholic acid into deoxycholic acid, an exclusively microbe-derived molecule with major implications for human health. While this enzyme family has also been implicated in a limited number of other catalytic transformations, little is known about the extent to which it is of more global importance in gut microbial metabolism. Here, we perform a large-scale computational genomic analysis to show that this enzyme superfamily has undergone a remarkable expansion in Clostridiales, and occurs throughout a diverse array of >1000 different families of putative metabolic gene clusters. Analysis of the enzyme content of these gene clusters suggests that they encode pathways with a wide range of predicted substrate classes, including saccharides, amino acids/peptides and lipids. Altogether, these results indicate a potentially important role of this protein superfamily in the human gut, and our dataset provides significant opportunities for the discovery of novel pathways that may have significant effects on human health.

Objective: Regio- and stereoselective hydroxylation of lithocholic acid (LCA) using CYP107D1 (OleP), a cytochrome P450 monooxygenase from the oleandomycin synthesis pathway of Streptomyces antibioticus., Results: Co-expression of CYP107D1 from S. antibioticus and the reductase/ferredoxin system PdR/PdX from Pseudomonas putida was performed in Escherichia coli whole cells. In vivo hydroxylation of LCA exclusively yielded the 6β-OH product murideoxycholic acid (MDCA). In resting cells, 19.5% of LCA was converted to MDCA within 24 h, resulting in a space time yield of 0.04 mmol L -1 h -1 . NMR spectroscopy confirmed the identity of MDCA as the sole product., Conclusions: The multifunctional P450 monooxygenase CYP107D1 (OleP) can hydroxylate LCA, forming MDCA as the only product.

Bile acids (BAs) are bioactive molecules that have potential therapeutic interest and their derived salts are used in several pharmaceutical systems. BAs have been associated with tumorigenesis of several tissues including the mammary tissue. Therefore, it is crucial to characterize their effects on cancer cells. The objective of this work was to analyse the molecular and cellular effects of the bile salts sodium cholate and sodium deoxycholate on epithelial breast cancer cell lines. Bile salts (BSs) effects over breast cancer cells viability and proliferation were assessed by MTS and BrdU assays, respectively. Activation of cell signaling mediators was determined by immunobloting. Microscopy was used to analyze cell migration, and cellular and nuclear morphology. Interference of membrane fluidity was studied by generalized polarization and fluorescence anisotropy. BSs preparations were characterized by transmission electron microscopy and dynamic light scattering. Sodium cholate and sodium deoxycholate had dual effects on cell viability, increasing it at the lower concentrations assessed and decreasing it at the highest ones. The increase of cell viability was associated with the promotion of AKT phosphorylation and cyclin D1 expression. High concentrations of bile salts induced apoptosis as well as sustained activation of p38 and AKT. In addition, they affected cell membrane fluidity but not significant effects on cell migration were observed. In conclusion, bile salts have concentration-dependent effects on breast cancer cells, promoting cell proliferation at physiological levels and being cytotoxic at supraphysiological ones. Their effects were associated with the activation of kinases involved in cell signalling.

Campylobacter is a recommended reference pathogen for the verification and validation of water recycling schemes in Australia and globally. In a larger study investigating the efficacy of pathogen removal in waste stabilization ponds (WSP), we cultivated bacteria from wastewater samples on modified charcoal-cefoperazone-deoxycholate agar (mCCDA) targeting the growth of Campylobacter. A high number of colonies characteristic of Campylobacter grew on this selective medium, but this did not correlate with qPCR data. Using primers targeting the 16S rRNA gene, and additional confirmatory tests to detect VS1, ompA , bla OXA-51-like , bla OXA-23-like genes, we tested 80 random colonies from 10 WSP samples. All 80 were identified as Acinetobacter baumannii. Wastewater grab samples taken three times over 6 months throughout the WSP system showed removal of A. baumannii in the WSP at rates similar to that of Escherichia coli . Our study suggests that mCCDA agar is not a suitable medium for isolating Campylobacter from environmental samples and that A. baumannii can be used as an indicator for removal of pathogens in WSPs.

This work presents the outcomes of a comparative study of molecular interactions of polymyxin B (PMB) and F12 and F13 formulations in the mole ratios of 1:2 and 1:3 of PMB:sodium deoxycholate sulfate (SDCS), respectively, and a commercial PMB formulation (CPMB) with lipopolysaccharides (LPS). Several spectroscopic and interfacial studies were performed to obtain LPS-peptide interactions at a molecular level. The fluorescence titrimetry method revealed that the F12 formulation (325 nM) exhibited a lower number of binding sites to the LPS compared to CPMB and F13 as well as PMB alone (537 nM). Similarly, in the presence of LPS, the F12 formulation (88 nm) exhibited smaller particle sizes in the dynamic light scattering study compared to PMB (116 nm), CPMB, and the F13 formulation. An interfacial study and circular dichroism spectroscopy revealed PMB and CPMB insertion into the LPS micelles to destabilize and disrupt the LPS membrane, whereas the F12 and F13 formulations may induce pseudo-aggregation. The NMR and IR studies showed that the presence of SDCS, the hydrophobicity of PMB increased by hydrogen bonding and electrostatic interactions and formed stabilized PMB-SDCS micelles. The PMB-SDCS formulation is likely to release PMB for easy penetration into the lipid membrane and cause disruption of the complex LPS micelles. Furthermore, the PMB-SDCS formulations neutralized and detoxified the LPS micelles with minimal toxicity to normal kidney tubular cells as well as an immortalised kidney cell line. The antimicrobial properties of PMBloaded SDCS nanomicelles were effective against a resistant strain of Pseudomonas aeruginosa., (Copyright © 2019 Elsevier B.V. All rights reserved.)

At the post-synaptic density (PSD), large protein complexes dynamically form and dissociate in response to synaptic activity, comprising the biophysical basis for learning and memory. The use of detergents to isolate the PSD and release its membrane-associated proteins complicates studies of these activity-dependent protein interaction networks, because detergents can simultaneously disrupt the very interactions under study. Despite widespread recognition that different detergents yield different experimental results, the effect of detergent on activity-dependent synaptic protein complexes has not been rigorously examined. Here, we characterize the effect of three detergents commonly used to study synaptic proteins on activity-dependent protein interactions. We first demonstrate that SynGAP-containing interactions are more abundant in 1% Deoxycholate (DOC), while Shank-, Homer- and mGluR5-containing interactions are more abundant in 1% NP-40 or Triton. All interactions were detected preferentially in high molecular weight complexes generated by size exclusion chromatography, although the detergent-specific abundance of proteins in high molecular weight fractions did not correlate with the abundance of detected interactions. Activity-dependent changes in protein complexes were consistent across detergent types, suggesting that detergents do not isolate distinct protein pools with unique behaviors. However, detection of activity-dependent changes is more or less feasible in different detergents due to baseline solubility. Collectively, our results demonstrate that detergents affect the solubility of individual proteins, but activity-dependent changes in protein interactions, when detectable, are consistent across detergent types.

Despite reducing the prevalent foodborne pathogen Campylobacter jejuni in chickens decreases campylobacteriosis, few effective approaches are available. The aim of this study was to use microbial metabolic product bile acids to reduce C. jejuni chicken colonization. Broiler chicks were fed with deoxycholic acid (DCA), lithocholic acid (LCA), or ursodeoxycholic acid (UDCA). The birds were also transplanted with DCA modulated anaerobes (DCA-Anaero) or aerobes (DCA-Aero). The birds were infected with human clinical isolate C. jejuni 81-176 or chicken isolate C. jejuni AR101. Notably, C. jejuni 81-176 was readily colonized intestinal tract at d16 and reached an almost plateau at d21. Remarkably, DCA excluded C. jejuni cecal colonization below the limit of detection at 16 and 28 days of age. Neither chicken ages of infection nor LCA or UDCA altered C. jejuni AR101 chicken colonization level, while DCA reduced 91% of the bacterium in chickens at d28. Notably, DCA diet reduced phylum Firmicutes but increased Bacteroidetes compared to infected control birds. Importantly, DCA-Anaero attenuated 93% of C. jejuni colonization at d28 compared to control infected birds. In conclusion, DCA shapes microbiota composition against C. jejuni colonization in chickens, suggesting a bidirectional interaction between microbiota and microbial metabolites., Competing Interests: The authors have declared that no competing interests exist.

The extent of human intestinal absorption (HIA) for a drug is considered to be an important pharmacokinetic parameter which must be determined for orally administered drugs. Traditional experimental methods relied upon animal testing and are renowned for being time consuming and expensive as well as being ethically unfavourable. As a result, the development of alternative methods to evaluate a drug's pharmacokinetics is crucial. Micellar liquid chromatography is considered to be one of these methods that can replace the use of animals in the prediction of HIA. In this study, the combination of an aminopropyl column with the biosurfactant sodium deoxycholate bile salt was used in the experimental determination of micelle-water partition coefficients (log P mw ) for a group of compounds. Multiple linear regression was then used for the prediction of HIA using the experimentally determined log P mw along with other molecular descriptors, leading to the construction of a model equation of R 2  = 85% and a prediction power represented by R 2 Pred. = 72%. The use of micellar liquid chromatography with an aminopropyl column in combination with sodium deoxycholate was found to be a good method for the prediction of human intestinal absorption, providing data for a far wider range of compounds compared with previous studies., (© 2019 John Wiley & Sons, Ltd.)

Spectral-fluorescent properties of polymethine dye probes anionic 3,3'-di(sulfopropyl)-4,5,4',5'-dibenzo-9-ethylthiacarbocyanine-betaine (DEC) and cationic 3,3',9-trimethylthiacarbocyanine iodide (Cyan 2) in the presence of biological surfactants, bile salts sodium cholate (NaC), sodium deoxycholate (NaDC) and sodium taurocholate (NaTC), as well as sodium dodecyl sulfate (SDS), have been studied in a wide range of surfactant concentrations. When a surfactant is introduced into a solution of DEC, changes of the spectral-fluorescent properties are observed due to decomposition of dye dimers into cis-monomers and cis-trans conversion of the resulting monomers. In the presence of SDS, both processes occur in parallel, caused by noncovalent interaction of dye monomers with micelles, and mainly occur near the critical micelle concentration (CMC). In contrast, upon the introduction of increasing concentrations of bile salts, decomposition of dye dimers into the monomers begins at lower concentrations than cis-trans conversion. The former process is almost completed at concentrations close to CMC of secondary micelles (CMC2), while the latter process occurs even at concentrations of bile salts much higher than CMC2. Hence, DEC can serve as a probe that permits estimating the value of CMC2 and is indicative of reorganization of secondary micelles upon an increase in bile salt concentration. Aggregation of DEC and Cyan 2 on bile salts is also observed. Since it is observed at relatively low concentrations of bile salts (

The primary bile acids (BAs) synthesized from cholesterol in the liver are converted to secondary BAs by gut microbiota. It was recently disclosed that the major secondary BA, deoxycholate (DCA) species, is stereoselectively oxidized to tertiary BAs exclusively by CYP3A enzymes. This work subsequently investigated the in vitro oxidation kinetics of DCA at C-1 β , C-3 β , C-4 β , C-5 β , C-6 α , C-6 β , and C-19 in recombinant CYP3A enzymes and naive enzymes in human liver microsomes (HLMs). The stereoselective oxidation of DCA fit well with Hill kinetics at 1-300 μ M in both recombinant CYP3A enzymes and pooled HLMs. With no contributions or trace contributions from CYP3A5, CYP3A7 favors oxidation at C-19, C-4 β , C-6 α , C-3 β , and C-1 β , whereas CYP3A4 favors the oxidation at C-5 β and C-6 β compared with each other. Correlation between DCA oxidation and testosterone 6 β -hydroxylation in 14 adult single-donor HLMs provided proof-of-concept evidence that DCA 19-hydroxylation is an in vitro marker reaction for CYP3A7 activity, whereas oxidation at other sites represents mixed indicators for CYP3A4 and CYP3A7 activities. Deactivation caused by DCA-induced cytochrome P450-cytochrome P420 conversion, as shown by the spectral titrations of isolated CYP3A proteins, was observed when DCA levels were near or higher than the critical micelle concentration (about 1500 μ M). Unlike CYP3A4, CYP3A7 showed abnormally elevated activities at 500 and 750 μ M, which might be associated with an altered affinity for DCA multimers. The disclosed kinetic and functional roles of CYP3A isoforms in disposing of the gut bacteria-derived DCA may help in understanding the structural and functional mechanisms of CYP3A., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

Bile acids and microbiota differ significantly in the gut of children and adults. In the first 3 yr of life, intestinal bile consists mostly of two primary bile acids, cholic acid (CA) and chenodeoxycholic acid (CDCA); however, in adults, primary bile acids are transformed into the secondary bile acids, deoxycholic acid (DCA) and lithocholic acid. This difference has a major impact on the gut microbiome, especially on anaerobic spore-forming bacteria. CA augments germination of spores in the terminal ileum. On the other hand, DCA curtails the number of germinated anaerobes entering the cecum from the terminal ileum. The control mechanism that exists in the adult cecum is absent in the young child and results in unrestrained proliferation of anaerobes, such as Clostridium difficile , in the cecum. A similar situation may develop during antibiotic therapy when an antibiotic eradicates the anaerobic population capable of converting primary bile acids into secondary bile acids.

Clostridium difficile is a major cause of nosocomial infections. Bacterial persistence in the gut is responsible for infection relapse; sporulation and other unidentified mechanisms contribute to this process. Intestinal bile salts cholate and deoxycholate stimulate spore germination, while deoxycholate kills vegetative cells. Here, we report that sub-lethal concentrations of deoxycholate stimulate biofilm formation, which protects C . difficile from antimicrobial compounds. The biofilm matrix is composed of extracellular DNA and proteinaceous factors that promote biofilm stability. Transcriptomic analysis indicates that deoxycholate induces metabolic pathways and cell envelope reorganization, and represses toxin and spore production. In support of the transcriptomic analysis, we show that global metabolic regulators and an uncharacterized lipoprotein contribute to deoxycholate-induced biofilm formation. Finally, Clostridium scindens enhances biofilm formation of C. difficile by converting cholate into deoxycholate. Together, our results suggest that deoxycholate is an intestinal signal that induces C. difficile persistence and may increase the risk of relapse., Competing Interests: The authors declare no competing interests.

Liver fibrosis is a serious liver disease associated with high morbidity and mortality. The activation of hepatic stellate cells (HSCs) and the overproduction of extracellular matrix proteins are key features during disease progression. In this work, chondroitin sulfate nanomicelles (CSmicelles) were developed as a delivery system targeting HSCs for the treatment of liver fibrosis. CS-deoxycholic acid conjugates (CS-DOCA) were synthesized via amide bond formation. Next, retinoic acid (RA) and doxorubicin (DOX) were encapsulated into CSmicells to afford a DOX+RA-CSmicelles codelivery system. CSmicelles were selectively taken up in activated HSCs and hepatoma (HepG2) cells other than in normal hepatocytes (LO2), the internalization of which was proven to be mediated by CD44 receptors. Interestingly, DOX+RA-CSmicelles preferentially accumulated in the Golgi apparatus, destroyed the Golgi structure, and ultimately downregulated collagen I production. Following tail-vein injection, DOX+RA-CSmicelles were delivered to the cirrhotic liver and showed synergistic antifibrosis effects in the CCl 4 -induced fibrotic rat model. Further, immunofluorescence staining of dissected liver tissues revealed CD44-specific delivery of CS derivatives to activated HSCs. Together, our results demonstrate the great potential of CS based carrier systems for the targeted treatment of chronic liver diseases.

In the human gut, Clostridium scindens ATCC 35704 is a predominant bacterium and one of the major bile acid 7α-dehydroxylating anaerobes. While this organism is well-studied relative to bile acid metabolism, little is known about the basic nutrition and physiology of C. scindens ATCC 35704. To determine the amino acid and vitamin requirements of C. scindens , the leave-one-out (one amino acid group or vitamin) technique was used to eliminate the nonessential amino acids and vitamins. With this approach, the amino acid tryptophan and three vitamins (riboflavin, pantothenate, and pyridoxal) were found to be required for the growth of C. scindens In the newly developed defined medium, C. scindens fermented glucose mainly to ethanol, acetate, formate, and H 2. The genome of C. scindens ATCC 35704 was completed through PacBio sequencing. Pathway analysis of the genome sequence coupled with transcriptome sequencing (RNA-Seq) under defined culture conditions revealed consistency with the growth requirements and end products of glucose metabolism. Induction with bile acids revealed complex and differential responses to cholic acid and deoxycholic acid, including the expression of potentially novel bile acid-inducible genes involved in cholic acid metabolism. Responses to toxic deoxycholic acid included expression of genes predicted to be involved in DNA repair, oxidative stress, cell wall maintenance/metabolism, chaperone synthesis, and downregulation of one-third of the genome. These analyses provide valuable insight into the overall biology of C. scindens which may be important in treatment of disease associated with increased colonic secondary bile acids. IMPORTANCE C. scindens is one of a few identified gut bacterial species capable of converting host cholic acid into disease-associated secondary bile acids such as deoxycholic acid. The current work represents an important advance in understanding the nutritional requirements and response to bile acids of the medically important human gut bacterium, C. scindens ATCC 35704. A defined medium has been developed which will further the understanding of bile acid metabolism in the context of growth substrates, cofactors, and other metabolites in the vertebrate gut. Analysis of the complete genome supports the nutritional requirements reported here. Genome-wide transcriptomic analysis of gene expression in the presence of cholic acid and deoxycholic acid provides a unique insight into the complex response of C. scindens ATCC 35704 to primary and secondary bile acids. Also revealed are genes with the potential to function in bile acid transport and metabolism., (Copyright © 2019 American Society for Microbiology.)

The gut microbiota modifies endogenous primary bile acids (BAs) to produce exogenous secondary BAs, which may be further metabolized by cytochrome P450 enzymes (P450s). Our primary aim was to examine how the host adapts to the stress of microbe-derived secondary BAs by P450-mediated oxidative modifications on the steroid nucleus. Five unconjugated tri-hydroxyl BAs that were structurally and/or biologically associated with deoxycholate (DCA) were determined in human biologic samples by liquid chromatography-tandem mass spectrometry in combination with enzyme-digestion techniques. They were identified as DCA-19-ol, DCA-6 β -ol, DCA-5 β -ol, DCA-6 α -ol, DCA-1 β -ol, and DCA-4 β -ol based on matching in-laboratory synthesized standards. Metabolic inhibition assays in human liver microsomes and recombinant P450 assays revealed that CYP3A4 and CYP3A7 were responsible for the regioselective oxidations of both DCA and its conjugated forms, glycodeoxycholate (GDCA) and taurodeoxycholate (TDCA). The modification of secondary BAs to tertiary BAs defines a host liver (primary BAs)-gut microbiota (secondary BAs)-host liver (tertiary BAs) axis. The regioselective oxidations of DCA, GDCA, and TDCA by CYP3A4 and CYP3A7 may help eliminate host-toxic DCA species. The 19- and 4 β -hydroxylation of DCA species demonstrated outstanding CYP3A7 selectivity and may be useful as indicators of CYP3A7 activity., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

Increased levels of intestinal bile acids (BAs) are a risk factor for colorectal cancer (CRC). Here, we show that the convergence of dietary factors (high-fat diet) and dysregulated WNT signaling (APC mutation) alters BA profiles to drive malignant transformations in Lgr5-expressing (Lgr5 + ) cancer stem cells and promote an adenoma-to-adenocarcinoma progression. Mechanistically, we show that BAs that antagonize intestinal farnesoid X receptor (FXR) function, including tauro-β-muricholic acid (T-βMCA) and deoxycholic acid (DCA), induce proliferation and DNA damage in Lgr5 + cells. Conversely, selective activation of intestinal FXR can restrict abnormal Lgr5 + cell growth and curtail CRC progression. This unexpected role for FXR in coordinating intestinal self-renewal with BA levels implicates FXR as a potential therapeutic target for CRC., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The human gut hosts trillions of microorganisms that exert a profound influence on human biology. Gut bacteria communicate with their host by secreting small molecules that can signal to distant organs in the body. Bile acids are one class of these signaling molecules, synthesized by the host and chemically transformed by the gut microbiota. Among bile acid metabolizers, bile acid 7-dehydroxylating bacteria are commensals of particular importance as they carry out the 7-dehydroxylation of liver-derived primary bile acids to 7-dehydroxylated bile acids. The latter represents a major fraction of the secondary bile acid pool. The microbiology of this group of gut microorganisms is understudied and warrants more attention. Here, we detail the bile acid transformations carried out by the 7-dehydroxylating bacterium Clostridium scindens in vitro and in vivo. In vitro, C. scindens exhibits not only 7α-dehydroxylating capabilities but also, the ability to oxidize other hydroxyl groups and reduce ketone groups in primary and secondary bile acids. This study revealed 12-oxolithocholic acid as a major transient product in the 7α-dehydroxylation of cholic acid. Furthermore, the in vivo study included complementing a gnotobiotic mouse line (devoid of the ability to 7-dehydroxylate bile acids) with C. scindens and investigating its colonization dynamics and bile acid transformations. Using NanoSIMS (Nanoscale Secondary Ion Mass Spectrometry), we demonstrate that the large intestine constitutes a niche for C. scindens , where it efficiently 7-dehydroxylates cholic acid to deoxycholic acid. Overall, this work reveals a novel transient species during 7-dehydroxylation as well as provides direct evidence for the colonization and growth of 7-dehydroxylating bacteria in the large intestine.