Infection with hepatitis delta virus (HDV) is one of the most severe hepatitis B virus (HBV) complications, with a more rapid progression to cirrhosis and an increased risk of hepatic decompensation and death. Data on HDV infection in Cuba are limited. The aims of our study were to determine the HDV prevalence in HBsAg carriers and to characterize the HDV strains circulating. The data were used to assess the possibility of HDV elimination in the Cuban HBV epidemiological setting.Five hundred and two serum samples from the same number of HBsAg carriers collected in the period 2006-2019 from all over the country were tested for anti-HDV total antibodies. If positive, the samples were analyzed for HDV-RNA using Real-Time RT-PCR targeting the ribozyme and HD antigen domains followed by genotyping based on phylogenetic analysis.Two samples were anti-HDV positive [0.39% (95% CI 0.11-1.44)]. One of them was also HDV-RNA positive. Clinically, the patient with active HDV infection had compensated liver cirrhosis. Phylogenetic analysis showed that the virus belonged to genotype 1 and thus clustered with contemporary strains from North America, Europe, Middle East, and Asia.This is the first HDV study, including molecular detection and virus characterization, done after the introduction of the universal childhood anti-hepatitis B vaccination. The very low prevalence of HDV infection in HBsAg carriers combined with the high HBV vaccination coverage of all newborn children, of previously identified risk groups, and of the general population currently under 40 years of age suggests that HDV elimination is feasible in Cuba if the success in HBV control is maintained.
Hepatitis B virus (HBV) infection remains a public health problem worldwide. Persistent HBV infection relies on active transcription of the covalently closed circular DNA (cccDNA) in hepatocytes, which is less understood at the single-cell level. In this study, we isolated primary human hepatocytes from liver-humanized FRG mice infected with HBV and examined cccDNA transcripts in single cells based on 5' end sequencing. Our 5' transcriptome sequencing (RNA-seq) analysis unambiguously assigns different viral transcripts with overlapping 3' sequences and quantitatively measures viral transcripts for structural genes (3.5 kb, 2.4 kb, and 2.1 kb) and the nonstructural X gene (0.7 kb and related) in single cells. We found that an infected cell either can generate all viral transcripts, signifying active transcription, or presents only transcripts from the X gene and its associated enhancer I domain and no structural gene transcripts. Results from cell infection assays with recombinant HBV show that nonproductive transcription of cccDNA can be activated by incoming virus through superinfection. Moreover, upon HBV infection, cccDNA apparently can be transcribed in the absence of HBx and produces HBx, needed for productive transcription of other viral genes. These results shed new light on cccDNA transcription at the single-cell level and provide insights useful for improving the treatment strategy against chronic HBV infection. IMPORTANCE Hepatitis B virus (HBV) infection can be effectively suppressed but rarely cured by available drugs. Chronic HBV infection is based on persistence of covalently closed circular DNA (cccDNA) and continuous infection and reinfection with HBV in the liver. Understanding transcriptional regulation of cccDNA will help to achieve permanent transcriptional silencing, i.e., functional cure of HBV. In our study, we found that an infected cell either can generate all viral transcripts, signifying active transcription, or presents only transcripts from the X gene and its associated enhancer I domain and no structural gene transcripts. The nonproductive transcription of cccDNA can be activated by incoming virus through superinfection. Upon an infection, cccDNA apparently can be transcribed in the absence of HBx to produce HBx, necessary for subsequent transcription of other HBV genes. Our studies shed new light on the mechanism of HBV infection and may have implications for a functional cure regimen for HBV.
Gene expression changes over the lifespan and varies among different tissues or cell types. Gene co-expression also changes by sex, age, different tissues or cell types. However, gene expression under the normal state and gene co-expression in the human brain has not been fully defined and quantified. Here we present a database named Brain EXPression Database (BrainEXP) which provides spatiotemporal expression of individual genes and co-expression in normal human brains. BrainEXP consists of 4567 samples from 2863 healthy individuals gathered from existing public databases and our own data, in either microarray or RNA-Seq library types. We mainly provide two analysis results based on the large dataset: (i) basic gene expression across specific brain regions, age ranges and sexes; (ii) co-expression analysis from different platforms.http://www.brainexp.org/.Supplementary data are available at Bioinformatics online.
Sodium taurocholate co-transporting polypeptide (NTCP, encoded by SLC10A1) is a bile acid transporter that also is known to serve as an entry receptor for both the hepatitis B virus and the hepatitis D virus.1Trauner M. Boyer J.L. Bile salt transporters: molecular characterization, function, and regulation.Physiol Rev. 2003; 83: 633-671Crossref PubMed Scopus (764) Google Scholar,2Yan H. Zhong G. Xu G. He W. Jing Z. Gao Z. Huang Y. Qi Y. Peng B. Wang H. Fu L. Song M. Chen P. Gao W. Ren B. Sun Y. Cai T. Feng X. Sui J. Li W. Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus.eLife. 2012; 1e00049Crossref PubMed Scopus (1241) Google Scholar The first reported NTCP knockout (Slc10a1-/-) mice showed a hypercholanemia phenotype to varying degrees (ie, increased serum bile acid concentrations), but did not show obvious liver injury or other anatomic abnormalities.3Slijepcevic D. Kaufman C. Wichers C.G. Gilglioni E.H. Lempp F.A. Duijst S. de Waart D.R. Oude Elferink R.P.J. Mier W. Stieger B. Beuers U. Urban S. van de Graaf S.F.J. Impaired uptake of conjugated bile acids and hepatitis b virus pres1-binding in na(+) -taurocholate cotransporting polypeptide knockout mice.Hepatology. 2015; 62: 207-219Crossref PubMed Scopus (87) Google Scholar In human beings, genetic variation in NTCP causes hypercholanemia in neonates, but their total bile acid (TBA) levels tend to decrease as age increases.3Slijepcevic D. Kaufman C. Wichers C.G. Gilglioni E.H. Lempp F.A. Duijst S. de Waart D.R. Oude Elferink R.P.J. Mier W. Stieger B. Beuers U. Urban S. van de Graaf S.F.J. Impaired uptake of conjugated bile acids and hepatitis b virus pres1-binding in na(+) -taurocholate cotransporting polypeptide knockout mice.Hepatology. 2015; 62: 207-219Crossref PubMed Scopus (87) Google Scholar, 4Deng M. Mao M. Guo L. Chen F.-P. Wen W.-R. Song Y.-Z. Clinical and molecular study of a pediatric patient with sodium taurocholate cotransporting polypeptide deficiency.Exp Ther Med. 2016; 12: 3294-3300Crossref PubMed Scopus (29) Google Scholar, 5Mao F. Liu T. Hou X. Zhao H. He W. Li C. Jing Z. Sui J. Wang F. Liu X. Han J. Borchers C.H. Wang J.-S. Li W. Increased sulfation of bile acids in mice and human subjects with sodium taurocholate cotransporting polypeptide deficiency.J Biol Chem. 2019; 294: 11853-11862Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 6Vaz F.M. Paulusma C.C. Huidekoper H. de Ru M. Lim C. Koster J. Ho-Mok K. Bootsma A.H. Groen A.K. Schaap F.G. Oude Elferink R.P. Waterham H.R. Wanders R.J. Sodium taurocholate cotransporting polypeptide (SLC10A1) deficiency: conjugated hypercholanemia without a clear clinical phenotype.Hepatology. 2015; 61: 260-267Crossref PubMed Scopus (122) Google Scholar Here, we made an overall pathologic examination of hypercolanemic Slc10a1-/- mice, and found that their gallbladders had multiple abnormalities throughout their life spans. Importantly, such gallbladder abnormalities also were detected in human infants and teenagers with the NTCP p.Ser267Phe variant. (Cell Mol Gastroenterol Hepatol 2021;11:831–839; https://doi.org/10.1016/j.jcmgh.2020.09.001) We examined organs including the liver, small intestine, large intestine, kidney, pancreas, and spleen in 4-week-old and aged Slc10a1-/- mice, and found that there were no obvious abnormalities in these organs in the Slc10a1-/- mice (Figure 1). However, we observed that Slc10a1 deficiency caused multiple abnormal phenotypes of the gallbladder: thickened gallbladder walls, enlarged gallbladders (with thickened walls), enlarged gallbladders with blackish green bile, Phrygian cap deformity in its fundus, and small rod-like gallbladders with thickened walls. Note that these phenotypes were present in both male and female Slc10a1-/- mice at 4 weeks of age (Figure 2A–D and Figure 3A and B). Histologic analysis showed that Slc10a1-/- mice had thickened gallbladder walls, enhanced proliferation of the epithelium, elongated epithelial folds, increased stromal granulocyte infiltration, and submucosal vasodilation (Figure 2E–L). Notably, the gallbladder weights were significantly higher in Slc10a1-/- mice than in littermate control wild-type (WT) mice (Figure 2M). Choi et al7Choi M. Moschetta A. Bookout A.L. Peng L. Umetani M. Holmstrom S.R. Suino-Powell K. Xu H.E. Richardson J.A. Gerard R.D. Mangelsdorf D.J. Identification of a hormonal basis for gallbladder filling.Nat Med. 2006; 12: 1253-1255Crossref PubMed Scopus (225) Google Scholar in 2006 reported that the fibroblast growth factor (FGF) family protein FGF15 stimulates the enlargement of gallbladders in mice. We thus investigated whether FGF15 may contribute to the observed gallbladder enlargement phenotype among others in the Slc10a1-/- mice. Ileal Fgf15 expression was induced significantly, and correlated positively with serum TBA levels in Slc10a1-/- mice and with gallbladder weight (Figure 3C–E). Therefore, increased ileal Fgf15 expression apparently contributes functionally to the gallbladder enlargement phenotype in hypercholanemic Slc10a1-/- mice, a finding that warrants further experimental investigations.Figure 2NTCP deficiency causes gallbladder abnormalities in mice. Representative photographs of gallbladders from fasted 4-week-old (A) male WT and (B–D) Slc10a1-/- mice. Scale bar: 1 mm. Representative gallbladder H&E staining of (E and I) WT mice related to panel A, and (F-H and J-L) Slc10a1-/- mice related to panels B-D, respectively. The arrow indicates elongated epithelial folds, arrowheads indicate stromal granulocyte infiltration, closed triangles indicate submucosal vasodilation, and open triangles indicate the presence of lipids in the gallbladder walls. (M) Gallbladder (GB) weights in WT (n = 20) and Slc10a1-/- mice (n = 19) at 4 weeks of age. (N) Representative photos of bile from WT and Slc10a1-/- mice. (O) BA profiling in bile from WT and Slc10a1-/- mice (WT, n = 5; Slc10a1-/-, n = 9) at 4 weeks of age. Inset: the significant decrease of taurine-conjugated β MCA (TβMCA). All mice were male. Values are shown as medians. Each symbol represents an individual mouse. Two-tailed Student t tests were used.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Slc10a1-/- mice did not develop cholesterol gallstone disease. Demonstrations of gallbladder abnormalities in 4-week-old (A) male and (B) female Slc10a1-/- mice. (C) Ileal Fgf15 expression in male WT (n = 8) and Slc10a1-/- mice (n = 10) at 4 weeks of age. (D) Correlation of ileal Fgf15 expression and TBA level in male Slc10a1-/- mice (n = 17). (E) Correlation of ileal Fgf15 expression and gallbladder weight in male Slc10a1-/- mice (n = 10). Concentrations of (F) bile acids, (G) cholesterol, and (H) phospholipids in GB bile, and (I) values of cholesterol saturation index in WT (n = 7) and Slc10a1-/- mice (n = 11) at 4 weeks of age. Values are given as medians. Each symbol represents an individual mouse. Two-tailed Student t tests and Spearman rank correlation analysis were used. mRNA, messenger RNA.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Slc10a1-/- mice had hypercholanemia at 4 weeks, however, there were no visible gallstones observed in the gallbladders (Figure 2B–D and Figure 3A and B). Although bile inside the gallbladders of a small proportion of the Slc10a1-/- mice was notably more turbid than gallbladder bile of WT mice (Figure 2N), a polarizing microscopy-based inspection of bile showed no obvious cholesterol monohydrate crystals (data not shown). We also found that the gallbladder levels of phospholipids increased significantly in Slc10a1-/- mice; no differences were observed for gallbladder cholesterol or bile acids levels (Figure 3F–H). Consistently, Slc10a1-/- mice had significantly lower bile cholesterol saturation index values compared with WT mice (P < .0001) (Figure 3I), suggesting that these animals are unlikely to suffer from cholesterol gallstone disease. Comprehensive bile acid (BA) profiling of 33 BA species in these mice was conducted by liquid chromatography tandem mass spectrometry (LC-MS/MS), and only concentration of taurine-conjugated β-muricholic acid (TβMCA) significantly decreased in Slc10a1-/- mice compared with WT mice (Figure 2O). We recently reported that the overall serum TBA levels tended to decrease gradually with increasing age in Slc10a1-/- mice.5Mao F. Liu T. Hou X. Zhao H. He W. Li C. Jing Z. Sui J. Wang F. Liu X. Han J. Borchers C.H. Wang J.-S. Li W. Increased sulfation of bile acids in mice and human subjects with sodium taurocholate cotransporting polypeptide deficiency.J Biol Chem. 2019; 294: 11853-11862Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar BA profiling in aged Slc10a1-/- mice showed that the concentrations of BAs, including cholic acid, αMCA, βMCA, ωMCA, chenodeoxycholic acid, ursodeoxycholic acid, and their taurine or glycine conjugates are similar to that of aged male or female WT mice, respectively (Figure 4A and B). However, the aged Slc10a1-/- mice still had thickened gallbladder walls, or even a completely filled gallbladder cavity by the age of 20 months (Figure 4C). Furthermore, no significant changes in bile flow and biliary excretion of BAs were observed between aged WT and Slc10a1-/- mice, although Slc10a1-/- mice showed a trend toward slightly increased bile flow (Figure 4D and E). To determine whether loss of NTCP function also can lead to gallbladder abnormalities in human beings, we conducted a retrospective analysis of 33 individuals with the NTCP p.Ser267Phe variant who we recently examined in a clinical study of young children showing abnormal TBA levels.5Mao F. Liu T. Hou X. Zhao H. He W. Li C. Jing Z. Sui J. Wang F. Liu X. Han J. Borchers C.H. Wang J.-S. Li W. Increased sulfation of bile acids in mice and human subjects with sodium taurocholate cotransporting polypeptide deficiency.J Biol Chem. 2019; 294: 11853-11862Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 11Yant Y.Y. Wang M.X. Gong J.Y. Liu L.L. Setchell K.D.R. Xie X.B. Wang N.L. Li W. Wang J.S. Abnormal bilirubin metabolism in patients with sodium taurocholate cotransporting polypeptide deficiency.J Pediatr Gastroenterol Nutr. 2020; 71: e138-e141PubMed Google Scholar Ultrasound examination of 2 teenagers (ages, 14–15 y) with this mutation showed obvious gallbladder polyps (Figure 5A and B); 2 infants (ages, 6–7 mo) also showed gallbladders containing either 1 large gallstone or multiple sand-like gallstones (Figure 5C and D). Beyond confirming our suppositions based on the gallbladder phenotypes of the Slc10a1-/- mice, these findings indicate that human beings with the NTCP p.Ser267Phe variant are at risk for developing gallbladder abnormalities. Indeed, a Fisher exact test–based analysis of the prospective cohorts showed that such individuals develop gallbladder abnormalities at significantly higher rates than 2 other inherited liver diseases: Wilson disease and glycogen storage disease (4 of 33 vs 0 of 174, respectively; P < .001) (Table 1).Table 1Information of Gallbladder Abnormalities in Inherited Liver DiseasesInherited liver diseasePatient IDSexDate of birth, month/day/yearDate of finding gallbladder abnormality, month/day/yearNTCP S267F (total, 33 patients)P1Male12/14/200008/11/2015P8Male06/11/201211/01/2013P19Male01/30/201709/04/2017P31Male12/14/200401/21/2019Wilson disease (total, 83 patients)0–––Glycogen storage disease (total, 91 patients)0––– Open table in a new tab The BA composition in plasma of NTCP p.Ser267Phe variants with gallbladder abnormalities is changed significantly (Figure 5E).5Mao F. Liu T. Hou X. Zhao H. He W. Li C. Jing Z. Sui J. Wang F. Liu X. Han J. Borchers C.H. Wang J.-S. Li W. Increased sulfation of bile acids in mice and human subjects with sodium taurocholate cotransporting polypeptide deficiency.J Biol Chem. 2019; 294: 11853-11862Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar However, no significant changes in serum levels of FGF19, the human ortholog of FGF15, were observed between healthy controls and NTCP p.Ser267Phe variants, although these variants showed a trend toward slightly increased serum FGF19 levels (P = .22) independent of the TBA levels (Figure 5F and G).6Vaz F.M. Paulusma C.C. Huidekoper H. de Ru M. Lim C. Koster J. Ho-Mok K. Bootsma A.H. Groen A.K. Schaap F.G. Oude Elferink R.P. Waterham H.R. Wanders R.J. Sodium taurocholate cotransporting polypeptide (SLC10A1) deficiency: conjugated hypercholanemia without a clear clinical phenotype.Hepatology. 2015; 61: 260-267Crossref PubMed Scopus (122) Google Scholar,8Zweers S.J.L.B. Booij K.A.C. Komuta M. Roskams T. Gouma D.J. Jansen L.M. Schaap F.G. The human gallbladder secretes fibroblast growth factor 19 into bile: Towards defining the role of fibroblast growth factor 19 in the enterobiliary tract.Hepatology. 2012; 55: 575-583Crossref PubMed Scopus (96) Google Scholar Thus, NTCP deficiency can result in dysregulated gallbladder wall morphology (ie, thickened gallbladder walls or gallbladder polyps) in both mouse and human beings but causes gallstone formation only in human beings. In conclusion, the present study suggests that deficiency of NTCP in hepatocytes leads to gallbladder abnormalities in both mice and human beings. In addition, and distinct from metabolism phenotypes such as dysregulated TBA levels in infants that later decrease, NTCP deficiency also can lead to gallbladder abnormalities that occur in children that may be irreversible throughout one's life. All of these findings point to NTCP deficiency as a potentially underappreciated cause for gallbladder diseases encountered by physicians in the clinic.9Dong C. Zhang B.-P. Wang H. Xu H. Zhang C. Cai Z.-S. Wang D.-W. Shu S.-N. Huang Z.-H. Luo X.-P. Clinical and histopathologic features of sodium taurocholate cotransporting polypeptide deficiency in pediatric patients.Medicine (Baltimore). 2019; 98e17305Crossref PubMed Scopus (12) Google Scholar Moreover, our study supports that potential gallbladder-related effects should be monitored carefully in ongoing efforts to develop therapies against hepatitis B virus/hepatitis D virus infections that are based on NTCP-related targets.10Schierwagen R. Klein S. Trebicka J. A new treatment for chronic hepatitis B and D offers novel insights into obesity and hepatic steatosis.Cell Mol Gastroenterol Hepatol. 2020; 10: 649-651Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar WT and Slc10a1-/- mice5Mao F. Liu T. Hou X. Zhao H. He W. Li C. Jing Z. Sui J. Wang F. Liu X. Han J. Borchers C.H. Wang J.-S. Li W. Increased sulfation of bile acids in mice and human subjects with sodium taurocholate cotransporting polypeptide deficiency.J Biol Chem. 2019; 294: 11853-11862Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar were housed in specific pathogen-free conditions at the animal facility of the National Institute of Biological Sciences (Beijing, China). Animal experiments were performed in accordance with the National Institute of Biological Sciences institutional regulations after approval by the Institutional Animal Care and Use Committee. To explore the gallbladder conditions in human beings, patients genetically diagnosed with NTCP p.Ser267Phe variant presenting to the Liver Center of Children's Hospital of Fudan University and the Department of Pediatrics at Jinshan Hospital of Fudan University from January 2012 to December 2018 were included for this study.11Yant Y.Y. Wang M.X. Gong J.Y. Liu L.L. Setchell K.D.R. Xie X.B. Wang N.L. Li W. Wang J.S. Abnormal bilirubin metabolism in patients with sodium taurocholate cotransporting polypeptide deficiency.J Pediatr Gastroenterol Nutr. 2020; 71: e138-e141PubMed Google Scholar Patients diagnosed with Wilson disease or glycogen storage disease were selected from the hospital's information system as age- and sex-matched controls. The information from gallbladder examination, abdominal ultrasonography and magnetic resonance imaging were picked up. Patients with hepatic failure, hyperuricemia, and nephrotic syndrome, or another disease that may cause edema or ascites, or patients who received a liver transplant were excluded. The study protocol was approved by the Children's Hospital of Fudan University and Jinshan Hospital of Fudan University Ethics Committees at both institutions. Written informed consent was obtained from parents, guardians, and/or patients. Each participant provided written informed consent before participation in accordance with the ethical guidelines of the 1975 Declaration of Helsinki. For H&E staining, fresh tissues were rinsed in phosphate-buffered saline and fixed in 4% paraformaldehyde overnight. Sections (5 μm) were deparaffinized, rehydrated, and stained with H&E. Slides were dehydrated, and then were cleared and mounted in neutral balsam. Images were obtained using a Zeiss Axio Imager M1 microscope with an AxioCam HRc camera controlled by AxioVision 4.6 software (Oberkochen, Germany) or Leica Aperio AT2 digital whole slide scanning (Vista, CA). Representative images of at least 3 sample replicates are presented in the figures (Figures 1, 2 and 4). Bile samples were collected using an infusion needle connected to a 1 mL syringe. Cholesterol and phospholipids were measured using enzymatic method kits (Applygen E1005, Beijing, China for cholesterol, and WAKO 296-63801, Osaka, Japan for phospholipids) following the product instructions. Measurement of bile acids from bile samples was conducted commercially by the Beijing Lawke Health Laboratory Center for Clinical Laboratory Development, Inc (Beijing, China). The cholesterol saturation index of bile was calculated as described by Carey.12Carey M.C. Critical tables for calculating the cholesterol saturation of native bile.J Lipid Res. 1978; 19: 945-955Abstract Full Text PDF PubMed Google Scholar Human BA profiling in plasma was performed by reverse-phase ultrahigh-performance liquid chromatography (UPLC)/multiple-reaction monitoring-mass spectrometry with negative ion detection.5Mao F. Liu T. Hou X. Zhao H. He W. Li C. Jing Z. Sui J. Wang F. Liu X. Han J. Borchers C.H. Wang J.-S. Li W. Increased sulfation of bile acids in mice and human subjects with sodium taurocholate cotransporting polypeptide deficiency.J Biol Chem. 2019; 294: 11853-11862Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar,13Han J. Liu Y. Wang R. Yang Juncong Ling V. Borchers C.H. Metabolic profiling of bile acids in human and mouse blood by LC–MS/MS in combination with phospholipid-depletion solid-phase extraction.analytical chemistry. 2014; 87: 1127-1136Crossref PubMed Scopus (98) Google Scholar,14Qiu Y.-L. Gong J.-Y. Feng J.-Y. eWang R.-X. Han J. Liu T. Lu Y. Li-Ting Li Zhang M.-H. Sheps J.A. Wang N.-L. Yan Y.-Y. Li J.-Q. Chen L. Borchers C.H. Sipos B. Knisely A.S. Long V. Xing Q.-H. Wang J.-S. Defects in myosin VB are associated with a spectrum of previously undiagnosed low γ-glutamyltransferase cholestasis.Hepatology. 2017; 65: 1655-1669Crossref PubMed Scopus (65) Google Scholar BAs were measured in bile samples that were previously stored at −80°C using a quantitative UPLC-MS/MS platform (ACQUITY UPLC-Xevo TQ-S; Waters Corp, Milford, MA).15Xie G. Wang Y. Wang X. Zhao A. Chen T. Ni Y. Wong L. Zhang H. Zhang J. Liu C. Liu P. Jia W. Profiling of serum bile acids in a healthy chinese population using UPLC–MS/MS.J Proteome Res. 2015; 14: 850-859Crossref PubMed Scopus (83) Google Scholar All bile samples were diluted 1000× before the test. All chromatographic separations were performed with an ACQUITY BEH C18 column VanGuard precolumn (2.1 × 5 mm) and analytical column (2.1 × 100 mm). The elution solvents were water with formic acid (pH, 3.25, A) and acetonitrile/methanol (8:2, B). The flow rate was 400 μL/min with the following mobile phase gradient: 0–0.3 minutes (5% B), 0.3–0.5 minutes (5%–10% B), 0.5–2 minutes (10%–15% B), 2–3 minutes (15%–30% B), 3–6 minutes (30% B), 6–8 minutes (30–35% B), 8–9 minutes (35%–40% B), 9–10 minutes (40% B), 10–15 minutes (40%–75% B), 15–15.5 minutes (75%–100% B), 15.5–16.2 minutes (100% B), 16.2–16.3 minutes (100–5% B), and 16.3–17 minutes (5% B). The cone and collision energy for each BA used the optimized settings from QuanOptimize application manager (Milford, CT). The raw data generated by UPLC-MS/MS then were processed using QuanMET software to perform peak integration, calibration, and quantitation for each BA. Testing services were provided by Metabo-Profile, Inc (Shanghai, China). Quantified BAs contained 33 species, including cholic acid, chenodeoxycholic acid, tauro ωMCA, tauro αMCA, tauro βMCA, taurocholic acid, glycocholic acid, glycochenodeoxycholate, taurochenodeoxycholate, deoxycholic acid, ursodeoxycholic acid, glycodeoxycholic acid, taurodeoxycholate, glycoursodeoxycholic acid, tauroursodeoxycholic acid, taurohyocholate, taurohyodeoxycholic acid, taurolithocholate, ursocholic acid, ωMCA, αMCA, βMCA, γ-muricholic acid/hyocholic acid, allocholic acid, nor cholic acid, glycohyocholate, glycohyodeoxycholate, 3β-ursodeoxycholic acid, β-hyodeoxycholic acid, α-hyodeoxycholic acid, 23-nordeoxycholic acid, 7-ketodeoxycholic acid, and 12-ketodeoxycholic acid. Bile secretion in fasted mice was performed by performing gallbladder cannulation and bile collection for 10 minutes after distal ligation of the common bile duct. Bile flow was determined gravimetrically assuming a density of 1 g/mL for bile. Biliary BA secretion was calculated as the product of the bile flow and the bile concentration in a 10-minute collection period.3Slijepcevic D. Kaufman C. Wichers C.G. Gilglioni E.H. Lempp F.A. Duijst S. de Waart D.R. Oude Elferink R.P.J. Mier W. Stieger B. Beuers U. Urban S. van de Graaf S.F.J. Impaired uptake of conjugated bile acids and hepatitis b virus pres1-binding in na(+) -taurocholate cotransporting polypeptide knockout mice.Hepatology. 2015; 62: 207-219Crossref PubMed Scopus (87) Google Scholar,16Oude Elferink R.P. Ottenhoff R. van Wijland M. Smit J.J. Schinkel A.H. Groen A.K. Regulation of biliary lipid secretion by mdr2 P-glycoprotein in the mouse.J Clin Invest. 1995; 95: 31-38Crossref PubMed Google Scholar Circulation FGF19 levels of the health controls and NTCP p.Ser267Phe variant were determined using a sandwich enzyme-linked immunosorbent assay according to the manufacturer's instructions (DF1900; R&D Systems, Inc, Minneapolis, MN).17Gomez-Ospina N. Potter C.J. Xiao R. Manickam K. Kim M.S. Kim K.H. Shneider B.L. Picarsic J.L. Jacobson T.A. Zhang J. He W. Liu P. Knisely A.S. Finegold M.J. Muzny D.M. Boerwinkle E. Lupski J.R. Plon S.E. Gibbs R.A. Eng C.M. Yang Y. Washington G.C. Porteus M.H. Berquist W.E. Kambham N. Singh R.J. Xia F. Enns G.M. Moore D.D. Mutations in the nuclear bile acid receptor FXR cause progressive familial intrahepatic cholestasis.Nat Commun. 2016; 7: 10713Crossref PubMed Scopus (148) Google Scholar Freshly isolated ileum samples were washed with phosphate-buffered saline to flush out their content. The cleaned samples were chopped into homogenates on the ice, treated with TRIzol reagent (Carlsbad, CA), and vortexed immediately. Samples in TRIzol were incubated at room temperature (∼25°C) for 5 minutes and stored at -80°C. Extraction of RNA followed the TRIzol product instructions. Quantitative polymerase chain reaction was performed on a 7500 fast instrument (Applied Biosystems, Foster City, CA) using SYBR Green.18He W. Ren B. Mao F. Jing Z. Li Y. Liu Y. Peng B. Yan H. Qi Y. Sun Y. Guo J.-T. Sui J. Wang F. Li W. Hepatitis D virus infection of mice expressing human sodium taurocholate co-transporting polypeptide.PLOS Pathogens. 2015; 11e1004840Crossref PubMed Scopus (79) Google Scholar Medians are presented in Figures 2, 3 and 5) unless otherwise stated. Inferential statistical significance between 2 groups was examined using 2-tailed Student t tests. Correlation between 2 groups of measured parameters was analyzed using Spearman rank correlation analysis implemented in GraphPad Prism (version 6.0, San Diego, CA). The Fisher exact test was performed to compare the difference in gender and disease groups. P values less than .05 were considered to indicate statistical significance. All authors had access to all of the data and reviewed and approved the final manuscript. The authors thank their patients for their participation in this study.
'/%3e%3cuse xlink:href='%23a' transform='rotate(23.036 .093 25.536)'/%3e%3cuse xlink:href='%23a' transform='rotate(45.87 1.273 16.18)'/%3e%3cuse xlink:href='%23a' transform='rotate(69.945 .996 12.078)'/%3e%3cuse xlink:href='%23a' transform='rotate(20.66 -19.689 31.932)'/%3e%3c/svg%3e)
