Affinity-matured homotypic interactions induce spectrum of PfCSP structures that influence protection from malaria infection.

The generation of high-quality antibody responses to Plasmodium falciparum (Pf) circumsporozoite protein (PfCSP), the primary surface antigen of Pf sporozoites, is paramount to the development of an effective malaria vaccine. Here we present an in-depth structural and functional analysis of a panel of potent antibodies encoded by the immunoglobulin heavy chain variable (IGHV) gene IGHV3-33, which is among the most prevalent and potent antibody families induced in the anti-PfCSP immune response and targets the Asn-Ala-Asn-Pro (NANP) repeat region. Cryo-electron microscopy (cryo-EM) reveals a remarkable spectrum of helical antibody-PfCSP structures stabilized by homotypic interactions between tightly packed fragments antigen binding (Fabs), many of which correlate with somatic hypermutation. We demonstrate a key role of these mutated homotypic contacts for high avidity binding to PfCSP and in protection from Pf malaria infection. Together, these data emphasize the importance of anti-homotypic affinity maturation in the frequent selection of IGHV3-33 antibodies and highlight key features underlying the potent protection of this antibody family.

An Atypical Presentation of Serotonin Syndrome.

The purpose of this paper is to highlight an uncommon presentation of serotonin syndrome and discuss important points such as causes, the manifestation of symptoms, and available treatments. The report highlights the importance of recognizing typical signs and symptoms in order to uncover an atypical presentation of serotonin syndrome. Serotonin toxicity can become life-threatening if not identified early in its course and the offending agents discontinued. This can be achieved by educated physicians and careful prescribing of these agents.

Post-Bariatric Abdominoplasty: Analysis of 406 Cases With Focus on Risk Factors and Complications.

BACKGROUND: Post-bariatric patients present a surgical challenge within abdominoplasty because of residual obesity and major comorbidities. In this study, we analyzed complications following abdominoplasty in post-bariatric patients and evaluated potential risk factors associated with these complications. OBJECTIVES: The authors sought to determine the complications and risk factors following abdominoplasty in post-bariatric patients. METHODS: A retrospective study of patients who underwent abdominoplasty was performed from January 2009 to December 2018 at our institution. Variables analyzed were sex, age, body mass index (BMI), smoking, surgical technique, operative time, resection weight, drain output, and complications. RESULTS: A total of 406 patients were included in this study (320 female and 86 male) with a mean age of 44.4 years and a BMI of 30.6 kg/m2. Abdominoplasty techniques consisted of traditional (64.3%), fleur-de-lis technique (27.3%), and panniculectomy without umbilical displacement (8.4%). Overall complications recorded were 41.9%, the majority of these being wound-healing problems (32%). Minor and major complications were found in 29.1% and 12.8% of patients, respectively. A BMI value of ≥30 kg/m2 was associated with an increased risk for wound-healing problems (P = 0.001). The frequency of total complications was significantly related to age (P = 0.007), BMI (P = 0.004), and resection weight (P = 0.001). Abdominoplasty technique tended to influence total complications. CONCLUSIONS: This study demonstrates in a fairly large sample of post-bariatric patients (n = 406) that abdominoplasty alone can be performed safely, with an acceptable complication rate. Age, BMI, and resection weight are shown to be significant risk factors for total complications. The role of surgical technique needs to be evaluated further.Level of Evidence: 4.

Indwelling Tunneled Pleural Catheters for the Management of Hepatic Hydrothorax. A Pilot Study.

RATIONALE: Hepatic hydrothorax is a complication of cirrhosis in which hydrostatic imbalances result in fluid accumulation within the pleural space. Although uncommon, this may cause significant morbidity, resulting in dyspnea requiring repeated pleural drainage procedures. Liver transplantation is curative, but it is rarely immediately available to qualified patients, presenting the clinical challenge of managing recurrent pleural effusions. Indwelling tunneled pleural catheters (ITPCs) have been used successfully to palliate dyspnea associated with recurrent malignant pleural effusions. OBJECTIVES: This study was performed to evaluate the feasibility of using ITPCs for the management of hepatic hydrothorax. METHODS: A single-center prospective feasibility study was performed to evaluate the use of ITPCs for the management of recurrent hepatic hydrothorax in patients who were eligible for liver transplant evaluation. MEASUREMENTS AND MAIN RESULTS: Twenty-five ITPCs were placed in 24 patients. The mean number of pleural drainage procedures before ITPC placement was 1.9, with no further pleural drainages required in any patient after ITPC placement. Spontaneous pleurodesis occurred in 8 of 24 patients (33%). All eight catheters were successfully removed without pleural fluid reaccumulation. Mean time to pleurodesis was 131.8 days. Pleural fluid infection occurred in 4 of 24 patients (16.7%), requiring catheter removal in 3 of the 4 patients. CONCLUSIONS: ITPCs may be successfully and safely used to control symptoms associated with hepatic hydrothorax. The rate of spontaneous pleurodesis that occurs is similar to that observed with ITPCs placed for malignant pleural effusion, although the infection rate may be higher. Clinical trial registered with www.clinicaltrials.gov (NCT02595567).

Targeting myofibroblasts in model systems of fibrosis by an artificial alpha-smooth muscle-actin promoter hybrid.

Myofibroblasts are the main cell types producing extracellular matrix proteins in a variety of fibrotic diseases. Therefore, they are useful targets for studies of intracellular communication and gene therapeutical approaches in scarring diseases. An artificial promoter containing the -702 bp regulatory sequence of the alpha-smooth muscle actin (SMA) gene linked to the first intron enhancer sequence of the beta-actin gene and the beta-globin intron-exon junction was constructed and tested for myofibroblast-dependent gene expression using the green fluorescent protein as a reporter. Reporter expression revealed myofibroblast-specific function in hepatic and renal myofibroblasts, in vitro. In addition, differentiation-dependent activation of the SMA-beta-actin promoter hybrid was shown after induction of myofibroblastic features in mesangial cells by stretching treatment. Furthermore, wound healing experiments with SMA-beta-actin promoter reporter mice demonstrated myofibroblast-specific action, in vivo. In conclusion, the -702 bp regulatory region of the SMA promoter linked to enhancing beta-actin and beta-globin sequences benefits from its small size and is suggested as a promising tool to target myofibroblasts as the crucial cell type in various scarring processes.

Regulation of human liver delta-aminolevulinic acid synthase by bile acids.

UNLABELLED: Aminolevulinic acid synthase 1 (ALAS1) is the rate-limiting enzyme of heme synthesis in the liver and is highly regulated to adapt to the metabolic demand of the hepatocyte. In the present study, we describe human hepatic ALAS1 as a new direct target of the bile acid-activated nuclear receptor farnesoid X receptor (FXR). Experiments in primary human hepatocytes and in human liver slices showed that ALAS1 messenger RNA (mRNA) and activity is increased upon exposure to chenodeoxycholic acid (CDCA), the most potent natural FXR ligand, or the synthetic FXR-specific agonist GW4064. Moreover, overexpression of a constitutively active form of FXR further increased ALAS1 mRNA expression. In agreement with these observations, an FXR response element was identified in the 5' flanking region of human ALAS1 and characterized in reporter gene assays. A highly conserved FXR binding site (IR1) within a 175-bp fragment at -13 kilobases upstream of the transcriptional start site was able to trigger an FXR-specific increase in luciferase activity upon CDCA treatment. Site-directed mutagenesis of IR1 abolished this effect. Binding of FXR/retinoid acid X receptor heterodimers was demonstrated by mobility gel shift experiments. CONCLUSION: These data strongly support a role of bile acid-activated FXR in the regulation of human ALAS1 and, consequently, hepatic porphyrin and heme synthesis. These data also suggest that elevated endogenous bile acids may precipitate neuropsychiatric attacks in patients with acute hepatic porphyrias.

FXR agonists and FGF15 reduce fecal bile acid excretion in a mouse model of bile acid malabsorption.

Bile acid malabsorption, which in patients leads to excessive fecal bile acid excretion and diarrhea, is characterized by a vicious cycle in which the feedback regulation of bile acid synthesis is interrupted, resulting in additional bile acid production. Feedback regulation of bile acid synthesis is under the control of an endocrine pathway wherein activation of the nuclear bile acid receptor, farnesoid X receptor (FXR), induces enteric expression of the hormone, fibroblast growth factor 15 (FGF15). In liver, FGF15 acts together with FXR-mediated expression of small heterodimer partner to repress bile acid synthesis. Here, we show that the FXR-FGF15 pathway is disrupted in mice lacking apical ileal bile acid transporter, a model of bile acid malabsorption. Treatment of Asbt-/- mice with either a synthetic FXR agonist or FGF15 downregulates hepatic cholesterol 7alpha-hydroxylase mRNA levels, decreases bile acid pool size, and reduces fecal bile acid excretion. These findings suggest that FXR agonists or FGF15 could be used therapeutically to interrupt the cycle of excessive bile acid production in patients with bile acid malabsorption.

Analysis of bile acid-induced regulation of FXR target genes in human liver slices.

Information about the role of nuclear receptors has rapidly increased over the last decade. However, details about their role in human are lacking. Owing to species differences, a powerful human in vitro system is needed. This study uses for the first time precision-cut human liver slices in the nuclear receptor field. The farnesoid X receptor (FXR) was chosen as a model. We were able to demonstrate that human liver slices efficiently take up bile acids and show a stable expression of a wide variety of genes relevant for bile acid metabolism, including bile acid transporters, cytochrome P450 enzymes and transcription factors. Treatment with chenodeoxycholate induced small heterodimer partner, bile salt export pump and p-glycoprotein, ABCB4 and repressed cholesterol 7alpha hydroxylase, hepatocyte nuclear factor (HNF)1, HNF4 and organic anion transporting peptide (OATP)1B1. OATP1B3, FXR, HNF3beta and cytochrome P450 enzyme remained relatively constant. In contrast to what has been observed in mice and rat studies, SHP induction did not result in repression of sodium-dependent bile acid cotransporter expression. Further, regulation of genes seemed to be dependent on concentration and time. Taken together, the study shows that the use of liver slices is a powerful technique that enables to study nuclear receptors in the human liver.

Genetic variability, haplotype structures, and ethnic diversity of hepatic transporters MDR3 (ABCB4) and bile salt export pump (ABCB11).

Biliary excretion of bile salts and other bile constituents from hepatocytes is mediated by the apical (canalicular) transporters P-glycoprotein 3 (MDR3, ABCB4) and the bile salt export pump (ABCB11). Mutations in ABCB4 and ABCB11 contribute to cholestatic diseases [e.g., progressive familial intrahepatic cholestasis 2 (PFIC2), PFIC3, and intrahepatic cholestasis of pregnancy], and our objective was to establish genetic variability and haplotype structures of ABCB4 and ABCB11 in healthy populations of different ethnic backgrounds. All coding exons, 5 of 6 noncoding exons, 50 to 300 base pairs of the flanking intronic regions, and 2.5 to 2.8 kilobase pairs of the promoter regions of ABCB4 and ABCB11 were sequenced in 159 and 196 DNA samples of Caucasian, African-American, Japanese, and Korean origin. In total, 76 and 86 polymorphisms were identified in ABCB4 and ABCB11, respectively; among them, 14 and 28 exonic polymorphisms, and 8 and 10 protein-altering variants, of which 4 were predicted to have functional consequences. Both genes showed substantial ethnic differences with respect to allele number, frequency of common and population-specific sites, and patterns of linkage disequilibrium. Population genetic analysis suggested some selective pressure against changes in the protein, supporting the important endogenous role of these transporters. Haplotype variability was greater in ABCB11 than in ABCB4. An ABCB11 promoter haplotype was associated with significant decrease of activity compared with wild type. Our results contribute to a better understanding of the molecular basis and of ethnic differences in drug response, and provide a valuable tool for future research on the heredity of cholestatic liver injury.

Pregnane X receptor is a target of farnesoid X receptor.

The pregnane X receptor (PXR) is an essential component of the body's detoxification system. PXR is activated by a broad spectrum of xenobiotics and endobiotics, including bile acids and their precursors. Bile acids in high concentrations are toxic; therefore, their synthesis is tightly regulated by the farnesoid X receptor, and their catabolism involves several enzymes regulated by PXR. Here we demonstrate that the expression of PXR is regulated by farnesoid X receptor. Feeding mice with cholic acid or the synthetic farnesoid X receptor (FXR) agonist GW4064 resulted in a robust PXR induction. This effect was abolished in FXR knock-out mice. Long time bile acid treatment resulted in an increase of PXR target genes in wild type mice. A region containing four FXR binding sites (IR1) was identified in the mouse Pxr gene. This region was able to trigger an 8-fold induction after GW4064 treatment in transactivation studies. Deletion or mutation of single IR1 sites caused a weakened response. The importance of each individual IR1 element was assessed by cloning a triple or a single copy and was tested in transactivation studies. Two elements were able to trigger a strong response, one a moderate response, and one no response to GW4064 treatment. Mobility shift assays demonstrated that the two stronger responding elements were able to bind FXR protein. This result was confirmed by chromatin immunoprecipitation. These results strongly suggest that PXR is regulated by FXR. Bile acids activate FXR, which blocks synthesis of bile acids and also leads to the transcriptional activation of PXR, promoting breakdown of bile acids. The combination of the two mechanisms leads to an efficient protection of the liver against bile acid induced toxicity.

The human Na+-taurocholate cotransporting polypeptide gene is activated by glucocorticoid receptor and peroxisome proliferator-activated receptor-gamma coactivator-1alpha, and suppressed by bile acids via a small heterodimer partner-dependent mechanism.

Na+-taurocholate cotransporting polypeptide (NTCP) is the major bile acid uptake system in human hepatocytes. NTCP and the ileal transporter ASBT (apical sodium-dependent bile acid transporter) are two sodium-dependent transporters critical for the enterohepatic circulation of bile acids. The hASBT gene is known to be activated by the glucocorticoid receptor (GR). Here we show that GR also induces the endogenous hNTCP gene and transactivates the reporter-linked hNTCP promoter, in the presence of its ligand dexamethasone. Mutational analysis of the hNTCP promoter identified a functional GR response element, with which GR directly interacts within living cells. The GR/dexamethasone activation of endogenous hNTCP expression was suppressed by bile acids, in a manner dependent on the bile acid receptor farnesoid X receptor. Overexpression of the farnesoid X receptor-inducible transcriptional repressor small heterodimer partner also suppressed the GR/dexamethasone-activation of the hNTCP promoter. The peroxisome proliferator-activated receptor-gamma coactivator-1alpha enhanced the GR/dexamethasone activation of the hNTCP promoter. In conclusion, the hNTCP promoter is activated by GR in a ligand-dependent manner, similarly to the hASBT promoter. Thus, glucocorticoids may coordinately regulate the major bile acid uptake systems in human liver and intestine. The GR/dexamethasone activation of the hNTCP promoter is counteracted by bile acids and small heterodimer partner, providing a negative feedback mechanism for bile acid uptake in human hepatocytes.

Sequence analysis of bile salt export pump (ABCB11) and multidrug resistance p-glycoprotein 3 (ABCB4, MDR3) in patients with intrahepatic cholestasis of pregnancy.

Intrahepatic cholestasis of pregnancy (ICP) is a liver disorder associated with increased risk of intrauterine fetal death and prematurity. There is increasing evidence that genetically determined dysfunction in the canalicular ABC transporters bile salt export pump (BSEP, ABCB11) and multidrug resistance protein 3 (MDR3, ABCB4) might be risk factors for ICP development. This study aimed to (i). describe the extent of genetic variability in BSEP and MDR3 in ICP and (ii). identify new disease-causing mutations. Twenty-one women with ICP and 40 women with uneventful pregnancies were recruited between April 2001 and April 2003. Sequencing of BSEP and MDR3 spanned 8-10 kb per gene and comprised the promoter region and 100-350 bp of the flanking intronic region around each exon. DNA sequencing of polymerase chain reaction fragments was performed on an ABI3700 capillary sequencer. MDR3 promoter activity of promoter constructs carrying different ICP-specific mutations was studied using reporter assays. A total of 37 and 51 variant sites were detected in BSEP and MDR3, respectively. Three non-synonymous sites in codons for evolutionarily conserved amino acids were specific for the ICP collective (BSEP, N591S; MDR3, S320F and G762E). Furthermore, four ICP-specific splicing mutations were detected in MDR3 [intron 21, G(+1)A; intron 25, G(+5)C and C(-3)G; and intron 26, T(+2)A]. Activity of the mutated MDR3 promoter was similar to that observed for the wild-type promoter. Our data further support an involvement of MDR3 genetic variation in the pathogenesis of ICP, whereas analysis of BSEP sequence variation indicates that this gene is probably less important for the development of pregnancy-associated cholestasis.

The human organic anion transporting polypeptide 8 (SLCO1B3) gene is transcriptionally repressed by hepatocyte nuclear factor 3beta in hepatocellular carcinoma.

BACKGROUND/AIMS: The organic anion transporting polypeptides (OATPs) mediate the uptake of numerous amphipathic compounds into hepatocytes. Our aim was to study the expression and regulation of OATP8 (OATP1B3, SLC21A8/SLCO1B3) and OATP-C (OATP1B1, SLC21A6/SLCO1B1) in hepatocellular carcinomas (HCC). METHODS: RNA and protein levels in 13 paired HCC and adjacent non-tumor liver samples were quantified by real-time polymerase chain reaction or Western blot, respectively. The OATP8 and OATP-C gene promoters were characterized by luciferase reporter assays and electrophoretic mobility shift assays (EMSA). RESULTS: The expression of OATP8 was decreased in 60% of HCC compared to surrounding non-tumor liver tissue, on both the mRNA and protein levels. Expression of the liver-enriched transcription factor hepatocyte nuclear factor 3beta (HNF3beta) was increased in 70% of HCC and correlated inversely with OATP8 mRNA (r=-0.75, P<0.05) and protein. In contrast to OATP8, expression of OATP-C was not significantly decreased in HCC. In transfected Huh7 cells, OATP8 promoter activity was inhibited by 70% when HNF3beta was cotransfected. An HNF3beta binding site was located at nt -39/-23 by EMSA. The OATP-C promoter was not inhibited by HNF3beta. CONCLUSIONS: HNF3beta represses transcription of the OATP8 but not the OATP-C gene, providing a mechanism for reduced expression of OATP8 in HCC.

Role of liver-enriched transcription factors and nuclear receptors in regulating the human, mouse, and rat NTCP gene.

Hepatic uptake of bile acids is mediated by the Na(+)-taurocholate cotransporting polypeptide (NTCP; SLC10A1) of the basolateral hepatocyte membrane. Several cis-acting elements in the rat Ntcp gene promoter have been characterized. However, little is known about the mechanisms that control the expression of the human or mouse NTCP/Ntcp. We, therefore, compared the transcriptional regulation of the human and mouse NTCP/Ntcp gene with that of the rat. By computer alignment, a sequence in the 5'-regulatory region that is conserved between species was identified near the transcription start site. Huh7 cells were transfected with luciferase constructs containing the conserved region from each species. The hepatocyte nuclear factors (HNF)1alpha and -4alpha and the retinoid X receptor/retinoic acid receptor dimer (RXRalpha/RARalpha) bound and transactivated the rat but not the human or mouse NTCP/Ntcp promoters. In contrast, activation by the CCAAT/enhancer binding protein-beta was specific for human and mouse NTCP/Ntcp. The only consensus motif present in all three species was HNF3beta. HNF3beta formed a specific DNA-protein complex in electrophoretic mobility shift assays and inhibited NTCP/Ntcp promoter activity in cotransfection assays. Finally, a minor repressive effect of bile acids was only found for rat Ntcp. The transcriptional repressor small heterodimer partner (SHP) did not affect NTCP/Ntcp promoter activity. We conclude that 1) the transcriptional regulation of the conserved NTCP/Ntcp 5'-regulatory region differs considerably among human, mouse, and rat; and 2) the conserved NTCP/Ntcp regulatory region is not directly regulated by SHP. Bile acids may regulate NTCP/Ntcp indirectly by modulating the capacity of nuclear factors to activate gene expression.

Hepatocyte nuclear factor 1 alpha: a key mediator of the effect of bile acids on gene expression.

Bile acids regulate the expression of genes involved in cholesterol homeostasis. They are ligands of the farnesoid X receptor, which induces small heterodimer partner (SHP)-1, a transcriptional repressor of bile acid synthetic enzymes. In cholestatic liver disease, hepatic bile acid concentrations are elevated and expression of the major Na+-independent bile acid uptake system, organic anion transporting polypeptide (OATP)-C (solute carrier gene family SLC21A6), is markedly decreased. Because the OATP-C gene is transcriptionally dependent on the hepatocyte nuclear factor (HNF) 1 alpha, we hypothesized that bile acids decrease OATP-C expression through direct repression of HNF1 alpha. To test this hypothesis, we studied the regulation of the human HNF1 alpha gene by bile acids. HNF1 alpha expression in cultured hepatoma cells was decreased approximately 50% after 12 hours' exposure to 100 micromol/L chenodeoxycholic acid (CDCA). Characterization of the human HNF1 alpha gene promoter identified a consensus bile acid response element that binds and is activated by HNF4 alpha. Mutagenesis of the HNF4 alpha site abolished baseline HNF1 alpha promoter activity. The central mechanism by which bile acids repress HNF1 alpha is decreased activation by HNF4 alpha. SHP directly inhibits HNF4 alpha-mediated transactivation of the HNF1 alpha promoter in cotransfection assays. In addition, HNF4 alpha nuclear binding activity is decreased by CDCA and the human HNF4 alpha gene promoter is repressed by CDCA through an SHP-independent mechanism. In conclusion, we show that repression of HNF1 alpha is an important new mechanism by which bile acids regulate the expression of HNF1 alpha-dependent genes in man. This explains the suppressive effect of bile acids on the OATP-C gene promoter, leading to decreased expression in cholestatic liver disease.

Human apical sodium-dependent bile salt transporter gene (SLC10A2) is regulated by the peroxisome proliferator-activated receptor alpha.

The apical sodium-dependent bile salt transporter (ASBT/SLC10A2), also called the ileal bile acid transporter, mediates the intestinal absorption of bile salts. The efficiency of this transport process is a determinant of hepatic bile salt synthesis from cholesterol and of serum triglyceride levels. Our aim was to characterize the human ASBT gene promoter with respect to regulatory mechanisms that coordinately affect ASBT expression and hepatic lipid and bile salt metabolism. The minimal construct that confers full promoter activity contains three functional hepatocyte nuclear factor 1alpha (HNF1alpha) recognition sites, explaining the dependence of ASBT gene expression upon HNF1alpha. A nuclear receptor binding site arranged as a direct hexanucleotide repeat (DR1 motif) is localized approximately 1.6 kb upstream of the transcription initiation site. Constructs containing this element were transactivated by WY14643 and ciprofibrate, ligands of the peroxisome proliferator-activated receptor alpha (PPARalpha), in Caco2 cells. The DR1 element was shown to bind the PPARalpha/9-cis-retinoic acid receptor heterodimer, and targeted mutagenesis of the DR1 motif abolished PPARalpha responsiveness. Ciprofibrate treatment of SK-ChA cholangiocytes increased ASBT mRNA levels, suggesting a physiologic role for PPARalpha-mediated ASBT gene regulation. This study identifies PPARalpha as a novel link between ileal bile salt absorption and hepatic lipid metabolism.

Human organic anion transporting polypeptide 8 promoter is transactivated by the farnesoid X receptor/bile acid receptor.

BACKGROUND & AIMS: OATP8 (gene symbol: SLC21A8) is a multispecific uptake system for organic anions, xenobiotics, and peptides expressed at the basolateral (sinusoidal) membrane of human hepatocytes. We investigated whether OATP8 gene expression is regulated by the nuclear receptors farnesoid X receptor/bile acid receptor (FXR/BAR; NR1H4), pregnane X receptor (PXR), or liver X receptor (LXR). METHODS: OATP8 promoter function was studied in reporter assays. OATP8 expression in cells was quantitated by real-time polymerase chain reaction. RESULTS: The bile acid chenodeoxycholic acid (CDCA), a ligand of FXR/BAR, but not clotrimazole or 25-hydroxycholesterol, ligands of PXR or LXR, respectively, induced OATP8 promoter activity. An inverted hexanucleotide repeat motif (IR-1 element) in the promoter sequence was shown by electrophoretic mobility shift assays to bind the FXR (9-cis-retinoic acid receptor [RXRalpha]) heterodimer. Targeted mutagenesis of the IR-1 element abolished inducibility of the OATP8 promoter by CDCA, confirming its role as a bile acid response element. CDCA treatment increased OATP8 messenger RNA levels in human hepatoma cells, suggesting a physiologic role for FXR-mediated OATP8 gene regulation. CONCLUSIONS: OATP8 gene expression is regulated by bile acids via FXR/BAR. Induction of OATP8 could serve to maintain hepatic extraction of xenobiotics and peptides in conditions of increased intracellular bile acids.