Importance of pseudoaneurysms after TAVI - a retrospective analysis of 2063 patients.

Background: Bifemoral arterial access is common in patients undergoing transcatheter aortic valve implantation (TAVI), with a primary treatment access (TAVI access) and a secondary non-TAVI access. Pseudoaneurysm (PSA) is an important complication of femoral arterial puncture. Major vascular complications after TAVI are well described, but little is known about PSA. Patients and methods: A total of 2063 patients underwent transfemoral TAVI between January 2014 and January 2020. Vascular ultrasound of the common femoral artery was assessed before and after TAVI. We compared patient characteristics, periprocedural risk scores, procedural characteristics, and access site bleeding events according to Valve Academic Research Consortium 3 (VARC-3) criteria, length of stay (LOS), and all-cause mortality at one year between patients with (46) and without (2017) PSA. Results: The incidence of PSA after TAVI was 2.2% (46/2063). All PSA were successfully treated with ultrasound-guided manual compression (UGMC) or thrombin injection (UGTI) without complications. Patients with PSA had lower platelet counts (210×1000/µl vs. 234×1000/µl; p<0.05), more heart failure symptoms on admission (91% vs. 25%; p<0.05), were more often treated with (N)OACs for atrial fibrillation (AF; 54% vs. 38%; p <0.05), and were less often treated with aspirin (35% vs. 51%; p<0.03). Multivariate analysis identified secondary access site (odds ratio [OR] 8.11; p<0.001) and (N)OAC therapy (OR 1.31; p = 0.037) as risk factors for PSA development. PSA is associated with VARC-3 type 1-3 access site bleeding and longer LOS (15.2 ± 11.3 d vs. 11.6 ± 8.9 d; p<0.01), but this did not affect one year mortality (17% vs. 14%; p = 0.53). Conclusions: Pseudoaneurysms are an important complication after TAVI and are associated with access site bleeding and prolonged hospital stay. (N)OAC therapy and secondary access are important risk factors. Pseudoaneurysms can be safely and effectively treated with thrombin injection and do not affect one-year mortality.

Hepatic Amyloid Beta-42-Metabolizing Proteins in Liver Steatosis and Metabolic Dysfunction-Associated Steatohepatitis.

Amyloid beta (Aß) plays a major role in the pathogenesis of Alzheimer's disease and, more recently, has been shown to protect against liver fibrosis. Therefore, we studied Aß-42 levels and the expression of genes involved in the generation, degradation, and transport of Aß proteins in liver samples from patients at different stages of metabolic dysfunction-associated liver disease (MASLD) and under steatotic conditions in vitro/in vivo. Amyloid precursor protein (APP), key Aß-metabolizing proteins, and Aß-42 were analyzed using RT-PCR, Western blotting, Luminex analysis in steatotic in vitro and fatty liver mouse models, and TaqMan qRT-PCR analysis in hepatic samples from patients with MASLD. Hepatocytes loaded with palmitic acid induced APP, presenilin, and neprilysin (NEP) expression, which was reversed by oleic acid. Increased APP and NEP, decreased BACE1, and unchanged Aß-42 protein levels were found in the steatotic mouse liver compared to the normal liver. Aß-42 concentrations were low in MASLD samples of patients with moderate to severe fibrosis compared to the livers of patients with mild or no MASLD. Consistent with the reduced Aß-42 levels, the mRNA expression of proteins involved in APP degradation (ADAM9/10/17, BACE2) and Aß-42 cleavage (MMP2/7/9, ACE) was increased. In the steatotic liver, the expression of APP- and Aß-metabolizing proteins is increased, most likely related to oxidative stress, but does not affect hepatic Aß-42 levels. Consistent with our previous findings, low Aß-42 levels in patients with liver fibrosis appear to be caused by the reduced production and enhanced non-amyloidogenic processing of APP.

Saturated Fat-Mediated Upregulation of IL-32 and CCL20 in Hepatocytes Contributes to Higher Expression of These Fibrosis-Driving Molecules in MASLD.

Metabolic dysfunction-associated steatotic liver disease (MASLD) comprises a spectrum of liver diseases, ranging from liver steatosis to metabolic dysfunction-associated steatohepatitis (MASH), increasing the risk of developing cirrhosis and hepatocellular carcinoma (HCC). Fibrosis within MASLD is critical for disease development; therefore, the identification of fibrosis-driving factors is indispensable. We analyzed the expression of interleukin 32 (IL-32) and chemokine CC ligand 20 (CCL20), which are known to be linked with inflammation and fibrosis, and for their expression in MASLD and hepatoma cells. RT-PCR, ELISA and Western blotting analyses were performed in both human liver samples and an in vitro steatosis model. IL-32 and CCL20 mRNA expression was increased in tissues of patients with NASH compared to normal liver tissue. Stratification for patatin-like phospholipase domain-containing protein 3 (PNPLA3) status revealed significance for IL-32 only in patients with I148M (rs738409, CG/GG) carrier status. Furthermore, a positive correlation was observed between IL-32 expression and steatosis grade, and between IL-32 as well as CCL20 expression and fibrosis grade. Treatment with the saturated fatty acid palmitic acid (PA) induced mRNA and protein expression of IL-32 and CCL20 in hepatoma cells. This induction was mitigated by the substitution of PA with monounsaturated oleic acid (OA), suggesting the involvement of oxidative stress. Consequently, analysis of stress-induced signaling pathways showed the activation of Erk1/2 and p38 MAPK, which led to an enhanced expression of IL-32 and CCL20. In conclusion, cellular stress in liver epithelial cells induced by PA enhances the expression of IL-32 and CCL20, both known to trigger inflammation and fibrosis.

Hepatic Expression of the Na+-Taurocholate Cotransporting Polypeptide Is Independent from Genetic Variation.

The hepatic Na+-taurocholate cotransporting polypeptide NTCP/SLC10A1 is important for the uptake of bile salts and selected drugs. Its inhibition results in increased systemic bile salt concentrations. NTCP is also the entry receptor for the hepatitis B/D virus. We investigated interindividual hepatic SLC10A1/NTCP expression using various omics technologies. SLC10A1/NTCP mRNA expression/protein abundance was quantified in well-characterized 143 human livers by real-time PCR and LC-MS/MS-based targeted proteomics. Genome-wide SNP arrays and SLC10A1 next-generation sequencing were used for genomic analyses. SLC10A1 DNA methylation was assessed through MALDI-TOF MS. Transcriptomics and untargeted metabolomics (UHPLC-Q-TOF-MS) were correlated to identify NTCP-related metabolic pathways. SLC10A1 mRNA and NTCP protein levels varied 44-fold and 10.4-fold, respectively. Non-genetic factors (e.g., smoking, alcohol consumption) influenced significantly NTCP expression. Genetic variants in SLC10A1 or other genes do not explain expression variability which was validated in livers (n = 50) from The Cancer Genome Atlas. The identified two missense SLC10A1 variants did not impair transport function in transfectants. Specific CpG sites in SLC10A1 as well as single metabolic alterations and pathways (e.g., peroxisomal and bile acid synthesis) were significantly associated with expression. Inter-individual variability of NTCP expression is multifactorial with the contribution of clinical factors, DNA methylation, transcriptional regulation as well as hepatic metabolism, but not genetic variation.

Aortic valve calcification is subject to aortic stenosis severity and the underlying flow pattern.

Sex- and flow-related aortic valve calcification (AVC) studies are still limited in number, and data on the exact calcium quantity and distribution are scarce. Therefore, we aimed to (1) re-define the best threshold of AVC load to distinguish severe from moderate aortic stenosis (AS) in common AS entities and to (2) evaluate differences in the aortic annulus and left ventricular outflow tract (LVOT) calcium load. Nine hundred and thirty-eight patients with contrast-enhanced cardiac MSCT and moderate-to-severe aortic stenosis (AS) were retrospectively enrolled. Patients with severe AS ≤ 1.0 cm2 (n = 841) were further separated into three AS entities: high gradient (HGAS, n = 370, 44.0%), paradoxical low gradient (pLGAS, n = 333, 39.6%), and classical low gradient (LGAS, n = 138, 16.4%). AVC, leaflet, and LVOT calcification were quantified. Aortic valve calcification scores were highest in severe HGAS, and lower in severe pLGAS and classical LGAS. In all severity and AS entities, the non-coronary cusp (NCC) was the most calcified one. LVOT calcification was consistently comparable between gender and AS entities. Accuracy of logistic regression was the highest in HGAS (male vs. female: AVC > 2156 Agatston units (AU), c-index 0.76; vs. AVC > 1292 AU, c-index 0.85; or AVC density > 406 AU/cm2, c-index 0.82; vs. > 259 AU/cm2, c-index 0.86; each p < 0.0001*) to diagnose severe AS. AVC could only be used in men to differentiate between severe LGAS and moderate AS. Data from this retrospective analysis indicate that the NCC is subject to pre-dominant degeneration throughout gender, AS severity, and several AS entities. AVC was consistently comparable in severe pLGAS and classical LGAS, but only AVC in severe LGAS could sufficiently distinguish from moderate AS in men. LVOT calcification failed to be a reliable indicator of accelerating AS.

Copy number variation profiling in pharmacogenes using panel-based exome resequencing and correlation to human liver expression.

Structural variants including copy number variations (CNV) have gained widespread attention, especially in pharmacogenomics but for several genes functional relevance and clinical evidence are still lacking. Detection of CNVs in next-generation sequencing data is challenging but offers widespread applications. We developed a cohort-based CNV detection workflow to extract CNVs from read counts of targeted NGS of 340 genes involved in absorption, distribution, metabolism and excretion (ADME) of drugs. We applied our method to 150 human liver tissue samples and correlated identified CNVs to mRNA expression levels. In total, we identified 445 deletions (73%) and 167 duplications (27%) in 36 pharmacogenes including all well-known CNVs of CYPs, GSTs, SULTs, UGTs, numerous described rare CNVs of CYP2E1, SLC16A3 or UGT2B15 as well as novel observations, e.g., for SLC22A12, SLC22A17 and GPS2 (G Protein Pathway Suppressor 2). We were able to fine-map complex CNVs of CYP2A6 and CYP2D6 with exon resolution. Correlation analysis confirmed known expression patterns for common CNVs and suggested an influence on expression variability for some rare CNVs. Our straightforward CNV detection workflow can be easily applied to any NGS coverage data and helped to analyze CNVs in an ADME-NGS panel of 340 pharmacogenes to improve genotype-phenotype correlations.

Factors Affecting Interindividual Variability of Hepatic UGT2B17 Protein Expression Examined Using a Novel Specific Monoclonal Antibody.

Accurate quantification of the metabolic enzyme uridine diphospho-glucuronosyltransferase (UGT) UGT2B17 has been hampered by the high sequence identity with other UGT2B enzymes (as high as 94%) and by the lack of a specific antibody. Knowing the significance of the UGT2B17 pathway in drug and hormone metabolism and cancer, we developed a specific monoclonal antibody (EL-2B17mAb), initially validated by the lack of detection in liver microsomes of an individual carrying no UGT2B17 gene copy and in supersomes expressing UGT2B enzymes. Immunohistochemical detection in livers revealed strong labeling of bile ducts and variable labeling of hepatocytes. Expression levels assessed by immunoblotting were highly correlated to mass spectrometry-based quantification (r = 0.93), and three major expression patterns (absent, low, or high) were evidenced. Livers with very low expression were carriers of the functional rs59678213 G variant, located in the binding site for the transcription factor forkhead box A1 (FOXA1) of the UGT2B17 promoter. The highest level of expression was observed for individuals carrying at least one rs59678213 A allele. Multiple regression analysis indicated that the number of gene copies explained only 8% of UGT2B17 protein expression, 49% when adding rs59678213, reaching 54% when including sex. The novel EL-2B17mAb antibody allowed specific UGT2B17 quantification and exposed different patterns of hepatic expression. It further suggests that FOXA1 is a key driver of UGT2B17 expression in the liver. The availability of this molecular tool will help characterize the UGT2B17 level in various disease states and establish more precisely the contribution of the UGT2B17 enzyme to drug and hormone metabolism.

Direct Quantification of Cytochromes P450 and Drug Transporters-A Rapid, Targeted Mass Spectrometry-Based Immunoassay Panel for Tissues and Cell Culture Lysates.

The quantification of drug metabolizing enzymes and transporters has recently been revolutionized on the basis of targeted proteomic approaches. Isotope-labeled peptides are used as standards for the quantification of the corresponding proteins in enzymatically fragmented samples. However, hurdles in these approaches are low throughput and tedious sample prefractionation steps prior to mass spectrometry (MS) readout. We have developed an assay platform using sensitive and selective immunoprecipitation coupled with mass spectrometric readout allowing the quantification of proteins directly from whole cell lysates using less than 20,000 cells per analysis. Peptide group-specific antibodies (triple X proteomics antibodies) enable the enrichment of proteotypic peptides sharing a common terminus. These antibodies were employed to establish a MS-based immunoassay panel for the quantification of 14 cytochrome P450 (P450) enzymes and nine transporters. We analyzed the P450 enzyme and transporter levels in genotyped liver tissue homogenates and microsomes, and in samples from a time course induction experiment in human hepatocytes addressing different induction pathways. For the analysis of P450 enzymes and transporters only a minute amount of sample is required and no prefractionation is necessary, thus the assay platform bears the potential to bridge cell culture model experiments and results from whole organ tissue studies.

Methyleugenol DNA adducts in human liver are associated with SULT1A1 copy number variations and expression levels.

Methyleugenol is a rodent hepatocarcinogen occurring in many herbs and spices as well as essential oils used for flavoring. Following metabolic activation by cytochromes P450 (CYPs) and sulfotransferases (SULTs), methyleugenol can form DNA adducts. Previously, we showed that DNA adduct formation by methyleugenol in mouse liver is dependent on SULT1A1 expression and that methyleugenol DNA adducts are abundant in human liver specimens. In humans, SULT1A1 activity is affected by genetic polymorphisms, including single-nucleotide polymorphisms (SNPs) and copy number variations (CNVs). Here we investigated the relationship between individual methyleugenol DNA adduct levels and SULT1A1 in human liver samples. Using isotope-dilution ultraperformance liquid chromatography coupled with tandem mass spectrometry, we quantified methyleugenol DNA adducts in 121 human surgical liver samples. Frequent CNVs, including deletions (f = 3.3%) and duplications (f = 36.4%) of SULT1A1, were identified using qPCR and TaqMan assays in the donors' genomic DNA. SULT1A1 mRNA and protein levels were quantified using microarray data and Western blot analysis, respectively. Methyleugenol DNA adducts were detected in all 121 liver samples studied. Their levels varied 122-fold between individuals and were significantly correlated to both mRNA and protein levels of SULT1A1 (r s = 0.43, and r s = 0.44, respectively). Univariate and multivariate statistical analysis identified significant associations of SULT1A1 CNVs with mRNA (p = 1.7 × 10-06) and protein (p = 4.4 × 10- 10) levels as well as methyleugenol DNA adduct levels (p = 0.003). These data establish the importance of SULT1A1 genotype for hepatic methyleugenol DNA adducts in humans, and they confirm a strong impact of SULT1A1 CNVs on SULT1A1 hepatic phenotype.

How Formaldehyde Inhibits Hydrogen Evolution by [FeFe]-Hydrogenases: Determination by ¹³C ENDOR of Direct Fe-C Coordination and Order of Electron and Proton Transfers.

Formaldehyde (HCHO), a strong electrophile and a rapid and reversible inhibitor of hydrogen production by [FeFe]-hydrogenases, is used to identify the point in the catalytic cycle at which a highly reactive metal-hydrido species is formed. Investigations of the reaction of Chlamydomonas reinhardtii [FeFe]-hydrogenase with formaldehyde using pulsed-EPR techniques including electron-nuclear double resonance spectroscopy establish that formaldehyde binds close to the active site. Density functional theory calculations support an inhibited super-reduced state having a short Fe-(13)C bond in the 2Fe subsite. The adduct forms when HCHO is available to compete with H(+) transfer to a vacant, nucleophilic Fe site: had H(+) transfer already occurred, the reaction of HCHO with the Fe-hydrido species would lead to methanol, release of which is not detected. Instead, Fe-bound formaldehyde is a metal-hydrido mimic, a locked, inhibited form analogous to that in which two electrons and only one proton have transferred to the H-cluster. The results provide strong support for a mechanism in which the fastest pathway for H2 evolution involves two consecutive proton transfer steps to the H-cluster following transfer of a second electron to the active site.

Human sterol regulatory element-binding protein 1a contributes significantly to hepatic lipogenic gene expression.

BACKGROUND/AIMS: Sterol regulatory element-binding protein (SREBP) 1, the master regulator of lipogenesis, was shown to be associated with non-alcoholic fatty liver disease, which is attributed to its major isoform SREBP1c. Based on studies in mice, the minor isoform SREBP1a is regarded as negligible for hepatic lipogenesis. This study aims to elucidate the expression and functional role of SREBP1a in human liver. METHODS: mRNA expression of both isoforms was quantified in cohorts of human livers and primary human hepatocytes. Hepatocytes were treated with PF-429242 to inhibit the proteolytic activation of SREBP precursor protein. SREBP1a-specifc and pan-SREBP1 knock-down were performed by transfection of respective siRNAs. Lipogenic SREBP-target gene expression was analyzed by real-time RT-PCR. RESULTS: In human liver, SREBP1a accounts for up to half of the total SREBP1 pool. Treatment with PF-429242 indicated SREBP-dependent auto-regulation of SREBP1a, which however was much weaker than of SREBP1c. SREBP1a-specifc knock-down also reduced significantly the expression of SREBP1c and of SREBP-target genes. Regarding most SREBP-target genes, simultaneous knock-down of both isoforms resulted in effects of only similar extent as SREBP1a-specific knock-down. CONCLUSION: We here showed that SREBP1a is significantly contributing to the human hepatic SREBP1 pool and has a share in human hepatic lipogenic gene expression.

Multiplexed Targeted Quantitative Proteomics Predicts Hepatic Glucuronidation Potential.

Phase II metabolism is prominently governed by UDP-glucuronosyltransferases (UGTs) in humans. These enzymes regulate the bioactivity of many drugs and endogenous small molecules in many organs, including the liver, a major site of regulation by the glucuronidation pathway. This study determined the expression of hepatic UGTs by targeted proteomics in 48 liver samples and by measuring the glucuronidation activity using probe substrates. It demonstrates the sensitivity and accuracy of nano-ultra-performance liquid chromatography with tandem mass spectrometry to establish the complex expression profiles of 14 hepatic UGTs in a single analysis. UGT2B7 is the most abundant UGT in our collection of livers, expressed at 69 pmol/mg microsomal proteins, whereas UGT1A1, UGT1A4, UGT2B4, and UGT2B15 are similarly abundant, averaging 30-34 pmol/mg proteins. The average relative abundance of these five UGTs represents 81% of the measured hepatic UGTs. Our data further highlight the strong relationships in the expression of several UGTs. Most notably, UGT1A4 correlates with most measured UGTs, and the expression levels of UGT2B4/UGT2B7 displayed the strongest correlation. However, significant interindividual variability is observed for all UGTs, both at the level of enzyme concentrations and activity (coefficient of variation: 45%-184%). The reliability of targeted proteomics quantification is supported by the high correlation between UGT concentration and activity. Collectively, these findings expand our understanding of hepatic UGT profiles by establishing absolute hepatic concentrations of 14 UGTs and further suggest coregulated expression between most abundant hepatic UGTs. Data support the value of multiplexed targeted quantitative proteomics to accurately assess specific UGT concentrations in liver samples and hepatic glucuronidation potential.

The truncated splice variant of peroxisome proliferator-activated receptor alpha, PPARα-tr, autonomously regulates proliferative and pro-inflammatory genes.

BACKGROUND: The peroxisome proliferator-activated receptor alpha (PPARα) controls lipid/energy homeostasis and inflammatory responses. The truncated splice variant PPARα-tr was suggested to exert a dominant negative function despite being unable to bind consensus PPARα DNA response elements. METHODS: The distribution and variability factor of each PPARα variant were assessed in the well-characterized cohort of human liver samples (N = 150) on the mRNA and protein levels. Specific siRNA-mediated downregulation of each transcript as well as specific overexpression with subsequent qRT-PCR analysis of downstream genes was used for investigation of specific functional roles of PPARα-wt and PPARα-tr forms in primary human hepatocytes. RESULTS: Bioinformatic analyses of genome-wide liver expression profiling data suggested a possible role of PPARα-tr in downregulating proliferative and pro-inflammatory genes. Specific gene silencing of both forms in primary human hepatocytes showed that induction of metabolic PPARα-target genes by agonist WY14,643 was prevented by PPARα-wt knock-down but neither prevented nor augmented by PPARα-tr knock-down. WY14,643 treatment did not induce proliferative genes including MYC, CDK1, and PCNA, and knock-down of PPARα-wt had no effect, while PPARα-tr knock-down caused up to 3-fold induction of these genes. Similarly, induction of pro-inflammatory genes IL1B, PTGS2, and CCL2 by IL-6 was augmented by knock-down of PPARα-tr but not of PPARα-wt. In contrast to human proliferative genes, orthologous mouse genes were readily inducible by WY14,643 in PPARα-tr non-expressing AML12 mouse hepatocytes. Induction was augmented by overexpression of PPARα-wt and attenuated by overexpression of PPARα-tr. Pro-inflammatory genes including IL-1ß, CCL2 and TNFα were induced by WY14,643 in mouse and human cells and both PPARα forms attenuated induction. As potential mechanism of PPARα-tr inhibitory action we suggest crosstalk with WNT/ß-catenin pathway. Finally, treatment with WY14,643 in the presence of PPARα-tr resulted in the significant reduction of cell viability of AML12 and human ovarian cancer cell line, SKOV3. CONCLUSIONS: Our data suggest that the truncated PPARα splice variant functions as an endogenous inhibitor of proliferative and pro-inflammatory genes in human cells and that its absence in mouse may explain species-specific differences in fibrate-induced hepatocarcinogenesis.

Pregnane X receptor activation and silencing promote steatosis of human hepatic cells by distinct lipogenic mechanisms.

In addition to its well-characterized role in the regulation of drug metabolism and transport by xenobiotics, pregnane X receptor (PXR) critically impacts on lipid homeostasis. In mice, both ligand-dependent activation and knockout of PXR were previously shown to promote hepatic steatosis. To elucidate the respective pathways in human liver, we generated clones of human hepatoma HepG2 cells exhibiting different PXR protein levels, and analyzed effects of PXR activation and knockdown on steatosis and expression of lipogenic genes. Ligand-dependent activation as well as knockdown of PXR resulted in increased steatosis in HepG2 cells. Activation of PXR induced the sterol regulatory element-binding protein (SREBP) 1-dependent lipogenic pathway via PXR-dependent induction of SREBP1a, which was confirmed in primary human hepatocytes. Inhibiting SREBP1 activity by blocking the cleavage-dependent maturation of SREBP1 protein impaired the induction of lipogenic SREBP1 target genes and triglyceride accumulation by PXR activation. On the other hand, PXR knockdown resulted in up-regulation of aldo-keto reductase (AKR) 1B10, which enhanced the acetyl-CoA carboxylase (ACC)-catalyzed reaction step of de novo lipogenesis. In a cohort of human liver samples histologically classified for non-alcoholic fatty liver disease, AKR1B10, SREBP1a and SREBP1 lipogenic target genes proved to be up-regulated in steatohepatitis, while PXR protein was reduced. In summary, our data suggest that activation and knockdown of PXR in human hepatic cells promote de novo lipogenesis and steatosis by induction of the SREBP1 pathway and AKR1B10-mediated increase of ACC activity, respectively, thus providing mechanistic explanations for a putative dual role of PXR in the pathogenesis of steatohepatitis.

Function and localization dynamics of bifunctional penicillin-binding proteins in Caulobacter crescentus.

The peptidoglycan cell wall of bacteria is a complex macromolecule composed of glycan strands that are cross-linked by short peptide bridges. Its biosynthesis involves a conserved group of enzymes, the bifunctional penicillin-binding proteins (bPBPs), which contain both a transglycosylase and a transpeptidase domain, thus being able to elongate the glycan strands and, at the same time, generate the peptide cross-links. The stalked model bacterium Caulobacter crescentus possesses five bPBP paralogs, named Pbp1A, PbpC, PbpX, PbpY, and PbpZ, whose function is still incompletely understood. In this study, we show that any of these proteins except for PbpZ is sufficient for growth and normal morphogenesis when expressed at native or elevated levels, whereas inactivation of all five paralogs is lethal. Growth analyses indicate a central role of PbpX in the resistance of C. crescentus against the noncanonical amino acid d-alanine. Moreover, we show that PbpX and PbpY localize to the cell division site. Their recruitment to the divisome is dependent on the essential cell division protein FtsN and likely involves interactions with FtsL and the putative peptidoglycan hydrolase DipM. The same interaction pattern is observed for Pbp1A and PbpC, although these proteins do not accumulate at midcell. Our findings demonstrate that the bPBPs of C. crescentus are, to a large extent, redundant and have retained the ability to interact with the peptidoglycan biosynthetic machineries responsible for cell elongation, cytokinesis, and stalk growth. Nevertheless, they may preferentially act in specific peptidoglycan biosynthetic complexes, thereby facilitating the independent regulation of distinct growth processes.

Isoniazid mediates the CYP2B6*6 genotype-dependent interaction between efavirenz and antituberculosis drug therapy through mechanism-based inactivation of CYP2A6.

Efavirenz is commonly used to treat patients coinfected with human immunodeficiency virus and tuberculosis. Previous clinical studies have observed paradoxically elevated efavirenz plasma concentrations in patients with the CYP2B6*6/*6 genotype (but not the CYP2B6*1/*1 genotype) during coadministration with the commonly used four-drug antituberculosis therapy. This study sought to elucidate the mechanism underlying this genotype-dependent drug-drug interaction. In vitro studies were conducted to determine whether one or more of the antituberculosis drugs (rifampin, isoniazid, pyrazinamide, or ethambutol) potently inhibit efavirenz 8-hydroxylation by CYP2B6 or efavirenz 7-hydroxylation by CYP2A6, the main mechanisms of efavirenz clearance. Time- and concentration-dependent kinetics of inhibition by the antituberculosis drugs were determined using genotyped human liver microsomes (HLMs) and recombinant CYP2A6, CYP2B6.1, and CYP2B6.6 enzymes. Although none of the antituberculosis drugs evaluated at up to 10 times clinical plasma concentrations were found to inhibit efavirenz 8-hydroxylation by HLMs, both rifampin (apparent inhibition constant [Ki] = 368 µM) and pyrazinamide (Ki = 637 µM) showed relatively weak inhibition of efavirenz 7-hydroxylation. Importantly, isoniazid demonstrated potent time-dependent inhibition of efavirenz 7-hydroxylation in both HLMs (inhibitor concentration required for half-maximal inactivation [KI] = 30 µM; maximal rate constant of inactivation [kinact] = 0.023 min(-1)) and recombinant CYP2A6 (KI = 15 µM; kinact = 0.024 min(-1)) and also formed a metabolite intermediate complex consistent with mechanism-based inhibition. Selective inhibition of the CYP2B6.6 allozyme could not be demonstrated for any of the antituberculosis drugs using either recombinant enzymes or CYP2B6*6 genotype HLMs. In conclusion, the results of this study identify isoniazid as the most likely perpetrator of this clinically important drug-drug interaction through mechanism-based inactivation of CYP2A6.

Targeting nuclear receptors with lentivirus-delivered small RNAs in primary human hepatocytes.

BACKGROUND: RNA interference (RNAi) has tremendous potential for investigating gene function and for developing new therapies. Primary human hepatocytes (PHH) are the "gold standard" for studying the regulation of hepatic metabolism in vitro. However, application of RNAi in PHH has some technical hurdles. The objective of this study was to develop effective and robust protocol for transduction of PHH with lentiviral vectors. METHODS: We used lentiviral vectors to transduce PHH for introduction of short hairpin RNAs (shRNAs) targeting constitutive androstane receptor (CAR), peroxisome proliferator activated receptor alpha (PPARα), and microRNA, miR-143. Infection efficiency was quantitatively analyzed by flow cytometry and microscopy. Target gene expression was assessed using quantitative real-time (qRT-PCR) method. RESULTS: Lentiviral vector transduction resulted in ≥95% of infected cells at low multiplicity of infection (MOI) of 3, which did not impair cellular viability. We demonstrated the feasibility of this technique in studies on targeting nuclear receptors, PPARα and CAR, with shRNAs as well as in lentivirus-mediated overexpression and knock-down of miRNA-143 experiments. CONCLUSIONS: We developed an efficient and robust protocol with standardized procedures for virus production, method of titer determination, and infection procedure for RNAi in primary human hepatocytes based on delivery of shRNAs, microRNAs or anti-microRNAs in different laboratory settings. This approach should be useful to study not only the regulation via nuclear receptors but also other biological, pharmacological, and toxicological aspects of drug metabolism.

The choice of ultra-low attachment plates impacts primary human and primary canine hepatocyte spheroid formation, phenotypes, and function.

Organotypic three-dimensional liver spheroid cultures in which hepatic cells retain their molecular phenotype and functionality have emerged as powerful tools for preclinical drug development. In recent years a multitude of culture systems have been developed; however, a thorough side-by-side benchmarking of the different methods is lacking. Here, we compared the performance of ten different 96- and 384-well microplate types to support spheroid formation and long-term culture. Specifically, we evaluated differences in spheroid formation kinetics, viability, functionality, expression patterns, and their utility for hepatotoxicity assessments using primary human hepatocytes (PHH) and primary canine hepatocytes (PCH). All 96-well plates enabled formation of PHH liver spheroids, albeit with differences between plates in spheroid size, geometry, and reproducibility. Performance of different 384-wells was less consistent. Only 6/10 microplates supported the formation of PCH aggregates. Interestingly, even if PCH aggregates in these six microplates were more loosely packed than PHH spheroids, they maintained their function and were compatible with long-term pharmacological and toxicological assays. Overall, Corning and Biofloat plates showed the best performance in the formation of both human and canine liver spheroids with highest viability, most physiologically relevant phenotypes, superior CYP activity and lowest coefficient of variation in toxicity assays. The presented data constitutes a valuable resource that demonstrates the impacts of current ultra-low attachment plates on liver spheroid metrics and can guide evidence-based plate selection. Combined, these results have important implications for the cross-comparison of different studies and can facilitate the standardization and reproducibility of three-dimensional liver culture experiments.

In silico and biological analyses of missense variants of the human biliary efflux transporter ABCC2: effects of novel rare missense variants.

BACKGROUND AND PURPOSE: The ATP-dependent biliary efflux transporter ABCC2, also known as multidrug resistance protein 2 (MRP2), is essential for the cellular disposition and detoxification of various xenobiotics including drugs as well as endogenous metabolites. Common functionally relevant ABCC2 genetic variants significantly alter drug responses and contribute to side effects. The aim of this study was to determine functional consequences of rare variants identified in subjects with European ancestry using in silico tools and in vitro analyses. EXPERIMENTAL APPROACH: Targeted next-generation sequencing of the ABCC2 gene was used to identify novel variants in European subjects (n = 143). Twenty-six in silico tools were used to predict functional consequences. For biological validation, transport assays were carried out with membrane vesicles prepared from cell lines overexpressing the newly identified ABCC2 variants and estradiol ß-glucuronide and carboxydichlorofluorescein as the substrates. KEY RESULTS: Three novel rare ABCC2 missense variants were identified (W227R, K402T, V489F). Twenty-five in silico tools predicted W227R as damaging and one as potentially damaging. Prediction of functional consequences was not possible for K402T and V489F and for the common linked variants V1188E/C1515Y. Characterisation in vitro showed increased function of W227R, V489F and V1188E/C1515Y for both substrates, whereas K402T function was only increased for carboxydichlorofluorescein. CONCLUSION AND IMPLICATIONS: In silico tools were unable to accurately predict the substrate-dependent increase in function of ABCC2 missense variants. In vitro biological studies are required to accurately determine functional activity to avoid misleading consequences for drug therapy.

Echocardiographic-Fluoroscopic Fusion Imaging Improves Interventionalists' Learning Curve for Percutaneous Left Atrial Appendage Closure-Initial, Single-Center, Retrospective Observations.

Due to the complex and variable anatomy of the left atrial appendage, percutaneous left atrial appendage closure (LAAC) can be challenging. In this study, we investigated the impact of fusion imaging (FI) on the LAAC learning curve of two interventionalists. The first interventionalist (IC 1) was initially trained without FI and continued his training with FI. The second interventionalist (IC 2) performed all procedures with FI. We compared the first 36 procedures without FI of IC 1 (group 1) with his next 36 interventions with FI (group 2). Furthermore, group 1 was compared to 36 procedures of IC 2 who directly started his training with FI (group 3). Group 1 demonstrated that the learning curve without FI has a flat course with weak correlations for fluoroscopy time, contrast volume, and procedure time, but not for dose area product. Group 2 with FI showed improvement with a steep course and strong correlations for all four parameters. In group 3, we also saw a steep progression with strong correlations. Furthermore, the mean measurements of the parameters in the groups with FI decreased significantly as an indicator of procedural efficacy. We demonstrated that FI may improve the learning curve of experienced and non-experienced ICs.