Description: Enzymatic conversion of most xenobiotic compounds is accomplished by hepatocytes in the liver, which are also an important target for the manifestation of the toxic effects of foreign compounds. Most cell lines derived from hepatocytes lack important toxifying or detoxifying enzymes or are defective in signaling pathways that regulate expression and activity of these enzymes. On the other hand, the use of primary human hepatocytes is complicated by scarce availability of cells and high interdonor variability. Thus, analyses of drug metabolism and hepatotoxicity in vitro are a difficult task. The cell line HC-AFW1 was isolated from a pediatric hepatocellular carcinoma and so far has been used for tumorigenicity and chemotherapy resistance studies. Here, a comprehensive characterization of xenobiotic metabolism in HC-AFW1 cells is presented along with studies on the functionality of the most important transcriptional regulators of drug-metabolizing enzymes. Results from HC-AFW1 cells were compared with commercially available HepaRG cells and cultured primary human hepatocytes. Data show that the nuclear receptors and xenosensors AHR (aryl hydrocarbon receptor), CAR (constitutive androstane receptor), PXR (pregnane-X-receptor), NRF2 [nuclear factor (erythroid-derived 2)–like 2], and PPAR α (peroxisome proliferator–activated receptor α ) are functional in HC-AFW1 cells, comparable to HepaRG and primary cells. HC-AFW1 cells possess considerable activities of different cytochrome P450 enzymes, which, however, are lower than corresponding enzyme activities in HepaRG cells or primary hepatocytes. In summary, HC-AFW1 are a new promising tool for studying the mechanisms of the regulation of drug metabolism in human liver cells in vitro.

Description: Genes involved in detoxification of foreign compounds exhibit complex spatiotemporal expression patterns in liver. Cytochrome P450 1A1 ( CYP1A1 ), for example, is restricted to the pericentral region of liver lobules in response to the interplay between aryl hydrocarbon receptor (AhR) and Wnt/β-catenin signaling pathways. However, the mechanisms by which the two pathways orchestrate gene expression are still poorly understood. With the help of 29 mutant constructs of the human CYP1A1 promoter and a mathematical model that combines Wnt/β-catenin and AhR signaling with the statistical mechanics of the promoter, we systematically quantified the regulatory influence of different transcription factor binding sites on gene induction within the promoter. The model unveils how different binding sites cooperate and how they establish the promoter logic; it quantitatively predicts two-dimensional stimulus-response curves. Furthermore, it shows that crosstalk between Wnt/β-catenin and AhR signaling is crucial to understand the complex zonated expression patterns found in liver lobules. This study exemplifies how statistical mechanical modeling together with combinatorial reporter assays has the capacity to disentangle the promoter logic that establishes physiological gene expression patterns.

Description: Spider venoms are a rich source of ion channel modulators with therapeutic potential. Given the analgesic potential of subtype-selective inhibitors of voltage-gated sodium (Na V ) channels, we screened spider venoms for inhibitors of human Na V 1.7 (hNa V 1.7) using a high-throughput fluorescent assay. Here, we describe the discovery of a novel Na V 1.7 inhibitor, μ -TRTX-Tp1a (Tp1a), isolated from the venom of the Peruvian green-velvet tarantula Thrixopelma pruriens . Recombinant and synthetic forms of this 33-residue peptide preferentially inhibited hNa V 1.7 〉 hNa V 1.6 〉 hNa V 1.2 〉 hNa V 1.1 〉 hNa V 1.3 channels in fluorescent assays. Na V 1.7 inhibition was diminished (IC 50 11.5 nM) and the association rate decreased for the C-terminal acid form of Tp1a compared with the native amidated form (IC 50 2.1 nM), suggesting that the peptide C terminus contributes to its interaction with hNa V 1.7. Tp1a had no effect on human voltage-gated calcium channels or nicotinic acetylcholine receptors at 5 μ M. Unlike most spider toxins that modulate Na V channels, Tp1a inhibited hNa V 1.7 without significantly altering the voltage dependence of activation or inactivation. Tp1a proved to be analgesic by reversing spontaneous pain induced in mice by intraplantar injection in OD1, a scorpion toxin that potentiates hNa V 1.7. The structure of Tp1a as determined using NMR spectroscopy revealed a classic inhibitor cystine knot (ICK) motif. The molecular surface of Tp1a presents a hydrophobic patch surrounded by positively charged residues, with subtle differences from other ICK spider toxins that might contribute to its different pharmacological profile. Tp1a may help guide the development of more selective and potent hNa V 1.7 inhibitors for treatment of chronic pain.

Description: The tumor-suppressive let-7 microRNA family targets various oncogene-encoding mRNAs. We identify the let-7 targets HMGA2, LIN28B and IGF2BP1 to form a let-7 antagonizing self-promoting oncogenic triangle. Surprisingly, 3'-end processing of IGF2BP1 mRNAs is unaltered in aggressive cancers and tumor-derived cells although IGF2BP1 synthesis was proposed to escape let-7 attack by APA-dependent (alternative polyadenylation) 3' UTR shortening. However, the expression of the triangle factors is inversely correlated with let-7 levels and promoted by LIN28B impairing let-7 biogenesis. Moreover, IGF2BP1 enhances the expression of all triangle factors by recruiting the respective mRNAs in mRNPs lacking AGO proteins and let-7 miRNAs. This indicates that the downregulation of let-7, largely facilitated by LIN28B upregulation, and the protection of let-7 target mRNAs by IGF2BP1-directed shielding in mRNPs synergize in enhancing the expression of triangle factors. The oncogenic potential of this triangle was confirmed in ovarian cancer (OC)-derived ES-2 cells transduced with let-7 targeting decoys. In these, the depletion of HMGA2 only diminishes tumor cell growth under permissive conditions. The depletion of LIN28B and more prominently IGF2BP1 severely impairs tumor cell viability, self-renewal and 2D as well as 3D migration. In conclusion, this suggests the targeting of the HMGA2-LIN28B-IGF2BP1 triangle as a promising strategy in cancer treatment.

Description: SUMMARY We propose a class of spherical wavelet bases for the analysis of geophysical models and for the tomographic inversion of global seismic data. Its multiresolution character allows for modelling with an effective spatial resolution that varies with position within the Earth. Our procedure is numerically efficient and can be implemented with parallel computing. We discuss two possible types of discrete wavelet transforms in the angular dimension of the cubed sphere. We describe benefits and drawbacks of these constructions and apply them to analyse the information in two published seismic wave speed models of the mantle, using the statistics of wavelet coefficients across scales. The localization and sparsity properties of wavelet bases allow finding a sparse solution to inverse problems by iterative minimization of a combination of the ℓ 2 norm of the data residuals and the ℓ 1 norm of the model wavelet coefficients. By validation with realistic synthetic experiments we illustrate the likely gains from our new approach in future inversions of finite-frequency seismic data.

Description: Histone post-translational modifications play an important role in regulating chromatin structure and gene expression in vivo . Extensive studies investigated the post-translational modifications of the core histones H3 and H4 or the linker histone H1. Much less is known on the regulation of H2A and H2B modifications. Here, we show that a major modification of H2B in Drosophila melanogaster is the methylation of the N-terminal proline, which increases during fly development. Experiments performed in cultured cells revealed higher levels of H2B methylation when cells are dense, regardless of their cell cycle distribution. We identified dNTMT (CG1675) as the enzyme responsible for H2B methylation. We also found that the level of N-terminal methylation is regulated by dART8, an arginine methyltransferase that physically interacts with dNTMT and asymmetrically methylates H3R2. Our results demonstrate the existence of a complex containing two methyltransferases enzymes, which negatively influence each other’s activity.

Description: We evaluated the impact of staging procedures to detect asymptomatic distant metastases (DM) in the management of women with operable invasive breast cancer (BC, entire cohort: n = 866). Out of 472 patients with lymph node (LN)-negative disease (pN0), DM were found in four cases (detection rate: 0.8%). All four patients presented with established risk factors: hormone receptor (HR)-negative status, HER2-positive status, n = 3; ‘triple-negative’ disease, n = 1. Considering the subgroup of LN-negative patients whose tumors showed the risk factor ‘negative HR status’ ( n = 66), the detection rate of DM was 6%. The detection rates of DM in higher pN categories were as follows: pN1:1.7%; pN2:9.5%; pN3:13.5%. We generally support the international guidelines, including those published by the European Society for Medical Oncology (ESMO) which emphasize that patients with early-stage BC do not profit from radiological staging for the detection of DM and recommend refraining from this. However, we would expand these guidelines and propose that screening should be carried out in node-negative patients whose tumors show established tumor-related risk factors (e.g. HR-negative and HER2-positive status), since in this particular subcohort, the detection rate of DM is with 6% similarly high as that of patients with four to nine positive LNs.

Description: The National Cancer Institute (NCI) Symptom Management and Health-Related Quality of Life Steering Committee convened four working groups to recommend core sets of patient-reported outcomes to be routinely incorporated in clinical trials. The Prostate Cancer Working Group included physicians, researchers, and a patient advocate. The group’s process included 1) a systematic literature review to determine the prevalence and severity of symptoms, 2) a multistakeholder meeting sponsored by the NCI to review the evidence and build consensus, and 3) a postmeeting expert panel synthesis of findings to finalize recommendations. Five domains were recommended for localized prostate cancer: urinary incontinence, urinary obstruction and irritation, bowel-related symptoms, sexual dysfunction, and hormonal symptoms. Four domains were recommended for advanced prostate cancer: pain, fatigue, mental well-being, and physical well-being. Additional domains for consideration include decisional regret, satisfaction with care, and anxiety related to prostate cancer. These recommendations have been endorsed by the NCI for implementation.

Description: Background Peripheral neuropathy is the major dose-limiting side effect of cisplatin and oxaliplatin, and there are currently no effective treatments available. The aim of this study was to assess the pharmacological mechanisms underlying chemotherapy-induced neuropathy in novel animal models based on intraplantar administration of cisplatin and oxaliplatin and to systematically evaluate the analgesic efficacy of a range of therapeutics. Methods Neuropathy was induced by a single intraplantar injection of cisplatin or oxaliplatin in C57BL/6J mice and assessed by quantification of mechanical and thermal allodynia. The pharmacological basis of cisplatin-induced neuropathy was characterized using a range of selective pharmacological inhibitors. The analgesic effects of phenytoin, amitriptyline, oxcarbazepine, mexiletine, topiramate, retigabine, gabapentin, fentanyl, and Ca 2+/ Mg 2+ were assessed 24 hours after induction of neuropathy. Results Intraplantar administration of cisplatin led to the development of mechanical allodynia, mediated through Na v 1.6-expressing sensory neurons. Unlike intraplantar injection of oxaliplatin, cold allodynia was not observed with cisplatin, consistent with clinical observations. Surprisingly, only fentanyl was effective at alleviating cisplatin-induced mechanical allodynia despite a lack of efficacy in oxaliplatin-induced cold allodynia. Conversely, lamotrigine, phenytoin, retigabine, and gabapentin were effective at reversing oxaliplatin-induced cold allodynia but had no effect on cisplatin-induced mechanical allodynia. Oxcarbazepine, amitriptyline, mexiletine, and topiramate lacked efficacy in both models of acute chemotherapy-induced neuropathy. Conclusion This study established a novel animal model of cisplatin-induced mechanical allodynia consistent with the A-fiber neuropathy seen clinically. Systematic assessment of a range of therapeutics identified several candidates that warrant further clinical investigation.

Description: The WNT/ β -catenin signaling pathway has been identified as an important endogenous regulator of hepatic cytochrome P450 (P450) expression in mouse liver. In particular, it is involved in the regulation of P450 expression in response to exposure to xenobiotic agonists of the nuclear receptors constitutive androstane receptor (CAR), aryl hydrocarbon receptor (AhR), and Nrf2. To systematically elucidate the effect of the WNT/ β -catenin pathway on the regulation and inducibility of major human P450 enzymes, HepaRG cells were treated with either the WNT/ β -catenin signaling pathway agonist, WNT3a, or with small interfering RNA directed against β -catenin, alone or in combination with a panel of activating ligands for AhR [2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD)], CAR [6-(4-chlorophenyl)imidazo[2,1- b ][1,3]thiazole-5-carbaldehyde- O -(3,4-dichlorobenzyl)oxime (CITCO)], pregnane X receptor (PXR) [rifampicin], and peroxisome proliferator–activated receptor (PPAR) α [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid (WY14,643)]. Assessment of P450 gene expression and enzymatic activity after downregulation or activation of the WNT/ β -catenin pathway revealed a requirement of β -catenin in the AhR-, CAR-, and PXR-mediated induction of CYP1A, CYP2B6 and CYP3A4 (for CAR and PXR), and CYP2C8 (for PXR) gene expression. By contrast, activation of the WNT/ β -catenin pathway prevented PPAR α -mediated induction of CYP1A, CYP2C8, CYP3A4, and CYP4A11 genes, suggesting a dominant-negative role of β -catenin in PPAR α -mediated regulation of these genes. Our data indicate a significant effect of the WNT/ β -catenin pathway on the regulation of P450 enzymes in human hepatocytes and reveal a novel crosstalk between β -catenin and PPAR α signaling pathways in the regulation of P450 expression.