Background: The liver-derived plasma protein fetuin-A is a systemic inhibitor of ectopic calcification. Fetuin-A stabilizes saturated mineral solutions by forming colloidal protein-mineral complexes called calciprotein particles (CPP). CPP are initially spherical, amorphous and soft, and are referred to as primary CPP. These particles spontaneously convert into secondary CPP, which are larger, oblongate, more crystalline, and less soluble. CPP mediate excess mineral transport and clearance from circulation.Methods: We studied by intravital two-photon microscopy the clearance of primary vs. secondary CPP by injecting i.v. synthetic fluorescent CPP in mice. We analyzed CPP organ distribution and identified CPP endocytosing cells by immunofluorescence. Cellular clearance was studied using bone marrow-derived mouse wildtype and scavenger receptor A (SRA)-deficient macrophages, as well as human umbilical cord endothelial cells (HUVEC), monocyte-derived macrophages (hMDM), and human aortic endothelial cells (haEC). We employed mouse wildtype and mutant immortalized macrophages to analyze CPP-induced inflammasome activation and cytokine secretion.Results: In live mice, only primary CPP were rapidly cleared by liver sinusoidal endothelial cells (LSEC), whereas primary and secondary CPP were cleared by Kupffer cells. Scavenger receptor A (SRA)-deficient bone marrow macrophages endocytosed secondary CPP less well than did wildtype macrophages. In contrast, primary CPP endocytosis did not depend on the presence of SRA, suggesting involvement of an alternative clearance pathway. CPP triggered TLR4 dependent TNFα and IL-1β secretion in cultured macrophages. Calcium content-matched primary CPP caused twice more IL-1β secretion than did secondary CPP, which was associated with increased calcium-dependent inflammasome activation, suggesting that intracellular CPP dissolution and calcium overload may cause this inflammation.Conclusions: Secondary CPP are endocytosed by macrophages in liver and spleen via SRA. In contrast, our results suggest that primary CPP are cleared by LSEC via an alternative pathway. CPP induced TLR4-dependent TNFα and inflammasome-dependent IL-1β secretion in macrophages suggesting that inflammation and calcification may be considered consequences of prolonged CPP presence and clearance.

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Recently, epidemiological studies have suggested that fluoride is a human developmental neurotoxicant that reduces measures of intelligence in children, placing it into the same category as toxic metals (lead, methylmercury, arsenic) and polychlorinated biphenyls. If true, this assessment would be highly relevant considering the widespread fluoridation of drinking water and the worldwide use of fluoride in oral hygiene products such as toothpaste. To gain a deeper understanding of these assertions, we reviewed the levels of human exposure, as well as results from animal experiments, particularly focusing on developmental toxicity, and the molecular mechanisms by which fluoride can cause adverse effects. Moreover, in vitro studies investigating fluoride in neuronal cells and precursor/stem cells were analyzed, and 23 epidemiological studies published since 2012 were considered. The results show that the margin of exposure (MoE) between no observed adverse effect levels (NOAELs) in animal studies and the current adequate intake (AI) of fluoride (50 µg/kg b.w./day) in humans ranges between 50 and 210, depending on the specific animal experiment used as reference. Even for unusually high fluoride exposure levels, an MoE of at least ten was obtained. Furthermore, concentrations of fluoride in human plasma are much lower than fluoride concentrations, causing effects in cell cultures. In contrast, 21 of 23 recent epidemiological studies report an association between high fluoride exposure and reduced intelligence. The discrepancy between experimental and epidemiological evidence may be reconciled with deficiencies inherent in most of these epidemiological studies on a putative association between fluoride and intelligence, especially with respect to adequate consideration of potential confounding factors, e.g., socioeconomic status, residence, breast feeding, low birth weight, maternal intelligence, and exposure to other neurotoxic chemicals. In conclusion, based on the totality of currently available scientific evidence, the present review does not support the presumption that fluoride should be assessed as a human developmental neurotoxicant at the current exposure levels in Europe.

BackgroundPartially methylated domains are extended regions in the genome exhibiting a reduced average DNA methylation level. They cover gene-poor and transcriptionally inactive regions and tend to be heterochromatic. We present a comprehensive comparative analysis of partially methylated domains in human and mouse cells, to identify structural and functional features associated with them.ResultsPartially methylated domains are present in up to 75% of the genome in human and mouse cells irrespective of their tissue or cell origin. Each cell type has a distinct set of partially methylated domains, and genes expressed in such domains show a strong cell type effect. The methylation level varies between cell types with a more pronounced effect in differentiating and replicating cells. The lowest level of methylation is observed in highly proliferating and immortal cancer cell lines. A decrease of DNA methylation within partially methylated domains tends to be linked to an increase in heterochromatic histone marks and a decrease of gene expression. Characteristic combinations of heterochromatic signatures in partially methylated domains are linked to domains of early and middle S-phase and late S-G2 phases of DNA replication.ConclusionsPartially methylated domains are prominent signatures of long-range epigenomic organization. Integrative analysis identifies them as important general, lineage- and cell type-specific topological features. Changes in partially methylated domains are hallmarks of cell differentiation, with decreased methylation levels and increased heterochromatic marks being linked to enhanced cell proliferation. In combination with broad histone marks, partially methylated domains demarcate distinct domains of late DNA replication.Electronic supplementary materialThe online version of this article (10.1186/s13059-018-1510-5) contains supplementary material, which is available to authorized users.

Bile duct ligation (BDL) is an experimental procedure that mimics obstructive cholestatic disease. One of the early consequences of BDL in rodents is the appearance of so-called bile infarcts that correspond to Charcot-Gombault necrosis in human cholestasis. The mechanisms causing bile infarcts and their pathophysiological relevance are unclear. Therefore, intravital two photon-based imaging of BDL mice was performed with fluorescent bile salts (BS) and non-BS organic anion analogues. Key findings were followed up by matrix-assisted laser desorption ionization imaging, clinical chemistry, immunostaining, and gene expression analyses. In the acute phase, 1-3 days after BDL, BS concentrations in bile increased and single-cell bile microinfarcts occurred in dispersed hepatocytes throughout the liver caused by the rupture of the apical hepatocyte membrane. This rupture occurred after loss of mitochondrial membrane potential, followed by entry of bile, cell death, and a "domino effect" of further death events of neighboring hepatocytes. Bile infarcts provided a trans-epithelial shunt between bile canaliculi and sinusoids by which bile constituents leaked into blood. In the chronic phase, ≥21 days after BDL, uptake of BS tracers at the sinusoidal hepatocyte membrane was reduced. This contributes to elevated concentrations of BS in blood and decreased concentrations in the biliary tract. Conclusion: Bile microinfarcts occur in the acute phase after BDL in a limited number of dispersed hepatocytes followed by larger infarcts involving neighboring hepatocytes, and they allow leakage of bile from the BS-overloaded biliary tract into blood, thereby protecting the liver from BS toxicity; in the chronic phase after BDL, reduced sinusoidal BS uptake is a dominant protective factor, and the kidney contributes to the elimination of BS until cholemic nephropathy sets in.

Aberrations within the PI3K/AKT signaling axis are frequently observed in numerous cancer types, highlighting the relevance of these pathways in cancer physiology and pathology. However, therapeutic interventions employing AKT inhibitors often suffer from limitations associated with target selectivity, efficacy, or dose-limiting effects. Here we present the first crystal structure of autoinhibited AKT1 in complex with the covalent-allosteric inhibitor borussertib, providing critical insights into the structural basis of AKT1 inhibition by this unique class of compounds. Comprehensive biological and preclinical evaluation of borussertib in cancer-related model systems demonstrated a strong anti-proliferative activity in cancer cell lines harboring genetic alterations within the PTEN, PI3K, and RAS signaling pathways. Furthermore, borussertib displayed antitumor activity in combination with the MEK inhibitor trametinib in patient-derived xenograft models of mutant KRAS pancreatic and colon cancer. Significance: Borussertib, a first-in-class covalent-allosteric AKT inhibitor, displays antitumor activity in combination with the MEK inhibitor trametinib in patient-derived xenograft models and provides a starting point for further pharmacokinetic/dynamic optimization.

Interferons are crucial for adaptive immunity and play an important role in the immune landscape of breast cancer. Using microarray-based gene expression analysis, we examined the subtype-specific prognostic significance of interferon-γ (IFN-γ) as a single gene as well as an IFN-γ signature covering the signaling pathway in 461 breast cancer patients. Prognostic significance of IFN-γ, as well as the IFN-γ signature for metastasis-free survival (MFS), were examined using Kaplan–Meier as well as univariate and multivariate Cox regression analyses in the whole cohort and in different molecular subtypes. The independent prognostic significance of IFN-γ as a single gene was limited to basal-like breast cancer (hazard ratio (HR) 2.779, 95% confidence interval (95% CI) 1.117–6.919, p = 0.028). In contrast, the IFN-γ-associated gene signature was an independent prognostic factor in the whole cohort (HR 2.287, 95% CI 1.410–3.633, p < 0.001) as well as in the basal-like (HR 3.458, 95% CI 1.154–10.359, p = 0.027) and luminal B (HR 2.690, 95% CI 1.416–5.112, p = 0.003) molecular subtypes. These results underline the subtype-dependent prognostic influence of the immune system in early breast cancer.