Insulin Determines Transforming Growth Factor ß Effects on Hepatocyte Nuclear Factor 4α Transcription in Hepatocytes.

Loss of hepatocyte nuclear factor 4α (HNF4α) expression is frequently observed in end-stage liver disease and associated with loss of vital liver functions, thus increasing mortality. Loss of HNF4α expression is mediated by inflammatory cytokines, such as transforming growth factor (TGF)-ß. However, details of how HNF4α is suppressed are largely unknown to date. Herein, TGF-ß did not directly inhibit HNF4α but contributed to its transcriptional regulation by SMAD2/3 recruiting acetyltransferase CREB-binding protein/p300 to the HNF4α promoter. The recruitment of CREB-binding protein/p300 is indispensable for CCAAT/enhancer-binding protein α (C/EBPα) binding, another essential requirement for constitutive HNF4α expression in hepatocytes. Consistent with the in vitro observation, 67 of 98 patients with hepatic HNF4α expressed both phospho-SMAD2 and C/EBPα, whereas 22 patients without HNF4α expression lacked either phospho-SMAD2 or C/EBPα. In contrast to the observed induction of HNF4α, SMAD2/3 inhibited C/EBPα transcription. Long-term TGF-ß incubation resulted in C/EBPα depletion, which abrogated HNF4α expression. Intriguingly, SMAD2/3 inhibitory binding to the C/EBPα promoter was abolished by insulin. Two-thirds of patients without C/EBPα lacked membrane glucose transporter type 2 expression in hepatocytes, indicating insulin resistance. Taken together, these data indicate that hepatic insulin sensitivity is essential for hepatic HNF4α expression in the condition of inflammation.

Intra- and extracellular real-time analysis of perfused fibroblasts using an NMR bioreactor: A pilot study.

INTRODUCTION: Metabolomic discrimination of different mitochondrial defects is challenging. We describe an NMR-based bioreactor allowing real-time intra- and extracellular metabolic investigation of perfused fibroblasts. OBJECTIVES: The objective of this study is (I) determining whether metabolic investigations of perfused fibroblasts overall and separated for intra- and extracellular contributions by real-time NMR allows for discrimination of different representative mitochondrial defects in a feasibility study and (II) gaining insight into physiological consequences of mitochondrial dysfunction in basal condition and during glycolysis inhibition. METHODS: Overall, intra- and extracellular metabolomes of malate dehydrogenase 2 (MDH2), pyruvate dehydrogenase (PDH), complex I (CI) deficient fibroblasts, and control fibroblasts were investigated under standard culture conditions and under glycolysis inhibition. In addition to "overall" metabolite quantification, intra- and extracellular metabolic contributions were separated based on diffusion rate differences. RESULTS AND DISCUSSION: Overall metabolites: Chemometric analysis of the entire metabolome revealed good separation between control, PDH and MDH2, while CI was less well separated. However, mixed intra- and extracellular changes complicated interpretation of the cellular metabolism. Intra- and extracellular metabolites: Compartment specific chemometrics revealed possibly augmenting metabolomic separation between control and deficient cell lines under basal and inhibition condition. All mitochondrial defects exhibited upregulation of glycolytic metabolism compared to controls. Inhibition of glycolysis resulted in perturbations of other metabolic pathways such as glutaminolysis, alanine, arginine, glutamate, and proline metabolism. MDH2 showed upregulation of alanine and glutamate metabolism, while the CI defect revealed lower intracellular arginine and downregulation of glutamate and arginine-dependent proline synthesis. CONCLUSION: Discrimination of intra- and extracellular metabolic contributions helps understanding the underlying mechanisms of mitochondrial disorders, uncovers potential metabolic biomarkers, and unravels metabolic pathway-specific adaptations in response to metabolic perturbations.

Complex I, V, and MDH2 deficient human skin fibroblasts reveal distinct metabolic signatures by 1 H HR-MAS NMR.

In this study, we investigated the metabolic signatures of different mitochondrial defects (two different complex I and complex V, and the one MDH2 defect) in human skin fibroblasts (HSF). We hypothesized that using a selective culture medium would cause defect specific adaptation of the metabolome and further our understanding of the biochemical implications for the studied defects. All cells were cultivated under galactose stress condition and compared to glucose-based cell culture condition. We investigated the bioenergetic profile using Seahorse XFe96 cell analyzer and assessed the extracellular metabolic footprints and the intracellular metabolic fingerprints using NMR. The galactose-based culture condition forced a bioenergetic switch from a glycolytic to an oxidative state in all cell lines which improved overall separation of controls from the different defect groups. The extracellular metabolome was discriminative for separating controls from defects but not the specific defects, whereas the intracellular metabolome suggests CI and CV changes and revealed clear MDH2 defect-specific changes in metabolites associated with the TCA cycle, malate aspartate shuttle, and the choline metabolism, which are pronounced under galactose condition.

FOXA2 prevents hyperbilirubinaemia in acute liver failure by maintaining apical MRP2 expression.

OBJECTIVE: Multidrug resistance protein 2 (MRP2) is a bottleneck in bilirubin excretion. Its loss is sufficient to induce hyperbilirubinaemia, a prevailing characteristic of acute liver failure (ALF) that is closely associated with clinical outcome. This study scrutinises the transcriptional regulation of MRP2 under different pathophysiological conditions. DESIGN: Hepatic MRP2, farnesoid X receptor (FXR) and Forkhead box A2 (FOXA2) expression and clinicopathologic associations were examined by immunohistochemistry in 14 patients with cirrhosis and 22 patients with ALF. MRP2 regulatory mechanisms were investigated in primary hepatocytes, Fxr -/- mice and lipopolysaccharide (LPS)-treated mice. RESULTS: Physiologically, homeostatic MRP2 transcription is mediated by the nuclear receptor FXR/retinoid X receptor complex. Fxr-/- mice lack apical MRP2 expression and rapidly progress into hyperbilirubinaemia. In patients with ALF, hepatic FXR expression is undetectable, however, patients without infection maintain apical MRP2 expression and do not suffer from hyperbilirubinaemia. These patients express FOXA2 in hepatocytes. FOXA2 upregulates MRP2 transcription through binding to its promoter. Physiologically, nuclear FOXA2 translocation is inhibited by insulin. In ALF, high levels of glucagon and tumour necrosis factor α induce FOXA2 expression and nuclear translocation in hepatocytes. Impressively, ALF patients with sepsis express low levels of FOXA2, lose MRP2 expression and develop severe hyperbilirubinaemia. In this case, LPS inhibits FXR expression, induces FOXA2 nuclear exclusion and thus abrogates the compensatory MRP2 upregulation. In both Fxr -/- and LPS-treated mice, ectopic FOXA2 expression restored apical MRP2 expression and normalised serum bilirubin levels. CONCLUSION: FOXA2 replaces FXR to maintain MRP2 expression in ALF without sepsis. Ectopic FOXA2 expression to maintain MRP2 represents a potential strategy to prevent hyperbilirubinaemia in septic ALF.

Methods for Oxygen Determination in an NMR Bioreactor as a Surrogate Marker for Metabolomic Studies in Living Cell Cultures.

Nuclear magnetic resonance (NMR) approaches have been described as a powerful method for measuring oxygen in tissue cultures and body fluids by using relaxation time dependencies of substances on pO2. The present NMR study describes methods to longitudinally monitor global, in situ intracellular, and spatially resolved oxygen tension in culture media and 3D cell cultures using relaxation times of water without the need to use external sensors. 1H NMR measurements of water using a modified inversion recovery pulse scheme were employed for global, i.e., intra- and extracellular oxygen estimation in an NMR-bioreactor. The combination of 1H relaxation time T1 and diffusion measurements of water was employed for in situ cellular oxygen content determination. Spatially selective water relaxation time estimations were used for spatially resolved oxygen quantification along the NMR tube length. The inclusion in a study protocol of the presented techniques for oxygen quantification, as a surrogate marker of oxidative phosphorylation (OXPHOS), provides the possibility to measure mitochondrial respiration and metabolic changes simultaneously.

A hierarchical regulatory network ensures stable albumin transcription under various pathophysiological conditions.

BACKGROUND AND AIMS: It remains unknown how patients with liver failure maintain essential albumin levels. Here, we delineate a hierarchical transcription regulatory network that ensures albumin expression under different disease conditions. APPROACH AND RESULTS: We examined albumin levels in liver tissues and serum in 157 patients, including 84 with HCC, 38 decompensated cirrhosis, and 35 acute liver failure. Even in patients with liver failure, the average serum albumin concentrations were 30.55 g/L. In healthy subjects and patients with chronic liver diseases, albumin was expressed in hepatocytes. In patients with massive hepatocyte loss, albumin was expressed in liver progenitor cells (LPCs). The albumin gene (ALB) core promoter possesses a TATA box and nucleosome-free area, which allows constitutive RNA polymerase II binding and transcription initiation. Chromatin immunoprecipitation assays revealed that hepatocyte nuclear factor 4 alpha (HNF4α), CCAAT/enhancer-binding protein alpha (C/EBPα), and forkhead box A2 (FOXA2) bound to the ALB enhancer. Knockdown of either of these factors reduced albumin expression in hepatocytes. FOXA2 acts as a pioneer factor to support HNF4α and C/EBPα. In hepatocytes lacking HNF4α and C/EBPα expression, FOXA2 synergized with retinoic acid receptor (RAR) to maintain albumin transcription. RAR nuclear translocation was induced by retinoic acids released by activated HSCs. In patients with massive hepatocyte loss, LPCs expressed HNF4α and FOXA2. RNA sequencing and quantitative PCR analyses revealed that lack of HNF4α and C/EBPα in hepatocytes increased hedgehog ligand biosynthesis. Hedgehog up-regulates FOXA2 expression through glioblastoma family zinc finger 2 binding to the FOXA2 promoter in both hepatocytes and LPCs. CONCLUSIONS: A hierarchical regulatory network formed by master and pioneer transcription factors ensures essential albumin expression in various pathophysiological conditions.

Follistatin-controlled activin-HNF4α-coagulation factor axis in liver progenitor cells determines outcome of acute liver failure.

BACKGROUND AND AIMS: In patients with acute liver failure (ALF) who suffer from massive hepatocyte loss, liver progenitor cells (LPCs) take over key hepatocyte functions, which ultimately determines survival. This study investigated how the expression of hepatocyte nuclear factor 4α (HNF4α), its regulators, and targets in LPCs determines clinical outcome of patients with ALF. APPROACH AND RESULTS: Clinicopathological associations were scrutinized in 19 patients with ALF (9 recovered and 10 receiving liver transplantation). Regulatory mechanisms between follistatin, activin, HNF4α, and coagulation factor expression in LPC were investigated in vitro and in metronidazole-treated zebrafish. A prospective clinical study followed up 186 patients with cirrhosis for 80 months to observe the relevance of follistatin levels in prevalence and mortality of acute-on-chronic liver failure. Recovered patients with ALF robustly express HNF4α in either LPCs or remaining hepatocytes. As in hepatocytes, HNF4α controls the expression of coagulation factors by binding to their promoters in LPC. HNF4α expression in LPCs requires the forkhead box protein H1-Sma and Mad homolog 2/3/4 transcription factor complex, which is promoted by the TGF-ß superfamily member activin. Activin signaling in LPCs is negatively regulated by follistatin, a hepatocyte-derived hormone controlled by insulin and glucagon. In contrast to patients requiring liver transplantation, recovered patients demonstrate a normal activin/follistatin ratio, robust abundance of the activin effectors phosphorylated Sma and Mad homolog 2 and HNF4α in LPCs, leading to significantly improved coagulation function. A follow-up study indicated that serum follistatin levels could predict the incidence and mortality of acute-on-chronic liver failure. CONCLUSIONS: These results highlight a crucial role of the follistatin-controlled activin-HNF4α-coagulation axis in determining the clinical outcome of massive hepatocyte loss-induced ALF. The effects of insulin and glucagon on follistatin suggest a key role of the systemic metabolic state in ALF.

Synthesis of Multiple Bispecific Antibody Formats with Only One Single Enzyme Based on Enhanced Trypsiligase.

Bispecific antibodies (bsAbs) were first developed in the 1960s and are now emerging as a leading class of immunotherapies for cancer treatment with the potential to further improve clinical efficacy and safety. Many different formats of bsAbs have been established in the last few years, mainly generated genetically. Here we report on a novel, flexible, and fast chemo-enzymatic, as well as purely enzymatic strategies, for generating bispecific antibody fragments by covalent fusion of two functional antibody Fab fragments (Fabs). For the chemo-enzymatic approach, we first modified the single Fabs site-specifically with click anchors using an enhanced Trypsiligase variant (eTl) and afterward converted the modified Fabs into the final heterodimers via click chemistry. Regarding the latter, we used the strain-promoted alkyne-azide cycloaddition (SPAAC) and inverse electron-demand Diels-Alder reaction (IEDDA) click approaches well known for their fast reaction kinetics and fewer side reactions. For applications where the non-natural linkages or hydrophobic click chemistry products might interfere, we developed two purely enzymatic alternatives enabling C- to C- and C- to N-terminal coupling of the two Fabs via a native peptide bond. This simple system could be expanded into a modular system, eliminating the need for extensive genetic engineering. The bispecific Fab fragments (bsFabs) produced here to bind the growth factors ErbB2 and ErbB3 with similar KD values, such as the sole Fabs. Tested in breast cancer cell lines, we obtained biologically active bsFabs with improved properties compared to its single Fab counterparts.

Determination of Intra- and Extracellular Metabolic Adaptations of 3D Cell Cultures upon Challenges in Real-Time by NMR.

NMR flow devices provide longitudinal real-time quantitative metabolome characterisation of living cells. However, discrimination of intra- and extracellular contributions to the spectra represents a major challenge in metabolomic NMR studies. The present NMR study demonstrates the possibility to quantitatively measure both metabolic intracellular fingerprints and extracellular footprints on human control fibroblasts by using a commercially available flow tube system with a standard 5 mm NMR probe. We performed a comprehensive 3D cell culture system characterisation. Diffusion NMR was employed for intra- and extracellular metabolites separation. In addition, complementary extracellular footprints were determined. The implemented perfused NMR bioreactor system allowed the determination of 35 metabolites and intra- and extracellular separation of 19 metabolites based on diffusion rate differences. We show the reliability and sensitivity of NMR diffusion measurements to detect metabolite concentration changes in both intra- and extracellular compartments during perfusion with different selective culture media, and upon complex I inhibition with rotenone. We also demonstrate the sensitivity of extracellular footprints to determine metabolic variations at different flow rates. The current method is of potential use for the metabolomic characterisation of defect fibroblasts and for improving physiological comprehension.

Dysregulated paired related homeobox 1 impacts on hepatocellular carcinoma phenotypes.

BACKGROUND: Hepatocellular carcinoma (HCC) is a major cause of cancer-related death. Paired related homeobox 1 (PRRX1) is a transcription factor that regulates cell growth and differentiation, but its importance in HCC is unclear. METHODS: We examined the expression pattern of PRRX1 in nine microarray datasets of human HCC tumour samples (n > 1100) and analyzed its function in HCC cell lines. In addition, we performed gene set enrichment, Kaplan-Meier overall survival analysis, metabolomics and functional assays. RESULTS: PRRX1 is frequently upregulated in human HCC. Pathway enrichment analysis predicted a direct correlation between PRRX1 and focal adhesion and epithelial-mesenchymal transition. High expression of PRRX1 and low ZEB1 or high ZEB2 significantly predicted better overall survival in HCC patients. In contrast, metabolic processes correlated inversely and transcriptional analyses revealed that glycolysis, TCA cycle and amino acid metabolism were affected. These findings were confirmed by metabolomics analysis. At the phenotypic level, PRRX1 knockdown accelerated proliferation and clonogenicity in HCC cell lines. CONCLUSIONS: Our results suggest that PRRX1 controls metabolism, has a tumour suppressive role, and may function in cooperation with ZEB1/2. These findings have functional relevance in HCC, including in understanding transcriptional control of distinct cancer hallmarks.

Optimizing bat bioacoustic surveys in human-modified Neotropical landscapes.

During the last decades, the use of bioacoustics as a non-invasive and cost-effective sampling method has greatly increased worldwide. For bats, acoustic surveys have long been known to complement traditional mist-netting, however, appropriate protocol guidelines are still lacking for tropical regions. Establishing the minimum sampling effort needed to detect ecological changes in bat assemblages (e.g., activity, composition, and richness) is crucial in view of workload and project cost constraints, and because detecting such changes must be reliable enough to support effective conservation management. Using one of the most comprehensive tropical bat acoustic data sets, collected in the Amazon, we assessed the minimum survey effort required to accurately assess the completeness of assemblage inventories and habitat selection in fragmented forest landscapes for aerial insectivorous bats. We evaluated a combination of 20 different temporal sampling schemes, which differed regarding number of hours per night, number of nights per site, and sampling only during the wet or dry season, or both. This was assessed under two different landscape scenarios: in primary forest fragments embedded in a matrix of secondary forest and in the same forest fragments, but after they had been re-isolated through clearing of the secondary forest. We found that the sampling effort required to achieve 90% inventory completeness varied considerably depending on the research aim and the landscape scenario evaluated, averaging ~80 and 10 nights before and after fragment re-isolation, respectively. Recording for more than 4 h per night did not result in a substantial reduction in the required number of sampling nights. Regarding the effects of habitat selection, except for assemblage composition, bat responses in terms of richness, diversity, and activity were similar across all sampling schemes after fragment re-isolation. However, before re-isolation, a minimum of four to six sampling hours per night after dusk and three to five nights of sampling per site were needed to detect significant effects that could otherwise go unnoticed. Based on our results, we propose guidelines that will aid to optimize sampling protocols for bat acoustic surveys in the Neotropics.

Predicting biodiversity change and averting collapse in agricultural landscapes.

The equilibrium theory of island biogeography is the basis for estimating extinction rates and a pillar of conservation science. The default strategy for conserving biodiversity is the designation of nature reserves, treated as islands in an inhospitable sea of human activity. Despite the profound influence of islands on conservation theory and practice, their mainland analogues, forest fragments in human-dominated landscapes, consistently defy expected biodiversity patterns based on island biogeography theory. Countryside biogeography is an alternative framework, which recognizes that the fate of the world's wildlife will be decided largely by the hospitality of agricultural or countryside ecosystems. Here we directly test these biogeographic theories by comparing a Neotropical countryside ecosystem with a nearby island ecosystem, and show that each supports similar bat biodiversity in fundamentally different ways. The island ecosystem conforms to island biogeographic predictions of bat species loss, in which the water matrix is not habitat. In contrast, the countryside ecosystem has high species richness and evenness across forest reserves and smaller forest fragments. Relative to forest reserves and fragments, deforested countryside habitat supports a less species-rich, yet equally even, bat assemblage. Moreover, the bat assemblage associated with deforested habitat is compositionally novel because of predictable changes in abundances by many species using human-made habitat. Finally, we perform a global meta-analysis of bat biogeographic studies, spanning more than 700 species. It generalizes our findings, showing that separate biogeographic theories for countryside and island ecosystems are necessary. A theory of countryside biogeography is essential to conservation strategy in the agricultural ecosystems that comprise roughly half of the global land surface and are likely to increase even further.

A global risk assessment of primates under climate and land use/cover scenarios.

Primates are facing an impending extinction crisis, driven by extensive habitat loss, land use change and hunting. Climate change is an additional threat, which alone or in combination with other drivers, may severely impact those taxa unable to track suitable environmental conditions. Here, we investigate the extent of climate and land use/cover (LUC) change-related risks for primates. We employed an analytical approach to objectively select a subset of climate scenarios, for which we then calculated changes in climatic and LUC conditions for 2050 across primate ranges (N = 426 species) under a best-case scenario and a worst-case scenario. Generalized linear models were used to examine whether these changes varied according to region, conservation status, range extent and dominant habitat. Finally, we reclassified primate ranges based on different magnitudes of maximum temperature change, and quantified the proportion of ranges overall and of primate hotspots in particular that are likely to be exposed to extreme temperature increases. We found that, under the worst-case scenario, 74% of Neotropical forest-dwelling primates are likely to be exposed to maximum temperature increases up to 7°C. In contrast, 38% of Malagasy savanna primates will experience less pronounced warming of up to 3.5°C. About one quarter of Asian and African primates will face up to 50% crop expansion within their range. Primary land (undisturbed habitat) is expected to disappear across species' ranges, whereas secondary land (disturbed habitat) will increase by up to 98%. With 86% of primate ranges likely to be exposed to maximum temperature increases >3°C, primate hotspots in the Neotropics are expected to be particularly vulnerable. Our study highlights the fundamental exposure risk of a large percentage of primate ranges to predicted climate and LUC changes. Importantly, our findings underscore the urgency with which climate change mitigation measures need to be implemented to avert primate extinctions on an unprecedented scale.

Confounding influence of tamoxifen in mouse models of Cre recombinase-induced gene activity or modulation.

Tamoxifen (TAM) is commonly used for cell type specific Cre recombinase-induced gene inactivation and in cell fate tracing studies. Inducing a gene knockout by TAM and using non-TAM exposed mice as controls lead to a situation where differences are interpreted as consequences of the gene knockout but in reality result from TAM-induced changes in hepatic metabolism. The degree to which TAM may compromise the interpretation of animal experiments with inducible gene expression still has to be elucidated. Here, we report that TAM strongly attenuates CCl4-induced hepatotoxicity in male C57Bl/6N mice, even after a 10 days TAM exposure-free period. TAM decreased (p < 0.0001) the necrosis index and the level of aspartate- and alanine transaminases in CCl4-treated compared to vehicle-exposed mice. TAM pretreatment also led to the downregulation of CYP2E1 (p = 0.0045) in mouse liver tissue, and lowered its activity in CYP2E1 expressing HepG2 cell line. Furthermore, TAM increased the level of the antioxidant ascorbate, catalase, SOD2, and methionine, as well as phase II metabolizing enzymes GSTM1 and UGT1A1 in CCl4-treated livers. Finally, we found that TAM increased the presence of resident macrophages and recruitment of immune cells in necrotic areas of the livers as indicated by F4/80 and CD45 staining. In conclusion, we reveal that TAM increases liver resistance to CCl4-induced toxicity. This finding is of high relevance for studies using the tamoxifen-inducible expression system particularly if this system is used in combination with hepatotoxic compounds such as CCl4.

BMP-9 interferes with liver regeneration and promotes liver fibrosis.

OBJECTIVE: Bone morphogenetic protein (BMP)-9, a member of the transforming growth factor-ß family of cytokines, is constitutively produced in the liver. Systemic levels act on many organs and tissues including bone and endothelium, but little is known about its hepatic functions in health and disease. DESIGN: Levels of BMP-9 and its receptors were analysed in primary liver cells. Direct effects of BMP-9 on hepatic stellate cells (HSCs) and hepatocytes were studied in vitro, and the role of BMP-9 was examined in acute and chronic liver injury models in mice. RESULTS: Quiescent and activated HSCs were identified as major BMP-9 producing liver cell type. BMP-9 stimulation of cultured hepatocytes inhibited proliferation, epithelial to mesenchymal transition and preserved expression of important metabolic enzymes such as cytochrome P450. Acute liver injury caused by partial hepatectomy or single injections of carbon tetrachloride (CCl4) or lipopolysaccharide (LPS) into mice resulted in transient downregulation of hepatic BMP-9 mRNA expression. Correspondingly, LPS stimulation led to downregulation of BMP-9 expression in cultured HSCs. Application of BMP-9 after partial hepatectomy significantly enhanced liver damage and disturbed the proliferative response. Chronic liver damage in BMP-9-deficient mice or in mice adenovirally overexpressing the selective BMP-9 antagonist activin-like kinase 1-Fc resulted in reduced deposition of collagen and subsequent fibrosis. CONCLUSIONS: Constitutive expression of low levels of BMP-9 stabilises hepatocyte function in the healthy liver. Upon HSC activation, endogenous BMP-9 levels increase in vitro and in vivo and high levels of BMP-9 cause enhanced damage upon acute or chronic injury.

Delta-Like Ligand 4 Modulates Liver Damage by Down-Regulating Chemokine Expression.

Disrupting Notch signaling ameliorates experimental liver fibrosis. However, the role of individual Notch ligands in liver damage is unknown. We investigated the effects of Delta-like ligand 4 (Dll4) in liver disease. DLL4 expression was measured in 31 human liver tissues by immunohistochemistry. Dll4 function was examined in carbon tetrachloride- and bile duct ligation-challenged mouse models in vivo and evaluated in hepatic stellate cells, hepatocytes, and Kupffer cells in vitro. DLL4 was expressed in patients' Kupffer and liver sinusoidal endothelial cells. Recombinant Dll4 protein (rDll4) ameliorated hepatocyte apoptosis, inflammation, and fibrosis in mice after carbon tetrachloride challenge. In vitro, rDll4 significantly decreased lipopolysaccharide-dependent chemokine expression in both Kupffer and hepatic stellate cells. In bile duct ligation mice, rDll4 induced massive hepatic necrosis, resulting in the death of all animals within 1 week. Inflammatory cell infiltration and chemokine ligand 2 (Ccl2) expression were significantly reduced in rDll4-receiving bile duct ligation mice. Recombinant Ccl2 rescued bile duct ligation mice from rDll4-mediated death. In patients with acute-on-chronic liver failure, DLL4 expression was inversely associated with CCL2 abundance. Mechanistically, Dll4 regulated Ccl2 expression via NF-κB. Taken together, Dll4 modulates liver inflammatory response by down-regulating chemokine expression. rDll4 application results in opposing outcomes in two models of liver damage. Loss of DLL4 may be associated with CCL2-mediated cytokine storm in patients with acute-on-chronic liver failure.

A frequent misinterpretation in current research on liver fibrosis: the vessel in the center of CCl4-induced pseudolobules is a portal vein.

Carbon tetrachloride-induced liver injury is a thoroughly studied model for regeneration and fibrosis in rodents. Nevertheless, its pattern of liver fibrosis is frequently misinterpreted as portal type. To clarify this, we show that collagen type IV+ "streets" and α-SMA+ cells accumulate pericentrally and extend to neighbouring central areas of the liver lobule, forming a 'pseudolobule'. Blood vessels in the center of such pseudolobules are portal veins as indicated by the presence of bile duct cells (CK19+) and the absence of pericentral hepatocytes (glutamine synthetase+). It is critical to correctly describe this pattern of fibrosis, particulary for metabolic zonation studies.

Caveolin-1 in the regulation of cell metabolism: a cancer perspective.

Caveolin-1 (CAV1) is an oncogenic membrane protein associated with endocytosis, extracellular matrix organisation, cholesterol distribution, cell migration and signaling. Recent studies reveal that CAV1 is involved in metabolic alterations - a critical strategy adopted by cancer cells to their survival advantage. Consequently, research findings suggest that CAV1, which is altered in several cancer types, influences tumour development or progression by controlling metabolism. Understanding the molecular interplay between CAV1 and metabolism could help uncover druggable metabolic targets or pathways of clinical relevance in cancer therapy. Here we review from a cancer perspective, the findings that CAV1 modulates cell metabolism with a focus on glycolysis, mitochondrial bioenergetics, glutaminolysis, fatty acid metabolism, and autophagy.

Selective Coupling of Click Anchors to Proteins via Trypsiligase.

The combination of pure chemical methods with enzymatic approaches offers a kit system with maximum flexibility for site-specifically tagging proteins with a broad variety of artificial structures. Trypsiligase, a recently introduced designer enzyme for both N- and C-terminal site-specific labeling of peptides and proteins, has been used to introduce click anchors into the human protein cyclophilin 18 and the antibody Fab fragments anti-TNFα and anti-Her2. The subsequent click reactions with tetrazine or norbornene moieties lead to quantitative conversions to the corresponding dihydropyridazine products, thereby forming a stable covalent linkage between the label and the protein of interest. With this technology, cyclophilin 18 has been efficiently modified with the fluorescent dansyl moiety and the pharmaceutically relevant polymer PEG exclusively at its N-terminus. With the same methodology, the Fab fragments of anti-TNFα and anti-Her2 were derivatized exclusively at their C-terminal ends with PEG and the fluorescent dye carboxyfluorescein in the case of anti-TNFα or with the cytotoxic payload DM1 in the case of anti-Her2, to form a homogeneous antibody-drug conjugate (ADC).

Gene network activity in cultivated primary hepatocytes is highly similar to diseased mammalian liver tissue.

It is well known that isolation and cultivation of primary hepatocytes cause major gene expression alterations. In the present genome-wide, time-resolved study of cultivated human and mouse hepatocytes, we made the observation that expression changes in culture strongly resemble alterations in liver diseases. Hepatocytes of both species were cultivated in collagen sandwich and in monolayer conditions. Genome-wide data were also obtained from human NAFLD, cirrhosis, HCC and hepatitis B virus-infected tissue as well as mouse livers after partial hepatectomy, CCl4 intoxication, obesity, HCC and LPS. A strong similarity between cultivation and disease-induced expression alterations was observed. For example, expression changes in hepatocytes induced by 1-day cultivation and 1-day CCl4 exposure in vivo correlated with R = 0.615 (p < 0.001). Interspecies comparison identified predominantly similar responses in human and mouse hepatocytes but also a set of genes that responded differently. Unsupervised clustering of altered genes identified three main clusters: (1) downregulated genes corresponding to mature liver functions, (2) upregulation of an inflammation/RNA processing cluster and (3) upregulated migration/cell cycle-associated genes. Gene regulatory network analysis highlights overrepresented and deregulated HNF4 and CAR (Cluster 1), Krüppel-like factors MafF and ELK1 (Cluster 2) as well as ETF (Cluster 3) among the interspecies conserved key regulators of expression changes. Interventions ameliorating but not abrogating cultivation-induced responses include removal of non-parenchymal cells, generation of the hepatocytes' own matrix in spheroids, supplementation with bile salts and siRNA-mediated suppression of key transcription factors. In conclusion, this study shows that gene regulatory network alterations of cultivated hepatocytes resemble those of inflammatory liver diseases and should therefore be considered and exploited as disease models.