Megalin (LRP2) is an endocytotic transmembrane receptor of about 600 kDa and belongs to the low density lipoprotein receptor family. It is located at the apical surface of several epithelia, where it binds and internalizes many physiologically relevant molecules. Recently, we identified megalin-expressing macrophages in fibrotic liver of mice [1]. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of liver homogenates under reducing conditions and immunoblotting using an antibody directed against the cytoplasmic tail of megalin revealed only a single 35 kDa-band corresponding to the megalin C-terminal fragment (MCTF), whereas in homogenates of kidney, both the 600 kDa-megalin band as well as the MCTF, which results from ectodomain shedding, were visualized. In contrast, gene expression studies using RNA from isolated liver macrophages revealed transcription of the complete megalin gene. We could now substantiate translation of the entire megalin transcript in liver. SDS-PAGE of liver homogenates under non-reducing conditions and immunoblotting using the antibody directed against the cytoplasmic tail of megalin demonstrated a protein band of 600 kDa in addition to the protein band corresponding to the MCTF. These findings suggest that megalin in liver macrophages, contrary to expression as a single molecule in kidney, consists of two polypeptides, the ectodomain and the MCTF, which are joined by disulfide bridges. We speculate that the heterodimeric structure of megalin in liver macrophages is associated with its likely function in the regulation of inflammation.

MicroRNAs (miRNAs), short non-coding RNAs of ~ 21 to 23 nucleotides in length, regulate target mRNAs post-transcriptionally. They play an important role in many different cellular, developmental, and physiological processes including hematopoiesis. It was the aim of this study to characterize the miRNA expression in human hematopoietic stem and progenitor cells. Hematopoietic stem cells (HSCs) have the ability to generate all different kinds of blood cells and express a number of specific surface markers such as CD34 or CD133. As CD133+ cells appear to be ancestral to CD34+ cells, the miRNA profile of these cells could broaden the knowledge of the miRNA role in the differentiation of CD133+ cells. To analyze the function of miRNAs in HSCs, a miRNA microarray platform (miRXploreTM) was developed. The platform was used, inter alia, to validate sequencing data of Small RNA libraries. Standard array experiments measure relative expression levels. However, absolute expression levels in terms of copy numbers per cell are also highly relevant. Therefore, a method for absolute quantification of miRNAs that relies on a universal reference – an equimolar pool of about 1000 synthetic miRNAs of known concentration – was developed. To characterize the role of miRNAs in hematopoiesis, different bone marrow subpopulations, namely CD133+, CD34+CD133– and CD34–CD133– cells, were isolated by magnetic cell separation. The analysis revealed 18 significantly differentially expressed miRNAs between CD133+ and CD34+CD133– cells, that could be validated via qRT-PCR and Solexa sequencing. To further analyze the role of the differentially expressed miRNAs in CD133+ stem cells, mRNA expression profiles were generated and the coexpression of bioinformatically predicted miRNA-mRNA pairs was examined. Luciferase assays were established to validate the predicted targets under physiological conditions. The miRNA-mRNA interactions that could be validated were miR 142-3p and CD133, miR-29a and FZD5 as well as miR-29a and TPM1. TPM1, an actin binding protein, and FZD5, a receptor of the Wnt-signaling pathway, play a role in the remodelling of the cytoskeleton. Further analysis of the predicted miRNA targets revealed that the miRNA targets are enriched for Gene Ontoloy categories related to stem cell-relevant processes. The differentially expressed miRNAs probably prevent differentiation of CD133+ cells and have an anti-apoptotic effect. Furthermore, first experiments were performed to analyze the influence of the differentially expressed miRNAs on the cultivation of CD133+ cells. In vitro expansion of CD133+ cells has turned out to be difficult as most of the tested culture supplements can induce proliferation but are unable to prevent differentiation. The addition of miRNAs to the culture medium could lead to an expansion of CD133+ cells without losing the primitive phenotype. Therefore, the influence of miRNA transfections on CD133+ cells was analyzed in a first step. In conclusion, the generated miRNA signature of CD133+ is the first comprehensive characterization of hematopoietic progenitor cells on miRNA level and will be highly relevant for the application of miRNAs in the field of regenerative medicine.

OBJECTIVES: Gene expression analysis by quantitative PCR is a standard laboratory technique for RNA quantification with high accuracy. In particular real-time PCR techniques using SYBR Green and melting curve analysis allowing verification of specific product amplification have become a well accepted laboratory technique for rapid and high throughput gene expression quantification. However, the software that is applied for quantification is somewhat circuitous and needs actually above average manual operation. DESIGN AND METHODS: We here developed a novel, simple to handle open source software package (i.e., MAKERGAUL) for quantification of gene expression data obtained by real time PCR technology. RESULTS: The developed software was evaluated with an already well characterized real time PCR data set and the performance parameters (i.e., absolute bias, linearity, reproducibility, and resolution) of the algorithm that are the basis of our calculation procedure compared and ranked with those of other implemented and well-established algorithms. It shows good quantification performance with reduced requirements in computing power. CONCLUSIONS: We conclude that MAKERGAUL is a convenient and easy to handle software allowing accurate and fast expression data analysis.

Fructose is one of the key dietary catalysts in the development of non-alcoholic fatty liver disease (NAFLD). NAFLD comprises a complex disease spectrum, including steatosis (fatty liver), non-alcoholic steatohepatitis, hepatocyte injury, inflammation, and fibrosis. It is also the hepatic manifestation of the metabolic syndrome, which covers abdominal obesity, insulin resistance, dyslipidemia, glucose intolerance, or type 2 diabetes mellitus. Commensal bacteria modulate the host immune system, protect against exogenous pathogens, and are gatekeepers in intestinal barrier function and maturation. Dysbalanced intestinal microbiota composition influences a variety of NAFLD-associated clinical conditions. Conversely, nutritional supplementation with probiotics and preobiotics impacting composition of gut microbiota can improve the outcome of NAFLD. In crosstalk with the host immune system, the gut microbiota is able to modulate inflammation, insulin resistance, and intestinal permeability. Moreover, the composition of microbiota of an individual is a kind of fingerprint highly influenced by diet. In addition, not only the microbiota itself but also its metabolites influence the metabolism and host immune system. The gut microbiota can produce vitamins and a variety of nutrients including short-chain fatty acids. Holding a healthy balance of the microbiota is therefore highly important. In the present review, we discuss the impact of long-term intake of fructose on the composition of the intestinal microbiota and its biological consequences in regard to liver homeostasis and disease. In particular, we will refer about fructose-induced alterations of the tight junction proteins affecting the gut permeability, leading to the translocation of bacteria and bacterial endotoxins into the blood circulation.

Aims: Vitamin D binding protein (DBP) is a highly expressed, multifunctional and polymorphic serum protein, which also serves as the major transporter for vitamin D sterols. The present study was performed to analyze the pathophysiological interaction between DBP and hepatocytes (PC) or hepatic stellate cells (HSC), the most important fat-/retinol- storing cells in the liver that spontaneously transdifferentiate to myofibroblasts (MFB) in culture.

There is growing interest in measuring plasma fibroblast growth factor 23 (FGF23) concentrations in a number of clinical settings. However, a reliable assay with acceptable performance is lacking. Plasma samples of healthy adults and patients with different stages of chronic kidney disease (CKD) were used to compare the precision, recovery, linearity and the pre-analytical stability characteristics of a new fully automated FGF23 (intact) assay with a commercially available FGF23 (intact) ELISA. Method agreement was evaluated, reference and stage-specific ranges for kidney disease were established. Other biomarkers relevant for CKD were measured and compared with the FGF23 assays. The fully automated FGF23 (intact) assay demonstrated superior performance compared with the ELISA. A marked positive proportional bias was detected relative to the ELISA assay readout, especially in samples of higher concentration of patients undergoing hemodialysis. Overall, the method comparison revealed a poor degree of correlation. A significant inverse correlation was found between the glomerular filtration rate and both FGF23 assays (both p < .001). Regression analysis revealed that both assays are suitable to predict progression of CKD. A positive correlation was found between FGF23 and phosphate, parathyroid hormone (PTH) and vitamin D, 25(OH)D and 1,25(OH)2D-total assays, respectively. Cutoff points between different stages of CKD were calculated by receiver operator characteristic analysis. The fully automated assay displayed an improved discrimination compared with the ELISA, especially in mild to moderate kidney disease. The new fully automated FGF23 (intact) assay demonstrates excellent analytical performance data and represents a robust, fast and precise alternative to manual FGF23 testing.

Context: Resveratrol is a natural occurring stilbene present in a limited number of dietary food products and beverages. It is assumed that resveratrol has health-promoting effects by lowering low-density lipoprotein (LDL) cholesterol, improving microcirculation, and inhibiting platelet aggregation. Altogether, these beneficial effects lower the risk of cardiovascular disease. The “French paradox” assumes that a diet with moderate consumption of red wine lowers the risk of cancer and cardiovascular disease. Based on its healthful activities, a large number of studies were performed during the last decades investigating potential biological effects of resveratrol on liver homeostasis and its potential application as a hepatoprotective drug. Objectives: The current study aimed at discussing the proposed therapeutic attributes of resveratrol on liver health and its postulated mode of activity. Evidence Acquisition: To conduct the current study, the PubMed full-text archive depository for articles presenting data on resveratrol in liver health and disease was searched. Results: Out of the 9268 published articles on resveratrol, a total of 742 articles focused on liver. Among them, 352 articles investigated potential therapeutic activities. Although some of the reported in vitro and in vivo benefits of resveratrol were highly encouraging, well-designed clinical studies were missing. Conclusions: Presently, it is still premature to advise nutritional supplementation of resveratrol to cure hepatic diseases. Moreover, uncritical recommendation to drink wine as a liver health-promoting beverage should be avoided.

AIM: To examine the activation of the Nalp3 inflammasome and its downstream targets following lipopolysaccharide (LPS)-induced stimulation in the liver.METHODS: Six-to-eight-week-old C57BL/6 chow fed mice were injected intraperitoneally with 0.5 μg/g bodyweight LPS and sacrificed 2, 4, 6, 18 or 24 h later. LPS-induced liver damage was confirmed by a biochemical assay to detect alanine aminotransferase (ALT) levels. To determine if LPS stimulation in the liver led to activation of the inflammasome, real-time quantitative polymerase chain reaction was used to evaluate the mRNA expression of components of the Nalp3 inflammasome. Enzyme-linked immunosorbent assays were used to determine the protein expression levels of several downstream targets of the Nalp3 inflammasome, including caspase-1 and two cytokine targets of caspase-1, interleukin (IL)-1β and IL-18.RESULTS: We found that LPS injection resulted in liver damage as indicated by elevated ALT levels. This was associated with a significant increase in both mRNA and protein levels of the proinflammatory cytokine tumor necrosis factor (TNF)-α in the liver, as well as increased levels of TNFs in serum. We showed that LPS stimulation led to upregulation of mRNA levels in the liver for all the receptor components of the inflammasome, including Nalp3, Nalp1, pannexin-1 and the adaptor molecule apoptosis-associated speck-like, caspase recruitment domain-domain containing protein. We also found increased levels of mRNA and protein for caspase-1, a downstream target of the inflammasome. In addition, LPS challenge led to increased levels of both mRNA and protein in the liver for two cytokine targets of caspase-1, IL-1β and IL-18. Interestingly, substantial baseline expression of pre-IL-1β and pre-IL-18 was found in the liver. Inflammasome and caspase-1 activation was indicated by the significant increase in the active forms of IL-1β and IL-18 after LPS stimulation.CONCLUSION: Our results show that the Nalp3 inflammasome is upregulated and activated in the liver in response to LPS stimulation. PMID: 22147977 Funding information This work was supported by: NIDDK NIH HHS, United States Grant ID: R01 DK075635

Leaf dark respiration (Rdark) is often measured in artificially dark-adapted samples at a single time point during the day, with temperature-normalised rates often assumed to be constant throughout a 24-hour cycle. However, the extent to which the 24-hour cycle influences leaf Rdark and respiratory metabolic profile remains unclear, particularly in C4 plants. We quantified O2-based leaf Rdark and metabolites at six time points over a diel (i.e. 24-hour) cycle in leaves of four grass species (one C3 and three C4 species). We found that Rdark and leaf metabolites varied among species, and between dark-adapted day-sampled and night-sampled leaves. In general, C4 plants contained a relatively higher content of organic acids and soluble sugars than C3 plants. Across the four species, variations in Rdark were associated with changes in the abundance of metabolites involved in the mitochondrial tricarboxylic acid pathway (malate, fumarate, succinate and citrate), amino acid metabolism (alanine and asparagine) and sugar interconversion (lactose and mannose). Multivariate statistics suggested that Rdark of the examined species is influenced more by the relative contribution of multiple concurrent metabolic pathways across the diel cycle than by C3 and C4 photosynthetic types. We suggest that Rdark and metabolite profiles measured during daytime on dark-adapted leaves are not good surrogates for nighttime respiratory properties. Understanding how the supply of respiratory substrates varies during the diel cycle would lead to a more accurate prediction of Rdark over the course of a day.