Repolarization Precedes Oval Cell-mediated Hepatocytic Regeneration in the CDE Diet Mouse Model.

The liver has a unique ability to recover from injury unlike any other organ. A poorly understood aspect of liver regeneration is the role of hepatocellular polarization. Neighbor of Punc E11 (Nope) is an oncofetal stem/progenitor cell marker, which is expressed by depolarized adult hepatocytes after cholestatic liver injury and in hepatocellular carcinoma. Liver injury induced by a choline-deficient and ethionine-supplemented diet is reversible if followed by an additional dietary stop interval and enabled us to study the expression of Nope during the induction of chronic liver injury and during subsequent liver regeneration. We could show by quantitative RT-PCR, Western blotting, and immunohistochemistry that the expression of Nope is induced in depolarized adult hepatocytes during injury. However, after another 2 weeks of a normal diet, the polarization of hepatocytes was almost completely restored and the expression of Nope remained limited to bile ducts and oval cells. Using an inducible CK19-lineage tracing model, we could demonstrate that oval cell-mediated hepatocyte regeneration is rare and was preceded by repolarization of hepatocytes. In conclusion, polarization of hepatocytes is an important part of liver regeneration and precedes oval cell-mediated regeneration of the liver. This process can be visualized by a characteristic expression pattern of Nope.

Expression of Neighbor of Punc E11 (NOPE) in early stage esophageal adenocarcinoma is associated with reduced survival.

Current recommendations suggest neoadjuvant treatment in node-positive esophageal cancer or tumors staged T3 and upwards but some T2 N0 patients might benefit from neoadjuvant therapy. It is of clinical relevance to identify this subgroup. Loss of epithelial apicobasal polarity is a key factor in the development of invasive capabilities of carcinoma. The oncofetal stem/progenitor cell marker NOPE is expressed in adult depolarized murine hepatocytes and in murine/human hepatocellular carcinoma. We analyzed NOPE expression in 363 patients with esophageal adenocarcinoma using an RNA Scope Assay on a tissue microarray and correlated results with clinical data. Median follow-up was 57.7 months with a 5-year survival rate of 26.6%. NOPE was detectable in 32 patients (8.8%). In pT1/2 stages, NOPE expression was associated with a significantly reduced median OS of 6.3 months (95% CI 1.2-19.4 months), the median OS is not reached in the NOPE-negative group (calculated mean OS 117.1 months) (P = 0.012). In advanced tumor stages, a NOPE dependent survival difference was not detected. This is the first report of NOPE expression demonstrating a prognostic value in esophageal cancer. Early stage, NOPE positive patients are at a high risk of tumor progression and may benefit from neoadjuvant treatment analogous to advanced stage cancer.

First evaluation of Neighbor of Punc E11 (NOPE) as a novel marker in human hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is the second most common cause of cancer death worldwide and the search for clinically useful biomarkers is ongoing. Neighbor of Punc E11 (NOPE) is an established biomarker of murine HCC that remains undetectable in normal liver and at preneoplastic stages. OBJECTIVE: The aim of our study was to evaluate the presence of NOPE in human HCC. METHODS: Histologically confirmed HCC and corresponding non-tumor liver samples from 20 patients were analyzed for expression of NOPE using qRT-PCR and mRNA-in-situ technology in a conserved tissue context. RESULTS: In our cohort, 30% of HCC samples were expressing NOPE which proved particularly useful in non-cirrhotic HCC samples with up to 155-fold higher expression than in adult liver. Using mRNA-in-situ technology, NOPE was clearly identified within epithelial tumor cells of NOPE positive human HCCs. In our analyzed cohort, the combination of AFP with NOPE did not reach more than 40% sensitivity while GPC-3 and NOPE were complementary to each other reaching a combined sensitivity of 85.7%. CONCLUSIONS: This is the first characterization of NOPE as a potential biomarker for human HCC. Our results underline the value of NOPE as a complementing biomarker for human HCC.

Zelkovamycin is an OXPHOS Inhibitory Member of the Argyrin Natural Product Family.

Natural products (NPs) are an important inspirational source for developing drugs and chemical probes. In 1999, the group of Omura reported the constitutional elucidation of zelkovamycin. Although largely unrecognized so far, this NP displays structural similarities as well as differences to the argyrin NP family, a class of peptidic NPs with promising anticancer activities and diverse mode-of-action at the molecular level. By a combination of structure elucidation experiments, the first total synthesis of zelkovamycin and bioassays, the zelkovamycin configuration was determined and its previously proposed molecular structure was revised. The full structure assignment proves zelkovamycin as an additional member of the argyrins with however unique OXPHOS inhibitory properties. Zelkovamycin may therefore not only serve as a new starting point for chemical inhibitors of the OXPHOS system, but also guide customized argyrin NP isolation and biosynthesis studies.

Depolarized Hepatocytes Express the Stem/Progenitor Cell Marker Neighbor of Punc E11 After Bile Duct Ligation in Mice.

There is a medical need of biomarkers for disease stratification in cholestatic liver diseases that come along with changes in hepatocyte polarity. Neighbor of Punc E11 (Nope) is an oncofetal marker that is lost after final differentiation and polarization of hepatocytes. We analyzed the expression pattern of Nope and connexin (Cx) 26 as markers of hepatocyte polarization during murine liver development as well as in adult liver with or without bile duct ligation (BDL) by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), western blotting (WB), and immunohistochemistry. Nope is highly expressed in fetal and postnatal liver but barely detectable thereafter. Cx26, however, is much higher expressed in adult than in fetal liver. Postnatally, Nope is directed to the sinusoidal membrane of early hepatocytes while Cx26 remains distributed over the whole membrane indicating limited polarization. In the adult liver, only Cx26 is detectable and restricted to the bile canalicular domain indicating fully polarized hepatocytes. After BDL, Nope is again >300-fold upregulated while Cx26 is reduced rapidly. By immunohistochemistry, Nope identifies a subset of hepatocytes with randomly distributed Cx26. In summary, Nope identifies depolarized adult hepatocytes after cholestatic liver injury resembling early postnatal hepatocytes. Therefore, Nope might be a valuable histochemical biomarker allowing stage-specific stratifications in cholestatic liver diseases.

Activity-based protein profiling as a robust method for enzyme identification and screening in extremophilic Archaea.

Archaea are characterized by a unique life style in often environmental extremes but their thorough investigation is currently hampered by a limited set of suitable in vivo research methodologies. Here, we demonstrate that in vivo activity-based protein profiling (ABPP) may be used to sensitively detect either native or heterogeneously expressed active enzymes in living archaea even under these extreme conditions. In combination with the development of a genetically engineered archaeal screening strain, ABPP can furthermore be used in functional enzyme screenings from (meta)genome samples. We anticipate that our ABPP approach may therefore find application in basic archaeal research but also in the discovery of novel enzymes from (meta)genome libraries.

Activity-Based Protein Profiling with Natural Product-Derived Chemical Probes in Human Cell Lysates.

Bioreactive natural products represent versatile starting points for the development of structurally unique activity-based probes. In the present protocol, we describe the workflow for an activity-based protein profiling (ABPP) experiment with an alkyne-tagged natural product derivative. Our protocol includes experimental procedures for in vivo labeling, sample preparation and 2-step (click chemistry) visualization and sample preparation for mass spectrometry-based target identification.

Chemoproteomic Evaluation of the Polyacetylene Callyspongynic Acid.

Polyacetylenes are a class of alkyne-containing natural products. Although potent bioactivities and thus possible applications as chemical probes have already been reported for some polyacetylenes, insights into the biological activities or molecular mode of action are still rather limited in most cases. To overcome this limitation, we describe the application of the polyacetylene callyspongynic acid in the development of an experimental roadmap for characterizing potential protein targets of alkyne-containing natural products. To this end, we undertook the first chemical synthesis of callyspongynic acid. We then used in situ chemical proteomics methods to demonstrate extensive callyspongynic acid-mediated chemical tagging of endoplasmic reticulum-associated lipid-metabolizing and modifying enzymes. We anticipate that an elucidation of protein targets of natural products may serve as an effective guide to the development of subsequent biological assays that aim to identify chemical phenotypes and bioactivities.

Peptidyl succinimidyl peptides as taspase 1 inhibitors.

Taspase 1 is an N-terminal threonine protease implicated in leukemia and other cancers. Despite intensive efforts in recent years, only a limited number of Taspase 1 inhibitors are currently available, and they lack general applicability. Here we present a novel class of Taspase 1 inhibitors based on a peptidyl succinimidyl peptide motif. These inhibitors were obtained from the substrate cleavage sequence and mechanistic considerations involving the previously proposed asparaginase-type cleavage mechanism. We anticipate that this class of Taspase 1 inhibitor will find wide application in further biochemical and structural studies, for example for better investigating the molecular details of the unusual enzymatic cleavage mechanism of Taspase 1.