Expression of proteins associated with hypoxia and Wnt pathway activation is of prognostic significance in hepatocellular carcinoma

In the development and progression of hepatocellular carcinoma, tumor hypoxia plays an important role, as does activation of the Wnt pathway. The aim of this study was to characterize the expression and interrelationship between hypoxia and Wnt-pathway-associated proteins as prognostic factors for hepatocellular carcinoma. Expression of HIF-1α, CA-IX, E-cadherin, β-catenin, and Ki-67 was assessed by immunohistochemistry in 179 primary hepatocellular carcinoma cases. Univariate and multivariate analyses were performed to assess the relationship between the clinicopathological factors, protein expression, overall survival (OS), and recurrence-free survival (RFS). By univariate analysis, tumor stage, size, satellitosis, and vascular invasion were confirmed as prognostic factors for worse OS and RFS. High expression of HIF-1α, CA-IX, β-catenin, Ki-67, and E-cadherin was observed in 60, 15, 64, 8, and 64 % of tumors, respectively, and this was significantly associated with poor OS. CA-IX, HIF-1α, and E-cadherin were independent predictors of poor prognosis. We stratified 169 patients into four groups according to the expression level of hypoxia and Wnt pathway markers. The group with high expression of both hypoxia and Wnt-pathway-associated proteins showed worst OS. The poor survival of this group was also significant in patients with early stage disease and tumor size of less than 5 cm (p < 0.05). We identified a subgroup of hepatocellular carcinoma patients with high expression of both hypoxia and Wnt pathway proteins and found this predictive of poor survival. The therapeutic options for this group might need to be revisited.

The sarcolemmal calcium pump inhibits the calcineurin/nuclear factor of activated T-cell pathway via interaction with the calcineurin A catalytic subunit

The calcineurin/nuclear factor of activated T-cell (NFAT) pathway represents a crucial transducer of cellular function. There is increasing evidence placing the sarcolemmal calcium pump, or plasma membrane calcium/calmodulin ATPase pump (PMCA), as a potential modulator of signal transduction pathways. We demonstrate a novel interaction between PMCA and the calcium/calmodulin-dependent phosphatase, calcineurin, in mammalian cells. The interaction domains were located to the catalytic domain of PMCA4b and the catalytic domain of the calcineurin A subunit. Endogenous calcineurin activity, assessed by measuring the transcriptional activity of its best characterized substrate, NFAT, was significantly inhibited by 60% in the presence of ectopic PMCA4b. This inhibition was notably reversed by the co-expression of the PMCA4b interaction domain, demonstrating the functional significance of this interaction. PMCA4b was, however, unable to confer its inhibitory effect in the presence of a calcium/calmodulin-independent constitutively active mutant calcineurin A suggesting a calcium/calmodulin-dependent mechanism. The modulatory function of PMCA4b is further supported by the observation that endogenous calcineurin moves from the cytoplasm to the plasma membrane when PMCA4b is overexpressed. We suggest recruitment by PMCA4b of calcineurin to a low calcium environment as a possible explanation for these findings. In summary, our results offer strong evidence for a novel functional interaction between PMCA and calcineurin, suggesting a role for PMCA as a negative modulator of calcineurin-mediated signaling pathways in mammalian cells. This study reinforces the emerging role of PMCA as a molecular organizer and regulator of signaling transduction pathways.

Geochronological database and classification system for age uncertainties in Neotropical pollen records.

The newly updated inventory of palaeoecological research in Latin America offers an important overview of sites available for multi-proxy and multi-site purposes. From the collected literature supporting this inventory, we collected all available age model metadata to create a chronological database of 5116 control points (e.g. 14C, tephra, fission track, OSL, 210Pb) from 1097 pollen records. Based on this literature review, we present a summary of chronological dating and reporting in the Neotropics. Difficulties and recommendations for chronology reporting are discussed. Furthermore, for 234 pollen records in northwest South America, a classification system for age uncertainties is implemented based on chronologies generated with updated calibration curves. With these outcomes age models are produced for those sites without an existing chronology, alternative age models are provided for researchers interested in comparing the effects of different calibration curves and age–depth modelling software, and the importance of uncertainty assessments of chronologies is highlighted. Sample resolution and temporal uncertainty of ages are discussed for different time windows, focusing on events relevant for research on centennial- to millennial-scale climate variability. All age models and developed R scripts are publicly available through figshare, including a manual to use the scripts.

Probing a 3,4'-bis-guanidinium diaryl derivative as an allosteric inhibitor of the Ras pathway

Mutations in the Ras-pathway occur in 40–45% of colorectal cancer patients and these are refractory to treatment with anti-EGFR-targeted therapies. With this in mind, we have studied novel guanidinium- based compounds with demonstrated ability to inhibit protein kinases. We have performed docking stud- ies with several proteins involved in the Ras-pathway and evaluated 3,40-bis-guanidinium derivatives as inhibitors of B-Raf. Compound 3, the most potent in this series, demonstrated strong cytotoxicity in WTB-Raf colorectal cancer cells and also cells with V600EB-Raf mutations. Cell death was induced by apop- tosis, detected by cleavage of PARP. Compound 3 also potently inhibited ERK1/2 signalling, inhibited EGFR activation, as well as Src, STAT3 and AKT phosphorylation. Mechanistically, compound 3 did not inhibit ATP binding to B-Raf, but direct assay of B-Raf activity was inhibited in vitro. Summarizing, we have identified a novel B-Raf type-III inhibitor that exhibits potent cellular cytotoxicity

Replicability of data collected for empirical estimation of relative pollen productivity

The effects of repeated survey and fieldwork timing on data derived from a recently proposed standard field methodology for empirical estimation of Relative Pollen Productivity have been tested. Seasonal variations in vegetation and associated pollen assemblages were studied in three contrasting cultural habitat types; semi-natural ancient woodlands, lowland heaths, and unimproved, traditionally managed hay meadows. Results show that in woodlands and heathlands the standard method generates vegetation data with a reasonable degree of similarity throughout the field season, though in some instances additional recording of woodland canopy cover should be undertaken, and differences were greater for woodland understorey taxa than for arboreal taxa. Large differences in vegetation cover were observed over the field season in the grassland community, and matching the phenological timing of surveys within and between studies is clearly important if RPP estimates from these sites are to be comparable. Pollen assemblages from closely co-located moss polsters collected on different visits are shown to be variable in all communities, to a greater degree than can be explained by the sampling error associated with pollen counting, and further study of moss polsters as pollen traps is recommended.