On the use of weather data in ecological studies along altitudinal and latitudinal gradients

Global warming has created a need for studies along climatic gradients to assess the effects of temperature on ecological processes. Altitudinal and latitudinal gradients are often used as such, usually in combination with air temperature data from the closest weather station recorded at 1.5–2 m above the ground. However, many ecological processes occur in, at, or right above the soil surface. To evaluate how representative the commonly used weather station data are for the microclimate relevant for soil surface biota, we compared weather station temperatures for an altitudinal (500–900 m a.s.l.) and a latitudinal gradient (49–68°N) with data obtained by temperature sensors placed right below the soil surface at five sites along these gradients. The mean annual temperatures obtained from weather stations and adjusted using a lapse rate of ?5.5°C km^?1 were between 3.8°C lower and 1.6°C higher than those recorded by the temperature sensors at the soil surface, depending on the position along the gradients. The monthly mean temperatures were up to 10°C warmer or 5°C colder at the soil surface. The within‐site variation in accumulated temperature was as high as would be expected from a 300 m change in altitude or from a 4° change in latitude or a climate change scenario corresponding to warming of 1.6–3.8°C. Thus, these differences introduced by the decoupling are significant from a climate change perspective, and the results demonstrate the need for incorporating microclimatic variation when conducting studies along altitudinal or latitudinal gradients. We emphasize the need for using relevant temperature data in climate impact studies and further call for more studies describing the soil surface microclimate, which is crucial for much of the biota.     

Adrenomedullin disulfide bond mimetics uncover structural requirements for AM1 receptor activation

Adrenomedullin (ADM) is a vasoactive peptide hormone of 52 amino acids and belongs to the calcitonin peptide superfamily. Its vasodilative effects are mediated by the interaction with the calcitonin receptor‐like receptor (CLR), a class B G protein‐coupled receptor (GPCR), associated with the receptor activity modifying protein 2 (RAMP2) and functionally described as AM‐1 receptor (AM₁R). A disulfide‐bonded ring structure consisting of six amino acids between Cys¹⁶ and Cys²¹ has been shown to be a key motif for receptor activation. However, the specific structural requirements remain to be elucidated. To investigate the influence of ring size and position of additional functional groups that replace the native disulfide bond, we generated ADM analogs containing thioether, thioacetal, alkane, and lactam bonds between amino acids 16 and 21 by Fmoc/t‐Bu solid phase peptide synthesis. Activity studies of the ADM disulfide bond mimetics (DSBM) revealed a strong impact of structural parameters. Interestingly, an increased ring size was tolerated but the activity of lactam‐based mimetics depended on its position within the bridging structure. Furthermore, we found the thioacetal as well as the thioether‐based mimetics to be well accepted with full AM₁R activity. While a reduced selectivity over the calcitonin gene‐related peptide receptor (CGRPR) was observed for the thioethers, the thioacetal was able to retain a wild–type‐like selectivity profile. The carbon analog in contrast displayed weak antagonistic properties. These results provide insight into the structural requirements for AM₁R activation as well as new possibilities for the development of metabolically stabilized analogs for therapeutic applications of ADM.     

Characterization of cullin-based E3 ubiquitin ligases in intact mammalian cells--evidence for cullin dimerization

Cullin-based E3 ligases are a large family of ubiquitin ligases with diverse cellular functions. They are composed of one of six mammalian cullin homologues, the Ring finger containing protein Roc1/Rbx1 and cullin homologue-specific adapter and substrate recognition subunits. To be active, cullin-based ligases require the covalent modification of a conserved lysine residue in the cullin protein with the ubiquitin-like protein Nedd8. To characterize this family of E3 ligases in intact cells, we generated a cell line with tetracycline-inducible expression of a dominant-negative mutant of the Nedd8-conjugating enzyme Ubc12, a reported inhibitor of cullin neddylation. Using this cell line, we demonstrate that the substrate recognition subunit Skp2 and the adaptor protein Skp1 are subject to Ubc12-dependent autoubiquitination and degradation. In contrast, cullin protein stability is not regulated by neddylation in mammalian cells. We also provide evidence that Cul1 and Cul3, as well as their associated substrate recognition subunits Skp2 and Keap1, respectively, homooligomerize in intact cells, suggesting that cullin-based ligases are dimeric. Cul3, but not Cul1 homooligomerization is dependent on substrate recognition subunit dimer formation. As shown for other E3 ubiquitin ligases, dimerization may play a role in regulating the activity of cullin-based E3 ligases.     

The role of neuronal synchronization in selective attention

Attention selectively enhances the influence of neuronal responses conveying information about relevant sensory attributes. Accumulating evidence suggests that this selective neuronal modulation relies on rhythmic synchronization at local and long-range spatial scales: attention selectively synchronizes the rhythmic responses of those neurons that are tuned to the spatial and featural attributes of the attended sensory input. The strength of synchronization is thereby functionally related to perceptual accuracy and behavioural efficiency. Complementing this synchronization at a local level, attention has recently been demonstrated to regulate which locally synchronized neuronal groups phase-synchronize their rhythmic activity across long-range connections. These results point to a general computational role for selective synchronization in dynamically controlling which neurons communicate information about sensory inputs effectively.     

Limited Impact of 6-Mercaptopurine on Inflammation-Induced Chemokines Expression Profile in Primary Cultures of Enteric Nervous System

Increasing evidences indicate that the enteric nervous system (ENS) and enteric glial cells (EGC) play important regulatory roles in intestinal inflammation. Mercaptopurine (6-MP) is a cytostatic compound clinically used for the treatment of inflammatory bowel diseases (IBD), such as ulcerative colitis and Crohn’s disease. However, potential impacts of 6-MP on ENS response to inflammation have not been evaluated yet. In this study, we aimed to gain deeper insights into the profile of inflammatory mediators expressed by the ENS and on the potential anti-inflammatory impact of 6-MP in this context. Genome-wide expression analyses were performed on ENS primary cultures exposed to lipopolysaccharide (LPS) and 6-MP alone or in combination. Differential expression of main hits was validated by quantitative real-time PCR (qPCR) using a cell line for EGC. ENS cells expressed a broad spectrum of cytokines and chemokines of the C-X-C motif ligand (CXCL) family under inflammatory stress. Induction of Cxcl5 and Cxcl10 by inflammatory stimuli was confirmed in EGC. Inflammation-induced protein secretion of TNF-α and Cxcl5 was partly inhibited by 6-MP in ENS primary cultures but not in EGC. Further work is required to identify the cellular mechanisms involved in this regulation. These findings extend our knowledge of the anti-inflammatory properties of 6-MP related to the ENS and in particular of the EGC-response to inflammatory stimuli.

     

Identification of Novel Biomarker Candidates for the Immunohistochemical Diagnosis of Cholangiocellular Carcinoma

The aim of this study was the identification of novel biomarker candidates for the diagnosis of cholangiocellular carcinoma (CCC) and its immunohistochemical differentiation from benign liver and bile duct cells. CCC is a primary cancer that arises from the epithelial cells of bile ducts and is characterized by high mortality rates due to its late clinical presentation and limited treatment options. Tumorous tissue and adjacent non-tumorous liver tissue from eight CCC patients were analyzed by means of two-dimensional differential in-gel electrophoresis and mass-spectrometry-based label-free proteomics. After data analysis and statistical evaluation of the proteins found to be differentially regulated between the two experimental groups (fold change ≥ 1.5; p value ≤ 0.05), 14 candidate proteins were chosen for determination of the cell-type-specific expression profile via immunohistochemistry in a cohort of 14 patients. This confirmed the significant up-regulation of serpin H1, 14-3-3 protein sigma, and stress-induced phosphoprotein 1 in tumorous cholangiocytes relative to normal hepatocytes and non-tumorous cholangiocytes, whereas some proteins were detectable specifically in hepatocytes. Because stress-induced phosphoprotein 1 exhibited both sensitivity and specificity of 100%, an immunohistochemical verification examining tissue sections of 60 CCC patients was performed. This resulted in a specificity of 98% and a sensitivity of 64%. We therefore conclude that this protein should be considered as a potential diagnostic biomarker for CCC in an immunohistochemical application, possibly in combination with other candidates from this study in the form of a biomarker panel. This could improve the differential diagnosis of CCC and benign bile duct diseases, as well as metastatic malignancies in the liver.Cholangiocellular carcinoma (CCC)¹ is a malignant neoplasm that arises from the cholangiocytes, the epithelial cells lining the bile ducts. The tumors, consisting of a significant amount of fibrous stroma, are classified as intrahepatic, extrahepatic, or hilar according to their anatomic location. Most common are the Klatskin tumors, originating from the confluence of the right and left hepatic ducts (1). Compared with other types of cancer, CCC is a relatively rare disease, accounting for about 3% of all gastrointestinal malignancies (2). However, its incidence is increasing, and as a result of poor patient outcomes it has overtaken hepatocellular carcinoma as the main cause of death from a primary hepatobiliary tumor (3). Reasons for the high mortality rate (5-year survival rate of about 5%) (4) are the difficult diagnosis and limited treatment options. At present, extensive surgical resection of the extrahepatic bile ducts and parts of the liver or liver transplantation remain the only potentially curative treatment options, although most patients are considered inoperable at the time of diagnosis (5).In general, the diagnosis of CCC is made based on histomorphological evaluation of core biopsies or cytological specimens. However, distinction between CCC and benign diseases such as reactive bile ductules or bile duct adenomas can be challenging when based on conventional histology alone. Additionally, it may be difficult to distinguish CCC from metastatic adenocarcinoma in the liver, especially when it originates from the pancreas like pancreatic ductal adenocarcinoma. Therefore, specific immunohistochemical tissue markers for CCC would be highly beneficial for further validation of the diagnosis. In routine immunohistochemical diagnosis of CCC, so far, the detection of p53 (a product of a tumor suppressor gene) has proven useful, although its application is limited because of low sensitivity (6). The cytokeratins Ck7, Ck8, Ck18, and Ck19 have been reported to have sensitivities of between 80% and 97% for CCC cells, but at low specificities and a similar expression as in non-tumorous cholangiocytes (7). In addition, the tumor marker carcinoembryonic antigen, which is a commonly applied serum marker, has been used for immunohistochemical staining of CCC tissue. Although this was reported to be positive in 100% of the tested CCC sections, it also was immunoreactive in 60% of hepatocellular carcinomas (8). Recently, it has been shown that the polycomb group protein EZH2 may be useful for differential diagnosis of cholangiolocellular carcinoma (a subtype of CCC), bile duct adenomas, and ductular reaction. This, however, applies only to this certain type of CCC (9). Establishing reliable immunohistochemical tumor markers specific for CCC therefore remains a challenge.Several proteomic studies using different sample types and various techniques have been performed in order to identify CCC-specific proteins. The analysis of CCC cell lines, for example, has led to the identification of potential diagnostic and prognostic biomarker candidates (10–12). In addition, cell lines have been used to discover proteins predictive of the response to chemotherapy (13). Because results from cell culture experiments do not always reflect the actual conditions in the tumor, the use of patient samples can be advantageous. The most appropriate source of tumor-specific signals is tumor tissue, which in the past has been analyzed via two-dimensional electrophoresis (14) and mass-spectrometry-based proteomic approaches such as histology-directed MALDI-TOF-MS (15), Surface-enhanced laser desorption/ionization (SELDI) TOF-MS (16), or LC-MS/MS (17). So far, however, none of the potential biomarkers have been successfully implemented into clinical routine.Recently, we demonstrated that the application of two complementary techniques, two-dimensional differential in-gel electrophoresis (2D-DIGE) and mass-spectrometry-based label-free LC-MS/MS, is an auspicious tactic for the discovery of novel biomarker candidates in hepatocellular carcinoma tissue (18). Here, we applied this well-established workflow as the initial step for the discovery of tissue markers that improve the differential diagnosis of intrahepatic CCC from benign bile duct diseases. In these experiments, CCC tumor tissue was compared with non-tumorous liver tissue (n = 8). Because this does not allow discrimination among different cell types such as hepatocytes, cholangiocytes, and tumor cells, an immunohistochemical determination of the cell-type-specific expression was subsequently performed for the most promising biomarker candidates. Stress-induced phosphoprotein 1, the protein showing the greatest specificity and sensitivity for CCC tumor cells, was verified as a suitable biomarker candidate for CCC in a larger patient cohort (n = 60).     

Fusion of Protein Aggregates Facilitates Asymmetric Damage Segregation

Asymmetric segregation of damaged proteins at cell division generates a cell that retains damage and a clean cell that supports population survival. In cells that divide asymmetrically, such as Saccharomyces cerevisiae, segregation of damaged proteins is achieved by retention and active transport. We have previously shown that in the symmetrically dividing Schizosaccharomyces pombe there is a transition between symmetric and asymmetric segregation of damaged proteins. Yet how this transition and generation of damage-free cells are achieved remained unknown. Here, by combining in vivo imaging of Hsp104-associated aggregates, a form of damage, with mathematical modeling, we find that fusion of protein aggregates facilitates asymmetric segregation. Our model predicts that, after stress, the increased number of aggregates fuse into a single large unit, which is inherited asymmetrically by one daughter cell, whereas the other one is born clean. We experimentally confirmed that fusion increases segregation asymmetry, for a range of stresses, and identified Hsp16 as a fusion factor. Our work shows that fusion of protein aggregates promotes the formation of damage-free cells. Fusion of cellular factors may represent a general mechanism for their asymmetric segregation at division.