Assessing the Environmental Impact of Water Consumption by Energy Crops Grown in Spain

The environmental impact of the water consumption of four typical crop rotations grown in Spain, including energy crops, was analyzed and compared against Spanish agricultural and natural reference situations. The life cycle assessment (LCA) methodology was used for the assessment of the potential environmental impact of blue water (withdrawal from water bodies) and green water (uptake of soil moisture) consumption. The latter has so far been disregarded in LCA. To account for green water, two approaches have been applied: the first accounts for the difference in green water demand of the crops and a reference situation. The second is a green water scarcity index, which measures the fraction of the soil‐water plant consumption to the available green water. Our results show that, if the aim is to minimize the environmental impacts of water consumption, the energy crop rotations assessed in this study were most suitable in basins in the northeast of Spain. In contrast, the energy crops grown in basins in the southeast of Spain were associated with the greatest environmental impacts. Further research into the integration of quantitative green water assessment in LCA is crucial in studies of systems with a high dependence on green water resources.     

Direct Fluorination of Phenolsulfonphthalein: A Method for Synthesis of Positron-Emitting Indicators for In Vivo pH Measurement

We report a reaction of direct electrophilic fluorination of phenolsulfonphthalein at mild conditions. This reaction affords the synthesis of novel positron-emitting ¹⁸F-labeled pH indicators. These compounds are useful for non-invasive in vivo pH measurement in biological objects.     

Differences in mRNA Expression,Protein Content,and Enzyme Activity of Superoxide Dismutases in Type II Pneumocytes of Acute and Chronic Lung Injury

The lung is protected against oxidative stress by a variety of antioxidants and type II pneumocytes seem to play an important role in antioxidant defense. Previous studies have shown that inhalation of NO₂ results in acute and chronic lung injury. How the expression and enzyme activity of antioxidant enzymes are influenced in type II cells of different inflammatory stages has yet not been studied. To elucidate this question, we exposed rats to 10 ppm NO₂ for 3 or 20 days to induce acute or chronic lung injury. From these and air-breathing rats, type II pneumocytes were isolated. The mRNA expression and protein content of CuZnSOD and MnSOD as well as total SOD-specific enzyme activity were determined. For the acute lung injury (3 d NO₂), the expression of CuZnSOD mRNA was significantly increased, while MnSOD expression was significantly reduced after 3 days of NO 2 exposure. For the chronic lung injury (20 d NO₂), CuZnSOD expression was still enhanced, while MnSOD expression was comparable to control. In parallel to CuZnSOD mRNA expression, the protein amount was significantly increased in acute and chronic lung injury however MnSOD protein content exhibited no intergroup differences. Total SOD enzyme activity showed a significant decrease after 3 days of NO₂ exposure and was similar to control after 20 days. We conclude that during acute and chronic lung injury in type II pneumocytes expression and protein synthesis of CuZnSOD and MnSOD are regulated differently.     

Chemical genetic screen in fission yeast reveals roles for vacuolar acidification,mitochondrial fission,and cellular GMP levels in lifespan extension

The discovery that genetic mutations in several cellular pathways can increase lifespan has lent support to the notion that pharmacological inhibition of aging pathways can be used to extend lifespan and to slow the onset of age‐related diseases. However, so far, only few compounds with such activities have been described. Here, we have conducted a chemical genetic screen for compounds that cause the extension of chronological lifespan of Schizosaccharomyces pombe. We have characterized eight natural products with such activities, which has allowed us to uncover so far unknown anti‐aging pathways in S. pombe. The ionophores monensin and nigericin extended lifespan by affecting vacuolar acidification, and this effect depended on the presence of the vacuolar ATPase (V‐ATPase) subunits Vma1 and Vma3. Furthermore, prostaglandin J₂ displayed anti‐aging properties due to the inhibition of mitochondrial fission, and its effect on longevity required the mitochondrial fission protein Dnm1 as well as the G‐protein‐coupled glucose receptor Git3. Also, two compounds that inhibit guanosine monophosphate (GMP) synthesis, mycophenolic acid (MPA) and acivicin, caused lifespan extension, indicating that an imbalance in guanine nucleotide levels impinges upon longevity. We furthermore have identified diindolylmethane (DIM), tschimganine, and the compound mixture mangosteen as inhibiting aging. Taken together, these results reveal unanticipated anti‐aging activities for several phytochemicals and open up opportunities for the development of novel anti‐aging therapies.     

Gastro-intestinal transport of calcium and cadmium in fresh water and seawater acclimated trout (Oncorhynchus mykiss)

Transport of calcium (Ca) and cadmium (Cd) was examined along the gastro-intestinal tract (GIT) of freshwater and seawater Oncorhynchus mykiss irideus (FWT and SWTies respectively) using in vitro and in vivo experiments. Based on known physiological differences between FWT and SWT which aid in regulating ion levels and osmolarity, we hypothesized that SWT would have lower rates of Ca uptake. Also, we predicted that Cd rates would also be lower because Cd is known to share a common transport mechanism with Ca. Kinetics of Ca and Cd transport were determined using mucosal salines of varying concentrations [1, 10, 30, 60, and 100 (mmol L^− 1 for Ca, μmol L^− 1 for Cd)]. Linear and saturating relationships were found for Ca for FWT and SWT, but overall SWT had lower rates. Linear and/or saturating relationships were also found for Cd uptake, but rates varied little between fish types. Elevated Ca had no inhibitory effect on Cd transport, and Ca channel blockers nifedipine and verapamil had little effect on Ca or Cd uptake. However, lanthanum reduced Ca transport into some compartments. A 21 day in vivo feeding experiment was also performed where FWT and SWT were exposed to control diets or Cd-spiked diets (552 μg Cd g^− 1 food). Whole body Cd uptake between fish types was similar, but the majority of Cd in SWT remained in the posterior intestine tissue, while FWT transported more Cd through their gut wall. Overall it appears that large differences in Ca and Cd uptake between FWT and SWT exist, with SWT generally having lower rates.     

Failure-to-Thrive Syndrome Associated with Tumor Formation by Madin–Darby Canine Kidney Cells in Newborn Nude Mice

Tumors that formed in newborn nude mice that were inoculated with 10⁷ Madin–Darby canine kidney (MDCK) cells were associated with a failure-to-thrive (FTT) syndrome consisting of growth retardation, lethargy, weakness, and dehydration. Scoliosis developed in 41% of affected pups. Pups were symptomatic by week 2; severely affected pups became moribund and required euthanasia within 3 to 4 wk. Mice with FTT were classified into categories of mild, moderate, and severe disease by comparing their weight with that of age-matched normal nude mice. The MDCK-induced tumors were adenocarcinomas that invaded adjacent muscle, connective tissue, and bone; 6 of the 26 pups examined had lung metastases. The induction of FTT did not correlate with cell-line aggressiveness as estimated by histopathology or the efficiency of tumor formation (tumor-forming dose 50% endpoint range = 10^2.8 to 10^7.5); however, tumor invasion of the paravertebral muscles likely contributed to the scoliosis noted. In contrast to the effect of MDCK cells, tumor formation observed in newborn mice inoculated with highly tumorigenic, human-tumor–derived cell lines was not associated with FTT development. We suggest that tumor formation and FTT are characteristics of these MDCK cell inocula and that FTT represents a new syndrome that may be similar to the cachexia that develops in humans with cancer or other diseases.Abbreviations: FTT, failure-to-thrive; MDCK, Madin–Darby canine kidney; TPD₅₀, tumor-producing dose log₁₀ 50% endpointThe Madin–Darby canine kidney (MDCK) cell line was established in 1958 from the kidney of a cocker spaniel.^6,16 Since 1962, this cell line has been an important reagent for the isolation and study of influenza viruses^8,22,31 and, more recently, for the development and manufacture of influenza virus vaccines.^3,7,19 MDCK cells are polarized, epithelial cells that exhibit properties of renal tubular epithelium and have been used as in vitro models to evaluate renal tubular functions.^24,36 Due to their apparent lack of expression of a tumorigenic phenotype in rodents,²⁵ MDCK cells have also been used to study neoplastic processes including epithelial-to-mesenchymal transition^23,27,28 and to assess the effects of viral oncogenes and chemical carcinogens on their phenotype.^13,32The results of studies that evaluate the ability of MDCK cells to form tumors in vivo have varied. Early studies found that these cells could produce tumors in chicken embryos but not in mature BALB/c nude mice.¹⁴ In contrast, MDCK cells formed progressively growing adenocarcinomas in newborn BALB/c nude mice, but tumor growth ceased as the pups approached maturity.²⁵ More recently, 2 different sublines of MDCK cells developed by independent groups were shown to be tumorigenic in athymic nude mice; but the incidence of tumor formation did not correlate with cell dose.^33-35As an initial approach to the study of neoplastic development in cells in culture, we evaluated the ability of MDCK cells to form tumors in athymic nude mice. We previously described the tumor-forming capacity of MDCK cells from different lots obtained from ATCC.²¹ That study revealed that MDCK cells from each of these lots formed tumors efficiently in adult and newborn nude mice, but the capacity of the cells to form tumors differed from lot to lot. During the initial experiments on MDCK cell tumor-forming efficiency in newborn nude mice, we observed what appeared to be a syndrome whose symptoms included tumor formation and disrupted growth leading to a failure-to-thrive (FTT) condition manifested by morbidity that required euthanasia of those pups most severely affected. During the study on the development of FTT, we found that the FTT syndrome occurred in newborn nude mice inoculated with 3 different sublines of MDCK cells. The current report describes an FTT syndrome associated with the formation of tumors by 10⁷ MDCK cells in newborn, athymic, nude mice.     

Proteomic Differences Between Hepatocellular Carcinoma and Nontumorous Liver Tissue Investigated by a Combined Gel-based and Label-free Quantitative Proteomics Study

Proteomics-based clinical studies have been shown to be promising strategies for the discovery of novel biomarkers of a particular disease. Here, we present a study of hepatocellular carcinoma (HCC) that combines complementary two-dimensional difference in gel electrophoresis (2D-DIGE) and liquid chromatography (LC-MS)-based approaches of quantitative proteomics. In our proteomic experiments, we analyzed a set of 14 samples (7 × HCC versus 7 × nontumorous liver tissue) with both techniques. Thereby we identified 573 proteins that were differentially expressed between the experimental groups. Among these, only 51 differentially expressed proteins were identified irrespective of the applied approach. Using Western blotting and immunohistochemical analysis the regulation patterns of six selected proteins from the study overlap (inorganic pyrophosphatase 1 (PPA1), tumor necrosis factor type 1 receptor-associated protein 1 (TRAP1), betaine-homocysteine S-methyltransferase 1 (BHMT)) were successfully verified within the same sample set. In addition, the up-regulations of selected proteins from the complements of both approaches (major vault protein (MVP), gelsolin (GSN), chloride intracellular channel protein 1 (CLIC1)) were also reproducible. Within a second independent verification set (n = 33) the altered protein expression levels of major vault protein and betaine-homocysteine S-methyltransferase were further confirmed by Western blots quantitatively analyzed via densitometry. For the other candidates slight but nonsignificant trends were detectable in this independent cohort. Based on these results we assume that major vault protein and betaine-homocysteine S-methyltransferase have the potential to act as diagnostic HCC biomarker candidates that are worth to be followed in further validation studies.Hepatocellular carcinoma (HCC)¹ currently is the fifth most common malignancy worldwide with an annual incidence up to 500 per 100,000 individuals depending on the geographic region investigated. Whereas 80% of new cases occur in developing countries, the incidence increases in industrialized nations including Western Europe, Japan, and the United States (1). To manage patients with HCC, tumor markers are very important tools for diagnosis, indicators of disease progression, outcome prediction, and evaluation of treatment efficacy. Several tumor markers have been reported for HCC, including α-fetoprotein (AFP) (2), Lens culinaris agglutinin-reactive fraction of AFP (AFP-L3) (3), and des-γ-carboxyl prothrombin (DCP) (4). However, none of these tumor markers show 100% sensitivity or specificity, which calls for new and better biomarkers.To identify novel biomarkers of HCC, many clinical studies using “omics”-based methods have been reported over the past decade (5–6). In particular, the proteomics-based approach has turned out to be a promising one, offering several quantification techniques to reveal differences in protein expression that are caused by a particular disease. In most studies, the well-established 2D-DIGE technique has been applied for protein quantification followed by identification via mass spectrometry (7–15). Even if the quantification is very accurate and sensitive in this gel-based approach, the relatively high amount of protein sample necessary for protein identification is the major disadvantage of this technique. Several mass-spectrometry-based quantitative studies using labeling-techniques like SILAC (stable isotope labeling by amino acids in cell culture) or iTRAQ (isobaric tags for relative and absolute quantification) have also been carried out for biomarker discovery of HCC (16–18). Here, the concomitant protein quantification and identification in a mass spectrometer allows high-throughput analyses. However, such experiments imply additional labeling reactions (in case of iTRAQ) or are limited to tissue culture systems (in case of SILAC). In the latter case, one can overcome the limitation by using the isotope-labeled proteins obtained from tissue culture as an internal standard added to a corresponding tissue sample. This approach is known as CDIT (culture-derived isotope tags) and was applied in a HCC study, very recently (19). Label-free proteomics approaches based on quantification by ion-intensities or spectral counting offer another possibility for biomarker discovery. These approaches are relatively cheap compared with the labeling approaches, because they do not require any labeling reagents and furthermore they allow for high-throughput and sensitive analyses in a mass spectrometer. A quantitative study of HCC using spectral counting has been reported (20), whereas to our knowledge an ion-intensity-based study has not been performed yet. Apart from these quantification strategies, protein alterations in HCC have been studied by MALDI imaging, as well. Here, the authors could show that based on its proteomic signature, hepatocellular carcinoma can be discriminated with high accuracy from liver metastasis samples or other cancer types (21) as well as liver cirrhosis (22). Based on these results, it could be assumed that MALDI imaging might be a promising alternative to standard histological methods in the future.Here, we report a quantitative proteomic study that combines two different techniques, namely the well-established 2D-DIGE approach and a label-free ion-intensity-based quantification via mass spectrometry and liquid chromatography. To our knowledge this is the first time such a combined study was performed with regard to hepatocellular carcinoma. By comparing the results of both studies, we aim to identify high-confident biomarker candidates of HCC, as gel- and LC-MS-based techniques are complementary. To verify the differential protein expressions detected in our proteomic studies we performed additional immunological verifications for selected proteins within two different sample sets (Fig. 1).Open in a separate windowFig. 1.Schematic representation of the applied workflow.     

Microarray analysis of miRNA expression profiles following whole body irradiation in a mouse model

Abstract

Context: Accidental exposure to life-threatening radiation in a nuclear event is a major concern; there is an enormous need for identifying biomarkers for radiation biodosimetry to triage populations and treat critically exposed individuals.

Objective: To identify dose-differentiating miRNA signatures from whole blood samples of whole body irradiated mice.

Methods: Mice were whole body irradiated with X-rays (2?Gy–15?Gy); blood was collected at various time-points post-exposure; total RNA was isolated; miRNA microarrays were performed; miRNAs differentially expressed in irradiated vs. unirradiated controls were identified; feature extraction and classification models were applied to predict dose-differentiating miRNA signature.

Results: We observed a time and dose responsive alteration in the expression levels of miRNAs. Maximum number of miRNAs were altered at 24-h and 48-h time-points post-irradiation. A 23-miRNA signature was identified using feature selection algorithms and classifier models. An inverse correlation in the expression level changes of miR-17 members, and their targets were observed in whole body irradiated mice and non-human primates.

Conclusion: Whole blood-based miRNA expression signatures might be used for predicting radiation exposures in a mass casualty nuclear incident.     

Identification of the binding site of Rlp7 on assembling 60S ribosomal subunits in Saccharomyces cerevisiae

Eukaryotic ribosome assembly requires over 200 assembly factors that facilitate rRNA folding, ribosomal protein binding, and pre-rRNA processing. One such factor is Rlp7, an essential RNA binding protein required for consecutive pre-rRNA processing steps for assembly of yeast 60S ribosomal subunits: exonucleolytic processing of 27SA₃ pre-rRNA to generate the 5′ end of 5.8S rRNA and endonucleolytic cleavage of the 27SB pre-rRNA to initiate removal of internal transcribed spacer 2 (ITS2). To better understand the functions of Rlp7 in 27S pre-rRNA processing steps, we identified where it crosslinks to pre-rRNA. We found that Rlp7 binds at the junction of ITS2 and the ITS2-proximal stem, between the 3′ end of 5.8S rRNA and the 5′ end of 25S rRNA. Consistent with Rlp7 binding to this neighborhood during assembly, two-hybrid and affinity copurification assays showed that Rlp7 interacts with other assembly factors that bind to or near ITS2 and the proximal stem. We used in vivo RNA structure probing to demonstrate that the proximal stem forms prior to Rlp7 binding and that Rlp7 binding induces RNA conformational changes in ITS2 that may chaperone rRNA folding and regulate 27S pre-rRNA processing. Our findings contradict the hypothesis that Rlp7 functions as a placeholder for ribosomal protein L7, from which Rlp7 is thought to have evolved in yeast. The binding site of Rlp7 is within eukaryotic-specific RNA elements, which are not found in bacteria. Thus, we propose that Rlp7 coevolved with these RNA elements to facilitate eukaryotic-specific functions in ribosome assembly and pre-rRNA processing.