Trade‐offs between land and water requirements for large‐scale bioenergy production

Bioenergy is expected to play an important role in the future energy mix as it can substitute fossil fuels and contribute to climate change mitigation. However, large‐scale bioenergy cultivation may put substantial pressure on land and water resources. While irrigated bioenergy production can reduce the pressure on land due to higher yields, associated irrigation water requirements may lead to degradation of freshwater ecosystems and to conflicts with other potential users. In this article, we investigate the trade‐offs between land and water requirements of large‐scale bioenergy production. To this end, we adopt an exogenous demand trajectory for bioenergy from dedicated energy crops, targeted at limiting greenhouse gas emissions in the energy sector to 1100 Gt carbon dioxide equivalent until 2095. We then use the spatially explicit global land‐ and water‐use allocation model MAgPIE to project the implications of this bioenergy target for global land and water resources. We find that producing 300 EJ yr^?1 of bioenergy in 2095 from dedicated bioenergy crops is likely to double agricultural water withdrawals if no explicit water protection policies are implemented. Since current human water withdrawals are dominated by agriculture and already lead to ecosystem degradation and biodiversity loss, such a doubling will pose a severe threat to freshwater ecosystems. If irrigated bioenergy production is prohibited to prevent negative impacts of bioenergy cultivation on water resources, bioenergy land requirements for meeting a 300 EJ yr^?1 bioenergy target increase substantially (+ 41%) – mainly at the expense of pasture areas and tropical forests. Thus, avoiding negative environmental impacts of large‐scale bioenergy production will require policies that balance associated water and land requirements.     

Evaluation of the DNA damaging effects of amitraz on human lymphocytes in the Comet assay

Amitraz is formamidine pesticide widely used as insecticide and acaricide. In veterinary medicine, amitraz has important uses against ticks, mites and lice on animals. Also, amitraz is used in apiculture to control Varroa destructor. It this study, the alkaline Comet assay was used to evaluate DNA damaging effects of amitraz in human lymphocytes. Isolated human lymphocytes were incubated with varying concentrations of amitraz (0.035, 0.35, 3.5, 35 and 350 μg/mL). The Comet assay demonstrated that all concentrations of amitraz caused statistically significant increase in the level of DNA damage, thus indicating that amitraz possesses genotoxic potential. The concentration of amitraz that produced the highest DNA damage (3.5 μg/mL) was chosen for further analysis with the antioxidant catalase. The obtained results showed that co-treatment with antioxidant catalase (100 IU/mL or 500 IU/mL) significantly reduced the level of DNA damage, indicating the possible involvement of reactive oxygen species in DNA damaging effects of amitraz. Flow cytometric analysis revealed increase of the apoptotic index following treatment with amitraz. However, co-treatment with catalase reduced the apoptotic index, while treatment with catalase alone reduced the percentage of apoptotoc cells even in comparison with the negative control. Therefore, catalase had protective effects against ROS-mediated DNA damage and apoptosis.     

Two preferentially expressed proteins protect vascular endothelial cells from an attack by peptide-specific CTL

Vascular endothelial cells (EC) are an exposed tissue with intimate contact with circulating Ag-specific CTL. Experimental in vitro and clinical data suggested that endothelial cells present a different repertoire of MHC class I-restricted peptides compared with syngeneic leukocytes or epithelial cells. This endothelial-specific peptide repertoire might protect EC from CTL-mediated cell death. The HLA-A*02-restricted peptide profile of human EC and syngeneic B lymphoblastoid cells was biochemically analyzed and compared. For EC selective peptides, source protein expression, peptide binding affinity, and peptide-HLA-A*02 turnover were measured. The significance of abundant peptide presentation for target cell recognition by immunodominant CTL was tested by small interfering RNA treatment of EC to knock down the source proteins. High amounts of two peptides, PTRF(56-64) and CD59(106-114), were consistently detected in EC. This predominance of two endothelial peptides was explained by cell type-specific source protein expression that compensated for poor HLA-A*02 binding affinity and short half-live of peptide/HLA-A*02 complexes. Knocking down the source proteins containing the abundant endothelial peptide motifs led to a nearly 100-fold increase of surface expression of SMCY(311-319), an immunodominant minor histocompatibility Ag, as detected by cytotoxicity assays using SMCY(311-319)-specific CTL. We conclude that EC express and present preferentially two distinct HLA-A*02-restricted peptides at extraordinary high levels. These abundant self-peptides may protect EC from CTL-mediated lysis by competing for HLA-A*02 binding sites with immunodominant scarcely expressed antigenic peptides.     

Identification of naturally processed hepatitis C virus-derived major histocompatibility complex class I ligands

Fine mapping of human cytotoxic T lymphocyte (CTL) responses against hepatitis C virus (HCV) is based on external loading of target cells with synthetic peptides which are either derived from prediction algorithms or from overlapping peptide libraries. These strategies do not address putative host and viral mechanisms which may alter processing as well as presentation of CTL epitopes. Therefore, the aim of this proof-of-concept study was to identify naturally processed HCV-derived major histocompatibility complex (MHC) class I ligands. To this end, continuous human cell lines were engineered to inducibly express HCV proteins and to constitutively express high levels of functional HLA-A2. These cell lines were recognized in an HLA-A2-restricted manner by HCV-specific CTLs. Ligands eluted from HLA-A2 molecules isolated from large-scale cultures of these cell lines were separated by high performance liquid chromatography and further analyzed by electrospray ionization quadrupole time of flight mass spectrometry (MS)/tandem MS. These analyses allowed the identification of two HLA-A2-restricted epitopes derived from HCV nonstructural proteins (NS) 3 and 5B (NS3_1406–1415 and NS5B_2594–2602). In conclusion, we describe a general strategy that may be useful to investigate HCV pathogenesis and may contribute to the development of preventive and therapeutic vaccines in the future.     

Efficacy of Imiquimod-Based Transcutaneous Immunization Using a Nano-Dispersed Emulsion Gel Formulation

Background

Transcutaneous immunization (TCI) approaches utilize skin associated lymphatic tissues to elicit specific immune responses. In this context, the imidazoquinoline derivative imiquimod formulated in Aldara applied onto intact skin together with a cytotoxic T lymphocyte (CTL) epitope induces potent CTL responses. However, the feasibility and efficacy of the commercial imiquimod formulation Aldara is limited by its physicochemical properties as well as its immunogenicity.

Methodology/Principal Findings

To overcome these obstacles, we developed an imiquimod-containing emulsion gel (IMI-Gel) and characterized it in comparison to Aldara for rheological properties and in vitro mouse skin permeation in a Franz diffusion cell system. Imiquimod was readily released from Aldara, while IMI-Gel showed markedly decreased drug release. Nevertheless, comparing vaccination potency of Aldara or IMI-Gel-based TCI in C57BL/6 mice against the model cytotoxic T-lymphocyte epitope SIINFEKL, we found that IMI-Gel was equally effective in terms of the frequency of peptide-specific T-cells and in vivo cytolytic activity. Importantly, transcutaneous delivery of IMI-Gel for vaccination was clearly superior to the subcutaneous or oral route of administration. Finally, IMI-Gel based TCI was at least equally effective compared to Aldara-based TCI in rejection of established SIINFEKL-expressing E.G7 tumors in a therapeutic setup indicated by enhanced tumor rejection and survival.

Conclusion/Significance

In summary, we developed a novel imiquimod formulation with feasible pharmaceutical properties and immunological efficacy that fosters the rational design of a next generation transcutaneous vaccination platform suitable for the treatment of cancer or persistent virus infections.     

Disulfiram moderately restores impaired hepatic redox status of rats subchronically exposed to cadmium

Examination of cadmium (Cd) toxicity and disulfiram (DSF) effect on liver was focused on oxidative stress (OS), bioelements status, morphological and functional changes. Male Wistar rats were intraperitoneally treated with 1?mg CdCl₂/kg BW/day; orally with 178.5?mg DSF/kg BW/day for 1, 3, 10 and 21 days; and co-exposed from 22nd to 42nd day. The co-exposure nearly restored previously suppressed total superoxide dismutase (SOD), catalase (CAT) and increased glutathione peroxidase (GPx) activities; increased previously reduced glutathione reductase (GR) and total glutathione-S-transferase (GST) activities; reduced previously increased superoxide anion radical (O₂^·?) and malondialdehyde (MDA) levels; increased zinc (Zn) and iron (Fe), and decreased copper (Cu) (yet above control value), while magnesium (Mg) was not affected; and decreased serum alanine aminotransferases (ALT) levels. Histopathological examination showed signs of inflammation process as previously demonstrated by exposure to Cd. Overall, we ascertained partial liver redox status improvement, compared with the formerly Cd-induced impact.     

Seasonal variation in the activity of selected antioxidant enzymes and malondialdehyde level in worker honey bees

The recent decline in managed honey bee populations, Apis mellifera L. (Hymenoptera: Apidae), has caused scientific, ecological, and economic concern. Research into the formation of reactive oxygen species (ROS), antioxidative defense mechanisms, and oxidative stress can contribute to our understanding of bee survival and conservation of this species. Activities of superoxide dismutase (SOD), catalase (CAT), and glutathione S‐transferase (GST) enzymes together with levels of malondialdehyde (MDA) were measured in summer and winter honey bees sampled from three colonies. One colony was stationary (C1), entering the winter period having accumulated Robinia pseudoacacia L. (Fabaceae) honey, and two were migratory (C2 and C3), entering the winter period with mainly Tilia (Malvaceae) and Brassica (Brassicaceae) honey, respectively. Compared to summer workers, winter worker bees had decreased SOD and GST activity, and MDA level, whereas CAT activity increased in all three colonies. We also demonstrated that seasonality is the main factor responsible for changes in antioxidant enzymes and MDA levels in worker honey bees. Overall, our results indicate a difference between summer and winter worker bees, pointing at a reduced level of antioxidant enzyme defenses during overwintering which may be due to a decrease in production of ROS. The decreased levels of MDA measured in winter honey bees confirm this. As ROS are actively used by insects as a defense mechanism to fight pathogens, we suggest that reduced production of ROS contributes to higher susceptibility of winter honey bees to infections and reduced overwinter survival.     

High Immune Response Rates and Decreased Frequencies of Regulatory T Cells in Metastatic Renal Cell Carcinoma Patients after Tumor Cell Vaccination

Our previously reported phase I clinical trial with the allogeneic gene–modified tumor cell line RCC-26/CD80/IL-2 showed that vaccination was well tolerated and feasible in metastatic renal cell carcinoma (RCC) patients. Substantial disease stabilization was observed in most patients despite a high tumor burden at study entry. To investigate alterations in immune responses that might contribute to this effect, we performed an extended immune monitoring that included analysis of reactivity against multiple antigens, cytokine/chemokine changes in serum and determination of the frequencies of immune suppressor cell populations, including natural regulatory T cells (nTregs) and myeloid-derived suppressor cell subsets (MDSCs). An overall immune response capacity to virus-derived control peptides was present in 100% of patients before vaccination. Vaccine-induced immune responses to tumor-associated antigens occurred in 75% of patients, demonstrating the potent immune stimulatory capacity of this generic vaccine. Furthermore, some patients reacted to peptide epitopes of antigens not expressed by the vaccine, showing that epitope-spreading occurred in vivo. Frequencies of nTregs and MDSCs were comparable to healthy donors at the beginning of study. A significant decrease of nTregs was detected after vaccination (p = 0.012). High immune response rates, decreased frequencies of nTregs and a mixed T helper 1/T helper 2 (T_H1/T_H2)-like cytokine pattern support the applicability of this RCC generic vaccine for use in combination therapies.     

Genetic diversity and relationships among species of the genus Thymus L. (section Serpyllum)

Amplified fragment length polymorphism (AFLP) markers were used to assess genetic relations of 32 Thymus populations belonging to seven species of genus Thymus, section Serpyllum, from Serbia. The collected species belong to three subsections: (1) Alternantes Klokov (Th. pulegioides L.), (2) Isolepides (Borbás) Halácsy (Th. glabrescens Willd., Th. marschallianus Willd., Th. pannonicus All.), and (3) Pseudomarginati (Braun ex Borbás) Jalas (Th. balcanus Borbás, Th. moesiacus Velen., Th. praecox Opiz). Analysis of molecular variance (AMOVA) revealed that most of the genetic diversity was attributable to differences among individuals within populations, while the remaining variability was almost equally distributed among species and among populations within species. The pairwise AMOVA analyses further showed that species differentiation was significant in all cases, except between Th. marschallianus and Th. pannonicus. All the individuals belonging to Th. pulegioides (subsection Alternantes) formed a well-supported clade, the most divergent from all other species in this study. This genetic differentiation of Th. pulegioides is supported by its morphological features because it is the only analyzed taxon having hairs only on the stem edges, and can easily be recognized by this character. According to our results, the subsection Isolepides is polyphyletic. Th. glabrescens is clearly separated from the other two species of this subsection (Th. marschallianus, Th. pannonicus). Although morphologically distinguishable by leaf indumentum, individuals belonging to Th. marschallianus and Th. pannonicus could not be differentiated at the molecular level. The species belonging to subsection Pseudomarginati formed a monophyletic clade, although not supported by high bootstrap value. The clade is further divided into three well-supported clades representing distinct species (Th. balcanus, Th. praecox and Th. moesiacus). In our study, the AFLP markers demonstrated that they could be suitable for studying complex genetic relationships, including frequent interspecies hybridization events, although strict reticulate evolutionary history could not be determined. All the population genetic parameters in the section Serpyllum suggest that the reproductive incompatibility between the species is very weak, which indicates a strong reticulating system, especially in the case when two or more species occur sympatrically.     

Selective ultrasound-assisted extractions of lipophilic constituents from Betula alleghaniensis and B. papyrifera wood at low temperatures

Betula alleghaniensis and B. papyrifera are widely distributed in the province of Québec (Canada) and, since these trees are valuable exports for the local lumber industry, large amounts of their residual ligneous biomass are available for further exploitation. Betula species are well known for their significant concentrations of triterpenes, some of which were recently discovered to present promising bioactivity. The secondary transformation of birch biomass could therefore become important for many industries, particularly the pharmaceutical industry. In the present study, extracts from birch sawdust were obtained using an optimised ultrasound-assisted extraction in which the careful choice of temperature permitted a selective extraction of the targeted triterpenes. Moreover, compared with the classical Soxhlet method, higher extraction yields were obtained in a shorter time. The lipophilic extracts obtained using dichloromethane as a solvent were analysed by GC-MS and the major compounds identified as lupane-type cyclic triterpenoids accompanied by the non-cyclic triterpene squalene. Numerous aliphatic long-chain fatty acids were also found in the extracts together with phytosterols. Betulonic acid and squalene, the major extract constituents for both B. alleghaniensis and B. papyrifera, are both bioactive molecules.