Environmental changes affect ecosystem services of the intermittent Lake Cerknica

Lake Cerknica is an intermittent wetland with seasonal water level fluctuations. This paper discusses the possible changes of ecosystem services due to altered ecosystem functions caused by regional climate change. For this purpose, the lake's water regime was analysed and biomass production of common reed (Phragmites australis), the prevailing species, was related to ambient temperatures and water level. The effect of the latter was also examined for reed transpiration rate and plant diversity. The results revealed a gradual loss of seasonality of floods and droughts. High water level in winter months and temperatures at the beginning as well as the end of vegetation period were found to have a significant influence on reed biomass. The plant diversity research in six subsequent years in three different aquatic habitats revealed trends of a decrease due to increased water depth during the vegetation period. The complexity of the system and consequently ecosystem services might also be affected due to changes of other processes, which revealed to be related to water level; namely transpiration rate, plant mycorrhizal colonisation and soil mineralisation.     

Scarless excision of an insertion sequence restores capsule production and virulence in Acinetobacter baumannii

We identify a new mechanism mediating capsule production and virulence in the WHO and CDC priority ESKAPE pathogen Acinetobacter baumannii. Non-capsulated and avirulent bacteria can revert into a capsulated and virulent state upon scarless excision of an ISAba13 insertion sequence under stress conditions. Reversion events fully restore capsule production and in vivo virulence. This increases our knowledge about A. baumannii genome dynamics, and the regulation of capsule production, virulence and resistance.Subject terms: Bacterial genetics, Bacterial pathogenesis     

Cross-presentation of Listeria-derived CD8 T cell epitopes requires unstable bacterial translation products

Presentation of bacteria-derived CD8 T cell epitopes by dendritic cells (DC) requires either their direct infection or that DC acquire and cross-present Ags from other infected cells. We found that cross-presentation of Listeria monocytogenes-derived CD8 T cell epitopes was much stronger than direct Ag presentation by infected murine DC. Cross-presentation of Listeria-derived CD8 T cell epitopes showed unique physiological requirements. It was dependent upon the delivery of unstable bacterial translation products by infected, but still viable, Ag donor cells. Cross-presentation was enhanced both when unstable translation products in infected Ag donor cells were protected from proteasomal degradation and when the production of misfolded bacterial proteins was increased. The requirement of unstable translation products for cross-presentation may represent a novel pathway that functions to focus the CD8 T cell response toward epitopes derived from newly synthesized proteins.     

A comparative genomic analysis of the calcium signaling machinery in Neurospora crassa, Magnaporthe grisea, and Saccharomyces cerevisiae

A large number of Ca2+ -signaling proteins have been previously identified and characterized in Saccharomyces cerevisiae but relatively few have been discovered in filamentous fungi. In this study, a detailed, comparative genomic analysis of Ca2+ -signaling proteins in Neurospora crassa, Magnaporthe grisea, and S. cerevisiae has been made. Our BLAST analysis identified 48, 42, and 40 Ca2+ -signaling proteins in N. crassa, M. grisea, and S. cerevisiae, respectively. In N. crassa, M. grisea, and S. cerevisiae, 79, 100, and 13% of these proteins, respectively, were previously unknown. For N. crassa, M. grisea, and S. cerevisiae, respectively, we have identified: three Ca2+ -permeable channels in each species; 9, 12, and 5 Ca2+/cation-ATPases; eight, six, and four Ca2+ -exchangers; four, four, and two phospholipase C's; one calmodulin in each species; and 23, 21, and 29 Ca2+/calmodulin-regulated proteins. Homologs of a number of key proteins involved in the release of Ca2+ from intracellular stores, and in the sensing of extracellular Ca2+, in animal and plant cells, were not identified. The greater complexity of the Ca2+ -signaling machinery in N. crassa and M. grisea over that in S. cerevisiae probably reflects their more complex cellular organization and behavior, and the greater range of external signals which filamentous fungi have to respond to in their natural habitats. To complement the data presented in this paper, a comprehensive web-based database resource (http://www.fungalcell.org/fdf/) of all Ca2+ -signaling proteins identified in N. crassa, M. grisea, and S. cerevisiae has been provided.     

Numerical optimization of gene electrotransfer into muscle tissue

Background  

Electroporation-based gene therapy and DNA vaccination are promising medical applications that depend on transfer of pDNA into target tissues with use of electric pulses. Gene electrotransfer efficiency depends on electrode configuration and electric pulse parameters, which determine the electric field distribution. Numerical modeling represents a fast and convenient method for optimization of gene electrotransfer parameters. We used numerical modeling, parameterization and numerical optimization to determine the optimum parameters for gene electrotransfer in muscle tissue.     

Novel thrombin inhibitors incorporating non-basic partially saturated heterobicyclic P1-arginine mimetics

The design, synthesis and biological activity of non-covalent thrombin inhibitors incorporating 4,5,6,7-tetrahydroindazole, 2-methyl-4,5,6,7-tetrahydroindazole, 4,5,6,7-tetrahydroisoindole, 5,6,7,8-tetrahydroquinazoline and 5,6,7,8-tetrahydroquinazolin-2-amine as novel, partially saturated, heterobicyclic P(1)-arginine side-chain mimetics is described. The binding mode of the most potent candidate in the series co-crystallized with human alpha-thrombin, which exhibited an in vitro K(i) of 140nM and more that 478-fold selectivity against trypsin, is discussed.     

Antiarrhythmic drug amiodarone displays antifungal activity,induces irregular calcium response and intracellular acidification of Aspergillus niger – Amiodarone targets calcium and pH homeostasis of A. niger

The rapidly developing resistance of fungi to antifungal drugs is a serious health problem. Today’s drugs mainly target cell membrane composition and synthesis. Moreover, some of them have serious side effects. New antifungal drugs targeting different molecular pathways are necessary. Amiodarone, an FDA approved antiarrhythmic drug displays antifungal activity. It targets calcium and pH homeostasis. In concentrations above 25 μM, it inhibits the growth of the filamentous fungi Aspergillus niger. It triggers a biphasic calcium response accompanied by a high [Ca²⁺]_c resting level and an intracellular acidification from 7.5 to 6.0, both of which are concentration dependent. Both extracellular calcium and calcium from intracellular organelles are sources of the transient second cytosolic calcium peak, whose amplitude is 0.12 μM for cells treated with 0.1 mM amiodarone. In P-type ATPase deficient A. niger strains pmrAΔ and pmcAΔ, the [Ca²⁺]_c resting level after amiodarone treatment is at least twice as high as that of the wild type, which correlates with fungal viability and hypersensitivity to amiodarone. A combination of amiodarone and amphotericin B is additive in terms of cell viability and cytosolic calcium influx. In contrast, a combination of azole drugs and amiodarone has a synergistic effect on the viability of fungi.     

The 4a/4a genotype of the VNTR polymorphism for endothelial nitric oxide synthase (eNOS) gene predicts risk for proliferative diabetic retinopathy in Slovenian patients (Caucasians) with type 2 diabetes mellitus

Thus far only a limited number of studies examined the association between endothelial nitric oxide synthase (eNOS) polymorphisms and proliferative diabetic retinopathy (PDR). In this report, two polymorphisms in the eNOS gene have been investigated, namely the 894G>T (Glu298Asp) and a 27 bp VNTR (4b/4a), to assess their possible relationships to PDR among Slovenian (Caucasians) type 2 diabetic patients. This cross-sectional case–control study enrolled 577 unrelated Slovenian subjects (Caucasians) with type 2 diabetes mellitus. The case group consisted of 172 patients with PDR and the control group had 405 patients who had no clinical signs of diabetic retinopathy (DR) but did have type 2 diabetes for more than 10 years’ duration. Genotyping of eNOS polymorphisms was carried out with conventional and real-time PCR assays. A significantly higher frequency of the eNOS minor “4a” allele was found in patients with PDR than in controls (23.6 versus 17.7%, p = 0.01). Moreover, the univariate analysis showed a significant association of the 27 bp VNTR 4a/4a genotype and PDR in the recessive model. The odds ratio (OR) of PDR for the 4a/4a genotype to 4b/4a plus 4b/4b was 2.9 (95% CI 1.3–6.2, p = 0.005). Further, the presence of 4a/a genotype was associated with a 3.4-fold (95% CI 1.4–8.6, p = 0.009) increased risk for PDR while adjusted for other risk factors. This is the first study to implicate eNOS 4a/4a homozygous deletion, and hence the “4a” allele, as the genetic risk factors for PDR in Caucasians.     

Noradrenergic stimulation of BDNF synthesis in astrocytes: mediation via alpha1- and beta1/beta2-adrenergic receptors

Brain-derived neurotrophic factor (BDNF) synthesis in astrocytes induced by noradrenaline (NA) is a receptor-mediated process utilizing two parallel adrenergic pathways: beta1/beta2-adrenergic/cAMP and the novel alpha1-adrenergic/PKC pathway. BDNF is produced by astrocytes, in addition to neurons, and the noradrenergic system plays a role in controlling BDNF synthesis. Since astrocytes express various subtypes of alpha- and beta-adrenergic receptors that have the potential to be activated by synaptically released NA, we focused our present study on the mediatory role of adrenergic receptors in the noradrenergic up-regulation of BDNF synthesis in cultured neonatal rat cortical astrocytes. NA (1 microM) elevates BDNF levels by four-fold after 6 h of incubation. Its stimulation was partly inhibited by either the beta1-adrenergic antagonist atenolol, the beta2-adrenergic antagonist ICI 118,551, or by the alpha1-adrenergic antagonist prazosin, while the alpha2-adrenergic antagonist yohimbine showed no effect. BDNF levels in astrocytes were increased by the specific beta1-adrenergic agonist dobutamine and the beta2-adrenergic agonist salbutamol, as well as by adenylate cyclase activation (by forskolin) and PKA activation (by dBcAMP). However, none of the tested agonists or mediators of the intracellular beta-adrenergic pathways were able to reach the level of NA's stimulatory effect. BDNF cellular levels were also elevated by the alpha1-adrenergic agonist methoxamine, but not by the alpha2-adrenergic agonist clonidine. The increase in intracellular Ca2+ by ionophore A23187 showed no effect, whereas PKC activation by phorbol 12-myristate 13-acetate (TPA) potently stimulated BDNF levels in the cells. The methoxamine-stimulated BDNF synthesis was inhibited by desensitizing pretreatment with TPA, indicating that the alpha1-stimulation was mediated via PKC activation. In conclusion, the synthesis of astrocytic BDNF stimulated by noradrenergic neuronal activity is an adaptable process using multiple types (alpha1 and beta1/beta2) of adrenergic receptor activation.