uPAR Expression Pattern in Patients with Urothelial Carcinoma of the Bladder – Possible Clinical Implications

The objective of the present study was to confirm the expression and localisation pattern of the urokinase-type plasminogen activator receptor (uPAR) focusing on its possible clinical relevance in patients with urothelial neoplasia of the bladder. uPAR is a central molecule in tissue remodelling during cancer invasion and metastasis and is an established prognostic marker in various cancer diseases other than bladder cancer. Formalin-fixed and paraffin-embedded tumour-tissue blocks from 186 patients treated with radical cystectomy were analysed. uPAR expression was scored as either negative or positive as well as by the actual score. Separate scores were obtained for cancer cells, macrophages and myofibroblasts at the invasive front and in tumour core. We were able to confirm, in an independent patient cohort, the tissue expression and localisation pattern of uPAR as investigated by Immunohistochemistry as well as a significant association between uPAR positivity and increasing tumour stage and tumour grade. This demonstrates the robustness of our previous and current findings. In addition the association between uPAR positive myofibroblasts and poor survival was reproduced. The highest hazard ratios for survival were seen for uPAR positive myofibroblasts both at the invasive front and in tumour core. Evaluating uPAR expression by the actual score showed a significant association between uPAR positive myofibroblasts in tumour core and an increased risk of cancer specific mortality. Our investigations have generated new and valuable biological information about the cell types being involved in tumour invasion and progression through the plasminogen activation system.     

Resuscitation with supplementary oxygen induces oxidative injury in the cerebral cortex

Isoprostanes, neuroprostanes, isofurans, and neurofurans have all become attractive biomarkers of oxidative damage and lipid peroxidation in brain tissue. Asphyxia and subsequent reoxygenation cause a burst of oxygen free radicals. Isoprostanes and isofurans are generated by free radical attacks of esterified arachidonic acid. Neuroprostanes and neurofurans are derived from the peroxidation of docosahexanoic acid, which is abundant in neurons and could therefore more selectively represent oxidative brain injury. Newborn piglets (age 12-36h) underwent hypoxia until the base excess reached -20mmol/L or the mean arterial blood pressure dropped below 15mm Hg. They were randomly assigned to receive resuscitation with 21, 40, or 100% oxygen for 30min and then ventilation with air. The levels of isoprostanes, isofurans, neuroprostanes, and neurofurans were determined in brain tissue (ng/g) isolated from the prefrontal cortex using gas chromatography-mass spectrometry (GC/MS) with negative ion chemical ionization (NICI) techniques. A control group underwent the same procedures and observations but was not submitted to hypoxia or hyperoxia. Hypoxia and reoxygenation significantly increased the levels of isoprostanes, isofurans, neuroprostanes, and neurofurans in the cerebral cortex. Nine hours after resuscitation with 100% oxygen for 30min, there was nearly a 4-fold increase in the levels of isoprostanes and isofurans compared to the control group (P=0.007 and P=0.001) and more than a 2-fold increase in neuroprostane levels (P=0.002). The levels of neuroprostanes and neurofurans were significantly higher in the piglets that were resuscitated with supplementary oxygen (40 and 100%) compared to the group treated with air (21%). The significance levels of the observed differences in neuroprostanes for the 21% vs 40% comparison and the 21% vs 100% comparison were P<0.001 and P=0.001, respectively. For neurofurans, the P values of the 21% vs 40% comparison and the 21% vs 100% comparison were P=0.036 and P=0.025, respectively. Supplementary oxygen used for the resuscitation of newborns increases lipid peroxidation in brain cortical neurons, a result that is indicative of oxidative brain damage. These novel findings provide new knowledge regarding the relationships between oxidative brain injury and resuscitation with oxygen.     

Synergistic Effect of Bimetallic MOF Modified Separator for Long Cycle Life Lithium-Sulfur Batteries

Severe polysulfide dissolution and shuttling are the main challenges that plague the long cycle life and capacity retention of lithium-sulfur (Li-S) batteries. To address these challenges, efficient separators are designed and modified with a dual functional bimetallic metal-organic framework (MOF). Flower-shaped bimetallic MOFs (i.e., Fe-ZIF-8) with nanostructured pores are synthesized at 35 °C in water by introducing dopant metal sites (Fe), which are then coated on a polypropylene (PP) separator to provide selective channels, thereby effectively inhibiting the migration of lithium polysulfides while allowing homogeneous transport of Li-ions. The active sites of the Fe-ZIF-8 enable electrocatalytic conversion, facilitating the conversion of lithium polysulfides. Moreover, the developed separator can prevent dendrite formation due to the uniform pore size and hence the even Li-ion transport and deposition. A coin cell using a Fe-ZIF-8/PP separator with S-loaded carbon cathode displayed a high cycle life of 1000 cycles with a high initial discharge capacity of 863 mAh g⁻¹ at 0.5 C and a discharge capacity of 746 mAh g⁻¹ at a high rate of 3 C. Promising specific capacity has been documented even under high sulfur loading of 5.0 mg cm⁻² and electrolyte to the sulfur ratio (E/S) of 5 µL mg⁻¹.     

Genetic variability and transgenerational regulation of investment in sex in the monogonont rotifer Brachionus plicatilis

In cyclical parthenogens such as aphids, cladocerans and rotifers, the coupling between sexual reproduction and the production of resting stages (diapausing eggs) imposes strong constraints on the timing of sex. Whereas induction of sex is generally triggered by environmental cues, the response to such cues may vary across individuals according to genetic and nongenetic factors. In this study, we explored genetic and epigenetic causes of variation for the propensity for sex using a collection of strains from a Spanish population of monogonont rotifers (Brachionus plicatilis) in which variation for the threshold population density at which sex is induced (mixis threshold) had been documented previously. Our results show significant variation for the mixis threshold among 20 clones maintained under controlled conditions for 15 asexual generations. The effect of the number of clonal generations since hatching of the diapausing egg on the mixis ratio (proportion of sexual offspring produced) was tested on 4 clones with contrasted mixis thresholds. The results show a negative correlation between the mixis threshold and mixis ratio, as well as a significant effect of the number of clonal generations since fertilization, sex being repressed during the first few generations after hatching of the diapausing egg.     

Dynamic coating for fast and reproducible determination of basic drugs by capillary electrophoresis with diode-array detection and mass spectrometry

The double coating principle of CEofix buffers was evaluated for the analysis of some basic drugs by capillary electrophoresis-diode-array detection (CE-DAD) and capillary electrophoresis-mass spectrometry (CE-MS). The involatile phosphate present in original low pH CEofix, was replaced with formic acid for hyphenation of CE with MS. The double coating produces a substantial and highly reproducible electroosmotic flow (EOF), even at low pH. The rinsing procedure and electrolyte composition were optimized for both CE-DAD and CE-MS. The system was evaluated with the analysis of a mixture of basic drugs and a spiked urine sample enriched by solid-phase extraction (SPE). The R.S.D. values on the migration time and peak area measured for 28 analyses with CE-DAD were below 0.25 and 2.40%, respectively. For CE-MS, the R.S.D. on the migration time was 0.85% or less and the area precision ranged from 5.65 to 14.33% (for seven injections). The LOD with the developed CE-MS method was below 50 ppb for all five drug standards tested.     

Resistance of the dopamine D4 receptor to agonist-induced internalization and degradation

Dopamine receptors are G-protein-coupled receptors involved in the control of motivation, learning, and fine-tuning of motor movement, as well as modulation of neuroendocrine signalling. Stimulation of G-protein-coupled receptors normally results in attenuation of signalling through desensitization, followed by internalization and down-regulation of the receptor. These processes allow the cell to regain homeostasis after exposure to extracellular stimuli and offer protection against excessive signalling.Here, we have investigated the agonist-mediated attenuation properties of the dopamine D4 receptor.We found that several hallmarks of signal attenuation such as receptor phosphorylation, internalization and degradation showed a blunted response to agonist treatment. Moreover, we did not observe recruitment of β-arrestins upon D4 receptor stimulation. We also provide evidence for the constitutive phosphorylation of two serine residues in the third intracellular loop of the D4 receptor.These data demonstrate that, when expressed in CHO, HeLa and HEK293 cells, the human D4 receptor shows resistance to agonist-mediated internalization and down-regulation. Data from neuronal cell lines, which have been reported to show low endogenous D4 receptor expression, such as the hippocampal cell line HT22 and primary rat hippocampal cells, further support these observations.     

Spatial variability in seed predation in Primula farinosa: local population legacy versus patch selection

Spatio-temporal variation in seed predation may strongly influence both plant population dynamics and selection on plant traits. The intensity of seed predation may depend on a number of factors, but the relative importance of previous predator abundance (“local legacy”), spatial distribution of the host plant, environmental factors and plant characteristics has been explored in few species. We monitored seed predation in the perennial herb Primula farinosa, which is dimorphic for scape length, during 5 consecutive years, in a 10-km × 4-km area comprising 79 P. farinosa populations. A transplant experiment showed that the seed predator, the oligophagous tortricid moth Falseuncaria ruficiliana, was not dispersal limited at the spatial scale corresponding to typical distances between P. farinosa populations. Correlations between population characteristics and incidence and intensity of seed predation varied among years. The incidence of the seed predator was positively correlated with host population size and mean number of flowers, while intensity of seed predation in occupied patches was positively related to the frequency of the long-scaped morph in 2 years and negatively related to host population size in 1 year. In both scape morphs, predation tended to increase with increasing frequency of the long morph. There was no evidence of a local legacy; incidence and intensity of seed predation were not related to the abundance of the seed predator in the population in the previous year. Taken together, the results indicate that among-population variation in seed predation intensity is determined largely by patch selection and that the seed predator’s preference for tall and many-flowered inflorescences may not only affect selection on plant traits within host plant populations, but also the overall intensity of seed predation.     

KLHL12-mediated ubiquitination of the dopamine D4 receptor does not target the receptor for degradation

In previous studies, we identified KLHL12 as a novel interaction partner of the dopamine D4 receptor that functions as an adaptor in a Cullin3-based E3 ubiquitin ligase complex to target the receptor for ubiquitination. In this study, we show that KLHL12 promotes poly-ubiquitination of the receptor by performing ubiquitination assays in eukaryotic cells. Furthermore, we demonstrate that KLHL12 not only interacts with both immature, ER-associated and mature, plasma membrane-associated D4 receptors, but also promotes ubiquitination of both receptor subpools. Unexpectedly, however, KLHL12-mediated receptor ubiquitination does not promote proteasomal degradation of newly synthesized receptors through the ER-associated degradation pathway or lysosomal degradation of mature receptors. Moreover, our data reveal that D4 receptors do not undergo agonist-promoted ubiquitination or degradation, in contrast to many other G-protein-coupled receptors (GPCRs) indicating that ubiquitination of GPCRs does not defaultly lead to receptor degradation. Interestingly, KLHL12 does also interact with β-arrestin2 but this has no effect on the ubiquitination or localization of β-arrestin2 nor on the internalization of the D4 receptor.