ROS-challenged keratinocytes as a new model for oxidative stress-mediated skin diseases

In the current study, the effects of the reactive oxygen species (ROS) generator 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) on extracellular and intracellular ROS production in human keratinocytes (HACAT) were studied. AAPH is a water-soluble compound able to generate ROS at known and constant rates at 37°C. The short treatment (2 h) with AAPH brought a significant dose-dependent increase in NADPH oxidase activity in intact keratinocytes. The long-term treatment (24 h) with AAPH led to a persistent increase in NADPH oxidase activity for up to 48 hour following the AAPH removal from cell incubation medium. ROS and nitric oxide levels, lipoperoxidation, intracellular calcium, mitochondrial superoxide production, and membrane potential were significantly modified in AAPH-treated HACAT. Superoxide dismutase (SOD) and/or catalase addition to HACAT revealed that untreated keratinocytes produce mostly superoxide anion (O ₂ ⁻), while AAPH-treated keratinocytes overproduce hydrogen peroxide (H ₂O ₂) in extracellular medium. H ₂O ₂ is particularly stable and plays important roles in several cell signaling pathways. Taken together, our findings suggest a cost-effective and easily reproducible in vitro model of stressed human keratinocytes releasing significantly elevated ROS amounts in extracellular medium with respect to control keratinocytes. The possible application of the proposed model for keratinocytes-melanocytes cross-talk studies is also suggested. The model of AAPH-stressed human keratinocytes described here can represent a useful tool for redox cross-talk studies between keratinocytes and other skin cell types, and applied for researches regarding skin pathologies associated with oxidative stress.     

Studies on lipid peroxidation in the rat brain

The aim of this study was to set up a simple procedure for assessing lipid peroxidation (L.P.) and testing the activity of antioxidant compounds. L. P. was determined in rat brain homogenates by measuring the endogenous and stimulated accumulation of malonaldehyde (MDA). MDA was assayed by an HPLC method. Homogenates spontaneously formed appreciable amounts of MDA. The addition of increasing concentrations of FeCl₂ resulted in a linear accumulation of MDA, up to 16.6-fold at 50 M. An organic form of iron (Fe-saccharate) was less active on MDA formation (11.4-fold increase at 100 M). The addition of xanthine-xanthine oxidase resulted in only a 2.4-fold increase in MDA formation. Various antioxidant or chelating compounds effectively inhibited L.P., with IC₅₀ between 0.1 M (phenoxazine) and 4–50 M (-tocopherol). Their potencies depended on the iron concentration and time of preincubation with the homogenates. In conclusion, this is a simple and reliable procedure for studying L.P. and inhibiting agents, provided that the experimental conditions are carefully assessed.     

Neurological disease in wild loggerhead sea turtles Caretta caretta

Beginning in October 2000, subadult loggerhead sea turtles Caretta caretta showing clinical signs of a neurological disorder were found in waters off south Florida, USA. Histopathology indicated generalized and neurologic spirorchiidiasis. In loggerhead sea turtles (LST) with neurospirorchiidiasis, adult trematodes were found in the meninges of the brain and spinal cord of 7 and 3 affected turtles respectively, and multiple encephalic intravascular or perivascular eggs were associated with granulomatous or mixed leukocytic inflammation, vasculitis, edema, axonal degeneration and occasional necrosis. Adult spirorchiids were dissected from meningeal vessels of 2 of 11 LST brains and 1 of 10 spinal cords and were identified as Neospirorchis sp. Affected LST were evaluated for brevetoxins, ciguatoxins, saxitoxins, domoic acid and palytoxin. While tissues from 7 of 20 LST tested positive for brevetoxins, the levels were not considered to be in a range causing acute toxicosis. No known natural (algal blooms) or anthropogenic (pollutant spills) stressors co-occurred with the turtle mortality. While heavy metal toxicosis and organophosphate toxicosis were also investigated as possible causes, there was no evidence for their involvement. We speculate that the clinical signs and pathologic changes seen in the affected LST resulted from combined heavy spirorchiid parasitism and possible chronic exposure to a novel toxin present in the diet of LST.     

Niche breadth of invertebrate shredders in tropical forest streams: which taxa have restricted habitat preferences?

Despite the importance of shredders in organic matter processing in streams, information about the habitat specificities of different taxa is scarce. Herein, we evaluated the habitat preferences of invertebrate shredders in tropical forest streams. A total of 72 leaf patches were sampled in three Atlantic Forest streams (SE Brazil). For each sample, 20 environmental variables were measured, including water properties and characteristics of the leaf patch. We used an Outlying Mean Index analysis to evaluate the niche breadth and overlap of each taxon found as well as habitat preferences. In total, we found 14 shredder taxa in 68 leaf patches, and the gradient of environmental conditions influenced the distribution of most taxa. Considering taxa that occurred in atypical habitats in the study sites, Blaberidae (semiaquatic cockroaches) and Trichodactylus fluviatilis (Decapoda) showed niche segregation, while Macrobrachium potiuna (Decapoda), Tupiperla (Plecoptera), and Nectopsyche (Trichoptera) showed niche overlap. On the other hand, Heterelmis (Coleoptera), Stenochironomus (Diptera), Phylloicus major, and Triplectides gracilis (Trichoptera) occurred in typical habitats. These results showed that shredder taxa had different habitat preferences. The observed differences in niche breadths and habitat specificities among taxa probably result in different leaf processing rates across leaf patches in Atlantic Forest streams.

     

Contrasting trait assembly patterns in plant and bird communities along environmental and human‐induced land‐use gradients

Human‐driven environmental changes can induce marked shifts in the functional structure of biological communities with possible repercussion on important ecosystem functions and services. At the same time it remains unclear to which extent these changes may differently affect various types of organisms. We investigated species richness and community functional structure of species assemblages at the landscape scale (1 km² plots) for two contrasting model taxa, i.e. plants (producers and sessile organisms) and birds (consumers and mobile organisms), along topography, climate, landscape heterogeneity, and land‐use (agriculture and urbanization) gradients in a densely populated region of Switzerland. Our study revealed that agricultural and urban land uses drove marked shifts in the functional structure of biological communities compared to changes along climate and topography gradients, especially for plants, while for birds these changes were comparable. Agricultural and urban land uses enhanced divergence in traits related to resource use for birds (diet and nesting), growth forms, dispersal, and reproductive traits for plants, while it induced convergence in vegetative plant traits (plant height and leaf dry matter content). These results suggest that contrasting assembly patterns may arise within and across taxonomic groups along the same environmental gradients as result of distinct underlying processes and ‘organism‐specific’ environmental perceptions. Our results further suggest a potential homogenization of biological communities, as well as low functional diversity and redundancy levels of bird assemblages in our human‐dominated study region. This might potentially compromise the maintenance of key ecological processes under future environmental changes.     

Estimating minke whale (Balaenoptera acutorostrata) boing sound density using passive acoustic sensors

Density estimation for marine mammal species is performed primarily using visual distance sampling or capture‐recapture. Minke whales in Hawaiian waters are very difficult to sight; however, they produce a distinctive “boing” call, making them ideal candidates for passive acoustic density estimation. We used an array of 14 bottom‐mounted hydrophones, distributed over a 60 × 30 km area off Kauai, Hawaii, to estimate density during 12 d of recordings in early 2006. We converted the number of acoustic cues (i.e., boings) detected using signal processing software into a cue density by accounting for the false positive rate and probability of detection. The former was estimated by manual validation, the latter by applying spatially explicit capture‐recapture (SECR) methods to a subset of data where we had determined which hydrophones detected each call. Estimated boing density was 130 boings per hour per 10,000 km² (95% CI 104–163). Little is known about the population's acoustic behavior, so conversion from boing to animal density is difficult. As a demonstration of the method, we used a tentative boing rate of 6.04 boings per hour, from a single animal tracked in 2009, to give an estimate of 21.5 boing‐calling minke whales per 10,000 km².     

Sam68 regulates translation of target mRNAs in male germ cells,necessary for mouse spermatogenesis

Sam68 is a KH-type RNA-binding protein involved in several steps of RNA metabolism with potential implications in cell differentiation and cancer. However, its physiological roles are still poorly understood. Herein, we show that Sam68^−/− male mice are infertile and display several defects in spermatogenesis, demonstrating an essential role for Sam68 in male fertility. Sam68^−/− mice produce few spermatozoa, which display dramatic motility defects and are unable to fertilize eggs. Expression of a subset of messenger mRNAs (mRNAs) is affected in the testis of knockout mice. Interestingly, Sam68 is associated with polyadenylated mRNAs in the cytoplasm during the meiotic divisions and in round spermatids, when it interacts with the translational machinery. We show that Sam68 is required for polysomal recruitment of specific mRNAs and for accumulation of the corresponding proteins in germ cells and in a heterologous system. These observations demonstrate a novel role for Sam68 in mRNA translation and highlight its essential requirement for the development of a functional male gamete.     

Monitoring of ochratoxin A and ochratoxin-producing fungi in traditional salami manufactured in Northern Italy

Fungi have a crucial role in the correct maturation of salami, but special attention should be addressed to the production of the nephrotoxic, immunotoxic, and carcinogenic mycotoxin ochratoxin A (OTA). In a monitoring study conducted in Northern Italy, OTA was detected by liquid chromatography coupled with mass spectrometry in 13 out 133 samples of traditional salami (9.8% of the total count). Mycological analysis of these samples yielded 247 fungal isolates which were identified to species level. The most frequent species were Penicillium nalgiovense, P. solitum, and P. chrysogenum. P. nordicum, an OTA-producing species commonly found in proteinaceous food, was not found in these samples. Three isolates were found to be Aspergillus westerdijkiae, an OTA-producing species. In order to check the results of the microbiological identification, 19 different strains of Aspergillus and 94 of Penicillium were tested for the presence of a sequence common to OTA-producing fungi by real-time PCR. None of the studied isolates, including the three A. westerdijkiae, possessed the otanpsPN target which is common to OTA-producing strains. Two out of three isolates of the A. westerdijkiae were also PCR-negative for the otanpsPN gene and did not produce OTA in culture. Conversely, this target sequence was amplified from the DNA purified from 14 salami casings including three casings harboring A. westerdijkiae. The amplification of sequences specific for OTA-producing strains performed on total genomic DNA extracted directly from salami casings provided a more suitable approach than PCR analysis of isolates from salami for the OTA-related otanpsPN gene to evaluate the risk of OTA contamination.