Two-color far-field fluorescence nanoscopy

We demonstrate two-color fluorescence microscopy with nanoscale spatial resolution by applying stimulated emission depletion on fluorophores differing in their absorption and emission spectra. Green- and red-emitting fluorophores are selectively excited and quenched using dedicated beam pairs. The stimulated emission depletion beams deliver a lateral resolution of <30 nm and 65 nm for the green and the red color channel, respectively. The approximately 5 nm alignment accuracy of the two images establishes the precision with which differently labeled proteins are correlated in space. Colocalized nanoscopy is demonstrated with endosomal protein patterns and by resolving nanoclusters of a mitochondrial outer membrane protein, Tom20, in relation with the F(1)F(0)ATP synthase. The joint improvement of resolution and colocalization demonstrates the emerging potential of far-field fluorescence nanoscopy to study the spatial organization of macromolecules in cells.     

Formation of reactive oxygen species in L929 cells after exposure to 900 MHz RF radiation with and without co-exposure to 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone

The aim of this study was to investigate the induction of reactive oxygen species in murine L929 fibrosarcoma cells exposed to radiofrequency (RF) radiation at 900 MHz, with or without co-exposure to 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a potent environmental carcinogen produced during chlorination of drinking water. Both continuous-wave and GSM mobile phone signals were applied for 10 or 30 min at specific absorption rates of 0.3 and 1 W/kg. Simultaneous sham exposures were performed for each exposure condition. MX treatment was performed at a subtoxic level of 500 microM, and the RF-field exposure was carried out during the first 10 or 30 min of the chemical treatment. The formation of reactive oxygen species was followed soon after the exposure and at different harvesting times until 1 h after RF-field treatment. The studied provided no indication that 900 MHz RF-field exposure, either alone or in combination with MX, induced formation of reactive oxygen species under any of the experimental conditions investigated. In contrast, exposure to MX resulted in a statistically significant increase in the formation of reactive oxygen species for all the treatment durations investigated, confirming that MX is an inductor of oxidative stress in L929 cells.     

Inhibition of Pin1 reduces glutamate-induced perikaryal accumulation of phosphorylated neurofilament-H in neurons

Under normal conditions, the proline-directed serine/threonine residues of neurofilament tail-domain repeats are exclusively phosphorylated in axons. In pathological conditions such as amyotrophic lateral sclerosis (ALS), motor neurons contain abnormal perikaryal accumulations of phosphorylated neurofilament proteins. The precise mechanisms for this compartment-specific phosphorylation of neurofilaments are not completely understood. Although localization of kinases and phosphatases is certainly implicated, another possibility involves Pin1 modulation of phosphorylation of the proline-directed serine/threonine residues. Pin1, a prolyl isomerase, selectively binds to phosphorylated proline-directed serine/threonine residues in target proteins and isomerizes cis isomers to more stable trans configurations. In this study we show that Pin1 associates with phosphorylated neurofilament-H (p-NF-H) in neurons and is colocalized in ALS-affected spinal cord neuronal inclusions. To mimic the pathology of neurodegeneration, we studied glutamate-stressed neurons that displayed increased p-NF-H in perikaryal accumulations that colocalized with Pin1 and led to cell death. Both effects were reduced upon inhibition of Pin1 activity by the use of an inhibitor juglone and down-regulating Pin1 levels through the use of Pin1 small interfering RNA. Thus, isomerization of lys-ser-pro repeat residues that are abundant in NF-H tail domains by Pin1 can regulate NF-H phosphorylation, which suggests that Pin1 inhibition may be an attractive therapeutic target to reduce pathological accumulations of p-NF-H.     

Taxonomic,functional, and phylogenetic β‐diversity patterns of stream fish assemblages in tropical agroecosystems

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Genetic trials improve the transfer of Douglas‐fir distribution models across continents

Climate change is likely to result in novel conditions with no analogy to current climate. Therefore, the application of species distribution models (SDMs) based on the correlation between observed species’ occurrence and their environment is questionable and calls for a better understanding of the traits that determine species occurrence. Here, we compared two intraspecific, trait‐based SDMs with occurrence‐based SDMs, all developed from European data, and analyzed their transferability to the native range of Douglas‐fir in North America. With data from 50 provenance trials of Douglas‐fir in central Europe multivariate universal response functions (URFs) were developed for two functional traits (dominant tree height and basal area) which are good indicators of growth and vitality under given environmental conditions. These trials included 290 North American provenances of Douglas‐fir. The URFs combine genetic effects i.e. the climate of provenance origin and environmental effects, i.e. the climate of planting locations into an integrated model to predict the respective functional trait from climate data. The URFs were applied as SDMs (URF‐SDMs) by converting growth performances into occurrence. For comparison, an ensemble occurrence‐based SDM was developed and block cross validated with the BIOMOD2 modeling platform utilizing the observed occurrence of Douglas‐fir in Europe. The two trait based SDMs and the occurrence‐based SDM, all calibrated with data from Europe, were applied to predict the known distribution of Douglas‐fir in its introduced and native range in Europe and North America. Both models performed well within their calibration range in Europe, but model transfer to its native range in North America was superior in case of the URF‐SDMs showing similar predictive power as SDMs developed in North America itself. The high transferability of the URF‐SDMs is a testimony of their applicability under novel climatic conditions highlighting the role of intraspecific trait variation for adaptation planning in climate change.     

MICAL2 is expressed in cancer associated neo-angiogenic capillary endothelia and it is required for endothelial cell viability,motility and VEGF response

The capacity of inducing angiogenesis is a recognized hallmark of cancer cells. The cancer microenvironment, characterized by hypoxia and inflammatory signals, promotes proliferation, migration and activation of quiescent endothelial cells (EC) from surrounding vascular network. Current anti-angiogenic drugs present side effects, temporary efficacy, and issues of primary resistance, thereby calling for the identification of new therapeutic targets.MICALs are a unique family of redox enzymes that destabilize F-actin in cytoskeletal dynamics. MICAL2 mediates Semaphorin3A-NRP2 response to VEGFR1 in rat ECs. MICAL2 also enters the p130Cas interactome in response to VEGF in HUVEC. Previously, we showed that MICAL2 is overexpressed in metastatic cancer. A small-molecule inhibitor of MICAL2 exists (CCG-1423).Here we report that 1) MICAL2 is expressed in neo-angiogenic ECs in human solid tumors (kidney and breast carcinoma, glioblastoma and cardiac myxoma, n = 67, were analyzed with immunohistochemistry) and in animal models of ischemia/inflammation neo-angiogenesis, but not in normal capillary bed; 2) MICAL2 protein pharmacological inhibition (CCG-1423) or gene KD reduce EC viability and functional performance; 3) MICAL2 KD disables ECs response to VEGF in vitro. Whole-genome gene expression profiling reveals MICAL2 involvement in angiogenesis and vascular development pathways.Based on these results, we propose that MICAL2 expression in ECs participates to inflammation-induced neo-angiogenesis and that MICAL2 inhibition should be tested in cancer- and noncancer-associated neo-angiogenesis, where chronic inflammation represents a relevant pathophysiological mechanism.     

Proteomic analysis of the inflamed intestinal mucosa reveals distinctive immune response profiles in Crohn's disease and ulcerative colitis

Although Crohn's disease (CrD) and ulcerative colitis (UC) share several clinical features, the mechanisms of tissue injury differ. Because the global cellular function depends upon the protein network environment as a whole, we explored changes in the distribution and association of mucosal proteins to define key events involved in disease pathogenesis. Endoscopic biopsies were taken from CrD, UC, and control colonic mucosa, and Multi-Epitope-Ligand-Cartographie immunofluorescence microscopy with 32 different Abs was performed. Multi-Epitope-Ligand-Cartographie is a novel, highly multiplexed robotic imaging technology which allows integrating cell biology and biomathematical tools to visualize dozens of proteins simultaneously in a structurally intact cell or tissue. In CrD, the number of CD3+CD45RA+ naive T cells was markedly increased, but only activated memory, but not naive, T cells expressed decreased levels of Bax, active caspase-3 or -8. In UC, only CD4+ T cells coexpressing NF-kappaB were caspase-8 and poly(ADP-ribose)-polymerase positive. Furthermore, the number of CD4+CD25+ T cells was elevated only in UC, whereas in CrD and controls, the number of these cells was similar. By using hub analysis, we also identified that the colocalization pattern with NF-kappaB+ and poly(ADP-ribose)-polymerase+ as base motifs distinguished CrD from UC. High-content proteomic analysis of the intestinal mucosa demonstrated for the first time that different T cell populations within the intestinal mucosa express proteins translating distinct biological functions in each form of inflammatory bowel disease. Thus, topological proteomic analysis may help to unravel the pathogenesis of inflammatory bowel disease by defining distinct immunopathogenic profiles in CrD and UC.     

TLR3- and Th2 cytokine-dependent production of thymic stromal lymphopoietin in human airway epithelial cells

Thymic stromal lymphopoietin (TSLP) is elevated in asthma and triggers dendritic cell-mediated activation of Th2 inflammatory responses. Although TSLP has been shown to be produced mainly by airway epithelial cells, the regulation of epithelial TSLP expression has not been extensively studied. We investigated the expression of TSLP in cytokine- or TLR ligand-treated normal human bronchial epithelial cells (NHBE). The mRNA for TSLP was significantly up-regulated by stimulation with IL-4 (5.5-fold) and IL-13 (5.3-fold), weakly up-regulated by TNF-alpha, TGF-beta, and IFN-beta, and not affected by IFN-gamma in NHBE. TSLP mRNA was only significantly up-regulated by the TLR3 ligand (dsRNA) among the TLR ligands tested (66.8-fold). TSLP was also induced by in vitro infection with rhinovirus. TSLP protein was detected after stimulation with dsRNA (120 +/- 23 pg/ml). The combination of TNF-alpha and IL-4 produced detectable levels of TSLP protein (40 +/- 13 pg/ml). In addition, TSLP was synergistically enhanced by a combination of IL-4 and dsRNA (mRNA; 207-fold, protein; 325 +/- 75 pg/ml). The induction of TSLP by dsRNA was dependent upon NF-kappaB and IFN regulatory factor 3 (IRF-3) signaling via TLR3 as indicated by a study with small interfering RNA. The potent topical glucocorticoid fluticasone propionate significantly suppressed dsRNA-dependent TSLP production in NHBE. These results suggest that the expression of TSLP is induced in airway epithelial cells by stimulation with the TLR3 ligand and Th2 cytokines and that this response is suppressed by glucocorticoid treatment. This implies that respiratory viral infection and the recruitment of Th2 cytokine producing cells may amplify Th2 inflammation via the induction of TSLP in the asthmatic airway.     

Survival of atherosclerotic patients as related to oxidative stress and gene polymorphisms

A prospective molecular epidemiology study was implemented in a cohort of 98 subjects suffering from severe atherosclerotic lesions requiring removal of an abdominal aorta fragment. We previously published the results relative to detection, in the aorta medium layer, of bulky DNA adducts and fluorescent polycyclic aromatic hydrocarbon-related DNA adducts, oxidative DNA damage, and mitochondrial DNA 4977 common deletion, as well as GSTM1 and GSTT1 gene polymorphisms. We report herein new data, relative to oxidative stress biomarkers, including oxidative DNA damage in both inner and medium aorta layers, malondialdehyde in the medium layer, homocysteine and reduced glutathione in plasma, and those relative to additional gene polymorphisms, including NAT1, NAT2, OGG1, MTHFR, Leiden factor V, and prothrombin. The results of biochemical and molecular analyses were related to survival of the patients, whose average age was 70 at the start of the follow up. During the following 14 years, 71.4% of them died. The results obtained provide evidence for the crucial impact of oxidative stress and certain gene polymorphisms on clinical and biochemical patterns as well as on survival of patients. Survival was significantly affected not only by traditional risk factors for atherosclerosis but also by molecular end-points and adverse gene polymorphisms, and by their combinations.