Syndecan-1 regulates the biological activities of interleukin-34

IL-34 is a challenging cytokine sharing functional similarities with M-CSF through M-CSFR activation. It also plays a singular role that has recently been explained in the brain, through a binding to the receptor protein tyrosine phosphatase RPTPβ/ζ. The aim of this paper was to look for alternative binding of IL-34 on other cell types. Myeloid cells (HL-60, U-937, THP-1) were used as cells intrinsically expressing M-CSFR, and M-CSFR was expressed in TF-1 and HEK293 cells. IL-34 binding was studied by Scatchard and binding inhibition assays, using 125I-radiolabelled cytokines, and surface plasmon resonance. M-CSFR activation was analysed by Western blot after glycosaminoglycans abrasion, syndecan-1 overexpression or repression and addition of a blocking anti-syndecan antibody. M-CSF and IL-34 induced different patterns of M-CSFR phosphorylations, suggesting the existence of alternative binding for IL-34. Binding experiments and chondroitinase treatment confirmed low affinity binding to chondroitin sulphate chains on cells lacking both M-CSFR and RPTPβ/ζ. Amongst the proteoglycans with chondroitin sulphate chains, syndecan-1 was able to modulate the IL-34-induced M-CSFR signalling pathways. Interestingly, IL-34 induced the migration of syndecan-1 expressing cells. Indeed, IL-34 significantly increased the migration of THP-1 and M2a macrophages that was inhibited by addition of a blocking anti-syndecan-1 antibody. This paper provides evidence of alternative binding of IL-34 to chondroitin sulphates and syndecan-1 at the cell surface that modulates M-CSFR activation. In addition, IL-34-induced myeloid cell migration is a syndecan-1 dependent mechanism

Validating the RedMIT/GFP-LC3 Mouse Model by Studying Mitophagy in Autosomal Dominant Optic Atrophy Due to the OPA1Q285STOP Mutation

Background: Autosomal dominant optic atrophy (ADOA) is usually caused by mutations in the essential gene, OPA1. This encodes a ubiquitous protein involved in mitochondrial dynamics, hence tissue specificity is not understood. Dysregulated mitophagy (mitochondria recycling) is implicated in ADOA, being increased in OPA1 patient fibroblasts. Furthermore, autophagy may be increased in retinal ganglion cells (RGCs) of the OPA1Q285STOPmouse model. Aims: We developed a mouse model for studying mitochondrial dynamics in order to investigate mitophagy in ADOA. Methods: We crossed the OPA1Q285STOPmouse with our RedMIT/GFP-LC3 mouse, harboring red fluorescent mitochondria and green fluorescent autophagosomes. Colocalization between mitochondria and autophagosomes, the hallmark of mitophagy, was quantified in fluorescently labeled organelles in primary cell cultures, using two high throughput imaging methods Imagestream (Amnis) and IN Cell Analyzer 1000 (GE Healthcare Life Sciences). We studied colocalization between mitochondria and autophagosomes in fixed sections using confocal microscopy. Results: We validated our imaging methods for RedMIT/GFP-LC3 mouse cells, showing that colocalization of red fluorescent mitochondria and green fluorescent autophagosomes is a useful indicator of mitophagy. We showed that colocalization increases when lysosomal processing is impaired. Further, colocalization of mitochondrial fragments and autophagosomes is increased in cultures from the OPA1Q285STOP/RedMIT/GFP-LC3 mice compared to RedMIT/GFP-LC3 control mouse cells that were wild type for OPA1. This was apparent in both mouse embryonic fibroblasts (MEFs) using IN Cell 1000 and in splenocytes using ImageStream imaging flow cytometer (Amnis). We confirmed that this represents increased mitophagic flux using lysosomal inhibitors. We also used microscopy to investigate the level of mitophagy in the retina from the OPA1Q285STOP/RedMIT/GFP-LC3 mice and the RedMIT/GFP-LC3 control mice. However, the expression levels of fluorescent proteins and the image signal-to-background ratios precluded the detection of colocalization so we were unable to show any difference in colocalization between these mice. Conclusions: We show that colocalization of fluorescent mitochondria and autophagosomes in cell cultures, but not fixed tissues from the RedMIT/GFP-LC3, can be used to detect mitophagy. We used this model to confirm that mitophagy is increased in a mouse model of ADOA. It will be useful for cell based studies of diseases caused by impaired mitochondrial dynamics

Visualisation of fouling during microfiltration of natural brown water by using wavelets of ultrasonic spectra

The fouling of microfiltration membranes by natural brown-coloured surface water was studied in situ by ultrasonic time-domain reflectometry. An ultrasonic measurement system consisting of a 7.5 MHz ultrasonic transducer, a pulser-receiver, a digital oscilloscope and a computer was connected to a laboratory-scale, flat-bed filtration module. The ultrasonic spectra were presented by line graphs and then transformed into wavelets. The latter presents the frequency spectrum of ultrasonic signals as the same function of time as in the line graphs. The ultrasonic frequency data indicated the first change at the membrane surface at 10 s. This could be seen more clearly by subtracting the zero-time waveform from the subsequent waveforms. The entire fouling process and fouling layer growth could be more clearly and qualitatively monitored by applying wavelets to the ultrasonic reflection data, which normally gives less quantitative factual data. Vertical lines on the time axis or horizontal lines on the frequency axis can be drawn to intersect the fouling peak and this data can then be extracted to plot amplitude of the fouling peak versus operation time or versus frequency. Such new curves portraying the growth of the fouling peak, even at extremely short-fouling times, provide interesting data on the fouling process. These ultrasonic results were supported by flux measurements and scanning electron microscopy (SEM) examinations of the original and fouled membrane surfaces. © 2005 Elsevier B.V. All rights reserved.Articl

Assessing the performance of EU nature legislation in protecting target bird species in an era of climate change

International legislation forms a cornerstone of conservation, yet its efficacy is rarely quantified. We assess whether species listed on Annex I of the European Union (EU) Birds Directive, for which EU Member States are obliged to implement special conservation measures, differ systematically in their short-term (2001-2012) or long-term (1980-2012) population trends from those of non-Annex I species. In both periods, Annex I species had more positive trends than non-Annex I species, particularly in countries that joined the EU earlier. There were additional signatures of climate change and life history strategy in the trends of species in one or both periods. Within Annex I species, long-distance migrants fared significantly worse than other species, suggesting that enhanced protection on the breeding grounds alone may be insufficient for these species. We conclude that the EU's conservation legislation has had a demonstrably positive impact on target species, even during a period in which climate change has significantly affected populations

Precipitation Estimation: From the RAO to EURAINSAT and Beyond

The key objective of the project “Use of the MSG SEVIRI channels in a combined SSM/I, TRMM and geostationary IR method for rapid updates of rainfall” is the development of algorithms for rapid-update of satellite rainfall estimations at the geostationary (GEO) scale. The new channels available with the Spinning Enhanced Visible and Infrared Imager (SEVIRI) radiometer in the visible (VIS), near infrared (NIR) and infrared (IR) portions of the spectrum provide new insights into the microphysical and dynamic structure of precipitating clouds thus allowing for a more precise identification of precipitation intensities. Passive microwave (PMW) radiometers on board low Earth orbiting (LEO) satellites are used to determine information on the vertical cloud structure. Key features of the new method(s) are: 1. Microphysical characterization of precipitating clouds with VIS/IR sensors; 2. Creation of cloud microphysical and radiative databases from cloud model outputs and aircraft penetrations; 3. Tuning of PMW algorithms for different cloud systems (maritime, continental, convective, stratiform,...); 4. Combination of data from different algorithms and application to a rapid update cycle at the GEO scale. The project provided the background for EURAINSAT “European Satellite Rainfall Estimation and Monitoring at the Geostationary Scale”, a research project co-funded by the Energy, Environment and Sustainable Development Programme of the European Commission within the topic “Development of generic Earth observation technologies”. The project web site is accessible at http://www.isac.cnr.it/~eurainsat/. Moreover, it has represented the European framework for the launch of the International Precipitation Working Group (IPWG)