Nonclinical evaluation of chronic cardiac contractility modulation on 3D human engineered cardiac tissues.

INTRODUCTION: Cardiac contractility modulation (CCM) is a medical device-based therapy delivering non-excitatory electrical stimulations to the heart to enhance cardiac function in heart failure (HF) patients. The lack of human in vitro tools to assess CCM hinders our understanding of CCM mechanisms of action. Here, we introduce a novel chronic (i.e., 2-day) in vitro CCM assay to evaluate the effects of CCM in a human 3D microphysiological system consisting of engineered cardiac tissues (ECTs). METHODS: Cryopreserved human induced pluripotent stem cell-derived cardiomyocytes were used to generate 3D ECTs. The ECTs were cultured, incorporating human primary ventricular cardiac fibroblasts and a fibrin-based gel. Electrical stimulation was applied using two separate pulse generators for the CCM group and control group. Contractile properties and intracellular calcium were measured, and a cardiac gene quantitative PCR screen was conducted. RESULTS: Chronic CCM increased contraction amplitude and duration, enhanced intracellular calcium transient amplitude, and altered gene expression related to HF (i.e., natriuretic peptide B, NPPB) and excitation-contraction coupling (i.e., sodium-calcium exchanger, SLC8). CONCLUSION: These data represent the first study of chronic CCM in a 3D ECT model, providing a nonclinical tool to assess the effects of cardiac electrophysiology medical device signals complementing in vivo animal studies. The methodology established a standardized 3D ECT-based in vitro testbed for chronic CCM, allowing evaluation of physiological and molecular effects on human cardiac tissues.

Bringing synapses into focus: Recent advances in synaptic imaging and mass-spectrometry for studying synaptopathy.

Synapses are integral for healthy brain function and are becoming increasingly recognized as key structures in the early stages of brain disease. Understanding the pathological processes driving synaptic dysfunction will unlock new therapeutic opportunities for some of the most devastating diseases of our time. To achieve this we need a solid repertoire of imaging and molecular tools to interrogate synaptic biology at greater resolution. Synapses have historically been examined in small numbers, using highly technical imaging modalities, or in bulk, using crude molecular approaches. However, recent advances in imaging techniques are allowing us to analyze large numbers of synapses, at single-synapse resolution. Furthermore, multiplexing is now achievable with some of these approaches, meaning we can examine multiple proteins at individual synapses in intact tissue. New molecular techniques now allow accurate quantification of proteins from isolated synapses. The development of increasingly sensitive mass-spectrometry equipment means we can now scan the synaptic molecular landscape almost in totality and see how this changes in disease. As we embrace these new technical developments, synapses will be viewed with clearer focus, and the field of synaptopathy will become richer with insightful and high-quality data. Here, we will discuss some of the ways in which synaptic interrogation is being facilitated by methodological advances, focusing on imaging, and mass spectrometry.

GDNF Increases Inhibitory Synaptic Drive on Principal Neurons in the Hippocampus via Activation of the Ret Pathway.

Glial cell line-derived neurotrophic factor (GDNF) has been shown to counteract seizures when overexpressed or delivered into the brain in various animal models of epileptogenesis or chronic epilepsy. The mechanisms underlying this effect have not been investigated. We here demonstrate for the first time that GDNF enhances GABAergic inhibitory drive onto mouse pyramidal neurons by modulating postsynaptic GABAA receptors, particularly in perisomatic inhibitory synapses, by GFRα1 mediated activation of the Ret receptor pathway. Other GDNF receptors, such as NCAM or Syndecan3, are not contributing to this effect. We observed similar alterations by GDNF in human hippocampal slices resected from epilepsy patients. These data indicate that GDNF may exert its seizure-suppressant action by enhancing GABAergic inhibitory transmission in the hippocampal network, thus counteracting the increased excitability of the epileptic brain. This new knowledge can contribute to the development of novel, more precise treatment strategies based on a GDNF gene therapy approach.

Synaptic proteomics reveal distinct molecular signatures of cognitive change and C9ORF72 repeat expansion in the human ALS cortex.

Increasing evidence suggests synaptic dysfunction is a central and possibly triggering factor in Amyotrophic Lateral Sclerosis (ALS). Despite this, we still know very little about the molecular profile of an ALS synapse. To address this gap, we designed a synaptic proteomics experiment to perform an unbiased assessment of the synaptic proteome in the ALS brain. We isolated synaptoneurosomes from fresh-frozen post-mortem human cortex (11 controls and 18 ALS) and stratified the ALS group based on cognitive profile (Edinburgh Cognitive and Behavioural ALS Screen (ECAS score)) and presence of a C9ORF72 hexanucleotide repeat expansion (C9ORF72-RE). This allowed us to assess regional differences and the impact of phenotype and genotype on the synaptic proteome, using Tandem Mass Tagging-based proteomics. We identified over 6000 proteins in our synaptoneurosomes and using robust bioinformatics analysis we validated the strong enrichment of synapses. We found more than 30 ALS-associated proteins in synaptoneurosomes, including TDP-43, FUS, SOD1 and C9ORF72. We identified almost 500 proteins with altered expression levels in ALS, with region-specific changes highlighting proteins and pathways with intriguing links to neurophysiology and pathology. Stratifying the ALS cohort by cognitive status revealed almost 150 specific alterations in cognitively impaired ALS synaptic preparations. Stratifying by C9ORF72-RE status revealed 330 protein alterations in the C9ORF72-RE +ve group, with KEGG pathway analysis highlighting strong enrichment for postsynaptic dysfunction, related to glutamatergic receptor signalling. We have validated some of these changes by western blot and at a single synapse level using array tomography imaging. In summary, we have generated the first unbiased map of the human ALS synaptic proteome, revealing novel insight into this key compartment in ALS pathophysiology and highlighting the influence of cognitive decline and C9ORF72-RE on synaptic composition.

Array tomography: 15 years of synaptic analysis.

Synapses are minuscule, intricate structures crucial for the correct communication between neurons. In the 125 years since the term synapse was first coined, we have advanced a long way when it comes to our understanding of how they work and what they do. Most of the fundamental discoveries have been invariably linked to advances in technology. However, due to their size, delicate structural integrity and their sheer number, our knowledge of synaptic biology has remained somewhat elusive and their role in neurodegenerative diseases still remains largely unknown. Here, we briefly discuss some of the imaging technologies used to study synapses and focus on the utility of the high-resolution imaging technique array tomography (AT). We introduce the AT technique and highlight some of the ways it is utilised with a particular focus on its power for analysing synaptic composition and pathology in human post-mortem tissue. We also discuss some of the benefits and drawbacks of techniques for imaging synapses and highlight some recent advances in the study of form and function by combining physiology and high-resolution synaptic imaging.

Future hydrological constraints of the Montseny brook newt (Calotriton arnoldi) under changing climate and vegetation cover.

The Montseny brook newt (Calotriton arnoldi) is a critically endangered amphibian species which inhabits a small 20 km2 holm oak and beech forest area in NE Spain. Calotriton arnoldi strictly lives in running waters and might be highly vulnerable to hydrological perturbations expected to occur under climate and vegetation cover changes. Knowledge about the potential response of the species habitat to environmental changes can help assessing the actions needed for its conservation. Based on knowledge of the species and supported by observations, we proposed daily low and high streamflow event thresholds for the viability of C. arnoldi. We used the rainfall-runoff model PERSiST to simulate changes in the frequency and duration of these events, which were predicted under two climate and four vegetation cover scenarios for near-future (2031-2050) and far-future (2081-2100) periods in a reference catchment. All future scenarios projected a significant decrease in annual streamflow (from 21% to as much as 67%) with respect to the reference period. The frequency and length of low streamflow events will dramatically increase. In contrast, the risk of catastrophic drift linked to high streamflow events was predicted to decrease. The potential change in vegetation toward an expansion of holm oak forests will be more important than climate changes in determining threshold low flow conditions. We thus demonstrated that consideration of potential changes in vegetation and not only changes in climate variables is essential in simulating future streamflows. This study shows that future low streamflow conditions will pose a severe threat for the survival of C. arnoldi and may help taking management actions, including limiting the expansion of holm oak forest, for ameliorating the species habitat and help its conservation.

Joining empirical and modelling approaches to estimate dry deposition of nitrogen in Mediterranean forests.

In Mediterranean areas, dry deposition is a major component of the total atmospheric N input to natural habitats, particularly to forest ecosystems. An innovative approach, combining the empirical inferential method (EIM) for surface deposition of NO3- and NH4+ with stomatal uptake of NH3, HNO3 and NO2 derived from the DO3SE (Deposition of Ozone and Stomatal Exchange) model, was used to estimate total dry deposition of inorganic N air pollutants in four holm oak forests under Mediterranean conditions in Spain. The estimated total deposition varied among the sites and matched the geographical patterns previously found in model estimates: higher deposition was determined at the northern site (28.9 kg N ha-1 year-1) and at the northeastern sites (17.8 and 12.5 kg N ha-1 year-1) than at the central-Spain site (9.4 kg N ha-1 year-1). On average, the estimated dry deposition of atmospheric N represented 77% ±â€¯2% of the total deposition of N, of which surface deposition of gaseous and particulate atmospheric N averaged 10.0 ±â€¯2.9 kg N ha-1 year-1 for the four sites (58% of the total deposition), and stomatal deposition of N gases averaged 3.3 ±â€¯0.8 kg N ha-1 year-1 (19% of the total deposition). Deposition of atmospheric inorganic N was dominated by the surface deposition of oxidized N in all the forests (means of 54% and 42% of the dry and total deposition, respectively). The relative contribution of NO2 to dry deposition averaged from 19% in the peri-urban forests to 11% in the most natural site. During the monitoring period, the empirical critical loads provisionally proposed for ecosystem protection (10-20 kg N ha-1 year-1) was exceeded in three of the four studied forests.

Ecological impacts of atmospheric pollution and interactions with climate change in terrestrial ecosystems of the Mediterranean Basin: Current research and future directions.

Mediterranean Basin ecosystems, their unique biodiversity, and the key services they provide are currently at risk due to air pollution and climate change, yet only a limited number of isolated and geographically-restricted studies have addressed this topic, often with contrasting results. Particularities of air pollution in this region include high O3 levels due to high air temperatures and solar radiation, the stability of air masses, and dominance of dry over wet nitrogen deposition. Moreover, the unique abiotic and biotic factors (e.g., climate, vegetation type, relevance of Saharan dust inputs) modulating the response of Mediterranean ecosystems at various spatiotemporal scales make it difficult to understand, and thus predict, the consequences of human activities that cause air pollution in the Mediterranean Basin. Therefore, there is an urgent need to implement coordinated research and experimental platforms along with wider environmental monitoring networks in the region. In particular, a robust deposition monitoring network in conjunction with modelling estimates is crucial, possibly including a set of common biomonitors (ideally cryptogams, an important component of the Mediterranean vegetation), to help refine pollutant deposition maps. Additionally, increased attention must be paid to functional diversity measures in future air pollution and climate change studies to establish the necessary link between biodiversity and the provision of ecosystem services in Mediterranean ecosystems. Through a coordinated effort, the Mediterranean scientific community can fill the above-mentioned gaps and reach a greater understanding of the mechanisms underlying the combined effects of air pollution and climate change in the Mediterranean Basin.

Quantitative study on nitrogen deposition and canopy retention in Mediterranean evergreen forests.

To assess the impact of nitrogen (N) pollutants on forest ecosystems, the role of the interactions in the canopy needs to be understood. A great number of studies have addressed this issue in heavily N-polluted regions in north and central Europe. Much less information is available for the Iberian Peninsula, and yet this region is home to mountain forests and alpine grasslands that may be at risk due to excessive N deposition. To establish the basis for ecology-based policies, there is a need to better understand the forest response to this atmospheric impact. To fill this gap, in this study, we measured N deposition (as bulk, wet, and throughfall fluxes of dissolved inorganic nitrogen) and air N gas concentrations from 2011 to 2013 at four Spanish holm oak (Quercus ilex) forests located in different pollution environments. One site was in an area of intensive agriculture, two sites were influenced by big cities (Madrid and Barcelona, respectively), and one site was in a rural mountain environment 40 km north of Barcelona. Wet deposition ranged between 0.54 and 3.8 kg N ha-1 year-1 for ammonium (NH4+)-N and between 0.65 and 2.1 kg N ha-1 year-1 for nitrate (NO3-)-N, with the lowest deposition at the Madrid site for both components. Dry deposition was evaluated with three different approaches: (1) a canopy budget model based in throughfall measurements, (2) a branch washing method, and (3) inferential calculations. Taking the average dry deposition from these methods, dry deposition represented 51-67% (reduced N) and 72-75% (oxidized N) of total N deposition. Canopies retained both NH4+-N and NO3-N, with a higher retention at the agricultural and rural sites (50-60%) than at sites located close to big cities (20-35%, though more uncertainty was found for the site near Madrid), thereby highlighting the role of the forest canopy in processing N pollutant emissions.

Atmospheric deposition of inorganic nitrogen in Spanish forests of Quercus ilex measured with ion-exchange resins and conventional collectors.

Atmospheric nitrogen deposition is one of the main threats for biodiversity and ecosystem functioning. Measurement techniques like ion-exchange resin collectors (IECs), which are less expensive and time-consuming than conventional methods, are gaining relevance in the study of atmospheric deposition and are recommended to expand monitoring networks. In the present work, bulk and throughfall deposition of inorganic nitrogen were monitored in three different holm oak forests in Spain during two years. The results obtained with IECs were contrasted with a conventional technique using bottle collectors and with a literature review of similar studies. The performance of IECs in comparison with the conventional method was good for measuring bulk deposition of nitrate and acceptable for ammonium and total dissolved inorganic nitrogen. Mean annual bulk deposition of inorganic nitrogen ranged 3.09-5.43 kg N ha(-1) according to IEC methodology, and 2.42-6.83 kg N ha(-1) y(-1) using the conventional method. Intra-annual variability of the net throughfall deposition of nitrogen measured with the conventional method revealed the existence of input pulses of nitrogen into the forest soil after dry periods, presumably originated from the washing of dry deposition accumulated in the canopy. Important methodological recommendations on the IEC method and discussed, compiled and summarized.

Long-term effects of changing atmospheric pollution on throughfall, bulk deposition and streamwaters in a Mediterranean forest.

The abatement programs implanted in Europe to reduce SO2, NO2 and NH3 emissions are here evaluated by analyzing the relationships between emissions in Spain and neighboring countries and atmospheric deposition in a Mediterranean forest in the Montseny mountains (NE Spain) for the last 3decades. A canopy budget model was applied to throughfall data measured during a period of high emissions (1995-1996) and a period of lower emissions (2011-2013) to estimate the changes in dry deposition over this time span. Emissions of SO2 in Spain strongly decreased (77%) and that was reflected in reductions for nssSO4(2-) in precipitation (65% for concentrations and 62% for SO4(2)-S deposition). A lower decline was found for dry deposition (29%). Spanish NO2 emissions increased from 1980 to 1991, remained constant until 2005, and decreased thereafter, a pattern that was paralleled by NO3(-) concentrations in bulk precipitation at Montseny. This pattern seems to be related to a higher share of renewable energies in electricity generation in Spain in recent years. However, dry deposition increased markedly between 1995 and 2012, from 1.3 to 6.7 kg ha(-1) year(-)(1). Differences in meteorology between periods may have had a role, since the recent period was drier thus probably favoring dry deposition. Spanish NH3 emissions increased by 13% between 1980 and 2012 in Spain but NH4(+) concentrations in precipitation and NH4(+)-N deposition showed a decreasing trend (15% reduction) at Montseny, probably linked to the reduction ammonium sulfate and nitrate aerosols to be scavenged by rainfall. NH4(+)-N dry deposition was similar between the compared periods. The N load at Montseny (15-17 kg ha(-1)y ear(-1)) was within the critical load range proposed for Mediterranean sclerophyllous forests (15-17.5 kg ha(-1) year(-1)). The onset of N saturation is suggested by the observed increasing N export in streamwaters.

Atmospheric pollutants in peri-urban forests of Quercus ilex: evidence of pollution abatement and threats for vegetation.

Peri-urban vegetation is generally accepted as a significant remover of atmospheric pollutants, but it could also be threatened by these compounds, with origin in both urban and non-urban areas. To characterize the seasonal and geographical variation of pollutant concentrations and to improve the empirical understanding of the influence of Mediterranean broadleaf evergreen forests on air quality, four forests of Quercus ilex (three peri-urban and one remote) were monitored in different areas in Spain. Concentrations of nitrogen dioxide (NO2), ammonia (NH3), nitric acid (HNO3) and ozone (O3) were measured during 2 years in open areas and inside the forests and aerosols (PM10) were monitored in open areas during 1 year. Ozone was the only air pollutant expected to have direct phytotoxic effects on vegetation according to current thresholds for the protection of vegetation. The concentrations of N compounds were not high enough to directly affect vegetation but could be contributing through atmospheric N deposition to the eutrophization of these ecosystems. Peri-urban forests of Q. ilex showed a significant below-canopy reduction of gaseous concentrations (particularly NH3, with a mean reduction of 29-38%), which indicated the feasibility of these forests to provide an ecosystem service of air quality improvement. Well-designed monitoring programs are needed to further investigate air quality improvement by peri-urban ecosystems while assessing the threat that air pollution can pose to vegetation.

Changes in atmospheric deposition and streamwater chemistry over 25 years in undisturbed catchments in a Mediterranean mountain environment.

Surface water chemistry has changed in response to reduced atmospheric deposition of sulphur and acidity in many regions of Europe and North America. Most of these studies come from acidic or low-alkalinity surface waters under high acidic deposition. Mediterranean climates offer a different biogeochemical context, characterised by streamwaters of higher alkalinity and low acid inputs. In this paper, we use surveys of streamwater chemistry conducted in 1981-1984 and again in 2007 in the Montseny natural park (NE Spain) to test whether streamwaters of these well-buffered catchments respond to changes in atmospheric deposition, which has declined for S during the last decades in NE Spain while remaining about stable for nitrogen. The 23 sampled streams drained heathland, beech forests and evergreen oak forests in relatively undisturbed small catchments underlain by silicate bedrock. Bulk deposition of sulphate at Montseny decreased by 54% while nitrate bulk deposition increased (non-significantly) by 30% in this period. Total N deposition is estimated in the range 15-30 kg N ha(-1) y(-1) for NE Spain. This is well above threshold values (e.g. 10 kg N ha(-1) y(-1)) reported as starting nitrogen saturation symptoms in forest ecosystems in Europe. Baseflow sulphate concentrations decreased on average by 47 µeq L(-1) or 29% of early 1980s concentrations. Baseflow mean nitrate concentrations increased significantly but only from 5.5 to 8.9 µeq L(-1). Thus, despite decades of high N deposition, these ecosystems appear to be still far from N saturation. Baseflow alkalinity and base cation concentrations increased substantially, probably a combined result of decreased S deposition, enhanced silicate weathering under current higher temperatures, reduced plant cation uptake as vegetation matures, and slightly drier conditions in the survey of 2007. Overall, these well-buffered catchments have shown sizable changes in baseflow chemistry in response to changed atmospheric deposition and other environmental changes.

Nitrogen-doped anatase nanofibers decorated with noble metal nanoparticles for photocatalytic production of hydrogen.

We report the synthesis of N-doped TiO(2) nanofibers and high photocatalytic efficiency in generating hydrogen from ethanol-water mixtures under UV-A and UV-B irradiation. Titanate nanofibers synthesized by hydrothermal method are annealed in air and/or ammonia to achieve N-doped anatase fibers. Depending on the synthesis route, either interstitial N atoms or new N-Ti bonds appear in the lattice, resulting in slight lattice expansion as shown by XPS and HR-TEM analysis, respectively. These nanofibers were then used as support for Pd and Pt nanoparticles deposited with wet impregnation followed by calcination and reduction. In the hydrogen generation tests, the N-doped samples were clearly outperforming their undoped counterparts, showing remarkable efficiency not only under UV-B but also with UV-A illumination. When 100 mg of catalyst (N-doped TiO(2) nanofiber decorated with Pt nanoparticles) was applied to 1 L of water-ethanol mixture, the H(2) evolution rates were as high as 700 µmol/h (UV-A) and 2250 µmol/h (UV-B) corresponding to photo energy conversion percentages of ∼3.6 and ∼12.3%, respectively.

Source areas and long-range transport of pollen from continental land to Tenerife (Canary Islands).

The Canary Islands, due to their geographical position, constitute an adequate site for the study of long-range pollen transport from the surrounding land masses. In this study, we analyzed airborne pollen counts at two sites: Santa Cruz de Tenerife (SCO), at sea level corresponding to the marine boundary layer (MBL), and Izaña at 2,367 m.a.s.l. corresponding to the free troposphere (FT), for the years 2006 and 2007. We used three approaches to describe pollen transport: (1) a classification of provenances with an ANOVA test to describe pollen count differences between sectors; (2) a study of special events of high pollen concentrations, taking into consideration the corresponding meteorological synoptic pattern responsible for transport and back trajectories; and (3) a source-receptor model applied to a selection of the pollen taxa to show pollen source areas. Our results indicate several extra-regional pollen transport episodes to Tenerife. The main provenances were: (1) the Mediterranean region, especially the southern Iberian Peninsula and Morocco, through the trade winds in the MBL. These episodes were characterized by the presence of pollen from trees (Casuarina, Olea, Quercus perennial and deciduous types) mixed with pollen from herbs (Artemisia, Chenopodiaceae/Amaranthaceae and Poaceae wild type). (2) The Saharan sector, through transport at the MBL level carrying pollen principally from herbs (Chenopodiaceae-Amaranthaceae, Cyperaceae and Poaceae wild type) and, in one case, Casuarina pollen, uplifted to the free troposphere. And (3) the Sahel, characterized by low pollen concentrations of Arecaceae, Chenopodiaceae-Amaranthaceae, Cyperaceae and Poaceae wild type in sporadic episodes. This research shows that sporadic events of long-range pollen transport need to be taken into consideration in Tenerife as possible responsible agents in respiratory allergy episodes. In particular, it is estimated that 89-97% of annual counts of the highly allergenous Olea originates from extra-regional sources in southern Iberia and northern Africa.

Migration of the painted lady butterfly, Vanessa cardui, to north-eastern Spain is aided by African wind currents.

1. Thousands of records of migratory butterfly species such as Vanessa cardui flying just above ground-level on fixed compass bearings have led to the common belief that these insects migrate within the so-called 'flight-boundary layer', where movements are relatively independent of the wind. 2. Given the selective advantages of windborne migration and the existence of a number of observations of flights of V. cardui from the upper levels of the atmosphere, we tested the hypothesis that migration from North Africa to southern Europe in this species is influenced by synoptic-scale wind currents. 3. Even with modern technology, it is extremely difficult to observe high-altitude flights directly, so we rely on an indirect approach that examines whether or not arrival peaks in north-eastern Spain are associated with winds blowing from Africa. 4. Arrivals of V. cardui were determined for the spring period (1 March-27 June, 1997-2006) at 79 sites in the Catalan Butterfly Monitoring Scheme. Wind patterns were described on the basis of synoptic-scale maps, transport models and back-trajectories calculated for each day of the spring period. 5. We found a strong association between migration and winds from North Africa, both for the whole data set (1997-2006; chi(2) = 4.7, P = 0.03) and for a restricted data set that excludes years in which the species was very scarce (chi(2) = 7.26, P = 0.007). 6. Episodes of massive northward migration within the species' flight-boundary layer also coincided with spells of winds from North Africa, suggesting a connection between low-altitude (observational) and high-altitude flights (inferred from wind patterns). 7. Finally, on the assumption that migration in V. cardui is windborne, a source-receptor transport model applied to spring abundance data in north-eastern Spain enables us to identify the most probable population source areas in North Africa.