Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the gold-standard experimental model to study basic neurophysiological mechanisms involved in the formation and maintenance of neuronal cell assemblies. However, in vitro studies have been limited to the recording of the electrophysiological activity generated by bi-dimensional (2D) neural networks. Nonetheless, given the intricate relationship between structure and dynamics, a significant improvement is necessary to investigate the formation and the developing dynamics of three-dimensional (3D) networks. In this work, a novel experimental platform in which 3D hippocampal or cortical networks are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are realized by seeding neurons in a scaffold constituted of glass microbeads (30-40 µm in diameter) on which neurons are able to grow and form complex interconnected 3D assemblies. In this way, it is possible to design engineered 3D networks made up of 5-8 layers with an expected final cell density. The increasing complexity in the morphological organization of the 3D assembly induces an enhancement of the electrophysiological patterns displayed by this type of networks. Compared with the standard 2D networks, where highly stereotyped bursting activity emerges, the 3D structure alters the bursting activity in terms of duration and frequency, as well as it allows observation of more random spiking activity. In this sense, the developed 3D model more closely resembles in vivo neural networks.     

Design,Surface Treatment,Cellular Plating,and Culturing of Modular Neuronal Networks Composed of Functionally Inter-connected Circuits

The brain operates through the coordinated activation and the dynamic communication of neuronal assemblies. A major open question is how a vast repertoire of dynamical motifs, which underlie most diverse brain functions, can emerge out of a fixed topological and modular organization of brain circuits. Compared to in vivo studies of neuronal circuits which present intrinsic experimental difficulties, in vitro preparations offer a much larger possibility to manipulate and probe the structural, dynamical and chemical properties of experimental neuronal systems. This work describes an in vitro experimental methodology which allows growing of modular networks composed by spatially distinct, functionally interconnected neuronal assemblies. The protocol allows controlling the two-dimensional (2D) architecture of the neuronal network at different levels of topological complexity.A desired network patterning can be achieved both on regular cover slips and substrate embedded micro electrode arrays. Micromachined structures are embossed on a silicon wafer and used to create biocompatible polymeric stencils, which incorporate the negative features of the desired network architecture. The stencils are placed on the culturing substrates during the surface coating procedure with a molecular layer for promoting cellular adhesion. After removal of the stencils, neurons are plated and they spontaneously redirected to the coated areas. By decreasing the inter-compartment distance, it is possible to obtain either isolated or interconnected neuronal circuits. To promote cell survival, cells are co-cultured with a supporting neuronal network which is located at the periphery of the culture dish. Electrophysiological and optical recordings of the activity of modular networks obtained respectively by using substrate embedded micro electrode arrays and calcium imaging are presented. While each module shows spontaneous global synchronizations, the occurrence of inter-module synchronization is regulated by the density of connection among the circuits.     

The Acclimation of European Sea Bass (Dicentrarchus labrax) to Temperature: Behavioural and Neurochemical Responses

Studies on fish behavioural and neurophysiological responses to water temperature change may contribute to an improved understanding of the ecological consequences of global warming. We investigated behavioural and neurochemical responses to water temperature in European sea bass (Dicentrarchus labrax) acclimated to three temperatures (18, 22 and 28°C). After 21 d of acclimation, three groups of 25 fish each were exposed to four behavioural challenges (foraging, olfactory, aversive and mirror tests). The expression of choline acetyltransferase (ChAT) was then analysed by Western blotting in CNS homogenates (from a subset of the same fish) as a marker for cholinergic system activity. In both foraging and olfactory tests, fish acclimated to 28°C exhibited significantly higher arousal responses than fish acclimated to lower temperatures. All specimens showed fright behaviour in the aversive test, but the latency of the escape response was significantly less in the fish at 28°C. Finally, the highest mirror responsiveness was exhibited by the fish acclimated to 22°C. As in the case of cholinergic neurotransmission, significantly higher ChAT levels were detected in the telencephalon, diencephalon, cerebellum and spinal cord of fish acclimated to 22 or 28°C in comparison with those maintained at 18°C. Lower ChAT levels were detected in the mesencephalon (optic tectum) at 22 and 28°C than at 18°C. These data indicate that neuronal functions are affected by water temperature. Increases or decreases in ChAT expression can be related to the functional modulation of brain and spinal cord centres involved in behavioural responses to temperature change. Overall, the results of this study suggest that the environmental temperature level influences behaviour and CNS neurochemistry in the European sea bass.     

The combined effects of climate change and river fragmentation on the distribution of Andean Amazon fishes

Upstream range shifts of freshwater fishes have been documented in recent years due to ongoing climate change. River fragmentation by dams, presenting physical barriers, can limit the climatically induced spatial redistribution of fishes. Andean freshwater ecosystems in the Neotropical region are expected to be highly affected by these future disturbances. However, proper evaluations are still missing. Combining species distribution models and functional traits of Andean Amazon fishes, coupled with dam locations and climatic projections (2070s), we (a) evaluated the potential impacts of future climate on species ranges, (b) investigated the combined impact of river fragmentation and climate change and (c) tested the relationships between these impacts and species functional traits. Results show that climate change will induce range contraction for most of the Andean Amazon fish species, particularly those inhabiting highlands. Dams are not predicted to greatly limit future range shifts for most species (i.e., the Barrier effect). However, some of these barriers should prevent upstream shifts for a considerable number of species, reducing future potential diversity in some basins. River fragmentation is predicted to act jointly with climate change in promoting a considerable decrease in the probability of species to persist in the long‐term because of splitting species ranges in smaller fragments (i.e., the Isolation effect). Benthic and fast‐flowing water adapted species with hydrodynamic bodies are significantly associated with severe range contractions from climate change.     

In Silico Structural and Functional Characterization of the RSUME Splice Variants

RSUME (RWD-containing SUMO Enhancer) is a small protein that increases SUMO conjugation to proteins. To date, four splice variants that codify three RSUME isoforms have been described, which differ in their C-terminal end. Comparing the structure of the RSUME isoforms we found that, in addition to the previously described RWD domain in the N-terminal, all these RSUME variants also contain an intermediate domain. Only the longest RSUME isoform presents a C-terminal domain that is absent in the others. Given these differences, we used the shortest and longest RSUME variants for comparative studies. We found that the C-terminal domain is dispensable for the SUMO-conjugation enhancer properties of RSUME. We also demonstrate that these two RSUME variants are equally induced by hypoxia. The NF-κB signaling pathway is inhibited and the HIF-1 pathway is increased more efficiently by the longest RSUME, by means of a greater physical interaction of RSUME267 with the target proteins. In addition, the mRNA and protein levels of these isoforms differ in human glioma samples; while the shortest RSUME isoform is expressed in all the tumors analyzed, the longest variant is expressed in most but not all of them. The results presented here show a degree of redundancy of the RSUME variants on the SUMO pathway. However, the increased inhibition conferred by RSUME267 over the NF-κB signaling pathway, the increased activation over the HIF-1 pathway and the different expression of the RSUME isoforms suggest specific roles for each RSUME isoform which may be relevant in certain types of brain tumors that express RSUME, like human pituitary adenomas and gliomas.     

Hospitalization Rates and Post-Operative Mortality for Abdominal Aortic Aneurysm in Italy over the Period 2000–2011

Background

Recent studies have reported declines in incidence, prevalence and mortality for abdominal aortic aneurysms (AAAs) in various countries, but evidence from Mediterranean countries is lacking. The aim of this study is to examine the trend of hospitalization and post-operative mortality rates for AAAs in Italy during the period 2000–2011, taking into account the introduction of endovascular aneurysm repair (EVAR) in 1990s.

Methods

This retrospective cohort study was carried out in Emilia-Romagna, an Italian region with 4.5 million inhabitants. A total of 19,673 patients hospitalized for AAAs between 2000 and 2011, were identified from the hospital discharge records (HDR) database. Hospitalization rates, percentage of OSR and EVAR and 30-day mortality rates were calculated for unruptured (uAAAs) and ruptured AAAs (rAAAs).

Results

Adjusted hospitalization rates decreased on average by 2.9% per year for uAAAs and 3.2% for rAAAs (p<0.001). The temporal trend of 30-day mortality rates remained stable for both groups. The percentage of EVAR for uAAAs increased significantly from 2006 to 2011 (42.7 versus 60.9% respectively, mean change of 3.9% per year, p<0.001). No significant difference in mortality was found between OSR and EVAR for uAAAs and rAAAs.

Conclusions

The incidence and trend of hospitalization rates for rAAAs and uAAAs decreased significantly in the last decade, while 30-day mortality rates in operated patients remained stable. OSR continued to be the most common surgery in rAAAs, although the gap between OSR and EVAR recently declined. The EVAR technique became the preferred surgery for uAAAs since 2008.     

Natural fragmentation in river networks as a driver of speciation for freshwater fishes

Although habitat fragmentation fosters extinctions, it also increases the probability of speciation by promoting and maintaining divergence among isolated populations. Here we test for the effects of two isolation factors that may reduce population dispersal within river networks as potential drivers of freshwater fish speciation: 1) the position of subdrainages along the longitudinal river gradient, and 2) the level of fragmentation within subdrainages caused by natural waterfalls. The occurrence of native freshwater fish species from 26 subdrainages of the Orinoco drainage basin (South America) was used to identify those species that presumably arose from in‐situ cladogenetic speciation (i.e. neo‐endemic species; two or more endemic species from the same genus) within each subdrainage. We related subdrainages fish diversity (i.e. total, endemic and neo‐endemic species richness) and an index of speciation to our two isolation factors while controlling for subdrainages size and energy availability. The longitudinal position of subdrainages was unrelated to any of our diversity measures, a result potentially explained by the spatial grain we used and/or the contemporary connection between Orinoco and Amazon basins via the upstream Casiquiare region. However, we found higher neo‐endemic species richness and higher speciation index values in highly fragmented subdrainages. These results suggest that habitat fragmentation generated by natural waterfalls drives cladogenetic speciation in fragmented subdrainages. More generally, our results emphasize the role of history and natural waterfalls as biogeographic barriers promoting freshwater biodiversity in river drainage basins.     

Effects of nonnative species on the stability of riverine fish communities

Despite the increasing ubiquity of biological invasions worldwide, little is known about the scale-dependent effects of nonnative species on real-world ecological dynamics. Here, using an extensive time series dataset of riverine fish communities across different biogeographic regions of the world, we assessed the effects of nonnative species on the temporal variability and synchrony in abundance at different organizational levels (population, metapopulation, community and metacommunity) and spatial scales (stream reach and river basin). At the reach scale, we found that populations of nonnative species were more variable over time than native species, and that this effect scaled up to the community level – significantly destabilizing the dynamics of riverine fish communities. Nonnative species not only contributed to reduced community stability, but also increased variability of native populations. By contrast, we found no effect of nonnative species dominance on local interspecific synchrony among native species. At the basin scale, nonnative metapopulations were again more variable than the native ones. However, neither native metapopulations nor metacommunities showed differences in temporal variability or synchrony as nonnative species dominance increased basin-wide. This suggests a ‘dilution effect’ where the contribution to regional stability of local native populations from sites displaying low levels of invasion reduced the destabilizing effects of nonnative species. Overall, our results indicate that accounting for the destabilizing effect of nonnative species is critical to understanding native species persistence and community stability.     

Photocytotoxic activity of a nitrosyl phthalocyanine ruthenium complex--a system capable of producing nitric oxide and singlet oxygen

The synthesis, structural aspects, pharmacological assays, and in vitro photoinduced cytotoxic properties of [Ru(NO)(ONO)(pc)] (pc = phthalocyanine) are described. Its biological effect on the B16F10 cell line was studied in the presence and absence of visible light irradiation. At comparable irradiation levels, [Ru(NO)(ONO)(pc)] was more effective than [Ru(pc)] at inhibiting cell growth, suggesting that occurrence of nitric oxide release following singlet oxygen production upon light irradiation may be an important mechanism by which the nitrosyl ruthenium complex exhibits enhanced biological activity in cells. Following visible light activation, the [Ru(NO)(ONO)(pc)] complex displayed increased potency in B16F10 cells upon modifications to the photoinduced dose; indeed, enhanced potency was detected when the nitrosyl ruthenium complex was encapsulated in a drug delivery system. The liposome containing the [Ru(NO)(ONO)(pc)] complex was over 25% more active than the corresponding ruthenium complex in phosphate buffer solution. The activity of the complex was directly proportional to the ruthenium amount present inside the cell, as determined by inductively coupled plasma mass spectroscopy. Flow cytometry analysis revealed that the photocytotoxic activity was mainly due to apoptosis. Furthermore, the vasorelaxation induced by [Ru(NO)(ONO)(pc)], proposed as NO carrier, was studied in rat isolated aorta. The observed vasodilation was concentration-dependent. Taken together, the present findings demonstrate that the [Ru(NO)(ONO)(pc)] complex induces vascular relaxation and could be a potent anti-tumor agent. Nitric oxide release following singlet oxygen production upon visible light irradiation on a nitrosyl ruthenium complex produces two radicals and may elicit phototoxic responses that may find useful applications in photodynamic therapy.     

Impaired meiotic competence in putative primordial germ cells produced from mouse embryonic stem cells

There are still several unanswered questions and problems about the recently claimed possibility of producing functional germ cells in vitro from pluripotent embryonic stem cells (ESCs). In the present paper, we compared by single-cell analysis the capability of putative primordial germ cells (PGCs), produced in vitro from ESCs, and that of endogenous PGCs isolated from embryos, to enter and progress through meiotic prophase I. Using a protocol previously reported to be suitable to produce female germ cells from mouse ESC monolayers, we first identified putative PGCs by analysing the expression pattern of several markers such as SSEA1, APase, OCT4, NANOG, MVH and SCP3 of pre- and post migratory PGCs. Next, after isolation of such cells from culture, we tested their meiotic capability. The evaluation at 2-5 days of culture of the number of cells showing meiotic nuclear SCP3 staining in cytospreads showed that it remained nearly constant in the putative PGCs, whereas it increased markedly in endogenous PGCs. Moreover, we observed that in putative PGCs, the nuclear distribution or expression of SCP3 and other meiotic markers such as DMC1, gH2AX and SCP1 were always highly abnormal in comparison to that observed in endogenous cultured PGCs. We conclude that although the formation of cells showing characteristics of PGCs can occur efficiently from ESCs in vitro, these cells possess impaired capability to enter and progress through meiotic prophase I.