Dynamics of Intrinsic Dendritic Calcium Signaling during Tonic Firing of Thalamic Reticular Neurons

The GABAergic neurons of the nucleus reticularis thalami that control the communication between thalamus and cortex are interconnected not only through axo-dendritic synapses but also through gap junctions and dendro-dendritic synapses. It is still unknown whether these dendritic communication processes may be triggered both by the tonic and the T-type Ca²⁺ channel-dependent high frequency burst firing of action potentials displayed by nucleus reticularis neurons during wakefulness and sleep, respectively. Indeed, while it is known that activation of T-type Ca²⁺ channels actively propagates throughout the dendritic tree, it is still unclear whether tonic action potential firing can also invade the dendritic arborization. Here, using two-photon microscopy, we demonstrated that dendritic Ca²⁺ responses following somatically evoked action potentials that mimic wake-related tonic firing are detected throughout the dendritic arborization. Calcium influx temporally summates to produce dendritic Ca²⁺ accumulations that are linearly related to the duration of the action potential trains. Increasing the firing frequency facilitates Ca²⁺ influx in the proximal but not in the distal dendritic compartments suggesting that the dendritic arborization acts as a low-pass filter in respect to the back-propagating action potentials. In the more distal compartment of the dendritic tree, T-type Ca²⁺ channels play a crucial role in the action potential triggered Ca²⁺ influx suggesting that this Ca²⁺ influx may be controlled by slight changes in the local dendritic membrane potential that determine the T-type channels’ availability. We conclude that by mediating Ca²⁺ dynamic in the whole dendritic arborization, both tonic and burst firing of the nucleus reticularis thalami neurons might control their dendro-dendritic and electrical communications.     

Modulation of Protease Activated Receptor 1 Influences Human Metapneumovirus Disease Severity in a Mouse Model

Human metapneumovirus (hMPV) infection causes acute respiratory tract infections (RTI) which can result in hospitalization of both children and adults. To date, no antiviral or vaccine is available for this common viral infection. Immunomodulators could represent an interesting strategy for the treatment of severe viral infection. Recently, the role of protease-activated receptors (PAR) in inflammation, coagulation and infection processes has been of growing interest. Herein, the effects of a PAR1 agonist and a PAR1 antagonist on hMPV infection were investigated in BALB/c mice. Intranasal administration of the PAR1 agonist resulted in increased weight loss and mortality of infected mice. Conversely, the PAR1 antagonist was beneficial to hMPV infection by decreasing weight loss and clinical signs and by significantly reducing pulmonary inflammation, pro-inflammatory cytokine levels (including IL-6, KC and MCP-1) and recruitment of immune cells to the lungs. In addition, a significant reduction in pulmonary viral titers was also observed in the lungs of PAR1 antagonist-treated mice. Despite no apparent direct effect on virus replication during in vitro experiments, an important role for PAR1 in the regulation of furin expression in the lungs was shown for the first time. Further experiments indicated that the hMPV fusion protein can be cleaved by furin thus suggesting that PAR1 could have an effect on viral infectivity in addition to its immunomodulatory properties. Thus, inhibition of PAR1 by selected antagonists could represent an interesting strategy for decreasing the severity of paramyxovirus infections.     

Is coarse-to-fine strategy sensitive to normal aging?

Theories on visual perception agree that visual recognition begins with global analysis and ends with detailed analysis. Different results from neurophysiological, computational, and behavioral studies all indicate that the totality of visual information is not immediately conveyed, but that information analysis follows a predominantly coarse-to-fine processing sequence (low spatial frequencies are extracted first, followed by high spatial frequencies). We tested whether such processing continues to occur in normally aging subjects. Young and aged participants performed a categorization task (indoor vs. outdoor scenes), using dynamic natural scene stimuli, in which they resorted to either a coarse-to-fine (CtF) sequence or a reverse fine-to-coarse sequence (FtC). The results show that young participants categorized CtF sequences more quickly than FtC sequences. However, sequence processing interacts with semantic category only for aged participants. The present data support the notion that CtF categorization is effective even in aged participants, but is constrained by the spatial features of the scenes, thus highlighting new perspectives in visual models.     

Exploring the relationship between NDVI and African elephant population density in protected areas

Assessing the extent to which populations are limited by bottom‐up processes driven by food limitation is crucial to our understanding of how ecosystems should be managed. Using satellite‐derived Normalised Difference Vegetation Index (NDVI) as an index of resource availability, we investigated the relationships between greenness levels and African elephant (Loxodonta africana) population densities. Our results unveiled a positive relationship between NDVI and elephant densities in nonforest populations, but failed to capture any significant effects of survey area and type, number of years after the continental poaching ban and the IUCN category of the protected area in which the survey was undertaken. The number of forest elephant populations for which density estimates were available was very low, and no significant relationship between NDVI and forest elephant density could be established. Altogether, our study suggests that NDVI can successfully be linked to megaherbivore abundance across Africa and further highlights that a continental approach to energy–abundance relationships can be conducted in a relatively low‐cost manner and short timescale, supporting managers' efforts to identify future suitable areas for elephant populations.     

Evolutionary Dynamics of the Leucine-Rich Repeat Receptor-Like Kinase (LRR-RLK) Subfamily in Angiosperms

Gene duplications are an important factor in plant evolution, and lineage-specific expanded (LSE) genes are of particular interest. Receptor-like kinases expanded massively in land plants, and leucine-rich repeat receptor-like kinases (LRR-RLK) constitute the largest receptor-like kinases family. Based on the phylogeny of 7,554 LRR-RLK genes from 31 fully sequenced flowering plant genomes, the complex evolutionary dynamics of this family was characterized in depth. We studied the involvement of selection during the expansion of this family among angiosperms. LRR-RLK subgroups harbor extremely contrasting rates of duplication, retention, or loss, and LSE copies are predominantly found in subgroups involved in environmental interactions. Expansion rates also differ significantly depending on the time when rounds of expansion or loss occurred on the angiosperm phylogenetic tree. Finally, using a d_N/d_S-based test in a phylogenetic framework, we searched for selection footprints on LSE and single-copy LRR-RLK genes. Selective constraint appeared to be globally relaxed at LSE genes, and codons under positive selection were detected in 50% of them. Moreover, the leucine-rich repeat domains, and specifically four amino acids in them, were found to be the main targets of positive selection. Here, we provide an extensive overview of the expansion and evolution of this very large gene family.Receptor-like kinases (RLKs) constitute one of the largest gene families in plants and expanded massively in land plants (Embryophyta; Lehti-Shiu et al., 2009, 2012). For plant RLK gene families, the functions of most members are often not known (especially in recently expanded families), but some described functions include innate immunity (Albert et al., 2010), pathogen response (Dodds and Rathjen, 2010), abiotic stress (Yang et al., 2010), development (De Smet et al., 2009), and sometimes multiple functions (Lehti-Shiu et al., 2012). The RLKs usually consist of three domains: an N-terminal extracellular domain, a transmembrane domain, and a C-terminal kinase domain (KD). In plants, the KD usually has a Ser/Thr specificity (Shiu and Bleecker, 2001), but Tyr-specific RLKs were also described (e.g. BRASSINOSTEROID INSENSITIVE1; Oh et al., 2009). Interestingly, it was estimated that approximately 20% of RLKs contain a catalytically inactive KD (e.g. STRUBBELIG and CORYNE; Chevalier et al., 2005; Castells and Casacuberta, 2007; Gish and Clark, 2011). In Arabidopsis (Arabidopsis thaliana), 44 RLK subgroups (SGs) were defined by inferring the phylogenetic relationships between the KDs (Shiu and Bleecker, 2001). Interestingly, different SGs show different duplication/retention rates (Lehti-Shiu et al., 2009). Specifically, RLKs involved in stress responses show a high number of tandemly duplicated genes whereas those involved in development do not (Shiu et al., 2004), which suggests that some RLK genes are important for the responses of land plants to a changing environment (Lehti-Shiu et al., 2012). There seem to be relatively few RLK pseudogenes compared with other large gene families, and copy retention was argued to be driven by both drift and selection (Zou et al., 2009; Lehti-Shiu et al., 2012). As most SGs are relatively old and RLK subfamilies expanded independently in several plant lineages, duplicate retention cannot be explained by drift alone, and natural selection is expected to be an important driving factor in RLK gene family retention (Lehti-Shiu et al., 2009).Leucine-rich repeat-receptor-like kinases (LRR-RLKs), which contain up to 30 leucine-rich repeat (LRRs) in their extracellular domain, constitute the largest RLK family (Shiu and Bleecker, 2001). Based on the KD, 15 LRR-RLK SGs have been established in Arabidopsis (Shiu et al., 2004; Lehti-Shiu et al., 2009). So far, two major functions have been attributed to them: defense against pathogens and development (Tang et al., 2010b). LRR-RLKs involved in defense are predominantly found in lineage-specific expanded (LSE) gene clusters, whereas LRR-RLKs involved in development are mostly found in nonexpanded groups (Tang et al., 2010b). It was also discovered that the LRR domains are significantly less conserved than the remaining domains of the LRR-RLK genes (Tang et al., 2010b). In addition, a study of four plant genomes (Arabidopsis, grape [Vitis vinifera], poplar [Populus trichocarpa], and rice [Oryza sativa]) showed that LRR-RLK genes from LSE gene clusters show significantly more indications of positive selection or relaxed constraint than LRR-RLKs from nonexpanded groups (Tang et al., 2010b).The genomes of flowering plants (angiosperms) have been shown to be highly dynamic compared with most other groups of land plants (Leitch and Leitch, 2012). This dynamic is mostly caused by the frequent multiplication of genetic material, followed by a complex pattern of differential losses (i.e. the fragmentation process) and chromosomal rearrangements (Langham et al., 2004; Leitch and Leitch, 2012). Most angiosperm genomes sequenced so far show evidence for at least one whole-genome multiplication event during their evolution (Jaillon et al., 2007; D’Hont et al., 2012; Tomato Genome Consortium, 2012). At a smaller scale, tandem and segmental duplications are also very common in angiosperms (Arabidopsis Genome Initiative, 2000; International Rice Genome Sequencing Project, 2005; Rizzon et al., 2006). Although the most common fate of duplicated genes is to be progressively lost, in some cases they can be retained in the genome, and adaptive as well as nonadaptive scenarios have been discussed to play a role in this preservation process (for review, see Moore and Purugganan, 2005; Hahn, 2009; Innan, 2009; Innan and Kondrashov, 2010). Whole-genome sequences also revealed that the same gene may undergo several rounds of duplication and retention. These LSE genes were shown to evolve under positive selection more frequently than single-copy genes in angiosperms (Fischer et al., 2014). That study analyzed general trends over whole genomes. Here, we ask if, and to what extent, this trend is observable at LRR-RLK genes. As this gene family is very dynamic and large, and in accordance with the results of Tang et al. (2010b), we expect the effect of positive selection to be even more pronounced than in the whole-genome average.We analyzed 33 Embryophyta genomes to investigate the evolutionary history of the LRR-RLK gene family in a phylogenetic framework. Twenty LRR-RLK SGs were identified, and from this data set, we deciphered the evolutionary dynamics of this family within angiosperms. The expansion/reduction rates were contrasted between SGs and species as well as in ancestral branches of the angiosperm phylogeny. We then focused on genes whose number increased dramatically in an SG- and/or species-specific manner (i.e. LSE genes). Those genes are likely to be involved in species-specific cellular processes or adaptive interactions and were used as a template to infer the potential occurrence of positive selection. This led to the identification of sites at which positive selection likely acted. We discuss our results in the light of angiosperm genome evolution and current knowledge of LRR-RLK functions. Positive selection footprints identified in LSE genes highlight the importance of combining evolutionary analysis and functional knowledge to guide further investigations.     

Elaboration of an electroporation protocol for Bacillus cereus ATCC 14579

An electro-transformation procedure was established for Bacillus cereus ATCC 14579. Using early growth-stage culture and high electric field, the ectroporation efficiency was up to 2 x 10(9) cfu microg(-1) ml(-1) with pC194 plasmid DNA. The procedure was tested with three other plasmids, of various sizes, replication mechanisms and selection markers, and the transformation efficiencies ranged between 2 x 10(6) and 1 x 10(8) cfu microg(-1) ml(-)(1). The effects of two wall-weakening agents on electroporation rates were also evaluated. The transformation rate that was reached with our procedure is 10(3) times higher than that previously obtained with members of the Bacillus genus with similar plasmids, and 10(6) times superior than that achieved with available protocols for B. cereus. The proposed method is quick, simple, efficient with small rolling circle plasmids and large theta replicating plasmids with low copy number per cell, and suitable for many genetic manipulations that are not possible without high-efficiency transformation protocols.     

The Siva protein is a novel intracellular ligand of the CD4 receptor that promotes HIV-1 envelope-induced apoptosis in T-lymphoid cells

In addition to its positive signaling function in the antigen presentation process, CD4 acts as the primary receptor for HIV-1. Contact between CD4 and the viral envelope leads to virus entry, but can also trigger apoptosis of uninfected CD4⁺ T-cells through a mechanism that is poorly understood. We show that Siva-1, a death domain-containing proapoptotic protein, associates with the cytoplasmic domain of CD4. This interaction is mediated by the cysteine-rich region found in the C-terminal part of the Siva-1 protein. Expression of Siva-1 specifically increases the susceptibility of both T-cell lines and unstimulated human primary CD4⁺ T-lymphocytes to CD4-mediated apoptosis triggered by the HIV-1 envelope, and results in activation of a caspase-dependent mitochondrial pathway. The same susceptibility is observed in T-cells expressing a truncated form of CD4 that is able to recruit Siva-1 but fails to associate with p56^Lck, indicating that Siva-1 participates in a pathway independent of the p56^Lck kinase activity. Altogether, these results suggest that Siva-1 might participate in the CD4-initiated signaling apoptotic pathway induced by the HIV-1 envelope in T-lymphoid cells. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization of the role of the receptors PEX5 and PEX7 in the import of proteins into glycosomes of Trypanosoma brucei

Peroxins 5 and 7 are receptors for protein import into the peroxisomal matrix. We studied the involvement of these peroxins in the biogenesis of glycosomes in the protozoan parasite Trypanosoma brucei. Glycosomes are peroxisome-like organelles in which a major part of the glycolytic pathway is sequestered. We here report the characterization of the T. brucei homologue of PEX7 and provide several data strongly suggesting that it can bind to PEX5. Depletion of PEX5 or PEX7 by RNA interference had a severe effect on the growth of both the bloodstream-form of the parasite, that relies entirely on glycolysis for its ATP supply, and the procyclic form representative of the parasite living in the tsetse-fly midgut and in which also other metabolic pathways play a prominent role. The role of the two receptors in import of glycosomal matrix proteins with different types of peroxisome/glycosome-targeting signals (PTS) was analyzed by immunofluorescence and subcellular fractionation studies. Knocking down the expression of either receptor gene resulted, in procyclic cells, in the mislocalization of proteins with both a type 1 or 2 targeting motif (PTS1, PTS2) located at the C- and N-termini, respectively, and proteins with a sequence-internal signal (I-PTS) to the cytosol. Electron microscopy confirmed the apparent integrity of glycosomes in these procyclic cells. In bloodstream-form trypanosomes, PEX7 depletion seemed to affect only the subcellular distribution of PTS2-proteins. Western blot analysis suggested that, in both life-cycle stages of the trypanosome, the levels of both receptors are controlled in a coordinated fashion, by a mechanism that remains to be determined. The observation that both PEX5 and PEX7 are essential for the viability of the parasite indicates that the respective branches of the glycosome-import pathway in which each receptor acts might be interesting drug targets.     

Monitoring Rarity: The Critically Endangered Saharan Cheetah as a Flagship Species for a Threatened Ecosystem

Deserts are particularly vulnerable to human impacts and have already suffered a substantial loss of biodiversity. In harsh and variable desert environments, large herbivores typically occur at low densities, and their large carnivore predators occur at even lower densities. The continued survival of large carnivores is key to healthy functioning desert ecosystems, and the ability to gather reliable information on these rare low density species, including presence, abundance and density, is critical to their monitoring and management. Here we test camera trap methodologies as a monitoring tool for an extremely rare wide-ranging large felid, the critically endangered Saharan cheetah (Acinonyx jubatus hecki). Two camera trapping surveys were carried out over 2–3 months across a 2,551km² grid in the Ti-n-hağğen region in the Ahaggar Cultural Park, south central Algeria. A total of 32 records of Saharan cheetah were obtained. We show the behaviour and ecology of the Saharan cheetah is severely constrained by the harsh desert environment, leading them to be more nocturnal, be more wide-ranging, and occur at lower densities relative to cheetah in savannah environments. Density estimates ranged from 0.21–0.55/1,000km², some of the lowest large carnivore densities ever recorded in Africa, and average home range size over 2–3 months was estimated at 1,583km². We use our results to predict that, in order to detect presence of cheetah with p>0.95 a survey effort of at least 1,000 camera trap days is required. Our study identifies the Ahaggar Cultural Park as a key area for the conservation of the Saharan cheetah. The Saharan cheetah meets the requirements for a charismatic flagship species that can be used to “market” the Saharan landscape at a sufficiently large scale to help reverse the historical neglect of threatened Saharan ecosystems.     

Expression of the cystathionine beta synthase (CBS) gene during mouse development and immunolocalization in adult brain.

Hyperhomocysteinemia, caused by a lack of cystathionine beta synthase (CBS), leads to elevated plasma concentrations of homocysteine. This is a common risk factor for atherosclerosis, stroke, and possibly neurodegenerative diseases. However, the mechanisms that link hyperhomocysteinemia due to CBS deficiency to these diseases are still unknown. Early biochemical studies describe developmental and adult patterns of transsulfuration and CBS expression in a variety of species. However, there is incomplete knowledge about the regional and cellular expression pattern of CBS, notably in the brain. To complete the previous data, we used in situ hybridization and Northern blotting to characterize the spatial and temporal patterns of Cbs gene expression during mouse development. In the early stages of development, the Cbs gene was expressed only in the liver and in the skeletal, cardiac, and nervous systems. The expression declined in the nervous system in the late embryonic stages, whereas it increased in the brain after birth, peaking during cerebellar development. In the adult brain, expression was strongest in the Purkinje cell layer and in the hippocampus. Immunohistochemical analyses showed that the CBS protein was localized in most areas of the brain but predominantly in the cell bodies and neuronal processes of Purkinje cells and Ammon's horn neurons.