Changes in the miRNA-mRNA Regulatory Network Precede Motor Symptoms in a Mouse Model of Multiple System Atrophy: Clinical Implications

Multiple system atrophy (MSA) is a fatal rapidly progressive α-synucleinopathy, characterized by α-synuclein accumulation in oligodendrocytes. It is accepted that the pathological α-synuclein accumulation in the brain of MSA patients plays a leading role in the disease process, but little is known about the events in the early stages of the disease. In this study we aimed to define potential roles of the miRNA-mRNA regulatory network in the early pre-motor stages of the disease, i.e., downstream of α-synuclein accumulation in oligodendroglia, as assessed in a transgenic mouse model of MSA. We investigated the expression patterns of miRNAs and their mRNA targets in substantia nigra (SN) and striatum, two brain regions that undergo neurodegeneration at a later stage in the MSA model, by microarray and RNA-seq analysis, respectively. Analysis was performed at a time point when α-synuclein accumulation was already present in oligodendrocytes at neuropathological examination, but no neuronal loss nor deficits of motor function had yet occurred. Our data provide a first evidence for the leading role of gene dysregulation associated with deficits in immune and inflammatory responses in the very early, non-symptomatic disease stages of MSA. While dysfunctional homeostasis and oxidative stress were prominent in SN in the early stages of MSA, in striatum differential gene expression in the non-symptomatic phase was linked to oligodendroglial dysfunction, disturbed protein handling, lipid metabolism, transmembrane transport and altered cell death control, respectively. A large number of putative miRNA-mRNAs interaction partners were identified in relation to the control of these processes in the MSA model. Our results support the role of early changes in the miRNA-mRNA regulatory network in the pathogenesis of MSA preceding the clinical onset of the disease. The findings thus contribute to understanding the disease process and are likely to pave the way towards identifying disease biomarkers for early diagnosis of MSA.     

A Heterologous Multiepitope DNA Prime/Recombinant Protein Boost Immunisation Strategy for the Development of an Antiserum against Micrurus corallinus (Coral Snake) Venom

BackgroundEnvenoming by coral snakes (Elapidae: Micrurus), although not abundant, represent a serious health threat in the Americas, especially because antivenoms are scarce. The development of adequate amounts of antielapidic serum for the treatment of accidents caused by snakes like Micrurus corallinus is a challenging task due to characteristics such as low venom yield, fossorial habit, relatively small sizes and ophiophagous diet. These features make it difficult to capture and keep these snakes in captivity for venom collection. Furthermore, there are reports of antivenom scarcity in USA, leading to an increase in morbidity and mortality, with patients needing to be intubated and ventilated while the toxin wears off. The development of an alternative method for the production of an antielapidic serum, with no need for snake collection and maintenance in captivity, would be a plausible solution for the antielapidic serum shortage.ConclusionHere we describe that the genetic immunisation with a synthetic multiepitope gene followed by booster doses with recombinant protein is a promising approach to develop an alternative antielapidic serum against M. corallinus venom without the need of collection and the very challenging maintenance of these snakes in captivity.     

Exocyst SEC3 and Phosphoinositides Define Sites of Exocytosis in Pollen Tube Initiation and Growth

Polarized exocytosis is critical for pollen tube growth, but its localization and function are still under debate. The exocyst vesicle-tethering complex functions in polarized exocytosis. Here, we show that a sec3a exocyst subunit null mutant cannot be transmitted through the male gametophyte due to a defect in pollen tube growth. The green fluorescent protein (GFP)-SEC3a fusion protein is functional and accumulates at or proximal to the pollen tube tip plasma membrane. Partial complementation of sec3a resulted in the development of pollen with multiple tips, indicating that SEC3 is required to determine the site of pollen germination pore formation. Time-lapse imaging demonstrated that SEC3a and SEC8 were highly dynamic and that SEC3a localization on the apical plasma membrane predicts the direction of growth. At the tip, polar SEC3a domains coincided with cell wall deposition. Labeling of GFP-SEC3a-expressing pollen with the endocytic marker FM4-64 revealed the presence of subdomains on the apical membrane characterized by extensive exocytosis. In steady-state growing tobacco (Nicotiana tabacum) pollen tubes, SEC3a displayed amino-terminal Pleckstrin homology-like domain (SEC3a-N)-dependent subapical membrane localization. In agreement, SEC3a-N interacted with phosphoinositides in vitro and colocalized with a phosphatidylinositol 4,5-bisphosphate (PIP₂) marker in pollen tubes. Correspondingly, molecular dynamics simulations indicated that SEC3a-N associates with the membrane by interacting with PIP₂. However, the interaction with PIP₂ is not required for polar localization and the function of SEC3a in Arabidopsis (Arabidopsis thaliana). Taken together, our findings indicate that SEC3a is a critical determinant of polar exocytosis during tip growth and suggest differential regulation of the exocytotic machinery depending on pollen tube growth modes.Pollen tube growth provides a unique model system for studying the role of exocytosis in cell morphogenesis. Pollen tubes are characterized by a highly rapid polarized unidirectional tip growth. Given the relative simplicity of their structure, fast growth rates, haploid genome content, and ability to grow under in vitro culture conditions, pollen tubes provide an extremely attractive system for studying cell morphogenesis. Furthermore, the growth characteristics of pollen tubes resemble those of root hairs, moss protonema, and fungal hyphae and to some extent can be paralleled to neurite growth (Chebli and Geitmann, 2007; Cheung and Wu, 2008; Guan et al., 2013; Hepler and Winship, 2015).It is well established that oscillating polarized exocytosis is fundamental for pollen tube development and determines growth rate (Bove et al., 2008; McKenna et al., 2009; Chebli et al., 2013). Exocytosis is required for the delivery of membrane and cell wall components to the growing tip. Yet, the exact location where exocytosis takes place is under debate. Ultrastructural studies showing the accumulation of vesicles at the tip suggested that exocytosis takes place at the tip (Lancelle et al., 1987; Lancelle and Hepler, 1992; Derksen et al., 1995), which was further supported by studies on the dynamics of cell wall thickness (Rojas et al., 2011), secretion of pectin methyl esterase (PME) and PME inhibitor, and staining of pectin by propidium iodide (PI; Röckel et al., 2008; Rounds et al., 2014). Conversely, based on colabeling with FM1-43 and FM4-64, it was concluded that exocytosis takes place in a subapical collar located in the transition zone between the tip and the shank, as well as at the shank, but not at the tip (Bove et al., 2008; Zonia and Munnik, 2008). In agreement, the pollen tube-specific syntaxin GFP-SYP124 was observed in the inverted cone, 10 to 25 μm away from the tip (Silva et al., 2010), and fluorescence recovery after photobleaching experiments with FM dyes also have indicated that exocytosis takes place at the subapical region (Bove et al., 2008; Moscatelli et al., 2012; Idilli et al., 2013). Yet, based on pollen tube reorientation experiments in a microfluidics device, it was concluded that growth takes place at the tip rather than at a subapical collar located in the transition zone between the apex and the shank (Sanati Nezhad et al., 2014). The tip-based growth is in agreement with exocytosis taking place at the tip. Presumably, part of the disagreement regarding the site of exocytosis resulted from the lack of intracellular markers for exocytosis (Cheung and Wu, 2008; Hepler and Winship, 2015), and as a result, the relationship between the FM dye-labeled inverted cone and exocytotic events during pollen tube growth is not fully understood.In many cell types, the process of secretory vesicles tethering and docking prior to fusion with the plasma membrane is initially mediated by an evolutionarily conserved tethering complex known as the exocyst. The exocyst is a heterooligomeric protein complex composed of eight subunits, SEC3, SEC5, SEC6, SEC8, SEC10, SEC15, EXO70, and EXO84 (TerBush et al., 1996; Guo et al., 1999). Studies originally based on budding yeast (Saccharomyces cerevisiae) have shown that the exocyst functions as an effector of Rab and Rho small GTPases that specifies the sites of vesicle docking and fusion at the plasma membrane in both space and time (Guo et al., 2001; Zhang et al., 2001). Support for the function of the exocyst in vesicle tethering was demonstrated recently by ectopic Sec3p-dependent vesicle recruitment to the mitochondria (Luo et al., 2014).Land plants contain all subunits of the exocyst complex, which were shown to form the functional complex (Elias et al., 2003; Cole et al., 2005; Synek et al., 2006; Hála et al., 2008). Studies in Arabidopsis (Arabidopsis thaliana) and maize (Zea mays) have implicated the exocyst in the regulation of pollen tube and root hair growth, seed coat deposition, response to pathogens, cytokinesis, and meristem and stigma function (Cole et al., 2005; Synek et al., 2006; Hála et al., 2008; Fendrych et al., 2010; Kulich et al., 2010; Pecenková et al., 2011; Safavian and Goring, 2013; Wu et al., 2013; Safavian et al., 2015; Zhang et al., 2016). The growth arrest of pollen tubes in sec8, sec6, sec15a, and sec5a/sec5b single and double mutants (Cole et al., 2005; Hála et al., 2008) or following treatment with the EXO70 inhibitor ENDOSIDIN2 (Zhang et al., 2016), and of root hairs in maize root hairless1 (rth1) SEC3 mutant (Wen et al., 2005), the inhibition of seed coat deposition in the sec8 and exo70A1 mutants (Kulich et al., 2010), and stigmatic papillae function in exo70A1 mutant plants (Safavian and Goring, 2013; Safavian et al., 2015) have implicated the exocyst in polarized exocytosis in plants. Given their function, it was likely that exocyst subunits could be used as markers for polarized exocytosis. Furthermore, it could also be hypothesized that, by studying the mechanisms that underlie the association of the exocyst complex with the plasma membrane, it should be possible to identify mechanisms underlying the regulation of polarized exocytosis (Guan et al., 2013). Moreover, since the interaction of exocytotic vesicles with the exocyst is transient and marks the site(s) of active exocytosis in the membrane, fluorescently labeled exocyst subunits could be used as markers for exocytosis while avoiding potential imaging artifacts stemming from pollen tube tips densely populated with vesicles.We have shown previously that the ROP effector ICR1 can interact with SEC3a and that ROPs can recruit SEC3a-ICR1 complexes to the plasma membrane (Lavy et al., 2007). However, ICR1 is not expressed in pollen tubes, suggesting that SEC3a membrane binding in these cells is likely dependent on other factors. In yeast, the interaction of Sec3p and Exo70p subunits with the plasma membrane is critical for exocyst function (He and Guo, 2009). It has been shown that the membrane binding of both Sec3p and Exo70p is facilitated by their interaction with phosphatidylinositol 4,5-bisphosphate (PIP₂; He et al., 2007; Zhang et al., 2008). The yeast Exo70p interacts with PIP₂ via a number of positively charged residues distributed along the protein, with the highest number located at the C-terminal end (Pleskot et al., 2015). It has been suggested that yeast Sec3p interacts with PIP₂ through N-terminal basic residues (Zhang et al., 2008). These data were further corroborated by x-ray crystallography studies, which showed that the yeast Sec3p N-terminal region forms a Pleckstrin homology (PH) domain fold (Baek et al., 2010; Yamashita et al., 2010), a PIP₂ interaction motif (Lemmon, 2008).The localization of the exocyst subunits has been addressed in several studies. In Arabidopsis root hairs and root epidermis cells, SEC3a-GFP was observed in puncta distributed throughout the cell (Zhang et al., 2013). Studies on the Arabidopsis EXO70 subunits EXO70E2, EXO70A1, and EXO70B1 revealed them to be localized in distinct compartments that were termed exocyst-positive organelles (Wang et al., 2010). The exocyst-positive organelles, visualized mostly by ectopic expression, were shown to be cytoplasmic double membrane organelles that can fuse with the plasma membrane and secrete their contents to the apoplast in an exosome-like manner. It is not yet known whether other exocyst subunits also are localized to the same organelles and what might be the biological function of this putative compartment (Wang et al., 2010; Lin et al., 2015). In differentiating xylem cells, two coiled-coil proteins termed VESICLE TETHERING1 and VESICLE TETHERING2 recruit EXO70A1-positive puncta to microtubules via the GOLGI COMPLEX2 protein (Oda et al., 2015). Importantly, the functionality of the XFP fusion proteins used for the localization studies described above was not tested, and in most cases, the fusion proteins were overexpressed. Therefore, the functional localization of the exocyst is still unclear.Here, we studied the function and subcellular localization of the Arabidopsis exocyst SEC3a subunit using a combination of genetics, cell biology, biochemistry, and structural modeling approaches. Our results show that SEC3a is essential for the determination of pollen tube tip germination site and growth. Partial complementation of sec3a resulted in the formation of pollen with multiple pollen tube tips. In Arabidopsis growing pollen tubes, SEC3a localization is dynamic, and it accumulates in domains of polarized secretion, at or close to the tip plasma membrane (PM). Labeling of GFP-SEC3-expressing pollen with FM4-64 revealed the spatial correlation between polarized exocytosis and endocytic recycling. Furthermore, the association of SEC3a with PM at the tip marks the direction of tube elongation and positively correlates with the deposition of PI-labeled pectins and specific anti-esterified pectin antibodies in the cell wall. In tobacco (Nicotiana tabacum), the mechanisms underlying SEC3a interaction with the PM and its subcellular distribution depend on pollen tube growth mode and involve the interaction with PIP2 through the N-terminal PH domain. Collectively, our results highlight the function of SEC3a as a polarity determinant that links between polarized exocytosis and cell morphogenesis. The correlation between exocyst function and distribution in pollen tubes provides an explanation for some of the current discrepancies regarding the localization of exocytosis.     

Study of the Serum Copper Levels in Patients with Major Depressive Disorder

Copper may be involved in the pathophysiology of depression. Clinical data on this issue are very limited and not conclusive. The purpose of the study was to determine the copper concentration in the serum of patients with major depressive disorder and to discuss its potential clinical usefulness as a biomarker of the disease. A case–control clinical study included 69 patients with current depressive episode, 45 patients in remission and 50 healthy volunteers. Cu concentration was measured by electrothermal atomic absorption spectrometry (ETAAS). The mean serum copper level in depressed patients was slightly lower (by 11 %; not statistically significant) than in the control group. Furthermore, there was no significant difference in Cu²⁺ concentration between depressive episode and remission, nor between remission and control group. In the remission group were observed significant correlations between copper levels and the average number of relapses over the past years or time of remission. There was no correlation between serum copper and severity of depression, as measured by HDRS and MADRS. The obtained results showed no significant differences between the copper concentration in the blood serum of patients (both with current depressive episode and in remission) and healthy volunteers, as well as the lack of correlations between the copper level in the active stage of the disease and clinical features of the population. Our study is the first conducted on such a large population of patients, so the results may be particularly important and reliable source of knowledge about the potential role of copper in depression.     

Passive acoustic monitoring reveals group ranging and territory use: a case study of wild chimpanzees (<Emphasis Type="Italic">Pan troglodytes</Emphasis>)

Background

Assessing the range and territories of wild mammals traditionally requires years of data collection and often involves directly following individuals or using tracking devices. Indirect and non-invasive methods of monitoring wildlife have therefore emerged as attractive alternatives due to their ability to collect data at large spatiotemporal scales using standardized remote sensing technologies. Here, we investigate the use of two novel passive acoustic monitoring (PAM) systems used to capture long-distance sounds produced by the same species, wild chimpanzees (Pan troglodytes), living in two different habitats: forest (Taï, Côte d’Ivoire) and savanna-woodland (Issa valley, Tanzania).

Results

Using data collected independently at two field sites, we show that detections of chimpanzee sounds on autonomous recording devices were predicted by direct and indirect indices of chimpanzee presence. At Taï, the number of chimpanzee buttress drums detected on recording devices was positively influenced by the number of hours chimpanzees were seen ranging within a 1 km radius of a device. We observed a similar but weaker relationship within a 500 m radius. At Issa, the number of indirect chimpanzee observations positively predicted detections of chimpanzee loud calls on a recording device within a 500 m but not a 1 km radius. Moreover, using just seven months of PAM data, we could locate two known chimpanzee communities in Taï and observed monthly spatial variation in the center of activity for each group.

Conclusions

Our work shows PAM is a promising new tool for gathering information about the ranging behavior and habitat use of chimpanzees and can be easily adopted for other large territorial mammals, provided they produce long-distance acoustic signals that can be captured by autonomous recording devices (e.g., lions and wolves). With this study we hope to promote more interdisciplinary research in PAM to help overcome its challenges, particularly in data processing, to improve its wider application.

     

Interspecies Comparison of the Bacterial Response to Allicin Reveals Species‐Specific Defense Strategies

Allicin, a broad‐spectrum antimicrobial agent from garlic, disrupts thiol and redox homeostasis, proteostasis, and cell membrane integrity. Since medicine demands antimicrobials with so far unexploited mechanisms, allicin is a promising lead structure. While progress is being made in unraveling its mode of action, little is known on bacterial adaptation strategies. Some isolates of Pseudomonas aeruginosa and Escherichia coli withstand exposure to high allicin concentrations due to as yet unknown mechanisms. To elucidate resistance and sensitivity‐conferring cellular processes, the acute proteomic responses of a resistant P. aeruginosa strain and the sensitive species Bacillus subtilis are compared to the published proteomic response of E. coli to allicin treatment. The cellular defense strategies share functional features: proteins involved in translation and maintenance of protein quality, redox homeostasis, and cell envelope modification are upregulated. In both Gram‐negative species, protein synthesis of the majority of proteins is downregulated while the Gram‐positive B. subtilis responded by upregulation of multiple regulons. A comparison of the B. subtilis proteomic response to a library of responses to antibiotic treatment reveals 30 proteins specifically upregulated by allicin. Upregulated oxidative stress proteins are shared with nitrofurantoin and diamide. Microscopy‐based assays further indicate that in B. subtilis cell wall integrity is impaired.     

Microbial communities associated with phosphogenic sediments and phosphoclast‐associated DNA of the Benguela upwelling system

The processes that lead to the precipitation of authigenic calcium phosphate minerals in certain marine pore waters remain poorly understood. Phosphogenesis occurs in sediments beneath some oceanic upwelling zones that harbor polyphosphate‐accumulating bacteria. These bacteria are believed to concentrate phosphate in sediment pore waters, creating supersaturated conditions with respect to apatite precursors. However, the relationship between microbes and phosphorite formation is not fully resolved. To further study this association, we examined microbial community data generated from two sources: sediment cores recovered from the shelf of the Benguela upwelling region where phosphorites are currently forming, and DNA preserved within phosphoclasts recovered from a phosphorite deposit along the Benguela shelf. iTag and clone library sequencing of the 16S rRNA gene showed that many of our sediment‐hosted communities shared large numbers of phylotypes with one another, and that the same metabolic guilds were represented at localities across the shelf. Sulfate‐reducing bacteria and sulfur‐oxidizing bacteria were particularly abundant in our datasets, as were phylotypes that are known to carry out nitrification and the anaerobic oxidation of ammonium. The DNA extracted from phosphoclasts contained the signature of a distinct microbial community from those observed in the modern sediments. While some aspects of the modern and phosphoclast communities were similar, we observed both an enrichment of certain common microbial classes found in the modern phosphogenic sediments and a relative depletion of others. The phosphoclast‐associated DNA could represent a relict signature of one or more microbial assemblages that were present when the apatite or its precursors precipitated. While these taxa may or may not have contributed to the precipitation of the apatite that now hosts their genetic remains, several groups represented in the phosphoclast extract dataset have the genetic potential to metabolize polyphosphate, and perhaps modulate phosphate concentrations in pore waters where carbonate fluorapatite (or its precursors) are known to be precipitating.     

Development of LCA benchmarks for Austrian torrent control structures

The International Journal of Life Cycle Assessment - This study emerged from a research project that aimed to develop a Life Cycle Assessment (LCA) model for torrent control structures. This...     

Tropical rainforest flies carrying pathogens form stable associations with social nonhuman primates

Living in groups provides benefits but also incurs costs such as attracting disease vectors. For example, synanthropic flies associate with human settlements, and higher fly densities increase pathogen transmission. We investigated whether such associations also exist in highly mobile nonhuman primate (NHP) Groups. We studied flies in a group of wild sooty mangabeys (Cercocebus atys atys) and three communities of wild chimpanzees (Pan troglodytes verus) in Taï National Park, Côte d'Ivoire. We observed markedly higher fly densities within both mangabey and chimpanzee groups. Using a mark–recapture experiment, we showed that flies stayed with the sooty mangabey group for up to 12 days and for up to 1.3 km. We also tested mangabey‐associated flies for pathogens infecting mangabeys in this ecosystem, Bacillus cereus biovar anthracis (Bcbva), causing sylvatic anthrax, and Treponema pallidum pertenue, causing yaws. Flies contained treponemal (6/103) and Bcbva (7/103) DNA. We cultured Bcbva from all PCR‐positive flies, confirming bacterial viability and suggesting that this bacterium might be transmitted and disseminated by flies. Whole genome sequences of Bcbva isolates revealed a diversity of Bcbva, probably derived from several sources. We conclude that flies actively track mangabeys and carry infectious bacterial pathogens; these associations represent an understudied cost of sociality and potentially expose many social animals to a diversity of pathogens.