Incorporation of ethidium bromide in the Drosophila salivary gland approached by microspectrofluorometry: evidence for the presence of both free and bound dye in the nuclei of cells in viable conditions

The incorporation of 10^–6 M ethidium bromide (EB) was studied in viable Drosophila melanogaster salivary glands with a spatial resolution reaching a few µm³, using a confocal laser microspectrofluorometer designed for spectral analysis. Spectra were recorded with the 514 nm Argon laser line during excitation times of 1 second (20 µW on the preparation) at 5 min intervals for 30 or 60 min, either at points in determined cell sites or serially throughout the cells. The fluorescence intensity time-course indicated that the EB intake was not an all-or-none process, but rather a graded, sensitive indicator of the functional state of the cell. On the micrometer scale, the cytoplasm behaved as an homogeneous substrate with the fluorescence intensity depending on EB intake and intracellular diffusion. In the nucleus, however, localized enhancement of the emission intensity was observed. Spectral analysis allowed us to characterize the interactions. The mean values of λ _max in the cytoplasm (600 nm), in the nucleus (601 nm) and outside the glands (602 nm) were less than for free EB in aqueous solution (630 nm); values of full width at half maximum were between 92 and 96 nm, which is much lower than the 120 nm observed for free EB. The recorded spectra were analyzed using a linear combination of two spectral models, namely free and DNA intercalated EB. In the nucleus, the free EB model spectra was found to represent up to 10% of the recorded spectra whereas it was near zero in the cytoplasm. The present data suggest that the intranuclear concentration of free EB (allowing for its lower fluorescence quantum yield) might be at least equal to that of the bound EB.     

Interface processes between protected and unprotected areas: A global review and ways forward

Land‐use changes and the expansion of protected areas (PAs) have amplified the interaction between protected and unprotected areas worldwide. In this context, ‘interface processes' (human–nature and cross‐boundary interactions inside and around PAs) have become central to issues around the conservation of biodiversity and ecosystem services. This scientific literature review aimed to explore current knowledge and research gaps on interface processes regarding terrestrial PAs. At first, 3,515 references related to the topic were extracted through a standardized search on the Web of Science and analyzed with scientometric techniques. Next, a full‐text analysis was conducted on a sample of 240 research papers. A keyword analysis revealed a wide diversity of research topics, from ‘pure' ecology to sociopolitical research. We found a bias in the geographical distribution of research, with half the papers focusing on eight countries. Additionally, we found that the spatial extent of cross‐boundary interactions was rarely assessed, preventing any clear delimitation of PA interactive zones. In the 240 research papers we scanned, we identified 403 processes that were studied. The ecological effects of PAs were well documented and appeared to be positive overall. In contrast, the effects of PAs on local communities were understudied and, according to the literature focusing on these, were very variable according to local contexts. Our findings highlight key research advances on interface processes, especially regarding the ecological outcomes of PAs, the influence of human activities on biodiversity, and PA governance issues. In contrast, main knowledge gaps concern the spatial extent of interactive zones, as well as the interactions between local people and conservation actions and how to promote synergies between them. While the review was limited to terrestrial PAs, its findings allow us to propose research priorities for tackling environmental and socioeconomic challenges in the face of a rapidly changing world.     

A novel allele of the Arabidopsis thaliana MACPF protein CAD1 results in deregulated immune signaling

Immune recognition in plants is governed by two major classes of receptors: pattern recognition receptors (PRRs) and nucleotide-binding leucine-rich repeat receptors (NLRs). Located at the cell surface, PRRs bind extracellular ligands originating from microbes (indicative of “non-self”) or damaged plant cells (indicative of “infected-self”), and trigger signaling cascades to protect against infection. Located intracellularly, NLRs sense pathogen-induced physiological changes and trigger localized cell death and systemic resistance. Immune responses are under tight regulation in order to maintain homeostasis and promote plant health. In a forward-genetic screen to identify regulators of PRR-mediated immune signaling, we identified a novel allele of the membrane-attack complex and perforin (MACPF)-motif containing protein CONSTITUTIVE ACTIVE DEFENSE 1 (CAD1) resulting from a missense mutation in a conserved N-terminal cysteine. We show that cad1-5 mutants display deregulated immune signaling and symptoms of autoimmunity dependent on the lipase-like protein ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), suggesting that CAD1 integrity is monitored by the plant immune system. We further demonstrate that CAD1 localizes to both the cytosol and plasma membrane using confocal microscopy and subcellular fractionation. Our results offer new insights into immune homeostasis and provide tools to further decipher the intriguing role of MACPF proteins in plants.     

Arabidopsis RECEPTOR-LIKE PROTEIN30 and Receptor-Like Kinase SUPPRESSOR OF BIR1-1/EVERSHED Mediate Innate Immunity to Necrotrophic Fungi

Effective plant defense strategies rely in part on the perception of non-self determinants, so-called microbe-associated molecular patterns (MAMPs), by transmembrane pattern recognition receptors leading to MAMP-triggered immunity. Plant resistance against necrotrophic pathogens with a broad host range is complex and yet not well understood. Particularly, it is unclear if resistance to necrotrophs involves pattern recognition receptors. Here, we partially purified a novel proteinaceous elicitor called SCLEROTINIA CULTURE FILTRATE ELICITOR1 (SCFE1) from the necrotrophic fungal pathogen Sclerotinia sclerotiorum that induces typical MAMP-triggered immune responses in Arabidopsis thaliana. Analysis of natural genetic variation revealed five Arabidopsis accessions (Mt-0, Lov-1, Lov-5, Br-0, and Sq-1) that are fully insensitive to the SCFE1-containing fraction. We used a forward genetics approach and mapped the locus determining SCFE1 sensitivity to RECEPTOR-LIKE PROTEIN30 (RLP30). We also show that SCFE1-triggered immune responses engage a signaling pathway dependent on the regulatory receptor-like kinases BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE1 (BAK1) and SUPPRESSOR OF BIR1-1/EVERSHED (SOBIR1/EVR). Mutants of RLP30, BAK1, and SOBIR1 are more susceptible to S. sclerotiorum and the related fungus Botrytis cinerea. The presence of an elicitor in S. sclerotiorum evoking MAMP-triggered immune responses and sensed by RLP30/SOBIR1/BAK1 demonstrates the relevance of MAMP-triggered immunity in resistance to necrotrophic fungi.     

Contrasting effects of climate change in continental vs. oceanic environments on population persistence and microevolution of Atlantic salmon

Facing climate change (CC), species are prone to multiple modifications in their environment that can lead to extinction, migration or adaptation. Identifying the role and interplay of different potential stressors becomes a key question. Anadromous fishes will be exposed to both river and oceanic habitat changes. For Atlantic salmon, the river water temperature, river flow and oceanic growth conditions appear as three main stressing factors. They could act on population dynamics or as selective forces on life‐history pathways. Using an individual‐based demo‐genetic model, we assessed the effects of these factors (1) to compare risks of extinction resulting from CC in river and ocean, and (2) to assess CC effects on life‐history pathways including the evolution of underlying genetic control of phenotypic plasticity. We focused on Atlantic salmon populations from Southern Europe for a time horizon of three decades. We showed that CC in river alone should not lead to extinction of Southern European salmon populations. In contrast, the reduced oceanic growth appeared as a significant threat for population persistence. An increase in river flow amplitude increased the risk of local extinction in synergy with the oceanic effects, but river temperature rise reduced this risk. In terms of life‐history modifications, the reduced oceanic growth increased the age of return of individuals through plastic and genetic responses. The river temperature rise increased the proportion of sexually mature parr, but the genetic evolution of the maturation threshold lowered the maturation rate of male parr. This was identified as a case of environmentally driven plastic response that masked an underlying evolutionary response of plasticity going in the opposite direction. We concluded that to counteract oceanic effects, river flow management represented the sole potential force to reduce the extinction probability of Atlantic salmon populations in Southern Europe, although this might not impede changes in migration life history.     

Why Are Babies Dying in the First Month after Birth? A 7-Year Study of Neonatal Mortality in Northern Ghana

Objectives

To determine the neonatal mortality rate in the Kassena-Nankana District (KND) of northern Ghana, and to identify the leading causes and timing of neonatal deaths.

Methods

The KND falls within the Navrongo Health Research Centre’s Health and Demographic Surveillance System (HDSS), which uses trained field workers to gather and update health and demographic information from community members every four months. We utilized HDSS data from 2003–2009 to examine patterns of neonatal mortality.

Results

A total of 17,751 live births between January 2003 and December 2009 were recorded, including 424 neonatal deaths 64.8%(275) of neonatal deaths occurred in the first week of life. The overall neonatal mortality rate was 24 per 1000 live births (95%CI 22 to 26) and early neonatal mortality rate was 16 per 1000 live births (95% CI 14 to 17). Neonatal mortality rates decreased over the period from 26 per 1000 live births in 2003 to 19 per 1000 live births in 2009. In all, 32%(137) of the neonatal deaths were from infections, 21%(88) from birth injury and asphyxia and 18%(76) from prematurity, making these three the leading causes of neonatal deaths in the area. Birth injury and asphyxia (31%) and prematurity (26%) were the leading causes of early neonatal deaths, while infection accounted for 59% of late neonatal deaths. Nearly 46% of all neonatal deaths occurred during the first three postnatal days. In multivariate analysis, multiple births, gestational age <32 weeks and first pregnancies conferred the highest odds of neonatal deaths.

Conclusions

Neonatal mortality rates are declining in rural northern Ghana, with majority of deaths occurring within the first week of life. This has major policy, programmatic and research implications. Further research is needed to better understand the social, cultural, and logistical factors that drive high mortality in the early days following delivery.     

The Effect of Vaccination Coverage and Climate on Japanese Encephalitis in Sarawak,Malaysia

Background

Japanese encephalitis (JE) is the leading cause of viral encephalitis across Asia with approximately 70,000 cases a year and 10,000 to 15,000 deaths. Because JE incidence varies widely over time, partly due to inter-annual climate variability effects on mosquito vector abundance, it becomes more complex to assess the effects of a vaccination programme since more or less climatically favourable years could also contribute to a change in incidence post-vaccination. Therefore, the objective of this study was to quantify vaccination effect on confirmed Japanese encephalitis (JE) cases in Sarawak, Malaysia after controlling for climate variability to better understand temporal dynamics of JE virus transmission and control.

Methodology/principal findings

Monthly data on serologically confirmed JE cases were acquired from Sibu Hospital in Sarawak from 1997 to 2006. JE vaccine coverage (non-vaccine years vs. vaccine years) and meteorological predictor variables, including temperature, rainfall and the Southern Oscillation index (SOI) were tested for their association with JE cases using Poisson time series analysis and controlling for seasonality and long-term trend. Over the 10-years surveillance period, 133 confirmed JE cases were identified. There was an estimated 61% reduction in JE risk after the introduction of vaccination, when no account is taken of the effects of climate. This reduction is only approximately 45% when the effects of inter-annual variability in climate are controlled for in the model. The Poisson model indicated that rainfall (lag 1-month), minimum temperature (lag 6-months) and SOI (lag 6-months) were positively associated with JE cases.

Conclusions/significance

This study provides the first improved estimate of JE reduction through vaccination by taking account of climate inter-annual variability. Our analysis confirms that vaccination has substantially reduced JE risk in Sarawak but this benefit may be overestimated if climate effects are ignored.