Real-time monitoring of PtaHMGB activity in poplar transactivation assays

Precise control of gene expression is essential to synchronize plant development with the environment. In perennial plants, transcriptional regulation remains poorly understood, mainly due to the long time required to perform functional studies. Transcriptional reporters based on luciferase have been useful to study circadian and diurnal regulation of gene expression, both by transcription factors and chromatin remodelers. The high mobility group proteins are considered transcriptional chaperones that also modify the chromatin architecture. They have been found in several species, presenting in some cases a circadian expression of their mRNA or protein. Results: Transactivation experiments have been shown as a powerful and fast method to obtain information about the potential role of transcription factors upon a certain reporter. We designed and validated a luciferase transcriptional reporter using the 5? sequence upstream ATG of Populus tremula × alba LHY2 gene. We showed the robustness of this reporter line under long day and continuous light conditions. Moreover, we confirmed that pPtaLHY2::LUC activity reproduces the accumulation of PtaLHY2 mRNA. We performed transactivation studies by transient expression, using the reporter line as a genetic background, unraveling a new function of a high mobility group protein in poplar, which can activate the PtaLHY2 promoter in a gate-dependent manner. We also showed PtaHMGB2/3 needs darkness to produce that activation and exhibits an active degradation after dawn, mediated by the 26S proteasome. Conclusions: We generated a stable luciferase reporter poplar line based on the circadian clock gene PtaLHY2, which can be used to investigate transcriptional regulation and signal transduction pathway. Using this reporter line as a genetic background, we established a methodology to rapidly assess potential regulators of diurnal and circadian rhythms. This tool allowed us to demonstrate that PtaHMGB2/3 promotes the transcriptional activation of our reporter in a gate-dependent manner. Moreover, we added new information about the PtaHMGB2/3 protein regulation along the day. This methodology can be easily adapted to other transcription factors and reporters

Novice nurse's transitioning to emergency nurse during COVID-19 pandemic: A qualitative study

To explore the experiences and perceptions of recent nursing graduates working in emergency departments during the COVID-19 outbreak

Uncovering cold disruption of the circadian clock in poplar

Dormancy is an adaptive mechanism that allows woody plants to survive at low temperatures during the winter. Disruption of circadian clock genes in winter or under low temperatures, both in long days as in short days, were described in our group few years ago (Ramos et al., 2005). Basic mechanisms of the circadian clock function are similar in herbaceous as well as in woody plants although there are differences in their response to low temperatures (Bieniawska et al., 2008). Woody plants growing in daylight conditions should have a specific transcriptional control above the circadian clock genes, which is responsible of their constitutive transcriptional activation observed under low temperatures conditions. In order to understand this regulatory process, we are analyzing the behavior of a circadian clock gene in poplar. To this aim, we have isolated its promoter region and fused to the luciferase reporter gene. This construct has been transformed into Populus tremula x P. alba 717-1B4 INRA clone. Here we present the characterization of these transgenic lines under different conditions of light and temperature

Area Freedom in Mexico from Mediterranean Fruit Fly (Diptera: Tephritidae): A Review of Over 30 Years of a Successful Containment Program Using an Integrated Area-Wide SIT Approach

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Novel winter-associated regulators of the circadian clock in poplar

Background Winter dormancy is an adaptive mechanism that allows trees from temperate and cold regions to survive the harsh conditions of this season. Critical steps of this process are strongly influenced by environmental cues, mainly daylength and temperature. The mechanism that integrates these signals is the circadian clock. Despite the importance of the correct functioning of the clock for the healthy state of the plant [1], low temperatures cause the disruption of the circadian clock in trees, which consists in a transcriptional activation followed by an arrhythmic expression [2-5]. In this work we uncover winter-associated regulators of the circadian clock in poplar. Methods Firstly, we made a transcriptional fusion with the promoter of LHY2, a circadian clock gene, and the luciferase gene. This construct was used to generate transgenic poplars (717-1B4, INRA clone). With these events we characterized the expression of this promoter under different conditions of photoperiod and temperature. To this aim we have set up a circadian luminiscence assay registering luciferase activity from leaf discs with a luminometer. Then we carried out a Yeast One Hybrid (Y1H) screening with a library enriched in winter-associated factors and using this promoter as bait. Candidate regulators are tested in vivo using Golden Braid technology [6] and transient assays in poplar, by which we overexpressed and silenced the candidate genes. Results and Conclusions Here we present the characterization of the Populus tremula x alba LHY2 promoter under three different photoperiod conditions. Our results indicate the selected promoter region contains the circadian elements as well as the luciferase activity shows the expected expression under both long and short days. In the Y1H screening, we found several candidates that are classified either as transcription factors or chromatin remodelers. We will discuss the possible role of these proteins as regulators of the poplar circadian clock

Epidemiology of Candidemia in Latin America: A Laboratory-Based Survey

Background: the epidemiology of candidemia varies depending on the geographic region. Little is known about the epidemiology of candidemia in Latin America.Methods: We conducted a 24-month laboratory-based survey of candidemia in 20 centers of seven Latin American countries. Incidence rates were calculated and the epidemiology of candidemia was characterized.Results: Among 672 episodes of candidemia, 297 (44.2%) occurred in children (23.7% younger than 1 year), 36.2% in adults between 19 and 60 years old and 19.6% in elderly patients. the overall incidence was 1.18 cases per 1,000 admissions, and varied across countries, with the highest incidence in Colombia and the lowest in Chile. Candida albicans (37.6%), C. parapsilosis (26.5%) and C. tropicalis (17.6%) were the leading agents, with great variability in species distribution in the different countries. Most isolates were highly susceptible to fluconazole, voriconazole, amphotericin B and anidulafungin. Fluconazole was the most frequent agent used as primary treatment (65.8%), and the overall 30-day survival was 59.3%.Conclusions: This first large epidemiologic study of candidemia in Latin America showed a high incidence of candidemia, high percentage of children, typical species distribution, with C. albicans, C. parapsilosis and C. tropicalis accounting for the majority of episodes, and low resistance rates.independent medical grant from Pfizer Inc.Univ Fed Rio de Janeiro, Univ Hosp, Rio de Janeiro, BrazilUniv Fed Parana, Hosp Clin, BR-80060000 Curitiba, Parana, BrazilHosp Escuela Tegucigalpa, Tegucigalpa, HondurasHosp Clin Jose San Martin, Buenos Aires, DF, ArgentinaUniv Nacl Colombia, Dept Internal Med, Bogota, ColombiaPontificia Univ Catolica Ecuador, Fac Med, Hosp Vozandes, Quito, EcuadorHosp Vargas de Caracas, Caracas, VenezuelaCtr Med Caracas, Caracas, VenezuelaUniv Chile, Fac Med, Dept Pediat, Hosp Luis Calvo Mackenna, Santiago 7, ChileUniv Desarrollo, Clin Alemana, Dept Med, Infect Dis Unit, Santiago, ChileInst Nacl Ciencias Med & Nutr Salvador Zubiran, Mexico City, DF, MexicoUniv Peruana Cayetano Heredia, Dept Med, Lima, PeruUniversidade Federal de São Paulo, Escola Paulista Med, Div Infect Dis, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Div Infect Dis, São Paulo, BrazilWeb of Scienc

The involvement of 5-methyl cytosine DNA Demethylases in the dormant-growth transition in poplar

Background Woody species are highly adapted to their habitats. In response to environmental cues woody perennials trigger self-protective developmental programmes, in which signal transduction, transcriptional reprogramming and epigenetic regulation could participate in defining the winter dormancy state. Winter dormancy is the mechanism used by perennial plants to survive the harsh conditions of winter in temperate and cold regions and determines the geographical distribution of tree species (Chuine and Beaubien 2001; Horvath et al. 2003; Allona et al. 2008). Epigenetic control of winter dormancy in woody plants is barely known. Among the important epigenetic marks, 5-methyl cytosine (5mC) regulates gene expression in animals and plants. Global changes in 5mC DNA methylation have been shown in the transition of developmental stages in plants such as chestnut bud set and burst, flowering in azalea, aging in pine trees among other. However, the mechanism and the enzymes involved in the modification of the methylome and its control over those development processes remain to be identified. Our previous results showed higher DNA methylation and less acetylated Lys 8 of histone H4 global levels in poplar stem during winter dormancy compared to active growing season (Conde et al. 2013). In this study we focus in the understanding of the molecular mechanism behind these changes in DNA methylation profile and their role in the control of winter dormancy. Methods Analysis of the 5-methyl cytosine levels by the application of the immunofluorescence-based method set up in our lab, in stem vibratome sections cut from hybrid poplar (Populus tremula x alba) growing in the field at different stages of winter dormancy process. To develop a protocol for buds paraffin wax embedding to analyze the level of 5-methyl cytosine by applying our immunofluorescence-based method in poplar apex microtome sections in diferents stages of winter dormancy. RT-PCR analysis to determine the profile of gene expresion at diferent stages of winter dormancy involved in modification of DNA methylation profile. Hybrid poplar transformation to obtain transgenic lines with modified expression of a demethylase and phenological experiments with selected lines. Results and Conclusions The immunolocalization assays performed in poplar stem sections showed that DNA methylation leves fall suddenly when trees coming from the dormant state are near to restore the growing season. We have determined the spatial distribution of DNA methylation changes in this organ. We have identified two poplar homologs to Arabidopsis DME gene: PtaDML8/PtaDML10. The DME protein promotes global DNA demethylation along the genome during endosperm development. Our RT-PCR analyses indicate that the expression of PtaDML8/PtaDML10 genes increases significantly when trees are near to restart growing after winter dormancy. The phenologycal assays showed that PtaDML8/PtaDML10 knockdown plants have a delayed in resuming of growth after dormancy. Taken together, we hypothesize that an active control of the 5mC DNA methylation might play a key role in winter dormancy and that 5mC demethylases would be crucial in this process

Estimation of the real population and its impact on the utilisation of healthcare services in Mediterranean resort regions: an ecological study

BACKGROUND: The demographic structure has a significant influence on the use of healthcare services, as does the size of the population denominators. Very few studies have been published on methods for estimating the real population such as tourist resorts. The lack of information about these problems means there is a corresponding lack of information about the behaviour of populational denominators (the floating population or tourist load) and the effect of this on the use of healthcare services. The objectives of the study were: a) To determine the Municipal Solid Waste (MSW) ratio, per person per day, among populations of known size; b) to estimate, by means of this ratio, the real population in an area where tourist numbers are very significant; and c) to determine the impact on the utilisation of hospital emergency healthcare services of the registered population, in comparison to the non-resident population, in two areas where tourist numbers are very significant. METHODS: An ecological study design was employed. We analysed the Healthcare Districts of the Costa del Sol and the island of Menorca. Both are Spanish territories in the Mediterranean region. RESULTS: In the two areas analysed, the correlation coefficient between the MSW ratio and admissions to hospital emergency departments exceeded 0.9, with p < 0.001. On the basis of MSW generation ratios, obtained for a control zone and also measured in neighbouring countries, we estimated the real population. For the summer months, when tourist activity is greatest and demand for emergency healthcare at hospitals is highest, this value was found to be double that of the registered population. CONCLUSION: The MSW indicator, which is both ecological and indirect, can be used to estimate the real population in areas where population levels vary significantly during the year. This parameter is of interest in planning and dimensioning the provision of healthcare services

Improved lignocellulosic biomass yield of RAV1 engineered poplars in a SRC field trial

Background Plantations of Populus spp, Salix spp. or Eucalyptus spp. are established to produce wood in a reduced space and a short time. Poplars are cultivated with cycles of 15-18 years to obtain saw timber and peeler logs, and when grown for biomass production as short-rotation coppice (SRC), cutting back/coppicing cycles are reduced to 2?5-years intervals. Syllepsis is among the valuable traits that can be targeted to enhance biomass yield of SRCs. Syllepsis, i.e. the outgrowth of lateral buds into branches the same season in which they form without an intervening rest period, increases carbon fixation and allocation in the shoot and hence the general growth of the tree. A high degree of sylleptic branching is known to be positively correlated with biomass yield when these plantations are grown under optimal conditions [1]. In 2012 we established in Madrid (Spain) a SRC field trial with genetically engineered poplars, previously shown to develop sylleptic branches when cultivated in growth chambers, under optimal conditions [2]. The aim of starting up this field trial was to test whether a plastic trait as syllepsis was maintained over time under natural conditions and eventually resulted in an enhanced biomass production Methods In vitro culture rooted cuttings were initially potted in 3.5L containers with blond peat and grown in the greenhouse as previously described [2]. The field trial was established in July 2012 in the experimental plot, and included five groups of hybrid poplar Populus tremula x P. alba INRA clone 717 1B, the wild-type genotype as control, transgenic events #37 and #60 carrying the 35S::3xHA:CsRAV1 cassette (3xHA:CsRAV1 OX), and events #1 and #22 carrying the 35S::PtaRAV1-hpiRNA cassette (PtaRAV1&2 KD). 30 individuals per group were planted into three blocks of 10 plants each. The experimental plot area was 204 m2 , and the plantation density 10000 trees/ha. It consisted of 12 x 17 rows with a tree spacing 2 x 0.5 m. The border rows were occupied by P. x euramericana clone I-214 individuals, planted as 25 cm-long cuttings. Irrigation and weed/pests control were applied, and the first coppicing cycle was done after the second growing season [3]. Several productivity determinants (stem height and diameter, syllepsis and phenology) were monitored, wood anatomy and chemistry analyzed, and aerial biomass yield and calorific value determined. Results and Conclusions CsRAV1 over-expressing event #60 showed an advantageous performance in the field regarding stem diameter and biomass production after the first coppicing cycle. In this event, sylleptic branches grew from the main shoot during the first growing seasons, after the plantation establishment and after coppicing. None of the other traits under study such as phenology, wood anatomy and chemistry were noticeably altered when compared to the wild type genotype. These results show that in woody species RAV1 is a highly valuable target gene that can be used as biotechnological tool to enhance biomass yield of poplar SRC plantations without detrimental side-effects in tree development and characteristics