Silencing of MicroRNA-21 Confers Radio-Sensitivity through Inhibition of the PI3K/AKT Pathway and Enhancing Autophagy in Malignant Glioma Cell Lines

Radiation is a core part of therapy for malignant glioma and is often provided following debulking surgery. However, resistance to radiation occurs in most patients, and the underlying molecular mechanisms of radio-resistance are not fully understood. Here, we demonstrated that microRNA 21 (miR-21), a well-known onco-microRNA in malignant glioma, is one of the major players in radio-resistance. Radio-resistance in different malignant glioma cell lines measured by cytotoxic cell survival assay was closely associated with miR-21 expression level. Blocking miR-21 with anti-miR-21 resulted in radio-sensitization of U373 and U87 cells, whereas overexpression of miR-21 lead to a decrease in radio-sensitivity of LN18 and LN428 cells. Anti-miR-21 sustained γ-H2AX DNA foci formation, which is an indicator of double-strand DNA damage, up to 24 hours and suppressed phospho-Akt (ser473) expression after exposure to γ-irradiation. In a cell cycle analysis, a significant increase in the G₂/M phase transition by anti-miR-21 was observed at 48 hours after irradiation. Interestingly, our results showed that anti-miR-21 increased factors associated with autophagosome formation and autophagy activity, which was measured by acid vesicular organelles, LC3 protein expression, and the percentage of GFP-LC3 positive cells. Furthermore, augmented autophagy by anti-miR-21 resulted in an increase in the apoptotic population after irradiation. Our results show that miR-21 is a pivotal molecule for circumventing radiation-induced cell death in malignant glioma cells through the regulation of autophagy and provide a novel phenomenon for the acquisition of radio-resistance.     

QTL-seq identifies an early flowering QTL located near Flowering Locus T in cucumber

Key message

Next-generation sequencing enabled a fast discovery of a major QTL controlling early flowering in cucumber, corresponding to the FT gene conditioning flowering time in Arabidopsis.

Abstract

Next-generation sequencing technologies are making it faster and more efficient to establish the association of agronomic traits with molecular markers or candidate genes, which is the requirement for marker-assisted selection in molecular breeding. Early flowering is an important agronomic trait in cucumber (Cucumis sativus L.), but the underlying genetic mechanism is unknown. In this study, we identified a candidate gene for early flowering QTL, Ef1.1 through QTL-seq. Segregation analysis in F₂ and BC₁ populations derived from a cross between two inbred lines “Muromskij” (early flowering) and “9930” (late flowering) suggested quantitative nature of flowering time in cucumber. Genome-wide comparison of SNP profiles between the early and late-flowering bulks constructed from F₂ plants identified a major QTL, designated Ef1.1 on cucumber chromosome 1 for early flowering in Muromskij, which was confirmed by microsatellite marker-based classical QTL mapping in the F₂ population. Joint QTL-seq and traditional QTL analysis delimited Ef1.1 to an 890 kb genomic region. A cucumber gene, Csa1G651710, was identified in this region, which is a homolog of the FLOWERING LOCUS T (FT), the main flowering switch gene in Arabidopsis. Quantitative RT-PCR study of the expression level of Csa1G651710 revealed significantly higher expression in early flowering genotypes. Data presented here provide support for Csa1G651710 as a possible candidate gene for early flowering in the cucumber line Muromskij.     

Genome-wide promoter methylation analysis in neuroblastoma identifies prognostic methylation biomarkers

Background

Accurate outcome prediction in neuroblastoma, which is necessary to enable the optimal choice of risk-related therapy, remains a challenge. To improve neuroblastoma patient stratification, this study aimed to identify prognostic tumor DNA methylation biomarkers.

Results

To identify genes silenced by promoter methylation, we first applied two independent genome-wide methylation screening methodologies to eight neuroblastoma cell lines. Specifically, we used re-expression profiling upon 5-aza-2''-deoxycytidine (DAC) treatment and massively parallel sequencing after capturing with a methyl-CpG-binding domain (MBD-seq). Putative methylation markers were selected from DAC-upregulated genes through a literature search and an upfront methylation-specific PCR on 20 primary neuroblastoma tumors, as well as through MBD- seq in combination with publicly available neuroblastoma tumor gene expression data. This yielded 43 candidate biomarkers that were subsequently tested by high-throughput methylation-specific PCR on an independent cohort of 89 primary neuroblastoma tumors that had been selected for risk classification and survival. Based on this analysis, methylation of KRT19, FAS, PRPH, CNR1, QPCT, HIST1H3C, ACSS3 and GRB10 was found to be associated with at least one of the classical risk factors, namely age, stage or MYCN status. Importantly, HIST1H3C and GNAS methylation was associated with overall and/or event-free survival.

Conclusions

This study combines two genome-wide methylation discovery methodologies and is the most extensive validation study in neuroblastoma performed thus far. We identified several novel prognostic DNA methylation markers and provide a basis for the development of a DNA methylation-based prognostic classifier in neuroblastoma.     

Influence of Soil Physical Properties on Plants of the Mussununga Ecosystem, Brazil

Distribution ranges of plant species are related to physical variables of ecosystems that limit plant growth. Therefore, each plant species response to physical factors builds up the functional diversity of an ecosystem. The higher the species richness of an ecosystem, the larger the probability of maintaining functions and the higher the potential number of plant functional groups (FGs). Thus, the richness potentially increases the number of functions of the highly diverse Atlantic Rainforest domain in Brazil. Severe plant growth limitations caused by stress, however, decrease species richness. In the Spodosols of the Mussununga, an associated ecosystem of Atlantic Rainforest, the percentage of fine sand is directly related to water retention. Moreover, the depth of the cementation layer in the Mussununga??s sandy soil is a physical factor that can affect the plants?? stress gradients. When a shallow cementation layer depth is combined with low water retention in soils and with low fine sand percentage, the double stresses of flooding in the rainy season and water scarcity in the dry season result. This study aimed to identify FGs among Mussununga plant species responding to water stress gradients of soil and to verify the effects of the gradients on plant species richness of the Mussununga. A canonical correspondence analysis (CCA) of species abundance and soil texture variables was performed on 18 plots in six physiognomies of the Mussununga. Species richness rarefactions were calculated for each vegetation form to compare diversity. The two main axes of the CCA showed two FGs responding to soil texture and cementation layer depth: stress tolerator species and mesic species. Physical variables affect plant diversity, with species richness rising as the fine sand proportion also rises in the Mussununga. The effect of the cementation layer is not significantly related to species richness variation.     

Role of histidine for charge regulation of unstructured peptides at interfaces and in bulk

Histidine‐rich, unstructured peptides adsorb to charged interfaces such as mineral surfaces and microbial cell membranes. At a molecular level, we investigate the adsorption mechanism as a function of pH, salt, and multivalent ions showing that (1) proton charge fluctuations are—in contrast to the majority of proteins—optimal at neutral pH, promoting electrostatic interactions with anionic surfaces through charge regulation and (2) specific zinc(II)‐histidine binding competes with protons and ensures an unusually constant charge distribution over a broad pH interval. In turn, this further enhances surface adsorption. Our analysis is based on atomistic molecular dynamics simulations, coarse grained Metropolis Monte Carlo, and classical polymer density functional theory. This multiscale modeling provides a consistent picture in good agreement with experimental data on Histatin 5, an antimicrobial salivary peptide. Biological function is discussed and we suggest that charge regulation is a significant driving force for the remarkably robust activity of histidine‐rich antimicrobial peptides. Proteins 2014; 82:657–667. © 2013 Wiley Periodicals, Inc.     

Land Use and Land Cover Change Dynamics across the Brazilian Amazon: Insights from Extensive Time-Series Analysis of Remote Sensing Data

Throughout the Amazon region, the age of forests regenerating on previously deforested land is determined, in part, by the periods of active land use prior to abandonment and the frequency of reclearance of regrowth, both of which can be quantified by comparing time-series of Landsat sensor data. Using these time-series of near annual data from 1973–2011 for an area north of Manaus (in Amazonas state), from 1984–2010 for south of Santarém (Pará state) and 1984–2011 near Machadinho d’Oeste (Rondônia state), the changes in the area of primary forest, non-forest and secondary forest were documented from which the age of regenerating forests, periods of active land use and the frequency of forest reclearance were derived. At Manaus, and at the end of the time-series, over 50% of regenerating forests were older than 16 years, whilst at Santarém and Machadinho d’Oeste, 57% and 41% of forests respectively were aged 6–15 years, with the remainder being mostly younger forests. These differences were attributed to the time since deforestation commenced but also the greater frequencies of reclearance of forests at the latter two sites with short periods of use in the intervening periods. The majority of clearance for agriculture was also found outside of protected areas. The study suggested that a) the history of clearance and land use should be taken into account when protecting deforested land for the purpose of restoring both tree species diversity and biomass through natural regeneration and b) a greater proportion of the forested landscape should be placed under protection, including areas of regrowth.     

Conformation and structure of 2-amino-8-(β-d-ribofuranosyl)imidazo [1,2-a]-s-triazin-4-one (5-aza-7-deaza guanosine), a potent antiviral nucleoside

The crystal and molecular structure of one imidazo[1,2-a]-s-triazine nucleoside and its antiviral activity are described. The crystal structure of 2-amino-8-(β-d-ribofuranosyl)imidazo-[1,2-a]-s-triazin-4-one monohydrate (C₁₀H₁₃N₅O₅·H₂O) was solved by X-ray counter data. The compound crystallizes in the monoclinic space group P2₁ with cell dimensions a = 7.353 (1), b = 6.465 (1), c = 13.701 (1) Å, β = 104.64 (1)°. The structure was solved by direct methods and refined by full matrix least-squares technique to a final value of the conventional R-factor of 0.049 using 1998 observed intensities. The orientation of the base relative to the sugar ring defined in terms of rotation about the C(1′)-N(8) glycosyl bond is anti (47.8°). The ribose moiety exhibits C(2′)-endo, ²E conformation. The conformation around C(4′)-C(5′) is gauche^?. Molecular packing is dominated by hydrogen bonds. Base stacking occurs long the b axis. 5-Aza-7-deazaguanosine has shown a marked antiviral activity in vitro against herpes simplex virus despite the fact that N(3) is effective as the hydrogen acceptor only.     

Water quality parameters and tipping points of dragonfly diversity and abundance in fishponds

Fishponds are often enriched with nutrients in order to increase phytoplankton and zooplankton populations to support fish production. This eutrophication often leads to a global decrease of biodiversity. This biodiversity shift may be identified by a tipping point, the value of an environmental parameter above which a significant change of species richness and abundance occurs. A total of 110 eutrophic to highly eutrophic fishponds were studied in two areas in France to investigate parameters governing dragonfly species richness and species abundance by determining tipping points. Parameters investigated were chlorophyll a (CHL), water transparency, total N (TN), total P (TP), aquatic plant richness and coverage, adult dragonfly richness and abundance, and fish harvest. A high species richness of dragonflies was found in fishponds, with a total of 34 species, including six species of conservation concern. Dragonfly richness and abundance was shown to be negatively influenced by higher degrees of eutrophication. A high diversity of dragonflies occurred in the fishponds with CHL concentrations below 127 µg/l, water transparency above 67 cm, TN concentrations below 2.30 mg/l, and a fish harvest smaller than 253 kg/ha. A minimum of 5% of aquatic plant cover and the presence of a minimum 9 aquatic plant species seem to promote the richness and abundance of dragonflies. According to tipping points, 19 dragonfly species could be determined as indicator species for water quality in fishponds.