High levels of arbuscular mycorrhizal fungus colonization on Medicago truncatula reduces plant suitability as a host for pea aphids (Acyrthosiphon pisum)

This study sheds light on a poorly understood area in insect-plant-microbe interactions,focusing on aphid probing and feeding behavior on plants with varying levels of arbuscular mycorrhizal(AM)fungus root colonization.It investigates a commonly occurring interaction of three species:pea aphid Acyrthosiphon pisum,barrel medic Medicago truncatula,and the AM fungus Rhizophagus irregularis,examining whether aphid-feeding behavior changes when insects feed on plants at different levels of AM fungus colonization(42% and 84% root length colonized).Aphid probing and feeding behavior was monitored throughout 8 h of recording using the electrical penetration graph(EPG)technique,also,foliar nutrient content and plant growth were measured.Summarizing,aphids took longer to reach their 1st sustained phloem ingestion on the 84% AM plants than on the 42% AM plants or on controls.Less aphids showed phloem ingestion on the 84% AM plants relative to the 42% AM plants.Shoots of the 84% AM plants had higher percent carbon(43.7%)relative to controls(40.5%),and the 84% AM plants had reduced percent nitrogen(5.3%)relative to the 42% AM plants(6%).In conclusion,EPG and foliar nutrient data support the hypothesis that modifications in plant anatomy(e.g.,thicker leaves),and poor food quality(reduced nitrogen)in the 84% AM plants contribute to reduced aphid success in locating phloem and ultimately to differences in phloem sap ingestion.This work suggests that M.truncatula plants benefit from AM symbiosis not only because of increased nutrient uptake but also because of reduced susceptibility to aphids.     

The strength of SMAD1/5 activity determines the mode of stem cell division in the developing spinal cord

The different modes of stem cell division are tightly regulated to balance growth and differentiation during organ development and homeostasis. However, the mechanisms controlling such events are not fully understood. We have developed markers that provide the single cell resolution necessary to identify the three modes of division occurring in a developing nervous system: self-expanding, self-renewing, and self-consuming. Characterizing these three modes of division during interneuron generation in the developing chick spinal cord, we demonstrated that they correlate to different levels of activity of the canonical bone morphogenetic protein effectors SMAD1/5. Functional in vivo experiments showed that the premature neuronal differentiation and changes in cell cycle parameters caused by SMAD1/5 inhibition were preceded by a reduction of self-expanding divisions in favor of self-consuming divisions. Conversely, SMAD1/5 gain of function promoted self-expanding divisions. Together, these results lead us to propose that the strength of SMAD1/5 activity dictates the mode of stem cell division during spinal interneuron generation.     

Phylobetadiversity among Forest Types in the Brazilian Atlantic Forest Complex

Phylobetadiversity is defined as the phylogenetic resemblance between communities or biomes. Analyzing phylobetadiversity patterns among different vegetation physiognomies within a single biome is crucial to understand the historical affinities between them. Based on the widely accepted idea that different forest physiognomies within the Southern Brazilian Atlantic Forest constitute different facies of a single biome, we hypothesize that more recent phylogenetic nodes should drive phylobetadiversity gradients between the different forest types within the Atlantic Forest, as the phylogenetic divergence among those forest types is biogeographically recent. We compiled information from 206 checklists describing the occurrence of shrub/tree species across three different forest physiognomies within the Southern Brazilian Atlantic Forest (Dense, Mixed and Seasonal forests). We analyzed intra-site phylogenetic structure (phylogenetic diversity, net relatedness index and nearest taxon index) and phylobetadiversity between plots located at different forest types, using five different methods differing in sensitivity to either basal or terminal nodes (phylogenetic fuzzy weighting, COMDIST, COMDISTNT, UniFrac and Rao’s H). Mixed forests showed higher phylogenetic diversity and overdispersion than the other forest types. Furthermore, all forest types differed from each other in relation phylobetadiversity patterns, particularly when phylobetadiversity methods more sensitive to terminal nodes were employed. Mixed forests tended to show higher phylogenetic differentiation to Dense and Seasonal forests than these latter from each other. The higher phylogenetic diversity and phylobetadiversity levels found in Mixed forests when compared to the others likely result from the biogeographical origin of several taxa occurring in these forests. On one hand, Mixed forests shelter several temperate taxa, like the conifers Araucaria and Podocarpus. On the other hand, tropical groups, like Myrtaceae, are also very representative of this forest type. We point out to the need of more attention to Mixed forests as a conservation target within the Brazilian Atlantic Forest given their high phylogenetic uniqueness.     

Contamination level and human health hazard assessment of heavy metals and polycyclic aromatic hydrocarbons (PAHs) in street dust deposited in Mahshahr,southwest of Iran

Mahshahr has a strategic position and is considered as industrial hub of Iran. Selected heavy metals and polycyclic aromatic hydrocarbons (PAHs) contamination and health risk, and the mineralogical composition of street dust from Mahshahr were investigated. Results indicated that geology is the main determinant of the dust mineralogical composition. Calculated enrichment factor (EF) and principal component analysis (PCA) showed that Pb, Hg, Zn, and Cu accumulations are greatly influenced by anthropogenic sources including traffic and industry. High heavy metals content poses great ecological risk in the study area and exposure doses revealed that ingestion is the main exposure route to street dust, especially for children in residential/commercial areas. It was found that the total amount of PAHs (∑PAHs) varies from 161 to 1996 µg/kg, dominated by four-ring PAHs. Diagnostic ratios and PCA showed that both petrogenic and pyrogenic sources of PAHs in Mahshahr street dust and traffic play important roles in this respect. Furthermore, toxic equivalents and incremental lifetime cancer risk of PAHs in street dust indicated a high potential carcinogenic risk for inhabitants mainly via dermal contact and ingestion pathways particularly for outdoor workers in industrial use scenario. Finally, distribution maps of total hazard index of heavy metals and cancer risk of PAHs indicated the most impacted zones for different groups and use scenarios.     

A Pilot Study Identifying a Set of microRNAs As Precise Diagnostic Biomarkers of Acute Kidney Injury

In the last decade, Acute Kidney Injury (AKI) diagnosis and therapy have not notably improved probably due to delay in the diagnosis, among other issues. Precocity and accuracy should be critical parameters in novel AKI biomarker discovery. microRNAs are key regulators of cell responses to many stimuli and they can be secreted to the extracellular environment. Therefore, they can be detected in body fluids and are emerging as novel disease biomarkers. We aimed to identify and validate serum miRNAs useful for AKI diagnosis and management. Using qRT-PCR arrays in serum samples, we determined miRNAs differentially expressed between AKI patients and healthy controls. Statistical and target prediction analysis allowed us to identify a panel of 10 serum miRNAs. This set was further validated, by qRT-PCR, in two independent cohorts of patients with relevant morbi-mortality related to AKI: Intensive Care Units (ICU) and Cardiac Surgery (CS). Statistical correlations with patient clinical parameter were performed. Our results demonstrated that the 10 selected miRNAs (miR-101-3p, miR-127-3p, miR-210-3p, miR-126-3p, miR-26b-5p, miR-29a-3p, miR-146a-5p, miR-27a-3p, miR-93-3p and miR-10a-5p) were diagnostic biomarkers of AKI in ICU patients, exhibiting areas under the curve close to 1 in ROC analysis. Outstandingly, serum miRNAs estimated before CS predicted AKI development later on, thus becoming biomarkers to predict AKI predisposition. Moreover, after surgery, the expression of the miRNAs was modulated days before serum creatinine increased, demonstrating early diagnostic value. In summary, we have identified a set of serum miRNAs as AKI biomarkers useful in clinical practice, since they demonstrate early detection and high diagnostic value and they recognize patients at risk.     

Purification and characterization of the enantioselective esterase from Kluyveromyces marxianus CBS 1553

An intracellular esterase from the yeast Kluyveromyces marxianus CBS 1553 with interesting enantioselective hydrolytic activity towards racemic esters of 1,2-O-isopropylidene glycerol (IPG) was purified and characterized. Optimal culture conditions for the obtainment of the enantioselective esterase on a 5 l-fermentation scale were investigated. Two esterase activities (EST1 and EST2) in the crude cell extract were identified by native PAGE with specific activity staining and separated from each other by anion-exchange chromatography. EST1 showed higher activity and enantioselectivity than EST2 in the resolution of racemic IPG acetate and was further purified by hydrophobic interaction chromatography and preparative electrophoresis (final specific activity approximately = 300 U mg(-1), showing a main protein band with a molecular mass of 29 kDa. EST1 showed optimal activity between pH 8.0 and 10.0 and was stable in the pH range 7.0-10.0. Moreover, it was rather thermostable and active up to 80 degrees C, and retained most of its activity in the presence of 15% (v/v) of various organic solvents. The enzyme showed similar Vmax in the hydrolysis of the acetate esters of IPG, whereas the Km value towards (S)-IPG acetate was significantly lower than the one towards the (R)-enantiomer (5.3 and 70 microM, respectively). Finally, comparison of EST1 activity in the presence of different glycerol esters and synthetic substrates with different chain lengths showed a strong preference of this biocatalyst for short-chain substrates.     

Ubiquitin over-expression promotes E6AP autodegradation and reactivation of the p53/MDM2 pathway in HeLa cells

It has been established that intracellular ubiquitin pools are subject to regulatory constrains. Less certain is the mechanism by which the pool of conjugated ubiquitin shift in parallel with total ubiquitin, and how this type of regulation affects the flux of substrates through the pathway. In this study we demonstrate that ubiquitin over-expression promotes the destabilization of the ubiquitin protein ligase E6AP, by a mechanism involving self-ubiquitination, and the stabilization of p53. These results represent the very first evidence that the levels of a ubiquitin ligase can be regulated in vivo by ubiquitin abundance, supporting the idea that a strict interrelationship between pathway component activities and ubiquitin pool size exists. Interestingly, ubiquitin-induced p53 accumulation did not induce cell-cycle arrest, suggesting that although fluctuations of the intracellular ubiquitin content may actively modulate the level of regulatory proteins, this event is not per se sufficient to elicit a cellular response in terms of proliferation.     

MAPK p38 and JNK have opposing activities on TRAIL-induced apoptosis activation in NSCLC H460 cells that involves RIP1 and caspase-8 and is mediated by Mcl-1

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce both caspase-dependent apoptosis and kinase activation in tumor cells. Here, we examined the consequences and mechanisms of TRAIL-induced MAPKs p38 and JNK in non-small cell lung cancer (NSCLC) cells. In apoptosis sensitive H460 cells, these kinases were phosphorylated, but not in resistant A549 cells. Time course experiments in H460 cells showed that induction of p38 phosphorylation preceded that of JNK. To explore the function of these kinases in apoptosis activation by TRAIL, chemical inhibitors or siRNAs were employed to impair JNK or p38 functioning. JNK activation counteracted TRAIL-induced apoptosis whereas activation of p38 stimulated apoptosis. Notably, the serine/threonine kinase RIP1 was cleaved following TRAIL treatment, concomitant with detectable JNK phosphorylation. Further examination of the role of RIP1 by short hairpin (sh)RNA-dependent knockdown or inhibition by necrostatin-1 showed that p38 can be phosphorylated in both RIP1-dependent and -independent manner, whereas JNK phosphorylation occurred independent of RIP1. On the other hand JNK appeared to suppress RIP1 cleavage via an unknown mechanism. In addition, only the activation of JNK by TRAIL was caspase-8-dependent. Finally, we identified Mcl-1, a known substrate for p38 and JNK, as a downstream modulator of JNK or p38 activity. Collectively, our data suggest in a subset of NSCLC cells a model in which TRAIL-induced activation of p38 and JNK have counteracting effects on Mcl-1 expression leading to pro- or anti-apoptotic effects, respectively. Strategies aiming to stimulate p38 and inhibit JNK may have benefit for TRAIL-based therapies in NSCLC.     

Biodistribution of sodium borocaptate (BSH) for boron neutron capture therapy (BNCT) in an oral cancer model

Boron neutron capture therapy (BNCT) is based on selective accumulation of ¹⁰B carriers in tumor followed by neutron irradiation. We previously proved the therapeutic success of BNCT mediated by the boron compounds boronophenylalanine and sodium decahydrodecaborate (GB-10) in the hamster cheek pouch oral cancer model. Based on the clinical relevance of the boron carrier sodium borocaptate (BSH) and the knowledge that the most effective way to optimize BNCT is to improve tumor boron targeting, the specific aim of this study was to perform biodistribution studies of BSH in the hamster cheek pouch oral cancer model and evaluate the feasibility of BNCT mediated by BSH at nuclear reactor RA-3. The general aim of these studies is to contribute to the knowledge of BNCT radiobiology and optimize BNCT for head and neck cancer. Sodium borocaptate (50 mg ¹⁰B/kg) was administered to tumor-bearing hamsters. Groups of 3–5 animals were killed humanely at nine time-points, 3–12 h post-administration. Samples of blood, tumor, precancerous pouch tissue, normal pouch tissue and other clinically relevant normal tissues were processed for boron measurement by optic emission spectroscopy. Tumor boron concentration peaked to therapeutically useful boron concentration values of 24–35 ppm. The boron concentration ratio tumor/normal pouch tissue ranged from 1.1 to 1.8. Pharmacokinetic curves showed that the optimum interval between BSH administration and neutron irradiation was 7–11 h. It is concluded that BNCT mediated by BSH at nuclear reactor RA-3 would be feasible.     

Mammals have two twinfilin isoforms whose subcellular localizations and tissue distributions are differentially regulated

Twinfilin is a highly conserved actin monomer-binding protein that regulates cytoskeletal dynamics in organisms from yeast to mammals. In addition to the previously characterized mammalian twinfilin-1, a second protein with approximately 65% sequence identity to twinfilin-1 exists in mouse and humans. However, previous studies failed to identify any actin binding activity in this protein (Rohwer, A., Kittstein, W., Marks, F., and Gschwendt, M. (1999) Eur. J. Biochem. 263, 518-525). Here we show that this protein, which we named twinfilin-2, is indeed an actin monomer-binding protein. Similar to twinfilin-1, mouse twinfilin-2 binds ADP-G-actin with a higher affinity (KD = 0.12 microM) than ATP-G-actin (KD = 1.96 microM) and efficiently inhibits actin filament assembly in vitro. Both mouse twinfilins inhibit the nucleotide exchange on actin monomers and directly interact with capping protein. Furthermore, the actin interactions of mouse twinfilin-1 and twinfilin-2 are inhibited by phosphatidylinositol (4,5)-bisphosphate. Although biochemically very similar, our Northern blots and in situ hybridizations show that these two proteins display distinct expression patterns. Twinfilin-1 is the major isoform in embryos and in most adult mouse non-muscle cell-types, whereas twinfilin-2 is the predominant isoform of adult heart and skeletal muscles. Studies with isoform-specific antibodies demonstrated that although the two proteins show similar localizations in unstimulated cells, they are regulated by different mechanisms. The small GTPases Rac1 and Cdc42 induce the redistribution of twinfilin-1 to membrane ruffles and cell-cell contacts, respectively, but do not affect the localization of twinfilin-2. Taken together, these data show that mammals have two twinfilin isoforms, which are differentially expressed and regulated through distinct cellular signaling pathways.