Temporal changes in population genetic diversity and structure in red and white clover grown in three contrasting environments in northern Europe

Backgound and Aims

Extending the cultivation of forage legume species into regions where they are close to the margin of their natural distribution requires knowledge of population responses to environmental stresses. This study was conducted at three north European sites (Iceland, Sweden and the UK) using AFLP markers to analyse changes in genetic structure over time in two population types of red and white clover (Trifolium pratense and T. repens, respectively): (1) standard commercial varieties; (2) wide genetic base (WGB) composite populations constructed from many commercial varieties plus unselected material obtained from germplasm collections.

Methods

At each site populations were grown in field plots, then randomly sampled after 3–5 years to obtain survivor populations. AFLP markers were used to calculate genetic differentiation within and between original and survivor populations.

Key Results

No consistent changes in average genetic diversity were observed between original and survivor populations. In both species the original varieties were always genetically distinct from each other. Significant genetic shift was observed in the white clover ‘Ramona’ grown in Sweden. The WGB original populations were more genetically similar. However, genetic differentiation occurred between original and survivor WGB germplasm in both species, particularly in Sweden. Regression of climatic data with genetic differentiation showed that low autumn temperature was the best predictor. Within the set of cold sites the highest level of genetic shift in populations was observed in Sweden.

Conclusions

The results suggest that changes in population structure can occur within a short time span in forage legumes, resulting in the rapid formation of distinct survivor populations in environmentally challenging sites.     

Inhibition of EIF-5A prevents apoptosis in human cardiomyocytes after malaria infection

In this study, a determination of Troponin I and creatine kinase activity in whole-blood samples in a cohort of 100 small infants in the age of 2–5 years from Uganda with complicated Plasmodium falciparum malaria suggests the prevalence of cardiac symptoms in comparison to non-infected, healthy patients. Troponin I and creatine kinase activity increased during infection. Different reports showed that complicated malaria coincides with hypoxia in children. The obtained clinical data prompted us to further elucidate the underlying regulatory mechanisms of cardiac involvement in human cardiac ventricular myocytes. Complicated malaria is the most common clinical presentation and might induce cardiac impairment by hypoxia. Eukaryotic initiation factor 5A (eIF-5A) is involved in hypoxia induced factor (HIF-1α) expression. EIF-5A is a protein posttranslationally modified by hypusination involving catalysis of the two enzymes deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase. Treatment of human cardiomyocytes with GC7, an inhibitor of DHS, catalyzing the first step in hypusine biosynthesis led to a decrease in proinflammatory and proapoptotic myocardial caspase-1 activity in comparison to untreated cardiomyocytes. This effect was even more pronounced after co-administration of GC7 and GPI from P. falciparum simulating the pathology of severe malaria. Moreover, in comparison to untreated and GC7-treated cardiomyocytes, co-administration of GC7 and GPI significantly decreased the release of cytochrome C and lactate from damaged mitochondria. In sum, coadministration of GC7 prevented cardiac damage driven by hypoxia in vitro. Our approach demonstrates the potential of the pharmacological inhibitor GC7 to ameliorate apoptosis in cardiomyocytes in an in vitro model simulating severe malaria. This regulatory mechanism is based on blocking EIF-5A hypusination.     

Male and female recombination landscapes of diploid Arabidopsis arenosa

The number and placement of meiotic crossover events during meiosis have important implications for the fidelity of chromosome segregation as well as patterns of inheritance. Despite the functional importance of recombination, recombination landscapes vary widely among and within species, and this can have a strong impact on evolutionary processes. A good knowledge of recombination landscapes is important for model systems in evolutionary and ecological genetics, since it can improve interpretation of genomic patterns of differentiation and genome evolution, and provides an important starting point for understanding the causes and consequences of recombination rate variation. Arabidopsis arenosa is a powerful evolutionary genetic model for studying the molecular basis of adaptation and recombination rate evolution. Here, we generate genetic maps for 2 diploid A. arenosa individuals from distinct genetic lineages where we have prior knowledge that meiotic genes show evidence of selection. We complement the genetic maps with cytological approaches to map and quantify recombination rates, and test the idea that these populations might have distinct patterns of recombination. We explore how recombination differs at the level of populations, individuals, sexes and genomic regions. We show that the positioning of crossovers along a chromosome correlates with their number, presumably a consequence of crossover interference, and discuss how this effect can cause differences in recombination landscape among sexes or species. We identify several instances of female segregation distortion. We found that averaged genome-wide recombination rate is lower and sex differences subtler in A. arenosa than in Arabidopsis thaliana.     

Physiological shifts in the microbial community drive changes in enzyme activity in a perennial agroecosystem

Perennial agroecosystems have the potential to promote plant–microbial linkages by increasing the quantity of root carbon entering the soil. However, an understanding of how perennial cropping systems affect microbial communities remains incomplete. The objective of this study was to determine the potential for a fertilized perennial bioenergy cropping system to impact microbial growth and enzyme activity. Three times throughout the growing season we examined the activity of four enzymes involved in decomposition (ß-glucosidase, ß-xylosidase, cellobiohydrolase, and N-acetyl glucosaminidase) in replicated plots of an annual (corn) and perennial-based (switchgrass) cropping system. We also took simultaneous measurements of microbial biomass and potential rates of microbial respiration and net N mineralization. Microbial biomass was unaffected by cropping system. Mid-summer, however, we observed increases in enzyme activity and potential microbial respiration in the perennial system that were independent of microbial biomass, likely in response to labile carbon inputs. Further, we observed lower net N mineralization, higher microbial biomass nitrogen and higher activity of nitrogen liberating enzymes, which are indicative of a community with high nitrogen demands. Overall, our research demonstrates that perennial agroecosystems can affect the physiological capacity of the microbial community, yielding communities with greater nitrogen retention and greater rates of decomposition as a result of allocation of resources towards enzyme production and nitrogen mining. These results can inform biogeochemical models with respect to the importance of temporally dynamic changes in carbon and nitrogen availability and microbial carbon use efficiency as drivers of enzyme production.     

Double-blind, placebo-controlled randomized trial with adalimumab for treatment of juvenile onset ankylosing spondylitis (JoAS): significant short term improvement

Introduction

While adalimumab is licensed for ankylosing spondylitis (AS), open uncontrolled studies suggest therapeutic efficacy of TNF-inhibitors in juvenile onset AS (JoAS).

Methods

A total of 32 patients aged 12 to 17 years with severe, active and refractory JoAS were enrolled in a multicenter, randomized, double-blind, placebo-controlled parallel study of 12 weeks, followed by open-label adalimumab until week 24 for all patients. ASAS40 was used as the primary, and ASAS20, PedACR and single items were used as the secondary outcome measures for the intention to treat population.

Results

A total of 17 patients were randomized to receive adalimumab 40 mg/2 weeks and 15 patients received placebo. Two patients (one of each group) discontinued prematurely due to insufficient efficacy and were labeled as non-responders. In the double-blind part, more patients on adalimumab achieved an ASAS40 at week 4 (41%), week 8 (53%) and week 12 (53%) than on placebo (20%, 33%, 33%), while differences at week 8 only reached borderline significance (P = 0.05). Also, at 4, 8 and 12 weeks ASAS20/PedACR30/70 response rates were higher in the adalimumab group (53%/53%/29%; 59%/76%/41%; 53%/65%/53%) compared to placebo (27%/27%/7%; 27%/33%/13%; 33%/40%/27%). In the adalimumab group a significant decrease of all disease activity parameters was noted at week 12 and was even more pronounced at week 24. At week 12 the Bath Ankylosing Spondylitis Disease activity spinal inflammation score decreased by 65% (P <0.001), the back pain score decreased by 50% (P <0.005), the Bath AS Functional Index (BASFI) score decreased by 47% (P <0.02), while the Childhood Health Assessment Questionnaire-Disability Index (CHAQ-DI) score improved by 65% (P <0.005). ANCOVA analysis demonstrated superiority of adalimumab over placebo for the physician global assessment of disease activity, parents' global assessment of subject's overall well-being, active joint count (all P <0.05) and erythrocyte sedimentation rate (ESR) (P <0.01). During the 12-week controlled phase, 29 AEs occurred in 10 patients on placebo compared to 27 AEs in 11 patients on adalimumab. Injection site reactions were the most common adverse events. There were 17 various infections occurring in the double-blind phase, 8 on placebo, 9 on adalimumab and a further 19 in the open label period.

Conclusions

Adalimumab was well tolerated and highly effective in a double-blind randomized trial in patients with JoAS. Treatment effects rapidly occurred and persisted for at least 24 weeks of treatment.

Trial registration

EudraCT 2007-003358-27.     

N:P Ratio and the Nature of Nutrient Limitation in Calluna-Dominated Heathlands

There is growing evidence from different sources that prolonged high N deposition causes a shift from nitrogen (N) limitation to nitrogen and phosphorus (P) co-limitation or even P limitation in many terrestrial ecosystems. However, the number of ecosystems where the type of limitation has been directly tested by longer-term full-factorial field experiments is very limited. We conducted a 5-year fertilization experiment with N and P in the Lüneburger Heide (NW Germany) to test the hypothesis that, following decades of elevated atmospheric N inputs, plant growth in dry lowland heaths may have shifted from N to N–P co-limitation or P limitation. We also tested whether the plant tissue N:P ratio reflects the type of nutrient limitation in a continental lowland heathland. Experimental plots dominated by Calluna vulgaris received regular additions of N (50 kg N ha⁻¹ y⁻¹), P (20 kg P ha⁻¹ y⁻¹), a combination of both, or water only (control) from 2004 to 2008. Over the whole study period, a highly significant positive N effect on shoot length was found, thus indicating N limitation. We conclude that a clear shift from N limitation to N–P co-limitation or P limitation has not yet occurred. Tissue N:P ratios showed a high temporal variability and no relationship between tissue N:P ratio and the shoot length response of Calluna to nutrient addition was found. The N:P tool is thus of limited use at the local scale and within the range of N:P ratio observed in this study, and should only be used as a rough indicator for the prediction of the type of nutrient limitation in lowland heathland on a larger geographical scale with a broader interval of N:P ratio.     

Functional surfaces of the human immunodeficiency virus type 1 capsid protein

The human immunodeficiency virus type 1 initially assembles and buds as an immature particle that is organized by the viral Gag polyprotein. Gag is then proteolyzed to produce the smaller capsid protein CA, which forms the central conical capsid that surrounds the RNA genome in the mature, infectious virus. To define CA surfaces that function at different stages of the viral life cycle, a total of 48 different alanine-scanning surface mutations in CA were tested for their effects on Gag protein expression, processing, particle production and morphology, capsid assembly, and infectivity. The 27 detrimental mutations fall into three classes: 13 mutations significantly diminished or altered particle production, 9 mutations failed to assemble normal capsids, and 5 mutations supported normal viral assembly but were nevertheless reduced more than 20-fold in infectivity. The locations of the assembly-defective mutations implicate three different CA surfaces in immature particle assembly: one surface encompasses helices 4 to 6 in the CA N-terminal domain (NTD), a second surrounds the crystallographically defined CA dimer interface in the C-terminal domain (CTD), and a third surrounds the loop preceding helix 8 at the base of the CTD. Mature capsid formation required a distinct surface encompassing helices 1 to 3 in the NTD, in good agreement with a recent structural model for the viral capsid. Finally, the identification of replication-defective mutants with normal viral assembly phenotypes indicates that CA also performs important nonstructural functions at early stages of the viral life cycle.     

Protection of fruit against infestation by apple maggot and blueberry maggot (Diptera: Tephritidae) using compounds containing spinosad

Two insecticide formulations containing the naturalyte insecticide spinosad, GF-120 Fruit Fly Bait and SpinTor 2 SC, were compared for control of apple maggot, Rhagoletis pomonella (Walsh), and blueberry maggot, Rhagoletis mendax Curran. In 2002 and 2003, larval infestation in blueberries and apples was significantly lower in plots treated with GF-120 (spinosad bait) or SpinTor than in untreated control plots. Fruit fly infestation in apples was reduced by 67% in 2002 after weekly application of GF-120 for 6 wk. Six weeks of GF-120 treatment reduced infestation in blueberries by 85% in 2002 and 98% in 2003. Plots treated weekly with the bait component of GF-120 for 6 wk had significantly higher infestation of blueberry maggot larvae compared with untreated plots in 2002. Observations of wild R. mendax flies revealed that similar numbers of flies landed on blueberry foliage treated with spinosad bait, the bait component alone, or water droplets. However, flies on spinosad bait and bait treated plants spent significantly more time within 5 cm of the treatment droplets compared with control (water) droplets. Overall, the results demonstrate a high degree of efficacy of baited spinosad formulations against these key pests of temperate fruit and suggest that GF-120 is an arrestant for foraging flies.     

Randomized Trial of Piperaquine with Sulfadoxine-Pyrimethamine or Dihydroartemisinin for Malaria Intermittent Preventive Treatment in Children

Background

The long terminal half life of piperaquine makes it suitable for intermittent preventive treatment for malaria but no studies of its use for prevention have been done in Africa. We did a cluster randomized trial to determine whether piperaquine in combination with either dihydroartemisin (DHA) or sulfadoxine-pyrimethamine (SP) is as effective, and better tolerated, than SP plus amodiaquine (AQ), when used for intermittent preventive treatment in children delivered by community health workers in a rural area of Senegal.

Methods

Treatments were delivered to children 3–59 months of age in their homes once per month during the transmission season by community health workers. 33 health workers, each covering about 60 children, were randomized to deliver either SP+AQ, DHA+PQ or SP+PQ. Primary endpoints were the incidence of attacks of clinical malaria, and the incidence of adverse events.

Results

1893 children were enrolled. Coverage of monthly rounds and compliance with daily doses was similar in all groups; 90% of children received at least 2 monthly doses. Piperaquine combinations were better tolerated than SP+AQ with a significantly lower risk of common, mild adverse events. 103 episodes of clinical malaria were recorded during the course of the trial. 68 children had malaria with parasitaemia >3000/µL, 29/671 (4.3%) in the SP+AQ group, compared with 22/604 (3.6%) in the DHA+PQ group (risk difference 0.47%, 95%CI −2.3%,+3.3%), and 17/618 (2.8%) in the SP+PQ group (risk difference 1.2%, 95%CI −1.3%,+3.6%). Prevalences of parasitaemia and the proportion of children carrying Pfdhfr and Pfdhps mutations associated with resistance to SP were very low in all groups at the end of the transmission season.

Conclusions

Seasonal IPT with SP+PQ in children is highly effective and well tolerated; the combination of two long-acting drugs is likely to impede the emergence of resistant parasites.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00529620","term_id":"NCT00529620"}}NCT00529620     

Role of ATM and the Damage Response Mediator Proteins 53BP1 and MDC1 in the Maintenance of G2/M Checkpoint Arrest

ATM-dependent initiation of the radiation-induced G₂/M checkpoint arrest is well established. Recent results have shown that the majority of DNA double-strand breaks (DSBs) in G₂ phase are repaired by DNA nonhomologous end joining (NHEJ), while ∼15% of DSBs are slowly repaired by homologous recombination. Here, we evaluate how the G₂/M checkpoint is maintained in irradiated G₂ cells, in light of our current understanding of G₂ phase DSB repair. We show that ATM-dependent resection at a subset of DSBs leads to ATR-dependent Chk1 activation. ATR-Seckel syndrome cells, which fail to efficiently activate Chk1, and small interfering RNA (siRNA) Chk1-treated cells show premature mitotic entry. Thus, Chk1 significantly contributes to maintaining checkpoint arrest. Second, sustained ATM signaling to Chk2 contributes, particularly when NHEJ is impaired by XLF deficiency. We also show that cells lacking the mediator proteins 53BP1 and MDC1 initially arrest following radiation doses greater than 3 Gy but are subsequently released prematurely. Thus, 53BP1^−/− and MDC1^−/− cells manifest a checkpoint defect at high doses. This failure to maintain arrest is due to diminished Chk1 activation and a decreased ability to sustain ATM-Chk2 signaling. The combined repair and checkpoint defects conferred by 53BP1 and MDC1 deficiency act synergistically to enhance chromosome breakage.DNA double-strand breaks (DSBs) activate the DNA damage response (DDR), a coordinated process that functions to enhance survival and maintain genomic stability. The DDR includes pathways of DSB repair and a signal transduction response that activates apoptosis and cell cycle checkpoint arrest and influences DSB repair (15). DNA nonhomologous end joining (NHEJ) and homologous recombination (HR) represent the major DSB repair mechanisms, NHEJ being the major mechanism in G₀/G₁, while both processes function in G₂ (9, 32). Ataxia telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) are related phosphoinositol 3-kinase-like kinases (PIKKs) that regulate the DNA damage signaling response. ATM is activated by DSBs, while ATR is activated at single-strand (ss) regions of DNA via a process that involves ATRIP-replication protein A (RPA)-ssDNA association. Ionizing radiation (IR) induces DSBs, base damage, and ss nicks. Since neither base damage nor ss nicks activate ATR, IR-induced signaling in the G₁ and G₂ phases is predominantly ATM dependent (3, 29). In S phase, ATR can be activated by both endogenous and exogenously induced lesions following replication fork stalling/collapse (8).Recent work has shown that in G₂ phase, DSBs can undergo resection via an ATM-dependent process generating ssDNA regions that can activate ATR following RPA association (11). ATR activation at resected DSBs is coupled to loss of ATM activation (11). Although ATM and ATR share overlapping substrates, there is specificity in their signaling to the transducer kinases; ATM uniquely phosphorylates Chk2, while ATR phosphorylates Chk1. Phosphorylation of either Chk1 or Chk2 causes their activation. Critical targets of Chk1/Chk2 are the Cdc25 phosphatases, which regulate the cyclin-dependent kinases (Cdks), including Cdk1, the regulator of mitotic entry (18). Collectively, these studies suggest that two components of ATM-dependent signaling to the G₂/M checkpoint machinery can occur: ATM-Chk2 signaling at unresected DSBs and ATM-ATR-Chk1 signaling at resected DSBs.Although much is known about the mechanism leading to G₂/M checkpoint activation, few studies have addressed how arrest is maintained and how release coordinates with the status of DSB repair. We examine here the maintenance of checkpoint arrest during the immediate phase of DSB repair. We do not address the issue of checkpoint adaptation, a distinct phenomenon which occurs after prolonged checkpoint arrest (22). Further, we focus on the process maintaining arrest in irradiated G₂-phase cells and do not consider how arrest is maintained in irradiated S-phase cells that progress into G₂ phase. (Previous studies have shown that while G₂/M arrest is ATM dependent at early times post-IR, at later times it becomes ATR dependent as S-phase cells progress into G₂ phase [2, 33].) To focus on mechanisms maintaining ATM-dependent signaling in G₂-phase cells, we use aphidicolin (APH) to prevent S-phase cells from progressing into G₂ during analysis. We, thus, examine checkpoint maintenance in cells irradiated in G₂ phase and do not evaluate arrest regulated by ATR following replication fork stalling. The basis for our work stems from two recent advances. First, we evaluate the impact of ATM-mediated ATR activation in the light of recent findings that resection occurs in G₂ phase (11). Second, we consider the finding that NHEJ represents the major DSB repair mechanism in G₂ and that a 15 to 20% subset of DSBs, representing those that are rejoined with slow kinetics in an ATM-dependent manner, undergo resection and repair by HR (3, 25). Thus, contrary to the notion that HR represents the major DSB repair pathway in G₂ phase, it repairs only 15 to 20% of X- or gamma-ray-induced DSBs and represents the slow component of DSB repair in G₂ phase. Given these findings, several potential models for how checkpoint arrest is maintained in G₂ can be envisaged. A simple model is that the initial signal generated by IR is maintained for a defined time to allow for DSB repair. Such a model appears to explain the kinetics of checkpoint signaling in fission yeast after moderate IR (17). In mammalian cells, the duration of arrest depends on dose and DSB repair capacity (6). Thus, it is possible that the status of ongoing repair is communicated to the checkpoint machinery to coordinate timely release with the process of DSB repair. Here, we consider the impact of resection leading to ATM-ATR-Chk1 signaling versus ATM-Chk2 signaling from nonresected DSBs and how they interplay to maintain rather than initiate checkpoint arrest.Mediator proteins, including 53BP1 and MDC1, assemble at DSBs in an ATM-dependent manner, but their roles in the DDR are unclear. Cells lacking 53BP1 or MDC1 are proficient in checkpoint initiation after moderate IR doses, leading to the suggestion that these proteins are required for amplification of the ATM signal after exposure to low doses but are dispensable after high doses, when a robust signal is generated, even in their absence (7, 16, 28, 31). Despite their apparent subtle role in ATM signaling, cells lacking these mediator proteins display significant genomic instability (19). We thus also examine whether the mediator proteins contribute to the maintenance of checkpoint arrest.We identify two ATM-dependent processes that contribute to the maintenance of checkpoint arrest in G₂-phase cells: (i) ATR-Chk1 activation at resected DSBs and (ii) a process that involves sustained signaling from ATM to Chk2 at unrepaired DSBs. Further, we show that 53BP1 and MDC1 are required for maintaining checkpoint arrest, even following exposure to high radiation doses due to roles in ATR-Chk1 activation and sustained ATM-Chk2 signaling, and that this contributes to their elevated genomic instability.