Structural and functional insights into the modulation of the activity of a flax cytokinin oxidase by flax rust effector AvrL567-A

During infection, plant pathogens secrete effector proteins to facilitate colonization. In comparison with our knowledge of bacterial effectors, the current understanding of how fungal effectors function is limited. In this study, we show that the effector AvrL567-A from the flax rust fungus Melampsora lini interacts with a flax cytosolic cytokinin oxidase, LuCKX1.1, using both yeast two-hybrid and in planta bimolecular fluorescence assays. Purified LuCKX1.1 protein shows catalytic activity against both N6-(Δ2-isopentenyl)-adenine (2iP) and trans-zeatin (tZ) substrates. Incubation of LuCKX1.1 with AvrL567-A results in increased catalytic activity against both substrates. The crystal structure of LuCKX1.1 and docking studies with AvrL567-A indicate that the AvrL567 binding site involves a flexible surface-exposed region that surrounds the cytokinin substrate access site, which may explain its effect in modulating LuCKX1.1 activity. Expression of AvrL567-A in transgenic flax plants gave rise to an epinastic leaf phenotype consistent with hormonal effects, although no difference in overall cytokinin levels was observed. We propose that, during infection, plant pathogens may differentially modify the levels of extracellular and intracellular cytokinins.     

Limited influence of local and landscape factors on finescale gene flow in two pond‐breeding amphibians

Dispersal and gene flow within animal populations are influenced by the composition and configuration of the landscape. In this study, we evaluated hypotheses about the impact of natural and anthropogenic factors on genetic differentiation in two amphibian species, the spotted salamander (Ambystoma maculatum) and the wood frog (Lithobates sylvaticus) in a commercial forest in central Maine. We conducted this analysis at two scales: a local level, focused on factors measured at each breeding pond, and a landscape level, focused on factors measured between ponds. We investigated the effects of a number of environmental factors in six categories including Productivity, Physical, Land Composition, Land Configuration, Isolation and Location. Embryos were sampled from 56 spotted salamander breeding ponds and 39 wood frog breeding ponds. We used a hierarchical Bayesian approach in the program GESTE at each breeding pond and a random forest algorithm in conjunction with a network analysis between the ponds. We found overall high genetic connectivity across distances up to 17 km for both species and a limited effect of natural and anthropogenic factors on gene flow. We found the null models best explained patterns of genetic differentiation at a local level and found several factors at the landscape level that weakly influenced gene flow. This research indicates multiscale investigations that incorporate local and landscape factors are valuable for understanding patterns of gene flow. Our findings suggest that dispersal rates in this system are high enough to minimize genetic structuring and that current forestry practices do not significantly impede dispersal.     

Zebrafish Caudal Fin Angiogenesis Assay—Advanced Quantitative Assessment Including 3-Way Correlative Microscopy

Background

Researchers evaluating angiomodulating compounds as a part of scientific projects or pre-clinical studies are often confronted with limitations of applied animal models. The rough and insufficient early-stage compound assessment without reliable quantification of the vascular response counts, at least partially, to the low transition rate to clinics.

Objective

To establish an advanced, rapid and cost-effective angiogenesis assay for the precise and sensitive assessment of angiomodulating compounds using zebrafish caudal fin regeneration. It should provide information regarding the angiogenic mechanisms involved and should include qualitative and quantitative data of drug effects in a non-biased and time-efficient way.

Approach & Results

Basic vascular parameters (total regenerated area, vascular projection area, contour length, vessel area density) were extracted from in vivo fluorescence microscopy images using a stereological approach. Skeletonization of the vasculature by our custom-made software Skelios provided additional parameters including “graph energy” and “distance to farthest node”. The latter gave important insights into the complexity, connectivity and maturation status of the regenerating vascular network. The employment of a reference point (vascular parameters prior amputation) is unique for the model and crucial for a proper assessment. Additionally, the assay provides exceptional possibilities for correlative microscopy by combining in vivo-imaging and morphological investigation of the area of interest. The 3-way correlative microscopy links the dynamic changes in vivo with their structural substrate at the subcellular level.

Conclusions

The improved zebrafish fin regeneration model with advanced quantitative analysis and optional 3-way correlative morphology is a promising in vivo angiogenesis assay, well-suitable for basic research and preclinical investigations.     

Chitosan induces different L-arginine metabolic pathways in resting and inflammatory macrophages

Chitosan is a linear polymer of N-acetyl-D-glucosamine and deacetylated glucosamine widely used as a wound-healing accelerator in clinical and veterinary medicine. Chitosan enhances the functions of inflammatory cells such as macrophages (Mphi), inducing the production of cytokines as well as the expression of activation markers, Fc receptors and mannose receptor. In this work we studied the effects of chitosan on the arginine metabolic pathways of both resident and inflammatory (proteose-peptone elicited) rat Mphi. Our results show that low molecular weight (LMW) chitosan activated moderately both the inducible nitric oxide synthase (iNOS) and arginase pathways in resident Mphi. In inflammatory Mphi treated with chitosan instead, the arginase activity was strongly enhanced. Supernatants of chitosan-stimulated Mphi enhanced the proliferation of the rat cell line C6. These findings suggest that the healing activity of chitosan could rely on the enhanced arginase activity observed in a wound-associated inflammatory milieu.     

Unequal meiotic crossover: a frequent cause of NF1 microdeletions

Neurofibromatosis type 1 is a common autosomal dominant disorder caused by mutations of the NF1 gene on chromosome 17. In only 5%-10% of cases, a microdeletion including the NF1 gene is found. We analyzed a set of polymorphic dinucleotide-repeat markers flanking the microdeletion on chromosome 17 in a group of seven unrelated families with a de novo NF1 microdeletion. Six of seven microdeletions were of maternal origin. The breakpoints of the microdeletions of maternal origin were localized in flanking paralogous sequences, called "NF1-REPs." The single deletion of paternal origin was shorter, and no crossover occurred on the paternal chromosome 17 during transmission. Five of the six cases of maternal origin were informative, and all five showed a crossover, between the flanking markers, after maternal transmission. The observed crossovers flanking the NF1 region suggest that these NF1 microdeletions result from an unequal crossover in maternal meiosis I, mediated by a misalignment of the flanking NF1-REPs.     

High initiation and long duration of breastfeeding despite absence of early skin-to-skin contact in Karen refugees on the Thai-Myanmar border: a mixed methods study

Background

Early skin-to-skin contact (SSC) after birth is recommended as part of the United Nations Children’s Fund (UNICEF) baby friendly health initiative to promote optimum breastfeeding. This paper reports rates of breastfeeding initiation and duration in a low resource environment, where early SSC is not practised, and explores views of pregnant women and midwives surrounding breastfeeding and swaddling.

Methods

Data from records from a single hospital on the Thai-Myanmar border where refugee women gave birth during a one-year period (2010) were used to determine breastfeeding initiation rates and the time of the first breastfeed, and duration of breastfeeding of the previous alive child in multigravidae. Focus group discussions (FGD) were conducted to obtain information from pregnant women attending antenatal care about their intended or previous duration of breastfeeding and views on breastfeeding. Interviews with local midwives explored reasons for high rates of breastfeeding in this setting and the practice of newborn swaddling.

Results

Of 1404 live births in 2010 in Maela refugee camp there were 982 evaluable mother-newborn pairs, including 80 infants born before 37 weeks gestation. Initiation of breastfeeding within the first hour after birth and exclusive breastfeeding at discharge in term mother-newborn pairs was 91.2% (823/902) and 99.3% (896/902); and before 37 weeks gestation, 48.8% (39/80) and 98.8% (79/80). Reported duration of previous breastfeeding was 19 (range 2 to 72) months.During FGD all primigravidae (n?=?17) intended to breastfeed and all multigravidae (n?=?33) had previously breastfed; expected or previous duration of feeding was for more than one year or longer. The major theme identified during FGD was breastfeeding is “good”. Women stated their intention to breastfeed with certainty. This certainty was echoed during the interviews with midwifery staff. SSC requires a delay in early swaddling that in Karen people, with animistic beliefs, could risk loss of the spirit of the newborn or attract malevolent spirits.

Conclusions

In a population with a strong culture of breastfeeding and robust breastfeeding practices, high rates of initiation and duration of breastfeeding were found despite a lack of early skin-to-skin contact. Local preferences, traditions and practices that protect, support and maintain high rates of breastfeeding should be promoted.

     

应用气-质联用仪测定三种螺旋藻中的甾醇成分

应用GLC/MS联用仪对室内培养的钝顶螺旋藻(Spirulina platensis (Nordstedt) Geitler)、极大螺旋藻(S.maxima (Stechell & Gardiner) Geitler)和盐泽螺旋藻(S.subsalsa Oerst)的甾醇成分进行了测定。从钝顶螺旋藻和盐泽螺旋藻中共分出11个相同的甾醇组分:胆甾醇、胆甾烷醇、芸苔甾醇、麦角甾醇、海绵甾醇、菜子甾醇、豆甾醇、24-乙基-Δ~(5,7,22)-胆甾醇、β-谷甾醇、异岩藻甾醇和4α,23,24-三甲基Δ~(5,22)-胆甾醇;从极大螺旋藻中只分离出8个甾醇组分。其中胆甾醇含量最高。4α,23,24-三甲基-Δ~(5,22)-胆甾醇为蓝藻中首次报导。     

Visual-Olfactory Integration in the Human Disease Vector Mosquito Aedes aegypti

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