Receptor protein tyrosine phosphatase mu expression as a marker for endothelial cell heterogeneity; analysis of RPTPmu gene expression using LacZ knock-in mice

The receptor-like protein tyrosine phosphatase mu (RPTPmu) belongs to the subfamily of meprin, A5, RPTPmu (MAM) domain-containing RPTPs, which are thought to play an important role in cell-cell adhesion mediated processes. The current study was designed to examine the expression pattern of RPTPmu in mice. We have generated RPTPmu-LacZ knock-in mice that express the beta-galactosidase (LacZ) reporter gene under the control of the RPTPmu promoter. LacZ expression patterns were analysed in embryos and adult mice by whole mount LacZ staining. Analysis of beta-galactosidase activity of heterozygous embryos and adult tissues revealed RPTPmu expression in endothelial cells of arteries and capillaries. In contrast, expression was virtually absent in endothelial cells of veins and in fenestrated endothelial cells in the adult liver and spleen. Moreover, RPTPmu expression was found in endothelial cells from the endocardium and the aorta in embryos, but not in adult mice. In addition to heterogeneous expression in endothelial cells, RPTPmu expression was found in cardiac muscle cells but not in skeletal muscle cells or smooth muscle cells. Expression was also found in Type II pneumonocytes in the lung alveoli and in Purkinje cells and other neurons in the brain. The specific expression of RPTPmu in arterial endothelial cells and in cardiac myocytes suggests that RPTPmu may play a role in the regulation of cardiovascular functions.     

Effects of carbon dioxide and nitrogen fertilization on phenolic content in Poa annua L

Different but partially overlapping hypotheses have been developed to predict the allocation of phenolics in elevated atmospheric CO(2). The carbon-nutrient balance hypothesis predicts increased allocation to phenolics due to reduced relative availability of nitrogen. The growth-differentiation balance hypothesis states that allocation will depend on source and sink strength, while the protein competition model predicts that allocation will remain unchanged. We grew Poa annua at two CO(2) concentrations in soils of three different nutrient levels. Although plant-tissue nitrogen levels were reduced in high CO(2) and photosynthetic rate increased, phenolic concentration and biomass allocation remained unchanged. We discuss these data in the context of the three models' predictions of phenolic allocation in conditions of elevated CO(2).     

Detailed imaging and genetic analysis reveal a secondary BRAFL505H resistance mutation and extensive intrapatient heterogeneity in metastatic BRAF mutant melanoma patients treated with vemurafenib

Resistance to treatment is the main problem of targeted treatment for cancer. We followed ten patients during treatment with vemurafenib, by three‐dimensional imaging. In all patients, only a subset of lesions progressed. Next‐generation DNA sequencing was performed on sequential biopsies in four patients to uncover mechanisms of resistance. In two patients, we identified mutations that explained resistance to vemurafenib; one of these patients had a secondary BRAF L505H mutation. This is the first observation of a secondary BRAF mutation in a vemurafenib‐resistant patient‐derived melanoma sample, which confirms the potential importance of the BRAF L505H mutation in the development of therapy resistance. Moreover, this study hints toward an important role for tumor heterogeneity in determining the outcome of targeted treatments.     

Indirect Short- and Long-Term Effects of Aboveground Invertebrate and Vertebrate Herbivores on Soil Microarthropod Communities

Recognition is growing that besides ungulates, small vertebrate and invertebrate herbivores are important drivers of grassland functioning. Even though soil microarthropods play key roles in several soil processes, effects of herbivores—especially those of smaller body size—on their communities are not well understood. Therefore, we progressively excluded large, medium and small vertebrate and invertebrate herbivores for three growing seasons using size-selective fences in two vegetation types in subalpine grasslands; short-grass and tall-grass vegetation generated by high and low historical levels of ungulate grazing. Herbivore exclusions generally had few effects on microarthropod communities, but exclusion of all herbivore groups resulted in decreased total springtail and Poduromorpha richness compared with exclusion of only ungulates and medium-sized mammals, regardless of vegetation type. The tall-grass vegetation had a higher total springtail richness and mesostigmatid mite abundance than the short-grass vegetation and a different oribatid mite community composition. Although several biotic and abiotic variables differed between the exclusion treatments and vegetation types, effects on soil microarthropods were best explained by differences in nutrient and fibre content of the previous year’s vegetation, a proxy for litter quality, and to a lesser extent soil temperature. After three growing seasons, smaller herbivores had a stronger impact on these functionally important soil microarthropod communities than large herbivores. Over longer time-scales, however, large grazers created two different vegetation types and thereby influenced microarthropod communities bottom-up, e.g. by altering resource quality. Hence, both short- and long-term consequences of herbivory affected the structure of the soil microarthropod community.     

Post-Prandial Protein Handling: You Are What You Just Ate

Background

Protein turnover in skeletal muscle tissue is highly responsive to nutrient intake in healthy adults.

Objective

To provide a comprehensive overview of post-prandial protein handling, ranging from dietary protein digestion and amino acid absorption, the uptake of dietary protein derived amino acids over the leg, the post-prandial stimulation of muscle protein synthesis rates, to the incorporation of dietary protein derived amino acids in de novo muscle protein.

Design

12 healthy young males ingested 20 g intrinsically [1-¹³C]-phenylalanine labeled protein. In addition, primed continuous L-[ring-²H₅]-phenylalanine, L-[ring-²H₂]-tyrosine, and L-[1-¹³C]-leucine infusions were applied, with frequent collection of arterial and venous blood samples, and muscle biopsies throughout a 5 h post-prandial period. Dietary protein digestion, amino acid absorption, splanchnic amino acid extraction, amino acid uptake over the leg, and subsequent muscle protein synthesis were measured within a single in vivo human experiment.

Results

55.3±2.7% of the protein-derived phenylalanine was released in the circulation during the 5 h post-prandial period. The post-prandial rise in plasma essential amino acid availability improved leg muscle protein balance (from -291±72 to 103±66 μM·min^-1·100 mL leg volume^-1; P<0.001). Muscle protein synthesis rates increased significantly following protein ingestion (0.029±0.002 vs 0.044±0.004%·h^-1 based upon the muscle protein bound L-[ring-²H₅]-phenylalanine enrichments (P<0.01)), with substantial incorporation of dietary protein derived L-[1-¹³C]-phenylalanine into de novo muscle protein (from 0 to 0.0201±0.0025 MPE).

Conclusion

Ingestion of a single meal-like amount of protein allows ~55% of the protein derived amino acids to become available in the circulation, thereby improving whole-body and leg protein balance. About 20% of the dietary protein derived amino acids released in the circulation are taken up in skeletal muscle tissue following protein ingestion, thereby stimulating muscle protein synthesis rates and providing precursors for de novo muscle protein synthesis.

Trial Registration

trialregister.nl 3638     

Impact of Anti-Retroviral Treatment and Cotrimoxazole Prophylaxis on Helminth Infections in HIV-Infected Patients in Lambaréné, Gabon

Background

Foci of the HIV epidemic and helminthic infections largely overlap geographically. Treatment options for helminth infections are limited, and there is a paucity of drug-development research in this area. Limited evidence suggests that antiretroviral therapy (ART) reduces prevalence of helminth infections in HIV-infected individuals. We investigated whether ART exposure and cotrimoxazole preventive therapy (CTX-P) is associated with a reduced prevalence of helminth infections.

Methodology and Principal Findings

This cross-sectional study was conducted at a primary HIV-clinic in Lambaréné, Gabon. HIV-infected adults who were ART-naïve or exposed to ART for at least 3 months submitted one blood sample and stool and urine samples on 3 consecutive days. Outcome was helminth infection with intestinal helminths, Schistosoma haematobium, Loa loa or Mansonella perstans. Multivariable logistic regression was used to assess associations between ART or CTX-P and helminth infection. In total, 408 patients were enrolled. Helminth infection was common (77/252 [30.5%]). Filarial infections were most prevalent (55/310 [17.7%]), followed by infection with intestinal helminths (35/296 [11.8%]) and S. haematobium (19/323 [5.9%]). Patients on CTX-P had a reduced risk of Loa loa microfilaremia (adjusted odds ratio (aOR) 0.47, 95% CI 0.23-0.97, P = 0.04), also in the subgroup of patients on ART (aOR 0.36, 95% CI 0.13-0.96, P = 0.04). There was no effect of ART exposure on helminth infection prevalence.

Conclusions/Significance

CTX-P use was associated with a decreased risk of Loa loa infection, suggesting an anthelminthic effect of antifolate drugs. No relation between ART use and helminth infections was established.     

Smoking in Relation to Coronary Atherosclerotic Plaque Burden,Volume and Composition on Intravascular Ultrasound

Background

This study aimed to evaluate the relationship between cigarette smoking and coronary atherosclerotic burden, volume and composition as determined in-vivo by grayscale and virtual histology (VH) intravascular ultrasound (IVUS).

Methods and Results

Between 2008 and 2011, (VH-)IVUS of a non-culprit coronary artery was performed in 581 patients undergoing coronary angiography. To account for differences in baseline characteristics, current smokers were matched to never smokers by age, gender and indication for catheterization, resulting in 280 patients available for further analysis. Coronary atherosclerotic plaque volume, burden, composition (fibrous, fibro-fatty, dense calcium and necrotic core) and high-risk lesions (VH-IVUS derived thin-cap fibroatheroma (TCFA), plaque burden ≥70%, minimal luminal area ≤4.0 mm²) were assessed. Cigarette smoking showed a tendency towards higher coronary plaque burden (mean±SD, 38.6±12.5% in current versus 36.4±11.0% in never smokers, p = 0.080; and odds ratio (OR) of current smoking for plaque burden above versus below the median 1.69 (1.04–2.75), p = 0.033). This effect was driven by an association in patients presenting with an acute coronary syndrome (ACS) (current smokers, plaque burden 38.3±12.8% versus never smokers, plaque burden 35.0±11.2%, p = 0.049; OR 1.88 (1.02–3.44), p = 0.042). Fibrous tissue tended to be lower in current smokers (mean±SD, 57.7±10.5% versus 60.4±12.6%, p = 0.050) and fibro-fatty tissue was higher in current smokers (median[IQR], 9.6[6.0–13.7]% versus 8.6[5.8–12.2]%, p = 0.039). However, differences in percentage necrotic core and dense calcium could not be demonstrated. Also, no differences were found with regard to high-risk lesions.

Conclusions

An association between smoking and degree of coronary atherosclerosis was present in patients undergoing coronary angiography who presented with ACS. Although smoking was associated with higher fibro-fatty percentage, no associations could be demonstrated with percentage necrotic core, nor with VH-IVUS derived TCFA lesions. Since the magnitude of the differences in both degree and composition of atherosclerosis was modest, clinical relevance of the findings may be questioned.     

ERECTA controls low light intensity-induced differential petiole growth independent of phytochrome B and cryptochrome 2 action in Arabidopsis thaliana

Plants can respond quickly and profoundly to changes in their environment. Several species, including Arabidopsis thaliana, are capable of differential petiole growth driven upward leaf movement (hyponastic growth) to escape from detrimental environmental conditions. Recently, we demonstrated that the leucine-rich repeat receptor-like Ser/Thr kinase gene ERECTA, explains a major effect Quantitative Trait Locus (QTL) for ethylene-induced hyponastic growth in Arabidopsis. Here, we demonstrate that ERECTA controls the hyponastic growth response to low light intensity treatment in a genetic background dependent manner. Moreover, we show that ERECTA affects low light-induced hyponastic growth independent of Phytochrome B and Cryptochrome 2 signaling, despite that these photoreceptors are positive regulators of low light-induced hyponastic growth.Key words: hyponastic growth, petiole, Arabidopsis, low light, ERECTA, differential growth, phytochrome B, cryptochrome 2Plants must adjust growth and reproduction to adverse environmental conditions. Among the strategies that plants employ to escape from unfavorable conditions is differential petiole growth-driven upward leaf movement, called hyponastic growth. Arabidopsis thaliana is able to exhibit a marked hyponastic response upon flooding, which is triggered by endogenous accumulation of the gaseous phytohormone ethylene.¹ Moreover, a similar response is triggered upon low light intensity perception and in response to supra-optimal temperatures.^2–5 By tilting the leaves to a more vertical position during submergence and shading, the plants restore contact with the atmosphere and light, respectively. The kinetics of the hyponastic growth response induced by the various stimuli is remarkably similar. This led to the hypothesis that shared functional genetic components may be employed to control hyponastic growth. Yet, at least part of the signaling cascades is parallel, as the hormonal control of the response differs between the stimuli. Low light-induced hyponastic growth for example does not require ethylene action.² Whereas the response to heat is antagonized by this hormone.⁵ The abiotic stress hormone abscisic acid (ABA) antagonizes ethylene-induced hyponastic growth and stimulates heat-induced hyponastic growth.^5,6 Moreover, ethylene-induced hyponasty does not involve auxin action⁷ whereas both heat- and low light-induced hyponasty require functional auxin signaling and transport components.^2,5In our recent paper, published in The Plant Journal,⁸ we employed Quantitative Trait Locus (QTL) analysis to identify loci involved in the control of ethylene-induced hyponastic petiole growth. By analyzing induced mutants and by complementation analysis of naturally occurring mutant accessions, we found that the leucine-rich repeat receptor-like Ser/Thr kinase gene ERECTA (ER) is a positive regulator of ethylene-induced hyponastic growth and most likely is causal to one of the identified QTLs. In addition, we demonstrated that the ER dependency is not via ER mediated control of ethylene production or sensitivity.Since low light-induced hyponasty does not require ethylene action,² ER may be part of the proposed shared signaling cascade leading to hyponastic growth where ethylene and low light signals meet. Therefore, we studied low light intensity-induced hyponasty in various erecta mutants. Moreover, natural occurring er mutant accessions complemented with a functional, Col-0 derived, ER allele were tested. The response of Lan-0 (Lan-0; with functional ER) to low light was indistinguishable from the response of Landsberg erecta (Ler) (Fig. 1A). However, complemented Ler (ER-Ler) showed an enhanced response compared to Ler (Fig. 1B). The response of mutant er105 was slightly attenuated compared to the wild type Columbia-0 (Fig. 1C). Mutant er104, however, showed an indistinguishable hyponastic growth phenotype to low light compared to the wild type Wassilewskija-2 (Ws-2) (Fig. 1D). Complementation of the natural occurring erecta mutant accession Vancouver-0 (Van-0) resulted in an enhanced hyponastic growth response to low light (Fig. 1E), whereas this was not the case for Hiroshima-1 (Hir-1) (Fig. 1F). Together, these data suggest that ER acts as positive regulator of low light-induced hyponastic growth and therefore may be part of the shared signaling cascade towards differential petiole growth. Yet, the effect is strongly dependent on the genetic background since the effects were not observed in every accession tested.Open in a separate windowFigure 1ERECTA involvement in low light-induced hyponasty. Effect of exposure to low light (spectral neutral reduction in light intensity from 200 to 20 µmol m⁻² s⁻¹) on the kinetics of hyponastic petiole growth in Arabidopsis thaliana. (A) mutant (circles) Ler and wild type (dashed line) Lan-0, (B) Ler and Ler complemented (ER-; squares) with the Col-0 ERECTA allele (ER-Ler), (C) er105 and Col-0 wild type, (D) er104 and Ws-2 wild type, (E) natural mutant Van-0 and Van-0 complemented with the Col-0 ER allele (ER-Van-0), (F) natural mutant Hir-1 and Hir-1 complemented with the Col-0 ER allele (ER-Hir-1). Petiole angles were measured using time-lapse photography and subsequent image analysis. Data is pairwise subtracted, which corrects for diurnal petiole movement in control conditions. For details on this procedure, growth conditions and materials, transformation protocol, treatments, data acquirement and all analyses see.^1,8 Error bars represent standard errors; n ≥ 12.Phytochrome B (PhyB) and Cryptochrome 2 (Cry2) photoreceptor proteins are required for a full induction of low light-induced hyponastic growth.² We transformed the phyb5 cry2 mutant9 (Ler genetic background) with Col-0 derived ER. This complementation did not restore the ability of phyb5 cry2 to induce hyponastic growth to neither ethylene (data not shown) nor low light conditions (Fig. 2A). Mutant phyb5 cry2 plants have a typical constitutive shade avoidance phenotype, reflected by severely elongated organs. This includes enhanced inflorescence and silique length and thin inflorescences (Fig. 2B-D). Complementation with ER resulted in a significant additional effect on these parameters (Fig. 2B-D). Together, this suggests that ER is not an integral part of PhyB nor Cry2 signaling with respect to (hyponastic) growth. Moreover, PhyB and Cry2 control of plant architecture does not require ER action. Rather, ER seems to mediate growth via genetic interaction with light-reliant growth mechanisms, instead of being downstream of photoreceptor action. Studies on the effects of ER on shade avoidance responses and various hormone responses, including cytokinin and auxin, led to the similar conclusion, suggesting a possible role for ER as a molecular hub coordinating light- and hormone-mediated plant growth.^10,11 One could speculate that ER fine-tunes other (than light) environmental clues with light signaling components. A comparable conclusion was drawn previously for gibberellin (GA) reliant growth mechanisms, as er enhanced the negative effect on plant size of the short internode (shi) mutation12 and er represses the positive effect of the spindly mutation in a GA independent manner.¹³Open in a separate windowFigure 2Effects of ERECTA on light signaling. (A) Effect of exposure to low light (spectral neutral reduction in light intensity from 200 to 20 µmol m⁻² s⁻¹) on the kinetics of hyponastic petiole growth of Ler (dashed lines), the photoreceptor double mutant phyb5 cry2 (circles) and this mutant complemented with the Col-0 ERECTA (ER-phyb cry2; squares). For details see legend Figure 1. (B) Plant height, (C) silique length and (D) inflorescence stem thickness of the above mentioned lines. These parameters were measured when the last flower on the plant developed a silique. Plant height was measured from root/shoot junction to inflorescence top. Stem thickness was measured ∼1 cm above the root/shoot junction with a caliper and silique lengths were measured from representative pedicels in the top ∼10 cm of the main inflorescence stem. Error bars represent standard errors; n ≥ 12. Significance levels; *p < 0.05; **p < 0.01; ***p < 0.001; ns = non significant, by Students t-test.