Putting the P in Ptilotus: a phosphorus-accumulating herb native to Australia

Background and Aims

Ptilotus polystachyus (green mulla mulla; ptilotus) is a short-lived perennial herb that occurs widely in Australia in arid and semi-arid regions with nutrient poor soils. As this species shows potential for domestication, its response to addition of phosphorus (P) and nitrogen (N) was compared to a variety of the domesticated exotic perennial pasture herb Cichorium intybus (chicory), ‘Puna’.

Methods

Pots were filled with 3 kg of an extremely nutrient-deficient sterilized field soil that contained 3 mg kg⁻¹ mineral N and 2 mg kg⁻¹ bicarbonate-extractable P. The growth and P and N accumulation of ptilotus and chicory in response to seven rates of readily available phosphorus (0–300 mg P pot⁻¹) and nitrogen (N) (0–270 mg N pot⁻¹) was examined.

Key Results

Ptilotus grew extremely well under low P conditions: shoot dry weights were 23, 6 and 1·7 times greater than for chicory at the three lowest levels of P addition, 0, 15 and 30 mg P pot⁻¹, respectively. Ptilotus could not downregulate P uptake. Concentrations of P in shoots approached 4 % of dry weight and cryo-scanning electron microscopy and X-ray microanalysis showed 35–196 mm of P in cell vacuoles in a range of tissues from young leaves. Ptilotus had a remarkable tolerance of high P concentrations in shoots. While chicory exhibited symptoms of P toxicity at the highest rate of P addition (300 mg P pot⁻¹), no symptoms were present for ptilotus. The two species responded in a similar manner to addition of N.

Conclusions

In comparison to chicory, ptilotus demonstrated an impressive ability to grow well under conditions of low and high P availability. Further study of the mechanisms of P uptake and tolerance in ptilotus is warranted.Key words: Phosphorus, nitrogen, hyperaccumulation, X-ray microanalysis, cell vacuole, Australian native plant, toxicity, domestication, phytoremediation, Ptilotus polystachyus, Cichorium intybus     

Tree litter and forest understorey vegetation: a conceptual framework to understand the effects of tree litter on a perennial geophyte, Anemone nemorosa

Background and Aims

Litter is a key factor in structuring plant populations, through positive or negative interactions. The litter layer forms a mechanical barrier that is often strongly selective against individuals lacking hypocotyle plasticity. Litter composition also interacts with plant growth by providing beneficial nutrients or, inversely, by allowing harmful allelopathic leaching. As conspicuous litter fall accumulation is often observed under deciduous forests, interactions between tree litter and understorey plant populations are worthy of study.

Methods

In a 1-year ex-situ experiment, the effects of tree litter on the growth of Anemone nemorosa, a small perennial forest geophyte, were investigated. Three ‘litter quantity’ treatments were defined, representative of forest floor litter (199, 356·5 and 514 g m⁻²), which were crossed with five ‘litter composition’ treatments (Quercus petraea, Fagus sylvatica, Carpinus betulus, Q. petraea + F. sylvatica and Q. petraea + C. betulus), plus a no-litter control. Path analysis was then used to investigate the pathways linking litter characteristics and components of adult plant growth.

Key Results

As expected, the heavier the litter, the longer the petiole; rhizome growth, however, was not depreciated by the litter-induced petiole lengthening. Both rhizome mass increment and number of initiated buds marginally increased with the amount of litter. Rhizome mass increment was in fact determined primarily by leaf area and leaf life span, neither of which was unequivocally correlated with any litter characteristics. However, the presence of litter significantly increased leafing success: following a late frost event, control rhizomes growing in the absence of litter experienced higher leaf mortality before leaf unfolding.

Conclusions

The study questions the role of litter as a physical or chemical barrier to ground vegetation; to better understand this role, there is a need for ex-situ, longer-term experiments coupled with in-situ observations in the forest.     

Linking carbon supply to root cell-wall chemistry and mechanics at high altitudes in Abies georgei

Background and Aims

The mobile carbon supply to different compartments of a tree is affected by climate, but its impact on cell-wall chemistry and mechanics remains unknown. To understand better the variability in root growth and biomechanics in mountain forests subjected to substrate mass movement, we investigated root chemical and mechanical properties of mature Abies georgei var. smithii (Smith fir) growing at different elevations on the Tibet–Qinghai Plateau.

Methods

Thin and fine roots (0·1–4·0 mm in diameter) were sampled at three different elevations (3480, 3900 and 4330 m, the last corresponding to the treeline). Tensile resistance of roots of different diameter classes was measured along with holocellulose and non-structural carbon (NSC) content.

Key Results

The mean force necessary to break roots in tension decreased significantly with increasing altitude and was attributed to a decrease in holocellulose content. Holocellulose was significantly lower in roots at the treeline (29·5 ± 1·3 %) compared with those at 3480 m (39·1 ± 1·0 %). Roots also differed significantly in NSC, with 35·6 ± 4·1 mg g⁻¹ dry mass of mean total soluble sugars in roots at 3480 m and 18·8 ± 2·1 mg g⁻¹ dry mass in roots at the treeline.

Conclusions

Root mechanical resistance, holocellulose and NSC content all decreased with increasing altitude. Holocellulose is made up principally of cellulose, the biosynthesis of which depends largely on NSC supply. Plants synthesize cellulose when conditions are optimal and NSC is not limiting. Thus, cellulose synthesis in the thin and fine roots measured in our study is probably not a priority in mature trees growing at very high altitudes, where climatic factors will be limiting for growth. Root NSC stocks at the treeline may be depleted through over-demand for carbon supply due to increased fine root production or winter root growth.     

Floral thermogenesis of three species of Hydnora (Hydnoraceae) in Africa

Background and Aims

Floral thermogenesis occurs in at least 12 families of ancient seed plants. Some species show very high rates of respiration through the alternative pathway, and some are thermoregulatory, with increasing respiration at decreasing ambient temperature. This study assesses the intensity and regulation of respiration in three species of African Hydnora that represent the Hydnoraceae, an unusual family of holoparasitic plants from arid environments.

Methods

Long-term respirometry (CO₂ production) and thermometry were carried out on intact flowers of H. africana, H. abyssinica and H. esculenta in the field, and short-term measurements were made on floral parts during the protogynous flowering sequence.

Key Results

For H. africana, there was no temperature elevation in either the osmophores or the gynoecial chamber in any phase, and mass-specific respiration rates of the flower parts were low (maximum 8·3 nmol CO₂ g⁻¹ s⁻¹ in osmophore tissue). Respiration tracked ambient and floral temperatures, eliminating the possibility of the inverse relationship expected in thermoregulatory flowers. Hydnora abyssinica flowers had higher respiration (maximum 27·5 nmol g⁻¹ s⁻¹ in the osmophores) and a slight elevation of osmophore temperature (maximum 2·8 °C) in the female stage. Respiration by gynoecial tissue was similar to that of osmophores in both species, but there was no measurable elevation of gynoecial chamber temperature. Gynoecial chamber temperature of H. esculenta could reach 3·8 °C above ambient, but there are no respiration data available. Antheral tissue respiration was maximal in the male phase (4·8 nmol g⁻¹ s⁻¹ in H. africana and 10·3 nmol g⁻¹ s⁻¹ in H. abyssinica), but it did not raise the antheral ring temperature, which showed that thermogenesis is not a by-product of pollen maturation or release.

Conclusions

The exceptionally low thermogenesis in Hydnora appears to be associated with scent production and possibly gynoecial development, but has little direct benefit to beetle pollinators.Key words: Pollination biology, Hydnora, thermogenesis, respiration rate, temperature, flowers, insects     

Predicted changes in vegetation structure affect the susceptibility to invasion of bryophyte-dominated subarctic heath

Background and Aims

A meta-analysis of global change experiments in arctic tundra sites suggests that plant productivity and the cover of shrubs, grasses and dead plant material (i.e. litter) will increase and the cover of bryophytes will decrease in response to higher air temperatures. However, little is known about which effects these changes in vegetation structure will have on seedling recruitment of species and invasibility of arctic ecosystems.

Methods

A field experiment was done in a bryophyte-dominated, species-rich subarctic heath by manipulating the cover of bryophytes and litter in a factorial design. Three phases of seedling recruitment (seedling emergence, summer seedling survival, first-year recruitment) of the grass Anthoxanthum alpinum and the shrub Betula nana were analysed after they were sown into the experimental plots.

Key Results

Bryophyte and litter removal significantly increased seedling emergence of both species but the effects of manipulations of vegetation structure varied strongly for the later phases of recruitment. Summer survival and first-year recruitment were significantly higher in Anthoxanthum. Although bryophyte removal generally increased summer survival and recruitment, seedlings of Betula showed high mortality in early August on plots where bryophytes had been removed.

Conclusions

Large species-specific variation and significant effects of experimental manipulations on seedling recruitment suggest that changes in vegetation structure as a consequence of global warming will affect the abundance of grasses and shrubs, the species composition and the susceptibility to invasion of subarctic heath vegetation.     

Protection of thylakoids against combined light and drought by a lumenal substance in the resurrection plant Haberlea rhodopensis

Background and Aims

Haberlea rhodopensis is a perennial, herbaceous, saxicolous, poikilohydric flowering plant that is able to survive desiccation to air-dried state under irradiance below 30 µmol m⁻² s⁻¹. However, desiccation at irradiance of 350 µmol m⁻² s⁻¹ induced irreversible changes in the photosynthetic apparatus, and mature leaves did not recover after rehydration. The aim here was to establish the causes and mechanisms of irreversible damage of the photosynthetic apparatus due to dehydration at high irradiance, and to elucidate the mechanisms determining recovery.

Methods

Changes in chloroplast structure, CO₂ assimilation, chlorophyll fluorescence parameters, fluorescence imaging and the polypeptide patterns during desiccation of Haberlea under medium (100 µmol m⁻² s⁻¹; ML) irradiance were compared with those under low (30 µmol m⁻² s⁻¹; LL) irradiance.

Key Results

Well-watered plants (control) at 100 µmol m⁻² s⁻¹ were not damaged. Plants desiccated at LL or ML had similar rates of water loss. Dehydration at ML decreased the quantum efficiency of photosystem II photochemistry, and particularly the CO₂ assimilation rate, more rapidly than at LL. Dehydration induced accumulation of stress proteins in leaves under both LL and ML. Photosynthetic activity and polypeptide composition were completely restored in LL plants after 1 week of rehydration, but changes persisted under ML conditions. Electron microscopy of structural changes in the chloroplast showed that the thylakoid lumen is filled with an electron-dense substance (dense luminal substance, DLS), while the thylakoid membranes are lightly stained. Upon dehydration and rehydration the DLS thinned and disappeared, the time course largely depending on the illumination: whereas DLS persisted during desiccation and started to disappear during late recovery under LL, it disappeared from the onset of dehydration and later was completely lost under ML.

Conclusions

Accumulation of DLS (possibly phenolics) in the thylakoid lumen is demonstrated and is proposed as a mechanism protecting the thylakoid membranes of H. rhodopensis during desiccation and recovery under LL. Disappearance of DLS during desiccation in ML could leave the thylakoid membranes without protection, allowing oxidative damage during dehydration and the initial rehydration, thus preventing recovery of photosynthesis.Key words: Haberlea rhodopensis, resurrection plant, electron microscopy, blue–green fluorescence, chlorophyll fluorescence     

Efficient 2,3-butanediol production from cassava powder by a crop-biomass-utilizer, Enterobacter cloacae subsp. dissolvens SDM

Background

2,3-Butanediol (BD) is considered as one of the key platform chemicals used in a variety of industrial applications. It is crucial to find an efficient sugar-utilizing strain and feasible carbon source for the economical production of BD.

Methodology/Principal Findings

Efficient BD production by a newly isolated Enterobacter cloacae subsp. dissolvens SDM was studied using crop-biomass cassava powder as substrate. The culture conditions and fermentation medium for BD production were optimized. Under the optimal conditions, 78.3 g l⁻¹ of BD was produced after 24 h in simultaneous saccharification and fermentation (SSF), with a yield of 0.42 g BD g⁻¹ cassava powder and a specific productivity of 3.3 g l⁻¹ h⁻¹. A higher BD concentration (93.9 g l⁻¹) was produced after 47 h in fed-batch SSF.

Conclusions/Significance

The results suggest that strain SDM is a good candidate for the BD production, and cassava powder could be used as an alternative substrate for the efficient production of BD.     

Protective Roles of DMP1 in High Phosphate Homeostasis

Purpose

Dmp1 (dentin matrix protein1) null mice (Dmp1^−/−) display hypophosphatemic rickets with a sharp increase in fibroblast growth factor 23 (FGF23). Disruption of Klotho (the obligatory co-receptor of FGF23) results in hyperphosphatemia with ectopic calcifications formed in blood vessels and kidneys. To determine the role of DMP1 in both a hyperphosphatemic environment and within the ectopic calcifications, we created Dmp1/Klotho compound deficient (Dmp1^−/−kl/kl) mice.

Procedures

A combination of TUNEL, immunohistochemistry, TRAP, von Kossa, micro CT, bone histomorphometry, serum biochemistry and Scanning Electron Microscopy techniques were used to analyze the changes in blood vessels, kidney and bone for wild type control, Dmp1^−/−, Klotho deficient (kl/kl) and Dmp1^−/−kl/kl animals.

Findings

Interestingly, Dmp1^−/−kl/kl mice show a dramatic improvement of rickets and an identical serum biochemical phenotype to kl/kl mice (extremely high FGF23, hyperphosphatemia and reduced parathyroid hormone (PTH) levels). Unexpectedly, Dmp1^−/−kl/kl mice presented elevated levels of apoptosis in osteocytes, endothelial and vascular smooth muscle cells in small and large blood vessels, and within the kidney as well as dramatic increase in ectopic calcification in all these tissues, as compared to kl/kl.

Conclusion

These findings suggest that DMP1 has an anti-apoptotic role in hyperphosphatemia. Discovering this novel protective role of DMP1 may have clinical relevance in protecting the cells from apoptosis in high-phosphate environments as observed in chronic kidney disease (CKD).     

MFGE8 does not influence chorio-retinal homeostasis or choroidal neovascularization in vivo

Purpose

Milk fat globule-epidermal growth factor-factor VIII (MFGE8) is necessary for diurnal outer segment phagocytosis and promotes VEGF-dependent neovascularization. The prevalence of two single nucleotide polymorphisms (SNP) in MFGE8 was studied in two exsudative or “wet” Age-related Macular Degeneration (AMD) groups and two corresponding control groups. We studied the effect of MFGE8 deficiency on retinal homeostasis with age and on choroidal neovascularization (CNV) in mice.

Methods

The distribution of the SNP (rs4945 and rs1878326) of MFGE8 was analyzed in two groups of patients with “wet” AMD and their age-matched controls from Germany and France. MFGE8-expressing cells were identified in Mfge8 ^+/− mice expressing ß-galactosidase. Aged Mfge8 ^+/− and Mfge8 ^−/− mice were studied by funduscopy, histology, electron microscopy, scanning electron microscopy of vascular corrosion casts of the choroid, and after laser-induced CNV.

Results

rs1878326 was associated with AMD in the French and German group. The Mfge8 promoter is highly active in photoreceptors but not in retinal pigment epithelium cells. Mfge8^−/− mice did not differ from controls in terms of fundus appearance, photoreceptor cell layers, choroidal architecture or laser-induced CNV. In contrast, the Bruch''s membrane (BM) was slightly but significantly thicker in Mfge8^−/− mice as compared to controls.

Conclusions

Despite a reproducible minor increase of rs1878326 in AMD patients and a very modest increase in BM in Mfge8^−/− mice, our data suggests that MFGE8 dysfunction does not play a critical role in the pathogenesis of AMD.     

Gene therapy in a humanized mouse model of familial hypercholesterolemia leads to marked regression of atherosclerosis

Background

Familial hypercholesterolemia (FH) is an autosomal codominant disorder caused by mutations in the low-density lipoprotein receptor (LDLR) gene. Homozygous FH patients (hoFH) have severe hypercholesterolemia leading to life threatening atherosclerosis in childhood and adolescence. Mice with germ line interruptions in the Ldlr and Apobec1 genes (Ldlr^−/−Apobec1^−/−) simulate metabolic and clinical aspects of hoFH, including atherogenesis on a chow diet.

Methods/Principal Findings

In this study, vectors based on adeno-associated virus 8 (AAV8) were used to deliver the gene for mouse Ldlr (mLDLR) to the livers of Ldlr^−/−Apobec1^−/− mice. A single intravenous injection of AAV8.mLDLR was found to significantly reduce plasma cholesterol and non-HDL cholesterol levels in chow-fed animals at doses as low as 3×10⁹ genome copies/mouse. Whereas Ldlr^−/−Apobec1^−/− mice fed a western-type diet and injected with a control AAV8.null vector experienced a further 65% progression in atherosclerosis over 2 months compared with baseline mice, Ldlr^−/−Apobec1^−/− mice treated with AAV8.mLDLR realized an 87% regression of atherosclerotic lesions after 3 months compared to baseline mice. Immunohistochemical analyses revealed a substantial remodeling of atherosclerotic lesions.

Conclusions/Significance

Collectively, the results presented herein suggest that AAV8-based gene therapy for FH may be feasible and support further development of this approach. The pre-clinical data from these studies will enable for the effective translation of gene therapy into the clinic for treatment of FH.     

LDL receptor knock-out mice are a physiological model particularly vulnerable to study the onset of inflammation in non-alcoholic fatty liver disease

Background & Aims

Non-alcoholic steatohepatitis (NASH) involves steatosis combined with inflammation, which can progress into fibrosis and cirrhosis. Exploring the molecular mechanisms of NASH is highly dependent on the availability of animal models. Currently, the most commonly used animal models for NASH imitate particularly late stages of human disease. Thus, there is a need for an animal model that can be used for investigating the factors that potentiate the inflammatory response within NASH. We have previously shown that 7-day high-fat-high-cholesterol (HFC) feeding induces steatosis and inflammation in both APOE2ki and Ldlr^−/− mice. However, it is not known whether the early inflammatory response observed in these mice will sustain over time and lead to liver damage. We hypothesized that the inflammatory response in both models is sufficient to induce liver damage over time.

Methods

APOE2ki and Ldlr^−/− mice were fed a chow or HFC diet for 3 months. C57Bl6/J mice were used as control.

Results

Surprisingly, hepatic inflammation was abolished in APOE2ki mice, while it was sustained in Ldlr^−/− mice. In addition, increased apoptosis and hepatic fibrosis was only demonstrated in Ldlr^−/− mice. Finally, bone-marrow-derived-macrophages of Ldlr^−/− mice showed an increased inflammatory response after oxidized LDL (oxLDL) loading compared to APOE2ki mice.

Conclusion

Ldlr^−/− mice, but not APOE2ki mice, developed sustained hepatic inflammation and liver damage upon long term HFC feeding due to increased sensitivity for oxLDL uptake. Therefore, the Ldlr^−/− mice are a promising physiological model particularly vulnerable for investigating the onset of hepatic inflammation in non-alcoholic steatohepatitis.     

Internalization of modified lipids by CD36 and SR-A leads to hepatic inflammation and lysosomal cholesterol storage in Kupffer cells

Background & Aims

Non-alcoholic steatohepatitis (NASH) is characterized by steatosis and inflammation, which can further progress into fibrosis and cirrhosis. Recently, we demonstrated that combined deletion of the two main scavenger receptors, CD36 and macrophage scavenger receptor 1 (MSR1), which are important for modified cholesterol-rich lipoprotein uptake, reduced NASH. The individual contributions of these receptors to NASH and the intracellular mechanisms by which they contribute to inflammation have not been established. We hypothesize that CD36 and MSR1 contribute independently to the onset of inflammation in NASH, by affecting intracellular cholesterol distribution inside Kupffer cells (KCs).

Methods & Results

Ldlr^−/− mice were transplanted with wild-type (Wt), Cd36^−/− or Msr1^−/− bone marrow and fed a Western diet for 3months. Cd36^−/−- and Msr1^−/−- transplanted (tp) mice showed a similar reduction in hepatic inflammation compared to Wt-tp mice. While the total amount of cholesterol inside KCs was similar in all groups, KCs of Cd36^−/−- and Msr1^−/−-tp mice showed increased cytoplasmic cholesterol accumulation, while Wt-tp mice showed increased lysosomal cholesterol accumulation.

Conclusion

CD36 and MSR1 contribute similarly and independently to the progression of inflammation in NASH. One possible explanation for the inflammatory response related to expression of these receptors could be abnormal cholesterol trafficking in KCs. These data provide a new basis for prevention and treatment of NASH.     

Glucose-dependent insulinotropic polypeptide prevents the progression of macrophage-driven atherosclerosis in diabetic apolipoprotein E-null mice

Aim

We recently reported that glucose-dependent insulinotropic polypeptide (GIP) prevents the development of atherosclerosis in apolipoprotein E-null (Apoe ^−/−) mice. GIP receptors (GIPRs) are found to be severely down-regulated in diabetic animals. We examined whether GIP can exert anti-atherogenic effects in diabetes.

Methods

Nondiabetic Apoe ^−/− mice, streptozotocin-induced diabetic Apoe ^−/− mice, and db/db mice were administered GIP (25 nmol/kg/day) or saline (vehicle) through osmotic mini-pumps for 4 weeks. The animals were assessed for aortic atherosclerosis and for oxidized low-density lipoprotein-induced foam cell formation in exudate peritoneal macrophages.

Results

Diabetic Apoe ^−/− mice of 21 weeks of age exhibited more advanced atherosclerosis than nondiabetic Apoe ^−/− mice of the same age. GIP infusion in diabetic Apoe ^−/− mice increased plasma total GIP levels by 4-fold without improving plasma insulin, glucose, or lipid profiles. GIP infusion significantly suppressed macrophage-driven atherosclerotic lesions, but this effect was abolished by co-infusions with [Pro³]GIP, a GIPR antagonist. Foam cell formation was stimulated by 3-fold in diabetic Apoe ^−/− mice compared with their nondiabetic counterparts, but this effect was halved by GIP infusion. GIP infusion also attenuated the foam cell formation in db/db mice. In vitro treatment with GIP (1 nM) reduced foam cell formation by 15% in macrophages from diabetic Apoe ^−/− mice, and this attenuating effect was weaker than that attained by the same treatment of macrophages from nondiabetic counterparts (35%). While GIPR expression was reduced by only about a half in macrophages from diabetic mice, it was reduced much more dramatically in pancreatic islets from the same animals. Incubation with high glucose (500 mg/dl) for 9–10 days markedly reduced GIPR expression in pancreatic islet cells, but not in macrophages.

Conclusions

Long-term infusion of GIP conferred significant anti-atherogenic effects in diabetic mice even though the GIPR expression in macrophages was mildly down-regulated in the diabetic state.     

CO2 efflux from cleared mangrove peat

Background

CO₂ emissions from cleared mangrove areas may be substantial, increasing the costs of continued losses of these ecosystems, particularly in mangroves that have highly organic soils.

Methodology/Principal Findings

We measured CO₂ efflux from mangrove soils that had been cleared for up to 20 years on the islands of Twin Cays, Belize. We also disturbed these cleared peat soils to assess what disturbance of soils after clearing may have on CO₂ efflux. CO₂ efflux from soils declines from time of clearing from ∼10 600 tonnes km⁻² year⁻¹ in the first year to 3000 tonnes km² year⁻¹ after 20 years since clearing. Disturbing peat leads to short term increases in CO₂ efflux (27 umol m⁻² s⁻¹), but this had returned to baseline levels within 2 days.

Conclusions/Significance

Deforesting mangroves that grow on peat soils results in CO₂ emissions that are comparable to rates estimated for peat collapse in other tropical ecosystems. Preventing deforestation presents an opportunity for countries to benefit from carbon payments for preservation of threatened carbon stocks.     

In the absence of Sonic hedgehog, p53 induces apoptosis and inhibits retinal cell proliferation, cell-cycle exit and differentiation in zebrafish

Background

Sonic hedgehog (Shh) signaling regulates cell proliferation during vertebrate development via induction of cell-cycle regulator gene expression or activation of other signalling pathways, prevents cell death by an as yet unclear mechanism and is required for differentiation of retinal cell types. Thus, an unsolved question is how the same signalling molecule can regulate such distinct cell processes as proliferation, cell survival and differentiation.

Methodology/Principal Findings

Analysis of the zebrafish shh ^−/− mutant revealed that in this context p53 mediates elevated apoptosis during nervous system and retina development and interferes with retinal proliferation and differentiation. While in shh ^−/− mutants there is activation of p53 target genes and p53-mediated apoptosis, an increase in Hedgehog (Hh) signalling by over-expression of dominant-negative Protein Kinase A strongly decreased p53 target gene expression and apoptosis levels in shh ^−/− mutants. Using a novel p53 reporter transgene, I confirm that p53 is active in tissues that require Shh for cell survival. Proliferation assays revealed that loss of p53 can rescue normal cell-cycle exit and the mitotic indices in the shh ^−/− mutant retina at 24, 36 and 48 hpf. Moreover, generation of amacrine cells and photoreceptors was strongly enhanced in the double p53 ^−/− shh ^−/− mutant retina suggesting the effect of p53 on retinal differentiation.

Conclusions

Loss of Shh signalling leads to the p53-dependent apoptosis in the developing nervous system and retina. Moreover, Shh-mediated control of p53 activity is required for proliferation and cell cycle exit of retinal cells as well as differentiation of amacrine cells and photoreceptors.     

Targeted deletion of neuropeptide Y (NPY) modulates experimental colitis

Background

Neurogenic inflammation plays a major role in the pathogenesis of inflammatory bowel disease (IBD). We examined the role of neuropeptide Y (NPY) and neuronal nitric oxide synthase (nNOS) in modulating colitis.

Methods

Colitis was induced by administration of dextran sodium sulphate (3% DSS) or streptomycin pre-treated Salmonella typhimurium (S.T.) in wild type (WT) and NPY (NPY^−/−) knockout mice. Colitis was assessed by clinical score, histological score and myeloperoxidase activity. NPY and nNOS expression was assessed by immunostaining. Oxidative stress was assessed by measuring catalase activity, glutathione and nitrite levels. Colonic motility was assessed by isometric muscle recording in WT and DSS-treated mice.

Results

DSS/S.T. induced an increase in enteric neuronal NPY and nNOS expression in WT mice. WT mice were more susceptible to inflammation compared to NPY^−/− as indicated by higher clinical & histological scores, and myeloperoxidase (MPO) activity (p<0.01). DSS-WT mice had increased nitrite, decreased glutathione (GSH) levels and increased catalase activity indicating more oxidative stress. The lower histological scores, MPO and chemokine KC in S.T.-treated nNOS^−/− and NPY^−/−/nNOS^−/− mice supported the finding that loss of NPY-induced nNOS attenuated inflammation. The inflammation resulted in chronic impairment of colonic motility in DSS-WT mice. NPY –treated rat enteric neurons in vitro exhibited increased nitrite and TNF-α production.

Conclusions

NPY mediated increase in nNOS is a determinant of oxidative stress and subsequent inflammation. Our study highlights the role of neuronal NPY and nNOS as mediators of inflammatory processes in IBD.     

Is the simple auger coring method reliable for below-ground standing biomass estimation in Eucalyptus forest plantations?

Background and Aims

Despite their importance for plant production, estimations of below-ground biomass and its distribution in the soil are still difficult and time consuming, and no single reliable methodology is available for different root types. To identify the best method for root biomass estimations, four different methods, with labour requirements, were tested at the same location.

Methods

The four methods, applied in a 6-year-old Eucalyptus plantation in Congo, were based on different soil sampling volumes: auger (8 cm in diameter), monolith (25 × 25 cm quadrate), half Voronoi trench (1·5 m³) and a full Voronoi trench (3 m³), chosen as the reference method.

Key Results

With the reference method (0–1m deep), fine-root biomass (FRB, diameter <2 mm) was estimated at 1·8 t ha⁻¹, medium-root biomass (MRB diameter 2–10 mm) at 2·0 t ha⁻¹, coarse-root biomass (CRB, diameter >10 mm) at 5·6 t ha⁻¹ and stump biomass at 6·8 t ha⁻¹. Total below-ground biomass was estimated at 16·2 t ha⁻¹ (root : shoot ratio equal to 0·23) for this 800 tree ha⁻¹ eucalypt plantation density. The density of FRB was very high (0·56 t ha⁻¹) in the top soil horizon (0–3 cm layer) and decreased greatly (0·3 t ha⁻¹) with depth (50–100 cm). Without labour requirement considerations, no significant differences were found between the four methods for FRB and MRB; however, CRB was better estimated by the half and full Voronoi trenches. When labour requirements were considered, the most effective method was auger coring for FRB, whereas the half and full Voronoi trenches were the most appropriate methods for MRB and CRB, respectively.

Conclusions

As CRB combined with stumps amounted to 78 % of total below-ground biomass, a full Voronoi trench is strongly recommended when estimating total standing root biomass. Conversely, for FRB estimation, auger coring is recommended with a design pattern accounting for the spatial variability of fine-root distribution.     

A comprehensive evaluation of potential lung function associated genes in the SpiroMeta general population sample

Rationale

Lung function measures are heritable traits that predict population morbidity and mortality and are essential for the diagnosis of chronic obstructive pulmonary disease (COPD). Variations in many genes have been reported to affect these traits, but attempts at replication have provided conflicting results. Recently, we undertook a meta-analysis of Genome Wide Association Study (GWAS) results for lung function measures in 20,288 individuals from the general population (the SpiroMeta consortium).

Objectives

To comprehensively analyse previously reported genetic associations with lung function measures, and to investigate whether single nucleotide polymorphisms (SNPs) in these genomic regions are associated with lung function in a large population sample.

Methods

We analysed association for SNPs tagging 130 genes and 48 intergenic regions (+/−10 kb), after conducting a systematic review of the literature in the PubMed database for genetic association studies reporting lung function associations.

Results

The analysis included 16,936 genotyped and imputed SNPs. No loci showed overall significant association for FEV₁ or FEV₁/FVC traits using a carefully defined significance threshold of 1.3×10⁻⁵. The most significant loci associated with FEV₁ include SNPs tagging MACROD2 (P = 6.81×10⁻⁵), CNTN5 (P = 4.37×10⁻⁴), and TRPV4 (P = 1.58×10⁻³). Among ever-smokers, SERPINA1 showed the most significant association with FEV₁ (P = 8.41×10⁻⁵), followed by PDE4D (P = 1.22×10⁻⁴). The strongest association with FEV₁/FVC ratio was observed with ABCC1 (P = 4.38×10⁻⁴), and ESR1 (P = 5.42×10⁻⁴) among ever-smokers.

Conclusions

Polymorphisms spanning previously associated lung function genes did not show strong evidence for association with lung function measures in the SpiroMeta consortium population. Common SERPINA1 polymorphisms may affect FEV₁ among smokers in the general population.