The Chloroplast-Localized Phospholipases D a4 and a5 Regulate Herbivore-Induced Direct and Indirect Defenses in Rice

The oxylipin pathway is of central importance for plant defensive responses. Yet, the first step of the pathway, the liberation of linolenic acid following induction, is poorly understood. Phospholipases D (PLDs) have been hypothesized to mediate this process, but data from Arabidopsis (Arabidopsis thaliana) regarding the role of PLDs in plant resistance have remained controversial. Here, we cloned two chloroplast-localized PLD genes from rice (Oryza sativa), OsPLDα4 and OsPLDα5, both of which were up-regulated in response to feeding by the rice striped stem borer (SSB) Chilo suppressalis, mechanical wounding, and treatment with jasmonic acid (JA). Antisense expression of OsPLDα4 and -α5 (as-pld), which resulted in a 50% reduction of the expression of the two genes, reduced elicited levels of linolenic acid, JA, green leaf volatiles, and ethylene and attenuated the SSB-induced expression of a mitogen-activated protein kinase (OsMPK3), a lipoxygenase (OsHI-LOX), a hydroperoxide lyase (OsHPL3), as well as a 1-aminocyclopropane-1-carboxylic acid synthase (OsACS2). The impaired oxylipin and ethylene signaling in as-pld plants decreased the levels of herbivore-induced trypsin protease inhibitors and volatiles, improved the performance of SSB and the rice brown planthopper Nilaparvata lugens, and reduced the attractiveness of plants to a larval parasitoid of SSB, Apanteles chilonis. The production of trypsin protease inhibitors in as-pld plants could be partially restored by JA, while the resistance to rice brown planthopper and SSB was restored by green leaf volatile application. Our results show that phospholipases function as important components of herbivore-induced direct and indirect defenses in rice.

Canopy cover mediates interactions between a specialist caterpillar and seedlings of a neotropical tree

1 Light availability may be crucial for understanding dynamics of plant–herbivore interactions in temperate and tropical forest communities. This is because local light availability can influence both tree seedling tolerance and susceptibility to herbivory – yet how they mediate levels of insect herbivory that vary with the density of host population is virtually unknown. Here we tested predictions of three key, non-mutually exclusive hypotheses of plant–herbivore interactions: the Limiting Resource Model (LRM), the Plant Vigour Hypothesis (PVH), and the Janzen-Connell Mechanism (JCM). 2 In an Amazonian forest, we planted Swietenia macrophylla seedlings (c. 5 months old) into natural canopy gaps and the shaded understorey and simulated the damage patterns of the specialist herbivore moth, Steniscadia poliophaea, by clipping seedling leaves. Over the next 8 months, we monitored seedling performance in terms of growth and survivorship and also quantified herbivory to new young leaves on a seasonal basis. 3 In support of the LRM, severe leaf damage (≥ 50%) was lethal for Swietenia macrophylla seedlings in the understorey, but in gaps only reduced seedling growth. In support of the PVH, gap seedlings suffered greater post-simulated herbivory (up to 100% defoliation) by S. poliophaea caterpillars than their understorey counterparts. 4 Adding a novel dimension to the Janzen–Connell hypothesis, we found that early wet season herbivory of seedlings in gaps increased with conspecific adult density within a 125-m radius; whereas in the understorey only those seedlings within 50 m of a Swietenia tree were attacked by caterpillars. 5 Synthesis. These results suggest lepidopterans that need young leaves for food may forage more widely in forests to find seedlings in light-rich canopy gaps. Moths may achieve this successfully by being first attracted to gaps, and then searching within them for suitable hosts. A conceptual model, integrating conspecific adult tree density with light-driven changes in seedling tolerance/vigour and their susceptibility to herbivory and mortality, is presented. Spatial variation in the light available to tree seedlings often affects their tolerance and vigour, which may have important consequences for leaf-chewing insects and the scale of density-dependent herbivory in forests.

Intervertebral Disc Repair using Fleece-Membrane Composite Silk Material and Genipin-enhanced Fibrin Hydrogel

Introduction: Treating low back pain (LBP) has become an increasing challenge, as it is one of the main factors causing pain and is accompanied by high costs for the individual and the society. LBP can be caused by trauma of the intervertebral disc (IVD) or IVD degeneration. In the case of disc herniation the inner gelatinous part of the IVD, called nucleus pulposus, is pressed through the fibrous, annulus fibrosus that forms the outer part of the IVD. Today’s gold standard for treatment is extensive surgery as removal of the IVD and fusion of the vertebrae. In order to find a more gentle way to treat LBP and restore the native IVD we use a novel silk fleece-membrane composite from genetically modified silk worms whose silk contains a growth factor (GDF-6) that is associated with pushing stem cells towards a disc like phenotype (1). By combining it with a genipin-enhanced fibrin hydrogel we tested its suitability in organ culture on prior injured bovine IVD in our custom built two-degree of freedom bioreactor to mimic natural loading conditions. Material & Methods: Bovine IVDs of 12-17 months old animals were isolated by first removing all surrounding tissue followed by cutting out the IVDs as previously described (2). Culturing of discs occurred in high glucose Dulbecco's Modified Eagle Medium (HG-DMEM) supplemented with 5% serum as previously described (2). On the next day injury was induced using a 2mm biopsy punch (Polymed, Switzerland). The formed cavity was filled with (0.4%) genipin-enhanced human based fibrin hydrogel (35-55mg/mL human fibrinogen, Baxter, Austria) and sealed with a silk fleece-membrane composite (Spintec Engineering, Germany). Different culture conditions were applied: free swelling, static diurnal load of 0.2MPa for 8h/d and complex loading at 0.2MPa compression combined with ± 2° torsion at 0.2Hz for 8h/d (2). After 14 days of culture cell activity was determined with resazurin assay. Additionally, glycosaminoglycan (dimethyl-methylene blue), DNA (Hoechst) and collagen content (hydroxy- proline) were determined. Finally, real-time qPCR of major IVD marker and inflammation genes was performed to judge integrity of IVDs. Results: The fibrin hydrogel is able to keep the silk seal in place throughout the 14 days of in organ culture under all conditions. Additionally, cell activity showed optimistic results and we could not confirm negative effects of the repaired discs regarding overexpression of inflammation markers. Conclusions: The genipin-enhanced fibrin hydrogel in combination with the silk fleece- membrane composite seems to be a promising approach for IVD repair. Currently we assess the capability of GDF-6 incorporated in our silk composites on human mesenchymal stem cells and later on in organ culture. References 1. Clarke LE, McConnell JC, Sherratt MJ, Derby B, Richardson SM, Hoyland JA. Growth differentiation factor 6 and transforming growth factor-beta differentially mediate mesenchymal stem cell differentiation, composition and micromechanical properties of nucleus pulposus constructs. Arthritis Res Ther 2014, Mar 12;16(2):R67. 2. Chan SC, Gantenbein-Ritter B. Preparation of intact bovine tail intervertebral discs for organ culture. J Vis Exp 2012, Feb 2;60(60):e3490. Acknowledgements. This work is funded by the Gebert Rüf Foundation, project number GRS-028/13.

Independent at heart: persistent association of altitude with ischaemic heart disease mortality after consideration of climate, topography and built environment.

BACKGROUND Living at higher altitude was dose-dependently associated with lower risk of ischaemic heart disease (IHD). Higher altitudes have different climatic, topographic and built environment properties than lowland regions. It is unclear whether these environmental factors mediate/confound the association between altitude and IHD. We examined how much of the altitude-IHD association is explained by variations in exposure at place of residence to sunshine, temperature, precipitation, aspect, slope and distance to main road. METHODS We included 4.2 million individuals aged 40-84 at baseline living in Switzerland at altitudes 195-2971 m above sea level (ie, full range of residence), providing 77 127 IHD deaths. Mortality data 2000-2008, sociodemographic/economic information and coordinates of residence were obtained from the Swiss National Cohort, a longitudinal, census-based record linkage study. Environment information was modelled to residence level using Weibull regression models. RESULTS In the model not adjusted for other environmental factors, IHD mortality linearly decreased with increasing altitude resulting in a lower risk (HR, 95% CI 0.67, 0.60 to 0.74) for those living >1500 m (vs<600 m). This association remained after adjustment for all other environmental factors 0.74 (0.66 to 0.82). CONCLUSIONS The benefit of living at higher altitude was only partially confounded by variations in climate, topography and built environment. Rather, physical environment factors appear to have an independent effect and may impact on cardiovascular health in a cumulative way. Inclusion of additional modifiable factors as well as individual information on traditional IHD risk factors in our combined environmental model could help to identify strategies for the reduction of inequalities in IHD mortality.

Role of electrostatic interactions for ligand recognition and specificity of peptide transporters.

BACKGROUND Peptide transporters are membrane proteins that mediate the cellular uptake of di- and tripeptides, and of peptidomimetic drugs such as β-lactam antibiotics, antiviral drugs and antineoplastic agents. In spite of their high physiological and pharmaceutical importance, the molecular recognition by these transporters of the amino acid side chains of short peptides and thus the mechanisms for substrate binding and specificity are far from being understood. RESULTS The X-ray crystal structure of the peptide transporter YePEPT from the bacterium Yersinia enterocolitica together with functional studies have unveiled the molecular bases for recognition, binding and specificity of dipeptides with a charged amino acid residue at the N-terminal position. In wild-type YePEPT, the significant specificity for the dipeptides Asp-Ala and Glu-Ala is defined by electrostatic interaction between the in the structure identified positively charged Lys314 and the negatively charged amino acid side chain of these dipeptides. Mutagenesis of Lys314 into the negatively charged residue Glu allowed tuning of the substrate specificity of YePEPT for the positively charged dipeptide Lys-Ala. Importantly, molecular insights acquired from the prokaryotic peptide transporter YePEPT combined with mutagenesis and functional uptake studies with human PEPT1 expressed in Xenopus oocytes also allowed tuning of human PEPT1's substrate specificity, thus improving our understanding of substrate recognition and specificity of this physiologically and pharmaceutically important peptide transporter. CONCLUSION This study provides the molecular bases for recognition, binding and specificity of peptide transporters for dipeptides with a charged amino acid residue at the N-terminal position.

Brain-borne IL-1 adjusts glucoregulation and provides fuel support to astrocytes and neurons in an autocrine/paracrine manner.

It is still controversial which mediators regulate energy provision to activated neural cells, as insulin does in peripheral tissues. Interleukin-1β (IL-1β) may mediate this effect as it can affect glucoregulation, it is overexpressed in the 'healthy' brain during increased neuronal activity, and it supports high-energy demanding processes such as long-term potentiation, memory and learning. Furthermore, the absence of sustained neuroendocrine and behavioral counterregulation suggests that brain glucose-sensing neurons do not perceive IL-1β-induced hypoglycemia. Here, we show that IL-1β adjusts glucoregulation by inducing its own production in the brain, and that IL-1β-induced hypoglycemia is myeloid differentiation primary response 88 protein (MyD88)-dependent and only partially counteracted by Kir6.2-mediated sensing signaling. Furthermore, we found that, opposite to insulin, IL-1β stimulates brain metabolism. This effect is absent in MyD88-deficient mice, which have neurobehavioral alterations associated to disorders in glucose homeostasis, as during several psychiatric diseases. IL-1β effects on brain metabolism are most likely maintained by IL-1β auto-induction and may reflect a compensatory increase in fuel supply to neural cells. We explore this possibility by directly blocking IL-1 receptors in neural cells. The results showed that, in an activity-dependent and paracrine/autocrine manner, endogenous IL-1 produced by neurons and astrocytes facilitates glucose uptake by these cells. This effect is exacerbated following glutamatergic stimulation and can be passively transferred between cell types. We conclude that the capacity of IL-1β to provide fuel to neural cells underlies its physiological effects on glucoregulation, synaptic plasticity, learning and memory. However, deregulation of IL-1β production could contribute to the alterations in brain glucose metabolism that are detected in several neurologic and psychiatric diseases.Molecular Psychiatry advance online publication, 8 December 2015; doi:10.1038/mp.2015.174.

Cellular and molecular effects of the mTOR inhibitor everolimus.

mTOR (mechanistic target of rapamycin) functions as the central regulator for cell proliferation, growth and survival. Up-regulation of proteins regulating mTOR, as well as its downstream targets, has been reported in various cancers. This has promoted the development of anti-cancer therapies targeting mTOR, namely fungal macrolide rapamycin, a naturally occurring mTOR inhibitor, and its analogues (rapalogues). One such rapalogue, everolimus, has been approved in the clinical treatment of renal and breast cancers. Although results have demonstrated that these mTOR inhibitors are effective in attenuating cell growth of cancer cells under in vitro and in vivo conditions, subsequent sporadic response to rapalogues therapy in clinical trials has promoted researchers to look further into the complex understanding of the dynamics of mTOR regulation in the tumour environment. Limitations of these rapalogues include the sensitivity of tumour subsets to mTOR inhibition. Additionally, it is well known that rapamycin and its rapalogues mediate their effects by inhibiting mTORC (mTOR complex) 1, with limited or no effect on mTORC2 activity. The present review summarizes the pre-clinical, clinical and recent discoveries, with emphasis on the cellular and molecular effects of everolimus in cancer therapy.

A fungal endophyte helps plants to tolerate root herbivory through changes in gibberellin and jasmonate signaling

Plant–microbe mutualisms can improve plant defense, but the impact of root endophytes on below-ground herbivore interactions remains unknown. We investigated the effects of the root endophyte Piriformospora indica on interactions between rice (Oryza sativa) plants and its root herbivore rice water weevil (RWW; Lissorhoptrus oryzophilus), and how plant jasmonic acid (JA) and GA regulate this tripartite interaction. Glasshouse experiments with wild-type rice and coi1-18 and Eui1-OX mutants combined with nutrient, jasmonate and gene expression analyses were used to test: whether RWW adult herbivory above ground influences subsequent damage caused by larval herbivory below ground; whether P. indica protects plants against RWW; and whether GA and JA signaling mediate these interactions. The endophyte induced plant tolerance to root herbivory. RWW adults and larvae acted synergistically via JA signaling to reduce root growth, while endophyte-elicited GA biosynthesis suppressed the herbivore-induced JA in roots and recovered plant growth. Our study shows for the first time the impact of a root endophyte on plant defense against below-ground herbivores, adds to growing evidence that induced tolerance may be an important root defense, and implicates GA as a signal component of inducible plant tolerance against biotic stress.

Expression and processing of Plasmodium berghei SERA3 during liver stages.

Cysteine proteases mediate liberation of Plasmodium berghei merozoites from infected hepatocytes. In an attempt to identify the responsible parasite proteases, we screened the genome of P. berghei for cysteine protease-encoding genes. RT-PCR analyses revealed that transcription of four out of five P. berghei serine repeat antigen (PbSERA) genes was strongly upregulated in late liver stages briefly before the parasitophorous vacuole membrane ruptured to release merozoites into the host cell cytoplasm, suggesting a role of PbSERA proteases in these processes. In order to characterize PbSERA3 processing, we raised an antiserum against a non-conserved region of the protein and generated a transgenic P. berghei strain expressing a TAP-tagged PbSERA3 under the control of the endogenous promoter. Immunofluorescence assays revealed that PbSERA3 leaks into the host cell cytoplasm during merozoite development, where it might contribute to host cell death or activate host cell proteases that execute cell death. Importantly, processed PbSERA3 has been detected by Western blot analysis in cell extracts of schizont-infected cells and merozoite-infected detached hepatic cells.